Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols [2207, 1 ed.] 9781071609194, 9781071609200

Table of contents :
Preface
Contents
Contributors
Chapter 1: Application of Neutralization and Freeze-Drying Technique for the Preparation of the Beneficial in Drug Delivery 2-Hydroxypropyl-β-Cyclodextrin Complexes with Bioactive Molecules
1 Introduction
2 Materials
3 Methods
3.1 Steps to Follow for the Preparation of Solid-State Irbesartan–2-HP-β-CD Inclusion Complex [25, 26]
3.2 Steps to Follow for the Preparation of Solid-State LOS–2-HP-β-CD Inclusion Complex
3.3 Steps to Follow for the Preparation of Solid-State CAN–2-HP-β-CD and CC–2-HP-β-CD Inclusion Complexes [27]
3.4 Steps to Follow for the Preparation of Solid-State RA–2-HP-β-CD and CA–2-HP-β-CD Inclusion Complexes
3.4.1 CA–2-HP-b-CD Inclusion Complex
3.4.2 RA–2-HP-b-CD Inclusion Complex
3.5 Steps to Follow for the Preparation of Solid State SLB–2-HP-β-CD Inclusion Complex [28]
3.6 Steps to Follow for the Preparation of Solid-State CRM–2-HP-β-CD Inclusion Complex
3.7 Steps to Follow for the Preparation of Solid-State QUE–2-HP-β-CD and QUE–Me-β-CD Inclusion Complexes [29–31]
3.7.1 QUE–2-HP-β-CD Inclusion Complex [29, 30]
3.7.2 QUE–Me-β-CD Inclusion Complex [31]
4 Notes
5 Conclusions
References
Chapter 2: Functionalized Carbon Nanohorns as Drug Delivery Platforms
1 Introduction
2 Materials
2.1 Preparation of CNHs
2.2 Drying Solvents
2.3 Synthesis of BOC-Aniline Derivative
2.4 Synthesis of BOC-CNHs and the Corresponding NH2-CNHs
2.5 Oxidation of CNHs
2.6 Conjugation of Drugs/Biomolecules
2.7 Nanomesh CNHs
2.8 Encapsulation of Drugs/Biomolecules
3 Methods
3.1 Preparation of CNHs
3.2 Procedures for Drying Solvents
3.3 Covalent Incorporation of Amine Moieties on CNHs
3.4 Incorporation of Carboxylic Acid Moieties on CNHs
3.5 Conjugation of Drugs/Biomolecules on Pre-modified CNHs
3.6 Encapsulation of Drugs/Biomolecules Within CNHs
4 Notes
5 Conclusions
References
Chapter 3: Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole Organs
1 Introduction
2 Materials
2.1 Reagents Used for Exosome Isolation
2.2 Reagents Used for the Characterization of Isolated Exosomes
2.2.1 Exosomal Protein Quantification
2.2.2 Western Blot Analysis
2.2.3 Transmission Electron Microscopy Imaging
2.2.4 RNA Isolation
3 Methods
3.1 Isolation of Exosomes from Brain, Heart, and Liver
3.2 Protein Quantification of Exosomes Using BCA Test
3.3 Exosome Particle Counting with CD63 ELISA Kit
3.4 Western Blot Analysis
3.5 Preparation of TEM Samples
3.6 RNA Isolation and Quantification
4 Notes
References
Chapter 4: Aggregate Determination by Permeation Technique
1 Introduction
2 Materials
2.1 Tested Solutions or Donor Solutions: Saturated Guest/Host Aggregate Solution
2.2 Self-Aggregate or Cluster Solutions
2.3 Receptor Solutions
2.4 Semipermeable Membrane
3 Methods
3.1 Permeation Studies
3.1.1 Franz Diffusion Cells
3.1.2 Micro-Equilibrium Dialysis Device
3.1.3 Cuplike MINI Dialysis Device
3.1.4 Determination of Permeation Profiles
3.2 Evaluation of Guest/Host Aggregate Profiles
3.3 Evaluation of Critical Aggregation Concentration (cac)
4 Notes
References
Chapter 5: Study of Candesartan Cilexetil: 2-Hydroxypropyl-β-Cyclodextrin Interactions: A Computational Approach Using Steered Molecular Dynamics Simulations
1 Introduction
2 Software
3 Methods
3.1 System Preparation
3.1.1 Manually Create the Drug: CD Complex
3.1.2 Minimize Structure
3.1.3 Create the Topology Files for Both Drug and Host
3.1.4 Create the Gromacs Input Files
3.1.5 Manually Create the Topology File for the Run
3.1.6 Create the Simulation Box
3.1.7 Solvate the System
3.2 Energy Minimization
3.2.1 Create Input
3.2.2 Run the Energy Minimization Simulation
3.3 Equilibration
3.3.1 NVT Ensemble Equilibration
Create Input
Create Input
Run the Equilibration Simulation
3.3.2 NPT Ensemble Equilibration
Create Input
Run the Equilibration Simulation
3.4 Steered Molecular Dynamics Simulation
3.4.1 Create Input for the Pulling Simulation
3.4.2 Run the Pulling Simulation
3.4.3 Collect Frames
3.4.4 Choose the Starting Configurations
3.5 Umbrella Sampling
3.5.1 Equilibration
3.5.2 Umbrella Sampling Simulations
3.6 Analysis
3.6.1 Create Input
3.6.2 Run the WHAM Analysis
4 Notes
References
Chapter 6: Drug Delivery: Hydrophobic Drug Encapsulation into Amphiphilic Block Copolymer Micelles
1 Introduction
2 Materials
3 Methods
3.1 Solution of Pluronic F-127 with 50% Curcumin Encapsulated (Organic Cosolvent Protocol)
3.2 Solution of  Pluronic F-127 with 50% Curcumin Encapsulated (Thin-Film Protocol)
3.3 Solution of PEO-b-PCL with 20% IND Encapsulated (Organic Solvent Protocol)
3.4 Solutions of PEO-b-PCL with 20% IND Encapsulated (Thin-Film Protocol)
3.5 UV-Vis Spectroscopy
3.6 Fourier Transform Infrared Spectroscopy (ATR-FTIR)
3.7 Dynamic Light Scattering (DLS)
4 Notes
References
Chapter 7: Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation
1 Introduction
2 Materials
2.1 Evaluation of Drug Loading and Release
2.1.1 Buffer Solutions [19–21]
2.1.2 Drug-Loading Suspensions
2.1.3 Drug-Release Suspensions
2.2 Biological Evaluation
2.2.1 Culture Media
2.2.2 MTT Solution Preparation
2.2.3 Other Reagents and Materials
3 Methods
3.1 Drug Loading and Release
3.1.1 Drug Loading in Nanocontainers
3.1.2 Drug Release from Nanocontainers
3.2 Hyperthermia Measurements
3.3 Cytotoxicity and Biocompatibility Evaluation
3.3.1 Cell Culture
3.3.2 MTT Cell Proliferation Assay
3.3.3 In Vitro Cytotoxicity Studies Under Hyperthermia
3.4 Scratch-Wound Healing
3.5 Imaging via Fluorescent Confocal Microscopy
4 Notes
References
Chapter 8: Drug Incorporation in the Drug Delivery System of Micelles
1 Introduction
2 Materials
3 Methods
4 Results and Discussion
5 Notes
References
Chapter 9: Molecular Dynamics Protocols for the Study of Cyclodextrin Drug Delivery Systems
1 Introduction
2 Materials
3 Methods
3.1 Molecular Modeling
3.1.1 Structure Retrieval
3.1.2 Molecular Docking Calculations
3.2 Structure Preparation for Molecular Dynamics
3.3 Molecular Dynamics Simulation of the Complex
3.3.1 Energy Minimization
3.3.2 Heating
3.3.3 Density Equilibration
3.3.4 MD Production Simulation
3.4 Energetic Analysis with the  MM–PBSA Method
3.5 Conformational Analysis
4 Notes
References
Chapter 10: Drug Delivery Through Multifunctional Polypeptidic Hydrogels
1 Introduction
2 Materials
3 Methods
3.1 Instrumentation
3.2 Synthesis of Monomers
3.2.1 Synthesis of ε-tert-Butoxycarbonyl-l-Lysine N-Carboxy Anhydride (Νε BOC-l-LYS NCA)
3.2.2 Synthesis of Nim-trityl-l-Histidine-N-Carboxy Anhydride (Trt-HIS-NCA) [4]
3.2.3 Synthesis of γ-Benzyl-l-Glutamate N-Carboxy Anhydride (BLG-NCA)
3.2.4 Synthesis of l-Leucine N-Carboxy Anhydride (LEU-NCA)
3.3 Synthetic Approach of Polypeptides PLys-b-(PHIS-co-PBLG (or PLEU))-b-PLys-b-(PHIS-co-PBLG (or PLEU))-b-PLys (See Fig. 2)
3.4 Formation of Hydrogels
3.5 Formation of Hydrogels Loaded with Gemcitabine [5, 6]
4 Notes
5 Results
References
Chapter 11: Polymersomes from Hybrids-Polypeptides for Drug Delivery Applications
1 Introduction
2 Materials
3 Methods
3.1 Instrumentation
3.2 Synthesis of Monomers
3.2.1 Synthesis of ε-tert-Butoxycarbonyl-l-Lysine N-Carboxy Anhydride, (Νε BOC-l-LYS NCA)
3.2.2 Synthesis of γ-Benzyl-l-Glutamate N-Carboxy Anhydride (BLG-NCA)
3.3 Synthesis of the Triblock Copolypeptide PLL-b-PBLG-d7-b-PLL
3.4 Synthesis of the Novel Triblock Copolypeptide PEO-b-PBLG-b- PLL
3.5 Drug Loading (See Fig. 3) [4, 5]
3.5.1 Doxorubicin Loading
3.5.2 Pacl Loading
4 Notes
5 Results
5.1 Synthesis and Characterization of the Polymers and Determination of the Concentration of Pacl in Polymersomes [6]
5.2 Loading and Stability
5.3 Polymersome Characteristics
5.4 In Vitro Release and Activity
5.5 In Vivo Toxicity Study
6 Conclusions
References
Chapter 12: Drug Delivery Systems Based on Modified Polysaccharides: Synthesis and Characterization
1 Introduction
2 Materials
3 Methods
3.1 Synthesis of PMMA@HPC@CS@CH Nanospheres (NCs)
3.2 Drug Encapsulation
3.3 Characterization of Vehicles
3.3.1 SEM Preparation
3.3.2 TEM Preparation
3.3.3 AT-IR Preparation
4 Notes
References
Chapter 13: Differential Scanning Calorimetry (DSC) on Sartan/Cyclodextrin Delivery Formulations
1 Introduction
2 Materials
2.1 Lyophilization of Raw Materials
2.2 Complex of Sartan with 2-HP-β-CD
2.3 Differential Scanning Calorimetry (DSC)
3 Methods
3.1 Lyophilization of Raw Materials
3.2 Complexation of the Sartans with 2-HP-β-CD
3.3 DSC Sample Preparation
3.4 DSC Analysis
3.5 DSC Diagram and Thermodynamic Parameter Extraction
3.6 DSC Profile Analysis of the Cyclodextrin:Sartan Systems
4 Notes
References
Chapter 14: Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability, and Therapeutic Efficacy Against Glioblastoma Multiforme
1 Introduction
2 Materials
2.1 Synthesis and Characterization of Encapsulated TMZ
2.2 UV-Vis Spectroscopy
2.3 Liquid Chromatography and Mass Spectrometry
2.4 In Vitro Assays of TMZ@PSC4 Activity
3 Methods
3.1 Synthesis, Characterization, and Quantification of the Encapsulated TMZ
3.1.1 Synthesis and Purification of Nanocapsule
3.1.2 Characterization of the Nanocapsule
3.1.3 Quantification of Encapsulated TMZ Using UV-Vis Spectroscopy
Preparation of the Calibration Curve Using UV-Vis Spectroscopy
Quantification of Encapsulated Drug Using UV-Vis Spectroscopy
3.2 Determination of Chemical Stability of TMZ and ΤΜΖ@PSC4 in Buffer Solutions Using UV-Vis, 1H-NMR, and LC-MS/MS
3.2.1 Determination of Chemical Stability of ΤΜΖ@PSC4 by UV-Vis
3.2.2 Determination of Chemical Stability of ΤΜΖ@PSC4 Using 1H-NMR
3.2.3 Determination of Chemical Stability of ΤΜΖ@PSC4 Using Liquid Chromatography and Mass Spectrometry
Method Development and Optimization
Buffer Chemical Stability Assay
3.3 Cell Culture
3.4 In Vitro Cytotoxicity: Sulforhodamine B (SRB) and Cell Counting Kit 8 (CCK8) Assays
3.5 In Vivo Pharmacokinetic Analysis
4 Notes
References
Chapter 15: Unveiling the Thermodynamic Aspects of Drug-Cyclodextrin Interactions Through Isothermal Titration Calorimetry
1 Introduction
2 Materials
2.1 Samples
2.2 Instrumentation
3 Methods
3.1 Experimental Design
3.2 Sample Preparation and Loading
3.3 Run Parameters
3.4 Data Analysis
4 Notes
References
Chapter 16: Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG and Rosin Esters
1 Introduction
1.1 Sphingolipid Metabolism and Cancer
1.2 Ceranib-2 as Potent Ceramidase Inhibitor
1.3 Therapeutic Limitation of Ceranib-2 as Anticancer Drug
1.4 Nanostructure Material in Anticancer Drug Delivery System
1.5 Polymeric Nanoparticle Drug Delivery Systems
1.6 Rosin Ester Nanoparticles in Drug Formulation
1.7 Improving the Structure
1.8 PEGylation
1.9 Preparation Methods for Nanoparticle Drug Delivery System
1.10 Solvent Evaporation
1.11 Ultrasonic Cavitation
2 Materials and Methods
2.1 Synthesis of PREC-2 NPs
2.2 Cell Culture
2.3 In Vitro Release Assessment
2.4 Preparation MTT Assay Stock Solution
2.5 Equipment and Instruments
2.6 Statistical Analysis
3 Methods
3.1 Preparation of Dispersion Phase
3.2 Preparation of Continuous Phase
3.3 Synthesis of PREC-2 NPs
3.4 Preparation of Physiological Reception Solution for In Vitro Release Experiment
3.5 In Vitro Drug-Release Profile of PREC-2 NPs
3.6 Preparation of DMEM Culture Media for Cell Line
3.7 Determination of the Efficacy and Therapeutic Effect of PREC-2 NPs
4 Notes
References
Chapter 17: Association of the Thermodynamics with the Functionality of Thermoresponsive Chimeric Nanosystems
1 Introduction
2 Materials
2.1 Preparation of Thermoresponsive Chimeric Bilayers
2.2 Differential Scanning Calorimetry (DSC)
2.3 Preparation of Thermoresponsive Chimeric Liposomes
2.4 Dynamic Light Scattering (DLS) and Heating Protocol
3 Methods
3.1 Preparation of Thermoresponsive Chimeric Bilayers
3.2 DSC Sample Preparation
3.3 DSC Analysis
3.4 DSC Profile and Thermodynamic Parameter Extraction
3.5 DSC Profile Analysis of the Thermoresponsive Chimeric Systems
3.6 Preparation of Thermoresponsive Chimeric Liposomes Through the Thin-Film Hydration Method
3.7 Dynamic Light Scattering (DLS)
3.8 Heating of the Thermoresponsive Chimeric Liposomes
3.9 Physicochemical Properties of  the Thermoresponsive Chimeric Liposomes
4 Notes
References
Chapter 18: 2D DOSY NMR: A Valuable Tool to Confirm the Complexation in Drug Delivery Systems
1 Introduction
2 Materials
3 Methods
3.1 Preparation of the Complex
3.1.1 Preparation of the Que-HP-β-CD Complex
3.1.2 Preparation of the TMZ-Calixarene Complex
3.2 NMR Spectroscopy
3.2.1 1H NMR Spectrum
3.2.2 2D DOSY NMR Spectrum
4 Notes
References
Chapter 19: Drug-Encapsulated Cyclodextrin Nanosponges
1 Introduction
2 Encapsulated Cyclodextrin Nanosponges
2.1 Cyclodextrin Nanosponges for Anticancer Drugs
2.2 Cyclodextrin Nanosponges for Anti-inflammatory Drugs
2.3 Cyclodextrin Nanosponges for Antiviral Drugs
2.4 Cyclodextrin Nanosponges for Antifungal Drugs
2.5 Cyclodextrin Nanosponges for Antioxidants
2.6 Cyclodextrin Nanosponges for Polyphenols
2.7 Cyclodextrin Nanosponges for Antibacterial Drug
2.8 Cyclodextrin Nanosponges for Anti-Zika
2.9 Molecularly Imprinted Cyclodextrin Nanosponges for Parkinson’s Disease
2.10 Cyclodextrin Nanosponges for Hypertension Medication
2.11 Cyclodextrin Nanosponges for Anti-seizure
2.12 Cyclodextrin Nanosponges for Sleep Disorder
2.13 Cyclodextrin Nanosponges for Gastric Reflux
2.14 Cyclodextrin Nanosponges for Gas Delivery
2.15 Cyclodextrin Nanosponges for Antihyperglycemic Agent
2.16 Cyclodextrin Nanosponges for Calcium Delivery
2.17 Cyclodextrin Nanosponges for Peptides and Steroids
2.18 Cyclodextrin Nanosponges with Photosensitizing/Photooxidation Agents
2.19 Cyclodextrin Nanosponges for Miscellaneous Uses
3 Conclusion
References
Chapter 20: Electrochemistry Investigation of Drugs Encapsulated in Cyclodextrins
1 Introduction
1.1 The CD-Drug Complex Is Oxidized at the Surface of Electrode and the Formation of a Complex Between CD and Oxidated Drug Is Observed
1.2 The CD-Drug Complex Dissociates and Afterwards the Free Drug Molecule Is Oxidized at the Surface of Electrode
2 Materials
2.1 Reagents
2.2 Cyclic Voltammetry
2.3 Electrolysis
2.4 UV–Vis Spectro-electrochemistry
2.5 Gas Chromatography
2.6 HPLC-DAD Chromatography
2.7 HPLC-MS/MS Chromatography
3 Methods
3.1 Cyclic Voltammetry
3.2 The Generation of Oxidation Products Electrochemically
3.2.1 In Situ UV–Vis Spectroelectrochemistry
3.2.2 The Generation of Oxidation Products by Potential Controlled Coulometry (Exhaustive Electrolysis)
3.3 Identification of Products
3.3.1 GC-MS Chromatography
3.3.2 HPLC-DAD Chromatography
3.3.3 HPLC-MS/MS Chromatography
4 Notes
References
Chapter 21: A Differential Scanning Calorimetry (DSC) Experimental Protocol for Evaluating the Modified Thermotropic Behavior of Liposomes with Incorporated Guest Molecules
1 Introduction
2 Materials
2.1 Preparation of  Liposomes with Incorporated Guest Molecules
3 Methods
3.1 Differential Scanning Calorimetry (DSC) Measurements
4 Notes
References
Chapter 22: Applications of NMR in Drug:Cyclodextrin Complexes
1 Introduction
2 Materials
2.1 General Practical Aspects
2.2 Materials for Liquid NMR Experiments
2.3 Materials for ssNMR Experiments
3 Methods
3.1 Investigation of the Structural Properties of Irbesartan and Irbesartan–2-Hydroxypropyl-β-Cyclodextrin Complex in Micelles
3.2 The Application of ssNMR Spectroscopy to Study Drug:Membrane Interactions
4 Notes
4.1 Notes Related to Subheading 3.1
4.2 Notes Related to Subheading 3.2 (Figs. 4 and 5)
References
Chapter 23: Construction of Peptide-Drug Conjugates for Selective Targeting of Malignant Tumor Cells
1 Introduction
2 Materials and Equipment
2.1 Synthesis of PDCs
2.1.1 Synthesis of PDCs Using Succinic Acid as Linker (Ester and Amide Bonds)
2.1.2 Synthesis of PDCs Using Carbamate Bond in the Linker
2.1.3 Synthesis of PDCs Using Aminooxy-PEG4-CH2CO2H as Linker (Amide and Bond)
2.2 Purification and Characterization of Intermediates and Final Conjugates
2.2.1 Column Purification
2.2.2 RP-HPLC Purification
2.2.3 Characterization with Mass Spectrometry and/or NMR Spectroscopy
3 Methods
3.1 Synthesis of PDCs
3.1.1 Synthesis of PDCs Using Succinic Acid as Linker (Ester and Amide Bonds) (Scheme 1)
Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Hemisuccinate)
Preparation of the Final Conjugate (Gemcitabine-Hemisuccinate-D-Lys6- GnRH)
3.1.2 Synthesis of PDCs Using Carbamate Bond in the Linker (Scheme 2)
Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Bis (4-Nitrophenyl)Carbonate)
Preparation of the Final Conjugate (Gemcitabine-carbamate-D-Lys6-GnRH)
3.1.3 Synthesis of PDC Using Aminooxy-PEG4-CH2CO2H as Linker (Amide and Oxime Bond) (Scheme 3)
Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Boc-Aminooxy-PEG4-CH2CO2H)
Attachment of Aldehyde Group on D-Lys6-GnRH, as previously reported [14], and described below (D-Lys6-GnRH aldehyde)
Preparation of the Final Conjugate (Gemcitabine-PEG4CH2CO2H-D-Lys6-GnRH)
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2207

Thomas Mavromoustakos Andreas G. Tzakos Serdar Durdagi Editors

Supramolecules in Drug Discovery and Drug Delivery Methods and Protocols

Methods

in

M o l e c u l a r B i o lo g y

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

Supramolecules in Drug Discovery and Drug Delivery Methods and Protocols Edited by

Thomas Mavromoustakos Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece

Andreas G. Tzakos Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, Ioannina, Greece

Serdar Durdagi Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey

Editors Thomas Mavromoustakos Department of Chemistry National and Kapodistrian University of Athens Zografou, Greece Serdar Durdagi Computational Biology and Molecular Simulations Laboratory Department of Biophysics, School of Medicine Bahcesehir University Istanbul, Turkey

Andreas G. Tzakos Department of Chemistry Section of Organic Chemistry and Biochemistry University of Ioannina Ioannina, Greece

ISSN 1064-3745     ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-0919-4    ISBN 978-1-0716-0920-0 (eBook) https://doi.org/10.1007/978-1-0716-0920-0 © Springer Science+Business Media, LLC, part of Springer Nature 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Amidst the sadness of endless mediocrity, which suffocates us everywhere, I am comforted that somewhere, in an arch, some stubborn people are struggling to neutralize corruption.—“The Little Epsilon”, Odysseus Elytis

This drug delivery systems volume is dedicated to drugs that are unable to reach the desired pharmaceutical drug target as a result of several limitations, mainly due to the lack of selectivity, poor targeting capacity, or undesired pharmacokinetic profile. The utilization of proper “delivery vehicles” for such drugs can tailor their physical chemical properties and enhance their efficacy, retaining their structures intact. Techniques for preparing such systems are described in Chapters 1–3. Drug delivery platforms are outlined in Chapters 4–13. Such platforms include cyclodextrins, micelles, liposomes, polymeric, and nanotubes. These nanotechnology systems are studied using different biophysical techniques such as DSC, ITC, solid and liquid NMR spectroscopy, and electrochemistry (Chapters 14–23). All accumulated chapters aim toward one target: to provide readers with critical information to accomplish (a) the synthesis of nanosystems, thus supramolecular entities complexing with drugs; (b) their characterization; as well as (c) studying their physical-chemical interactions that govern their stability and properties. Experimental details and notes not found in the scientific literature to cover these three aspects of drug delivery systems are provided by experts in the field. This volume is thus very useful for all scientists who study or plan to study such systems. We wish to thank all authors whose effort provided manuscripts that overcome “the sadness of endless mediocrity” and build a foundation of solid knowledge on this growing field. Rational design is restrained not only to novel structures but also to old ones which decorate themselves with supramolecules that convert their disadvantages to advantages. We also wish to thank Prof. John Walker for his valuable impact and all personnel of Springer who “are struggling to neutralize corruption.” Zografou, Greece Ioannina, Greece  Istanbul, Turkey 

Thomas Mavromoustakos Andreas G. Tzakos Serdar Durdagi

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Application of Neutralization and Freeze-Drying Technique for the Preparation of the Beneficial in Drug Delivery 2-Hydroxypropyl-β-Cyclodextrin Complexes with Bioactive Molecules����������������  1 Eirini Christodoulou, Dimitrios Ntountaniotis, Georgios Leonis, Thomas Mavromoustakos, and Georgia Valsami 2 Functionalized Carbon Nanohorns as Drug Delivery Platforms ��������������������������� 13 Anastasios Stergiou and Nikos Tagmatarchis 3 Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole Organs������������������������������������������������������������������������� 25 Burak Derkus and Emel Emregul 4 Aggregate Determination by Permeation Technique��������������������������������������������� 35 Phennapha Saokham and Thorsteinn Loftsson 5 Study of Candesartan Cilexetil: 2-Hydroxypropyl-β-­Cyclodextrin Interactions: A Computational Approach Using Steered Molecular Dynamics Simulations������������������������������������������������������������������������� 45 Sofia Kiriakidi and Thomas Mavromoustakos 6 Drug Delivery: Hydrophobic Drug Encapsulation into Amphiphilic Block Copolymer Micelles��������������������������������������������������������������������������������������������� 71 Angeliki Chroni, Varvara Chrysostomou, Athanasios Skandalis, and Stergios Pispas 7 Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation������������������������������������������������������������������������������������������� 85 Maria Theodosiou, Theodora Koutsikou, and Eleni K. Efthimiadou 8 Drug Incorporation in the Drug Delivery System of Micelles������������������������������� 99 Evangelia Soumelidou, Simona Golič Grdadolnik, and Thomas Mavromoustakos 9 Molecular Dynamics Protocols for the Study of Cyclodextrin Drug Delivery Systems���������������������������������������������������������������������������������������� 109 Georgios Leonis, Dimitrios Ntountaniotis, Eirini Christodoulou, and Thomas Mavromoustakos 10 Drug Delivery Through Multifunctional Polypeptidic Hydrogels������������������������ 127 Hermis Iatrou, Panagiota G. Fragouli, Dimitra Stavroulaki, and Barbara Athanasiou 11 Polymersomes from Hybrids-Polypeptides for Drug Delivery Applications���������� 139 Hermis Iatrou, Panagiota G. Fragouli, Dimitris Skourtis, and Ioanna Stavropoulou

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12 Drug Delivery Systems Based on Modified Polysaccharides: Synthesis and Characterization���������������������������������������������������������������������������� 151 Aikaterini-Foteini Metaxa, Eleni Vrontaki, Eleni K. Efthimiadou, and Thomas Mavromoustakos 13 Differential Scanning Calorimetry (DSC) on Sartan/Cyclodextrin Delivery Formulations���������������������������������������������������������������������������������������� 163 Nikolaos Naziris, Maria Chountoulesi, Dimitrios Ntountaniotis, Thomas Mavromoustakos, and Costas Demetzos 14 Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability, and Therapeutic Efficacy Against Glioblastoma Multiforme ������������������ 175 Antonis D. Tsiailanis, Alexander Renziehausen, Serdar Karakurt, Tim Crook, Nelofer Syed, and Andreas G. Tzakos 15 Unveiling the Thermodynamic Aspects of Drug-­Cyclodextrin Interactions Through Isothermal Titration Calorimetry�������������������������������������� 187 Maria V. Chatziathanasiadou, Thomas Mavromoustakos, and Andreas G. Tzakos 16 Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG and Rosin Esters ������������������������������������������������������������������������������������������������ 199 Ali Ben Taleb, Selcan Karakuş, Ezgi Tan, Merve Ilgar, Özlem Kutlu, Devrim Gözüaçık, Hatice Mehtap Kutlu, and Ayben Kilislioğlu 17 Association of the Thermodynamics with the Functionality of Thermoresponsive Chimeric Nanosystems������������������������������������������������������ 221 Nikolaos Naziris, Athanasios Skandalis, Thomas Mavromoustakos, Stergios Pispas, and Costas Demetzos 18 2D DOSY NMR: A Valuable Tool to Confirm the Complexation in Drug Delivery Systems������������������������������������������������������������������������������������ 235 Christos M. Chatzigiannis, Sofia Kiriakidi, Andreas G. Tzakos, and Thomas Mavromoustakos 19 Drug-Encapsulated Cyclodextrin Nanosponges �������������������������������������������������� 247 Maria Tannous, Fabrizio Caldera, Gjylije Hoti, Umberto Dianzani, Roberta Cavalli, and Francesco Trotta 20 Electrochemistry Investigation of Drugs Encapsulated in Cyclodextrins�������������� 285 Romana Sokolová and Ilaria Degano 21 A Differential Scanning Calorimetry (DSC) Experimental Protocol for Evaluating the Modified Thermotropic Behavior of Liposomes with Incorporated Guest Molecules�������������������������������������������������������������������� 299 Maria Chountoulesi, Nikolaos Naziris, Thomas Mavromoustakos, and Costas Demetzos 22 Applications of NMR in Drug:Cyclodextrin Complexes�������������������������������������� 313 Dimitrios Ntountaniotis, Georgios Leonis, Eirini Christodoulou, and Thomas Mavromoustakos 23 Construction of Peptide-Drug Conjugates for Selective Targeting of Malignant Tumor Cells ���������������������������������������������������������������������������������� 327 Eirinaios I. Vrettos and Andreas G. Tzakos Index �������������������������������������������������������������������������������������������������������������������������339

Contributors Barbara Athanasiou  •  Department of Chemistry, Industrial Chemistry Laboratory, National and Kapodistrian University of Athens, Zografou, Greece Fabrizio Caldera  •  Dipartimento di Chimica, Università di Torino, Torino, Italy Roberta Cavalli  •  Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino, Italy Maria V. Chatziathanasiadou  •  Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Christos Μ. Chatzigiannis  •  Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Maria Chountoulesi  •  Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Zografou, Greece Eirini Christodoulou  •  Laboratory of Biopharmaceutics-Pharmacokinetics, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Zografou, Greece; Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece Angeliki Chroni  •  Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Varvara Chrysostomou  •  Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Tim Crook  •  Department of Oncology, St Luke’s Cancer Centre, Royal Surrey County Hospital, Guildford, UK Ilaria Degano  •  Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy Costas Demetzos  •  Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Zografou, Greece Burak Derkus  •  Chemistry Department, Faculty of Science, Ankara University, Ankara, Turkey Umberto Dianzani  •  Dipartimento di Scienze della Salute, Università del Piemonte Orientale, Torino, Italy Eleni K. Efthimiadou  •  Inorganic Chemistry Laboratory, Chemistry Department, National and Kapodistrian University of Athens, Zografou, Greece; NCSR “Demokritos”, Sol-Gel Laboratory, Institute of Nanoscience and Nanotechnology, Agia Paraskevi, Attikis, Greece Emel Emregul  •  Chemistry Department, Science Faculty, Ankara University, Ankara, Turkey Panagiota G. Fragouli  •  Laboratory of Dyeing, Finishing, Dyestuffs and Advanced Polymers, DIDPE, University of West Attica, Athens, Greece

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Contributors

Devrim Gözüaçık  •  Koç University Hospital, School of Medicine and Koç University Research Center for Translational Medicine (KUTTAM), Koç University, Zeytinburnu, Istanbul, Turkey Simona Golič Grdadolnik  •  Laboratory for Molecular Structural Dynamics, National Institute of Chemistry, Ljubljana, Slovenia Gjylije Hoti  •  Dipartimento di Chimica, Università di Torino, Torino, Italy Hermis Iatrou  •  Department of Chemistry, Industrial Chemistry Laboratory, National and Kapodistrian University of Athens, Zografou, Greece Merve Ilgar  •  Department of Chemistry, Faculty of Engineering, Istanbul University-­ Cerrahpasa, Istanbul, Turkey Serdar Karakurt  •  Department of Biochemistry, Faculty of Science, Selcuk University, Konya, Turkey Selcan Karakuş  •  Department of Bio and Nanotechnology, Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey; Department of Chemistry, Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey Ayben Kilislioğlu  •  Department of Bio and Nanotechnology, Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey; Department of Chemistry, Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey Sofia Kiriakidi  •  Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece Theodora Koutsikou  •  Inorganic Chemistry Laboratory, Chemistry Department, National and Kapodistrian University of Athens, Zografou, Greece; NCSR “Demokritos”, Sol-Gel Laboratory, Institute of Nanoscience and Nanotechnology, Agia Paraskevi, Attikis, Greece Hatice Mehtap Kutlu  •  Department of Biology, Faculty of Science, Eskişehir Technical University, Eskişehir, Turkey Özlem Kutlu  •  Nanotechnology Research and Application Center (SUNUM), Sabanci University, Istanbul, Turkey Georgios Leonis  •  Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece Thorsteinn Loftsson  •  Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland Thomas Mavromoustakos  •  Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece Aikaterini-Foteini Metaxa  •  Sol–Gel Laboratory, Institute of Nanoscience & Nanotechnology, Agia Paraskevi, Attikis, Greece Nikolaos Naziris  •  Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Zografou, Greece Dimitrios Ntountaniotis  •  Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece Stergios Pispas  •  Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Alexander Renziehausen  •  John Fulcher Neuro-Oncology Laboratory, Imperial College London, Hammersmith Hospital, London, UK Phennapha Saokham  •  College of Pharmacy, Rangsit University, Pathum Thani, Thailand

Contributors

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Athanasios Skandalis  •  Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Romana Sokolová  •  J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Prague, Czech Republic Evangelia Soumelidou  •  Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece Dimitra Stavroulaki  •  Department of Chemistry, Industrial Chemistry Laboratory, National and Kapodistrian University of Athens, Zografou, Greece Anastasios Stergiou  •  Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Nelofer Syed  •  John Fulcher Neuro-Oncology Laboratory, Imperial College London, Hammersmith Hospital, London, UK Nikos Tagmatarchis  •  Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece Ali Ben Taleb  •  Department of Bio and Nanotechnology, Faculty of Engineering, Istanbul University-Cerrahpasa, Istanbul, Turkey Ezgi Tan  •  Department of Chemistry, Faculty of Engineering, Istanbul University-­ Cerrahpasa, Avcilar, Istanbul, Turkey Maria Tannous  •  Dipartimento di Chimica, Università di Torino, Torino, Italy; Department of Chemistry, University of Balamand, Tripoli, Lebanon Maria Theodosiou  •  Inorganic Chemistry Laboratory, Chemistry Department, National and Kapodistrian University of Athens, Zografou, Greece; NCSR “Demokritos”, Sol-Gel Laboratory, Institute of Nanoscience and Nanotechnology, Agia Paraskevi, Attikis, Greece Francesco Trotta  •  Dipartimento di Chimica, Università di Torino, Torino, Italy Antonis D. Tsiailanis  •  Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Andreas G. Tzakos  •  Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Georgia Valsami  •  Laboratory of Biopharmaceutics-Pharmacokinetics, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Zografou, Greece Eirinaios I. Vrettos  •  Laboratory of Organic Chemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece Eleni Vrontaki  •  Division of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Zografou, Greece

Chapter 1 Application of Neutralization and Freeze-Drying Technique for the Preparation of the Beneficial in Drug Delivery 2-Hydroxypropyl-β-Cyclodextrin Complexes with Bioactive Molecules Eirini Christodoulou, Dimitrios Ntountaniotis, Georgios Leonis, Thomas Mavromoustakos, and Georgia Valsami Abstract Bioavailability of active substances is of great importance for the formulation of a drug product, as it actually reflects drug absorption and achievement of the optimum pharmacological effect. A great number of chemical compounds with excellent pharmacological properties possess low solubility and permeability values, ending in low bioavailability in the human body after administration (especially after per os administration). CDs are oligosaccharides that possess biological properties similar to their linear counterparts, but some of their physicochemical properties differ. They are enhancing bioavailability and solving problems of absorption for poorly soluble lipophilic drugs by forming water-soluble inclusion complexes. For this reason, they are widely used in drug delivery systems (Carrier et al. J Control Release 123:78–99, 2007; Kurkov and Loftsson, Int J Pharm 453:167–80, 2013). The main purpose of this chapter is to show a protocol for the preparation of drug:CDcomplex delivery systems. Key words Cyclodextrins, 2-Hydroxypropyl-β-CD, Methyl-β-CD, Bioavailability, Lyophilization, Inclusion complex

1  Introduction Cyclodextrins (CDs) are a-(1,4)-linked cyclic oligosaccharides containing glucopyranose units. The glucopyranose units possess chair conformation, due to which CDs are not cylindrical, and exhibit torus-like shape (truncated cone), in which the primary hydroxyl groups are present on narrow edge and secondary hydroxyl groups are on the wider edge of the cone in the outer surface. In the interior of the CD molecule, skeletal carbons with hydrogen atoms and oxygen bridges are present leading to hydrophilic outer surface and lipophilic central cavity of CD [1–3]. Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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One of the most remarkable features of CDs is their tendency to form solid inclusion complexes with a wide variety of solid, liquid and gaseous compounds through the process of molecular complexation. The formed complexes are called host–guest type of complexes. Such type of complexes involves a guest molecule (drug) which is properly fitted into the lipophilic cavity of a host (CD molecule) [4]. There is no breakage or formation of covalent bonds during the formation of an inclusion complex [5, 6]. When the CD is added to an aqueous solution, the polar water molecules fit into the lipophilic cavity of the CD, but these are immediately replaced by the more favored guest molecule, which is less polar than the water molecules [7]. The main driving force behind this complex formation is the replacement of highenthalpy water molecules with the guest moiety which adopts van der Waals interactions, hydrogen bondings and charge transfer interactions [6]. The ratio in which host and guest molecules bind together is generally 1:1 [5], even though in many cases complexes at different molecular ratios are formed. The equilibrium reaction of complexation is shown below:

Drug free + CDfree



Drug : CDcomplex



The binding strength of the formed complex depends on the ability of the guest molecule to bind suitably to the host in order to form a stable complex. The formation of an inclusion complex depends upon the following factors: (a) Size of the CD to the size of the guest molecule or some main functional groups within the guest moiety: If the guest is of inappropriate size, it will not fit properly into the host cavity. (b) Thermodynamic interactions between the various components of the system (CD, guest, solvent; [4]). (c) Structure of added substituent to the CD derivative. (d) Location of substituent within the CD molecule. (e) Number of substituents per CD molecule. CDs play an important role in the chemical stability of drugs. The assumptions made after studying the interaction between CDs and labile compounds are as follows [8]: (a) CDs retard or accelerate degradation (b) CDs have no effect on reactivity The most commonly used methods for the preparation of drug-­ cyclodextrin inclusion complexes include the following [4, 9–13]: (a) Coprecipitation It is the most commonly used method of complex preparation in laboratory scale. It is simple and easy to apply, with no exceptional laboratory equipment needed. The necessary

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amount of cyclodextrin is dissolved in water and the guest active ingredient, dissolved in an organic solvent, is gradually added in the solution under continuous stirring. The solution is cooled and complex precipitate as a final product is formed, collected washed with another solvent or water and dried. (b) Slurry complexation In order to prepare the complex, it is not necessary for the cyclodextrin to be completely dissolved. Cyclodextrin can be added to water at a concentration of 50–60% w/v in solid form and then stirred. The aqueous phase will be saturated with cyclodextrin in solution. Guest molecules will complex with the cyclodextrin in solution and, as the cyclodextrin complex saturates the water phase, the complex will be crystallized or precipitate out of the aqueous phase. The cyclodextrin crystals will be dissolved and continue to saturate the aqueous phase to form the complex and precipitate or crystallize out of the aqueous phase. The complex can be collected in the same manner as with the coprecipitation method. (c) Kneading method: paste complexation A small amount of water is added to the cyclodextrin along with the active ingredient in order to form a paste, using a mortar and pestle or (on a large scale) using a kneader. The time required is dependent on the guest molecule. The resulting complex can be dried directly or washed with a small amount of water to remove the non-complexed particles that are adsorbed on the cyclodextrin surface and collected by filtration or centrifugation to be dried under vacuum. Pastes will sometimes get dry forming a hard mass instead of a fine powder. This is dependent on the guest molecule and the amount of water used in the paste. (d) Damp mixing and heating in a sealed container The guest and cyclodextrin molecules are thoroughly mixed and after adsorbing a definite amount of water vapor are placed in a sealed container. The amount of water used in the method can range between the amount of water of hydration in the cyclodextrin and added guest to up to 20–25% water on a dry basis. The sealed container and its contents are heated to about 100  °C and then the contents are removed and dried. The amount of water added, the degree of mixing and the heating time have to be optimized for each guest. (e) Extrusion Extrusion is a variation of the heating and mixing method and is a continuous system. Cyclodextrin, guest molecule and water can be premixed or mixed as added to the extruder. Degree of mixing and amount of heating and time can be controlled in the barrel of the extruder. Depending upon the

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amount of water, the extruded complex may dry as it cools or the complex may be placed in an oven to dry. (f) Dry mixing Some guest molecules can be complexed by simply adding them to the cyclodextrin and mixing. This works best with oils or liquid guest molecules. The amount of mixing time required is variable and depends on the guest. Generally, this method is performed at ambient temperature and is an alternative to the paste method. In the following section a detailed guide is given for the preparation of solid-state inclusion complexes of a number of poorly water-­soluble lipophilic drug molecules with 2-hydroxypropyl-β-­ cyclodextrin (2-HP-β-CD). The drug molecules of interest are irbesartan, losartan, candesartan and candesartan cilexetil, as well as five bioactive molecules of plant origin, namely caffeic acid, rosmarinic acid, silibinin, curcumin, and quercetin. Irbesartan (IRB), losartan (LOS), and candesartan (CAN) or candesartan cilexetil as prodrug (CC) belong to the family of angiotensin II receptor blockers and are used to treat high blood pressure, while beyond their ability to lower blood pressure, they also confer cardiovascular and renal protective effects [14–16]. IRB and LOS belong to class II of the Biopharmaceutics Classification System, BCS [17, 18], meaning that their poor aqueous solubility is the rate-limiting step for drug absorption. CAN is classified as a BCS Class IV drug, meaning that it shows both low solubility and low permeability in vitro. CC is the prodrug form of the active substance CAN and is classified as a BCS Class II drug (low solubility) [16]. As a result, enhancement of the aqueous solubility of all three drug molecules can lead to improved oral bioavailability. Caffeic acid (CA) is an antioxidant molecule both in vitro and in vivo [19]. Caffeic acid also shows immunomodulatory and anti-­ inflammatory activities [20, 21]. Chemically, rosmarinic acid (RA) is a caffeic acid ester of 3-(3,4-dihydroxyphenyl)-lactic acid and can act as a prodrug for caffeic acid. CA is poorly soluble only in hot water whereas RA is insoluble in water and soluble only in organic solvents, not biologically compatible with the human organism. As a result, the inclusion of both molecules in 2-HP-β-CD is extremely useful in order to enhance oral bioavailability. Silibinin (SLB) is the main active component of the flavonoid mixture silymarin extracted from the plant Silybum marianum (milk thistle) and is mainly known for its hepatoprotective properties. More specifically, there is some clinical evidence for the use of SLB as a supportive element in alcoholic and child grade “A” liver cirrhosis [22]. SLB is one of the most characteristic examples of poorly water-soluble biomolecules, as its bioavailability is extremely low when administered orally.

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Curcumin (CRM) is a bright yellow chemical compound produced by Curcuma longa plants. It is the principal curcuminoid of turmeric (Curcuma longa), a member of the ginger family, Zingiberaceae. CRM has a surprisingly wide range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity, but its low aqueous solubility, physical instability and low bioavailability make it difficult to study [23]. Quercetin, (QUE) a plant flavonol from the flavonoid group of polyphenols, is found in many fruits, vegetables, leaves and grains; red onions and kale are common foods containing appreciable content of QUE [24]. It has a bitter flavor and is used as an ingredient in dietary supplements, beverages and foods. Due to its antioxidant properties, QUE has been considered as a potent molecule against cancer, while recently it is considered protective agent against early inflammatory stages of Alzheimer’s disease. Its low solubility, and thus limited bioavailability, is the primary reason for formulating the molecule into a 2-HP-β-CD inclusion complex.

2  Materials 1. Use purified water and analytical grade guest molecules. 2. Store all guest molecules at room temperature (unless indicated otherwise). 3. Prepare ammonium hydroxide solution by diluting 20  mL of ammonia solution (25% v/v concentrated solution) to a final volume of 100 mL with purified water and mix gently. The concentration of the obtained NH4OH solution is 5% v/v. This solution is used for all preparations described below. 4. Use 2-HP-β-CD as the host molecule and drug carrier for solubility amelioration through a neutralization and freeze-drying procedure in the following preparations.

3  Methods 3.1  Steps to Follow for the Preparation of Solid-State Irbesartan–2-HP-β-CD Inclusion Complex [25, 26]

1. Accurately weigh 60  mg of IRB and 408  mg of 2-HP-β-CD and transfer them in a 100 mL beaker. 2. Suspend with 50 mL of water. 3. Add small amounts of ammonium hydroxide solution 5% v/v under continuous stirring and pH monitoring until complete dissolution. The optimum pH value is approximately 10.5 for the complexation to take place (see Notes 1–3). 4. When complete dissolution is achieved (visual observation to obtain clear colorless solution) fix the volume at 60  mL with purified water.

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5. The resulting solution at a molar ratio of 1:2 (IRB:2-HP-β-CD) (see Notes 4–6) is thereafter frozen at −80 °C and freeze-dried [15] to remove the water and obtain the white solid irbesartan– 2-HP-β-CD lyophilized product (see Note 7). 3.2  Steps to Follow for the Preparation of Solid-State LOS–2-­ HP-­β-CD Inclusion Complex

1. Accurately weigh 3445.6 mg of 2-HP-β-CD. 2. Transfer in a glass vessel with 300 mL of purified water and set under magnetic stirring. 3. When the cyclodextrin is completely dissolved, add 500 mg of LOS potassium (accurately weighed) in the vessel and continue the magnetic stirring (LOS:CD molar ratio 1:2, see Notes 4–6). 4. Adjust the pH at approximately 10.5 with ammonium hydroxide solution 5% v/v to facilitate the reaction of inclusion complex formation (see Notes 1–3). 5. When the mixture turns to a clear and colorless solution, fix the final volume at 500 mL with purified water. 6. Immediately freeze the solution at −80 °C for lyophilization to follow and obtain the white solid LOS-2-HP-β-CD inclusion complex (see Note 7).

3.3  Steps to Follow for the Preparation of Solid-State CAN–2-­ HP-­β-CD and CC–2-­ HP-­β-CD Inclusion Complexes [27]

1. Accurately weigh 30  mg of CAN or CC and 198.85  mg or 143.37 mg of 2-HP-β-CD, respectively. 2. Mix the weighed amounts of CAN (or CC) and 2-HP-β-CD in a glass beaker along with 20 mL of purified water. 3. Add small amounts of the ammonium hydroxide solution 5% v/v under continuous magnetic stirring. 4. Adjust pH at approximately 10.5 (see Notes 1–3). 5. Adjust the final volume at 30 mL with purified water and mix thoroughly. 6. Freeze the resulting solution immediately after preparation at −80 °C (see Note 7). 7. Freeze-dry the iced preparation in a lyophilizer (e.g., Kryodos-­50 model Telstar) to obtain a white amorphous solid.

3.4  Steps to Follow for the Preparation of Solid-State RA–2-­ HP-­β-CD and CA–2-­ HP-­β-CD Inclusion Complexes

1. Weigh accurately 60 mg of CA and 0.964 g of 2-HP-β-CD.

3.4.1  CA–2-HP-b-CD Inclusion Complex

4. Continue magnetic stirring under pH monitoring until a clear solution is obtained.

2. Transfer the weighed amounts of CA and 2-HP-β-CD in a 100 mL beaker and suspend with 50 mL of water. 3. Add small amounts of ammonium hydroxide 5% v/v aqueous solution and adjust the pH at approximately 9–10 under magnetic stirring (see Notes 1–3).

5. Fix the final volume at 60 mL with purified water.

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6. The resulting solution has a 1:2 molar ratio of CA:2-HP-β-CD (see Notes 4–6). 7. Immediately transfer this clear colorless solution at a freezer at −80 °C (see Note 7). 8. Freeze-dry the resulting iced product to obtain a white fluffy solid-state CA–2-HP-β-CD lyophilized product. 3.4.2  RA–2-HP-b-CD Inclusion Complex

1. Accurately weigh 120  mg of RA and 964  mg of 2-HP-β-CD and transfer in a 200 mL beaker. 2. Suspend with 100 mL of purified water. 3. Add small amounts of 5% v/v ammonium hydroxide solution under magnetic stirring and pH monitoring (see Notes 1–3). 4. Keep pH at approximately 9-10. 5. After complete dissolution of the solids, fix the final volume at 120 mL with purified water. 6. Immediately transfer the resulting clear colorless solution (at RA:2-HP-β-CD molar ratio of 1:2, see Notes 4–6) at a freezer at −80 °C (see Note 7). 7. When it turns iced, freeze-dry to a lyophilizer to obtain the final solid-state RA–2-HP-β-CD lyophilized product.

3.5  Steps to Follow for the Preparation of Solid State SLB–2-­ HP-­β-CD Inclusion Complex [28]

1. Accurately weigh 300 mg of SLB and 1860 mg of 2-HP-β-CD. 2. Transfer the weighed amounts of SLB and 2-HP-β-CD in a glass vessel and suspend with 200 mL of purified water under magnetic stirring. 3. Add small amounts of 5% v/v ammonium hydroxide solution under continuous stirring and pH monitoring (pH should be adjusted at approximately 10–10.5, see Notes 1–3). 4. After complete dissolution a slight pink clear solution should be obtained. 5. Fix the final volume of the solution with purified water at 300 mL. 6. Freeze the resulting clear, pink solution (at a 1:2 SLB:2-HP-­ β-CD molar ratio, see Notes 4–6) at −80 °C (see Note 7). 7. Freeze-dry the iced product to obtain the solid-state inclusion complex of SLB–2-HP-β-CD.

3.6  Steps to Follow for the Preparation of Solid-State CRM–2-­ HP-­β-CD Inclusion Complex

1. 150 mg of CRM and 1197 mg 2-HP-β-CD, accurately weighed, should be mixed in a glass vessel containing 120 mL of purified water (see Notes 4–6). 2. Then, add small amounts of 5% v/v ammonium hydroxide solution to adjust the pH at approximately 10–10.5, under continuous stirring and pH monitoring (see Notes 1–3).

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3. After complete dissolution a yellow solution is obtained. 4. Fix the final volume of the obtained solution at 150 mL with purified water. 5. Immediately freeze this solution at −80 °C (see Note 7). 6. Freeze-dry the iced product to obtain a yellow solid state of the CRM–2-HP-β-CD inclusion complex. 3.7  Steps to Follow for the Preparation of Solid-State QUE–2-­ HP-­β-CD and QUE– Me-β-CD Inclusion Complexes [29–31] 3.7.1  QUE–2-HP-β-CD Inclusion Complex [29, 30]

1. For the preparation of QUE–2-HP-β-CD aqueous solutions for freeze-drying in a molar ratio of 1:2, mix 30 mg of QUE and 306 mg of 2-HP-β-CD, accurately weighed in a 50 mL beaker (see Notes 4–6). 2. Suspend the mixture of solids with 20 mL of purified water. 3. The pH value should be adjusted at approximately 9–9.5 with the help of ammonium hydroxide solution (5% v/v) under continuous magnetic stirring (see Notes 1–3). 4. After complete dissolution fix the volume of the obtained solution (1:2 QUE:CD molar ratio) to 60 mL. 5. Freeze the obtained orange-colored solution at −80  °C (see Note 7). 6. Freeze-dry the final iced product to remove water and obtain the final solid-state yellowish QUE–2-HP-β-CD inclusion complex.

3.7.2  QUE–Me-β-CD Inclusion Complex [31]

1. For the preparation of QUE–Me-β-CD aqueous solution for freeze-drying in a molar ratio of 1:1, mix 2200 mg of Me-β-CD and 500 mg of QUE, accurately weighed, into a glass vessel (see Notes 4–6). 2. Suspend the mixture of solids with 400 mL of purified water. 3. The pH value should be adjusted at approximately 9–9.5 with the help of ammonium hydroxide solution (5% v/v) (see Notes 1–3). 4. After complete dissolution, fix the volume of the obtained solution (1:1 QUE:CD molar ratio) to 500 mL. 5. Freeze the obtained orange-colored solution at −80  °C (see Note 7). 6. Freeze-dry the final iced product to remove water and obtain the yellow solid-state QUE–Me-β-CD inclusion complex.

4  Notes 1. The neutralization method [13] is applied prior to lyophilization (freeze-drying) to facilitate drug dissolution. 2. pH monitoring is important during the preparation of the drug-CD aqueous solution, to maintain up to 10.5 and avoid CD decomposition.

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3. Note that all drug (bioactive) molecules described in this chapter are acidic in nature and therefore neutralization takes place at an alkaline pH between 9 and 10.5. 4. For the preparation of inclusion complexes in solid state using the neutralization and freeze-drying technique, aqueous solutions of drug and CD should be prepared at the optimum molar ratio for freeze-drying to follow. 5. In the described examples the optimum drug:CD molar ratio is 1:2 for the preparation of the aqueous solutions. 6. Aqueous solutions of drug and CD at a different drug:CD molar ratio than 1:2 can also be used if needed. For example:

(a) Prepare SLB–2-HP-β-CD aqueous solutions for freeze-­ drying in molar ratios of 1:1 and 1:4 following the steps described in Subheading 3.5 but weigh accurately 930 mg and 3720 mg of 2-HP-β-CD, respectively, and mix with 300 mg of SLB at a final volume of 300 mL [28].



(b) For the preparation of CAN− or CC−2-HP-β-CD aqueous solutions for freeze-drying in a molar ratio of 1:1 follow the procedure described in Subheading 3.3 but weigh accurately 99.28 mg or 71.69 mg of 2-HP-β-CD and mix with 30  mg of CAN or CC, respectively (accurately weighed), at a final volume of 30 mL.

7. Freeze the prepare aqueous solution immediately at ≤−70 °C, to avoid layering during freezing.

5  Conclusions The neutralization and freeze-drying (or lyophilization) technique can be successfully applied for the preparation of drug:CD inclusion complexes [25–31]. All the abovementioned example complexes prepared were confirmed to be adequately complexed with CD through NMR and thermal analysis procedures in order to evaluate the strength of inclusion and possible molecular structure, while in all cases in vitro bioactivity was confirmed [25–31]. In parallel, in vitro solubility and dissolution experiments were conducted to compare the complex developed against either the pure drug or the reference marketed products and evaluate their possible bioavailability when orally administered [32–34].

Acknowledgments This work was financially supported by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, K.Ε. 14995, Preparation and Study of Novel Forms of

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Administration of Pharmaceutical Molecules with the aim to improve their pharmacological properties, PI Prof Th. Mavromoustakos). References 1. Carrier RL, Miller AL, Ahmed I (2007) The utility of cyclodextrins for enhancing oral bioavailability. J Control Release 123:78–99 2. Kurkov S, Loftsson T (2013) Cyclodextrins. Int J Pharm 453(1):167–180 3. Cal K, Centkowska K (2008) Use of cyclodextrins in topical formulations: practical aspects. Eur J Pharm Biopharm 68(3):467–478 4. Del Valle EMM (2004) Cyclodextrins and their uses: a review. Process Biochem 39:1033–1046 5. Loftsson T, Jarho P, Másson M, Järvinen T (2005) Cyclodextrins in drug delivery. Expert Opin Drug Deliv 2(2):335–351 6. Loftsson T, Hreinsdóttir D, Másson M (2005) Evaluation of cyclodextrin solubilization of drugs. Int J Pharm 302(1–2):18–28 7. Iványi R, Jicsinszky L, Juvancz Z, Roos N, Otta K, Szejtli J (2004) Influence of (hydroxy)alkylamino substituents on enantioseparation ability of single-isomer amino-beta-­ cyclodextrin derivatives in chiral capillary electrophoresis. Electrophoresis 25(16):2675–2686 8. Loftsson T, Brewster ME (1996) Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J Pharm Sci 85(10):1017–1025 9. Saenger W (1980) Cyclodextrin inclusion compounds in research and industry. Angew Chem Int Ed Engl 19:344–362 10. Hirayama F, Uekama K (1987) Methods of investigating and preparing inclusion compounds. In: Duchene D (ed) Cyclodextrins and their industrial uses. Les editions de Sante, Paris, pp 131–172 11. Duchene D, Wouessidjewe D (1990) Pharmaceutical uses of cyclodextrins and derivatives. Drug Dev Ind Pharm 16:2487–2499 12. Jacob S, Nair A (2018) Cyclodextrin complexes: perspective from drug delivery and formulation. Drug Dev Res 79:201–217 13. Figueiras A, Carvalho RA, Ribeiro L, Torres-­ Labandeira JJ, Veiga FJ (2007) Solid-state characterization and dissolution profiles of the inclusion complexes of omeprazole with native and chemically modified beta-cyclodextrin. Eur J Pharm Biopharm 67:531–539 14. Irbesartan Monograph for Professionals. Drugs.com. American Society of Health-­ System Pharmacists. Retrieved 3 March 2019

15. Losartan Potassium. The American Society of Health-System Pharmacists. Retrieved 8 December 2017 16. Candesartan label (PDF). FDA. February 2016 17. Amidon GL, Lennernäs H, Shah VP, Crison JR (1995) A theoretical basis for a biopharmaceutic drug classification: the correlation of in  vitro drug product dissolution and in  vivo bioavailability. Pharm Res 12(3):413–420 18. Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-­ Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System Guidance for Industry, U.S.  Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER), December 2017 19. Olthof MR, Hollman PC, Katan MB (2001) Chlorogenic acid and caffeic acid are absorbed in humans. J Nutr 131(1):66–71 20. Hirose M, Takesada Y, Tanaka H, Tamano S, Kato T, Shirai T (1998) Carcinogenicity of antioxidants BHA, caffeic acid, sesamol, 4-methoxyphenol and catechol at low doses, either alone or in combination, and m ­ odulation of their effects in a rat medium-term multi-­ organ carcinogenesis model. Carcinogenesis 19(1):207–212 21. Basu Mallik S, Mudgal J, Nampoothiri M, Hall S, Dukie SA, Grant G, Rao CM, Arora D (2016) Caffeic acid attenuates lipopolysaccharide-­induced sickness behaviour and neuroinflammation in mice. Neurosci Lett 632:218–223 22. Saller R, Brignoli R, Melzer J, Meier R (2008) An updated systematic review with meta-­ analysis for the clinical evidence of silymarin. Forsch Komplementmed 15(1):9–20 23. Hatcher H, Planalp R, Cho J, Torti FM, Tortic SV (1631–1652) Curcumin: from ancient medicine to current clinical trials. Cell Mol Life Sci 65(11):2008 24. Flavonoids (Review). Micronutrient Information Center, Linus Pauling Institute, Oregon State University, Corvallis, OR. November 2015. Retrieved 1 April 2018 25. Leonis G, Ntountaniotis D, Christodoulou E, Mavromoustakos T Effects of the complex HP-β-CD with irbesartan on lipid bilayers containing cholesterol. Pharm J. https://www.

Neutralization and Freeze-Drying Technique to Prepare Drug: CD Complexes hsmc.gr/wp-content/uploads/2019/02/ issue_4_2018.pdf 26. Liossi A, Ntountaniotis D, Kellici T, Chatziathanasiadou M, Megariotis G, Mania M, Becker-Baldus J, Kriechbaum M, Krajnc A, Christodoulou E, Glaubitz C, Rappolt M, Amenitsch H, Mali G, Theodorou D, Valsami G, Pitsikalis M, Iatrou H, Tzakos A, Mavromoustakos T (2017) Exploring the interactions of irbesartan and irbesartan–2-­ hydroxypropyl-­β-cyclodextrin complex with model membranes. Biochim Biophys Acta 1859:1089–1098 27. Ntountaniotis D, Andreadelis I, Kellici TF, Karageorgos V, Leonis G, Christodoulou E, Kiriakidi S, Becker-Baldus J, Stylos EK, Chatziathanasiadou MV, Chatzigiannis CM, Damalas DE, Aksoydan B, Javornik U, Valsami G, Glaubitz C, Durdagi S, Thomaidis NS, Kolocouris A, Plavec J, Tzakos AG, Liapakis G, Mavromoustakos T (2019) Host-guest interactions between candesartan and its prodrug candesartan cilexetil in complex with 2-hydroxypropyl-β-cyclodextrin: on the biological potency for Angiotensin II. Mol Pharm 16(3):1255–1271 28. Kellici TF, Ntountaniotis D, Leonis G, Chatziathanasiadou M, Chatzikonstantinou AV, Becker-Baldus J, Glaubitz C, Tzakos AG, Viras K, Chatzigeorgiou P, Tzimas S, Kefala E, Valsami G, Archontaki H, Papadopoulos MG, Mavromoustakos T (2015) Investigation of the interactions of silibinin with 2-­hydroxypropylβ-cyclodextrin through biophysical techniques and computational methods. Mol Pharm 12(3):954–965 29. Kellici TF, Chatziathanasiadou MV, Diamantis D, Chatzikonstantinou AV, Andreadelis I, Christodoulou E, Valsami G, Mavromoustakos T, Tzakos AG (2016) Mapping the interactions and bioactivity of quercetin–

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(2-hydroxypropyl)-cyclodextrin complex. Int J Pharm 511(1):303–311 30. Diamantis DA, Ramesova S, Chatzigiannis C, Degano I, Gerogianni P, Karadima K, Perikleous S, Rekkas D, Gerothanassis I, Galaris D, Mavromoustakos T, Valsami G, Sokolova R, Tzakos A (2018) Exploring the oxidation and iron binding profile of a cyclodextrin encapsulated quercetin complex unveiled a controlled complex dissociation through a chemical stimulus. BBA-General Subjects 1862:1913–1924 31. Papakyriakopoulou P, Manta K, Spaneas D, Christodoulou E, Colombo G, Rekkas D, Dallas P, Valsami G (2019) Ex vivo transport study on quercetin-cyclodextrin inclusion complexes as candidates for nose-to-brain delivery. AAPS PharmSci 360:3–6 32. Christodoulou E, Kechagia IA, Kostomitsopoulos N, Balafas E, Archontaki E, Dokoumetzidis A, Valsami G (2015) Pharmacokinetics of silibinin after peros and intravenous administration to mice, as a HP-β-CD lyophilized product. Int J Pharm 493:366–373 33. Tsaroucha A, Valsami G, Kostomitsopoulos N, Lambropoulou M, Anagnostopoulos C, Christodoulou E, Falidas E, Betsou A, Kakazanis Z, Pitiakoudis M, Simopoulos C (2018) Silibinin effect on Fas/FasL, HMGB1, and CD45 expressions in a rat model subjected to liver ischemia-reperfusion injury. J Invest Surg 31:491–502 34. Kyriakopoulos G, Tsaroucha A, Valsami G, Lambropoulou M, Kostomitsopoulos N, Christodoulou E, Kakazanis Z, Anagnostopoulos C, Tsalikidis C, Simopoulos C (2018) Histological assessment and tissue expression of TNF-α and M30  in the kidney of an experimental rat model after hepatic ischemia/reperfusion and silibinin administration. J Investig Surg 31:201–209

Chapter 2 Functionalized Carbon Nanohorns as Drug Delivery Platforms Anastasios Stergiou and Nikos Tagmatarchis Abstract Carbon nanohorns (CNHs) resembling a single-layered graphene sheet wrapped in a conical shape can be chemically modified in order to immobilize, carry, and release biologically active molecules. Here, we describe the major routes for the preparation of CNH-based drug delivery platforms, via covalent coupling and encapsulation, proficient to facilitate the design of sophisticated drug nanocarriers. Key words Carbon nanohorns, Functionalization, Encapsulation, Hybrids, Biomaterials, Drug delivery

1  Introduction Drug delivery system technology is considered as a major area of research and innovation on disease treatment. As a result, a plethora of delivery platforms have been developed and tested for their efficacy, with some traditional formulations long being on the market, others only recently approved, while numerous being under screening and investigation. In addition, the flexibility in biomaterial synthesis and the extensive knowledge on self-assembly mechanisms compel researchers to combine simple structures and/or nanomaterials to generate more sophisticated drug nanocarriers. Carbon nanohorns (CNHs), within the larger carbon allotrope family and composed of sp2 hybridized carbon atoms, possess a conical structure 2–5  nm in diameter and 40–50  nm in length. Notably, CNHs aggregate in larger spherical superstructures of around 100 nm [1] and can be economically produced in industrial quantities without requiring the involvement of any toxic metal catalyst. The latter is a significant advantage and key difference of CNHs as compared to carbon nanotubes [2]. In addition, the dissimilarity in shape and size of CNHs as compared to the micrometer-long single-walled carbon nanotubes affects their properties and applications. However, CNHs lack solubility in Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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aqueous media, which is critical for manipulation and processing for the envisaged applications. To overcome that handicap, chemical functionalization of CNHs is imperative and to this end a plethora of strategies have been developed broadly based on the covalent addition of organic species onto the outer skeleton of CNHs [3– 17], as well as on the non-covalent/supramolecular interactions through electrostatic association [18–20] with charged species and/or π − π stacking forces [21–23] with planar aromatic molecules. In addition, oxidation of the conical tips of CNHs, introducing carboxylic units suitable for further chemical alteration, is another widely employed methodology for effectively modifying CNHs [18, 24–33]. A typical process for the oxidation of CNHs involves heat treatment in the presence of oxygen, while the light-­ assisted oxidation of CNHs in the presence of hydrogen peroxide is comparatively a much milder technique [33, 34]. Hence without surprise, a variety of biomolecules were attached through carbodiimide-­mediated condensation reactions on oxidized CNHs. For example, bovine serum albumin (BSA) was covalently incorporated at the carboxylic units of light-assisted oxidized CNHs. The BSA-CNH material formed homogeneous dispersions into phosphate buffer saline, allowing incorporation inside mammalian cells through endocytosis [33]. A significant benefit of CNHs as compared to carbon nanotubes is that they do not have the high aspect ratio issues related to toxicity observed in longer nanotubes. Furthermore, the irregular structure of CNHs permits the opening of nanowindows at the tips and sidewalls of CNHs, from which incorporation and release of bioactive molecules are feasible and easier. Hence, CNHs as a highly pure and easily available material have been successfully tested as drug carriers [35–38]. Summarizing, the capability for large-scale production of CNHs accompanied by their handy chemical manipulation, as illustrated in Fig.  1, makes this unique carbon nanostructure a promising biocompatible drug delivery platform.

2  Materials 2.1  Preparation of CNHs

1. A chamber equipped with a high-power CO2 laser source (wavelength 10.6 μm, maximum power 5 kW, variable pulse duration from 10  ms to continuous illumination, and beam

Fig. 1 (continued)  at the edge of the conical tips of CNHs, which can then be converted to the corresponding acyl chlorides for subsequent coupling with hydroxyl- or amine-terminated drugs/biomolecules. Right: Harsh oxidative treatment of CNHs, followed by reduction of the introduced oxygen functionalities, generates pores at the edge of the cone and onto the sidewalls of CNHs, thereby enabling the release of the drugs/biomolecules encapsulated by nanoprecipitation into CNHs

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Fig. 1 Illustration of the three general methods for the preparation of CNH-based drug/biomolecule delivery systems. Left: Pristine CNHs are modified with a BOC-aniline derivative via diazonium chemistry, followed by cleavage of the BOC-protecting group and covalent coupling of the free amine terminus with carboxylic acid-­ terminated drugs/biomolecules. Middle: Sequential oxidation of CNHs introduces carboxylic acid functionalities

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diameter of 10  mm and a plastic-resin reaction chamber 30 × 30 × 25 cm3) equipped with a vacuum pumping system, with inlet and outlet gas valves, gas pressure and flow controllers, and a ZnSe lens system for adjusting the beam intensity. 2. A 50 mm long graphite target rod with a 30 mm diameter and purity 99.99%, with no catalytic metal inclusions. 3. Cylindrical filters, 50 mm in diameter and 150 mm long. 2.2  Drying Solvents

1. Tetrahydrofuran (THF). 2. Dichloromethane (DCM). 3. Nitrogen gas. 4. Metal sodium. 5. Benzophenone. 6. Calcium chloride.

2.3  Synthesis of BOC-Aniline Derivative

1. p-Nitrobenzoyl chloride. 2. tert-Butyl 2-(2-(2 aminoethoxy)ethoxy)-ethylcarbamate. 3. NaOH pellets. 4. Anhydrous Na2SO4. 5. Pt/C 10 wt%. 6. H2 gas. 7. Celite®.

2.4  Synthesis of BOC-CNHs and the Corresponding NH2-CNHs

1. CNHs powder. 2. BOC-aniline derivative. 3. Isoamyl nitrite. 4. 1,2-Dichlorobenzene. 5. N2 gas. 6. Acetonitrile. 7. Dimethyl formamide. 8. Dichloromethane. 9. HCl gas. 10. Polytetrafluoroethylene (PTFE) membrane filters with pore size of 0.2 μm. 11. Glass apparatus for vacuum filtration. 12. Vacuum pump. 13. A microwave reactor up to 300 W power (optional—see Note 3).

2.5  Oxidation of CNHs

1. CNH powder. 2. A high-temperature (600  °C) pressurized furnace equipped with gas inlet.

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3. O2 gas. 4. 30% w/w H2O2. 5. Halogen lamp 500 W. 6. Deionized water. 7. Methanol. 8. PTFE membrane filters with pore size of 0.2 μm. 2.6  Conjugation of Drugs/Biomolecules

1. Oxidized CNHs. 2. Thionyl chloride. 3. Dry tetrahydrofuran. 4. Amine (or hydroxyl)-terminated drug/biomolecule. 5. PTFE membrane filters with pore size of 0.2 μm.

2.7  Nanomesh CNHs

1. CNH powder. 2. A high-temperature (1200 °C) furnace with gas inlet. 3. O2 gas. 4. H2 gas.

2.8  Encapsulation of Drugs/Biomolecules

1. Nanomesh CNHs. 2. Drugs/biomolecules. 3. Dimethyl formamide. 4. PTFE membrane filters with pore size of 0.2 μm.

3  Methods All experiments are carried out under inert atmosphere conditions unless otherwise stated. Solvents, chemicals and CNHs are used as received unless otherwise stated. 3.1  Preparation of CNHs

The CNHs are produced in a plastic chamber by CO2 laser ablation of graphite in an Ar (760 Torr) atmosphere at room temperature. The inside of the chamber is evacuated, Ar gas is introduced and flowed through it, while the gas pressure is kept constant, typically at 760 Torr. The gas flow rate is 40 L/min, which is required to move the produced CNHs immediately from the reaction chamber to the collection filter. The graphite target rod is located in the middle of the reaction chamber. The graphite rod is rotated around its axis at 6 rpm, and advanced along its axis so that a fresh target is continually exposed to the laser beam. The rod is illuminated by the laser beam vertically at its cylinder-wall surface. All laser ­ablation experiments are conducted at room temperature, although the actual target temperature rises during the ablation. Carbonaceous products are collected by cylindrical filters located in a pumping

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line between the reaction chamber and the vacuum pump. Each filter can collect up to 500 mg of CNHs produced before filtering efficiency is deteriorated. 3.2  Procedures for Drying Solvents

1. Tetrahydrofuran (THF): THF (100 mL) is placed in a round-­ bottom flask, followed by the addition of metal sodium flakes (3–4 flakes of 2 × 2 cm2) soaked in oil. Caution: Handle metal sodium lumps and flakes with special care. Do not wash the covering oil and contact of metal sodium with atmosphere moisture must be avoided. Then add 2–3 flakes of benzophenone (1  ×  1  cm2), acting as indicator. Equip the flask with a condenser and a collection flask. Boil the mixture gently under nitrogen atmosphere until the solution turns deep blue in color. Always check that there is sufficient amount of metal sodium in the solution. If the mixture has a greenish color, then add 1–2 more flakes of benzophenone. Then collect the dry THF solvent and transfer to the reaction flask under nitrogen. 2. Dichloromethane (DCM): DCM is placed in a round-bottom flask, followed by the addition of 10–20% powder w/w of anhydrous calcium chloride. Let the mixture stand overnight. Equip the system with a condenser and a collection flask. Boil the mixture gently, under nitrogen atmosphere, for 2 h. Then collect dry DCM and transfer it to the reaction flask under nitrogen.

3.3  Covalent Incorporation of Amine Moieties on CNHs

1. Add pristine CNHs (5 mg) and 1,2-dichlorobenzene, oDCB, (10 mL), in a round-bottom flask under nitrogen atmosphere and bath-sonicate for 10 min (see Note 1). 2. Dissolve the tert-butoxy carbamate (BOC)-protected aniline derivative (2.6 mmol) in acetonitrile (5 mL) (see Note 2). 3. Add the solution of the BOC-protected aniline derivative in the reaction mixture. 4. Add isoamyl nitrite (4.0 mmol) and stir the reaction mixture at 60 °C for 18 h (see Note 3). 5. After cooling down to room temperature, dilute the reaction mixture with N,N-dimethylformamide, DMF (30 mL). 6. Filter the reaction mixture over a polytetrafluoroethylene (PTFE) membrane filter with pore size of 0.2 μm (see Note 4). 7. Wash the solid residue obtained on top of the PTFE filter with DMF and dichloromethane (50 mL) (see Note 5). 8. Recover the BOC-modified CNHs as powder and store it at room temperature in the dark. 9. Disperse the BOC-modified CNHs (5  mg) in dry dichloromethane (10 mL).

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10. Bubble gaseous HCl for 30 s in the dispersion of the BOC-­ modified CNHs (see Note 6). 11. Stir the reaction mixture under an inert atmosphere of nitrogen for 12 h. 12. Evaporate the solvent under reduced pressure (see Note 7). 13. Add fresh dichloromethane (10  mL), and filter the mixture over a PTFE membrane filter with pore size of 0.2 μm. 14. Wash the solid residue obtained on top of the PTFE filter with DMF and dichloromethane (50 mL). 15. Recover the amine-modified CNHs as powder and store it at room temperature in the dark. 3.4  Incorporation of Carboxylic Acid Moieties on CNHs

1. Treat pristine CNHs (50  mg) with molecular oxygen at 0.1 MPa for 10 min at 580° C. 2. Transfer the oxidized CNHs in a round-bottom flask and add 30% w/w solution of H2O2 (60 mL). 3. Apply light irradiation and heat the reaction mixture at 120° C for 3 h (see Note 8). 4. Filter the dispersion of the CNHs through a PTFE membrane filter with pore size of 0.2 μm. 5. Wash the solid residue obtained on top of the PTFE filter with a large amount of deionized water and methanol (50 mL). 6. Recover the carboxylic acid-modified CNHs as powder and store it at room temperature in the dark.

3.5  Conjugation of Drugs/Biomolecules on Pre-modified CNHs

1. Treat the carboxylic acid-modified CNHs (50 mg) with thionyl chloride, SOCl2 (50 mL), at 70° C for 8 h under nitrogen atmosphere (see Note 9). 2. Evaporate the SOCl2 under reduced pressure. Then, add dry THF (20 mL), bath-sonicate the mixture for 5 min, and then evaporate the solvent to dryness. Repeat the latter twice and keep the dry solid residue (acyl chloride-modified CNHs) under nitrogen atmosphere (see Note 10). 3. Add dry THF (10 mL) to the acyl chloride-modified CNHs and keep it under nitrogen atmosphere. Dissolve the hydroxylor amino-terminated drug/biomolecule (100  mg) in dry THF (20 mL) and add dropwise to the dispersion of the acyl chloride-­modified CNHs. Stir all the mixture at room temperature for 24 h. 4. Filter the dispersion of the drug/biomolecule-functionalized CNHs through a PTFE membrane filter with pore size of 0.2 μm.

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5. Wash the solid residue obtained on top of the PTFE filter with THF and dichloromethane (50  mL) to remove any physisorbed drug/biomolecule (see Note 11). 6. Recover the drug/biomolecule-functionalized CNHs as powder and store it at room temperature in the dark. 3.6  Encapsulation of Drugs/Biomolecules Within CNHs

1. Treat pristine CNHs (50 mg) in oxygen gas at 570–580 °C for 15 min to create holes on their walls. Then, heat the oxidized material at 1200° C under hydrogen atmosphere to remove the oxygen functionalities attached to the hole edges (see Note 12). 2. Dissolve the drug/biomolecule (400 mg) in DMF (100 mL) in a glass container and add the as-prepared meshed CNHs (50  mg). Bath-sonicate the mixture for 30  min. Subject the mixture to slow evaporation of the DMF with the aid of dry air over 5 s (see Note 13). 3. Collect the black material (CNHs filled with the drug/biomolecule) from the bottom of the container. 4. Wash the obtained CNHs filled with the drug/biomolecule on top of a PTFE membrane filter with DMF and dichloromethane (50 mL) to remove any physisorbed drug/biomolecule (see Note 14). 5. Recover the drug/biomolecule encapsulated within CNH material as powder and store it at room temperature in the dark.

4  Notes 1. Bath sonication allows dispersion of insoluble CNHs and facilitates reaction. Add the solvent in small portions to wet the solid. CNH is a very light powder; thus fast incorporation of the solvent may produce airstream inside the flask pushing the light powder out. 2. The BOC-protected aniline derivative, namely tert-butyl 2-(2-(2-(4-aminobenzamido)ethoxy)ethoxy) ethylcarbamate, is prepared according to the following procedure: a solution of p-nitrobenzoyl chloride (464 mg, 2.5 mmol) in DCM (30 mL) was added to a cold solution (ice bath) of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (500  mg, 2.0 mmol) in 0.2 N NaOH solution (50 mL). The reaction mixture was stirred vigorously at 0°  C for 3  h. The organic layer was then separated, washed successively with 0.5  N NaOH and water and then dried over anhydrous Na2SO4. Evaporation of the solvent under reduced pressure affords the nitro-derivative as a white solid with 85% yield. The aforementioned nitro-derivative (170 mg, 0.427 mmol) was dissolved in dry ethanol (20 mL) and Pt/C 10 wt% (18 mg) was added.

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Tip: Degassing the mixture with the aid of bath sonication and a vacuum pump, followed by flushing with hydrogen gas, will enhance the hydrogen adsorption in the porous catalyst. The reaction mixture was stirred under hydrogen atmosphere for 24  h. The catalyst was removed by filtration on Celite®, the filter pad was washed excessively with DCM and the filtrate was evaporated under reduced pressure to furnish the BOC-­ protected aniline derivative in 91% yield. Tip: It is suggested to prepare fresh BOC-protected aniline derivatives. 3. Alternatively, microwave irradiation can be employed for reducing reaction time and amount of solvents, especially when reaction is performed at larger scale. In a typical procedure, add pristine CNHs (30  mg), BOC aniline derivative (350 mg, 0.95 mmol), and oDCB (2 mL) in a glass vial under an inert nitrogen atmosphere and bath-sonicate for 15  min. Then, add isoamyl nitrite (5.7 mmol), seal the vial with a septum cap, heat at 150 °C, and apply microwave irradiation with 100 Watts for 60 min. 4. Filtration over PTFE membrane filter with the particular 0.2 μm or smaller pore size allows retaining covalently functionalized CNHs on top of the filter. If PTFE filter with bigger pore size is employed, modified CNHs shall pass through the filter or block/chock the pores. 5. Washing modified CNHs onto the PTFE filter allows to completely remove organic material physisorbed onto the surface of CNHs. Furthermore, examination of the filtrate via UV-Vis allows monitoring the purification. 6. Removal of the BOC-protecting group with gaseous HCl yields a cleaner material as compared to the one obtained upon the corresponding cleavage of BOC with trifluoroacetic acid (TFA). Caution: Work with gaseous HCl only in a well-­ ventilated fume hood. 7. During the evaporation of the solvent the dissolved HCl gas will also be released. 8. A conventional 500 W halogen (or Xe) lamp is sufficient. 9. This treatment will convert the carboxylic acids to the corresponding acyl chlorides allowing the subsequent reaction of the modified CNHs with hydroxyl- and amino-terminated drugs/biomolecules. 10. The objective is to remove the traces of SOCl2. The use of dry solvent and nitrogen atmosphere is essential for the stability of the acyl chlorides, since they are susceptible to hydrolysis by traces of moisture. Caution: Work with SOCl2 only in a well-­ ventilated fume hood. Contact of SOCl2 with moisture results in hydrolysis affording SOx and HCl gas. Do not store the acyl chloride-modified CNHs and use them instantly.

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11. Examination of the filtrate via UV-Vis allows monitoring the purification of the solid material. 12. The objective is to create pores across the sidewalls of CNHs allowing the release of the drug/biomolecule via diffusion from the interior of the CNH to the target. 13. The selection of the solvent is related to the property of DMF to stabilize well the CNHs. Further, the drug/biomolecule must be well dissolved prior to the addition of the opened CNHs. Therefore, if the mentioned concentration (4  mg/ mL) of the drug/biomolecule is not achievable, increase the amount of the required solvent. The method is based on the slow evaporation of the solvent inducing the aggregation of the drug/biomolecule in the interior of CNHs. 14. Examination of the filtrate via UV-Vis allows monitoring the purification of the solid material.

5  Conclusions Although the synthesis of CNHs requires advanced infrastructure, the overall procedure is simple and straightforward, since no posttreatment or purification is needed. Alternatively, CNHs can be purchased in the market. As presented in detail, the synthesis of CNH-based nanocarriers exploits simple methods adopted from organic chemistry and chemistry of materials and the required purification/isolation is handy and capable of scale-up. It should be underlined that for all CNH-based materials complementary characterization is necessary in order to evaluate the success of the described functionalization procedures. Namely, spectroscopic (UV-Vis, Raman, ATR-IR), thermogravimetric (TGA), and microscopy (AFM, SEM, TEM) techniques are mandatory to verify the generation of added functionalities or the presence of covalently grafted drugs/biomolecules, the incorporation of holes on the sidewalls of CNHs, the presence of drugs/biomolecules into the cavity of CNHs, etc. Concluding, the research on CNH-based delivery platforms is based on simple chemical protocols and requires wellequipped laboratories for synthesis and characterization and most importantly interdisciplinary research collaborations in order to realize CNHs as nanocarriers in living cells and organisms.

Acknowledgments Partial support by the project Nanotechnology, Advanced Nanoelectronics” (MIS 5002772) the “Reinforcement of the

“National Infrastructure in Materials and Micro-/ which is implemented under Research and Innovation

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Infrastructures,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund), is acknowledged. References 1. Karousis I, Martinez IS, Ewels CP, Tagmatarchis N (2016) Structure, properties, functionalization, and applications of carbon nanohorns. Chem Rev 116:4850–4883 2. Kostarelos K (2008) The long and short of carbon nanotube toxicity. Nat Biotechnol 26:774–776 3. Tagmatarchis N, Maigne A, Yudasaka M, Iijima S (2006) Functionalization of carbon nanohorns with azomethine ylides: towards solubility enhancement and electron-transfer processes. Small 2:490–494 4. Cioffi C, Campidelli S, Brunetti FG, Meneghetti M, Prato M (2006) Functionalisation of carbon nanohorns. Chem Commun 20:2129–2131 5. Pagona G, Karousis N, Tagmatarchis N (2008) Aryl diazonium functionalization of carbon nanohorns. Carbon 46:604–610 6. Nakamura E, Koshino M, Tanaka T, Niimi Y, Harano K, Nakamura Y et al (2018) Imaging of conformational changes of biotinylated triamide molecules covalently bonded to a carbon nanotube surface. J Am Chem Soc 130:7808–7809 7. Lacotte S, Garcia A, Decossas M, Al-Jamal WT, Li S, Kostarelos K et al (2008) Interfacing functionalized carbon nanohorns with primary phagocytic cells. Adv Mater 20:2421–2426 8. Economopoulos SP, Pagona G, Yudasaka M, Iijima S, Tagmatarchis N (2009) Solvent-free microwave-assisted Bingel reaction in carbon nanohorns. J Mater Chem 19:7326–7331 9. Karousis N, Ichihashi T, Yudasaka M, Iijima S, Tagmatarchis N (2011) Microwave-assisted functionalization of carbon nanohorns via [2+1] nitrenes cycloaddition. Chem Commun 47:1604–1606 10. Pagona G, Katerinopoulos HE, Tagmatarchis N (2011) Synthesis, characterization, and photophysical properties of a carbon nanohorn-­ coumarin hybrid material. Chem Phys Lett 516:76–81 11. Vizuete M, Gomez-Escalonilla MJ, Fierro JLG, Yudasaka M, Iijima S, Vartanian M et al (2011) A soluble hybrid material combining carbon nanohorns and C60. Chem Commun 47:12771–12773 12. Karousis N, Sato Y, Suenaga K, Tagmatarchis N (2012) Direct evidence for covalent func-

tionalization of carbon nanohorns by high-­ resolution electron microscopy imaging of C60 conjugated onto their skeleton. Carbon 50:3909–3914 13. Pagona G, Zervaki GE, Sandanayaka AD, Ito O, Charalambidis G, Hasobe T et  al (2012) Carbon nanohorn-porphyrin dimer hybrid material for enhancing light-energy conversion. J Phys Chem C 116:9439–9449 14. Chronopoulos D, Karousis N, Ichihashi T, Yudasaka M, Iijima S, Tagmatarchis N (2013) Benzyne cycloaddition onto carbon nanohorns. Nanoscale 5:6388–6394 15. Miyako E, Russier J, Mauro M, Cebrian C, Yawo H, Menard-Moyon C et  al (2014) Photofunctional nanomodulators for bioexcitation. Angew Chem Int Ed 53:13121–13125 16. Chronopoulos DD, Liu Z, Suenaga K, Yudasaka M, Tagmatarchis N (2016) [3 + 2] cycloaddition reaction of azomethine ylides generated by thermal ring opening of aziridines onto carbon nanohorns. RSC Adv 6:44782–44787 17. Pagona G, Sandanayaka ASD, Maigne A, Fan J, Papavassiliou GC, Petsalakis ID et al (2007) Photoinduced electron transfer on aqueous carbon nanohorn–pyrene–tetrathiafulvalene architectures. Chem Eur J 13:7600–7607 18. Vizuete M, Gomez-Escalonilla MJ, Fierro JLG, Sandanayaka ASD, Hasobe T, Yudasaka M et  al (2010) A carbon nanohorn porphyrin supramolecular assembly for photoinduced electron-transfer processes. Chem Eur J 16:10752–107563 19. Jiang BP, Shen LFXC, Ji SC, Shi Z, Liu CJ, Zhang L et  al (2014) One-step preparation of a water-soluble carbon nanohorn/phthalocyanine hybrid for dual-modality photothermal and photodynamic therapy. ACS Appl Mater Interfaces 6:18008–18017 20. Pagona G, Sandanayaka ASD, Araki Y, Fan J, Tagmatarchis N, Yudasaka M et  al (2006) Electronic interplay on illuminated aqueous carbon nanohorn-porphyrin ensembles. J Phys Chem B 110:20729–20732 21. Pagona G, Fan J, Maigne A, Yudasaka M, Iijima S, Tagmatarchis N (2007) Aqueous carbon nanohorn-pyrene-porphyrin nanoensembles: controlling charge-transfer interactions. Diam Relat Mater 16:1150–1153

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22. Mountrichas G, Ichihashi T, Pispas S, Yudasaka M, Iijima S, Tagmatarchis N (2009) Solubilization of carbon nanohorns by block polyelectrolyte wrapping and templated formation of gold nanoparticles. J Phys Chem C 113:5444–5449 23. Pagona G, Tagmatarchis N, Fan J, Yudasaka M, Iijima S (2006) Cone-end functionalization of carbon nanohorns. Chem Mater 18:3918–3920 24. Sandanayaka ASD, Pagona G, Tagmatarchis N, Yudasaka M, Iijima S, Araki Y et al (2007) Photoinduced electron-transfer processes of carbon nanohorns with covalently linked pyrene chromophores: charge-separation and electron-migration systems. J Mater Chem 17:2540–2546 25. Pagona G, Sandanayaka ASD, Araki Y, Fan J, Tagmatarchis N, Charalambidis G et al (2007) Covalent functionalization of carbon nanohorns with porphyrins: nanohybrid formation and photoinduced electron and energy transfer. Adv Funct Mater 17:1705–1711 26. Cioffi C, Campidelli S, Sooambar C, Marcaccio M, Marcolongo G, Meneghetti M et al (2007) Synthesis, characterization, and photoinduced electron transfer in functionalized single wall carbon nanohorns. J Am Chem Soc 129:3938–3945 27. Pagona G, Sandanayaka ASD, Hasobe T, Charalambidis G, Coutsolelos AG, Yudasaka M et  al (2008) Characterization and photoelectrochemical properties of nanostructured thin film composed of carbon nanohorns covalently functionalized with porphyrins. J Phys Chem C 112:15735–15741 28. Rotas G, Sandanayaka ASD, Tagmatarchis N, Ichihashi T, Yudasaka M, Iijima S et  al (2008) (Terpyridine)copper(II)-carbon nanohorns: metallo-nanocomplexes for photoinduced charge separation. J Am Chem Soc 130:4725–4731 29. Zhang M, Murakami T, Ajima K, Tsuchida K, Sandanayaka ASD, Ito O et  al (2008) Fabrication of ZnPc/protein nanohorns for double photodynamic and hyperthermic can-

cer phototherapy. Proc Natl Acad Sci U S A 105:14773–14778 30. Sandanayaka ASD, Ito O, Zhang M, Ajima K, Iijima S, Yudasaka M et al (2009) Photoinduced electron transfer in zinc phthalocyanine loaded on single-walled carbon nanohorns in aqueous solution. Adv Mater 21:4366–4371 31. Karousis N, Ichihashi T, Chen S, Shinohara H, Yudasaka M, Iijima S et al (2010) Imidazolium modified carbon nanohorns: switchable solubility and stabilization of metal nanoparticles. J Mater Chem 20:2959–2964 32. Vizuete M, Gomez-Escalonilla MJ, Fierro JLG, Ohkubo K, Fukuzumi S, Yudasaka M et al (2014) Photoinduced electron transfer in a carbon nanohorn-C60 conjugate. Chem Sci 5:2072–2080 33. Zhang MF, Yudasaka M, Ajima K, Miyawaki A, Iijima S (2007) Light-assisted oxidation of single-wall carbon nanohorns for abundant creation of oxygenated groups that enable chemical modifications with proteins to enhance biocompatibility. ACS Nano 1:265–272 34. Xu J, Zhang M, Nakamura M, Iijima S, Yudasaka M (2010) Double oxidation with oxygen and hydrogen peroxide for hole-­ forming in single wall carbon nanohorns. Appl Phys A Mater Sci Process 100:379–383 35. Ajima K, Yudasaka M, Murakami T, Maigne A, Shiba K, Iijima S (2005) Carbon nanohorns as anticancer drug carriers. Mol Pharm 2:475–480 36. Jianxun X, Yudasaka M, Kouraba S, Sekido M, Yamamoto Y, Iijima S (2008) Single wall carbon nanohorn as a drug carrier for controlled release. Chem Phys Lett 461:189–192 37. Guerra J, Herrero MA, Vazquez E (2014) Carbon nanohorns as alternative gene delivery vectors. RSC Adv 4:27315–27321 38. Li N, Zhao Q, Shu C, Ma X, Li R, Shen H et al (2014) Targeted killing of cancer cells in vivo and in  vitro with IGF-IR antibody-directed carbon nanohorns based drug delivery. Int J Pharm 478:644–654

Chapter 3 Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole Organs Burak Derkus and Emel Emregul Abstract Exosomes, natural and nanovesicular structures surrounded by a lipid membrane, tend to be secreted toward extracellular environments by almost all cell types. Late studies have shown them to be effective in several complex biological processes like cancer development and metastasis, immune system regulation, cellular signal transduction, stem cell differentiation, and regeneration of damaged tissues. Although there are many studies dealing with the role of exosomes in the aforementioned fields, the mechanisms remained largely unknown. There is therefore a need for further study on exosome isolation from different sources. While researchers mostly use serum, plasma, urine, and cell culture media as a source for exosome isolation, there are no studies dealing with direct isolation of exosomes from whole organs in literature. In this study, we propose a protocol for effective isolation of exosomes from whole organs. Mouse brain, heart, and liver were chosen as the sources of exosomes in this study. Isolated exosomes were successfully characterized with BCA test, western blot, transmission electron microscopy and ELISA. Key words Extracellular vesicles, Nanovesicles, Exosomes, Isolation, Ultrasonication

1  Introduction Exosomes, which are released by almost all kinds of cells to the extracellular environment, are lipid-involved natural nanovesicles with a diameter of 50–150 nm. Although they were evaluated as cellular wastes in the early studies, their potential of use in diagnosis and treatment of diseases was recognized after 2000s. Owing to their rich microRNA (miRNA) content, which is 60 times higher than regular biological fluids, exosomes today are utilized in diagnostics [1], cancer treatment [2], drug delivery [3] and stem cell-­ based technologies [4]. Exosomes are mainly isolated from cell culture wastes [5], plasma [6], serum [7], urine [8], and semen [9]. While they are mostly and practically isolated from cell culture-­ conditioned media (CM), it is thought that exosomal content might be unsteady due to genotypic and phenotypic differences Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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between cell lines [10]. Moreover, the cellular content does not fully reflect the molecular composition of tissues. Since the exosomes isolated from secondary cell lines do not fully reflect the properties of the primary cells or original tissue [11], it is difficult to judge the molecular contents of exosomes. It is important to know the exact molecular content of the exosomes during secretion from the tissue so that the diagnostic and therapeutic approaches can be properly examined. In addition, in order to fully illuminate the mechanisms of exosomes in cellular communication, attempted in various publications [12], isolation from natural environments namely living tissues and organs would be preferred instead of cell culture CM. However, it can be seen in the literature that there are only a couple studies dealing with the isolation of exosomes from whole tissues/organs [13]. This study mainly focuses on the effective isolation of exosomes from heart, brain and liver. Ultrasonication was for the first time applied in this study for exosome isolation that loosened the extracellular space of organs and enabled us to perform a more efficient isolation process. Bicinchoninic acid (BCA) and enzyme-linked immunosorbent assay (ELISA) tests (calibration graphs have been presented in Fig. 1) revealed high protein contents (Table 1) indicating the presence of exosomes. The calculated protein content varied between 2.7 and 4.1 mg.mL−1, and the exosome count was found to be 108–109 particles, which was quite good for downstream analyses. Exosomal RNA, one of the most important elements of exosomes, seemed suitable (Table 1) for cDNA synthesis and for performing a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) or transcriptomics study. Western blot images revealed the existence of CD63 and CD81 for brain- and heart-derived exosomes, whereas only CD81 expression was seen in the liver-derived exosomes (Fig. 2). Considering the fact that the presence of one of the three (CD63 CD81 and Alix) exosomal markers is sufficient for confirming the exosomes,

Fig. 1 Calibration graph for BCA test (a), and CD63 ELISA assay (b)

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Table 1 Protein content, particle count and RNA content of isolated exosomes BCA, O.D.

Protein conc., μg.mL−1

ELISA CD63, Number of CD63+ O.D. exosomes

RNA content, ng.μL−1

Brain exosomes

3093

3781

0.195

2.6 × 109

45.5

Heart exosomes

2217

2663

0.099

6.0 × 108

6.3

Liver exosomes

3349

4107

0.305

4.9 × 109

143.0

Fig. 2 Western blot analysis of CD63 and CD81 exosomal markers for brain-, heart- and liver-derived exosomes

regarding the blotting results, we can conclude that the suggested methodology is appropriate for exosome isolation from tissues and organs. Finally, the transmission electron microscope (TEM) images in Fig.  3 for the brain (A)-, heart (B)-, and liver (C)-derived exosomes further demonstrated the success of the ultrasonication-­ assisted isolation protocol. The exosomes obtained had a diameter between 50 and 150 nm. The blue arrows seen in the figure clearly show the stained membranes proving exosomal character.

2  Materials 2.1  Reagents Used for Exosome Isolation

1. Freshly removed mouse brain, heart and liver. One-mouse hemi brain was used for exosome isolation. 2. 70% Ethanol. 3. Sulfuric acid (H2SO4). 4. Sterile scissors, tweezers/forceps, and razor blade for dissecting and mincing the organs.

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Fig. 3 Transmission electron microscopy images of brain (A)-, heart (B)- and liver (C)-derived exosomes

5. Sterile and cold phosphate-buffered saline (1× PBS, pH 7.4). 6. 50 mL Conical tubes. 7. Polyethylene glycol 4000 (PEG 4000). 8. 0.2  μm Filter adapters with surfactant-free cellulose acetate membrane and 10 mL plunge syringes. 2.2  Reagents Used for the Characterization of Isolated Exosomes

1. 1× RIPA lysis buffer (1× lysis buffer, 200 mM PMSF, inhibitor cocktail, and sodium orthovanadate) (Santa Cruz sc-24948).

2.2.1  Exosomal Protein Quantification

4. CD63 and CD81 ELISA kits (System Bioscience EXOEL-­ CD63A-­1 and EXOEL-CD81A-1).

2.2.2  Western Blot Analysis

1. Acrylamide/bis-acrylamide, 30% solution (Sigma).

2. BCA kit (Thermo Scientific 23227). 3. Flat-bottom 96-well microplate and adhesive covers.

2. Tris (pH 8.8, 0.5 M).

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3. Tris (pH 6.8, 0.5 M). 4. 10% Sodium dodecyl sulfate (SDS, Sigma). 5. 10% Ammonium persulfate (APS, AppliChem), freshly prepared. 6. Tetramethylethylenediamine (TEMED, Merck). 7. Isopropanol. 8. 2× Laemmli buffer (0.125 mM Tris–HCl, 4% SDS, 20% glycerol, 0.04% bromophenol blue, 10% β-mercaptoethanol, pH 6.8). 9. 5× Running buffer (125 mM Tris base, 0.96 M glycine, 0.5% SDS, pH 8.3). 10. 5× Transfer buffer (250 mM Tris base, 192 mM glycine, 0.1% SDS, pH 9.2). 11. Tris-buffered saline with Tween 20 (TBST) buffer (20  mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20). 12. 0.45  μm Polyvinylidene (Immobilon, Millipore).

fluoride

membrane

(PVDF)

13. Methanol for hydrophilization of membrane. 14. Blocking buffer (5% nonfat milk powder) in TBS-T. 15. Monoclonal primary antibodies anti-CD63 (Abnova MAB0931) and anti-CD81 (Abnova MAB6435) in TBS-T (1:2000). 16. Anti-rabbit IgG secondary antibody in PBS (1:5000) (System Bioscience). 17. ECL Western Blotting Substrate and SuperSignal West Femto Substrate (Thermo Scientific) for luminescence-based imaging. 2.2.3  Transmission Electron Microscopy Imaging

1. 4% Paraformaldehyde (Santa Cruz). 2. 1% Glutaraldehyde, EM grade (Sigma). 3. 0.5% Uranyl acetate, pH 4.0 (Polysciences). 4. PBS kept at 4 °C. 5. 200 mesh Formvar carbon-coated EM grids (Electron Microscopy Sciences, FCF300). 6. Parafilm. 7. Clean forceps. 8. Whatman filter paper. 9. 0.22-μm-pore-sized syringe filter.

2.2.4  RNA Isolation

1. Promega SV total RNA isolation kit. 2. 60% Ethanol.

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3  Methods 3.1  Isolation of Exosomes from Brain, Heart, and Liver

1. Sacrifice a mouse sticking to the rules constructed by your animal care committee. 2. Remove the brain, heart and liver, and be sure that the organs are clean of hair and other wastes. Olfactory bulbs of brain are removed and the brain is divided into two hemi brains. 3. The sacrificed organs are washed with sterile and cold PBS, transferred to separate tubes containing 15 mL sterile PBS and then ultrasonicated for 3 min under 750 W power and 20 kHz frequency ultrasonication conditions (Sonics VCX 750) in order to dissociate the tissue and release the extracellular vesicles (see Notes 1 and 2). 4. Centrifuge the tissue homogenates at 300  × g, 2000  × g and 13,000 × g for 15 min at 4 °C, respectively, and remove the cellular debris, microvesicles and apoptotic bodies. 5. Transfer 10 mL of the supernatant into 50 mL tubes and add 10 mL 20% PEG 4000 solution. 6. Incubate the tissue homogenate-PEG mixture overnight at 4 °C. 7. Centrifuge the mixture at 13,000 × g at 4 °C and discard the supernatant without disturbing the exosome pellet. 8. Resuspend the pellet in 100 μL PBS kept at 4 °C and store at −20 °C for further use.

3.2  Protein Quantification of Exosomes Using BCA Test

Quantification of the exosomal protein content is performed following the manufacturer’s instruction. Briefly: 1. Exosomes are diluted 1:2 in PBS. 2. Exosome lysate is obtained by adding an equal volume of RIPA buffer including protease inhibitors. 3. Pipette 25  μL of each bovine serum albumin (BSA) standard with concentrations between 12.5  μg.mL−1 and 2.5  mg.mL−1 and organ-derived exosome samples into a 96-well microplate. The procedure is carried out in triplicate. 4. Add 200 μL of the working reagent into each well and shake the plate on a platform shaker for 30 s. 5. Cover the plate and incubate at 37 °C for 30 min. 6. Measure the absorbance at 562  nm on a spectrophotometer (Perkin Elmer 1420 Multilabel Counter).

3.3  Exosome Particle Counting with CD63 ELISA Kit

Determination of exosome particle count is performed following the manufacturer’s instruction of CD63 ExoElisa Kit (System Bioscience). Briefly:

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1. Add 50  μL of freshly prepared protein standards (108– 1.35 × 1010 exosome.mL−1) and exosome samples to the appropriate well of the microtiter plate. 2. Incubate the plate at 37 °C for 2 h. 3. Wash the plate three times for 5 min each with 100 μL wash buffer. 4. Add 50 μL of 1:100 diluted CD-63 primary antibody to each well and incubate while shaking at room temperature for 1 h. 5. Wash the plate three times for 5 min each with 100 μL wash buffer. 6. Add 50 μL of 1:5000 diluted secondary antibody to each well and incubate with shaking at room temperature for 1 h. 7. Add 50 μL of supersensitive TMB ELISA substrate and incubate with shaking at room temperature for 15–45 min. 8. Add 50  μL of stop buffer and read the absorbance values at 450 nm immediately (Lambda Scan Instrument). 3.4  Western Blot Analysis

1. Add 2× Laemmli buffer to the exosome suspensions (18 μg), and then boil the samples at 96 °C for 3 min. 2. Centrifuge the exosomal protein solutions at 13,000  ×  g for 3 min; take the supernatant into clean microtubes. 3. Load 25 μL of each sample into each well (5–12% gel system is used) and then run proteins at 100 V, 35–40 mA, for about 2 h (Bio-Rad Wet/Tank Blotting System). 4. Transfer the proteins electrophoretically (100 V, 400 mA) for 1 h onto immobilon PVDF membrane. 5. Block the membrane with 5% nonfat milk powder in TBS-T for 1 h on a platform shaker. 6. Incubate the membrane with CD63 and CD81 antibodies overnight at 4 °C. 7. Following the washing step with TBS-T, incubate the membrane with secondary antibody diluted in PBS for 1 h at 4 °C. 8. After washing with PBS, the membrane is treated with ECL substrate and imaged on an Odyssey imaging system.

3.5  Preparation of TEM Samples

1. The exosome suspension is treated with 4% paraformaldehyde for fixation. 2. Deposit 10  μL of the fixed exosomes onto Formvar carbon-­ coated grids and wait for 20 min for adequate absorption. 3. Put 100 μL of PBS onto a piece of parafilm and place the grid on PBS drop with a clean forceps. Repeat the washing step three times.

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4. Remove the excess water using filter paper. 5. Transfer the grids onto 1% glutaraldehyde drop placed on parafilm and leave the exosomes for further fixation for a period of 5 min. 6. Repeat the washing step three times and remove the excess water using filter paper. 7. Put the grids onto 0.5% uranyl acetate drops and incubate for 10 min for adequate staining (see Note 3). 8. Observe the samples under the electron microscope (Joel USA JEM 2100-F). 3.6  RNA Isolation and Quantification

1. Put 175 μL RNA lysis buffer into an Eppendorf tube in which 30 mg of exosome suspension is situated. 2. Following homogenization by vortexing, pipetting is done for efficient lysis. 3. Add 350 μL of RNA dilution buffer to 175 μL of lysate. Mix by inverting the tube 3–4 times. Place in a heating block at 70 °C for 3 min. 4. Centrifuge at 13.000 × g for 10 min at 20–25 °C. 5. Transfer the lysate into a clean tube and add 200 μL 95% ethanol. Mix by pipetting 3–4 times. 6. Transfer the mixture to the spin column assembly and centrifuge at 13,000 × g for 1 min. 7. Discard the liquid in the collection tube and add 600 μL of RNA wash solution to the spin column assembly. Centrifuge at 13,000 × g for 1 min. 8. Apply 50 μL of the DNase incubation mix onto the column membrane and incubate for 15 min at room temperature. Add 200 μL of DNase stop solution and centrifuge at 13,000 × g for 1 min. 9. Add 600 μL of wash solution and centrifuge at 13,000 × g for 1 min. 10. Discard the liquid in the collection tube and add 250 μL RNA wash solution into the column. Centrifuge at 13,000 × g for 2 min. 11. Add 100 μL of nuclease-free water into the column fitted with an elution tube and centrifuge for 1 min. 12. Quantify the amount of exosomal RNA using a Nanodrop (Thermo Fisher) by dropping 5 μL of RNA solution onto the pedestal (see Note 4).

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4  Notes 1. The ultrasonic probe should be washed with H2SO4, ultrapure water and ethanol, respectively. 2. The ultrasonication step should be carried out on ice in order to prevent heating of the tissues due to high-frequency sonication. 3. Uranyl acetate solution should be filtered from 0.22 μm pore-­ sized syringe filter in order to prevent accumulation of large or aggregated particles onto the grid. 4. A260/280 ratio for RNA quantification should be around 2, indicating the purity of RNA.  If this ratio is over/lower, a protein-­caused impurity is a question that requires an extensive purification step.

Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.” References 1. Melo SA, Luecke LB, Kahlert C et  al (2015) Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523:177–182 2. Malla B, Zaugg K, Vassella E et  al (2017) Exosomes and exosomal microRNAs in prostate cancer radiation therapy. Int J Radiat Oncol Biol Phys 98(5):982–995 3. Agrawal AK, Aqil F, Jeyabalan J et  al (2017) Milk-derived exosomes for oral delivery of paclitaxel. Nanomedicine 13(5):1627–1636 4. Chen B, Li Q, Zhao B et al (2017) Stem cell-­ derived extracellular vesicles as a novel potential therapeutic tool for tissue repair. Stem Cells Transl Med 6:1753–1758. https://doi. org/10.1002/sctm.16-0477 5. Leblanc P, Arellano-Anaya ZE, Bernard E et al (2017) Isolation of exosomes and microvesicles from cell culture systems to study prion transmission. Methods Mol Biol 1545:153–176 6. Garcia-Contreras M, Shah SH, Tamayo A et  al (2017) Plasma-derived exosome charac-

terization reveals a distinct microRNA signature in long duration type 1 diabetes. Sci Rep 7(1):5998 7. Helwa I, Cai J, Drewry MD et  al (2017) A comparative study of serum exosome isolation using differential ultracentrifugation and three commercial reagents. PLoS One 12(1):e0170628 8. Miranda KC, Bond DT, Levin JZ et al (2014) Massively parallel sequencing of human urinary exosome/microvesicle RNA reveals a predominance of non-coding RNA. PLoS One 9(5):e96094 9. Vojtech L, Woo S, Hughes S et  al (2014) Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res 42(11):7290–7304 10. Perkins EJ, Bao W, Guan X et  al (2006) Comparison of transcriptional responses in liver tissue and primary hepatocyte cell cultures after exposure to hexahydro-1, 3, 5-trinitro-1,

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3, 5-triazine. BMC Bioinformatics 7(Suppl 4):S22 11. Katayama S, Skoog T, Jouhilahti EM et  al (2015) Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control. BMC Genomics 16:476

12. Derkus B, Emregul KC, Emregul E (2017) A new approach in stem cell research-exosomes: their mechanism of action via cellular pathways. Cell Biol Int 41(5):466–475 13. Pérez-González R, Gauthier SA, Kumar A et al (2017) Method for isolation of extracellular vesicles and characterization of exosomes from brain extracellular space. Methods Mol Biol 1545:139–151

Chapter 4 Aggregate Determination by Permeation Technique Phennapha Saokham and Thorsteinn Loftsson Abstract Permeation technique is used to study molecular aggregation in aqueous solutions including formation of cyclodextrin guest/host aggregates. Since only guest molecules, host molecules and guest/host aggregates that are smaller than the pore size of a given semipermeable membrane are able to permeate through the membrane, negative deviation of permeation profiles indicates formation of guest/host aggregates or self-aggregates. This chapter describes how the method is used to detect formation of nano-sized aggregates and to determine the critical aggregation concentration (cac) from permeation profiles of a guest molecule. Key words Inclusion complexes, Critical aggregation concentration (cac), Permeation, Aggregates

1  Introduction Clear aqueous solutions can contain molecular aggregates with diameter below the wavelength of visible light. Frequently these aggregates and clusters are transient; that is, they are constantly being formed and dissembled [1]. Thus, it can be difficult to detect them through conventional methods like dynamic light scattering (DLS). Molecular membrane permeation is a passive diffusion process as described by Fick’s law [2]. Herein, we demonstrate how the permeation technique through semipermeable membranes is used to study the formation of transient guest/host aggregates (e.g., drug/cyclodextrin complex aggregates) and clusters (i.e., loosely connected molecular structures). This method is based on permeation of aggregating molecules through semipermeable membranes of different molecular weight cutoffs (MWCOs) [3– 9]. Donor solutions (i.e., the test solutions) containing various concentrations of, for example, guest/host aggregates or self-­ aggregates are placed in the donor chamber of Franz diffusion cells (Fig.  1) [3–8] or miniature dialysis devices such as micro-­ equilibrium dialysis device (Fig. 2) [10] or cuplike MINI dialysis Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Phennapha Saokham and Thorsteinn Loftsson

Fig. 1 Jacketed (left) and unjacketed (right) Franz diffusion cells consist of donor and receptor compartments separated by semipermeable membrane

Fig. 2 A 96-well micro-equilibrium dialysis device which places semipermeable membrane between two Teflon bars to form donor and receptor compartments

device (Fig. 3) [9, 11]. The donor and receptor compartments are separated by a single-layer semipermeable membrane. In general, the studies are performed at room temperature. After fluxes from various MWCO membranes have been obtained, plots of fluxes against sampling time (i.e., permeation profiles) are designed. A negative deviation of permeation profile for Fick’s first law provides quantitative analysis of formation of nano-sized aggregates that are unable to permeate through a given MWCO semiperme-

Aggregate Determination by Permeation Technique

37

Fig. 3 A cuplike MINI dialysis device attached with specific MWCO semipermeable membrane and 1.5 mL Eppendorf tube as receptor compartment

able membrane [3–5] and the critical aggregation concentration (i.e., the lowest concentration of host molecule that forms guest/ host aggregates and clusters) is determined [6–9, 11].

2  Materials 2.1  Tested Solutions or Donor Solutions: Saturated Guest/Host Aggregate Solution

Prepare saturated guest molecule (e.g., drug) in various concentrations of host molecule (e.g., cyclodextrin) using ultrapure water, purifying deionized water with 18 MΩ-cm at 25 °C and analytical grade reagents. Add excess amount of guest to aqueous solution of host molecule (see Note 1). Then heat, sonicate or autoclave suspensions to promote aggregate formation. Continue to add small amount of the guest until precipitation is observed after equilibration. Equilibrate at room temperature (or some other temperature) for 3–7 days (see Note 2) under constant agitation using, for example, orbital laboratory shaker. After equilibrium, filtrate the suspensions through 0.45  μm cellulose acetate or comparable membrane filter. Analyze the amount of guest permeating through given membrane by an appropriate analytical method such as high-­ performance liquid chromatography (HPLC).

2.2  Self-Aggregate or Cluster Solutions

Prepare aqueous solutions containing various concentrations of aggregating molecules (e.g., drug or cyclodextrin) using ultrapure water and analytical grade reagents. Filtrate through 0.45 μm cellulose acetate or comparable membrane filter and analyze the amount of compound permeating through membrane by an appropriate analytical method.

2.3  Receptor Solutions

Prepare aqueous solutions containing identical composition as saturated guest/host solution without guest compound or medium in case of cluster solutions. Filtrate through 0.45 μm cellulose acetate membrane filter or sonicate to deaerate prior to use.

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2.4  Semipermeable Membrane

Cut single-layer semipermeable cellulose ester membrane to a suitable size to cover the diffusion area of the Franz cells or dialysis devices. Soak in degassed receptor solution for at least 6 h or overnight. Membrane must be in equilibrium with the receptor solution prior to the test. Do not let membrane become dry.

3  Methods 3.1  Permeation Studies 3.1.1  Franz Diffusion Cells

1. Mount receptor compartments of vertical Franz diffusion cells on stage magnetic stirring apparatus or suitable supportive apparatus to hold each cell in place. Point the sampling ports at an angle out to the side so samples can be easily collected. 2. Place a magnetic stirring bar in each cell. Ensure that suitable size of magnetic stirring bars is used. 3. Pre-fill each receptor compartments with deaerated receptor solution until the cell is about 90% filled (see Note 3). 4. Start magnetic stirring apparatus at constant speed (see Note 4) to ensure that there are no bubbles in the cell especially on the glass wall of receptor compartment. If bubbles are noticed, run the stirrer at high speed for a few seconds or use a sampling needle to release bubbles. 5. Continue to fill receptor compartment (with receptor solution) until there is a positive meniscus covering the top of the cell. 6. Carefully mount a single-layer-soaked membrane using a pair of tweezers (see Note 5). Inspect underneath the mounted membrane for bubbles. Always ensure that the stirring is off before placing the soaked membrane on the cells. 7. Assemble donor and receptor compartments, pinch together with pinch clamp and ensure that there is no leakage. Soaked membrane must completely cover the diffusion area of cell and the two compartments aligned. 8. Start the stirrers and pipette tested solution into donor compartment. Start the timer for the experiment and cover donor compartment with adhesive seal film (see Note 6). It is recommended to run each test solution at least triplicate. 9. At the sampling time point (see Note 7), withdraw an appropriate volume of receptor medium from close to the bottom of receptor compartment through sampling port using syringe connected with suitable length needle. Replace immediately with an equal volume of degassed receptor solution. Confirm that there are no bubbles underneath membrane during sampling and refilling. 10. Analyze the content of guest compound in sampling solutions with an appropriate analytical method (e.g., HPLC).

Aggregate Determination by Permeation Technique 3.1.2  Micro-Equilibrium Dialysis Device

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1. Place perpendicularly Teflon bar with two connecting rods at each edge of the bar. 2. Place precut soaked membrane on the Teflon bar. Ensure that membrane below the top edge of the bar and the lower edge of membrane covers the bottom of all wells. 3. Repeat layering soaked membrane and Teflon bar until the device is fully assembled (see Note 8 and Fig. 2). Flatten membrane before the next Teflon bar is put in place. 4. Insert the fully assembled Teflon block into the base of dialysis device and then tighten them. 5. Immediately add receptor solution to the dialysis side of the wells, assigned as receptor compartment, to prevent dehydration of soaked membranes. Ensure that no bubbles or leakage is noticed. 6. Add equal volume of test solutions to the other side of the dialysis well (as donor compartment) using appropriate pipetting device (see Note 9) and start timer. 7. Cover the top surface of dialysis device with an adhesive sealing film to prevent evaporation. 8. At equilibrium time (see Note 10), discard solutions from receptor compartment to analyze the amount of aggregating molecule with an appropriate analytical method.

3.1.3  Cuplike MINI Dialysis Device

1. Add 1.2 mL of receptor medium to 1.5 mL conical tube and set aside. 2. Load ultrapure water into dialysis device and observe for at least 5 min to moisture membrane and check the integrity of device. If droplets are noticed from across the membrane, leakage occurs and the device should not be used. 3. Decant ultrapure water and shake dialysis device to completely remove water. Do not touch the wetted membrane with ungloved hands. Once the membrane is wet, do not let it become dry. 4. Immediately pipette a 0.5 mL of tested solution and then cap the dialysis device to prevent evaporation. 5. Slowly place the filled dialysis device into conical tube containing receptor (i.e., dialysis cell). Ensure that the membrane contacts with receptor solution and does not introduce any bubbles. 6. Place individually dialysis cell on microcentrifuge tube rack or suitable supportive apparatus. 7. Shake gently on an orbital shaker (i.e., 100 rpm). Experiment is performed at room temperature (unless indicated otherwise).

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8. At sampling time, remove dialysis device and collect sample from conical tube. 9. Analyze the amount of interested molecule permeating through membrane with an appropriate analysis method. 3.1.4  Determination of Permeation Profiles



1. Plot the amount of permeated compound over sampling time point and calculate the steady-state flux (J) following the equation J=

dq A × dt

dq is the slope of the linear section of the amount of the dt guest in the receptor compartment (q) versus time (t) (see Note 11) and A is the surface area of mounted membrane. where

2. Plot steady-state flux of guest compound against concentration of host compound for permeation profile (see sample in Fig. 4) or steady-state flux vs. initial concentration of interested molecule, in case of clusters. 3.2  Evaluation of Guest/Host Aggregate Profiles



1. Perform permeation profile of each MWCO of membrane separately. 2. Calculate the fraction of aggregates (fA) (see Note 12), aggregates with molecular weight larger than pore size of membrane and unable to penetrate through that membrane from permeation profile, according to the following equation: fA = 1−

J exp J theo



where Jexp is the experimental flux and Jtheo is the theoretical flux when the free guest compounds and aggregates are able to permeate through the membrane. 3. Analyze the aggregation process presented as aggregation population (fD) and defined as

f D = f Ai − f Aj where i and j are incremental MWCO values and i  0 pull_coord1_rate = 0.01 ; 0.01 nm per ps = 10 nm per ns pull_coord1_k = 1000 ; kJ mol^-1 nm^-2 In the above code we ask the program to perform a pulling simulation (pull=yes) and we impose one reaction coordinate. We define two different groups (CC and 2-HP-B-CD in this case) by imposing a harmonic potential (pull_coord1_type=umbrella). We choose to pull as per distance (different options such as direction are available; for further alternatives see  Note 6). The next code line (pull_coord1_dim= N N Y) specifies that the pulling will take place only to the z-direction with N standing for No and Y standing for Yes. The reaction coordinate connects groups 1 and 2 and the first COM distance is the reference distance for the first frame.

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Fig. 10 A model protocol file for the pulling process in the isothermal-isobaric ensemble (NPT)

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The simulation will take place with a pulling rate of 0.01 nm/ps (meaning that during the 500 ns of simulation, the drug will be pulled for 5 nm) and the force constant will be 1000 kl/mol. 1. Create the binary input file using the following command: gmx_d grompp -f md_pull.mdp –n index.ndx -c npt.gro -p topol.top -r npt.gro -t npt.cpt -o pull.tpr 3.4.2  Run the Pulling Simulation

The simulation will be run using a similar command, but in this case the –px and –pf commands will be used additionally, in order to save the pull COM coordinates and forces: gmx_d mdrun -deffnm pull -pf pullf.xvg -px pullx.xvg

3.4.3  Collect Frames

Now a series of configurations will be extracted from the trajectory in order to use some of them as the starting configuration for the umbrella sampling procedure, as it is schematically illustrated in Fig. 3. In order to extract the frames from the pulling trajectory (a file will have been created and named as pull.xtc), use the trjconv module of gromacs. When prompted, choose to save the whole system. The terminal command for the frame extraction is gmx_d trjconv -s pull.tpr -f pull.xtc -o conf.gro –sep A series of coordinate files (conf0.gro, conf1.gro, etc.) will be produced, corresponding to each of the frames saved in the continuous pulling simulation. The COM distance between CC and 2-HP-B-CD will be calculated by the distance module of gromacs. To iteratively call the module distance on all of these frames that were generated, use the bash script (get_distances.sh) provided by Assist. Prof. Lemkul at his website (http://www.mdtutorials. com/gmx/umbrella/05_pull.html) and change it as follows. First comment out or delete the line that starts with “echo 0” since it contains the command that you just did, i.e., separate the trajectory in frames. Then, alter the number of iterations (for ((i=0; i99.5%), Milli-Q H2O. Prepare all solutions using Milli-Q H2O (alternatively water for injection can be used) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise).

3  Methods Carry out all procedures at room temperature unless otherwise specified. In the case of curcumin, experiments for the encapsulation of 50% and 100% w/w drug relative to the hydrophobic core were attempted, while in the case of indomethacin experiments for the encapsulation of 20%, 50%, and 100% w/w drug relative to the hydrophobic core were attempted (see Note 1). 3.1  Solution of Pluronic F-127 with 50% Curcumin Encapsulated (Organic Cosolvent Protocol)

1. In a glass vial weigh 10  mg Pluronic F-127 and add 1  mL acetone. Wait for the polymer to dissolve (as inspected visually). 2. In a second vial, weigh 1.5  mg curcumin and add 200  μL acetone. Wait for the drug to dissolve (as inspected visually).

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3. Mix the Pluronic F-127 solution with the curcumin solution in one vial. 4. In a pre-weighed vial, add 10 mL filtered Milli-Q water and a magnetic stirrer, place it over a hot plate, and turn on the stirring as much as needed in order to see a vortex. 5. Inject fast the mixed Pluronic F-127/curcumin acetone solution in the stirred water and wait until the solution becomes clear (approximately 5–10 min). 6. Transfer the solution in a 100 mL round-bottom flask. 7. Place the flask in a rotary evaporator, set the water bath temperature at 40 °C and the stirring at 600 rpm. 8. Leave the flask in the rotary evaporator until the whole amount of acetone is evaporated. 9. When acetone is evaporated, remove the flask and put the solution back in the vial. 10. Weigh the solution in a pre-weighed vial. The volume must be 10 mL in order to achieve a concentration of 1 × 10−3g/mL (see Note 2). If it is less, it means that part of the water was also evaporated during the evaporation step. 11. Fill out with filtered Milli-Q H2O till 10  mL final volume accordingly (see Note 3). 3.2  Solution of  Pluronic F-127 with 50% Curcumin Encapsulated (Thin-­Film Protocol)

1. In a glass vial weigh 10  mg Pluronic F-127 and add 3  mL acetone. Wait for the polymer to dissolve (as inspected visually). 2. In a second vial, weigh 1.5  mg curcumin and add 200  μL acetone. Wait for the drug to dissolve (as inspected visually). 3. Mix the Pluronic F-127 solution with the curcumin solution in a vial. 4. Transfer the solution in a 50 mL round-bottom flask. 5. Place the flask in a rotary evaporator, set the water bath temperature at 40 °C and the stirring at 600 rpm. 6. Leave the flask in the rotary evaporator until the whole amount of acetone is evaporated and a thin film of polymer and curcumin will be formed around the inner part of the flask. 7. When the thin film is formed, wait for 20 more minutes before removing the flask from the rotary evaporator in order to make sure that there is no acetone left. 8. Remove the flask and add 10 mL filtered Milli-Q H2O. 9. Gently shake the flask with your hand until the entire thin film is dissolved. 10. Transfer the solution to a clean vial.

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3.3  Solution of PEO-­ b-­PCL with 20% IND Encapsulated (Organic Solvent Protocol)

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1. Weigh 10 mg PEO-b-PCL in a vial and add 3 mL THF. Wait for the polymer to dissolve (as inspected visually). 2. Weigh 0.6 mg IND in another vial and add 200 μL THF. Wait for the drug to dissolve (as inspected visually). 3. Mix IND solution with PEO-b-PCL solution. 4. In a pre-weighed vial add 10 mL filtered Milli-Q H2O and a magnetic stirrer. Put the vial over a hot plate and turn on the stirring as much as needed in order to see a vortex (see Note 4). 5. Inject fast the PEO-b-PCL/IND mixture in the stirring water using a syringe and wait until the solution becomes clear (appx. 5–10 min) (see Note 5). 6. Transfer the solution in a 100 mL round-bottom flask. 7. Place the flask in a rotary evaporator, set the water bath temperature at 40 °C and the stirring at 600 rpm. 8. Leave the flask in the rotary evaporator until the whole amount of THF is evaporated. 9. When THF is evaporated, remove the flask and put the solution back in the vial. 10. Weigh the solution. The volume must be 10 mL. If it is less, it means that part of the water was also evaporated during the evaporation. 11. Fill out with filtered Milli-Q H2O till 10 mL final volume.

3.4  Solutions of PEO-b-PCL with 20% IND Encapsulated (Thin-Film Protocol)

1. In a glass vial weigh 10 mg PEO-b-PCL and add 3 mL THF. 2. In a second vial, weigh 0.6 mg IND and add 200 μL THF. 3. Mix the PEO-b-PCL solution with the solution of IND. 4. Transfer the solution in a 50 mL round-bottom flask. 5. Place the flask in a rotary evaporator, set the water bath temperature at 40 °C and the stirring at 600 rpm. 6. Leave the flask in the rotary evaporator until the whole amount of THF is evaporated and a thin film of polymer and IND will be formed around the inner part of the flask. 7. When the thin film is formed, wait for 20 more minutes before removing the flask from the evaporator in order to ensure that there is no THF left. 8. Remove the flask and add 10 mL filtered Milli-Q H2O. 9. Gently shake the flask with your hand until the entire thin film is dissolved. 10. Transfer the solution to a clean vial.

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3.5  UV-Vis Spectroscopy

UV-Vis spectroscopy is used for the confirmation of the successful encapsulation of the drugs into the polymeric micelles. Since it is known that the copolymers do not absorb in the UV-Vis region, the wavelength of each peak observed from the drug/copolymer solutions is referred to as the drug characteristic UV-Vis absorption. UV-Vis measurements were performed using a Perkin Elmer (Lambda 19) UV-Vis-NIR spectrophotometer (Waltham, MA, USA). The wavelength of the measurements ranged from 200 to 800 nm. It should be noted that absorption observed in all cases comes from the encapsulated drugs and not from the copolymers. 1. Select the desired measurement settings. 2. Perform a reference measurement without sample. 3. In a quartz cuvette (H × D × W: 48 mm × 12.5 mm × 12.5 mm, from Sigma-Aldrich) add 3 mL of the solution and place it in the instrument. 4. Perform the measurement. 5. We used 200 μL of the initial solution diluted with 2.8 mL H2O (see Note 6). 6. The Pluronic F-127 solution does not show absorption in the UV spectrum. On the contrary, the Pluronic F-127/curcumin solutions show the characteristic peaks of curcumin at approximately 420 nm. This observation, together with the absence of any precipitation, proves the successful encapsulation of curcumin in the polymeric micelles (Fig. 3a). 7. Again, there are no peaks observed for the PEO-b-PCL micellar solution in the UV-Vis region before the encapsulation of indomethacin. On the contrary, after the encapsulation of indomethacin with both protocols, the characteristic peak of indomethacin can be observed at approximately 260 nm, implying the existence of the hydrophobic drug in polymeric micelles (Fig. 3b). Solutions containing the micelle-encapsulated drugs show no hint of precipitation.

3.6  Fourier Transform Infrared Spectroscopy (ATR-FTIR)

Attenuated total reflection-Fourier transform infrared (ATR-­ FTIR) spectroscopy is used for the identification of the block copolymers/drug chemical structure, as well as for the confirmation of the successful drug loading into the hydrophobic cores of the micelles (appearance of new absorption peaks that correspond to the characteristic functional groups of the drug). Fourier transform infrared spectroscopy (FTIR) measurements are performed in order to examine drug encapsulation into the polymeric micelles core, using a Fourier transform instrument (Bruker Equinox 55), equipped with a single-bounce attenuated total reflectance (ATR) diamond accessory (Dura-Samp1IR II by SensIR Technologies).

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Fig. 3 UV-Vis spectra of (a) Pluronic F-127 and Pluronic F-127/curcumin solutions. The characteristic absorption peak of curcumin at 420 nm indicates its successful encapsulation in the polymeric micelles and (b) PEO-­b-PCL/20% indomethacin prepared by thin-film and organic solvent protocols. The observation of the characteristic peak of indomethacin at 260 nm confirms the successful drug loading into the hydrophobic core of the block copolymer

1. Select the preferable measurement options, e.g., spectral range from 5000 to 500 cm−1, wavenumber resolution of 4 cm−1 and number of scans 64. 2. Firstly, take a background before sample measurement. 3. Using a micropipette, add 20 μL of polymer solution directly on the surface of the flat diamond crystal. 4. Using nitrogen gas flow, the polymer solution is purged at 100 ml/min flow rate to evaporate the solvent and the recording of the spectrum starts simultaneously. 5. FTIR-ATR spectra are recorded in real-time mode until solvent evaporation occurs and a polymeric thin film is formed on the diamond’s flat surface. 6. Remove the formed thin film from the surface of the flat diamond crystal by cleaning carefully the surface with pure H2O. 7. Perform again a new measurement without the deposition of sample to clarify that the surface is clean as the obtained ATR-­ FTIR spectrum indicates. 8. For the measurements of polymeric micelles with the encapsulated drug, add 20 μL solution directly on the surface of the flat diamond crystal. 9. Follow steps 4–8 and repeat the procedure with the same measurement conditions. 10. The spectra obtained by ATR-FTIR spectroscopy certify the expected chemical structure of the block copolymers and the successful encapsulation of the drug into polymeric micelles (see Note 7).

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11. The analysis of Pluronic F-127 ATR-FTIR spectrum is accomplished as illustrated in Fig. 4a. It contains characteristic bands of C-H stretching vibrations at 2882  cm−1, C-H bending vibrations at 1468 cm−1, and one strong characteristic band at 1114 cm−1 due to the C-O-C stretching vibration of the ether bonds. Spectral fingerprints are the same for all Pluronic F-127 samples prepared with thin-film or organic solvent protocol. 12. In the case of Pluronic F-127 with encapsulated curcumin, the ATR-FTIR spectrum in Fig. 4a depicts the appearance of new absorption peaks at 1627 cm−1 due to aromatic moiety C=C stretching vibrations at 1586 cm−1 corresponding to benzene ring stretching vibrations and at 1516 cm−1 due to C=O and C=C stretching vibrations (see Note 8) [20]. 13. In the case of PEO-b-PCL polymeric micelles, the ATR-FTIR spectrum is presented in Fig.  4b. One of the characteristic absorption peaks of PEO-b-PCL appears at 2885 cm−1 which corresponds to C-H stretching vibrations. The peak at 1722 cm−1 is attributed to the C=O stretching vibration, while the peak at 1468 cm−1 indicates the C-H bending vibrations. Finally, the peak at 1110 cm−1 corresponds to C-O-C stretching vibrations. 14. The ATR-FTIR spectrum in Fig.  4b shows the PEO-b-­ PCL  polymeric micelles with encapsulated indomethacin. In this case, there is a contribution of PEO-b-PCL and ­indomethacin in the absorption of characteristic peaks, such as at 2885 cm−1, at 1722 cm−1, at 1468 cm−1, and at 1110 cm−1. The appearance of new characteristic peaks is hardly observed. However, there are new peaks recorded at 1686 cm−1 due to

Fig. 4 ATR-FTIR spectra of (a) Pluronic F-127 and Pluronic F-127/ 50% curcumin, (b) PEO-b-PCL and PEO-b-­ PCL/20% indomethacin. All samples were prepared by thin-film protocol. The appearance of new characteristic absorption peaks confirms the existence of the model drug in the polymer-drug mixed solutions

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the amide group of IND, as well as at 1595 cm−1 ascribed to C-C stretching modes of the aromatic rings. Finally, the peak at 760 cm−1 may be attributed to C-Cl stretching modes [21]. The spectral fingerprint is the same for all samples prepared with both protocols. Concluding, the appearance of new peaks indicates the existence of indomethacin in the formulations. The peaks are hardly observed due to the low percentage of the encapsulated drug (20%). 3.7  Dynamic Light Scattering (DLS)

Dynamic light scattering (DLS) measurements were conducted before and after the encapsulation of the drugs into the polymeric micelles in order to determine possible differences between the two encapsulation protocols regarding the scattering intensity, size, and polydispersity index values of the empty/loaded micelles. Size is one of the most important parameters/characteristics of nanocarriers used in drug delivery [1, 2]. DLS measurements were conducted on an ALV/CGS-3 compact goniometer system (ALVGmbH), equipped with an ALV 5000/EPP multi-τ digital correlator with 288 channels and an ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. A JDS Uniphase 22  mW He-Ne laser (λ  =  632.8  nm) was used as the light source.  All solutions were measured five times at each angle and the average was taken.  The solutions were filtered through 0.45  μm hydrophilic PVDF filters (Millex-LCR from Millipore) before measurements. The angular range for the measurements was 30–150°. Obtained correlation functions were analyzed by the cumulant method and the CONTIN software. The size data and figures shown below are from measurements at 90° (see Note 9). 1. Adjust a 0.45 μm filter to a plastic syringe and add 4 mL of the solution (see Note 10). 2. Take a RIA test tube (dimension: diameter 10  mm, height 75 mm) and remove the dust from its interior using rigorous N2 gas flow. 3. In the dust-free test tube, put 1 ml of filtered solution and shake it in order to remove possible impurities or remaining dust in the test tube. Repeat this procedure one more time. 4. Add 1 mL of the solution in the test tube and place it in the instrument for measurement. 5. Apply the above-mentioned settings in the measurement setup. 6. Measure the polymeric solutions both before and after the encapsulation of the hydrophobic drugs. 7. Size distribution graphs from CONTIN analysis for Pluronic F-127 solutions prepared with organic solvent protocol show the existence of a single peak before and after the encapsulation

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of curcumin. The size of the micelles is ca. 7  nm before the encapsulation and ca. 13  nm after the encapsulation of curcumin. However, it seems that in the presence of curcumin the size distributions become more narrow (Fig. 5a). The micelles prepared with the thin-film protocol are also monomodal and with the addition of curcumin the size distribution becomes more narrow and much more symmetrical. The size increases from ca. 21  nm before the encapsulation of curcumin to ca. 31 nm after the encapsulation. As far as size distributions of PEO-b-PCL solutions are concerned, it seems that there are no significant changes in the size of the micelles after the encapsulation of indomethacin when the thin-­ film protocol is utilized. Yet, the existence of the drug seems to increase the polydispersity index of the nanoparticles in solution (Fig. 5b). Taking into consideration all the results, it seems that each protocol used plays an important role regarding the size and polydispersity of polymeric nanocarriers obtained (see Note 11).

4  Notes 1. Higher amounts of curcumin and indomethacin encapsulation (ca. 100%) led to precipitation phenomena, when the drug/ copolymer solutions were added in the aqueous media. These observations indicate that larger amount of the hydrophobic drug results in colloidally unstable solutions/formulations.

Fig. 5 Size distribution graphs from CONTIN analysis for (a) Pluronic F-127 and Pluronic F-127/curcumin aqueous solutions prepared by the organic solvent protocol and (b) PEO-b-PCL and PEO-b-PCL/indomethacin aqueous solutions prepared using the thin-film protocol. In all cases, there are significant changes in the hydrodynamic radii and size polydispersities before and after the encapsulation. All measurements were performed at pH = 7 and 90°

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2. For reasons of comparability, all final solutions prepared by thin-film hydration and organic solvent evaporation protocols had the same polymer concentration (1x10−3 g.mL−1). 3. In all cases, Milli-Q water was filtered before each dissolution to ensure its clearance from dust and bacteria. 4. In the case of organic cosolvent protocol, when placing the vial of filtered Milli-Q water on the hot plate, the stirring speed should be enough to develop a vortex. This allows for efficient and rapid mixing of the solutions resulting in more stable and finely dispersed nanoparticles. 5. After the injection of drug/copolymer solution into the vial containing the filtered Milli-Q water, the solution mixture turned turbid due to the mixing of the solvents used and the formation of drug-loaded micelles. 6. The absorbance of solutions in the UV-Vis range should be below or equal to 1.0 in order for the Beer-Lambert law to be applicable. If not, the solutions must be diluted as much as needed in order for their absorbance to be within this range. 7. Baselines of all ATR-FTIR spectra were corrected after the measurement by subtracting the baseline in air. 8. From the ATR-FTIR spectrum of Pluronic F-127 with encapsulated curcumin the existence of a peak at 3312 cm−1 corresponding to O-H stretching is observed (Fig.  4a). This particular peak is attributed to internal moisture of the sample. 9. The size distributions obtained from DLS are from measurements at 90°, 25 °C, and pH = 7. 10. It is recommended that syringes should not have Luer Lock in order to minimize the risk of contamination. 11. It must be noted that the solutions prepared via the thin-film protocol present better colloidal stability since precipitation was observed in the solutions prepared by the organic solvent method after several hours or days depending on the copolymer/drug mixture.

Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.”

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Macromol Rapid Commun 26(24):1918– 1924. https://doi.org/10.1002/ marc.200500591 11. Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Maitra A, Maitra A (2007) Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin”): a novel strategy for human cancer therapy. J Nanobiotechnol 5(1):3. https:// doi.org/10.1186/1477-3155-5-3 12. Sharma RA, Steward WP, Gescher AJ (2007) Pharmacokinetics and pharmacodynamics of curcumin. In: The molecular targets and therapeutic uses of curcumin in health and disease. Springer, New York, NY, pp 453–470. https:// doi.org/10.1007/978-0-387-46401-5_20 13. Liu M, Teng CP, Win KY, Chen Y, Zhang X, Yang DP, Li Z, Ye E (2019) Polymeric encapsulation of turmeric extract for bioimaging and antimicrobial applications. Macromol Rapid Commun 40(5):e1800216. https://doi. org/10.1002/marc.201800216 14. Nalamachu S, Wortmann R (2014) Role of indomethacin in acute pain and inflammation management: a review of the literature. Postgrad Med 126(4):92–97. https://doi. org/10.3810/pgm.2014.07.2787 15. Dupeyron D, Kawakami M, Ferreira AM, Caceres-Velez PR, Rieumont J, Azevedo RB, Carvalho JC (2013) Design of indomethacin-­ loaded nanoparticles: effect of polymer matrix and surfactant. Int J Nanomedicine 8:3467– 3477. https://doi.org/10.2147/IJN.S47621 16. Khadka P, Ro J, Kim H, Kim I, Kim JT, Kim H, Cho JM, Yun G, Lee J (2014) Pharmaceutical particle technologies: an approach to improve drug solubility, dissolution and bioavailability. Asian J Pharm Sci 9(6):304–316. https://doi. org/10.1016/j.ajps.2014.05.005 17. Wei Z, Hao J, Yuan S, Li Y, Juan W, Sha X, Fang X (2009) Paclitaxel-loaded Pluronic P123/F127 mixed polymeric micelles: formulation, optimization and in  vitro characterization. Int J Pharm 376(1–2):176–185. https:// doi.org/10.1016/j.ijpharm.2009.04.030 18. Dabholkar RD, Sawant RM, Mongayt DA, Devarajan PV, Torchilin VP (2006) Polyethylene glycol-phosphatidylethanolamine conjugate (PEG-PE)-based mixed micelles: some properties, loading with paclitaxel, and modulation of P-glycoprotein-mediated efflux. Int J Pharm 315(1–2):148–157. https://doi. org/10.1016/j.ijpharm.2006.02.018 19. Kabanov AV, Batrakova EV, Alakhov VY (2002) Pluronic® block copolymers as novel polymer

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Chapter 7 Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation Maria Theodosiou, Theodora Koutsikou, and Eleni K. Efthimiadou Abstract This chapter focuses on the in vitro biological evaluation of multisensitive nanocontainers as drug delivery systems for cancer treatment. Cancer tissues possess some unique characteristics such as increased temperature due to inflammation, thermal vulnerability (40–45 °C), low cellular pH, and redox instabilities. The employment of polymers bearing pH, thermo, and/or redox sensitivities in the synthesis of hollow polymeric nanostructures has led to the formulation of a variety of drug delivery vehicles that are capable of targeted delivery and trigger specific drug release. The cavity in the structure allows for the encapsulation of anticancer drugs as well as other moieties with anticancer activity, like iron oxide magnetic nanoparticles. The drug loading and release capability of the nanocontainers is evaluated prior to biological studies in order to determine the concentration of the drug in the structure. The in  vitro assessment includes cytotoxicity studies, quantitatively through the colorimetric MTT assay as well as qualitatively via the scratch-wound healing assay, on both cancer and healthy cell lines. The cellular localization of the studied drug-loaded and unloaded nanocontainers is determined through confocal fluorescence microscopy. Key words Polymeric nanocontainers, Nanomedicine, MTT assay, Scratch-wound healing assay, Multisensitive drug delivery systems, Triggered drug release

1  Introduction Nanomedicine is the field of medicine where organic, inorganic, and/or hybrid materials at the nanoscale (nanomaterials) can be used in therapeutic and diagnostic (theranostic) applications for various diseases [1]. Great focus has been given in the application of nanomaterials as site-specific and sustainable drug delivery systems (DDS) [2, 3]. One of the main nanomaterials in DDS research are the stimuli-“sensitive” or -“responsive” polymers [4]. Polymers, depending on their physicochemical characteristics, display structural changes upon endogenous or exogenous triggers and can be classified accordingly into three main categories: (i) pH-sensitive polymers incorporating weak acidic or basic ionic functional groups which respond to endogenous tissue pH variations [5], (ii) therThomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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mosensitive polymers that exhibit sol-gel transitions in response to both endogenous and exogenous thermal fluctuations [6], and (iii) redox-sensitive polymers commonly containing labile disulfide linkages that respond to endogenous redox alterations [7]. Other emerging types of responsive polymers for targeted drug delivery have also been reported and can be separated based on the origin of stimuli. For internally triggered DDS, biologically, enzyme- and inflammation-responsive polymers have been developed while externally triggered DDS include polymers that are photo, ultrasound, magnetically, or electrically sensitive [8]. Recent trends in polymer DDS account for the combination of two or more of the aforementioned characteristics in one polymeric structure which can result in a dual or multisensitive polymer, thus leading to a multifunctional material that can act in a more holistic approach for treatment of the targeted disease [9]. The morphology of these kinds of polymers at the nanoscale can result in various moieties like polymersomes, nanocontainers, hydrogels, polymeric micelles, nanospheres, and dendritic nanocarriers [10]. The use of stimuli-responsive polymers as DDS is based on their ability to interact with a drug, bind covalently or electrostatically, and release it upon specific trigger, which depends on the type of the targeted disease [11]. For example, in cancer theranostics the rationale behind the design of a responsive polymeric nanomaterial is based on the physiology of cancer tissues, acidic environment, thermal liability (40–45  °C), redox instabilities, specificity of growth factors, and rise of the enhanced permeation and retention (EPR) effect [12]. Bearing this into consideration by loading an anticancer drug in a single- or multisensitive polymeric nanocarrier which is further functionalized with targeting agents for cancer cells, prolonged circulation of the drug, selective recognition, and targeted release of the drug can be achieved. In this way chemotherapy becomes more efficient and less toxic for the patient [13]. Toniolo et  al. synthesized a triple-sensitive (pH, thermo, redox) polymeric nanocontainer, loaded with the model anticancer drug daunorubicin hydrochloride. These nanocarriers exhibited a drug-loading capacity and an encapsulation efficiency of 85% and 68% approximately whereas the drug release profile was significantly enhanced at acidic pH, increased temperature, and presence of glutathione [14]. Inorganic nanomaterials can also be incorporated in the structure of polymeric nanocarriers. For the purpose of this study, iron oxide nanoparticles are presented. Iron oxide nanoparticles are superparamagnetic (mNPs) and thus under the application of an alternating magnetic field they dissipate thermal energy due to Néel and Brown relaxations. One of the most studied applications of mNPs in cancer treatment is through magnetic hyperthermia treatment [15]. Polymeric nanocontainers containing iron oxide nanoparticles (mNCs) can be used in magnetic hyperthermia and cause a synergistic effect of temperature increase in the tumor area

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that can inherently lead to regression as well as promote the release of anticancer drugs loaded in the mNCs. Efthimiadou et al. fabricated pH-responsive hollow microspheres by using emulsion polymerization in a two-step procedure and further modified their surface with iron oxide superparamagnetic nanoparticles (mNPs). The mNPs added an extra functionality to the microspheres that could be used as a bifunctional DDS for controlled drug release in combination with magnetic hyperthermia. The inner cavity formed during shell fabrication aims in the accommodation of guest molecules like the drug doxorubicin hydrochloride and iron oxide nanoparticles. Microspheres’ drug-release behavior was evaluated under different pH conditions as well as under magnetic hyperthermia, which induced controlled release [16–18]. Herein we present the basic strategies of testing the drug loading and release behavior of a polymeric nanostructure as well as the in vitro biological evaluation protocols followed to assess their impact on cancer theranostics.

2  Materials 2.1  Evaluation of Drug Loading and Release 2.1.1  Buffer Solutions [19–21]

All solutions must be prepared in double-distilled water at room temperature and stored as indicated. 1. Phosphate-buffered solution (PBS) 0.1  M, pH  7.4: Mix 40.5 mL of solution A, 0.2 M sodium phosphate and dibasic dihydrate (Na2HPO4•2H2O), with 9.5  mL of solution B, sodium phosphate, monobasic, and monohydrate (NaH2PO4•H2O), and dilute to 100 mL. Store at 4 °C. 2. Citrate buffer solution (CBS) 0.1 M, pH 4.6: Mix 44.5 mL of solution A, 0.1  M citric acid monohydrate (C6H8O7•H2O), with 55.5 mL of solution B, 0.1 M trisodium citrate and dihydrate (C6H5O7Na3•2H2O). Store at 4 °C.

2.1.2  Drug-Loading Suspensions

Suspend the desired amount of nanocontainers in PBS with the anthracycline model drug in a ratio of 1:1 (w/w) under gentle agitation for 24–72 h depending on the interaction between the selected drug and the nanocontainers. Detailed procedure is found in Subheading 3.1.1.

2.1.3  Drug-Release Suspensions

Drug-loaded nanocontainers are injected in semipermeable cellulose membrane dialysis bag with a cutoff molecular weight that allows the drug to be diffused.

2.2  Biological Evaluation

The growth medium is high-glucose Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2  mM l-glutamine, and antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin).

2.2.1  Culture Media

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2.2.2  MTT Solution Preparation

Dissolve 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in PBS at a concentration of 1 mg/mL by using vortexing or sonication and filtration. Store the solution at −20 °C for 4 months and at −4 °C for few days. MTT is photosensitive so use foil to protect it.

2.2.3  Other Reagents and Materials

1. Dimethyl sulfoxide (DMSO). 2. Dulbecco’s phosphate-buffered saline (sterile PBS, 10 mM). 3. Sonicator. 4. Cellulose membrane dialysis bag of specific molecular weight cutoff. 5. Magnetic hyperthermia apparatus. 6. EDTA-trypsin. 7. L-Poly lysine. 8. p-Formaldehyde. 9. Antiphotobleacher: b-Mercaptoethanol. 10. 96-Well plates, 6-well plates. 11. UV-vis microplate reader. 12. UV-vis spectrophotometer. 13. Glass coverslips. 14. Hematocytometer.

3  Methods 3.1  Drug Loading and Release

Tumoral tissues exhibit pH, thermal, and redox instabilities which are taken into consideration when designing a polymeric nanocontainer (NCs) as a DDS. In more detail, cancer cells proliferate in a more acidic environment; they are very sensitive to temperatures between 40 and 45 °C and they tend to thrive in a hypoxic environment. These three basic characteristics can be exploited by fabricating a model triple-sensitive polymeric nanocontainer. For example, as a temperature-sensitive polymer PNIPAm is commonly used and has a lower critical solution temperature (LCST) of 32 °C where it shows a reversible volume-phase transition. Another commonly used polymer is methacrylic acid (MAA) which is pH sensitive and under acidic environment it is fully protonated, facilitating the release of an electrostatically bound drug and cellular uptake through adsorptive endocytosis. Finally, polymers containing glutathione labile bonds like N,N′-(disulfanediylbis(ethane-2,1-diyl)) bis(2-methylacrylamide) (DSBMA) are employed in order to allow site-specific cleavage in the reductive tumoral environment. Nanocontainers synthesized with these kind of polymers can serve as a model platform for intracellular release of anticancer drugs and

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of enhanced therapeutic efficacy due to their stimulus-sensitive nature. Above their volume-phase transition temperatures they shrink; in acidic pH they are protonated and at high intracellular glutathione (GSH) levels they suffer from disulfide bond collapse. Finally, multisensitive polymeric nanocontainers modified with iron oxide nanoparticles (mNCs) possess an extra sensitivity related to heat in the concept of inducing cancer cell apoptosis and enhancement of drug release under the application of magnetic hyperthermia. Combination of the aforementioned materials promotes drug release and in general targeted intracellular drug delivery. 3.1.1  Drug Loading in Nanocontainers

1. Create a standard curve at pH 7.4 based on the concentration-­ dependent UV-vis absorption peak of the employed model drug (e.g., doxorubicin hydrochloride is at 480  nm) (see Note 1). 2. In a container of known mass, disperse 5 mg of hollow NCs or mNCs in 5  mL of phosphate-buffered solution at pH  7.4 (PBS) (see Note 2). 3. Add 5 mg of drug and sonicate for 5 min at 25 °C. 4. Stir for 24–72 h under gentle agitation at 25 °C. 5. Centrifuge the mixture in order to remove the unloaded drug. Resuspend the material by vortexing and centrifuge at 6080 × g until supernatant is clear or measured absorption is close to zero. 6. Measure the absorption of centrifugations supernatants containing the unloaded drug. 7. Freeze-dry, weight, and store the precipitate at 25 °C for a few days until the release study. 8. Determine the amount of loaded drug via standard curve methodology. 9. Calculate the loading content using the equations below: Loading capacity  %  

3.1.2  Drug Release from Nanocontainers

Weight of the drug in NCs 100 Total Weight of the NCs

Encapsulation efficiency  %  



Weight of the drug in NCs 100 Weight of the feeding drug

Generally in vitro release studies are performed at 37 °C (physiological temperature), though in some cases testing is performed at elevated temperatures for exploring and characterizing drug release using a variety of dosage forms. The most renowned and versatile method of assessing drug release from nano-sized dosage forms is the dialysis method. Using a dialysis membrane, which is permeable by the desired drug, physical separation will take place through diffusion. The protocol for this is as follows:

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1. Create a standard curve at pH  7.4 and 4.6 based on the concentration-­ dependent UV-vis absorption peak of the employed model drug (e.g., doxorubicin hydrochloride is at 480 nm). 2. Redisperse × mg of loaded NCs in water. 3. Introduce the dispersion into the dialysis bag and seal it. 4. Place the dialysis bag in a vessel containing the desired buffer solution (pH 7.4 and/or 4.6) at different temperatures. 5. Remove aliquots of 0.5 mL at different time points and add 0.5 mL to maintain the total volume. 6. Measure characteristic absorption wavelength of released drug at each aliquot removed. 7. Quantify the release percentage by the standard curve equation created in step 1. 8. Investigate the mechanism of release according to Korsmeyer– Peppas equation:

R % t   kt n



R%(t) is the percent of drug released at time t, while k and n are the kinetics constant and the release exponent, respectively. 3.2  Hyperthermia Measurements

In order to assess the optimum frequency related to the appropriate concentration for in vitro and in vivo experiments, first the mNCs are tested in aqueous solutions ex vitro. 1. Create 1  mL dispersions of consecutive concentrations of mNCs in ddH2O in glass vials of the same dimensions. 2. Insert glass vials in the coil of the magnetic hyperthermia apparatus (see Note 3). 3. Apply alternating magnetic field of various frequencies for 30 min. 4. Record temperature fluctuations. The optimum sample and frequency to be used for further biological evaluation should have a thermal response plateau between 40 and 45 °C at the shortest time interval.

3.3  Cytotoxicity and Biocompatibility Evaluation

Comparative studies should be performed where the tested sample should include NCs and/or mNCs, drug-loaded NCs, and/or drug-loaded mNCs and pure drug. The concentrations at which all the samples will be evaluated are tested in a range around the IC50 of the pure drug used for drug loading in order to assess the novelty of the DDS compared to the free drug.

3.3.1  Cell Culture

In order to perform an in vitro biological evaluation proper human cell lines should be cultured according to American Tissue Type

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Collection (ATTC) guidelines. Cells are commonly stored in liquid nitrogen (−195.8 °C) in special vials and the thawing process is as follows: 1. Prepare water bath at 37 °C. 2. Submerge the vials for 2–3 min and gently agitate. 3. Disperse them in growth medium. 4. Transfer to cell culture flask. 5. Incubate until it becomes confluent. The incubation environmental conditions should be at 37 °C in a humidified atmosphere with 5% CO2 supply. The cryo-storage medium is FBS and it is common to be enriched by DMSO which is used in order to avoid the formation of ice crystals during freezing that would be detrimental for the cells. In this case the growth medium where cells were incubated should be removed as soon as they start to grow in order to avoid contamination from DMSO. When cell cultures reach confluency on the first passage the cells can be detached and used for the biological evaluations discussed below. The detachment protocol involves the following steps: 1. Remove supernatant growth medium. 2. Gentle rinse with PBS of a volume enough to cover the flask. 3. Remove PBS. 4. Add trypsin of known volume just enough to cover the flask. 5. Incubate for a time depending on the specification of each cell line to achieve detachment. Caution! Do not exceed the time limit as trypsin might cause cell damage. 6. Agitate the flask gently. 7. Add triple volume of growth medium based on the added trypsin volume and cell suspension. 8. Count alive cells with hematocytometer according to published protocol (see Note 4). 9. Create dilution to reach the desired cellular population for the applied assay. For most assays a confluency of 70 or 80% is required after a 24-h incubation. 10. Seed the cell-containing growth medium of known population in the appropriate plate for the experiment. 3.3.2  MTT Cell Proliferation Assay

ΜΤΤ assay is a colorimetric methodology which is based on the reduction of yellow tetrazolium to violet formazan crystals for assessing quantitative cell viability when the cells are incubated with materials, drugs, small molecules, nanoparticles, proteins, and

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metal complexes. The assay is based on enzymatic reduction of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan which is catalyzed by mitochondrial succinate dehydrogenase. MTT assay is dependent on mitochondrial respiration and indirectly assesses the cellular energy capacity of a cell [22]. Cytotoxicity of the NCs can be evaluated by the MTT assay on different human cell lines. In order to perform the MTT assay (Fig. 1) the protocol is as follows: Following step 8 of the cell culture procedure (Subheading 3.3.1) described previously, 1. Seed 100 μL of 8 × 103 cells in each well in 96-well plate, flat-­ bottomed microplates. 2. Incubate the cells 24 h before the experiment. 3. Disperse the NCs/mNCs, free drug, and loaded NCs/mNCs in ddH2O or growth medium as stock solution. 4. Create serial dilutions of comparable concentration for each sample in growth medium of at least six different concentrations. 5. After 24  h (70% confluency), remove supernatant growth medium. 6. Remove cellular debris by adding and quickly but gently removing 100 μL PBS. 7. Add 100  μL of each concentration in row triplicate wells as well as 3 columns of control wells in each plate containing pure growth medium. 8. Incubate the cells for different time intervals (e.g., 4, 24, 48, and 72 h). 9. Remove the supernatant and wash twice with PBS. 10. Replace with 100 μL MTT solution. 11. Incubate for 4 h, or until the formazan crystals have formed.

Fig. 1 Schematic representation of MTT assay procedure

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12. Remove MTT solution. 13. Solubilize the formazan crystals by the addition of 100  μL DMSO or isopropanol. 14. Record the absorbance using a UV-vis microplate reader at a specific wavelength (see Note 5). 15. Average control wells’ absorption readings and calculate standard deviation. 16. Average the treated triplicate wells’ absorption readings and standard deviation (SD). 17. Subtract control average from each treated average absorption value. 18. Convert to percentile values that express cellular proliferation, aka cell viability (see Note 6). 19. Plot the % viability (y-axis) versus the tested sample concentrations (x-axis) in a column graph expressing the SD with error bars. 3.3.3  In Vitro Cytotoxicity Studies Under Hyperthermia

The induced cytotoxicity when cells are treated with mNCs drug loaded or unloaded can be assessed by performing the MTT assay after the application of magnetic hyperthermia in treated cell samples. According to our protocol the steps are briefly described below: 1. Treat the appropriate number of cells with mnps or magnetic NCs in desired concentrations. 2. Incubate the cells for 24 h (see Subheading 3.2, step 2). 3. Wash with PBS twice to remove the non-internalized material. 4. Add the appropriate amount of trypsin to suspend the cells. 5. Suspend cells to growth medium in the appropriate concentration in sterile tube. 6. Treat the solution with alternating magnetic field for 30 min in agreement to hyperthermia measurements ex vitro. 7. Seed the treated cells of different concentrations of drugloaded and -unloaded mNCs or mNPs in a 96-well plate. 8. Apply MTT assay (see Subheading 3.2, step 2). 9. In this way induced cytotoxicity from the treated samples can be indirectly calculated from the % viability.

3.4  Scratch-Wound Healing

The scratch-wound healing assay [23] is performed usually in 6-,12-, or 24-well plates with plastic or glass slides in each well, where the cells are seeded in the appropriate growth medium (Fig.  2). The same number of cells should be seeded (3 × 10^5 cell/mL) in each well in order to have comparable cell culture monolayers. As soon as confluency is reached (approx. 24 h) the assay can begin:

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Fig. 2 Schematic illustration of the scratch by a tip and compound treatment for the scratch-wound healing assay

1. Remove supernatant from each well. 2. Add a small portion of PBS to remove cellular debris, then gently shake the plate by hand, and remove it with a pipette. When removing medium or PBS with a pipette do not touch the formed cellular monolayer to avoid any unwanted scratches that will impede the assay. 3. Create the scratch. There are two methods that can be followed: One is by hand with a sterilized tip of a pipette which is the cost-effective way. Apply medium pressure as overpressing the tip might harm the slide surface. Try to create consistent scratches with gaps of the same width in all wells. The other is by adding an insert before the cell seeding and removing it after cells have reached confluency. Different shapes and sizes of inserts are commercially available like linear or circular. Inserts guarantee reproducibility of the wound. 4. Wash again with PBS to remove the extracted cells. 5. Add growth medium with and without the tested substances. Always use one well in each plate as control (plain growth medium). The other wells should be treated with growth medium containing the final product and separately all the added moieties like drugs, targeting agents, and nanoparticles. 6. Observe and acquire image under microscope at time point zero (t = 0 s/min/h/days). This should be the 0% gap width coverage or 100% gap width. 7. Continue the observations and image acquisitions at the desired time intervals, e.g., at 24, 48, and 72 h, depending on the tested substances. 8. You can conduct image analysis through different software where the gap width at each time interval can be calculated as the cells migrate or proliferate to close the gap.

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9. You can perform quantitative analysis to calculate the percentage of wound closure by the equation [24]

Wound closure%   At 0h  At  xh / At 0h   100,



where A is the wound area at different time points t = 0 h and xh. 3.5  Imaging via Fluorescent Confocal Microscopy

Confocal fluorescent microscopy can be employed in order to identify the localization of the tested samples and interpret their interaction with the cells. The standard protocol used for this is illustrated in Fig. 3. In more detail: 1. Cultivate the cells in a 6-well plate containing coverslips with poly-l-lysine to improve adhesion. 2. Incubate for 24 h. 3. Remove growth medium. 4. Wash with PBS. 5. Add growth medium containing the sample. 6. Incubate for a desired amount of time. 7. Remove the growth medium. 8. Wash with PBS. 9. Add 4% p-formaldehyde for 8  min to fixate the cells on the coverslips. 10. Wash them again with PBS. 11. If necessary, add appropriate a fluorescent dye to do co-­ localization (DAPI blue fluorescent molecule that stains the nucleus). If the tested samples are autofluorescent it might not be needed to add another dye. 12. Mount the cells by adding an anti-bleaching compound. If the tested samples are autofluorescent and need to be tested for the efficacy of their fluorescence this step can be omitted. 13. Observe under confocal fluorescent microscope.

Fig. 3 Preparation of samples for confocal fluorescent microscopy experiment

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4  Notes 1. Standard curve preparation: Measure the absorbance of the desired drug in different concentrations at its characteristic concentration-­dependent absorption wavelength. Then plot the absorbance values and create a linear equation. This equation will be used to determine the concentration of the loaded and released amount of drug during the treatment. Standard curve should be prepared for all tested pH values. 2. The vials commonly used in loading and release studies are polypropylene Eppendorf or Falcon tubes depending on the sample volume being used. 3. There are many different types of magnetic hyperthermia apparatus commercially available. In general, they consist of a coil—or a set of coils of different dimensions—connected to a monitor that allows changing the value of the applied magnetic field and/or the frequency. The sample is placed in the center of the coil to ensure homogeneous magnetization of the sample. The temperature is monitored and recorded through various apparatus like electronic thermometers, fiberoptic temperature detectors, and thermal cameras. 4. In order to determine the number of alive cells in the suspension and be able to seed the appropriate population required to perform any of the biological assays, alive cells should be counted with a hematocytometer after treatment with trypan blue. It is a standard procedure which has already been published by Springer Protocols [25]. 5. The absorption measurement and recording of the MTT plate can be in the range of 500–600 nm depending on the solvent and reference filter should be over 600  nm. Control wells should give values close to zero (± 0.1). 6. Treatment with biocompatible NCs shows higher absorption values indicating increased viability whereas drug-loaded NCs show lower absorption values indicating decreased viability.

Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.”

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References 1. Patra JK, Das G, Fraceto LF et al (2018) Nano based drug delivery systems: recent developments and future prospects. J Nanobiotechnol 16(1):71 2. Mirza AZ, Siddiqui FA (2014) Nanomedicine and drug delivery: a mini review. Int Nano Lett 4(1) 3. Li Z, Tan S, Li S et  al (2017) Cancer drug delivery in the nano era: An overview and perspectives (Review). Oncol Rep 38(2):611–624 4. Liechty WB, Kryscio DR, Slaughter BV et  al (2010) Polymers for drug delivery systems. Annu Rev Chem Biomol Eng 1:149–173 5. Reyes-Ortega F (2014) pH-responsive polymers: properties, synthesis and applications, pp 45–92 6. Teotia AK, Sami H, Kumar A (2015) Thermo-­ responsive polymers, pp 3–43 7. Guo X, Cheng Y, Zhao X et al (2018) Advances in redox-responsive drug delivery systems of tumor microenvironment. J Nanobiotechnols 16(1):74 8. Indermun S, Govender M, Kumar P et  al (2018) Stimuli-responsive polymers as smart drug delivery systems: classifications based on carrier type and triggered-release mechanism. Woodhead Publishing, Sawston, pp 43–58 9. Pasparakis G, Vamvakaki M (2011) Multiresponsive polymers: nano-sized assemblies, stimuli-sensitive gels and smart surfaces. Polymer Chem 2:1234–1248 10. Iyisan, B. and K.  Landfester, Polymeric Nanocarriers. 2019: 53–84. 11. Petros RA, DeSimone JM (2010) Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 9(8):615–627 12. Danhier F, Feron O, Preat V (2010) To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release 148(2):135–146 13. Ganta S, Devalapally H, Shahiwala A et  al (2008) A review of stimuli-responsive nanocarriers for drug and gene delivery. J Control Release 126(3):187–204 14. Toniolo G, Efthimiadou EK, Kordas G et  al (2018) Development of multi-layered and

multi-sensitive polymeric nanocontainers for cancer therapy: in  vitro evaluation. Sci Rep 8(1):14704 15. Laurent S, Dutz S, Hafeli UO et  al (2011) Magnetic fluid hyperthermia: focus on superparamagnetic iron oxide nanoparticles. Adv Colloid Interface Sci 166(1–2):8–23 16. Efthimiadou EK, Tziveleka LA, Bilalis P et  al (2012) Novel PLA modification of organic microcontainers based on ring opening polymerization: synthesis, characterization, biocompatibility and drug loading/release properties. Int J Pharm 428(1–2):134–142 17. Tapeinos C, Efthimiadou EK, Boukos N et al (2013) Microspheres as therapeutic delivery agents: synthesis and biological evaluation of pH responsiveness. J Mater Chem B 1(2):194–203 18. Efthimiadou EK, Tapeinos C, Chatzipavlidis A et al (2014) Dynamic in vivo imaging of dual-­ triggered microspheres for sustained release applications: synthesis, characterization and cytotoxicity study. Int J Pharm 461(1–2):54–63 19. Stoll VS, Blanchard JS (2009) Chapter 6. Buffers, vol 463. Academic Press Inc, Cambridge, MA, pp 43–56 20. Good NE, Winget GD, Winter W et al (1966) Hydrogen ion buffers for biological research. Biochemistry 5(2):467–477 21. Arduengo PM (2010) Sloppy technicians and the progress of science. Promega Connections 22. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63 23. Jonkman JE, Cathcart JA, Xu F et  al (2014) An introduction to the wound healing assay using live-cell microscopy. Cell Adh Migr 8(5):440–451 24. Grada A, Otero-Vinas M, Prieto-Castrillo F et  al (2017) Research techniques made simple: analysis of collective cell migration using the wound healing assay. J Invest Dermatol 137(2):e11–e16 25. Louis KS, Siegel AC (2011) Cell viability analysis using trypan blue: manual and automated methods. Methods Mol Biol 740:7–12

Chapter 8 Drug Incorporation in the Drug Delivery System of Micelles Evangelia Soumelidou, Simona Golič Grdadolnik, and Thomas Mavromoustakos Abstract Micelles is a system frequently used for drug delivery. Drugs are incorporated and protected in micelles before being delivered. Nuclear magnetic resonance is a suitable technique to detect the localization and incorporation of drugs into the micelle system. Free radicals are used to further facilitate the probing of the interactions between drug and micelles. This information is critical because drug-micelle interactions determine how easily the drug will be released from micelles and therefore how easily will be delivered to the target. Key words Micelles, Captopril, NMR, 5-DSA spin label, Sodium dodecyl sulfate (SDS)

1  Introduction It is well known that conventional drugs possess many shortcomings in clinical applications such as inactivity in drug delivery, toxic metabolization, and high hydrophobicity. Nanopharmaceutics has been developed for optimizing the drug delivery to the target and addressing the above mentioned shortcomings. Various vehicles are well known to be developed to transfer the drugs selectively and control their release. Micelles are among the loading systems used especially for delivering drugs and controlling their release. In this matrix-loading system, the drug is embedded in the nanocarrier micelle and its release is determined by the degradation of the carrier or diffusion [1]. In our study micelles are composed by the surfactant sodium dodecyl sulfate (SDS) which is an amphiphilic molecule containing hydrophobic segment [CD3(CD2)11] and hydrophilic SO3−Na+. When its concentration exceeds the critical micelle concentration (CMC) it agglomerates in D2O because its solubility becomes very low. The hydrophobic tails of SDS squeeze together in the core in an attempt to minimize their contacts with water. The hydrophilic heads from the other aspect are exposed to water in order to decrease the system energy and provide stable forms [2]. Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Captopril (1-(2S)-3-mercapto-2-methyl-propionyl]-1-proline) is an angiotensin-converting enzyme (ACE) inhibitor that has been extensively used for the treatment of hypertension and congestion heart failure. According to the drug bank https://www. drugbank.ca/drugs/DB01197 it is water soluble (4.52 mg/mL) with logP to range between 0.34 and 1.02 based on the sources and becomes unstable as the pH becomes greater than 1.2. This fact decreases the therapeutic effect of captopril [3]. For this reason there have been important advances in the area of pharmananotechnology and the controlled release of drugs, destined to circumvent many limitations of conventional therapies for the treatment of diseases such as hyperlipidemia, hypertension, myocardial infarction, stroke, and thrombosis [4]. Captopril, according to Biopharmaceutical Classification System (BCS), is a class II drug, with high solubility but poor permeability. Thus, it is bioconjugated with a light subunit of Agaricus bisporus mushroom tyrosinase, a drug carrier, and for oral delivery [5]. Biodegradable hydrogels for its controlled delivery are used [6]. Optimization of self-nanoemulsifying orodispersible films (SNEODF) of captopril for hypertension was studied [7]. Captopril was coated with magnetic nanoparticles (MNPs) as a new dual-mode agent for simultaneous MRI contrast and drug delivery system [8]. Gastro-retentive captopril-loaded alginate beads were prepared by an ionotropic gelation method using sodium alginate in combination with natural gums containing galactomannans (Senna tora, seed gum, guar gum, and locust bean gum) in the presence of calcium chloride. The objective of this work is to develop successful formulation of gastro-retentive mucoadhesive alginate beads of captopril with galactomannan [9]. Captopril-polyethyleneimine (CP) containing low-molecular-weight polyethyleneimine and antiangiogenesis drug captopril conjugated via an amide bond was fabricated to modify gold nanoparticles and complex with siRNA to construct siRNA/CP/GNP complexes for the co-delivery of drug and siRNA in antiangiogenesis breast cancer therapy [10]. Captopril was engulfed in a cyclodextrin-based nanosponge for studying as a potential delivery system [11]. Due to its narrow absorption window, captopril has to be administered to the upper parts of the intestine in order to maintain sustained therapeutic levels. Thus, it was examined if this could be achieved by gastro-retentive dosage form (GRDF) which consists of a drug-loaded bilayer polymeric film, folded into a hard gelatin capsule [12]. Systematic studies were achieved with captopril-loaded polyester fiber mats [13] and poly(L-lactic acid/captopril) composite monofiber membranes prepared by electrospinning and in order to increase its delivery [14]. The intercalation of captopril (CP) into the interlayers of montmorillonite (MMT) affords an intestine-selective drug ­delivery system [15]. Methocel and Eudragit RS were used in captopril-­ loaded microspheres as release-controlling factors to

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evaluate its release kinetic models [16]. Silica-captopril formulations were tested and found to be attractive controlled-release systems, potentially offering better bioavailability and other benefits, compared to the commercial formulation [17, 18]. It is noted by Wang et al. [1] that there is a lack of a comprehensive understanding of the compositions or structural characteristics of nanocarriers and this leads to limited designed ideas and selectivity and makes it difficult to integrate advantages and achieve optimal design. This notion triggered our interest to study the interactions of captopril in SDS micelles using high-resolution 1H NMR spectroscopy and to provide some evidence on its molecular interactions. High-resolution NMR spectroscopy is the most suitable and powerful technique to prove at molecular level the interactions of nanocarriers with drugs. 5-DOXYL-stearic acid (5-DSA) free radical was used to further facilitate the probing of the interactions between the quest molecule captopril and SDS micelles (Fig. 1). 5-DSA spin label is a nitroxide radical localized near the head of the stearate and induces relaxation of the NMR signals in the proximity of the micelle surface. It was added to the NMR solutions in a ratio of 1:4 with respect to the captopril [19]. In specific, the aim of this chapter is to provide experimental details and notions on the procedure used, as well as to explain the useful information that can be derived.

2  Materials 1. Deuterated solvents SDS-d25 (Fig.  1), D2O, and CD3OD are obtained pure 99%+. 2. Vials for the mixing of the drug with CHCl3 were of appropriate diameter and height to accept ca 4 mL solvent. 3. NMR tubes must be appropriate for the magnetic field used. The experiments are performed in 600  MHz Varian Innova instrument but of course can be run in any higher or lower magnetic field NMR spectrometer. 4. Use 5-DSA as a probe in SDS micelles. 5. Mix 125.41 mg of dry SDS-d25 lipid with 2.20 mg captopril.

Fig. 1 Structures of captopril, SDS-d25, and 5-DOXYL-stearic acid

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3  Methods 1. Mix 2.5 mg of SDS in 1 mL CD3OD/D2O and sonicate it for 15 min (see Note 1) in order to form micelles. 2. SDS concentration (400 mM) must exceed the critical micelle concentration of 8.2 mM in order to ensure micelle formation and the applied molar ratio of 5 mM captopril/400 mM SDS-­ D25 (1:80) must be used to establish the immersion of at least one drug molecule in the micellar aggregate, assuming an SDS aggregation number of at least 60 monomers. The sample is then transported to a 600 μL NMR tube. 3. Use increasing quantities of 5-DSA in the sample in order to examine its concentration-dependent effects. Every addition contains 5 μL and after four additions, the molar ratio of drug to the spin label is 1:1 (see Note 2). 4. NMR data were collected on a Varian INOVA 600 MHz spectrometer (Slovenian NMR Centre at National Institute of Chemistry) using pulse sequences and phase cycling routines provided in spectrometer library of pulse programs (see Note 3). The 1H spectral width was set to 5679.8 Hz according to the range of 1H resonances of captopril. 5. The ROESY experiment was recorded with 4096 data points in t2, 238 complex points in t1, 32 scans, a relaxation delay of 1.5 s, and a 4 kHz spin-locking field strength. A mixing time of 150  ms was used according to our previous studies in SDS micelle environment [19–22] (see Note 4). 6. Experiments were run right after the preparation and at stable temperature (see Notes 5 and 6).

4  Results and Discussion In Fig. 2 is shown the 1H NMR spectrum of captopril in SDS-d25/ CD3OD/D2O. One approach for structure identification of captopril-observed resonance peaks is of course to search if similar or identical experiment is performed in the literature. This will save time and accelerate the search in understanding the effects of captopril in SDS-d25. 1H NMR results for captopril in D2O are reported by Casy and Dewar [19] and are described in Table  1. Based on the D2O-­reported results the 1H NMR chemical shifts obtained with ­D2O/CΗ3ΟD/ SDS-d25 sample are easily elucidated and are shown in Table 1.

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Fig. 2 1H NMR spectrum of captopril in SDS-d25/CD3OD/D2O obtained at 600 MHz and 25 °C Table 1 1 H NMR chemical shifts of captopril in D2O/CΗ3ΟD/SDS-d25 obtained at 600 MHz and 25 °C Chemical shift δ(ppm) Protons in D2O [REF]

Chemical shift δ(ppm) in D2O/CΗ3ΟD/SDS-d25

Number of protons

Multiplicity

2′-Me

M 1.14 m 1.10

1.21 1.19

3

d (J = 6.7 Hz)a d(J = 6.82 Hz)b

3–4

1.95–2.05 and 2.25–2.35

1.90–2.13 and 2.25–2.45

4

m

2′

3.01–3.08

3.01–3.08

1

m

3′

2.45–2.7

2.58–2.80

2

m

5

M 3.62–3.80 m 3.62–3.38

M 3.74–3.88 m 3.51–3.68

2

m

2

M 4.42 m 4.61

M 4.51 m 4.68

dd (J = 8.7, 4.1)a dd = (J = 2.18, 2.15)b

Refer to the sample captopril in D2O Refer to the sample captopril in D2O/CΗ3ΟD/SDS-d25

a

b

Important features of the spectrum are the appearance of a major (M) and minor (m) conformation according to the equilibrium shown below (Fig. 3). The 2D ROESY experiment is shown in Fig.  4 and a model of the captopril which can explain all the

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Fig. 3 Captopril exists in two forms. The major and minor. These forms are clearly depicted in the 1H NMR spectrum both in D2O and in D2O/CΗ3ΟD/SDS-d25. The protons adjacent to amide bond due to the different surrounding environment clearly show different chemical shifts

Fig. 4 Spatial correlations of captopril in D2O/CΗ3ΟD/SDS-d25 performed at 600 MHz and 37° C

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Fig. 5 This model satisfies the two critical ROEs 2′-5 and 2–5. The average distances of these two critical ROEs are 2.49 Å and 3.82 Å

ROE data is shown in Fig. 5. The most critical ROE observed is between the protons 5 and 2′ which defines the relative orientation of the ring with respect to the chain of the major conformer. The gradual addition of 5-DSA in the D2O/CΗ3ΟD/SDS-d25 sample is shown in Fig. 6. There are two major characteristics of the spectra obtained after the four subsequent additions of DSA. (a) The chemical shifts due to the captopril are not affected and (b) all the peaks decrease in intensity and are broadened which results in losing the multiplicity information. For example, after the second addition of DSA, the peaks resonated at 1.21  ppm and 1.19 ppm merge only to the one at 1.21 ppm. Thus, the information for the minor conformer depicted at 1.19 ppm is lost. Clearly, the results show that 5-DSA affects significantly all peaks of captopril. This means that DSA which is localized in intermediate polarity region (interface) of SDS is in spatial vicinity with captopril. Thus, captopril is localized in the interface of SDS. In Fig.  7, a cartoon is presented in which are shown the interactions of captopril with SDS-d25 micelles.

5  Notes 1. During the sonication heat is generated, so the sample should be in an ice bath; otherwise there is a danger of breaking the glass vial. 2. Spectral precaution of the sonicator tip is given in order not to touch the bottom or sides of the vial.

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Fig. 6 Gradual addition of DSA in D2O/CΗ3ΟD/SDS-d25 sample. (a) No addition. (b) Addition of 5 μL DSA (1:1 DSA/ captopril molar ratio). (c) Addition of another 5 μL DSA (2:1 DSA/captopril molar ratio). (c) Addition of another 5 μL DSA (3:1 DSA/captopril molar ratio). (c) Addition of another 5 μL DSA (4:1 DSA/captopril molar ratio)

Fig. 7 The picture depicts the interactions of captopril with SDS-d25 micelles. The carboxylate group of captopril orients to the positively charged sodium while the rest of the molecule is localized in the intermediate polar and hydrophobic region (interface) in order to maximize its interactions. This scheme explains the reason that captopril is affected significantly by the spin label 5-DOXYL-stearic acid as it is in its spatial vicinity

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3. All companies that sell spectrometers generally have a library of pulse programs and stored default parameter sets for common 1D and 2D NMR experiments. 4. There is no special precaution for the handling of free radicals, which are spin labels and used for investigating structural changes in lipids. 5. Although micelles are relatively stable, the noise of the spectrum is deteriorated if sample is not fresh. 6. It is essential the NMR spectrometer room temperature be stable during the experiment as the quality of the spectrum is affected by temperature instability.

Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.” SGG acknowledges support from the Slovenian Research Agency (Grant No. J1-8145). References 1. Wang N, Cheng X, Li N, Hong W, Chen H (2019) Nanocarriers and their loading strategies (review article). Adv Healthcare Mater 8(6). 1801002 (1–26) 2. Wang Y, He J, Liu C, Chong WH, Chen H (2015) Thermodynamics versus kinetics in nanosynthesis. Angew Chem Int Ed 54(7):2022–2051 3. Mehta TJ, Motihal M, Patel MR et al (2011) Optimization of granulation and compression process variables of atenolol tablets using Box-­ Behnken design. Pharm Letter 3(3):103–109 4. Virna M, Gimenez M, Kassuha DE, Manucha W (2017) Nanomedicine applied to cardiovascular diseases: latest developments. Ther Adv Cardiovasc Dis 11(4):133–142 5. Diana D, Wangsa TI, Vincencius FM, Olivia MT, Raymond RT, Heni R (2018) Bioconjugation of captopril–light subunit of Agaricus bisporus mushroom tyrosinase: characterization and potential use as a drug carrier for oral delivery. Biol Pharm Bull 41:1837–1842 6. Shahid N, Samiullah K, Umar F et  al (2018) Biocompatible hydrogels for the controlled delivery of anti-hypertensive agent: develop-

ment, characterization and in vitro evaluation. Des Monomers Polym Ed 21:18–32 7. Sawani DT, Rahul VH, Rutesh HD (2019) Evaluation of self-nanoemulsifying drug delivery systems using multivariate methods to optimize permeability of captopril oral films. Eur J Pharm Sci 130:215–224 8. Sajjad AP, Hamid RS (2018) Captopril-loaded superparamagnetic nanoparticles as a new dual-­ mode contrast agent for simultaneous in vitro/ in vivo MR imaging and drug delivery system. Pharm Chem J 10:852–862 9. Harshal AP, Lalitha KG, Ruckmani K (2015) Beads of captopril using galactomannan containing Senna tora gum, guar gum and locust bean gum. Int J Biol Macromol 76:119–131 10. Manhong L, Li Y, Xiaohui H, Xizhi L (2015) Captopril-polyethyleneimine conjugate modified gold nanoparticles for co-delivery of drug and gene in anti-angiogenesis breast cancer therapy. J Biomater Sci Polym Ed 26(13):813–827 11. Oltenanu AA, Arama CC, Bleotu C, Lupuleasa D, Monciu CM (2015) Investigation of cyclodextrin based nanosponges complexes with

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angiotensin I converting enzyme inhibitors. Farmacia 63:492–503 12. Dharani S, Subedari H, Panakanti PK, Yamsani MR (2013) Preparation and evaluation of novel expandable drug delivery system with captopril current drug therapy. Curr Drug Ther 8:206–214 13. Hua Z, Shaofeng L, Gareth RW et al (2012) A systematic study of captopril-loaded polyester fiber mats prepared by electrospinning. Int J Pharm 439:100–108 14. Wei A, Wang J, Wang X et  al (2012) Morphology and surface properties of poly (L-lactic acid)/captopril composite nanofiber membranes. J Engineered Fibers Fabrics 7(1):129–135 15. Suguna LM, Joseph SW, Mandal AB et  al (2012) Intestine-specific, oral delivery of captopril montmorillonite: formulation and release kinetics. Nanoscale Res Lett 7(15):1–8 16. Khamanga SM, Walker RB (2012) In vitro dissolution kinetics of captopril from microspheres manufactured by solvent evaporation. Dissolution Technologies, February 42–51

17. Propovici RF, Alexa IF, Novac O, Vinceanu N et al (2011) Pharmacokinetics study on mesoporous silica-captopril controlled release systems. Dig J Nanomater Biostruct 6(4):1619–1630 18. Popovici RF, Seftel EM, Mihai GD (2011) Controlled drug delivery system based on ordered mesoporous silica matrices of captopril as angiotensin-converting enzyme inhibitor drug. J Pharm Sci 100(2):704–714 19. Grdadolnik SG, Pristovšek P, Mierke DF (1997) Vancomycin: interactions with a cell model membrane. Biopolymers 42:627–632 20. Casy AF, Dewar GH (1994) Captopril and its probable contaminants: NMR and MS features of analytical value. J Pharm Biomed Anal 12(7):855–861 21. Griesinger C, Ernst RR (1987) Frequency offset effects and their elimination in NMR rotating-frame cross-relaxation spectroscopy. J Magn Reson 75:261–271 22. Kyrikou I, Grdadolnik SG, Tatari M, Poulos C, Mavromoustakos T (2003) Structural elucidation and conformational properties of the toxin paralysin b-Ala/Tyr. J Pharm Biomed Anal 31:713–721

Chapter 9 Molecular Dynamics Protocols for the Study of Cyclodextrin Drug Delivery Systems Georgios Leonis, Dimitrios Ntountaniotis, Eirini Christodoulou, and Thomas Mavromoustakos Abstract Hypertension treatment is a current therapeutic priority as there is a constantly increasing part of the population that suffers from this risk factor, which may lead to cardiovascular and encephalic episodes and eventually to death. A number of marketed medicines consist of active ingredients that may be relatively potent; however, there is plenty of room to enhance their pharmacological profile and therapeutic index by improving specific physicochemical properties. In this work, we focus on a class of blood pressure regulators, called sartans, and we present the computational scheme for the pharmacological improvement of irbesartan (IRB) as a representative example. IRB has been shown to exert increased pharmacological action compared with other sartans, but it appears to be highly lipophilic and violates Lipinski rule (MLogP >4.15). To circumvent this drawback, proper hydrophilic molecules, such as cyclodextrins, can be used as drug carriers. This chapter describes the combinatory use of computational methods, namely molecular docking, quantum mechanics, molecular dynamics, and free energy calculations, to study the interactions and the energetic contributions that govern the IRB:cyclodextrin association. We provide a detailed computational protocol, which aims to assist the improvement of the pharmacological properties of sartans. This protocol can also be applied to any other drug molecule with diminished hydrophilic character. Key words Sartans, Irbesartan, 2-Hydroxypropyl-β-cyclodextrin, Blood pressure regulation, Hypertension, Molecular modeling, Molecular dynamics, Pharmacological enhancement

1  Introduction The pharmaceutical industry aims at the development of effective medications with the lowest safety risk. However, this is not an easy task, since efficacy is often compromised for diminished side effects. Several marketed medicines contain active compounds, which possess specific physicochemical properties that deviate from an optimal pharmacological profile. The main reasons for poor drug performance are the low solubility and permeability, fast metabolism and excretion, and also undesired side effects [1, 2]. It is noted Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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that at least 30–35% of marketed drugs suffer from low solubility [3]. Sartans are a class of chemical compounds [angiotensin II type 1 receptor blockers (ARBs)] that hinder the detrimental hypertensive effects of angiotensin II at the AT1 receptor in a pathological state [4]. Despite their proven beneficial action against hypertension, sartans are characterized by high lipophilicity and violate the Lipinski rule (MLogP >4.15). This feature negatively impacts the absorbance, tissue penetration, and bioavailability of these molecules. To improve the pharmacological properties of such chemicals, one may incorporate them into the structures of hydrophilic molecules, which form inclusion complexes with the drug and may act as carriers for selective drug release to the active target. Cyclodextrins have been shown to constitute proper transport systems for sartans, as they can easily accommodate these molecules into their binding region, thus inducing favorable interactions within the inclusion complex [5]. Computational techniques are widely used for the study of biomolecular systems, mainly because they combine reliable predictions with high speed at a minimal cost. Computational chemistry has been successfully applied to the study of proteins, nucleic acids, lipid bilayers, and transport systems and in the drug design process. Therefore, it is evident that such approaches can offer significant help in the rational design of new drug formulations [6–10]. In this work, we present the detailed computational methodology for: 1. Modeling the inclusion of sartans into cyclodextrins 2. Monitoring and analyzing the conformational evolution of the complexes 3. Predicting the energetic properties of the complexes In particular, we apply computational methods [i.e., molecular docking, quantum chemistry (QM), molecular dynamics (MD), and binding free energy calculations] for the study of conformational properties and interactions between drug irbesartan (IRB) and hydrophilic 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) as a representative example (Fig. 1). We note that the same methodology can also be applied to any other drug:cyclodextrin system. Besides its effectiveness on blood pressure regulation, IRB exerts poly-pharmacologic actions against other conditions, such as diabetes and cancer, and it can be considered superior to other sartans [11–13]. However, IRB deviates from an excellent pharmaceutical profile as it presents minimal hydrophilicity and low bioavailability (approx. 65–70%) [14].

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Fig. 1 The structures of drug irbesartan (IRB) and 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) used in this work. Hydrogen atoms of IRB are not shown for simplicity. The structure of 2-HP-β-CD is presented as a surface

2  Materials Within this protocol, the following platforms, software suites, and programs were used: 1. The Cambridge Structural Database (CSD): A compound library, which contains >1 million crystal structures of organic small molecules (free access). The CSD was used to retrieve the initial structure of IRB (https://www.ccdc.cam.ac.uk/solutions/csd-system/components/csd/). 2. PubChem (U.S.  National Library of Medicine): A database of chemical structures, which also contains biological activity information (free access). PubChem was used for 2-HP-β-CD structure retrieval (https://pubchem.ncbi.nlm.nih.gov/compound/HP-beta-CD). 3. ArgusLab: [15] Software for molecular docking applications (free of charge). ArgusLab was used for the inclusion of IRB into 2-HP-β-CD (http://www.arguslab.com/arguslab.com/ ArgusLab.html). 4. AMBER 16: [16] A suite, which contains a number of programs for computational applications to biomolecular systems (license fee required). AMBER was used to perform MD simulations and molecular mechanics Poisson-Boltzmann surface area (MM–PBSA) free energy calculations for IRB:2-β-CD complexes. We note that AMBER operates on a Linux/Unix environment. The unfamiliar user may consult relevant Unix tutorials and can acquire AMBER documentation and instructions through http://ambermd.org/.

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5. Gaussian 09: [17] A program for electronic structure calculations (license fee required). Gaussian was used to perform IRB optimization and RESP charge derivation for IRB and 2-HP-β-CD (https://gaussian.com/). 6. UCSF CHIMERA 1.12: [18] A program for visualization and analysis of biomolecules (free of charge). CHIMERA was used for structure visualization, and figure generation (https:// www.cgl.ucsf.edu/chimera/).

3  Methods 3.1  Molecular Modeling 3.1.1  Structure Retrieval

3.1.2  Molecular Docking Calculations

Obtain the crystal structures of IRB and 2-HP-β-CD from the CSD and PubChem, respectively. The reference codes correspond to CCDC: 130127 for IRB and PubChem CID: 14049689 for 2-HP-β-CD.  Save the structures in PDB formats (IRB.pdb and 2-HP-β-CD.pdb). Molecular docking of IRB into 2-HP-β-CD: IRB can be included into the cavity of 2-HP-β-CD in two different orientations. The first orientation considers the tetrazole moiety of IRB near the HP end of 2-HP-β-CD, while the opposite orientation associates the butyl alkyl chain of IRB with the HP group (Fig. 2). The optimal placement of IRB in its two orientations into 2-HP-β-CD and the binding strength was predicted with ArgusLab. It is noted that any other suitable docking software can also be applied. For the sake of simplicity, hereafter we may occasionally refer only to one orientation. Open the main panel in ArgusLab and perform the following steps: 1. Open molecule (browse and upload ligand). 2. The name of selected ligand (IRB.pdb) appears on the left-side panel. Click on the cross to expand; on the extended menu click Residues and then Misc. Next, select the molecule that appears (i.e., 1 MOL) and right-click. On the new menu, select Make a Ligand Group from this Residue. 3. Repeat the procedure for the receptor: open molecule in the main panel and browse to upload 2-HP-β-CD.pdb. 4. Repeat step 2 and after clicking Misc, select all 2-HP-β-CD subunits that appear. On the new menu, select Make a Group from this Residue. Next select Binding Site. 5. In the main window, press Calculation  →  Dock a Ligand. A new menu appears with a list of docking parameters. Most of the default selections will suffice for a rough estimation. The user should carefully experiment and choose among various

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Fig. 2 The two possible orientations of IRB in 2-HP-β-CD as obtained by molecular docking calculations. 2-HP-β-CD is depicted as a transparent surface. The HP groups of 2-HP-β-CD are at the bottom. Hydrogen atoms of IRB are not shown for simplicity

options that closely depend on the system under study. However, based on personal experience, we indicate the following modifications for optimal performance. 6. Binding Site Bounding Box: Box sizes that range between 15 and 25Å (depending on the system’s size) are preferable. Docking Engine: GADock may be preferred. The other options are generally safe to be left as default. 7. In the same menu, click Advanced and increase Max. Generations to 10,000. Press OK. 8. In the main Docking menu press Start to begin the docking calculation. 9. After the calculation is over, you are provided with a docking score (in kcal/mol) and a PDB structure of the complex in its most favorable conformation (save it as complex.pdb). The two optimally docked conformations of IRB:2-HP-β-CD complexes are next prepared for MD simulations. 3.2  Structure Preparation for Molecular Dynamics

A general scheme of IRB, 2-HP-β-CD, and IRB:2-HP-β-CD complex treatment is presented in Fig.  3. More detailed instructions are provided below. IRB preparation: The structure of IRB is geometrically optimized at the HF/6-31G* level of theory with Gaussian. 1. Convert IRB file format from PDB to com with the ANTECHAMBER subroutine of AMBER for subsequent Gaussian calculations. Install and compile (i) ANTECHAMBER

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Fig. 3 Schematic workflow for systems’ preparation for MD simulations

from the AmberTools program (free of charge) and (ii) AMBER program; then run the command: antechamber –i IRB.pdb –fi pdb –o IRB.com –fo gcrt Note: For the required calculations, the use of AMBER and Gaussian is not restrictive; other appropriate molecular modeling and QM packages may be used as well. 2. Install and compile Gaussian; run Gaussian (on Linux/Unix environment) for geometry optimization of IRB and for electrostatic potential (ESP) generation: g09 IRB.com > IRB.log The input file (IRB.com) for Gaussian calculations is shown in Note 1. Output IRB.log contains the optimized structure of IRB and information regarding ESP. Note: Most of the output files produced by Gaussian and AMBER regarding this case study are very large and therefore are not presented here. 3. Generate charges according to the restrained electrostatic potential (RESP) method with ANTECHAMBER. The geometry of IRB and the RESP charges are included in the IRB.mol2 output file: antechamber –i IRB.log –fi gout –o IRB.mol2 –fo mol2 –c resp 4. Generate missing parameters for IRB with the parmchk routine of ANTECHAMBER:

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parmchk –i IRB.mol2 –f mol2 –o IRB.frcmod Missing parameters are included in the IRB.frcmod output file. 5. Assign force field and construct a parameter library for IRB with the tLEaP module of AMBER. The general AMBER force field (GAFF) was used to assign force field parameters to IRB [19]:

(i) Open tLEaP program:

tleap 

(ii) Load force field:

source leaprc.gaff 

(iii)  Load mol2 file for IRB:

MOL = loadmol2 IRB.mol2 

(iv)  Load missing parameter file for IRB:

loadamberparams IRB.frcmod 

(v) Save library for IRB: saveoff MOL IRB.lib 

The generated output IRB.lib and the IRB.frcmod will be used later for complex construction. 2-HP-β-CD preparation: RESP charges for all atoms of 2-HP-β-CD, missing parameters (2-HP-β-CD.frcmod), and library files only for the 2-HP part of cyclodextrin (2-HP.lib) are generated similarly as in steps 1–5 above during IRB preparation. AMBER employs a well-tested force field for the treatment of sugars (i.e., cyclodextrin); however, modified structures cannot be supported. Therefore, one may treat the 2-HP part of 2-HP-β-CD as a small molecule with GAFF and has to manually generate a library file (2-HP.lib) as commented above. IRB:2-HP-β-CD complex preparation: Construct topology and coordinate files, which will be used as inputs for MD simulations. The force field GLYCAM 06 [20] is used to treat the CD part of 2-HP-β-CD while GAFF is used for the 2-HP part. The TIP3P water model is employed for the treatment of solvation [21]. (i) Open tLEaP program: tleap (ii) Load force fields: source leaprc.gaff source leaprc.GLYCAM_06j-1 (iii) Assign explicit model for water molecules:

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source leaprc.water.tip3p (iv) Load missing parameters file for IRB and 2-HP loadamberparams IRB.frcmod loadamberparams 2-HP-β-CD.frcmod (v) Load library files for IRB and 2-HP: loadoff IRB.lib loadoff 2-HP.lib (vi) Load IRB:2-HP-β-CD complex file: a = loadpdb complex.pdb (vii) Assign pre-calculated RESP atomic charges to 2-HP-β-CD (see Note 2). (viii) Add a truncated octahedral box containing TIP3P water molecules around the complex. The edge of the periodic box is set to be at least 16 Å away from each complex atom: solvateoct a TIP3PBOX 16 (ix) Save topology (complexparm.top), coordinates (complexparm.crd), and PDB (complex_final.pdb) files of the complex: saveamberparm a complexparm.top complex parm.crd savepdb a complex_final.pdb 3.3  Molecular Dynamics Simulation of the Complex

The detailed preparatory steps for MD and the actual production MD run are described below. The MD calculations are performed with the GPU version of PMEMD [22] from AMBER 16.

3.3.1  Energy Minimization

Complexes are minimized in three stages for 10,000 cycles each. Minimization is performed with the steepest descent method for the first 5000 steps and the conjugate gradient algorithm follows for the next 5000 steps. The first step considers the complex practically fixed with the application of a harmonic force constant of 500 kcal mol−1 Å−2, thus allowing the structures of the water molecules to relax. During the second step, the restraint was reduced to 10 kcal mol−1 Å−2, and finally all atoms were totally unrestrained to move. A nonbonded cutoff of 10.0 Å is applied under constant volume. The three input files are provided and explained in Note 3. An example of the command to perform minimization is srun /amber16/bin/pmemd -O -i Min1.in -o complex_min1.out -p complexparm.top -c complexparm.crd -r complex_min1.rst Note the use of complexparm.top/crd files, which were generated from Subheading. 3.2; the complex_min1.rst file will be used as the input for the second stage of minimization and so on. Output file complex_min1.out contains the energy information for every step of the run.

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The next procedure involves the gradual heating of the complexes from 0 to 310 K using the Langevin thermostat [23] for temperature regulation. The simulation is performed under constant volume for 400 ps and the collision frequency is set at 2 ps−1. Positional restraints of 10  kcal  mol−1  Å−2 were applied to the atoms of the complex. The algorithm SHAKE is enabled to keep hydrogen atoms at their equilibrium position and a 2 fs time step was used [24]. The corresponding input file is given in Note 4. Execute the following command for heating MD run: srun /amber16/bin/pmemd.cuda_SPFP -O -i Heat. in -o complex_heat.out -p complexparm.top -c complex_min3.rst -r complex_heat.rst -x complex_heat.nc Note the use of complex_min3.rst from the minimization process as a restart file for heating; the complex_heat.rst file will be used as the input for the next simulation step. The complex_heat. nc file contains the trajectory generated by the run.

3.3.3  Density Equilibration

Equilibration of the complexes was performed under constant pressure in two steps of 400 ps each. In the first step, constraints of 10 kcal mol−1 Å−2 were applied to the solute, and all restraints were removed in the last step. The two input files are provided in Note 5. The corresponding command lines are Step1: srun /amber16/bin/pmemd.cuda_SPFP -O -i Density.in -o complex_density.out -p complexparm.top -c complex_heat.rst -r complex_density.rst -x complex_density.nc Step2: srun /amber16/bin/pmemd.cuda_SPFP -O -i Eq.in -o complex_eq.out -p complexparm.top -c complex_density.rst -r complex_eq.rst -x complex_eq.nc

3.3.4  MD Production Simulation

This is the final step for the generation of the MD trajectory for further analysis. Two unrestrained, constant-pressure MD simulations for IRB in the two binding orientations are performed at 310 K for 3 μs each. Additional details of the simulation are provided in Table 1 and the corresponding input file is shown in Note 6. Execute the following command: srun /amber16/bin/pmemd.cuda_SPFP -O -i MD.in -o complex_md.out -p complexparm.top -c complex_ eq.rst -r complex_md.rst -x complex_md.nc

3.4  Energetic Analysis with the  MM–PBSA Method

The resulting trajectories are subjected to MM–PBSA analysis for the estimation of the Gibbs free energy based on enthalpy and entropy contributions. Enthalpy estimation: The enthalpy of binding can be predicted with application of the MM–PBSA or the molecular mechanics–

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Table 1 Simulation parameters for production of MD simulations of IRB:2-HP-­ β-CD complexes Parameters

Value

Total simulation time

3 μs

Time step

2 fs

Periodic boundaries

Yes (constant pressure)

Pressure scaling

Isotropic position scaling

Pressure relaxation time

2.0 ps

Nonbonded cutoff

10.0

Restrained atoms

None

Bonds constrained

Hydrogens involving (SHAKE)

Temperature control

Langevin thermostat

Collision frequency

2.0 ps−1

Average temperature

310 K

generalized born surface area (MM–GBSA) method; [25–27] details on the underlying theory can be found in other publications [28, 29]. The input (mmpbsa_enthalpy.in) is presented in Note 7. Execute the following Python script in AMBER: /amber16/bin/MMPBSA.py -O -i mmpbsa_enthalpy. in -o RESULTS_MMPBSA.dat -sp complexparm. top -cp complex_only.parm.top -rp receptor_only. parm.top -lp ligand_only.parm.top -y complex_ md.nc Note that the topology (complexparm.top) and trajectory (complex_md.nc) files from Subheading 3.3 are used as inputs for the MM–PBSA calculations. Additional input files are the complex_only.parm.top, receptor_only.parm.top, and l­ igand_only. parm.top, which are topology files for the complex, 2-HB-β-CD, and IRB, respectively, and do not include any water molecules. The RESULTS_MMPBSA.dat file contains the enthalpy estimation in kcal/mol and also provides individual energy components, such as electrostatic, van der Waals, and nonpolar contributions. Entropy estimation: The entropy estimation is obtained with normal mode analysis. The execution is the same as for the enthalpy calculations, with the only difference being the use of input mmpbsa_entropy.in instead of mmpbsa_enthalpy.in (see differences in Note 7).

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Conformational analysis: Analysis of the resulting trajectories is performed with the cpptraj module [30] of AMBER. The cpptraj functionality is able to calculate a variety of properties, such as distances, angles, torsions, RMSD, hydrogen bonds, fluctuations, J-coupling, surface areas, radii of gyration, average structures, diffusion, and radial distribution functions, among many others. Here, we provide a representative example of an input (ptraj.in), which includes distance, RMSD, and fluctuation calculations (see Note 8). Execute in AMBER: cpptraj complexparm.top ptraj.in Output files are not included in the above command line as they are generated from cpptraj after they have been included in the ptraj.in file.

4  Notes 1. A simplified Gaussian input file for the geometry optimization and ESP generation of IRB (IRB.com) is shown below: IRB.com --Link1- %chk=molecule #HF/6-31G* SCF=tight Test Pop=MK iop(6/33=2) iop(6/42=6) opt nosymm remark line goes here 0 1 ##Author’s note: The initial X-­ Y-­ Z coordinates of IRB follow here (not shown). 2. The RESP charges for 2-HP-β-CD are manually assigned to each atom in tLEaP. The following command is an example for charge assignment to the oxygen atom, which is labeled “O2” and belongs to the first residue (HP group) of 2-HP-β-CD: set a.1.O2 charge −0.631696 Similar command lines should be included in tLEaP for each 2-HP-β-CD atom and its respective RESP charge. 3. Input files for complex minimization with AMBER.  Next to each command, a short explanation is offered in italics: Min1.in Minimization of system keeping complex fixed &cntrl imin=1, Perform minimization maxcyc=10000, The total number of cycles per formed ncyc=5000, First 5000 cycles with steepest descent before switching to conjugate gradient cut=10.0, Cutoff for nonbonded interactions ntb=1, Constant volume

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ntr=1, Obtain a restart file / Keep complex fixed 500.0 Positional restraints for the solute (residues 1 to 15 of 2-HP-β-CD) RES 1 15 END END Min2.in

Minimization of system keeping complex fixed &cntrl imin=1, maxcyc=5000, ncyc=5000, cut=10.0, ntb=1, ntr=1, / Keep complex fixed 10.0 RES 1 15 END END Min3.in

Minimization of system with complex unrestrained &cntrl imin=1, maxcyc=10000, ncyc=5000, cut=10.0, ntb=1, ntr=0, /

Note: The input files demonstrate the basic commands of each procedure and they should be merely considered as simplified examples. The command choices and their values may vary and depend on the particular system under study. Also, for simplicity, some less important keywords were omitted.

4. Input file for complex heating with AMBER: Heat.in Equilibration with restraints on complex &cntrl imin=0, irest=0, cut=10.0, ntb=1, ntr=1, ntc=2, ntf=2, SHAKE algorithm applied tempi=0, temp0=310.0, Target temperature ntt=3, Langevin thermostat gamma_ln=2.0, Collision frequency nstlim=200000, # of MD steps dt=0.002, time-step /

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Keep complex fixed 10.0 RES 1 15 END END / Keep complex fixed 10.0 RES 1 15 END END

5. Input files for density equilibration with AMBER: Density.in Molecular Dynamics on whole system &cntrl imin=0, irest=1, cut=10.0, ntb=2, pres0=1.0, Constant pressure ntp=1, taup=2.0, Pressure relaxation time (in ps) ntc=2, ntf=2, tempi=310.0, temp0=310.0, ntt=3, gamma_ln=2.0, nstlim=200000, dt=0.002, / Keep complex fixed 10.0 RES 1 15 END END Eq.in

Molecular Dynamics on whole system &cntrl imin=0, irest=1, cut=10.0, ntb=2, pres0=1.0, ntp=1, taup=2.0, ntr=0, ntc=2, ntf=2, tempi=310.0, temp0=310.0, ntt=3, gamma_ln=2.0, nstlim=200000, dt=0.002, /end 6. Input file for production MD run with AMBER: MD.in Molecular Dynamics on whole system for 3 μs &cntrl imin=0, irest=1,

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cut=10.0, ntb=2, pres0=1.0, ntp=1, taup=2.0, ntr=0, ntc=2, ntf=2, tempi=310.0, temp0=310.0, ntt=3, gamma_ln=2.0, nstlim=1500000000, dt=0.002, /end 7. Input files for MM–PBSA enthalpy and entropy calculations: mmpbsa_enthalpy.in Input file for running PB and GB &general endframe=1000, startframe=1, interval=10,Calculate from trajectory frame 1 to 1000 every 10 / &gb Perform MM–GBSA calculation igb=5, Use a modified GB model[31] saltcon=0.100 Salt concentration (in M) / &pb Perform MM–PBSA calculation istrng=0.100, Ionic strength (in M) / mmpbsa_entropy.in Input file for running entropy calculations using NMode &general endframe=1000, / &nmode Perform normal mode analysis nmstartframe=100, nmendframe=1000, nminterval=10, nmode_igb=1, Default GB model [32] nmode_istrng=0.1, Ionic strength (in M) / 8. Example of a simple input file for conformational analysis with cpptraj: ptraj.in trajin complex_md.nc Read and analyze each frame of MD trajectory distance :2@O1 :3@H2 out distance_2-­ 3_complex. out Calculate the distance between oxygen atom labeled O1 in residue 2 and hydrogen atom labeled H2 in residue 3. Report the results for every frame in output file named distance_2-3_ complex.out rms first mass out rms_CD.out :1-14 Calculate RMSD for all 2-HP-β-CD residues (1 to 14) and report the results in file rms_CD.out

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rms first mass out rms_IRB.out :15 Calculate RMSD for all atoms of IRB (residue 15) and report the results in file rms_IRB.out atomicfluct out fluct_CD.out :1-­ 14 Calculate RMS fluctuations for the 2-HP-β-CD residues

Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.” References 1. Hodgson J (2001) ADMET—turning chemicals into drugs. Nat Biotechnol 19(8):722– 726. https://doi.org/10.1038/90761 2. Kalepu S, Nekkanti V (2015) Insoluble drug delivery strategies: review of recent advances and business prospects. Acta Pharm Sin B 5(5):442–453. https://doi.org/10.1016/j. apsb.2015.07.003 3. Loftsson T, Brewster ME (2010) Pharmaceutical applications of cyclodextrins: basic science and product development. J Pharm Pharmacol 62(11):1607–1621. https://doi. org/10.1111/j.2042-7158.2010.01030.x 4. Kellici TF, Tzakos AG, Mavromoustakos T (2015) Rational drug design and synthesis of molecules targeting the angiotensin II type 1 and type 2 receptors. Molecules 20(3):3868– 3897. https://doi.org/10.3390/ molecules20033868 5. Hirlekar R, Kadam V (2009) Preformulation study of the inclusion complex irbesartan-­ beta-­ cyclodextrin. AAPS PharmSciTech 10(1):276–281. https://doi.org/10.1208/ s12249-009-9206-5 6. Karplus M, Petsko GA (1990) Molecular dynamics simulations in biology. Nature 347(6294):631–639. https://doi. org/10.1038/347631a0 7. Karplus M, McCammon JA (2002) Molecular dynamics simulations of biomolecules. Nat Struct Biol 9(9):646–652. https://doi. org/10.1038/nsb0902-646

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Chapter 10 Drug Delivery Through Multifunctional Polypeptidic Hydrogels Hermis Iatrou, Panagiota G. Fragouli, Dimitra Stavroulaki, and Barbara Athanasiou Abstract Over the last two decades, remarkable progress has been made to the discovery of novel drugs as well as their delivery systems for the treatment of cancer, the major challenge in medicine. Pharmaceutical scientists are trying to shift from traditional to novel drug delivery systems by applying nanotechnology and, in particular, polymeric carriers to medicine. In complex diseases, very sophisticated nanocarriers should be designed to encapsulate a significant quantity of drugs and bypass biological barriers with minimum cargo loss to effectively and directly deliver the encapsulated drug to the desired pathological site. One of the most promising classes of polymeric materials for drug delivery applications is polypeptides, combining the properties of the traditional polymers with the 3D structure of natural proteins, i.e., a-helices and β-sheets. In this chapter, we present the recent progress in the synthesis of polymers that form hydrogels in aqueous solutions, based on polypeptides prepared through ring-opening polymerization of N-carboxy anhydrides and which have been loaded with anticancer drugs and studied for their functionality. Advancements in drug design and improvement of multifunctional nanocarriers from the combination of well-defined macromolecular architectures and smart materials are the future for the successful treatment of numerous lethal diseases. Key words Polypeptides, Ring-opening polymerization, Hydrogels, pH- and enzyme stimuli-­ responsive, Pancreatic cancer, Gemcitabine

1  Introduction A novel, multifunctional hydrogel that exhibits a unique set of properties for the effective treatment of pancreatic cancer (PC) is presented. The material is comprised of a pentablock terpolypeptide of the type PLys-b-(PHIS-co-PBLG)-PLys-b-(PHIS-co-­ PBLG)-b-PLys which is a noncytotoxic polymer [1]. It can be implanted via the least invasive route and selectively delivers gemcitabine to efficiently treat PC.  Mixing the novel terpolypeptide with an aqueous solution of gemcitabine within a syringe results in the facile formation of a hydrogel that has the ability to become Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 Illustration of chemical treatment of pancreatic cancer: hydrogel with encapsulated drug is implanted in least invasive way and melts only in the vicinity of cancerous tissue due to lower pH, leading to targeted and directional delivery of PLys-b-(PHIS-co-PBLG)-PLys-b-(PHIS-co-PBLG)-b-PLys

liquid under the shear rate of the plunger. Upon injection in the vicinity of cancer tissue, it immediately reforms into a hydrogel due to the unique combination of its macromolecular architecture and secondary structure. Because of its pH responsiveness, the hydrogel only melts close to PC; thus, the drug can be delivered directionally toward the cancerous rather than healthy tissues in a targeted, controlled, and sustained manner (Fig. 1).

2  Materials 1. Boc-HIS(Trt)−OH (>99%). 2. Triphosgene (99%). 3. Triethylamine (>99%) (see Note 1). 4. Thionyl chloride (>99%). 5. l-Lysine (>99%). 6. γ-Benzyl-l-glutamate. 7. H-Leu-OH (>99%). 8. Diethyl ether. 9. Fluorescamine. 10. Trypsin. 11. Tetrahydrofuran (THF) (dried, max 0.005% water) (see Note 2). 12. Dichloromethane. 13. Trifluoroacetic acid (TFA) (>99%). 14. Ethyl acetate (>99.5%) (see Note 3).

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15. Hexane (>99%) (see Note 4). 16. Dimethylformamide (DMF) (99.9+%, special grade for peptide synthesis with less than 50 ppm of active impurities) (see Note 5) is the polymerization solvent. 17. 1,6-Diaminohexane (98%) is the initiator of the polymerization (see Note 6).

3  Methods 3.1  Instrumentation

1. Use size-exclusion chromatography (SEC) to determine the Mn and Mw/Mn values. Perform the analysis using a SEC equipment composed of 600 high-pressure liquid chromatographic pump, Ultrastyragel columns, a differential refractometer detector, and a precision PD light scattering detector at 60 °C featuring two detectors at 15° and 90° (TALLS). Use a DMF solution containing 0.1 N LiBr as an eluent at a rate of 1 mL/min, operating at 60 °C. 2. Perform nuclear magnetic resonance spectroscopy (1H NMR), 300  MHz. Take the spectra of the N-carboxyanhydrides (NCAs) in CDCl3 at room temperature. 3. Perform Fourier transform infrared (FTIR) spectroscopy measurements in KBr pellets at room temperature, in the range of 450–4000 cm−1. 4. Perform circular dichroism with an instrument containing thermo-stabilizing system. Use cell of 1 mm Quartz Suprasil. The aqueous solution concentration of the PBLG/PHIS polymer with a ratio of the monomeric units 50/50 is 3.3 × 10−4 g/mL, while the aqueous solution concentration of the PBLG/PHIS polymer with a ratio of the monomeric units 70/30 at every pH is 2.5 × 10−4 g/mL. Perform the adjustment of pH through addition of diluted HCl or NaOH. 5. Perform scanning electron microscopy (SEM)-EDS of the hydrogels using carbon grids. Prepare the samples first by quick deep freezing of a small amount of the hydrogels with liquid nitrogen, followed by freeze-drying to remove water. Make sputter coating of the samples with gold prior to the measurements. 6. Perform enzymatic degradation-fluorescence measurements using 10  M multimode microplate reader and 96-well black microplates. To measure the enzymatic degradation of the hydrogel by either trypsin or leucine aminopeptidase (LAP), mix 2 mg of hydrogel with 0.1 nM trypsin or 100 nM LAP in PBS and incubate at 37 °C for 1 or 24 h, respectively. At the end of the incubation, mix a sample of 10 μL from the reaction with 90  μL of a solution of fluorescamine containing

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1 mg/mL fluorescamine in acetonitrile, 15 μL of 0.1 M borate buffer at pH 8.0, and 145 μL of Milli-Q water. After 5 min, measure the fluorescence using excitation at 405 nm and emission at 485 nm. 3.2  Synthesis of Monomers

3.2.1  Synthesis of ε-tert-Butoxycarbonyl-lLysine N-Carboxy Anhydride (Νε BOC-l-LYS NCA)

Perform the synthesis using high-vacuum techniques [2]. The purity of the NCA monomers is crucial for their successful living polymerization utilizing ROP through primary amine difunctional initiator and confirms by FTIR along with 1H NMR spectroscopy analysis [3]. 1. Add Nα,Nε-Di-(tert-butoxycarbonyl)-l-lysine into a flask, place it on the vacuum line, and pump overnight. 2. Distill purified ethyl acetate, followed by argon insertion, in order to reach atmospheric pressure and by the addition of triphosgene. Leave the mixture to react for 10 min. 3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and immerse the solution in an ice-water bath for 6 h. 4. Filter the precipitate, in order to remove the HCl salt of triethylamine, and immerse the clear solution in an ice bath. 5. Extract the NCA repeatedly with Milli-Q water, until neutral pH of the aqueous phase is achieved. 6. Recrystallize the purified NCA three times under high vacuum in a custom-made apparatus, with ethyl acetate/hexane (1/5 v/v) pair at −20 °C. The yield is 60%.

3.2.2  Synthesis of Nim-trityl-l-Histidine-NCarboxy Anhydride (Trt-HIS-NCA) [4]

1. Add in a 500  mL round-bottom flask 20  g (40.2  mmol) of Boc-His(Trt)-OH and dry overnight under high vacuum. 2. Distill 150  mL of THF in the flask, giving a clear yellowish solution. 3. In an ice bath place the reaction flask, filled with argon. 4. Dilute 3.25 mL (44.2 mmol) of thionyl chloride in 20 mL of THF and add dropwise in a period of 10 min. 5. After 2 h, pour the solution in 2 L of cold (Et)2O with precipitation of Trt-His-NCA.HCl as the major product. 6. Finally, filter the solid (glass sintered filter 3) and then transfer it to a round-bottom flask of 500  mL.  Drying in HV gives 17.2 g. 7. Recrystallize the above solid mixture, containing the HCl salt, free anhydride, and the initial substrate by distilling 300 mL of ethyl acetate under HV. 8. Remove the round-bottom flask of 500  mL, containing the suspension, from HV and place it into a water bath, at 45 °C for 1 h, resulting in dissolution.

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9. Cool the solution to 0 °C with an ice bath and form Trt-His NCA.HCl as a precipitate which is isolated as the only product after filtration. 10. Transfer the NCA salt to another flask of 500  mL and dry overnight under HV (12.5 g = 28 mmol). 11. Subsequently, distill 200  mL of EtAc into the flask; remove the flask from HV, fill with Ar, and place to an ice bath. 12. Add slowly dropwise under vigorous stirring (duration of additional 1 h), at 0 °C, 3.5 mL (28 mmol) of the stoichiometric amount of triethylamine dissolved in 50  mL of the same solvent. 13. Filter off the resulted triethylamine hydrochloride and pour the filtrate into 1.5 L of non-solvent hexane in order to recrystallize the Trt-His-NCA. 14. Make a second recrystallization with a mixture of solvent/ non-solvent EtAc/hexane (1:5) and isolate by filtration the white solid precipitate Trt-His-NCA. 15. Finally, dry Trt-HIS-NCA under HV overnight and transfer it into glove box resulting in 11.05 g (27 mmol, 67% yield). 3.2.3  Synthesis of γ-Benzyl-l-Glutamate N-Carboxy Anhydride (BLG-NCA)

1. Suspend γ-benzyl-l-glutamate in dry ethyl acetate followed by addition of triphosgene. 2. Heat the mixture at 70 °C until the solution becomes clear, indicating the formation of the NCA. 3. Distill off the solvent in the vacuum line, and distill fresh dry ethyl acetate in the flask, to dissolve the crude NCA, followed by removal of the solvent by distillation. 4. Repeat twice this procedure in order to remove the excess phosgene that sublimes under high vacuum. 5. Remove the unreacted species, such as free amino acids along with the HCl salts of the amino acids produced during the synthesis, by extraction with an alkali solution in water. 6. Purify further the resulted BLG-NCA by three recrystallizations from dry ethyl acetate/hexane (1/5  v/v) under high vacuum at −20 °C, leading to a 65% yield of the BLG-NCA formation.

3.2.4  Synthesis of l-Leucine N-Carboxy Anhydride (LEU-NCA)

1. Add in a flame-dried 1 L two-neck round-bottom flask 15 g (114  mmol) of l-leucine and degas under high vacuum overnight.

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2. Distill 250 mL of highly dry THF; remove the flask from the vacuum line and brink to atmospheric pressure by the careful addition of dry argon. The flask is equipped with a condenser and an inlet for Ar streaming. 3. Add under stirring 29.6 mL (183 mmol, 1.6 eq.) of purified (+) (−) limonene and allow the suspension to warm under vigorous stirring at 50–55 °C. At this time, add 13.6 g (46 mmol, 1.2/3 eq.) of triphosgene. Allow the reaction mixture to stir at this temperature until a clear dark orange solution is formed (after 1–2 h). 4. Stir the clear solution for another 1 h under Ar flow and transfer the solution by filtration to the crystallization apparatus. Attach the apparatus to the vacuum line and pump out the solvent to dryness. Then, distill a small amount of highly dry THF (~20 mL) capable to dissolve the solid monomer and a clear solution is formed. 5. Slowly distill, under vigorous stirring, n-hexane (~500 mL) in order for the NCA to precipitate in the form of a fine powder. Keep the apparatus at −20 °C overnight. 6. Next day, filter off the orange supernatant and dry the solid in vacuo for 1 h. 7. Perform three additional crystallizations and finally dissolve the white solid product in dry THF and cannula transfer it in a sealed flask. Attach the flask to the vacuum line, remove the solvent, and dry overnight the final product. 8. Finally, move the flask to the glove box and weigh solid yielding 16.6 g (95 mmol) of Leu-NCA (83%). 3.3  Synthetic Approach of Polypeptides PLys-b(PHIS-co-PBLG (or PLEU))-b-PLys-b(PHIS-co-PBLG (or PLEU))-b-PLys (See Fig. 2)

1. Add an α,ω-hexamethylenediamine solution in DMF, in a solution of ε-tert-butoxycarbonyl-l-lysine N-carboxy anhydride in DMF (Lys-NCA) (see Note 7). 2. After completion of polymerization (middle block), dissolve in DMF appropriate amounts of Nim-trityl-protected l-histidine N-carboxy anhydride (HIS-NCA) and γ-benzyl-l-glutamate N-carboxy anhydride (BLG-NCA) (molar ratio 1:1) and add to the reaction flask. Allow the polymerization to continue for 6 days (two hydrophobic blocks). 3. Add then Lys-NCA, followed by precipitation, in diethyl ether after the completion of the polymerization (see Note 8). 4. Mix the deprotected pentablock with Milli-Q water and dialyze (membrane molecular weight cutoff 3500  Da) against dilute HCl solution (pH = 3) for 2 days, dilute NaOH solution (pH = 8) for 2 days, and DI water for 2 days (for each step change the water every 12 h).

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Fig. 2 Schematic illustration of pentablock terpolypeptide of the type PLys-b(PHIS-co-PBLG (or PLEU))-b-PLys-b-(PHIS-co-PBLG (or PLEU))-b-PLys

5. Finally, lyophilize the solution to obtain the final pentablock terpolypeptide (Fig.  3) (see Note 9) as a white powder (see Note 10). 3.4  Formation of Hydrogels 

1. Prepare pentablock terpolypeptide hydrogels very simply by adding Milli-Q water in a vial containing the solid polypeptide. 2. After 2–3 h, the hydrogel is ready without requiring any other treatment. The water swells the polypeptide, which keeps the general shape of the initial solid.

3.5  Formation of Hydrogels Loaded with Gemcitabine [5, 6]

1. Prepare pentablock terpolypeptide hydrogels loaded with gemcitabine (Hydrogem) by first dissolving the appropriate amount of gemcitabine in pyrogen-free Milli-Q water (gemcitabine:polypeptide ratio 1:1 (w/w)), at 35 °C, followed by addition of the warm gemcitabine solution to the polypeptide. Keep the temperature at 35 °C until complete swelling of the polypeptide and formation of homogeneous hydrogel, about 3–4 h (see Note 11). 2. The hydrogel is stable at room temperature for several days, while the same gemcitabine solution in Milli-Q water precipitates after 0.5 h when the temperature is lowered to 20 °C.

4  Notes 1. Dry triethylamine over calcium hydride for 1  day and then distill and store in the vacuum line over sodium. The appropriate quantity needed is freshly distilled in a vacuum line.

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Fig. 3 Reactions used for the synthesis of polypeptide PLys-b-(PHIS-co-PBLG)-b-PLys-b-(PHIS-co-PBLG) -b-PLys

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2. Perform the purification of tetrahydrofuran using standard high-vacuum techniques. THF is refluxed over Na under N2 atmosphere in the presence of benzophenone, until a bright deep purple color is attained, before being collected in a round-bottom flask containing fresh finely grounded CaH2. Connect this flask to the vacuum line; degas the solvent and distill in a flask containing a Na/K (1/3 by weight) alloy. The bright blue color, which develops after stirring for some time, indicates that the solvent is free from impurities. 3. Ethyl acetate is fractionally distilled over phosphorus pentoxide. 4. Hexane is fractionally distilled over sodium. 5. Purify dimethylformamide by short-path fractional distillation under vacuum in a custom-made apparatus. Use always the middle fraction. 6. Distill fractionally 1,6-diaminohexane under high vacuum and treat it with sodium at room temperature for 1 day. Then subsequently distill it into precalibrated ampoules with break seals. Dilute it then with DMF to the appropriate concentration in a sealed apparatus equipped with precalibrated ampoules and keep away from light. 7. Synthesize the polypeptides using high-vacuum techniques. Design the polymerization reactors to have volumes at least three times larger than that of the CO2 generated by each polymerization. Carry out polymerizations with 1,6-­diaminohexane as the initiator. 8. The selective cleavage of trityl and tert-butoxycarbonyl groups from poly (l-histidine) and poly(l-lysine) segments, respectively, is achieved by treating with trifluoroacetic acid (TFA), followed by addition of triethyl silane. The solution is precipitated in diethyl ether, and the white solid is filtered and dried. 9. These terpolypeptides are composed of one middle hydrophilic block (PLys), two hydrophobic blocks poly(l-histidine-­ co-γ-benzyl-l-glutamate (or l-leucine)), followed by two hydrophilic blocks of PLys, each one connected at the outer sides of the hydrophobic blocks. The middle hydrophilic block of poly(l-lysine) should have a high molecular weight to form strong hydrogels at very low concentrations, that is, 3.33% (w/w) in water. 10. A similar procedure is followed for the synthesis of the terpolypeptides exhibiting poly(l-leucine) (PLEU) instead of PBLG. 11. To ensure the formation of a homogeneous mixture, the hydrogel is inserted into a syringe and passed between two syringes multiple times using a syringe connector, prior to the in vivo tests.

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5  Results Over the last 40 years, impressive advancements toward the discovery of novel drugs as well as responsive multifunctional drug delivery systems (DDS) have been reported. As a consequence, out of more than 200 different cancers, some highly lethal cancers are now chronic diseases. Unfortunately, only limited progress has been made for some specific forms of cancer such as PC. For this complex disease, very sophisticated carriers should be designed to bypass biological barriers with minimum cargo loss, and effective and selective delivery to the desired pathological site, as demonstrated by the material described in this work. An amphiphilic pentablock terpolypeptide was developed by optimizing the macromolecular architecture and composition as well as the 3D structure of the hydrophobic blocks. The final pentablock terpolypeptide exhibits a unique combination of properties in the same molecule, which has not been achieved so far. Simply mixing the polypeptide and gemcitabine dissolved in water results in the facile formation of an injectable and quickly self-healing hydrogel which forms in situ and can be injected in the least invasive way close to cancer tissue. These properties rely on the secondary structure of the hydrophobic part of the amphiphilic terpolypeptide. After implantation, the hydrogel becomes liquid only close to the cancer tissue, mainly due to the lower pH of the pathological site, thus releasing the drug only in the vicinity of cancer tissue. The ability of these polypeptides to form hydrogels by adding Milli-Q water depended on two parameters: their molecular and compositional homogeneity and the PHIS/PBLG ratio. It was found that the pentablocks should be well defined with a high degree of molecular and compositional homogeneity in order to form strong hydrogels. If the polydispersity was high by intentionally mixing two different polypeptides, or by intentionally omitting one block, they either formed very weak hydrogels at higher polypeptide concentrations or did not gel at all. This obliges the directional release of the drug toward the cancer rather than healthy tissue, as shown by in vivo experiments. It was found that the delivery of only 40% of gemcitabine in one dose directed by the hydrogel could slow down the development of the cancer tissue to the same extent with the delivery of 100% of pure gemcitabine in two doses, the typical chemotherapy used so far in clinics. Therefore, we achieved the same or slightly better deceleration of tumor growth with fewer drugs and less number of doses due to the guided delivery through the Hydrogem. The hydrogel also responds to enzymes, rendering it biodegradable, thus not requiring removal through resection following drug delivery. Regarding the PHis/PBLG ratio, PHis 100 and PHis 70 formed a weak hydrogel at 20  °C, while its strength was highly temperature dependent, and at 37 °C it was transformed into liquid. PHis 50 and PHis 30 formed strong hydrogels depending on

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the polypeptide concentration and the pH of water provided that at pH  =  6.6 the polymer was transformed into liquid. A well-­ ordered 3D structure of interconnected nanofibers and thin nanosheets was maintained by the terpolypeptide PHis 50 at pH = 7.4, while at pH = 6.5 the structure was akin to collapsed thick sheets with interconnecting thick fibers and larger voids, resembling a liquid-like structure. The presence of the α-helix conformation that the increased BLG content gives to the terpolypeptide is critical for the hydrogel formation. The synthesized material is modular since its properties can be modified by its molecular characteristics to deliver other drugs, rendering it very useful for a variety of biological applications such as bone regeneration and catheters for treatment of coronary artery disease. We intend to improve the hydrogel effectiveness by incorporating more stimuli, such as temperature and redox, to increase selectivity for targeting cancer cells. We believe that the synergy of material and pharmaceutical scientists, biologists, and clinical oncologists is imperative to produce efficient DDS that possess advanced properties and required functionalities to fight cancer. This work is a testimony of this important function.

Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.” References 1. Bilalis P, Skoulas D, Karatzas A, Marakis J, Stamogiannos A, Tsimblouli C, Sereti E, Stratikos E, Dimas K, Vlassopoulos D, Iatrou H (2018) Self-healing pH- and enzyme stimuli-­responsive hydrogels for targeted delivery of gemcitabine to treat pancreatic cancer. Biomacromolecules 19:3840–3852 2. Hadjichristidis N, Iatrou H, Pispas S, Pitsikalis M (2000) Anionic polymerization: high vacuum techniques. J Polymer Sci Part A-Polymer Chem 38:3211–3234 3. Aliferis T, Iatrou H, Hadjichristidis N (2004) Living polypeptides. Biomacromolecules 5:1653–1656 4. Mavrogiorgis D, Bilalis P, Karatzas A, Skoulas D, Fotinogiannopoulou G, Iatrou H (2014)

Controlled polymerization of histidine and synthesis of well-defined stimuli responsive polymers. Elucidation of the structure–aggregation relationship of this highly multifunctional material. Polym Chem 5:6256–6278 5. Altunbas A, Pochan DJ (2012) Peptide-based and polypeptide-based hydrogels for drug ­delivery and tissue engineering. Top Curr Chem 310:135–167 6. Huang J, Hastings CL, Duffy GP, Kelly HM, Raeburn J, Adams DJ, Heise A (2013) Supramolecular hydrogels with reverse thermal gelation properties from (oligo)tyrosine containing block copolymers. Biomacromolecules 14:200–206

Chapter 11 Polymersomes from Hybrids-Polypeptides for Drug Delivery Applications Hermis Iatrou, Panagiota G. Fragouli, Dimitris Skourtis, and Ioanna Stavropoulou Abstract Recently, the explosion of progress of materials at the nanoscale level has paved the way for a new category of healthcare technologies termed nanomedicine. Nanomedicine involves materials at the nanometer level for products that can improve the currently used technologies for biomedical applications. While traditional therapeutics have allowed for limited control of their distribution in the body and clearing times, engineering at the nanoscale level has allowed for significant advances in biocompatibility, biodistribution, and pharmacokinetics. Among all materials, polymers have dominated the nanomedicine world, due to their ability to manipulate their properties by combining different materials in a wide variety of macromolecular architectures. The development of novel polymeric materials is guided by the goal of improving patient survival and quality of life by increasing the bioavailability of drug to the site of disease, targeting delivery to the pathological tissues, increasing drug solubility, and minimizing systemic side effects. Polymersomes (vesicles) are the only type of polymeric nanocarriers that can physically encapsulate at the same nanoparticle hydrophilic drugs in their aqueous interior and/or hydrophobic agents within their lamellar membranes. Polymersomes have been shown to possess superior biomaterial properties compared to liposomes, including greater stability and storage capabilities, as well as prolonged circulation time. Key words Nanoparticles, Nanotechnology, Pancreatic cancer, Polymersomes, Polypeptides, Ring-­ opening polymerization

1  Introduction Well-defined amphiphilic polymers of the ABA and ABC type are synthesized, where A is poly (l-lysine hydrochloride) (PLL), B is poly(γ-benzyl-(d7) l-glutamate) (PBLG(-d7)), and C is poly(ethylene oxide) (PEO) [1] (Fig.  1). Both polymers form polymersomes in water. The polymersomes are loaded with doxorubicin or paclitaxel. It is found that in the ABC, due to asymmetry of the two hydrophilic blocks, PEO is always on the outer periphery and the dimensions of the vesicles are smaller. The release of the vesicles is temperature and pH dependent. In vivo, the empty Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 Reactions used for the synthesis of the amphiphilic triblock-co-polypeptides poly(l-lysine hydrochloride)-b-poly(γ-benzyl-d7-l-glutamate)-b-poly(l-lysine hydrochloride)

vesicles are not toxic. In vitro activity of the loaded vesicles against human pancreatic cancer cell lines reveals comparable activity to Myocet for the ABA loaded with doxorubicin, while lower activity is observed for the ABC.

2  Materials 1. γ-Benzyl-l-glutamate (>99%). 2. l-Lysine (>99%). 3. Poly(ethylene oxide) end-functionalized monoamine used as monofunctional macroinitiator. 4. N,N-dimethylformamide (DMF) (99.9+%, special grade for peptide synthesis with less than 50 ppm of active impurities) is the polymerization solvent (see Note 1). 5. 1,6-Diaminohexane (99.9%) serves as the initiator for the triblock copolypeptide (see Note 2). 6. Benzene (99%, thiophen free grade) (see Note 3). 7. Triphosgene (99%) was used as purchased.

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8. Triethylamine (Et3N >99%) (see Note 4). 9. Purification of tetrahydrofuran (THF; dried, max 0.005% water) was performed using standard high-vacuum techniques and dried over sodium potassium alloy. 10. Ethyl acetate (>99.5%) was fractionally distilled over phosphorous pentoxide. 11. Hexane (>99%, Merck) was fractionally distilled over sodium and subsequently distilled over n-BuLi. 12. Solid Dox (98%) and Pacl (98%) were used as purchased (see Note 5).

3  Methods 3.1  Instrumentation

1. Use size-exclusion chromatography (SEC) to determine the Mn and Mw/Mn values. Use two SEC systems, one in order to obtain the molecular weights of the protected polymers and the other for the determination of the amount of Pacl contained in the vesicles. The first system is composed of a 600 high-pressure liquid chromatographic pump, columns for high temperatures, a differential refractometer detector, and a two-angle (15°, 19°) light scattering detector at 60  °C.  A 0.1  N LiBr solution in DMF is used as an eluent at a rate of 1 mL.min−1. The second system is composed of a 600 high-pressure liquid chromatographic pump, columns, and a differential refractometer detector at 25 °C. The system runs using a mixture of CHCl3/Et3N as a mobile phase in a ratio of 95/5 (v/v). 2. Achieve the 1H NMR spectra of the polymers and NCAs in CDCl3 at room temperature, and that of the triblock polymers in dimethyl sulfoxide (DMSO). 3. Carry out Fourier transform infrared measurements (FT-IR) in KBr pellets at room temperature, in the range of 450–4000 cm−1. Use less than 1 mg of samples for each spectrum. 4. Perform UV spectroscopy from 190 to 500 nm, at room temperature with cells requiring 120 mL. 5. Conduct dynamic light scattering measurements with a system composed of a goniometer with a stepper motor controller, a variable power Ar+ laser operating at 690 nm and with 10 mW power, a temperature control unit, and a pump/filtering unit. Analyze correlation functions by the cumulant method and the Contin software. The correlation function is collected at 90°. Perform all measurements in isotonic Tris buffer (0.010 M) at pH = 7.4. Measure in the concentration range between 5 × 10−5 and 1 × 10−6 g.mL−1.

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6. Measure the electrophoretic mobility of the empty and drug-­ loaded vesicle dispersion. All the measurements are at least the average of three runs performed at 25 °C. Carry out all measurements in isotonic (0.150 M NaCl) Tris buffer (0.010 M) at pH = 7.4. 7. Cell cultures: Human pancreatic adenocarcinoma cell lines AsPC1 and BxPC-3, obtained from the American Type Culture Collection. Propagate the cells in medium containing 5% fetal calf serum and antibiotics (penicillin, streptomycin) in 5% CO2 atmosphere in a 37 °C incubator. 3.2  Synthesis of Monomers

3.2.1  Synthesis of ε-tert-Butoxycarbonyl-lLysine N-Carboxy Anhydride, (Νε BOC-l-LYS NCA)

Perform the synthesis using high-vacuum techniques [2]. The purity of the NCA monomers is crucial for their successful living polymerization utilizing ROP through primary amine difunctional initiator and is confirmed by FTIR along with 1H NMR spectroscopy analysis [3]. 1. Add Nα,Nε-Di-(tert-butoxycarbonyl)-l-lysine into a flask, place it on the vacuum line, and pump overnight. 2. Distill purified ethyl acetate, followed by argon insertion in order to reach atmospheric pressure and by the addition of triphosgene. Leave the mixture to react for 10 min. 3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and immerse the solution in an ice-water bath for 6 h. 4. Filter the precipitate, in order to remove the HCl salt of triethylamine, and immerse the clear solution in an ice bath. 5. Extract the NCA repeatedly with Milli-Q water, until neutral pH of the aqueous phase is achieved.

3.2.2  Synthesis of γ-Benzyl-l-Glutamate N-Carboxy Anhydride (BLG-NCA)

6. Recrystallize the purified NCA three times under high vacuum in a custom-made apparatus, with ethyl acetate/hexane (1/5 v/v) pair at −20 °C. The yield is 60%. 1. Suspend γ-benzyl-l-glutamate in dry ethyl acetate followed by addition of triphosgene. 2. Heat the mixture at 70  °C until the solution becomes clear, indicating the formation of the NCA. 3. Distill off the solvent in the vacuum line, and distill fresh dry ethyl acetate in the flask, to dissolve the crude NCA, followed by removal of the solvent by distillation. 4. Repeat twice this procedure in order to remove the excess phosgene that sublimes under high vacuum. 5. Remove the unreacted species, such as free amino acids along with the HCl salts of the amino acids produced during the synthesis, by extraction with an alkali solution in water.

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6. Purify further the resulted BLG-NCA by three recrystallizations from dry ethyl acetate/hexane (1/5  v/v) under high ­vacuum at −20  °C, leading to a 65% yield of the BLG-NCA formation. 3.3  Synthesis of the Triblock Copolypeptide PLL-b-PBLG-d7-b-PLL

1. Dissolve the copolypeptides in a mixture of trifluoroacetic acid and dichloromethane (TFA/DCM 1:1, v/v) containing a few mL of anisole, for 1.5 h (see Note 6).

3.4  Synthesis of the Novel Triblock Copolypeptide PEO-b-PBLG-b- PLL

The reactions used for the synthesis of the hybrid triblock terpolymer PEO-b-PBLG-b-PLL are shown in Fig. 2.

2. Distill TFA and DCM, and remove the low-molecular-weight salts and reagents, using dialysis in aqueous HCl (pH = 5.0) (see Note 7).

1. Pump to dryness in the HV line the amino end monofunctional PEO (1.0 g, 0.112 mmol of C-NH2 groups) (see Note 8). 2. Distill purified benzene (100 mL) (see Note 9). 3. Distill off the solvent and leave the polymer to dry overnight. 4. Distill DMF (10 mL) to dissolve the polymer. 5. Filtrate the solution in an ampoule (see Note 10). 6. Pump in the high-vacuum line to dryness for 1  day, 1.27  g BLG-NCA (4.86 mmol). 7. Dissolve the monomer in 20 mL of freshly distilled DMF (see Note 11). 8. Rupture the glass magnet of the macroinitiator ampoule, allowing the ring-opening polymerization of BLG-NCA to occur under vigorous stirring for 2  days, with occasional degassing. 9. Remove an aliquot of the solution after the completion of the polymerization for characterization of the PEO-b-PBLG diblock copolymer. 10. Finally, add via cannula 1.22 g (4.50 mmol) of Boc-L-lysine-­ NCA solution in DMF (see Note 12). 11. Transfer the PEO-b-PBLG solution in the Ar/vacuum line, and leave under vigorous stirring for 3 days, with occasional degassing, ultimately yielding PEO-b-PBLG-b-PBocLL (see Note 13). 12. In order to obtain the PEO-b-PBLG-b-PLL: Dissolve 2.95 g of the terpolymer in 15 mL of DCM. 13. Add TFA (DCM/TFA  =  1/1 (v/v), ca. 10% w/w polymer concentration) and leave it to react for 3 h. 14. Distill off the excess organic acid along with the solvent in the HV line.

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Fig. 2 Reactions used for the synthesis of the amphiphilic triblock hybrid terpolymer poly(ethylene oxide)-b-­ poly(γ-benzyl-l-glutamate)-b-poly(l-lysine hydrochloride)

15. Dissolve the polymer in Milli-Q water in the presence of HCl, to reach a pH = 5.0. 16. Dialyze six times against Milli-Q water with pH  =  5.0 (see Note 14). 17. Freeze-dry the final product.

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Fig. 3 Schematic representation of the vesicles formed with the encapsulated drugs and the curvature of the interphase. The lower asymmetry in the hydrophilic blocks results in larger dimensions of the vesicles and lower curvature of the interphase

3.5  Drug Loading (See Fig. 3) [4, 5] 3.5.1  Doxorubicin Loading

1. Dissolve 10 mg of the triblocks in 4 mL of DMSO (see Note 15). 2. Add 5 mg of Dox (HCl salt) after obtaining a clear solution (in the case of PEO-b-PBLG-b-PLL) or as lightly turbid solution (in the case of PLL-b-PBLG-b-PLL). 3. Leave for half an hour to be dissolved. 4. Place the solution in a dialysis bag (see Note 16). 5. Dialyze against isotonic Tris buffer at pH = 7.4 (0.150 M NaCl, 0.010 M Tris) or isotonic carbonate buffer at pH = 10.5. 6. Take out the dialysis bag at pH = 10.5, to a 2 L solution of Tris buffer at pH = 7.4 (see Note 17).

3.5.2  Pacl Loading

1. Dissolve 10 mg of polymer in 4 mL of DMSO (see Note 18). 2. Add 2 mg of Pacl, and leave for 1 h to be dissolved. 3. Place the solution in a dialysis bag (see Note 19).

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4. Dialyze against isotonic Tris buffer at pH = 7.4 (0.150 M NaCl, 0.010 M Tris). 5. Place a small stir bar in the dialysis bag and vigorously stir during the exchange of the solutions (600 rpm). 6. Remove excess of insoluble Pacl in the buffer by centrifugation at 112 × g at 20 °C for 10 min. 7. Separate the supernatant very carefully from the solid (Pacl). 8. Repeat the centrifugation three times (see Note 20).

4  Notes 1. N,N-dimethylformamide is further purified by short-path fractional distillation under high vacuum in custom-made apparatus. Use always the middle fraction. 2. 1,6-Diaminohexane is a highly hygroscopic compound, leave it to dry over a sodium mirror for 24 h, then dilute with purified DMF, subdivide into ampoules, and store under high vacuum (HV) at room temperature. 3. Benzene is purified by stirring over concentrated sulfuric acid for a week. It is subsequently washed with an aqueous solution of NaOH and then with water many times until it becomes neutral and finally dry with CaCl2. The dry material is then transferred into a round-bottom flask containing fresh finely grounded CaH2 and a magnetic stirring bar, and is attached to the vacuum line and degassed. Leave the flask for reaction of CaH2 with moisture overnight, degas again, and distill in a calibrated cylinder containing n-BuLi and styrene. 4. Triethylamine is dried over calcium hydride for 1 day and then distilled and stored in the vacuum line over sodium. The appropriate quantity needed is freshly distilled in a vacuum line prior to use. 5. For in vitro and in vivo experiments use the injectable forms of Dox (adriamycin 2 mg.mL−1) and Pacl (taxol 6 mg.mL−1). 6. Since the benzyl groups are deuterated, the composition of the protected copolypeptides cannot be obtained by 1H NMR spectroscopy, but is verified using UV spectroscopy in DMF, since the PBLG-d7 block absorbs at 267 nm. Anisole is used as a scavenger of the tert-butyl cations. It is well established that Boc and benzyl groups can be orthogonally deprotected: the former under acidic and the latter under basic conditions. Confirm the complete removal of the Boc groups by 1H NMR spectroscopy. 7. The Boc groups are selectively deprotected. 8. Use a custom-made 100 mL glass apparatus (equipped with a filter), which has been previously flame dried several times.

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9. Benzene is used in order to azeotropically remove traces of water. 10. The solution in the ampoules will be used as the macroinitiator. 11. The dissolution of the monomer takes place in another specially designed custom made glass apparatus containing the macroinitiator equipped with a breakseal. 12. Custom-made 100 mL glass reactor. 13. The triblock copolymer is precipitated in cold ether and dried in vacuo. The amount of polymer obtained is 2.95 g (96%). 14. Adjust the pH by adding drops of concentrated HCl, 1 day each. The amount of polymer obtained is 2.40 g (88%). 15. The polymer PEO-b-PBLG-b-PLL loaded with Pacl will be indicated as PEOPacl, the same polymer loaded with Dox as PEODox, while the PLL-b-PBLG-d7-b-PLL loaded with Dox will be indicated as PLLDox (Fig. 4). Load Dox at different pH, i.e., pH  =  7.4 and pH  =  10.5, and at different initial concentrations. 16. Spectrapor, molecular weight cutoff (MWCO): 3500 Da, 30 °C. 17. Initially deprotonate the Dox (hydrochloride), to become hydrophobic Dox free base. This would then be condensed in the hydrophobic block and finally be dissolved in the interior of the vesicle. Vesicle concentration obtained is 1.67 mg.mL−1. Dox loading will be given through the loading efficiency (%) (mass of Dox in vesicles/Dox mass in the initial solution) and loading content (%) (mass of Dox in vesicles/polymer mass). 18. Pacl is loaded at pH  =  7.4 at 20  °C and at different concentrations. 19. Spectrapor MWCO 3500 Da, 20 °C.

Fig. 4 The synthesized PLL-b-PBLG-d7-b-PLL

polymers

PEO-b-PBLG-b-PLL

and

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20. Obtain the optimum conditions for the centrifugation by SEC analysis of the supernatant and the solid (free Pacl) in the system running CHCl3/Et3N. Under these conditions only free Pacl is precipitated leaving the vesicles with the encapsulated Pacl in solution. The amount of Pacl in the vesicles is obtained by SEC analysis, using an appropriate set of columns in order to separate the polymer from the drug. Quantification is performed by setting calibration curves of Pacl in CHCl3/Et3N, where the drug is soluble.

5  Results 5.1  Synthesis and Characterization of the Polymers and Determination of the Concentration of Pacl in Polymersomes [6]

A special set of size-exclusion columns was used in order to characterize the polymer and determine the concentration of Pacl in polymersomes. As Pacl is a rather big molecule, compared to other side species of the solvent, it can be clearly separated and quantified, by making a calibration curve. The polymer can also be quantified, since it can be separated from Pacl that has a much smaller hydrodynamic volume. It is also obvious that Pacl has smaller polydispersity compared to the polymeric material.

5.2  Loading and Stability

The highest concentration achieved for Dox was 0.89  mg Dox mL−1 at pH = 7.4. The highest Dox loading efficiency achieved was 33.0% and loading content of 19.5%. The PLLDox vesicles remained stable in storage for at least 12 h in a 10 vol% fetal calf serum at 37 °C, while the PEODox vesicles in a 10 vol% fetal calf serum at 37 °C were stable only for 2 h. The highest Pacl concentration achieved was 0.4  mg Pacl mL−1. The highest Pacl loading efficiency achieved was 25.0% and a loading content of 13.0%. The PEOPacl nanoparticles remained stable over storage at 37 °C for at least 12 h containing 10 vol% fetal calf serum.

5.3  Polymersome Characteristics

In all polymersomes formed by the PEO-b-PBLG-b-PLL (blank and loaded), the zeta potential measured was between 3.2 and 5.4 mV, revealing that PEO is always in the outer periphery and PLL is in the interior of the polymersome, since if PLL was in the outer periphery, the zeta potential would be positive and much higher. The dimensions of the nanoparticles were determined by DLS, with an average diameter ranging from 100 to 300 nm.

5.4  In Vitro Release and Activity

The release is temperature and pH dependent. In case of PEOPacl, the release is higher at higher pH values and temperatures. In case of Dox-loaded polymers, the release increases at lower pH values and higher temperatures. In all cases the release profiles show a rather quick release in the beginning and a slower release after 15–20  h that continued for more than 3  days, revealing a slow sustainable (controlled) drug release.

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In cultures where polymers loaded with Dox were added, slight sediments were observed 24  h upon their addition at the highest concentration tested. However, cultures with lower concentrations of the Dox-loaded polymers as well as those where Pacl-loaded polymers were added were clear of sediments. Pacl-­ loaded polymers (i.e., PEOPacl) were found to be the most active among the three loaded polymers tested. From the two Dox-­ loaded polymers, PEODox-loaded polymer (PEODox) was found to be the less active as it showed only marginal antiproliferative activity against BxPC-3 cells. The PLL polymer (i.e., PLLDox) exhibited higher activity, which in fact was very close to that of Myocet in regard to its antiproliferative and cytostatic activity. The significantly lower activity of the PEODox might be due to the lower stability that these nanoparticles showed. 5.5  In Vivo Toxicity Study

Empty PEO and PLL polymers were also tested for acute toxicity in immunocompromised (SCID) mice. Both polymersomes were administered intraperitoneally in a single injection to the animals at the following doses: 200, 133, 100, and 67 mg.kg−1. Animals were subsequently weighted and observed for a period of 7  days for signs of toxicity or changes in their routine. The only side effect observed during this period was a light sedation starting 5 min and progressing until 15 min after the administration of the 200 mg.kg−1 dose for both PEO-b-PBLG-b-PLL and PLL-b-PBLG-d7-b-PLL polymers. All animals recovered 24 h later and no further sign of toxicity was recorded until the end of the observation period.

6  Conclusions Comparing the ABA triblock copolypeptide and ABC terpolymer, both formed polymersomes in water, due to the macromolecular architecture of the polymers along with the use of a rod middle block. It was found that the hydrophilic block with the higher volume fraction in the ABC architecture remains in the outer periphery of the formed vesicle. Therefore, it is possible to select between two different hydrophilic blocks to be at the outer periphery just by choosing their molecular characteristics. In case one of these is PEO, it is preferred to be in the shell due to its properties. Positively charged nanoparticles are incorporated faster into the cell due to the presence of negatively charged species on the cell membranes. This would result in higher accumulation of drug within the cells for the ABA copolypeptide rather than the ABC, and consequently higher activity. It is expected to further elucidate the structure-delivery relationship in order to achieve more efficient complex structures in terms of activity and selective delivery of multiple anticancer drugs and genes.

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Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.” References 1. Iatrou H, Dimas K, Gkikas M, Tsimblouli C, Sofianopoulou S (2014) Polymersomes from polypeptide containing triblock co- and terpolymers for drug delivery against pancreatic cancer: asymmetry of the external hydrophilic blocks. Macromol Biosci 14:1222–1238 2. Hadjichristidis N, Iatrou H, Pispas S, Pitsikalis M (2000) Anionic polymerization: high vacuum techniques. J Polymer Sci Part A-Polymer Chem 38:3211–3234 3. Aliferis T, Iatrou H, Hadjichristidis N (2004) Living polypeptides. Biomacromolecules 5:1653–1656

4. Chécot F, Brûlet A, Oberdisse J, Gnanou Y, Mondain-Monval O, Lecommandoux S (2005) Structure of polypeptide-based diblock copolymers in solution: stimuli-responsive vesicles and micelles. Langmuir 21:4308–4315 5. Matsumura Y, Kataoka K (2009) Preclinical and clinical studies of anticancer agent-­ incorporating polymer micelles. Cancer Sci 100:572–579 6. Disher DE, Ahmed F (2006) Polymersomes. Annu Rev Biomed Eng 8:323–341

Chapter 12 Drug Delivery Systems Based on Modified Polysaccharides: Synthesis and Characterization Aikaterini-Foteini Metaxa, Eleni Vrontaki, Eleni K. Efthimiadou, and Thomas Mavromoustakos Abstract Common chemotherapeutic drugs exhibit no specificity for cancer cells and destroy simultaneously healthy cells exhibiting high toxicity and reduced efficacy. The use of nanotechnology, especially of drug delivery systems to the size of the nanoscale, provides rational drug design solutions. Such nanomaterials may have a range of desired characteristics (lack of toxicity, response to certain characteristics of the cancer cells, antimicrobial properties, specific activity, etc.) in order to achieve targeted cancer therapy. In this chapter, polymeric systems with core-shell structure are synthesized, characterized, and studied as potent drug delivery devices for targeted cancer therapy. These polymeric systems are based on natural polysaccharides like cellulose, chitosan, and their derivatives, in combination with synthetic polymer. Polymethylmethacrylate (PMMA) nanospheres are used as a core in order to coat the surface with multiple layers of polysaccharides via layer-by-layer deposition. This design is advantageous due to the use of water as the appropriate solvent. Fabricated polymeric carriers are characterized structurally by AT-IR spectroscopy and morphologically by transmission (TEM) and scanning electron microscopy (SEM). Finally, daunorubicin, an anticancer agent, was encapsulated as a drug model into the carriers. Key words Drug delivery systems, Polysaccharides, Chitosan, Cellulose, Drug vehicles

1  Introduction In recent years, scientific research has focused on investigating new systems for early diagnosis and therapy against different types of cancer. Although a broad variety of chemotherapeutic agents are developed, these suffer from a lack of specificity [1]. Due to this, the chemotherapeutic agents along with the tumor cells destroy also the healthy cells and therefore exhibit high toxicity [2]. To reduce the toxicity, dose administration is minimized and at the same time their activity and their effectiveness are reduced [3]. These problems can be solved with the use of drug delivery systems at the nanoscale, aimed directly at the pathogenic area [4, 5]. Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Natural polysaccharides and their derivatives represent a group of polymers widely used in pharmaceuticals and biomedicine for the controlled release of drugs. Polysaccharides have several advantages over synthetic biopolymers to be used as a raw material for the synthesis of new drug delivery systems. These materials are nontoxic, and have good biocompatibility and low production cost. Furthermore, polysaccharides are used for nanoparticle coating and are attractive candidates for biomedical applications because of their biocompatibility and biodegradability. In addition, the nanoparticles coated with polysaccharides are not recognized by the phagocytic system. Since polysaccharides are hydrophilic polymers, they induce system stability in the bloodstream and they can lengthen the life span in the body and therefore increase the absorption of encapsulated drugs. Thus, a combined use of polysaccharides with synthetic water-soluble polymers leads to the production of materials with improved biochemical and mechanical properties [6–9]. With about 1011 tons of cellulose growing and disappearing annually, cellulose is the most common organic polymer in earth, a poly-dispersed linear homopolymer consisting of region- and enantioselective β-1,4 glycosidic-linked D-glucose units. In our work, we use two derivatives of cellulose: cellulose succinate (CS) and hydroxypropyl cellulose (HPC). CS is a pH-sensitive polymer that is synthesized in solid phase at 398 K, without the use of solvents, only by using the melting point of succinic anhydride [10, 11]. HPC is a nonionic biodegradable polysaccharide, derivative of cellulose which is a thermosensitive polymer, with a low critical solution temperature in water (LCST) observed at 41 °C [12]. Chitosan is compatible with the biological tissues and does not cause allergic reactions. It is biodegradable as it is cleaved by enzymes in harmless products (amino sugar) which are completely absorbed by the human body without causing side effects [13]. Chitosan possesses pharmacological properties such as hypocholesterolemic action, enhances healing of wounds, helps in the treatment of stomach ulcer, and has antimicrobial action [14, 15]. In the last years, searching the literature it can be realized that there is an increase of publications related to drug delivery systems based on polysaccharides. However, only few of the publications are focusing on systems based on cellulose. In addition these publications do not focus particularly on pharmaceutical formulations for specific diseases with in vitro and/or in vivo applications.

2  Materials 1. Polysaccharides: Powdered cellulose, MW  =  78,000– 98,000 g/mol, was purchased from Riedel de Haen Ag Seelze— Hannover. The medium molecular weight chitosan,

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deacetylated, and hydroxypropyl cellulose MW = 370,000 g/ mol were purchased from Aldrich. Methyl methacrylate (99% MMA) was purchased from Aldrich and purified by distillation before use. Succinic anhydride was purchased from Sigma-­ Aldrich and purified with recrystallization. Daunorubicin hydrochloride (DNR) was provided by Pharmacia & Upjohn and used as received. 2. Solvents: Deionized and distilled water was used in all processes.

3  Methods The synthetic procedure of the PMMA@HPC@CS@CH microspheres consists of four steps: (a) synthesis of PMMA nanospheres (NCs), (b) synthesis of PMMA@HPC NCs, (c) synthesis of PMMA@HPC@CS NCs, and (d) synthesis of PMMA@HPC@CS@ CH NCs (see Note 1, Fig. 1). Initially, PMMA nanospheres are synthesized by random radical emulsion polymerization of methyl methacrylate:

3.1  Synthesis of PMMA@HPC@CS@ CH Nanospheres (NCs)

1. In a round-bottom flask, add: (a) 28 mL H2O (see Note 2).

(b) 3 mL Methyl methacrylate (MMA) (see Note 3).



(c) 0,015 g KPS initiator (K2S2O8, t1/2 = 8 h at 70 °C).

2. Leave the reaction overnight for completion. 3. Centrifuge the milky emulsion for nanospheres’ isolation (see Note 4). Continuously, PMMA nanospheres are used as core for the polysaccharides’ coating by sequential deposition technique of cortices (layer-by-layer deposition). Forming of the first layer by deposition of hydroxypropyl cellulose (HPC): 4. Dissolve 0.4  g of hydroxypropyl cellulose (HPC) in 50  mL acidic solution (see Note 5). 5. Add and disperse 0.4 g of PMMA spheres in 50 mL aqueous solution (see Note 6). 6. Leave the mixture under stirring for 24 h at 50 °C. 7. Centrifuge the emulsion (see Note 7). Forming of the second layer by succinic acid-modified cellulose (CS, see Note 8): 8. Dissolve 0.1 g of cellulose succinate in 50 mL of basic solution (see Note 6). 9. Add and disperse 0.4 g of HPC-coated spheres in 50 mL of acidic aqueous solution (see Note 5).

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MMA Core MMA

HPC

HPC

CS CS

CHIT

EDC/NHS DAUNORUBICIN

CHIT

crosss lin cro linkk

Fig. 1 Synthetic scheme of the present study

10. Add the suspended microspheres to the aqueous solution of cellulose. 11. Leave the mixture under stirring at room temperature for 24 h. 12. Centrifuge the emulsion (see Note 9). Forming of the third layer by chitosan deposition (CS) (see Note 10): 13. Dissolve 0.05 g of chitosan in 25 mL aqueous acidic solution (see Note 5). 14. Add and disperse 0.4 g of modified spheres in 50 mL of basic aqueous solution (see Note 6). 15. Add the dispersion to the aqueous solution of chitosan. 16. Leave the mixture under continuous stirring for 24 h at room temperature. 17. Centrifuge the emulsion (see Note 9). Aiming at cross-linking the three layers are deposited on vehicle’s surface: 18. Disperse 0.4 g of coated PMMA@HPC@CS@CH spheres in 2 mL water. 19. Add aqua solution of EDC (10  mg, 0.06  mmol) and NHS (5 mg, 0.04 mmol) in the resulting emulsion. 20. Leave the mixture for 30 min under vigorous stirring. 21. Centrifuge the final mixture (see Note 9) and purify after suspended in water.

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3.2  Drug Encapsulation

Drug encapsulation or drug loading is the process of incorporating the pharmaceutical compound into a polymeric network or capsule. The release of the drug in a controlled manner is the inverse process by which the drug molecules leave the solid phase of the polymeric network. Loading and drug release are closely related processes that depend on the physicochemical properties of the polymeric network, the physicochemical properties of the drug, and the environmental impact [16]. The drug can be trapped in the system by intermolecular interactions with the polymeric network, such as hydrogen bonds, ionic interactions, and intramolecular dipole-dipole interactions. It can also be trapped on the surface by adsorption. In most drug delivery systems, encapsulation occurs through different mechanisms at the same time [16]. In the present study, the anticancer drug daunorubicin was used as a standard pharmaceutical compound (Fig. 1). 1. In a beaker disperse 10.0  mg of synthesized nanospheres in 10 mL of isotonic solution (0.9 wt% NaCl). 2. Add 4 mg of daunorubicin hydrochloride, DNR. 3. Leave the mixture to stir gently for 48 h at 25 °C. 4. Centrifuge (see Note 11) and keep the isolated supernatant to determine the amount of daunorubicin not entrapped in the spheres through standard curve method (Scheme 1, see Note 12). UV–vis absorption spectra in the wavelength range of 200–800  nm were obtained on a Jasco V-650 spectrometer, with UV–vis at 480 nm spectrometer. An ultrasonic bath was used for sonication. Using the reference curve (Fig. 2), the amount of drug trapped can be calculated. The absorbance of isolated supernatant (1 mL) was A = 2.38, so we can calculate the concentration of daunorubicin from the equation y = 0.0157x + 0.0619 (y = A and x = c (μg/ mL)). The concentration of the non-loaded drug was 147.65 μg/ mL, and as we know the total volume of the supernatant solution, we can easily calculate the mass of the non-loaded (10  mL  ×  147.65  μg  =  1.4765  mg) and loaded drug (4  mg  – 1.4765 mg = 2.5235 mg). Also, the encapsulation efficiency and the ability to capture it from the microspheres (loading capacity) are calculated according to the following equations (see Table 1):



Encapsulation capacity ( % ) = Encapsulation efficiency ( % ) =



Weight of the loaded drug in vehicles ×100 Total weight of the vehicles



Weight of the loaded drug in vehicles ×100 Weight of the starting amount of drug



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y = 0,0157x + 0,0619 R2 = 0,9958

2, 5 A

2 1, 5 1 0, 5 0

0

50

100

150

c(μg/ml)

200

250

Fig. 2 The reference curve

Table 1 Drug encapsulation results of the present study Weight of vehicles (mg) Absorbance (A)

2.38

Concentration of drug (c) at supernatant (μg/mL)

147.65

Non-loaded drug (mg)

1.4765

Starting amount of drug (mg) Loaded drug (mg)

3.3  Characterization of Vehicles

10

4 2.5235

Encapsulation efficiency (%)

63%

Encapsulation capacity (%)

25%

The fabricated vehicles can be characterized structurally by FT-IR spectroscopy and morphologically by scanning (SEM) and transmission electron microscopy (TEM) techniques (see Notes 12–15). Microscopy is one of the most powerful techniques and can provide valuable information about the size, shape, and morphology of nanoparticles. Electron microscopy provides high-­resolution images and is the only technique that provides reliable information about the shape on this scale. However, scientists need to ensure that enough particles are examined to have a statistically valid representation of the size and shape distribution. This can be very difficult and time consuming and may require the analysis of the image of literally thousands of individual particles. However, scientists need to ensure that enough particles are examined to have a

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statistically valid representation of the size and shape distribution. This can be very difficult and time consuming and may require the analysis of the image of literally thousands of individual particles. There are many commercially automated image analysis systems and software packages used for this purpose. The quality of the images presented in these systems is critical to their performance. It should also be noted that electron microscopy normally provides two-dimensional images, so care must be taken to avoid bias [17, 18]. 3.3.1  SEM Preparation

1. Convert microspheres into powder. 2. Press a small amount of powder on a conductive adhesive tape and mount on a holder. 3. For conventional imaging in the SEM, the samples must be electrically conductive, at least on the surface, to prevent the accumulation of electrostatic charge. Nonconductive materials are usually coated with electrically conductive material deposited on the sample. The conductive material used as a coating was gold. 4. The sample is irradiated with a high-energy electron beam. The surface characteristics of the nanoparticles are illustrated by the secondary electrons emitted from the surface of the sample. 5. Take images after every stage of the procedure to ensure the deposition of each layer. SEM images of microspheres in different stages of synthesis are presented in Fig. 3.

3.3.2  TEM Preparation

The principle of transmission electron microscopy is based on principles different from SEM, although it often provides the same results. 1. The microspheres are dispersed in an appropriate solvent and deposited on thin films. 2. The specimen characteristics are represented by a thin electron beam that permeates the sample and interacts with it. TEM images of PMMA@HPC@CS@CH microspheres are presented in Fig. 4.

3.3.3  AT-IR Preparation

1. Convert microspheres into powder. 2. Place a small amount of powder on the instrument and measure. 3. Take samples for each stage of the synthesis and compare the differences in spectra. The most important peaks of FT-IR spectra at various stages of the synthesis are summarized in Table 2.

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Fig. 3 SEM images of microspheres in different stages of synthesis. A. PMMA spheres’ diameter ranges at 200 ± 15 nm with low polydispersity. B. PMMA@HPC spheres’ diameter is increased confirming the successful deposition. C. PMMA@HPC@CS microspheres’ diameter is increased to 300 ± 30 nm. D. PMMA@HPC@ CS@CH microspheres’ diameter is increased to 350–370 nm

Fig. 4 TEM images of PMMA@HPC@CS@CH microspheres. The core of polymethyl methacrylate is distinguished with dark color and the deposited layers of polysaccharides are distinguished with a light gray layer. Typical configurations of spheres are due to the intermolecular hydrogen bonds that grow between the spheres. It is also observed that the coating is relatively uneven and varies forming core-shell morphology

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Table 2 AT-IR characteristic peaks of synthesized multi-sensitive microspheres Wavenumber (cm−1)

Vibration

Synthesis of PMMA@HPC@CS microspheres 3669

C-H bond of hydroxypropyl cellulose’s propyl group

2980, 2893

Aliphatic bonds C-H

1726 (sharp peak)

-C=O of methyl methacrylate and succinic acid segment of modified cellulose

1059

C-O-C pyranose ring (see Note 16)

892

β-Glycosidic bonds between the structural units of the cellulose

Synthesis of PMMA@HPC@CS@CH microspheres 1640

N-H bond of chitosan

After cross-linking 1631

-C=O of N-H (see Note 17)

4  Notes 1. This composition is advantageous in comparison to the literature, due to the fact that in all synthetic steps, water is used as solvent, avoiding the use of organic solvents. 2. Warm to 70 °C for 30 min with vigorous stirring in a nitrogen flow. 3. Leave the mixture for 30 min. 4. The emulsion is centrifuged by various cycles of resuspension in water (3 × 8000 rpm (7491 × g) for 5 min). 5. Aqueous solution of HCl, pH = 3. 6. Aqueous solution of NaOH, pH = 10. 7. The resulting emulsion is purified via various resuspension cycles in water (4 × 10,000 rpm (11704 × g) for 5 min). 8. The intermediate layer is selected to be formed by use of modified cellulose which functions as a cross-linker for stabilization purposes between the other two layers. This stabilization has been performed by cross-linking between the modified cellulose and hydroxypropyl cellulose via ester bonds and between chitosan through amide bond formation.

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9. The resulting emulsion is centrifuged and resuspended in water for purification (3 × 10,000 rpm (11704 × g) for 5 min). 10. The last coating aims at improving the microsphere-protein interactions. 11. Centrifuge for 5 min at 10,000 rpm. 12. The curve is constructed by absorption measurements of standard solutions of known concentration, since according to the Lambert-Beer law, A = ε × b × c, the concentration of drug is proportional to absorption. 13. SEM and TEM images are obtained on a FEI Inspect microscope with W (tungsten) filament operating at 25  kV and a FEI CM20 microscope operating at 200 kV, respectively. 14. The spectra are scanned over the range of 4000–500  cm−1. UV–vis absorption spectra in the wavelength range of 200– 800 nm were obtained on a Jasco V-650 spectrometer, with UV–vis at 480 nm spectrometer. 15. An ultrasonic bath was used for sonication (Elma Sonic, S. 30H). 16. Structural units of cellulose. 17. The appearance of the peak at 1631 cm−1 due to the absorption of the carbonyl amide bond and the absence of the peak at 3669 cm−1 due to vibration of O-H group of HPC is confirmed by the successful cross-linking. The absence of peak attributed to hydroxyl groups of hydroxypropyl cellulose and carboxylic acids of the CS confirms the ester bond formation. References 1. Carr C, Ng J, Wigmore T (2008) The side effects of chemotherapeutic agents. Curr Anaesth Crit Care 19(2):70–79. https://doi. org/10.1016/j.cacc.2008.01.004 2. Kim GNS (2005) Targeted cancer nanotherapy. Nano Today 8:28–33 3. Arruebo M, Fernández-Pacheco R, Ibarra MR, Santamaría J (2007) Magnetic nanoparticles for drug delivery. Nano Today 2(3):22–32. https://doi.org/10.1016/ s1748-0132(07)70084-1 4. Torchilin VP (2012) Multifunctional nanocarriers. Adv Drug Deliv Rev 64:302–315. https:// doi.org/10.1016/j.addr.2012.09.031 5. Torchilin VP (2007) Targeted pharmaceutical nanocarriers for cancer therapy and imaging. AAPS J 9(2):E128–E147. https://doi. org/10.1208/aapsj090201 6. Alvarez-Lorenzo C, Blanco-Fernandez B, Puga AM, Concheiro A (2013) Crosslinked ionic polysaccharides for stimuli-sensitive drug

delivery. Adv Drug Deliv Rev 65(9):1148– 1171. https://doi.org/10.1016/j. addr.2013.04.016 7. Posocco B, Dreussi E, de Santa J, Toffoli G, Abrami M, Musiani F, Grassi M, Farra R, Tonon F, Grassi G, Dapas B (2015) Polysaccharides for the delivery of antitumor drugs. Materials 8(5):2569–2615. https://doi.org/10.3390/ ma8052569 8. Park JH, Saravanakumar G, Kim K, Kwon IC (2010) Targeted delivery of low molecular drugs using chitosan and its derivatives. Adv Drug Deliv Rev 62(1):28–41. https://doi. org/10.1016/j.addr.2009.10.003 9. Zhang J, Chen XG, Li YY, Liu CS (2007) Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin. Nanomedicine 3(4):258–265. https://doi.org/10.1016/j. nano.2007.08.002

Design of Drug Delivery Systems Based on Polysaccharides 10. Metaxa AF, Efthimiadou EK, Boukos N, Kordas G (2012) Polysaccharides as a source of advanced materials: cellulose hollow microspheres for drug delivery in cancer therapy. J Colloid Interface Sci 384(1):198–206. https://doi.org/10.1016/j.jcis.2012.04.073 11. Metaxa A-F, Efthimiadou EK, Kordas G (2014) Cellulose-based drug carriers for cancer therapy: cytotoxic evaluation in cancer and healthy cells. Mater Lett 132:432–435. https://doi. org/10.1016/j.matlet.2014.06.134 12. Metaxa AF, Efthimiadou EK, Boukos N, Fragogeorgi EA, Loudos G, Kordas G (2014) Hollow microspheres based on  - folic acid modified  - Hydroxypropyl cellulose and synthetic multi-responsive bio-copolymer for targeted cancer therapy: controlled release of daunorubicin, in  vitro and in  vivo studies. J Colloid Interface Sci 435C:171–181. https:// doi.org/10.1016/j.jcis.2014.08.001 13. Kean T, Thanou M (2010) Biodegradation, biodistribution and toxicity of chitosan. Adv Drug Deliv Rev 62(1):3–11. https://doi. org/10.1016/j.addr.2009.09.004

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14. Felt O, Buri P, Gurny R (1998) Chitosan: a unique polysaccharide for drug delivery. Drug Dev Ind Pharm 24(11):979–993. https://doi. org/10.3109/03639049809089942 15. Sinha VR, Singla AK, Wadhawan S, Kaushik R, Kumria R, Bansal K, Dhawan S (2004) Chitosan microspheres as a potential carrier for drugs. Int J Pharm 274(1–2):1–33. https:// doi.org/10.1016/j.ijpharm.2003.12.026 16. De Villiers MM (2009) Nanotechnology in drug delivery. Springer, New York 17. Dhawan A, Sharma V (2010) Toxicity assessment of nanomaterials: methods and challenges. Anal Bioanal Chem 398(2):589–605. https:// doi.org/10.1007/s00216-010-3996-x 18. Powers KW, Brown SC, Krishna VB, Wasdo SC, Moudgil BM, Roberts SM (2006) Research strategies for safety evaluation of nanomaterials. Part VI Characterization of nanoscale particles for toxicological evaluation. Toxicol Sci 90(2):296–303. https://doi.org/10.1093/ toxsci/kfj099

Chapter 13 Differential Scanning Calorimetry (DSC) on Sartan/ Cyclodextrin Delivery Formulations Nikolaos Naziris, Maria Chountoulesi, Dimitrios Ntountaniotis, Thomas Mavromoustakos, and Costas Demetzos Abstract Differential scanning calorimetry (DSC) is a widely utilized method for the interactions of drug molecules with drug delivery systems (DDSs). Herein is described a protocol for studying the interactions and entrapment efficiency of the prototype sartan losartan and the polydynamic, structurally similar irbesartan inside the nontoxic 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD). The thermal scan properties of both sartan molecules have been studied when physically mixed or complexed with the cyclodextrin. The thermograms indeed showed significant differences between the mixtures and complexes, establishing DSC as a valuable method to characterize the state of the drugs in these pharmaceutical formulations. Key words Differential scanning calorimetry, Irbesartan, Losartan, 2-Hydroxypropyl-β-cyclodextrin, Mixing, Lyophilization, Interactions, Complexation

1  Introduction Thermal analysis (TA) techniques are widely used in order to study solid, semisolid, or liquid substances. Some of the commonly studied materials are foods, electronic materials, polymers, organic or inorganic compounds, biological organisms, and pharmaceuticals [1]. In pharmaceutical sciences, the most frequently used TA method is differential scanning calorimetry (DSC), where the heat flow rate difference between a reference and a sample material is measured. It is primarily applied on crystalline solids, solid dispersions, and polymeric dosage forms, for the characterization of polymorphic forms, study of the effects of lyophilization, as well as kinetics of various phenomena, such as decomposition and accelerated aging [2–4]. There is the heat-flux DSC and the power-compensation DSC, the main difference of the two being that the first uses one furnace heater for both samples, applying the same temperature to both Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Fig. 1 Heat-flux DSC

sample and reference, while the second uses two individual heaters, allowing it to measure the change in power or energy that is required for sample and reference to have the same temperature [5]. In the herein described work, the heat-flux type was utilized for the analysis of the cyclodextrin:sartan formulations (Fig. 1). Concerning pharmaceutical development, thermal analysis techniques facilitate the study of phase transitions and changes in the heat capacity of pharmaceutical formulations, including drug delivery systems (DDSs), such as cyclodextrins (CDs). That way, information about the purity, polymorphism and interactions of final pharmaceutical forms, as well as properties of packaging materials, is provided, giving the opportunity for assessment of their lifetime physical stability. For this reason, pharmaceutical thermal analysis is a useful tool for the drug development process, ensuring the physical stability of the final pharmaceutical product, by studying the bioactive molecules, excipients, as well as compatibility between them [6, 7]. DSC can be utilized for the analysis of thermal phenomena in materials and biomaterials, like melting, crystallization, glass transition, evaporation, decomposition, and dehydration. A typical DSC thermogram or thermal scan of an amorphous solid is given in Fig. 2 [8]. DSC has been extensively utilized for the analysis and study of drug-cyclodextrin complexes. In particular, these include hydroxypropyl-β-cyclodextrin (HP-β-CD) or 2-HP-β-CD complexes with thalidomide, paracetamol, or meclizine HCl, where the thermal analysis of free forms, physical mixture, and complex between the drug and the CD allows for the evaluation of the association between them [9–11]. The method of complexation, as well as the physical state of each component, affects their degree of association. For example, it has been demonstrated that the physical mixing/grinding or the preparation of complex through the kneading, freeze-drying, or coprecipitation method may all yield different degrees of interaction, which are reflected on the DSC

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Fig. 2 Thermogram of an amorphous compound showing glass transition (Tg), crystallization (Tc), melting (Tm) and degradation

profiles [10, 11]. Other DSC studies on drug-CD complexes include methyl-β-cyclodextrin (M-β-CD) with lidocaine and 2-HP-β-CD with oxicams [12, 13]. In all these cases, each component and each complex or mixture were analyzed and through comparison, an estimation of the nature and degree of interactions was obtainable. DSC generally provides an insight into the system thermodynamics, which complements classic pharmaceutical formulation tests, such as phase solubility, dissolution studies, and in vitro permeability. Sartans and specifically irbesartan and losartan have been characterized by DSC analysis [14–17]. In addition, the complex between valsartan and β-cyclodextrin (β-CD) was prepared by various methods, i.e. solid dispersion, freeze-drying, kneading and physical mixture, and studied by DSC [18]. The reduction of crystallinity of the system is an indication of the complexation between the two materials and is reflected on the observed peaks in the calorimetric profile, where the melting transition of the drug molecule is diminished or vanished.

2  Materials The materials described below are referred to the DSC analysis of sartan-2-HP-β-CD mixtures or complexes or the components

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alone. Herein, complexes are formed by either dissolving and ­suspending the molecules in aqueous medium and freeze-drying or physically mixing raw materials and lyophilized materials in all possible combinations. Lyophilization is applied on frozen solutions of the materials in purified water. All the final samples that are utilized for DSC analysis, whether they are single materials or mixtures or complexes, are in dry powder form. 2.1  Lyophilization of Raw Materials

1. Losartan potassium (Mw = 461.01). 2. 2-HP-β-CD (Mw = 1460 (average)). 3. Purified water. 4. NH3 5 N.

2.2  Complex of Sartan with 2-HP-β-CD

1. Irbesartan (Mw = 422.91) or losartan potassium. 2. 2-HP-β-CD. 3. Purified water. 4. NH3 5 N.

2.3  Differential Scanning Calorimetry (DSC)

1. DSC aluminum pans with O-ring (40 μL). 2. Pure indium (Tm = 156.6 °C). 3. Samples: ∙∙

Irbesartan or losartan raw material

∙∙

Lyophilized losartan

∙∙

2-HP-β-CD raw material

∙∙

Lyophilized 2-HP-β-CD

∙∙

Raw material physical mixture

∙∙

Lyophilized form mixture (see Note 1)

∙∙

Raw material 2-HP-β-CD mixture with lyophilized losartan

∙∙

Lyophilized 2-HP-β-CD mixture with raw material losartan

∙∙

Complex of sartan with 2-HP-β-CD (see Note 2)

3  Methods Heat-flux DSC is herein applied on samples containing the cyclodextrin 2-HP-β-CD and irbesartan or losartan in various states. The samples that are subjected to DSC analysis contain the raw material forms of the cyclodextrin and sartans; their lyophilized forms, except for irbesartan, which cannot be lyophilized; the lyophilized complexes between the cyclodextrin and the drugs; the mixtures of the raw materials; the mixtures of the lyophilized forms, whenever possible; as well as all possible mixture combinations. Lyophilization

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was applied on the raw materials of cyclodextrin and losartan, in order to analyze them alone or in mixtures, as well as on a complexed form between the cyclodextrin and each sartan. 3.1  Lyophilization of Raw Materials

1. Dissolve 2-HP-β-CD and losartan potassium in purified water separately, both at a concentration of 8 mg mL−1. 2. Freeze the solutions at −80 °C overnight (see Note 3). 3. Lyophilize samples with any appropriate lyophilizer, under the following conditions: condenser temperature −50 °C; vacuum 8.2 × 10−2 mb; and duration 24 h. 4. Store the lyophilized powders at 4 °C.

3.2  Complexation of the Sartans with 2-HP-β-CD

1. Transfer 6783.62  mg of 2-HP-β-CD in a glass vessel with 800 mL of purified water and set under magnetic stirring. 2. When the cyclodextrin is totally dissolved, add irbesartan or losartan in the vessel and continue magnetic stirring (see Note 4). The molar ratio between cyclodextrin and either drug is 3.6:1 and usually the drug used is ca 1000 mg. 3. Adjust pH at approximately 10.5 with NH3 5 N and the reaction of enclosure takes place. 4. When the solution is clear, fix the final volume at 1000 mL with purified water and immediately freeze the solution of the complex at −80 °C for lyophilization to follow.

3.3  DSC Sample Preparation

1. Prepare each sample for DSC analysis by weighting roughly 3  mg of dry powder that contains one or more components inside a 40 μL aluminum crucible with O-ring (see Note 5). 2. Seal each crucible by using a sealing press, in order to make a hermetic pan, and leave it to rest for a 15-min period, in order to achieve equilibration of the sample (see Note 6).

3.4  DSC Analysis

1. Obtain the DSC thermogram of each sample by utilizing any appropriate DSC calorimeter. 2. Calibrate the calorimeter with pure indium (Tm  =  156.6  °C) before analyses, by applying a single heating cycle and checking the characteristic transition temperature Tm and enthalpy change ΔH to be within specifications. 3. Include for each analysis a 5-min isotherm at 10 °C (see Note 7) and a heating process from 10  °C to 230  °C for irbesartan-­ containing samples and 10 °C to 300 °C for losartan-­containing samples (see Note 8), at a heating rate of 10 °C min−1 (see Note 9), under constant nitrogen gas flow rate of 50 mL min−1 (see Note 10).

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3.5  DSC Diagram and Thermodynamic Parameter Extraction

1. Analyze the obtained calorimetric data by using the software installed in the thermometer. 2. Normalize each analysis per total sample weight (see Note 11). 3. Choose the desired thermodynamic parameters for each thermodynamic phenomenon, i.e. endothermic or exothermic. These are the characteristic transition temperatures Tonset and T, enthalpy change ΔH, and width at half peak height of the Cp profiles ΔT1/2 (see Note 12). 4. During peak integration, manually set the baseline (see Notes 13–15).

3.6  DSC Profile Analysis of the Cyclodextrin:Sartan Systems

The profiles of all samples containing 2-HP-β-CD and irbesartan or losartan are presented in Fig. 3. The following observations can be made on the thermograms: ∙∙

2-HP-β-CD and its lyophilized form exhibited a melting transition at around 170 °C, with the first being more crystalline than the second, resulting in a sharper peak and slightly higher transition enthalpy (Fig. 3a, b) [12]. The two drugs gave transition peaks at around 185 °C and 270 °C for irbesartan and losartan, respectively, with these values being very close to the bibliography (Fig.  3c, d). In addition, a pre-transition was recorded for losartan [14, 16, 17]. The lyophilized form of losartan led to the appearance of another form of the molecule that melts at 165 °C (Fig. 3e).

∙∙

The nature of the raw material physical mixture between 2-HP-β-CD and losartan was very different from that with irbesartan (Fig.  3f, g). Though the molar ratio of cyclodextrin:sartan was the same in the two cases (3.6:1), the mixture with losartan led to an endothermic peak at 175 °C, suggesting the existence of crystalline cyclodextrin in the mixture and the loss of crystallinity of losartan, due to intermolecular interactions. On the contrary, for irbesartan, there was a low enthalpy peak at 170 °C and another at 200 °C, the first owed to the cyclodextrin and the second to the drug, which suggests that part of the drug has not been complexed and only a small amount of cyclodextrin is in the crystalline state.

∙∙

The physical mixture of the drugs with lyophilized 2-HP-β-CD led to similar profiles, where a small peak was observed for irbesartan at 180 °C and for losartan at 240 °C. For losartan, this should be attributed to the drug; however, in the case of irbesartan, it could be due to the cyclodextrin, since the melting peaks of the two are very close.

∙∙

The complex of 2-HP-β-CD with irbesartan or losartan resulted in different DSC heating profiles. For the first com-

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Fig. 3 The DSC thermograms of (a) 2-HP-β-CD raw material, (b) 2-HP-β-CD lyophilized, (c) irbesartan raw material, (d) losartan raw material, (e) losartan lyophilized, (f) mixture of irbesartan raw material with 2-HP-β-CD raw material, (g) mixture of losartan raw material with 2-HP-β-CD raw material, (h) mixture of irbesartan raw material with 2-HP-β-CD lyophilized, (i) mixture of losartan raw material with 2-HP-β-CD lyophilized, (j) mixture of losartan lyophilized with 2-HP-β-CD raw material, (k) mixture of losartan lyophilized with 2-HP-β-CD lyophilized, (l) lyophilized complex of irbesartan with 2-HP-β-CD, and (m) lyophilized complex of losartan with 2-HP-β-CD

plex, it led to a wide peak that started from 20 °C and ended at 240 °C, while for the second, it also absorbed energy from low temperatures; however, it gave an endothermic peak at 170 °C that resembles the one of lyophilized 2-HP-β-CD. This suggests that an excess of crystalline cyclodextrin exists after complexation in the case of losartan. From all these observations, we can assume that the amount of 2-HP-β-CD necessary to complex losartan may be lower than 3.6 molecules for every molecule of drug, while for irbesartan, it seems

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that it is ideal; however, the physical mixture process does not yield 100% complexation. ∙∙

The physical mixture of lyophilized losartan with raw material 2-HP-β-CD led to interaction between the two molecules and their existence in amorphous state.

∙∙

Finally, the physical mixture of lyophilized losartan with lyophilized 2-HP-β-CD was thermodynamically very close to their complex, which indicates the high degree of interactions between the molecules by utilizing these methods.

4  Notes 1. The raw materials are first subjected to lyophilization and then physically mixed, in order to be analyzed. Lyophilization is only possible for losartan in its salt form. 2. The complex is formed by dissolving the molecules and then freeze-drying. 3. For losartan, this can be achieved because of the drug being available in salt form with potassium, while this does not apply for irbesartan. 4. The process of stirring inside the cyclodextrin solution aims to increase the insertion of the drugs inside the cavity and lead to complexation. 5. Single-component samples are neat 2-HP-β-CD, irbesartan, losartan, or their lyophilized forms. Samples containing mixture of raw materials, their lyophilized forms, or combinations of those are prepared by weighting raw material or lyophilized 2-HP-β-CD and raw material or lyophilized sartan, in 3.6:1 molar ratio, for a total of approximately 3  mg. Cyclodextrin:sartan complex preparations have been described previously. 6. Equilibration is considered necessary for samples that have been previously hydrated with aqueous media; however, the same applies for solids, because of the pressure applied during the crucible sealing. 7. The isotherm at the analysis starting point is necessary to ensure that the sample is at equilibrium. 8. Typically, the analysis temperature range depends on the melting points and the various thermodynamic alterations, endothermic or exothermic, that are expected for every component that is analyzed. As a result, the range should be the same for all samples, in order to be able to assess all types of interactions between the components. Based on the bibliography, the expected melting points for irbesartan and losartan, respec-

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tively, are 185  °C and 270  °C, while 2-HP-β-CD has been reported to melt at a broad range, from around 20  °C to 150 °C [9, 14, 15]. 9. A high heating rate may not allow for certain kinetic phenomena to appear and as a result should not be too high. Based on the bibliography, 10  °C  min−1 is appropriate for cyclodextrin:sartan system analysis [9, 12, 15, 16]. 10. Constant pressure is essential in this type of calorimetry, in order to monitor the heat capacity of the sample under constant pressure (Cp). 11. Each analysis may be normalized per total sample, per cyclodextrin, or per sartan mass, depending on which DSC peak is studied. For example, in a physical mixture of 2-HP-β-CD and irbesartan, a certain amount of drug is probably not complexed and will melt because of its crystalline state. In cases of complexes and mixtures, certain peaks may reflect one material or both materials in their complexed form and as a result should be analyzed appropriately (Fig. 4). 12. A lot of other parameters that concern the calorimetric profiles are available to extract; however, these are the most utilized in the bibliography [11, 14, 16]. 13. The baseline may vary each time it is set. For this reason, three different attempts should be made, in order to extract a statistical sample for the enthalpy change ΔH. This does not apply on other thermodynamic parameters that are more accurate. 14. During analysis, the baseline might deviate from its original horizontal, due to kinetically slow endothermic or exothermic phenomena or because of the measurement itself. In case we wish to include this deviation during peak integration, we have to set the baseline accordingly. In this way, comparisons between peaks and areas that contain these peaks are attainable. An example is given below (Fig. 5). 15. The enthalpy change ΔH is considered negative for an endothermic process and positive for an exothermic process.

Acknowledgments We gratefully thank Prof. G.  Valsami and E.  Christodoulou for their help to prepare the complexes. This work has been co-­ financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.”

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Fig. 4 The DSC profiles of (a) physical mixture of irbesartan raw material with 2-HP-β-CD raw material, (b) irbesartan raw material with 2-HP-β-CD lyophilized, and (c) complex of irbesartan with 2-HP-β-CD

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Fig. 5 In this example of 2-HP-β-CD lyophilized, by setting the baseline of analysis at different points, the integration will vary, including wider areas of endothermic or exothermic processes References 1. Cheng SZD, Li CY, Calhoun BH et al (2000) Thermal analysis: the next two decades. Thermochim Acta 355:59–68 2. Mura P, Moyano JR, Gonzalez-Rodriguez ML et  al (2005) Characterization and dissolution properties of ketoprofen in binary and ternary solid dispersions with polyethylene glycol and surfactants. Drug Dev Ind Pharm 31:425–434 3. Aceves-Hernandez JM, Nicola-Varquez I, Aceves FJ et  al (2009) Indomethacin polymorphs: experimental and conformational analysis. J Pharm Sci 98:2448–2463 4. Rameau A, Chevalier A, Neves C (2004) Lyophilization optimization using DSC and MT-DSC. American Pharm Rev 7:50–60 5. Hatakeyama T, Quinn FX (1999) Thermal analysis: fundamentals and applications to polymer science. John Wiley & Sons, New York 6. Clas SD, Dalton CR, Hancock BC (1999) Differential scanning calorimetry: applications in drug development. Pharm Sci Technolo Today 2:311–320 7. Demetzos C (2008) Differential scanning calorimetry (DSC): a tool to study the thermal behavior of lipid bilayers and liposomal stability. J Liposome Res 18:159–173 8. Jenkins HDB (2008) Chemical thermodynamics at a glance. Blackwell, London 9. Kratz JM, Teixeira MR, Ferronato K et  al (2011) Preparation, characterization, and

in  vitro intestinal permeability evaluation of thalidomide-hydroxypropyl-β-cyclodextrin complexes. AAPS PharmSciTech 13:118–124 10. Talegaonkar S, Khan AY, Khar RK et  al (2007) Development and characterization of paracetamol complexes with hydroxypropyl-β-­ cyclodextrin. Iran J Pharm Res 6:95–99 11. George SJ, Vasudevan DT (2012) Studies on the preparation, characterization, and solubility of 2-HP-β-cyclodextrin-meclizine HCl inclusion complexes. J Young Pharm 4:220–227 12. da Silva LFJS, do Carmo FA, de Almeida Borges VR et al (2011) Preparation and evaluation of lidocaine hydrochloride in cyclodextrin inclusion complexes for development of stable gel in association with chlorhexidine gluconate for urogenital use. Int J Nanomedicine 6:1143–1154 13. Suta LM, Vlaia L, Fulias A et  al (2013) Evaluation study of the inclusion complexes of some oxicams with 2-hydroxypropyl-­ β-cyclodextrin. Rev Chim (Bucharest) 64:1279–1283 14. Soma D, Attari Z, Reddy MS et  al (2017) Solid lipid nanoparticles of irbesartan: preparation, characterization, optimization and pharmacokinetic studies. Braz J Pharm Sci 53:e15012 15. Khandai M, Chakraborty S, Ghosh AK (2014) Losartan potassium loaded bioadhesive micro-­ matrix system: an investigation on effects of

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hydrophilic polymeric blend on drug release. Pharm Anal Acta S8:001 16. Aglawe Sachin B, Gayke AU, Metkar PS et  al (2017) Formulation and evaluation of losartan potassium microspheres by solvent evaporation method. Indo Amer J Pharm Res 7:735–743 17. Al-Dmour NS, Abu-Dahab RMN, Evstigneev MP et  al (2019) Interaction of pseudo-

ephedrine and azithromycin with losartan: Spectroscopic, dissolution and permeation studies. Spectrochim Acta A Mol Biomol Spectrosc 221:117194 18. Jensen CE, dos Santos RA, Denadai AM (2010) Pharmaceutical composition of valsartan: beta-cyclodextrin: physico-chemical characterization and anti-hypertensive evaluation. Molecules 15:4067–4084

Chapter 14 Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability, and Therapeutic Efficacy Against Glioblastoma Multiforme Antonis D. Tsiailanis, Alexander Renziehausen, Serdar Karakurt, Tim Crook, Nelofer Syed, and Andreas G. Tzakos Abstract Cancer occupies a high rank in the global morbidity and mortality scale with glioblastoma multiforme (GBM) accounting for almost 80% of all primary tumors of the brain. Despite the increasing availability of targeted and immunotherapeutic agents, chemotherapy still plays an important role in the treatment of neoplastic diseases. Limitations to the effective use of chemotherapy such as low aqueous solubility and high toxicity have directed the scientific community’s interest to the development of new therapeutic agents with enhanced efficacy and limited toxicity. Supramolecular chemistry has offered an alternative way on the design and development of new therapeutic agents as a result of their unique properties. Supramolecules can be used as drug carriers since their cavities can host a wide range of small drugs and surpass in this way the drawbacks of current therapeutic agents. Herein, we present the principles that should be followed for the encapsulation of small drugs in supramolecules with enhanced physicochemical properties and increased efficacy against glioblastoma multiforme. Key words Glioblastoma multiforme, Temozolomide, Supramolecule, p-sulfonatocalix[4]arene, Encapsulation, 1H-NMR spectroscopy, Mass spectrometry, Liquid chromatography, LC-MS/MS

1  Introduction Cancer is one of the leading global causes of morbidity and mortality. Although targeted and immunotherapeutic agents are increasingly available, systemic chemotherapy continues to have an important role in the management of the neoplastic disease. Despite obvious benefits of chemotherapy, its utility is frequently limited, particularly in metastatic disease, by innate and/or acquired drug resistance (the latter an inevitable consequence of tumor heterogeneity) and by toxicity of drugs [1]. Further limitations to the effective use of chemotherapy arise due to poor aqueous solubility, instability, and low drug-loading capacity. The ability Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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to enhance efficacy and reduce toxicity, i.e., to increase the ­therapeutic index of anticancer drugs, would have a significant impact on facilitating the optimal use of chemotherapy and serving as an important driver in improving clinical outcomes [2, 3]. A large pool of bioactive compounds and approved drugs has evolved from multiple medicinal chemistry projects in the pharma industry and academia. Supramolecular chemistry has served as a useful tool in biomedical applications [4, 5]. Due to their unique properties, supramolecules can be used as alternative drug carriers, based on their ability to encapsulate small drugs in their cavity. The encapsulation of small molecules into the cavity is the result of noncovalent interactions, such as van der Waals interaction, hydrogen bonding, and hydrophobic interactions. According to recent literature, macromolecules such as cucurbit[n]urils, cyclodextrins, calix[n]arenes, and their derivatives have drawn the attention of the scientific community, since they can surpass problems that current cytotoxic agents present [6, 7]. Recently, our group developed a new formulation that demonstrated enhanced activity against glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, by encapsulation of temozolomide (TMZ) into a supramolecular host [8]. TMZ is an alkylating agent used as first-line chemotherapy for GBM [9, 10]. However, under slightly alkaline conditions it is rapidly hydrolyzed to MTIC, which in turn rapidly degrades to the methyl diazonium cation and the metabolite 5-amino-imidazole-­ 4-carboxamide (AIC), resulting in reduced passage across the BBB and therefore reduced clinical efficacy [11, 12] (Fig.  1). Hence, high doses of TMZ are administrated to achieve therapeutic concentrations in the central nervous system (CNS), resulting in numerous side effects including myelosuppression. To abrogate these issues, we used p-sulfonatocalix[4]arene (PSC4) nanocapsule as host. PSC4 contains a hydrophilic external surface and a hydrophobic cavity that can entrap small drugs inside. PSC4 has been widely used as host due to its high aqueous solubility and low toxicity [13, 14]. The encapsulation of TMZ in PSC4 greatly improved the stability of TMZ and its efficacy against GBM in vitro and in vivo (Scheme 1). Here, we summarize the principles that influence the development of new formulations and, in particular, the encapsulation of small molecules into PSC4. We describe the protocols that have been developed for the determination of the encapsulated drug and the stability of the complex. After the synthesis of the formulate, LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@PSC4 in vivo. Finally, the cytotoxicity of the analog was evaluated in vitro in patient-­ derived primary lines that express MGMT and are normally highly resistant to TMZ and in a mouse orthotopic model.

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Fig. 1 Reaction scheme for the base-catalyzed decomposition of TMZ to its active metabolite MTIC and then to 5-amino-imidazole-4-carboxamide (AIC) and methyl diazonium cation O N N

NH2

O N

N

HN N

N N

NH2

O

N N H

TMZ@PSC4

MTIC

Temozolomide

O N N

O N N

NH2

O

N

N O

NH N

N

Temozolomide

N

HN N

NH2 N

NH2

N H

MTIC

O N

pt

s osi

o Ap

N

NH N

N H

Scheme 1 Schematic representation of the degradation of TMZ to 5-(3-methyl-triazen-1-yl) imidazole-4-carboxamide (MTIC) outside of the cell and the mechanism of stabilization of TMZ through complexation with PSC4

2  Materials 2.1  Synthesis and Characterization of Encapsulated TMZ

1. Temozolomide (T2577) and p-sulfonatocalix[4]arene (55523) were purchased from Sigma-Aldrich. 2. Methanol and water (both LC-MS grade).

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3. PBS buffer (10 mM, pH 7.0): Both interactants should be dissolved in the same buffer. 4. Deuterated dimethyl sulfoxide 99.8% (DMSO-d6) and deuterium oxide 99.9% (D2O). 5. Bruker 400  MHz Avance spectrometer equipped with a z-­gradient unit (Bruker BioSpin, Rheinstetten, Germany): The NMR system is controlled by the software TopSpin 3.1. 2.2  UV-Vis Spectroscopy

1. PBS buffer (10  mM, pH  7.0), water (double distilled), methanol. 2. 1 mL Cuvettes. 3. Water bath. 4. 37 °C Shaker and incubator. 5. UV-Vis spectrometer (slit = 1, speed 240 nm/min).

2.3  Liquid Chromatography and Mass Spectrometry

1. Theophylline (internal standard). 2. Regenerated cellulose membrane syringe filters 0.2 μm. 3. Cellulose nitrate filters, 0.2  μm pore size, for mobile-phase filtering. 4. Acetonitrile, water, and formic acid (LC-MS grade). 5. Mobile phase A: Water containing 0.1% (v/v) formic acid. 6. Mobile phase B: Acetonitrile containing 0.1% (v/v) formic acid. 7. C18 column 100 mm × 2.1 mm, 2.6 um, with ProGuard column 2.1 mm. 8. Triple-quadrupole mass spectrometer coupled to ultrahigh-­ performance liquid chromatography (Bruker’s EVOQ Elite ER-UHPLC).

2.4  In Vitro Assays of TMZ@PSC4 Activity

1. Patient-derived primary GBM cell cultures (GBM31, GBM59, GBM77) were established from fresh tumor tissue obtained from first surgical debulking or stereotactic biopsies at Charing Cross Hospital as described previously. 2. Tissue samples were provided by the Imperial College Healthcare NHS Trust Tissue Bank, which is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London.

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3  Methods 3.1  Synthesis, Characterization, and Quantification of the Encapsulated TMZ 3.1.1  Synthesis and Purification of Nanocapsule

1. Dissolve p-sulfonatocalix[4]arene in phosphate buffer pH 7.0 (10 μM). 2. Dilute TMZ in MeOH. 3. Add dropwise TMZ in the aqueous solution and magnetically stir at room temperature for 1–2 h (see Note 1). 4. Remove MeOH under low pressure to precipitate the unencapsulated drug. 5. Filter contents through a nylon filter with 0.45 μm pore size. 6. In order to obtain the complex evaporate aqueous phase under high vacuum.

Fig. 2 1H-NMR spectra of PSC4 (green), TMZ (blue), and TMZ@PSC4 complex (red). The significant alteration observed in the chemical shift of the Hb proton of the imidazotetrazine ring of TMZ in the NMR spectrum of the TMZ@calix complex concerning the native TMZ pinpoints that the imidazotetrazine ring of TMZ is incorporated deep inside the hydrophobic cavity of calix

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Fig. 3 Calibration curve for the determination of TMZ by UV-Vis

3.1.2  Characterization of the Nanocapsule

1. Perform the characterization of compounds via 1H-NMR and 13 C-NMR spectroscopy. 2. Dissolve 2  mg of each compound in 500  uL D2O in an Eppendorf. 3. Transfer samples to 5 mm NMR tubes. 4. Load the sample into the Bruker AV-400 spectrometer. 5. Lock, tune, and match the sample according to the manufacturer’s guidelines (Fig. 2).

3.1.3  Quantification of Encapsulated TMZ Using UV-Vis Spectroscopy Preparation of the Calibration Curve Using UV-Vis Spectroscopy

1. Dilute TMZ in a proper volume of MeOH to obtain the desired concentration (seeNote 2). 2. Prepare aliquots of stock solutions from the initial stock of TMZ (5–30 μM). 3. Scan samples by UV-Vis spectrophotometer in the range of 200–450 nm, using MeOH as blank. 4. Analyze samples for their respective absorbance at 330 nm λmax. 5. Plot the calibration curve (seeNote 3) (Fig. 3).

Quantification of Encapsulated Drug Using UV-Vis Spectroscopy

1. Dissolve samples in double-distilled H2O at 100 μM and incubate under shaking at 25 ± 0.1 οC at 600 rpm. 2. Centrifuge samples for 5  min and filter off the precipitated TMZ through RC syringe filters of 0.2 μm pore size. 3. Scan the solution in UV region (200–440 nm) in triplicates.

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Fig. 4 Stability of TMZ and TMZ@PSC4 at pH 7.1 and 37 °C as monitored by UV–Vis. TMZ is rapidly transformed to AIC while TMZ@PSC4 is stable for at least 6 h

4. Calculate the drug content of the sample using the standard calibration curve. 3.2  Determination of Chemical Stability of TMZ and ΤΜΖ@ PSC4 in Buffer Solutions Using UV-Vis, 1H-NMR, and LC-MS/MS

To validate the hypothesis that encapsulations could enhance the stability of parent drug, time-dependent studies of the degradation of parent drug have to be conducted. The stability studies should be performed in aqueous buffer solutions under various pH conditions according to the FDA and European Medicines Agency (EMA) guidelines [15]. Stability could be monitored in a time-­ dependent manner utilizing various analytical techniques such as UV-Vis, NMR, and mass spectrometry.

3.2.1  Determination of Chemical Stability of ΤΜΖ@PSC4 by UV-Vis

1. Dilute TMZ@PSC4 in phosphate buffer (pH 7.1) to a final volume of 3 mL (100 μM). 2. Incubate at 37 οC in a shaking bath. 3. Remove the samples from the bath at time intervals of 0, 2, 4, 6, and 8 h. 4. Transfer the samples to cuvette for analysis. 5. Conduct the same experiments for TMZ in order to compare the stability of TMZ@PSC4 and TMZ. 6. Study all samples in triplicate (Fig. 4).

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Fig. 5 1H-NMR spectra of the time-dependent degradation of native TMZ and TMZ@PSC4 to MTIC at deuterated phosphate buffer in D2O (10 μm). The highlighted gray peak at 7.23 ppm represents the 7-H proton of the MTIC form. The highlighted peaks at 3.52 ppm represent the 1-H proton of the MTIC form and its adduct

3.2.2  Determination of Chemical Stability of ΤΜΖ@PSC4 Using 1 H-NMR

1. Dissolve TMZ and complex in 500  μL phosphate buffer pH 7.1 in D2O. 2. Add tetramethylsilane (TMS) as internal standard. 3. Incubate samples under shaking at 37 °C ± 0.1. 4. Remove the samples from the bath at time intervals of 0, 2, 4, 6, 8, 16, and 24 h. 5. Transfer samples to 5 mm NMR tubes to analyze. 6. Load the sample into the Bruker AV-400 spectrometer. 7. Lock, tune, and match the sample according to the manufacturer’s guidelines (Fig. 5).

3.2.3  Determination of Chemical Stability of ΤΜΖ@PSC4 Using Liquid Chromatography and Mass Spectrometry Method Development and Optimization

1. Prepare a stock solution of each compound at a concentration of 1 mg/mL (seeNote 4). 2. Dilute stock solutions at a concentration of 500  ng/mL for direct infusion in the MS (seeNote 5). 3. Use the mass spectrometry software to calculate the most prevalent daughter ions of the parent compounds. 4. Dilute stock solutions at an appropriate concentration (1 μM) for LC-MS/MS analysis. 5. Vortex-mix and filter the samples with 0.2  μm RC syringe filters. 6. Transfer samples to LC-MS vials, load them onto auto-sampler, and perform LC-MS runs to determine the optimal chromatographic conditions (seeNote 6).

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Fig. 6 Representative chromatogram of (a) TMZ and internal standard along with the optimal transitions and most abundant daughter ion of (b) TMZ and (c) theophylline

7. Maintain the column and auto-sampler temperature stable (seeNote 7). 8. On the same chromatographic runs, optimize the various ESI parameters of mass spectrometer like spray voltage, heated probe temperature, gas, and nebulizer. 9. Build up the method in mass spectrometer software, utilizing multiple reaction monitoring (MRM) modes for the detection and quantification of the compounds (Fig. 6). Buffer Chemical Stability Assay

1. Add 10  μL of TMZ@PSC4  in 280  μL hydrochloric acid (pH 2.1) and incubate at 37 °C in a shaking bath. 2. Remove the samples from the bath at time intervals of 0, 2, 4, 6, 8, 16, and 24 h. 3. Add 10 μL of IS, vortex-mix, and transfer to LC-MS vials for analysis. 4. Follow the same procedure for the samples incubated in ammonium formate (pH 4.5) and phosphate buffer (pH 7.1). 5. Conduct the same experiments for TMZ in order to compare the stability of TMZ@PSC4 and TMZ. 6. Study all samples in triplicate and plot the % fraction remaining of parent TMZ against incubation time.

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Fig. 7 The degradation rate of TMZ and TMZ@PSC4 in mice plasma. TMZ@PSC4 is more stable 4 h post-dosage in comparison to TMZ 3.3  Cell Culture

Established GBM cell lines (U87, 8MG) and primary cultures were cultured in DMEM or DMEM/F12, respectively, supplemented with 10% FBS and maintained at 37 °C in a 5% CO2-humidified incubator. Established lines were purchased from ATCC and primary cells were generated in-house from fresh brain tumor tissue as described previously by Renziehausen et al. [8].

3.4  In Vitro Cytotoxicity: Sulforhodamine B (SRB) and Cell Counting Kit 8 (CCK8) Assays

1. Seed the cells in 96-well plates at 2 × 103 cells per well in their respective culture medium supplemented with 2% FBS. 2. Treat 24 h post-plating cells with TMZ, PSC4, a physical mixture of TMZ and PSC4, or TMZ@PSC4 complex in culture medium supplemented with 2% FBS. 3. To determine the IC50 of native TMZ, treat cells with 2, 4, 8, 16, 32, 64, 128, 256, and 512 μM TMZ and analyze for proliferation using SRB assay 9  days posttreatment as previously described [16]. 4. For experiments comparing the efficacy of the PSC4 complex with native TMZ, use doses just below the IC50 of native TMZ (5 μM and 10 μM for U87 and 8MG, respectively; 100 μM and 200 μM for the primary lines). 5. Calculate and use the equivalent equimolar concentrations of the complex.

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3.5  In Vivo Pharmacokinetic Analysis

185

1. Separate C57BL/6 female mice 6 weeks old into two groups (n  =  3) and inject them intraperitoneally either with a single dose of TMZ or a single dose of TMZ@PSC4 complex. 2. Collect the blood by cardiac puncture before treatment (day 0) and then at 0.5, 2, and 4 h posttreatment in tubes containing heparin. 3. Centrifuge blood samples at 2862 × g for 10 min to separate the plasma. 4. Acidify plasma (pH 99% (TLC) (DPPC—Fig. 1). 3. Deuterium-depleted water (99.99 atom% 16O). 4. 2-HP-β-CD (average Mw ~1460).

3  Methods 3.1  Investigation of the Structural Properties of Irbesartan and Irbesartan–2-­ Hydroxypropyl-­β-­ Cyclodextrin Complex in Micelles

1. Obtain 1H NMR spectra of the samples: (a) Irbesartan dissolved in D2O; (b) 2-HP-β-CD dissolved in D2O D2O; (c) irbesartan dissolved in SDS micelles; (d) IRB complexed with 2-HP-β-ΟΗ and dissolved in D2O; (e) IRB complexed with 2-HP-β-ΟΗ and dissolved in SDS micelles. Pulse sequences are provided in spectrometer libraries. 2. Dissolve 9.8 mg of the complex irbesartan–2-hydroxypropyl-β-­ cyclodextrin in D2O in order to obtain NMR spectra. Τhe pulse sequences included in the libraries of pulse programs were used in order to acquire 1D 1H and 13C NMR spectra. 3. For the preparation of 5  mM of irbesartan in 400  mM SDS-­ d25/D2O, subject the mixture to sonication in order to provide a transparent solution. 4. The preparation of the complex irbesartan–2-hydroxypropyl-β-­ cyclodextrin is carried out during a freeze-drying procedure as it is described in [33]. Specifically, the neutralization method is applied for the preparation of IRB-2-HP-β-CD aqueous solutions for freeze-drying in a molar ratio (irbesartan/2-HP-­ β-CD) 1:2. Transfer 408  mg of 2-HP-β-CD and 60  mg of irbesartan in a 100 μL beaker and suspend them with 50 mL of water. Subsequently, add small amounts of ammonium hydroxide under stirring, while pH is monitored until complete dissolution and pH adjustment to a value of 9–10. Then, freeze the resulting solution at −80  °C and freeze-dry using a Kryodos-­50 model Telstar lyophilizer.

3.2  The Application of ssNMR Spectroscopy to Study Drug:Membrane Interactions

1. Fully hydrate DPPC to form multilamellar bilayers. 2. Record a 13C cross polarization/magic-angle spinning (13C CP/MAS) solid-state NMR spectra on a 600 MHz spectrometer equipped with a 3.2  mm HX MAS probe. Pack approximately 20 mg of each sample tightly into a zirconia rotor and spin at 5 kHz. The duration of the CP block in the CP/MAS experiment should be 5  ms, repetition delay between scans should be 2 s, and the number of scans 400. The 13C chemical shift axis should be referenced to as tetramethylsilane. For the sample DPPC/HP-β-CD (x  =  0.20), record both 13 C CP/MAS and MAS spectra at a sample rotation frequency of 15  kHz. Record the spectra on a 600  MHz spectrometer

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319

equipped with a 4  mm HX MAS probe. Pack approximately 50 mg of the sample tightly into a zirconia rotor. Apply cross polarization for 13C excitation using an 80% linear ramp. During 13 C detection, 100 kHz Spinal-64 proton decoupling should be applied [34–36]. The number of scans in the experiment should be 1000. 3. Dissolve weighted amounts of dry lipid and IRB powder in chloroform in order to prepare DPPC and IRB stock solutions. The drug concentration should be 20 mol%. Prepare the mixtures by appropriate amounts of drug in a pure or complexed form with 2-HP-β-CD. Evaporate the organic DPPC/IRB or DPPC/complex IRB–2-HP-β-CD at room temperature under a gentle stream of nitrogen and thereafter place them under vacuum for 12 h and consequently a thin lipid film at the bottom of glass vials should be formed. The obtained mixtures should be then fully hydrated (50% w/w deuterium-depleted water) and this procedure will result in multilamellar vesicles (MLVs). Alternatively, add complexed IRB–2-HP-β-CD compounds to the aqueous phase of readily formed DPPC MLV dispersions. 4. Perform 13C CP/MAS spectra at three temperatures (25, 35, 45 °C) to cover all mesomorphic states of DPPC bilayers.

4  Notes 4.1  Notes Related to Subheading 3.1

1. Irbesartan’s alkyl chemical shifts are not modified significantly when it is complexed in D2O while in micelles a clear and significant downfield chemical shift is observed. Interestingly, the quintet at ca 1.43 ppm is better resolved when the complex is in micelles than in D2O. The same trend is observed with cyclopentane ring. The differences in the aromatic region are very interesting. Downfield, but also upfield, shifts are observed in the micelle environment compared to that of D2O. 2. (a) The complexation of IRB with 2-HP-β-CD induces some chemical shift changes to IRB. (b) The micelle environment induces many chemical shift changes to IRB. (c) Protons 6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 19, 20, 21, 22, and 28 are affected most when IRB is complexed with 2-HP-β-CD and (d) when the complex is transferred to SDS environment most of the chemical shifts are affected significantly (Table 1, Figs. 2 and 3). The 1H NMR spectra of (a) irbesartan dissolved in D2O; (b) 2-HP-β-CD dissolved in D2O; (c) irbesartan dissolved in SDS micelles; (d) IRB complexed with 2-HP-β-CD and dissolved in D2O; and (e) IRB complexed with 2-HP-β-CD in SDS micelles are presented in Figs. 2 and 3.

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Table 1 Chemical shifts of irbesartan alone or complexed with 2-HP-β-CD in D2O and in SDS micelles

Hydrogen atoms

IRB in D2O [ppm (multiplicity)]

IRB in SDS micelles [ppm (multiplicity)]

Complex IRB-2-HP-­ β-CD in D2O [ppm (multiplicity)]

Complex IRB-2-HP-­ β-CD in SDS micelles [ppm (multiplicity)]

31

0.78 (triplet)

0.84 (triplet)

0.76 (triplet)

0.86 (triplet)

30

1.25 (sextet)

1.32 (sextet)

1.23 (sextet)

1.32 (sextet)

29

1.43 (pentet)

1.52 (pentet)

1.41 (pentet)

1.54 (pentet)

6a, 6b 9a, 9b

1.82 1.93–1.99 (multiplet and multiplet)

1.91 1.97–2.01 (multiplet and multiplet)

1.91–1.96 (multiplet and multiplet)

1.97 (multiplet and multiplet)

7a,7b

1.93–1.99 (multiplet)

1.91, 2.03–2.06 (multiplet)

1.77–2.02 (multiplet) 1.80, 1.98 (multiplet)

8a, 8b

1.93–1.99 (multiplet)

2.03–2.06 (multiplet)

1.77–2.02 (multiplet) 1.80, 1.98 (multiplet)

28

2.43 (triplet)

2.56 (triplet)

2.37 (triplet)

2.45 (triplet)

10

4.78 (singlet)

4.84 (triplet)

4.76 (singlet)

4.77 (singlet)

12/16

7.10 (doublet)

7.10 (doublet)

7.09 or 7.14 (doublet)

7.12 or 7.13 (doublet)

13/15

7.10 (doublet)

7.16 (doublet)

7.09, or 7.14 (doublet)

7.12 or 7.13 (doublet)

22

7.53–7.55 (multiplet)

7.41 (doublet)

7.48 (multiplet)

7.39 (doublet)

20

7.53–7.55 (multiplet)

7.52 (triplet)

7.49 (multiplet)

7.46 (triplet)

19

7.59 (doublet)

7.84 (doublet)

7.62 (doublet)

7.78 (doublet)

21

7.61 (triplet)

7.57 (triplet)

7.57 (triplet)

7.53 (triplet)

4.2  Notes Related to Subheading 3.2 (Figs. 4 and 5)

1. The peaks of 2-HP-β-CD are barely observable. The absence of cross polarization between 2-HP-β-CD and phospholipid DPPC presumably shows that the former molecule approaches the surface of the lipid bilayers and that the distances between the 1H and 13C nuclei of the two species are thus sufficiently large or that the dynamics of the 2-HP-β-CD is sufficiently fast, so that the 1H–13C (residual) dipolar interaction is practically negligible. 13C MAS experiments lead to the conclusion that this preposition as the peaks attributed to 2-HP-β-CD is eminent at low temperatures. 2. The presence of the drug in the lipid bilayers does not modify significantly DPPC’s carbon chemical shifts.

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Fig. 2 Part of 1H NMR spectra (0.7–2.5 ppm) of (a) irbesartan dissolved in D2O, (b) 2-HP-β-CD dissolved in D2O, (c) irbesartan dissolved in SDS micelles, (d) IRB complexed with 2-HP-β-CD and dissolved in D2O, (e) IRB complexed with 2-HP-β-CD in SDS micelles

3. The observation of 13C NMR spectrum of the drug is a proof that the butyl chain of irbesartan and its cyclopentyl ring are embedded in the more flexible hydrophobic core of DPPC bilayers. 4. After irbesartan complex is inserted into DPPC bilayers, signals of the drug are very broad (CP experiments). On the contrary, 2-HP-β-CD is not observed giving the information that this CD is localized on the surface of DPPC bilayers.

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Fig. 3 Part of 1H NMR spectra (3.3–7.9 ppm) of (a) irbesartan dissolved in D2O, (b) 2-HP-β-CD dissolved in D2O, (c) irbesartan dissolved in SDS micelles, (d) IRB complexed with 2-HP-β-CD and dissolved in D2O, (e) IRB complexed with 2-HP-β-CD in SDS micelles

Acknowledgments This work has been co-financed by the European Union and Greek national funds through the program “Support for Researchers with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ 14995) and under the research title “Preparation and study of innovative forms of administration of pharmaceutical molecules targeting at improved pharmacological properties.”

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C2

DPPC

C2*** C1

x2

c1’,c1”

C1***

(CH2)*10,(CH2)**10

N(CH3)3

C15*,C15** C14*,C14** C3*,C3**

X4

C3

C16*,C16**

C2*,C2*

DPPC/irbesartan 80:20

Cirb430 25

Cirb4

20 Cirb31

CHP6

70

60

DPPC/2-HP-β-CD 80:20

DPPC/ complex irbesartan-2-HP-β-CD 80:20

DPPC/ complex irbesartan-2-HP-β-CD(added from outside) 80:20

190

170

150

130

90

80

70

60

50

40

30

20

10 (ppm)

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14. Bendeby B, Kenne L, Sandström C (2004) 1 H-NMR studies of the inclusion complexes between α-cyclodextrin and adamantane derivatives using both exchangeable hydroxy protons and non-exchangeable aliphatic protons. J Incl Phenom Macrocycl Chem 50:173–181 15. Hakkarainen B, Fujita K, Immel S et al (2005) 1 H-NMR studies on the hydrogen-bonding network in mono-altro-β-cyclodextrin and its complex with adamantane-1-carboxylic acid. Carbohydr Res 340(8):1539–1545 16. Wójcik J, Ejchart A, Nowakowski M (2019) Shape adaptation of quinine in cyclodextrin cavities: NMR studies. Phys Chem Chem Phys 21:6925–6934 17. Fielding L (2000) Determination of association constants (Ka) from solution NMR data. Tetrahedron 56:6151–6170 18. Monticelli C, Fantin G, Carmine G et al (2019) Inclusion of 5-mercapto-1-phenyl-tetrazole into β-cyclodextrin for entrapment in silane

NMR Techniques Applied to Drug: Cyclodextrin Complexation coatings: an improvement in bronze corrosion protection. Coatings 9(8):508 19. Fernandes A, Ivanova G, Bras NF et al (2014) Structural characterization of inclusion complexes between cyanidin-3-O-­ glucoside and β-cyclodextrin. Carbohydr Polym 102(1):269–277 20. Greatbanks D, Pickford R (1987) Cyclodextrins as chiral complexing agents in water, and their application to optical purity measurements. Magn Reson Chem 25:208–215 21. Armstrong DW, Ward DJ, Armstrong RD et al (1986) Separation of drug stereoisomers by the formation of β-cyclodextrin complexes. Science 232(4754):1132–1135 22. Fronza G, Mele A, Redenti E et  al (1996) 1 H NMR and molecular modeling study on the inclusion complex β-cyclodextrin-­ indomethacin. J Org Chem 61(3):909–914 23. Gardner KH, Zhang X, Gehring K et al (1998) NMR studies of a 42 KDa Escherichia coli maltose binding protein/β-Cyclodextrin complex: chemical shift assignments and analysis. J Am Chem Soc 120:11738–11748 24. Murray DT, Das N, Cross TA (2013) Solid state NMR strategy for characterizing native membrane protein structures. Acc Chem Res 46(9):2172–2181 25. Priotti J, Ferreira MJ, Lamas MC et al (2015) First solid-state NMR spectroscopy evaluation of complexes of benznidazole with cyclodextrin derivatives. Carbohydr Polym 131:90–97 26. Nevzorov AA, Opella SJ (2007) Selective averaging for high-resolution solid-state NMR spectroscopy of aligned samples. J Magn Reson 185:59–70 27. Dvinskikh SV, Yamamoto K, Ramamoorthy A (2006) Heteronuclear isotopic mixing separated local field NMR spectroscopy. J Chem Phys 125:034507

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Chapter 23 Construction of Peptide-Drug Conjugates for Selective Targeting of Malignant Tumor Cells Eirinaios I. Vrettos and Andreas G. Tzakos Abstract Cancer constitutes a major threat to humanity, while its incidence and mortality rates are increasing rapidly worldwide. To tackle cancer, numerous strategies have been exploited, including the development of peptide–drug conjugates (PDCs), which are considered an appealing approach to selectively populate malignant tumors with toxic substances. The general architecture of a PDC usually includes three parts: the tumor-targeting peptide, the cytotoxic drug, and the biodegradable linker. Due to the fact that peptides possess fast renal clearance, affecting the bioavailability of the PDC, a nanodrug formation concept can be exploited to ameliorate this pitfall. Herein, we present methodologies to develop PDCs, along with certain basic principles governing such constructs. In addition, we highlight possible problems that may appear during the synthesis of PDCs, as also solutions to overcome them. Key words Cancer, Drug delivery, Bioconjugates, Peptides, Peptide-drug conjugates (PDCs), Targeted drug delivery

1  Introduction Cancer is a rapidly increasing global threat and the rate of appearance is continuously growing. The 12.7 million cancer cases and 7.6 million deaths in 2008 have mounted up to 18.1 million cases and 9.6 million deaths in 2018 [1, 2]. The most common types of cancer are lung, breast, colorectal, and prostate, contributing to 12.3%, 12.3%, 10.6%, and 7.5% of the total number of new cases in 2018, respectively [1]. These statistics indicate that the current therapeutics in oncology are not effective against the evolving complexity of cancer, often due to their unspecific toxicity, reduced solubility, permeability, and drug resistance of cancer cells. Current cancer treatments usually include a combination of surgery, radiation, immunotherapy, and chemotherapy. Chemotherapeutic drugs utilized for this purpose are toxic in order to kill cancer cells, inevitably affecting the healthy cells and thereThomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_23, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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fore deteriorating the patient’s health. For instance, gemcitabine, an anticancer agent used against a variety of solid tumors even in the latter stages, shows higher toxicity for healthy cells, after long-­ term administration, due to the drug resistance that cancer cells develop [3]. An appealing strategy to surpass this hurdle is by offering to chemotherapeutic drugs selective delivery to malignant cells (Fig. 1). Moreover, chemotherapy is often ineffective due to the unfavorable ADME (absorption, distribution, metabolism, excretion) properties of the utilized drugs, associated with their poor water solubility, low bioavailability, and rapid metabolic inactivation. Therefore, it is of interest to invest in enhancing the biological profile of existing cytotoxic drugs by transforming them into targeted chemotherapeutics. Along these lines, drug delivery formulations that could improve drug potency and be specifically tailored for every type of cancer are of ultimate interest. Peptide-drug conjugates (PDCs) could serve as ideal candidates for this purpose, as they fulfill the requested features. Different PDCs can be constructed for each type of cancer and conform with each patient’s needs (personalized medicines), due to the fact that plentiful combinations can be used. The usual building blocks of a PDC include the cytotoxic agent, the tumor-­ targeting peptide, and the linker tethering them (Fig. 2). The efficacy of a PDC is mainly associated with the cytotoxicity of the utilized drug and the targeting efficacy of the peptide. The drug should be highly potent, with a known mechanism of metabolism and action, and should possess conjugatable functional groups. The tumor-homing peptide should bind selectively and with high

Fig. 1 Conventional chemotherapy versus targeted chemotherapy. Black color  =  solid malignant tumor; green = conventional untargeted cytotoxic agent; blue = targeted cytotoxic agent. Reprinted from [4] with permission

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Fig. 2 The general architecture of a peptide-drug conjugate

affinity to a certain receptor, overexpressed or uniquely expressed on the surface of the targeted cells. Also, the peptide should possess conjugatable groups (usually lysine and cysteine), but the conjugation site should be carefully selected since it could lead even to the total abolishment of its binding affinity/selectivity to the certain receptor. The linker should be selected rationally as it plays a crucial role in the properties of the final assembly, especially in the stability of the conjugate and the release rate of the drug. Notably, an improper linker could demolish the overall activity of the bioconjugate. Peptides are considered as an inextricable part of the armamentarium against cancer as they display high affinity and selectivity for certain receptors overexpressed on malignant tumor cells, they possess low inherent toxicity, and they are facile to synthesize. However, peptides also display a very short half-life during the blood circulation mainly due to excretion by the kidneys, because of their small size, and to a lesser extent caused by their susceptibility to proteases. As a result, PDCs display low bioavailability, rendering them inefficient against cancer. An intriguing approach for extending the half-lives of PDCs is by conjugating them to nanoparticles [5]. For instance, gold nanoparticles (AuNPs) have been utilized for enhancing the halflife of certain PDCs from ~10 min, when administered alone, up to ~20 h when administrated as conjugates on AuNPS, while retaining their cytotoxicity profiles [6]. In addition, certain PDCs may self-assemble into supramolecular structures like nanofibers, nanotubes, and hydrogels [7], offering two functions: They can act as nanocarriers to selectively deliver the drug to malignant tumor cells and/or can enhance the pharmacokinetic properties of the utilized drug [8]. Although the common syntheses of PDCs are conducted in a rapid and facile manner, various problems may arise. The most usual ones appear during the synthesis and/or the purification of the tumor-homing peptide as summarized below:

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1. The peptide might be difficult to synthesize, especially when the target peptide consists of more than 30 amino acids. The interactions of the side chains of the amino acids cause problems during the synthesis, as the new amino acids to be attached require higher reaction times, and the final peptide is usually impure. An effective way to surpass this hurdle is by shortening the sequence and/or substituting the hydrophobic residues. The aforementioned problem is usually encountered during the manual solid-phase peptide synthesis; in some cases it can be addressed by utilizing microwave-assisted synthesis. 2. The synthesized peptide might possess low aqueous solubility, which renders its purification extremely hard. Insolubility issues can be surpassed by shortening the sequence through elimination of hydrophobic residues or by lengthening the sequence through the addition of polar amino acids. Another effective way to face the low solubility is by altering the C-/Nterminus and/or substituting specific hydrophobic residues. 3. During the conjugation between the peptide and the drug or the linker, some problems may arise, derived from the utilized coupling reagents. For instance, HATU is known to react with the N-terminus of the peptide and recently, its participation in the formation of side products on the side chains of specific peptide residues has been described [9]. In this chapter, we provide the procedure to assemble a peptide-­ drug conjugate consisting of D-Lys6-GnRH (peptide) and gemcitabine (anticancer agent), tethered via various bonds (ester, amide, carbamate and oxime) derived from the utilization of different linkers (succinyl, carbamate, and PEG-aminooxy). The utilized peptide (D-Lys6-GnRH-II) is gonadotropin-releasing hormone that binds selectively on type II GnRH-receptor (GnRH-R), which is overexpressed in various cancer types including prostate, lung, and breast [10]. D-Lys6-GnRH possesses a lysine that can be utilized for orthogonal coupling in liquid phase with the linker and consequently the drug. Gemcitabine belongs to the antimetabolite family of anticancer agents and is active against various solid malignant tumors including ovarian, prostate, lung, breast, and pancreatic. Gemcitabine possesses three possible conjugation sites: (1) a primary –OH that is the site of intracellular phosphorylation leading to the active metabolites difluorodeoxycytidine diphosphate (dFdCDP) and difluorodeoxycytidine triphosphate (dFdCTP), (2) a secondary –OH, and (3) a primary –NH2 which mediates its metabolic inactivation toward 2′,2′-difluorodeoxyuridine (dFdU) via the cytidine deaminase [11]. Herein, it will be described the synthetic procedure concerning the primary  or  the secondary –OH, where different linkers can be incorporated.

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We present the synthesis of three PDCs with linkers consisting of different lengths, bonds, and stability (they include various mixtures between ester, amide, carbamate, and oxime bonds), focusing on the description of the synthetic procedures to obtain them.

2  Materials and Equipment Materials used are commercially available and of the best quality, unless otherwise stated. 2.1  Synthesis of PDCs 2.1.1  Synthesis of PDCs Using Succinic Acid as Linker (Ester and Amide Bonds)

1. D-Lys6-GnRH (synthesized via the classical solid-phase peptide synthesis on Rink amide resin, as previously described [12]) (see Note 1). 2. Gemcitabine base (gemcitabine is Boc-protected on the –NH2 and the secondary –OH prior to usage, as previously described [13]) (see Note 1). 3. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU). 4. 1-hydroxybenzotriazole (HOBt). 5. Succinic anhydride. 6. Triisopropylsilane (TIS). 7. N,N-diisopropylethylamine (DIPEA). 8. Trifluoroacetic acid (TFA). 9. Anhydrous N,N-dimethylformamide (DMF). 10. Anhydrous dichloromethane (CH2Cl2).

2.1.2  Synthesis of PDCs Using Carbamate Bond in the Linker

1. D-Lys6-GnRH (synthesized via the classical solid-phase peptide synthesis on Rink amide resin, as previously described [12]). 2. Gemcitabine base (gemcitabine is Boc-protected on the –NH2 and the secondary –OH prior to usage, as previously described [13]). 3. Bis(4-nitrophenyl)carbonate. 4. Triisopropylsilane (TIS). 5. N,N-diisopropylethylamine (DIPEA). 6. Trifluoroacetic acid (TFA). 7. Anhydrous N,N-dimethylformamide (DMF). 8. Anhydrous acetonitrile.

2.1.3  Synthesis of PDCs Using Aminooxy-PEG4-­ CH2CO2H as Linker (Amide and Bond)

1. D-Lys6-GnRH (synthesized via the classical step-by-step solid-­ phase peptide synthesis on Rink amide resin, as previously described [12]) (see Note 1).

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2. Gemcitabine base (gemcitabine is Boc-protected on the –NH2 and the primary –OH prior to usage, as previously described [13]) (see Note 1). 3. 1-hydroxybenzotriazole (HOBt). 4. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU). 5. t-Boc-Aminooxy-PEG4-CH2CO2H. 6. 4-dimethylaminopyridine (DMAP). 7. Triisopropylsilane (TIS). 8. N,N-diisopropylethylamine (DIPEA). 9. Trifluoroacetic acid (TFA). 10. Fmoc-Ser(tBu)-OH. 11. Piperidine. 12. Imidazole. 13. Sodium periodate (NaIO4). 14. Ethylene glycol. 15. Anhydrous N,N-dimethylformamide (DMF). 16. Anhydrous acetonitrile. 2.2  Purification and Characterization of Intermediates and Final Conjugates 2.2.1  Column Purification 2.2.2  RP-HPLC Purification

1. Column chromatography performed using SiO2. 2. Thin-layer chromatography (TLC) plates, silica gel coated with fluorescent indicator F254 (see Note 2). 3. Dichloromethane, analytical grade. 4. Methanol, analytical grade. 5. Acetone, analytical grade. 1. HPLC solvent delivery system with binary gradient capability and a UV detector. 2. Reversed-phase Jupiter® C18 column (25 cm × 21.2 mm) at a flow rate of 20 mL/min and 10 μm particle size as stationary phase. 3. Sample filters, 0.2 μm pore size. 4. Acetonitrile, water, and trifluoroacetic acid (TFA), HPLC grade. 5. Mobile phase A: TFA 0.1% (v/v) in water. 6. Mobile phase B: TFA 0.1% (v/v) in acetonitrile.

2.2.3  Characterization with Mass Spectrometry and/or NMR Spectroscopy

1. Acetonitrile, water, and formic acid (LC-MS grade). 2. Mass spectrometer instrumentation with C18 column 100 mm × 2.1 mm, 2.6 μm, with ProGuard column 2.1 mm. 3. Mobile phase A: Water containing 0.1% (v/v) formic acid.

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4. Mobile phase B: Acetonitrile containing 0.1% (v/v) formic acid. 5. NMR spectrometer for 1H and 13C spectroscopy and the corresponding software. 6. DMSO-d6 (>99.8%) (see Note 3).

3  Methods 3.1  Synthesis of PDCs 3.1.1  Synthesis of PDCs Using Succinic Acid as Linker (Ester and Amide Bonds) (Scheme 1) Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-­ Hemisuccinate) Preparation of the Final Conjugate (Gemcitabine-­ Hemisuccinate-­D-­ Lys6- GnRH)

1. Dissolve Boc-protected gemcitabine (8 eq) and succinic anhydride (1 eq) in CH2Cl2. 2. Add DIPEA (10 eq) at 0 °C. 3. Stir the reaction for 12 h at room temperature. 4. Distill the solvent under reduced pressure. 5. Purify the residue by RP-HPLC to receive gemcitabine-linker (diBoc-Gemcitabine-Hemisuccinate). 6. Characterize the compound with 1H/13C NMR spectroscopy and mass spectrometry. 1. Dissolve the gemcitabine linker (1 eq), HATU (1 eq) in anhydrous DMF under inert atmosphere (see Note 4). 2. Add DIPEA (2 eq) at 0 °C. 3. After 10  min, add D-Lys6-GnRH (1  eq) dissolved in anhydrous DMF dropwise and stir the reaction for 12 h at room temperature. 4. Distill the solvent. 5. Dissolve the residue in TFA-H2O-TIS (95:2.5:2.5) and stir for 30 min (see Note 5). 6. Distill the solvent. 7. Purify the residue with RP-HPLC and lyophilize the peak to receive the final conjugate in pure form. 8. Characterize the compound with mass spectrometry (see Note 6).

3.1.2  Synthesis of PDCs Using Carbamate Bond in the Linker (Scheme 2) Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Bis (4-­Nitrophenyl)Carbonate)

1. Dissolve Boc-protected gemcitabine (1  eq) in anhydrous acetonitrile under an inert atmosphere. 2. Add DIPEA (40 eq) at 0 °C. 3. After 5  min, add a solution of bis(4-nitrophenyl)carbonate (8  eq) in anhydrous acetonitrile dropwise and stir at room temperature for 4 h. 4. Distill the solvent.

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Scheme 1 Synthesis of PDCs using carbamate bond as linker. Reagents and conditions: (a) Succinic anhydride, DIPEA, CH2Cl2, rt., 12 h; (b) D-Lys6-GnRH, HATU, DIPEA, DMF, rt., 12 h; (c)TFA-H2O-TIS (95:2.5:2.5), rt., 30 min

5. Purify the residue via column chromatography (eluent: CH2Cl2/acetone 9/1) to afford gemcitabine linker (diBocgemcitabine-­bis(4-nitrophenyl)carbonate). 6. Characterize with 1H and spectrometry. Preparation of the Final Conjugate (Gemcitabine-­ carbamate-­D-Lys6-GnRH)

C NMR spectroscopy and mass

13

1. Dissolve D-Lys6-GnRH (1 eq) in anhydrous DMF under inert atmosphere. 2. Add DIPEA (3 eq) at 0 °C dropwise. 3. After 5 min, add gemcitabine-linker (1 eq) dissolved in anhydrous DMF dropwise and stir the reaction for 12 h at room temperature. 4. Distill the solvent. 5. Dissolve the residue in TFA-H2O-TIS (95:2.5:2.5) and stir for 12 h (see Note 5). 6. Distill the solvent. 7. Purify the residue with RP-HPLC and lyophilize the peak to receive the final conjugate in pure form. 8. Characterize the compound with mass spectrometry (see Note 6).

3.1.3  Synthesis of PDC Using Aminooxy-PEG4-­ CH2CO2H as Linker (Amide and Oxime Bond) (Scheme 3) Preparation of Gemcitabine-Linker (diBoc-Gemcitabine-Boc-­ Aminooxy-PEG4-CH2CO2H)

1. Dissolve Boc-protected gemcitabine (1 eq), t-Boc-Aminooxy-­ PEG4-CH2CO2H (1.5  eq), DCC (1.5  eq), and DMAP (0.5  eq) in anhydrous dichloromethane under inert atmosphere. 2. Add DIPEA (4 eq) at 0 °C and stir the reaction for the required time at room temperature (monitor the reaction progress with TLC). 3. Distill the solvent. 4. Purify the residue with column chromatography (eluent: CH2Cl2/acetone 9/1) to afford gemcitabine-linker (diBoc-Gemcitabine-Boc-Aminooxy-PEG4-CH2CO2H).

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Scheme 2 Synthesis of PDCs using carbamate bond in the linker. Reagents and conditions: (a) Bis(4nitrophenyl)carbonate, DIPEA, acetonitrile, rt., 6 h; (b) D-Lys6-GnRH, DIPEA, DMF, rt., 12 h; (c) TFA/H2O/TIS (9.5/0.25/0.25, v/v), rt., 12 h

5. Characterize the compound with 1H and 13C NMR spectroscopy and mass spectrometry. Attachment of Aldehyde Group on D-Lys6-GnRH, as previously reported [14], and described below (D-Lys6-GnRH aldehyde)

1. Dissolve Fmoc-Ser(tBu)-OH (1.5 eq), HATU (1.5 eq), and HOBt (1.5 eq) in anhydrous DMF under inert atmosphere. 2. Add DIPEA (5 eq) at 0 °C. 3. After 5 min, add D-Lys6-GnRH (1 eq) dissolved in anhydrous DMF dropwise and stir the reaction for the required time at room temperature (monitor the reaction progress with TLC). 4. Distill the solvent. 5. Wash the residue with 6 Fmoc-Ser(tBu)-D-Lys -GnRH.

acetonitrile

to

afford

6. Dissolve Fmoc-Ser(tBu)-D-Lys6-GnRH in 20% piperidine in DMF at 0 °C and stir at room temperature for the required time (monitor the reaction progress with TLC). 7. Distill the solvent and wash the residue with acetonitrile to afford Ser(tBu)-D-Lys6-GnRH. in TFA/TIS/H2O 8. Dissolve Ser(tBu)-D-Lys6-GnRH (95/2.5/2.5, v/v) and stir at room temperature for the required time (monitor the reaction progress with TLC). 9. Distill the solvent, and wash the residue with acetonitrile. 10. Purify the compound with RP-HPLC and lyophilize the fraction to afford Ser-D-Lys6-GnRH. 11. Dissolve Ser-D-Lys6-GnRH (1 eq) in H2O. 12. Add imidazole (5 eq) and sodium periodate (1.2 eq) and stir at room temperature for 5–10 min. 13. Quench the reaction with ethylene glycol (2 eq). 14. Purify the mixture with RP-HPLC and lyophilize the fraction to afford D-Lys6-GnRH aldehyde in pure form. 15. Characterize the compound with mass spectrometry (see Note 6).

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Scheme 3 Synthesis of PDCs using Aminooxy-PEG4-CH2CO2H as linker (amide and oxime bond). Reagents and conditions: (a) DCC, DMAP, DIPEA, CH2Cl2, rt., 12 h; (b) CH2Cl2:TFA 95:5, rt.; (c) D-Lys6-GnRH aldehyde, appropriate conditions, 1-24 h, rt.

Preparation of the Final Conjugate (Gemcitabine-­ PEG4CH2CO2H-­D-­Lys6GnRH)

1. Dissolve Gemcitabine-Linker (diBoc-Gemcitabine-Boc-­ Aminooxy-PEG4-CH2CO2H) (1 eq) in CH2Cl2/TFA (95/5) at 0 °C and stir the resulting mixture at room temperature for the required time (monitor the reaction progress with TLC). 2. Distill the solvent. 3. Dissolve the residue in the appropriate solvent system depending on the substrates, including 0.2  M NaOAc buffer (pH 3-5), or 0.2 M NH4OAc buffer (pH 4-5), or 0.5 M Phosphate buffer/acetonitrile 3:1 (pH 4.5) (see Note 7). 4. Add D-Lys6-GnRH aldehyde (1 eq) and stir at room temperature for 1–24 h. 5. Lyophilize the reaction to afford the final conjugate in crude form and then purify with RP-HPLC. 6. Collect, lyophilize and  characterize the final  conjugate with mass spectrometry (see Note 6).

4  Notes 1. In the specific projects we used gemcitabine as the anticancer agent and D-Lys6-GnRH as the tumor-homing peptide. All the reactions can be performed with other drug and tumorhoming peptide that have the necessary conjugatable groups. 2. TLC plates are used for two purposes: to assist the column purification and also to monitor the reactions. The reaction times mentioned in the synthetic part are based on progress according to TLC.

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3. In the specific reactions we used DMSO-d6 to characterize the purity of the compounds due to solubility. In other cases, other deuterated solvents can be used as well. 4. HATU should be used in a ratio of 1:1 with the aminoacid since excess quantity can form unwanted side products [9]. 5. TFA cleavage cocktail is used to cleave the Boc-groups of gemcitabine. If another drug is used that is not Boc-protected, this step is not required. 6. If more peaks appear during HPLC purification, collect and lyophilize all and then characterize all starting from the major one, until you identify the expected product. 7. Usually the addition of DMSO or aniline-based catalysts are mandatory for the reaction to proceed.

Acknowledgments This research was co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Program “Human Resources Development, Education and Lifelong Learning 2014–2020” in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research  second Cycle” (MIS 5000432). The research work was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “First Call for H.F.R.I.  Research Projects to support Faculty members and Researchers and the procurement of high-­ cost research equipment grant” (Project Number: 991, acronym PROTECT). References what can they really do in vivo? F1000 Res 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, 6:681 Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence 6. Kalimuthu K, Lubin B-C, Bazylevich A, and mortality worldwide for 36 cancers in 185 Gellerman G, Shpilberg O, Luboshits G, countries. CA Cancer J Clin 68:394–424 Firer MA (2018) Gold nanoparticles stabilize peptide-­drug-conjugates for sustained targeted 2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, drug delivery to cancer cells. J Nanobiotechnol Forman D (2011) Global cancer statistics. CA 16:34–34 Cancer J Clin 61:69–90 3. Szakacs G, Paterson JK, Ludwig JA, Booth-­ 7. Fan Q, Ji Y, Wang J, Wu L, Li W, Chen R, Chen Z (2018) Self-assembly behaviours of Genthe C, Gottesman MM (2006) Targeting peptide-drug conjugates: influence of multiple multidrug resistance in cancer. Nat Rev Drug factors on aggregate morphology and potenDiscov 5:219–234 tial self-assembly mechanism. R Soc Open Sci 4. Vrettos EI, Mezo G, Tzakos AG (2018) On the 5:172040–172040 design principles of peptide-drug conjugates for targeted drug delivery to the malignant 8. Chang R, Zou Q, Xing R, Yan X (2019) Peptide-based supramolecular nanodrugs as tumor site. Beilstein J Org Chem 14:930–954 a new generation of therapeutic toolboxes 5. Wang YF, Liu L, Xue X, Liang XJ (2017) against cancer. Adv Ther 2:1900048 Nanoparticle-based drug delivery systems:

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9. Vrettos EI, Sayyad N, Mavrogiannaki EM, Stylos E, Kostagianni AD, Papas S, Mavromoustakos T, Theodorou V, Tzakos AG (2017) Unveiling and tackling guanidinium peptide coupling reagent side reactions towards the development of peptide-drug conjugates. RSC Adv 7:50519–50526 10. Gründker C, Emons G (2017) The role of gonadotropin-releasing hormone in cancer cell proliferation and metastasis. Front Endocrinol (Lausanne) 8:187–187 11. de Sousa Cavalcante L, Monteiro G (2014) Gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur J Pharmacol 741:8–16 12. Sayyad N, Vrettos EI, Karampelas T, Chatzigiannis CM, Spyridaki K, Liapakis

G, Tamvakopoulos C, Tzakos AG (2019) Development of bioactive gemcitabineD-­ L ys(6)-GnRH prodrugs with linker-­ controllable drug release rate and enhanced biopharmaceutical profile. Eur J Med Chem 166:256–266 13. Karampelas T, Argyros O, Sayyad N, Spyridaki K, Pappas C, Morgan K, Kolios G, Millar RP, Liapakis G, Tzakos AG, Fokas D, Tamvakopoulos C (2014) GnRHGemcitabine conjugates for the treatment of androgen-­ independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery. Bioconjug Chem 25:813–823 14. Spears RJ, Fascione MA (2016) Site-selective incorporation and ligation of protein aldehydes. Org Biomol Chem 14(32):7622–7638

Index A Affinity������������������������������� 188, 190, 191, 194, 202, 206, 329 Agglomeration������������������������������������������������������������������231 Aggregations�������22, 37, 40, 41, 102, 203, 229, 231, 264, 274 Amorphous state������������������������������������������������������ 170, 273 Amphiphilic block copolymers������ 71–81, 222, 225, 228, 303 Amphiphilic diblock copolymer���������������������������������������222 Amphiphilic triblock hybrid terpolymer���������������������������144 Angiotensin converting enzyme (ACE)���������������������������100 Angiotensin II type 1 receptor blockers���������������������������110 Aniline derivatives�������������������������������������������������� 18, 20, 21 Antibacterial����������������������������������������������������� 205, 266–267 Anticancer������������������������������������72, 214, 248, 249, 252, 255 Anticancer agents��������������������������������������199, 328, 330, 336 Anticancer drugs���������������������������86–88, 149, 176, 199, 200, 202–204, 206, 248, 250, 253, 254 Antifungal����������������������������������������������������������������262–264 Anti-hypertensive drugs��������������������������������������������� 45, 190 Anti-inflammatory��������������� 4, 5, 72, 204, 256, 258, 259, 275 Antioxidants����������������������������������4, 5, 42, 72, 252, 264–266, 270, 285, 286 Anti-seizure����������������������������������������������������������������������269 Antiviral�������������������������������������������������������������������259–262 Anti-zika��������������������������������������������������������������������������267 Aqueous solutions����������������������������� 2, 6, 8, 9, 35, 37, 80, 90, 127, 129, 146, 153, 154, 159, 179, 191, 258 Association constant (Kα)�������������������������������������������������189 Asymmetry��������������������������������������������������������������� 139, 145 AT1 receptor��������������������������������������������������������������������110 Attenuated total reflection-Fourier transform infrared (ATR-FTIR)���������������������������������������� 76–78, 81

B γ-benzyl-l-glutamate������������������������� 128, 131, 140, 142, 144 Bilayers������������������������������������� 100, 222–224, 230, 299–301, 303–306, 308, 309, 318 Bioactive molecules�������������������������������������1–9, 14, 164, 278 Bioavailability������������������������������������ 4, 5, 9, 45, 72, 101, 110, 188, 190, 200, 202, 204, 207, 208, 222, 236, 238, 248, 249, 251, 252, 256, 260–266, 268–270, 272, 273, 275, 287, 300, 316, 328, 329 Biocompatibility����������������������������������������152, 203–205, 260 Biodegradable����������������������������������������������71, 100, 136, 152 Biomaterials����������������������������������13, 164, 221, 227, 231, 300

Biomolecules����������������������������������� 4, 14–15, 17, 19–22, 112 Biopharmaceutical classification system (BCS)����������� 4, 100, 255, 261, 263, 264, 266, 268, 269 Block copolymers������������������������������ 71, 72, 76, 77, 300, 303 Blood-brain barrier (BBB)��������������������������������������� 236, 237 Blood pressure regulators��������������������������������������������������110 Bovine serum albumin (BSA)�����������������������14, 30, 273, 274 Brain exosomes�������������������������������������������������������������������27

C Caffeic acid (CA)�����������������������������������������������������������������4 Calixarenes�����������������������������������������������������������������������238 Cancer�������������������������������������������5, 25, 72, 86–89, 100, 110, 127, 128, 136, 137, 140, 151, 175, 199–203, 214, 216, 248–250, 252–256, 271, 327–330 Cancer cells�������������������������������72, 86, 88, 89, 137, 201–203, 214, 216, 249, 250, 253–255, 327, 328 Candesartan (CAN)������������������������������������������4, 6, 9, 45–69 Candesartan cilexetil������������������������������������������������� 4, 45–69 Captopril������������������������������������������������������������������100–106 Carbon nanohorns��������������������������������������������������������13–22 13 C cross polarization/magic-angle spinning (13C CP/MAS)������������������������������� 318, 319, 323 Cell cultures��������������������������������������� 25, 26, 90–93, 142, 178 Cell growth������������������������������������������������������� 200, 201, 254 Ceramidase inhibitors������������������������������������������������������202 Ceranib-2 (C-2)�������������������������������������������������������199–217 Characteristic transition temperatures�������167, 168, 225, 302 Characterizations������������������������������������ 22, 25–33, 143, 148, 151–160, 163, 180, 210, 266, 271, 332–333 Chemical shift changes�����������������������������������������������������319 Chemical stability��������������������������������������������������� 2, 71, 316 Chimeric nanosystems����������������������������������������������221–232 Circular dichroism������������������������������������������������������������129 CO2 laser���������������������������������������������������������������������� 14, 17 Complexation���������������������������������� 2, 3, 5, 46, 164, 165, 167, 169, 170, 177, 189–191, 235–245, 250, 251, 258, 262, 264, 270, 271, 273, 314, 319 Complexation energy���������������������������������������������������������46 Complexes������������������������� 1–9, 35, 46, 49–52, 59, 66–68, 92, 100, 102, 110, 111, 113, 115–119, 136, 149, 164–172, 176, 179, 182, 184, 185, 188, 189, 194, 201, 238, 239, 241, 242, 244, 248, 249, 251, 252, 255, 256, 259–262, 264–266, 268, 272, 273, 278, 287–289, 292–296, 304–306, 313–322

Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols, Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0 © Springer Science+Business Media, LLC, part of Springer Nature 2021

339

upramolecules in Drug Discovery and Drug Delivery: Methods and Protocols 340       ISndex

Compositional homogeneity��������������������������������������������136 Compositions��������������������������������������� 26, 37, 101, 136, 146, 159, 203, 227, 288, 308 Compounds����������������������������������������� 2, 5, 37, 38, 40, 42, 94, 95, 109–111, 146, 155, 163, 165, 176, 180, 182, 183, 185, 191, 204, 238, 239, 249, 251, 254, 255, 271, 274, 278, 285–288, 292, 293, 313–315, 319, 333–335, 337 Computational chemistry������������������������������������������� 54, 110 Computational protocol�����������������������������������������������45–69 Concentrations������������������������������������������3, 5, 22, 30, 35, 37, 40, 41, 69, 74, 81, 88, 90, 92, 93, 96, 99, 102, 129, 135–137, 141, 143, 147–149, 155, 156, 160, 167, 176, 180, 182, 184, 189, 191, 194, 195, 212, 214, 217, 227, 230, 231, 237, 249–253, 256, 258, 266–268, 276–278, 286, 288, 289, 292, 293, 301, 309, 319 Confocal��������������������������������������������������������95–96, 254, 261 Conjugation������������������������������������� 17, 19–20, 235, 329, 330 Constant pressure�������������������������������� 57, 117, 118, 171, 230 Controlled drug release�������������������������������������� 87, 203, 204 Cooperativity������������������������������������� 300, 303, 305, 307, 309 Co-precipitation������������������������������������������������������� 2, 3, 164 Covalent coupling���������������������������������������������������������14–15 Critical micelle concentration (CMC)��������������� 99, 102, 274 Crystalline state������������������������������������������������ 168, 171, 231 Curcumin (CRM)��������������������4, 5, 7, 72–74, 76–78, 80, 251 Cyclodextrin (CD)������������������������������������1–4, 6, 8, 9, 35, 37, 42, 46, 49, 50, 53, 59, 61, 64, 68, 109–123, 164, 167–171, 188, 190, 191, 194, 195, 247–279, 287–289, 294–296, 304, 305, 313–322 Cyclodextrin inclusion����������������������������������������������������������2 Cytotoxicity���������������������������������������72, 90, 92, 93, 176, 184, 238, 249, 251, 256, 266, 328, 329

D Damp mixing and heating����������������������������������������������������3 Degree of substitution (DS)����������������������188, 194, 236, 272 Dendrimers��������������������������������299, 300, 303, 305, 308, 309 Deuterated solvents������������������������������������������ 101, 317, 337 Diagnosis������������������������������������������������������������ 25, 151, 221 Dialysis�������������������������������� 35–40, 42, 87–90, 143, 145, 146, 206, 209–211 1,6-Diaminohexane�����������������������������������129, 135, 140, 146 Differential power (DP)����������������������������189, 192, 195, 196 Differential scanning calorimetry (DSC)���������������� 163–172, 222–227, 229, 230, 248, 250, 251, 253, 273, 299–309 Diffusion coefficients������������������238, 239, 244, 245, 288, 289 Diffusion- ordered NMR spectroscopy����������������������������239 Difunctional initiator������������������������������������������������ 130, 142 3D structure������������������������������������������������������������� 136, 137 Dipalmitoyl-phosphatidylcholine (DPPC)������������� 222–229, 231, 304–308, 316, 318–321, 323

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)�������������������������� 222–224, 227–229, 231, 304–308, 316, 318–320 Dispersion phase��������������������������������������������������������������210 Dispersions���������������������������������� 14, 19, 20, 72, 90, 142, 154, 163, 165, 206, 207, 245, 259, 264, 267, 276, 301, 303, 304, 306, 319 Doxorubicin������������������������������������87, 89, 139, 140, 145, 253 5-doxyl-stearic acid (5-DSA)��������������������101, 102, 105, 106 DPPC bilayers����������������������������������� 225, 304, 306, 319, 321 Drug absorption������������������������������������4, 45, 76–78, 89, 90, 96, 100, 160, 315, 328 CD complexes, 288 delivery........................................1–9, 13–22, 25, 71–81, 85–96, 99, 100, 109–123, 139–149, 151, 152, 155, 164, 188, 203–204, 206, 207, 221, 222, 231, 235–245, 248, 252, 254, 258, 267, 268, 271, 279, 300, 313, 315, 316, 328 encapsulations............................................238, 248, 271 formulations.......................13, 73, 79, 80, 100, 101, 110, 188, 190, 202–205, 244, 276, 328 loading, 76, 77, 86, 87, 90, 99, 155, 175, 192, 260 localization................................................................. 95 molecules............................... 4, 5, 37, 46, 48–52, 57, 62, 64, 68, 87, 99, 102, 155, 165, 204–206, 222, 267, 269, 287, 288, 299, 300, 303, 315, 317 membrane interactions............................................. 318 Drug delivery system (DDS)����������������������������13, 85–88, 90, 99–107, 136, 137, 199, 202–207, 211, 251, 269, 308 Dry mixing ��������������������������������������������������������������������������4 Dynamic light scattering (DLS)��������������������� 35, 79, 81, 141, 148, 222, 224, 227, 228, 231, 249

E Electron microscopy��������������������������������������28, 29, 156, 157 Electrostatic association������������������������������������������������������14 Electrostatic interactions���������������������������������� 189, 260, 274 Encapsulated curcumin������������������������������������������������ 78, 81 Encapsulation of indomethacin������������������������������������ 76, 80 Encapsulation processes�����������������������������������������������������71 Encapsulation protocols����������������������������������������� 72, 73, 79 Encapsulations���������������������������������� 20, 71–81, 86, 155, 156, 175–185, 188, 191, 248, 252, 256, 258, 259, 265–267, 271, 273, 274, 276, 277 Endocytosis������������������������������������������������������������������ 14, 88 Endothermic peak�������������������������������������������� 168, 169, 303 Endothermic process������������������������������������������������ 171, 196 Energetics����������������������������������������������������46, 110, 117–118 Enhancement of the aqueous solubility��������������������������������4 Enthalpy������������������������� 2, 117, 118, 122, 168, 189, 307, 308 Enthalpy changes����������������������������� 167, 168, 171, 189, 192, 195, 225, 302 Entrapment efficiency����������������������� 258, 269, 272, 315, 324

Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols   341 Index    Entropy��������������������������������������������� 117, 118, 122, 189, 190 Equilibrium state������������������������������������������������ 47, 230, 231 Exosome during secretion from the tissue��������������������������26 Exosome from whole organs����������������������������������������25–33 Exosome isolation��������������������������������������������������������26–28 Exosomes����������������������������������������������������������������������25–33 Exothermic processes��������������������������������������� 171, 173, 192 External potential����������������������������������������������46, 47, 57, 64 Extracellular vesicles�����������������������������������������������������������30 Extra sensitivity������������������������������������������������������������������89 Extrusion���������������������������������������������������������������������� 3, 253

F Fluorescence������������������������������������������������95, 130, 188, 261 Fourier transform infrared spectroscopy�����������������������������76 Free energy calculations�������������������������������������������� 110, 111 Freeze-drying����������������������������������� 1–9, 164–166, 170, 258, 289, 318 Functionalities������������������������������������ 14–15, 20, 22, 87, 119, 137, 221–232, 300 Functionalization�������������������������������������������������� 14, 22, 270 Functional phase����������������������������������������������� 229, 231, 232

G Gas delivery��������������������������������������������������������������271–272 Gastric reflux������������������������������������������������������������270–271 Gemcitabine������������������������������ 127, 133, 136, 328, 330–337 Gibbs free energy (ΔG)������������������46, 47, 117, 189, 194, 195 Glioblastoma multiforme�����������������������������������������175–185 Glove box����������������������������������������������������������������� 131, 132 Glutathione�����������������������������������������������������86, 88, 89, 252 Graphite����������������������������������������������������������������������� 16, 17 Gromacs������������������������������� 48, 50–52, 54, 57, 61, 64, 68, 69

H Half peak height����������������������������������������������� 168, 225, 302 Heart exosomes������������������������������������������������������������������27 Heat capacity���������������������������������������������164, 171, 196, 230 Heat-flux DSC������������������������������������������������� 163, 164, 166 Heating rate������������������������������������������������������������� 167, 171 Heating scans������������������������������������ 225–227, 230, 231, 307 High energy states��������������������������������������������������������������46 High vacuum techniques���������������������������130, 135, 141, 142 1H-NMR spectroscopy������������������������������������ 101, 142, 146 Host-guest interactions������������������������������������������������ 2, 188 Host-guest complex��������������������������������������������������������������2 Hybrids��������������������������������������������������������85, 139–149, 278 Hydrogels���������������������������� 86, 100, 127–137, 258, 262, 263, 271, 277, 278, 329 Hydrogen bonds�������������������������������� 119, 155, 158, 189, 286 Hydrophilic blocks������������������������������������135, 139, 145, 149 Hydrophobic cores������������������������������������������73, 76, 77, 321 Hydrophobic drugs����������������������������������������������� 71–81, 306

Hydrophobic interactions��������������������������176, 189, 194, 229 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) ������������������������������������ 4, 110, 111 Hyperlipidemia associated diseases����������������������������������272 Hypertension���������������������������������������������100, 110, 268–269 Hyperthermia��������������������������������������������������� 86–90, 93, 96

I Imaging�����������������������������������������������������������29, 31, 95, 157 Improvement of the pharmacological properties�������������������������������������10, 70, 110, 152 In vitro��������������������������������������������� 4, 9, 87, 89, 90, 140, 146, 148–149, 152, 165, 176, 202, 209–214, 249–256, 261, 262, 264–266, 268, 270–274 In vivo������������������������������������� 4, 90, 135, 136, 139, 146, 149, 152, 176, 202, 250, 252–254, 256, 261, 264, 265, 267, 270, 273, 275 In vivo pharmacokinetic analysis��������������������������������������185 Inclusion complexes����������������������������������������������2, 4–9, 110, 189, 190, 248, 251, 253, 256, 259, 265–268, 271, 288, 314 Indomethacin������������������������������������������� 72, 73, 76–80, 306, 308, 314 Inorganic����������������������������������������85, 86, 163, 203, 260, 278 Interactions�����������������������2, 14, 45–69, 87, 95, 101, 105, 106, 110, 155, 160, 164, 165, 168, 170, 176, 187–196, 244, 245, 251–253, 258, 267, 269, 274, 277, 288, 299, 303, 305, 308, 309, 314, 318–320, 330 Interdigitation������������������������������������������������������������������308 Interfaces���������������������������������������������������105, 106, 286, 291 Irbesartan (IRB)������������������������������� 4–6, 110–119, 165–172, 304–306, 316–322 Irbesartan2-HP-β-CD association�����������������������������������110 Isothermal Titration Calorimetry (ITC)���������� 188, 190, 191 Isotherms�������������������� 167, 170, 189, 193, 225, 230, 302, 305

K Kinetic phenomena����������������������������������������������������������171 Kneading method�����������������������������������������������������������������3

L LCST����������������������������������������������������������������������� 152, 308 Lipid bilayers����������������������110, 300, 303–305, 309, 310, 320 Lipophilic drugs����������������������������������������������4, 46, 269, 277 Lipophilicity/Hydrophilicity���������������������������� 110, 264, 272 Liposomes������������������������� 221–224, 227–230, 299–310, 315 Liver exosomes�������������������������������������������������������������������27 Losartan (LOS)������������������������� 4, 6, 165–170, 192–194, 196 Losartan potassium�����������������������������������166, 167, 190–192 Lower critical solution temperature (LCST)������������������������������88, 222, 226, 229–231 Lyophilization������������������������ 6, 8, 9, 163, 166, 167, 170, 276 Lyotropic effect����������������������������������������������������������������225

upramolecules in Drug Discovery and Drug Delivery: Methods and Protocols 342       ISndex

M Macroinitiator�������������������������������������������������� 140, 143, 147 Macromolecular architecture���������������������������� 128, 136, 149 Main transition temperature����������������������225, 231, 301, 307 Major (M) and minor (m) conformation��������������������������103 Melting temperature�������������������152, 164, 165, 168, 170, 253 Metastable phases����������������������������������������������������� 300, 308 Methyl-β-cyclodextrin (M-β-CD)�����������������������������������165 MGMT expression�������������������������������������������������� 176, 236 Micellar systems���������������������������������������������������������������316 Mice plasma���������������������������������������������������������������������184 Micelles�������������������������������������������71–81, 99–107, 316–322 MicroRNA (miRNA)��������������������������������������������������������25 Microscope���������������������������������������������� 32, 94, 95, 160, 254 Microwave irradiation��������������������������������������������������������21 Milli-Q water�����������������������������130, 132, 133, 136, 142, 144 Mixed nanosystems����������������������������������������������������������221 Modifications���������������������������� 113, 203–206, 258, 275, 287 Molar ratios�������������������������������������� 6–9, 102, 106, 132, 167, 168, 170, 192–194, 224, 227, 228, 230, 241, 258, 259, 266, 267, 276, 300, 304, 307, 310, 318 Molecular docking����������������������������������������������������110–113 Molecular dynamics (MD)��������������������������� 45–69, 109–123 Molecular homogeneity����������������������������������������������������136 Molecular interactions���������������������������������������������� 101, 313 Molecular Mechanics Poisson-Boltzmann Surface Area (MM–PBSA) method������ 111, 117, 118, 122 Molecular modeling�������������������������������������������������112–114 Molecular structures���������������������������������� 9, 35, 50, 251, 313 MTT assays������������������������������������������� 92, 93, 209, 214, 255 Multi-component solutions����������������������������������������������238 Multifunctional drug delivery systems������������������������������136

N Nanocarriers�������������������������� 13, 22, 71, 79, 86, 99, 101, 221, 250, 252, 255, 260 Nanoencapsulation�����������������������������������������������������������207 Nanoparticles (NPs)������������������������������80, 81, 86, 87, 89, 91, 94, 100, 148, 149, 152, 156, 157, 203–208, 210–215, 221, 224, 229, 260, 275, 300, 329 Nanospheres������������������������������������������������86, 153, 155, 204 Nanosponges (NSs)������������������������������������������ 100, 247–279 Nanosystems����������������������������������������������������� 221, 231, 300 Nanotechnology��������������������������������������������������������� 22, 203 Nanotherapeutics��������������������������������������������������������������279 Nanovesicles�����������������������������������������������������������������������25 Natural products��������������������������25, 100, 152, 190, 203, 204, 247, 251, 254, 265 N-carboxy Anhydride��������������������������������������� 130–132, 142 Neutralization method������������������������������������������������� 8, 318 Nim–trityl-l-histidine��������������������������������������������������������130 Nitroxide radical���������������������������������������������������������������101

NMR chromatography��������������������������������������������� 238, 239 NMR spectroscopy��������������������������� 101, 130, 241–245, 305, 314, 315, 317, 332–335 Non-toxic�������������������������������������������������������������������������190 Non-reversible Phase��������������������������������������������������������231 Nuclear magnetic resonance (NMR)��������������������������� 9, 129, 178–182, 188, 229, 313–321, 333 Nuclear Overhauser effect (NOE)�����������������������������������314

O Organic solvent evaporation����������������������������������������� 73, 81 Organic solvent method�����������������������������������������������������81 Oxidation������������������������������ 14–17, 277, 285–288, 292–296

P Paclitaxel���������������������������������������������������������� 139, 248, 249 Pancreatic cancer (PC)������������������������������������� 127, 128, 140 Parkinson’s disease (PD)�������������������������������������������267–268 Paste complexation���������������������������������������������������������������3 PEGylation��������������������������������������������������������������205–206 Pentablock terpolypeptide�������������������������������� 127, 133, 136 Peptides�������������������������������������129, 140, 273–276, 315, 316, 328–331, 336 Perturbation�������������������������������������������������� 46–47, 303–305 pH������������������������������������������� 5–9, 28, 29, 80, 81, 85–90, 96, 128–130, 132, 136, 137, 139, 141––148, 152, 159, 167, 178, 179, 181–183, 185, 190, 193, 194, 209, 210, 212, 214, 222, 223, 227, 230, 237, 241, 249, 251, 252, 254, 255, 262, 268, 274–276, 278, 286, 289, 292, 293, 295, 303, 318, 336 Pharmaceutical applications����������������������204–206, 248, 265 Pharmaceutical formulations������152, 164, 165, 204, 206, 208 Pharmaceutical systems����������������������������������������������������314 Pharmacological actions���������������������������������������������������264 Phase transitions������������� 88, 89, 164, 222, 227, 301, 303, 305 Phospholipids����������������������������������� 222, 226, 227, 230, 231, 299, 300, 304, 308, 320 Physical mixtures��������������� 164–166, 168, 170–172, 184, 266 Physicochemical characterization����������������������� 73, 224, 231 Physicochemical properties������� 109, 155, 188, 222, 228–229 Physisorption��������������������������������������������������������������������271 Plasma stability�����������������������������������������������������������������176 Pluronic F-127������������������������������������������72–74, 76–81, 264 Poly caprolacton (PLC)����������������������������������������������������200 Poly(ethylene oxide) (PEO)���������������������������71–73, 88, 100, 139, 140, 143, 148, 149 Poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL)���������������������������72, 73, 75–78, 80 Poly(ethylene oxide)-b-poly(propylene oxide)-b-­ poly(ethylene oxide) (PEO-b-PPO-b-PEO)������������������������������ 71, 72 Poly(lactic-co-glycolic) acid (PLGA) copolymer�������������203 Poly(lauryl acrylate) (PLA)������������������������������ 225, 229, 230 Poly(N-isopropylacrylamide) (PNIPAM)������������������������222

Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols   343 Index    Poly(N-isopropylacrylamide)-b-poly(lauryl acrylate) (PNIPAM-b-PLA)��������������������������������223–230 Polyacrylates (PA)�����������������������������������������������������������203 Polyalacticle (PL)�������������������������������������������������������������203 Polydispersities�����������������������79, 80, 136, 148, 204, 227–230 Polyethylene glycol (PEG)������������ 28, 30, 199–217, 259, 261 Polymer therapeutics����������������������������������������������������������71 Polymeric micelles�������������������������������������������������� 76–79, 86 Polymeric nanocarriers������������������������������������������������� 80, 86 Polymerizations�����������������������������73, 87, 129, 130, 132, 135, 140, 142, 143, 153, 229 Polymers������������������������������������ 42, 73–75, 77, 81, 85, 86, 88, 127, 129, 137, 139, 141, 143–145, 147–149, 152, 163, 203–206, 221, 222, 225–227, 229–232, 247, 251, 252, 258, 260, 264, 267, 269–272, 277–279, 299, 300, 305, 307, 308 Polymersomes�����������������������������������������������������������139–149 Polypeptides��������������������������������������������� 132–133, 135–137 Polyphenols���������������������������������������������������������� 5, 265, 266 Poorly water-soluble molecules����������������������������������������248 Precipitation�������������������������������37, 76, 80, 81, 130, 132, 258 Pretransition����������������������������������������������226, 303, 304, 308 Proliferation����������������������������������� 91–93, 184, 200, 202, 255 p-sulfonatocalix[4]arene��������������������������� 176, 177, 179, 237, 241, 242, 244 PTFE membrane filters����������������������������������������� 17, 19–21

Q Quantum mechanics���������������������������������������������������������110 Quercetin (QUE)��������������������������4, 5, 8, 235, 236, 241–244, 264, 265, 287–289, 293–296

R Recrystallizations���������������������������������������������� 131, 143, 249 Redox����������������������������� 86, 88, 137, 285–288, 292, 294, 295 Release��������������������������������������� 14–15, 22, 30, 38, 46, 48, 71, 86–90, 96, 99–101, 110, 136, 139, 148–149, 152, 155, 190, 203, 205, 206, 209–213, 222, 249–260, 264–266, 268–274, 276, 278, 279, 303, 308, 316, 317, 329 Ring opening polymerization������������������������������������� 73, 143 Rosin esters��������������������������������������������������������������199–217 Rosmarinic acid (RA)������������������������������������������������������� 4, 7

S Sartans�������������������������������������������������������������� 110, 164–171 Scanning electron microscopy (SEM)������������������������������ 129, 156–158, 160, 249 Scratch������������������������������������������������������������������ 93, 94, 163 Secondary structure�������������������������������������������������� 128, 136 Self-assembly properties�����������������������������������������������������71 Self-healing hydrogel��������������������������������������������������������136 Silibinin (SLB)������������������������������������������������������������� 4, 7, 9 Size distributions��������������������������� 79–81, 204, 207, 227, 229

Size exclusion chromatography (SEC)������������� 129, 141, 148 Sleep disorders������������������������������������������������������������������270 Slurry complexation��������������������������������������������������������������3 Sodium dodecyl sulfate (SDS)��������������������29, 99, 101, 102, 105, 316–322 Solid state NMR and solution state NMR������� 258, 305, 318 Solubility�������������������������� 4, 5, 9, 13, 42, 45, 72, 99, 100, 109, 110, 165, 175–185, 187, 188, 190, 191, 200, 202–205, 207, 235, 236, 238, 248, 249, 251, 254–256, 258, 259, 261–266, 268, 269, 271, 272, 276, 278, 279, 287, 316, 327, 330, 337 Solvent evaporation�������������������������������������77, 206, 207, 268 Solvents�������������������������������������������������������������4, 17, 21, 152 Sonication�������������������������������� 20, 33, 88, 105, 155, 160, 210, 216, 227, 231, 259, 301, 318 Spectroscopic�������������������������������������������������������������� 22, 188 Sphingolipids (SPL)�������������������������������������������������200–202 Spin labels�������������������������������������������������101, 102, 106, 107 Stabilizers�������������������������������������������������������������������������277 Steered MD������������������������������������������������������������������45–69 Steroids���������������������������������������������������������������������273–276 Stimuli responsive����������������������������������������������������� 221, 252 Stoichiometry��������������������������������������������191, 195, 196, 314 Structural properties���������������������������������������������������������318 Supramolecular carriers����������������������������������������������������238 Supramolecular chemistry������������������������������������������������176 Synthesis of polypeptides�������������������������������������������������134

T Targeted Drug Delivery�����������������������������������������������������86 Temozolomide (TMZ)��������������������� 176, 177, 179–185, 236, 237, 241, 242, 244, 250, 251 Temperatures��������������������������� 4, 5, 16–20, 31, 32, 36, 37, 39, 42, 55, 57, 73–75, 86–90, 96, 102, 107, 117, 118, 129, 132, 133, 135–137, 139, 141, 146, 148, 152, 154, 163, 164, 167, 169, 170, 179, 183, 185, 188, 189, 192, 195, 196, 206, 207, 210, 217, 222, 227, 229–232, 241, 245, 252, 253, 259, 267, 271, 274, 277, 289–291, 302–305, 319, 320, 333–336 ε-tert-butoxycarbonyl-l-lysine�������������������130, 132, 135, 142 Theranostics��������������������������������������������������������� 85–87, 221 Therapies�����������������������������������100, 151, 205, 221, 254, 255, 262, 268, 269 Thermal analysis (TA)������������������������������������������ 9, 163, 164 Thermo�������������������������������������������������������������28, 29, 32, 86 Thermodynamic parameters��������������������� 168, 171, 188, 194, 195, 225, 302, 303 Thermodynamics���������������������2, 46, 165, 168, 170, 187–196, 221–232 Thermograms����������������������������164, 165, 167–169, 225, 302, 303, 309, 310 Thermoresponsive�������������������������������������������� 221–232, 306 Thermotropic behavior������������������������������������� 225, 299–310 Thin film hydration������������������72, 73, 81, 224, 227, 300, 303

upramolecules in Drug Discovery and Drug Delivery: Methods and Protocols 344       ISndex

Thin-film hydration method (TFHM)����������������������������������224, 227, 300, 303 Thin film protocol��������������������������������� 72, 74, 75, 78, 80, 81 Transporters������������������������������������������������������� 46, 266, 269 Transport systems����������������������������������������������������� 110, 269 Triple quadrupole mass spectrometry�������������������������������178 Two-dimensional diffusion-ordered NMR spectroscopy (2D DOSY)�������������������������������������������235–245

U U87, 8MG������������������������������������������������������������������������184 Ultra High Performance Liquid Chromatography���������������������������������������������178 Ultrasonication������������������������������������������������������� 26, 30, 33 Ultrasonic cavitation����������������������������������������� 206–208, 211 Umbrella sampling��������������������������������������46, 48, 61–67, 69 UV-Vis spectroscopy����������������������������������������������������������76

V Van der Walls forces������������������������������������������������� 189, 259 Vesicles������������������������������139–142, 145, 147–149, 223, 224, 231, 301, 319

W Water���������������������������� 2–8, 17, 19, 20, 32, 33, 37, 39, 53, 68, 72–75, 81, 87, 90, 91, 99, 100, 115, 116, 118, 128–133, 135–137, 139, 141, 142, 146, 147, 149, 152–154, 159, 160, 166, 167, 177, 178, 188, 190, 195, 209, 210, 212–214, 216, 217, 222, 227, 241, 251, 260, 262, 264–266, 268, 270, 276, 278, 287, 289, 291–293, 295, 301, 303, 304, 318, 319, 332 Water solubility������������������ 190, 206, 250, 272, 315, 316, 328 Wound closure��������������������������������������������������������������������95 Wound healing�������������������������������������������������������������������94