Nanotechnology Based Approaches for Tuberculosis Treatment [1 ed.] 0128198117, 9780128198117

Table of contents :
Chapter 1 - Pathogenesis, biology, and immunology of tuberculosis
1 - Introduction
2 - Mycobacterium tuberculosis and its transmission
2.1 - Scientific classification [11]
2.2 - Transmission
3 - Factors responsible for its transmission
3.1 - Patient-related risk factor
3.1.1 - Proximate risk factor
3.1.2 - Links between proximate risk factors and socioeconomic status
3.1.3 - Role of urbanization
3.2 - Bacteriological factors
3.2.1 - Case Study 1
3.2.2 - Case Study 2
4 - Pathogenesis
4.1 - Survival mechanisms of Mycobacterium tuberculosis
4.1.1 - The uniqueness in cell wall structure
4.1.2 - Advantages due to presence of lipid at the cell wall
4.1.3 - Intercellular growth
4.1.4 - Function of phagocytic cells
4.1.5 - Stage of granuloma
4.1.6 - Inhibition of phagosome-lysosome fusion
4.1.7 - Role of eukaryotic like protein kinases in mycobacterial cell
4.1.8 - Seizing of calcineurin pathway
4.1.9 - The after stages of macrophage activation
4.1.9.1 - Role of cytokines
4.1.9.2 - Role of TLR ligands
4.1.9.3 - Role of mycobacterial glycolipids
4.1.9.4 - Modulation of bacterial metabolic pathway
4.1.9.5 - Transfer of protein
5 - Mycobacterium tuberculosis capsule
5.1 - Cellular structure of Mycobacterium tuberculosis capsule
5.2 - Host-pathogen interaction from the capsule point of view
5.3 - Pharmaceutical methodology to target capsule
6 - Immunology—Introduction
6.1 - Immunology of tuberculosis
6.1.1 - Immunology of upper respiratory track
6.2 - Innate immune system
6.2.1 - The Mycobacterial cell wall
6.2.2 - Innate immune recognition of Mycobacterium tuberculosis
6.2.3 - Bacterial factors that evade these innate immune responses
6.2.4 - Other mechanisms of recognition of Mycobacterium tuberculosis by innate immune system
6.2.5 - Effector functions of macrophages against engulfed Mycobacterium tuberculosis
6.3 - Inflammatory responses
6.4 - Adaptive immunity
6.5 - Granuloma formation
6.6 - Conclusion
References
Chapter 2 - Tuberculosis: introduction, drug regimens, and multidrug-resistance
1 - Introduction
2 - Drug regimens for the treatment of tuberculosis
3 - First line drugs for tuberculosis
4 - Isoniazid
5 - Rifampin
6 - Pyrazinamide
7 - Ethambutol
8 - Streptomycin
9 - Second-line antituberculosis drugs
10 - Mechanisms of drug resistance
11 - Conclusions
References
Chapter 3 - Nanotechnology as a potential tool against drug- and multidrug-resistant tuberculosis
1 - Tuberculosis as an infectious disease
2 - Nanotechnology-based systems and the administration of drugs against tuberculosis
2.1 - Solid-lipid forms
2.1.1 - Solid–lipid microparticles (SLM)
2.1.2 - Solid–lipid nanoparticles
2.1.3 - Nanostructured lipid carrier
2.2 - Emulsion-based systems
2.2.1 - Microemulsion (ME)
2.2.2 - Nanoemulsions (NE)
2.3 - Vesicular drug-delivery systems
2.3.1 - Liposomes (LPS)
2.3.2 - Niosomes (NIOs)
2.3.3 - Lipospheres (LIPs)
2.4 - Miscellaneous NPs
2.4.1 - Dendrimers
2.4.2 - Nano/micro particles (NMPs)
2.4.3 - Microspheres
2.4.4 - Carbon nanotubes (CNTBs)
2.4.5 - Nanosuspension (NSP)
2.4.6 - Nanomicelles (NMCs)
2.4.7 - Polymersomes
2.4.7.1 - Implications of nanotechnology in MDR-TB and XDR-TB treatment
3 - Factors affecting NPs properties
3.1 - Potential benefits and risks in the use of NPs
3.1.1 - Final considerations
References
Chapter 4 - Translational research for therapy against tuberculosis
1 - Research for tuberculosis elimination
2 - Advances in the therapy for tuberculosis
3 - New drugs for tuberculosis or new regimens
3.1 - The issues
3.2 - Recent advances
3.3 - Future challenges
4 - Drugs repurposed for tuberculosis
4.1 - The issues
4.2 - Recent advances
4.3 - Future challenges
5 - Host-directed therapy for tuberculosis
5.1 - The issues
5.2 - Recent advances
5.3 - Future challenges
6 - Tuberculosis research and care biomarkers. The OMICs of tuberculosis
6.1 - Genomics
6.2 - Transcriptomics
6.3 - Proteomics
6.4 - Metabolomics
7 - The impasse of translational medicine in tuberculosis and future challenges
References
Chapter 5 - Vaccine delivery systems against tuberculosis
1 - Introduction
2 - TB vaccine candidates in the pipeline
2.1 - Viral vectored TB vaccines
2.2 - Adjuvanted subunit TB vaccine
2.3 - DNA TB vaccine
2.4 - Whole-cell and live Mycobacteria TB vaccine
3 - Vaccine administration routes for TB vaccine
3.1 - Intradermal route of administration
3.2 - The intramuscular route of administration
3.3 - Subcutaneous route of administration
3.4 - Intranasal (mucosal, sublingual) route of administration-
4 - Advanced TB vaccine delivery systems and their related immune responses
4.1 - Nanoparticles-based TB vaccine delivery systems
4.2 - Cationic nanoparticle-based TB vaccine delivery
4.3 - Chitosan-based nanoparticle TB vaccine delivery
4.4 - Polymeric/polyester-based nanoparticle as a TB vaccine delivery system
4.5 - Liposome-based TB vaccine delivery
4.6 - Dendrimer-based TB vaccine delivery system
4.7 - Immune stimulating complexes (ISCOMs) as a TB vaccine delivery system
4.8 - Virus-like particles (VLPs)-based TB vaccine delivery system
4.9 - Virosomes-based TB vaccine delivery system
4.10 - Role of adjuvants in TB vaccine formulation and their delivery
References
Chapter 6 - Inhalable polymeric dry powders for antituberculosis drug delivery
Abbreviations
1 - Introduction
2 - Challenges with current anti-TB therapies
3 - Rationale of pulmonary drug delivery in TB
4 - Feasibility of lung as a portal for delivery of ATD
5 - Pulmonary delivery of ATD
6 - Formulations for DPIs
7 - Drug carriers for pulmonary delivery
7.1 - Polymeric nanoparticles
7.2 - Hybrid nano-in-microparticles
7.3 - Solid-lipid nano particles
7.4 - Liposomes
7.5 - Microparticles
8 - Inhalation delivery devices for DPI
9 - Clinical trials
10 - Future of polymeric powder-based drug development for TB
11 - Conclusions
References
Chapter 7 - Liposomes-and niosomes-based drug delivery systems for tuberculosis treatment
1 - Introduction
2 - Epidemiology
3 - Nature of causative agent
4 - Emergence of MDR and XDR TB
5 - Drug regimens
6 - Need for novel and sustained delivery systems
7 - Nanodelivery systems
7.1 - Introduction
7.2 - Types of nanocarriers
7.3 - Advantages of nanotechnology-based drug delivery system
8 - Liposomes
8.1 - Definition of liposomes
8.2 - Types and uses of liposomes
8.3 - Pulmonary TB and the importance of liposomal drugs
8.4 - Si-RNA liposomes
8.5 - Targeting of liposomes
9 - Niosomes
9.1 - Definition of niosomes
9.2 - Advantages of niosomes
9.3 - Various types of niosomes
9.4 - Niosomes versus liposomes; which is superior?
9.5 - Application of niosomes in drugs
9.6 - Niosomes in the treatment of TB
9.7 - Niosomal drug delivery system role in cerebral, drug-resistant TB
10 - Pulmonary delivery of nanoparticle encapsulated antitubercular drugs
11 - The future of combating TB
References
Chapter 8 - Polymer-based nanoparticles as delivery systems for treatment and vaccination of tuberculosis
1 - Polymer-based nanoparticles as drug delivery systems of tuberculosis
1.1 - Nanocarriers based on natural polymers
1.1.1 - Polysaccharide-based carriers
1.1.1.1 - Chitosan-based carriers
1.1.1.2 - Alginate-based carriers
1.1.1.3 - Guar gum-based carriers
1.1.2 - Polypeptide and protein-based carriers
1.1.2.1 - Gelatin-based carriers
1.1.2.2 - Albumin-based carriers
1.2 - Nanocarriers based on synthetic polymers
1.2.1 - PLGA-based nanocarriers
2 - Nanoparticle-based delivery systems for vaccination against tuberculosis
2.1 - Tuberculosis vaccines
2.1.1 - BCG
2.1.2 - Preexposure vaccines
2.1.3 - Postexposure vaccines
2.1.4 - Therapeutic vaccines
2.1.5 - Current vaccines weaknesses
2.2 - Adjuvants
2.3 - Vaccine delivery systems
2.3.1 - Natural polymer-based nanoparticles
2.3.2 - Biodegradable synthetic polymer-based nanoparticles
2.3.3 - Nonbiodegradable synthetic polymers
2.4 - The future challenges
References
Chapter 9 - Nanotechnology-based approaches for tuberculosis treatment
1 - Drug delivery systems
2 - Tuberculosis: the need for antitubercular drug delivery systems
3 - Nanomedicine and tuberculosis
4 - Oral ATD-nanomedicine
5 - Ligand-appended oral ATD-nanomedicine
6 - Pulmonary delivery of ATD-nanomedicine
7 - Injectable ATD-nanomedicine
8 - Alginate-based ATD-nanomedicine
9 - Lipid-based ATD-nanomedicine
9.1 - Liposome-based drug delivery systems
9.2 - Microemulsions as potential ATD delivery systems
9.3 - Niosomes-based ATD delivery system
9.4 - Solid lipid nanoparticles-based ATD-nanomedicine
10 - ATD-nanomedicine for special situations: cerebral TB, drug-resistant TB, and latent TB
11 - Potential toxicity of ATD-nanomedicine
12 - ATD-nanomedicine: unresolved and upcoming issues
13 - Conflict of interest
References
Chapter 10 - Dendrimer-based drug delivery systems for tuberculosis treatment
1 - Introduction
2 - Dendrimers
3 - PAMAM dendrimers for tuberculosis treatment
4 - PPI dendrimers for tuberculosis treatment
5 - Melamine, PEHAM, and PEA dendrimers for tuberculosis treatment
6 - Conclusion
References
Chapter 11 - Polymeric micelle-based drug delivery systems for tuberculosis treatment
1 - Introduction
2 - The structure of polymeric micelle
2.1 - Corona of miceller structure
2.2 - Core of miceller structure
3 - Commonly used polymers in polymeric micelle
3.1 - Commonly used amphiphilic block copolymers
4 - Polymeric micelles for enhanced permeability and retention effect
4.1 - Factors affecting EPR of miceller deliveries
4.1.1 - Bradykinin and permeability of cells
4.1.2 - Free radicals in cell permeability
4.1.3 - Permeability and prostaglandin with other factors
5 - Tuberculosis and urge of novel delivery approaches
6 - Recent advances of polymeric micelles in tuberculosis
7 - Conclusion
Acknowledgment
Conflict of interest
References
Chapter 12 - Nanostructured lipid carrier-based drug delivery systems for tuberculosis treatment
1 - Methods of production
1.1 - Hot homogenization method
1.2 - Cold homogenization method
2 - Excipients used in lipid nanocarriers
2.1 - Lipids
2.2 - Emulsifiers
3 - Challenges for current tuberculosis therapy
4 - Lipid nanoparticles for TB treatment
5 - Solid lipid nanoparticles and nanostructured lipid carriers as alternative nanomedicines for TB treatment
6 - Lipid nanoparticles functionalization
References
Chapter 13 - DNA markers and nano-biosensing approaches for tuberculosis diagnosis
List of abbreviations
1 - Introduction
2 - DNA structure
3 - Carbonaceous nanomaterials-based DNA biosensors
3.1 - Graphene derivatives
3.2 - Carbon nanotubes
4 - Nanoparticles based-DNA biosensors
4.1 - Noble metal nanoparticles
4.2 - Metal oxide nanoparticles
4.3 - Magnetic beads
4.4 - Quantum dots
5 - Conclusion
References
Chapter 14 - Recent advancement and future perspective for the treatment of multidrug-resistant tuberculosis
1 - Multidrug-resistant tuberculosis (MDR-TB): the emergence of new global threat to tuberculosis (TB) eradication
2 - The progression of treatment guidelines for MDR-TB: the past and present
3 - The advancement in MDR-TB treatment: the recent, on-going, and future direction
3.1 - The promising new all-oral drugs versus the extensively use injectable drugs
3.1.1 - Bedaquiline
3.1.2 - Delamanid
3.1.3 - Pretomanid—the newly approved oral drugs for the treatment of highly resistant TB
3.2 - Other ongoing trials for new treatment regimen for MDR-TB/XDR-TB
4 - Host-directed therapies as a future option in treating MDR-TB
5 - Conclusion
References
Chapter 15 -
Nanotechnology approach in conquering anti-TB resistance
Abbreviations
1 - Mycobacterium: pathogenesis and its problem in the resistant
2 - Antituberculosis and the mechanism of antituberculosis resistant
3 - Nanoparticle and its use to conquer tuberculosis infection
4 - Function nanoparticle for overcoming resistance tuberculosis treatment
5 - Nanoparticle for diagnose tuberculosis
References

Citation preview

NANOTECHNOLOGY BASED APPROACHES FOR TUBERCULOSIS TREATMENT Edited by

Prashant Kesharwani Assistant Professor & Ramanujan Fellow, Department of Pharmaceutics, School of Pharmaceutical Education and Research Jamia Hamdard (Hamdard University) New Delhi, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-819811-7 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisitions Editor: Erin Hill-Parks Editorial Project Manager: Pat Gonzalez Production Project Manager: Maria Bernard Designer: Miles Hitchen Typeset by Thomson Digital

Contributors

Suneera Adlakha  Institute of Nano Science and Technology (INST), Mohali, Punjab, India

Ashish Kumar Garg  Akal College of Pharmacy and Technical Education, Mastuana Sahib Barnala Road, Mastuana Sahib, Punjab, India

Zahoor Ahmad  Infectious Diseases PK/PD Lab, Life Science Block, Indian Institute of Integrative Medicine, Srinagar, Jammu and Kashmir, India

Yolanda Gonzalez  Department of Research in Microbiology, National Institute of Respiratory Diseases, Mexico City, Mexico

Ravi Bandaru  Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indianoil Odisha Campus, Bhubaneswar, India

Bapi Gorain  School of Pharmacy, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Selangor, Malaysia

Simone Pinto Carneiro  Laboratory of Phytotechnology, School of Pharmacy, Federal University of Ouro Preto (UFOP), Ouro Preto, Minas Gerais, Brazil

Silvia Guzmán-Beltrán  Department of Research in Microbiology, National Institute of Respiratory Diseases, Mexico City, Mexico

Laura E. Carreto-Binaghi  Department of Research in Microbiology, National Institute of Respiratory Diseases, Mexico City, Mexico

Mayank Handa  Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research—Raebareli, Lucknow, India

Hira Choudhury  Department of Pharmaceutical Technology, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia

Ali Ibrahim Bekraki  Faculty of Pharmacy, Jinan University, Tripoli, Lebanon

Rambabu Dandela  Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indianoil Odisha Campus, Bhubaneswar, India

Esmeralda Juárez Department of Research in Microbiology, National Institute of Respiratory Diseases, Mexico City, Mexico

Neelam Dhankar  Department of Pharmaceutical Sciences, Starex University, Gurugram, Haryana, India

Prashant Kesharwani Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Hamdard University), New Delhi, India

Orlando David Henrique dos Santos  Laboratory of Phytotechnology, School of Pharmacy, Federal University of Ouro Preto (UFOP), Ouro Preto, Minas Gerais, Brazil

Farzad Khademi Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran

Damián Eduardo Pérez-Martínez  Health Sciences Doctoral Program, Health Sciences Institute, University of Veracruz, Xalapa, Veracruz, México

Mradul Mohan  ICMR-National Institute of Malaria Research, New Delhi, India

Zohreh Firouzi  Department of Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Nagashekhara Molugulu School of Pharmacy, Monash University, Jalan Lagoon Selatan, Bandar Sunway, Selangor, Malaysia

ix

x Contributors Mohamad Mosa Mubarak  Infectious Diseases PK/ PD Lab, Life Science Block, Indian Institute of Integrative Medicine, Srinagar, Jammu and Kashmir, India

Deviprasad Sahoo  Department of Engineering and Materials Physics, Institute of Chemical Technology, Indianoil Odisha Campus, Bhubaneswar, India

Ramakanta Naik  Department of Engineering and Materials Physics, Institute of Chemical Technology, Indianoil Odisha Campus, Bhubaneswar, India

Aakriti Sethi  Department of Chemistry and Applied Biosciences, ETH Zurich, Zürich, Switzerland Noorsuzana Mohd Shariff︎  Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia

Bernadette Dian Novita  Department of Pharmacology and Therapy, Faculty of Medicine, Widya Mandala Catholic University Surabaya, Surabaya, Indonesia

Ankur Sharma  Institute of Nano Science and Technology (INST), Mohali, Punjab, India

Rupal Ojha  Department of Biochemistry, Central University of Rajasthan, Ajmer, Rajasthan, India

Farideh shiehzadeh  Department of Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Rajan Kumar Pandey  Department of Biochemistry, Central University of Rajasthan, Ajmer, Rajasthan, India

Rahul Shukla  Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research—Raebareli, Lucknow, India

Sreenivas Patro Sisinthy  School of Pharmacy, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Selangor, Malaysia

Sima Singh Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa

Vijay Kumar Prajapati  Department of Biochemistry, Central University of Rajasthan, Ajmer, Rajasthan, India

Mohsen Tafaghodi  Department of Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences; Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

Amal Rabti  Sensors and Biosensors Group, Laboratory of Analytical Chemistry and Electrochemistry (LR99ES15), Department of Chemistry, University of Tunis El Manar, Tunis, Tunisia Amal Raouafi  Sensors and Biosensors Group, Laboratory of Analytical Chemistry and Electrochemistry (LR99ES15), Department of Chemistry, University of Tunis El Manar, Tunis, Tunisia

Pushpendra K. Tripathi  Rameshwaram Institute of Technology and Management, Lucknow, India

Noureddine Raouafi Sensors and Biosensors Group, Laboratory of Analytical Chemistry and Electrochemistry (LR99ES15), Department of Chemistry, University of Tunis El Manar, Tunis, Tunisia

Kalpesh Vaghasiya  Institute of Nano Science and Technology (INST), Mohali, Punjab, India Rahul Kumar Verma  Institute of Nano Science and Technology (INST), Mohali, Punjab, India

Eupa Ray  Institute of Nano Science and Technology (INST), Mohali, Punjab, India

Roberto Zenteno-Cuevas  Public Health Institute, University of Veracruz, Xalapa, Veracruz, México

 

Preface Tuberculosis (TB) is a leading chronic bacterial infection. Despite potentially curative pharmacotherapies being available for over 50 years, the length of the treatment and the pill burden can hamper patient lifestyle. Prolonged treatment, high pill burden, low compliance, and stiff administration schedules are factors that are responsible for the emergence of multidrugresistant strains. According to WHO reports, 53 million TB patients died from 2000 to 2016. Therefore, early diagnosis of the disease is of great importance for global health care programs. Various unique antibodies have been developed to overcome drug resistance, reduce the treatment regimen, and elevate the compliance to treatment. Therefore, we need an effective and robust system to subdue technological drawbacks and improve the effectiveness of therapeutic drugs which remains a major challenge for pharmaceutical technology. Regarding TB treatment, nanoparticles can be a useful strategy for two distinct applications: (1) for their intrinsic antimycobacterial activity and (2) as vehicles for known antitubercular drugs to allow the reduction of dose- and drugassociated side-effects and administration via user-friendly administration routes such as pulmonary or oral ones.

This book will summarize the types of nanodrugs, their synthesis, formulation, characterization, and applications, with the most important administration routes. Thus, this book will discuss various nanotechnology-based approaches which may help overcome persisting limitations of conventional/traditional treatment. Also, recent advances and achievements regarding therapeutic efficacy provide possible future applications in this field. In this scenario, this book will directly address all translational aspects and clinical perspectives of TB nanomedicine from a comprehensive and multidisciplinary perception. This book is thus (1) an unrivalled, comprehensive summary of the field and (2) rationally conceived clinical stage of TB nanomedicines. The editor and contributors (authors) cover a wide range of expertise in the nanomedicine and TB and all of them are already proven their international acclaim. We thank all the authors for their valuable and timely contributions. We believe that the book, with its comprehensive coverage of fundamental and applied aspects of the subject, will prove immensely useful to its readers and stimulate further interest. Prashant Kesharwani

xi

C H A P T E R

1

Pathogenesis, biology, and immunology of tuberculosis Ravi Bandarua, Deviprasad Sahoob, Ramakanta Naikb, Prashant Kesharwanic and Rambabu Dandelaa a

Department of Industrial and Engineering Chemistry, Institute of Chemical Technology, Indianoil Odisha Campus, Bhubaneswar, India; bDepartment of Engineering and Materials Physics, Institute of Chemical Technology, Indianoil Odisha Campus, Bhubaneswar, India; cDepartment of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Hamdard University), New Delhi, India

1 Introduction

showcase well defined phenotypic properties and host range. The Mycobacterium tuberculosis genome shows 250 times higher than in the United States [11]. The burden of TB is also borne predominantly by the poorest people in countries. The prevalence of active TB disease is about twice as high in men as in women, and about 10% of all new cases occur in children worldwide [12]. The incidence of global TB is estimated to decline slowly by 1.6% per year, far from the estimated 4%–5% required to achieve the objectives of the WHO End TB strategy targets [13]. Mortality, on the other hand, is dropping more rapidly at 4.1% per year. Global burden of diseases, injuries, and risk factors data for TB (1990–2016) indicates that if current incidence trends continue, few countries are likely to meet the UN sustainable development goals objective of ending the epidemic by 2030 [14].

Treatment of TB disease involves several months of multiple drugs. Such long drug regimes are problematic for patients and healthcare systems, especially in low- and middleincome countries (LMICs), where the disease burden often goes far beyond local resources [15]. The incidence of drug-resistant TB in some areas is increasing, requiring even longer treatment schemes with drugs that are more expensive and difficult to tolerate. There are three primary ways of controlling TB: avoiding infection, stopping progression from infection to active disease, and managing active disease. The key elements of the public health response to TB are early and reliable diagnosis techniques and an effective treatment therapy for TB are key pillars of TB controls.

2  Drug regimens for the treatment of tuberculosis

 

M. tuberculosis has never been considered a colonizer on any substrate, or in any body or fluid, nor is it considered to be part of the normal human flora. In addition, active TB still requires multidrug treatment [16]. The mechanisms of action for several of the TB drugs are poorly understood compared to drugs used for bacterial infections, although this field continues to progress [17]. Treatment for TB disease can typically be divided into two parts. Typically, the initial, intensive phase uses at least four drugs to rapidly reduce the overall body burden of TB [18]. Next, the continuation stage is usually designed to eliminate the “persists” after 2 months of intensive care. Next, after 2 months of intensive care, the continuation stage is usually designed to eliminate the “persistent”. Otherwise, 6 months of isoniazid with ethambutol or thiacetazone may be the continuation phase [19]. In 1952, with the advent of isoniazid, an active, well-tolerated and cheap medication, anti-TB chemotherapy became a reality. TB



29

4 Isoniazid

chemotherapy is one of the most cost-effective treatments. Chemotherapy has the two goals of rapidly destroying a bacterial population in order to prevent the development of drug resistance and to shorten the treatment time [20]. There are currently 12 drugs approved by the US Food and Drug Administration as shown in Table 2.1.

3  First line drugs for tuberculosis

drug that requires conversion to its active form by the catalase-peroxidase enzyme system in M. tuberculosis. The medication has a profound early bactericidal effect when activated in susceptible species that decreases bacillary populations by about 2 logs within 48 h [22]. Most strains of M. tuberculosis are inhibited by isoniazid concentrations between 0.05 and 0.20 g/mL and changed the landscape of treatment of TB. Since then, the mode of action of isoniazid has been the subject of intensive studies and the drug appears to have temporally distinct pleiotropic effects. The drug penetrates well into all body fluids and cavities, creating similar concentrations to those present in serum. Isoniazid primarily performs its effect by inhibiting mycolic acid synthesis of the cell wall [23]. Half-life of serum varies, depending on whether the person is a quick or slow acetylator; slow acetylators take 2–4 h and quick acetylates 0.5–1.5 h [24]. In patients treated with isoniazid monotherapy, resistant strains were isolated shortly following the introduction of isoniazid. This was shown in an early clinical study in which, after 1, 2, and 3 months of treatment, 11%, 52%, and 71% t of patients with isoniazid alone devel-

The standard TB treatment regime follows the WHO’s directly observed therapy short course strategy. Standard treatment for TB comprises four first-line antimicrobials: isoniazid, rifampicin, pyrazinamide, streptomycin, and ethambutol.

4 Isoniazid Two Czech biochemists synthesized isoniazid for the first time in 1912. The potent, anti-TB activity was detected in 1951 and only effective against M. tuberculosis [21]. Isoniazid is a pro-

TABLE 2.1 List of US-FDA approved drugs for treatment of tuberculosis. Category

First line drugs

Second line drugs

Drug

Pathway

Target

Streptomycin

AG synthesis

Arabinosyl transferase

Isoniazid

Mycolic acid pathway

InhA

Pyrazinamide

Mycolic acid pathway

FAS-I

Ethambutol

Protein synthesis

30S ribosomal subunit

Rifampin

RNA synthesis

RNA polymerase

Fluoroquinolones

DNA synthesis

DNAgyrase

PAS

Folate synthesis

DHPS

Kanamycin

Mycolic acid pathway

InhA

Ethionamide

Protein synthesis

30S ribosomal subunit

Capreomycin

Protein synthesis

16SrRNA

Amikacin

Protein synthesis

30S ribosomal subunit

Cycloserine

Alanine metabolism

L-ala racemase

 

30

2.  Tuberculosis: introduction, drug regimens, and multidrug-resistance

6 Pyrazinamide

oped resistant strains [25]. Some of the highly resistant strains (MIC > 50 IJ.g/ml) were found to have lost their catalase-peroxidase activity and to have shown attenuated virulence in the guinea pig model [22]. The most common of these occurs in the gene, kat G, which codes for the development of catalase-peroxidase, resulting in decreased or absent enzyme function that converts isoniazide to its active form [26]. Again, however, it was found that single drug treatment with isoniazid was inadequate and that resistance developed quickly.

5 Rifampin

Pyrazinamide’s antituberculous activity was detected in 1952 but the dramatic entry of isonazide chemotherapy overshadowed its description [31]. Pyrazinamide is effective in an acidic pH against M. tuberculosis, which indicates activation of the drug under these conditions. The drug is particularly active against dormant or semi-dormant M. tuberculosis in macrophages or in the acidic environment in the caseation areas and is rapidly bacteriostatic but only slowly bacterial [32]. The most reliably absorbed TB drug is pyrazinamide. Peak levels of serum occur about 2 h after ingestion. Pyrazinamide normally hits the Cmax 1–2-h postdose and is present in the serum for many hours with its long half-life (about 9 h). Therefore, specimens of 2 and 6 h are well suited for pyrazinamide TDM [33]. The pyrazinamide mechanism is supposed to disturb the metabolism of membrane energy [34]. The genetic origin of mycobacterial pyrazinamide resistance appears to be one of these pncA mutations, encoding the pyrazinamidase enzyme. A dose-related hepatotoxicity is the major adverse effect on pyrazinamide [35]. Pyrazinamide and its metabolites inhibit uric acid secretion in the kidneys and possibly other drugs [36].

Rifampin, a lipophilic ansamycin, was used in 1967 in TB therapy and is highly active against mycobacteria because it spreads rapidly across the hydrophobic cell membrane. Rifampin is also a bactericidal agent similar to isoniazid for M. tuberculosis. Rifampin is a key component of antiTB, and its use has significantly reduced the length of chemical therapy required for the therapeutic TB of drugs. Rifampicin absorption may be the most variable among the TB drugs. It is quickly absorbed from the gastrointestinal tract; serum concentrations of 6–7 µg/mL occur 1.5–2 h after ingestion. The half-life in the blood is 3–3.5 h, although this may be shortened for people who take the drug for several weeks [27]. High-fat meals lower Cmax and delay Cmax (Tmax) time. Rifampicin should be supplied on an empty stomach, or if necessary with a light snack [28]. Rifampicin absorption is modestly reduced by fixed-drug combinations (FDCs) with isoniazid and pyrazinamide [29]. Rifampicin, as indicated earlier, clearly shows concentrationdependent killing, so higher doses (1200 mg or more daily) may be tolerable and more effective. Among the wild strains of M. tuberculosis, the rate of rifampin resistance, conferring mutations is approximately 10 × 1010 per bacterium per generation [30].

7 Ethambutol

 

Ethambutol is an active and precise medication, which is part of the standard TB treatment regimens. It is bacteriostatic and has no impact on cell viability and metabolism. Ethambutol has a static effect on M. tuberculosis in normal doses of 15 mg/kg body weight [37]. It is mainly used in patients with TB caused by strains that have primary resistance to isoniazid to reduce the risk of rifampin resistance. The mechanisms of action described the effects on nucleic acid metabolism of mycolic acid synthesis, ;metabolism and arabinogalactan synthesis [38]. At doses of



9  Second-line antituberculosis drugs

15 mg/kg, the peak concentration is approximately 4 µg/mL [39]. With increasing doses, the concentration increases proportionally. Typically, ethambutol achieves 2- to 3-h postdose Cmax. Nevertheless, the absorption of ethambutol is highly variable and sometimes incomplete [40]. Minimum inhibitory concentrations of the drug for M. tuberculosis range from 1 to 5 µg/mL [41]. Mutations in the embB gene, which codes for arabinosyl transferases together with embA and embC, are found in about 70% of ethambutolresistant strains. The level of mutations confirming immunity to ethambutol is 0.5– 104 [42]. The main adverse effect of ethambutol is retrobulbar neuritis. Blurred hearing, central scotomata, and blindness with red and green color are all signs [41].

31

and rpsL) were found in 65%–77% of resistant strains [47]. The rate of mutations conferred by resistance is one in 3.8 X106 generations [30]. The most common adverse effect of streptomycin is ototoxicity [48].

9  Second-line antituberculosis drugs

8 Streptomycin

Managing drug-resistant TB in clinical and public health is complicated. The therapeutic approach and the prognosis are closely linked to the pattern of resistance [49]. The presence of drug resistance, contraindications, or intolerance to antituberculous agents of the first line requires the use of second-line agents. Fluoroquinolones (FQs), amikacin, kanamycin, and linezolid have anti-TB effects and are used in the treatment of patients with TB due to drug-resistant or first-line drug intolerance. In addition to this, the FDA has approved four new agents for TB treatment: p-Aminosalicylic acid (PAS), ethionamide, cycloserine, and capreomycin [50]. These drugs, overall, are considerably more toxic than front-line drug use and are used only to treat cases that are resistant to one or more of the front-line drugs, particularly those that cause multidrug-resistant TB infections [51]. FQs may be the most effective and thus the most commonly used drug in the secondary line. TB is currently being treated by levofloxacin, moxifloxacin, and gatifloxacin [52]. They exhibit behavior dependent on concentration against most species. The cellular target of FQs is DNA gyrase- a type II topoisomerase consisting of two A and two B subunits encoded by gyrA and gyrB genes, respectively. In turn, gyrase inhibition inhibits the synthesis of DNA. Mutations in the gyrA and gyrB genes that encode for DNA gyrase mediate resistance to these agents [53]. As a group, FQs, like caffeine-like effects and insomnia, can induce CNS excitation [54]. PAS was one of the first antibiotics to show anti-TB activity in combination with isoniazid

Streptomycin is the first drug to be used in the treatment of TB. It is a broad-based aminoglycoside antibiotic. Today, however, its use is limited due to increased opposition and the need for parental administration. The drug is quickly bactericidal, although an acid pH inhibits its effectiveness [43]. Streptomycin is given intramuscularly or intravenously, so there is generally no concern about malabsorption. Peak serum concentrations occur after an intramuscular dose of approximately 1 h. The peak concentration is in the range of 40 µg/mL at a dose of 15 mg/kg [44]. The blood half-time is about 5 h. Sensitive strains of M. tuberculosis are inhibits at a concentration of 8 µg/mL of streptomycin [45]. The drug has strong tissue penetration, but only in the case of meningeal inflammation it enters the cerebrospinal fluid [46]. Streptomycin functions by interfering with the synthesis of ribosomal proteins. The site of streptomycin action is the ribosome subunit small or 30S, in particular the ribosomal protein S 12 and 16S rRNA. Mutations in the genes coding for 16S rRNA (rrs

 

32

2.  Tuberculosis: introduction, drug regimens, and multidrug-resistance

and streptomycin in the 1960s. Later on, it was replaced by ethambutol [55]. Due to the limited use over the past 3 decades, the majority of TB isolates remain susceptible to PAS, making it useful for MDR-TB patients. A high frequency of gastrointestinal upset is associated with PAS administration. In 5%–10% of patients taking the drug, hypersensitivity reactions occur. Furthermore, the usual dose of 10–12 g/day requires 20–24 tablets to be ingested. In various countries, including the United States, the granule form of PAS is the only form available. The use of a granular formulation of the drug has made administration much simpler. PAS granules are enteric coated and sustained release. Samples for Cmax should therefore be collected approximately 6-h postdose [56]. Cycloserine (CS) is an antibiotic used only to treat TB. The specific mechanism of action of cycloserin is unspecified, but it is thought to prohibit TB bacteria from generating substances called peptidoglycans that are needed to form a bacterial cell wall. This results in the weakening of the cell wall of the bacteria, which kills the bacteria [57,58]. CS induces psychological problems in a significant number of patients taking the medication. CS remains a second-line TB drug due to its frequent CNS effects [59]. The most frequently reported patients are unfocused or lethargic. These complaints appear in the low end of the normal range (20–35 µg/ml) even when serum levels are present. Food modestly reduces cycloserine absorption, so it is best to give this medication on an empty stomach [60].

10  Mechanisms of drug resistance

in 1948 [61]. These can be roughly summarized into three categories: (1) barrier mechanisms (decreased permeability and efflux pumps); (2) degrading or inactivating enzymes-for example, β-lactamases; and (3) drug target modificationsgenetic resistance and resistance to phenotypes [62]. Mycobacteria are fundamentally different from many other bacteria. First, mycobacterial cell membrane is structurally distinguishable from the gram-positive and gram-negative bacteria. Because of its thick lipid-rich cell wall, M. tuberculosis is intrinsically recalcitrant to small molecule permeation. It appears that passive diffusion accounts for only a fraction of total product permeation. The membrane-associated energy-driven efflux, which plays a major role in drug resistance, especially in combination with the permeation barrier, prevents the access of drugs to targets [63]. Second, Mycobacteria are usually β-lactam resistant. Mycobacteria produce enzymes that are degrading such as β-lactamases. This may be due in part to β-lactamase, as recorded on mycobacteria such as M. tuberculosis [64]. Within the cell wall, β-lactams bind many enzymes with various peptidoglycan-synthetic and -lytic roles. Inhibiting these enzymes can lead to cell death through multiple mechanisms that interfere with the balance of synthetic and lethal activity. Essentially, two different mechanisms are used to create resistance by β-lactams. The first wideclass includes those mechanisms that reduce effective β-lactam concentrations at the general location of the action, periplasm, or wall area of the cell. This class includes changes in permeability of the outside membrane, changes to activity of β-lactamase and changes in activity of efflux pumps. The second diverse class of β-lactam resistance mechanisms is to modify the cell’s penicillin-binding protein (PBP) profile in such a way that there is still sufficient transpeptidase activity to allow survival even in the presence of βlactam. This can be accomplished by modifying the target site where an individual PBP acquires

Analysis of the mechanisms of action and resistance of antituberculous drugs provides useful insights for managing patients with MDRTB. A wide number of antibiotics are known to resist members of the genus Mycobacterium for many years. The first step of human TB diagnosis identified as the emergence of drug resistance

 

References 33

a mutation that changes its affinity to β-lactams or by acquiring or activating previously unused PBPs with low β-lactam affinity [65]. Thus, the acquisition of resistance in M. tuberculosis derives from chromosomal mutational events in particular single-nucleotide polymorphisms. M. tuberculosis begins to develop drug resistance through mutations (resistance developed by the acquisition of new DNA is not reported). Genetic drug resistance in growing bacteria is mainly due to chromosomal gene mutations. Susceptibility to drugs effective against M tuberculosis is caused by chromosomal defects in the absence of horizontally transmitted susceptibility determinants. These chromosomal defects may result in drug resistance by altering or overexpressing the target drug as well as preventing prodrug activation [66]. MDR-TB represents the gradual accumulation of causal, stepping-stone, compensatory, or corresponding human mutations. In other words, the mutation described may not simply trigger drug resistance [1]. WHO estimated that 80% of patients diagnosed with active TB every year are completely susceptible to all current antibiotics and 20% of the remaining diagnosed with drug resistant strains M tuberculosis strains. Extrapolating from these figures, in 2014, nearly 1.9 million people developed active drug-resistant TB disease—a huge burden. Drug resistance needs patient longer and more severe treatment strategies [49].

first line: isoniazid, rifampicin, pyrazinamide, and ethambutol. There may be resistance to all drugs. Like most other pathogens, the M. tuberculosis complex has a noteworthy ability to adapt and adapt to the presence of antimicrobials, develop virulence or resistance to frequently used drugs and essentially make them ineffective. Extensively drug-resistant TB infection, causing even more serious symptoms of disease, is not only resistant to isoniazid and rifampicin, but also to any FQ and any of the three secondline aminoglycosides that can be administered. For active TB disease and drug-sensitive and drug-resistant TB disease, diagnostic and therapeutic options vary. Hence, there are several areas of work that continue to be needed for the battle against drug resistance to TB from the programmatic and clinical perspective. From a programmatic point of view, it is important to develop strategies to increase antimicrobial use and ways to achieve enforcement. From a therapeutic point of view, clinical trials are critical for validating the use of existing and new drug and regimes and for improving surveillance and monitoring methods.

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11 Conclusions

[1] L. Christoph, B. Kalsdorf, F.P. Maurer, J. Heyckendorf, Tuberculosis, Internist 60 (2019) 1155–1175, doi: 10.1007/s00108-019-00685-z. [2] B.A. Forbes, Mycobacterial infections, in: Molecular Pathology in Clinical Practice, second ed., 2016 , Springer Science+ Business Media, LLC., doi:10.1007/978-3-31919674-9_53. [3] S. Ahmad, Pathogenesis, immunology, and diagnosis of latent Mycobacterium tuberculosis infection, Clin. Dev. Immunol. 2011 (8) 814943 https://doi: org/10.1155/2011/814943. [4] M. Biot, D. Chandramohan, J.D.H. Porter, Tuberculosis treatment in complex emergencies: Are risks outweighing benefits? Trop, Med. Int. Health 8 (2003) 211–218, doi: 10.1046/j.1365-3156.2003.01025.x. [5] C.E. Barry, H.I. Boshoff, V. Dartois, T. Dick, S. Ehrt, J. Flynn, et al. The spectrum of latent tuberculosis: rethinking the biology and intervention strategies, Nat. Rev. Microbiol. 7 (2009) 845–855, doi: 10.1038/nrmicro2236.

The products of a number of comprehensive efforts to identify research priorities for TB were published over the past decade. The ancient and lethal human disease M. tuberculosis encounters local environmental factors during the complex process of its infection. M tuberculosis pathogenesis depends on a heterogeneous, complex and immuno-regulatory cell surface. Standard TB treatment consists of four antimicrobials of the

 

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2.  Tuberculosis: introduction, drug regimens, and multidrug-resistance

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C H A P T E R

3

Nanotechnology as a potential tool against drug- and multidrugresistant tuberculosis Damián Eduardo Pérez-Martíneza and Roberto Zenteno-Cuevasb a

Health Sciences Doctoral Program, Health Sciences Institute, University of Veracruz, Xalapa, Veracruz México; bPublic Health Institute, University of Veracruz, Xalapa, Veracruz, México

1  Tuberculosis as an infectious disease

been used for decades, but their percentages of sensitivity and specificity range from 60% to 95% and require from hours to weeks to give results. Based on these and other aspects, these procedures are not useful for eliminating TB as a public health problem by 2030 [2]. In response, a new generation of diagnostic procedures based on DNA analysis has been developed. The loop-mediated isothermal amplification test (TB-LAMP), the line probe assays, GenXpert, and recently the whole genome sequencing, are some of the most successful examples [3]. These systems are actually printing a new dynamic not only in the diagnostic of TB but also the diagnostic of resistance against firstand second-line drugs, with sensitivities and specificities higher than 90%, positively impacting the evaluation of programs fighting TB.

Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis, which is transmitted from person to person via droplets released by the infected person. According to the annual 2018 report of the World Health Organization, there were an estimated of 10.5 million of new TB cases and 1.5 million of deaths, placing TB as the most important infectious disease worldwide [1]. The diagnosis of TB is made mainly by sputum smear. The culture and isolation of the bacterium is considered as the gold standard diagnostic process. The third most common method is the chest radiography, in which radiographic lesions highly suggestive of the presence of TB infection are evidenced. These three assays have

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00003-5

37

Copyright © 2020 Elsevier Inc. All rights reserved.

38

3.  Nanotechnology as a potential tool against drug- and multidrug-resistant tuberculosis

TB is a treatable and curable disease, the standard international treatment scheme is also known as directly observed therapy, which involves the combined use of four antibiotics: isoniazid (H), rifampicin (R), pyrazinamide (Z), and etambutol (E), in a strictly supervised period of four to six months, if no cure is observed, then a second line panel of drugs is administered [4] (Table 3.1). The administration of inadequate drug regimens, poor adherence, or abandonment of treatment and comorbidity of type 2 diabetes mellitus are important factors related to treatment failure and development of drug resistance TB. In this sense, WHO has classified the types of pharmacological resistance into four types: (1) Mono-drug resistance; (2) poly-drug resistance, simultaneous resistance to more than one drug (other combinations beside H and R); (3) multidrug resistance (MDR-TB), simultaneous resistance to H and R drugs; (4) extensive-drug resistance (XDR-TB), a MDR-TB strain with resistance to flouroquinolone and a second-line injectable drug [4]. According to the 2018 global TB report, of the ten millions of new cases reported in 2017, close to 200,000 have some type of resistance, 161,000 were rifampicin resistance (RR-TB) and MDR-TB, and close to 14,000 showed XDR-TB [1]. These figures reveal the severity of the drug resistance, and the urgent need to include new and more effective drugs in order to remove TB as a global public health problem by 2030 [5]. In response to this, a new group of drugs such as bedaquiline, delamanid, pretomanid, and linezolid has been approved for use in special cases (Table 3.1).

2  Nanotechnology-based systems and the administration of drugs against tuberculosis

points out that “Ensure healthy lives and promote well-being for all at all ages.” The point 3.3 makes the specific mention to end the epidemics of TB by 2030, by reducing 90% in deaths and 80% incidence in TB by 2030 [2]. The third pillar that supports this End TB strategy says that “intensified research and innovation, focused in the discovery, development and rapid uptake of new tools, interventions and strategies. Likewise, research to optimize implementation and impact, and promote innovations.” This call to action helps to explain the dynamics observed in the development of diagnostic procedures, vaccines and drugs against TB in the last years [2]. Considering the above, Nanotechnology (NT) is an emergent technology that has been making its apparition on the TB scenario during the last years [6–14]. This technology makes reference to the development, creation, and use of nanoparticles (NPs), which are particles with a 10–100 nm size and in some cases up to 1000 nm. The major advantage of these NPs is that they are very useful for transportation and delivery of a broad range of molecules. Actually, NPs have important implications in several fields including pharmacy and medicine [15]. The NPs focused on the administration of drugs used for immunization or therapeutic intentions are classified into five major groups: (1) solid-lipid delivery systems, (2) emulsion-based system, (3) vesicular systems, and 4) miscellaneous structures. NPs had different structural and biological properties, modifiable according to polymers, solutions and additives used to make them. These formulations confer diverse properties such as protection to the capsule and content, increase of solubility, uptake and bioavailability of molecules transported, controlled content release and target to a specific cell o tissue type. In addition, NPs have a high loading capacity, ability to incorporate lipophilic/hydrophilic substances and feasibility of administration through different routes such as oral, topical, parenteral, and mucosal. Here are described the main characteristics of the most outstanding

Of the 13 sustainable development goals adopted by the United Nations in 2015, the third

 



2  Nanotechnology-based systems and the administration of drugs against tuberculosis

39

TABLE 3.1 Drugs used against tuberculosis infection. Drug

Introduction Symbol date Gen

Mechanism

First line drugs Isoniazid

H

1952

katG

Code a catalase-peroxidase enzyme, inhibit the synthesis of micolic acid

inhA

Encodes synthesis of enoyl ACP reductase protein, block the production of fatty acids

Rifampicin

R

1965

rpoB

Interferes with bacterial DNA synthesis blocking the ß-subunit of RNA polymerase

Ethambutol

E

1961

embB

Blocks arabinosyltransferase, inhibits permeability and entry of other medications

Pyrazinamide

Z

1952

pncA

Encodes pyrazinamidase enzyme, produces pyrazinoic acid, and decrease acidic pH

Second line drugs Levofloxacin Moxifloxacin

Lfx

1988

gyrA

Block the DNA-gyrase type A

Mxf

1996

gyrB

Block the DNA-gyrase type B

Streptomycin

S

1945

rpsL

Encodes the RNAr 12S, related with inhibition of protein synthesis

gidB

N/D

rrS

Encodes the RNAr 16S, related with inhibition of protein synthesis

Amikacin

Am

1973

rrS

Kanamycin

Km

1957

Capreomicin

Cm

1962

rrs eis tlyA

Encodes the RNAr 16S, related with inhibition of protein synthesis Encodes for an aminoglicosil transferase, mechanisms are not well known Encodes a RNAr metiltransferase, the action of the enzyme is modified

Para AminoPas salicylic acid

1949

thyA, folC, ribD

N/D

Cycloserin

1954

alr

Codes for an enzyme related to riboflavin biosynthesis

ddlA

Encodes for a D-alanine ligase, incorporates Alanines to the synthesis of peptidoglycans

ethA

Encodes for a monooxygenase enzyme, which process the prodrug

mabA

Encodes for a 3-ketoacil reductase related to the synthesis of micolic acids.

inhA

Encodes for an enoyl-ACP reductase, necessary for micolic acid synthesis

Ethionamide

Cs

Eto

1956

Rifabutin

Rfb

1980

rpoB

DNA-dependent RNA polymerase inhibitor

Oxofloxacin

Ofx

1982

gyrA, gyrB

DNA replication inhibitors

Ciprofloxacin

Cfx

1983

gyrA, gyrB

DNA replication inhibitors

Linezolid

Lz

1996

Rrl

Modification of RNA ribosomal 23S

rplC

Modification of ligase L3, ribosomal 50S Inhibit MA synthesis

Proteonamide

Ptn

2000

ddn

Bedaquiline

Bdq

2005

Delamanid

Dlm

2005

atpE Encode for an ATP synthase, and modify the ATP synthesis fgd1, fbiC, fbiA, Inhibit MA synthesis; F420 biosynthesis genes fbiB, dnn  

40

3.  Nanotechnology as a potential tool against drug- and multidrug-resistant tuberculosis

NPs and how they actually are used as emergent technologies against TB.

2.1  Solid-lipid forms

ministration of drugs is due to the diversity of their physicochemical properties and ability to cross through several anatomical barriers. They are effective in drug-targeting purposes; high drug-loading efficiency, possibility to include lipophilic and hydrophilic drugs, high bioavailability, and lower toxicity. Table 3.2 shows the approaches of these NPs for the transportation of drugs against TB [16–29].

2.1.1  Solid–lipid microparticles (SLM) These NPs are conformed by 10–1000 nm size range colloidal particles, comprised by melted biodegradable lipids in water or in aqueous solution of surfactant. SLMs mainly possess a central solid–lipid core matrix that can solubilize lipophilic molecules stabilized by different surfactants (Fig. 3.1). Their success in the ad-

2.1.2  Solid–lipid nanoparticles These NPs contains a concentration up to 2.5% of lipids. These lipids are commonly found

FIGURE 3.1  Variety of nanoparticles drug delivery systems.

 



2  Nanotechnology-based systems and the administration of drugs against tuberculosis

41

2.2.2  Nanoemulsions (NE) These are emulsions with drop diameters size ranging from 50 to 1000 nm (Fig. 3.1). NEs contain a double phase, one oil and other aqueous and also makes use of cosurfactants that helps to form a thermodynamically and kinetically stable structure. The preparation of NE can be made readily, with low energy consumption, making the procedure cheap and easy to achieve. Considering these characteristics, NEs could have applications in various healthcare fields including the experimental evaluation of some drugs against TB (Table 3.3) [33].

in biological membranes such as triglycerides, fatty acids are mainly used for its preparation. Other compounds are also frequently used, being such as lecithin or soya lecithin, and nonionics compounds like ethylene oxide or propylene oxide copolymers. Because of the physiological lipids used in the elaboration, these structures have a high tolerability, stability, and ability to incorporate highly stable hydrophobic or hydrophilic drugs, with apparent no cytotoxic effects. Using this structure as carrier for TB drugs can be observed in Table 3.2 [16–29]. 2.1.3  Nanostructured lipid carrier These structures were developed as an alternative to carriers derivate from SLM, with the intention to obtain blends of particles that made a matrix conformed only by solid lipids combined with melted lipids in proportion of 70:30. Thanks to this formulation is possible to incorporate lipophilic and hydrophilic drugs and can be administered by different routes such as oral, ocular, pulmonary and intravenous. Table 3.2 shows the diversity of works where these structures are being evaluated as carriers [30,31].

2.3  Vesicular drug-delivery systems

2.2  Emulsion-based systems

2.3.1  Liposomes (LPS) These NPs are vesicles in which an aqueous solution is enclosed by lipid bilayer membrane, size range between 0.05 and 5.0 mm in diameter and can be formulated and processed by making structures with different size, composition and charge (Fig. 3.1). The lipidic components are phospholipids which include natural and synthetic phospholipids and sphingolipids. The biological composition provides important biological advantages such as biocompatibility, easy degradation, nontoxic, and nonimmunogenic, and ability to encapsulate hydrophilic and lipophilic molecules. Perhaps, the most important property of LPS is that they can be taken and internalized by phagocytic cells, fused with lysosomes, and further degraded, releasing the content inside phagocytic cells. This makes LPS highly effective against intracellular pathogens, such as TB. Attempts to use these properties when administering drugs against TB are described in Table 3.4 [34–43].

2.2.1  Microemulsion (ME) This NPs are mainly composed of aqueous, oily and emulsifying agents and their diameter usually ranges 10–100 nm (Fig. 3.1). The different compositions and combination of compounds used for elaborating this structure makes them thermodynamically stable, improving solubilization, increasing absorption, permeability, and the protection of the drugs carried against enzymatic hydrolysis. These characteristics provide ME with a wide application in colloidal drug delivery systems. Three major types of microemulsions are recognized depending on its composition; oil-inwater (o/w), bicontinuous, and water-in-oil. Table 3.3 lists the most outstanding examples of these structures when carrying drugs against TB [32].

2.3.2  Niosomes (NIOs)

 

These NPs are vesicles conformed by a bilayered membrane able to encapsulate hydrophilic and lipophilic drug molecules in a lipid vesicular

42

3.  Nanotechnology as a potential tool against drug- and multidrug-resistant tuberculosis

TABLE 3.2 Characteristics of soli-lipid, lipid, and lipid-polymer nanoparticles used as transporters of anti-TB drugs. Drug

Admin route

Invitro/ preclinical

Comments

Reference

[16]

Solid-lipid micro and nanoparticles R

Oral

+/−

High binding efficiency to porcine mucin.

R

Oral

+/+

Enhanced oral bioavailability after a single dose. The [17] drug release profile from the lipid matrix denoted a triphasic behavior. Plasma concentration over the MIC90 was observed during 5 days.

R

Respiratory

+/−

High binding efficiency to mucin. Greater in vitro absorp- [18] tion efficiency of mannosylated coupled Nanocarriers (NCs) in macrophages.

R

Oral

+/−

Rifampicin solid-lipid nanoparticles degradation in acid medium was reduced in combination of isoniazid solid-lipid NPs (up to 12.35%).

[19]

R

ND

+/−

Enhanced in vitro uptake of the NPs (methyl α-Dmannopyranoside surface) by murine macrophages

[20]

H

ND

+/−

Mannose-functionalized solid lipid NPs are effective in targeting alveolar macrophages.

[21]

Rfb

ND

ND

Mannose-coated NCs are more suitable for alveolar macrophages, sustained drug release and reduced side effects.

[22]

R

ND

ND

Rifampicin-loaded NCs were internalized more selective- [23] ly in alveolar macrophages than in alveolar epithelial.

R

Dry powder

ND

Good release kinetics and good flow properties of dry powder of Rifampicin.

H

ND

−/+

Enhanced bioavailability in plasma and brain after giving [25] isoniazid loaded NCs and free drug solution at the same dose.

R

ND

ND

The antimycobacterial activity was improved against M. fortuitum. MIC of drug loaded NCs was eight times fewer than free rifampin solution administered.

[26]

H

ND

ND

Use of polysorbate80 and sonication was effective for entrapment efficiency and size reduction of NP.

[27]

+/+

Deep lung deposition of NCs loaded with spray-dried drugs. More than half of the administered dose was in the lungs after 30 min. High accumulation of drugs in the lungs for 24 h.

[28]

−/+

MIC values of Bedaquiline were not modified after drug encapsulation.

[29]

+/+

R-loaded cationic lipid NPs showed tissue selectivity and [31] lung accumulation of R. Improved mannosylated NCs uptake in alveolar macrophages versus mannose-free NCs.

[24]

Lipid-polymer hybrid nanoparticles Cfx

Respiratory

Lipid nanoparticles Bdq

Intravenous

Nanostructured lipid carrier R

ND

ND, No data.

 



43

2  Nanotechnology-based systems and the administration of drugs against tuberculosis

TABLE 3.3 Characteristics micro and nanoemulsions nanoparticles used as transporters of anti-TB drugs. Drug

Admin route

In vitro/ preclinical

Comments

Reference

Micro/nano emulsion R, H, and Z

ND

ND

The microemulsion has been tested using different dye solutions: [32] Rifampicin has strong interaction with Nile red dye and Pyrazinamide/Isoniazid have interaction with ruthenium dichloride.

R, H, and Z

ND

ND

Antitubercular drugs in single and mixed drug formulations were followed nonFickian release behavior except for rifampicin in pH 7.4 release medium.

[33]

ND, no data.

membrane, similar to LPS (Fig. 3.1). Nevertheless, NIOs have additional advantages. The materials required to prepare NIOs are cheaper, more stable and less toxic. They improve the therapeutic index of the drug being carried and can be targeted against specific cells and tissues. They do not need special storage conditions. Considering this, NIOs have a great potential to be used as carriers for different types of drugs against TB (Table 3.4) [44–48].

receptors and increasing solubility, reactivity, and miscibility. These structures have a large interior void space that may be used to encapsulate or incorporate small drug molecules. The toxicity is low, and the functional groups help to improve and control drug release. Examples of DNs used as carriers of anti-TB drugs are shown in Table 3.5A [50].

2.3.3  Lipospheres (LIPs) This NPs are lipid microspheres with size ranging between 0.01 and 100 mm in diameter (Fig. 3.1). Constituted by hydrophobic triglycerides with outer monolayer of phospholipids embedded on the surface, the solid core is used for carrying drug molecules in a solid fat matrix. This composition makes them ideal for oral, parenteral, and topical drug-delivery. LIPs have been evaluated for transportation of rifampicin (Table 3.4) [49].

2.4.2  Nano/micro particles (NMPs) These circular structures are made with different kinds of polymers, where poly lactide-coglycolide (PLGA) (Fig. 3.1) is the most common. NMPs can be easily transformed depending on the surfactants, organic solvents, and the structural moiety of drugs or ligands used for their elaboration or attached. Conjugation with lectins, guar gum, and other biomolecules increase half-life and improve mucoadhesion or bio-recognition by bacterial cell wall. Due to this biological versatility and available in sizes, NMPs have important advantages over other structures, such as the effective drug delivery system that uses several routes of administration. A description of these structures used as carrier for drugs against TB can be found in Table 3.5A and B [51–67].

2.4  Miscellaneous NPs 2.4.1 Dendrimers This NPs are highly branched polymeric systems, with a diameter ranging between 2 and 10 nm (Fig. 3.1). They are made with synthetic nanomaterials. Functional groups can be attached on the external surface and modify the structure, modifying the interaction with target

 

2.4.3 Microspheres These NPs are spherical particles, with diameters in range of 1–1000 mm structures made up of one or more miscible polymers, in which

44

3.  Nanotechnology as a potential tool against drug- and multidrug-resistant tuberculosis

TABLE 3.4 Characteristics of Vesicular drug-delivery nanoparticles used as transporters of anti-TB drugs. Admin route

In vitro/ preclinical

R

Respiratory

R

Drug

Comments

Reference

+/−

Liposomes cryoprotected with mannitol demonstrated deep lung deposition

[34]

Respiratory

+/−

Liposomes with cholesterol demonstrated the best nebulization properties and highest cellular uptake.

[35]

H

Respiratory

+/−

Showed antimycrobacterial activity. Controlled and sustained release of H from liposomes was observed over 1 day.

[36]

Car and Lfx

ND

+/−

Improved the antimicrobial effect of Levofloxacin versus free drug against TB resistant-strain

[37]

H

Nebulizer

ND

The formulation is hemo-compatible and cytocompatible. [38]

R

Dry powder

ND

Liposomes within breathable size range enhance of drug permeation in alveolar epithelium.

[39]

R and H

ND

ND

Encapsulated drugs be less toxic than free drugs in cell lines

[40]

R, H, Z, and E

ND

ND

Liposomal formulation had a sustained and prolonged release of the drug, and with better absorption

[41]

Z

ND

ND

Proliposomes loaded with were developed and up to 45% encapsulation efficiency was observed

[42]

R

ND

ND

(Pre-formed and in situ formed liposomes) Prolonged drug release was achieved to the in situ formulations.

[43]

Z

Subcutaneous

+/+

Lower bacterial count in lungs of infected guinea pigs. Biphasic release behavior of Pyrazinamide from NCs up to 96 H. Highest percentage of pyrazinamideloading efficiency using surfactant span-60.

[44]

E

Subcutaneous

+/+

Lower bacterial count in lungs for the EthambutolLoaded nanocarriers than the free drug in an infected model. Higher Ethambutol lung deposition for the drug-loaded niosomes

[45]

R, H, and Z

ND

+/−

Isoniazid and Pyrazinamide release profiles demonstrated a biphasic behavior. Rifampicin had a sustained release over 5 h

[46,47]

H

ND

ND

Cholesterol affects drug entrapment and encapsulation efficiency. Loaded with H are less toxic, and can be provided in lower dose and frequency

[48]

ND

ND

The lipospheres loaded with Rifampicin had a size of 247 nm and were stable for 4 weeks (storage at 4-25 C)

[49]

Liposomes

Niosomes

Lipospheres R

ND, no data; Car, Cardiolipin.

 



45

2  Nanotechnology-based systems and the administration of drugs against tuberculosis

TABLE 3.5A Characteristics of miscellaneous nanoparticles used as transporters of anti-TB drugs. Drug

Admin route

In vitro/ preclinical

ND

Comments

Reference

ND

Increases encapsulation capacity of the drug. Suitable for sustained and controlled release of rifampicin

[50]

Dendrimers R

Nano/micro particles Eto

Oral

−/+

The nanoparticles had a loading capacity of 35% and improved pharmacodynamics

[51]

R

Dry powder

−/+

Successfully deposited in the lungs. Drug is released in a sustained manner >8 h in the lungs

[52]

H

Oral

+/−

The in vitro muco adhesive test showed greater adhesion forces as the size of the nanocarriers decreased.

[53]

R

ND

+/−

Improved drug absorption in macrophages. Demonstrated bactericidal against M. fortuitum.

[54]

H

Oral

+/+

Improve bioavailability of H, maintains liberation until 124 h

[55]

Mxf

Oral

+/+

Coated have low plasma protein binding and improve drug bioavailability. Important decrement of Mxf in the liver.

[56]

R and Mxf

ND

+/−

Drug-loaded nanocarriers promoted a cellular immune response in murine alveolar macrophages.

[57]

Bdq

ND

+/−

Sustained release of Bedaquiline for 3 days.

[58]

H

ND

ND

Effective in directing the H to alveolar region with good bactericidal results, decrease liver toxicity.

[59]

Eto

ND

ND

The nanoparticle has a controlled release of ethionamide for 6 days.

[60]

R

ND

ND

Conjugated lactose nanocarriers have a slower drug release and greater absorption in lung tissue

[61]

Lfx

Dry powder

ND

The bactericidal activity is maintained after spray drying.

[62]

Lfx, Cf, Ofx

ND

ND

The hybrid levofloxacin nanoparticle showed a rapid release in the first 5 h and a slow release in the next 20 h

[63]

Z

ND

ND

The optimized formulation was stable for 2 months.

[64]

ND, no data; Ofx, Ofloxacin

2.4.4  Carbon nanotubes (CNTBs) This NPs are have become more important as carriers in the recent years. They have several micrometers in length with diameter in the range of 1–100 nm (Fig. 3.1). CNTBs can be classified as single- or multiwalled and can be easily functionalized with different proteins, peptides,

drug particles are dispersed at the molecular or macroscopic level. MCs can be made with various natural and synthetic materials which are biodegradable. Some reports describe the use of MCs for delivering antitubercular drugs in both blood plasma and organ tissues [68–70], (Table 3.5B).

 

46

3.  Nanotechnology as a potential tool against drug- and multidrug-resistant tuberculosis

TABLE 3.5B Characteristics of miscellaneous nanoparticles used as transporters of anti-TB drugs. Drug

Admin route

In vitro/ preclinical

Comments

Reference

Nano/micro particles R

Nebulizer

ND

The nanoparticles had aerodynamic diameters in the respirable range [65] and a prolonged release of the drug for 3 days.

H

Dry-powder ND

Isoniazid loaded chitosan/tripolyphosphate NPS have a rapid initial release of up to 4 h and sustained for 6 days.

[66]

R and H

Nebulizer

ND

The nanocharger decreases cytotoxicity and has good bactericidal function in the lungs versus the free drug.

[67]

Microspheres R

ND

ND

Rifampicin loaded PLGA microspheres were effectively taken up by the NR-8383 cells were found localize in phagolysosomes.

[68]

R

ND

ND

Rifampicin-loaded microspheres had a drug release for almost 5 days.

[69]

R

ND

ND

The nanometric particles are more retained in the lungs compared to micrometer-sized particles.

[70]

ND

Carbon nanotubes can be directed to cells or organs by different types of coating (bioactive peptides, proteins or nucleic acids).

[71]

Carbon nanotubes —

ND

Nanosuspensions Clo

Intravenous −/+

High concentration of the drug in lungs, spleen and liver, and bactericidal effect for M. avium. lyophilized nanocarriers can be stored for a long time.

[72]

Cfx

Dry powder ND

High loading efficiency of the drug (81% -96%). The dissolution capacity of the drug is improved, a quality useful for investigating new drugs that presented good bactericidal capacity but with problems of solubility.

[73]

ND, no data; Ofx, Ofloxacin; Clo, Clofazimine.

and nucleic acids coupled to their surface to makes them easily available to cells or address target against specific organ or tissue, increasing their biochemical utility. Table 3.5B shows some examples of these CNTBs as carrier of drugs against TB [71].

to transport drugs through different routes like oral, topical, parenteral etc. Specifically, they have been used as carriers for rifampicin (Table 3.5B) [72,73].

2.4.5  Nanosuspension (NSP) These are solid NPs dispersed in an aqueous vehicle forming a colloid. The size of particles in the NSP range between 200 and 600 nm and can be stabilized with different surfactants (Fig. 3.1). These structures have been tested

2.4.6  Nanomicelles (NMCs) These NPs are polymeric micelles raging between 10–200 nm, with a hydrophobic internal core and a hydrophilic external surface (Fig. 3.1). Due to their biocompatibility, structural stability, high drug capacity in the external surface and to solubilize hydrophobic drugs in the internal core, NMCs have important advantages

 



47

2  Nanotechnology-based systems and the administration of drugs against tuberculosis

TABLE 3.5C Characteristics of miscellaneous nanoparticles used as transporters of antiTB drugs. Drug

Admin route

In vitro/ preclinical

Comments

Reference

Nanomicelles R

Oral

+/−

The Rifampicin self-aggregation in aqueous medium was minimized by its encapsulation within polymeric Micelles

[74]

R

ND

+/−

The release of Rifampicin was greater when changing an acidic pH (5.3) due to the swelling of the micelles. Micelles were absorbed by the A549 cell lines.

[75]

R and H ND

+/−

Rifampicin-loaded/ Isoniazid-conjugated micelles showed a lower [76] hemolytic toxicity than the free drug. These micelles were more effective against M. tuberculosis than the administration of free drugs.

R

ND

+/−

“flower-shaped” polymeric micelles increase the intracellular concentration of rifampicin in macrophages.

[77]

R

ND

+/−

Ligand micelles increase intracellular concentration of rifampicin versus free drug.

[78]

Rifampicin-loaded polymersomes improved the drug intracellular concentration in murine macrophages.

[79]

Polymersomes R

Respiratory +/−

ND, no data; Ofx, Ofloxacin.

over other types of NPs. Information of assays where this structure is used for transportation of drugs against TB is found in Table 3.5C [74–78]. 2.4.7 Polymersomes They are vesicles surrounded by a bilayer membrane composed of hydrophobic polymer domains, surrounded by self-assembled hydrophilic polymers. They have an aqueous core inside and can carry a wide variety of drugs (hydrophobic, hydrophilic, and amphiphilic) with high colloidal stability (Table 3.5C) [79].

proved drugs against TB and the new shortened therapies are mainly addressed to the management of these types of cases, with highly positives results [80]. In this sense, NT can be used as an element that could be incorporated to fight against TB, transporting just one a combination of the most common first and second line drugs, or even those that are recently included such as bedaquiline, delamanid, etc. considering new or different administration routs, and addressing the development of individualized treatments, administrating the drugs according to the specific drug resistant profile of the patient. However, as can be seen in Tables 3.2–3.5, reports describing the affectivity of these NPs in animal models or in clinical trials are scarce or practically unreported. Considering the above, NT opens a whole new field for the development of new treatments and drug administration schemes against resistant forms of TB and improve treatment compliance and success in its cure.

2.4.7.1  Implications of nanotechnology in MDR-TB and XDR-TB treatment

As previously described, TB multidrug resistant (MDR) and XDR-TB are emerging threats in management and treatment of TB. Two are the most important elements that explain the annual increase of these types of strains: first, the administration of the same drugs in the last 40 years and second, poor management of disease course or therapy. The most recently ap-

 

48

3.  Nanotechnology as a potential tool against drug- and multidrug-resistant tuberculosis

3  Factors affecting NPs properties The structural properties of NPs influence the efficiency as carriers. It has been described that the shape has an important impact in the cellular uptake, spherical NPs have a more uptake efficiency than rod-shaped structures [81]. In addition, the charge of the surface also determines the capacity of aggregation of the NPs. This could have important biological implications, such as the cellular uptake and the potential aggregation in the bloodstream [82]. The composition of the elements that conforms these structures also could have important implications in the bioavailability, for example, the use of polyethylene glycol increases the bioavailability and stability, reducing the interaction with enzymes and mitigates adverse environmental conditions, maintaining the integrity of the drug transported. These factors show the important diversity of functions and properties that can be considered in the formulation and modification of NPs that improve the potential impact as carriers of drugs against TB.

a controlled manner with the aim of reducing the dose and dose frequency. Considering that one of the main causes of treatment failure is the nonadherence of the patient. All these previously described properties of the NPs would be very useful and help to improve the patient compliance to the therapy. Besides, depending on the chosen nanosystem, the development of nanoformulations loaded with one or more anti-TB drug would be less expensive and more accessible to the patients from low and middle income countries. On the other hand, some disadvantages need to be taken in consideration. One of the most important has to do with elaboration of some types of NPs; this could be cumbersome, and the scale-up processes have several difficulties, and finally the need for areas with sterile conditions. By last, it has to be considered that, due to the usual aggregating tendency of NPs this could have collateral unwanted effects, such as vascular thrombosis or dermatological and cardiovascular affectations, considering the administration route, this has been frequently observed when some particles are also used as carriers of antigens [14,83]. These need to be carefully evaluated in preclinical models, previous to the evaluation of these technologies in humans. By last, the related cost for the elaboration and production of some NPs is an important characteristic that should be considered, especially in countries with highest prevalences of TB.

3.1  Potential benefits and risks in the use of NPs Of the most outstanding benefits of NPs is that depending of the structure, this can be targeted against one specific organ or tissues. This remark especial considerations in TB, considering that this disease could be located specifically in the lung, causing the traditional disease or can be present in a different organ, giving place to the extrapulmonar form of TB. The possibility of address one specific NP specifically where the TB infection is located will increase the efficiency and specificity of the NP. In addition, some NPs have the ability to carry hydrophilic and hydrophobic drugs and be administered in different ways via keeping the integrity of the drug and improve the efficiency in the treatment. In addition, some nanocarriers could also be targeted and release the content in

 

3.1.1  Final considerations As can be seen in this chapter, most of the trials related with the use of NPs against TB have been carried out in in vitro and in a lesser proportion in animal assays. In this review, it was not possible to find a clinical assay describing the evaluation of one of these nanocarriers transporting any specific drug against TB directly in humans. It is evident that previous to this direct use in humans, further studies are necessary in order to evaluate the real impact and benefits of these new technologies.



3  Factors affecting NPs properties

Nevertheless, these NPs are potential carriers of drugs and new drugs against TB, with different properties and administration routes. This open the possibility to use these NPs depending on conducting further studies that determine the possibility and feasibility of using these NPs by considering the results yielded by clinical assays and interaction with other drugs used in comorbidities such as type 2 DM and HIV. There is no doubt that research in this field will become intensified in the near future, bringing new solutions against TB.

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3  Factors affecting NPs properties

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C H A P T E R

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Translational research for therapy against tuberculosis Yolanda Gonzalez, Silvia Guzmán-Beltrán, Laura E. Carreto-Binaghi and Esmeralda Juárez Department of Research in Microbiology, National Institute of Respiratory Diseases, Mexico City, Mexico

1  Research for tuberculosis elimination

research and patient-oriented research for the development of diagnostic tests, new drugs and regimens for the treatment of all clinical forms of TB, and effective vaccines. Also, optimization and implementation of new tools and interventions are needed to develop new medical technologies for the different areas of interest. In this chapter, we will discuss the dire need for new therapies and approaches for improving treatment in the fight against TB in the context of translational research. Clinical research is performed in human subjects and is patient-oriented. Clinical research contributes to the understanding or improvement of mechanisms of human disease, therapeutic interventions, or development of new technologies. It also covers epidemiologic and behavioral studies and health services research. Translational research, however, goes beyond. Translational research is the process of making the knowledge and discoveries from basic biology and clinical trials readily transformed into

The ability of Mycobacterium tuberculosis to survive within the macrophages and switch between varied physiological states in the lungs poses a challenge for current therapeutic regimens. In addition, there has been a significant rise of antimicrobial resistance. Existing control measures, including treatment regimens require reinvention. In view of the current state of the global tuberculosis (TB) epidemic and the need for revolutionary interventions to accelerate the rate of decline of TB, research is a crucial component of the World Health Organization (WHO) End TB Strategy. Intensified research and innovation conform a single item and are the third pillar of the End TB Strategy. Their objective is to increase the effectiveness of existing tools and to develop revolutionary new technologies to transform the way TB is diagnosed, treated, and prevented [1]. TB elimination requires an intensive effort to stimulate outcome-oriented

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00004-7

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Copyright © 2020 Elsevier Inc. All rights reserved.

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4.  Translational research for therapy against tuberculosis

techniques and tools that solve critical medical issues and enhance the implementation of best practices in the community. Cost-effectiveness of prevention and treatment strategies is also an important part of translational science [2,3]. Typically, the term translational research is applied to the research whose findings are moved from the researcher’s bench to the patient’s bedside and community, and it is frequently described in phases of translation, or “T-phases.” In early descriptions, translational research comprised two stages; the first stage (termed T1) transfers knowledge from basic patient-oriented research to clinical research, and the second stage (termed T2) transfers findings from clinical studies or clinical trials to practice settings and communities to improve health [4]. Because the concept of translational research is continually evolving, different phases have been proposed covering a continuum from gaining knowledge from the basic sciences to its application in clinical and community settings. For the purposes of this chapter, we settled for T0-T4 phases. T1 research covers from basic research to initial testing in humans; thus it moves basic discovery into a candidate health application. T2 goes from establishing the effectiveness of an intervention to the development of evidencebased guidelines. T3 focuses on implementation of evidence-based guidelines into health practice through delivery, dissemination, and diffusion research. T4 evaluates outcomes of population health practice. Because translational phases imply continuity, T0 closes the research cycle back to T1 by identifying opportunities, and new approaches to health problems [5,6]. The five phases of translational research do not necessarily move sequentially; they often interact with each other through the entire spectrum in no particular order. Fig. 4.1 illustrates the flux of translational research and provides examples of the type of research that belongs to each phase. The burden of TB disease is falling globally; yet TB is one of the top 10 causes of death worldwide and all indicators related to treatment ex-

 

hibit shortcomings [7]. Despite pharmacological advances and the undeniable benefits of current antituberculous drugs, TB treatment remains suboptimal. According to the 2018 WHO Tuberculosis Report, the TB global treatment success rate is 82%, and the multidrug-resistant TB (MDR-TB) treatment success remains lower, at 55% [7]. Because such indicators have consistently fallen below expectations, the WHO developed a Global Action Framework for TB Research, to adopt high-quality national and global TB research toward 2025, whose fundamental objectives are to promote, enhance, and intensify TB research and innovation at country level, with a focus on low- and middle-income countries, and also at a global level [8]. In addition, the World Health Assembly adopted WHO’s “Global strategy and targets for tuberculosis prevention, care and control after 2015” with ambitious targets of 95% reduction in TB mortality and 90% reduction in TB incidence by 2035, and a more long-term goal of eliminating the disease as a public health concern by 2050 [9]. Many challenges remain in several areas of TB treatment. Novel drugs are required to develop optimal TB treatment regimens either of shorter course, more effective, safer, or better tolerated. Immune-based treatments and host-directed therapies are required as adjunct therapy to enhance the host capabilities of eliminating M. tuberculosis infection, shortening the duration of treatment, preventing permanent lung injury, and avoiding the development of new drug resistance. Repurposed drugs are required to overcome the costly process of developing new drugs and the low treatment success rates (for both drug-susceptible and MDR-TB). Preventive therapy with safer drugs is required to face the threat that latent TB poses. Consequently, to enhance research for TB elimination, as well as to develop and implement novel strategies for optimal TB control around the globe, research must be strictly patient-oriented. That is why translational research is much needed.



2  Advances in the therapy for tuberculosis

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FIGURE 4.1  Phases of translational research. Translational research is a continuum. Each phase goes from the final point of the previous one to its final goal. The types of research that occur during each phase (bulleted) can be overlapping, and each phase can interact with the others in no particular order.

2  Advances in the therapy for tuberculosis The current standard treatment for drug-susceptible pulmonary TB consists of an intensive phase (two months) with four drugs: rifampin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (EMB), followed by a longer phase (4 months) of RIF and INH to completely eradicate the remaining bacilli. This regimen is highly effective, with a success rate of 82% [7]. Unfortunately, both noncompliance and late diagnosis largely contribute to TB drug resistance that is difficult to manage with the current drugs. An additional problem of drug-resistant TB is the duration of treatment. The typical treatment for

drug-susceptible TB goes from 6 to 12 months, while patients with drug-resistant TB must endure 24 months of treatment or longer, with severe side effects, high cost, and a low chance of cure [10]. Drug-resistant TB is a public health crisis, in particular because TB is the leading cause of death related to antimicrobial resistance. In 2017 an estimated 558,000 people developed TB resistant to rifampin (RR-TB), the most effective first line drug, in the world. Of these patients, 82% had MDR-TB. Only 25% of the estimated cases were enrolled on treatment with a secondline regimen. Among cases of MDR-TB in 2017, 8.5% were estimated to have extensively drugresistant TB (XDR-TB), where resistance to two

 

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4.  Translational research for therapy against tuberculosis

of the key second line drugs is also present [7]. Regardless of the alleged reasons for this rise of resistance such as patient habits, incorrect diagnoses, and failure of national healthcare systems, the issue of the lack of efficient drugs persists. Researchers around the globe are working to overcome the major issues of TB therapy and the recent advances are discussed later.

3  New drugs for tuberculosis or new regimens

prove the efficacy and safety of potential antibiotics in patients and, eventually, to elucidate the most appropriate dosage and regimen. Clinical trials are classified in phases I, II, III, and IV; they are typically performed with a limited sample of patients that enlarges as the phases progress to make inferences about how treatment should be directed in the general population that will need treatment in the future [12]. Phase I: This phase evaluates the pharmacology and toxicity. The new drug is tested in healthy volunteers for safety evaluation. It is acceptable to use a single dose. This phase also evaluates the metabolism of the drug and its bioavailability. Further studies using various doses will be necessary to determine regimens for the next phase. Phase I trials require a sample of 20–80 subjects [13]. Examples of new drugs for TB that are in phase I are described in Table 4.1. Phase II: In this phase the clinical investigation searches for therapeutic effects in patients. This comprises small-scale investigations to establish the effectiveness and safety of a drug and requires close monitoring of each patient. In this phase a few drugs with authentic potential are selected from a larger number of drugs, which proved inactive or toxic for the patients to proceed to the next phase trial. Phase II evaluates 100–200 patients [13]. Examples of new drugs for TB currently on phase II are described in Table 4.2. Phase III: This is a full-scale evaluation of a new treatment. Once the effectiveness has been proven, it is essential to compare the new drug with the current standard treatment. This phase is the most important, rigorous, and extensive and requires hundreds or thousands of volunteers [13]. The drugs for TB that are currently on phase III are described in Table 4.3. Clinical trials in the phase I and II of development belong to T1 of translational research, and phase III trials belong to T2. In phase IV of development the drug is tested in the real world for effectiveness, adverse reactions, and the collection of clinical data relevant to the use of the

3.1  The issues There is an urgent need for development of new TB drugs that are effective against drugresistant M. tuberculosis strains, as well as new strategies to reduce duration of treatment regimens to reduce the side effects. The development of drugs follows well-established paths to make sure that antibiotics are safe and effective when they reach the population. The process to approve a new drug consists of two main stages: drug discovery and drug development. The discovery stage includes the design of the new drug, the unraveling of its possible action mechanisms, and the validation of its activity against specific pathogens. Further, the new compound should be tested for safety and efficacy in animal studies. The development stage includes clinical phases and manufacturing that comprises three steps: (1) conducting of clinical trials dedicated to the formulation, drug delivery, and human clinical studies, (2) manufacturing, that involves chemical synthesis, and (3) optimizing doses [11]. The complete development is strenuous and highly expensive, which demands the commitment of agencies and countries, not only pharmaceutical laboratories.

3.2  Recent advances Several new drugs for TB are currently under clinical trials. The objective of clinical trials is to

 



57

3  New drugs for tuberculosis or new regimens

TABLE 4.1 New drugs for TB in clinical development phase I. Drug

Description

Reference

Contezolid (MRX-4/MRX-1)

It is a potent oxazolidinone antibiotic against Gram-positive pathogens including M. tuberculosis. It inhibits the protein biosynthesis by binding to the V region of 23S rRNA.

[14]

TBA-7371 (AZ 7371)

It is an azaindole, noncovalent inhibitor of decaprenylphosphoryl-β-Dribose 2’-epimerase (DprE1). It is sate and well tolerated in healthy adults.

[15]

GSK 656 (GSK 070)

It is an oxaborole, a novel molecule that inhibits the enzyme leucyltRNA synthetase (LeuRS), which is essential for protein synthesis.

[16]

Macozinone (MCZ, PBTZ-169)

It is a piperazine-containing benzothiazinone that inhibits DprE1, an enzyme essential for the biosynthesis of the cell wall. It is highly potent against MDR M. tuberculosis strains and it has been tested in healthy male volunteers.

[17,18]

Clofazimine (TBl-166)

It is a riminophenazine. It is effective against drug-resistant clinical isolates but not against nonreplicating M. tuberculosis in vitro. It is a mycobacterial DNA intercalating agent.

[19,20]

BTZ-043

It is a benzothiazinone that inhibits the enzyme DprE1 and its active against drug-susceptible and XDR-TB.

[21]

SPR720 (VXc-100, VXc-486)

It is an aminobenzimidazole that inhibits the gyrase B. It has broadspectrum antibacterial activity and is a potential candidate for the treatment of infections caused by nontuberculous mycobacteria.

[15]

TABLE 4.2 New drugs for TB in clinical development phase II. Drug

Description

Reference

OPC-167832

It is a newly synthesized 3,4-dihydrocarostyril derivative which inhibits the enzyme DprE1. It is bactericidal for replicating and intracellular mycobacteria and synergizes with delamanid.

[22]

Telacebec (Q203)

It is an imidazo[1,2-a]pyridine-3-carboxamide (IPA) that inhibits the cytochrome bc1 complex leading to the depletion of ATP. Q203 was well tolerated in phase I studies and currently under phase IIA with drug susceptible TB patients.

[23]

Delpazolid (LCB01-0371)

It is a new oxazolidinone with acyclic amidrazone that inhibits protein synthesis by binding to 23S rRNA and preventing the initiation complex. It is active against mycobacterial MDR- and XDR·TB isolates.

[24]

Sutezolid (PNU100480)

It is a thiomorpholine analogue of linezolid that inhibits the protein synthesis by binding the bacterial 23S rRNA.

[25]

Sequella (SQ109)

It is a novel 1,2-ethylene diamine related to ethambutol that inhibits trehalose dimycolate (TDM) production and attachment to arabinogalactan. SQ109 probably inhibits MmpL3, a transporter of trehalose monomycolate (TMM). SQ109 is effective, well tolerated in MDR-TB patients, and kills MDR-·and XDR-·TB clinical strains.

[26,27]

Macozinone (PBTZ-169, MCZ)

It is a piperazinobenzothiazinone derivative optimized from BT2043. This compound binds covalently to DprE1 preventing the cell wall biosynthesis. PBTZ169 forms an adduct with the active site of Cys387 and generates enzyme inactivation.

[17]

 

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4.  Translational research for therapy against tuberculosis

TABLE 4.3 New drugs for TB in clinical development phase III. Drug

Description

Reference

Bedaquiline (TMC207, Sirturo)

It is a diarylquinoline that inhibits the adenosine triphosphate (ATP) synthase enzyme of mycobacteria. The ATP-synthase is essential to generate its energy supply. This molecule is active against drug-susceptible and resistant strains with strong bactericidal and sterilizing properties.

[28,29]

Delamanid (OPC-67683)

It is a nitro-dihydro-imidazooxazole that inhibits mycolic acid biosynthesis, leading to defective cell wall formation ultimately leading to bacterial death. Delamanid is relatively well tolerated in humans. The regimen consists of 100 mg twice a day for 2 months followed by 200 mg once a day for 4 months in combination with an optimized background regimen.

[30,31]

Pretomanid (PA-824)

It is a bicyclic nitroimidazole that causes cell wall damage and respiratory poisoning. This pro-drug is effective against replicating and hypoxic and nonreplicating M. tuberculosis, and also against drug-resistant clinical isolates. It is a potential cornerstone for future TB and drug-resistant TB regimens.

[32,33]

new drug [34]. This phase belongs to T3, the implementation phase of translational research.

3.3  Future challenges

of drug development, there has been considerable interest in the repurposing of drugs, which means finding drugs that are already approved by the Food and Drug Administration that have unexpectedly been found to have activity against TB. The most likely candidates for repurposing are those with known antibiotic activity, but other drugs can be useful as well.

Most of the antibiotics in any of the phases of clinical trials are focused in MDR ant XDR mycobacterial strains and the replicative phase. However, despite the great advances in TB treatment, new drugs to combat latent TB, which is estimated to affect one third of the world’s population, are much needed. It is also essential to find new drugs to treat pulmonary TB caused by nontuberculous mycobacteria, which are usually resistant to conventional drugs.

4.2  Recent advances

4  Drugs repurposed for tuberculosis

An excellent example of translational research is the use of beta-lactams for the treatment of TB. Beta-lactams are any of a large class of natural and semisynthetic antibiotics (such as the penicillins and cephalosporins) with a lactam ring in their molecular structure and are the most widely used antibiotics. Beta-lactams are known to be inactive against TB because they are inactivated by a beta-lactamase intrinsically produced by M. tuberculosis, and therefore are not normally prescribed for TB [36]. In the phase of discovery (T0-T1), beta-lactams such as amoxicillin and ofloxacin were observed to have some anti-TB activity when coadministered with clavulanic acid (a beta-lactamase inhibitor), suggesting this combination had the potential to help XDR-TB patients [37,38].

4.1  The issues Although finding new drugs that have improved activity against the persistent forms of TB bacilli is necessary, the problem with this approach is the expensive journey to develop new drugs specific for TB and the perceived lack of financial return that would follow the successful development of a new TB drug, which is expected to be affordable [35]. To reduce the cost

 



4  Drugs repurposed for tuberculosis

Moving along T1, various carbapenems (meropenem, doripenem, faropenem, ertapenem, and imipenem), a class of beta-lactams highly resistant to beta-lactamase and widely used in hospital-acquired infections, were found to effectively kill virulent strains of M. tuberculosis in microbiological cultures and also intracellular mycobacteria in vitro alone or in combination with clavulanic acid. Carbapenems were also found to be effective for improving survival and decreasing bacterial loads in vivo. However, only modest results were observed when using mouse models, and negligible benefits when rabbits or macaques were used which can be explained by differences in the metabolism of carbapenems in those species which motivated research in small cohorts of TB patients with good success [39–41]. Here are some examples of the studies in small groups of MDR-TB and XDR-TB patients (less than 300 patients). Imipenem proved to be useful when administered in combination with second-line antibiotics [39]. Treatment with meropenemclavulanic acid increased the proportion of microbiological converters when added to linezolid-containing regimens [42]. The administration of meropenem in a bolus infusion plus oral amoxicillin–clavulanic acid reduced the sputum mycobacterial load by 1.5 orders of magnitude in the first 14 days of treatment; the same was observed in patients treated with INH, RIF, PZA, and EMB [43]. Observational studies (T2) produced interesting remarks that culminated with the publication of practice guidelines. Imipenem, meropenem, ertapenem, faropenem, and to a lower extent doripenem, biapenem, and tebipenem were effective for killing reference strains, and some clinical isolates in vitro, and more so when administered with clavulanic acid. Mouse models revealed that only imipenem and meropenem combined with clavulanic acid exhibit modest benefits in vivo. Clinical studies demonstrated that patients have clini-

59

 

cal improvement equivalent to that observed for conventional drug treatments when using imipenem and ertapenem in combinations with other drugs, but meropenem-clavulanic acid is the most promising carbapenem-based approach because it reached great success even in XDR-TB and MDR-TB patients [44]. Currently, the use of meropenem, amoxicilinclavulanic acid, and imipenem-cilastatin are suggested for the long regimen treatment of MDR-TB patients that are not eligible for the short regimen according to the WHO treatment guidelines for drug-resistant TB [45]. Implementing these practice guidelines moves research along T3. The use of beta-lactams is moving toward T4, but recent discoveries point to T0, to the visualization of new therapeutic approaches. For example, dual beta-lactam combinations are more effective than single beta-lactam regimens to kill mycobacteria of clinical interest [46]. Furthermore, the recent identification of synergistic activity between beta-lactams and rifampin suggests beta-lactam and rifampin combinations as potential treatment-shortening approaches for drug-susceptible TB [47]. Biapenem, which is highly resistant to hydrolysis and thus more stable, is effective to kill virulent M. tuberculosis as well as rifampin-resistant strains when coadministered with rifampin in vivo [48]; cephalosporins alone or in combination with rifampin are also bactericidal and sterilizing for drug resistant strains of M. tuberculosis [49]. Other antibiotic not originally prescribed for TB has gone through the T0-T4 translational research phases. Moxifloxacin, a fluoroquinolone with broad-spectrum antimicrobial activity, has already been trialed in humans showing promise for shortening treatment time and reducing toxicity; it is now in clinical phase III trials in several drug combinations for MDR-TB [50–52]. Currently, after an outcome research (T4) the WHO treatment guidelines for drug-resistant TB suggests a new short MDR-TB regimen stating that

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4.  Translational research for therapy against tuberculosis

“in patients with RR-TB or MDR-TB who were not previously treated with second-line drugs and in whom resistance to fluoroquinolones and second-line injectable agents was excluded or is considered highly unlikely, the shorter MDR-TB regimen of 9–12 months may be used” [45]. The shorter MDR-TB treatment regimen consists of an intensive phase of four months with gatifloxacin (or moxifloxacin), kanamycin, prothionamide, clofazimine, high-dose INH, PZA, and EMB. This is followed by a continuation phase of 5 months with gatifloxacin (or moxifloxacin), clofazimine, PZA, and EMB [53]. Going from T4 to T0 to T1, moxifloxacin’s ability to kill M. tuberculosis moved beyond suggestion to be administered as a first-line drug and is now under evaluation to determine its utility in drug-susceptible TB [54,55]. Both the short and long regimens in the WHO treatment guidelines for MDR-TB treatment include clofazimine, a riminophenazine originally used to treat leprosy. After showing antibacterial effects against M. tuberculosis and its drugresistant strains together with potential benefits for patients, clofazimine was repurposed for TB [56,57]. The lack of extensive T3-T4 research with this drug, and the scarcity of treatment options make the use of clofazimine a suggestion with cautionary remarks. A variety of drugs that are not categorized as antibiotics have been proposed for the treatment of TB or MDR-TB or even nontuberculous mycobacteria, but none of them have gone beyond the discovery phase. Among these, we have anticancer drugs [58–60]. Because anticancer agents target the host, they are less likely to generate resistance compared to conventional antibiotics, but more likely to increase the side effects. Other drugs with demonstrated ability to kill M. tuberculosis, and its drug-resistant strains include antiparasite medicines [61–63]; nonsteroidal antiinflammatory drugs such as diclofenac, ibuprofen, and oxyphenbutazone, which have been found to act upon replicating, dormant and also drug-resistant clinical

isolates of M. tuberculosis [64]; antipsychotics [65,66]; asthma drugs [67]; and efflux pump inhibitors normally used for hypertension [68]. Several of these studies lack detailed description of the mechanism or host pathway affected by the new therapeutic approach and most of them have been demonstrated only in vitro, which makes the advancement toward the next phases of translational research essential. Bactericidal and synergistic activities of these drugs with conventional antibiotics might be useful for future promising combination therapy against M. tuberculosis to shorten the duration of regimens or to secure an early microbiological cure.

4.3  Future challenges

 

Although great advances have been made in the search for TB drugs, either new or repurposed, most of the studies are conducted using collection strains or in TB patients regardless of the actual strain that ails them, but there are pathogen-associated features which also contribute to drug resistance in TB that need to be taken into account. Some lineages are predominantly transmissible to susceptible hosts and appear to acquire resistance very successfully [69,70]. Individuals infected with such strains deserve immediate attention, as treatment options remain limited, and also require susceptibility tests, even for the new therapeutics, and long-term follow up to monitor the emerging of resistance. Regarding the variety of drugs that seem promising, the wide range of mechanisms of action requires a comprehensive analysis that would help us prioritize drugs for further development. Bioinformatics facilitates this approach and would help accelerate the process of TB drug development by incorporating structural analysis, molecular modeling, and protein-drug interaction network analyses to predict standalone drugs for TB treatment and to identify the drugs for combination treatments with the



5  Host-directed therapy for tuberculosis

additional advantage that safety profiles and known clinical outcomes can be incorporated in the modeling [71,72]. Although the bioinformatics analyses would predict the most promising drug, the efficacy of the predicted drugs needs to be experimentally validated, and the incorporation of treatment outcome research is highly desirable.

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5  Host-directed therapy for tuberculosis

nary TB patients [75]. Further revisions revealed that corticosteroids were useful for improving intermediate and long-term determinants, such as hospital stay duration, clinical progress, and weight gain [76,77]. Steroids inhibit cytokines production, mainly IFN-γ, TNF-α, and IL-1β [78], thus preventing the inflammation-associated tissue damage [79]. Other modulators of inflammation have been used successfully to treat mycobacterial infections such as leprosy and TBM. The production of drugs that inhibit phosphodiesterases, such as thalidomide, control cAMP production, and reduce TNF-α levels, is stimulated by mycobacterial adenylyl cyclases [80–83]. However, the safety issues of thalidomide limit its use; analogs would be preferable, yet they have only been evaluated in animal models [84,85]. The inflammatory response can also be controlled by eicosanoids, and drugs that target eicosanoids production are good candidates for TB. Zileuton, a drug initially approved for asthma, is a lipoxin-blocker that improves mycobacterial control through the modulation of the 5-lipooxygenase [86], enhancing prostaglandin E2 (PGE2) production and, consequently, controlling the excess in type I IFN production [87], which has been related to a more severe disease. In the field of enhancing bactericidal mechanisms of the host, a wide range of drugs have been proposed. M. tuberculosis elimination depends highly on phagosome maturation. Imatinib is a tyrosine kinase inhibitor that increases mycobacterial destruction within the phagosome, resulting in lower bacterial loads and decreased granuloma formation in mice infected with M. tuberculosis or M. marinum, particularly when added to the classical antituberculous drugs [59,88]. Phagosome maturation can be stimulated after reducing cholesterol levels with statins [89]. Host cholesterol represents a source of carbon for mycobacteria upon the activation of the IFN-γ pathway and the limitation of this molecule diminishes bacterial survival

5.1  The issues Antituberculous drugs have severe hepatotoxic adverse effects. Host-directed therapies represent a difficult but promissory approach because of the safety and tolerability that drugs whose target is the human should meet [73]. Studies on the host immune response to M. tuberculosis infection have revealed new pathways for the treatment of this pulmonary disease, either for mycobacterial elimination or for damage control, that are reachable with adjunct therapies [74]. Several aspects of the host immune response have been evaluated, from innate mycobacterial recognition to autophagy and other adaptive mechanisms for mycobacterial killing [74]. For example, the equilibrium between the host pro and antiinflammatory mediators is feeble, it shapes the immune response against M. tuberculosis, and the pathological aspects of inflammation have a direct impact on tissue damage.

5.2  Recent advances Modulation of inflammation is expected to benefit tissue restoration. The most broadly used drugs for antiinflammatory therapies are corticosteroids, which have been particularly used in TB meningitis (TBM) or pericarditis. A recent meta-analysis showed a reduction in the overall TB mortality when using corticosteroids, although few studies were performed on pulmo-

 

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4.  Translational research for therapy against tuberculosis

5.3  Future challenges

[90,91]. Another drug related to the lipolytic pathway is the mepenzolate bromide, which inhibits cAMP signaling through a G-proteincoupled receptor called GPR109A, reducing bacterial burden during murine M. tuberculosis infection, and promoting a foamy phenotype in macrophages [92]. These foamy cells abundantly express the peroxisome proliferator-activated receptor gamma (PPAR-γ), a nuclear receptor/ transcriptional factor that regulates fatty acid metabolism in macrophages, primarily stimulated by the lipoarabinomannans (LAM) from M. tuberculosis cell wall [93]. This receptor enhances the production of PGE2 and induces a TLR-independent IL-8 response, through the recognition of mannose-capped LAM by the mannose receptor, during M. tuberculosis [94] or M. bovis infection [95], leading to mycobacterial control within macrophages. PPAR-γ is the target of two hypoglycemic (pioglitazone, rosiglitazone) and one antihypertensive drug (treprostinil) [93,96], suggesting a potential activity of these agents as adjunct TB therapies. The concomitant administration of drugs that potentiates phagosome activity to kill mycobacteria may accelerate the bactericidal effects of the conventional antituberculous treatment and shorten the regimens; however, no studies in humans have been conducted. Vitamin D has been profusely studied as a possible immune modulator in TB. The main activity described for the bioactive form of vitamin D is the generation of antimicrobial peptides after macrophage TLR stimulation [97], as well as the enhancement of the IFN-γ or IL-1β pathways [98]. Moreover, vitamin D has been suggested to regulate the excessive inflammation associated with TB, which defines the clinical outcomes of the disease [99]. Several clinical trials have been conducted with vitamin D within different populations with inconclusive results [100,101]; these findings suggest that an individual approach to a specific population is needed for the implementation of vitamin D as an adjunct therapy for TB.

Targeting the host immune response seems the best alternative when treating TB, considering the multiple mechanisms that mycobacteria have developed to resist antituberculous drugs. However, clinical trials must consider the consequences of altering host cell processes, particularly if the molecule to be used is not specific for the cell types directly related with TB, such as macrophages or alveolar cells. The side effects of modulating the host immune response should also be thoroughly reviewed in order to prevent opportunistic infections or autoimmune diseases. Another issue for further discussion will be how to choose between different therapies for an individual patient, because host-directed therapies need to address the specific necessities of each patient.

6  Tuberculosis research and care biomarkers. The OMICs of tuberculosis

 

The difficulty to monitor the therapy efficacy and make early treatment interventions, and the lack of a marker that predicts relapse or outcome of a given treatment curtails the progress in TB care. The gold standard for microbiological diagnosis of TB relies on identification of the M. tuberculosis bacilli in clinical specimens. In total, 2 or 3 months after chemotherapy, the bacillus is not detected outside the lungs and the efficacy of the therapy is uncertain. Consequently, many scientists are intently looking for a useful biomarker for TB treatment monitoring. According to the National Institutes of Health Biomarkers, a biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” [102]. A large number of genes or molecules are under study looking for a biomarker for early



63

6.1 Genomics

FIGURE 4.2  Biomarkers for TB. The OMICs technology has advanced TB research contributing with biomarkers. The potential application of these biomarkers ranges from diagnosis to personalized medicine.

infection detection, disease progression, diagnosis, immunization and treatment efficacy for TB. The collective technologies in life sciences used to explore the roles, relationships, and actions of the various types of molecules that focus on large-scale data/information to understand life with a sense of wholeness or completion are termed “OMES” and “OMICS” such as proteomics, genomics, metabolomics, etc. [103]. The OMICs technology involves different areas of science: immunologists, biologists, computational biologists, biostatisticians, biomathematicians, and physicists. The OMICs technology applied to research in TB generates datasets, which will be useful to comprehend the disease,

make a diagnosis, and predict the outcome of treatment [104]. The OMICs belong to the T1 phase of translational research and are summarized in Fig. 4.2.

6.1 Genomics

 

Genomics is the study of organisms’ whole genomes. The human genome consists of 3 billion DNA base pairs, encoding approximately 20,000 genes, and many variants exist, the majority of which are benign, some are protective, and others confer susceptibility to diseases [105]. The research based in Genome-wide association

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4.  Translational research for therapy against tuberculosis

6.2 Transcriptomics

studies have typically focused on the analysis of single markers, which often lacks the power to uncover the relatively small size effects conferred by most genetic variants [106]. The genetic variants among people of the same race or among people of different races are influenced by the environment, and their association with the state of disease or health is not necessarily causal. Genomics is a data-rich and diverse subfield within modern quantitative biology. The analysis of genomics unravels how a genetic variant can alter the sequence or expression of a single gene, which in turn influences a pathway of interconnected genes, which then influences the overall cell or individual characteristics, all within a particular environment or disease stage [106,107]. Numerous candidate gene studies have been conducted to assess the host genetic factors that confer risk in TB. Recently, 15 genes were identified as associated to TB disease (CD209/ DC-SIGN, HLA-A, HLA-DQB1, HLA-DRB1, IFNG, IFNGR1, IL-10, MBL2, MCP1, NRAMP1/ SLC11A1, TLR2, TLR4, TLR9, TNFA, VDR) [108]. However, this association with TB susceptibility was observed in a specific population and not globally. In these studies, the coevolution of host and pathogen is an important issue to consider [108]. The construction of genomic profiles is currently the main product of genomics, which makes genomics an example of T1 research. To move toward T2, it would be necessary to produce genetic tests and establish their analytic validity by means of clinical evaluations. The consequent step would be the implementation of genomic applications in routine clinical practice and only a few studies have reached this stage; most of them are related to cancer [6]. Genomics research in TB are in the first stage of translational research, and the knowledge is in the process of being translated to clinical trials. Still, genomics gives hope for the development of personalized medicine.

 

The transcriptome is the complete set of RNA transcripts in a cell or tissue. In humans, 1.5%–2% of the genome is represented in the transcriptome as protein-coding genes [104]. The dynamics of cellular and tissue metabolism impact on the transcriptome profiles and affect health and disease. Changes in gene expression for the presence/absence of disease or stimuli and the quantification of a transcript, the evaluation of alternative/differential splicing to predict protein isoforms and the quantitative assessment of genotype influence on gene expression are fundamental phenomena described with transcriptomics [105]. In TB patients, the blood transcriptomic profile describes a large number of genes that are induced after the infection with M. tuberculosis. Some of them are useful for treatment follow up, others for diagnosis. A recent review on human transcriptomics concentrated a large list of genes potentially useful for TB diagnosis. Three independent studies reported the expression of DUSP3, FCGR1A, GBP5, and SEPT4; two independent studies reported the expression of ANKRD22, BATF2, FCGR1B, FCGR1C, GAS6, GBP1, GBP6, LHFPL2, S100A8, SCARF1, and SERPING1; and also 93 varied genes have been associated to TB development [109]. Using a mathematical framework, Singhania et al. suggested a promising signature of 20–27 genes capable of discriminating between active TB and latent TB and 16 genes’ expression as biomarkers of risk for TB progression [109]. Using a multitarget gene expression in response to M. tuberculosis specific antigens, the Th1-type cytokines (TNF-α and IL-2R) and IFNγ–induced chemokines (CXCL9 and CXCL10) mRNA could be used to detect infection and to differentiate between the active form of pulmonary TB, latent TB, and healthy controls in a cohort from Korea [110]. In total, 11 transcripts (IL2, IP10, IFN-γ, IL13, MIG, SCF, b-NGF, IL12-p40,



6.3 Proteomics

TRAIL, IL2Ra, LIF) have been found to discriminate between non-TB and latent TB groups, and 14 transcripts (IL2, IP10, IFN-γ, MIG, SCF, bNGF, IL12-p40, TRAIL, IL2Ra, MIF, TNF-β, IL3, IFN-α2, LIF) discriminated between non-TB and active TB groups in a cohort from Italy [111]. In a cohort of 120 TB patients and 80 healthy donors where 360 target genes were analyzed, a 4-gene model (GBP1, IFITM3, P2RY14, and ID3) displayed the best performance for discriminating between TB patients and healthy individuals from South Africa and the Gambia [112]. The transcriptomics research in TB for diagnosis or for monitoring treatment outcome is in the first phase of translational research but moving forward in the translational phases is still far away. One of the reasons is because most of the existing studies cover local communities and not a wide range of populations. A recent review of proteomics and transcriptomics demonstrated that only a few of the identified biomarkers are validated in clinical settings. While the literature comprises more than 150,000 papers documenting thousands of claimed biomarkers, fewer than 100 have been validated for routine. The lack of reproducibility, which is the main reason for the delay in setting actionable tools, is partly due to the specimen handling and storage, which can dramatically affect the levels of the biomarkers detected [113].

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6.3 Proteomics

other molecules. This complexity challenges the development of proteomics-based tests [104]. For reference, a consortium of proteomic databases denominated The Proteome Xchange was established to sustain a collection of proteomic experiments, and the Protein Data Bank has a collection of >30,000 structures for human proteins and is currently the main structural proteome repository [105]. Proteomics of TB research mainly focuses on immune-related proteins, particularly cytokines and chemokines that may find a signature of active TB in a wide range of experimental settings yielding heterogeneous results. For example, EGF, fractalkine, IFN-γ, IL-4, MCP-3, and IP-10 discriminate active TB cases from latently infected TB contacts, and IFN-γ, IL-4, MCP-3, MIP-1β, and IP-10 are also useful for monitoring antituberculous treatment [114]. Another study including 161 patients with TB and 50 healthy controls, analyzed 1011 proteins and identified 153 proteins differentially expressed in patients with severe TB, and four proteins (ORM2, S100A9, IL36α, and SOD1) were associated with the development of TB, suggesting potential biomarkers to distinguish between different stages of TB [115]. A group of 215 active TB patients were followed for 2, 4, and 6 months posttreatment, and the serum amounts of IL-1, IL-2, IL-12P70, and soluble CD62E protein levels were found to be significantly higher, whereas the IL-4, IL-5, IL7, IL-8, IL-10, IL-17, IL-21, soluble CD54, MIG, and TGF-β levels were lower at the beginning of recruitment. After treatment completion, IFN-γ, IL-2, IL-7, and soluble CD54 protein levels increased [116]. Another research revealed that IP10 is present in the urine of TB adults and correlates with the efficacy of therapy [117]. Not only the human proteome, but also the proteome of M. tuberculosis provides identification of target proteins with therapeutic value [118]. The potential use of proteomics as biomarkers for TB diagnosis, treatment follow-up, identification of stages of TB disease, and efficacy of

The proteome is the complete set of proteins expressed in cells, tissues, or organisms under defined conditions. The proteome is inherently quite complex because proteins can undergo posttranslational modifications (glycosylation, phosphorylation, acetylation, ubiquitylation, and many other modifications to the amino acids constituting the proteins), have different spatial configurations and intracellular localizations, and interact with other proteins as well as

 

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4.  Translational research for therapy against tuberculosis

therapy, open new advances in diagnosis and treatment for the control of TB. However, the complexity of pathways, interactions between bacteria and host cells, and the interaction between host and environment, make it very difficult to extrapolate the putative biomarkers to patients around the globe. Proteomics mostly belong to the first level of translational research.

6.4 Metabolomics

tion of active TB, and identified key parameters existing 12 months before TB diagnosis, proposing a tool to make timely interventions to prevent disease progression and transmission [122]. Metabolomics is promissory for TB control; however, the environmental influence is the principal challenge for the translation of this knowledge to clinical practice, and most of the reports are in the T1 phase of translational research.

7  The impasse of translational medicine in tuberculosis and future challenges

Metabolomics technology analyzes the metabolites produced during biochemical reactions. Metabolomic databases such as the human metabolome database, METLIN and MetaboLights collect information on metabolites identified in biological samples through chromatography, nuclear magnetic resonance, and mass spectrometry paired with associated metadata. The changes in the production of metabolites reflect a particular combination of an individual’s genetics and environmental exposures [105]. TB-associated changes in the metabolite profile have been examined in blood and other clinical specimens such as urine, sputum, cerebrospinal fluid, or breath with variable results [119]. When analyzing 23,241 metabolites in 34 subjects by mass spectrometry, 61 metabolites were identified, including the resolvins (RvD1 and RvD2) that distinguish TB patients from their household contacts [120]. Another study identified four metabolites of 4α-Formyl-4βmethyl-5α-cholesta-8-en-3β-ol combined with 12(R)-HETE or cholesterol sulfate that differentiated TB patients from controls. These novel plasma biomarkers, especially 12(R)HETE and 4α-formyl-4β-methyl-5α-cholesta8-en-3β-ol, alone or in combination, are potentially useful for the diagnosis of TB [121]. A study with a large cohort of 4462 healthy household contacts of 1098 index TB cases identified a signature that predicts development of subclinical disease prior to manifesta-

 

The main goal of translational research in TB is to improve tools and approaches for effective diagnosis, treatment, and prevention. To ensure success, synergy among researchers, research institutions, health systems, and patients is required [123]. However, despite many years in research and advancements in technology, there is still a gap between what is known and what is acted upon. Research is expected to rapidly transfer, introduce, and adapt new methods and strategies to local contexts. However, several matters that were not discussed above contribute to the lack of completion of the translational research cycle in TB research. One difficulty for going from the discovery phase to a health application (T1) is that studies in humans and animals are often focused on only one or two factors at a time (reductionist approach), but what is needed is a more global approach. Single component studies in animal models, or even in humans, are necessary and have been very informative, but it can be difficult to place these studies in the context of a very complex disease. Another challenge is that, in humans, the primary compartment available for study is blood, while in TB, immune and pharmacological determinants that control or exacerbate disease are concentrated in the lungs.



7  The impasse of translational medicine in tuberculosis and future challenges

Neglection of associated comorbidities also challenges the progress toward T2 research. The rates of TB co-infection in HIV positive patients are rising [124]. Diabetes mellitus predisposes the patient to develop active TB and severely affects the treatment outcome [125]. Patients with active pulmonary TB have increased chances of developing mycosis such as coinfections with Cryptococcus spp., Coccidioides spp., and Histoplasma spp. [126,127]. The main issues in this respect are the worsening of symptoms and increasing of side effects because of possible interactions between the drugs used for each medical condition. The focus of this chapter is limited to strategies for the treatment of pulmonary TB, but other forms of TB are neglected as well. Tubercular infection of the central nervous system, often known as TBM is a serious and life-threatening condition. It accounts for up to 5%–10% of all TB cases worldwide but is responsible for more than 40% of the deaths due to TB. Patients with TBM are difficult to treat because the disease persists even with antituberculous therapy, and invasive treatment can lead to severe complications [128]. For a health application design to move toward approved practice guidelines (T2) several concerns must be addressed. Unknown drug interactions are one of the reasons why a “one for all” treatment regimen does not work; thus drug interactions must be investigated during T1 research. More precise animal models are required to help prioritize the new or repurposed drugs that have the highest potential in achieving sterilizing cure. In addition, a big challenge is ensuring that the treatments/applications are affordable, effective, safe, and reach the people who need them. To reach implementation, diffusion and dissemination (T3) of a treatment or application so that it becomes common health practice, reporting in peer-reviewed journals is not enough. Translational research must provide actionable tools for ready implementation, such as guide-

67

 

lines, decision aid tools, service specification, clinical decision aid, patient decision aid, algorithms for clinical decision making, risk assessment tool, local protocols, national protocols, or guidelines for policy makers and practitioners [129]. Such actionable tools should disseminate the knowledge in a format that meets the end users’ needs. An additional factor that reduces the effectiveness, depth, quality, and impact of research that causes implementation failures is the lack of interaction between biomedical TB researchers and those involved in areas such as health systems, health economics, and social and behavioral research. Assessing the population health impact of a treatment or application (T4) requires effective access of the population to the products and an accurate reporting system. Also, the optimal coordination among partners, institutions, programs, and individuals involved in TB research is necessary to improve the relevance, quality, and efficiency of research. The research needs and priorities should be adopted at national and international levels. At the country level, it is essential to create a research-enabling environment that implements high-quality research, and to develop country-specific, prioritized TB research agendas. The main considerations a research needs to cover to secure its movement through the phases of translational research are summarized in Fig. 4.3. Finally, moving forward to the discovery phase (T0) to close the cycle, a useful approach would include not only to analyze the population health impact, but to look beyond data. Translational systems biology, which is the use of experimental findings combined with mathematical modeling and/or engineering principles to understand a biological process for the purpose of “optimizing clinical practice,” will be leading this area of TB research by building bridges and closing gaps within and across the disciplines [130,131].

68

4.  Translational research for therapy against tuberculosis

FIGURE 4.3  Conditions to warrant the moving forward on the phases of translational research. The main goal of translational research in TB is to improve tools and approaches for effective diagnosis, treatment, and prevention. However, despite many years in research and advancements in technology, there is a gap between what is known and what is acted upon. For a research to move through the phases of translational research these conditions should be complied with.

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[88] H. Bruns, F. Stegelmann, M. Fabri, K. Döhner, G. van Zandbergen, M. Wagner, et al. Abelson tyrosine kinase controls phagosomal acidification required for killing of mycobacterium tuberculosis in human macrophages, J. Immunol. 189 (8) (2012) 4069 LP–4078. [89] S.P. Parihar, R. Guler, R. Khutlang, D.M. Lang, R. Hurdayal, M.M. Mhlanga, et al. Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation, J. Infect. Dis. 209 (5) (2013) 754–763. [90] A.K. Pandey, C.M. Sassetti, Mycobacterial persistence requires the utilization of host cholesterol, Proc. Natl. Acad. Sci. 105 (11) (2008) 4376 LP–4380. [91] J.E. Griffin, A.K. Pandey, S.A. Gilmore, V. Mizrahi, J.D. Mckinney, C.R. Bertozzi, et al. Cholesterol catabolism by mycobacterium tuberculosis requires transcriptional and metabolic adaptations, Chem. Biol. 19 (2) (2012) 218–227. [92] V. Singh, S. Jamwal, R. Jain, P. Verma, R. Gokhale, K.V.S. Rao, Mycobacterium tuberculosis-driven targeted recalibration of macrophage lipid homeostasis promotes the foamy phenotype, Cell Host Microbe. 12 (5) (2012) 669–681. [93] M. Kiss, Z. Czimmerer, L. Nagy, The role of lipid-activated nuclear receptors in shaping macrophage and dendritic cell function: from physiology to pathology, J. Allergy Clin. Immunol. 132 (2) (2013) 264–286. [94] M.V.S. Rajaram, M.N. Brooks, J.D. Morris, J.B. Torrelles, A.K. Azad, L.S. Schlesinger, Mycobacterium tuberculosis activates human macrophage peroxisome proliferator-Activated Receptor γ linking mannose receptor recognition to regulation of immune responses, J. Immunol. 185 (2) (2010) 929 LP–942. [95] P.E. Almeida, A.R. Silva, C.M. Maya-Monteiro, D. Töröcsik, H. D′Ávila, B. Dezsö, et al. Mycobacterium bovis Bacillus Calmette-Guérin infection induces TLR2-dependent peroxisome proliferator-activated receptor γ expression and activation: functions in inflammation, lipid metabolism, and pathogenesis, J. Immunol. 183 (2) (2009) 1337 LP–1345. [96] M. Rask-Andersen, S. Masuram, H.B. Schiöth, The druggable genome: evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication, Annu. Rev. Pharmacol. Toxicol. 54 (1) (2014) 9–26. [97] P.T. Liu, S. Stenger, H. Li, L. Wenzel, B.H. Tan, S.R. Krutzik, et al. Toll-like receptor triggering of a vitamin d-mediated human antimicrobial response, Science 311 (5768) (2006) 1770–1773. [98] M. Verway, M. Bouttier, T.-T. Wang, M. Carrier, M. Calderon, B.-S. An, et al. Vitamin D induces interleukin-1β expression: paracrine macrophage epithelial signaling controls M. tuberculosis infection, PLOS Pathog. 9 (6) (2013) e1003407.  

[99] A.K. Coussens, R.J. Wilkinson, Y. Hanifa, V. Nikolayevskyy, P.T. Elkington, K. Islam, et al. Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment, Proc. Natl. Acad. Sci. 109 (38) (2012) 15449 LP–15454. [100] C. Wejse, V.F. Gomes, P. Rabna, P. Gustafson, P. Aaby, I.M. Lisse, et al. Vitamin D as supplementary treatment for tuberculosis, Am. J. Respir. Crit. Care Med. 179 (9) (2009) 843–850. [101] A.R. Martineau, P.M. Timms, G.H. Bothamley, Y. Hanifa, K. Islam, A.P. Claxton, et al. High-dose vitamin D3 during intensive-phase antimicrobial treatment of pulmonary tuberculosis: A double-blind randomised controlled trial, Lancet 377 (9761) (2011) 242–250. [102] K. Strimbu, J.A. Tavel, What are biomarkers? Curr. Opin., HIV AIDS 5 (6), 2010, 463–466, doi:10.1097/ COH.0b013e32833ed177. [103] S.P. Yadav, The wholeness in suffix -omics, -omes, and the word om, J. Biomol. Tech. 18 (5) (2007) 227–1227. [104] C.M. Micheel, S.J. Nass, G.S. Omenn, Board on Health Sciences Policy, Institute of Medicine, Evolution of Translational Omics: Lessons Learned and the Path Forward, The National Academies Press, Washington, DC, 2012, https://doi.org/10.17226/13297. [105] C. Manzoni, D.A. Kia, J. Vandrovcova, J. Hardy, N.W. Wood, P.A. Lewis, et al. Genome, transcriptome and proteome: the rise of OMICs data and their integration in biomedical sciences, Brief Bioinform. 19 (2) (2018) 286–302 10.1093/BIB/BBW114. [106] K. Wang, M. Li, H. Hakonarson, Analysing biological pathways in genome-wide association studies, Nat. Rev. Genet. 11 (12) (2010) 843–854 10.1038/nrg2884. [107] M.C. Schatz, Biological data sciences in genome research, Genome Res. 25 (10) (2015) 1417–1422 10.1101/ gr.191684.115. [108] C.M. Stein, L. Sausville, C. Wejse, R.S. Sobota, N.M. Zetola, P.C. Hill, et al. Genomics of human pulmonary tuberculosis: from genes to pathways, Curr. Genet. Med. Rep. 5 (2017) 149–166 10.1007/s40142-017-01309. [109] A. Singhania, R.J. Wilkinson, M. Rodrigue, P. Haldar, A. O’Garra, The value of transcriptomics in advancing knowledge of the immune response and diagnosis in tuberculosis, Nat. Immunol. 19 (11) (2018) 1159–1168. [110] S. Kim, H. Lee, H. Kim, Y. Kim, J.E. Cho, H. Jin, et al. Diagnostic performance of a cytokine and IFN-γinduced chemokine mRNA assay after mycobacterium tuberculosis-specific antigen stimulation in whole blood from infected individuals, J. Mol. Diagnostics. 17 (1) (2015) 90–99 10.1016/j.jmoldx.2014.08.005. [111] M.P. La Manna, V. Orlando, P. Li Donni, G. Sireci, P. Di Carlo, A. Cascio, et al. Identification of plasma biomarkers for discrimination between tuberculosis infection/disease and pulmonary non tuberculosis disease, PLoS One 13 (3) (2018) e0192664.

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for diagnosis, J. Clin. Microbiol. 53 (12) (2015) 3750– 3759 10.1128/JCM. 01568-15. [122] J. Weiner, J. Maertzdorf, J.S. Sutherland, F.J. Duffy, E. Thompson, S. Suliman, et al. Metabolite changes in blood predict the onset of tuberculosis, Nat. Commun. 9 (2018) 5208 10.1038/s41467-018-07635-7. [123] C. Lienhardt, K. Lönnroth, D. Menzies, M. Balasegaram, J. Chakaya, F. Cobelens, et al. Translational research for tuberculosis elimination: priorities, challenges, and actions, PLoS Med. 13 (3) (2016) 1–11. [124] S.S.A. Karim, G.J. Churchyard, Q.A. Karim, S.D. Lawn, HIV infection and tuberculosis in South Africa: an urgent need to escalate the public health response, Lancet 374 (9693) (2009) 921–933. [125] M.A. Baker, M.B. Murray, K. Lönnroth, C.Y. Jeon, J.E. Hart, S.-E. Ottmani, et al. The impact of diabetes on tuberculosis treatment outcomes: a systematic review, BMC Med. 9 (1) (2011) 81. [126] J. Cadena, A. Hartzler, G. Hsue, R.N. Longfield, Coccidioidomycosis and tuberculosis coinfection at a tuberculosis hospital, Medicine (Baltimore) 88 (1) (2009) 66–76. [127] G.D. Brown, D.W. Denning, N.A.R. Gow, S.M. Levitz, M.G. Netea, T.C. White, Hidden killers: human fungal infections, Sci. Transl. Med. 4 (165) (2012) 165rv13165rv13. [128] M. Wasay, S. Farooq, Z.A. Khowaja, Z.A. Bawa, S.M. Ali, S. Awan, et al. Cerebral infarction and tuberculoma in central nervous system tuberculosis: frequency and prognostic implications, J. Neurol. Neurosurg. Psychiatry. 85 (11) (2014) 1260–1264. [129] S. Hampshaw, J. Cooke, and L. Mott, What is a research derived actionable tool, and what factors should be considered in their development? A Delphi study, BMC Health Serv Res. 18 (1) (2018) 740. [130] Y. Vodovotz, M. Csete, J. Bartels, S. Chang, G. An, Translational systems biology of inflammation, PLoS Comput. Biol. 4 (4) (2008) e1000014 10.1371/journal. pcbi.1000014. [131] D.E. Kirschner, D. Young, J.A.L. Flynn, Tuberculosis: Global approaches to a global disease, Curr. Opin. Biotechnol. 21 (4) (2010) 524–531.

C H A P T E R

5

Vaccine delivery systems against tuberculosis Rupal Ojha, Rajan Kumar Pandey and Vijay Kumar Prajapati Department of Biochemistry, Central University of Rajasthan, Ajmer, Rajasthan, India

1 Introduction

Later, it was also reported that TB and HIV coinfection is more dangerous and is the foremost cause of death in HIV positive people [4]. In 2017 a significant increase (62%) in the number of new TB cases was reported in western Pacific and south-east Asia, while 25% cases were reported from the African region (https:// www.who.int/news-room/fact-sheets/detail/ tuberculosis). There are mainly three forms of TB—active, miliary, and latent form of infection. In the active form of TB infection, the Mycobacterium divides quickly in the host body and affect different parts or organs. This deadly form of TB transmits from person to person via droplets shed during coughing or sneezing. The symptoms, which are associated with an active form of TB, are fever, headache, the cough along with sputum, chest pain, paleness, sweating, and weight loss. The second form, miliary TB, affects the multiple organs at once. The symptoms, which are associated with this condition, are fever, headache, cough, along with sputum,

Tuberculosis (TB) is an ancient infection but still has left its presence as the most challenging diseased condition, which severely distressing the world population. The causal organism for this infectious condition is Mycobacterium tuberculosis, a gram-positive bacterium. It primarily affects the macrophages and hepatocytes cells of humans belonging to all age groups but mostly adults [1,2]. As per the global burden survey report (the 1990s), TB was included in the list of top 10 deadly infection leads to severe mortality and casualty, worldwide because it affects around one-third of the worldwide population [3], and hence World Health announced the disease as a global crisis. As per the World Health Organization (WHO) report 2018, approximately 1.6 million people died from this disease, and 10 million are at the risk of infection (https://www.who.int/tb/publications/ global_report/en/).

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00005-9

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Copyright © 2020 Elsevier Inc. All rights reserved.

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5.  Vaccine delivery systems against tuberculosis

chest pain, paleness, sweating, and weight loss. The situation gets more worsen when the bacteria enters into the host via nasal route, during inhalation. Here, bacteria directly inter into the alveoli results into the granuloma formation. This granuloma formation occurs due to the cytokine induction, which further leads to the Th1-type of the immune response. These granulomas cross the blood–brain barrier and result in the CNS TB or tuberculous meningitis. In this condition, the immunostimulatory molecules released incessantly, where TNF plays the leading role in the inflammatory response[5]. Later, in the latent form of TB infection, the immune system activates the M. tuberculosis antigens without the previous epidemic of active TB. There are 5%–10% chances of disease reactivation based on risk factors from moderate to severe in case of LTBI. The diagnostic methods which are associated with this form of TB are IFN-gamma includes QFT-G (QuantiFERON-TB gold) test and skin test include tuberculin tests (Mantoux and Heaf test) (https://www.who.int/tb/ areas-of-work/preventive-care/ltbi_faqs/en/). When coming to the treatment part, many first-line drugs have been reported like isoniazid, ethambutol, pyrazinamide, and rifampicin [6]. As per literature, it was also found that streptomycin has been used to treat TB. However, this drug does not prescribe generally by the doctors due to its side effects and inability to enter the blood-brain barrier, which ultimately becomes a cause of nephrotoxicity and ototoxicity (toxic to the ear)[7]. The causative agent progressively shown multidrug resistance against the antibiotic therapies and hence, recognized as a new form of TB, that is, multidrug resistance TB (MDR-TB). The rationale behind the resistance may be due to unbalanced consumption of medications by patients, improper supervision, and inappropriate awareness programs. The diagnostics methods available for the disease identification are sputum smear microscopy and Xpert MTB/RIF assay, but

 

these two diagnostic methods are unable to diagnose the MDR-TB [8]. By considering all these drawbacks, there is an immense requirement of a lively or potential regimen and advanced delivery systems for the prevention of disease. After the drugs resistance scenario, vaccines came into the picture, as they are standardized as the most effective and protective treatment for public health. Effective vaccines provide prolonged immunity against many infectious diseases. Because of vaccination, some lifethreatening conditions have been exterminated, and a significant reduction in mortality and fatality rate was observed. For TB, a vaccine bacillus Calmette-Guerin (BCG) was introduced and used to immunize the young ones in the profoundly affected provinces [9]. This vaccine was developed by Albert Calmette, and Camille Guerin, by using a live-attenuated bacterial strain of Mycobacterium Bovis, because of its ability to induce the immune responses in the host [10]. This vaccine delivery through the intradermal path, hence, possesses the fewer chances of neurovascular damage (https://www.who.int/ biologicals/areas/vaccines/bcg/en/). However, the outcomes of the vaccines showed that it was unable to cure the chronic form of infection ad have the prospects of disease reoccurrence. Additionally, apart from this single available TB vaccine, many other vaccine options are available but are either in the pipeline or in the different stages of clinical trials. According to the clinical trial.gov (https://clinicaltrials.gov/) data, a total of 149 studies of TB have been reported with their status in different phases of clinical trials. Among them, 105 have shown the status as completed while 15 studies are under the recruitment process (Fig. 5.1). Note: There are total 15 TB vaccine trials reported, which showed that vaccine candidate VMP001 is in phase 2 and 3 while MTBVAC is in phase 1 and 2, so we have considered the phases accordingly. Apart from this data, two clinical trials are recruited for epidemiological TB analysis and infant vaccination evaluation study;



2  TB vaccine candidates in the pipeline

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FIGURE 5.1  Graphical representation of the status of TB vaccines candidates in the pipeline.

hence, the phase does not apply to them. However, many successfully developed vaccines are come across with some limitations, including reduced ability to generate humoral and cellmediated immunity. To overcome these downsides, we require a promising immunization delivery system united with some novel preparations and have the capability to retain the effectiveness of poorly immunized vaccines. This chapter mainly focuses on vaccine delivery systems against TB. The vaccine delivery systems can be ranging from nanoparticles, liposomes, virus-like particles, virosomes, and dendrimers to ISCOMs. Vaccine protein encapsulated with vaccine delivery system can be administered via different routes to elicit the robust immune responses against the pathogen. These advanced delivery systems help to improve the quality and effectiveness of the vaccine by providing long-term immunity. Their biocompatible, nanosize, nontoxic, and targeted delivery made them unique in comparison with the traditional delivery approaches. Apart from this, adjuvants are also known to play a vital role in the improvement of vaccine immunogenicity. They can work as immunopotentiators (for the initiation of innate immunity) as well as delivery systems (via

nanoparticles or other methods). The adjuvants conjugated with the subunit vaccines help the targeted movement of vaccine protein towards the antigen-presenting cells where the vaccine protein got processed and displayed by MHC class molecules for the generation of both cells mediated and humoral immune responses. As we have discussed, the Mycobacterium enters the host body via nasal route and then spreads the infection in the lungs. So, it would be a great approach to deliver the vaccine via the nasal or mucosal route so that it can directly target the lungs and helps in the elimination of the pathogen. For TB, many vaccines are in the pipeline by using next-generation vaccine approaches, and in the upcoming years, the advanced delivery systems and their associated administration routes will bring a revolutionary change in the field of vaccinology by reducing the economic burden of the diseases.

2  TB vaccine candidates in the pipeline

 

Vaccination is the most efficient and costeffective means to prevent infectious diseases and associated morbidity and mortality. As per

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5.  Vaccine delivery systems against tuberculosis

the WHO, immunization has helped to save the lives of over 2.5 million children each year globally. This biological preparation used to generate enhanced immunity against a particular pathogen-associated diseased condition. Vaccines can be further classified into the traditional one and modern vaccine. The former includes live-attenuated or heat-killed microbes, subunit vaccine, toxoid, and polysaccharides vaccine. While the later include recombinant vector and DNA vaccine. Instead of good immunogenicity of a vaccine candidate, the selection of an appropriate vaccine delivery system is the key element to decide the fate and success of the vaccine. As we have already discussed, BCG is the only existing and licensed TB vaccine for human use. However, due to the low immunogenicity and incapability to cure the immunocompromised people coinfected with HIV, researchers are in search of new potential vaccine candidates for the prevention of this contagious disease. After the literature survey, it was found that many TB vaccine candidates are in the development of a vaccine against TB (Table 5.1) [11,12]. The processed vaccine candidates belong to the different classes of vaccines as follows.

are TB/FLU-01L (https://clinicaltrials.gov/ ct2/show/NCT03017378) (combination of replication-deficient influenza virus A expressing ESAT-6 antigen of pathogen), FP85A [17] (fowl pox virus expressing secretory antigenic protein 85A of pathogen), ChAdox185A [18] (chimpanzee adenovirus showing antigen 85A of pathogen), MVA85A-IMX313 [19], AERAS-456 (H56:IC31) [20], and Ad5Ag85A [21] (adenovirus 5-based booster vaccine expressing antigen Ag85A of pathogen). These novel vaccine candidates are in the different phases of clinical trial study and the recent studies at somewhat extent they are showing the immunogenicity against the TB condition.

2.2  Adjuvanted subunit TB vaccine

2.1  Viral vectored TB vaccines

Subunit vaccines comprise the antigenic protein part of the pathogen, having the capability to induce the immunogenicity actively. There are 3 TB subunit vaccines which are undergoing the clinical trials namely ID93+GLA-SE is a recombinant polyprotein fusion vaccine which contains 4 antigenic proteins Mtb Rv1813, Rv2608, Rv3619, and Rv3620 delivered with GLA-SE, a synthetic adjuvant, to enhance the long-term immunogenicity [22]. It was reported that this vaccine mainly targets pulmonary TB, a latent form of TB. Other vaccine candidates who are in the pipeline are Mtb72F/AS02 (Mtb fusion protein in combination with AS02 adjuvant) [23] and GamTBVac [24] (BCG booster multi-subunit vaccine fused with 2 antigens ESAT6-CFP10 and Ag385A). This combination of antigens and adjuvants will provide protective and long-lasting immunity in contrast to the severe infection.

These vaccines are the combination of avirulent virus and potential antigen of the pathogen and also function as the prime booster. This viral vector helps in the expression of the antigenic protein of pathogen, which further induces the cell-mediated or humoral immunity in the host [13,14]. Many viral vectored vaccines for TB are in the clinical trial processing, for instance, MVA85A is a phase 1 viral vectored vaccine of Modified Ankara virus which does not replicate in the humans and hence can express the mycobacterial antigens [15]. The clinical trial data showed that the vaccine MVA85A was given as the prime booster to enhance the activity of BCG vaccine [16]. Other recombinant viral vectored vaccines candidates in the clinical trial process

2.3  DNA TB vaccine

 

Vaccination by using DNA vaccines is the most common form of immunization. The DNA vaccines can induce both humoral and cellmediated immune responses in the preclinical models. In the DNA vaccines, the gene of



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2  TB vaccine candidates in the pipeline

TABLE 5.1 Pipeline vaccine candidates proposed for antiTB immunization. S. No.

Vaccine candidates

Classification of vaccine

1

MVA85A

Viral vector (booster vaccine)

2

ID93+GLA-SE

3

Number of studies registered

Phases of clinical trial

Route of administration

Tuberculosis and TB HIV co-infection

Phase 1

Aerosol, intradermal, intravenous, and intramuscular intervention

28

Protein adjuvanted (subunit vaccine)

Pulmonary tuberculosis

Phase 1 and Phase 2a

Intramuscular

7

TB/FLU-01L

Viral vector

Tuberculosis

Phase 1

Intranasal and sublingual aerosol

1

4

FP85A

Viral vector

Tuberculosis

Phase 1

Intradermal

1

5

DAR-901

Inactivated whole-cell Tuberculosis Mycobacterial vaccine

Phase 1 and Phase 2

Intradermal

3

6

ChAdox1 85A

Viral vector

Tuberculosis

Phase 1

Intramuscular

2

7

RUTI

Fragmented MTB

Latent tuberculosis infection

Phase 2a

Subcutaneous

3

8

MVA85A1MX313

Viral vector with a carrier protein

Tuberculosis

Phase 1

Intradermal

1

9

ID93+AP10-602 Protein adjuvanted (subunit vaccine)

Tuberculosis

Phase 1

Intramuscular

2

10

AERAS-456 (H56: IC31)

Viral vector

Latent tuberculosis infection

Phase 1 and Phase 2

Intramuscular

3

11

Ag85B-ESAT6

Subunit vaccine

Tuberculosis

Phase 1

Intranasal

5

12

Mtb72F/AS02

Protein adjuvanted (subunit vaccine)

Tuberculosis

Phase 1 and Phase 2b

Intramuscular

4

13

Ad5Ag85A

Adenovirus-based vaccine (Booster vaccine)

Pulmonary tuberculosis

Phase 1

Aerosol

2

14

GC3107

Live attenuated M. tuberculosis

Tuberculosis

Phase 3

Intradermal

2

15

MTBVAC

Live attenuated M. tuberculosis

Tuberculosis

Phase 2

Intranasal and mucosal

5

16

Gam TB Vac

Subunit vaccine

Tuberculosis

Phase 1 and Phase 2

Subcutaneous

2

17

VPM1002

Live recombinant BCG (rBCG) (BCG replacement)

Tuberculosis

Phase 2 and Phase 3

Intradermal

5

18

ESAT6-CFP10

Recombinant fusion protein

Tuberculosis

Phase 3

Intradermal

8

19

SRL 173

Inactivated whole-cell Mtb

Tuberculosis and Phase2 (TB) and Intradermal HIV infections Phase 3 (HIV infection)

1

20

Vaccae

Whole-cell M. vaccae (BCG replacement)

Tuberculosis

4

Condition

 

Phase 3

Intramuscular

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5.  Vaccine delivery systems against tuberculosis

interest (antigen) is cloned into a plasmid vector and injected directly (via intramuscular path) into the animal models, which leads to translation of proteins in the cytoplasm. The antigenspecific immune responses were generated in the host. If the gene encoding antigens show similarity with the host proteins, it may lead to the generation of cross-reactive antibodies. By using Mtb Ag85A antigens, the first DNA vaccine against TB was developed. The antigens administered into the mice via an intradermal or intramuscular path, and it was found to generate strong CD4+ Th1 immune response along with interferon-gamma and interleukin-2 in high amount. Further, it was observed that DNA encoding Ag85B, ESAT6, and Hsp65 Mtb antigens were also responsible for the induction of robust immune responses [25].

to come in the clinical trial and formulated as the replacement of existing BCG vaccine in the young ones of mice. This vaccine has been developed by deleting the virulent Mtb genes, phoP (Mtb transcription factor), and Fad26 (involved in biosynthesis pathways). The results obtained after the vaccination of MTBVAC has shown alike immunogenic, and efficacy as BCG displayed [29,30]. Next, VPM1002, the phase 2b, and phase-3 recombinant vaccine candidate were compared with BCG in terms of safety and immunogenicity, and the results concluded that VPM1002 showing good efficacy in immunocompromised population and can prevent TB reoccurrence [31].

2.4  Whole-cell and live Mycobacteria TB vaccine

3  Vaccine administration routes for TB vaccine

The whole-cell vaccines are the formulation of killed or inactivated bacteria, which are unable to cause disease. These vaccines are not associated with long-term immunity and require several doses to enhance the immune response. However, they considered being more stable than other type of vaccines with no risk of infection. Vaccae [26], phase-3, M. vaccae whole-cell vaccine candidates are in the clinical trial to prevent TB. The other whole-cell/extract vaccines which are the replacement of the existing BCG vaccine under the clinical trial process are Dar901 [27] (M. Obuense inactivated whole-cell vaccine) and RUTI [28] (made up from fragmented Mtb cells). Whereas, the live attenuated vaccines are weakened form of bacteria which exploits the protein or its component for the vaccine designing. Due to the generation of the robust immune response, this type of vaccines is widely preferred to prevent diseases. There are two live Mycobacteria vaccine which is in the different phases of a clinical trial, for instance, MTBVAC (phase 1), the first live Mycobacterium vaccine

Vaccination is an effective way for the prevention of severe infectious diseases. However, the efficacy of the vaccine very much depends on the delivery systems in the host. The delivery system decides the providence of vaccines; a suitable route of administration is crucial for the certification of active immunization. It was reported that the administration routes regulate the patterns of immune responses [32]. The parameters, which are associated with the vaccine administration route, include mainly the site of vaccination and the interval of doses. Further, if we talk about the rationale of immunization where APCs plays a pivotal role in the generation of immune reactions because they uptake the antigens via endocytosis and initiate the antigen processing to present them with MHC class molecules [33]. However, if the designed vaccine is incapable of reaching the immunologically privileged locations, there will be no immune response generation, which ultimately leads to the failure of the vaccine. Generally, most of the vaccines delivered intradermally, intramuscularly, and subcutaneously. Here is some detailed description of the

 



3  Vaccine administration routes for TB vaccine

parenteral delivery systems that are currently in use to administer the earlier-discussed vaccine candidates of TB.

81

3.1  Intradermal route of administration

protected responses with less reactogenicity against pulmonary TB. The TB vaccines which are under the clinical trials are ChAdox185A [18], MVA85A, ID93+GLA-SE, ID93+AP10-602 (clinicaltrials.gov registry no. NCT02508376), AERAS-456 (H56: IC31), and Mtb72F/AS02 [23]. The intramuscular path mainly preferred due to less pain and reaction at the injection site also the large volume of vaccine can be delivered via this path.

The intradermal route delivers the immunogenic vaccine candidate directly into the dermis, primarily the deltoid region of the upper arm with the help of needle or syringe of 15 mm, 26 gauge [34]. The use of short and narrow needle makes the intradermal route a safe mode of immunization. It is known that intradermal vaccination is responsible for Th1/Th2 immune responses, mainly Th2 response, and also provides adequate lung protection [35]. This delivery system is associated with the induction of active systemic as well as mucosal immune responses because of the presence of the higher number of antigen-presenting cells beneath the skin. The TB vaccines which are currently under the different phases of clinical trials and administered intradermal are MVA85A [30], DAR-901 [27], MVA85A-1MX313 [19], GC3107 (clinicaltrials.gov registry no. NCT03363178), and VPM1002 [31]. With the help of intradermal path, the 10-fold optimum range of dose or antigen was enough to induce the humoral and cellmediated immune responses.

3.3  Subcutaneous route of administration

3.2  The intramuscular route of administration

The administration route follows the delivery of vaccine into the subcutaneous layer (below the skin and above the muscle). The needle is of ⅝-inch, 23- to 25-gauge injected into the dermal tissue at 45-degree angle (https://www. cdc.gov/vaccines/pubs/pinkbook/vac-admin. html). Presently, only the measles vaccines delivered subcutaneously. In TB, there are only few vaccine candidates proposed for subcutaneous administration, but all of them are in the clinical trial. In 2009 Begam et al. reported that Mtb32a vaccine to deliver subcutaneously, and it had shown to induce CD8+ T-cell immune response in the lungs of mice [36]. Next, RUTI (phase 2), fragmented TB vaccine was introduced to reduce the chemotherapy, given during latent TB infection. Then vaccine administered subcutaneously after the one-month treatment with isoniazid drug in immunocompromised HIV+ individuals. The results showed that tolerability and efficacy of the vaccine were good enough in the individuals with the minor side effects [37]. However, vaccine candidates are still under clinical trials.

To induce immunogenicity against the pathogen, the respective vaccine must reach the lymph nodes when it first injected into the muscle tissue. With this intramuscular delivery, the vaccine will directly reach out to the lymph nodes very quickly. Here they get engulfed by APCs via endocytosis, and their processing started followed by MHC class-II molecules mediated presentation to the T-cells, which leads to the generation of cell-mediated (CD4+ and CD8+) immune responses [33]. This type of immunization is associated with the induction of innate

3.4  Intranasal (mucosal, sublingual) route of administration-

 

The intranasal path is the most painless, noninvasive delivery system with no chances of adverse events and toxicity in comparison to others and is highly recommended for the young ones.

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The mucosa present in the nasal protects against foreign particles. Vaccine delivery via mucosal route makes a path for the antigen to directly contact with the mucosal-associated lymphoid tissue [38], which in turn initiate both the local and systemic immunogenic effects. It has also been reported that this type of vaccination is associated with the production of IgA and IgG antibodies, which plays a unique role in the elimination of pathogens. As per published studies, nasal administration leads to the induction of T helper-17 CD4+ cells [39]. These cells are involved in the release of immunostimulatory molecules like interleukins IL-17A, IL-17F, IL-21, and IL-22 which lead to the increased cytokine level and hence caused IFN-γ stimulation which in response boost the Th1/Th-17 cells to trigger the macrophages and other immune cells. The TB vaccines that are under clinical trial and proposed to administer as intranasal or via aerosol are MVA85A, TB/FLU-01L, Ad5Ag85A, and MTBVAC. Among these vaccines, Ad5Ag85A and MTBVAC delivered via aerosol, which develops a passage to match the immunization route to the infection route [40]. The delivery of aerosolized vaccine directly into the lungs or respiratory mucosa offers a great way of immunization. In a recent study, MVA85A (phase I), given by aerosol (via nebulizer MicroAIR NE-U22) in a nonhuman primate, which was already BCG vaccinated, has shown a potential enhancement in mucosal as well as systemic immune responses [41]. The nebulizer used for the aerosolized delivery, inject the vaccine directly into the lungs. This device contains 6000 narrowing holes for spraying the aerosol liquid efficiently. Next, Apa Rv1860, the alanine and proline-rich antigen when delivered to the mice via an intranasal path it has shown the protective and significant immune responses against TB. The findings also showed that the intranasal vaccination of BCG generated a strong Th1 immune response in the lungs of the mice [42]. Furthermore, sublingual or mucosal route of vaccination is somewhat similar to the intranasal.

With this delivery system, the vaccine delivered under the tongue, which further able to elicits the strong immune responses and helps in impeding the local infections by inducing the cytokines production. It was believed that this type of delivery route mainly supports to cure allergic hypersensitivity. Further, it was found that the Mtb recombinant vaccines Ag858 and ESAT-6 (linked with adjuvant glycolipid alpha-galactosylceramide, for enhanced immunogenicity) has been delivered to the mice via sublingual route [43]. After vaccination, the results showed a robust antigen-specific T-cell immune response in the spleen, lymph nodes (cervical), and lungs of the mice model. The data regarding the TB vaccines, which are in the pipeline, have been presented in Table 5.1 along with their phases and route of administrations, and all this information has been collected from (https://clinicaltrials.gov/0).

4  Advanced TB vaccine delivery systems and their related immune responses Advanced or next-generation vaccines include the recombinant fusion proteins or DNA along with adjuvant added, to boost the immunogenicity against potential infectious diseases. For an efficient immune response, the successful vaccine delivery systems are essential which stimulate the uptake of vaccines quickly. Some of the advanced delivery systems have been discussed as follows.

4.1  Nanoparticles-based TB vaccine delivery systems

 

The nanoparticle-based modern delivery systems have the potential to improve the vaccine efficacy because they possess immunostimulatory effects and hence provide an immediate immune cell stimulation [44]. Their antigen depot formation property and high filling capacity of multiple antigen and adjuvant at a time make them a unique carrier for vaccine delivery [45].



4  Advanced TB vaccine delivery systems and their related immune responses

They also increase the dendritic cell-based direct antigen uptake, which ultimately leads to the stimulation of DCs and also upholds antigen cross-presentation. Nanoparticles protect the adjuvanted vaccines and their antigens from early proteolytic and enzymatic degradations. In comparison to other vaccines which administered in solution, nanoparticles defend themselves from the endosomal degradation and directly enter into the cytosol of the cells and deliver the antigen for further antigen processing and additional initiate the TLRs signaling cascades [46]. There are many nanoparticle-based TB vaccines that are under the clinical trial phases. Ag85A-HBHA, recombinant, and immunogenic Mtb specific antigen protein fused with heparin binding hemagglutinin adhesion protein [47] when encapsulated with carnauba wax (obtained from the plant Copernicia cerifera) nanoparticles and administered in BALB/c mice models (already BCG vaccinated) via intranasal/mucosal path, the mice showed vigorous immune responses (humoral and cell-mediated) along with the stimulation of various immunomodulatory molecules [48]. The immunomodulatory molecules are associated with the activation of innate as well as adaptive immunity. The designed vaccine candidate when entering the host body; it behaves like MTB itself. These two antigens Ag85A and HBHA with coated nanoparticles plays a vital role in mycobacterial adherence and its direct contact with the lung alveoli, which enhance the chances of antigens uptake by APCs efficiently. Apart from the carnauba wax, some environmentally friendly or biodegradable nanoparticles like carbon, silica, and gold are also used as nanocarriers for antigen delivery and are known as nanoimmunostimulatory inorganic molecules with the size of 20–100 nm. Hsp65, the Mtb specific antigen when enclosed with gold nanoparticles, and delivered to the host it showed a remarkable decrement in the bacterial growth (Mtb) in infected mice and provided a protective host immune response in contrast to TB. To induce the desired immune response against fatal

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infectious diseases like TB, there are various other types of nanoparticles-based vaccine delivery systems that mainly focused on vaccine formulation along with its efficient delivery for the generation of potent immune responses. Here are the types of some nanoparticles that are being used for the TB vaccine preparation.

4.2  Cationic nanoparticle-based TB vaccine delivery The cationic nanoparticles are environmentally friendly, that is, biodegradable and have cation ionic surface, which is suitable for the delivery of DNA vaccines. This type of nanoparticles was designed to increase the delivery of DNA vaccines directly to the dendritic and macrophages cells when administered intramuscularly. In comparison to naked DNA vaccine immunization, the cationic vaccines have shown an enhance immune responses and a substantial increase in the serum antibody production. Furthermore, the TB DNA vaccine candidate Ag85A (Mtb specific antigen)-ESAT-6 (Mtb secretory antigenic protein), and IL-21 (interleukin-21) fused and engineered with the Fe3O4Glu-polyethyleneimine nanoparticle to check the efficacy of the developed vaccine. After the efficacy evaluation of naked DNA vaccine in comparison to the nanoparticle conjugated one, they found that the DNA vaccine delivered without the nanoparticle was not able to induce the potential immune response. But when the nanoparticle engineered DNA vaccine was delivered to the infected mice, it has shown a high efficacy along with the durable and protective immune responses by inhibiting the growth of Mycobacterium in the mice models.

4.3  Chitosan-based nanoparticle TB vaccine delivery

 

The only existing vaccine which is presently available for the treatment of TB is BCG. The BCG vaccine alone has not shown effective

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immune responses against pulmonary disease in adults. However, to improve its efficacy, it has been modified with the chitosan-basednanoparticles which encompass poly I:C (polyinosinic–polycytidylic acid), a TLR-3 agonist synthetic adjuvant, to improve the immunogenicity of BCG vaccine in the macrophages isolated from the mice bone marrow. It was observed that the chitosan-based nanoparticles protect the poly I:C adjuvant from the nuclease degradation and enhance the uptake of vaccine into the mice. Apart from the existing BCG vaccine, the other multiepitope (T-cell epitopes), pPES (multigene family protein) DNA vaccine was also formulated by utilizing mannosylated chitosan (MCS) nanoparticles. The intranasal vaccination of this vaccine candidate, conjugated with nanoparticle MCS, induces a significant antigen-specific T-cell immune response including the stimulation of immunomodulatory molecules like IFN-γ, IL-2, and TNF-α in the spleen. The immune response generated by the MCS was much stronger in comparison to BCG vaccine and Chitosan-based vaccine. This mucosal mediated delivery of the vaccine proved to provide enhanced protection or immunity against the fatal pulmonary TB.

4.4 Polymeric/polyester-based nanoparticle as a TB vaccine delivery system

degradation in the host body environment when administered via mucosal immunization. For example, H1 antigen (Ag85A+ESAT6 protein) of Mtb enclosed with PLGA nanoparticles were delivered to the C57BL/6J mice (single dose), and it was found that after the immunization the serum antibody production (IgG) was very high in comparison to the non-conjugated H1. The results obtained after the vaccination of mice were significant and has shown enhanced Th1 immune response [51]. During TB infection, the bacteria get colonize in the respiratory tract mucosa. Therefore, intranasal delivery of vaccine candidate will directly able to generate the mucosal immunity. HspX/ EsxS, a recombinant Mtb specific antigens, adjuvanted with MPLA (monophosphoryl lipid A, synthetic, TLR4 agonist) and encapsulated with PLGA: DDA (poly lactic-co-glycolic acid: dimethyldioctadecylammonium) polymeric nanoparticles. After the preparation, the vaccine was administered intranasally have shown a high level of Th1 and Th17 immune response along with serum antibody generation (IgA, IgG1, and IgG2a). Therefore it was concluded that the designed vaccine candidate loaded with nanoparticles provoke mucosal as well as systemic immunity with or without prime booster BCG and could be beneficial for the treatment of TB. Other than this, researchers have developed polyester beads in such manner so that they can display the Mtb antigens (Rv1626, Rv1789, Rv2032,) on their surface [52]. These beads were then combined with the adjuvant DDA and then delivered into the mice via the subcutaneous delivery system. The results showed a significant increase of IgG1 antibody in the mice immunized with Rv1626 beads in comparison to Rv1789 polyester beads, whereas the antigen Rv2032 with beads have shown an immense IgG2 antibody/immune response. This study showed the importance of polyester beads in TB vaccine designing by displaying the Mtb antigens on the bead surface, which helped in the

Nowadays, the newly designed multiepitope subunit vaccines become accessible for the treatment of various infectious diseases. Polymeric nanoparticles were made by combining small monomeric units. They are mainly used in drug and vaccines delivery due to their small size, flexible, and biodegradable nature. poly lactic-co-glycolic acid (PLGA) is a decomposable polymer which is registered for human use and permitted by the US FDA (Food and Drug Administration) and EMA (European Medicines Agency) [49, 50]. Due to PLGA attractive properties, it can be used as nanoparticle for encapsulating the multiple antigens without their

 



4  Advanced TB vaccine delivery systems and their related immune responses

generation of a potent immune response against TB [53]. Further, by applying the same approach, PHB (polyhydroxy butyrate) a biopolyester were developed and used as nanoparticles for the treatment of TB. This designed nanoparticle-based vaccine when delivered to the mice, it was observed that the Mtb specific antigens Ag85A and ESAT-6, were displayed on the surface of the PHB biobeads and an enhanced Th1 and Th17 immune response was generated with the increased amount of IL-17, IL-6, IFN gamma, and TNF alpha immune-stimulatory molecules [54].

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4.5  Liposome-based TB vaccine delivery

which is a glycolipid and sited in the cell wall of Mtb and mainly reported as immune stimulator (humoral and cell-mediated) but the clinical trial with this vaccine composition is under process [57]. Further, the peptidyl-serine liposome, present on the surface of apoptotic cells, when united with the adjuvant PolyI:C, and encapsulated with Ag85B and ESAT6, Mycobacterium specific antigens, generated a mixed and robust immune response (Th1/Th17-Th2) for Ag85B but not for ESAT6 in the mice model. When the BCG immunized mice were vaccinated with the Lipo-AE recombinant vaccine formulation via aerosol/ mucosal path, it was found that the Lipo-AE composition, enhanced an abrupt inhibition in the growth of Mycobacterium, can induce the mucosal mediated immunity [58]. The other TB vaccine, which is liposome-based is RUTI, formulated by removal of toxic fragments from the Mtb cells. For the treatment of the latent form of TB, chemotherapy is the only option, but when the RUTI vaccine delivered through the liposome, it has shown a significant reduction in the growth of dormant bacilli and minimizes the use of chemotherapy [28].

The liposomes are the double-layered sphereshaped vesicles composed of phospholipids [55]. They are zwitterionic (ability to carry both hydrophilic and hydrophobic charge) make them different from the other delivery systems. The other fascinating feature of the liposome is its nontoxic nature and ability to work as adjuvant as well as a carrier. Liposomes mediated vaccine delivery has shown the great ability to induce both CD4+ and CD8+ T-cell and B-cell immune responses. Moreover, liposomes protect the antigens from host-mediated degradation and enhance their uptake by APCs. Previously, the liposomebased delivery was generally used for drug delivery, but nowadays it has been used as a successful vaccine delivery system. This delivery is widely used as a treatment for TB and has made a remarkable contribution. For example, the vaccine candidates Ag85A and ESAT 6 (early secretory antigenic protein of Mycobacterium) were fused and adjuvanted with IC31, which was delivered into a human during the clinical trial. With this study, it was found that the recombinant vaccine candidate generated an enduring Th1 immune response. But later on, for more enhanced and protective immune responses the vaccine was conjugated with cationic CAF01 [56] (cord factor or trehalose dimycolate) liposome,

4.6  Dendrimer-based TB vaccine delivery system

 

Dendrimers have well known for their chemical as well as biological properties. They are nanosized, synthetic; multilayered branched polymer. They are water-soluble and have substantial antigen encapsulation capability; this distinctiveness makes them a suitable carrier for the delivery of the drugs molecule or vaccine [59]. It has been known that dendrimers when conjugated with adjuvant generated an effective and long-lasting immunity. They are widely used for the delivery of antiretroviral drugs. Some dendrimers which were developed by different companies are L-DOPA (3,4-dihydroxy-l-phenylalanine), poly(amidoamine) polyamidoamine (PAMAM) which is registered

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5.  Vaccine delivery systems against tuberculosis

with Starbust, PPI [Poly (propylene imine)] and PEHAM [poly(etherhydroxylamine)] registered with Astramol and Priostar, respectively [60]. With the help of this delivery system, we can explore the different aspects of delivered therapeutics like the efficacy of the drug, its interaction with lipid bilayers, the pharmacokinetics characteristics including the drug internalization, its bioavailability and their retention time. Dendrimers have been used as a vehicle for the delivery of vaccines in various diseases like cancer, AIDS, rheumatic heart disease, etc. [61]. MAPs (multiple antigen peptides) are radiated peptides branched macromolecules which linked with the peptide bonds together. These polyamino acids generally used in the serological diagnosis and various biological assays. For the delivery of TB vaccine MAPs dendrimers have been used to prevent the diseased condition. This type of carrier system has been used to deliver the peptide-based vaccine, ESAT-6, and it was observed that the single-dose immunization with ESAT-6 epitope can generate a significant immune response and protection against the Mycobacterium [62]. To date, this is the only vaccine candidate of TB which has been reported to deliver via the dendrimerbased delivery system, and future application of dendrimers for delivery of TB vaccine is still awaited.

infectious diseases. With this delivery system, the vaccine (or antigens) can be administered via mucosal as well as intradermal pathways for the generation of effective cell-mediated and humoral immune responses. For instance, the TB antigen fusion protein Ag85B-ESAT-6 when adjuvanted with and CTA1-DD/ISCOM (mucosal vector) and delivered intranasally, it was found to induce strong Th1-specific immune response along with the generation of interferon-gamma which is responsible for both innate and adaptive immunity against the Mycobacterium. This combination of vaccine primarily boosted the prior BCG immunity and heavily contributed to the recruitment of antigen-specific cells to the infection site. Finally, it was concluded that this vaccine, when combined with the adjuvant vector and delivered via a mucosal route, provides the adequate and protective immunization against TB infection [64].

4.8  Virus-like particles (VLPs)-based TB vaccine delivery system

4.7  Immune stimulating complexes (ISCOMs) as a TB vaccine delivery system

It is well established that adjuvants containing vaccines are more productive, stable as compared to the classical vaccines. Viruslike particles (VLPs) exhibit excellent adjuvant properties and also imitates the structure of the virus from which they are derived as well. Viral antigens are presented by VLPs in more accurate conformation compare to monomeric structural proteins. They can activate both innate and cognate immune responses. Therefore recombinant VLPs are considered as a potent and effective vaccine delivery system [65]. Hepatitis B virus core protein-VLPs (HBc-VLPs) are nanosized particles and capable of inducing both cellular and humoral immune responses. In recent research, it has been shown that the use of HBc-VLPs bearing mycobacterial antigen CFP-10 fusion protein as vaccine delivery system helps in increased immune response compared to combinations of native antigen in Balb/c mice [66].

ISCOMs are the lipid-based immunostimulant complexes (ISCOMs) and possess the ability to work as adjuvant and vehicle for the antigens. These adjuvant particles are developed by combining the mixture of phospholipids, cholesterol, and saponin in the proper fraction [63]. They are about 40-60 nm in size and in the aqueous solution they look like as cage structures with a hollow center. ISCOMs widely used for the efficient delivery of the vaccine candidates in several

 



4  Advanced TB vaccine delivery systems and their related immune responses

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FIGURE 5.2  Schematic representation of vaccine delivery systems against tuberculosis.

4.9  Virosomes-based TB vaccine delivery system Virosomes are mainly used as a carrier for the delivery of vaccines or drugs. They have single layer outer covering made up of phospholipid and glycoproteins, which helps them to interact with the target cells. The interior part of the virosome is filled with a cavity, which clutches the macromolecules or drugs inside for their targeted delivery. Virosomes have been used for the vaccine delivery because of their noninfectious, adjuvant (for the immune system activation), biodegradable, and nonreplicative nature in the host. They are responsible for the induction of both B-cell, CD4+, and CD8+ immune responses. It was reported that the delivery of therapeutics would be more effective when the virosomes reconstituted in the buffers (saline) and delivered via intramuscular, mucosal, and parenteral routes [67]. There are many types of virosome, which have been used for vaccine delivery such as HIV, influenza, and noninfluenza

virus, hepatitis virus, and Sendai virus virosomes. The use of Sendai virus, also known as HVJ or hemagglutinating virus of Japan, as virosome has been reported for the treatment of TB [68]. The DNA encoding Ag85A antigen, conjugated with pAAVCMV plasmid, enclosed into the Sendai virosome and injected into mice via an intramuscular path. The outcome after the mice immunization showed an increased level of Th1 mediated cytokine response and activated the cytotoxic activity of CTL, and NK cells were observed. From this study, it was concluded that Sendai virus virosomes could be utilized as an efficient vehicle for the delivery of therapeutic agents against the TB infection [69] (Fig. 5.2).

4.10  Role of adjuvants in TB vaccine formulation and their delivery

 

Adjuvants are known to enhance the immunogenicity of the vaccines. They play a crucial role in the next-generation vaccine preparations because antigen alone could not be able to induce

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5.  Vaccine delivery systems against tuberculosis

the potential immune response for an extended period. There is a possibility that some antigens unable to reach the lymphatic regions and incapable of initiating the signaling cascades, in that scenario adjuvants show their capability to minimize these drawbacks. The adjuvants promote the uptake of antigen to DCs, and their maturation helps in the production of healthy immune responses (humoral and cell-mediated). In short, the injection of adjuvants at the immune privilege sites makes way for antigens to reach directly to the APCs. There are many few adjuvants that are approved by the FDA and licensed for human use. The highly immunogenic and synthetic approved adjuvants, which generally used for vaccine formulation are GLA (glucopyranosyl-lipid A), GLA-SE, MPL (monophosphoryl lipid A) [70], etc. The adjuvant GLA-SE, a TLR-4 agonist is known to activate both MyD88 and TRFI dependent signaling cascades. It has been used for the development of polyprotein vaccine candidates (ID93+GLA-SE), which is the combination of four Mycobacterium antigens. This combination of adjuvant and antigens, when administered intramuscularly, has shown significantly enhanced immunogenicity and protection in the infected mice models [71]. Further, polymers of the acrylic acid or polymeric carbomers have been designed and utilized by the pharma companies to regulate the release of drugs. Vaccination with this adjuvant helps to achieve both Th1 and Th2 immune responses along with the stimulation of vital immunostimulatory molecules like cytokines, interleukin-2, and 4 and interferon gamma [39, 72]. CpG 7909, TLR9 agonist has also been used as an adjuvant to improve the efficacy and immunogenicity of the vaccine candidates. This adjuvant was mainly used in the formulation of the Hepatitis B vaccine, but it was also used for TB vaccines. ESAT6-Ag85A recombinant fusion protein when given in combination with CpG7909 in vaccinated mice it demonstrated boosted immunogenicity efficacy but unable to provide a strong defense against TB infection. AS01E is the

other adjuvant which is used for the formulation of TB subunit vaccines such as M72: AS01E and ID93+AS01. According to the recent data, it was found that the intramuscular delivery of M72: AS01E during the latent TB and HIV coinfection (HIV-negative), more than 50% of the infected population has shown effectiveness by inducing a robust Th1 immune response [73].

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[45] A.K. Salem, Nanoparticles in vaccine delivery, AAPS J. 17 (2) (2015) 289–291. [46] S. Singha, et al. Nanoparticles for immune stimulation against infection, cancer, and autoimmunity, ACS Nano. 12 (11) (2018) 10621–10635. [47] D. Raze, C. Verwaerde, G. Deloison, E. Werkmeister, B. Coupin, M. Loyens, P. Brodin, C. Rouanet, C. Locht, Heparin-binding Hemagglutinin Adhesin (HBHA) is involved in intracytosolic lipid inclusions formation in mycobacteria, Front Microbiol. 9 (2018) 2258. [48] E. Stylianou, et al. Mucosal delivery of antigen-coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice, Eur. J. Immunol. 44 (2) (2014) 440–449. [49] J.-M. Lü, et al. Current advances in research and clinical applications of PLGA-based nanotechnology, Expert Rev. Mol. Diagn. 9 (4) (2009) 325–341. [50] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and gene delivery to cells and tissue, Adv. Drug. Deliv. Rev. 55 (3) (2003) 329–347. [51] A. Malik, et al. Single-dose Ag85B-ESAT6-loaded poly(lactic-co-glycolic acid) nanoparticles confer protective immunity against tuberculosis, Int. J. Nanomedicine 14 (2019) 3129–3143. [52] P.R. Reyes, N.A. Parlane, D.N. Wedlock, B.H. Rehm, Immunogencity of antigens from Mycobacterium tuberculosis self-assembled as particulate vaccines, Int. J. Med. Microbiol. 306 (8) (2016) 624–632. [53] S.C. Derrick, et al. Immunogenicity and protective efficacy of novel Mycobacterium tuberculosis antigens, Vaccine 31 (41) (2013) 4641–4646. [54] N.A. Parlane, et al. Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis, Clini. Vaccine Immunol. 19 (1) (2012) 37–44. [55] R.A. Schwendener, Liposomes as vaccine delivery systems: a review of the recent advances, Ther. Adv. Vaccines 2 (6) (2014) 159–182. [56] E.M. Agger, et al. Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements, PLoS One 3 (9) (2008) e3116. [57] R.B. Bekale, et al. Mycobacterium Tuberculosis and interactions with the host immune system: opportunities for nanoparticle based immunotherapeutics and vaccines, Pharmaceut. Res. 36 (1) (2018) 8. [58] G.R. Diogo, et al. Immunization with Mycobacterium tuberculosis antigens encapsulated in phosphatidylserine liposomes improves protection afforded by BCG, Front. Immunol. 10 (2019) 1349–11349.

 

[59] E. Abbasi, et al. Dendrimers: synthesis, applications, and properties, Nanoscale Res. Lett. 9 (1) (2014) 247–1247. [60] Q. Cai, et al. Synthesis and properties of star-shaped polylactide attached to poly(amidoamine) dendrimer, Biomacromolecules 4 (3) (2003) 828–834. [61] J. Yang, et al. Cell-penetrating peptides: efficient vectors for vaccine delivery, Curr. Drug Deliv. 16 (5) (2019) 430–443. [62] K. Sadler, J.P. Tam, Peptide dendrimers: applications and synthesis, Rev. Mol. Biotechnol. 90 (3) (2002) 195–229. [63] H.X. Sun, Y. Xie, Y.P. Ye, ISCOMs and ISCOMATRIX, Vaccine 27 (33) (2009) 4388–4401. [64] C.S. Andersen, et al. The combined CTA1-DD/ISCOMs vector is an effective intranasal adjuvant for boosting prior Mycobacterium bovis BCG immunity to Mycobacterium tuberculosis, Infect. Immun. 75 (1) (2007) 408–416. [65] D. Dhanasooraj, R.A. Kumar, S. Mundayoor, Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles, Int. J. Nanomed. 8 (2013) 835–843. [66] J. López-Vidal, et al. Improved production efficiency of virus-like particles by the baculovirus expression vector system, Plos One 10 (10) (2015) e0140039. [67] H. Liu, et al. Virosome, a hybrid vehicle for efficient and safe drug delivery and its emerging application in cancer treatment, Acta Pharmaceutica 65 (2) (2015) 105–116. [68] Y. Kaneda, et al. Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system, Molecul. Ther. 6 (2) (2002) 219–226. [69] I.-h. Song, Immunogenic responses by tuberculosis vaccination with Sendai-virosomal pDNA encoding mAg85A (Doctoral dissertation), Graduate School, Yonsei University. [70] F. Yu, et al. Nanoparticle-based adjuvant for enhanced protective efficacy of DNA vaccine Ag85A-ESAT-6IL-21 against Mycobacterium tuberculosis infection, Nanomedicine 8 (8) (2012) 1337–1344. [71] R.N. Coler, et al. The TLR-4 agonist adjuvant, GLA-SE, improves magnitude and quality of immune responses elicited by the ID93 tuberculosis vaccine: first-in-human trial, NPJ Vaccines 3 (1) (2018) 34. [72] G. Krashias, et al. Potent adaptive immune responses induced against HIV-1 gp140 and influenza virus HA by a polyanionic carbomer, Vaccine 28 (13) (2010) 2482– 2489. [73] O. Van Der Meeren, et al. Phase 2b controlled trial of M72/AS01E vaccine to prevent tuberculosis, N. Engl. J. Med. 379 (17) (2018) 1621–1634.

C H A P T E R

6

Inhalable polymeric dry powders for antituberculosis drug delivery Suneera Adlakha, Kalpesh Vaghasiya, Ankur Sharma, Eupa Ray and Rahul Kumar Verma Institute of Nano Science and Technology (INST), Mohali, Punjab, India

Abbreviations

TB (TDR-TB) cases has further exacerbated the problem. Hence, there is a strong demand for a much simpler, affordable, and effective medication approach. Due to extended treatment, there is poor patient adherence and appearance of severe side effects which eventually fuel the development of multidrug resistance (MDR). Pulmonary delivery of drugs for lung infections employing DPIs is a rapidly emerging approach to treat and cure TB completely [4]. As compared to conventional pulmonary delivery for ailments such as asthma and chronic obstructive pulmonary disease (COPD), TB requires a high dosage of drugs to be delivered to the lungs. However, such a high dose delivery requires and depends on the successful design of the inhaler device and the formulation of fine aerosolizable powders. Thus, particle engineering methods play a vital role in the development of dry powder formulations [5]. Delivery via pulmonary mode is noninvasive, evades the first-pass metabolism in the liver and enables targeting of therapeutic agents to the target site. DPI is suitable for highdose which may decrease dose requirement and thus accompanying side effects. A potent

Tuberculosis (TB) Dry powder inhalers (DPIs) M Tb: Mycobacterium tuberculosis

1 Introduction TB has plagued mankind for thousands of years [1] and to date remains one of the leading health problems in the world, with an estimated 8 million new cases and at least 2 million deaths occurring every year due to it [2]. M. tuberculosis is communicated mainly by small airborne droplets or nuclei produced by coughing or sneezing of a person already affected with pulmonary or laryngeal TB. Unlike common respiratorybased infections that usually require one or two weeks of antibiotics treatment, successful treatment of TB requires a minimum of six months of oral antibiotics administered on a daily basis. The long duration and unpleasant side effects of the drugs often lead to high amounts of noncompliance and an increased mortality rate [3]. The emergence of totally drug-resistant

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00006-0

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Copyright © 2020 Elsevier Inc. All rights reserved.

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6.  Inhalable polymeric dry powders for antituberculosis drug delivery

DPI with great fractions of respirable particles combined with a cost-effective inhaler device is an attractive platform for TB drug delivery. Noninvasive therapy using DPI also eludes the need for injections in the case of second- and third-line anti-TB drugs (ATDs), allows a high drug concentration in the lungs thus reducing the frequency of dosing, directly targets alveolar macrophages which harbor high concentration of M. tuberculosis. Direct absorption of active agents into the systemic circulation aids to bypasses the hepatic first-pass metabolism thus avoiding drug degradation in the acidic environment of the stomach [6].

regimen. It finally leads to treatment failure and relapse in most of the cases. Incomplete management also results in drug-resistance which further requires the use of more toxic, expensive, and less effective second- or third-line drugs. Adherence to long-term antituberculosis therapy is vital for preserving acceptable blood drug levels. Therapeutic approaches for handling and managing drug-resistant TB are quite tedious and burdensome, because of the poor, less-effective, and toxic alternatives available to the firstline drug therapies [7].

2  Challenges with current anti-TB therapies

3  Rationale of pulmonary drug delivery in TB

Current treatment is majorly based on the principles of combination chemotherapy. Multiple drugs are being used both to increase efficacy and to prevent the emergence of resistant strains of organisms. The greatest hurdle for optimal TB therapy with the currently available drugs is a long time required to achieve the desired cure. The requirement for this long duration is generally attributed to the physiologic heterogeneity of TB causing bacteria. It appears that though one or more of bacterial subpopulations, which are genetically drugsensitive, display phenotypic drug-resistance in response to the new environmental signals and thus able to survive long periods of treatment [4,5]. These bacteria are often called "persisters". These bacteria may or may not be in the same physiologic state as the mycobacteria found in the majority of individuals infected with M Tb infection. One of the main reasons for the emergence of drug-resistant strains is the contact of mycobacteria to subtherapeutic levels of one or even more antibiotics. The prolonged duration of therapy and administration of high doses of drugs often lead to toxicities, intolerance, and nonadherence to the complete treatment

More than 80% of reported TB cases are that of pulmonary TB where large drug dosages are essential to be directed because only a small portion of the total administered dose reaches the lungs after oral administration. Current injectable or oral ATDs regimens are well known, recognized, and are relatively inexpensive and affordable by the common population. One of the major constraints in the conventional treatment is that patients need to take large number of tablets, usually 10-15 per day for about 2 months (known as initial phase of treatment), followed by 3-9 tablets daily for 4-6 months (i.e., continuation phase). This small fraction of drug is cleared in few hours thus explaining the dire necessity to administer multiple ATDs on a regular interval basis, a regimen that today the bulk of TB patients find difficult and hard to adhere to. So, we require an effective, vigorous, and robust system to pacify technological drawbacks and advance the effectiveness of therapeutic drugs which remains a chief challenge for pharmaceutical technology. As lungs are the chief location and site of M Tb infection, administration by the pulmonary route could be an effective and targeted approach to increase the effectiveness of the medications employed. This strategy could provide less side effects as

 



4  Feasibility of lung as a portal for delivery of ATD

compared to other routes [8,9]. Administration of drugs by the pulmonary route to the lungs permits higher drug deposition in the vicinity of the affected lesions also thus more relief than anticipated from the normal administration of drug doses. Lung lesions contain enormous amounts of bacteria and are poorly vascularized and are fortified with thick fibrous tissue; conventional therapy either by the oral or parenteral routes helps to provide subtherapeutic levels of ATD drugs to these extremely sequestered organisms. Complementing conventional therapy with that of inhaled ATD therapy allows therapeutic concentrations of drugs to penetrate efficiently into the lung lesions and help to treat the residing mycobacteria. ATD delivery systems, which can be efficiently administered by the pulmonary route and also avoid daily drug dosing, would be a huge development because it would help in: (1) drug delivery directly to the diseased organ and tissue; (2) direct targeting to alveolar macrophages which are employed by the mycobacteria as a safe and secure site for prolonging their survival; (3) improve patient compliance; and (4) reduce systemic toxicity of the administered drugs. For inhaled TB treatment, drugs should target alveolar macrophages that majorly harbor microorganisms and maintain high drug concentration at the infected site. Inhaled drug delivery offers this prospect and has the potential to achieve high concentrations of pharmacologic agents in the lungs while producing adequate systemic concentrations through alveolar-capillary absorption to treat extrapulmonary sites of infection. It would seem that a localized delivery method would be attractive for a disease that is very often restricted to the lung [10]. Nanoparticle-based ideology has shown convincing and better treatment, promising outcomes for chronic infectious diseases. Inhalable particles which possess an improved capability of particle delivery, adherence to mucosa, and thus more amount of drug delivery to the lungs [11,12]. In a study conducted on the guinea pig TB preclinical model, a

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small number and dosage of administrations of aerosolized nanoparticles which were required to reach the same effect as those attained with ATD administered freely daily by the oral route. Intrapulmonary aerosol delivery of drugs is being considered as a promising alternative strategy able to reduce symptoms as well as doses, dose frequency and systemic side effects [13,14]. Using dry powders, reduces the number of tablets to be taken to as few as three or four per day for the course of the treatment. Consuming fewer pills per day to swallow makes the treatment easier, and minimizes the probability of splitting the doses. Used as a primary or adjunct to the current standard therapy, inhaled therapy holds the key as a potential new delivery mode and has improved health benefits. These likely to include reduced treatment duration and possibly limited drug resistance. Furthermore, existing treatment regimens already approved by the WHO having antimycobacterial activity but cannot be given systematically due to high toxicity, could be used if repurposed for the inhalation route. In the same way, big pharma companies are interested in developing and propagating inhaled antibiotics for different pathologies, paving the way for inhaled therapies for TB.

4  Feasibility of lung as a portal for delivery of ATD

 

Pulmonary route as a mode for drug delivery is gaining considerable prominence in the present day research arena as it helps in targeting the drug directly to lung both for local as well as systemic action. Large alveolar surface area, less thickness of the epithelium barrier, and a widespread vascularization area make the pulmonary route an ideal route for medication administration [15] (Fig. 6.1). Furthermore, the concentration of drug-metabolizing enzymes is present in a lesser amount in the lungs as compared to the liver and gastrointestinal tract (GIT). The architecture of the airways and its

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6.  Inhalable polymeric dry powders for antituberculosis drug delivery

FIGURE 6.1  Pulmonary delivery of nanoparticle carriers and alveolar targeting.

properties makes it well suited for efficient drug delivery, deposition, and absorption. The respiratory tract of lungs begins from the nose, which goes along with pharynx, larynx, trachea that divides into left and right bronchi each of which further divides into smaller bronchioles finish by terminal bronchioles with an alveolar sac. The delivery and deposition of drugs via pulmonary delivery is a complex process which depends upon the intricate lung geometry and its physiological condition. The movement and deposition of inhaled particles in the lungs are significantly influenced by the architecture of the airways. The divergence, branching, and change in diameters of cavity diameter of the airway tubes influence the mechanism of drug deposition and hence it affects therapeutic efficacy. Pathophysiological variations in respiratory tract induced by infections may change the delivery and deposition pattern of inhaled drugs. The therapeutic efficacy of inhaled drug gets affected by the diseased condition of the respiratory tract as it may change the width of the airway lumen which defines the portion inhalable dose deposited in different areas of the lungs. The aerodynamic and physicochemical properties of drug particles or formulations also influence the movement of the drug inside the lungs. Drug particles of a precise size and shape will deposit

in the particular region of the lungs [15,16]. Particle size, breathing pattern, airflow, etc., decide the mechanism of deposition of drug particles in the lungs, i.e., sedimentation, interception, impaction, or diffusion. Thus, it is important to predict the deposition of drug in lung airways and determine the physicochemical and aerodynamic characteristics of the drug to develop a suitable formulation for ideal delivery. The major portion of drug/formulation particles having size-range ≥5 µm gets accumulated in upper portion respiratory tract by inertial impaction, while smaller particles (≤1 µm) get exhaled during breathing (Fig. 6.2).

5  Pulmonary delivery of ATD

 

Historically, aerosols were employed for the treatment of obstructive and congestive airway diseases, such as that of asthma, COPD, but in the past few decades, their use has been expanded to treat lung-based infections associated with TB, cystic fibrosis, and other respiratory disorders [17]. Current inhaler devices that are commonly employed for inhaled therapies for treating humans effectively and rationally, they are often limited by the size of the doses they can deliver with one puff which is administered.



5  Pulmonary delivery of ATD

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FIGURE 6.2  Mechanism of dry particle deposition in lungs.

These small dose sizes are suitable for potent therapeutics and widely used for the treatment of asthma, but are significantly smaller for the doses required to deliver therapeutics for diseases such as TB. Thus, there are few reports for their potential and use in TB treatment or as vaccines. The first documented usage of inhaled therapy for TB in humans was the work of Paraf et al. in 1953 using liquid aerosols of antibiotics [18], while the first usage of the inhaled vaccine against TB was reported by Rosenthal et al. [19]. For the delivery of antibiotics and vaccines via inhalation, liquid aerosol formulations had been used to be generated by nebulizers. This efficacy was lowered significantly when nebulization was used to deliver aerosol to rodents, because of their smaller lung capacity and breathing rate. The use of nebulizers to deliver vaccines has also been reported to decrease the potency of vaccines because of the high shear force produced by the nebulizer to produce the aerosol and delivery to the lungs [20]. An additional limitation of utilizing solution-based formulation products to deliver therapeutics against TB is the formulation of the therapeutic compound by itself. The properties and characteristics of the

 

formulation are majorly limited by parameters such as solubility, stability, and sterility of drugs in the solution, as well as the condition requirements for storage. All these drawbacks and limitations commonly observed have encouraged researchers to find a substitute and alternate formulations therapeutics and strategies so as to deliver inhaled therapeutics efficiently. Dry powder formulations are the most stable from all inhaled dosage forms and can be delivered as DPIs. Their popularity is such that drugs which are previously formulated into metered dose inhalers are now being manufactured and sold as DPIs. Advantages of DPIs over nebulizers include those of low cost, portability, better control of the delivered dose. DPIs can also be classified as single-dose or multi-dose devices, depending on the number of doses that can be dispensed, or into passive or active, based on the mechanism of powder dispersion. The efficiency of delivery for passive DPIs is also greatly influenced by the mechanism of dispersion and the inspiratory pressure. The efficiency of delivery is more for active that is battery-driven DPIs since the powdered dispersion does not depend on the inspiratory pressure of the patients.

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6.  Inhalable polymeric dry powders for antituberculosis drug delivery

6  Formulations for DPIs DPIs contains dry powdered formulations of the drug, where drug/formulation particles (50%). SLN are usually taken up by the lymphatic system after inhalation which undergo phagocytosis by macrophages; hence, inhalation can be an effective route to deliver particles to the alveolar macrophages where mycobacterium harbors [33].

Pulmonary delivery of extremely small lowdensity nanoparticles might be exhaled during the breathing process. This problem can be resolved by processing nanoparticles into the inhalable hybrid nano-in-micro system with appropriate aerodynamic properties. Goyal and group developed inhalable ATD drugs loaded HPMC nano-aggregates for sustained delivery of drugs to the lung [27]. Sharma et al. developed micron-sized inhalable hybrid nano-inmicroparticles with nano-scale properties to improve the delivery of anti-TB-peptide to the lungs and enhance its stability [28]. Another study shows the inhalation delivery of human IgG using nano-in-micro prepared by spray drying technology, which showed a 35-day release [29]. Goyal and his research group designed nano-embedded microparticles for the pulmonary delivery of ATD. Rifampicin and isoniazid were loaded in guar gum polymer using the spray drying technique. Particles obtained were uniform in size, smooth surface, and appropriate aerodynamic properties. Efficacy studies showed almost five times reduction of the CFU bacteria as compared to the control group [30].

7.3  Solid-lipid nano particles Solid-lipid nano particles (SLN) are submicron drug delivery carriers ranging from 50 to 1000 nm, which are composed of solid lipid, dispersed in a surfactant solution. It consists of a surfactant-stabilized solid lipid core matrix that is used to solubilize that lipophilic drugs. It usually contains a hydrophobic solid core and phospholipid coating over it. It mainly comprises

7.4 Liposomes

 

Liposomes are spherical vesicles consisting of one or more phospholipid bilayers (unilamellar or multilamellar concentric bilayers) in which one part of the molecule is hydrophilic and the other hydrophobic. Patil et al. (2015)



8  Inhalation delivery devices for DPI

developed inhalable freeze-dried liposomes for poorly soluble rifampicin for improved lung delivery of the drug. Drug-loaded liposomal formulation was freeze-dried and consequently evaluated for aerodynamic characters, surface morphology, in vitro dissolution, in vitro antitubercular activity, and pharmacokinetic studies. This study revealed that pharmacokinetic profile of the liposome-encapsulated drug was better than the free drug [34]. Bhardwaj et al. designed and validated ligand-appended liposomes for the pulmonary delivery of ATD. Mannan ligand was conjugated on drug-loaded liposomes to enhance alveolar targeting in the lungs. Liposomes showed high drug entrapment efficiency and prolonged release of rifampicin. Aerodynamic properties demonstrated its suitability for pulmonary delivery.Vyas et al. designed inhalable ligand-decorated rifampicin-loaded liposomes for enhanced targeting of alveolar macrophages. Drug-loaded liposomes were coated maleylated bovine serum albumin and O-steroyl amylopectin which showed preferential uptake in the lung macrophages and enhanced cellular uptake by ∼3.5 times [35].

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treatment. The optimized microparticles were effective for the delivery of rifampicin to the lung and improved targeting to alveolar macrophages. The inhalation delivery of Rif-MP demonstrated higher concentrations of the drug found inside the alveolar macrophages compared to the systemic delivery of free drugs. Garcia-Contreras et al. developed porous microparticles of capreomycin for inhalation delivery against experimental TB in in a guinea pig model. These particles were prepared by spraydrying technique and characterized optimum aerodynamic parameters. The bioavailability of capreomycin after inhalation was higher compared to oral administration. The animals that received inhalation delivery of capreomycin significantly lowered the bacterial CFU count in the lungs and improved histopathology of animals [36]. We made attempt to fabricate nitric oxidereleasing microparticles for pulmonary delivery which can directly act on where the TB bacteria actually resides, validated formulation, analyzed their therapeutic efficacy in cell lines and laboratory animal models of TB. Further, we made an attempt to elucidate the biochemical and cellular mechanisms of antimicrobial action [37].

7.5 Microparticles 8  Inhalation delivery devices for DPI

The use of inhalable polymeric microparticles to deliver ATDs has been reported by several investigators. Due to their biodegradability, biocompatibility, and controlled release properties, poly (lactide-co-glycolide) has been a popular choice as a drug carrier. The use of polymeric microparticles to deliver ATDs by different routes (injectable, oral, and aerosol) has been reported by several investigators. Because of its biodegradability and biocompatibility, poly (lactide-co-glycolide). PLA, chitosan, alginates, etc. has been a popular choice as drug carriers for inhalation delivery. The inhalation delivery of rifampicin-loaded microspheres showed encouraging results against TB bacteria but it is essential to combine other ATD drugs as disease needs multidrug therapy for complete  

Selection of device for delivery of drugs into the lungs is an important and requisite factor in the designing of formulation. If the drug is planned to be delivered to a specific organ, i.e., lungs, then the selected device must be capable of generating and delivering the particles or droplets having a specific aerodynamic diameter. Devices commonly used for respiratory lung delivery are nebulizers, metered-dose inhalers, and DPIs. In recent years, substantial progresses have been made for designing of novel as well as advanced devices. However, still the devices are much less discovered and explored than that of powder type formulation [58]. A drug may also be delivered to the lungs directly, i.e., without

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6.  Inhalable polymeric dry powders for antituberculosis drug delivery

prior aerosolization, employing a device called an insufflator. Compared to that of a nebulizer, a DPI is more well organized, efficient in terms of delivery, and also less time consuming [59]. The selection of an appropriate inhalation device depends on parameters, such as the nature of the drug and its prepared formulation, the intended site of action, and the pathophysiology of the lungs. Aerosols based on mechanically generated vibration technologies have been used successfully to deliver drugs to lungs and are currently being used for protein- and peptidebased pharmaceuticals. In order to aerosolize the drug powder, particles are de-agglomerated using the impaction of external forces. The relative effect of air turbulence and mechanical impaction for controlling powder dispersion in the device is still quite unclear. Recent applications of computational fluid dynamics have been helpful in the designing and development of DPI devices. DPIs are popular devices used to deliver drugs. There is an extensive range of passive breath driven and active power-driven single or multiple dose DPIs currently available in the market for use. Most of the devices are passive breath-actuated devices, which depend upon the patient's inspiration rate to provide adequate airflow for de-aggregation and aerosol formation of the formulation. The major advantage of these devices is that it does not need coordination with the breathing of the patients. The available passive DPIs in the market are the Rotahaler(tm) and the Spinhaler(tm). Rotahaler(tm), is a single dose capsule-based device, where actuation pierces the capsule and releases the powder which is passively inhaled by the patient. In Spinhaler(tm) device, powderloaded capsule is opened in the device to release the formulation which is passively inhaled by the inspiratory flow. DPI are also be categorized into single-unit dose, multiunit dose, and multidose reservoirs. The single unit utilizes disposable discrete capsules while the multiunit DPI uses a strip of capsules sealed with individual doses of powder. Multidose reservoir devices hold the

dry powder in bulk, and individual actuation releases a specified amount of powder [60].

9  Clinical trials

 

Introduction of initial drugs for TB treatment around 50 years ago such as streptomycin, paraaminosalicylic acid led to optimism that the disease TB could be controlled, if not completely eradicated. The likelihood of any given product coming successfully through various phases of the drug development process is limited; thousands of compounds screened give only a handful which finally make it to the preclinical phase, and a fraction of those which finally enter human experimentation. As expected, the number of products finally entering clinical development is considerably low than those in earlier stages of the pipeline. Clinical trials conducted over the past 10 years, data pertaining to that only one paper has been published on a clinical trial using inhalation therapy formulated for targeting pulmonary TB treatment. Dharmadhikari and colleagues conducted a Phase I clinical study to examine the safety as well as tolerability of capreomycin in a total of 20 healthy subjects and measured its pharmacokinetic parameters. It was formulated by using spray drying along with leucine and administered single dosages of 25 mg, 75 mg, 150 mg, or 300 mg. Five subjects experienced mild/moderate cough, but overall, the inhaled drug formulation was well tolerated by the body. No major changes in pulmonary, renal, and liver functions were detected post inhalation of the formulation. Inhalation of 300 mg of powder permitted a systemic concentration of 2.3 µg/mL as compared with that of 32.0  µg/mL when only 1.0 g of the drug was administered by intramuscular route. A high drug concentration was expected in the lungs after inhalation than after intramuscular delivery. The half-life (t1/2) of capreomycin was also extended to 4.8 h as compared with that of 2-3 h after intramuscular administration.



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11 Conclusions

These findings are important and clearly demonstrate that inhaled capreomycin could achieve high drug concentrations at the infected site and longer drug retention in the systemic circulation, which would have the potential to reduce drug dose as well as dosing frequency, and consequently drug associated adverse events. However, the inhaled pharmacokinetic data should be interpreted with caution as the studies were performed on healthy volunteers and they may not necessarily be able to replicate in TB cases. There is still a gap of understanding on the respiratory physiology of TB patients, and this aspect needs further elaborate clinical investigations to address the fate of nano and other drug particles deposited in the lungs.

10  Future of polymeric powder-based drug development for TB

the drug aerosol in the lungs is desired. An indepth understanding of the lung and airways of patients and the correlation with the aerosol deposition is crucial to facilitate the success of administered inhaled ATD therapies. Currently, extended therapeutic regimens for the duration of at least 6 months are essential and necessary for optimal clinical outcomes and aid to prevent the emergence of drug-resistance. Inhaled antibiotics also play a role as adjuncts in oral therapy by providing a high concentration of drugs at the infected site. Multiple drug dosing may be required for an optimal pulmonary concentration; it also depends on the powder formulation and pharmacokinetics of the administered drug dosage. The frequency of drug dosing and its effects still need to be investigated extensively. Not all drugs or vaccines are ideal to be given as an inhaled therapy and there should be careful selection by testing. These candidates should be highly effective and able to reduce the dosage frequency when given by inhalation compared to the conventional route of administration. Cheap manufacturing and packaging procedures along with inexpensive inhaler devices will be advantageous. The major focus of current efforts in TB drug development is the identification and registration of shorter, and simpler treatment regimens. Development of a 2-3-month regimen with once-weekly dosing of three to four drugs would result in decreasing the duration of treatment from the currently recommended 28 weeks to 8-12 weeks, and from approximately 130 doses of a combination regimen to 10. Such changes should have a significant positive impact on the control and cure of the disease by improving patient adherence and inhibiting the development of drug resistance.

Current drug delivery systems are not much efficient from the perspective of their low and less efficient intracellular targeting ability, thus leading to a lack of and compromised therapeutic efficiency for TB, which mainly has its locus inside the macrophage cells. Thus, TB treatment research should be done in such a way that it maximizes the intracellular targeting. Promising results obtained from a plethora of in vivo studies and the Phase I human clinical trials give evidence for performing large-scale efficacy experiments and evaluating the safety of inhaled ATD. There are many challenges that timely need to be addressed and taken care of. There is a lack of complete and thorough understanding of lung and airways physiology of TB patients. The airflow of patients differs in the healthy population and affects the deposition of particles in the lungs. The intended region for powder accumulation and deposition is also determined by the formulation and device designs. Tuberculous granulomas may be in some cases distributed throughout the lung surface and therefore more diffused deposition of

11 Conclusions

 

The advantages of aerosol administration of drugs include higher drug loading at the site of infection thus reducing and limiting systemic

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6.  Inhalable polymeric dry powders for antituberculosis drug delivery

side effects, enhanced protection with the help of conventional drugs. These benefits are not only based in clinical but are also pharmaceutical thus offering stable formulations and dosage forms that can be stored for longer durations without requiring refrigeration and have a high potential for their usage in regions of the world where the burden of TB infection is quite high. TB has a very big impact on developing nations. In this scenario, for the effective control of the TB, drug-resistant TB acts as a major challenge. The aim is to find a solution to this by creating or producing better and more effective drugs that lessen the period of treatment, reduce drug toxicity, and have longer bioavailability. Till now, apart from a few drugs such as quinolones and rifamycins, no major contributions have been made to the ATD therapy and certainly an effective TB vaccine has also remained equally elusive. The goal is to find out a solution to eradicate the transmission of the causative organism but this is difficult, multifarious, and thorny due to the difficulty of diagnosis, MDR, and patients' low compliance to treatment. Advancements in the nanoparticle-based delivery systems represent a commercial, practical, and most promising substitute for potential TB chemotherapy. The superior drug bioavailability and therapeutic usefulness are even at low therapeutic doses of the formulation and period of chemotherapy can also be reduced. All these aspects are vital in substantially curtaining the expenditure of treatment, reducing interactions with anti-HIV drugs, and improved management of MDR-TB and latent TB. Pulmonary administration employing powdered aerosols is a new approach for the treatment and vaccination against, TB especially targeting for MDR cases. Optimized powder formulation coupled with a suitable inhaler device is critical for effective delivery and intended clinical outcomes. Substantial amount of powdered formulation works has been undertaken over the past few decades aimed at delivering inhaled TB therapeutics to the infected lung site. A common

theme is to develop carrier-free formulations so as to enhance the drug load required for effective high-dose antibiotics. It also helps to avoid any carrier-based toxicity. Combination drug formulations necessary for effective treatment and for reducing the emergence of drug resistant strains. Formulation of micro- or nano-particles with a slow-release profile promotes phagocytosis by M Tb-infected alveolar macrophages. An inhaler device having an "open-inhale-close" routine enhances patient's compliance, but both generation as well as administration of a high powder dose still remains a challenge. It is also necessary for the formulation and device to be cost-effective especially for countries having a high burden of TB cases. Though the formulation development and preclinical evaluations are still in their advanced stages, clinical trials are lagging behind, partly due to the safety concerns of administered inhaled drugs or vaccines. However, if the biosafety hurdles can be diminished and the TB drugs or vaccines efficiently delivered, then TB therapy has the potential to contribute to the global elimination of TB cases.

References

 

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C H A P T E R

7

Liposomes-and niosomes-based drug delivery systems for tuberculosis treatment Ali Ibrahim Bekraki Faculty of Pharmacy, Jinan University, Tripoli, Lebanon

1 Introduction

kidneys, brain, spine, liver, skin, genitourinary system, and intestine [3].

Tuberculosis (TB) caused predominantly by Mycobacterium tuberculosis remains a global health problem and a leading cause of deaths among adults in developing countries [1]. Despite the discovery of very effective and affordable drugs against human immunodeficiency virus (HIV) infection, TB still remains the primary factor of mortality and socioeconomic disaster for millions of people around the world, including both HIV negative and HIV positive people [2]. TB and HIV are closely associated with TB being the most common cause of AIDSrelated death [3]. Being an airborne infectious disease, TB is most commonly transmitted from a person with infectious pulmonary TB to others by droplet nuclei which are aerosolized by coughing, sneezing, or even speaking [4]. About one-third of the world’s population is estimated to have been exposed to TB bacteria and potentially affected [2]. It is important to remember that TB typically affects the lungs, but it also can affect other organs such as lymph nodes, bones,

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00007-2

2 Epidemiology TB occurs in every part of the world. After HIV/AIDS, TB is the most commonly occurring and fatal infectious disease [5]. In 2018, the largest number of new TB cases occurred in the South East Asia region, with 44% of new cases, followed by the African region, with 24% of new cases, and the Western Pacific with 18% [6]. In 2018, 87% of new TB cases occurred in the 30 high TB burden countries. Eight countries accounted for two-thirds of the new TB cases: India, China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh, and South Africa [6]. In 1993, World Health Organization (WHO) declared TB a global health emergency [7]. At present, more than 2 billion people are infected worldwide with M. tuberculosis, representing about 30% of the total population [5]. In 2018, WHO reported an estimated 10 million people developed TB and 1.5 million died from the

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disease worldwide, including 251,000 among people with HIV [6]. Though prevalent in building countries where elevated mortality indexes have been reported, howsoever, the infection has also resurged significantly in the urbanized countries [5]. Billions of dollars are spent each year and the governments all over the world stand committed to the eradication of TB; however, the disease still remains out of bound, infecting millions and killing thousands of infected population [5,6].

3  Nature of causative agent

the drugs used in the treatment of TB [8]. They include unavailability of anti-TB drugs, use of substranded drugs, lack of awareness programs, ignorance of health care workers, inadequate drug supply and lack of supervision, massive bacillary load, epidemic HIV infection, improper prescription regimens, interruption of chemotherapy, and ineffective TB control programs [8,10]. Multidrug resistant tuberculosis (MDR TB) is the TB strains in which the resistance mainly produced to first-line drugs such as isoniazid and rifampicin [4,11]. Whereas the extensively drug-resistant tuberculosis (XDR TB) is a new TB strain which is mainly resistant to fluoroquinolones agents and one or more second-line injectable antitubercular agents such as amikacin and kanamycin capreomycin [4,12]. In general, drug resistance is a man-made problem which results from the misuse of available antitubercular drugs and due to poor management of disease course or therapy [4,13]. In other words, mismanagement of first-line drugs results in the emergence and spread of MDR TB. However, when the second-line anti-TB drugs are misused XDR TB emerges, especially in HIV-positive individuals [5]. This makes the so-called cursed duet, the cure for which takes longer and require approximately 30% of the yearly income of an infected household in direct and indirect costs, thus becoming a real socioeconomic calamity for these families, and further hampering the efforts to control and manage the disease [5,14]. Since the emergence of MDR strains in the 1990s, the prevalence of MDR TB has constantly increased [1]. In 2018, WHO estimates that there were about 484,000 new cases with resistance to rifampicin, of which 78% had MDR TB. XDR TB is a nearly untreatable form of the disease and has been reported in 58 countries with an estimated rate of 15% among the MDR strains. The XDR TB prevalence among MDR TB cases ranged from 6.6% to 23.7% worldwide [6,7]. Traditionally, TB control efforts were focused mainly on the improvement of cure rates for drug-susceptible disease to reduce the number

M. tuberculosis is an acid-fast bacillus and intracellular pathogen, which has developed numerous strategies to avoid being killed by macrophages [8]. It is considered as the most successful pathogen capable of persisting in host for decades without causing the disease [8]. The Mycobacteria are plentiful in soil and water, but M. tuberculosis is mainly identified as a pathogen that lives in the host and several species of the M. tuberculosis complex have significantly adapted their genetic structure to infect human populations [5].

4  Emergence of MDR and XDR TB The critical problem with the current TB chemotherapy is that when the drug is taken intravenously or administered orally, it is distributed throughout the body via the systemic blood circulation, and a majority of molecules do not reach their targets [8]. Consequently, they stay in the body causing adverse side effects including liver injury, skin reactions, and gastrointestinal and neurological disorders in a proportion of patients [1,8]. On the other hand, drugs have short plasma-life and rapid clearance, which limits their effectiveness [9]. Many factors have been described to be responsible for the emergence of resistance against

 



6  Need for novel and sustained delivery systems

of drug-resistant cases arising from acquired resistance [6,15]. Recently, aiming to defeat all these challenges and to improve the clinical outcome and overcome MDR in the chemotherapy of TB, new anti-TB drugs as well as a drug delivery mechanism that would target directly to diseased cells specifically with little or no interaction with nontarget tissue has become of utmost importance and necessity [1,5,8]. To date, there has been increasing interest in the design and use of nanotechnology-based new delivery platforms with the potential for improving the efficacy and reducing the side effects associated with the treatment of newly diagnosed TB [1]. In other words, the alarm of the spread of the MDR and XDR strains and the lack of successful treatment options strengthen the need to develop new and effective anti-TB drugs to overcome the problem of drug resistance, shorten the treatment course, and achieve better compliance [8].

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5  Drug regimens

Aminoglycosides such as amikacin and kanamycin; Polypeptides such as capreomycin, viomycin, and enviomycin; Fluoroquinolones such as ciprofloxacin, levofloxacin and moxifloxacin; and Thiomides such as ethionamide, prothionamide, and cycloserine [2,3,10]. These drugs have lower antitubercular efficacy as well as higher toxicity than the first-line drugs [2]. They are more expensive treatment that may last much longer (up to 2 years), which causes patient noncompliance and higher failure rate [10]. A third-line anti-TB drug exists including rifabutin, thioridazine, arginine, vitamin D, and macrolides such as clarithromycin and thioacetazone [8]. Like other drugs for the treatment of TB, the thirdline drugs are not as effective or their efficacy has not been proven [8,10]. In some cases, more severe drug resistance can develop. Extensively drug-resistant XDR TB is a more serious form of MDR TB caused by bacteria that do not respond to the most effective second-line anti-TB drugs, often leaving patients without any further treatment options [3,5,16]. Therefore, new advanced technologies such as the design of carrier-based drug delivery systems are under the inspection for the treatment of TB [8]. Relevant to the needed doses of antitubercular drugs, most typical and alarming adverse effects comprise hepatic damage, nephrotoxicity, neurotoxicity, oculotoxicity, and ototoxicity [17,18]. These effects bring the substranded modes of drug administration which has become a challenge for drug discovery and development in particulate to release rate [17,18].

In general, TB is treated with the first-line drugs as a combination therapy with Isoniazid, Rifampin, Pyrazinamide, either Ethambutol or Streptomycin for several months [2,4,5,8]. These drugs are administered orally and have outstanding effectiveness against M. tuberculosis as well as low toxicity [2]. Various supportive treatments are Diet, as whole food diet including raw foods, fluids and particularly pears, Nutritional Therapy as vitamins (A, beta carotene, E, C, B complex), essential fatty acids, multiminerals and zinc, Herb Therapy as tincture of Echinacea, elecampane, and mullein, garlic capsules, Hydrotherapy, Juice Therapy as raw potato juice, carrot juice, Topical Treatment as eucalyptus packs, etc. [16]. When the strain becomes resistant to isoniazid and rifampin, two of the most powerful first-line drugs, it develops into a more complex form of TB known as MDR TB [5,8]. A combination of second-line drugs used to cure MDR TB is

6  Need for novel and sustained delivery systems

 

Paul Ehrlich, in 1909, initiated the era of development for targeted delivery when he envisaged a drug delivery mechanism that would target directly to diseased cells [2]. Drug targeting can be defined simply as the ability to direct a therapeutic agent specifically to the

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desired site of action with little or no interaction with nontarget tissue [1,2,5]. Nanotechnology science has been a boon to current pharmacology and biopharmaceutical enhancement of drug performance. It is possible to design drug delivery systems capable of targeting phagocytic cells which are infected by intracellular pathogens, such as Mycobacteria. Delivery systems based on nanotechnology offer wide opportunities for improving the therapy for a range of diseases including TB [5]. To extort a maximum therapeutic benefit, a drug must be formulated carefully and this forms the fundamental concept behind a drug delivery system. Four “D’s” are assigned to a drug delivery system, that is, drug, destination, disease, and delivery, out of which the last one is the only changeable factor [5,14].

7  Nanodelivery systems

nanoparticles), particulate drug delivery systems (nanoparticles, microparticles, and dendrimers), supramolecular drug delivery systems (polymeric micelles), specialized drug delivery systems (nanosuspensions, nanoemulsions, microemulsions, and drug powders), complex conjugate drug delivery systems (immunostimulating complex and cyclodextrin inclusion complexes), and other carrier-based drug delivery systems as nanotubes, nanobeads, nanofibers, quantum dots, etc. [1,8,17,20]. These nanometric delivery systems are expected to offer advantages over conventional systems by producing optimum effectiveness to the target site, enhanced therapeutic efficacy, uniform distribution of the drug throughout the target site, increased bioavailability and sustainability of anti-TB drugs, fewer side effects, and increased patient compliance [3,21]. The pharmaceutical technologists of today are aiming at improving the efficiency and reducing associated toxicity of anti-TB drugs by purposely targeting the site of infection [5]. The potential to develop more effective and compliant therapy with existing molecules seems to lie within nanotechnology [1,3,14,16]. Nanoparticles as drug carriers enable higher stability and carrier capacity along with immense improvement of drug bioavailability, which further leads to a reduction in dosage frequency [1,3,21]. In addition, the viability of various routes of administration such as oral delivery and inhalation make a nanoparticle-based drug delivery system ideal for the treatment of TB [8]. Among different carriers, liposomes and niosomes are well documented, effective, and safe drug carriers, and can be functionalized with a great variety of ligands for targeting [2,3,4,16,21].

7.1 Introduction In recent times, nanotechnology has emerged as highly sophisticated and advanced technology, referring to the nanoscale size range of atoms, molecules, and macromolecules with exclusive or significantly better physiochemical properties [5,19]. With the advent of nanotechnology, it may become possible to more effectively treat dreadful diseases such as TB and AIDS [2,5]. Frequent therapeutic failures and emergence of MDR strains, a need to curtail the treatment duration and more importantly to reduce drug interactions are the major factors which suggest the need for developing nanocarriers systems for drug agents used for these diseases [5,10].

7.2  Types of nanocarriers

7.3  Advantages of nanotechnology-based drug delivery system

Many types of nanocarriers have been tested against TB, including vesicular drug delivery systems (liposomes, niosomes, and solid lipid

1. The size and versatility of the nanoparticles makes the drug administration better over standard techniques [3].

 



8 Liposomes

2. Nano-capsules have been commonly shown to increase efficacy of the administered drugs, reduce degradation in the bowels, and increase uptake and bioavailability [3,4,8]. 3. Upon administration, nanoparticles are directly supplied to the bloodstream with all anti-TB drugs, in effect resulting in absolute bioavailability [3,5]. 4. Nanoparticles can use multiple synergistic paths to enhance the antimicrobial activity and overcome the antibiotic resistance [3,4].

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8 Liposomes

preferentially [1]. Incorporation of drugs in liposome enhances bactericidal activity as compared to free drug, especially for the treatment of monocytes and macrophages [16]. Pulmonary delivery has been improved and even tested in animals and human subjects. Drug distribution depends upon the drug release from the liposome which could serve to retain drugs in the lungs and minimize their distribution to other organs [16,22]. Application of liposomes include relatively low toxicity, prepared in wide size range (20–1 mm), ability to solubilize poorly water-soluble drugs, facilitating their nebulization, they serve as a biodegradable pulmonary reservoir with prolonged residence time, they decrease mucociliary clearance of drugs due to their surface viscosity, can be exploited as targeting device to individual population within the lung, specifically to the infected or impaired alveolar macrophages and the lung epithelium [1,16,23]. Novel delivery systems can be administered to the lungs by various modes of delivery, that is, nebulization, instillation, and insufflations. In the last decade, the use of liposomes as a vaccine carrier has increased [16].

8.1  Definition of liposomes Liposomes are concentric bilayer vesicles in a nano- to microrange composed of phospholipids bilayer surrounding an aqueous core encapsulating the desired drug [4,5,8,16]. These are the most extensively investigated systems for controlled delivery of drugs to the lungs, since they can be prepared with phospholipids endogenous to the lungs as surfactants [16]. Importantly, the bilayer of liposome can be utilized to combine a wide range of both hydrophilic and hydrophobic drugs through self-assembling [4,16,21]. The mechanism of delivering drugs is either by fusing with the cell membrane and releasing the drug or by the endocytosis mechanism of the cells [22]. The vesicles can be multilamellar, small unilamellar, and large unilamellar depending on the method of preparation. Unilamellar vesicles consist of a single phospholipid bilayer enclosing the aqueous solution, while multilamellar vesicles consist of concentric phospholipid spheres separated by water layers. For targeting, the surfaces of liposomes are attached with ligands specific to the target site [22,23,24]. Liposomes are biodegradable vesicles with low cytotoxicity and immunogenicity, and enter into mononuclear phagocytic macrophages

8.2  Types and uses of liposomes

 

Considering their wide applications, lipo­ somes have been extensively reviewed by many groups. Their reviews cover methods of preparation, physiochemical properties, such as particle size, charge, lamellarity, and their effects on drug or vaccine delivery and different conjugation chemistry [25]. Different types of liposomes exist for different applications. Conventional liposomes are used in targeted delivery to macrophages and in vaccines. Cationic liposomes are used for gene delivery. PH sensitive liposomes are used for targeting tumors and in endocytosis. There are temperature-sensitive liposomes that are used for site-specific delivery of solid tumors. On the other hand, Stealth liposomes are used for selective targeting to pathologic areas. Immuno liposomes are used

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in receptor-mediated endocytosis. Finally, magnetic liposomes are used in targeting antibodies to the brain [22]. Currently, stealth liposomes are preferred over conventional liposomes due to their stability in the bloodstream because they can escape the mononuclear phagocytic system, which engulfs and clears liposomes from circulation [23]. Stealth liposomes have been used successfully in the delivery of doxorubicin for the treatment of solid tumors [26]. Lung specific stealth liposomes composed of phosphatidylcholine, dicetyl-phosphate, O-steroyl amylopectin, cholesterol, and monosialogangliosides-distearylphosphatidylethanolaminepolyethylene glycol (PEG) were utilized for the targeted delivery of anti-TB drugs (isoniazid and rifampicin; entrapment 8%–10% and 44%–49%, respectively) to the lungs [5]. Significant accumulation of the nanocarriers in the lungs proved their prospective use in targeted drug delivery [27]. The liposomal drugs have also been shown to considerably decrease the bacterial load when compared to the free drug, improving the antimycobacterial efficacy and decreasing the toxicity of the encapsulated drug [28]. Moreover, coating liposomes with PEGs reduces their recognition by the reticuloendothelial system through steric inhibition of hydrophobic and electrostatic interactions with plasma proteins, hence halting their uptake by macrophages and ultimately favoring its prolonged presence in the bloodstream [29,30,31]. Several antibacterial drugs were formulated as liposomes for increasing bioavailability, antibacterial activity, entrapment, prolonging release and permeation. These liposomes exhibit high antibacterial activity against many microbes including Mycobacterium avium, pneumonia, Staphylococcus aureus, Klebsiella pneumonia, and many others, and can be delivered through ocular, intravenous, topical and inhalation routes depending on the area infected [22]. Treatment of pulmonary inflammation has been done successfully by injecting liposomes loaded with levofloxacin intravenously [22,32].

High lung targeting efficiency was achieved reducing the side effects such as hematotoxicity and neurotoxicity caused by direct injection of the drug. Similarly, antifungal drugs were formulated as liposomes and are used for treating infections caused by fungal organisms such as Candida albicans with great efficacy [22].

8.3  Pulmonary TB and the importance of liposomal drugs

 

M. tuberculosis affects all organs of the human body, but high incidences are reported in the lung because the primary route of infection is the inhalation of the microorganism from close contact with infected human subjects [23]. M. tuberculosis can reach the lung alveoli where the cells are phagocytized by alveolar macrophages [1]. M. tuberculosis resists macrophage-mediated bactericidal mechanisms by preventing phagolysosome formation [23]. Therefore M. tuberculosis can multiply and spread to other organs of the body, resulting in extrapulmonary TB [33]. Some conditions such as HIV and diabetes are associated with a high risk of susceptibility to M. tuberculosis infection. Apart from conventional drug delivery, nano-drug delivery systems will provide an opportunity to exploit the nasal delivery of anti-TB drugs directly to the lungs. This approach has the advantage of achieving pharmacologically effective drug concentrations in the alveolar macrophages, which ensures better treatment outcomes. Furthermore, the nano-drug delivery system reduces the adverse systemic side effects and frequency of drug administration, which eventually leads to better patient compliance [23,34]. Although the oral route is the most convenient and least expensive, repeated administration of high doses is required to achieve therapeutic levels of antiTB drugs due to rapid hepatic first-pass metabolism and reduced gastrointestinal absorption. Other disadvantages of the oral route are the high systemic exposure and adverse side effects. On the other hand, parenteral and pulmonary



8 Liposomes

routes have higher bioavailability due to bypassing the first-pass metabolism [34]. In this context, the inhalation route is advantageous for the pulmonary delivery of anti-TB drugs, and requires lower doses to achieve therapeutic effects [35]. The pulmonary delivery of drugs is a convenient and effective approach for the treatment of TB. In this context, the encapsulation of drugs into nano-drug delivery systems offers a number of potential benefits to penetrate and cross the biological barriers to reach the targeted sites in the lungs. Furthermore, the phagocytic nature of alveolar macrophages is an advantage for targeted delivery in the lungs [36]. The physiochemical properties of drugs encapsulated in nanoparticles are the most important characteristic for achieving a proper drug distribution in the lungs. A particle size below 1 µm is suitable to deliver the drugs to the pulmonary alveoli [37]. The use of neutral nanoparticles as drug delivery vehicles is the most common approach in anti-TB therapy [38]. On the other hand, targeted delivery of anti-TB drugs could also be achieved by using liposomes coated with macrophage-specific ligands [23]. This will facilitate the preferential accumulation of formulations in lung macrophages, which will further reduce systemic and local toxicity and provides better results than uncoated formulations [27]. The remarkable potential of liposomes in the direct lung delivery of anti-TB drugs is clear and obvious.

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8.4  Si-RNA liposomes

gene expression and investigating genes at the cellular level, promoting homologous mRNA degradation, and inducing specific gene deletion phenotype [40]. Potential applications of SiRNA have led to a great interest in harnessing this technology for therapeutic use in cancer and TB and other diseases as well [1]. For therapeutic applications, synthetic SiRNA is used for targeting oncogenes and genes that are involved in cancer cell proliferation, survival, invasion, angiogenesis, metastasis, and resistance to chemotherapy or radiotherapy in cancer and for targeting disease-causing genes in other pathologies [41]. The major limitations of the systemic use of Si-RNA based therapies include rapid degradation by nucleases and renal clearance following systemic administration. To enhance the stability of Si-RNA various chemical modifications have been used. However, poor cellular intake remains an important issue due to negatively charged cell membranes preventing efficient intracellular uptake of SiRNA molecules, which also have a negatively charged backbone, leading to electrostatic repulsion, requiring a carrier to increase the uptake into cancer cells [1,39,40]. Si-RNA with a negative charge is combined with positively charged liposomes to form stable nanoparticles. Here comes the importance of nanotechnology which holds promise for widespread clinical application of Si-RNA therapeutics [39,40]. These novel nanoparticle liposomes have a great potential to reduce Si-RNA related toxicities and prevent offtarget effects in normal tissues. As nanocarriers, liposomes were able to overcome most hurdles that prevent the systemic use of Si-RNA. Liposomal formulations are known to be the most popular delivery system and have been used extensively to enhance the efficiency of drug delivery by systemic administration due to their high degree of biocompatibility. The ability of liposomes to deliver a variety of payloads including Si-RNA has made them most the successful method for delivery of therapeutic agents [39,40,41].

Small interfering RNAs (Si-RNAs) have recently emerged as a new class of therapeutics with a great potential to revolutionize the treatment of cancer and other diseases including TB [39]. The discovery of RNA interference including micro RNA (mi-RNA) and Si-RNA–mediated gene silencing is considered one of the most important advancements in biology in the last decade [1,39]. Si-RNA is now commonly used as a powerful tool for silencing posttranscriptional

 

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In view of the key role of the immune response in TB development, targeting the inflammatory response in TB may represent an effective approach to control TB. Silencing of transforming growth factor-β1 (TGF-β1) by Si-RNA in macrophages results in a significant decrease in titers of the intracellular M. tuberculosis. TGF-β1 primarily produced by mononuclear phagocytes is known as the immunoregulatory cytokines, inhibiting T-cell responses and deactivating the macrophages in patients with TB. Overexpression of TGF-β1 results in tissue damage and fibrosis in TB patients. The reduction of TGF-β1 leads to a decrease in the tuberculous granuloma formation [1]. On the other hand, the diverse regulatory effects of the novel nanoparticle-SiRNA (NP-Si-RNA) liposomes on the generation of cytokines have been proven, and this may enhance the therapeutic effect in the treatment of TB [1]. Given the physiochemical properties of NPSi-RNA liposomes, the results have demonstrated that the liposomes can be used to encapsulate the anti-TB drugs. The novel NP-Si-RNA liposomes are facile to be prepared, stabilized, and controlled in a reasonable size. These physiochemical properties facilitate its internalization through the endocytosis by human macrophages [1]. They determine the pharmacological and toxicological features of the novel NP-Si-RNA liposomes, such as absorption, distribution, metabolism, excretion, and toxicity. The favorable physiochemical properties of the newly synthesized NP-Si-RNA liposomes ensure their pharmacological effect in the treatment of TB [1]. Indeed it has been shown that the novel NP-Si-RNA liposomes exhibit a slow drug release profile within the initial 12 h compared with the free drugs. Notably, the novel NP-Si-RNA liposomes showed a sustained drug release profile between 12 and 72 h, which has great clinical importance in terms of enhancing the therapeutic effect and minimizing the side effects in the treatment of TB. At the same time, it has been proven that NP-Si-RNA

liposomes were safe and nontoxic to the host cells. The novel NP-Si-RNA liposomes exhibit favorable physiochemical and biochemical properties that make it a promising therapy for TB treatment [1,39].

8.5  Targeting of liposomes

 

Delivering an effective concentration of antiTB drugs to sites where M. tuberculosis resides in the immune cells, deep within the lungs, is a huge feat, as often drugs dissolve rapidly and are absorbed by the blood [42,43]. Granulomas are clusters of M. tuberculosis-infected macrophages surrounded by many immune cells, fatty acids, and cholesterol, securing the microorganism [42]. Their function is to localize and contain the infection, preventing both its growth and replication; however, they remain unable to destroy all bacilli or prevent them from generating energy [44]. These structures are poorly vascularized, and therefore it is very difficult to target drugs to these high content bacilli constructs [14,45]. The literature on using nanomedicines to target TB is predominantly based on the use of polymers or liposomes with anti-TB drugs [46]. The use of nano-formulations for therapeutic purposes aims to lower drug dose administration to patients, while achieving better cure rates with fewer side effects and toxicity in a shorter treatment time [4]. This is accomplished through improved targeting of drug bearing nano-particles to the required target, therefore enhancing the drug concentration at specific sites while decreasing delivery to nontarget sites [42,47]. Targeting, which can be either active or passive, can be achieved by modifying nanoparticle surfaces with polymers and/ or through bio-conjugation of antibodies and specific ligands. This can prevent nano-particles from binding with nonspecific blood components and targets them to specific receptors [48]. This can also increase the blood circulating time of nanomedicines, which may be achieved



9 Niosomes

by reducing the phagocytic clearance of a drug [49]. The addition of the polymers, such as PEG, to the surface of nano-particles renders the nano-particles hydrophilic. This addition reduces the reticuloendothelial system uptake of nanoparticles especially by the liver and spleen, thus allowing it to stay longer in the circulation [50]. PEG is also reported to reduce the formation of aggregates. Additionally, if drugs are encapsulated by nano-particles as liposomes, they can be protected from enzymatic degradation in the blood and elsewhere, and this could also improve drug stability [42,50].

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9 Niosomes

stability [51,54]. Niosomes can be used for entrapping both hydrophilic and lipophilic drugs in the aqueous layer and vesicular membrane, respectively. The bilayers of niosomes have both inner and outer surfaces to be hydrophilic with a sandwiched lipophilic area in between. Thus a large number of drugs and other materials can be delivered using niosomes [55]. Unlike other nanoparticles, liposomes and niosomes have many similarities and can be loaded with both hydrophilic and hydrophobic drugs; therefore they could codeliver both hydrophilic and hydrophobic drugs in one vesicle. Compared with liposomes, niosomes have advantages such as good stability, low cost, easy formulation, and scaling up [56]. They are considered to be versatile, as it can be given by oral, parenteral, pulmonary, as well as topical routes. They can penetrate through the cornea and can be used for ocular drug delivery as well [57].

9.1  Definition of niosomes Niosomes are spherical nonionic surfactant vesicles having a bilayer structure formed by self-assembly of hydrated surfactant monomers [14,16,51]. The bilayer is multilamellar or unilamellar which enclose an aqueous solution of solutes and lipophilic components are in the bilayer itself [29,51]. Niosomes act as drug carriers to deliver drug to the site of action. These vesicular carriers have a size that ranges between 10 and 1000 nm, wherein aqueous phase is enclosed in a highly ordered bilayer of nonionic surfactants with or without cholesterol and dicetylphosphate [22]. Different types of surfactants at variable combinations and molar ratios are used to form niosomes [52]. Examples of surfactants include alkyl ethers, alkyl glyceryl ethers, sorbitan fatty acid esters, and polyoxyethylene fatty acid esters [52,53]. Addition of cholesterol maintains the rigidity of the bilayer, resulting in less leaky niosomes [52]. These nonionic surfactants give niosomes the advantage of being more stable when compared to liposomes, and thus overcoming the problems associated with liposomes, that is, susceptibility to oxidation, high price and difficulty in procuring high purity levels which influence size, shape, and

9.2  Advantages of niosomes

 

1. They can act as carriers for drugs which have a wide range of solubility as it has both hydrophilic and hydrophobic part [2,3,56,57]. 2. Surfactants used in niosomes are biodegradable, biocompatible and nonimmunogenic [3,57]. 3. Niosomes of desired property can be prepared by altering vesicle characteristics such as vesicle composition, size, lamellarity, yapped volume and surface charge [3,52,57]. 4. As niosomal suspension has hydrophilic tail, therefore, it can be considered to provide better patient compliance than oil-based formulation [56,57]. 5. Niosomes can also be used as a depot formulation, therefore, releasing the drug in a controlled manner [57]. 6. They can reduce drug toxicity because of their nonion nature, one of the most useful aspects of niosomes in their ability to target drugs [2,5,56].

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9.3  Various types of niosomes

9.5  Application of niosomes in drugs

Based on the vesicle size, niosomes can be divided into three groups:

The first niosome formulations were developed and patented by L’Oreal in 1975 in the field of cosmetics [4,56,62]. Niosomes were first utilized in drug delivery for anticancer agents. It was discovered that they were capable of altering the pharmacokinetic profile, drug distribution in organ, and even drug metabolism [56,62]. Being more stable and advantageous compared to liposomes, niosomes proved to have wider potential for clinical uses [20]. They emerged first in the field of cosmetics, but are attracting nowadays extensive attention as a vesicle delivery system in pharmaceutics. Due to their ability to entrap both hydrophilic and hydrophobic drugs, niosomes are reported as ideal carriers for the delivery of drugs such as doxorubicin, vaccines, insulin, Si-RNA, antiTB drugs, and so on [56]. Niosomes could prolong the half-life of drugs during circulation, reduce capture by the liver, and improve the uptake of the loaded drugs [63]. It is reported that niosomes could increase the uptake of methotrexate into the brain due to the possibility that niosome components could permeate the blood-brain barrier [64]. The therapeutic effects of niosomes are widely applicable (e.g., anti-Alzheimer, anticancer, antioxidant, diabetes, and antimicrobials) and can be administered via different methods such as intravenously, orally, and transdermally as well [56,65]. It is quite obvious that niosomes can be used as alternatives to liposomes as nano-vesicle-based delivery system for chemical drug delivery. They can also provide a way for the codelivery of two different kinds of drugs to achieve the desired therapeutic effects [56]. Some protein and peptides such as insulin and bacitracin may function as important therapeutic agents for the treatment of diseases, but their clinical application is hindered due to poor bioavailability and instability during storage and administration. To overcome these problems, niosomes may serve as good carriers for the delivery of various proteins and

1. Small unilamellar vesicles having a size ranging between 0.025 and 0.05 µm. 2. Multilamellar vesicles having a size larger than 0.05 µm. 3. Large unilamellar vesicles having a size larger than 0.10 µm [57].

9.4  Niosomes versus liposomes; which is superior? Niosomes possess a bilayer structure, which is similar to liposomes. However, the materials used to prepare niosomes confer better stability on them [58]. Niosomes are prepared from uncharged single-chain surfactants and cholesterols. By contrast, liposomes are prepared from neutral or charged double chain phospholipids. The concentration of cholesterol is higher in liposomes than in niosomes. As a result, the drug entrapment efficiency of liposomes is less than that of niosomes [52]. Niosomes are costeffective for industrial manufacture and do not require special storage conditions, which are essential while manufacturing liposomes. The cost of liposome preparation is high because of the unstable chemical ingredients (phospholipids), which undergo oxidative degradation. Liposomes, therefore, require special handling methods [59]. Niosomes are visualized as alternative delivery systems that can overcome the drawbacks associated with sterilization, high production costs, scale-up difficulties, and the instability of the phospholipidic components of liposomes upon light exposure even at room temperature [5,56]. Niosomes possess a longer shelf life than liposomes [60]. They prolong the circulation of encapsulated drugs and increase metabolic stability in an emulsified form, whereas liposomes have limited shelf life because of the rancidification of their lipid components [53,60,61].

 



9 Niosomes

peptide drugs, and also show good performance in vaccine formulation and application [66,67]. In gene therapy, which has emerged as a powerful tool in the treatment of several diseases recently, delivery remained a big problem for clinical application. Nonviral gene carriers have been employed for the delivery of gene materials but were associated with high toxicity and nonspecific attachment during the circulation in vivo [68,69]. Here emerged the importance of niosomes as oligonucleotide carriers that can be used for the delivery of gene materials with the advantage of good chemical and physical stability, relatively smaller sizes, and low costs [56]. Niosomes can also serve as a delivery system for targeting stem cells, and intracellular delivery of Si-RNA/mi-RNA used in specific gene silencing [70]. Applications of niosomes has covered areas of antineoplastic agents, nonsteroidal antiinflammatory drugs, antileishmanial agents, gene delivery, cosmetics, proteins, antibiotics, antifungal agents, antivirals, vaccines and immunization, vitamins, antiglaucoma agents. Hormones, muscle relaxants, anesthetics, antidiabetics, contraceptives proved suitable for a wide range of active pharmaceutical agents including anti-TB drugs [51].

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delivery to the target site. The use of niosomes overcame weakness of many drugs including conventional inhalation therapies [52]. Several niosomal formulations for the delivery of antiTB drugs have been developed to achieve effective treatment of TB, reduction of drug dosage, and toxicity as well as frequency, which should bring about improved patient compliance, while targeting specifically the macrophages where TB bacteria are harbored [14]. To date, several anti-TB drugs have been successfully delivered to their targeted sites with niosome carriers. Some of these include: 1. Niosomes of ethambutol HCL prepared by reverse-phase evaporation method [71,72]. 2. Tyloxapol niosome encapsulation of rifampicin, isoniazid, pyrazinamide, and antimycobacterium drugs [8,73]. 3. Niosomes containing rifampicin and gatifloxacin prepared by lipid hydration technique against logarithmic phase cultures of M. tuberculosis [74].

9.6  Niosomes in the treatment of TB

Levofloxacin in nano-niosomes has also been designed for achieving delayed release and longer duration of action against MDR-TB and reducing dosing frequency, thus improving patient compliance [29,75]. Niosomal encapsulation of isoniazid was found to effectively treat TB, with 62% of cellular uptake by macrophages. The additional advantages of the niosomal formulation are that it was site specific to where M. tuberculosis hide, and was able to maintain steady drug concentration for up to 30 h [76]. In vivo studies indicated that by regulating the size of the carrier, up to 65% localization of drug can be achieved in the lungs. Only 15% of the administered drug was found in the lungs when an equivalent amount of free drug was administered. The remaining drug was found to be distributed in the liver (20%), spleen (17%), kidney (12%), and blood (36%) [5]. When incorporated in appropriate niosomes, it has been proved that the isoniazid

Combination chemotherapy is a basic principle to cure TB patients. Multiple drug chemotherapy is effective in curing TB, but with challenges and limitations. The reason along with many drawbacks associated with conventional methods of TB treatment demands the development of novel lung-targeted drug delivery approaches [2]. Several attempts have been made to use niosomes for the delivery of antiTB drugs to achieve better therapeutic outcomes [16]. In this domain, niosomes offered many advantages including high drug encapsulation efficiency, strong mucus permeation, and sustained

 

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accumulation by niosomal drug delivery in visceral organs was lower than free drug indicating less incidence of toxicity of niosomal drug delivery system than free drug [76]. The intravenous and intraperitoneal administration of rifampicin encapsulated in niosomes prepared using surfactants of sorbitan ester class:cholesterol (50:50 percent mole fraction ratios) has been used effectively for the treatment of TB [77]. The rifampicin was found accumulated significantly in the lungs, indicating that niosomes are an enhanced drug delivery system for this therapy, and therefore offering the possibility of improved anti-TB drug management [78]. Niosomal formulations reached substantially higher rifampicin concentration, that is, 46.2% of the administered dose, in thoracic lymph nodes when administered via the intraperitoneal route as compared to 13.1% for the free drug [5]. Niosomes of rifampicin and gatifloxacin exhibited prolonged release of the drug up to 16 h, which could overcome the limitations such as frequent administration to patients, rheumatoid syndrome, and hepatotoxic disorders. This also gave better bactericidal activity against M. tuberculosis as that of the pure drug solutions [74]. It has been reported that niosomal encapsulation of pyrazinamide was done to achieve sufficient macrophage targeting and to overcome drug resistance [79]. Although there are several beneficial uses of niosomes, the demerits are associated with its physically unstable state, leakage of entrapped drugs, aggregation, fusion, and hydrolysis of the encapsulated drug which inhibit the shelf life of the dispersion [78]. There are some of the drawbacks that need to be addressed before the large scale application of niosomes as a potential drug carrier can be implemented.

high mortality [80]. Numerous reports support the feasibility of niosomes loaded with anti-TB drugs as very effective medications in the management of cerebral TB [20]. It is well known that the majority of active agents do not readily permeate into brain due to the presence of blood-brain barrier and blood-cerebrospinal fluid barrier [81]. Currently, the most innovative and promising noninvasive strategy in brain delivery is the design and preparation of nanocarriers, which can move through the brain endothelium [20,82]. Niosomes can perform brain delivery and are reported to show better cerebral uptake and a lesser risk of toxicity to the blood-brain barrier with both physiochemical and serum stability [83]. Another exhilarating development has been the nano-encapsulation of azole antifungals and fluoroquinolones in a suitable vesicular-like niosomes that are able to extend the presence of these drugs in the systemic circulation, thereby enhancing penetration into the target site with minimum toxicity. Azole antifungals as clotrimazole and econazole have shown potent antimycobacterial activity against drug-sensitive and drug-resistant strains of M. tuberculosis as well as the latent bacilli [84]. Fluoroquinolones especially moxifloxacin, also possess the strong antimycobacterial activity and achieve prolonged, high concentrations in alveolar macrophages predominantly following encapsulation. Moxifloxacin encapsulated in nanoparticles proved to accumulate in alveolar macrophages three times more efficiently than free moxifloxacin, and were detected intracellularly for six times longer period than the free drug, even at similar extracellular levels [85].

9.7  Niosomal drug delivery system role in cerebral, drug-resistant TB

10  Pulmonary delivery of nanoparticle encapsulated antitubercular drugs Nanoparticles possess the capability to attain a maximum drug filling, reduce the usage of polymers, barrier cross permeability, and better

Cerebral TB is perhaps the most horrible form of extrapulmonary TB because of the associated

 



11  The future of combating TB

therapeutic efficacy. Alveoli-targeted net drug delivery is easily approachable by their mucosal adherence by inhalation [86]. Poly-lactic-coglycolic acid (PLGA), a copolymer, is the most possibly investigated nanoparticulate drug transporter due to their degradability and biocompatibility [87]. Nebulizers or pressurized metered dose inhalers (pMDIs) and dry powder inhalers are the three techniques to inhaled drug delivery each with their exclusive potency and properties [88]. In order to conquer the downsides of pMDI encountered during their usage, dry powder inhalers DPIs were presented because of their high formulation stability alveolar deposits [89]. Their physical characteristics equipped them to avoid natural clearance from the lungs and allow alveolar drug release even [90]. Diseases such as TB, cystic fibrosis, and lung cancer were treated well due to combined benefits of nanoscale formulation and localized delivery of nanoparticles from DPIs [91]. The targeted pulmonary drug delivery of nanoparticles to the lower respiratory tract offers prospective use in several lung ailments. Inhalation therapy of antitubercular drugs is the most effective treatment for pulmonary TB with good systemic bioavailability also for extrapulmonary TB and endobronchial TB as well. Inhalation therapy proved to be an excellent mode of treating MDR TB that was not remedied with conventional drugs [17].

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drugs remains a challenge to the TB research community, especially for treating the latent form of the disease, where bacilli reside within granulomas [21]. On the other hand, drug resistance remains a constant menace because patients stop the medication once they start feeling better. New drugs are required to be explored which are effective against TB, especially drug-resistant TB [8]. On the other hand, upgraded researches for the development of novel drug delivery systems based on nanotechnology proved promising to revolutionize the treatment of TB, by reducing the particle size to nanoscale and improving the solubility of the poor soluble drugs. Nanomedicines as drug delivery carriers of anti-TB medications were able to achieve faster absorption of the drug, targeted interaction at a specific site, and its release in a controlled manner into the human body in a relatively short span of time with minimal side effects [17,92]. One of the biggest advantages of manufacturing such drugs is low cost, a very important factor to be considered in the management of TB as it affects people more in the developing countries [92]. Advancements in the nanoparticle-based delivery systems represent a commercial, practical, and most promising substitute for potential TB chemotherapy. Nanotechnology has led to the development of inhaled drug delivery system with potential merits such as direct drug delivery to the site of infection, avoiding the first pass metabolism, reducing the dose of the drug, and hence reducing its toxicity [10]. The nanoparticles get swamped by the alveolar macrophages, thus leading to the direct release of anti-TB drugs into the alveolar macrophages. This alveoli targeting strategy is playing a prominent role in attacking the TB bacilli. The various nano-formulations can be converted into dry powder to potentiate their merits. Oral and inhalational routes of drug administration are another reason why it makes nanoparticles more feasible in the treatment of TB [8]. Finally, it is worth to mention that there are new drugs such as bedaquiline, delamanid, and teixobactin evolving as potent medications

After 70 years drought, several drugs are looming from the pipeline to combat TB. They will serve as a boon to the field that has been burdened with primitive, inadequate treatments, and drug-resistant bacterial strains [10]. The first line drugs take about 6 months or more for the entire treatment. The second-line remedy for resistant TB requires daily injections which carry severe side effects. The intracellular delivery of

 

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which may serve a nice step forward with a better outcome, especially against resistant strains of M. tuberculosis [10].

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C H A P T E R

8

Polymer-based nanoparticles as delivery systems for treatment and vaccination of tuberculosis Mohsen Tafaghodia,b, Farzad Khademic, Farideh shiehzadeha and Zohreh Firouzia a

Department of Nanotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; bNanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran; c Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran

1  Polymer-based nanoparticles as drug delivery systems of tuberculosis On the path to disease eradication, studies focus on the prevention and early diagnosis of diseases on the one hand, and on the other, focus on discovering newer therapies or modifying existing therapies. Both approaches to treatment are to reduce side effects and increase effectiveness. Studies in tuberculosis treatment are not an exception. In addition to trying to synthesize new drugs, of course, a very time-based and costly way, modifying existing drugs such as using pharmaceutical carriers or changing routes of administration have been taken into consideration in recent years. In general, it can be said that drug carriers can move drug molecules more efficiently to

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00008-4

the main target site. This can be done by various events such as increasing the drug’s solubility or increasing carrier accumulation in comparison with drug molecules in the target tissue. The lung is the main target organ in tuberculosis. The pathogen is an intracellular agent and alveolar macrophages are the reservoir and the main target of the treatment [1]. In recent decades, various polymeric carriers with natural or synthetic origin have been used for drug targeting to the deep lung. The particle size of these carriers has been in the range of micro- to nanometer. Despite the advantages of micron-sized carriers, due to special potentials of nanoparticles, these carriers have recently been important in pharmaceutical science. At the first part of this chapter, polymeric nanoparticles that have been used to treat

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8.  Polymer-based nanoparticles as delivery systems for treatment and vaccination of tuberculosis

tuberculosis are classified in two parts: nanoparticles composed of natural polymers and synthetic polymers. At the second part of this chapter, the studies focused on tuberculosis vaccination using polymeric nanoparticles are investigated.

1.1  Nanocarriers based on natural polymers

lung tissue and also increased the presence time and local concentration of drugs compared to their free form. It has also been shown that the drug concentration in other organs, such as kidney and liver, has decreased compared with the free drug. The significant point in this study was the reduced MIC of nanoparticles for drug-resistant strains in comparison with the free drug [5]. Chitosan nanoparticles have mainly been used for encapsulating first-line anti-TB drugs, like isoniazid and rifampicin (Table 8.1). According to WHO guidelines for TB treatment, administration of multiple oral drugs regimens is preferable to prevent development of multidrug resistance (MDR). Therefore, containing the combination of anti-TB drugs in one formulation is considered an advantage. Rifampicin has a hydrophobic nature; however, due to the special position of the rifampicin in the treatment of tuberculosis, loading of this drug has also been investigated in chitosan carriers. In some studies, in order to obtain an enhanced loading of hydrophobic drugs, it was tried to increase the hydrophobicity of chitosan nanocarrier. In one study, one hydrophobic derivative of chitosan (octanoyl chitosan) was used for the preparation of nanoparticles containing rifampicin. This study showed that drug loading capacity improved compared to other studies. In addition, the system did not produce significant in vitro cytotoxicity. In vivo studies on the pulmonary administration of these nanoparticles using nebulizers have shown the potential of these nanoparticles to reach alveoli [12]. Also, by looking at the drugs that have been loaded in this nanocarrier, the remarkable point is the preparation of chitosan nanoparticles containing aminoglycosides which possess a positive charge. In order to overcome the repulsion between the carrier and the drug, an anionic substance, called dextran sulfate has been used in the preparation process. In the case of streptomycin, this modification could improve loading efficiency (58.8%). In this study, it was shown that after oral administration, chitosan

There are several advantages for natural polymers, among them could be referred to being inexpensive and available, the biocompatibility and hydrophilic nature that the importance of the latter is related to the reduction of the need to use organic solvents. 1.1.1  Polysaccharide-based carriers 1.1.1.1  Chitosan-based carriers

Chitosan is a naturally occurring polysaccharide derived from chitin and is highly positive in a neutral and acidic environment. It has the ability to attach to the physiological surfaces such as mucosal surfaces and the surface of the cells due to its positive charge. In pharmaceutical sciences, this polymer is used in many different formulations, due to its low toxicity and high biocompatibility and is known as a mucoadhesive excipient. Therefore, this carrier can be a good candidate for mucosal drug delivery to the lung and gastrointestinal tract. Among the natural polymers, chitosan is the most frequently used polymer for the preparation of antituberculosis drugs [2–4]. The main method of preparation of chitosan nanoparticles is ionotropic gelation. Generally, in this method, the chitosan solution containing the drug is prepared and then the TPP solution as a cross-linking polyanion is slowly added to the previous solution. Other cross-linker agents like genipin that has a natural origin have also been applied. Chitosan nanoparticles loaded with isoniazid and rifampicin were prepared as dry powder inhaler and were administered to an animal model. This formulation did not show any toxicity in the

 



TABLE 8.1 Studies on chitosan-based nanoparticles encapsulating anti-TB drugs. Targeting moiety Note

Reference

Streptomycin, gentamicin, tobramycin

Oral

–

• The positive charge of aminoglycosides is shielded by dextran sulfate to be incorporated in the chitosan carriers efficiently • Streptomycin loaded oral formulation was as iv vivo effective as subcutaneous injection

[2]

Chitosan nanoparticles

Isoniazid

Pulmonary

–

• The MIC of drug-loaded carriers against intracellular mycobacterium was reduced in comparison with the drug solution

[6]

Chitosan nanoparticles

Rifampicin

Oral

–

• The optimized formulation was prepared • pH-dependent and concentration-independent in vitro sustained release was shown

[7]

Chitosan-coated magnetic nanoparticles

Rifampicin, isoniazid

Pulmonary

–

• A potential theranostic agent • Positively charged drug was absorbed on the surface of the carrier • The MIC value was reduced in comparison with the unloaded carrier

[8]

Nano-embedded microparticles of chitosan and guar gum- coated chitosan nanoparticles

Rifampicin, isoniazid

Pulmonary (dry powder)

–

• Carrier mediated formulations (especially gum [9–11] coated chitosan) showed lower MIC, higher and more extended in vivo antibacterial effects in comparison with oral and pulmonary free drug

Chitosan nanoparticles

Rifampicin

Pulmonary (dry powder)

Chitosan nanoparticles

Loaded drug

Chitosan nanoparticles

  [4]

Prothionamide Pulmonary (dry powder)

• The in vivo residence time in the lung was extended in case of nanoparticles in comparison with free drug

[3]

Octanyl chitosan nanoparticles

Rifampicin

Pulmonary delivery – (dry powder)

• Suitable entrapment and in vitro aerodynamic behavior

[12]

Genipin-crosslinked carboxymethyl chitosan nano gel

Isoniazid and rifampicin

Pulmonary delivery – (dry powder)

• Effective against MDR-TB • Higher in vivo organ retention in compare with free drug

[5]

Chitosan/carbon nanotubes microspheres

Isoniazid

Intravenous

–

• More effective against in vivo extrapulmonary TB (tubercular ulcers) in compare with free drug and drug-loaded carbon nanotubes

[13]

Mannose-functionalized chitosan nanocarriers and chitosan nanocarriers

–

–

Mannose

• Manosylation helped the nanoparticles to interfere with the metabolic pathways of mycobacterium

[14]

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• Higher pulmonary bioavailability and extended residence time in comparison with oral and pulmonary free drug

1  Polymer-based nanoparticles as drug delivery systems of tuberculosis

Route of administration

Carrier

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8.  Polymer-based nanoparticles as delivery systems for treatment and vaccination of tuberculosis

nanoparticles encapsulating streptomycin have similar in vivo antibacterial effects to subcutaneously injected free drug. This result is very important given that the free form of streptomycin has no gastrointestinal absorption [2]. Apart from streptomycin, prothionamide is another second-line TB medicine which its encapsulation has been investigated in this carrier. Due to the irregular gastrointestinal absorption of the drug, the use of a nanocarrier for pulmonary administration of dry powder has been studied. Dried powder showed a very high in vitro capability to reach the alveoli (fine particle fraction (FPF) = 81%). In addition, the biodistribution study of the encapsulated drug has shown a longer presence in the lung and plasma than its free form [3]. In most of the studies, anti-TB drugs loaded chitosan nanoparticles were designed for oral and pulmonary administration. However, in one study on the therapeutic effect of isoniazid loaded chitosan coated carbon nanotubes (CNT), the intravenous administration of the drug was used to deliver the medicine to the location of tuberculosis wounds. In this study, it has been claimed that the use of chitosan, in addition to its previously mentioned benefits, could show potential bactericidal effects. This system had significantly reduced the inflammation of the wound compared to the drug-containing CNT and free drug [13]. Therefore, in addition to using chitosan as the core of the nanoparticle, it has also been used as the nanoparticle coating. Another example of this approach was the preparation of iron oxide nanoparticles with chitosan coating which provided a carrier with theranostic properties. A theranostic carrier, have simultaneous therapeutic and diagnostic properties. In this study, streptomycin was loaded on the chitosan surface. Although the amount of drug loading efficiency has not been reported, a reduced MIC was determined relative to the blank carriers [8]. Biological distribution, as well as biological efficacy of these systems, has been studied on

the in vivo models. Increasing the bioavailability of the drug due to its greater persistence in the body and reducing the need for recurrent drug use has been their remarkable characteristics [9–11]. Targeted drug delivery to macrophages surface markers is also possible. At the surface of these cells, there are specific receptors for mannose which can lead to better drug delivery. This idea has been investigated in one study. It has been shown in this study that although both types of particles are up taken by macrophages, the presence of this ligand can lead to metabolic changes in these cells, thereby contributing to the improved treatment index of TB [14]. In general, slower drug release, decreased MIC, and increased in vivo retention time than the free form of the drug were common findings in these studies. 1.1.1.2  Alginate-based carriers

 

Alginate is a naturally occurring, negatively charged hydrophilic polysaccharide. Alginate, like chitosan, has advantages such as being affordable, biocompatible, and biodegradable. It is also widely used in the pharmaceutical industry. Its characteristic feature is the formation of a gel-like structure in the presence of bivalent ions such as calcium ions. Its carriers have been used to provide continuous release of various drugs. Alginate nanocarriers have also been used to encapsulate anti-TB drugs (Table 8.2). While studies focused on this carrier are limited in comparison with those of chitosan carriers, their important feature is the simultaneous encapsulation of several anti-TB drugs and studies on their in vivo efficacy. In a group of studies conducted by one research team, the simultaneous encapsulation of first-line anti-TB drugs in alginate nanoparticles and the evaluation of their bioavailability and efficacy in an animal model were considered. In the first step, the nanoparticles containing the three drugs namely isoniazid, rifampicin and pyrazinamide were administered to the animal



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1  Polymer-based nanoparticles as drug delivery systems of tuberculosis

TABLE 8.2 Studies on alginate-based nanoparticles encapsulating anti-TB drugs. Route of administration

Targeting moiety

Isoniazid, Rifampicin, and pyrazinamide

Pulmonary

Alginate nanoparticles

Isoniazid, rifampicin, pyrazinamide, and ethambutol

Carrier

Loaded drug

Note

Reference

Alginate nanoparticles

–

• Higher relative bioavailability and higher in vivo efficacy compared with oral free drugs

[15]

Oral

–

• Higher relative bioavailability compared with oral free drugs

[16]

Alginate nanoparticles

Isoniazid, Oral rifampicin, pyrazinamide, ethambutol, and econazole

–

• Higher relative bioavailability and efficacy compared with oral free drugs • In vivo efficacy of econazole to replace rifampicin and isoniazid

[17]

Sodium alginate nano carrier coated with chitosan and Tween 80

Rifampicin and ascorbic acid

Pulmonary (not – tested yet)

model via the pulmonary root. In the second study, ethambutol was added to the previous drugs while in the third study, econazole was also added to the set of drugs (it has shown antiTB effects) and the bioavailability and efficacy of the drugs were evaluated after oral administration. In all of the studies on the in vivo model, there was a marked increase in the presence of the drug in the body compared to its free form. It consequently reduced the need for repeated doses to eliminate the pathogen [15,17,19]. In another study, a complex carrier of alginate core and chitosan coating was used to encapsulate rifampicin. This formulation has shown better antimicrobial effects than the free drug in the in vitro [18].

• Reduced MIC in compare with free [18] drug • Synergic effects of rifampicin and ascorbic acid

previously noted, be important for the active delivery to macrophage cells. A study on guar gum-coated chitosan nanoparticles has shown better in vivo efficacy compared with nanoparticles without this coating [9,11]. 1.1.2  Polypeptide and protein-based carriers 1.1.2.1  Gelatin-based carriers

1.1.1.3  Guar gum-based carriers

Gelatin is a biodegradable material from natural sources and has been extensively used for nanoparticular drug delivery systems for the last three decades. Gelatin nanoparticles have been evaluated for the encapsulation of first-line drugs including rifampicin and isoniazid for intravenous administration. The nanoparticles containing isoniazid were mannosylated to enhance macrophage targeting. The results of in vivo studies showed higher accumulation in organs and greater efficacy of these formulations than the free and nontargeted carrier [20,21]

Guar gum is a natural polysaccharide composed of sugars such as galactose and mannose. The particular importance of this polysaccharide in the delivery of antituberculosis drugs may be related to its mannose groups, which may, as

 

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8.  Polymer-based nanoparticles as delivery systems for treatment and vaccination of tuberculosis

In another study, posttreatment immune responses to gelatin nanoparticles containing rifampicin and moxifloxacin were investigated. In this study, it was shown that the treatment of alveolar macrophages with these nanoparticles would stimulate cellular immune responses and help eliminate pathogens, which would be enhanced by encapsulated anti-TB drug [22].

have allocated a greater proportion of studies. Among the synthetic polymers used in pharmaceuticals such as polyesters (lactones) and acrylates, PLGA has been the most popular type and most studies in the field of synthetic polymeric nanoparticles containing anti-TB drugs focused on this polymer. 1.2.1  PLGA-based nanocarriers Poly(d,l-lactide-co-glycolide) (PLGA) is a linear synthetic copolymer of lactic acid (LA) and glycolic acid (GA). Based on the percentage of hydrophobic to hydrophilic monomers, this polyester is divided into different types. The ability to change lipophilicity has made it a valuable carrier for a variety of medications. Among the PLGA used, the 50:50 lactic/glycolic acid ratio is the most commonly used type in drug delivery. The advantages of this polymer include: (1) high biocompatibility and biodegradability, (2) having U.S. Food and Drug Administration approval for clinical use, (3) having surface decoration potential; and (4) having potential to be designed for controlled release [26]. Because of favorable release kinetics, PLGA is one of the most widely used polymers for sustained release delivery systems and is considered as a “gold standard” among biodegradable polymers used for controlled delivery [27]. In the majority of studies, PLGA nanoparticles had an initial burst release. This occurs because of the diffusion of the drug from the surface of nanoparticles. The rest of the encapsulated drug is released from the particles over time as sustained-release [28–30]. Like many other materials, this polymer has the ability to be used for the preparation of nano-sized particles. PLGA nanoparticles are currently being used for therapeutic applications in several forms encompassing therapeutic agents encapsulated in PLGA nanoparticles or linked on the surface of nanoparticles. Depending on the preparation method, the size of this particulate system can be varying between about 100 nm to a few microns. This is a powerful property which enables them to be used for

1.1.2.2  Albumin-based carriers

Albumin is a naturally occurring protein that has recently been considered as a drug carrier for drugs of different nature due to the presence of hydrophobic and hydrophilic components as well as its positive and negative charges [23]. The potential of the bovine serum albumin to form nanoparticles which encapsulating streptomycin sulfate has been evaluated. In this study, the pulmonary dry powder containing albumin nanoparticles along with albumin microspheres and free drug were investigated. The results of in vivo study showed an increase in the presence time and concentration of the encapsulated drug in the target tissue, the lung. In this study, drug concentrations of carrier-based formulations in other tissues and blood were reported to be lower than that of the free drug, which in turn was anticipated to reduce systemic drug side effects [24].

1.2  Nanocarriers based on synthetic polymers Despite the aforementioned benefits of the natural polymeric carriers, these polymers encounter some limitations such as batch to batch structural heterogeneity and developing some allergic reactions in human [25]. To get access to the safe and efficient carriers, synthetic polymers have found their way to pharmaceutics. As natural polymeric nanocarriers, the use of these nanoparticles has not a long history in anti-TB drug delivery and has been taken into consideration in the past two decades. However, compared to the natural polymeric nanocarriers, they

 



1  Polymer-based nanoparticles as drug delivery systems of tuberculosis

different purposes. For instance, methods that give nanoparticles in the range of 100–200 nm can be used in drug delivery for injectable formulation such as immunotherapy and also for targeting. With property and size manipulation, these nanoparticles can also be used in nasal and mucosal drug delivery [31]. As mentioned earlier, according to WHO protocols, the use of a multidrug regimen of tuberculosis reduces the incidence of MDR. Accordingly, in preliminary studies, the PLGA nanocarriers which simultaneously encapsulate several first-line drugs have been studied by an Indian research group. They include rifampicin, isoniazid, pyrazinamide [32–35], and in some cases, ethambutol [36,37]. The administration of these systems has been studied in three routes including subcutaneous [34], oral [33], and pulmonary [32]. Evaluation of the bioavailability of the drugs in the animal models as well as their effectiveness revealed the increase in the presence of drugs in the body for several days and the reduced need for frequent doses. Furthermore, this group reported a good therapeutic effect by the similar nanoparticles containing a reduced dose of drugs compared to their previous studies [36]. Histopathology and toxicity studies have shown the lack of significant biotoxicity of these carriers [37]. Because the most common form of tuberculosis is pulmonary TB, the delivery of drugs through the lungs is very important. Pulmonary dry powders are one of the formulations that have a high potential for prescribing nanoparticles to the lung [38]. Due to problems with nano-sized formulations in pulmonary drug delivery, for proper deposition of the nanoparticles, they must be placed in the particles with appropriate size for deposition in the deep lung (1–5 µm) [38]. In one study, a slower release rate of rifampicin from PNAP (porous nanoparticleaggregate particle) has been shown compared to the free drug administered orally and intravenously [28]. In addition, in another study, the fate of nanoparticles loaded with rifampicin

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and administered as dry powder to the lungs, was studied. In this study, the PLGA microparticles showed a higher in vitro uptake by macrophages, as compared with in vivo uptake of nanoparticles. This finding was attributed to a more prolonged presence of nanoparticles in the alveoli and more time for being up taken with macrophages [39]. In another study, nanoparticles containing rifampicin could accumulate in phagolysosomes, after they enter the macrophages, and they release their medicine slowly [40]. The mechanism of nanoparticle interactions with Bacillus was also studied. It was found that nanoparticles loaded with a hydrophobic analog of isoniazid can directly interact with pathogens and were effective on both the intra- and extracellular bacilli [41]. In addition to the use of common animal models, in some studies, a zebrafish embryo has been used to investigate the efficacy of these nanoparticles. Because of zebrafish inherent susceptibility, the toxic effects of the formulation can easily be detected. Nanoparticles containing rifampicin alone [42] or in combination with thioridazine [43], which is a bacterial efflux pump inhibitor and its free molecular form is supposed to induce a high in vivo toxicity, showed an improved efficacy and animal survival, compared to the free drug. As mentioned earlier, with the change in the polymer structure, the drugs with different natures can be encapsulated inside the carrier. For example, in a study to enhance the encapsulation of isoniazid, the PLGA-PEG-PLGA three-block copolymer was used. Oral administration of this system was associated with more bioavailability of the drug, compared with the free form [30]. On the other hand, in order to increase the isoniazid encapsulation efficiency in the PLGA nanoparticles, a hydrophobic derivative (modified as isoniazid benz-hydrazone) with the same antibiotic effects as isoniazid has been loaded in this carrier. Nanoparticles containing this drug and rifampicin had lower MIC compared to the free drugs [44].

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2  Nanoparticle-based delivery systems for vaccination against tuberculosis

Another fascinating topic is the active targeting of macrophages that are the main source of bacteria. As for targeting moieties, mycolic acid [45], 1-β-glucans [46,47], and lipotuftsin [48] were coupled to PLGA nanoparticles, which led to a better uptake. Due to the high toxicity of second-line antiTB drugs and need to parenteral administration, carriers-based formulations that could reduce the frequency of administration and toxicity, and thus enhance the patient’s compliance are also of particular importance. Among these drugs, ethionamide (PLGA nanoparticles in the form of pulmonary dry powder) [49], levofloxacin (oral PLGA nanoparticles) [50], ciprofloxacin (PLGA-lipid nanoparticles in the form of pulmonary dry powder) [51], moxifloxacin loaded in PEG-chitosan coated PLGA nanoparticle (stealth nanoparticles) [52], and moxifloxacin/econazole [53] and amikacin/moxifloxacin (alginate-PLGA nanoparticles) [54] can be pointed out. In the last case, the uptake of PLGA nanoparticles by macrophages was also studied using confocal microscopy. The modified PLGA nanoparticles were easily internalized into the macrophages. The effectiveness of drug-loaded PLGA nanoparticles has also been studied on MDR TB. In a study, the in vivo effectiveness of orally administered levofloxacin, encapsulated with PLGA nanoparticles, was shown in the treatment of resistant TB [55]. The in vivo efficacy and improved pharmacokinetics of orally administered PLGA nanoparticles loaded with ethionamide on the MDR in vivo model was also observed [55]. In general, polymer-based nanocarriers are suitable candidates for anti-TB drug delivery. These nanoparticles can release the cargo slowly, increase the therapeutic index of the drugs, and reduce their side effects. However, further studies and clinical trials are needed to explain the advantages and disadvantages of the polymerbased particles.

2.1  Tuberculosis vaccines The use of vaccines as a cost-effective medical treatment for the prevention of diseases was initiated since 1796 by Edward Jenner, who introduced the first vaccine against cowpox virus [56]. Nowadays, many vaccines developed against bacterial and viral infections which estimated they can save 8 million lives annually [57]. These designed vaccines are based on liveattenuated or killed pathogens or their subunit antigens [56]. Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) which its vaccine was developed in 1921 [58]. According to the World Health Organization (WHO) reports, TB disease remains as a major deadly global epidemic with approximately 1.67 million deaths in 2016 which was higher than 1 million and 445,000 deaths, respectively, associated with HIV/AIDS- and malaria-related diseases in the same year [59]. Regarding the emerging antibiotic resistance in M. tuberculosis strains, especially MDR, extensively drug-resistant (XDR), or totally drug-resistant (TDR) strains of M. tuberculosis, development of TB vaccines is a global health priority. Recently WHO released an international plan called the Stop TB Partnership with the aim of TB elimination as a public health problem by 2050 [60,61]. To achieve these global targets, the successful research and development for new TB vaccines is one of the most important methods. In this section, types of TB vaccines from the oldest to the newest, advantages and disadvantages of each of them as well as the role of nanoparticles as delivery systems/adjuvants for new TB vaccines will be discussed.

 

2.1.1 BCG The only vaccine available against human TB is a live-attenuated bacterial vaccine called Bacilli Calmette-Guérin (BCG) which was derived



2  Nanoparticle-based delivery systems for vaccination against tuberculosis

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FIGURE 8.1  TB controlling ways.

from Mycobacterium bovis in 1902 after cultured for 13 years by Albert Calmette and Camille Guérin [58,62,63]. The first BCG was used in humans in 1921 through the intradermal administering of 0.05 mL vaccine at birth, infants aged 1 year [58,64]. In addition to TB protection, BCG vaccination can also be used for the treatment of bladder cancer, primary prevention against leprosy and some protection against Buruli ulcer and NTM [65]. BCG is a safe and effective vaccine against severe forms of the disease including childhood TB meningitis and miliary TB disease and recommended especially in countries with a high incidence of TB [66,67]. However, the protection rate of BCG is for a restricted time and controversial in all age groups and against all forms of TB. For example, induced immunity by BCG vaccine is not able to prevent or eliminate TB infection; protective efficacy of BCG in adolescents and adults pulmonary TB is between 0% and 80%; inability in prevention of reactivation of latent TB infection

(LTBI); and there are concerns about safety and effectiveness in human immunodeficiency virus (HIV)-infected children [63,65,67,68]. Overall, three steps proposed for control of TB, including the use of BCG vaccine in healthy individuals at birth, prevention from changing of latent TB to an active form and stopping TB transmission to susceptible individuals (Fig. 8.1) [69]. Therefore, developing new TB candidate vaccines for reducing TB transmission is valuable. For these aims, there are different vaccines such as postexposure, preexposure, and prophylactic vaccines.

 

2.1.2  Preexposure vaccines Similar to current BCG vaccine, there are several in development TB vaccine candidates including the recombinant viable vaccines, such as rBCG and rMtb deletion mutant, and subunit vaccines which considered as preexposure or prophylactic vaccines and target infants and designed for administering prior to TB infection (Fig. 8.2) [63]. Preexposure vaccines are considered to boost BCG, subunit vaccines, or to replace

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TABLE 8.3 Preexposure TB vaccines in clinical trials. Clinical phase (https://clinicaltrials.gov)

Vaccine

Type

Description

Ad5Ag85A (aerosol)

Viral vector

Used antigen: Ag85A Delivery system: adenovirus 5 vector

I

MVA85A (aerosol)

Viral vector

Used antigen: Ag85A Delivery system: modified vaccinia Ankara

I

H4:IC31

Protein/adjuvant Subunit vaccine

Used antigens: Ag85B, TB10.4 Delivery system: IC31

IIa

H56:IC31

Protein/adjuvant Subunit vaccine

Used antigens: Ag85B, ESAT-6, Rv2660c Delivery system: IC31

IIb

ID93/GLASE

Protein/adjuvant Subunit vaccine

Used antigens: Rv2608, Rv3619, Rv3620, Rv1813 Delivery system: GLA-SE

IIa

MTBVAC

Live genetically attenuated MTB

Delivery system: Live mycobacteria

IIa

M72/ AS01E

Protein/adjuvant Subunit vaccine

Used antigens: Rv1196, Rv0125 Delivery system: AS01E

IIb

VPM1002

Live rBCG strain

II/III

Source: TB prevention pipeline report 2018.

BCG, the recombinant viable vaccines and subunit vaccines [63]. As shown in Table 8.3, the VPM1002 and the MTBVAC are two recombinant viable TB vaccine candidates that are entered in different steps in clinical trials. In VPM1002, the immunogenicity of live vaccine improved due to expressing listeriolysin gene (hly) of Listeria monocytogenes and deletion of the urease gene (ureC) [63]. This vaccine currently completed a phase II study in newborn infants with characteristics including BCG-naïve, HIV-exposed, and HIV-unexposed, is beginning a phase III prevention of disease study in individuals with household contact with TB and is initiating a phase II/III prevention of recurrence study in HIV-negative adults (https://clinicaltrials.gov). The MTBVAC is another Mtb-based live vaccine without two virulence genes encoding PhoP and FadD26 [63]. The MTBVAC completed a phase I safety/immunogenicity in adults and preparing to begin a phase IIa dose-defining safety/immunogenicity study in infants (https://clinicaltrials.gov). Subunit vaccines and viral-vectored

vaccines are other preexposure TB vaccine candidates which entered clinical studies for boosting BCG-induced immunity (Table 8.3). Fusion protein M72, consisting of Mtb antigens Rv1196 and Rv0125, along with AS01E adjuvant is a subunit vaccine which currently completing a phase IIb prevention of disease study in HIV-negative and MTB-infected adults. Hybrid 4 (H4) fusion protein, Ag85B, and TB10.4, formulated in the IC31 adjuvant and ID93/GLA-SE other adjuvanted subunit vaccines.

 

2.1.3  Postexposure vaccines Based on the WHO estimate, approximately 1.7 billion of the world’s population are latently infected with TB infection and 5%–10% of them develop active TB [70]. On the other hand, it has been proved that the protective efficacy of BCG vaccine would decline with time after primary infant BCG vaccination due to advancing age and inadequate immunological memory [67]. Additionally, BCG revaccination in adolescents and adults is not recommended due to none positive



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FIGURE 8.2  Three different stages of TB infection/disease for administering of novel vaccines.

effectiveness for the prevention of TB infection [71]. Therefore, BCG is not a good choice for posexposure prophylaxis and in preventing latent TB [72]. A possible reason may be missing some late-stage antigen gene segments in BCG strains compare with M. tuberculosis which are associated with the dormant state such as hypoxia and starvation and weak induction of immune responses against late-stage antigens [73]. Therefore, it seems that the most successful TB vaccines should target the latent stage of TB disease such as postexposure or prophylactic vaccines. Postexposure vaccines will be administered in adolescents and adults with latent infection to eliminate latent TB and prevent reactivation (Fig. 8.2) [67]. However, developing a postexposure vaccine is still in the early steps. There are postexposure TB

vaccines in clinical trial which are based on protein/adjuvant subunit vaccine including ID93/ GLA-SE and M72/AS01E (Table 8.4). These postexposure vaccines can also be used as preexposure TB vaccines and ID93/GLA-SE target TB infection as a therapeutic vaccine. 2.1.4 Therapeutic vaccines After the first attempt by Robert Koch for the treatment of TB patients using semipurified culture supernatants of M. tuberculosis, which called tuberculin, as a therapeutic vaccine, there are many endeavors to use of therapeutic vaccine candidates in combination with conventional chemotherapy in order to prevent TB recurrence or TB treatment especially in TB active patients who suffering from HIV co-infection or MDR-,

TABLE 8.4 Postexposure TB vaccines in clinical trials. Clinical phase (https://clinicaltrials.gov)

Vaccine

Type

Description

ID93/GLA-SE

Protein/adjuvant Subunit vaccine

Used antigens: Rv2608, Rv3619, Rv3620, Rv1813 Delivery system: GLA-SE

IIa

M72/AS01E

Protein/adjuvant Subunit vaccine

Used antigens: Rv1196, Rv0125 Delivery system: AS01E

IIb

Source: TB prevention pipeline report 2018.

 

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TABLE 8.5 Therapeutic TB vaccines in clinical trials. Clinical phase (https://clinicaltrials.gov)

Vaccine

Type

Description

ID93/GLA-SE

Protein/adjuvant Subunit vaccine

Used antigens: Rv2608, Rv3619, Rv3620, Rv1813 Delivery system: GLA-SE

RUTI

Whole fragmented MTB

MIP

Whole-cell M. indicus pranii

Whole killed mycobacteria

III

M. vaccae

Whole-cell M. vaccae

Whole killed mycobacteria

III

IIa

IIa

Source: TB prevention pipeline report 2018.

XDR- or TDR-M. tuberculosis strains [63,74]. Similar to tuberculin failure in TB treatment, due to disease exacerbation via tissue destruction and inducing systemic dissemination of bacterium, there is an intense debate on the safety and therapeutic efficiency of current developing vaccines [63,73,74]. Today, there are four TB vaccine candidates as therapeutic vaccines in clinical trials. These vaccines are based on the killed or attenuated mycobacteria or protein/adjuvant subunit vaccine. ID93/GLA-SE is a therapeutic TB vaccine composed of a synthetic MPL formulated in a glucopyranosyl lipid stable emulsion (GLA-SE) and TB antigens and its function was hopeful in terms of reducing the duration of treatment and mortality rate and protective efficiency in preclinical studies [74]. The ID93/ GLA-SE vaccine target latent and active TB at all age groups and completed phase IIa safety/ immunogenicity study in HIV-negative adults and is planning for phase IIb prevention of recurrence trial (https://clinicaltrials.gov) [63,74]. The RUTI vaccine is a semipurified preparation of killed M. tuberculosis which is produced under stress and starvation conditions with the aim of increasing the expressing dormancy antigens [63]. The vaccine is undergoing phase IIa therapeutic vaccination study in adult patients with MDR-TB. Two other therapeutic TB vaccines, that is, MIP and M. vaccae, that do not have many of the harmful components of M. tuberculosis, are preparing to begin (MIP) or completing

a phase III prevention of disease trials, M. vaccae (Table 8.5).

 

2.1.5  Current vaccines weaknesses Killed whole or live-attenuated organisms or inactivated toxins or subunit proteins are the main ways to develop the new vaccines. They have some advantages such as inducing humoral and cellular immunity only with one administration dose for example in live attenuated vaccines. However, the effectiveness of the most of this new generation of vaccine candidates is faced with challenges due to naturally low immunogenicity, inherent inability to deliver antigens to appropriate sites in order to stimulate both innate and adaptive type of immune system, require to multiple doses and conversion to previous virulent form [75–77]. M. tuberculosis is a bacterium with the intracellular life and therefore elimination of TB infection requires a potent cellular immunity, especially Th1 responses [75,76]. In healthy individuals, M. tuberculosis acquires through coughing or sneezing patients with the active form of TB. When contaminated air-borne droplets reach to the pulmonary alveoli in the human lung, the bacteria are phagocytosed and replicated within inactivated alveolar resident macrophages and other antigen-presenting cells (APCs) including dendritic cells (DCs) and pulmonary epithelial cells. APCs have an important role in establishing the relationship between the innate and the



2  Nanoparticle-based delivery systems for vaccination against tuberculosis

adaptive immune response [78]. They, in addition, to activate the innate immune cells and then eliminate the pathogen through interaction between mycobacterial cell wall components and their specific receptors on APCs, can migrate to draining lymph nodes, activate the adaptive immune responses through presenting antigenic peptides to helper and killer T lymphocytes [78,79]. Therefore, in addition to vaccine safety, an important issue about the new generation of TB vaccine candidates is generating a potent immune response through targeting DCs and following that initiating T-cell-mediated immunity [76]. To achieve the DCs targeting in developing new TB vaccines, the use of adjuvants and delivery systems are recommended.

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2.2 Adjuvants

models to reduce the bacterial load, protective efficacy against primary and LTBI and protect against MDR TB [67]. However, these excellent results of multistage TB-vaccines were obtained along with proper adjuvants because in contrary to live and whole inactivated vaccines, multistage vaccines inherently are not able to activate APCs [67]. A multistage subunit vaccine against TB which is currently in a clinical trial is ID93 in GLA-SE (IDRI) that combined the latency-associated antigen, encoded by Rv1813, along with other early-associated antigens and adjuvant. Adjuvants are enhancers of the immune responses to antigens and can act either as vehicles which efficiently storage, control the release and deliver vaccine antigens to the immune systems or act as immunostimulants [81]. Other adjuvant-based TB vaccines that are in progress are H1/H56/IC31, H4/IC31, and M72 [78]. Additionally, our research showed that the most common adjuvants used in combination with multistage TB subunit vaccines were including DDA, dimethyl dioctadecylammonium bromide, TDB, trehalose 6,6′-dibehenate, MPL, monophosphoryl lipid A, trehalose dimycolate, poly I:C, polyinosinic acid:polycytidylic acid, IC31, gelatin, CpG DNA, aluminum hydroxide, GLA-SE, and MF59 [67]. An appropriate adjuvant for TB infection must be able to induce a robust and lasting Th1 type response [81]. However, currently, only alum (aluminum salts), MF59, AS03 (Adjuvant System 03), Montanide ISA 51, AS04 (Adjuvant System 04), and virosomes are adjuvants which licensed for human vaccines and mostly against viral and extracellular bacterial diseases [82]. Therefore, they are not able to induce cellular immunity against intracellular pathogens such as M. tuberculosis, where Th1 type responses have an important role [81,82]. On the other hand, they have some other limitations such as inducing hypersensitivity reactions, weak stimulation of some immune responses, cytotoxic T-cell and mucosal IgA responses, weak uptake of some antigens, and require administration of multiple doses

One of the most common and promising strategies to replace or improve the BCG vaccine is subunit protein vaccines. These subunit vaccines have been shown several advantages in terms of specificity, safety, easy production, and induction of potent Th1 immune responses [67,80,81]. In recent years, selection of protein antigens, either alone or as a fusion of two or more proteins, from highly immunodominant M. tuberculosis-secreted antigens have been used for designing subunit TB vaccines. These vaccines were based on either early-stage antigens produced by replicating bacteria or late-stage TB antigens expressed from persistent bacilli [67]. However, the capacity of these types of protein-based subunit vaccines was inferior to multistage subunit vaccines [67]. A comprehensive study conducted by Khademi et al. on multistage TB-vaccines showed that they were able to induce potent immune responses, both humoral and cellular, comparable to BCG as well as could boost BCG-primed immunity [67]. In addition, multistage TB-vaccines can be administered as preexposure, postexposure, and therapeutic vaccines and had good ability in animal

 

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[67]. It has been recommended that nanoparticles can help to eliminate the above mentioned limitations of adjuvants. The following characteristics have been proved for nanoparticles including their ability in improving antigens immunogenicity and inducing both humoral- and cell-mediated responses, increasing in vivo antigen stability, and prolonged release of antigens for APCs [67].

2.3  Vaccine delivery systems

physicochemical characteristics to carry various antigens and adjuvants and improve antigen uptake by DCs. Following uptake, both humoral and cellular (CD4, both Th1 and Th2 and CD8) immune responses, as well as memory responses, are induced. These properties are due to their flexible size, charge, and encapsulation efficiency [58]. Therefore, they have extensive applications as a TB vaccine delivery system. Appropriate characteristics of liposomes-based carriers for delivery of TB subunit vaccines have been proved in our research. We observed that liposome-forming lipid such as DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and DDA containing fusion proteins, either alone or along with other biomaterials were able to induce Th1 responses [68,70,84]. On the other hand, lipid-based nanostructures can also show immunostimulant characteristics. We showed that negatively charged ISCOMATRIX nanoadjuvant which is composed of phospholipid, cholesterol, and saponins as well as positively charged PLUSCOM nano-adjuvant which is composed of a phospholipid, cationic lipids and saponins are other lipid-based nanostructures which can be used as TB vaccine adjuvants. Our research in the animal model showed that immunogenicity of a multistage fusion protein of M. tuberculosis was considerably increased (high levels of IFN-γ and IgG2a immune responses) after co-administration with ISCOMATRIX and PLUSCOM nano-adjuvants (unpublished data). Micron-sized particles, 1–10 µm, which called microparticles/microspheres, are capable to be used as vaccine adjuvants as well as antigen delivery systems. Microspheres can induce antigen-specific immune responses particularly via subcutaneous or intramuscular routes and depot the antigens at the injection site and target the DCs and macrophages. They can save encapsulated antigens from clearance, decrease formulation loss as well as induce both immune responses depending on MHC class I and II molecules, as compared to the administration of free antigen which is presented on

One of the most important properties of an effective and ideal vaccine is the induction of life-long immunity, which is not observed in the BCG vaccine [83]. Other good characteristics are stable formulation and easy administration [83]. Achievement of this ideal TB vaccine is highly dependent on antigen delivery systems, in addition to immunodominant antigens and potent immune potentiators [83]. Vaccine delivery systems can classify into virus-like particles, liposomes, microspheres, and nanoparticles. Viral vectors such as vaccinia virus and adenoviruses are two most common replication-deficient systems that their immunogenic properties have proved for boosting the BCG vaccine, especially when administered via the mucosal route [74]. The most important TB vaccine candidates based on the viral vectors, which are in a clinical phase I trial, are Ad5Ag85A and MVA85A. Ad5Ag85A is an adenoviral system and MVA85A is a modified vaccinia virus Ankara which both of them express the Ag85A antigen of M. tuberculosis. They administered as preexposure TB vaccine. ChAdOx1.85A with and without MVA85A is other viral-based vaccine which is completed phase I trial in terms of the safety and immunogenicity. Liposomes are spherical lipid-based nanostructures that have widely used since 1965 as vesicles for drug, gene and cell delivery [58]. However, their immunostimulant and carrier characteristics in order to promote the immune responses were also introduced in 1974 and 1976. These liposomes have shown appropriate

 



2  Nanoparticle-based delivery systems for vaccination against tuberculosis

MHC class II molecules [85]. In addition, they have a good ability for B cell activation through antigen presentation on their surfaces. Several microparticles have been used as a TB vaccine delivery system including chitosan, sodium alginate, PLGA, and polystyrene [79]. Immunogenicity of the smaller particles is higher than the larger particles, therefore, one of the most important reasons to use the nanoparticles as vaccines adjuvant or vaccine delivery system is the particle size [79]. The ability of the nanoparticles with a size range between 300 and 600 nm for uptake by APCs is more efficient than micron-sized particles. Therefore, they can induce necessary immune responses against TB infection, that is, Th1, while the particles in the range of 2–8 µm induce Th2 responses [86]. Additionally, easy preparation and better antigen adsorption due to higher surface area are other advantages of nanoparticles [87]. In this section, we focus on the properties of the natural or synthetic polymer-based nanoparticles. There are several kinds of polymer-based particles which have been used as a TB vaccine adjuvants or delivery system.

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2.3.1  Natural polymer-based nanoparticles Natural polymeric nanoparticles classified into proteins and polysaccharides. Protein-based natural polymeric nanoparticles are including albumin, collagen, and gelatin and polysaccharides-based nanoparticles including chitosan/ chitin and alginate [88]. Among protein-based natural polymeric nanoparticles, the potential of albumin polymer particles as vaccine delivery systems/adjuvants against TB infection was evaluated and reported that can induce mucosal and systemic immune responses [88]. However, the most common natural carbohydrate polymer used for TB vaccine development is a derivative of chitin, chitosan, composed of repeating units of d-glucosamine and N-acetyl-dglucosamine [75,88]. This hydrophilic linear polysaccharide showed several benefits for vaccine delivery including biodegradable and

biocompatible characteristics, less toxic properties, high affinity to mucosal surfaces of epithelial cells due to mucoadhesive characteristics and positive surface charge and the paracellular transport via penetrating tight intercellular connections, controlled release properties, potent stimulation of the immune system due to efficient uptake by APCs and protect from in vivo antigen degradation [75,89]. Additionally, they showed good physicochemical characteristics in terms of size, shape, surface charge, hydrophilicity, and lipophilicity. Chitosan and its derivatives are positively charged particles that can efficiently interact with subunit vaccines and also the mucosal surface of different tissues. Therefore, they can promote immune responses by increasing loaded vaccine antigens stability and encapsulation efficiency and efficient cellular uptake. They can act either as an adjuvant system or a delivery system. In both conditions, they can potently induce both humoral and cell-mediated immune responses [75]. A comprehensive systematic review on chitosan natural polymers showed that there is promising results about the potential of chitosan and its derivatives in parenteral and nonparenteral immunization of TB vaccines [75]. The results of this study reported that chitosan and its derivatives along with TB vaccine candidates that are in developing can enhance immune responses, improve BCG-primed immunity and protect TB-challenged mice against infection [75]. Similar results have been observed in our studies when chitosan and trimethyl chitosan nanoparticles were used as adjuvant/delivery system for M. tuberculosis antigen after parenteral and nasal administration [62]. Positively charged chitosan nanoparticles can act as twoedged sword. They can facilitate efficient cellular uptake of vaccine candidates and on the other hand, cellular toxicity associated with cationic particles can be a problem [75]. For solving this problem, positively charged chitosan nanoparticles can coat with negatively charged particles. We will report that adding alginate

 

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8.  Polymer-based nanoparticles as delivery systems for treatment and vaccination of tuberculosis

can decrease the surface charge of chitosan nanoparticles from +29 ± 2 to −36 ± 2. Additionally, we observed that alginate-coated chitosan nanoparticles exhibited more stability, long-lasting antigen release and strong humoral and cellular immune responses after subcutaneous and intranasal administration in mice (unpublished data). Furthermore, an unpublished study by the same group showed PLGA particles coated by trimethylchitosan could induce higher levels of IFN-γ response through both subcutaneous and nasal routes in BCG-primed vaccinated mice and probably can boost BCG-primed immunity. Other protein-based natural polymeric nanoparticle, that is, alginate was also loaded with TB vaccines. Studies showed that alginatebased TB vaccines when administered subcutaneously and orally, they can induce the mucosal and systemic immune responses [89]. 2.3.2  Biodegradable synthetic polymer-based nanoparticles Biodegradable polymeric nanoparticles can carry a wide spectrum of drugs, proteins, and nucleic acids. They have also good properties in terms of being nontoxic and cell and tissue biocompatibility, stable in blood, controlled/ sustained release, noninflammatory and resistant to the reticuloendothelial system (RES) [89]. One of the most common synthetic biodegradable and biocompatible polymers, which has frequently been used as a delivery system in the development of new TB vaccines, is PLGA (poly(lactide-co-glycolide)) [88]. PLGA is a biodegradable nanosystem with minimal systemic toxicity because of its hydrolysis in the body to LA and GA [89]. PLGA nanoparticles along with other synthetic polymers including PLA (d,l-lactic acid), PGA (poly(glycolic acid)), PCL (poly(ε-caprolactone)), and PMMA (poly(methyl methacrylate)) are approved by the U.S. Food and Drug Administration (FDA) for human administration [90]. Based on a systematic review performed on 14 studies, PLGA polymers, either as nanoparticle or microparticle,

and in combination with various antigens and adjuvants have been used to develop new TB vaccine candidates and illustrated the high levels of inducing humoral and cellular (Th1 and Th2) immunity when administered from different routes in animal model [88]. PLGA nanoparticles are negatively charged. However, a good characteristic of PLGA particles is their structural diversity and possibility to coat their surface with different biomaterials [86]. For example, in one study to prepare the cationic PLGA nanoparticles, we added a cationic lipid/adjuvant (DDA) to the PLGA matrix. These nanoparticles were loaded with a M. tuberculosis fusion protein (HspX/EsxS) and their immunological efficiency was studied. This PLGA:DDA hybrid nanoparticles loaded with HspX/EsxS protein ± MPLA (monophosphoryl lipid A) adjuvant were able to induce Th1 responses after nasal and subcutaneous administration in an animal model [68,70]. These cationic lipid-modified PLGA nanoparticles also showed good properties in terms of stability, better antigen release and then uptake by DCs. Furthermore, the addition of cationic lipids decreased the particle size, while showed a negative effect on the encapsulation efficiency, yield, and morphology [86]. On the other hand, modifications of PLGA nanoparticles with mannose can promote antigen presentation to DCs. In a study, we modified the surface of PLGA with mannose and loaded it with a multistage TB antigen (HspX-Ppe44EsxV) in order to antigen targeting to DCs and inducing immune responses after subcutaneous delivery in an animal model. Compared to plain PLGA nanoparticle, the results were as follows: increase in the particle size, better antigen release properties, better DCs uptake of antigen, and a significant increase in the level of IFN-γ (unpublished data). Another biocompatible and biodegradable polymeric nanoparticle which similar to PLGA particles can be converted into natural monomeric units of LA in the body is PLA [89]. In two conducted studies, the FDA approved PLA which carrying the TB subunit

 

References 139

vaccines was able to robust inducing Th1-type responses in an animal model [88]. 2.3.3  Nonbiodegradable synthetic polymers Polyvinylpyrrolidone, poloxamers, and poly(methyl methacrylates) are nonbiodegradable synthetic polymers which some of them used for TB vaccines [88]. Among them, only poloxamer polymer showed good potentials to induce humoral and cell-mediated immune responses after pulmonary administration in an animal model [88].

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2.4  The future challenges Subunit TB vaccines especially multistage subunit vaccines showed a good potential to develop a new TB vaccine except for their weak immunogenicity. It seems that nanoparticle-based adjuvants/delivery systems are a promising strategy for overcoming this problem. However, for achievement to an excellent formulation for TB vaccine candidates, many changes are needed in both TB vaccines and adjuvants/delivery systems. As assessed, adjuvants/delivery systems give advantages to conventional TB vaccine candidates. However, their ability is doubted in terms of toxicity, safety, crossing from the biological membranes and the slow biodegradability, synthesis costs, and removing from the circulation by the RES. Therefore, further studies are needed to overcome such limitations.

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C H A P T E R

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Nanotechnology-based approaches for tuberculosis treatment Mohamad Mosa Mubarak and Zahoor Ahmad Infectious Diseases PK/PD Lab, Life Science Block, Indian Institute of Integrative Medicine, Srinagar, Jammu and Kashmir, India

1  Drug delivery systems In current times, drug designing is flourishing notably not only as a science but also as an art. This can be credited to advances in knowledge of disease pathogenesis, especially at the molecular level. Drugs are usually administered in a formulated state. A drug dosage formulation comprises one or more active ingredients (components that are actually effective against disease) and their excipients. Modern research has unraveled that the excipients are as important as the drug itself and are no longer considered inert substances. This is due to the fact that these excipients facilitate the preparation, administration, increase the consistent release of the drug, and prevent its degradation. These are also keys to influence the rate and/or extent of drug absorption and thus directly influence the bioavailability of the drug. Bioavailability of a drug can be defined as the amount of the drug available to the systemic circulation out of the total drug administered to a patient. Bioavailability of a drug is an important consideration in developing its pharmaceutical Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00009-6

dosage formulations, as considerable presence of the drug in the systemic circulation is essential to reach its target site and exert its maximum therapeutic benefit. The Four D’s are keys in designing an efficient drug delivery system: drug, destination, disease, and delivery; of these, delivery is the only variable parameter [1]. A modified release system is defined as a drug formulation that is designed to alter the rate and/or place of drug release. Other terms used to define such a formulation include sustained/controlled/slow/ extended/prolonged release systems, and so on. This is usually achieved by encapsulation, which finds extensive applications in the pharmaceutical industry for the abovementioned purposes. Polymers are exclusively used as both conventional excipients and in the form of vehicles in controlled drug delivery. These are classified into natural and synthetic polymers (Table 9.1). Synthetic polymers used as drug delivery vehicles include poly(dl-lactide-co-lactide) (PLG), poly lactic acid (PLA), poly glycolic acid (PGA), poly anhydrides, poly methyl acrylates, carbomers, alginic acid, chitosan, gelatin, dextrans, and so

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TABLE 9.1 Types of polymeric carriers used in drug delivery systems A. Synthetic carriers

B. Natural carriers

1. Aliphatic polyesters and hydroxy acids

1. Proteins and polypeptides

• Polylactic acid • Polyglycolic acid • Poly(lactide-co-glycolide) • Polyhydroxy butyric acid • Polycaprolactone 2. Polyanhydrides

• Albumin • Fibrinogen/fibrin • Collagen • Gelatin • Casein 2. Polysaccharides

3. Polyorthoesters 4. Polyalkylcyanoacrylate 5. Polyamino acids

• Alginic acid • Starch • Dextrans/dextrin

6. Polyacrylamides

• Hyaluronic acid

7. Polyalkylcarbonates

• Chitin • Chitosan

TABLE 9.2 Variables influencing drug bioavailability that are amenable to improvement by using an appropriate delivery system Parameters

Examples of drugs

Low shelf-life

Ethambutol

Unpalatability Extremes of pH Interaction with food Interaction with other drugs Poor solubility in intestinal fluid

Metronidazole Rifampicin in presence of isoniazid in acidic pH Rifampicin Rifampicin Danazol

Poor intestinal absorption

Streptomycin

Extensive first pass metabolism Subtherapeutic levels in plasma Short duration of stay

Propranolol Clotrimazole Azathioprine

Distribution to nontarget organs

Anticancer drugs

2  Tuberculosis: the need for antitubercular drug delivery systems

on are some of the examples of natural polymers [1]. PLGs, alginic acid, and chitosan are flexible to work with and are thus commonly used. Drug carriers from non-polymeric sources such as lipids in the form of solid lipid nanoparticles (SLNs) are also gaining importance. The ultimate aim in choosing a particular carrier system is to circumvent one or several possible factors known to affect the bioavailability of a drug and improve its uptake (Table 9.2).

 

Tuberculosis (TB) is the major cause of morbidity and mortality worldwide among infectious diseases. It is one of the top 10 causes of death and the leading most cause from a single-infectious agent, Mycobacterium tuberculosis (M. tb). In 2017, 1.3 million deaths were estimated due to TB in HIV-negative people and



2  Tuberculosis: the need for antitubercular drug delivery systems

300,000 deaths were recorded among HIV-positive people. An estimated 10 million new cases of TB occur yearly (133 cases in 100,000 population). Additionally and importantly, one-third of the world population harbors M. tb in latent state with chances of getting active disease. There are several antibiotics that are known to have anti-TB activity, including natural products such as the aminoglycosides, the congeners (SM, kanamycin, amikacin [AMK], viomycin, capreomycin), and cycloserine; synthetic compounds such as the nicotinamide analogs (isoniazid [INH], pyrazinamide [PZA], and ethionamide), PAS, thiacetazone, and Ethambutol (EMB); and semisynthetic compounds derived from natural substances such as the rifamycins (rifampicin [RIF], rifabutin [RBT], and rifapentine [RPT]). These antituberculosis drugs (ATDs) are classified as “first-line” and “second-line” based on their activity and toxicity. Drugs (INH, RIF, PZA, EMB, and SM) that exhibit potent anti-TB activity and limited toxicity are called first line, whereas those (kanamycin, AMK, viomycin, capreomycin, cycloserine, ethionamide, PAS, thiacetazone, etc.) that have lesser activity and higher toxicity are known as second-line drugs. Modern recommended standard TB chemotherapy for drug susceptible TB, called directly observed treatment, short-course (DOTS), is a 6-month regimen consisting of an initial bactericidal or intensive phase of treatment with four drugs (INH, RIF, PZA, and EMB) and a continuation or sterilizing phase with INH and RIF for another 4 months. Most of the bacteria are killed during the intensive phase, resulting in reduction of clinical symptoms, risk of transmission, and emergence of resistance. Remaining drug-tolerant persisters are sterilized during continuation phase, thereby reducing relapse rates. Though DOTS can cure TB, owing to long duration and complex treatment, it most often leads to patient noncompliance (nonadherence). Majority of the patients either fail or are unable

145

 

to follow this multidrug regiment-based therapy because of such a long duration. Indeed, it is one of the major reasons behind the fact that among prevalent active cases significant numbers estimated not to receive appropriate anti-TB treatment. Inappropriate treatment happens owing to (1) wrong prescribed treatments in terms of drugs, dosing intervals, and durations; and (2) improper intake by patients. This poor patient compliance has been identified as the principal cause for the generation of drug-resistant TB, which is a major public health concern in many countries. Indeed, this misuse and mismanagement of these drugs has led to the emergence of multidrug-resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB), and total drugresistant TB (TDR-TB). MDR-TB is defined as the TB that is resistant to the most potent ATDs, that is, RIF and INH, and requires treatment with a second-line regimen. XDR-TB is MDR-TB with resistance to at least to one fluoroquinolone and one injectable agent (AMK, capreomycin, or kanamycin) [1a]. Both XDR-TB and TDR-TB raise concerns of a future TB epidemic with limited therapeutic options and wreck the achievements and progress made in TB control. Many countries lack the infrastructure in accurately diagnosing MDR-TB and XDR-TB, which has led to an alarming rise in TB mortality. Additionally, drug–drug interactions, especially between the rifamycins and antiretroviral agents (e.g., protease inhibitors and non-nucleoside reverse transcriptase inhibitors) also complicate TB treatment. Further, drug toxicity has been in focus owing to the rates of adverse reactions and by the hepatotoxicity of some regimens for latent TB infection in HIV-seronegative patients. The administration of ATDs as such causes their dilution and premature metabolic degradation. Therefore, net drug content that reaches the target site is low, which might result in the primary drug resistance. Importantly, current treatment strategies have limited scope in areas where there is high incidence of MDR-TB as one-third of the patients missed the treatment.

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9.  Nanotechnology-based approaches for tuberculosis treatment

In conclusion, for successful treatment, a number of conditions must be met, including (1) multidrug therapy is utmost as combinations prevent the selection of resistant mutants, (2) duration of treatment must be long at least 6 months to prevent post-treatment relapse, and (3) clinician and patient compliance should be supervised to ensure proper administration and intake of antibiotics. These requirements are often practically impossible to meet, especially in developing countries and HIV-TB epidemics. Inadequate therapy leads to poor clinical responses, continued disease transmission, and the emergence and expansion of drug resistance. Therefore, new regimens that require shorter duration or could be administered with longer intermittent duration (once weekly or even less frequently) without sacrificing therapeutic outcome will make treatment more widely available and more acceptable. At present, it is difficult to see how existing ATDs could be used more effectively in this regard and no new drugs are used in the later stages of the drug development pipeline. For this practical difficulty, nanotechnology-based drug delivery systems have the tremendous potential to fulfill this need of TB therapy. Early attempts toward preparing a nanotechnology-based ATD formulation involved the encapsulation of INH in three different polymers, viz., polymethyl methacerylate, polyvinyl chloride, and carbomer [2]. The study was focused to achieve sustained plasma INH levels in fast acetylators of the drug. INH encapsulation and its detailed release kinetics were also studied using another polymer Eudragit RS 100 in the early 1990s [3]. This formulated spherical microcapsule was not further evaluated due to limited advantage. Polyesters such as PLA, PGA, and PLG (also abbreviated as PLGA, Fig. 9.1) are known to possess excellent biodegradability and mechanical strength [4]. The use of PLGAs is approved by the U.S. Food and Drug Administration (FDA) for drug delivery. One advantage of using these PLGAs is the

FIGURE 9.1  Structure of poly(lactide-co-glycolide) (PLG or PLGA)

ease to formulate these into various devices for carrying a wide range of macromolecules. Thus, the use of PLG as a carrier of INH started the era of aliphatic polyesters as sustained-release ATD carriers [4a] Considerable progress has been reported in this field in the past 20 years [5–9]). After laying the foundation for sustainedrelease ATD delivery systems, several pertinent issues were to be addressed, including higher drug encapsulation capacity, higher drug loading, more improved bioavailability, retention time in organs, and further reduction in dosing frequency. The progresses made in nanomedicine-based drug delivery systems addressed all these issues.

3  Nanomedicine and tuberculosis

 

Nanotechnology bridges the barriers between biological and physical sciences by applying nanostructures and nanophases in various fields of science [10], especially in nanomedicine-based drug delivery systems, where such particles are of major interest [11]. The importance of polymeric nanoparticles (NPs) and their role in the development of drug delivery systems is well established (Table 9.3). Nanomaterials are defined as materials with sizes ranging between 1 and 1000 nm. Nanomaterials have influenced all the frontiers of nanomedicine, including biosensors, microfluidics, drug delivery, microarray tests, and tissue engineering [12]. Nanotechnology uses curative agents at the nanoscale in developing nanomedicine [13]. NPs are used as nanospheres; these are designed to act at the atomic or molecular level [14]. Several methods have been reported to obtain particles in the nano-range



3  Nanomedicine and tuberculosis

147

TABLE 9.3 Some important drugs reported to be incorporated in synthetic polymeric nanoparticles Drug

Category

Digitoxin Rolipram Heparin

Cardiac glycoside Antiinflammatory Anticoagulant

Betamethazone Enalaprilat Octylmethoxycinnamate Cyclosporine Insulin Praziquantel Clotrimazole, Econazole Moxifloxacin Gentamicin

Corticosteroid Antihypertensive Sunscreen Immunosuppressant Hormone Antihelminthic Antifungal Quinolone Aminoglycoside

TABLE 9.4 Various preparation techniques for PLG-nanoparticles Techniques

Merits/demerits

Emulsion/evaporation Double emulsion/evaporation Salting out Emulsification-diffusion Solvent displacement/nanoprecipitation Emulsification-diffusion-evaporation

Poor entrapment of hydrophilic drugs Good entrapment of hydrophilic/hydrophobic drugs Lengthy purification process Quick process Poor entrapment of hydrophilic drugs Better reproducibility of size/shape of nanoparticles

[15] (Table 9.4). Nanospheres exhibit unique structural, chemical, mechanical, magnetic, electrical, and biological properties. This enables them to move freely in human body compared to bigger materials. Nanomedicine uses nanostructures to deliver various agents by encapsulating or attaching therapeutic drugs and delivering them to target tissues more precisely with a controlled release [16]. Nanomedicine thus employs the use of knowledge and techniques of nanoscience in medical biology for disease prevention and remediation. Additionally, the implication of nanorobots; nanosensors for diagnosis, delivery, and sensory purposes; and actuate materials in live cells is innate to nanomedicine. Nanostructures are also reported to aid in prevention of drugs from getting tarnished in the gastrointestinal region. These are also shown to help in the delivery of sparingly water-soluble drugs to their target location. Nanodrugs exhibit higher

oral bioavailability as they utilize absorptive endocytosis for uptake. Nanostructures last out in the blood circulatory system for an extended period, enabling the release of amalgamated drugs as per the specified dose. Thus, they cause fewer plasma fluctuations with reduced adverse effects [17]. Nanosize of the structures makes them able to penetrate in the tissue system, thus facilitating easy uptake of the drug by the cells, permit an efficient drug delivery, and ensure action at the targeted location. The uptake of nanostructures by cells has been reported to be much higher than microparticles, and so on. [18]. NPs are thus able to directly interact with diseased cells with enhanced efficiency and negligible side effects. NPs are also useful in acquiring useful information as they are a key to numerous novel assays to diagnose diseases. Benefits of NPs are due to their surface properties, as numerous proteins are appended on the surface.

 

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9.  Nanotechnology-based approaches for tuberculosis treatment

TABLE 9.5 The important variables influencing the encapsulation of ATDs in PLGnanoparticles prepared by the double emulsion/evaporation technique.

For example, gold NPs are used as biomarkers and tumor labels for diagnosis procedural assays. Physiochemical features of NPs are basis for their use in drug delivery. The blended use of NPs with bioactive natural compounds is very attractive and is trendy in recent times. Other nanomaterials used for enhanced target-specific drug delivery include metallic, organic, inorganic, and polymeric nanostructures, including dendrimers, micelles, and liposomes. Drugs having poor solubility with less absorption ability are frequently tagged with these NPs [19]. However, the effectiveness of these nanostructures as drug delivery vehicles changes depending on the size, shape, and other inherent biophysical/ chemical characteristics. For example, polymeric nanomaterials with diameters ranging from 10 to 1000 nm exhibit characteristics that is ideal for an efficient delivery vehicle [8]. Polyvinyl alcohol (PVA), poly-l-lactic acid, polyethylene glycol, poly(lactic-co-glycolic acid), and natural polymers, such as alginate and chitosan, are frequently uses owing to their high biocompatibility and biodegradability properties [20]. Polymeric NPs, nanospheres, and nanocapsules are also excellent drug delivery systems. Similarly, compact lipid nanostructures and phospholipids including liposomes and micelles are very useful in targeted drug delivery. An ideal nanodrug delivery system is primarily based on the biophysical and biochemical properties of the targeted drugs being selected for the treatment [21]. Thus, nanotechnology offers perfect and promising benefits in treating chronic human diseases by site-specific and target-oriented delivery of medicines.

Variable

Drug encapsulation efficiency (%)

1. Drug:polymer  1:0.5  1:0.8  1:1  1:1.5  1:2

40–48 44–56 60–70 60–70 60–70

2. Concentration of polyvinyl alcohol (%w/v)  0.5  1.0  1.5  2.0

45–55 60–70 60–70 54–66

3. Polyvinyl alcohol: dichloromethane (v/v)  100:10  50:10  10:10  8:10  5:10

40–50 55–65 60–70 60–70 45–65

4  Oral ATD-nanomedicine

reported [22]. The particle size of these NPs ranged from 186 to 290 nm, and the drug encapsulation efficiency was recorded to be 60%–70% for all the drugs. Polydispersity index of this formulation was 0.38, which indicated homogeneous nature of these particles with respect to size. Several variables were found to influence the drug encapsulation procedure and their details are summarized in Table 9.5. The formulation was evaluated in vivo for its pharmacokinetics and pharmacodynamic potential at therapeutic drug doses, viz., RIF 12 mg/ kg + INH 10 mg/kg + PZA 25 mg/kg of body weight [23]. The drug levels were maintained above their minimum inhibitory concentrations (MICs) for 6–9 days in the plasma, after a single oral dose of drug-loaded PLG-NP to mice. However, there were no detectable levels of drug beyond 12 h following the oral

The achievement of NP-based encapsulation and controlled delivery of ATDs began almost 25 years back. PLG-NPs prepared by the double emulsion and solvent evaporation technique co-encapsulating with RIF, INH, and PZA were

 



5  Ligand-appended oral ATD-nanomedicine

administration of free drugs (RIF/INH/PZA) [22]. The homogenates of the lungs, liver, and spleen also showed the drug levels above the MIC values for up to Day 9. Therefore, the therapeutic schedule implied requires the administration of the PLG-NP–encapsulated dose every 10 days to infected mice. Such a frequent dosage might cause dose-dependent toxicity because to possible drug accumulation due to sustained release. To evaluate this concern, studies were carried out in which tissue drug levels were monitored on every 10th day following the repeated administration of the formulation. The results indicated that there was no evidence of any drug accumulation in the lungs, liver, and spleen. The chemotherapeutic evaluation of free drugs administered daily (46 doses) and drugloaded PLG-NPs administered every 10 days (5 doses orally to M. tb-infected mice) showed no detectable bacilli compared with a high bacterial load in the lungs/spleen of untreated mice [22]. The biodistribution, pharmacokinetics, and chemotherapeutic efficiency of this formulation in mice were similar when carried out in guinea pigs [23]. Besides reducing the overall duration of chemotherapy, reduction in drug dosage and dosing frequency are key issues to improve the patient’s compliance during ATD treatment. Thus, WHO rightly recommends the use of bacteriostatic drug EMB in the intensive phase of chemotherapy, as it is known to fasten the rate of sputum conversion [24]. A formulation based on PLG-NP–encapsulated EMB was coadministered with the three first-line encapsulated ATDs via oral route to mice and their chemotherapeutic potential was evaluated. It was observed that the drug concentrations were maintained in the plasma of mice for 3, 6, and 8 days in the case of EMB, RIF, and INH/PZA, respectively [25]. On the other hand, free drugs were not detectable in the plasma beyond 12 h of intravenous/oral administration. Bioavailability of the encapsulated drugs was 10- to 20-fold higher than free drugs. The lung, liver, and spleen homogenates

149

showed a detectable EMB level until Day 7. There was also no detectable CFU of M. tb after 4 weeks postchemotherapy in those groups of mice where EMB had been supplemented to the three-drug regimen [25]. These results imply that (1) 3–4 doses of ATD-nanomedicine could replace 28 doses of conventional free drugs and (2) addition of EMB in the treatment regimen yielded a state of undetectable bacilli in just 4 weeks. Thus, the combination of RIF, INH, PZA, and EMB in PLG-NPs improves the drug availability and reduces the dosing frequency (daily to every 10 days) and dose number (28 to 3–4). In the above mentioned studies, experimental infection had been established in mice via the intravenous route. To confirm the conclusions made, it was important to evaluate the efficacy of the PLG-NP formulations in animals infected via the aerosol route as it is the natural mode of acquiring TB. Five oral doses of ATD-loaded PLG-NP and 46 doses of free drugs were found to be equi-efficacious [26]. Similar encouraging results have been reported with ATDs loaded in other synthetic polymer-based nanoparticulate systems, such as poly(butylcyanoacrylate) [27] and poly(ε-caprolactone) [28].

5  Ligand-appended oral ATD-nanomedicine

 

The PLG-based nanomedicine was further improved by the addition of mucosaligand-lectin. The maximum duration of addition of a drug carrier is limited in a mucosal surface and usually lasts only for a few hours. To overcome this problem, polymeric drug carriers can be ligated to certain cytoadhesive ligands that exhibit specific receptor-mediated interactions with epithelial cells of various mucous membranes. Lectins compromise a structurally diverse class of proteins found in organisms, ranging from viruses to plants and humans [29,30]. Lectins are highly specific and are resistant to

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9.  Nanotechnology-based approaches for tuberculosis treatment

proteolytic degradation. These features make lectins an appropriate ligand candidate for drug delivery systems. Wheat germ agglutinin (WGA), a plant-based lectin, is reported to be least immunogenic [31] and it has a number of applications in drug delivery. WGA receptors are distributed on intestinal and alveolar epithelium, potentiating its use for oral as well as aerosol drug delivery [32]. The lectin-coated ATD-nanomedicine regimen was detectable in tissue homogenates until Day 15 compared to Day 11 of the uncoated formulation, indicating a highly significant improvement in the sustained-release profile of all the ATDs using this approach. In guinea pigs infected with M. tb, the dose number decreased to 3 oral doses spaced 15 days for lectin PLG-NP–encapsulated ATDs as compared to 46 in case of unencapsulated ATDs.

6  Pulmonary delivery of ATDnanomedicine

of such a formulation when administered to guinea pigs was able to maintain the therapeutic concentration of the drug in the plasma for 6–9 days and in the lungs for 9–12 days. The half-life, mean residence time, and bioavailability of the encapsulated drugs showed a striking improvement compared to free drugs. Although the pulmonary deposition of ATDs is the main aim in this strategy of drug delivery, a significant improvement in systemic bioavailability is still advantageous following inhalation. The reason is that the enhanced bioavailability would lead to an increase in drug flow toward the lungs via circulation. In this way, the systemic spillover would not be considered drug wastage. Five nebulized doses of the nanomedicine spaced 10 days apart resulted in undetectable CFU in the lungs of M. tb-infected guinea pigs replacing 46 conventional doses [34]. Inhalable PLG-NPs showed an advantage over inhalable microsphere in many ways. Unlike microspheres, it was possible to coadminister multiple ATDs encapsulated in NPs. Similarly, the repeated administration of the NP-based formulation did not show any hepatoxicity [34]. These also elicited a better therapeutic response [32a]. RIF-encapsulated PLG-NP showed remarkable success in targeting drug delivery to alveolar macrophages. RIF formulated into spray-dried PLG-NP and administered to guinea pigs via the intratracheal route was reported to maintain the pulmonary drug levels for 8 h, which is significantly higher than free RIF. The systemic levels in this method were also reported to be 6–8 h [35]. Another method involved encapsulation of RIF into mannitol microspheres. Following inhalation, the alveolar microphages in rats showed a higher uptake of these microspheres as compared to simple RIF-PLG microspheres. The in vivo imaging study showed that these NPs were cleared and more slower than microparticles, leading to an increase in pulmonary retention [36].

The respiratory route of delivering ATDs to the lungs remains the most effective method of drug delivery in TB, as pulmonary TB is the most common form of the disease. NPs that can be inhaled stand a better chance of mucosal adherence, particle delivery, and increase the content of drugs delivered to the lungs [32a]. NPs are also known to be efficiently phagocytized by alveolar macrophages, a board of M. tb, and thus are efficient in releasing their payload at the site of infection [33]. RIF, INH, and PZA were co-encapsulated in PLG-NPs, which upon aerosolization obtained a mass median aerodynamic diameter (MMAD) of 1.88 µm, which is suitable for deep lung delivery. Since high surface hydrophobicity can cause particle aggregation during nebulization on a jet nebulizer, the use of PLG-NP was stabilized by PVA. The use of PVA imparted hydrophilicity to the nanomedicine and hence prevented particle aggregation. A single nebulization

 



8  Alginate-based ATD-nanomedicine

151

7  Injectable ATD-nanomedicine

In other study, lectin was coupled to PLGNP in a formulation for pulmonary delivery of ATDs. Since the lectin receptors are widely distributed in the respiratory tract, the chemotherapeutic potential of this lectin-functionalized PLG-NP was expected to be effective upon nebulization to guinea pigs, the therapeutic drug concentrations were observed to be maintained in the plasma for 6–10 days and in the liver/ spleen/kidney for 15 days. This lectin-coated formulation showed an enhanced bioavailability as compared to uncoated PLG-NP and free drugs. The dose frequency was reduced to 3 doses every 2 weeks to TB-infected guinea pigs, leading to an undetectable CFU in the lungs and spleen. It was also concluded that 46 conventional doses could be reduced to 5 nebulized doses of PLG-NP and just 3 doses with lectincoated PLG-NP [36a]). Nebulization as a mode of ATD delivery is a challenging task. A nebulized dose may be actually swallowed rather than inhaled, making it a confounding variable. Similarly, the net drug delivery in this method depends on the quality of nebulizer and the inspiratory efforts of the subject. Other mean of delivering ATDs to the lungs is insufflation. This involves spray-drying of drug-loaded particles to form inhalable powders. This is followed by administering these powders via the pulmonary route. Insufflation of PA-824 (a nitroimidazopyran) to guinea pigs resulted in sustained drug levels in the lungs up to 32 h, which was significantly higher than oral delivery of the same drug. The CFU and tissue damage levels in the infected guinea pigs were lower, which received high doses of aerosolized PA-824 [37]. Insufflation was also reported to be effective in lower animals such as mice where nebulization procedures are difficult. It was also useful in effective delivery of other ATDs such as capreomycin as well as vaccines [38–40]. Insufflation represents a stepping stone toward quick, safe, and efficient means of pulmonary drug delivery of ATDs.

Instant availability of the drugs to the systemic circulation results in an absolute bioavailability. This is achieved by the intravenous administration of the drugs. Other roots with similar bioavailability profiles are subcutaneous and intramuscular. PLG-NPs are reported to possess high chemotherapeutic efficiency upon administration via the subcutaneous route. A sustained drug level in the blood plasma for 32 days was observed after a single injection of drug-loaded PLG-NP. Significant levels of drugs were also reported from the organs for 36 days. Absolute bacterial clearance from the lungs/spleen of the TB-infected mice was observed upon subcutaneous administration of a single shot of the formulation [41]. These results demonstrated better efficacy of these drugs compared to daily oral-free drug treatment. This system of subcutaneous administration of ATDs proved to be better than injectable PLG microparticles as the later was unable to result in complete tissue sterilization [6]. Table 9.6 summarizes some of the advantages of nanoparticulate system over microparticulate systems of injectable ATD delivery systems based on same polymer. In a study, INH was encapsulated in gelatin NPs conjugated with mannose, using a two-step desolvation method. The particle size ranged from 260 to 380 nm. This formulation was successful in achieving lung targeting in TB-infected mouse when administered via the intravenous route. A significant reduction in CFU was observed with a minimal hepatotoxicity [42].

8  Alginate-based ATD-nanomedicine

 

Alginic acid is a co-polymer of mannuronic acid and guluronic acid (Fig. 9.2). It is approved by FDA for oral usage; oral route is best for patient compliance. It is employed clinically for supportive treatment in reflux esophagitis. It is also in use as a suspending and a thickening

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9.  Nanotechnology-based approaches for tuberculosis treatment

TABLE 9.6 Merits of PLG-nanoparticles over PLG-microparticles as ATD carriers. Parameters

PLG microparticles

PLG nanoparticles

1. Particle size 2. Type of formulation 3. Drug encapsulation (%) 4. Drug:polymer 5. Polymer consumption 6. Sustained drug release in plasma following oral administration to experimental animals 7. Feasibility of nebulization 8. Increase in drug bioavailability 9. Schedule of oral therapy in experimental TB models 10. Therapeutic benefit

10–12 µm Separate formulations for each drug 8%–40% Varies with the drug High 3–4 days No 7–9 fold Weekly Partial

186–290 nm Single formulation encapsulating multiple drugs 55%–73% 1:1 for each drug Low 6–9 days Yes 9–53 fold Every 10–15 days Complete

FIGURE 9.2  Structure of alginic acid.

requires a mild room temperature for drug encapsulation process that is free of any organic solvents. It has high gel porosity, allowing higher diffusion rates of macromolecules, and this porosity can be controlled with simple coating procedures involving the use of polycations such as chitosan or poly (l-lysine) (PLL). The dissolution/biodegradation of alginate-based systems is done under normal physiological conditions [43–45]. All these features have made alginate a carrier of choice for controlled release of various molecules of clinical importance, for example, Indomethacin [46], sodium diclofenac [47], nicardipine [48], diacoumarol [49], gentamicin [50], ketoconazole [51], amoxycillin [52], insulin [53], anticancer drugs [54], and ATDs [55–57]. Alginate-based drug delivery systems are classified as membrane- and matrixbased systems. The membrane-reservoir system involves the release of drug from the core of the inner reservoir and is controlled by the polymer encapsulating the membrane with specific permeability. The release rate is inversely proportional to the thickness of the coat/membrane. The co-encapsulation with nonpolar substances leads to a further decrease in the release rate. This property was exploited in a formulation involving indomethacin which is highly irritant to gastrointestinal mucosa and, as such, the sudden release of drug is highly undesirable [46]. The matrix system is also referred to as the

agent in water-miscible gels/lotions/creams. Alginic acid is also used as a stabilizer for emulsions, and as a binding and disintegrating agent in tablets. Table 9.7 summarizes the various features of alginate as an ideal drug delivery vehicle. Alginate has a high aqueous environment within the matrix. It undergoes adhesive interactions with intestinal epithelium. Alginate TABLE 9.7 Alginate- advantages and favorable properties for use in drug delivery • A natural polymer • Large-scale production economically • Aqueous based technology • Compatible with a variety of substances • Simple drug encapsulation process • Mucoadhesive • Biodegradable • Nontoxic • Formulation of different delivery systems • Sustained drug release • Enhanced drug bioavailability • Applications in biotechnology

 



9  Lipid-based ATD-nanomedicine

TABLE 9.8 Factors influencing drug encapsulation/ release from alginate-based systems

153

• pH of the surrounding medium • Relative proportion of G and M residues • Molecular weight and viscosity of alginate • Drug-polymer ratio • Ionic nature of the drug • Nature and amount of cross-linker • Gelling time • Variation in particle size • Addition of regulatory molecules

that such formulations allow for more loading of the drug with lower consumption of the polymer, as drug-to-polymer ratio in PLG-NP is 1:1. This formulation is reported to exhibit a sustained release for 7–11 days in the plasma and 15 days in the organs, the lung/liver/spleen, after a single oral dose [60]. Fortnightly administered 3 oral doses of this alginate-based formulation was observed to cause complete bacterial clearance in mice infected with M. tb, compared to 45 doses of free drugs [61]. Oral as well as respiratory administration of this alginatebased formulation to guinea pigs gave comparable results [62,63]. Alginate-based NPs are thus considered to be better ATD-nanomedicine when compared to PLG-NP [64]. Other natural carrier-based nanoparticulate systems have also shown similar results with ATDs (particularly RIF). Gelatin NPs encapsulating RIF prepared by two-step desolvation process have shown improved pharmacokinetics and pharmacodynamics in TB-infected mice [64a].

swelling-dissolution-erosion system. It employs a system wherein the drug molecules are dispersed in a rate-controlling polymer matrix. Drug release is modulated by the matrix swelling as well as dissolution/erosion occurring concomitantly at the matrix [58]. A variety of factors influences the drug encapsulation and its sustained release from an alginate-based system and are summarized in Table 9.8. Since the concentration of divalent cations is key in the gelification of alginate, critical adjustments in proportions of Ca2+/alginate allow the formation of microdomains, which exhibit cation-induced rearrangement. These microdomains formed are the NPs referred to as the pre-gel and can be recovered by high-speed centrifugation. Such formulations have been studied via the intravenous route of ATD delivery and have been reported to be effective [59]. This is explained by the facts that alginate exhibits hemocompatibility and avoids rapid clearance from blood by the mononuclear phagocytes due to its hydrophilic nature. These also lead to a longer half-life for alginate-based NPs. Thus, alginate provides a suitable alternative to encapsulate neutral polymers or liposomes, which otherwise require hydrophilic copolymers or PEG fatty acid derivatives to improve their hydrophilicity. RIF/INH/PZA/EMB were encapsulated in alginate NPs and stabilized with PLL/chitosan. Different drug : polymer ratio were tested but optimum drug encapsulation and loading were observed at 7.5:1 and therefore all the studies were carried at this ratio. This ratio suggests

9  Lipid-based ATD-nanomedicine 9.1  Liposome-based drug delivery systems

 

Liposomes are composed of closed vesicles consisting of phospholipid bilayer enfolding an aqueous section. These have been studied as a promising drug delivery model for bioactive compounds because of their exclusive ability to encapsulate both hydrophilic and hydrophobic drugs. When administered into the human body, phagocytic cells promptly recognize these carriers and vacant them from the blood stream. Liposomes were also seen to enhance vigorous take-up by alveolar macrophages and were seen to be effective against intracellular pathogens [65]. Liposomes have been evaluated as better chemotherapeutic agents for delivering ATDs in animal models like mice. Drugs like amphotericin B and doxorubicin have been approved

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9.  Nanotechnology-based approaches for tuberculosis treatment

TABLE 9.9 Mechanism of improved oral bioavailability by Microemulsion Systems

for human use [66]. Gentamicin, a drug used to treat infection by Mycobacterium avium was incorporated into liposomes and the antimicrobial activity was compared to that of the free drug in a mouse model [67]. It was observed that the encapsulated drug considerably reduced the bacterial load in the liver and spleen; however, total sterilization was not achieved. Similar outcome was obtained with diverse liposomeentrapped second-line antibiotics [68]. Owing to their susceptibility to intestinal lipases, liposomes are to be administered by respiratory means or intravenous route. Inclusion of PEG in the liposomal formulations reduces their nonspecific uptake by mononuclear phagocyte system (MPS) of the liver and spleen [69]. Similarly, RIF or INH enclosed in liposomes upon administering into M. tb-infected mice twice a week for 6 weeks was more powerful in clearing mycobacterial infection when compared to the free drugs. Upon coadministration in liposomes of these two first-line ATDs, dose was fruitfully reduced to 1 weekly administration for 6 weeks. No hepatotoxicity was observed upon histopathological examination, and the levels of serum albumin, alanine aminotransferase, and alkaline phosphatase supported the claim. [70].

• Protection of drugs (such as peptides and proteins) from degradation by the metabolizing enzymes. • Increasing the gastrointestinal membrane permeability. • Enhancing the solubility and dissolution of lipophilic drugs. • Increasing the translymphatic drug transport for lipophilic drugs. • Inhibition of the P-glycoprotein (P-gp) efflux pump, which is responsible for secretion of the absorbed molecules entering the enterocytes back to the gastrointestinal lumen.

9.2  Microemulsions as potential ATD delivery systems

[74]. The droplet diameter is usually within the range of 10–100 nm; hence, these systems are also termed as nanoemulsions [75]. For drug targeting and controlled release, microemulsions are widely used in colloidal drug delivery systems (Table 9.9, (2) bicontinuous, and (3) water-in-oil (w/o) microemulsion [76]. Tween-based microemulsion systems have been studied for potential application as a drug carrier for the ATD RIF [77]. Formulation contained oleic acid + phosphate buffer (PB) + Tween 80 + ethanol. The system was analyzed using various physiochemical methods, such as electron microscopy, NMR, optical microscopy, and dissolution and release kinetics. It was concluded that microemulsions containing Tween 80 were effective, since they encapsulated the ATDs (RIF, INH, and PZA) in different combinations. Besides, no precipitation or phase separation was observed [77]. In another formulation, INH was incorporated in o/w microemulsion or w/o microemulsion comprising TX100:AcOH (1:1), followed by cetyltrimethylammonium dichromate (CTADC), chloroform, and water. Such a system was shown to possess the opportunity of sustained release, thus increasing drug solubility and bioavailability [78]. Castor oil and ethylene oxide–based microemulsion system was studied as a means of concentrating RIF for oral drug delivery [79]. This study established that such a formulation was effective and may likely prevail over the problem, since lowering the dose lessens the toxicity.

Hoar and Schulman were the first to introduce the concept of microemulsions in 1943 [71]. Microemulsions are defined as “a system of water, oil, and an amphiphile (surfactant and cosurfactant) which is a single optically isotropic and thermodynamically stable liquid solution” [72]. Due to their thermodynamic stability, high diffusion and absorption rates, ease of preparation, and high solubility, these have gained a lot of attention for the development and design of new drug delivery [72a]. Improved drug bioavailability [73], resistance against enzymatic hydrolysis, and reduced toxicity are some of the features of using microemulsion systems

 



9  Lipid-based ATD-nanomedicine

9.3  Niosomes-based ATD delivery system

155

Niosomes are described as “thermodynamically stable liposome like colloidal particles formed by self-assembly of nonionic surfactants and hydrating mixture of cholesterol in aqueous medium resulting in multilamellar systems, unilamellar systems, and polyhedral structures” [80]. Such systems are analogous to liposomes and can be used as carriers of amphiphilic and lipophilic drugs. Niosomes differ from liposomes in that they are composed of a surfactant bilayer with its hydrophilic ends exposed on the outside and inside of the vesicle to the aqueous phase, whereas hydrophobic chains face each other within the bilayer [81]. The bilayer system in niosomes consists of uncharged single-chain nonionic surface-active agents, while the liposomal structures include the double-chain phospholipids (neutral or charged). The particle size of niosomes ranges from 10 to 100 nm; thus, these lie in nanometric scale and are microscopic. Niosomes exhibit the properties of biodegradability, biocompatibility, chemical stability, low production cost, easy storage and handling, and low toxicity and thus have drawn a lot of interest in the field of modern drug delivery systems [82]. These features of niosomes to deliver drugs in a controlled/sustained manner in different applications and therapies have led to bioavailability development and continuous therapeutic effect over a longer phase of time [83]. INH-based noisome drug delivery system was observed to possess an exceptional potential for development into a low dose performed with effective treatment for TB [84]. Niosomes have also been studied as orally controlled-release systems. Tyloxapol niosome membrane enhanced the drug release of ATDs of hydrophilic origin, such as INH and PZA, which are orally active [85]. Niosomes of RIF and gatifloxacin prepared by lipid hydration technique showed inhibition and reduced growth index when studied the by the BACTEC radiometric technique using the

resistant strain (RF 8554) and sensitive strain (H37Rv) of M. tb [86]. This implied that RIF and gatifloxacinniosomes provided extensive release of drugs, which was optimum for a decrease in dose, lesser treatment days, and increased patient compliance [86]. Brij-35-, Tween 80-, and Span-80-based formulations with PZA niosomes were concluded to help in avoiding hepatotoxicity by limiting the cholesterol content. Drug and polymer compatibility was established by Fourier transform infrared (FTIR) results. Span80-based formulation was observed to possess highest percentage release as compared to others [87].

9.4  Solid lipid nanoparticles-based ATD-nanomedicine

 

SLNs are suspensions of nanocrystalline in water. These are prepared from lipids, which are solid at room temperature [88]. These are relatively new as compared to other forms of lipid-based NPs, including liposomes, lipid emulsions, and polymeric NPs. These exhibit excellent tolerability as they are derived from physiological lipids. SLNs processing and scaling up feasibility is also good. These also have the ability to incorporate hydrophobic/hydrophilic drugs with an enhanced stability that is conferred to the incorporated drugs. SLNs are unique in combining the features of all traditional forms of NPs while eliminating their major shortcomings [89]. SLNs have been studied for formulation development and for drug incorporation with improved bioavailability and targeted drug delivery [90–93]. A variety of methods have been developed for SLN preparation [88]. For example, in the o/w emulsions, the lipophilic core material of SLNs is dissolved in water-immiscible organic solvent that is emulsified in an aqueous phase. In another approach, these are prepared from warm o/w microemulsions. This involves mixing of water, surfactant, and co-surfactant, from which the microemulsion is quenched

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and dispersed in cold aqueous medium. Upon solidification, lipid forms the NPs. High-pressure homogenization has also been used to develop SLNs [88]. RIF/INH/PZA has been co-incorporated in the SLNs using the emulsion diffusion technique. Upon administration via the respiratory route in guinea pigs, it was observed that SLNs were able to maintain the drug release in plasma for upto 5 days and for 7 days in the organs. Seven doses of this formulation were reported to result in undetectable bacilli in the organs of guinea pigs infected with M. tb as opposed to 46 conventional doses [94]. Similar positive results have been reported for the SLNs upon administration via the oral route [95].

10  ATD-nanomedicine for special situations: cerebral TB, drug-resistant TB, and latent TB

the meninges of M. tb-infected mice, as indicated by CFU counting and histopathology [101]. It has been mentioned that poor patient compliance is one of the reasons behind the failure in TB chemotherapy as well as in the emergence of drug resistance, including MDR/XDR/TDR forms of TB [102]. In view of the threat of resistant form of TB and its fatal alliance with HIV [103,103a], nanomedicines were developed using second-line ATDs. Ethionamide (ETH) was co-encapsulated and the formulation was characterized in early 2000s [104]. ETH NPs were prepared by encapsulation in PLG-NP [104a]. A single dose of this formulation was observed to produce a sustained release in plasma for 6 days as compared to 6 h for free drug. ETH was detected in organs for 5–7 days. The results indicated a weekly therapeutic regimen of this formulation. Azoles and fluoroquinolones have also been used in nanomedicine [105,106]. Azole antifungals (clotrimazole and econazole) have been reported to possess some antimycobacterial activity against drug-sensitive [107,108] and drug-resistant [109] strains of M. tb as well as latent bacilli [110]. Similarly, fluoroquinolones (moxifloxacin) are also reported to possess antimycobacterial activity. These activities were strengthened and their concentrations increased and prolonged in alveolar macrophages using NP encapsulation, Moxifloxacin upon encapsulation by poly(butylcyanoacrylate) NPs was observed to exhibit 3 times higher accumulation in macrophages than free drug. The extracellular levels of this formulation were also observed to be 6 times higher than the free moxifloxacin. To inhibit the mycobacterial growth, 0.1 µg/mL of the encapsulated moxifloxacin was compared to 1.0 µg/mL of free drug [106]. A combined formulation of moxifloxacin, econazole, and RIF was also studies upon encapsulation with PLG-NP. Eight weekly doses of this formulation was observed to lead to complete bacterial clearance from the organs of the infected animals [111]. Econazole NPs prepared with alginate

Tuberculosis associated with central nervous system (CNS-TB) is the most feared form of extrapulmonary TB as it is associated with increased mortality and morbidity [96]. Onethird of the patients affected by CNS-TB and the survivors of CNS-TB are known to be left with a lifelong disability. NP-based drugs have been shown to be feasible for localization and effective drug delivery in the brain [97–99]. This is influenced by the targeting process [99a] as well as by the surface charge on the NPs. Neutral NPs such as PVA-stabilized PLG are expected to have better chances of cerebral uptake with low risk of toxicity to the blood–brain barrier (BBB) [100]. This was explored using ATDs encapsulated in PLG-NPs, to check their localization in brain tissue postoral administration when most of the NPs are distributed in extrapulmonary sites including the liver and spleen [22,23]. A single dose of such a formulation was observed to attain sustainable levels in the brain for 9 days. By giving a dose of 5 regular oral doses every 10 days, undetectable bacilli were observed in

 



12  ATD-nanomedicine: unresolved and upcoming issues

have been reported to exhibit an edge over PLGNPs in terms of PK-PD properties as well as in reduced dose frequency by onefold [105,111]. Streptomycin carrying NPs have also been studied as ATD delivery vehicles via the oral route [112]. Streptomycin lost its popularity as an ATD owing to its need to be administered via the parenteral route as well as its potential nephrotoxicity. It is still considered one of the most cost-effective ATDs and is recommended in relapse/treatment failure, withdrawal of INH and RIF, TB meningitis, and co-treatment with HIV protease inhibitors and certain cases of MDR-TB (WHO, 2018). Nanomedicine based on co-encapsulation of streptomycin in PLG-NPs was orally administered to mice, which is not possible in case of free streptomycin. This formulation also exhibited an enhanced bioavailability, increased therapeutic efficacy, and no nephrotoxicity [112].

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11  Potential toxicity of ATD-nanomedicine

therapeutic dose. The PLG formulation examined was found not to be toxic even at 150 times the therapeutic dose. This formed the basis for subacute and chronic toxicity evaluation, wherein PLG formulation was administered every 10th day. These studies also confirmed that there were no adverse effects with drug-loaded and drug-free NPs in mice and rats. These results were confirmed by analysis of the liver, kidneys, pancreas, and bone marrow. Similar results were confirmed by histopathological and biochemical analysis [113]. This confirmed that polymeric NPs in use for medicinal purposes are less toxic as compared to industrial produce/exposure to NPs [114,115]. These data should encourage further exploration of PLG safety profile that may lead to its approval for oral usage. This should also lead to evaluation of PLG in higher animal models as a prelude to clinical studies. Similar studies are also undergoing for other forms of ATD-nanomedicine.

The use of standard ATDs and PLG in surgical implants and sutures has been popular for several decades [4]. However, PLG and drug combination leads to a formulation that combines two different new chemical entities. Thus, PLG-encapsulated ATDs require stringent toxicological assessments for both short-term and long-term chronic uses. Short-term use involves administering a single dose, and its toxicological assessment involves determining median lethal dose, LD50, that is, a dose that would produce mortality in 50% of the animals within 14 days. Long-term toxicological assessment involves administering multiple doses and determining subacute toxicity for 28 days and 90 days for chronic use. The single-dose acute toxicity assessment in mice showed safety as the LD50 could not be determined in comparison to free drugs, which were found to be toxic at 80 their regular

12  ATD-nanomedicine: unresolved and upcoming issues

 

Plenty of experimental evidences have established the fact that nanotechnology-based ATD-nanomedicine is significantly better way to go compared to current TB chemotherapy practices. Careful analysis shows that there are yet unachieved milestones in this domain that hinder human trials for ATD-nanomedicine (Table 9.10). For example, the removal of organic solvents (such as dichloromethane used in the preparation of PLG-NP) in large-scale production of synthetic polymer-based ATD-nanomedicine could be a potential problem. This could be solved by temperature-controlled vacuum drying, which, in turn, raises the cost of production and hence needs attention for more research. A natural polymer-based delivery system such as alginate could serve as a suitable alternative to this issue. Considering the fact that nanosized particles are involved better-quality control

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13  Conflict of interest

TABLE 9.10 Shortfalls hindering ATD-nanomedicine from reaching human trials

The authors declare no conflict of interest, financial or otherwise.

• Removal of residual organic solvents • Cost of polymer/drug carrier • Large-scale optimization of batch-to-batch drug loading • Long-term stability studies • Safety/toxicity profile of new chemical entities • Efficient pulmonary delivery of a suitable dosage form (nebulization vs. insufflation)

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C H A P T E R

10

Dendrimer-based drug delivery systems for tuberculosis treatment Rahul Shuklaa, Aakriti Sethib, Mayank Handaa, Mradul Mohanc, Pushpendra K. Tripathid and Prashant Kesharwanie a

Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research—Raebareli, Lucknow, India; bDepartment of Chemistry and Applied Biosciences, ETH Zurich, Zürich, Switzerland; cICMR-National Institute of Malaria Research, New Delhi, India; dRameshwaram Institute of Technology and Management, Lucknow, India; eDepartment of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, Delhi, India

1 Introduction World Health Organization (WHO) report of 2018 for tuberculosis, extending over 216 countries, including its member states, generated data for the year 2017, proposing around 1.3 million deaths worldwide amongst the HIV negative individuals [1]. Analyzing the census report for HIV positive patients, around 300,000 deaths were reported due to tuberculosis [2]. Further statistical glance asseverated that approximately 10 million new cases of tuberculosis were diagnosed in the year 2017, where the maximum burden was denoted in the Southeast Asian, sub-Saharan African, and Western Pacific regions [3,4]. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, has shown

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00010-2

its evolution over generations, where it was once found in soil and is now it inhabits mammals [5]. Apart from M. tuberculosis, the Mycobacteriaceae family includes varied other species and the most familiar six species are as follows: M. canetti, M. bovis, M. caprae, M. africanum, M. microti, and M. pinnipedii [6]. It is an aerosol-based transmissible disease, with aerosol particles being the most standard mode of transmission. The categorization of the particle size varies from 0.65 to 7 µm, where the former is considered to be smaller while the latter is contemplated as large-sized droplets and carrying along with bacterial viability [7]. The certainty of not developing a disease, even after getting infected with the causative agent, has been found to be around 90%, where

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FIGURE 10.1  Various lines of drugs used for the treatment of TB.

the individuals may encounter one or the other outcomes as mentioned below: • the infection might not have been registered [8], • rapid clearing up of infection after becoming infected [9], • latent infection [10], • positive TB test [11].

capreomycin, cycloserine, p-aminosalicylic acid, fluoroquinolones, and ethionamide. The latter treatment is found to have serious side effects compared to the former [12–14]. Fig. 10.1 summarizes the medication treatment available, the mechanism of action and the side effects associated with the drugs. However, the present-day treatment options have been confronted with varied issues such as:

Isoniazid (INH), rifampicin (rifampin/R), pyrazinamide (PZA/Z), and ethambutol (EMB/E) are the most effective and established as first-line treatment against tuberculosis, administered for a duration of 6 months. However, if this therapy fails due to resistance exhibited by the bacteria against the drug, then the transposition is toward the second-line treatment, which includes streptomycin, kanamycin,

 

• Extensive treatment may arrest patient compliance, leading to minimal response against the treatment [15]. • TB being an opportunistic infection is found to highly affect people having weakened immune system (HIV), where drug treatments such as protease inhibitors,



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nonnucleoside reverse-transcriptase inhibitors, and rifampicin have been found to interact with Cyt P-450 3A4, thus, posing a hindrance in the treatment [16]. • Advancing number of multidrug resistance and extensive-drug resistance cases has further aggravated the current treatment available [17]. • Serious adverse events reported such as peripheral neuritis (ethionamide, ethambutol, and isoniazid), hepatotoxicity, and optic neuritis (ethambutol) have further led to nonadherence to the present treatment [18,19].

therapeutic window, would diminish the associated side effects, and would make the treatment simpler [21–23]. This chapter will mainly focus on dendrimers as a drug delivery system in tuberculosis.

2 Dendrimers

In order to overcome all the above-mentioned complications, a nanotechnology-based approach as a drug delivery system has exhibited an edge over conventional treatment, showing significant improvement in lessening of side effects, elevation in the bioavailability of drug substance, and ameliorated carrier capability by allowing the encapsulation of multiple drugs [20]. Liposomes, solid lipid nanoparticles, microemulsions, liquid-crystalline systems, and dendrimers are some of the nanotechnologybased drug delivery approaches. It is expected that an ideal drug delivery system will be able to deliver the active moieties at the target site, would reduce the dose without affecting the

Dendrimers are globe-shaped macromolecules with a number of branches radiating from a central nucleus [24–27]. It has a precise uniform-sized structure, consisting of numerous adjustable surface functionalities [28,29]. Also, their internal core cavity can be modified as well. All these attributive features, accompanying high solubility in water, magnetize the researchers for its significance in drug delivery [30]. Fig. 10.2 represents the dendrimer interaction with the drug. Augmentation of solubility is one of the proven characteristic features of dendrimers, which has endorsed the researchers to study the interactions between dendrimers and drugs [31– 33]. Though numerous interactions have been examined between dendrimer and drug, they have been mainly converged into two broad categories which are as follows:

FIGURE 10.2  Dendrimer interaction with the drug.

 

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FIGURE 10.3  Diagrammatic representation of various components of dendrimer and its generation.

3  PAMAM dendrimers for tuberculosis treatment

• Drug or the active pharmaceutical ingredient (API) gets encapsulated or entrapped within the dendrimer attributable to the hydrophobic interactions between them or hydrogen bonding or electrostatic force (noncovalent interactions). • Drug interacts to the periphery of the dendrimer due to the formation of covalent interactions between them [34–36]. The incorporation of the hydrophobic drug within the internal core of dendrimer controls the release of the drug because of the variety of noncovalent interactions between them [37]. Apart from acting as a carrier, they can deliver the molecules intracellularly by passing through the cells and the biological membrane [38–40]. Through paracellular and transcellular routes, penetration of dendrimers across the epithelial tissues becomes feasible, which divulges them as potential carriers for oral delivery of drugs [41,42]. Although a wide-ranging list of polymers is available for the preparation of dendrimers, the most widely used are PAMAM (poly(amidoamine)) and PPI (poly(propylene imine)) dendrimers [43–45]. Other dendrimers include melamine, PEA (poly(esteramine)), PEHAM (poly(ether hydroxylamine)), and polyglycerol [46,47]. Fig. 10.3 represents various components of dendrimer along with its generation.

Rifampicin, first-line antituberculosis drug, has been found to encounter solubility issues within the aqueous media and is thus categorized as BCS class II drug [48]. One of the major issues with rifampicin is the conversion of rifampicin into less solubilize form, that is, 3-formyl rifampicin in acidic conditions. In order to overcome this solubility concern, rifampicin was conjugated with the G4 generation PAMAM dendrimer and the stability of the complex was studied at acidic and neutral pH. The complex was found to be quite stable at neutral pH, resembling a sustained release formulation mediately. However, at acidic low pH, the release was discerned to be more simultaneous [49,50]. Simulation studies with molecular dynamics (MD) by Bellini et  al., showed the association of 4th generation PAMAM dendrimer with the first-line treatment drug for tuberculosis, that is, rifampicin. The study results showed that maximally, 20 rifampicin molecules could be loaded in the 4th generation PAMAM dendrimers. According to the docking results of rifampicin with the 4th generation PAMAM dendrimer, a sensibly stable complex was observed at neutral pH while on the contrary, at low pH, rifampicin was observed to be rapidly discharged in the solvent mixture. Thus, this proves that rifampicin loaded

 



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TABLE 10.1 Comparison of PK characteristics of rifampicin and rifampicin loaded PEGylated PAMAM dendrimer.

into PAMAM dendrimers could be used as potential carriers in order to deliver drugs at the acidic sites. Another attempt was to prepare the microspheres of rifampicin utilizing three different generations of PAMAM dendrimer for administration via the pulmonary route [51]. For generations G1, G2, and G3 of PAMAM dendrimer, the pharmacokinetic features were evaluated and compared at pH 5.4, which is the pH of the fluid present in alveoli and also at pH 7.4 (cytoplasmic pH). The complex between drug and G3 PAMAM dendrimer upon evaluation showed prolonged sustained release in comparison to the other two generations [52,53]. Following reasons could be accounted for the same:

Characteristics

Rifampicin encapsulated in the 5th generation EDA-PAMAM dendrimer

Rifampicin alone

AUC

71,451 µg/L h

1154 µg/L h

Cmax

47.8 µg/mL

19.8 µg/mL

• Exponential increase in size and also the molecular weight from generation G1 and G2 PAMAM dendrimers to G3 generation PAMAM dendrimer [54]. • High density of PAMAM G3 dendrimers compared to the other two generations [55]. • Rifampicin might get entrapped within the branches of generation G3 PAMAM dendrimer [51].

delivery of the drug rifampicin [61]. Kumar et al, developed recently rifampicin loaded 5th generation PEGylated EDA-PAMAM dendrimers. Fourier transform infrared spectrophotometry (FTIR) and NMR spectra were used to confirm the PEGylation of 5th generation PAMAM dendrimers. Upon observation, it was being found that the rifampicin entrapped within the PEGylated form of dendrimer had an efficiency of around 98%. Also, the rate of drug release was observed in both PEGylated and non-PEGylated forms of dendrimer, where the former showed the drug release rate of 81% while the latter exhibited the drug release rate of 98%. The study also compared the percentage of hemolytic toxicity in the case of PEGylated and non-PEGylated and a 2.5% decrease was observed in the former from the 11.6%–25.3% of levels observed in the latter [62]. The 5th generation PEGylated PAMAM dendrimer was studied for its PK (pharmacokinetic) characteristics in the Wistar albino rats and the study compared the Cmax (µg/mL), Tmax (h), AUC (µg/L h), t1/2, and MRT (h) between rifampicin and rifampicin loaded PEGylated PAMAM dendrimer. The results are shown in Table 10.1 and Fig. 10.4. Singh and coworkers loaded 1.5-generation PAMAM dendrimer with Isoniazid with the help of the dialysis method, which was further confirmed with FTIR and UV spectroscopy. It was being observed that approximately 93.25% of isoniazid was continuously released for around 24 h, following zero-order kinetics [63].

The negative charge on the cell membrane interacts strongly with the positive charge on PAMAM dendrimer, leading to cell membrane distortion (lysis) and also hemolysis, which is reported as one of the major disadvantages of using PAMAM dendrimers as drug delivery system [55–57]. Upon reduction of positive charge on PAMAM cationic dendrimer either by the addition of lipid or by PEGylation, has shown to decrease the toxicities stated above [58,59]. Furthermore, when rifampicin was loaded with PAMAM dendrimer G5, whose PEGylation was done using polyethylene glycol 2000, where epichlorohydrin was used to link both the polymers, led to a decrease in toxicity [60]. Also, an increase in the loading capacity of the drug was noted with the above-mentioned structural design, hence, proved suitable for sustained

 

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FIGURE 10.4  Comparison of PK characteristics of rifampicin and rifampicin loaded PEGylated PAMAM dendrimer.

4  PPI dendrimers for tuberculosis treatment PEGylation of 5G PPI dendrimer, similar to PAMAM dendrimer, not only increases the biodistribution of the antituberculosis drug but also plays a considerable role in enhancing the capacity of the dendrimer to load the drug and minimizes the toxicity issues encountered with the PPI dendrimers [64]. Further, the drug binds to the PEGylated dendrimer via a combination of hydrogen bond and other hydrophobic interactions behaving as a system from where the release of the drug occurs in a slow manner [65]. Also, due to PEGylation, the 4th and the 5th generation PPI dendrimers, that is, 4G and 5G PEGylated PPI dendrimers not only attained a sustained delivery administration but have also shown an increase in the entrapment of drug rifampicin, where initial loading in 4G PEG-PPI dendrimer was 28% which increased to 39%

with the encapsulation, while in case of 5G PEGPPI dendrimer, it enhanced to 47% [66]. Vijayaraj Kumar et al. prepared 5th generation EDA-PPI dendrimers (where the central core is ethylenediamino), which were mannosylated to deliver the drug selectively at the target site, that is, macrophages in the alveolus. Rifampicin, the first-line antituberculosis drug, was selected and loaded into the above-mentioned complex [67]. The concentration of drug at the target site, that is, macrophages in the alveolus is more than in the plasma, almost in the ratio of 16:1. Thus, this would be appropriate to target for treatment in tuberculosis [68]. For mannosylation, the 5th generation EDAPPI dendrimers were coated with mannose (30D-mannose molecules) and loading of the drug was carried out. The selection of mannose was based on the fact that it is easily recognized by the phagocytes lectin receptors, which resides on the surface of the cells. As stated earlier, the

 



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4  PPI dendrimers for tuberculosis treatment

stability of the complex between the drug and the dendrimer was found to be predominantly dependent on hydrogen bonding, other interactions are supposed to be hydrophobic interactions and electrostatic forces [67]. With the help of a conventional and familiar dissolution protocol, approximately 37 molecules of rifampicin were incorporated into the PPI dendrimers. The shape of the mannosylated PPI dendrimers was characterized using SEM studies and it was found to be irregular and agglomerated, having a diameter of less than 5  µm. The solubility levels of the drug rifampicin was compared in both nonmannosylated and mannosylated PPI dendrimers, where the former exhibited the solubility levels of approximately 50 mg/mL while the latter showed a decrease in solubility by approximately 5 mg/ mL in contrast, although these levels were twice in comparison to the solubility of rifampicin alone in an aqueous solution. The solubility of rifampicin, upon mannosylation, showed expansion by a factor of 2 because of the interactions between the drug and the dendrimer complex stated above. Also, mannosylation led to a further reduction in the hemolytic toxicity of EDA-PPI dendrimer of 5th generation from a value of 15.6% to 2.2% approximately. The reduction is observed because of the fact that the mannosylation prevents the interaction of NR4+ (quaternary ammonium ions) with the cells [67]. The cytotoxicity was further confirmed against the Vero cell line, where mannosylated PPI dendrimer encapsulating rifampicin showed higher

viability of approximately 85% compared to rifampicin alone, which showed viability of 50% when the concentration of both was same. Mannosylation further increased the availability of the drug at the target site. The free solution of rifampicin, which did not participate in any of the complex formations, when compared with the drug at the target site complexed with mannosylated EDA-PPI dendrimer (5G), the latter showed an increase in the concentration of drug at the target site [69,70]. The evaluation of drug release was carried out at two different pH, 5 and 7.4. At pH 7.4, the formulation mimicked the sustained release/ delayed release because of the presence of 3° amine group within the cavities of the dendrimer micelles, which deprotonates the drug rifampicin, leading to the binding of 3°N with the carboxylate ions of the drug molecule. However, at pH 5, comparatively faster release rate was observed because of the protonated rifampicin molecules within the cavities of dendrimers. An increment in the uptake of mannosylated PPI dendrimer encapsulating rifampicin was observed in the macrophages of the alveoli of rat lungs was observed in comparison to the drug alone [67]. Carrying the same approach further, 4th and 5th generation PEGylated PPI dendrimers were looked upon and results stated that they exhibited effects similar to the sustained release form of rifampicin. The PEGylation further increased the entrapment capability as well as the release of rifampicin from PPI dendrimer as shown in Table 10.2.

TABLE 10.2 Comparison of entrapment capability and release rate of rifampicin from 4th and 5th generation PEGylated PPI dendrimers. Characteristics

4th generation PEGylated PPI dendrimers

5th generation PEGylated PPI dendrimers

Rifampicin entrapment

Before PEGylation

After PEGylation

Before PEGylation

After PEGylation

28%

39%

57%

61%

Cumulative release rate of rifampicin (after 36 h)

97.3%

 

46.3%

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10.  Dendrimer-based drug delivery systems for tuberculosis treatment

5  Melamine, PEHAM, and PEA dendrimers for tuberculosis treatment Melamine dendrimers/melamine-based dendrimers are synthesized by using either the convergent approach or the divergent approach, both discussed later in this chapter in detail [71–73]. The PEG group, when attached to the melamine dendrimers, exhibits the same formulation principle as shown by the sustained release formulation when it encapsulates the antituberculosis drug. One of the examples of this composition is 3rd generation PEGmelamine dendrimers [74]. Similar to other dendrimer structures, the presence of cationic groups impart hemolysis and cytotoxicity and addition of PEG groups lead to a reduction in the same [75]. In addition, the drugs encapsulated into melamine dendrimers have exhibited to have improved solubility [76]. All the abovementioned approaches for the melamine dendrimers could be found beneficial in improving the availability, solubility, and the efficacy of antituberculosis drugs. Melamine-based dendrimers have pioneered in the field of cancer by improving the solubility of anticancer drugs and also lowered the toxicity [77]. PEHAM dendrimers tend to improve the aqueous solubility of some poorly soluble hydrophobic drugs. Hence, PEHAM dendrimers could be used as one of the approaches to escalate the bioavailability of antituberculosis drugs by enhancing their solubility [78,79]. The active drug substance could be encapsulated within the core of the dendritic micelle, or could be associated with one or the other surface groups of the dendrimers or could be both because of the electrostatic, hydrophobic, and hydrogen bond interaction between the drug and the dendrimer molecule [80,81]. The presence of cationic groups on the surface of PEHAM dendrimers tends to elevate the toxicity towards the red blood cells [46,82]. Thus, approaches to overcome the toxicity issues associated with PEHAM dendrimers encapsulating

antituberculosis drug could be glycosylation and acetylation, apart from PEGylation [83,84]. Similar to the other classes of dendrimers, PEA polymers can also encapsulate drugs and thus enhance the t½ (half-life) of the molecule, attaining sustained release approach. Hence, PEA polymers could be an exemplary approach for the antituberculosis drug to upsurge their availability at the target site [85].

6 Conclusion

 

Tuberculosis is one of the most infectious diseases in developing countries. The present treatment involves an extensive regimen for the cure of a disease that may disturb patient compliance and thus, could lead to withdrawal from the DOTS therapy. Though a number of first-line antituberculosis treatments are available, the therapy usually fails due to resistance exhibited against the drugs by the bacteria. Even if a shift to a different regimen is made, say second-line treatment option, serious side effects are usually observed, which may again lead to withdrawal from the present treatment therapy. However, a shift to the nanotechnology-based approach as a wiser option for the administration of the drug is made, which has shown impressive results in not only lessening the side effects but also shown improvements to enhance the bioavailability of the drugs. Further, this approach as a drug delivery system could be administered via different routes, be it oral or pulmonary administration. The drug can be loaded in the matrix (single or multiple drugs) and thus its carrier ability is improved. The chapter highlights the use of dendrimers as a novel drug delivery system for the treatment of tuberculosis. The problems encountered with the drugs associated with the DOTS treatment, such as low solubility, poor bioavailability, and minimized efficacy have been overcome using dendrimers. The administration of the drug directly at the target site, mimicking sustained



6 Conclusion

release formulation, maintaining high efficacy, all of these have been achieved exquisitely with the use of dendrimers. Also, dendrimers have managed to let the drug remain in the skin layers and hence, not entering into the systemic circulation. Oral, parenteral, intraocular, and nasal routes could be accessed for drug administration with the help of dendrimers. The dendrimer-based drug delivery system has also advanced in varied other fields of bioscience, be it genetics, where it used as a vector for therapeutic applications in gene therapy. Commonly used dendrimers are PAMAM and PPI. Also, these have been found useful in the diagnosis and treatment of cancer, where it has been found to overcome the issues associated with conventional anticancer drugs. Hopefully, the future may rise with the new drug delivery options and somehow, could also reduce the side effects associated with the use of dendrimers in treatment therapy. Also, research is going on to improve the tolerability of the dendrimers in the big pharma companies and one can expect them to be one of the pioneer methods of administration.

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C H A P T E R

11

Polymeric micelle-based drug delivery systems for tuberculosis treatment Bapi Goraina, Hira Choudhuryb, Sreenivas Patro Sisinthya and Prashant Kesharwanic a

School of Pharmacy, Faculty of Health and Medical Sciences, Taylor’s University, Subang Jaya, Selangor, Malaysia; bDepartment of Pharmaceutical Technology, School of Pharmacy, International Medical University, Kuala Lumpur, Malaysia; cDepartment of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Hamdard University), New Delhi, India

1 Introduction Delivery of therapeutics with the desired effectivity and safety to the site of action is a great challenge to the formulation scientists. Nanotechnological platform helped to develop different nanocarriers for therapeutic and diagnostic applications of several drug molecules targeting to several diseases to obtain desired efficacy [1–4]. Basically the hydrophobic nature of the therapeutics creates the hurdle to deliver the drugs using conventional drug delivery platform. Several polymeric approaches, such as nanoparticles, dendrimers, soluble complexes, and polymeric micelles, are widely used to solubilize the hydrophobic therapeutic agents and

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00011-4

deliver them effectively and safely to the target site [5–8]. Among these polymeric nano-deliveries, polymeric micelles have gained tremendous interest in treatment and diagnosis of several complicated diseases. The amphiphilic polymers are used to develop such potential nanocarrier to overcome the solubility issues of hydrophobic drugs. The sizes of these polymeric micelles are ranged between 10 and 200 nm [9–11], where the structure of the carrier is formed at a certain concentration of the polymer in the aqueous environment, that is, critical micelle concentration (CMC). The inner hydrophobic core of the miceller delivery allows to solubilize the drugs, which is further completely separated from the exterior hydrophilic environment by the external

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hydrophilic membrane of the polymeric micelle. Possibilities are inclusive to improve external architecture of the nanocarrier by specific ligand, which when delivered to the systemic circulation distribute the incorporated drug at the site of action, thus prevents unfavorable exposure of the therapeutics to the normal healthy cells [5]. Researches on miceller deliveries since last three decades has brought several approaches to improve the solubility, as well as pharmacokinetic profile of the lipophilic therapeutics [12,13]. Entrapment of the drugs within the miceller structure have shown to improve in vivo behavior of the drug when compared with free drug, where the formulation allows site-specific delivery of the therapeutics, thereby able to reduce the toxicities associated with the therapeutic agents [12–14]. This versatile nanocarrier can have the possibility to deliver through oral, ocular, parenteral, and intranasal administration to achieve the desired site-specific delivery of the therapeutics [15,16]. Sustained release of the entrapped drug from the miceller structure allows to maintain a constant concentration for a prolong period [17]. Their nanometric range of size capable to prevent from recognition by the reticuloendothelial system (RES), allow them to travel through the blood capillaries, at the same time these are not allowed to excrete prematurely via glomerular filtration [18]. Furthermore, these structures allowed rapid penetration of the drug from the cell membrane, may be by improving uptake of the drug loaded miceller structures or by taking an alternate pathway of internalization (endosomes) [11]. Thus, by translating the advantages of miceller deliveries, we can achieve longer mean residence time of the therapeutics in systemic circulation with altered pharmacokinetics profile, reduction of required dose of administration, improved bioavailability of the drug, and decreased unfavorable toxicities because of precise delivery of the therapeutics at the diseased site [14,19–22]. Polymeric micelle plays an important role in the search of advance therapy due to their

hydrophobic core, which can accommodate hydrophobic drugs; whereas, hydrophilic corona of polymeric micelle can be modified with polyethylene glycol (PEG) to prolong their circulation time, further enhanced cellular uptake and can support both active and passive targeting. Interestingly, various polymeric micelles of chemotherapeutics reached in clinical study [23]. Few of the preclinical application of polymeric micelles in the improved therapy of different critical health problems have been summarized in Table 11.1.

2  The structure of polymeric micelle The structure of the polymeric micelles is found to be similar to those proposed in several miceller theories so far. As mentioned earlier, the structure of the micelles are composed of an interior hydrophobic section, “core,” while the exterior part of the micelles are called as “corona,” the hydrophilic area of the amphiphilic polymer structure (Fig. 11.1). Several different amphiphilic polymers could be employed to obtain such core-corona structure of the micelles. The details of the amphiphilic polymers with examples, explaining their advantages in conforming miceller structure, have been demonstrated in subsequent section of the chapter.

2.1  Corona of miceller structure

 

This part of the micelle represents the hydrophilic surface of the structure. In most of the cases in literature, represent PEG as the building block for the hydrophilic surface of polymeric micelles [5]. Because of its justifiable advantages as pharmaceutical excipient, it has been approved by FDA and consequently been incorporated extensively as component in hydrophilic corona in polymeric miceller deliveries [5]. Different properties of PEG have been described below, which make this excipient as



2  The structure of polymeric micelle

177

TABLE 11.1 Application of polymeric micelles in the treatment of various therapy. Disease target

Entrapped medication

Polymer used

Source

Breast cancer, squamous carcinoma

Paclitaxel

Methoxy PEG-b-(N-(2-benzoyloxypropyl) methacrylamide) block copolymers

[24]

Breast cancer

Docetaxel

Amphiphilic block copolymers PEG methyl etherblock-poly(d,l lactide)

[25]

Breast cancer, ovarian cancer, and multidrug resistant cancers

Quercetin

Pluronic P123 and P407, TPGS

[26]

Subcutaneous and metastatic breast tumor

Doxorubicin

Methoxy PEG-P(AAm-co-AN-co-VIm) copolymer

[27]

Small cell and nonsmall cell lung cancer

Etoposide and cisplatin prodrug

Poly(2-oxazoline)s poly(2-methyl-2-oxazoline-block2-butyl-2-oxazoline-block-2-methyl-2-oxazoline)

[28]

Tuberculosis

Mycolic acid

PEG-b1-poly(propylene sulfide)

[29]

Tuberculosis

Rifampicin, Pyrazinamide and Isoniazid

Polyvinylpyrrolidone–polycaprolactone

[30]

Tuberculosis

Rifampicin and ferulic acid

Chitosan-modified-polycaprolactone

[31]

Tuberculosis

Rifampicin

Poly(ε-caprolactone)–b-PEG-poly(ε-caprolactone)

[32]

Tuberculosis

Rifampicin

Chitosan or hydrolyzed galatomannan/chitosan modified poly(ε-caprolactone)-b-PEG-bpoly(epsilon-caprolactone) polymeric micelle

[33]

Tuberculosis

Rifampicin

Poly(ε-caprolactone)-b-PEG-b-poly(ε-caprolactone)

[34]

Tuberculosis

Rifampicin/or pyrazinamide individually and combination rifampicin and isoniazid combination

PEG-poly(dioxanone-co-methyl dioxanone) block copolymer

[35]

Rheumatoid arthritis

Methotrexate, nimesulide

RGD peptide-PEG3400-poly(lactic acid)2000

[36]

—

Alpinumisoflavone

mPEG-b-poly(d,l-lactide)

[37]

an important component in pharmaceutical preparations: 1. chemical structure of PEG consists of large number of hydroxyl groups; 2. molecular weight of the compound is low; 3. benefit in forming stealth carriers, which can easily be escaped from the RES; 4. compatible to the biological system; 5. nontoxic nature.

the hydrophobic therapeutics or any bioactive materials. Hydrophilicity of PEG at the surface of the micelle prevents several unwanted consequences of the formulations, such as precipitation of the micelles or their aggregation, adhesion to the cells or absorption of proteins for clearing from the system [39,40]. PEG has been widely used in the incorporation of several therapeutics when formulated using polymeric micelle platform, including chemotherapeutics such as paclitaxel, doxorubicin, camphothecin, and β-lapachone [41–44]. Among the varieties of

These properties of PEG insist miceller corona formation by solubilizing and encapsulating

 

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11.  Polymeric micelle-based drug delivery systems for tuberculosis treatment

FIGURE 11.1  Schematic representation for the micellization of diblock copolymers and drug encapsulation in polymeric micelle. CMC, critical micelle concentration [38].

available PEG, most of the researchers adopted low molecular weight PEG, within the range of around 2000–5000 Da for their amphiphilic block copolymeric formulation development; however, evidence of utilizing high molecular weight PEG in miceller formulation development is also evident in literature [45–47]. Change in molecular weight of PEG may alter the CMC to formulate the polymeric micelle, at the same time the loading capacity of the formulated micelle is also impacted [5]. In a single study it has been clearly reported the effect of different molecular weight PEG when delivering a hydrophobic therapeutic agent. The authors incorporated methoxy derivative of PEG conjugated N-octyl-O-sulfate chitosan for the preparation of the polymeric micelles. In this context, they used three different molecular weights of methoxy PEG, that is, 1100, 2000, and 5000 Da. A stronger polymeric micelle was formulated with the higher molecular weight PEG, using the 2000 and 5000 Da, when compared to the polymeric micelle with 1000 Da PEG. Further analysis has brought the highest entrapment efficiency of the hydrophobic drug with 2000 Da PEG and it was more than 82%. Escaping by RES systems and simultaneous absorption of plasma proteins and formation proteins corona on hydrophobic core of nanocarriers was also decreased due to the presence of PEG [45]. Some other researchers have also introduced poly(ethylene oxide) as a corona forming unit with the similar advantages

of PEG [48]. Thus, presence of multiple hydroxyl groups, biocompatibility, and less toxicities of PEG contributes advantages of PEG in the formation of polymeric micelles. Apart, different ligand moieties can also be functionalized onto the surface of the hydrophilic corona in order to make a targeted miceller delivery specifically for a specific site. Several ligand molecules are thus can be attached to the surface of the micelles include monoclonal antibodies, folic acid, or monosaccharides to achieve active targeting of the entrapped therapeutic. Further, these surface modifications can also help to prepare temperature or pH responsive nanocarriers [49–51].

2.2  Core of miceller structure

 

This is the hydrophobic block of the miceller structure, use of which is immense in the literature [52–56]. The choice and design of this hydrophobic block can be altered based on the requirement, where a wide variety of lipophilicity can be obtained in the miceller delivery to incorporate drug of choice. Most of the researchers have incorporated poly(β-benzyll-aspartate), poly(dl-lactic-co-glycolic acid), poly(d,l-lactide), polycaprolactone, poly(l-lactic acid), etc., which were extensively discussed in the next section [57–61]. Different researchers have incorporated di-block copolymers and triblock copolymers in the preparation of core



3  Commonly used polymers in polymeric micelle

structure of the polymeric micelle to effectively deliver therapeutics to the site of action [5]. Therefore, there are large likelihoods to choose and select the polymers for the formation of hydrophobic core to encapsulate the hydrophobic therapeutics effectively through development of polymeric micelles.

179

3  Commonly used polymers in polymeric micelle

Some of the commonly used core forming blocks of polymeric micelles include poly(propylene oxide), poly(lactic acid), poly(amino acids), copolymers of lactic acid and glycolic acids, and poly(caprolactone). This component is responsible for imparting good stability, high drug loading capacity, and desired drug release profile. On the other hand, the hydrophilic block affects the stealth properties and influences the circulation kinetics the micellar structure. Poly(ethylene glycol) with a molecular weight of 1–15 kDa is the most commonly used hydrophilic segment of the block copolymers as it is nontoxic, provides steric stability, and FDA approved for biomedical applications in humans. The other frequently used PEG alternatives as hydrophilic blocks are poly(N-vinyl-2-pyrrolidone) and poly(acrylic acid).

Polymer micelles are made up of amphiphilic copolymers with distinctive hydrophilic and hydrophobic blocks. These copolymers may be amphiphilic diblock (hydrophilic-hydrophobic) or triblock (hydrophilic-hydrophobic-hydrophilic) polymers. The commonly used diblock copolymers used to design polymeric micelles are of A-B type where A represents a hydrophilic block and B represents a hydrophobic block. Triblock copolymers can be prepared from either two polymers (ABA) or three polymers (ABC). For drug-carrier applications, ABA type is commonly employed [62]. The defining characteristic of polymeric micelles is their ability to spontaneously self-aggregate into nanoscale structures. The formation of a micelle in an aqueous solution occurs when the concentration of the block copolymer increases above a certain concentration called as CMC. At the CMC, hydrophobic portion of block copolymer start to associate to form a core in order to minimize the contact with water molecules, surrounded by a hydrophilic shell leading to a coreshell structure. Mostly, polymeric micelles have a low CMC compared to low molecular weight surfactant micelles because of the greater interfacial free energy derived from the larger insoluble segments [9]. Apart from the ability to spontaneously self-assemble in water they should also improve the drug solubility, provide high loading efficiency, remain stable upon dilution in the GI tract, be biocompatible and nontoxic, and be easy to synthesize at large scale.

3.1  Commonly used amphiphilic block copolymers

 

Derivatives of poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers are the widely studied amphiphilic materials to prepare polymeric mixed micelles [63]. The commercially available grades of these block copolymers are Pluronics or Poloxamers which are linear triblocks and Tetronics or Poloxamines which are their branched counterparts. Poloxamines comparatively provide thermal stability and pH sensitiveness due to the presence of two tertiary amine groups in the center of the molecule [64]. Both Pluronics and Tetronics confer advantages like low toxicity and suitable biocompatibility because of their availability in a wide range of molecular weights and ethylene oxide/propylene oxide ratios [65]. Regardless of these advantages, these block copolymers usually exhibit high CMC, which makes the formulation less stable and the micelles tend to dissociate easily upon dilution in the bloodstream [66]. Other block copolymers used to prepare polymeric

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micelles are the ones formed by biodegradable hydrophobic blocks of polyesters such as poly(lactic acid), poly(lactic-co-glycolic acid), poly(ε-caprolactone), polyanhydrides which are often covalently bonded to hydrophilic blocks, mainly PEG [67]. Apart from above, other polymers that selfassemble into polymeric micelles are the PEGylated phospholipid-based block copolymer, such as PEG-distearoyl phosphatidyl ethanolamine. The advantage of using these polymers is that they are simple and more reproducible to prepare, provide longer circulation times, avoid macrophage phagocytosis system uptake, biocompatible, and nontoxic [63]. Recently, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus), a polymer with amphiphilic properties is studied for its successful solubilization potential for poorly water-soluble drugs. These polymers exhibit high stability upon dilution due to its low CMC value [68–70]. Similarly, Solutol HS 15, a mixture of PEG15 mono and di-esters of 12-hydroxystearic acid and free PEG a recently explored polymer is used for the preparation of mixed micelles [71] to improve the solubility of poorly soluble drug and is widely employed in both oral and intravenous drug delivery formulations [72]. Some of the polymeric micelles are also widely exploited for their pH-sensitivity and potential as pH-dependent release at tumor sites. Polymers from the poly(l-amino acid) (PAA) family covalently bonded to hydrophilic blocks of PEG have been studied for this purpose. The widely used polymers from PAA family include poly(l-histidine), poly(l-aspartic acid), poly(lglutamic acid), and poly(l-lysine). One of the requirements for this is that the PAA portion of the micelle must be either neutrally charged or conjugated to lipophilic fractions [73]. Another class of polymers is acrylic-based such as poly(methacrylic acid) (PMAA) which are also widely used in oral pH-sensitive drug delivery [74]. Copolymeric net-

work of poly(methacrylic acid) grafted with poly(ethylene glycol), shown a reversible pHdependent swelling behavior. The formation of interpolymer complexes between protonated acid groups and the etheric groups on the graft chain resulted in the formation a gel, which exhibited less swelling at low pH. PMAA and PAA block copolymers have also been exploited for their mucoadhesive properties for oral drug delivery applications [75]. The bioadhesion of the polymeric micelles to the mucosal lining lead to immobilization of the drug carrier and this prolonged contact will result in the enhancement of absorption and bioavailability. Polymeric micelles can also be modulated to overcome the drug-efflux by inhibiting Pglycoprotein (P-gp) transporter and subsequently enhance the oral bioavailability. Amphiphilic polymers inhibit of efflux transport by modifying the fluidity of the cellular membrane [76]. Polymeric micelles prepared with d-α-tocopheryl polyethylene glycol succinate (TPGS) and Pluronics have been widely studied for this purpose and if found to inhibit the P-gp efflux transporter system. In addition to this, other amphiphilic polymers that has been reported as P-gp inhibitors are PEG-phosphatidylethanolamine [77], mPEG-block-polycaprolactone [78], mPEG-poly(caprolactone-trimethylene carbonate) [79], PEG-b-poly(lactic acid) (PLA) [80], and N-octyl-O-sulfate chitosan [81]. Some of the selected amphiphilic block copolymers have been listed in Table 11.2 which are incorporated in different preclinical research to obtain improved delivery using the polymeric miceller platform.

4  Polymeric micelles for enhanced permeability and retention effect

 

Improved therapeutic approaches towards infections including tuberculosis largely falls into two categories; firstly, identifying new molecular target based on the molecular



TABLE 11.2 Selected examples of amphiphilic block copolymers and their applications. Drug

Pharmacological activity

Application

Source

1

Solutol HS15 and Poloxamer 188 (F68)

Baicalein

Antibacterial, antiviral, antitumor, antiinflammatory and antioxidant

Enhancement of oral bioavailability

[82]

2

PCL-PEG-PCL poly(epsilon-caprolactone) -b-poly(ethylene glycol)poly(epsilon-caprolactone)

Rifampicin

Antitubercular

Improvement of aqueous solubility

[13]

3

Methoxypoly(ethylene glycol) and N-(2-hydroxypropyl) methacrylamide (mPEG-HPMA)

Curcumin

Antiinflammatory, antioxidant, Antiarthritic, antiamyloid

Enhancement of solubility

[83]

4

PDEAEMA-PEG-PDEAEMA poly(N,N-diethylamino-2-ethylmethacrylate)poly(ethylene glycol)-poly(N,N-diethylamino-2ethylmethacrylate)

Doxorubicin

Anticancer

pH responsive drug delivery

[84]

5

DPA-MPC-DPA poly(2-(diisopropylamino)ethyl methacrylate)-b-poly(2methacryloyloxyethyl phosphorylcholine)-b-poly(2(diisopropylamino)ethyl methacrylate)

Ketoprofen Spironolactone

Antiinflammatory and analgesic Antihypertensive

pH-responsive Controlled release

[85]

6

Poloxamer 407/Pluronic P123

Nevirapine

Antiretroviral

Sustained drug release

[86]

7

HPMA-PLA N(2-hydroxypropyl) methacrylamide-poly(lactic acid)

Rifampicin Isoniazid

Antitubercular

Sustained drug release

[87]

8

Pluronic-g-poly(acrylic acid)

Camptothecin

Anticancer

Solubilization and stabilization

[88]

9

Pluronic F127 and L61

Magnolol

Anticancer antimicrobial

Enhanced aqueous solubility

[89]

10

PEG-PE-TPGS Polyethylene glycol-phosphatidylethanolamine-d-αtocopheryl polyethylene glycol 1000 succinate

Paclitaxel

Anticancer

P-gp Inhibition and enhancement of bioavailability.

[77]

4  Polymeric micelles for enhanced permeability and retention effect

Block copolymer

 

Sl. No.

181

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11.  Polymeric micelle-based drug delivery systems for tuberculosis treatment

physiology of the diseased condition, and secondly, delivering the existing therapies using modern drug delivery tools. Enhanced permeability and retention (EPR) effect of the nanocarrier inspired the predominant nanoformulations designing philosophy for the delivery of chemotherapeutics in tumor site via passive targeting. Increased vascularity and porosity of the vessels at the cancer microenvironment increase the propensity of nanocarriers to preferentially accumulate at the cancer site, to improve therapeutic efficacy [7,90]. Similarly, small molecular weight therapeutics can also be targeted to the infected cells and tissues to improve their efficacy and to reduce their systemic toxic manifestations [91,92]. EPR effect due to the miniaturized size of the nanocarrier allows them to stay in systemic circulation for proloned period, which effectually pay the major role delivering the drug to the diseased site. Polymeric nanocarriers, including nanoparticles, polymeric micelles, or polymerconjugated drugs has shown to be present in the systemic circulation for longer period and simultaneously accumulated in the disease site via passive targeting. Similar to tumor microenvironment [93], inflammation and infection also facilitate permeation of nanocarriers to the diseased site because of augmented angiogenesis and vascular density [94]. Vascular permeability enhancement is another consequence of tumor area, which further facilitate penetration of macromolecules because of abnormality of the tumor vessels with lack of smooth muscle and loose endothelial fenestration [95,96]. In a similar pattern, the permeability of the infected cells increases to permeate the macromolecules easily. The literatures are supporting the information that the infection to the respiratory system stimulates the immune system to synergistically act to increase in vascular permeability [97]. Similarly, Meignan and team also demonstrated that epithelial permeability of the lung tissues of infected patients as evident from the results of high cellularity and permeation of high

proportion of lymphocytes at the site of infection [98].

4.1  Factors affecting EPR of miceller deliveries There are several factors involved in the increased permeability scenario in infected cells and in tumor environment. 4.1.1  Bradykinin and permeability of cells One of the most significant pain-inducing peptides is bradykinin. It is a nonapeptide kinin, produced continuously from kininogen at the site of cancer and infection by the action of proteases, such as kallikrein [99]. Increased permeability is a consequence of kallikreinkinin cascade modulation, thus there is always extravasation of constituents from plasma to the site of cancer or infection to induce edema or inflammation [100,101]. Thus, stimulation of kallikrein-kinin cascade pathway to increase the kinin concentration at the site of disease results in increased permeability to facilitate EPR effect of the nanocarrier to permeate more at the diseased site.

 

4.1.2  Free radicals in cell permeability Production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are usually induced during the infected condition of bacteria and virus. A viral infected lung of experimental mice has shown to induce ROS production (O2•−) to 300 fold than a normal healthy animal, where the cause has been described by the induction of xanthine oxidase production at the site of infection [102,103]. Simultaneously, the production of RNS (nitric oxide: NO) was also found to increase at the site of infection parallel with the production of O2•− [104]. It has also been suggested that the expression of NO synthase (NOS) is increased in tumor environment, and thus production of NO. Increased NO has shown to facilitate EPR effect at the tumor site [99], and it can be hypothesized that increased



5  Tuberculosis and urge of novel delivery approaches

production of NO at the infected site simultaneously increase the EPR effect of nanocarriers.

183

4.1.3  Permeability and prostaglandin with other factors It has been demonstrated that prostaglandin and overexpression of vascular endothelial growth factor results in activation of RNS, particularly NO, and thus can facilitate the EPR effect of nanocarriers to the site of generation [93,105]. Similarly, other cytokines such as, interleukin-8, tumor necrosis factor-α, other inflammatory mediators and transforming growth factor-β inhibitors may also be involved in promoting EPR effect of the nanocarriers via different underlying mechanisms [93,106–108]. Therefore, connecting to antimicrobial therapy, EPR effect can further expedite delivery of the therapeutics to the target site. Such passive targeting of the therapeutics by this EPR effect will reveal delivery potentials of the polymeric nanocarriers, to selectively target the diseased tissues to act effectively and safely against the invaded infection.

just able to reduce the incidence of tuberculosis slightly, 1%–2% each year [109]. Poor treatment and improper diagnosis along with development of multidrug resistant (MDR) tuberculosis strains in the patients develops poor treatment outcomes [113]. Therefore, treatment strategies for tuberculosis remain a great challenge to the clinicians, and delivering the therapeutics to the site of actions effectively is also another challenge to the formulation scientists. Reason behind such difficulty is due to the high systemic doses of single antitubercular agent or combined treatments of them cause severe toxicities to the users because of high systemic exposure [114]. Therefore, despite of potential results in vitro by a variety of available therapeutics against tuberculosis pathogens, they are found ineffective when applied in vivo models, may be because of the incapability of these therapeutics in employing suitable activity within the biological environment. Isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin are the first line treatment options for the clinical management and treatment of tuberculosis, whereas some other antibiotics including amikacin and kanamycin are listed as second line treatment for tuberculosis [115,116]. Among these, for example, application of rifampicin is found to be limited in the effective treatment of the disease because of its poor aqueous solubility as well as low diffusion in biological liquids. These two issues limit its effectivity at cellular level, as required concentration of the drug could not reach to the site of action. Thus, in order to increase the concentration of the drug at the target site, the conventional treatment strategies results in increased risk of hepatic toxicity in many patients. Further, the treatment for tuberculosis need prolonged therapy, required to administer the medication for 6–9 months [115,117,118]. Continuing our previous discussion on formulation advancement under the umbrella of nanotechnology, the polymeric micelle-based drug delivery systems are found to be crucial

5  Tuberculosis and urge of novel delivery approaches Being an ancient disease, tuberculosis is found largely curable with the available and affordable treatments. However, current statistics reveal tuberculosis as one of the leading cause of death related to infectious disease [109]. It has been estimated that more than a million of tuberculosis patients are dying each year with the increase in load of 10 million patients per year globally. Available treatment of tuberculosis through the Directly Observed Treatment, Short Course (DOTS) strategy since 1990 has strengthened the distribution of quality treatment for this dangerous disease [110,111]. Thus, mortality rate in tuberculosis has been reduced via the nationally coordinated tuberculosis programmes [109,112]. These continuous programmes and efforts were

 

184

11.  Polymeric micelle-based drug delivery systems for tuberculosis treatment

in the designing and preparation of controlled release formulations with possibilities of modifying carrier surface based on the requirement to target specific site [29,87]. These drug delivery systems ideally dispense the entrapped medication to the target site for a specific period of period. Literature depicted several evidences on delivery of therapeutics in the effective treatment of tuberculosis with additional advantages of decreased systemic side effects. The connecting section of the chapter will be discussed on different polymeric miceller-based delivery systems in the safe and effective delivery of antitubercular therapeutics.

6  Recent advances of polymeric micelles in tuberculosis

instigated another research, where isoniazid and rifampicin were entrapped within the polymeric micelles constructed using HPMA-PLA copolymers. Similar to the previous approach of formulation development, the final rifampicin loaded isoniazid conjugated HPMA-PLA polymeric micelle was reported to be 87.64 ± 1.98 nm in size with 97.2 ± 1.56% of drug entrapment. The miceller formulation depicted significant decrease in hemolysis compared to free drug, with significant decrease in MIC against sensitive and resistant strains of M. tuberculosis [87]. Concurrently, another group of researchers reported the development of polymeric micelles of isoniazid and rifampicin using the ethylene oxide-propylene oxide tri-block copolymers, Pluronic®. They formulated the micelles of saturated Pluronic® using sonication and overnight mixing, which finally resulted in a biphasic in vitro release of the entrapped drugs. Initial burst release of the drugs from formulation was then followed by controlled release of the drug for longer period of time. Such release pattern of the drugs from the miceller formulation showed improved control against multidrug resistant strain of M. tuberculosis when compared with free drugs of solutions. Entrapment of the two antitubercular drugs within the miceller core was found to improve permeability of the drugs from epical side to basal side in Caco-2 monolayer. This formulation also found to decrease the MIC up to 10- or 20-fold of rifampicin and isoniazid, respectively [120]. In another research, the microwave assisted polymers was formulated, chitosan-graftpoly(caprolactone)/(ferulic acid), in order to formulate nano-miceller delivery for rifampicin. The size of the drug-loaded micelles was ranged from 100 to 210 nm, where the micelles were found to swell at pH 5.3, rather than pH 7.4. Thus, it can be assumed that the entrapped drug would not be released within the systemic circulation when the formulation will be transported, however, will release the drug at the acidic lysosomal microenvironments due to

Biodegradable polymers are widely applied in the delivery of antitubercular drugs via formation of miceller delivery for their promising results. These formulations are known to improve solubility of poorly soluble drugs and thus resulted in increased bioavailability, stability, prolonged circulation, controlled release, and simultaneously decrease in toxicity, antigenicity, and immunogenicity with improved targetability [5,119]. In a research by Rani and team attempted miceller delivery of isoniazid and rifampicin using the di-block polymer, poly-llactic acid/polyethylene glycol. Stepwise, the di-block polymer is formed to conjugate with isoniazid, which was finally loaded with rifampicin at a CMC of 8.9 ± 0.96 mg/L. The size of the micelle was less than 200 nm (187.9 ± 2.68 nm) with an entrapment efficiency more than 70%. The formulated drug loaded polymeric miceller delivery was reported to be safer when tested for RBC hemolysis test, whereas the formulation was reported to improve the efficacy through reduction of minimum inhibitory concentration (MIC) against sensitive Mycobacterium tuberculosis strains approximately up to eightfold [116]. Limitations of first line antitubercular drugs

 



185

7 Conclusion

FIGURE 11.2  Fluorescence micrographs of the uptake

succinylated derivative of the INVITE micelle showed superior antibacterial activity against Gram +ve bacteria. Results were indicating that formulated micelles could be used as an effective carrier for the hydrophobic antibiotics for lung infections [122]. This polymeric miceller drug delivery approach was further extended to develop pediatric formulation of rifampicin. Usually, rifampicin undergoes self-aggregation when formulated in aqueous formulation for pediatric use. In order to avoid such aggregation, Grotz and team had adopted miceller approach, where Kolliphor® HS 15 was used to formulate the polymeric micelles [123]. The self-aggregation problem of the drug was successfully avoided when the drug was entrapped within the hydrophobic core of miceller structure, which ultimately helped in improving the stability of the drug, also protected from the aqueous environment, whereas precipitation of the drug was also prevented [123]. Thus, this nano-engineered polymeric miceller delivery could be an interesting and promising platform to deliver antitubercular agents to avoid their solubility and toxicity issues.

of rifampicin-chitosan-graft-poly(caprolactone)/(ferulic acid) in A549 cells at (A) 12 h (B) 24 h (C) 48 h, and (D) 72 h [31].

rapid degradation of amide and ester bonds in micelles. Intracellular uptake of the formulation in in vitro A549 cells were analyzed using fluorescence microscopy, which indicated a timedependent uptake of the micelles by the living cells (Fig. 11.2A–D) [31]. Recent research concentration on natural components has brought novel research strategies to include natural polymers, where Yuan and team has introduced guar gum to formulate the interconnected micelles for the poorly aqueous soluble antitubercular agent rifampicin [121]. The authors formulated the hydrophobic outer and hydrophilic inner core using the guar gum/chitosan/polycaprolactone to obtain a superior activity against THP-1 cells, where the hydrogel-based miceller delivery was reported to be a potential delivery tool for intracellular macrophage therapy [121]. In a similar approach with natural polymers Tripodo and team has reported development of rifampicin micelles using inulin functionalized with vitamin E (INVITE), where the micelles are formed by selfassembling sustained by the interaction. These micelles showed strong miceller interaction for the favorable mucoadhesive interactions, where

7 Conclusion

 

Being a deadliest disease, tuberculosis instantaneously affects adults as well as children, where the conventional first line therapies are found to produce unfavorable side effects and delivery of rifampicin was also found to be limited because of its poor aqueous solubility as well as low diffusion in biological liquids. Thus, polymeric micelles are found to be a potential platform to deliver the antitubercular agents effectively, where different research has showed to improve the efficacy against MDR cell lines as well. This chapter has brought detailed description on polymeric micelles and different polymers which can be incorporated in formulation development with recent approaches of improved efficacy against tuberculosis.

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11.  Polymeric micelle-based drug delivery systems for tuberculosis treatment

Acknowledgment Dr. Gorain and Dr. Sisinthy would like to acknowledge School of Pharmacy, Taylor’s University, Malaysia and Dr. Choudhury would like to acknowledge School of Pharmacy, International Medical University, Malaysia for providing resources and support in completing this work.

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Conflict of interest The authors declare no conflict of interest, financial or otherwise.

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C H A P T E R

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Nanostructured lipid carrier-based drug delivery systems for tuberculosis treatment Simone Pinto Carneiro and Orlando David Henrique dos Santos Laboratory of Phytotechnology, School of Pharmacy, Federal University of Ouro Preto (UFOP), Ouro Preto, Minas Gerais, Brazil The success of any pharmacotherapy is not only related to the drug being used but also with the drug delivery system employed, the way the drug is encapsulated, its interaction with the excipients, and how it is delivered, considering the behavior of the formulation inside the body. The key objective of a drug delivery is effective and safe delivery of a drug to its target, which could be tissues, organs and/or cells. The transport of a drug to its specific targets consists one of the main challenges for effective drug delivery [1]. For systemic drug administration, conventional oral or parenteral drug delivery methods are usually employed, and include the formulation of a drug into an appropriate dosage form for gastrointestinal administration, as solutions, tablets, capsules, suspensions, syrups, among others or injectable sterile drug preparations, like solutions, suspensions, emulsions, or reconstituted lyophilized powders [2,3]. However, systemic drug delivery systems usually suffer

Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00012-6

from a series of limitations such as relatively low site-specific bioavailability, unfavorable biodistribution and, in most cases, low accumulation in the target site. These characteristics are responsible for the occurrence of adverse side effects that compromise the conventional pharmacotherapy due to limited patient compliance, loss of quality of life and, in the case of treatment of infections, higher incidence of microorganism resistance [4]. Special drug delivery systems, that escape from the problems of conventional medicines could be applied, particularly in personalized medicine and targeted therapies. These products make use of the interaction of the drug with different excipients where they are encapsulated, achieving a carrier-based drug delivery system that defines drug absorption, solubility in body fluids, affinity to the target (tissues, organs, or cells), degradation and stability, pharmacokinetics and metabolism and introduction of new

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Copyright © 2020 Elsevier Inc. All rights reserved.

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routes of administration, making the therapy more efficient, convenient and comfortable for the patient [5,6]. Pronounced progress has been made in the treatment of a series of diseases with the use of nanoparticulate drug delivery systems. Nanoparticles are dispersions in which particle size ranges from 10 to 1000 nm, where the drug molecules are absorbed, dispersed or dissolved, being entrapped inside or attacked on the surface of the particle. Usually, nanoparticles can be obtained using polymers or lipids as a carrier material. Lipid-based nanocarriers are composed of physiological or biocompatible lipids, making them well-tolerated, with the original molecule and their metabolites usually nontoxic. Recent research is new approaches for lipid-based nanocarriers that include liposomes, niosomes, ethosomes, transfersomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and lipid nanoemulsions for safe and effective delivery of drugs [7]. Investigation of novel lipid nanocarrier system was started in the early 1990s from the production of SLNs. SLN, also known as the first generation of lipid nanoparticles, has been developed as a new class of lipid-based nanocarrier and an alternative carrier to emulsions, liposomes, and polymer nanoparticles. They are colloidal drug delivery systems [8–11] very similar to nanoemulsions, with the difference of, instead of using liquid lipids, they employ another that is solid at room temperature [8,12–14]. The oil phase is dispersed in water and stabilized by surfactants, producing nanoparticles with diameter between 40 and 1000 nm and presenting characteristics of good stability, drug protection against degradation, low toxicity, enhanced cell penetration, and drug controlled release. Encapsulation of a drug into biocompatible or biodegradable lipids formulated at nanosize range has become a promising approach of drug delivery as lipid drug delivery system [15]. SLNs can be produced from natural or synthetic solid lipids, are nontoxic and versatile

nanocarriers for drug delivery that can transport both lipophilic or hydrophilic drugs [16]. SLNs have high drug loading ability and large surface area and can be designed to achieve controlled release and increased stability of encapsulated drugs [7,17]. The solid lipid is used as a matrix material for drug encapsulation and is usually selected from a variety of lipids, including monoglycerides, triglycerides, and fatty acids. They can be employed pure of as a mixture (as is common when natural lipids are applied, especially vegetable waxes and butters). SLNs present the benefits of physical stability, protection against labile drug degradation, possibility of controlled release and targeting of drugs [18] and easy preparation [19]. Furthermore, when compared to polymeric nanocarriers, due to the use of more biocompatible and biodegradable lipids, toxicity and acidity issues seem not to be observed in SLNs [16,20]. SLNs have been reported in various application routes [8]: • parenteral (intravenously, intramuscularly, or subcutaneously) [19,21], • oral [22–23], • rectal [24], • opthalmic [25,26], and • topical (in both cosmetics and dermatological preparations) [27–30].

 

There are two models of drug incorporation on SLNs: the solid solution model and the coreshell model (drug-enriched shell and drug-enriched core). For the solid solution model, the drug is solubilized in the lipid matrix when the particles are produced by cold homogenization, without using surfactant or any drug-solubilizing surfactant. In this case, the drug needs to have strong interaction with the lipid [31,32]. In the core-shell model, a nanoemulsion is produced at temperatures higher than the melting point of the lipid. When the drug is incorporated inside the SLN, according to the drug-enriched shell model, a solid lipid core forms when



Nanostructured lipid carrier-based drug delivery systems for tuberculosis treatment

the recrystallization temperature of the lipid is reached. With the reduction of temperature, the drug concentrates in the still liquid outer shell of the SLN [33,34]. Following the drug-enriched core model, with cooling the nanoemulsion the drug which is dissolved in the lipid melt at or close to its saturation solubility achieves a supersaturation state, leading to its precipitation preceding lipid recrystallization. Additional cooling induces the recrystallization of the lipid surrounding the precipitated drug as a shell [16]. Some factors that influence the drug loading capacity in a lipid are [16]:

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• solubility of drug in lipid melt, • miscibility of drug melt and lipid melt, • chemical and physical structure of the solid matrix lipid, and • polymorphic state of the lipid material.

degree of this modification, the number of crystal imperfections is reduced, culminating on the expulsion of encapsulated drug from the SLN. Usually, after this process, molecules of the lipid phase approximate themselves, reducing intermolecular spaces where the drug is dispersed, expelling them from the particle, thus reducing drug loading [14]. In 1999/2000, Muller developed a second generation of lipid nanocarrier named NLCs. NLCs were designed by exchanging part of solid lipids with liquid lipids, forming a drug incorporated matrix [37]. The presence of liquid lipids with different fatty acid C-chains produces NLC with less organized crystalline structure and provides better loading capacity for drug accommodation. NLCs are considered as potential drug carriers due to their biocompatibility and superior formulation properties over SLNs [38]. NLCs formulation is based on the idea of entrapment of the drug in the mixture of varying proportions of solid lipid and liquid lipid, planned to attain the less/no crystalline matrix with solidified core to overcome the limitations occurred due to recrystallization of SLNs core. Fig. 12.1 illustrates the lipid matrix of both nanoparticles, comparing their crystal structure and demonstrating the highest encapsulation of the drug in the NLC. Because of the presence of liquid lipids alongside solid lipid, the particle core does not form a perfect crystalline matrix. These imperfections and potential amorphous states allow higher

In general, there is an inverse relationship between solubility of the drug and loading capacity, showing an increased entrapment as more lipophilic is the drug, and the opposite for hydrophilic [35]. Some limitations of SLNs, especially related to the fact of being based on the use of only solid lipids, were observed by some investigators. Apart from the benefits of SLN as a drug nanocarrier, there are still some issues especially related to loss of stability and leakage of entrapped drug over time associated with the use of a solid lipid matrix and is explained by changings on physicochemical structure of solid lipids during storage [36]. Some of the lipids present polymorphism, with more than one solid-state organization. Depending on the method of production and formulation, a nonpreferred arrangement forms just after preparation and, after some time, the form that is more stable tends to predominated due to system organization. After SLN preparation, solid lipids organize into a high energy crystalline form, however, after a while, this organization transforms into a more orderly and low-energy crystalline state—the β modification. Due to the high order

FIGURE 12.1  Representation of both lipid nanoparti-

 

cles composition: SLN (on the left) and NLC (on the right).

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drug loading and avoidance of drug leakage. For drug that presents higher solubility in liquid lipid used, an oil-in-solid or fat-in-water (O/F/W) system is formed. Small oil droplet is incorporated into solid lipid core, thus the drug is slowly released from oil droplets towards the dispersing phase [15,34]. There are three types of NLCs [15]:

nique, emulsification-ultrasonication, film ultrasonication, and solvent emulsification are some of the commonly used methods for preparation of SLNs and NLCs: • High-pressure homogenization—HPH

1. NLC Type I: it can also be referred as imperfect type. It is formed by the replacement of a fraction of solid lipid by liquid lipid/oil leading to an imperfect crystal lattice/matrix. This phenomenon permits more space for accommodation of drug, allowing higher loading. Is also avoids the formation of highly structured or ordered matrix which could expulse the drug from the particle core. 2. NLC Type II: this is also known as amorphous/structureless type. It is produced employing solid lipids which remain in a stable α polymorph state after solidification and during storage along with liquid lipids, creating an amorphous core. This is advantageous over type I NLCs as no crystallization occurs and drug remains entrenched in amorphous matrix. 3. NLC Type III: this is multiple type and developed based on the concept of W/O/W double emulsion. It is basically O/F/W type NLC, which can be developed only by phase separation technique. When drug shows higher solubility in the liquid oil, this method can be used in order to improve drug loading and stability. Small droplets of oil are dispersed uniformly in solid lipid matrix and this system is dispersed in the aqueous medium [39].

HPH has been extensively used especially in large-scale production [40]. It is based on the application of high pressure promoting very high shear stress on the lipid, resulting in subdivision of particles down to the submicrometer or nanometer size. Scaling up problem is not usually an issue for the HPH [41–43]. There are two methods mainly used to prepare NLCs by HPH [44]: • hot homogenization [45] and • cold homogenization [46].

1.1  Hot homogenization method An emulsion is firstly produced with the molten lipid and the drug under constant stirring by a high shear device. Using a piston-gap homogenizer, the hot preemulsion is submitted to HPH forming a nanoemulsion that is cooled down to room temperature where the lipids solidify, forming lipid nanoparticles [47–50]. Some reports include the use of lipophilic surfactant in lipid melt [51]. This may be to improve the stability of preemulsion during homogenization. This method is suitable for drugs without heat instability.

1.2  Cold homogenization method

1  Methods of production

Cold homogenization comprises high-pressure processing of a suspension. The temperature is essential during HPH step where it should not exceed the temperature of the lipid melting point. Usually, a cold emulsifying agent solution is used to stabilize newly formed nanoparticles. This technique is very interesting when thermal sensitive drugs are used. Crystallization can be controlled with this method, being one of its major advantages. The desired crystal structure

Methods of production of SLNs and NLCs do not present significant differences [15]. Highpressure homogenization, microemulsion tech-

 



1  Methods of production

of NLCs can be obtained by the rapid cooling process. Larger particle size and wider size distribution of nanoparticles are usually obtained compared to other techniques [9].

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• Microemulsion technique

anol, ethanol, and acetone. The drug and lipids are dissolved with organic solvent and then sonicated and at high temperature to obtain clear lipid phase. The organic-lipid phase is added to an aqueous phase containing a suitable surfactant or stabilizer under mechanical stirring at elevated temperature. Solidification is reached by cooling the dispersion up to at room temperature under stirring, allowing the evaporation of organic solvent [58].

The drug is incorporated in molten lipid and mixed with water, the surfactant, co-surfactant(s) at the same temperature, forming a microemulsion. The components are used in the correct ratios for microemulsion formation, achieving a transparent and thermodynamically stable state. The microemulsion is dispersed under mild mechanical mixing in a cold aqueous medium with water in a ratio in the range 1:25–1:200 (microemulsion: cold aqueous medium, respectively). Microemulsion is taken into consideration to obtain the particles of the required size. Rapid recrystallization of the oil droplets occurs during the dispersion in cold aqueous medium [52–54]. Energy is not required to produce nanoparticles with this technique because the droplet structure is already present in the initial microemulsion. Rapid lipid crystallization and inhibition of aggregation are facilitated by hightemperature gradients. Due to the dilution step, the content of lipids is lower compared with the HPH-based formulations [55–57].

• Solvent emulsification evaporation method This technique differs from the previous one (solvent diffusion method) by the fact of instead of using water miscible organic solvent, water immiscible organic solvents such as chloroform and cyclohexane are used to dissolve drug and lipids [37]. Usually, low pressure or vacuum is applied to induce solvent evaporation and elimination. Use of organic solvents is the main drawback of solvent diffusion and evaporation method as some traces of it may remain in the formulations. • Film-ultrasonication method

• Emulsification-ultrasonication method

This process is adapted from the preparation methods of vesicular drug delivery systems, as liposomes and micelles. Lipids and drug are dissolved in an organic solvent. Organic phase is removed by applying vacuum using rotary evaporator. This forms a thin film of drug-lipid blend which is collected and dispersed in heated aqueous phase, consisted by water and surfactant, under sonication. This dispersion is cooled down at room temperature to obtain dispersed solidified nanoparticles [59].

This method is similar to HPH, differing on the source of high shear stress that breaks down the particles. Drug, liquid and solid lipids are mixed and melted above melting point of solid lipid. The surfactant is dissolved in distilled water and heated at same temperature and mixed with lipid phase, forming a preemulsion. Then, preemulsion is ultrasonicated for specific time and then added to specified volume of distilled water. Solidification is achieved by cooling down to room temperature [40].

• Supercritical fluid (SCF) technology

• Solvent diffusion method

Lipids are melted and solubilized with SCF (if possible carbon dioxide). This form either a gas saturated suspension or a solution depending, on the solubility of materials in the SCF. Thus, this dispersion is atomized through nozzle and sprayed into a chamber. During this phase, decompression and evaporation of gas occurs

Solvent diffusion method comprises the use of water miscible organic solvents such as meth-

 

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forming solid nanoparticles, usually in a dried state [15,60].

combination of emulsifiers which are used more efficiently [13,63,64].

2  Excipients used in lipid nanocarriers

3  Challenges for current tuberculosis therapy

2.1 Lipids

Tuberculosis (TB) therapy comprises the use of antituberculosis drugs, such as rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (EMB). The World Health Organization (WHO) established a treatment protocol over a minimum 6-month period and recommends the orally administration of these four drugs during the intense phase—the first part of therapy that normally takes around 2 months and the use of high amounts of anti-TB drugs is employed to quickly reduce the replicating capacity of mycobacteria [65]. After this period, the patient must continue treatment with rifampicin and isoniazid for around 4 months to sterilize lesions containing fewer and slow-growing bacilli, known as maintaining or continuation phase. The main drawback associated with TB treatment is the severe side effects reported by patients, such as cutaneous, gastrointestinal reactions and hepatotoxicity, which may even progress into hepatitis [66]. Because of that, patients early drop out therapy, culminating in a low patient compliance and this practice contributes to the emergency of bacteria multidrugresistant TB. Thus, advances in TB therapy with the views of reducing dose administered, overall treatment time and consequently undesired effects are still highly demanded.

The inner cores maybe consisted of both solid and liquid lipids. As solid lipid at room temperature, Glyceryl behenate, glyceryl palmitostearate, fatty acids (mainly stearic acid), triglycerides, tristearin, steroids, cetyl palmitate, and waxes are commonly used in lipid nanocarriers. Also, some digestible oils from natural sources were used in NLC. Medium chain triglyceride (MCT) (Miglyol 812), 2-octyl dodecanol, paraffin oil, isopropyl myristate, propylene glycol dicaprylocaprate, and squalene are used as liquid lipid. Oleic acid, linoleic acid, and decanoic acid, which are penetration enhancers were also used for topical delivery, not only to skin. α-Tocopherol and other tocopherols were used due to their stability and good solubility in lipophilic drugs. Apart from pure compounds, lipid nanocarriers can be produced with natural oils, butters and waxes, as olive, sunflower, soya bean, corn oils, coconut, cupuaçu, murumuru, sea butters and bee, carnauba, and candelila waxes [15,40]. Food graded MCT is obtained by esterification and fractionation methods, being recognized as safe by the USFDA, and presenting high stability against oxidation [14,16,39,46,61,62].

2.2 Emulsifiers 4  Lipid nanoparticles for TB treatment

Emulsifiers have been widely applied to stabilize the lipid dispersions. Pluronic F68 (poloxamer 188) and polysorbates (Tween) were identified as the most widely used hydrophilic emulsifiers. Lipophilic emulsifiers such as Span 80, lecithin and other phospholipids are widely utilized for fabrication of lipid nanocarriers. The particle aggregation can be prevented by the

 

TB is ranked among one of the leading causes of death produced by a single infectious agent [65], although treatable. As mentioned above, due to the long period of therapy, many severe adverse effects are related by patients resulting in a low patient compliance, thus, a major



5  Solid lipid nanoparticles and nanostructured lipid carriers as alternative nanomedicines for TB treatment

challenge for the academic community is to develop more effective and tolerable formulations [66-67]. In the last four decades no new drug with antituberculosis potential has been launched by pharmaceutical industries, despite some candidates designed especially for TB resistance cases are in clinical phase, but demand a longer period of testing until reach the market [69]. This scenario reinforces the necessity of innovative research on TB therapy, aiming to optimize therapy and improve the patient life quality during treatment. In this context, nanotechnology emerges as an alternative for the development of new drug carrier systems, intending to improve pharmacotherapy, performance and drug bioavailability. The most relevant technological advantages of nanoparticles as carrier systems are the possibility of encapsulating hydro or lipophilic drugs, greater stability, viability for different routes of administration and reduced dose administered [70]. Among nanocarriers, lipid nanoparticles hold many characteristics that make them suitable as nanoformulations to improve anti-TB-drugs performance. According to many authors, they can be prepared by many methods—easy to execute, without toxic organic solvent residues and low-cost ones. Moreover, lipid nanoparticles formulations present low toxicity, high encapsulation efficiency, drug controlled release, stabilization of drugs, improved bioavailability and, most relevant, these nanosystems are able to be chemically modified to insert molecules of interest aiming to target a specific cell.

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5  Solid lipid nanoparticles and nanostructured lipid carriers as alternative nanomedicines for TB treatment

advantageous properties, which make them suitable to deliver antituberculosis drugs and improve TB therapy. These nanosystems are related to present low-toxicity independently of administration route, since the use of biodegradable and biocompatible lipids and surfactants is feasible, culminating in less-toxic formulations [71]; NLC easily adhere to lungs superficial mucosa for a prolonged period, which increases the efficiency of these formulations in deliver antituberculosis drugs directly on target cells—the alveolar macrophages [72]; in addition, NLC is associated to the properties of retention, accumulation and controlled release of the drug, leading to a sustained therapeutic effect that results in a longer time interval between doses and consequently increases patient compliance [36,73]. Considering all this information, applications and potential of both SLN and NLC, many authors have already reported their use as antituberculosis drugs nanocarriers with encouraging findings aiming therapy optimization. Pandey and Khuller [74] published a relevant study, in which the chemotherapeutic potential of nebulized antituberculosis drugs (rifampicin, isoniazid, and pyrazinamide) loaded solid lipid particles (SLPs) was evaluated against experimental guinea pig tuberculosis model. SLPs were prepared as dry powder for pulmonary administration and formulations characterization detected an appropriate mass median aerodynamic diameter for bronchoalveolar drug delivery. Main findings demonstrated that nebulized SLPs were detected in plasma from 6 h onwards up to 120 h whereas free drugs were cleared by 1–2 days. Moreover, with a single nebulization, a sustained drug release for 7 days and 5 days was attained in the organs (liver, lungs, and spleen) and plasma, respectively. It has been found that after nebulization of drug-loaded SLPs on MTb H37Rv infected guinea pig, at every 7th day, no tubercle bacilli could be detected in the lungs/ spleen after seven doses of treatment, whereas 46 daily doses of orally administered drugs

Both SLNs and NLC are widely used in different pharmaceutical fields, covering cosmetics and therapeutics, due to their compatible and

 

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were required to obtain an equivalent therapeutic benefit. It was also remarked that there was not any evidence of hepatotoxicity. In conclusion, this work achieved promising in vivo results and demonstrated that nebulization of SLP-based ATDs improves bioavailability with reduction in dosing frequency, offering the possibility of decreasing side effects and better patient compliances [74]. In parallel, the same research group published another study describing the effects of SLNs encapsulated with RIF, INH and PZA after oral administration in MTb H37Rv infected mice and found out that therapeutic concentrations were maintained in the plasma and organs (lungs, liver, and spleen) for 10 and 8 days, respectively while free drugs were cleared by 1–2 days after a single oral administration to mice. To summarize the potential of these nanoformulations, the authors also reported that five oral doses of SLNs loaded anti-TB drugs had the same therapeutic effect than 46 daily oral doses of free-drugs administered in mice [74]. Table 12.1 summarizes many studies that have been reported in literature employing lipid nanoparticles based antituberculosis drug delivery systems, highlighting the main outcome of each work as a potential finding to improve TB treatment.

6  Lipid nanoparticles functionalization

potential to maximize efficacy and minimize toxicity. The main goal in TB therapy is to enhance the uptake of nanocarriers encapsulated with antiTB drugs by alveolar macrophages [88]. Thus, one strategy of functionalization is to attach cell penetrating peptides (CPP) on nanoparticles surfaces. CPP are cationic or amphipathic peptides composed of a small sequence of amino acids that have been distinguished by their role of cell release vectors, due to their intrinsic ability of increasing cell penetration and mediate the uptake of several macromolecular substances, such as genes, drugs or nanoparticles [89]. In this context, Carneiro and co-workers [90] developed functionalized RIF-loaded NLC and evaluated the suitability of in vitro properties to improve TB therapy. A CPP, known as tuftsin (composed of four amino acids) was synthesized and attached on nanoparticles surface to functionalize them. As there are many receptors to this peptide located on alveolar macrophages cells, the objective of employing this strategy was to enhance nanoparticles uptake by these cells, which was confirmed after cell culture assays. In addition, this nanoformulation did not present a toxic potential and improved antimycobacterial activity twofold comparing to free RIF solution. Another functionalization strategy evolving lipid nanoparticles and the optimization of TB treatment is to decorate surface nanoparticle with mannose. Mannose surface modification is normally done to take advantage of sugar receptors available in alveolar macrophages, aiming to improve cellular uptake with an active targeting strategy. Vieira et al. [91] designed RIF-loaded SLN using mannose as a lectin receptor-ligand conjugate for macrophage targeting and expecting an increase on therapeutic index of RIF. Results demonstrated that mannose-nanoparticles were more internalized by human macrophages cells than nonmannosylated SLN and they were not considered toxic for these cells, suggesting

As described in previous sections, lipid nanoparticles have many benefits in the treatment of pulmonary diseases, such as TB. However, they are not selective for any cellular type, which could hamper the reduction in dose administered and side effects. One alternative to overcome this drawback is to functionalize lipid nanoparticles, through the insertion of molecules of interest on nanoparticles surfaces, with the intention of their specific recognition by target cells. Delivery of antituberculosis drugs via nanoparticles directly to infected cells has the

 



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6  Lipid nanoparticles functionalization

TABLE 12.1 Published studies describing anti-TB drugs loaded lipid nanoparticles and the main findings in TB treatment. Formulation Drug

Main finding

Reference

SLN

INH

Higher plasma and brain bioavailability (six and fourfold, respectively) comparing to free drugs at the same dose, after oral administration of single doses on rats

[75]

SLN

RIF

RIF loaded SLN were administered in Wistar rats following a single dose of 50 mg/kg and plasma levels were around eightfold higher and maintained for 5 days while comparing with free RIF. Hepatotoxicity was reduced

[76]

SLN

RFB

Nanoparticles uptake by THP-1 differentiated in macrophages cells was 46% and 26% for glyceryl di-behenate and glyceryl tristearate SLN, respectively. Low cell cytotoxicity was reported

[77]

SLN

RIF and INH RIF and INH loaded SLN prevented their degradation from acidic gastric pH [78] and drug-drug interactions. RIF-SLN were able to bring down its extent of degradation up to 9% when present alone whereas individually encapsulation of drugs on SLN (RIF-SLN?SLN) reduced RIF degradation to 12.35% and INH to 2.7%

SLN

Ciprofloxacin Ciprofloxacin loaded SLN showed sustained drug release (Higuchi model) avoiding burst effect of the free drugs for up to 80h

SLN

RIF

RIF-loaded SLN sustained the drug release for 72 h and nanoparticles were [80] eightfold more effective against Mycobacterium fortuitum in vitro than free RIF, highlighting the relevant antimycobacterial potential of these lipid nanoparticles

SLN

RIF

Low toxicity of RIF-loaded SLN to alveolar macrophages and alveolar epithelial [81] type-II cells (cell viability over than 80% after treatment with nanoparticles)

SLM

RIF

RIF-loaded SLM presented 77% and 80% of drug release at 12 h in phosphate buffer (pH 6.8) and simulated intestinal fluid (pH 7.4), respectively. Nanoformulation also reduced RIF degradation, as SLM exhibited 48.51% of RIF degradation in simulated gastric fluid at 3 h, while pure RIF had 95.5% degradation

[82]

SLM

RIF

SLM exhibited aerodynamic diameter proper to be transported up to the alveolar epithelium and a low-cytotoxic potential. RIF-SLM were efficiently taken up by macrophages—confocal microscopy indicated the presence of nanoparticles inside the cell cytoplasm and, consequently, the activation of the endocytic process by macrophages cells

[83]

NLC

RFB

Rifabutin (RFB)-loaded NLC presented high storage stability (around 6 months) [84] and entrapment efficiency (above 80%). RFB release was pH-sensible: drug was faster released at acidic pH than at neutral one

LM

RIF

Intranasal administration of lipid microspheres containing RIF (LM-RIF) exhibited the bacteriostatic effect against Mycobacterium tuberculosis and high efficacy in immunodeficient BALB/c mice lungs, compared to the oral RIF administration

SLN

RIF

In vitro dissolution studies revealed a biphasic drug release profile for RIF[86] loaded SLN and the accelerated stability studies for 6 months did not demonstrate any significant change in characteristics of developed nanoformulation

SLN

EMB

Powder EMB-loaded SLN were considered suitable for pulmonary administration and showed biocompatibility and a nontoxicity profile

[79]

[85]

[87]

EMB, ethambutol hydrocloride; INH, isoniazid; LM, lipid microsphere; RFB, rifabutin; RIF, rifampicin; SLNs, solid lipid nanoparticles; SLM, solid lipid microparticles.

 

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that these nanocarriers could be used to safely deliver RIF with an enhanced cell uptake. Song et al. [92] developed mannosylated NLC to deliver RIF to targeted alveolar macrophages. The main findings showed that, compared to nonmannosylated NLC, RIF-mannosylatedNLC exhibited significantly higher uptake efficiency in NR8383 cells and alveolar macrophages, which achieved cell-specific targeting. In addition, this nanoformulation demonstrated to be safe with minimum toxicity and no inflammatory response. All studies mentioned in this chapter present a promising and innovative potential in improving TB treatment. Many of them require more investments in research to complement in vitro studies with in vivo ones, until achieve clinical research and, finally pharmaceutical market as nanomedicines to improve TB therapy.

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C H A P T E R

13 DNA markers and nano-biosensing approaches for tuberculosis diagnosis Amal Rabti, Amal Raouafi and Noureddine Raouafi Sensors and Biosensors Group, Laboratory of Analytical Chemistry and Electrochemistry (LR99ES15), Department of Chemistry, University of Tunis El Manar, Tunis, Tunisia

List of abbreviations acpcPNA  pyrrolidinyl peptide nucleic acid AgNPs  silver nanoparticles ALP  alkaline phosphatase AuNPs  gold nanoparticles CFU  colony-forming unit CNTs  carbon nanotubes DPV  differential pulse voltammetry dsDNA  double-strand DNA EC-SERS electrochemical surface-enhanced Raman spectroscopy E-DNA  electrochemical DNA Exo III  Exonuclease III Fc  ferrocenyl groups FRET  fluorescence resonance energy transfer GF  graphene flakes GOPS 3-glycidoxypropyltrimethoxysilane HAD  helicase-dependent DNA amplification HPV  human papillomavirus HRP  horseradish peroxidase HRP-Strep  streptavidin-horseradish peroxidase conjugate ITO  indium-doped tin oxide ITS  internal transcribed spacer LAMP  loop-mediated isothermal amplification MBA  mercaptobenzoic acid Nanotechnology Based Approaches for Tuberculosis Treatment http://dx.doi.org/10.1016/B978-0-12-819811-7.00013-8

MBs  magnetic beads MERS-CoV  Middle East respiratory syndrome coronavirus MGIT  mycobacteria growth indicator tube MPs  magnetic particles MSPQC  multichannel series piezoelectric quartz crystal Mtb  Mycobacterium tuberculosis MWCNTs  multiwalled carbon nanotubes OMPA oligo-methoxyphenylacetonitrile PAMAM poly(amidoamine) PANI polyaniline PCR  polymerase chain reaction PNA  peptide nucleic acid PPy polypyrrole QCM  quartz crystal microbalance QDs  quantum dots RCA  rolling circle amplification rGO  reduced graphene oxide rpoB  gene responsible for development of resistance to rifampicin SAM  self-assembled monolayers SPR  surface plasmon resonance SWV  square wave voltammetry TB tuberculosis tHDA thermophilic helicase-dependent isothermal amplification TMB 3,3′,5,5′-tetramethylbenzidine

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13.  DNA markers and nano-biosensing approaches for tuberculosis diagnosis

1 Introduction Tuberculosis is one of the most serious infectious diseases worldwide with an estimated 10 million new TB cases and a death toll of approximately 1.3 million in 2018 [1]. Despite vaccination and antibiotic treatment available today and that diagnosis and successful treatment of people with TB avert millions of deaths each year, the “end” of TB as an epidemic and a major public health problem remains an aspiration for most countries rather than a reality. Different techniques such as radiometric detection, immunoassays like enzyme-linked immunospot, polymerase chain reaction, TB rapid cultivation detection systems like MB/BacT system, Septi check and BACTEC MGIT system, have been developed in order to reduce the large and persistent gaps in TB detection and treatment [2]. However, these approaches are rather expensive and centralized in large stationary laboratories because they require complex instrumentation with highly qualified technical staff, which make it of paramount importance to construct portable, fast and highly sensitive realtime system for accurate diagnosis and screening of TB infection on time. As an appealing alternative to conventional techniques, nanotechnology triggered the development of new molecular nanodiagnostic tools with increased sensitivity, specificity, and speed at lower costs. In fact, a variety of nanomaterials-based biosensors have been developed for detection of TB [3,4]. Nano-biosensor is generally defined as a compact analytical device incorporating a biorecognition element with a physicochemical nanometric materials-sized transducer [5]. The biorecognition element, which can be an enzyme, antigen-antibody, nucleic acid, whole cell, etc., is immobilized tightly by different chemical or physical processes onto the transducer that can then measure the arising signals precisely [6]. Here we will provide a closer look into DNA markers used for TB nanodiagnostic and/or

Mycobacterium tuberculosis (Mtb) detection and characterization. Then, DNA nano-biosensing approaches based on the use of carbonaceous nanomaterials [i.e., graphene and carbon nanotubes (CNTs)] and nanoparticles such as noble metal nanoparticles, metal oxide nanoparticles, magnetic beads (MBs), and quantum dots (QDs) will be described.

2  DNA structure

 

In order to develop DNA nano-biosensors, the identification and validation of DNA biomarkers for specific sensitive diagnostic of tuberculosis is one of the great challenges to overcome. These biomarkers should be able to identify TB infection in different sample matrixes, such as sputum, plasma, and urine in detectable levels. Moreover, they should be capable to discriminate between infected patients and noninfected subjects [7]. To do so, a single pair of primers can be used as diagnostic markers to detect TB or Mtb at a single gene target resolution. Having a very specific target gene does promise high positive predictive values and low false-negative results. However, in terms of analytical sensitivity, a gene with high copy numbers, that is, IS6110 (up to 25 copies in Mtb genome), plays an important role in determining the limit of detection of an assay, and thus contributes to higher sensitive diagnostic tests [8]. Chin et al. nicely summarized all the relevant data about DNA targets used for tuberculosis diagnosis in their review [9]. Moreover, they explained the advantages and disadvantages of using each existing marker in Table 13.1. These targets include the rrs (16S rRNA), ITS (16S-23S rRNA), IS6110, groEL2 (hsp65), dnaJ, fbpA (32 kD protein), MPT64 (MPB64), devR, PPE24 (KS4), and lepA genes. Among them, the IS6110 is the most attractive one, as it demonstrates higher sensitivity and specificity due to the multiple copies present in the Mtb genome [10].



TABLE 13.1 DNA markers used for TB diagnosis: advantages and disadvantages. Advantages

Disadvantages

rrs (16S rRNA)

• Universally used for bacteria identification • Rapid identification of all Mycobacteria • Differentiate between Mycobacterium tuberculosis complex (MTBC) and tuberculous mycobacteria (NTM) groups • Useful for mutation studies associated with amikacin, kanamycin, and capreomycin resistance

• Insufficient accurate identification of NTM

ITS (16S23S rRNA)

• Identification of the genus Mycobacterium • Differentiate between MTBC and NTM • Greater sequence variations than 16S rRNA, which are useful for species differentiation, mainly for NTM

• Unable to differentiate MTBC members

IS6110

• Found exclusively in MTBC in multiple copies • Excellent element for strain genotyping, IS6110-RFLP • High sensitivity and specificity compared to culture and acid-fast bacilli staining in the diagnosis of pulmonary TB

• Low discriminatory power of IS6110-RFLP in isolates with ≤5 IS6110 copies • Unable to differentiate between MTBC members • False positive results with some nonmycobacterium species • False negative results in some MTB strains

groEL2 (hsp65)

• Detected in all mycobacterial strains • hsp65-RFLP can differentiate between MTBC and NTM • Higher accuracy in identification of NTM compared to 16S Rrna

• Lower discriminatory power than dnaJ and fbpA for NTM

dnaJ

• Found in all mycobacteria • Higher discriminatory power than 16S rRNA and hsp65 for NTM

• MTBC members have identical sequence: not suitable for species differentiation

fbpA (32 kDa protein)

• Detected in all mycobacterial strains • Better discriminatory power than hsp65 for NTM

• Identical among MTBC members, not suitable for species differentiation

MPT64 (MPB64)

• Specific for MTBC • Reported useful for TB diagnosis in sputum, ascitic fluid, cerebrospinal fluid, and urine samples • MPB64 PCR is a complement to IS6110 PCR in strains that do not have IS6110

• Might give false positive results in blood samples • False negative results due to mutations in the gene

devR

• Shorter fragments of the gene significantly increased the sensitivity of TB diagnosis

• Lower sensitivity compared to MPB64 and IS6110

PPE24 (KS4)

• High sensitivity and specificity detecting MTBC

• Cross-react with some NTM

lepA

• Useful to identify Mycobacterium caprae (specific substitution C690T)

2  DNA structure

 

Markers

Reprinted with permission from [9].

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13.  DNA markers and nano-biosensing approaches for tuberculosis diagnosis

3  Carbonaceous nanomaterials-based DNA biosensors

3.1  Graphene derivatives Since its discovery in 2004, graphene has been quickly established as a material of choice for many applications in different fields to outshine CNTs, particularly in biosensing applications due to its exceptional mechanical, electronic and optical properties [13]. Chiu et al. developed a novel surface plasmon resonance (SPR) biosensor using graphene nanocomposites-based bioships integrated loop-mediated isothermal amplification for TB biosensing [14]. In fact, SPR relies on the changes of refractive index at the sensor surface in detection applications. The increase in the refractive index is a consequence

Carbon nanomaterials, such as graphene and CNTs, have been booming for several decades in different fields of applications, especially in analytical and industrial electrochemistry for the investigation of many diseases and their therapy [11,12]. The high surface area of nanoscaled carbonaceous derivatives, their mechanical and electrical properties have made them materials of choice for the manufacture of sensitive, selective and low-cost biosensors for many diseases and especially tuberculosis (Table 13.2).

TABLE 13.2 Various types of carbon nanomaterials based-DNA biosensors for the detection and identification of tuberculosis. Signal transduction method

Nanomaterial identity

Samples analyzed

Limit of detection

Linear range

References

Surface plasmon Graphene resonance Graphene

IS6110 of M. tuberculosis

Not available

Not available

[14]

IS6110 of M. tuberculosis

28 fM

Not available

[15]

Electrochemical rGO response

DNA from M. tuberculosis complex

Not available

Not available

[16]

rGO

IS6110 of M. tuberculosis

50 fM

0.1 pM − 10 nM

[17]

Graphene

DNA of M. tuberculosis

0.015 ng µL−1

0.015–150 ng /µL−1

[18]

Graphene

DNA of M. tuberculosis

7.853 × 10  M

10

rGO/AuNPs

IS6110 of M. tuberculosis

1 fM

1.0 × 10

rGO/AuNPs

DNA of M. tuberculosis

0.1 fM

Not available

[21]

Graphene/Au nanorods

DNA of M. tuberculosis

10 fM

10 fM to 0.1 µM

[22]

GO/CdSQDs

DNA of M. tuberculosis

8.948 × 10−13 M

1 × 10−11 to 1 × 10−7 M

[23]

MWCNTs

rpoB gene of M. tuberculosis 0.3 fM

1 fM to 10 pM

[27]

MWCNTs

rpoB gene of M. tuberculosis Not available

Not available

[30]

MWCNTs

DNA of M. tuberculosis

Not available

Not available

[28]

CNTs

IS6110 gene of M. tuberculosis

0.33 fM

1 fM to 10 nM

[29]

CNTs/ZrO2

DNA of M. tuberculosis

0.01 nM

1 × 10−2 to 1 × 10−8 mM

[31]

IS6110 of M. tuberculosis

Not available

Not available

[24]

Fluorescence GO resonance energy transfer

 

−7

−6

[19]

to 10  M −9

−15

 − 1.0 × 10  M [20] −9



3  Carbonaceous nanomaterials-based DNA biosensors

of an increase in mass suggesting the occurrence of a binding event. The prepared nanocomposite was constructed of single-layer graphene film deposited onto self-assembled monolayer at a gold electrode. Owing to the strong interactions existing between graphene and DNA, graphene film enabled the detection of Insertion Sequence 6110 (IS611) gene of Mtb. The reported results showed high sensitivity and good reliability toward Mtb DNA which encouraged the applicability of the SPR-LAMP chip in disease detection. Recently, graphene was also used by Prabowo et al. for the elaboration of another SPR-based biosensor [15]. A few graphene layers were deposited on top of SPR sensing chip by simple drop casting and used as a platform for DNA probe immobilization. The gold nano urchin tagged-DNA probe was immobilized onto graphene surfaces through π–π stacking interactions. In presence of the target DNA (IS6110), the probe desorbed from the surface to hybridize with its target, which resulted in a variation in the SPR response. The biosensor achieved a detection limit around 28 fM of cssDNA target in salt buffer. A tremendous body of work has been done to design graphene-based electrochemical biosensor for TB detection. Nguyen et al. described a new approach based on the functionalization of reduced graphene oxide sheets with electroactive copolymer juglone [16]. The hybrid material showed good stability in a biocompatible buffer medium. Moreover, current increase upon hybridization (signal-on) evidenced that short DNA target as well as polymerase chain reaction related to different lineages of Mtb strains were successfully detected. The signal-on reached ∼40% with 1 nM of short DNA (25 mer) target, while PCR product produced a current change of ∼20%. Polyaniline is another polymer largely used with reduced graphene oxide for biosensing systems. In this context, Chen et al. developed a sandwich-type of Mtb DNA biosensor based on

211

 

the use of polyaniline-reduced graphene oxide, which was decorated with DNA label immobilized onto gold nanoparticles (Fig. 13.1) [17]. The hybridization of the target IS6110 DNA sequence of Mtb with its probe leaded to the appearance of a voltammetric signal change resulting from PANI-rGO redox probe. Moreover, the specific IS6110 DNA gene could be detected in the 0.1 pM to 10 nM concentration range, with a detection limit of 50 fM. As for Mukherjee et al., they have achieved the feat of preparing a 2-dimensional graphene flakes onto indium-doped tin oxide-coated glass plates by electrochemical exfoliation of graphite rods using in situ intercalation of potassium ions [18]. In this approach, the authors modified graphene flakes with a TB DNA probe via amide bond formation between –CO2H group of GF and –NH2 group of DNA probe. In presence of DNA target, the system responded in the 0.015– 150 ng µL−1 concentration range with a detection limit of 0.015 ng µL−1. Furthermore, Mohamad et al. achieved a larger concentration range from 10−6 to 10−9 M by developing a PANI/Graphene nanofiber platform [19]. The polymerization of aniline monomer and graphene was carried out in the presence of a poly(methyl vinyl etheralt-maleic acid) solution before the immobilization of a DNA probe related to tuberculosis. Hybridization of the target DNA was monitored by DPV of methylene blue as redox intercalator. The use of metal nanoparticles, such as gold, with graphene is a very useful way to enhance the biosensing platform metrics. A reduced graphene oxide decorated with AuNPs was exploited by Liu et al. as a sensing platform for the detection of the highly specific tuberculosis biomarker IS6110 in the presence of Au/PANI as a tracer label for signal amplification [20]. The biosensor provided a simple, versatile and powerful tool for reliable diagnosis of Mtb over a broad linear range between 10−15 and 10−9 M. In the approach developed by Mogha et al., the authors immobilized AuNPs on reduced

212

13.  DNA markers and nano-biosensing approaches for tuberculosis diagnosis

FIGURE 13.1  Schematic representation of the stepwise fabrication process of the electrochemical DNA biosensor. Reprinted with permission from [17].

graphene oxide nanoribbons before their functionalization with the DNA probe [21]. Cyclic voltammetry and chronoamperometry were used to quantify the Mtb target DNA showing good sensitivity and specificity. Not far from this concept, Perumal et al. prepared another graphene and gold nanocomposite-based biosensor [22]. A 3-D graphene grown by chemical vapor deposition has been modified by gold nanorods obtained from self-assembly of AuNPs on the graphene foam then conjugated to TB DNA probe. The target detection was monitored by electrochemical impedance spectroscopy over a wide detection linear range from 10 fM to 0.1 µM. Nanocomposite of reduced graphene oxide and CdS QDs were also exploited for the detection of Mtb. Zaid et al. introduced CdS QDs to a reduced graphene oxide bearing amine groups before the immobilization of peptide nucleic

 

acid probe [23]. The detection of TB DNA was followed by the DPV signal of methylene blue. The prepared biosensor showed good linearity ranging from 10−11 to 10−7 M with a detection limit of 8.95 × 10−13 M. Fluorescence-based biosensors have been attracted great attention from scientific committee these recent years. Usually, when the target concentration is so low, an enzymatic amplification by PCR is essential before or during the analytical process with fluorescence in order to bring the analyte concentration beyond the detection threshold. In this context, Hwang et al. have developed a simple PCR-GO-based system for IS6110 DNA sequence detection [24]. They showed that, in the presence of the target DNA, the results obtained with PCR-GO system using a simple fluorimeter were in agreement with a conventional real-time quantitative PCR results.



3  Carbonaceous nanomaterials-based DNA biosensors

3.2  Carbon nanotubes

213

CNTs were observed for the first time in 1976 by a group of Japanese researchers using a transmission electron microscope [25]. Since their identification in 1991 by Ijima [26], they aroused great interest in several areas thanks to their unique mechanical, thermal, and electronic properties. As an example, they were used by Korri-Youssoufi and collaborators to detect DNA of rpoB gene of Mtb [27]. For this purpose, they elaborated a multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole and redox PAMAM dendrimers composite serving

as a detection platform (Fig. 13.2). PPy coated MWCNTs acted as transducer and PAMAM dendrimers served to amplify the electrochemical signal. Ferrocenyl groups attached to the surface served as a redox marker. The MWCNTsPPy-PAMAM-Fc layer was proven as an efficient platform for DNA detection by monitoring the ferrocene redox marker. Cyclic voltammetry and square wave voltammetry demonstrated that the system can detect the target DNA in a large concentration range (1 fM to 100 nM) with low limit of detection. Later on, the same group prepared another MWCNTs-based TB biosensor

FIGURE 13.2  Schematic illustration of the biosensor elaboration. (A) electropolymerization of pyrrole on MWCNTs, (B) dendrimers attachment through electrochemical oxidation of PAMAM’s G4 amines, (C) covalent attachment of ferrocene, (D) covalent attachment of DNA probe, and (E) hybridization with DNA target. Reprinted with permission from [27].

 

214

13.  DNA markers and nano-biosensing approaches for tuberculosis diagnosis

using ferrocene modified oligo-methoxy-phenyl-acetonitrile as transducing polymer [28]. This nanocomposite showed high performance of DNA hybridization with a detection limit of 0.08 fM. The combination of a flower-like CNT with polyaniline was investigated by Chen et al. [29]. In their study, the authors used a rather complicated system with multiple signal amplification strategy in the diagnosis of tuberculosis through DNA detection. The CNTs-PANI nanohybrid was modified with a tracer label to generate the electrochemical signal. The DNA probe hybridization with the target DNA in the presence of an assistant probe yielded a recycling process based on nicking endonuclease-assisted three-way DNA junction process on modified glassy carbon electrode with functionalized fullerene C60. The electrochemical signal had been afforded after hybridization between the cleaved capture probe and the tracer label (Fig. 13.3). The developed biosensor was applicable in a wide linear

range (1 fM to 10 nM) with detection limit of 0.33 fM. Recent advances in electrochemical sensing techniques, combined to microfluidics and nanomaterials, have led to more sophisticated devices. The work of Zribi et al. highlighted the use of microfluidic platform based on MWCNTs modified by ferrocene moieties to detect pathogenic viral Hepatitis C and genomic Mtb DNA in clinical isolates [30]. The limit of detection of the reported electrochemical biosensor was enhanced from picomolar in bulk solution to femtomolar in the fluidic device with a wide linear range from 0.1 fM to 1 pM. Moreover, working under high flow, it allowed selective direct detection of rpoB gene of Mtb H37Rv from clinical isolate extracted DNA. Metal oxide nanoparticles were also coupled to CNTs for tuberculosis detection. In fact, Zirconia (ZrO2) grafted MWCNTs (crystallite size of ZrO2 ∼28nm), obtained via isothermal hydrolysis of zirconium oxychloride in presence

FIGURE 13.3  Schematic representation of the fabrication process of the biosensor. Reprinted with permission from [29].

 



4  Nanoparticles based-DNA biosensors

of CNTs, have been electrophoretically deposited onto indium-doped tin oxide-coated glass to develop an impedimetric nucleic acid biosensor for Mtb detection [31]. The elaborated nanocomposite applied for IS6110 detection displayed good performances in the concentration range from 10−2 to 10−8 M with improved detection limit of 0.01 nM.

215

4  Nanoparticles based-DNA biosensors

been extensively used due to their easy preparation, inert nature, favorable biocompatibility, high surface area, unique optical properties with their typical bright-red color in colloidal solutions associated with a well-defined SPR band in the visible region of the spectrum, and especially their suitability for binding to biomolecules via thiol-gold association [34]. Colorimetric sensing using gold nanoparticles has been extensively used to detect and characterize pathogens [35] including Mtb. These methods rely on the colorimetric changes of the colloidal solution upon aggregation either mediated by a change of the dielectric medium or by recognition of a specific target. The design of these systems is centered in the ability of complementary targets to balance and control inter-particle attraction and repulsion forces, which determine whether AuNPs are stabilized or aggregated and, consequently, the SPR band and color of the solution remains unaltered or changes, respectively. Baptista et al. reported the first application of AuNPs for the molecular diagnostics of Mtb by differential stabilization of gold nanoprobes in the presence of different DNA targets [36]. The gold nanoprobes were functionalized with thiol-modified oligonucleotides harboring a sequence derived from the Mtb RNA polymerase β-subunit gene sequence suitable for mycobacteria identification. The presence of a complementary target prevented nanoprobe aggregation and the solution remained red, because the nanoprobe solution had a SPR at an absorbance peak of 526 nm, while noncomplementary/mismatched targets or their absence do not prevent gold nanoprobe aggregation at high NaCl concentrations, resulting in a visible change of color from red to blue. The aggregation profile of Au-nanoprobes, and thus identification of specific sequences of Mtb complex, namely Mycobacterium bovis and Mtb in biological samples, was also achieved via the evaluation of the spectra acquired by traditional UV-visible spectrophotometry using a portable and low-cost optoelectronic platform integrating

Incorporation of nanomaterials and especially nanoparticles with unique properties in biosensing design provided more simple, swift, sensitive and hybrid nano-biosensor platforms with synergetic properties and functions [32]. The intelligent use of such nano-objects led to clearly enhanced performances with increased sensitivities and lowered detection limits of several orders of magnitude, which is due to their high specific surface thus enabling the immobilization of an enhanced amount of bioreceptor units. Moreover, owing to their small size (normally in the range of 1–100 nm), nanoparticles exhibit unique chemical, physical and electronic properties that are different from those of bulk materials, which were used to construct novel and improved biosensing devices. Many types of nanoparticles of different sizes and compositions are now commercially available, which facilitate their application in detection of infectious disease and especially tuberculosis. Table 13.3 summarizes the various types of nanoparticles based-DNA biosensors for the detection and identification of TB.

4.1  Noble metal nanoparticles Becoming a critical component in the development of nanotechnology-based detection of pathogens, noble metal NPs have attracted considerable attention in molecular diagnostic applications due to their simplicity and versatility [33]. Gold nanoparticles, in particular, have

 

216

TABLE 13.3 Various types of nanoparticles based-DNA for the detection and identification of tuberculosis. Nanoparticle identity

Samples analyzed

Detection limit

Linear range

Detection time References

Colorimetric sensing

AuNPs

DNA of M. tuberculosis

0.75 µg

Not available

2 h

[36]

AuNPs

DNA from M. tuberculosis complex

50 fmol µL

50–300 nM

3 h

[37]

AuNPs

178 bp of IS6110 of M. tuberculosis

5 pg

Not available

1 h

[38]

AuNPs

DNA of M. tuberculosis

90 ng

Not available

30 min

[39]

AuNPs

M. tuberculosis complex/rpoB

Not available

Not available

Not available

[40]

AuNPs

123 bp IS6110 of M. tuberculosis dsDNA

1.95 × 10  ng mL

−1

−2

−1

60 min 1.95 × 10 to 1.95 × 101 ng mL−1

[41]

−2

AgNPs

DNA of M. tuberculosis

1.27 nM

50–2500 nM

Not available

[52]

Surface plasmon resonance

AuNPs

DNA from M. tuberculosis complex

104 CFU mL−1

104 to 108 CFU mL−1

40 min

[43]

AuNPs

rpoB gene of M. tuberculosis

Not available

Not available

Not available

[44]

Quartz crystal microbalance

AuNPs

IS6110 of M. tuberculosis

5 pg

Not available

Not available

[45]

AuNPs

16S rDNA fragment from M. tuberculosis

20 CFU mL

10 to 10  CFU mL