Modern Biomedical Applications of Magnetic Nanoparticles (SpringerBriefs in Molecular Science) 981197103X, 9789811971037

Table of contents :
Preface
Contents
1 Traditional Applications of Magnetic Nanoparticles in Clinical Diagnostics and Therapeutics
1.1 Magnetic Resonance Imaging
1.1.1 Working Mechanism of Contrast Agents in MRI
1.1.2 SPIONs as T1-T2 Dual Modal Contrast Agents
1.2 Magnetic Cell Separation
1.2.1 Separation of Circulating Tumor Cells (CTCs)
1.2.2 Separation of Stem Cells
1.3 Magnetic Nanoparticles for Therapeutic Applications
1.3.1 Magnetic Hyperthermia and Drug Delivery
1.3.2 MRI and Drug/photosensitizer Delivery
1.3.3 MRI and Magnetic Manipulation
References
2 Modern Biomedical Applications of Magnetic Nanoparticles
2.1 Magnetic Particle Imaging (MPI) and Magnetic Particle Spectroscopy (MPS)
2.1.1 Brief Principle of MPI
2.1.2 MPI and MPS in Bio-Applications
2.2 Magnetic Nanoparticles for Designing Biomedical Sensors
2.2.1 Magneto-Optic Biosensors
2.2.2 Magnetic Nanoparticle-Based Microfluidic Sensors
2.2.3 Magnetoresistive Sensors and Non-magnetoresistive Sensors
2.3 Application of MNPs for Cancer Immunotherapy
2.3.1 The Roles of MNPs in Nanovaccine Formulation
2.3.2 MNP-Mediated Immune Cell Regulation
2.4 Gene Delivery and Therapy
2.4.1 Gene Delivery Using MNP-Incorporated Viral Vectors
2.4.2 Gene Delivery Using MNP-Incorporated Non-viral Vectors
2.5 Organelle Isolation for Proteomic Research
2.5.1 Isolation of Exosomes
2.5.2 Isolation of Mitochondria
2.5.3 Isolation of Lysosomes
References
3 Concluding Remarks and Future Prospects
3.1 Summary
3.2 Future Prospects
References

Citation preview

SpringerBriefs in Molecular Science Mari Takahashi · The Son Le · Shinya Maenosono

Modern Biomedical Applications of Magnetic Nanoparticles

SpringerBriefs in Molecular Science

SpringerBriefs in Molecular Science present concise summaries of cutting-edge research and practical applications across a wide spectrum of fields centered around chemistry. Featuring compact volumes of 50 to 125 pages, the series covers a range of content from professional to academic. Typical topics might include: • A timely report of state-of-the-art analytical techniques • A bridge between new research results, as published in journal articles, and a contextual literature review • A snapshot of a hot or emerging topic • An in-depth case study • A presentation of core concepts that students must understand in order to make independent contributions Briefs allow authors to present their ideas and readers to absorb them with minimal time investment. Briefs will be published as part of Springer’s eBook collection, with millions of users worldwide. In addition, Briefs will be available for individual print and electronic purchase. Briefs are characterized by fast, global electronic dissemination, standard publishing contracts, easy-to-use manuscript preparation and formatting guidelines, and expedited production schedules. Both solicited and unsolicited manuscripts are considered for publication in this series.

Mari Takahashi · The Son Le · Shinya Maenosono

Modern Biomedical Applications of Magnetic Nanoparticles

Mari Takahashi Japan Advanced Institute of Science and Technology Nomi, Ishikawa, Japan

The Son Le Japan Advanced Institute of Science and Technology Nomi, Ishikawa, Japan

Shinya Maenosono Japan Advanced Institute of Science and Technology Nomi, Ishikawa, Japan

ISSN 2191-5407 ISSN 2191-5415 (electronic) SpringerBriefs in Molecular Science ISBN 978-981-19-7103-7 ISBN 978-981-19-7104-4 (eBook) https://doi.org/10.1007/978-981-19-7104-4 © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are solely and exclusively licensed 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 Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Responding to the surge in nanotechnology in the late of twentieth and twenty-first centuries, various applications based on nanoparticles (NPs) have appeared. One of the fields which has seen a significant application of NPs is the biomedical field, which specifically makes use of iron oxide-based magnetic nanoparticles (MNPs). Iron oxide is the magnetic material which is the most familiar to humankind because it exists in abundance in nature as a mineral. Superparamagnetic iron oxide nanoparticles (SPIONs) are the most used MNPs for biomedical applications because of their biocompatibility and high chemical stability. There are many types of biomedical applications using MNPs (including SPIONs) including in vitro, in vivo, conventional and novel techniques. This book summarizes how MNPs are used in those different applications and discuses some of the latest research. We hope that this book will help readers who want a broad overview of the applications using MNPs. This book contains three chapters. In Chap. 1, the traditional applications using MNPs are summarized. Magnetic resonance imaging (MRI), magnetic separation, magnetic hyperthermia and drug delivery have been investigated for long time. Recent progress in those fields is summarized. In Chap. 2, relatively modern applications such as magnetic particle imaging and spectroscopy, sensors, gene delivery, therapy and organelle separation are introduced. In both chapters, research published mainly from 2019 to 2022 is included. A summary of Chaps. 1 and 2 and the future prospects of MNPs in biomedical applications are written in Chap. 3. Finally, we would like to thank to Mr. Shinichi Koizumi who is an editor with Spring Nature Publisher for his help in the release of this book. Also we would like to thank to Mr. Simon Moore for the English proofreading. Nomi, Ishikawa, Japan

Mari Takahashi The Son Le Shinya Maenosono

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Contents

1 Traditional Applications of Magnetic Nanoparticles in Clinical Diagnostics and Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Working Mechanism of Contrast Agents in MRI . . . . . . . . . . . 1.1.2 SPIONs as T 1 -T 2 Dual Modal Contrast Agents . . . . . . . . . . . . 1.2 Magnetic Cell Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Separation of Circulating Tumor Cells (CTCs) . . . . . . . . . . . . 1.2.2 Separation of Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Magnetic Nanoparticles for Therapeutic Applications . . . . . . . . . . . . . 1.3.1 Magnetic Hyperthermia and Drug Delivery . . . . . . . . . . . . . . . 1.3.2 MRI and Drug/photosensitizer Delivery . . . . . . . . . . . . . . . . . . 1.3.3 MRI and Magnetic Manipulation . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Modern Biomedical Applications of Magnetic Nanoparticles . . . . . . . . 2.1 Magnetic Particle Imaging (MPI) and Magnetic Particle Spectroscopy (MPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Brief Principle of MPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 MPI and MPS in Bio-Applications . . . . . . . . . . . . . . . . . . . . . . 2.2 Magnetic Nanoparticles for Designing Biomedical Sensors . . . . . . . . 2.2.1 Magneto-Optic Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Magnetic Nanoparticle-Based Microfluidic Sensors . . . . . . . . 2.2.3 Magnetoresistive Sensors and Non-magnetoresistive Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Application of MNPs for Cancer Immunotherapy . . . . . . . . . . . . . . . . 2.3.1 The Roles of MNPs in Nanovaccine Formulation . . . . . . . . . . 2.3.2 MNP-Mediated Immune Cell Regulation . . . . . . . . . . . . . . . . . 2.4 Gene Delivery and Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1 Gene Delivery Using MNP-Incorporated Viral Vectors . . . . . 2.4.2 Gene Delivery Using MNP-Incorporated Non-viral Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 3 9 9 13 14 14 17 18 21 25 25 26 28 34 35 42 48 52 53 58 61 62 66

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Contents

2.5 Organelle Isolation for Proteomic Research . . . . . . . . . . . . . . . . . . . . . 2.5.1 Isolation of Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2 Isolation of Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.3 Isolation of Lysosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 73 76 78 85

3 Concluding Remarks and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93 93 96 97

Chapter 1

Traditional Applications of Magnetic Nanoparticles in Clinical Diagnostics and Therapeutics

Abstract Superparamagnetic iron oxide nanoparticles (SPIONs) have been used for a variety of applications in biological fields. Taking advantage of their biocompatibility and magnetic properties, the applications of SPIONs have developed over the years and each has achieved a sophisticated state. In this chapter, traditional applications using SPIONs and other magnetic nanoparticles (MNPs) will be introduced. It covers magnetic resonance imaging (MRI), magnetic separation of cells, magnetic hyperthermia (MH) and drug delivery, all of which are representative applications of MNPs. The purpose of this chapter is to introduce a general picture of the traditional applications to readers and explain the most recent research in each field. Keywords SPIONs · MRI · Magnetic hyperthermia · Drug delivery and cell separation

1.1 Magnetic Resonance Imaging MRI is one of the most frequently used clinical techniques in medicine. MRI visualizes the environmental difference of protons which constitute a variety of body tissues. MRI is obtained as a cross sectional image of a body. Since the proton environment in normal tissue and in a tumor is different, MRI is applied to both early detection and ongoing assessment. MRI provides two types of images (T 1 - or T 2 -weighted images) depending on the different relaxation processes of a proton’s excited magnetic moment. In order to increase their respective image contrasts, T 1 or T 2 contrast agents are often used in clinical practice. So far, several contrast agents such as Gd-based contrast agents [1] for T 1 images and SPIONs [2] for T 2 images have been approved by the US Food and Drug Administration (FDA). In this section, the current progress regarding the development of contrast agents will be summarized. Although, recently manganese ferrite [3] and manganese-zinc ferrite [4] have been investigated as T 2 contrast agents due to their high potential, SPIONs have been extensively studied owing to their characteristic superparamagnetic properties. Recently, ultrasmall SPIONs have also been studied as both T 1 and T 2 contrast

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Takahashi et al., Modern Biomedical Applications of Magnetic Nanoparticles, SpringerBriefs in Molecular Science, https://doi.org/10.1007/978-981-19-7104-4_1

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agents. Here, recent research around SPIONs for T 1 and weighted dual images will be introduced after briefly explaining the mechanism of T 1 and T 2 contrast agents.

1.1.1 Working Mechanism of Contrast Agents in MRI The brief principle of MRI is summarized here. Most clinical MRI machines utilize protons (1 H). Under a large magnetic field, the magnetic moment in a proton aligns parallel or antiparallel with the direction of the magnetic field. Then, a radiofrequency (RF) is applied to excite the magnetic moment, aligning the direction of the magnetic moment orthogonally to the magnetic field. When the RF is turned off, relaxation of the magnetic moment occurs, and it returns to its original equilibrium energy state through precessional motion under the magnetic field (Fig. 1.1). MRI visualizes two different relaxation processes: T 1 relaxation (also known as longitudinal relaxation or spin–lattice relaxation) and T 2 relaxation (also known as transverse relaxation or spin–spin relaxation) [5]. T 1 relaxation takes place by transferring the energy of the excited proton to the surrounding lattice. The longitudinal magnetization (M z ) increases over time. T 1 represents the time required for a group of protons to regain 63% of their original M z . On the other hand, T 2 relaxation represents the loss of magnetization in the plane (M xy ) transverse to the direction of the main magnetic field. Dipole–dipole interactions between protons results in the randomization of the magnetic moment due to thermal energy. M xy gradually decreases over time and T 2 represents the time required to achieve 63% of the original M xy of the excited state. Contrast agents alter those relaxation times, and we are able to differentiate tumors from normal tissue based on the different efficiency of introducing the SPIONs to those tissues. There are six characteristic mechanisms of contrast agents: (1) the change in tumbling time, (2) the aggregation state, (3) the chemical exchange rate with water, (4) the water accessibility, (5) the ligand proximity and (6) the electronic state [6]. Since T 1 relaxation is caused by the transfer of excess energy through a dipole–dipole interaction; tumbling time, electron spin state and the distance between the two dipoles are the main mechanisms. A change in the distance between water molecules and the contrast agent also influences the ability of the contrast agent. In order to obtain clear T 1 -weighted images, desirable T 1 contrast agents are composed of paramagnetic material to enhance the T 1 relaxation process [7]. Gd complexes are typical examples of T 1 contrast agents used clinically. However, the potential harm of Gd3+ -based contrast agents has recently been reported and is currently under investigation by the FDA [7]. In the case of T 2 contrast agents, the dephasing process of coherent magnetic moments accounts for the decay of the T 2 signal. Therefore, the creation of locally inhomogeneous magnetic fields by T 2 contrast agents can enhance the T 2 signal decay. In this sense, superparamagnetic SPIONs are frequently used owing to their superparamagnetic properties and biocompatibility. It is known that when the size of SPIONs becomes extremely small, the SPIONs start to show a T 1 enhancement capability overwhelming that of their T 2 contrast enhancement [7]. The optimal size of SPIONs as T 1 contrast agents was reported to be 3.6 nm [8].

1.1 Magnetic Resonance Imaging

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Fig. 1.1 Simplified schematic illustration of the mechanism of MRI. Top: the direction of magnetic moment (black arrows) set up in protons (blue circles) randomize in the absence of a magnetic field. Under a magnetic field (B), the magnetic moments align in the same direction as B. Applied RF excites the protons and the magnetic moments align orthogonally to B. Stopping RF allows the magnetic moments to relax to the original direction through precessional movement. Middle: T 1 and T 2 relaxation curves as a function of time. Bottom: Schematic illustration of relaxation process of magnetic moments of protons. Blue arrows and red arrows represent the increase of M z corresponding to the T 1 signal and decrease of M xy signal corresponding to T 2 signal

1.1.2 SPIONs as T1 -T2 Dual Modal Contrast Agents It is worth remembering that the size of SPIONs should be small in order to extract maximum performance as T 1 contrast agents while an inhomogeneous magnetic field is required to enhance contrast in T 2 -weighted images, which can be achieved by the assembly of SPIONs. Keeping this in mind, many researchers proposed switchable contrast agents for T 1 - and T 2 -weighted images by controlling their association. Several recent investigations are introduced here. Bai et al. synthesized SPIONs with a size of 5 nm and coated them with bull serum albumin (BSA@Fe3 O4 ) in order to give colloidal stability and biocompatibility [9]. The hydrodynamic size of BSA@Fe3 O4 was about 19 nm. Hepatocellular carcinoma (HCC) is one of most common cancer-related causes of death, and so early detection of hepatic tumors is required. A rabbit which was implanted with a hepatic tumor was used as a model in this study. The BSA@Fe3 O4 were intravenously administered to the hepatic tumor rabbit (5 mg of Fe/kg of body weight) and time dependent T 1 - and T 2 -weighted images were obtained. The administered BSA@Fe3 O4 were phagocytosed by reticuloendothelial cells such as Kupffer cells in

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the liver. It is known that hepatic lesions show less phagocytosis ability than normal liver tissue. Due to the different accumulation rates of BSA@Fe3 O4 in the normal and the tumorous parts, which resulted from the difference in the phagocytosis ability, BSA@Fe3 O4 were likely to be accumulated in the normal liver tissue site rather than the tumor. Figure 1.2a shows T 1 -weighted images at different times after the injection of BSA@Fe3 O4 . The signal at the tumor shown in the red circle gradually increased and achieved its highest signal at 90 min after the injection while the signal in the normal tissue site quickly decreased. The contrast-to-noise ratio (CNR) (Fig. 1.2b) indicated that the best contrast enhancement was obtained at 90 min after the injection. A large number of administered BSA@Fe3 O4 were quickly phagocytosed by Kupffer cells and likely accumulated in the normal liver tissue site. Hence, the T 1 signal in the normal liver tissue decreased. Simultaneously, BSA@Fe3 O4 were also gradually delivered to the tumor site by an enhanced permeability and retention (EPR) effect (Fig. 1.2c), resulting in an increase of the T 1 signal. On the other hand, the brightness of T 2 signal at the tumor site quickly increased while the normal tissue site showed a dark image after 4 h (Fig. 1.2d). This is because BSA@Fe3 O4 in the normal liver tissue site aggregated and showed a T 2 enhancement effect. The best CNR was obtained within 5 min after administration. Different accumulation rates of BSA@Fe3 O4 in the normal liver tissue and the tumor were able to produce different spatial distributions of BSA@Fe3 O4 resulting in a greater contrast in both T 1 - and T 2 -weighted images. Thus, uniform and small-sized BSA@Fe3 O4 were successfully utilized as a T 1 contrast agent. There are several studies utilizing the acidic microenvironment of tumors or lysosomes as a trigger for the assembly of SPIONs. He et al. designed a pH responsive system for T 1 to T 2 switchable contrast agents responsive to acidic pH by utilizing a combination of polyzwitterion and a negatively charged polymer as the surface ligands of extremely small SPIONs (ESIONP) [10]. First of all, poly(ethylene glycol) (PEG) modified-SPIONs with a size of 3 nm were prepared. Then, poly(N-{N’-[N”(2-carbox aminoethyl)]-2-aminoethyl}glutamide) (PDC) or poly (L-glutamic acid) (PGA) was conjugated the end of the PEG chains. Here, PDC was a polyzwitterion and showed a neutral charge at pH 7.4 and a positive charge at pH 6.5, while PGA exhibited a negative charge at both pH 6.5 and pH 7.4. Therefore, under acidic conditions, those two ESIONPs (i.e. ESIONP-PEG-PGA and ESIONP-PEG-PDC) were expected to aggregate resulting in the appearance of a T 2 signal enhancement. Furthermore, thanks to the size increase, the retention time of ESIONP in the tumor could be prolonged. To evaluate their ability to switch from T 1 to T 2 , both T 1 - and T 2 -weighted MRI images were obtained at pH 6.5 and 7.4 by changing the concentration of ESIONPs (converted and denoted as Fe concentration). Figure 1.3 shows reciprocal plots of T 1 and T 2 to calculate relaxivity, r 1 and r 2 , at different pH and their MRI images. The ratio of r 2 /r 1 is used to determine the preference of contrast agents. In general, when the r 2 /r 1 ratio is smaller than 3, it is used as a T 1 contrast agent, while when the ratio is larger than 8, it is suitable for use as a T 2 contrast agent. The r 2 /r 1 ratios of the ESIONP system were found to be 1.59 and 10.87 at pH 7.4 and 6.5 respectively, suggesting the switchable ability of the ESIONP system from a T 1 to a

1.1 Magnetic Resonance Imaging

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◄Fig. 1.2 a MR images of rabbit hepatic tumors (the red dashed circles represent orthotopic tumors) at different times before and after the intravenous administration of BSA@Fe3 O4 under an MRI scanner with a T 1 -weighted sequence. b Quantification of the T 1 signal changes of the tumor-to-liver CNR at the corresponding times. c Schematic distribution of BSA@Fe3 O4 in the tumor and normal liver tissues before injection and after 90 min. d MR images of rabbit hepatic tumors (the red dashed circles represent orthotopic tumors) at different times before and after the intravenous administration of BSA@Fe3 O4 under an MRI scanner with a T 2 -weighted sequence. e Quantification of the T 2 signal changes of the tumor-to-liver CNR at the corresponding times. f Schematic distribution of BSA@Fe3 O4 in the tumor and normal liver tissues before injection and after 5 min. (The error bars represent ± standard deviation of three parallel experiments.) Reprinted with permission from reference [9]. Copyright 2020, American Chemical Society

T 2 contrast agent in an acidic environment. Finally, the ESIONP system (a combination of the two types of ESIONPs), ESIONP-PEG-PGA or ESIONP-PEG-PDC was injected into tumor-bearing mice. The results showed that the ESIONP system showed significantly darker T 2 -weighted images compared to control experiments. Since the ESIONP system has a small size in order to utilize the EPR effect, it can more easily reach tumor sites than conventional SPIONs. Furthermore, the slightly higher acidic environment in tumor tissues compared to normal tissues can enhance the T 2 signal by triggering the pH dependent electrostatic aggregation. Pan et al. designed hyaluronic acid (HA) decorated with multiple SPIONs and used them to distinguish vulnerable plaque based on the fact that the vulnerable plaque was more likely to accumulate SPIONs than stable plaque because of the large number of macrophages [11]. Development of a system for the early detection of vulnerable plaque is an urgent task to suppress cardiovascular and cerebrovascular diseases

Fig. 1.3 a Longitudinal relaxation rate (1/T 1 ), b transverse relaxation rate (1/T 2 ), c T 1 -weighted, and d T 2 -weighted images of ESIONP systems at various iron concentrations and at pH 7.4 and 6.5 (0.5 T, 35 °C). Reprinted with permission from [10]. Copyright 2020 American Chemical Society

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which are caused by arteriosclerosis. The rupture of vulnerable plaques directly links to fatality. However, the contrast of T 1 - and T 2 -weighted MRI images is not enough to accurately differentiate between vulnerable plaque and stable plaque. Dual modality with a clear contrast using a contrast agent is key to solving this clinical problem. In this study, SPIONs capped with polyacrylic acid were conjugated to aminefunctionalized HA through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling. The resulting particles had a HA/SPION-polyacrylic acid core/satellite structure (SPION-HP). The hydrodynamic diameter of SPION-HP was 153 nm and the size of the primary SPIONs was estimated to be 4.7 nm from transmission electron microscope (TEM) images. The SPION-HP particles were intravenously injected into a mouse which was grown to have vulnerable plaques. The authors expected that SPION-HP particles would accumulate in vulnerable plaque because of the larger number of macrophages compared to stable plaque. The phagocytosed SPION-HP particles were delivered to lysosomes which have an acidic environment and thus, the HA would be hydrolyzed. Hydrolysis of bulky HA resulted in the clustering of SPION-HP particles which was the trigger for the appearance of T 2 enhancement. The brightness of the vulnerable plaques (indicated by the yellow arrows in the T 1 weighted images) increased and the intensity achieved its highest point at 2 h after the injection (Fig. 1.4). On the other hand, the T 1 signal did not change significantly in the case of stable plaque, although the base signal level was high and showed slight enhancement of the T 1 signal. Regarding the T 2 -weighted images, no signal change in either the vulnerable or stable plaques was confirmed within the first few hours after injection. A slight decrease in the T 2 signal of the vulnerable plaque was observed after 6 h and could clearly be seen 9 h after the injection. At the same time, the T 1 signal also decreased in the vulnerable plaque. On the contrary, T 2 enhancement in later stages was not detected in the stable plaques even up to 24 h after the injection. These results show that the larger number of macrophages in vulnerable plaque compared to stable plaque engulfed the SPION-HP particles and showed T 1 enhancement at an early stage. Then the engulfed SPION-HP particles formed clusters in the lysosomes of macrophages resulting in T 2 enhancement at a later stage. In the case of stable plaques, neither T 1 nor T 2 signal changed dramatically as a function of time. So far, several studies dealing with the conversion of contrast agents from T 1 to T 2 enhancement have been introduced, however an opposite conversion approach exists. Liu et al. fabricated CaCO3 coated PEG modified ultrasmall SPIONs (USPIONs@CaCO3 ) [12]. The sizes of PEG modified primary SPIONs and USPIONs@CaCO3 were approximately 3 and 100 nm respectively. It is known that direct use of USPIONs would cause rapid excretion from the body due to their small size. The authors tried to avoid this problem by making a CaCO3 coating on agglomerated USPIONs. Another role of the CaCO3 coating was that it was able to release USPIONs through the dissolving of the coating in the acidic environment of lysosomes in tumors resulting in a T 1 signal enhancement (Fig. 1.5). Because of the EPR effect, USPIONs@CaCO3 are likely to be sent to the tumor. The authors performed a demonstration experiment using a mouse model bearing a subcutaneous 4T1 xenograft. First, the appearance of the T 1 signal resulting from the dissolving of

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Fig. 1.4 a T 1 - and b T 2 -weighted images and the corresponding color-coded images of an ApoE−/− mouse in the vulnerable plaque group at different time points before and after intravenous injection of SPION-HP. Scale bars: 5 mm. c The SNR curves of plaque in the vulnerable plaque mouse group from the T 1 - and T 2 -weighted images. Prussian blue staining of plaque in the vulnerable plaque mouse group at d 2 h and e 9 h post-intravenous injection of SPION-HP. Scale bars: 100 μm. Reprinted with permission from [11]. Copyright 2021 American Chemical Society of Chemistry

the CaCO3 and the releasing of the USPIONs was investigated in different pH buffer solutions. USPIONs@CaCO3 provided a clear T 1 signal within 5 min when the pH was less than 6. Even when the pH was 6.8, which corresponds to the pH of the tumor microenvironment, a significant T 1 enhancement was observed after 20 min of incubation. The mouse was injected with USPIONs@CaCO3 (25 mg/kg of body weight) in a vein and a time dependent MRI measurement was performed. The results showed that the tumor site showed a weak T 2 signal at 2 h after the injection. Then, the T 1 signal at the tumor site significantly increased at 4 h after the injection and the enhancement lasted until 8 h. The reason for the different enhancement rates of the T 1 signal in the mouse compared to the acidic buffer system was attributed to the idea that the dissociation of the shell and the release of the USPIONs was more difficult and slower in actual living tissue. Exploiting these USPIONs as long-term effective T 1 contrast agents could be beneficial because the traditional Gd3+ based contrast agents are under question due to their toxicity. As shown here, most cases utilize small SPIONs to express T 1 enhancement and–by utilizing the microenvironment of tumors–the association level of SPIONs can be tuned, resulting in the appearance of a T 2 enhancement effect. The way of controlling the association level differs in each study. The acidic environment of tumors and/or the different uptake efficiency of tumors compared to normal tissues are often utilized. Since MRI contrast agents target tumor sites, drugs are often conjugated to the agent, which will be explained in subsection 1.3.2. Multimodal imaging based on MNPs for combinations of MRI/photoacoustic/ultrasound imaging [13] or MRI/fluorescence/computed tomography imaging [14] has also been proposed

1.2 Magnetic Cell Separation

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Fig. 1.5 Schematic diagram of the synthesis and sensing principle of the PEG-USPIONs@CaCO3 nanoprobe. Reprinted with permission from [12]. Copyright 2021 American Chemical Society

recently, which can facilitate early diagnosis. We expect that a variety of MRI contrast agents based on MNPs will be proposed and some of them will be applied to clinical settings in the near future.

1.2 Magnetic Cell Separation The magnetic separation of cells has been performed for many years. The early stages of magnetic separation of cells appeared as early as 1975 [15]. At this time, the separation target was limited to material which already had natural magnetic properties. Therefore, most targets were blood cells. After that, magnetic separation of lymphoid cells using polymeric particles containing small MNPs was reported by several groups in the 1970s [16, 17]. As time passed, a variety of target materials including cells, pathogens, proteins and cellular organelles were separated for the purpose of diagnosis, collection and fundamental research. In this section examples of several magnetic separation methods will be summarized.

1.2.1 Separation of Circulating Tumor Cells (CTCs) CTCs are tumor cells which flow in the blood when the metastasis of cancer cells happens. Since more than 90% of cases of cancer death are attributed to metastasis

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[18], the investigation of CTCs is important in terms of prognosis. Flow cytometric detection of CTCs is popular and the CellSearch® system is the only FDA-cleared system for the separation and enrichment of CTCs from blood samples, which utilizes MNPs for the separation of CTCs. Since CTCs are rare cells and one CTC in 10 mL of blood is indicative of a metastatic patient [19], many researchers have spent a long time trying to increase detection sensitivity. Many types of MNPs, surface ligands and separation systems have been reported and we recommend readers refer to them elsewhere [20]. In this section, we would like to spotlight the separation of CTCs based on their phenotypes. CTCs are known to show a heterogeneous subpopulation and the analysis of phenotypic variability is considered to reflect metastasis progress [21]. Therefore, it is important to separate viable CTCs based on their phenotype for downstream analyses. Kelley et al. fabricated several systems for the separation of CTCs according to their surface molecular properties. One of their works separated CTCs based on their protein expression level using antibody-conjugated MNPs. They designed a microfluidic chip with a magnetic field gradient which allows for the deflection of a flow of CTCs depending on the number of MNPs attached to the cells. Specifically, they used anti-epithelial cell adhesion molecule (EpCAM) antibody-conjugated MNPs in order to separate CTCs according to the expression level of EpCAM on the CTCs [22]. It’s important to note that EpCAM is known to be expressed on most CTCs significantly more than on normal cells. That chip also contains a Hall sensor, which allows for the counting of the number of cells and the differentiation of a single cell from a cell cluster. The analysis of the ratio of single cells to cell clusters is important since it can offer prognostic value. The design of the chip is shown in Fig. 1.6. It has two inlets (one is for the sample and the other one is for a buffer), one outlet and deflection guides made of a nickel–cobalt based alloy. A neodymium magnet was placed underneath the chip when sample flowed. When a cell is labelled with MNPs, the flow of the cell is deflected by a combination of the magnetic force and the fluidic drag force (Fig. 1.6d). Because the number of MNPs and the protein expression level on the cells has a linear relationship, cells with more MNPs—that is a higher protein expression level—tend to be deflected more. Non-magnetized cells went through the center line and were collected by channel 1. In order to assess the ability of the chip, two different prostate cancer cell lines with different expression levels of EpCAM were used. VCaP cells are known to express high levels of EpCAM while PC-3 M cells express a low to moderate level. The two different cell lines were mixed in blood and flowed through the chip. The result showed that the chip was able to profile the surface properties of CTCs. The sorted cells showed viability, which is good for downstream analysis. The purity using this approach was very high. Also, the Hall sensor integrated into the chip was able to differentiate the number of cells making clusters. In conclusion, their chip utilized an external magnetic field to deflect a flow of CTC cells and collected viable CTCs separately according to their protein expression level with high purity. Protein expression levels on CTCs as well as the relative number of single cell to cluster

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Fig. 1.6 Design of the prism chip. a Prismatic deflection separates a continuous sample stream into discrete subpopulations based on surface marker expression. b Magnetic nanoparticles offer a more faithful representation of cell surface protein expression than larger magnetic microparticles. c Schematic of the Co-based ribbon prismatic deflection chip (not drawn to scale). Deflection guides are made up of distinct segments having angles ranging from 2 to 30°. d Forces acting on a cell in the horizontal plane (neglecting friction). Magnetically labelled cells will follow the deflection guides until the magnetic force pulling the cells toward the guide is overcome by the component of the drag force acting perpendicular to the guide. e Comparison of the magnetic field enhancement generated with nickel and Co-based deflection guides using COMSOL Multiphysics. f Qualitative comparison of factors that affect cell deflection. Co-based deflection requires less particle loading for efficient deflection than comparable nickel-based guides. Reprinted with permission from [22]. Copyright 2018, American Chemical Society

cells can provide insightful information for the understanding of cancer metastasis and thus link to appropriate medical treatment. The other work of Kelley and coworkers focused on single cell profiling [23]. They designed an integrated single-cell proteomics (SCP) chip as shown in Fig. 1.7. It has constriction channels aligned with cobalt-based magnetic guides which can capture magnetically labelled CTCs and allows for on-chip incubation. The SCP chip can provide temporal phenotypic information of CTCs by in situ cell-surface protein expression analysis. The SCP chip has a round shape and contains 32 inlets at the edge, 4 capture zones per channel, and 1 outlet positioned at the center of the chip so that a syringe pump can be used to create a flow from the outside to

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the inside. A neodymium magnet comprises the bottom of the SCP chip. When they used MNPs targeted to EpCAM, MCF7 cells with high EpCAM expression were selectively separated from a 1000 times higher number of Hela cells with low EpCAM expression. By passing through fresh medium, the SCP chip showed the ability to incubate captured cells. In order to profile protein expression using the SCP chip, they utilized genetically barcoded fragment antibodies (Fabs) displayed on phages. Six different Fab-phages were selected to monitor the expression levels of H460 cells (non-small lung carcinoma cells) at a single cell level. Briefly, after magnetic separation in the SCP chip, the cells were incubated with a mixture of the Fab-phages. The results showed that the expression level of frizzled receptor 2 (FZD2) on individual H460 cells fluctuated and was different from the bulk population of H460 cells. It is known that FZD2 is associated with tumor development and metastasis. Their results imply that FZD2 levels may affect critical processes during the transition of H460 cells. In summary, they showed the ability of the SCP chip to capture a single CTC and incubate the cell in situ for the profiling of its protein expression level using semi-quantitatively barcoded Fabs.

Fig. 1.7 Schematic of the SCP chip to study the CTC adhesion and metastasis. a Target cells are labelled with antibody-conjugated magnetic particles in buffer or processed blood samples. b Overall architecture of SCP chip. c Cell capture approach used in the SCP chip. d Calculated magnetic force distribution along X-direction when magnetically labelled cells flow through the constriction channel. The top graph shows the values of magnetic force as a function of distance and the bottom image displays this information as a heatmap. e Representative SEM image of the capture area. The scale bar is 10 μm. Reprinted with permission from [23]. Copyright 2021, American Chemical Society

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CTC separations in this chapter mainly focused on the separation based on their phenotype for downstream analyses. Other CTC separations focusing on diagnosis were summarized in subsection 2.2.2.2.

1.2.2 Separation of Stem Cells Magnetic separation is also used for the separation of stem cells. Pluripotent stem cells (PSCs) are known to differentiate into many types of cells which can constitute different tissues. Examples of PSCs are embryonic stem cells and induced pluripotent stem cells. On the other hand, multipotent stem cells (MSCs) exist in specialized tissue and differentiate into specific types of cells under an appropriate signal. The application of stem cells in regenerative medicine is one of the main columns supporting current clinical medicine. For example, CD133+ hematopoietic stem cells, one of the MSCs, have been shown to have great potential in the field of regenerative medicine by differentiating into various blood cells. The purification of CD133+ hematopoietic stem cells from other blood components is therefore important and so magnetic separation is utilized [24, 25]. It is known that when PSCs differentiate into desirable target cells, the heterogeneity of the cells derived from PSCs limits its study and application, therefore magnetic separation using immuno-MNPs are used for purification [26, 27]. Here, one example of such will be introduced. A multiple separation of heterogeneous stem cells utilizing magnetic separation was reported by Haam and coworkers [26]. A heterogeneous population of cells derived from PSCs contains different types of cells, not only MSCs and PSCs but also unnecessary stem cells which spontaneously differentiated. An efficient separation of MSCs from other cells is important. Fluorescence-activated cell sorting (FACS) is a widely used technique, however it takes a long time and is not suitable for a large number of cells. MACS® (Miltenyi Biotec), on the other hand, allows for rapid separation on a large scale. But MACS® can separate only single magnetically labelled samples. Since MSCs are needed for stem cell therapy, PSCs can be reused, and other unnecessary stem cells are to be discarded, the sorting of these three groups is required. In order to sort these cells, they utilized a combination of two different sizes of magnetic particles and two different antibodies. Then, magnetically labelled cells were sorted in a microfluid under an external magnetic field gradient. The trajectories of the cells were decided by the total magnetization of the particles attached to the cells. In detail, the diameters of the two different particles were 225 nm (200 MNPs) and 500 nm (500 MNPs) respectively. The magnetizations were 87.2 emu/g for 200 MNPs and 105.8 emu/g for 500 MNPs. Anti-S antibodies (Ab-S) were conjugated to the 200 MNPs and anti-CD44 antibodies were conjugated to the 500 MNPs. It is known that the antigen for Ab-S is highly expressed on the surface of PSCs while MSCs express the antigen at about half the level of PSCs. On the other hand, the CD44 antigen is expressed by MSCs at a high level. They used a rectangle-shaped microfluidic system which has 1 inlet and 5 exits at opposite sides. The length of the system was

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40 mm, and the width was 12 mm. Under an external magnetic field gradient, which was perpendicular to the sorting direction, the majority of either MSCs or PSCs were collected at different exits. The collected PSCs still showed pluripotent, and so can be reused, while the MSCs can be used for further applications. The separation of target cells from heterogeneous stem cells is of great importance. By changing the types of antibodies, this magnetophoresis-based system can be applied to different cell systems which will broaden the possible applications of stem cells.

1.3 Magnetic Nanoparticles for Therapeutic Applications There are many techniques based on MNPs in biomedical fields and there are wellorganized reviews about different therapeutic applications of MNPs to which readers can refer [28–30]. Among these applications, this section focuses on the therapeutic applications which utilize the multifunctionality of MNPs. By combining MNPs and functional polymers or other inorganic materials, the probe often shows multiple functions and thus, the usage of the probe can be expanded. When it comes to cancer treatment, combinations of treatments such as MH and chemotherapy work better than cases where only a single treatment was performed. This effect is known as the synergetic effect. Here, some recent studies where the multifunctionality of MNPs were used for therapeutic purposes are summarized.

1.3.1 Magnetic Hyperthermia and Drug Delivery MH utilizes the eddy current heating, hysteresis loss and magnetic moment relaxation in MNPs under alternative magnetic fields for heating a localized area at a tumor site [31]. Since tumor cells have little resistance to sudden temperature changes, an increase in the temperature from 42 to 44 ºC at a tumor site can cause tumor cell death. There are two main approaches to deliver SPIONs to tumor sites. One is direct local delivery by injection, and the other is systemic delivery. To increase therapeutic performance, MH is often combined with chemotherapy. So far, a large number of combinations of MNPs and chemicals have been reported [32]. It is known that when angiogenesis in tumors is immature, macromolecules tend to elute off from blood vessels and are retained there, which is known as the EPR effect. Therefore, small sized SPIONs which are loaded with drugs show the ability to be retained and subsequently release drugs (passively or actively) as well as increase the efficiency of MH therapy. Doxorubicin (DOX) is a typical anticancer drug which was approved by the FDA and a lot of research conjugates DOX to MNPs. Furthermore, drug release from MNPs can be activated using thermo-responsive or pH-responsive polymers. In this section, several recent examples of MH combined with chemotherapy will be introduced.

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SPIONs coated with a thermo-responsive polymer are often used to trigger drug release. For example, Ferjaoui et al. prepared SPION core thermo-responsive copolymer shell particles [33], as shown in Fig. 1.8. Here, 2-(2-methoxy) ethyl methacrylate (MEO2 MA) and oligo(ethylene glycol)methacrylate (OEGMA) moieties were used. Specifically, they fabricated Fe3 O4 core P shell (where P represents MEO2 MAx -OEGMA100-x ) particles. Then DOX was loaded into the polymer shell. The size of the NPs was 10.0 ± 1.5 nm. The hydrodynamic diameters of the SPIONs in water and PBS were around 50–80 nm in both cases. Considering the EPR effect, a size of between 10 and 200 nm is known to be optimal in order to accumulate SPIONs in a tumor site. Therefore, the size reported in this study seemed to be within the optimal range. When the hydrodynamic diameter of Fe3 O4 @P (MEO2 MA60 OEGMA40 ) was measured with increasing temperature, it showed a low critical solution temperature at around 41 ºC. On the other hand, Fe3 O4 @P (MEO2 MA65 OEGMA35 ) was found to have a lower critical solution temperature (LCST) at 38 ºC which is too close body temperature. A DOX release test showed that the release process at 37 ºC was much slower than that of at 42 ºC and most of the DOX was released after 52 h at 42 ºC while less than 70% of DOX was released after 52 h at 37 ºC. The core@shell SPIONs were found to have MH properties based on specific absorption rate (SAR) measurements. The SAR in physiological media for Fe3 O4 @P (MEO2 MA60 -OEGMA40 ) was 12.6 W/g while the value increased to 45.7 W/g for Fe3 O4 @P (MEO2 MA65 -OEGMA35 ). Here, the main heat generation factors are

Fig. 1.8 Schematic illustration of the synergetic effect against tumor cells using SPIONs coated with a thermo-responsive polymer. Reprinted with permission from reference [33]. Copyright 2019, American Chemical Society

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Brownian and/or Néel spin relaxation derived from SPIONs. Cell cytotoxicity was investigated against human colon adenocarcinoma (HT29) cells. Dose-dependent cytotoxicity and a major decrease in cell viability were observed when the amount of Fe3 O4 @P (MEO2 MA60 -OEGMA40 ) was higher than 12.5 μg/mL. Nevertheless, the cell viability after 24 h and 72 h was 85 ± 8 and 75 ± 55% respectively when 25 μg/mL of SPIONs were added and no cytotoxicity was observed for concentrations of up to 12 μg/mL. Finally, the cytotoxicity of different concentrations of DOXloaded SPIONs at different temperatures against ovarian cancer SKOV-3 cells was compared to those treated with free DOX. The cells were incubated in the presence of either SPIONs or free DOX (fixed at 0.78 μg/mL) at 37 ºC for 24 h or 41 ºC for 5 h. The results clearly showed that using SPIONs as drug carriers resulted in a decrease in cell viability compared to free DOX as the amount of DOX released from SPIONs increased. Furthermore, the increase in temperature was followed by the reduction of cell viability, which implies a synergetic effect of MH and chemotherapy can be expected using this probe in the future. Mai et al. utilized cubic shape-SPIONs, which have a higher heat performance than spherical SPIONs in MH [34]. They fabricated iron oxide nanocubes coated with a DOX-loaded thermo-responsive polymer in the aim of seeing a synergetic effect between MH and chemotherapy. To coat the cubes with the thermo-responsive polymer they performed a surface-initiated photoinduced copper-mediated radical polymerization. After coating the iron oxide cubes with catechol-based initiators, two monomers were used to polymerize on the surface of the cubes. One is diethylene glycol methyl ether methacrylate (DEGMEMA) and the other is oligoethylene glycol methyl ether methacrylate (OEGMEMA). Under UV light and with a copper-based catalyst, polymerization took place on the surface of the cubes. The existence of polymer shell was confirmed by TEM imaging. A change in transmittance shows that the cubes with polymer shell composed of 20% OEGMEMA and 80% DEGMEMA had an LCST of 41 ºC. The size of the cubes and their SAR values were found to have a linear trend. When 19 nm cubes were used, heat profile measurements showed that their heating performance was still maintained after a third MH cycle. The oligoethylene glycol component has a role of suppressing protein adsorption, which is favorable for avoiding opsonization. When sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis (SDS-PAGE) analysis of the cubes was performed, both in the presence and absence of serum proteins, the results showed less adsorption of proteins on the polymer coated cubes. DOX was adsorbed on the cube nonspecifically and its release profile revealed that the amount of DOX released was around 3 times higher under MH than that measured at room temperature. Then, the cubes with or without DOX were intratumorally injected into a xenograft mouse model induced with A431 cells. The mouse was subjected to three MH cycles (30 min each) for three days. The temperature difference between the tumor and the skin was around 10–15 ºC. It was found that the combination of MH and chemotherapy showed complete tumor suppression while neither sole injection of the cubes nor the DOX alone could suppress the tumor. They also investigated the clearance of the cubes after injection. When they intravenously injected a 1 mg portion of iron per mouse, the material was found to be

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accumulated in the liver based on MRI. Then, the cubes were eliminated from the body 1 week post-injection, and 5–8 months post-injection no MRI signal from the cubes was obtained. Their research clearly shows the importance of the synergetic effect of MH and chemotherapy. The combination of a biocompatible thermoresponsive polymer for controlling DOX release and the cubic shape of iron oxide shows potential as a nanoplatform in oncothermia.

1.3.2 MRI and Drug/photosensitizer Delivery MNPs tend to accumulate at tumor sites due to the EPR effect and thus, they are suited for use as drug carriers. A combination of MRI and either drug or photosensitizer delivery is also one of the major therapeutic applications, since imaging techniques help in understanding the position of drug-conjugated MNPs and the pathological process of the tumor. Although recent research about SPIONs as T 1 contrast agents was introduced in the first section in this book, SPIONs are mostly used in T 2 weighted imaging in MRI. In this section, research focusing on a combination of MRI and drug or photosensitizer delivery will be introduced. Many types of materials are under consideration as candidates for drug carriers. Hydrogels composed of natural biopolymers such as polysaccharide are a promising platform for drug carriers since they are biodegradable and their rheological properties can be tuned. Ribeiro et al. prepared biocompatible xanthan gum (XG)/Fe3 O4 based drug-loaded nanoparticles [35]. XG is a polymer produced by bacterium with a polysaccharide backbone. Thanks to the presence of hydroxyl and carboxyl groups in the molecule, functionalization can be done easily. SPIONs coated with polyacrylic acid (PAA) were synthesized using a hydrothermal method. Then, magnetic hydrogels were prepared through the incorporation of SPIONs into the XG based hydrogel matrix. After the incorporation of SPIONs, the maximum degree of swelling was found to be 240% which was higher than that of the hydrogel without SPIONs. The reason for the higher degree of swelling of the hydrogel with the SPIONs was attributed to the hydrophilic nature of the surface of the PAA-coated SPIONs. In their study the antifungal drug terbinafine was used. As a matter of fact, the hydrogel with SPIONs showed T 2 contrast enhancement and their temperature could increase up to 60 ºC under an alternating magnetic field. Furthermore, drug release under an alternating magnetic field was found to be threefold faster than that of passive release. The cell cytotoxicity test against human dermal fibroblasts revealed that they did not show cell cytotoxicity after a 72-h incubation time. Finally, the killing effect of the loaded drug against C. albicans biofilms was confirmed. Their study showed the potential of hydrogels as platforms for drugs and their effective release with combination of SPIONs under an alternating magnetic field. Photodynamic therapy (PDT) uses photosensitizers to kill cancer cells under illumination and is an FDA-approved cancer treatment. Photosensitizers need to be localized at a tumor site, where they are excited under light irradiation and produce singlet oxygen, which is harmful to tumor cells. Yan et al. utilized micelles containing

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SPIONs as photosensitizer carriers which can be traced using MRI [36]. Benzoporphyrin derivative monoacid ring A (BPD) was used as the photosensitizer in their study, where it was conjugated to dextran. Due to the amphiphilic properties of dextran-BPD, it self-assembles into micelles in water, and thus hydrophobic BPD could be transferred to tumor sites efficiently. The SPIONs were loaded into the micelles based on an oil-in-water microemulsion method. TEM image analysis showed that the sizes of the SPIONs and the emulsion were around 10 and 60 nm respectively. The packing of SPIONs in a micelle was also clearly observed. The hydrodynamic diameter was found to be 80 nm by dynamic light scattering (DLS). The difference in sizes between TEM and DLS could be explained by the presence of the dextran. SPION-loaded micelles showed a much higher transverse relaxation rate (r 2 ) than commercial dextran-coated SPIONs due to the packing of the SPIONs into micelles. Stability evaluation tests showed that SPION-loaded micelles did not show a significant change in hydrodynamic diameter after 1 week. When their cytotoxicity was examined against 4T1 cells, minimal cytotoxicity was observed in the absence of light when the concentration of the micelles was less than 1250 nM, while the cell viability decreased under PDT with increasing concentrations of the micelles. The half-maximal inhibitory concentration (IC50 ) with PDT was determined to be 100 nM, which shows the effectiveness of SPION-loaded micelles for PDT. T 2 -weighted MRI was also performed on mice bearing breast tumors before and 24 h after an injection of SPION-loaded micelles as shown in Fig. 1.9a and b (note that SPION-loaded micelles are referred to as SDBMs in the figure). The decrease in signal clearly showed the location of tumor. The signal-to-background ratio (SBR) measurement indicated that SBR taken after injection was about 3 times lower than before injection (Fig. 1.9c). Tumor bearing mice were divided into three groups: (1) control, (2) treated with SPION-loaded micelles under light, (3) treated by free BPD under light. The chemicals were intravenously injected into the mice and after 24 h, all mice were illuminated at the tumor site (690 nm, 75 mW/cm2 , 135 J/cm2 ). When the tumor size was measured, it was found that tumor growth was the lowest for the mice treated with SPION-loaded micelles (Fig. 1.9g). The mice treated with free BPD showed little therapeutic effect compared to the control group (Fig. 1.9g). This is because free BPD is likely to have a short circulation time in blood stream and a short retention time in the tumor. Their study showed the potential of SPION-carrying micelles as MRI contrast agents and photosensitizers as tumor killing chemicals.

1.3.3 MRI and Magnetic Manipulation The imaging and magnetic manipulation abilities of SPIONs were applied to the field of stem cell therapy. Chen et al. fabricated mesoporous silica decorated with SPIONs [37] for the purpose of heart disease therapy. The SPIONs were grown directly on the surface of pores of the silica particle. Since silica particles have a lot of pores, drugs are easily loaded. The pore size was 16.6 nm and the total size of

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◄Fig. 1.9 SDBMs-enhanced imaging and in vivo tumor response. a and b T 2 -weighted magnetic resonance images in the axial plane prior to injection (pre-contrast, a) and 24 h after intravenous injection (postcontrast, b) of the SDBMs. Tumor location is indicated by dotted red circle. c Quantitative analysis of MR images. Signal-to-background ratio (SBR) measurements were made using the tumor and the water as background. Pre-injection versus post-injection SBR measurements are shown (n = 3 mice; columns represent SBR with error bars as standard deviation). d In vivo fluorescent images of mice with 4T1 orthotopic tumor 24 h post administration of free BPD or the SDBMs at a BPD concentration of 2.5 mg kg–1 body weight (n = 3). e Fluorescent images of excised tumors 24 h post administration of free BPD or the SDBMs. f Semiquantitative analysis of tumor fluorescence from d and e. g In vivo tumor response study after IV injection of PBS (control), free BPD, and the SDBMs at a BPD concentration of 2.5 mg kg–1 body weight. The PDT conditions for the in vivo study were as follows: wavelength: λ = 690 nm. Fluence rate: 75 mW/cm2 . Fluence: 135 J/cm2 . e Body weight changes of tumor-bearing mice after different treatments. (m and i) h & e-stained tumor sections excised from 4T1 tumor bearing mice following 22 d treatment with (m) PBS, i the SDBMs. The images of tumor were obtained by a Zeiss microscope at low magnification (20×). Reprinted with permission from reference [36]. Copyright 2019, American Chemical Society

the silica-SPIONs (note that silica-SPIONs are described as SIO in the Fig. 1.10) was found to be 360 ± 166 nm. Then, human mesenchymal stem cells (hMSCs) were labelled with the silica-SPIONs. The labeling of hMSCs did not influence the cellular metabolism, viability, proliferation, differentiation, phenotypes or migration ability. When the retention of hMSCs labelled with SPIONs in a tube where a shear stress of 12.8 dyn/cm2 was applied under a magnetic field was investigated, 85% of the hMSCs could be retained. Also, the silica-SPION-labelled hMSCs could be used as a T 2 -weighted MRI contrast agent. The shear stress used corresponds to the mean wall shear stress in the human left ventricle. In their study, insulin-like growth factor (IGF), which improves cell viability, was loaded into the particle. Mice experiments in vivo were conducted. A murine ischemia reperfusion model was used here. 60 days after the injection of 0.1 million IGFloaded silica-SPION-labelled hMSCs, the heart functions such as the left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS) were significantly improved when they were compared to the case where only 0.1 million hMSCs without particles were injected (Fig. 1.10a). According to the average heart-to-body weight ratios, IGF-loaded silica-SPION-labelled hMSC transplantation was found to suppress cardiomegaly (Fig. 1.10e). Trichrome staining of heat slices revealed that the percentage of fibrosis tissue (blue color) was the smallest for the heart treated with IGF-loaded silica-SPION-labelled hMSCs (Fig. 1.10f). As described here, the combination of SPIONs and mesoporous silica in stem cell therapy can achieve a release of growth factor and retention in a high-pressure blood flow, which cannot be performed by stem cells alone.

References

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Fig. 1.10 Effects of IGF-loaded SIO-labelled hMSC treatment on cardiac functions and pathological evaluation. a Comparison of mean LVEF among four groups (n = 12) from baseline, day 30, and day 60. IGF-loaded SIO-labelled hMSCs significantly increased LVEF and absolute value of global longitudinal strain (GLS) on both day 30 and 60 compared to the baseline. Error bars are standard errors (n = 12); * p < 0.05, ** p < 0.005. b Change of LVEF on day 60 from baseline for individual subjects. Error bars are standard deviations (n = 12); ** p < 0.005 (two-tail and type-two t test was used). c Mean GLS of each group (n = 12) from baseline, day 30, and day 60. d Photos show frontal view (top row) and transverse view (bottom row) of a representative heart from each group. (e) Mean heart-to-body weight ratio of each group. Error bars are standard errors (n = 11 for hMSC control group and 12 for the other three groups); * p < 0.05, ** p < 0.005, *** p < 0.0005. f Representative heart slices stained with trichrome staining from each group. Blue indicates fibrosis, and red indicates myocardium. RV: right ventricle. g Quantitative analysis for fibrosis. Error bars are standard deviations for six animals. Reprinted with permission from reference [37]. Copyright 2019, American Chemical Society

References 1. Lee W, Jung KH, Park JA, Kim JY, Lee YJ, Chang Y et al (2021) In vivo evaluation of PEGylated-liposome encapsulating gadolinium complexes for gadolinium neutron capture therapy. Biochem Biophys Res Commun 568:23–29. https://doi.org/10.1016/j.bbrc.2021. 06.045 2. Hubert V, Dumot C, Ong E, Amaz C, Canet-Soulas E, Chauveau F et al (2019) MRI coupled with clinically-applicable iron oxide nanoparticles reveals choroid plexus involvement in a murine model of neuroinflammation. Sci Rep 9(1):10046. https://doi.org/10.1038/s41598-01946566-1 3. Diez-Villares S, Ramos-Docampo MA, da Silva-Candal A, Hervella P, Vazquez-Rios AJ, Davila-Ibanez AB et al (2021) Manganese Ferrite nanoparticles encapsulated into vitamin

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Chapter 2

Modern Biomedical Applications of Magnetic Nanoparticles

Abstract As illustrated in Chap. 1, the utilization of MNPs in biomedical applications is considered to have potential technical benefits since they can be manipulated by external magnetic fields. In this chapter, we highlight some recent advances of MNPs in designing advanced biomedical techniques including magnetic particle imaging, biomedical sensors, the developement of new theranostic methods for cancer treatment other than magnetic hyperthermia, the integration of MNPs in gene delivery systems, and the isolation of cellular organelles. Finally, a perspective of magnetic nanoparticles in future biomedical applications is also discussed. Keywords Magnetic particle imaging · Magnetic particle spectroscopy · Biomedical sensor · Gene delivery · Organelle isolation

2.1 Magnetic Particle Imaging (MPI) and Magnetic Particle Spectroscopy (MPS) Magnetic particle imaging (MPI) and magnetic particle spectroscopy (MPS) have attracted attention in the biological fields [1–5]. The underlying physics in both techniques is similar. MPI generates a mapping image of MNPs by detecting signals from the MNPs. The advantage of MPI as a modality when compared to MRI is that a high resolution and quantitative analysis can be achieved. The names MPI and MRI are similar and both modalities often use SPIONs, however, they rely on different principles. An MPI signal comes from the ensemble magnetization of SPIONs and the scanner in MPI directly images the distribution of SPIONs. In the case of MRI, it is impossible to avoid detection of 1 H in water and biological tissue. This means that MPI does not suffer from a background signal, unlike MRI, since native biological tissue does not contain any SPIONs. This makes MPI seem promising compared to MRI. The spatial resolution of MPI is about 1 mm or less, which is influenced by the properties of the SPIONs and the scanner geometry. Although using MRI for the lungs and bones is known to be challenging, MPI has the potential to image those tissues. The image contrast in MPI is known to be comparable to positron emission tomography (PET) which uses a radioactive © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Takahashi et al., Modern Biomedical Applications of Magnetic Nanoparticles, SpringerBriefs in Molecular Science, https://doi.org/10.1007/978-981-19-7104-4_2

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isotope, but MPI’s non-radioactive tracers make it a more convenient option. The type of surface ligands used on the SPIONs in MPI depends on the target imaging objects. For example, when immune or stem cells are to be labelled, a carboxydextran-based coating is suitable so that the SPIONs can be easily incorporated by those cells. On the other hand, when vascular or tumor targeting is required, a PEG-based coating is preferable in order to prolong circulation time in blood and the retention of the SPIONs in the tumor. The applications of MPI are not only limited to imaging. It has a good compatibility with MH because both techniques use alternating magnetic fields (AMFs). SPIONs can also act as drug carriers. Combinations of different techniques using SPIONs provide a variety of applications. When it comes to MPS, it was initially used to characterize the properties of MNPs for improving the performance of MNPs in MPI. An MPS system can be regarded as 0-dimensional MPI scanner. Recently a wide variety of applications of MPS have been explored, not only the characterization of SPIONs but also immunoassays, cellular uptake monitoring, temperature measurement, viscosity monitoring and so on. In this section, recent studies using MPI and MPS for bio-applications will be introduced.

2.1.1 Brief Principle of MPI The principle of MPI is briefly introduced in this section [3, 4, 6, 7]. The first MPI system was reported by Gleich and Weizenecker in 2005 in Germany [6]. In their study, the spatial resolution achieved was already less than 1 mm. The high spatial resolution of MPI is a result of the excitation of SPIONs at a local point or line by utilizing the superposition two magnetic fields. Both a static magnetic field and an AMF are used in the system. Static magnetic fields are symmetrically generated so that superposition of the magnetic fields makes a field free region (FFR) or field free line (FFL) (Fig. 2.1a). This field is called the selection field. The magnetization curve of SPIONs is expressed by the Langevin function and the response of SPIONs to the field is nonlinear. SPIONs which are located outside the FFR or FFL are magnetically saturated due to the time-independent static magnetic field. When an AMF is also applied to the system, only SPIONs in the FFR or FFL can respond to the AMF while SPIONs located in other regions cannot respond since they are already saturated by the static magnetic field. In other words, the magnetizations of SPIONs outside of the FFR or FFL are locked. This AMF is called the drive field. The time-dependent magnetic response is detected by a receiving coil and converted into an electric signal. The response of SPIONs to an AMF at the FFR or FFL produces an oscillatory signal. Since the magnetic response of SPIONs is nonlinear, the signal has distortions, and the Fourier transformation of the signal contains an odd number of higher harmonics (Fig. 1b). In contrast, the signal from SPIONs outside of the of FFR or FFL is weak, which allows the scanner to detect the signal mainly from the FFR or FFL (Fig. 1c). The fundamental harmonic contains

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Fig. 2.1 a Schematic illustration of the FFR and FFL. Black lines depict magnetic lines. b Magnetization curve of SPIONs. Including the response of magnetization of superparamagnetic nanoparticle to an AMF in the FFR or FFL and the Fourier transform of the signal. c The response of magnetization of SPIONs to an AMF outside of the FFR or FFL and the Fourier transform of the signal

information about the frequency of the drive field. Using higher harmonics, the position of the SPIONs can be determined by moving the FFR or FFL across a sample. Movement of the FFR can follow different trajectories such as raster scanning or a Lissajous trajectory. Furthermore, the amplitude of the harmonics is proportional to the number of SPIONs and thus, a quantification analysis is possible. Magnetic fields can penetrate a body without a depth limit, which means a 3-dimensional visualization is also available. To generate rapid magnetization changes in SPIONs, a frequency of around 20 kHz is often used for the AMF. The characteristic properties of the SPIONs such as their size, shape and saturation magnetization influence the MPI signal as well as the geometry of the coils in MPI scanner. Initially, MPS was developed to evaluate the performance of SPIONs as MPI tracers. Recently, lab scaled MPS systems using a similar principle to that used in MPI are used for different applications [1, 2]. When an AMF is applied to MNPs, the relaxation process occurs via Brownian and Néel relaxations. The slight change in those relaxations influences the response of the SPIONs to the AMF and can be detected as a signal. Utilizing this relationship, information of interest such as the concentration of target materials, temperature or viscosity surrounding the SPIONs can be obtained.

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2.1.2 MPI and MPS in Bio-Applications In this section, several recent studies utilizing MPI and MPS will introduced. There are review papers summarizing these studies in detail [2–5]. Readers who want to study further could refer to those well-summarized papers. MPI utilizes an AMF to cause an oscillatory signal which is derived from magnetic moment oscillatory reversal in SPIONs to construct images. It has potential for use in diagnostic imaging. Magnetic hyperthermia (MH) has been a prominent field in tumor treatment using SPIONs by applying an AMF and thus, many researchers have intensively investigated it. However conventional MH has problems in terms of the control of the position of the administered SPIONs in a tumor. If the SPIONs reside not only in a target tumor site but also other healthy tissues, they could cause severe side effects. In this sense, MPI has an advantage; MPI can excite SPIONs and generate heat only in the FFR or FFL. Therefore, MPI is considered compatible with MH where SPIONs are used for image-guided heating by changing the AMF frequencies. Tay et al. examined the capabilities of SPIONs as MPI tracers and MH probes [8]. While conventional MH could not avoid heating in off-target regions such as the excretory organs of the liver and the spleen due to systemically accumulated SPIONs, MPI could minimize collateral damage by setting the FFR at a chosen position. Figure 2.2 depicts the concept of their research. It is important to apply the AMF at different frequencies in MPI and MH in order to get MPI images and perform MH separately. A low frequency of AMF of around 20 kHz was used for the MPI so that the SPIONs did not generate heat during diagnosis. Then, a high frequency of AMF of around 354 kHz was applied to generate heat after locating the tumor area in the FFR. The in vitro results showed that when a field gradient of 2.35 T/m was applied, the heating spot size could be localized within a 7 mm radius and negligible heating was observed outside the spot. They then demonstrated the use of MPI for guidance to carefully control the MH heating spot in a mouse model. The results showed that heat damage could be localized in the target tumor while other off-target regions were spared. A good correlation between the MPI signal and the specific absorption rate (SAR) was obtained, which could be used in thermal dose control. Combining MPI-based temperature monitoring, heating by MH and drug release by stimulating temperature has the potential to be a powerful tumor treatment in the future. Monitoring drug release in vivo is important in terms of reducing side effects and increasing efficacy. However, conventional imaging techniques such as fluorescence imaging and MRI have limitations. For example, fluorescence imaging suffers from a low tissue penetration depth and MRI is not able to perform a quantification of the drug release rate. On the other hand, MPI has the advantages of a high spatial resolution, freedom from depth limitations and the ability to perform a quantification of the drug release [9].

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Fig. 2.2 Theranostic workflow demonstrated experimentally on a U87MG xenograft mouse model with superparamagnetic iron oxide nanoparticles (SPIONs) present in the liver and a tumor. Step 1: Magnetic particle imaging (MPI) scan at 20 kHz, 20 mT enables clear visualization with high contrast of the SPION biodistribution in regions of pathology (tumor) and also in healthy clearance organs (liver). Imaging parameters are such that SPIONs do not heat. Step 2: User selects a region, in this case the tumor, to localize the magnetic hyperthermia. Step 3: MPI gradients are shifted to center the field-free region (FFR) on the target. This magnetically saturates SPIONs away from the FFR to prevent heating. Step 4: Heat scan at 354 kHz, 13 mT is performed while the MPI gradients are on and held in position. Heating is experimentally localized in the FFR (centered at tumor) while minimizing collateral heat damage to the liver. Reprinted with permission from reference [8]. Copyright 2018, American Chemical Society

Zhu et al. prepared SPIONs core@poly (lactide-co-glycolide acid: PLGA) shell nanocomposites (represented by SPNC in Fig. 2.3). Then DOX was loaded into the PLGA shell (represented by SPNCD in Fig. 2.3). This nanocomposite, which contains multiple SPIONs in a core, acts as a drug delivery carrier and an MPI tracer. Under mild acidic conditions, the PLGA shell degrades and the drug is released. This degradation triggers the disassembly of the SPIONs resulting in a change in the MPI signal which allows for the quantification of the drug release. There are two types of relaxation processes in SPIONs: Brownian and Néel relaxations. Before the disassembly of the SPIONs, Brownian relaxation was limited in the nanocomposites. But after the degradation of the PLGA, Brownian relaxation in the SPIONs was enhanced resulting in an increase of the MPI signal. The size of the DOX-loaded nanocomposites was about 123 nm based on dynamic light scattering measurement. In order to evaluate the relationship between drug release and MPI signal, the DOXloaded nanocomposites were dispersed in a mild acidic buffer (pH = 6.2) for 48 h. Then the release of DOX and the MPI signals were quantified at each time point. The drug exhibited a fast release for the first 5 h and a slower release after 5 h. The MPI signal intensity also showed similar trends: a fast increase for the first 5 h and a slower increase after 5 h. When the DOX release and the MPI signals were compared, a linear correlation was obtained. Therefore, the DOX release process can be monitored by detecting the MPI signal change.

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Fig. 2.3 SPNCD for MPI-guided drug release monitoring in tumor-bearing mice. a MPI and Xray computed tomography (CT) merged images (CT is employed to overlay anatomic structures of the animal over the MPI image) of an MDA-MB-231 tumor-bearing nude mouse injected intratumorally with SPNCD. MPI signals are shown in pseudo-color. MPI signal intensity within the tumor gradually increased with time after injection of SPNCD into mice. b Quantification of MPI signal intensity from the tumor site in MDA-MB-231 tumor bearing nude mice at a series of time points from 0 to 48 h post-injection of the SPNCD nanocomposite (N = 3 mice). Error bars are presented as standard error of the mean (SEM). c Relative DOX release percentage in the tumor site of MDA-MB-231 tumor-bearing nude mice over time, calculated based on the MPI signal in b and the calibration curve. Error bars are presented as SEM. d Tumor sections of MDA-MB-231 tumorbearing nude mice injected intratumorally with saline (blank), SPNC (negative control), DOX only (positive control), and SPNCD 48 h post-injection. A TUNEL assay was used to evaluate apoptosis in tumors with the different treatment conditions, and representative images are shown from N = 3 mice per condition. Green signal (Alexa Fluor 488, AF488) highlights the apoptotic regions in the tumor, and red signal represents DOX fluorescence, which overlap. Reprinted with permission from reference [9]. Copyright 2019, American Chemical Society

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Finally, they injected DOX-loaded nanocomposites intratumorally into a murine breast cancer model. The MPI signal was observed to increase gradually after injection (Fig. 2.3a). Rapid (from 0 to 5 h) and slow increases (after 5 h) of the MPI signals were observed (Fig. 2.3b). Based on this result, the DOX release rate could be calculated according to the linear relationship (Fig. 2.3c). The results showed that about 67% of DOX molecules loaded in the nanocomposites were released within 48 h after the injection. When the breast tumor tissue was harvested 48 h after the injection, significant apoptosis was observed in the tumor sections, which was detected using Alexa Fluor 488 (Fig. 2.3d). DOX fluorescence was also observed within the same region. The size of the tumor was found to have shrunk in the mice treated with the nanocomposites while the weight of the mice increased, suggesting that their health was maintained. Their study clearly showed the capability of MPI signals for the monitoring and quantification of drug release in vivo. This strategy will prove beneficial in optimization of drug dosage for the reduction of side effects. MPI and MPS were used for the quantification of lipoprotein uptake in vivo [10]. Brown adipose tissue (BAT) is known to store lipids and produce heat in the body, and it has been investigated because of its key roles in several diseases such as obesity, diabetes, dyslipidemia and cardiovascular disease. The quantification of lipid uptake in BAT is valuable for the measurement of BAT activity and the understanding of lipid transport and storage. Chylomicrons (also known as ultra-lowdensity lipoprotein particles (ULDL)) have a size of between 100 and 1000 nm and contain dietary lipids acquired in the gut. BAT uptakes lipoprotein particles and stores their lipids. BAT activity has been measured by different techniques such as positron emissions tomography-computed tomography (PET-CT) and MRI. However, there remain problems in terms of health risks and the low specificity of the methods. Hildebrand et al. used SPION-loaded chylomicrons for the imaging and quantification of BAT activity in vivo using MPS. Artificial chylomicrons (ACM) with a hydrodynamic size of 387 nm in diameter were prepared. Then SPIONs with a size of 20 nm were loaded into the ACM. In their study, acute cold exposure was used to induce the activation of BAT to uptake the ACM. After the injection of SPION-loaded ACM in the tail vein of C57BL/6-J mice, the mice were exposed to cold temperatures (4 ºC). A series of experiments were done to show if the uptake of SPION-loaded ACM in BAT can be detected. However, when it comes to their application in MPI, due to the low amplitude in the higher harmonics in the spectra, the SPION-loaded ACM was considered inadequate. Therefore, Zn-doped SPIONs (ZnMNPs), which had a higher saturation magnetization than the non-doped SPIONs were fabricated to improve the MPI signal. The ZnMNPs had a size of 10 nm and a saturation magnetization of around 110 emu/g. Then, the ZnMNP-loaded ACM was injected into the mice as before and the mice were analyzed by MPI scanning. As a control experiment, Resovist® which is a commercially available MRI contrast agent using SPIONs was used. MRI and MPI were obtained for both groups (Fig. 2.4a, b). The results showed that after cold exposure, an MPI signal was obtained at the interscapular area when ZnMNP-loaded ACM was injected (Fig. 2.4c, d), while no signal could be detected in the case of Resovist® . Thus, suggesting ZnMNPs suitability as a tracer in MPI. The uptake

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of Fe in different organs was also studied using inductively coupled plasma mass spectrometry (ICP-MS). The measurement of Fe uptake using MPI was found to have similar sensitivity on a ppm scale when compared to the ICP-MS results. Overall, their study showed that ZnMNPs worked as an MPI tracer and probe for tracking lipoprotein metabolism in vivo with high sensitivity. MPS is also used for different applications, for example the analysis of cells and blood, monitoring viscosity and temperature, and the characterization of MNPs [2]. Here, the detection of the influenza (Inf) A virus subtype H1N1 will be introduced [11]. The Inf A virus is one of the notable viruses in the world and early diagnosis is required to prevent severe symptoms and suppress seasonal pandemics. Wu et al. applied MPS as a volumetric-based biosensing platform for the detection of H1N1. SPIONs with a size of 25 nm were coupled with Inf A nucleoprotein specific polyclonal IgG through EDC coupling. EDC coupling makes a covalent bond between carboxyl groups on the SPIONs and amino groups on the antibodies. They prepared different samples which contained SPIONs and varying concentrations of H1N1 from 4.42 μM down to 44 nM. Since H1N1 has multiple epitopes which the different polyclonal antibodies on the SPIONs can target, the hydrodynamic size of the SPIONs after adding H1N1 increased with the increasing concentration of H1N1. Both Brownian and Néel relaxation processes are responsible for the oscillational signal in MPS. In the case of SPIONs with a size of 25 nm, Brownian relaxation becomes dominant. Therefore, the degree of assembly of the SPIONs influences MPI signal. As the hydrodynamic size of the SPION and H1N1 aggregate increases, the amplitude of higher harmonics decreases due to the increased Brownian relaxation time, resulting in an increased phase lag between the excitation AMF and the response from the SPIONs. The third and the fifth harmonics were recorded by a lab based MPS system and the ratio of the third over the fifth harmonics (R35) was calculated. R35 was used as the SPIONs quantity-independent metric for the detection of the biomarkers. In fact, a clear trend was observed in that a smaller R35 value was detected for a higher concentration of H1N1 for all driving field frequencies (from 1 to 20 kHz) (Fig. 2.5). Based on their system, the detection limit was found to be as low as 44 nM. Their results showed that an MPS-based bioassay is feasible and a rapid diagnosis with high sensitivity can be performed. MPI and MPS are relatively new techniques compared to others which make use of MNPs. They use biocompatible and cheap MNPs, mostly SPIONs. The harmonic signals detected in both cases are sensitively influenced by the relaxation processes, magnetization, environment, temperature and so on. A combination of the signal and appropriate physical parameters can allow for customized applications. Particularly, MPI has great potential as a modality in clinical settings. MPI does not suffer from a background signal, which allows it to have a high spatial resolution of less than 1 mm. Because of the similar underlying physics in both MPI and MH, they have good compatibility. So far, drug delivery systems using SPIONs is in the early stages of research, however, setting the heating location using MPI and heating by MH to initiate a drug release using thermoresponsive polymers would have great impact in tumor treatment. The applications reported using MPI and MPS so far are just tip of the iceberg and there remains tremendous potential for future applications.

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Fig. 2.4 MPI-based quantification of ZnMNP-ACM uptake in BAT after cold exposure. a Representative whole-body MRI and MPI images of mice injected with ZnMNP-ACM after exposure to either 23 or 4 °C. b Representative whole-body MRI and MPI images of mice injected with Resovist after exposure to either 23 or 4 °C. c Representative MPI images of the interscapular area (red box) with MRI overlay. d ZnMNP-ACM uptake in BAT after 20 h of cold-exposure measured from whole-animal MPI scans (n = 4 mice per group). Data are presented as means ± SEM relative to uptake at 23 °C in each tissue, ** = p< 0.01, *** = p < 0.001, two-tailed Student’s t test, 23 °C versus 4 °C. Reprinted with permission from reference [10]. Copyright 2021, American Chemical Society

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Fig. 2.5 MPS measurements of the ratio of the third over the fifth harmonics (R35) from samples I–IX. a Harmonic ratios, R35, from samples I–IX as we vary the driving field frequencies from 400 Hz to 20 kHz. Insets (i)–(iv) highlight the R35 measured at 1 kHz, 5 kHz, 10 kHz, and 20 kHz, respectively. b Boxplot of R35 from samples I–IX. Reprinted with permission from reference [11]. Copyright 2020, American Chemical Society

2.2 Magnetic Nanoparticles for Designing Biomedical Sensors In recent decades, analytical chemistry has experienced a dramatic change as a result of various technical advancements in different fields. Specifically, the implementation of nanoparticles (NPs) and other nanotools in biomedical diagnostics has had a significant impact on analytical processes and performance. NPs could provide various tunable electronic, optical and magnetic properties for the creation of new detection techniques. Among those, MNPs have been extensively used in designing biosensors, since their magnetic properties could improve sensor specifications such as sensitivity, linearity and acquisition time. Currently, with the development of nanomaterial science, functionalized MNPs can be prepared in various configurations to exhibit different physicochemical properties. Therefore, the use of MNPs

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in biomedical sensors is not only limited to sample enrichment but also involved the improvement of detection processes. In this context, the aim of this section is to provide a snapshot of the current state of the use of MNPs in the creation of new analytical methods for biomedical applications.

2.2.1 Magneto-Optic Biosensors Biosensors are analytical devices designed to identify specific analytes with high accuracy. The working mechanism of a biosensor is based on the conversion of a biological interaction into physical signals such as thermal, optical, chemical and electrical using a transducer. Among various biosensor platforms, optical biosensors have received the greatest attention in recent years, owing to their distinct features including high sensitivity and a label-free concept. Optical biosensors based on absorbance, photoluminescence, surface-enhanced Raman scattering, and surface plasmon resonance are expected to be the next generation of sensing devices for daily use. The incorporation of MNPs into optical biosensor platforms is paving the way for the creation of new sensing systems that are more sensitive, faster, and more cost-effective. In addition, the creation of new multitasking devices could be achieved by employing the synergistic effects of their optical properties and magnetic properties in a single biosensing platform.

2.2.1.1

Surface Plasmon Resonance (SPR)-Based Biosensor

Surface plasmon resonance (SPR) is a well-known physical phenomenon in which the resonant oscillation of the conduction electrons in a metal surface is achieved by light stimulation under the condition of total internal reflection. Due to their high precision and sensitivity, optical biosensors based on SPR have been acclaimed as the most promising label-free biosensors for biomedical applications. In SPR methods, the refractive index change caused by the binding of target analytes on the biorecognition layer of the surface of the SPR sensors is detected through optical measurements. Despite their many advanced features, the performance of conventional SPR methods is often impeded during the detection of either a low concentration of analytes or low molecular-weight biomolecules since the binding process would only generate a trivial variation in the refractive index. In addition, the slow diffusion rate of biomolecules could require a long working time. To overcome these problems, the incorporation of MNPs—which could be used to capture target analytes from a complex matrix and amplify the SPR response signals due to their high refractive index—in SPR sensing systems is of interest [12]. For instance, Luo et al. has developed an SPR biosensor using nanoparticle-organic clusters (NOC) to detect the scrapie isoform of the prion protein (PrPSC ) that is a biomarker for prion diseases [13]. This protein has a low molecular weight (23 kDa), and only a trace amount is found in circulating blood. In a traditional 2D sandwich

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format (Fig. 2.6a), the SPR sensor consisted of a layer of AuNPs that capture prion proteins through a disulfide bond, followed by a layer of anti-PrPSC conjugated MNPs for the amplification of the SPR response. However, the quantitative capability of this design is limited by the number of active sites on the biosensor surface. Therefore, this work employed the anti-PrPSC conjugated MNPs to magnetically enrich PrPSC from a sample before introducing them to the SPR biosensor, which was later named the magnetic-NOC-enhanced SPR sensor (Fig. 2.6b). As a result, compared to direct SPR measurement, the traditional 2D sandwich format increased the SPR response by 65 times, while the new method amplified SPR signals by 215-fold. In addition, the detection limit was significantly lowered from 0.01 ng/mL using the 2D-sandwich format to 1 × 10−4 ng/mL in the magnetic-NOC-enhanced SPR sensor. Other than that, the linear range of the new method was also widened to a range of 1 × 10−4 to 1 × 105 ng/mL compared to a range of 0.01 − 500 ng/mL when using the 2D-sandwich format. In another study, Jia et al. designed a MNP-enhanced SPR sensor for the quantification of estradiol (E2 ) in food samples [14]. Specifically, a layer of chitosan was spin-coated onto an Au surface to immobilize E2 -BSA using glutaraldehyde. Following this, a mixture of E2 and E2 -mAb-MNPs with a known concentration was introduced to the sensing system. Since this sensor was designed in an indirect competition format, the concentration of E2 was inversely proportional to the SPR response. It’s worth noting that this design could also be an alternative design to overcome the limitations of the traditional 2D-sandwich format mentioned in previous example.

Fig. 2.6 Schematic illustration of a a conventional sandwich SPR sensor and b the layout of magnetic-NOC-enhanced SPR sensor. Insert: the representation of biomolecular interactions at the 2-D interfaces which happen in both SPR formats. Reprinted with permission from the reference [13]. Copyright 2017, American Chemical Society

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The results showed that the incorporation of MNPs into the detection process greatly enhanced the SPR signals. The limit of detection was 0.814 ng/mL with a linear range of 1.95–2000 ng/mL [14]. The two examples above are typical designs of SPR sensors with the assistance of MNPs in the detection process. It is clear that MNPs not only work to enrich the target analytes but also play a role in increasing the diffusion rate of small molecules and amplifying the refractive index changes to induce better SPR signals. Consequently, faster and more sensitive SPR sensors could be designed.

2.2.1.2

Surface Enhanced Raman Scattering (SERS)-Based Biosensors

Despite the fact that SPR sensors have been demonstrated to be a powerful quantitative approach in determining biomolecular interactions, the specificity of this technique that strongly depends on capture layers is of concern, which is limiting its wider application. Basically, discrimination of specific and non-specific binding to the sensor surface is a challenge. In contrast, surface-enhanced Raman scattering (SERS), a surface-sensitive phenomenon which possesses excellent specificity could provide details of the chemical information of the binding molecules based on their vibrational signatures. The SERS effect originates from the interaction of surface plasmons with the vibrational modes of the adsorbed molecules under the stimulation of electromagnetic radiation. Recently, MNPs have been used in SERS sensors to improve the reproducibility and stability of the detection of trace amounts of target analytes. Since SERS techniques are ultrasensitive, they can be easily affected by signals from impurities. A composite of MNPs and noble metals could improve the quality of the SERS signal by enabling the magnetic separation of SERS substrates from the sample matrix to avoid interference. Importantly, MNPs could be designed as a template that allows for the loading of multiple noble metal NPs in a single nanostructure and the magnetic manipulation of the interparticle distance in order to create plasmonic hotspots for the enhancement of SERS signals. In a recent study, Scaramuzza et al. proposed a strategy to prepare magneticplasmonic Fe-Ag nanotruffles using laser ablation in liquid for the creation of SERS substrates [15]. The colloidal stability of the nanostructures was maintained by using thiolated PEG, which also played the role of controlling interparticle distance according to the thickness and chain length of the PEG layer. The results indicated that aside from local hotspots generated by the irregular shape of the composite particles, additional hotspots formed by interparticle interactions at a nanoscale distance could be modulated using an external magnetic field (Fig. 2.7a, b). The shorter the interparticle distance, the stronger the SERS signal was. Additionally, thanks to the presence of the PEG layer, the analyte molecules adsorbed onto the surface of the SERS substrate could be washed with water multiple times in order to detect different cationic molecules sequentially (Fig. 2.7c). The detection limit of this magnetic-plasmonic probe was estimated to be about 60 pM with an acquisition time of 10 s [15]. Furthermore, it was demonstrated that this SERS substrate could enrich the target analyte using magnetic separation up to 10–50 times, which is an

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important feature when dealing with a highly diluted sample. This study is one of many examples illustrating the excellent controllability and enhancement capability of a SERS substrate based on magnetic-plasmonic NPs and shows its great potential in designing biomedical sensors. Currently, different configurations of magnetic-plasmonic NPs have been established for various biomedical applications including the detection of cancer cells, viruses and even for the investigation of cellular metabolism. The early detection of CTCs in blood is of particular interest in clinical diagnostics and SERS-based sensors have offered a simple and sensitive approach to detect CTCs [16].

Fig. 2.7 a Logarithmic graph of SERS enhancement factor (GSERS ) versus interparticle distance. b Illustration of hot-spot formation in a dispersion induced by an external magnetic field. c The regenerative capability of SERS substrates after sequential measurement of the Raman spectra of different analytes. Adapted with permission from reference [15]. Copyright 2019, Royal Society of Chemistry

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In one study, Xue et al. developed a multitask SERS-active magnetic probe using magnetic-plasmonic NPs for the isolation and detection of CTCs [17]. Typically, the positively charged Polyethylenimine (PEI) coated SPIONs were prepared using a solvothermal reaction, followed by the self-assembly of negatively charged AuNPs with a size of 30 nm onto the surface of the SPIONs, which later created hotspots to amplify SERS signals. The obtained particles were then encoded with 4-mercaptobenzoic acid (MBA, a Raman reporter) and labeled with folic acidconjugated reduced bovine serum albumin (FA-rBSA), resulting in the creation of SERS active magnetic probes SPION-PEI@AuNPs-MBA-rBSA-FA. It was shown that the folate-receptor (FR) positive CTCs could be selectively captured from blood samples via the FA-FR interaction with an efficiency of 91%, followed by detection by SERS measurements. The selectivity and specificity of the SERS active probes was characterized using blended cells and a blood sample. The probes showed a high specificity towards HeLa cells in the blended sample of 105 A549 cells and 10 HeLa cells, and in the blood sample containing HeLa cells with a concentration of 1–250 cells per mL. The limit of detection was estimated to be 1 cell per mL. Importantly, after the magnetic isolation of the cancer cells from the blood, the NPs could be detached by culturing them in an FA containing medium. The cells were then grown for the further characterization of molecular phenotypes. Additionally, the SPION-PEI@AuNPs-MBA-rBSA-FA were also demonstrated to be feasible in the detection of cervical cancer cells in blood samples of first-stage clinical patients [17]. Aside from the detection of the biomarkers of cancers, the SERS-based sensors have also received attention for their rapid detection of viral targets to prevent the spread of infections. In a recent work, Wang et al. proposed a strategy to prepare magnetic SERS nanotags Fe3 O4 @Ag for the multiplex detection of respiratory viruses including Inf A H1N1 and human adenoviruses (HAdVs) using a quantitative SERS-based lateral flow immunoassay (SERS-LFIA) [18]. Specifically, the PEI-coated Fe3 O4 NPs were used as the template for the seeding of AuNPs, followed by the growth of an Ag shell to form the SERS substrates. After this, 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) molecules were attached to the surface of the Ag shell via thiol groups in order to provide strong SERS signals. For selective targeting of H1N1 and HAdVs, either H1N1 antibodies or HAdVs antibodies were conjugated onto surface of the magnetic SERS substrates using an EDC coupling reaction. The simultaneous detection of viruses is illustrated in Fig. 2.8a. Owing to the strong magnetic responses of the 150 nm Fe3 O4 cores, the captured targets on the SERS substrates were quickly enriched using magnetic separation before being deployed on the LFIA strip. As a result, compared to the colorimetric LFIA strip using AuNPs, the sensitivity of SERS-LFIA was enhanced up to tenfold with naked-eye detection and 2000-fold with SERS signals (Fig. 2.8b). The two above examples have demonstrated that SERS-active magnetic substrates have excellent sensitivity, specificity, stability and controllability. These unique features have also drawn attention to the possibility of employing SERS magnetic nanoprobes in specific target imaging and separation. For instance, Wang et al. proposed a label-free SERS imaging technique using Fe3 O4 @Ag core-satellite NPs

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Fig. 2.8 a Schematic illustration of the procedure of multiplex detection of H1N1 and HAdVs viruses using a SERS-LIFA strip. b photographs of LFIA strips and SERS signal mapping of test lines using AuNPs and magnetic SERS probes. Reprinted with permission from reference [18]. Copyright 2019, American Chemical Society

in order to visualize H2 O2 released from living cells [19]. Notably, the level of H2 O2 in cells has a profound relation with cellular physiological processes [20], and is often used as a biomarker for the diagnosis of the progression of cancer cells and the evaluation of therapeutic effects [21, 22]. In this study, Fe3 O4 not only played the role of the template for the embedding of Ag NPs to create SERS hotspots, but also acted as the catalyst for the decomposition of H2 O2 released from cells after being stimulated by phorbol myristate acetate

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(Fig. 2.9a). The produced ·OH radicals oxidized 3,3’,5,5’-tetramethylbenzidine (TMB), a peroxidase substrate. Then, the oxidized TMB was detected by SERS signals and used to quantify the H2 O2 concentration. Moreover, in situ SERS imaging could be used to visualize H2 O2 levels (Fig. 2.9b), which could be valuable information for the development of clinical applications [19]. In another study, Kim et al. designed SERS probes composing of bifunctional magnetic-plasmonic NPs modified with cyclo(-Arg-Gly-Asp-D-Tyr-Lys) (cRGDyK) peptides to target cancer cells for SERS imaging and magnetic separation [23]. With the cRGDyK tags, the SERS probes could selectively bind to the surface of the cancer cells, followed by magnetic separation using an external magnetic field. The isolated cells were then visualized using SERS imaging (Fig. 2.9c).

Fig. 2.9 a Schematic illustration of the catalytic property of Fe3 O4 @Ag in H2 O2 decomposition and the oxidation of TMB. b bright field and SERS mapping images of single cells after treatment with phorbol myristate acetate (PMA) for stimulating H2 O2 production in living cells. Adapted with permission from reference [19]. Copyright 2022, Royal Society of Chemistry. c Illustration of SERS probes for targeting cancer cells, followed by magnetic separation. The isolated cells could be visualized using SERS imaging technique. Reprinted with permission from reference [23]. Copyright 2020, John Wiley and Sons

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2.2.2 Magnetic Nanoparticle-Based Microfluidic Sensors Since the introduction of the micro-total-analysis system (μTAS) in the 1990s, the development of miniaturized devices known as microfluidic sensors or the ‘lab-ona-chip’ (LOC) has revolutionized the application of high-throughput screening in biomedical fields, including cell culture and analysis, clinical diagnostics and pointof-care analysis. Compared to conventional analytical techniques, these microsystems have shown significant advantages such as portability, high throughput, high sensitivity, multiplex detection, a low amount of sample required and a shorter acquisition time. Moreover, microfluidic sensors also significantly simplify sample preparation and sensing, as well as reduce the number of steps involving the direct handling of hazardous materials. To present, MNPs have been employed to design the microfluidic components integrated in LOC systems owing to their unique superparamagnetic properties. Generally, the MNPs are used as actuators to manually manipulate the movement of a small sample droplet on an open surface using external magnetic fields, which enables a flexible fluidic operation. Furthermore, aside from their role as a droplet actuator, the surface of the MNPs can be also functionalized with biomolecules for labeling, capturing or concentrating target analytes in a micro channel [24, 25]. To date, two main approaches have been employed to incorporate MNPs into microchips. In the first approach, the functionalized MNPs are trapped or immobilized in a microfluidic chamber by an external magnet during the capture of the target analytes. Following this, a different buffer solution is then introduced to wash and elute the targets in a continuous flow-through mode. In contrast, the second approach utilizes positioned buffers that could be either static or continuous, and the MNPs are manipulated by an inhomogeneous magnetic field to make them move across different solutions for the capturing, washing and eluting steps. All in all, MNP-based microfluidic sensing platforms have been explored extensively as the next generation of sensing technologies in various fields including biomedicines, diagnostics, and environmental monitoring. In this subsection, we will focus on recent advancements in the incorporation of MNPs in LOC systems for some specific biomedical applications such as the detection of viruses, the isolation of CTCs and DNA extraction.

2.2.2.1

Viral Detection

Early detection of infected patients is a crucial step in the prevention of viral outbreaks. Developing a fast and reliable diagnostic method is essential in order to reduce the spread of viruses. At present, the reverse transcription polymerase chain reaction (RT-PCR) is the most popular method for viral detection, which often requires laborious procedures and well-trained personnel. Therefore, establishing alternative approaches with high sensitivity and specificity is still urgently required.

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Recently, MNP-based microfluidic sensors for rapid and accurate diagnosis of contagious viruses like influenza have appeared as a promising technique for preventing community transmission. In a recent study, Lu et al. demonstrated a new design of a magnetic digital microfluidic platform for the detection of the Inf A H1N1 (InfA/H1N1) [26]. The microfluidic chip was fabricated with multiple droplets containing surface energy traps (SETs) acting as the micro reaction chambers. The magnetic beads were employed as probes for the capture of viruses and as actuators driven by an electromagnet to mix, wash and position the droplet across the microfluidic channels (Fig. 2.10). Notably, the droplet containing the magnetic beads could be mixed efficiently using a magnetic flux, which did not require the equipping of an additional mixing component to the microchip. The diagnostic process of this microfluidic platform was designed to be similar to enzyme-linked immunosorbent assay (ELISA). Basically, InfA/H1N1 viruses were captured and purified in a droplet containing the H1N1 specific aptamer-coated magnetic beads with a concentration of 107 beads/μL. Afterwards, aa tyramide signal amplification (TSA) assay was used to generate fluorescent signals for the quantification of viral magnetic complexes. The sensitivity of the magnetic digital microfluidic sensors was determined to be 0.032 HAU per reaction with total analysis time of 40 min, which is a promisingly short time for use in clinical applications [26]. In another study, Shen et al. developed an integrated microfluidic system that could automatically perform multiple processes including sample treatment, RT-PCR and the detection of different Inf virus strains [27]. It’s worth noting that the mortality rate

Fig. 2.10 Magnetic manipulation of the droplet. a magnetic beads were extracted out of the left droplet and transferred to the right one under a magnetic field. This strategy was employed for the extraction and washing steps in the microfluidic system. b photograph of natural diffusion and electromagnetic mixing. Mixing with the assistance of an external magnetic field resulted in higher efficiency compared to natural diffusion after 60 s. Adapted with permission from reference [26]. Copyright 2022, Royal Society of Chemistry

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is highly dependent on the viral strain, therefore the determination of the subtype of influenza viruses has a significant impact on clinical management—such as treatment plans, and control and prevention strategies. In this work, the surfaces of magnetic beads were modified with glycans to magnetically isolate all Inf viruses from the sample, followed by viral thermolysis and a one-step RT-PCR process with different viral primers in the arrayed microchambers of the microfluidic sensor. Notably, the glycan-coated magnetic beads exhibited high viral capture efficiencies in the opentype micromixer under the mixing conditions of; a negative gauge pressure of − 13.3 kPa, an operating frequency of 2 Hz and a mixing time of 10 min. It should be emphasized that the high viral affinity of the probe is an important factor for enhancing the sensitivity of the sensor. The results indicated that the developed microfluidics system was successful in simultaneously detecting different Inf subtypes within 100 min. The limits of detection (LODs) of the microfluidic chip for different viral strains including InfA/H1N1, InfA/H3N2, InfA/H5N1, InfA/H5N2, InfA/H7N9, InfB/Victoria, and InfB/Yamagata were determined to be 4.3 × 10−1 , 8.5 × 10−2 , 50, 100, 200, 3.5, and 7.8 × 10−1 PFU·mL−1 , respectively, which is comparative to the commercially available microfluidic-microarray system [27].

2.2.2.2

CTC Isolation and DNA Extraction for Cancer Diagnostics

Despite breakthroughs in treatment, cancer remains amongst the leading causes of death in the modern world. Primarily, cancer-related death originates from tumor cell metastasis, in which cancer cells escape from the tumor and spread into the blood vessels, then invade distant tissues to form new tumors. These cancer cells are often known as CTCs, which could be a prognostic indicator for cancer diagnostics and progression as well as a prediction of therapeutic effectiveness. For these reasons, the development of new strategies for the isolation and detection of CTCs has received great attention in cancer research. However, the number of CTCs in blood is extremely low, which becomes a major challenge for either the separation or detection of CTCs in patient samples. Microfluidic devices are among the most promising CTC enrichment approaches because of their high efficiency and processing at low fluid pressures, which maximize the possibility of recovering functional CTCs [28]. Recently, magnetic microfluidics has appeared as a feasible, high-throughput sorting technique for CTC isolation from blood samples, owing to the low production cost, multiplexing capabilities and simple operating procedures. Generally, MNPs are used to label either target CTCs or other non-target blood cells, methods which are called positive selection and negative selection, respectively. Particularly, in positive selection techniques, MNPs are modified with the specific antigens for targeting CTCs via their specific surface proteins to then isolate them using a magnetic field. Meanwhile, in negative selection, the number of non-target cells is diminished using magnetic isolation while CTCs remain. Each approach has their own advantages. The positive selection strategy offers a high purity due to the specificity of the capturing process, while the negative selection approach could provide non-invasive isolation of CTCs because only

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non-targeted blood cells are interacted with. As a result, the isolated CTC could be intact and viable, which would benefit later clinical analysis. Furthermore, negativeselection platforms, once established, are often easily adapted to different cancer cells. In a recent study, Huang et al. developed a magnetically driven microchip device with a label-oriented approach to isolate and detect prostate cancer CTCs and protein biomarkers [29]. Specifically, streptavidin-coated 1 μm magnetic beads were functionalized using either biotinylated anti-PSMA (prostate-specific membrane antigen) for the targeting of free PSMA protein or anti-EpCAM for the targeting of prostate cancer CTCs (Fig. 2.11a). After the capturing process, the sample mixture was added into microfluidic devices which consisted of two chambers separated by an array of micro-apertures consisting of 2.6 × 105 holes each with a diameter of 6 μm. The mixture was circulated through the upper chamber at a rate of 2 mL·min−1 for 4 min. At the same time, a permanent magnet was placed beneath the microchip device to pull-down protein-bead complexes and bead-bound cells. Due to the difference in size, the protein-bead complexes and free beads could be drawn to the lower chamber, while the bead-bound cells were retained at the surface of the micro-apertures in upper chamber (Fig. 2.11b). Finally, a fluorescence-based assay was used to quantify the isolated cells and molecular targets. The results indicated that the recovery efficiency for CTCs was 85–90%, while the LOD for PSMA protein was about 2.7 ng·mL−1 [29]. With the aim of developing an ultra-high throughput approach based on a negative selection strategy for CTC enrichment, Mishra et al. proposed a microfluidic device which could remove tagged hematopoietic cells from blood samples at very high cell concentrations [30]. Since CTCs are very rare with one estimate giving a concentration of one CTC per billion blood cells, the screening of a large volume of blood is needed to obtain an adequate number of CTCs for clinical analysis. Because directly drawing large volumes of blood from a patient is prohibited, this work employed leukapheresis for blood sample preparation. Specifically, ~5 L of whole blood was processed by leukapheresis, which produces 65 mL of a product containing about 3–6 billion leukocytes (or white blood cells, WBCs) and 10–30 billion red blood cells. The number of CTCs in the product was estimated to be about 100 to 20,000. It’s worth noting that sorting a large number of cells for CTC isolation is still a major challenge, with the processing of 200 million cells being the highest threshold of current CTC isolation technologies. Thus, the potential of the leukapheresis technique in improving the CTC yield has not yet been fully realized. To overcome this problem, the authors designed a permeability enhanced magnetic sorter that could handle a leukapheresis product sample containing 100-fold more cells than that of the whole blood sample. First, the WBCs were tagged using a mixture of biotinylated antibodies including CD45, CD16, CD3, CD45RA, and CD66b. Afterwards, the red blood cells and platelets were depleted using an inertial separation array chip [31]. The remaining mixture consisting of tagged WBCs and CTCs was incubated with streptavidin-conjugated magnetic beads before being introduced to a magnetic sorter chip (Fig. 2.12a). After injecting the cell mixture into the inlet port, the suspension flowed through two filters with special geometric layouts to eliminate both large

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Fig. 2.11 Schematic illustration of the workflow of the microchip. a magnetic beads conjugated with antibodies capture free PSMA protein and prostate cancer CTCs. b samples are passed through the microfluidic device, protein-bead complexes are pulled through to the lower chamber while bead-bound cells were retained at surface of micro-apertures. The protein-beads were collected using a flow of washing buffer without the presence of the magnet. Reprinted with permission from reference [29]. Copyright 2022, Royal Society of Chemistry

and slender debris. Following this, the cell suspension flowed to the stage-1 sorting channel at 48 mL/h via the inertial focusing-based concentrator with the curved structure (Fig. 2.12a). A buffer flow of 120 mL/h was delivered to the center of the sorting channel to inertially keep the cells near the channel wall. The magneticallylabelled WBCs were deflected toward the center of the stage-1 channel where the magnetic field was highest, before being removed via the waste port. This process was repeated once in stage 2 to make sure that all the WBCs were eliminated. Next, the outlet cells from stage-1 flowed into stage 2 through filter 3 and cell concentrator units. At the end of the concentrators the cells were enriched ~20-fold, while the excess fluid was collected in a waste port. Afterwards, the concentrated cell suspension was introduced to the stage-2 sorting channel via six-feeder channels. Again, the cells labelled with the magnetic beads were deflected by a magnetic field to the stage 2 waste-port, while the untagged CTCs were focused close to the channel wall and collected in the product chamber. Overall, by using a spiked CTC sample, it was determined that this magnetic sorting chip could recover CTCs with a yield

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of 86%, while 99.97% of WBCs were successfully removed. Importantly, the CTC morphology was preserved during the separation process [30]. Recently, microfluidic platforms using magnetic fluidized beds have drawn attention as a potentially revolutionary toolbox for the extraction and purification of nucleic acids like DNA, which would significantly simplify the process of molecular diagnostics. Perez-Toralla et al. proposed a microfluidic device using a fluidized magnetic bed for the efficient extraction of circulating cell-free DNA (cfDNA) from human serum, which could be used for cancer diagnostics [32]. The microchip had a simple geometrical layout as shown in Fig. 2.12b (i). This design was first proposed by Pereiro et al. and was used for bacteria capture, detection and quantification [33]. The magnetic fluidized bed was formed by trapping magnetic beads with a size of

Fig. 2.12 a The schematic illustration of the workflow of the magnetic-sorting chip. b (i) The schematic illustration of a microfluidic device using a fluidized magnetic bed, and the micrograph of the fluidized bed before (ii) and during (iii) sample perfusion. (iv) The trajectory of magnetic particles during sample perfusion. Reprinted with permission from reference [32]. Copyright 2019, Elsevier

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850–900 nm in diameter in a diamond-shaped chamber using an NdFeB permanent magnet. The porosity of the bed could be tuned by adjusting the flow rate of a solution [Fig. 2.12b (ii), (iii)]. For cfDNA extraction, the human serum sample was pretreated with a lysis buffer and was then perfused through the fluidized bed at a rate of 0.4–0.5 μL/min via the inlet port. The cfDNA was captured on the surface of the beads by an electrostatic interaction. Following this, a washing buffer was introduced at a rate of 0.5–1 μL/min to remove any unbound materials. Notably, during the perfusion of sample and washing buffer, the magnetic beads were continuously recirculated due to the imbalance of the magnetic and drag forces [Fig. 2.12b (iv))], which enhanced the efficiency of both the capturing and washing processes. Finally, the cfDNA was collected by flowing an elution buffer at a rate of 0.2–1 μL/min through the magnetic bed. The cfDNA capture efficiencies in serum of this microchip were estimated to be above 63% and 48% for samples containing 100 and 200 ng of cfDNA, respectively [32]. In another study which used a similar concept of magnetic beads in a chamber, Cheng et al. presented an integrated microfluidic system that could perform both cfDNA extraction and quantitative PCR (qPCR) processes for the detection of the mutant BRCA1 gene, a marker for breast cancer [34]. In order to capture cfDNA, a MagMAX cfDNA isolation kit (Thermo Fisher, USA) was used, which consisted of magnetic beads coated with cfDNA probes. In this microfluidic platform, micropumps were used to transport reagents across chambers on the chip. The magnetic capture of cfDNA was performed in a microchamber equipped with a vortex-type micromixer. In addition, a magnet was used to immobilize magnetic beads and allow for the discarding of waste during washing or eluting the cfDNA supernatant. Afterwards, the eluted cfDNA was transported to a PCR chamber for the amplification and detection of BRCA1 genes. The results showed that the cfDNA recovery efficiency of this microfluidic chip was in the range of 60–80%, depending on the available concentration of cfDNA, which is comparative to the benchtop protocol. Furthermore, it was demonstrated that the microchip could be applied to the detection of wild-type and mutant genes in the spiked clinical samples.

2.2.3 Magnetoresistive Sensors and Non-magnetoresistive Sensors Magnetic field sensors are important tools for various biomedical applications due to their non-invasive nature, high sensitivity, and short acquisition time. With the advancement of nano-fabrication technology, the development of magnetic field sensors has advanced rapidly. To date, many types of magnetic sensors attributed to different physical origins have been developed for measuring small changes in magnetic fields, for example: direct current superconducting quantum interference

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devices (dc SQUIDs), Hall sensors, magnetoelastics, fluxgate sensors, magnetoresistive (MR) sensors and giant magnetoimpedance. Out of these, MR sensors, especially, giant MR (GMR) sensors, have been the favored choice for the fabrication of biomedical sensing platforms thanks to their compactness, low energy consumption and their capability to detect very weak magnetic fields at room temperature [35]. Additionally, GMR sensors could be mass-produced at low cost using the same technology established to produce silicon chips, as well as easily integrated into microfluidic systems. In the subsection 2.2.2, it was shown that MNPs could be employed in different roles in the development of biomedical sensors and microfluidic systems, for example: (1) for the capture, isolation and enrichment of target analytes using specific functionalized MNPs, (2) as magnetic actuators for transporting and mixing droplets in microfluidic systems. In addition, MNPs could also participate in developing next-generation diagnostic platforms by coupling with GMR chips to enhance their measurement speed and sensitivity. The general principle of these biosensors is to detect the change in magnetic stray fields that is associated with binding events or interactions of MNP-labelled target analytes with the functionalized surface of the magnetic sensors. Importantly, the detection of these magnetic signals is free from interference from the surrounding biological medium, which is important in order to provide highly reliable data. In this subsection, we will focus on recently developed applications of magneto-nanosensor platforms based on MNPs and GMR chips. The analysis of binding kinetics is one of the most important experiments in molecular biology. Currently, SPR-based biosensors are regarded as the gold standard for binding assays [36]. However, the sensitivity of these optical biosensors is reduced as the molecular weight of the target analytes becomes smaller [37]. As an alternative to optical sensors, magneto-nanosensors have been employed for the measurement of binding kinetics, owing to their better sensitivity. Another concern is that in the static solution, diffusion processes should be considered during the estimation of binding kinetics. As a result, a complex binding-diffusion model must be used to derive the kinetic parameters. To address these problems, in a recent report, Lee et al. introduced a magnetonanosensor integrated into a microfluidic chip for the determination of the dissociation constant of a micromolar affinity interaction in a multiplex manner [38]. In this biosensor platform, MNPs were employed as carriers for proteins as well as the detection probes. ‘Prey’ proteins conjugated to MNPs at a high-density were passed through a microchannel through magneto-nanosensors coated with different sensor-immobilized ‘bait’ proteins (Fig. 2.13), which imitated the natural receptor clustering on cells through the enabling of multivalent receptor interactions with bait proteins. Upon the interaction between prey and bait proteins, the change of magnetic signals could be monitored to establish the real-time binding curves. According to the design of this sensor, the flow rate in the microfluidic chip was sufficiently high, therefore, the kinetic parameters could be estimated using a simple Langmuir isotherm model. Also, thanks to the multiplex capabilities, this sensor was able to screen for multiple binding affinities between the prey protein and its ligands at the same time under the same conditions, which significantly enhanced the accuracy of

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the measurement. Furthermore, compared to traditional SPR sensors, the signal of the magneto-biosensor does not experience interference from the pH or salinity of the solution. Therefore, the investigation of protein–protein interactions in different environments can be conducted. In another study, this biosensor platform was used to discover the novel interaction between PD-L1 and PD-L2, two known ligands of human-programmed cell death proteins, an observation which had never been achieved by SPR sensors. Aside from binding kinetic analysis, the magneto-nanosensor platform has been demonstrated to be a promising technique for point-of-care (POC) diagnosis. The sandwich immunoassay format is the most common design for this purpose. Specifically, the surface of the GMR sensor is functionalized by antibodies, the target analytes are then captured on sensor surface. Subsequently, MNPs conjugated with detection antibodies are bound to the captured analytes to generate a variation in electrical resistance.

Fig. 2.13 Protein A-coated MNPs were conjugated with Fc-tagged proteins. The complexes were separated from the mixture, and serial dilutions of the complexes were flowed into four microfluidic channels where six different bait proteins were immobilized on the sensors. The dimensions of each magneto-nanosensor are 100 × 100 μm, and the microfluidic channel width is 200 μm. The solutions containing the complexes were delivered by syringe pumps individually connected to each channel. Reprinted with permission from reference [38]. Copyright 2022, Springer Nature

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Yao et al. proposed an automated and portable GMR sensor system for the early detection of hepatocellular carcinoma (HCC), a common type of liver cancer [39]. Of note, the current standard for screening HCC in high-risk patients is ultrasonographic examination which is less accessible and requires repeated checking every 6 months [40]. Thus, the development of an automated and POC sensing platform for the early diagnosis of HCC is in high demand. The working mechanism of the magnetonanosensors fabricated in this study is shown in Fig. 2.14. The target analytes were first captured on the sensor surface, followed by a layer of biotin-conjugated detection antibodies. Next, streptavidin-conjugated MNPs were then attached via a biotin-avidin interaction for the detection process. By integrating this system into a microfluidic chip, different relevant biomarkers for HCC diagnosis including alphafetoprotein and C-reactive protein could be screened at the same time. The total test time of this automated system was 28 min with only around 30 s of user operation required. The detection limit and dynamic range of this GMR biosensor could meet the standard lab levels. Importantly, the MNPs could be washed using an automated wash cycle for reuse. With the same design concept, Elaine et al. fabricated a miniaturized magnetonanosensor array platform for POC testing of HIV in saliva and leucocytosis in blood [41]. For each test, only 50 μL of either saliva or blood was added to the GMR biosensor. The working mechanism of the sensor was also the same as the illustration in Fig. 2.14. However, in order to improve the detection signal of viscous samples such as blood, the injection of MNPs was performed twice to enhance the binding activities as well as provide for a local mixing effect. The total analysis time was 16 min with accuracies for the detection of HIV in saliva and leucocytosis in blood of 80% and 90%, respectively.

Fig. 2.14 Illustration of the working mechanism of the GMR biosensor and the setup of GMR biosensing immunoassays. Reprinted from reference [39]. Copyright 2022, Elsevier

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Fig. 2.15 Contact Area for Spherical MNPs Compared to Cubic MNPs. Reprinted with permission from reference [42]. Copyright 2017, American Chemical Society

The examples above have demonstrated the development of magnetic field sensing devices using MNPs as labels in order to enhance analytical signals. Although most current research focuses on the structural design of devices and the integration of sensor chips into microfluidic systems, the role of MNPs is also extremely important in magnetoresistive detection. The ideal probes are MNPs with high magnetic moments and large susceptibilities. The stronger the magnetic properties of the MNPs are, the better the sensitivity of the GMR sensors becomes. Another factor that could affect the analytical sensitivity is the shape of MNPs. Arati et al. indicated that Fe3 O4 nanocubes could result in a higher sensitivity of GMR sensors compared to that of Fe3 O4 nanospheres of the same volume, which originates from the higher contact area and higher saturation magnetization (Fig. 2.15) [42]. Additionally, the MNPs must be biocompatible and monodispersed, as well as properly functionalized to avoid non-specific interaction, which is critical to improve the sensitivity of sensing platform. Furthermore, the MNPs are required to be stable in biological fluid, so that their interaction with the target analytes could be controlled.

2.3 Application of MNPs for Cancer Immunotherapy Immunotherapy, also known as biologic therapy, is a type of cancer treatment, which stimulates the hosts’ immune system to recognize and eliminate malignant growths. Recently, there has been a significant push toward the use of nanoparticles, especially MNPs, for the targeting of the immune cells that participate in the immunotherapy mechanism. Owing to their versatility, MNPs could be used for various purposes in immunotherapeutic applications. One of the most promising approaches is the utilization of MNPs as a nanocarriers, in which multiple components such as antigens, adjuvants, and drugs could be incorporated into or onto the MNP in a controlled manner for different therapeutic purposes. Furthermore, MNP-based cargo could

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be guided using an external magnetic field and tracked by non-invasive imaging techniques including MRI, PET/single-photon emission computerized tomography (SPECT) and MPI. Along with acting as a nanocarrier platforms, there is evidence that MNPs could enhance the immune response by serving as adjuvants, although the mechanism by which this happens has not been clarified. In addition, MNPs have the potential to be used in manipulating the accumulation and retention of different types of lymphocytes in a specific tumor area to maximize the immune response in cell therapy. This section will highlight the recent advances in MNPderived immunotherapies including cancer vaccine design and magnetic-guided cell therapy, in which MNPs could be used for various purposes such as nanocarriers, magnetic tracers, adjuvants and magnetic targeting.

2.3.1 The Roles of MNPs in Nanovaccine Formulation For over two hundred years, vaccines have proven their role in preventing infectious diseases caused by viruses and bacteria and saving millions of lives globally from some of the twentieth century’s deadliest diseases. By exposing people to a weakened or inactivated part of a particular organism, vaccines enable the immune system to recognize the threat (antigens) for their specific marker and trigger the response against them [43]. Now the success of vaccines is once again generating excitement around the possibility of developing a similar inoculation for cancer. The key points for designing an effective cancer vaccine are based on the cancer-immunity cycle developed by Dan Chen and Ira Mellman in 2013 [44]. This process is initiated by the release of neoantigens from tumors. Subsequently, the antigen-presenting dendritic cells (APCs) capture the tumor-associated antigens and process them to produce peptides on their surface with major histocompatibility complex (MHC) class I or II molecules, which later prime and activate T cells in lymph nodes. These effector cells migrate and infiltrate the tumor stroma, where they can induce an immune response to eliminate the cancer cells. Based on this mechanism, various strategies have been explored to elicit a controlled immune response through the regulation of the antigen uptake and the migration of APCs by way of a cancer vaccine. So, how can MNPs be of use in this system? First, MNPs could act as nanocarrier platforms. An MNP-based formulation of a cancer vaccine has considerable potential and could be designed to effectively target the immune cells by an appropriate surface modification. By incorporating them inside the MNPs, the vaccine components can be protected from premature enzymatic and proteolytic degradation during the delivery process. As a result, the potential risk of systemic side effects and augmented immune tolerance caused by the vaccine components could be reduced. For example, in order to enhance the antigen delivery to dendritic cells (DCs), a nanovaccine formulation of heat shock protein 70 (Hsp70) (known to chaperone antigenic peptides) coated SPIONs was developed [45]. After treatment with Hsp70-SPIONs, a dramatic boosting of antitumor immunity was observed though the increase of interferon-γ (IFNγ) secretion

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and the infiltration of CD45RO+, CD8+ T, and Ly-6c+ cells into tumor tissues when compared to cells treated only with free Hsp70. As a result, the survival of tumorbearing animals was significantly prolonged compared to the control group [45]. With a similar concept of nanovaccine formulation, another study showed the expression level of TNF-α, IL-6, and IFN-γ in DC2.4 cells was greatly enhanced in response to ovalbumin (OVA) loaded SPIONs stimulation, which later resulted in a significant tumor reduction in tumor-bearing mice. Meanwhile, the free OVA did not inhibit tumor growth [46]. In addition to the delivery of antigens, an adjuvant can also be included in MNPbased vaccine formulations in order to synergistically activate immune responses. Mareque-Rivas et al. developed a magnetic nanovaccine formulation consisting of phospholipid micelles-encapsulated zinc-doped iron oxide NPs (Zn-SPIONs), polyIC as Toll-like receptor (TLR)-3 and imiquimod (R837) as a TLR-7 agonist, in combination with OVA as the model antigen. A robust innate immune response was induced in the lymph nodes (LNs) as a result of the combination of polyIC and R837 without causing pro-inflammatory cytokines to be released throughout the body. The vaccines demonstrated outstanding effectiveness against OVA-expressing B16-F10 melanoma cells resulting in a significant reduction of tumor size. This was further enhanced by the PD-L1 blockade at the level of the cancer cells [47] (Fig. 2.16). Secondly, MNPs could act as a tool for manipulating retention time and revealing pharmacokinetics. The magnetic properties of MNP-based nanocarriers could be used to magnetically manipulate the retention time of cargo in the region of interest in order to boost the therapeutic efficacy. For instance, Li et al. fabricated a magnetosome which consisted of a magnetic Fe3 O4 core coated with CpG oligodeoxynucleotide as a vaccine adjuvant and a cancer cell membrane as a multiantigen reservoir via electrostatic interaction (Fig. 2.17a). Subsequently, the magnetosomes were decorated with anti-CD205 in order to be recognized by CD8+ dendritic cells and stimulate an anti-tumor immune response though CD8+ T-cell activation. Upon applying an external magnetic field, the retention half-life of magnetosomes in the lymph node was increased sixfold (Fig. 2.17b). As a result, the therapeutic and prophylactic efficacy against different tumor models was improved [49]. Additionally, the magnetic properties also enable in vivo imaging using non-invasive techniques such as MRI, PET/SPECT and MPI [47, 49–51]. Those imaging strategies could be used to elucidate the spatial heterogeneity during therapeutic delivery and track the status of active immune cells. This could reveal the pharmacokinetics, and correlation between therapeutic presence and efficacy in clinical trials, ultimately leading to the design of highly efficient and safe cancer vaccines [49, 52, 53]. Finally, asides from acting as a nanocarrier platform, MNPs like iron oxide nanoparticles (SPIONs) can also work independently as an adjuvant to enhance the therapeutic effects of SPION-based nanomedicine (Fig. 2.18) [46, 54–56]. It should be emphasized that although finding new adjuvants is one of the main focuses in vaccinology, only a few adjuvants have been licensed for human use since 1920 [57]. For a long time, MNPs have mainly been used as a vaccine delivery platform, however, their immunostimulatory capabilities are poorly understood. Exploring the immune system stimulation of MNPs not only means improving the efficiency of

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Fig. 2.16 Schematic illustration of nanovaccine design. Multiantigens and adjuvants could be incorporated in a single nanocarrier for efficient delivery to lymph nodes and interaction with antigen-presenting cells to trigger antitumor immunity through T cell differentiation. Reprinted with permission from reference [48]. Copyright 2017, American Chemical Society

nanovaccines, but also the prevention of potentially severe allergic reactions caused by uncontrolled immune system stimulation [58, 59]. Therefore, its investigation is of particular interest in nanovaccine design. The biological effects of SPIONs may be the result of intrinsic features of their iron oxide core, inducing reactive oxygen species (ROS) and modulating intracellular redox and iron metabolism. According to recent research, the iron content in SPIONs induces a phenotypic shift in M2 macrophages towards the M1 macrophage subtype characterized by upregulated CD86, TNFa, ferritin, and cathepsin L, which may be detrimental in microenvironment of plaque tissue [61]. In addition, it was indicated that this M1 polarization resulted in a Fenton reaction which induced cancer cell apoptosis [56]. The intrinsic immunostimulatory properties of the magnetic cores also can be changed by doping with other elements. For instance, by doping manganese into the magnetite structure to form manganese ferrite (MnFe2 O4 ) NPs, their adjuvant properties were found to induce the response of specific immune cells including Th1 (CD4+ IFN- γ), Th17 (CD4+ IL-17+) and TCD8+ cells [54].

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Fig. 2.17 a Schematic illustration of magnetosome fabrication. b The retention of magnetosomes in the lymph node could be manipulated using an external magnetic field to prolong immune system stimulation after injection. The magnetosome concentration in the lymph node could be monitored though MRI signal intensity. Reprinted with permission from reference [49]. Copyright 2019, American Chemical Society

Alternatively, the effect of metal-based NPs on the immune system is also driven by their physicochemical properties including size [62, 63], shape [64], and surface charge [65–67]. For instance, Liu et al. reported that different-sized SPIONs had different effects on the secretion of interleukin-1β (IL-1β), an inflammatory cytokine, from macrophages. Specifically, SPIONs of 30 nm in size significantly increased the secretion of IL-1β, while the larger size SPIONs (80 and 120 nm) had little impact on IL-1β release [68]. In another study, the effect of SPIONs on the inflammatory responses in human blood was found to be size-dependent. Particularly, human blood samples with different-sized SPIONs were incubated with Toll-like receptor ligands. Consequently, the secretion of TLR2/6, TLR4 and TLR8-induced cytokines was significantly enhanced in the presence of 10 nm SPIONs. In contrast, largersized SPIONs (30 nm) only increased TLR2/6 production, while TLR8-mediated cytokine production was decreased [69]. Along with particle size, the morphology of SPIONs was also known to induce distinct effects in the immune system. Wen et al. performed a systematic study of the morphological effect on SPION-induced

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Fig. 2.18 The physicochemical properties of MNPs including composition, shape, size, surface charge and coating layer strongly influence on the nanoparticle-cell interaction, which results in different adjutancy effects. Reprinted with permission from reference [60]. Copyright 2021, Elsevier

inflammasome activation in macrophages. By using four types of SPIONs including octapods, plates, cubes and spheres with the same aspect ratio and surface charge, it was indicated that SPION-induced IL-1β release and pyroptosis were influenced by the shape of the SPIONs. For example, octapod and plate shaped SPIONs had a more significant effect than cube and sphere SPIONs. Importantly, different SPIONs morphologies were found to have distinct influences on the mechanisms of inflammasome activation in macrophages including ROS production, lysosomal damage, and potassium efflux [64]. In addition, the surface charge of the MNP-carried cargo could also result in different immune system stimulation due to its effects on cellular uptake, and the interaction with cell membranes or cell surface receptors. For example, Saw et al. demonstrated that different surface charges of SPIONs with sizes of 15–25 nm would result in distinct impacts on the modulation of tumor associated macrophages (TAMs). Both negatively and positively charged SPIONs showed considerable cellular uptake and had an effect on driving the polarization of TAMs in vitro and in vivo, as evidenced by the enhanced expression of some characteristic proteins including TNF-α, iNOS, CD11b, and CD80. In this study, negatively

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charged SPIONs exhibited significantly higher cytotoxicity as the concentration of the SPIONs increased. Meanwhile, neutral SPIONs had both low cellular uptake and cytotoxicity, but did not induce macrophage polarization [70].

2.3.2 MNP-Mediated Immune Cell Regulation T cells are a type of lymphocytes which play an important role in the adaptive immune response, and can be categorized based on the T-cell receptors on their surface. To generate immune responses, T cells need to encounter and interact with their specific antigen, then differentiate into one of the various effector T cell subsets [71]. In cancer immunotherapy, these antigen-experienced T cells are programmed to find and eliminate tumor cells. However, the therapeutic efficiency of this approach strongly relies on the infiltration and locomotion of T cells in the tumor region. Generally, chemical signals have a major role in attracting these activated T cells to a solid tumor. However, the physical properties of the tumor stroma such as abnormal vasculature, solid stress and the extracellular matrix exert strong influences on the infiltration and distribution of T cells in the tumor. In order to enhance therapeutic effects, many consider that manipulating the accumulation and retention of the activated T cells in the tumor sites could be a potential solution. In a simple example, activated T cells could be loaded with MNPs and stimulated to migrate into the region of interest using an external magnetic field (Fig. 2.19). Thus, it could be expected that not only the therapeutic efficacy would be improved, but also the side effects to the body caused by T cell exposure could be reduced. To control cell locomotion by magnetic fields, the cells need to be magnetizable, which requires an appropriate modification using MNPs. This in turn would require either obtaining a sufficient uptake of MNPs into the cell while maintaining low cytotoxicity, or the binding of MNPs to the cell surface without altering the function of cell receptors [72]. In a preliminary study, Tietze et al. demonstrated that citrate-coated SPIONs had the potential to be a probe for manipulating T cell migration using external magnetic fields [73]. For the loading step, EL4 cells (T lymphocytes from mouse lymphoma) were incubated with citrate-coated SPIONs for 24 h at a concentration of 75 μg/mL.

Fig. 2.19 Illustration of magnetic field-assisted T cell targeting. SPION-loaded T cells were injected into the tumour supplying artery while applying an external magnetic field to the tumour region. The infiltration efficiency of SPION-loaded T cells was enhanced due to the attraction caused by the magnetic force. Reprinted with permission from reference [72]. Copyright 2019, Elsevier

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Under these specific conditions, the cell viability was above 80%. Then by applying a magnetic flux density of about 240 mT, the in vitro migration of MNP-loaded T cells due to the external magnetic field was demonstrated [73]. In another work, Baber et al. studied the locomotion and retention of T cells loaded with MNPs in the lymph nodes when directed by a magnetic field. Specifically, the MNPs were first coated with different macromolecules including dimercaptosuccinic acid (DMSA), 3-aminopropyl-triethoxysilane (APS) and Dextran (6 kDa). From microscopy analysis, it was confirmed that for all cases, most of MNPs remained on the surface of T cells in close contact with the cell membrane. Based on a quantification of iron content, the amount of APS-MNPs which associated with T cells was measured and found to be higher than for the other two combinations, probably owing to the positive charge of the APS. Importantly, the cells’ viability and the functioning of the T cells surface markers (including CD62L, CD44, CD111a and CCR7) were not affected by the presence of the MNPs or magnetic fields. Although the chemotactic response, which is responsible for guiding T cells migration was slightly decreased, this shortcoming could be later compensated for by magnetic field guidance. In an in vivo experiment, the migration of SPION-labeled T cells guided by an external magnetic field (EMF) was explored by applying a magnetic field near the popliteal lymph node and observing T cell retention. More of the CD4+ and CD8+ cells labelled with SPIONs were found to be retained than T cells without SPION labels (Fig. 2.20) [74]. Natural killer (NK) cells are another type of lymphocyte, which have also received great attention regarding the development of therapeutic methods against tumors and hematological malignancies in the past decade. Unlike cytotoxic T cells, NK cells were found to be able to eliminate tumor cells without prior immunization or activation [75]. In the presence of the cognate MHC I receptor on the surface of healthy cells, the inhibitory receptors on NK cells are triggered to deactivate the NK cells, thus preventing them from killing healthy cells. Meanwhile, cancer and infected cells often lose their MHC I receptor, which makes them vulnerable to attack by NK cells. Upon the detection of tumor cells, NK cells instigate their apoptosis using cytotoxic granules containing perforin and granzymes [76]. Due to their built-in ability to eliminate tumor cells by MHC-independent cytotoxicity, NK cells are considered to be a rising star in cancer immunotherapy. However, a number of issues which hinder their therapeutic efficacy need to be addressed, including their limited infiltration into solid tumors and their low persistence in vivo [75]. However, MNP-labelled NK cells could offer a potential solution to these issues by improving the accumulation and retention of the NK cells in tumor sites through the use of a magnetic field gradient. To improve the dispersion of in vivo administrated NK cells, Baber et al. attached APS-MNPs to the surface of NK cells. This study showed that the functionalities of different NK cell models such as proinflammatory cytokine production or cytolytic activity remained intact after the attachment of MNPs. In addition, the in vitro migration of MNP-loaded NK cells in a capillary system was successfully manipulated magnetically [77]. To enhance the therapeutic efficacy of NK cells in solid tumor treatment, Kim et al. developed a magnetic nanocomplex (called HAPF) composing

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Fig. 2.20 In vivo homing capacity of Jurkat and murine primary T cells after MNP treatment with and without an EMF. a Experimental set-up for determining the homing capacity of MNP-loaded cells compared to MNP-free cells. A mixture of differentially fluorescence-labelled MNP-free and MNP-loaded Jurkat or murine T cells (107 cells; ratio 1:1) was prepared and intravenously injected into nude (Jurkat) or C57BL/6 J (murine T cells) recipient mice. After 1 h, peripheral (PLN) and mesenteric (MLN) lymph nodes and the spleen were collected, processed and analyzed using flow cytometry. b, c The homing capacity of MNP-free and MNP-loaded b Jurkat and c murine T cells in the absence of an EMF, 1 h after cell injection. Data (mean ± SD) are representative of three independent experiments (n = 6). Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. d, e Ratio of MNP-free and MNP-loaded d Jurkat and e murine T cells in the LN when exposed to an EMF compared to a control LN with no EMF, 20 min after the intravenous injection of the cell mixture into the recipient mice, normalized to the input ratio. f Ratio of MNP-free murine T cells, administered alone as control, in the LN when exposed to an EMF compared to a control LN with no EMF after intravenous injection. Ratios of cell homing in magnet LN compared to no magnet LN (mean ± SD) are representative of three independent experiments [n = 4 (Jurkat cells), n = 6 (murine T cells)]. Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.00. Reprinted with permission form reference [74] under the term of the Creative Commons Attribution 4.0 International License. Copyright 2019, Springer Nature

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Fig. 2.21 From left to right. Schematic of HAPF-labelled NK cells that can address current challenges of NK cell therapy in solid tumors. NK cells were efficiently labelled with HAPF nanocomplexes. The HAPF labelling was utilized for NK cell magneto-activation and MRI imaging. The MRI visible and magneto-activated HAPF-labelled NK cells can be effective for MR image-guided local NK cell therapy to treat solid tumors. Reprinted with permission from reference [78]. Copyright 2021, American Chemical Society

of hyaluronic acid (HA), protamine (P) and ferumoxytol (F), that are all FDAapproved agents, to label the NK cells (Fig. 2.21) [78]. The results indicated that the labeling efficiency of NK cells by the HAPF nanocomplexes was considerably higher than that of the sample only using F and maintained a high cell viability. Based on confocal microscopy images, the number of HAPF nanocomplexes associated with NK cell membranes was significantly higher than that in the intracellular region. The NK cells were then magneto-activated using a sine-waved magnetic field generated by dual electromagnets, which promotes the secretion of cytotoxic granules. The accumulation of the HAPF-NK cells in the tumor region could be monitored using the contrast effect on MRI T 2 -weighted images. After being treated with a transcatheter intra-arterial infusion of magneto-activated HAPF-NK cells, the tumor size was measured using MRI, which confirmed that the tumor growth was suppressed in comparison with the control group. The results of this research have shown the great potential of MNPs in the development of cancer therapy using NK cells.

2.4 Gene Delivery and Therapy Gene therapy is a cutting-edge technique in which genetic materials (e.g. DNA or siRNA) are introduced to specific cells or tissues in order to edit or replace existing genetic codes for certain therapeutic effects. This approach has great potential in that it could significantly improve the treatment of many hitherto incurable diseases such as cancer and hereditary abnormalities. Although the history of gene therapy began in 1953 with the discovery of the structure of DNA, it is still mainly used in research or clinical trials due to safety concerns. In recent years, two in vivo gene therapies have been approved by the US FDA, including Luxturna® for the treatment of inherited retinal dystrophy, and Zolgensma® for the treatment of spinal muscular atrophy, which gives us hope for the development of gene therapy as a practical medical technique for the future. However, despite recent achievements, the currently available gene therapies on the market remain costly and risky [79].

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Therefore, developing a safe, efficient, precise, and cost-effective mechanism of therapeutic gene delivery into targeted cells is still a must to ensure the success of gene therapy without invoking severe immune response [80, 81]. Generally, for effective gene therapy, the genetic materials are often delivered to the target cells in either viral or non-viral vectors in order to prevent them from being degraded by the cell nucleuses. In a virus-mediated gene delivery system, the vectors are developed based on the natural ability of viruses to inject their DNA into host cells to replicate their own genetic materials. To reduce the risk of allergic reactions, only the blueprint of a virus was used to encase the genes. Although the delivery efficacy of viral vectors is high; large-scale production, high cost, and long-term safety continue to be significant barriers to its widespread clinical implementation. In comparison, non-viral gene delivery—which uses chemical compounds such as liposomes and polymers; or physical force such as needle injection, electroporation and magnetofection—to deliver genes into target cells is relatively safe, cost-effective, readily available, and scalable. However, the significantly lower effectiveness of nonviral methods compared to viral alternatives remains the primary unresolved issue. Recent advances in nanotechnology have presented the incorporation of MNPs like SPIONs as a potential solution in both viral and nonviral carriers using various assembly approaches including electrostatic absorption, encapsulation and chemical conjugation. Subsequently, an external magnetic field could be used to enhance the internalization of MNP-based gene carriers in vitro and the delivery efficiency to targeted cells in order to minimize side effects in vivo. Specifically, the presence of MNPs could synchronize and improve the virus infection process as well as accelerate the gene transfection kinetics through the use of magnetic gradients, while lowering the transfection doses to avoid potential overexposure of gene delivery vectors. This section reviews recent attempts in the incorporation of MNPs into gene carriers in order to enhance the gene delivery efficiency in both viral and non-viral vectors and their potential biomedical applications.

2.4.1 Gene Delivery Using MNP-Incorporated Viral Vectors To present, viral vectors remain the most frequently used method of gene delivery. However, safety concerns are considered the “Achilles’s heel” of this approach since all classes of viral vectors might be recognized as foreign invaders by the immune system, which may cause serious allergic reactions in a patient. Like druginduced toxicities, the severity of side effects and other detrimental immune-mediated reactions caused by in vivo administration of viral vectors was found to be dosedependent. Today the adeno-associated virus vector (AAV), which has an excellent safety profile, is the most popular choice in studies and clinical trials for viral therapeutic gene delivery. Nevertheless, dose-escalation studies conducted in mouse models showed that the toxicity of AAVs correlated with high administered doses, while no symptoms were observed at low doses [82]. Furthermore, the hypersensitive immune reactions could be even more complicated if the viral vector carriers start to

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disperse in the circulatory system. Because after local administration, the physical force generated by blood flow may hinder the efficient gene transfer of viral particles to targeted cells and instead spread them to other organs. As shown in one study, the liver and neurons in nonhuman primates and piglets suffered serious damage after overexposure to AVVs designed for treating spinal muscular atrophy [83]. Since the transfection efficiency of viral vector is often high due to the infectious nature of viruses, enhancing the targeting capability of viral carriers remains as a key issue in maximizing their therapeutic potential while minimizing administered doses to avoid the adverse effects. This objective could be accomplished by incorporating MNPs into the viral carrier formulation. In this section, we will highlight some strategies proposed to improve the targeting efficiency of in vivo gene delivery processes using MNP-incorporated viral vectors to reduce potential systemic toxicity. For instance, in order to enhance lentiviral transduction under nonpermissive conditions for viral infection, including hydrodynamic stress and low temperature, Hofmann et al. proposed a viral carrier formulation consisting of an MNP-incorporated lentiviral vector (LV/MNP) [84]. In this study, two types of commercial MNPs were used for incorporation into lentiviral vectors. Namely, polyethyleneimine coated MNPs [combiMAG (CM), positive charge] and starch coated MNPs [TranMAG (TM), negative charge] with phosphate terminals. It was found that LVs were efficiently bound to both oppositely charged particles to form the LV/MNP formulation by either electrostatic complexation between LV and CMMNPs (LV/CM MNP) or direct conjugation of LV on TM-MNPs (LV/TM MNP) through the phosphate terminals. It should be noted that the surface charge of the resultant nanocomplex is an important parameter, which may affect to the stability of MNPbased gene carriers in different biological contexts. Therefore, two types of particles were used for comparison. In in vitro experiments, the viral infection of both LV/MNPs to human umbilical vein endothelial cells (HUVECs) was investigated under hydrodynamic stress at room temperature and hypothermic conditions at 4 °C. The results showed that applying an external magnetic field considerably enhanced the transduction efficiency of both types of LV/MNP under the non-permissive conditions. Similar observations were made in different cell types from various species including rat endothelial cells, mouse embryonic fibroblasts and porcine skin fibroblasts. In ex vivo experiments, isolated mouse aortas were transduced with LV/MNP complexes and LV for comparison at 4 °C. Consequently, with the presence of a magnetic field, both LV/MNP types had significantly higher transduction efficiency than that of LV alone. Additionally, a flow-loop in aortas was constructed in order to mimic hydrodynamic stress and was placed into the magnetic field gradient. The fluorescent signal of gag-EGFP fusion proteins in the LV vectors was used to evaluate the retention of LV/MNP complexes under the magnetic field. After perfusion, about 96% of LV/MNP complexes were deflected from the flow and accumulated close to the magnetic field region, which indicated high targeting efficiency. After 6 days, the EGFP expression in the intima layer of aortas was confirmed by fluorescent imaging and the colocalization of CD31, an antibody in the endothelial layer, with EGFP proteins was observed.

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Importantly, it was also demonstrated that the biodistribution of LV/MNPs in different organs could be altered in vivo under the magnetic gradients. Specifically, LV/CM MNP complexes were injected into mice carotid arteries, while a magnet was placed at the right abdominal wall near the liver. After 6 days, a qPCR was used to determine the LV contents in different organs. Without the magnet, LV/CM MNP were mainly distributed in both the lungs and the liver. However, the application of an external magnetic field redistributed the LV/CM MNP towards the liver, while dramatically reducing the number of LV/CM MNP in the lungs. The result of this research clearly indicates the potential of MNP-based gene delivery, in which the targeting capabilities could be enhanced even under non-permissive conditions. The technique described above that involves enhancing transfection efficiency and site-specificity using an external magnetic field is generally known as magnetofection (Fig. 2.22). This concept was first described by Plank and Bergemann in 2002 [85]. Notably, magnetofection could be faster and more effective than other transfection techniques. In addition, magnetofection does not affect cellular uptake and forces gene carriers to cross the cell membrane, therefore, no immunomodulatory effects are induced. Although the in vivo targeting capability of magnetofection is significantly higher than that of traditional viral transfection, the weak magnetic response of a single MNP, its colloidal stability in blood plasma, and its rapid clearance from systemic circulation are the pitfalls of this technique in intravascular applications. Therefore, further refinement is still necessary before magnetofection could be widely applied. To enhance their magnetic moments and circulation, Mannell et al. developed a magnetic carrier composing of MNPs and microbubbles (MBs) for the targeted delivery of genes to specific sites through the systemic vasculature. This formulation of magnetic microbubbles (MMBs) not only enabled magnetic response for gene carriers due to the embedding of multiple MNPs on one MB, but also reduced biological clearance thanks to the intrinsic properties of MBs. In this technique, after MMBs carrying the gene of interest were injected intravenously, ultrasound (US) was applied to break the MB structures and trigger the magnetic accumulation of genetic vectors in the region of interest where the magnetic force was exerted, then initiate gene transfer (Fig. 2.23). Please note that the use of ultrasound and MBs in gene delivery has been demonstrated, in which the frequency and cycles per pulse not only influences the degree of MB rupturing but also induces cell permeability. In the first study published in 2012, PEI-coated MNPs (PEI-Mag MNP) were used as the magnetic component of the gene carrier. It was demonstrated that the plasmid DNA was transported using MMBs through the vascular system and specifically localized at sites where the magnetic field was applied [87]. The efficiency of gene delivery was significantly improved in comparison to the conventional MB approach. Although the feasibility of gene delivery using MBBs was demonstrated, further optimization of the MBB technique was recently performed to obtain more magnetizable gene carriers [88]. Because the magnetic properties are important parameters to attain successful in vivo therapeutic application. Since the naked MBs had a positive charge, silicon-oxide coated MNPs (SO-Mag) with a negative charge were used to coat the membranes of microbubbles (MB) [88]. Compared to PEI-Mag

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Fig. 2.22 Schematic illustration of magnetofection technique. Cells are treated with the MNPbased non-viral vectors. Following this, a magnetic field is applied to direct vector migration towards or enhance vector retention in target cells. Reprinted with permission from reference [86]. Copyright 2005, Elsevier

NPs possessing a positive charge, the average number of SO-Mag NPs embedded in one MB was almost double, while the lentivirus binding capacity of SO-Mag/MBs and PEI-Mag/MBs showed no difference. To demonstrate the targeting capabilities of SO-Mag/MBs carriers, the human vascular endothelial growth factor (VEGF)expressing lentivirus-coated magnetic microbubbles (MMB) were efficiently delivered to mouse aorta endothelium in an ex vivo experiment, under hydrodynamic stress and in the presence of US and magnetic fields (MF). At shear rates of 5 and 7.5 dyn/cm2 , the PEI-Mag/MBs showed a reduced transduction efficiency, while the gene delivery efficiency of SOMag/ MBs was still maintained, thanks to their higher magnetic properties. More importantly, in an in vivo experiment using mice models (C57BL/6) carrying a dorsal skinfold chamber (DSFC), lentiviral particles carrying luciferase coated SO-Mag/MB (lentiviral MMB) were successfully delivered to the designated DSFC site through intravenous administration by applying MF and US treatment simultaneously on opposite sides of the DSFC window. Under these conditions, transgene expression was achieved at the target site, while without MF and US treatment, gene delivery was not accomplished. Of note, by determining the non-specific transgene expression in the lungs and the liver, it was confirmed that the off-target delivery of lentiviral MMB was greatly reduced through MF and US treatment [88].

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Fig. 2.23 The delivery of the lentiviral MMB via intravascular administration. (1) MMB-carried pDNA was injected to into the arteria carotis. (2) MF was applied at the region of interest (DSFC) to accumulate lentiviral MMB. (3) Simultaneously, the lentiviral MMB was disrupted in the vessel upon US treatment, triggering the release of the cargo at the site of the MF for the initiation of gene transfer. Gene expression was observed after 48-72 hours. Reprinted with permission from reference [87]. Copyright 2012, Elsevier

2.4.2 Gene Delivery Using MNP-Incorporated Non-viral Vectors In recent years, non-viral vectors have emerged as a promising alternative gene delivery technique due to their inherently low toxicity and immunogenicity. The non-viral gene delivery technique employs either physical methods to deliver naked DNA, or biocompatible materials such as NPs, liposomes, polymers, and lipids as vehicles to ferry the therapeutic genes to the target cells. However, owing to the poor

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efficiency of transfer, the development of non-viral vectors has been ignored for a long time. Despite recent achievements, the clinically approved gene therapy techniques using viral vectors are still very expensive, up to millions of dollars per patient, and there always exists a small potential to activate an immunogenic reaction or produce a transgenic mis-insertion. This has drawn attention back to the development of nonviral gene delivery, which is not only biosafe but also cost-effective and has much more scalable production. Moreover, the practical advantage of non-viral vectors is the potential for repeat administration to attain progressive therapeutic effects. Unfortunately, it should be emphasized that the non-viral delivery method is simple in theory but challenging in practical implementation. The non-viral vectors currently under development are trying to fulfill the requirements of the ideal gene delivery system. Compared to their viral counterparts, the delivery of genetic materials by non-viral vector systems must overcome a higher number of biological barriers. For extracellular processes, the carriers should retain a proper circulation time after injection in order to accumulate at and penetrate the target tissues, and then achieve a rapid uptake of the active vectors by cells. For the intracellular process, active vectors should be able to resist cytosolic degradation or activate the endosomal escape mechanism if trapped in endosomes to avoid being delivered to lysosomes. These barriers are two of the most basic, but strongly affect the performance of nonviral vectors at the target tissues. Recently, the incorporation of MNPs into non-viral vectors for the employment of magnetofection has been demonstrated as an effective approach to enhance transfection kinetics and efficiency as well as the targeting capability of non-viral vector systems. Here, we will highlight some recent attempts to design different formulations of non-viral MNP-based gene delivery for some novel biomedical applications. Gene delivery holds huge potential for use in cancer treatment. However, due to the lack of an efficient gene delivery system, the number of clinical applications is still very limited. Recently, suicide gene therapy has raised interest in gene delivery cancer destruction due to its bystander effects [89]. However, its low therapeutic effect, low specificity, and the high dose required for administration hinder the translation to clinical application of suicide gene therapy. To overcome those limitations, Wang et al. demonstrated the delivery of the herpes simplex virus thymidine kinase (HSV-TK) gene using a nonviral MNP-based vector for the treatment of liver cancer in a mouse model [90]. Importantly, as a multimodal probe, the magnetic vectors could enable hyperthermia for a synergistic treatment effect and can be used with non-invasive imaging to evaluate the therapeutic performance. In this study, the nanoplatforms were prepared using carboxyl-functionalized magnetic mesoporous silica nanoparticles (M-MSNs) in spherical and rod shapes for a comparison of morphology-dependent antitumor effects. PEG-grafted-poly-Llysine (PEG-g-PLL) was then attached to the surface of the M-MSNs using carbodiimide crosslinker chemistry, followed by the loading of ganciclovir (GCV) and HSVTK, which was abbreviated as M-MSNs-P@GCV@pTK (Fig. 2.24). In order to have a low systemic toxicity in this non-viral magnetic vector the inorganic components chosen were iron oxides and silica, which are biocompatible materials approved by

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the FDA. In an in vivo experiment, mice with tumor sizes of 80–100 mm3 were divided into 7 groups (n = 6) to investigate the influence of EMF on cancer cell targeting and the synergistic effect of suicide gene therapy and hyperthermia. The magnetic gene carriers were injected into the tail vein at a dose of 25 mg/kg for 3 days. For EMF treatment, NdFeB magnets were placed at the tumor site for 2 h immediately after injection. The results indicated that without EMF treatment, both sphere-like and rod-like M-MSNs-P@GCV@pTK were mainly accumulated at the spleen and in the liver. In contrast, applying an EMF significantly increased the number of NPs at the tumor site. It was observed that the rod-like NPs had a higher accumulation efficiency than that of the sphere-like ones. Regarding the therapeutic effects, the groups which received magnetic targeted treatment had significant tumor size reduction compared to the saline control group. The mice group that received both EMF and hyperthermia treatment exhibited a faster decrease in tumor size compared to the mice group with only EMF treatment. Notably, the tumor inhibition rate of the rod-like M-MSNs group was also higher than for those treated with sphere-like M-MSNs. In this study, MRI imaging was employed to continuously monitor the tumor sizes after magnetic-assisted suicide gene therapy. It was observed that the accumulation of M-MSNs at the tumor site enhanced the contrast of the T 2 -weighted MR image of the tumor. Importantly, liver and kidney functions did not deteriorate after the combined treatment. In addition, the systemic cytotoxicity of the non-viral vector

Fig. 2.24 Schematic illustration of the different shaped M-MSNs for MRI-guided, magnetically targeted and hyperthermia-enhanced suicide gene therapy of hepatocellular carcinoma. Reprinted with permission form reference [90]. Copyright 2018, Elsevier

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formulation was also confirmed by hematoxylin and eosin (H&E) staining. The results indicated that no pathological changes in the heart, liver, spleen, lungs, or kidneys were observed in all treated groups. The above example is a typical formulation of a non-viral vector for magnetofection, in which the MNPs are coated with a cationic polymer. Genetic materials composed of nucleic acids are generally considered to be efficient cation binders. Therefore, cationic polymers such as polyethylenimine, chitosan, and poly-L-lysinePEGs are the most common choice. Of note, it should be mentioned that the interface, the region of contact with the cell, is one of the most important considerations in the formulation of non-viral MNP-based vectors, as it not only influences the loading capacity of the therapeutic gene but also affects the cytotoxicity of the carriers. Although the transfection efficiency of cationic polymers is high, their toxicity and non-specific uptake due to their positive charge limits their success. For that reason, it is still imperative to develop other surface modification strategies for MNPs in order to maintain a high loading of genetic materials and transfection efficiency but low cytotoxicity. To reduce the toxicity of cationic polymers, Lo et al. assembled magnetic non-viral vectors by coating chondroitin sulfate-polyethylenimine on the surface of SPIONs (CPIOs) via electrostatic interaction for the delivery of pDNA containing MicroRNA128 (miR-128) [91]. The presence of the chondroitin sulfate moiety not only reduces the cytotoxicity of the PEI but also provides the ability to target CD44, a surface receptor that is expressed in cancer cells. The cytotoxicity of CPIOs/pDNA in respect to different cell-lines including HEK293T, CRL-5802, and U87 cells showed that the viability of all cell-lines was above 90% after treatment with CPIOs/pDNA at concentrations ranging from 3-15wt% for 20 min under a magnetic field. Additionally, the transfection efficiency in vivo was enhanced significantly by employing the magnetofection technique. In an in vitro experiment, CPIOs/pDNA was injected into the tail-veins of mice models with U87-xenografted tumors. The accumulation of the nanocomplex in tumors with the magnet placed nearby was almost double when compared to that of the tumors in the absence of a magnet. Along with the modification of polymeric structure, exploring alternatives to cationic polymers is another approach in designing non-viral magnetic vectors. For instance, Gozuacik et al. recently introduced an approach to prepare non-cationic SPION-based gene carriers for the treatment of breast cancer [92]. In this research, Argonaute 2 protein (AGO2)-conjugated SPIONs are used to deliver microRNA MIR376B to inhibit the autophagy of breast cancer cells in both in vitro and in vivo applications. Since the upregulation of autophagy activity is involved in the survival rate of cancer cells under chemotherapy, the synergistic effect of chemotherapy and autophagy blocking is particularly appealing [93, 94]. To target HER2-positive breast cancer cells, the non-viral vectors were conjugated with fluorophore-labelled antiHER2 antibodies. The cytotoxicity of the as-prepared vectors was evaluated in vivo using different breast cancer cells, including MCF-7, SKBR3 and MDA-MB-453 and in vitro using murine models. The results showed that the non-viral vector NPs demonstrated a high biocompatibility at both cellular and organism levels. In addition, the MIR376B was successfully delivered into the cancer cells and tumors, and

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later had a strong inhibiting effect on autophagy. Importantly, in an in vivo experiment, it was demonstrated that the combination of the targeted delivery of MIR376B and chemotherapy using cisplatin (Cisp) resulted in a synergistic effect of their anticancer activity, which significantly reduced tumor volume after 6 days of treatment (Fig. 2.25). In short, this study indicated that AGO2 protein could be a potential alternative to cationic polymers in the modification of the surface of SPIONs used in non-viral vectors for cancer treatment. Biomimetic NPs are also a promising alternative in the design of safer non-viral vectors. Since the 1970s, calcium phosphate (CaP), a major component of bones, has been utilized to fabricate non-viral vectors, owing to its excellent biocompatibility and biodegradability [95]. Additionally, after cellular uptake, the CaP matrix can be easily dissolved in the acidic environment of endosomes for the quick release of the transported DNA. Despite these outstanding features, the main pitfall of

Fig. 2.25 The therapeutic effect of SP-AH/MIR376B non-viral vectors. a The viability of breast cancer cells SKBR3 and MDA after treatment with PBS (control sample), Cisp, SP-AH+ Cisp and SP-AH/MIR376B+ Cisp (n = 5, * p < 0.05). b The diagram of in vivo experimental setup. Tumors were first grown for 30 days. SP-AH/MIR376B (or PBS) was then injected to suppress autophagy, followed by treatment with Cisp (or PBS) on day 32. c In vivo fluorescence image of tumors in murine models after different treatment conditions. d The relative tumor volumes were recorded after each injection (n = 6, *** p < 0.01, ** p < 0.03). e The variation of tumor volume after different treatment condition on day 36 (n = 6). SP-AH: AGO2 protein-conjugated, anti-HER2 antibodylinked and fluorophore-tagged SPION nanoparticles. Reprinted with permission from reference [92] under the terms of the Creative Commons CC BY license. Copyright 2020, Springer Nature

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CaP carriers is their poor transfection efficiency. To overcome this disadvantage, employing magnetofection is an appealing approach in enhancing the therapeutic effects of gene delivery using CaP. For this purpose, Subhra et al. presented a onepot synthesis of a magneto-gene carrier composing of CaP, iron oxides and DNA (DNA-IO-CaP NPs) [96]. The source of the iron oxides used in this study was ferucarbotran, an FDA approved agent. In the in vitro tests, the DNA-IO-CaP NPs did not exhibit any significant cytotoxicity towards CHO-K1 cells, and using magnetofection considerably increased the transfection efficiency of luciferase, a reporter gene. In a preliminary in vivo study, the targeting capability of the established magneto-gene carriers was investigated using mouse models. The results indicated that the distribution of the delivered gene varied according to the gradient of the magnetic field. The gene expression level was higher closer to the magnet. Although a specific therapeutic application has not yet been shown, this study demonstrated the feasibility of this technique in designing magnetic non-viral vectors with high biocompatibility and transfection efficiency. In recent years, another biomimetic NP that has emerged as a potential gene carrier owing to its superior stealth effect (it’s ability to be retained in cells) is the membranecamouflaged MNP, also known as a magnetosome. Notably, this structure possesses excellent biocompatibility as the MNPs are covered with biomembranes. Recently, Zhang et al. proposed a strategy to prepare biomimetic magnetosomes composing of SPIONs coated with macrophage membranes in order to deliver siRNA for anticancer therapy [97]. Specifically, clusters of SPIONs were assembled in the presence of a PEI surfactant. Following this, siRNA was conjugated onto the magnetic clusters via electrostatic interaction, then further cloaked by fragments of macrophage membranes that were pre-modified with azide. Finally, arginylglycylaspartic acid (Arg-Gly-Asp) peptides known for mediating cell adhesion were attached to the surface of the magnetosomes using click chemistry in order to target the tumor. The in vivo experiment demonstrated that the obtained magnetosomes could efficiently deliver siRNA to the tumor and exhibit considerable tumor growth inhibition with few adverse effects, owing to its various excellent features including long circulation time, magnetofection and MRI capability. A similar gene carrier design was also reported by Mu et al. in which SPIONs were coated with polydopamine for conjugation with siRNA and then camouflaged using the membranes of mesenchymal stem cells [98]. The magnetosomes were injected into tumor bearing mice to investigate their anti-tumor effect (Fig. 2.26). By combining the therapeutic effects of siRNA and photothermal therapy, the volumes of the tumors were significantly reduced after 15 days of treatment in comparison with the control group.

2.5 Organelle Isolation for Proteomic Research The isolation of organelles is a key step to revealing their function through analysis of their contents. Traditionally, cell lysates consisting of a mixture of organelles are subjected to density-gradient centrifugation in order to acquire the isolated fraction of

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Fig. 2.26 Schematic illustration of the preparation of magnetosomes and the in vivo tumor treatment. Reprinted with permission from reference [98]. Copyright 2018, American Chemical Society

interest. However, the purity of the isolated fraction using traditional methods is often limited due to the overlap of organelle density. Recently, with the increasing demand for a method which lowers the contamination in the isolated organelle fraction and obtains a highly reliable downstream analysis, magnetic separation has become a popular choice for the isolation of different intracellular organelles from the complex cell lysate. The primary advantage of magnetic separation is its ability to preserve the integrity of the organelles during the separation process. Furthermore, it is also known to be quick, high-throughput, low-cost, and less energy-intensive. Basically, in order to magnetically isolate the organelles of interest from the cell lysate, they must be labeled by MNPs. To date, two common strategies have been employed for organelle separation using MNPs, which are: affinity interactions to capture organelles using MNPs coated with specific antibodies, or the delivery of MNPs to subcellular compartments through endocytosis for intravascular magnetization before performing the magnetic extraction. For magnetic affinity separations, two distinct approaches—direct and indirect capturing—have been proposed. In the direct method, suitable ligands such as the DNA aptamer, antibody and the biopolymer exhibiting specific affinity towards the target organelles are immobilized on the surface of the MNPs to create the affinity probes. After the addition of these magnetic probes to the cell homogenate, the organelles of interest would be captured by the MNPs. To enhance the capture efficiency, the cells could be also genetically modified to expose tag molecules on the organelles of interest. Meanwhile, in the indirect approach, the antibodies are added to the cell homogenate as free molecules, which then interact with target organelles. Following this, MNPs conjugated with the second antibodies or proteins were added to capture the target organelles. In both approaches, after the capturing step, the mixture is subjected to an external magnetic field for magnetic separation. Finally, a series of washing procedures could be easily performed to remove non-bounded magnetic materials and enhance the purity of the isolated fraction. Based on these concepts, a number of affinity-based strategies have been proposed to isolate various organelles including exosomes, mitochondria and lysosomes.

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For intravascular magnetization, the magnetic labeling process is carried out by employing the natural endocytosis process to deliver magnetic nanoparticles to the lumen of certain organelles such as the early endosomes and the lysosomes. In such a situation, the understanding of intracellular trafficking is crucial in order to obtain a high purity of isolated fraction. The advantage of this approach is its ability to preserve the intact structure of organelles because the nanoparticles remain inside the lumen. In this section, we focus on the recent advances in these magnetic isolation approaches to obtain the high purity isolations of exosomes, mitochondria and lysosomes, three popular organelles for various biomedical applications.

2.5.1 Isolation of Exosomes Extracellular vesicles (EVs) are cellular vesicles including apoptotic bodies, microvesicles and exosomes released by various cell types, which consist of proteins, nucleic acid and lipids. EVs play a vital role in cell to cell communication. Recently, there is growing evidence that EVs are involved in regulating the microenvironment of tumors [99]. Therefore, the concept of exploiting the role of EVs for the diagnosis and treatment of cancer has emerged. In order to investigate the function of EVs and their potential applications, the isolation of EVs from bodily fluids is required. For the immunomagnetic purification of exosomes, Fritsch et al. presented a technique utilizing MNPs (Streptavidine microbeads, Miltenyi Biotec) that were coated with exosome-recognizing antibodies including CD9, CD62 and CD81 in order to capture exosomes from human plasma and cell homogenates [100]. The Western blot (WB) results indicated that the exosomes were successfully isolated from two human plasma samples (Fig. 2.27a). Multiple MNPs were observed to be bound to the surface of the exosomes using TEM analysis (Fig. 2.27b). The yield of the magnetic isolation technique was found to be significantly higher than that of ultracentrifugation when applied to isolate exosomes from the cell homogenate (Fig. 2.27c). The direct coating of antibodies on the surface of MNPs was demonstrated to be effective in capturing exosomes from specific biological fluids. However, concern has arisen that the isolated exosomes are not able to be non-destructively released from MNPs because breaking the antigen–antibody bonds would require a low pH environment or detergents, which would later affect the functional analysis. It’s worth noting that non-destructive release is still one of the technical challenges that remain in traditional antibody-based magnetic separation. Therefore, developing a new magnetic isolation technique with an easy releasing capability is of great interest. To overcome this problem, appropriate engineering of the surface properties of MNPs is required. With the aim of magnetically isolating intact exosomes, Zhang et al. proposed a rapid magnetic isolation of EVs using an aptamer-based magnetic isolation system that could be able to release EVs without any damage [101]. In this study, the

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Fig. 2.27 Analysis of purified exosomes. a WB analysis of exosomes isolated from human plasma of two different healthy donors (#1 and #2). The markers CD63 and CD9 are clearly enriched compared to the input material. b Shows two representative electron micrographs from magnetically isolated exosomes. The surface of the exosomes appears decorated with electron dense magnetic particles. Scale bar: 200 nm. c WB analysis of magnetically isolated exosome cultures’ MDA-MB231 cells. The exosome markers CD63, CD9 and CD81 are enriched compared to the cell lysate and the cell culture supernatant (SNT) without ultracentrifugation (uc) after uc and also compared to the uc sediment. Equal amounts of protein (5 μg) were loaded per lane. Reprinted with the permission from the reference [100]. Copyright 2022, John Wiley and Sons

EVs were marked by the biotin-labelled anti-CD63 aptamer in solution, since transmembrane protein CD63 is in high abundance in EVs. Following this, streptavidincoated magnetic beads were introduced to capture EVs and magnetic separation was performed. After washing 3 times with PBS to further remove non-magnetic materials, the EVs were released from the magnetic beads by adding a complementary oligonucleotide for the CD63 aptamer. Then, to avoid non-specific interaction with magnetic beads, BSA blocking was carried out (Fig. 2.28a). Furthermore, the specificity of the as-synthesized CD63 aptamer was also investigated (Fig. 2.28c, d). The results indicated that the EVs recovery efficiency in this technique was around 78% within 90 min, and after releasing the structure of the EVs remained intact. In another study, Brambilla et al. also presented the capture and release of intact EVs using magnetic beads (Dynabeads, Thermo Fischer) [102]. Unlike the above example, anti-CD63 antibodies were conjugated onto the surface of MNPs using a DNA-directed immobilization strategy. Specifically, starting with streptavidincoated MNPs, biotinylated ssDNA was added to make an ssDNA-functionalized surface on the MNPs. Following this, complementary ssDNA-tagged anti-CD63 antibodies were introduced, which lead to the immobilization of the antibodies on the surface of the MNPs upon interaction between the two complementary ssDNA strands. Notably, the advantage of DNA-directed immobilization is that the DNA linker could enhance the flexibility of the antibodies while reducing steric hinderance to result in a higher capture efficiency. In addition, after the capture of the EVs, the DNA linker could be removed using DNase I in order to release the EVs from the MNPs without damaging the structure of the EVs. Generally, magnetic isolation using the intravascular magnetization approach is rarely applicable to exosomes due to the lack of an available transport pathway for the delivery of nanoparticles to exosomes. Nevertheless, Nemati et al. recently presented a novel magnetic isolation strategy in order to obtain tumor exosomes

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Fig. 2.28 Investigation of the nonspecific adsorption of magnetic beads and the specificity of the CD63 aptamer. a The EV adsorption efficiency of magnetic beads before and after BSA blocking (3 independent experiments, t-test, ** p < 0.01). b Quantification of the EVs captured by CD63 aptamer and representation of a random sequence, in comparison with the PBS group as the control experiment (3 independent experiments, t-test, *** p < 0.001). c Confocal microscopy of EVs captured by CD63 aptamer, random sequence (scale bar: 10 μm). Reprinted with permission from reference [101]. Copyright 2022, American Chemical Society

released by canine osteosarcoma cell lines using Fe/Au magnetic nanowires (MNWs) (Fig. 2.29) [103]. Specifically, MNWs were prepared by electrodeposition, and were then delivered to lysosomes through the endocytic pathway. At the lysosomes, the MNWs fragmented into smaller segments at 48 h after the cellular uptake. The authors also observed that the fragmentation process was probably affected by the coating of the MNWs. Although the mechanism was unclear, it was hypothesized that the fragments of the MNWs were then picked up by tumor exosomes and released into the medium. As a result, the tumor exosomes could be isolated by magnetic separation. The isolated exosomes were analyzed using NanoSight (Malvern Panalytical, USA) and compared with exosomes obtained from ultracentrifugation, which indicated the presence of exosomes in the isolated fraction. But this approach is still under development and further biochemical analysis of the isolated exosomes fraction is required. Despite this, it could be a promising alternative to magnetic affinity separation in order to obtain intact exosomes.

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Fig. 2.29 Illustration of the magnetic isolation of tumor exosomes: (1) MNWs were internalized into cancer cells, (2) MNWs were delivered to lysosomes where they were fragmented. (3) The fragments of MNWs were then packed inside tumor exosomes and released into the surrounding environment. (4) Exosomes were then isolated through use of an external magnetic field. Reprinted with the permission from the reference [103]. Copyright 2022, American Chemical Society

2.5.2 Isolation of Mitochondria Mitochondria are double membrane-bound organelles that have a crucial role in various cellular functions such as energy production, calcium homeostasis and programmed cellular death, as well as being the pathogenesis of many neurodegenerative disorders. To undercover the functionality and the involvement of mitochondria in the pathological process, the isolation of mitochondria with a high yield, preserved integrity and low contamination is mandatory for downstream analysis like proteomics. Currently, the magnetic isolation approach is regarded as one of the most reliable approaches for the assessment of mitochondrial function. This section will highlight some recent progress in mitochondria separation using MNPs. As mentioned in the previous subsection, the release of organelles after magnetic separation is often the biggest technical challenge. Therefore, designing a magnetic probe that could release the captured mitochondria is of significant interest. In a recent study, Liao et al. developed paramagnetic beads composing of nickel(II) nitrilotriacetic acid in order to isolate mitochondria from Saccharomyces cerevisiae (S. cerevisase) [104]. The illustration of the isolation protocol is shown in Fig. 2.30. Firstly, the S. cerevisiae strains were modified to insert 6 histidines (6 × His) into the TOM70 gene. HisPur™ Ni–NTA magnetic beads (Thermo Fisher) were added to the cell homogenate after removing any debris. The mitochondria captured on the magnetic beads were isolated using an external magnetic field, and the mitochondria were then released from beads by adding 500 mM of imidazole. The results showed that the obtained mitochondria fraction only contained a small amount of non-mitochondrial components. This technique was demonstrated to be faster than centrifugation-based techniques, while the integrity of the isolated fraction

2.5 Organelle Isolation for Proteomic Research Fig. 2.30 Scheme of mitochondrial isolation from yeast using magnetic beads. Yeast cells expressing Tom70-6xHis were converted to spheroplasts through incubation with Zymolyase and disrupted using a Dounce homogenizer. The resulting whole-cell lysate is subjected to low-speed centrifugation to remove intact cells, cellular debris and nuclei, followed by high-speed centrifugation of the supernatant to concentrate the mitochondria. The resuspended pellet from the high-speed centrifugation was referred to as the mitochondria-enriched fraction. The mitochondria-enriched fraction is the starting point for further purification of the mitochondria. For affinity purification, the mitochondria-enriched fraction is incubated with HisPur Ni–NTA magnetic beads. Bead-bound mitochondria are separated from debris and other organelles in a magnetic field and finally removed from the beads with 500 mM of imidazole. Reprinted from the reference [104] under CC BY license. Copyright 2018, Public Library of Science

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was similar. Compared to isolation approaches using antibody-coated MNPs, this technique was more cost-effective and it was possible to release intact mitochondria after separation. Notably, the magnetic beads used in this study were available from commercial source. In another piece of research, with a similar purpose of finding an alternative method for traditional immunomagnetic separation, Xiong et al. designed a new affinity isolation method using twin strep tag [105]. Firstly, lentiviral vectors carrying Mito-2Strep were transfected to cells for the fusion of twin strep tag to mitochondrial membranes. After obtaining a cell-line with a stable expression, the cells were collected and homogenized. The commercial streptavidin-conjugated magnetic beads (Dynabeads, Thermo Fisher) were then introduced, and the mixture was incubated for 5 min in order to capture the mitochondria, followed by the magnetic separation process. Importantly, after separation, the mitochondria could be released from beads by adding 20 mM of biotin solution. The isolated fraction had a very high purity, which was confirmed by WB analysis. The effect of the particle size was also investigated, and indicated that magnetic beads with a size of 1 μm had the highest performance in mitochondria isolation. Remarkably, it was shown that within 30 s of incubation, about 60% of mitochondria could be recovered. Aside from affinity-based techniques, the design of mitochondria-targeting NPs in intact cells for magnetic isolation were also investigated. In one report, Banik et al. demonstrated an isolation protocol that utilized MNPs, named mito-magneto NPs, to target mitochondria. They subsequently homogenized the cells and performed mitochondria isolation using an external magnet [106]. The isolation protocol and the preparation of the mito-magneto NPs are shown in Fig. 2.31. Specifically, to prepare the mito-magneto NPs, the oleic acid coating layer on the SPIONs was replaced with 6-carboxyhexyltriphenylphosphonium bromide [TPP-(CH2 )5 -COOH]Br. Note that targeting mitochondria by using a lipophilic cation like triphenylphosphonium (TPP +) is well-known, and is often employed in the delivery of drug molecules to the mitochondria [107]. The mito-magneto NPs did not show any significant toxicity or immunogenicity to the treated cells. To reach the mitochondria, the NPs at a concentration of 20 μg/mL were incubated with the cells for 12 h. The results showed that the isolated mitochondria fraction possessed intact structures with a high purity. Functional assays including: cytochrome c oxidase (COX) activity, ATP synthase activity, citrate synthase (a marker of the Krebs cycle) and ATP production confirmed that the functions of the isolated mitochondria were comparable to that of those obtained from centrifugation-based methods. Finally, it was also demonstrated that this technique was applicable to various cell types.

2.5.3 Isolation of Lysosomes Lysosomes are membrane-bound organelles that have recently been discovered to be central to various cellular metabolism processes [108]. The dysfunction of lysosomes is involved in a number of metabolic disorders known as lysosomal storage disorders

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Fig. 2.31 a Illustration of magnetic isolation of mitochondria using mito-magneto NPs. b The preparation of mito-magneto NPs. Adapted with permission from reference [106]. Copyright 2022, The Royal Society of Chemistry

(LSD). To date, proteosome analysis of lysosomes is considered an important tool to elucidate lysosomal functions and their involvement in LSD pathophysiology. Due to the low abundance of lysosomes in cells, the high-purity isolation of intact lysosomes is crucial in order to monitor the alteration in lysosomal protein composition and detect low-abundance metabolites. Among the established approaches for isolating lysosomes, magnetic separation has received great attention due to its quick and gentle nature. In this section, we highlight some recently established techniques which utilize MNPs for lysosome isolation. Like other cellular organelles, the immunomagnetic separation of lysosomes has been extensively investigated. Different protocols have been proposed to quicky isolate lysosomes while maintaining intact structures. In 2011, a version of the immunomagnetic separation method was developed based on magnetic beads coated with multiple antibodies targeting the A or B domain of the V1 subunit of the VATPase pump residing on the lysosomal membrane [109]. However, this approach had some drawbacks including requiring a long incubation time (up to 90 min) and a large amount of antibodies for coating the surface of magnetic beads to capture lysosomes. The reason for these drawbacks was the lack of lysosomal membrane proteins with cytosolic-facing epitopes, which leads to the low capture efficiency of magnetic beads towards lysosomes. To overcome this, a new strategy for the purification of lysosomes has been proposed to obtain a faster lysosome capture rate using magnetic beads. First introduced by Abu-Remaileh et al., the concept of this technique was the modification of the lysosomal membrane with tag molecules. Specifically, the transmembrane protein 192 (TMEM192) of the lysosomes was fused with three tandem human influenza virus hemagglutinin (HA) epitopes [110]. After this, the antibodies targeting HA conjugated to magnetic beads were used to capture lysosomes from the cell lysate. Remarkably, the intact lysosomes could be isolated within 10 min after cell homogenization. This approach was later named Lyso-IP (IP denotes immunoprecipitation). The biochemical analysis results indicated that the isolated fraction had a high purity. To date, the Lyso-IP method has been successfully employed for a number of analysis methods in order to uncover lysosomal functions such as:

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the dependence of V-ATPase and mTOR on the regulation of amino acids efflux [110], the mechanism of LSD diseases like Niemann-Pick Type C Disease [111], and autophagy-related mechanisms [112]. Although Lyso-IP greatly improved the efficiency and kinetics of lysosome capture compared to that of the traditional approaches, the elution of the lysosomes from the magnetic beads remained a concern. To further refine the Lyso-IP technique, Xiong et al. developed an alternative method using a twin strep tag (WSHPQFEK) to fuse with the C-terminus of monomeric GFP (mGFP)-fused LAMP1, where LAMP1 is an abundant lysosomal membrane [105]. Following this, the streptavidin coated magnetic beads were incubated with the cell lysate for 30 s in order to bind with the lysosomes. The lysosome capturing process was faster and more efficient than that of Lyso-IP because compared to HA or polypeptide protein (FLAG) epitope tags, the binding affinity between the twin strep tag and the streptavidin variants was much stronger due to the smaller dissociation rate constant. Furthermore, one important feature of this approach was that the lysosomes could be eluted from the magnetic beads after magnetic separation using with 20 mM of biotin. Overall, it took less than 10 min to obtain a lysosome fraction with high purity and intactness from the original cell sample. Along with Lyso-IP and its variant methods, another magnetic separation approach based on the intravascular magnetization of lysosomes has been successfully used for lysosomal proteome analysis. The first novel approach proposed in 1998 by Winchester et al. used SPIONs coated with dextran (FeDex) to isolate lysosomes from fibroblasts [113]. Specifically, the lysosomes were loaded with FeDex through the endocytic pathway. After this, the magnetized lysosomes could be isolated using MACS. This pioneering work of Winchester and coworkers inspired the later development of different versions of this technique. More recently, with the further development of nanotechnology, small MNPs with a high colloidal stability and advanced functionality could be prepared to target lysosomes via the endocytic pathways. Note that the colloidal stability of MNPs in a biological medium is an important parameter since their aggregation would have negative impact on cellular uptake. Additionally, after being delivered to the lysosomes, MNPs should remain in a fluid phase in the lysosomal lumens. This is due to the fact that the lysosomal membranes could be damaged if MNPs were to solidify [114]. The surface properties of the MNPs also need to be tailored to efficiently target the lysosomes. To date, various molecules such as dextran, peptides and dimercapto succinic acid (DMSA) have been used to functionalize the MNPs. In a recent study, Tharkeshwar et al. prepared DMSA-coated SPIONs to isolate lysosomes for the investigation of Niemann-Pick disease type C1 (NPC1) deficient cells [115]. First, the SPIONs were added to the cells for a pulse of 15 min. The excess SPIONs were removed before cells were re-incubated in a culture medium for a chasing period, t chase . It was demonstrated that for different values of t chase , the isolated fraction would contain different subcellular components (Fig. 2.32a–e). At t chase = 0 min, the main composition was plasma membrane. By prolonging the chasing time, the SPION-loaded components changed accordingly. At t chase = 4 h, late endosomes/lysosomes were dominant in the isolated fraction. This study also

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investigated the influence of the coating layer on the intracellular trafficking of the SPIONs. It was found that amino lipid-coated SPIONs would result in the localization of the SPIONs on the plasma membrane irrespective of the value of t chase . The main isolated fraction always consisted of plasma membrane as shown in Fig. 2.32f. SDSPAGE with silver staining analyses of the whole cell lysate, the isolated lysosome fraction and the isolated plasma membrane fraction revealed distinct protein profiles (Fig. 2.32g). Furthermore, TEM images of the isolated fraction confirmed the intact structure of isolated fractions as well as verified the specificity of the different coating layers on the SPIONs (Fig. 2.32h). This study is a typical example of a method of magnetic isolation of lysosomes based on intravascular magnetization. Since this technique does not rely on affinity interactions, there is no need to be concerned about the modification of the lysosomal membranes or the elution of the lysosomes from the magnetic beads. Importantly, a recent systematic comparison of different lysosome isolation methods conducted by Winter et al. showed that compared to density-gradient centrifugation and organelle-enriched pellets, magnetic separation using intravascular magnetization and Lyso-IP could result in a much better enrichment factor of the lysosomes [116]. Both techniques are excellently suited to label-free protein quantification. Furthermore, it was pointed out that Lyso-IP could yield more intact lysosomes but has a lower overall efficiency than the intravascular magnetization approach. Importantly, a greater fluctuation in protein abundance was observed in Lyso-IP due to the possible loss of lysosomal and luminal proteins during the purification process. Therefore, it was concluded that the magnetic isolation of lysosomes with intravascular magnetization yielded the maximum number of lysosomes relative to the starting material and retained the lysosomes’ integrity in the most effective manner [116].

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Fig. 2.32 a Quantitative immunoblot analysis (equal protein loading) for the indicated organelle marker proteins in isolated fractions using DMSA-SPIONs with increasing chase periods of a 0 min, b 1 h, c 2 h, d 3 h, e 4 h or in fractions isolated using amino lipid-SPIONs f as a fold increase relative to total cell lysate (mean ± SEM, n = 3). Na+ K+ (Na+ K+ -ATPase) is a PM-localized integral membrane protein, EEA1 marks the early endosomes, Rab7 the late endosomes and Lamp1 the lysosomes. RIB (Ribophorin) and GAPDH represent the endoplasmic reticulum and the cytosol, respectively. TCL—total cell lysate; UB—Unbound/non-magnetic fraction and B—Bound/magnetic fraction. g Silver staining of total cell lysate (TCL), bound/magnetic fraction isolated using SPIONs functionalized with DMSA (LYS) or with amino lipids (PM). The distinct protein profile in the bound fraction as observed by the lane scan underscores the enrichment of specific protein subsets (M—Marker, SeeBlue plus2 rainbow protein marker (Invitrogen)). TEM analysis of the fractions isolated using DMSA- (H) and amino lipid- (I) coated SPIONs. Scale bar = 0.5 μm. Reprinted with permission from reference [115] under the terms of the Creative Commons CC BY license

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As illustrated in Fig. 2.32a–e, the composition of the isolated fraction using intravescular magnetization was dependant on the chasing time. Therefore, understanding the kinetic transport of the MNPs to the lysosomes is the key step in obtaining an isolated fraction with the highest purity. Since this parameter is cell type-dependent, the investigation of intracellular trafficking is a prerequisite in order to adapt this procotol to different cell-types. TEM imaging and flourescent dye-based tecniques are the most common methods for the visualization of intracellular trafficking. However, TEM imaging is too laborious with a complicated sampling process, which is not well suited to trial and error experimentation. On the other hand, the conjugation of a flourescent dye onto the surface of the MNPs may affect the particle-cell interactions due to the change in surface properties, which would result in an alteration of the cellular uptake and intracellular trafficking processes of the MNPs [117, 118]. Furthermore, due to the digestive environment in the lysosomal lumen, the fluorescent signal could be quenched or distorted, which may affect the data interpretation [119]. To further refine magnetic separation methods using intravescular magnetization, a recent study introduced a multifunctional magnetic probe that had an intrinsic, plasmonic imaging capability for the visualization of intracellular trafficking and magnetic properties for lysosome isolation [120]. Typically, the magnetic-plasmonic NPs (Ag@FeCo@Ag core@shell@shell NPs; hereafter referred to as MPNPs) were prepared using a polyol method. Following this, the MPNPs were encapsulated in phospholipd micelles and coated with amino-dextran (aDxt-MPNPs) (Fig. 2.33a). The hydrodynamic size of aDxt-MPNPs was ~ 50 nm, which is small enough to be easily internalized by cells. Importantly, aDxt-MPNPs exhibited low cytotoxicity and an excellent colloidal stability in the culture medium. Before magnetic separation, the aDxt-MPNPs could be observed using confocal laser scanning microscopy, owing to their plasmonic properties, in order to track their locations (Fig. 2.33b). Finally, a lysosome fraction with a high purity could be isolated by magnetic separation. This study also demonstrated that this protocol could be easily adapted for different cell-types.

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Fig. 2.33 a Schematic illustration of the preparation of aDxt-MPNPs. b CLSM images showing colocalization of aDxt-MPNPs with LAMP1 (nucleus: blue, aDxt-MPNPs: green, lysosomes: red) (C NPs = 100 μg/mL). The aDxt-MPNPs were monitored through plasmonic scattering signals using CLSM. At chasing times (t chase ) = 4 and 7 h, the colocalization of aDxt-MPNPs with LAMP-1 is clearly observed. Scale bar is 20 μm. Adapted with the permission from reference [120]. Copyright 2022, American Chemical Society

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Chapter 3

Concluding Remarks and Future Prospects

Abstract The application of magnetic nanoparticles (MNPs) in medical and biological fields has attracted many researchers as mentioned in Chaps. 1 and 2. The traditional applications were summarized in Chap. 1 while relatively new research fields were summarized in Chap. 2. In this chapter, we will briefly summarize the contents of the current book from the point of view of the application of MNPs for imaging, therapy, separation and sensors. Then, we will close the book by looking at perspectives on the future potential applications of MNPs. Keywords Imaging · Therapy · Separation · Sensor

3.1 Summary Among useful magnetic materials in medicine, superparamagnetic iron oxide nanoparticles (SPIONs) are well known for being less toxic to the human body. In general the preparation of SPIONs utilizes the easy to perform coprecipitation method. Their ease of preparation and low toxicity are the great advantages of using SPIONs in biological applications. Traditional examples of their use in biomedical fields were introduced in Chap. 1. SPIONs as T 2 contrast agents for magnetic resonance imaging (MRI) are a United States Food and Drug Administration (FDA)-approved imaging agent. The agent utilizes the superparamagnetic properties of SPIONs to enhance randomization of a proton’s spin resulting in the shortening of the T 2 (transverse) relaxation time. In general, tumors have a higher permeability to SPIONs and will thus retain more SPIONs compared to normal tissue because the blood vessels in the tissue are not matured and are inclined to have large pores. This phenomenon is called the enhanced permeability and retention (EPR) effect. Due to their different uptake efficiency, SPIONs can enhance the T 2 -image contrast. Although most SPIONs are used as T 2 contrast agents, there is some research which uses SPIONs as T 1 contrast agents [1]. For the use of SPIONs as T 1 contrast agents, a smaller size of ~3.6 nm is considered to be optimal [1, 2]. Several examples of SPIONs as T 1 -T 2 dual contrast agents were introduced in subsection 1.1.2. The © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Takahashi et al., Modern Biomedical Applications of Magnetic Nanoparticles, SpringerBriefs in Molecular Science, https://doi.org/10.1007/978-981-19-7104-4_3

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fabrication of well-designed SPIONs responding to the microenvironment of tumor tissues such as pH allows for the control of different association levels of SPIONs, which enables the conversion from T 1 to T 2 (or T 2 to T 1 ) enhancement effect in MRI. Since MRI is a common imaging modality in hospitals, the improvement of SPION-based contrast agents will contribute to the early detection of cancer and disease. Although MRI is a standard imaging modality, it is always influenced by the background signal of water in the living tissues because it detects the spin of protons. Recently, magnetic particle imaging (MPI) has emerged and attracted attention as a potential next-generation imaging modality. MPI uses a different principle to MRI. The principle and some research examples of MPI were summarized in Sect. 2.1. MPI allows for the direct imaging of SPIONs and does not suffer from background signals originating from water molecules, thus providing a high-resolution image. The contrast obtained by MPI is known to be comparable to that obtained by positron emission tomography [3]. Since SPIONs are considered to be biocompatible and are already used as contrast agents in MRI, the feasibility of using MPI in clinical settings is high. Magnetic particle spectroscopy (MPS) is a derivative analysis of MPI. It is also called zero dimensional MPI because it does not have an imaging function but utilizes a similar principle to MPI. Both MPI and MPS can detect the difference in the Brownian and/or Néel relaxation processes of SPIONs or magnetic nanoparticles (MNPs) under an alternating magnetic field (AMF). By detecting the difference in the relaxation, other environmental information such as the presence of antigens, temperature, viscosity, and the cellular uptake of SPIONs can be obtained. Both MPI and MPS are regarded as relatively new techniques that will be put to practical use in the near future. Magnetic hyperthermia (MH) is a typical biomedical application of SPIONs for cancer therapy. It is known that cancers and tumors have a lower resistance to heat compared to normal tissues. MH utilizes SPIONs as a heat source for killing cancers or tumors by applying an AMF. In traditional MH, SPIONs are often conjugated to drugs in order to increase their therapeutic efficiency. Some examples were shown in subsection 1.3.1. Since the region of the AMF cannot be focused in a traditional MH setup, concern has been expressed about possible side effects where SPIONs in normal tissue also generate heat and cause damage. Both MH and MPI use an AMF to cause spin relaxation in MNPs, but since MPI can target a smaller region (1 mm to submillimeter [4] thanks to its confined AMF) it can suppress unwanted off-target heat generation and thus it is considered to be highly compatible with MH. In fact, by changing the excitation frequency of the AMF, the mode can be switched between MPI and MH [5], which helps in the precise targeting of the therapy site. Also, the local excitation of MNPs resulting in a temperature increase could enhance drug release for MNPs covered with a thermo-responsive polymer containing drugs. The combination of MH, MPI and drug delivery is expected to be a powerful therapeutic application in the future.

3.1 Summary

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Besides MH, several therapeutic applications using MNPs have been studied. For example, MNPs can be used as carriers for nanovaccines (as shown in subsection 2.3.1). By using MNPs as carriers, the retention time of vaccine components can be enhanced. Also, MNPs are known to regulate immune cells such as T cells and natural killer (NK) cells (described in subsection 2.3.2). The immunostimulatory activity under the presence of SPIONs, however, has not been completely investigated, and thus, the mechanism of nanovaccines using SPIONs must be clarified. Similarly to nanovaccines, gene delivery uses MNPs as the carriers of genetic materials for the expression or suppression of target proteins in tissues. The advantage of the utilization of MNPs as carriers in gene delivery is that it can enhance the insertion of genetic materials under a magnetic field. Some examples were introduced in Sect. 2.4. Magnetic separation is a powerful technique in order to separate target materials such as proteins, cells or cellular organelles for the purpose of disease diagnosis or fundamental research. Cell separation using SPIONs has a long history. In 1990, Miltenyi et al. applied dextran-coated SPIONs for cell separation, then their equipment (magnetic-activated cell sorting: MACS) became a de-facto standard in the field of cell separation [6]. One common application is the separation of circulating tumor cells (CTCs) which is used for checking the prognosis of cancer patients. Recently, not only the separation of CTCs, but also the analysis of the phenotypes of the separated CTCs has attracted attention. A detailed analysis of the phenotypes is thought to provide information on the metastasis progress. Some examples of the magnetic separation of CTCs for phenotype analysis were shown in subsection 1.2.1. When it comes to magnetic separation of cellular organelles, most techniques are based on immunoprecipitations in which antibody-conjugated MNPs were applied to a cellular lysate containing target organelles, and—using a magnetic column—the target organelles could be obtained. The important thing in the magnetic separation of cellular organelles is keeping the organelles as intact as possible. The analysis of proteins on the intact organelles will provide more accurate information on protein function because the quality of proteins is known to be degraded during long and harsh purification processes [7]. There is some research utilizing magnetic separation for endosomes, exosomes, mitochondria and lysosomes [7, 8]. Current examples of magnetic separation methods were shown in subsection 2.5.1. In order to separate small intracellular vesicles such as endosomes and lysosomes, MNPs have to be incorporated into those organelles. Therefore, a small size of MNPs is considered to be desirable for organelle separation. The incorporation of MNPs in optical sensors in order to detect biomolecules has advantages in enriching the target molecules and amplifying the output signal. Optical sensors utilizing surface plasmon resonance (SPR) are common, however, the detection of trivial changes in the refractive index limits the detectable concentration range. With the combination of MNPs in a SPR based sensor, the SPR response was enhanced resulting in a lowering of the detection limit (see division 2.2.1.1). Similarly, a surface enhanced Raman scattering (SERS) based sensor utilizes MNPs for the enrichment or separation of target analytes (described in division 2.2.1.2). In this case, an MNP also acts as a template for the loading of metal NPs in order to

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create hotspots for SERS signals. A microfluidic sensor or ‘lab-on-a-chip (LOC)’ can be used for the diagnosis of cancer and the early detection of viral infections. Many sophisticated designs of microfluidic sensors have been proposed. In most cases, MNPs are decorated with biomolecules such as antibodies or avidin which have an affinity to the target materials and can be used for enrichment, trapping and manipulation using a magnetic field. The combination of giant magnetoresistance (GMR) sensors and microfluidics can also enhance the speed and sensitivity of the measurement. Several examples were shown in division 2.2.2. So, here we briefly summarized the contents of the book in terms of the applications of MNPs for imaging, therapy, separation and sensors. Each field has a long historical development and details can be obtained elsewhere. The purpose of this book was to provide broad overview of the biomedical applications of MNPs and show recent research. Hence, we focused on the research published mainly from 2019 to 2022.

3.2 Future Prospects The amount of research relating to biomedical applications using MNPs has been increasing since the start of the twenty-first century. The types of applications are segmentalized, however, they all utilize the intrinsic properties of MNPs, such as spin relaxation and magnetophoresis. By designing appropriate systems, MNPs can exhibit multifunctionality. Furthermore, through combination with other functional materials (polymers, biomolecules or inorganic materials) a wide variety of functions can be achieved which goes beyond what is possible by the individual components. That is the reason, why this field has flourished and is still a hot topic. When the biomedical applications of MNPs are considered, they can be separated into two broad groups: applications using MNPs inside the body or outside the body. The incorporation of any materials from outside to inside always has higher barrier and takes a long time to evaluate and recognize the safety of the materials. Since SPIONs are FDA-approved MRI contrast agents, other applications using SPIONs inside the body are more feasible when compared with other types of MNPs. Hence, MPI which uses SPIONs in the body is expected to be a promising modality in the future. On the other hand, applications using MNPs outside of the body such as separation or sensors do not have any restrictions on the material components. Therefore, a wide array of application can be considered by designing multifunctional materials. Researchers have tried to improve the current applications through the combined use of MNP probes and microfluidic systems. In conclusion, biomedical applications using MNPs has been and will continue to be an attractive and exciting field full of possibilities.

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