Interdisciplinary Research of Magnetic Fields and Life Sciences 9782759825004

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Current Natural Sciences

Xin ZHANG and Junfeng WANG, Eds

Interdisciplinary Research of Magnetic Fields and Life Sciences

Xin Zhang and Junfeng Wang High Magnetic Field laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences Hefei, Anhui, 230031, P.R. China

This book was originally published in “Physics - Series of Magnetics and Magnetic Materials” series by Science Press, © Science Press, 2018.

Printed in France

EDP Sciences – ISBN(print): 978-2-7598-2491-5 – ISBN(ebook): 978-2-7598-2500-4 All rights relative to translation, adaptation and reproduction by any means whatsoever are reserved, worldwide. In accordance with the terms of paragraphs 2 and 3 of Article 41 of the French Act dated March 11, 1957, “copies or reproductions reserved strictly for private use and not intended for collective use” and, on the other hand, analyses and short quotations for example or illustrative purposes, are allowed. Otherwise, “any representation or reproduction – whether in full or in part – without the consent of the author or of his successors or assigns, is unlawful” (Article 40, paragraph 1). Any representation or reproduction, by any means whatsoever, will therefore be deemed an infringement of copyright punishable under Articles 425 and following of the French Penal Code. The printed edition is not for sale in mainland China. Customers in mainland China please order the print book from Science Press. ISBN of the China edition: Science Press ISBN: 978-7-03-059910-0 ©

Science Press, EDP Sciences, 2020

Foreword I am honored, to be invited, to write the foreword for the book "Interdisciplinary Research of Magnetic Fields and Life Sciences". The two authors of this book are outstanding young scientists I am familiar with, who have accomplished a lot in their fields. Dr. Xin Zhang has been studying the effects of magnetic field on cells and organisms, while Dr. Junfeng Wang has been using NMR to investigate the structure and function of proteins. This book is valuable because it brings together the works of young scientists currently engaged in related research in the mainland of China. Magnetic field is a ubiquitous physical field. Humans have long been aware that certain birds can use the Earth's magnetic field to navigate. Indeed, such phenomenon inspired people to explore the biological effects of magnetic field and the underlying mechanism. The Earth's magnetic field or geomagnetic field is a relatively weak static magnetic field. In the laboratories, people can generate static magnetic field as strong as one million times higher than the geomagnetic field. In addition, pulsed magnetic fields can be generated twice as higher as the highest static magnetic field. At the other extreme, the magnetic field in the outer space is usually very weak, which is estimated at less than thousandth of the Earth's magnetic field but can be simulated in the laboratories. As a result, the man-made ultra-high or ultra-weak magnetic fields provide excellent experimental conditions for material sciences and life sciences. Since the beginning of the 1990s, magnetic resonance imaging (MRI) has been widely introduced as a stand-alone diagnostic technique in hospitals. As the image quality of MRI is positively correlated with the strength of the magnetic field, MRI instrument equipped with ever stronger magnet has been built, from 1.5 to 3 Tesla and higher. FDA (Food and Drug Administration) of the United States has approved the clinical use high up to 7 Tesla MRI. However, pursuing for higher magnetic field strength never topped. For example, MRI with 9.4 Tesla has recently been employed for pre-clinical research. It seems that the competition of high magnetic field human

Foreword

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MRI will be fierce. Thus, there is an urgent need to understand the safety limit of the maximum magnetic field strength on human health. In comparison to the research in condensed matter physics or material science, life science research using magnetic field is at its infancy. At the same time, I believe that the life science research in magnetic fields should afford many opportunities and challenges. As such, I hope that the publication of this book will attract more young scientists to be engaged into this interdisciplinary field, which motivates me for this foreword. Chaohui Ye Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences Early summer of 2018

Preface This book was initiated as part of a research project on the development strategy of "Basic Scientific Questions in High Magnetic Field" led by Dr. Baogen Shen and Dr. Yuheng Zhang, which discusses about the progresses and future directions in physics, material sciences, chemistry as well as life sciences in high magnetic field in mainland China. This particular book mainly focuses on the progresses of interdisciplinary researches of high magnetic fields and life sciences by researchers from mainland China. The topics covered can be roughly divided into two major categories. One is to study the effects of high magnetic fields on biological samples, such as cell, humans and animals. The other is to utilize techniques that based on high magnetic field to study biological questions, such as using NMR (Nuclear Magnetic Resonance) and MRl (Magnetic Resonance Imaging) in structural biology and medical imaging. It should be noted that the exact definition of High Magnetic Field is currently obscure, which depends on specific research field. For physics and material sciences, magnetic fields of higher than 10 'Thsla are considered to be high. However, in most other cases, magnetic fields of higher than 1 Tesla can be considered as high. For human beings, animals and other biological samples, magnets of a few thousand Gausses are already strong enough to elicit some responses, especially when the magnets are used in a dynamic mode, and/or in combination with magnetic nanoparticles. In this book, the biological effects of static high magnetic fields with large gradient and no gradient are discussed, as well as magnetic field with nanoparticles. Furthermore, cellular studies of radiofrequency magnetic fields, as well as cellular, animal and some preliminary studies on human bodies of low frequency magnetic fields are also included. Overall, the researches about the biological effects of high magnetic fields are very interesting and inspiring, but still at a very initial stage. More studies are needed to promote the scientific development of this field, and their potential applications in medicine. Sharply different from the above section, the NMR and MRl related works are much more advanced. The successful applications of NMR and MRl have enabled

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Preface

numerous pioneering scientific findings in a wide range of life science subjects. In this book, recent progresses on protein-protein and protein-drug interactions, NMR spectroscopy in cell-like environment, NMR applications in measuring protein noncovalent interactions, and nanodiscs in examining protein-membrane interaction are presented. Topics on high-resolution NMR under inhomogeneous magnetic fields, 19

F NMR/MRI, ultrasensitive Xenon MRI and molecular imaging, are also included. We would especially thank Dr. Baogen Shen and Dr. Yuheng Zhang for their

support in organizing and publishing this book. We also want to thank Dr. Chaohui Ye and Dr. Yunyu Shi for their helpful suggestions. Moreover, we thank all authors and editor Jun Qian for their efforts. Due to time conflicts of the researchers and space limitations, we want to apologize for not being able to include the works of many outstanding researchers and their exceptional discoveries in the field of NMR and MRI, as well as the clinical applications of magnetic fields in Transcranial Magnetic Stimulation (TMS) and the recently emerged Magnetic Surgery. However, we hope this book will inspire more people to get involved in using high magnetic field in their life science researches, and we are looking forward to more groundbreaking findings in the near future! Dr. Xin Zhang & Dr. Junfeng Wang xinzhang©lhmfi.ac.cn; junfeng©lhmfl.ac.cn Life Science Division High Magnetic Field Laboratory Hefei Institutes of Physical Science Chinese Academy of Sciences, Hefei, China

Contents Foreword Preface

Section 1 Chapter 1

Magnetic Fields and Their Biological Effects

Medical Theranostics Based on Iron Oxide N anoparticles under Magnetic Fields · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 3

1.1 Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 3 1.2 Properties ofiONPs under magnetic fields · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 4 1.3 Pharmacokinetic and biocompability · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 5 1.4 Contrast agents of MR1· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 7 1.5 Hyperthermal agents· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 8 1.6 Drug delivery systems · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 12 1.7 Multimodality theranostics systems of magnetic microbubbles, microcapsules and liposomes · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 13 1.8 Other therapeutic potentials · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 17 1.9 Summary and future prospective· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 20 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 21 Chapter 2 Progress of Static Magnetic Fields and Cancer Research 2.1

in China · · · · · ·· · · · · · · · · ··· ···· · · · · ··· ···· · · · · ···· ··· · · · · ·· · · ·· ·31 Initial clinical studies showed that SMFs could inhibit cancer growth in

some patients· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 34 2.2 Cellular studies show that SMF inhibits multiple cancer cell growth but has minimum effects on non-cancer cells · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 36 2.3 Mechanisms of cancer inhibition by SMFs studied at molecular, cellular and animal levels · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 37 2.4 SMF in combination with chemodrugs · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 48 2.5 Parameters that directly affect the outcomes of SMF exposure········· 50 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 54

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Contents

Chapter 3

Study of SMF's Effects on Cells, Microorganisms, Animals and Plants in NPU · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 61

3.1

The effects of SMFs on cells · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 62

3.2 The effects of SMFs on microorganism· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 72 3.3 The effects of SMF on small animals··································· 74 3.4 The effects of SMFs on plants· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 78 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 80 Chapter 4 In Vitro Cellular Response to ELF-MF and RF-EMF· · · · 83 4.1

Gap junctional intercellular communication · · · · · · · · · · · · · · · · · · · · · · · · · · · · 85

4.2 Epidermal growth factor receptor (EGFR) clustering· · · · · · · · · · · · · · · · · · · 88 4.3 Stress kinases signaling in response to ELF-MF · · · · · · · · · · · · · · · · · · · · · · · · 91 4.4 Effect of ELF-MF and RF-EMF on gene expression···················· 92 4.5 Effect of ELF-MF and RF-EMF on protein expression················· 95 4.6 DNA damage response to EMF exposure······························ ·97 References· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 100 Chapter 5 Utilization of Magnetic Field for Protein 5.1

Crystallization· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·108 Introduction························································· ·109

5.2 Effects of magnetic field on protein crystallization····· · · · · · · · · · · · · · · · ·117 5.3 Quality of protein crystals obtained in the magnetic field······ · · · · · · · ·125 5.4 Toward applications in practical protein crystallization· · · · · · · · · · · · · · · ·133 5.5 Concluding remarks and future perspectives · · · · · · · · · · · · · · · · · · · · · · · · · · 135 References· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 136 Chapter 6 High Static Magnetic Fields (SMFs) on Reproduction and Development of an Intact Living Organism:

Caenorhabditiselegans · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 144 6.1

Caenorhabditis elegans (C. elegans) and its response to magnetic

field································································· ·146 6.2 Source of high SMF and exposure procedures························· 148 6.3 SMFs on the lifespan and development process of C. elegans · · · · · · · · · · 149 6.4 SMF on brood size and germline apoptosis in C. elegans · · · · · · · · · · · · · 151 6.5 SMF on mitochondrial damage and oxidative stress in C. elegans· · · · ·156 References· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 159

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Chapter 7 7.1

Low Frequency Magnetic Field Regulates Immunity and

Inhibits Cancer· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·164 Low frequency magnetic field (LF-MF) exposure system·············· 164

7.2 Screening for suitable tumor parameters of LF-MF · · · · · · · · · · · · · · · · · · · ·166 7.3

Tumor inhibition effect of LF-MF· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·167

7.4 LF-MF modulated the function of immune system··················· ·181 7.5

LF-MF inhibited adipogenesis of human mesenchymal stem cells····· ·192

7.6

Research progress of MF in China···································· 197

7. 7 Summary· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 202 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 202 Chapter 8 Moderate Intensity Rotating Low Frequency Magnetic

Fields and Their Effects on Human Bodies··············· ·210 8.1

MIR-LF-MF with different parameters······························· ·211

8.2

MIR-LF-MF effects on healthy volunteers···························· ·214

8.3

MIR-LF-MF and high-altitude polycythemia (HAPC) · · · · · · · · · · · · · · · · · 215

8.4 MIR-LF-MF effects on patients with chronic diseases················· 216 8.5

MIR-LF-MF effects on cancer patients· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 217

8.6 General effects of MIR-LF-MR on human bodies and working hypothesis· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 220 8. 7 Conclusions and perspectives · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 222 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 222 Chapter 9 Advances in Static Magnetic Field Safety Studies · · · · · · · · 224 9.1

Laboratory studies with cells········································· 225

9.2

Laboratory studies with animals · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 227

9.3 SMF studies with humans · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 229 9.4 Epidemiological studies··············································· 231 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 232

Section 2 Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) Chapter 10 10.1

Structural Basis of Biomolecular Interactions Studied by

NMR Spectroscopy · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 243 Experimental considerations of NMR-based structural studies···· · · · · 244

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10.2 Proteins related to regulation of gene expression····················· 245 10.3 Proteins in cell junction············································· 255 10.4 NMR methodology development for macromolecular structure and dynamics···························································· 257 10.5 NMR application in fragment-based lead discovery·················· ·259 10.6 Summary and outlook· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 261 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 261 Chapter 11 Protein NMR Spectroscopy in Cell-like Enviromnent · · · 268 11.1

The effect of macromolecular crowding on protein structure and

function· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 269 11.2 The effect of confinement on protein structure and function·········· 274 11.3 Protein enrichment in living cells···································· 278 11.4 Isotopic labeling for protein NMR in cells · · · · · · · · · · · · · · · · · · · · · · · · · · · · 279 11.5 Protein structure and conformation in living cells···················· 280 11.6 Protein dynamics and interactions in living cells····················· 287 11.7 Perspective· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 296 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 296 Chapter 12 High-resolution Nuclear Magnetic Resonance Techniques

under Inhomogeneous Magnetic Fields · · · · · · · · · · · · · · · · · · · 303 12.1 Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 304 12.2 IMQC-based high-resolution NMR· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·305 12.3 SQC-based high-resolution NMR · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 332 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 346 Chapter 13 19 F Nuclear Magnetic Resonance/Magnetic Resonance

Imaging in China · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 355 13.1 Introduction · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 356 13.2 Single-modality probes · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 356 13.3 Dual-modality probes··············································· ·363 13.4 Multi-modality probes· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 366 13.5 Others · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 369 13.6 Conclusions and future prospects · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 371 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 371

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Chapter 14 14.1

Ultrasensitive Xenon MRI and Molecular Imaging· · · · · · 375

IDtrasensitive pulmonary gas magnetic resonance imaging··········· 376

14.2 Ultrasensitive HP 129Xe NMR probes · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 381 14.3 Microenvironment responsive probes· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 391 14.4 Ultralow field magnetic resonance spectroscopy······················ 397 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 399 Chapter 15 NMR Applications in Measuring Protein Noncovalent 15.1

Interactions · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 402 h-bond interaction··················································· 403

15.2 C-H/n interaction··················································· 407 15.3 Electrostatic interaction· ........ · · · .... · · ........ · ................ · · 408 15.4 Electric field························································ ·411 References· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 415 Chapter 16 Application of Nanodiscs in Examining Protein-Membrane Interaction· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 418 16.1 Introduction to nanodiscs .. · · · ...... · · · · · · .. · · · · · · · · · ...... · · · · .. · .. ·418 16.2 Self-assembly of nanodiscs .... · .......... · · .... · .... · · .............. · 421 16.3 Properities of nanodiscs · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 423 16.4 Application of nanodiscs for studies of membrane proteins · · · · · · · · · · · 424 References · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 435 Index········································································ 442

Chapter 1 Medical Theranostics Based on Iron Oxide Nanoparticles under Magnetic Fields Qiwei Wang and Ning Gu• State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Sciences and Medical Engineering, Southeast University, Nanjing 210096, China. • Correspondence should be sent to: [email protected] The combination of imaging diagnosis and therapeutics allows large degrees for controlling the medical treatment efficiency, which is now generally referred to as "theranostics". Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as a unique platform for theranostic applications because of their responses to external magnetic fields and excellent biocompatibility. The tissue penetration ability of magnetic fields allows the enhancing of magnetic resonance imaging (MRI) sensitivity and hyperthermia efficiency, which makes SPIOs as promising candidates for future theranostics under magnetic fields. Here, we review recent advances of our laboratory in SPIOs-based theranostic platforms such as hyperthermia, drug delivery and other potential medical applications. What's more, we proposed future scientific issues to be solved in advancing the pre-clinical translations.

1.1

Introduction

In past decades, an emerging trend in the direction of biomedical nanotechnology is "theranostics", which including diagnosis and therapy of diseases through applications of nanoparticles

[l- 31.

Among various types of nanoparticles, greater attention

to iron oxide nanoparticles (IONPs) can be attributed to their unique magnetic

4

Chapter 1

Medical Theranostics Based on Iron Oxide N anoparticles under· · ·

properties [4•51. Indeed, IONPs present disparate characters under different types of magnetic fields, which has been exploited to render IONPs a diagnostic tool (in MRl), a treating agent (in tumor hyperthermia) or a targetable drug delivery system [6-81.

(DDS)

Currently, most work in this field has already been done in improving

the synthesis methods, biocompatibility, surface modification and quality control of these particles. Our investigations main focused in developing the theranostics platform of magnetic assemblies and multimodality imaging based on clinical-approved IONPs. In this chapter, we briefly review the recent progress in the development of IONPs for theranostics under different magnetic fields. Firstly, we introduce the key properties of IONPs under magnetic fields, which make them attractive for theranostics utilizations. We also highlighting recent applications with respect to disease diagnosis and therapy. Lastly, we conclude this chapter by discussing the future considerations and current challenges for pre-clinical or clinical translation of IONPs.

1.2

Properties of IONPs under magnetic fields

As the size ofiONPs is reduced to a few nanometers (< 30 nm), they are forced to possess a single domain and become superparamagnetic, which occurs when the particles size are small enough to cause randomization of magnetic moments [6 1. In the absence of an applied magnetic field, the orientation of randomized mag-

netic moments results in an average magnetization of zero (Figure 1.1). Owing to reorientation and magnetization of individual particles to the external fields, superparamagnetic IONPs (SPIOs) present greater magnetic susceptibility and magnetic saturation compared to paramagnetic materials, which is paramount in their function as contrast agents in MRl [91. The presence of attractive forces between neighboring ferromagnetic NPs could tend to large aggregates [101, which are more easily cleared by macrophages and pose greater risks for vascular embolism

[l11.

Thus, SPIOs are

ideal for in vivo because of they will lose their magnetization and become dispersed again when external magnetic fields in switched off (Figure 1.1). Due to the damaged vasculature, solid tumor tissues can capture more dispersed particles than normal ones through enhance permeability and retention (EPR) effect, terms as "passive targeting'' [12 •131. Moreover, when employing a gradient magnetic field after SPIOs administration, the driving force guides particles to their targeting tissue, and to

1.3

Phatmacokioeti.c and biocompability

5

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Ferromagnetic nanoparticles

Superparamagnetic na.noparticles

Figure 1.1 Illustration of magnetic moments orientations in ferromagnetic nanoparticles

and SPIOa. In the absence of magnetic field, the orientation of randomized magnetic moments reeulta in an average magnetization of zero. With magnetic field exposure, magnetic moments reorient with external fields, and SPIOs will randomized again when magnetic field in switched off.

The accumulation of SPIOs provides a great opportunity for SPIOs-based imaging and therapy 1151. For SPIOs-based hyperthermia, a general route includes distribution of SPIOs inside the targeted tumor region, and then followed by generation of heat using an external alternating current m.agnetic field (ACMF). The dynamic responses to ACMF of particles with its magnetic moments govern the transformation of magnetic energy into thermal :fluctuations [16). The heat generation mechanism can be attributed to dynamic hysteresis lo!18e8 induced by Neel and Brownian relax-

ations [t1]. Moreover, SPIO-based hyperthermia provides another great opportunity for controlled drug release through bond breaking or decapsulation of polymeric drug carrier 1181.

1.3

Pharmacokinetic and biocompability

SPIOs is generally delivered in three ways to tumor tissue: arterial injection (Figure 1.2, p8811ive target induced by EPR effect), active target (Figure 1.2, magnetic forced targeting, tumor specific biomarker targeting) , in situ injection or/and transplantation 1121. Until now, both passive and active target are not able to provide sufficiently concentration in tumor, becaUBe of the cellular uptake of normal cells is unavoidable 1131. Thus, the magnetic property of SPIOs and delivery effi.ciency

6

Chapter 1

Medical Ther&tLOsti.cs B!Uied on Iron Oxide Naoopatticles UDder···

have to be increased. Direct injection allows most of SPIOs retention in tumor tissue, which is desirable for optimal hyperthermia treatments with low side effect [191. However, this method could only be used in superficial tumors, like breast cancer

and melanoma.

e

Tumor cells

~ ~~ular endothelial SPIOs

Tumar specific tBI'geted SPIOs

Figure 1.2 Modes of tumor targeting patterns of SPIOa. Pasaive targeting, SPIOs reach

tumor regioos through the leaky vasculature sUlTOUII.di.ng the tumors. Active targeting,

specific ligands modified SPIOs bi.od to receptors overexpressed .i.o tumor ceUs. SPIOs delivery can also controlled remotely by external m.agnetic field.

In general, arterials injected n.anoparticles have to fare the physiological barriers, including blood and clearance organs (liver, spleen) 1201. As injected SPIOs interact with blood components immediately, SPIOs with dillerent parameters in size, surface charge and hydrophilicity show differences in blood circulation half-life and organ distribution 111•211. After intravenous administration, SPIOs larger than 800 nm are more taken up by liver and spleen, whereas SPIOs smaller than 8 nm tend to be filtrated through the kidneys 1221. Commonly, SPIOs with medium size between 10 and 300 nm accumulate in reticuloendothelial system (RES) organ due to their prolonged blood circulation time 1231. Moreover, SPIOs with neutral charge surface

tend to exhibit longer blood circulation and reduced uptake by RES 123•241. Recent studies demonstrated also the surface adsorption of proteins {like IgG, la.minin or complement proteins) to SPIOs with surface charges could help ma.crophages to recognize and uptake particles rapidly 123•251. FUrthermore, it was found that :MIM (missing in metastasis), a scaffold protein binds with intracytoplasmic membrane,

1.4

Contrast agents of MRl

7

play a positive role during the endocytosis of SPIOs in macrophages [261. Therefore, knock-down of MIM is significant to avoid cellular uptake of SPIOs by RAW264.7 cell lines, which may function to enhance the accumulation of SPIOs in target sites. Except the size and charge, polyethylene glycol (PEG) modifications can effectively inhibit protein adsorption and enhance circulation time [231. SPIOs with a longer circulation half-life have more accumulation to target tissues, which can improve the utility of particles [27]. Biodistribution is also important, the majority of SPIOs often distributed in RES organs. The significant accumulation in liver and spleen raised nonnegligible concerns of toxicity potential, though most developed SPIOs are well tolerated [28 •291. Liver functionality tests (LFTs), cytokine detection, histology and blood counts have all been useful in assessing toxicity in vivo [30 •311. Toxicity of administered SPIOs is highly dependent on these characteristics: synthesis procedure, type and charge of surface coating, route of administration and purity [321.

1.4

Contrast agents of MRl

The relaxation time and hydrogen proton density of different tissues are different under magnetic fields exposure, these different signals produce contrast among tissues [331. SPIOs have unique magnetic susceptibility with shortening effects of longitudinal relaxation (Tt) and transverse relaxation (T2) which could be used to enhance the tissue contrast [3 4-3 7]. Until now, several SPIOs-based MRl contrast agents are clinically approved [38 •391, including (Figure 1.3): ferumoxytol (Feraheme), ferumoxides (Feridex in USA, Endorem in Europe), ferucarbotran (Resovist), ferristene (Abdoscan) and ferumoxsil (Gastromark in US, Lumirem in Europe). Another two kinds of SPIOs are ferumoxtran-10 (Combidex in USA, Sinerem in Europe) and PEG-Feron (Clariscan), which are in clinical trials. Ferumoxytol is the most recently FDA approved SPIO (2009) for treatment of iron deficiency anemia (IDA) in adults with chronic kidney disease (CKD). It can be considered as a potential replacement for Gadolimium-based small molecular contrast agents especially in patients with kidney failure. At present, 17 cases of FDA-approved clinical trials using ferumoxytol are completed, include 11 cases of MRl and 6 case of IDA. The ferumoxytol contrasted MRl is performed in primary tumor, metastatic cancer lymph node and myocardial infarction. Ferumoxides was

8

Chapter 1

Medical Theranostics Based on hon Oxide Nanoparticles under···

approved in 1996 as a MRI contrast agent for detection of liver lesions, and discontinued in 2008 because of its less competitive in intravenous contrast agent markets. However, their application in cell tracking by MlU is recognized, which possess 4 in

5 approved MRl cell tracking clinical trials. Ferucarbotran was approved in 2001 also as a small focal. liver lesions specific MlU contrast agent. In clinical trials it

presented a safety profile, and discontinued in 2009 with the market introduction of ferumaxytol. Ferristene and reruiii.OX!Iil are oral SPIOs as intestinal MlU contrast agent, they were gradually knocked out from market due to the poor profit although they are effective and safe. Clinic.a.l approved SPIOs ar in clinic&! tria.lB Core component:

"t-~OsnanocrystaJ

r--, Coatings:

Size: 1~180 nm(Feridex) 60 nm(ReBovist)

i : CarbaxyQextra.u (Resovist)

•

..,_ ....- -

30 nm(Fera.heme) 300 nm(Abdosca.n,Lumirem) D-7 nm(ClariscB.D)

: Dextra.u(feridex,Combidex)

: Car~etbfladextran (Feraheme) : Siloxane(LUIIlli'em) : Sulfonated Styrene-di-vinglbenzene L_! copolymer(A6d011CBD.)

: : :

PEG-6ta.rcll(Cla.riscan)

20-40 nm(Combidex)

Figure 1.3 Clinically approved or in clinical trials SPIOs with biocompatible coating and iron oxide nanocryBtal core. Commercial products are list aside, for each size and coating

materials.

SPIOs-based MRl contrast agents provide excellent .imaging sensitivity and good biocompatibility for in vivo applications. Through summarization the current status of SPIOs from clinical trials, we concluded that the clinical use of SPIOs would add more opportunities in imaging of metastatic tumor and cell based therapy.

1.5

Hyperthermal agents

Magnetic mediated hyperthermia, more specifically, magnetic Huid hyperthermia, involves dispersing SP!Os throughout target tissue and then applying an ACMF with effective strength and frequency to generate internal heat in the particles by hysteresis losses and Nee!-Brownian rel.a.xations 1401. Compared with other hyperthermia modalities induced by microwave, laser or ultrasonic wave, magnetic fluid hyperthermia has the best potential to optimizing the targeting to tumor cells and reducing the toxic side effect

(41(.

'lb control the temperature 8.1ld the heat region precisely is also a challenging

1.5

Hyperthermal agents

9

task during magnetic fluid hyperthermia therapy. MRI-based location analysis or magnetic forced-targeting combined with hyperthermia can make it possible [421, both approaches need to employ static magnetic fields (SMF). It has been reported that SMF changes the nonlinear magnetization dynamics of SPIOs substantially, and a small value of bias in SMF can affect the dynamic magnetic hysteresis loop significantly [43, 441. AB we know, the heat energy generated by SPIOs is terms as specific absorption rate (SAR), it is possible for designing weak changes in the SMFs to control the heat production (SAR) effectively. The magnetically heating properties in a combination of ACMF and SMF was investigated [451. See in Figure 1.4(a), #1 and #2 inside the coil present identical heating efficiency with ACMF only. However, #2 generate more heat than #1 with a combination of SMF (Figure 1.4(b).-v(d)), which suggested that magnetic inductive hyperthermia can be restricted to a designated small region (15mm) by applying a designed SMF. This magnetic hyperthermia system could be further improved to control temperature and the heat region more precisely [461. Another meaningful example in controlling the magnetothermal performance actually is the magnetic anisotropic hydrogels [4 7]. Hydrogels attract growing interest in the area of organ regeneration and controlled drug release as an implantation biomaterial [48 ,491, and the presence of SPIOs brings out additional potentials in hyperthermal and stem cells fate regulation [50- 521. However, for the hydrogel with randomly distributed SPIOs fixed inside, the heat generation effect is very little because of the interaction among individual particles negligible. Nevertheless, if the SPIOs inside hydrogel can be anisotropically arranged into one-dimensional structures, the magnetic coupling among particles will be introduced [631. AB shown in Figure 1.5, the thermogenesis was increased while the orientation of assembled SPIOs chains with respect to ACMF altered from vertical to parallel (the same direction) [4 7]. By doing this, the magnetically hyperthermia can be regulated by controlling the orientation of the magnetic anisotropic hydrogels with respect to ACMF. Additionally, the heat generation efficiency of SPIOs assemblies can also be controlled by tuning the incident energy of ACMF [461. Extending the traditional concept of hyperthermia, target heating of engineered SPIOs was used to control cellular process and gene expression remotely

[54 ,56-581.

These inspiring reports were based on TRPV1, also known as capsaicin receptor, a kind of temperature-sensitive calcium channel expressed on the plasma membrane of

Chapter 1

10

Medical Theranosti.cs BBBed on Iron Oxide Naoop&Tticles under···

cells 1591. Engineered SPIOs targeted to this protein and heated by radiofrequency

magnetic fields, leading to the opening of TRPVl, then the cellular macltine is remotely controllable (Figure 1.6(a)) 1541. If the 5' regulatory region of desirable gene is engineered with Ca2+ response elements in cis (Figure 1.6(b)) I57J, gene expression can be remotely regulated by magnetic fields heating, for instance, insulin to treat diabetes and obesity 155•601. Interestingly, these provide an opportunity for using SPIOs with diverse magnetic properties to respond selectively to a range of parameters of oscillating magnetic fields in order to meet different therapeutic effect [CSl-63]. 120

a

~ 100

};80

········-

'

i9

~ 60

·- -·-·-·-· - ···-··· ..

~

~----·-

~

40

20

Permanent magnet

0

~

(a) 45

45

44

44

43

~

42 41

41

40

40 39 38 37

39

38 37 36

~ ~

35

34

34

t3 (c)

(d)

Figure 1.4 (a) Schematic of the experimental setup with a combination of ACMF and SMF for heating cell 8USPension8· (b) MTT assay values for ceUa treated in ao ACMF aod a combination of ACMF and SMF. Different letters denote statistically significant differences at p


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Z. Chen, J.J. Yin, Y.T. Zhou, Y. Zhang, L. Song, M. Song, S. Hu, and N. Gu, Dual enzyme-like activities of iron oxide nanoparticles and their implication for diminishing cytotoxicity. Acr3 Nano, 2012. 6(5): p. 4001-4012.

[96] M. Mahmoudi, H. Hosseinkhani, M. Hosseinkhani, S. Boutry, A. Simchi, W.S. Journeay, K. Subramani, and S. Laurent, Magnetic resonance imaging tracking of stem cells in vivo using iron oxide nanoparticles as a tool for the advancement of clinical regenerative medicine. Chemical Reviews, 2010. 111(2): p. 253-280. [97] F. Yang, M. Zhang, W. He, P. Chen, X. Cai, L. Yang, N. Gu, and J. Wu, Controlled release of Fe3 04 nanoparticles in encapsulated microbubbles to tumor cells via sonoporation and associated cellular bioeffects. Small, 2011. 7(7): p. 902-910. [98] F. Yang, M. Li, H. Cui, T. Wang, Z. Chen, L. Song, Z. Gu, Y. Zhang, and N. Gu, Altering the response of intracellular reactive oxygen to magnetic nanoparticles using ultrasound and microbubbles. Science China Materials, 2015. 58(6): p. 467-480. [99] D. Ye, Q. Wang, W. Zhang, J. Sun, and N. Gu, Recent progress in magnetic labeling for stem cell. Chinese Science Bulletin, 2017. 62(20): p. 2301-2311. [100] A. Uccelli, L. Moretta, and V. Pistoia, Mesenchymal stem cells in health and disease. Nature reviews. Immunology, 2008. 8(9): p. 726. [101] M.J. Dalby, N. Gadegaard, and R.O. Oreffo, Harnessing nanotopography and integrinmatrix interactions to influence stem cell fate. Nature materials, 2014. 13(6): p. 558. [102] D.M. Huang, J.K. Hsiao, Y.C. Chen, L.Y. Chien, M. Yao, Y.K. Chen, B.S. Ko, S.C. Hsu, L.A. Tai, and H.Y. Cheng, The promotion of human mesenchymal stem cell proliferation by superparamagnetic iron oxide nanoparticles. Biomaterials, 2009. 30(22): p. 36453651. [103] Q. Wang, B. Chen, M. Cao, J. Sun, H. Wu, P. Zhao, J. Xing, Y. Yang, X. Zhang, and M. Ji, Response of MAPK pathway to iron oxide nanoparticles in vitro treatment promotes osteogenic differentiation of hBMSCs. Biomaterials, 2016. 86: p. 11-20. [104]

Q. Wang, B. Chen, F. Ma, S. Lin, M. Cao, Y. Li, and N. Gu, Magnetic iron oxide nanoparticles accelerate osteogenic differentiation of mesenchymal stem cells via modulation of long noncoding RNA INZEB2. Nano Research, 2017. 10(2): p. 626-642.

[105] H.Y. Xu, and N. Gu, Magnetic responsive scaffolds and magnetic fields in bone repair and regeneration. Frontiers of Materials Science, 2014. 8(1): p. 20-31. [106] J. Sun, X. Lin, J. Huang, L. Song, Z. Chen, H. Lin, Y. Li, Y. Zhang, and N. Gu, Magnetic aaaembly-mediated enhancement of differentiation of mouse bone marrow cells cultured on magnetic colloidal assemblies. Scientific reports, 2014. 4.

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Medical Theranostics Based on Iron Oxide Nanoparticles under···

[107] X. Liu, J. Zhang, S. Tang, J. Sun, Z. Lou, Y. Yang, P. Wang, Y. Li, and N. Gu, Growth enhancing effect of LBL-assembled magnetic nanoparticles on primary bone marrow cells. Science China Materials, 2016. 59(11): p. 901-910. [108] H.M. Yun, S.J. Ahn, K.R. Park, M.J. Kim, J.J. Kim, G.Z. Jin, H.W. Kim, and E.C. Kim, Magnetic nanocomposite scaffolds combined with static magnetic field in the stimulation of osteoblastic differentiation and bone formation. Biomaterials, 2016. 85: p. 88-98. [109] J. Meng, Y. Zhang, X. Qi, H. Kong, C. Wang, Z. Xu, S. Xie, N. Gu, and H. Xu, Paramagnetic nanofibroua composite films enhance the osteogenic responses of pre-

osteoblast cells. Nanoscale, 2010. 2(12): p. 2565-2569. [110] J. Meng, B. Xiao, Y. Zhang, J. Liu, H. Xue, J. Lei, H. Kong, Y. Huang, Z. Jin, and

N. Gu, Super-paramagnetic responsive nanofibrous scaffolds under static magnetic field enhance osteogenesis for bone repair in vivo. Scientific reports, 2013. 3. [111] F. Xiong, H. Wang, Y. Feng, Y. Li, X. Hua, X. Pang, S. Zhang, L. Song, Y. Zhang, and N. Gu, Cardioprotective activity of iron oxide nanoparticles. Scientific reports, 2015.

5. [112] J. Han, B. Kim, J.Y. Shin, S. Ryu, M. Noh, J. Woo, J.S. Park, Y. Lee, N. Lee, and T. Hyeon, Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells' therapeutic efficacy for myocardial infarction. ACS nano, 2015. 9(3): p. 2805-2819. [113] M.W. Hentze, M.U. Muckenthaler, and N.C. Andrews, Balancing acts: molecular control of mammalian iron metabolism. cell, 2004. 117(3): p. 285-297. [114] N.C. Andrews, Disorders of iron metabolism. New England Journal of Medicine, 1999. 341(26): p. 1986-1995. [115] B.S. Spinowitz, A.T. Kausz, J. Baptista, S.D. Noble, R. Sothinathan, M.V. Bernardo, L. Brenner, and B.J. Pereira, Ferumoxytol for treating iron deficiency anemia in CKD. Journal of the American Society of Nephrology, 2008. 19(8): p. 1599-1605. [116] L. Chen, C. Chen, P. Wang, and T. Song, Mechanisms of Cellular Effects Directly Induced by Magnetic Nanoparticles under Magnetic Fields. Journal of Nanomaterials, 2017. 2017.

Chapter 2 Progress of Static Magnetic Fields and Cancer Research in China 1

Xin Zhang 1'2'* High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 230031; 2

Institute of Physical Science and Information Technology, Anhui University, 230601, Hefei, Anhui, P. R. China. *Correspondence should be sent to: [email protected]

Static magnetic field (SMF) is different from time-varying magnetic fields generated from cell phones, microwaves or power lines. For SMF, the magnetic field intensity and direction do not change over time. Various reports show that SMFs of different parameters could influence biological systems, but the exact effects still lack consensus. However, results from multiple studies including ours together demonstrate that SMF could inhibit the growth of some types of cancer, including cancer cell proliferation in vitro and tumor growth in vivo. Here in this chapter, I reviewed the research progress of SMF and cancer studies in China in recent few years. Experimental observations, potential mechanisms, as well as parameters that lead to the lack of consistencies in the literature are all discussed. Based on currently available data, although these researches are still at initial stage, enormous progress has been achieved to imply the potentials to apply SMF in cancer treatment in the future. We look forward to more experimental and theoretical work in both clinical and basic researches to facilitate the development of this field. Static magnetic field (SMF) is a type of magnetic field that does not change over time, including the magnetic field direction and intensity, which is different

32

Chapter 2

Progress of Static Magnetic Fields and Cancer Research in China

from the time-varying magnetic field generated by power lines or cellular phones. SMF belongs to non-ionizing electromagnetic field, which is in contrast to ionizing radiations, such as X-rays and gamma rays, which have enough energy to break molecular bonds. This chapter will only discuss about the research progress of SMF, while information about the time-varying electromagnetic field can be found in Chapter 4 of this book. Moreover, time-varying magnetic fields generated by rotating permanent magnets on cells, mice and human bodies can be found in Chapter 7 and Chapter 8 of this book.

Background It should be mentioned that over 2000 years ago, people have already started to notice magnetic fields could induce some biological effects. However, it was not really studied until the 1960's. From 1960s, there are over a thousand papers studying the biological effects of various magnetic fields. Then in 1970s, more and more related researches were carried out. Some of these studies were published in esteemed journals such as Science, Nature and PNAS [l-1]. For example, people found that for proteins, the diamagnetic anisotropy is largely due to the alpha helix, beta sheet, aromatic rings and even peptide bonds [5 •61. Although a single peptide bond has weak diamagnetic anisotropy [51but when they link together in a fixed and organized orientation in alpha helix or beta sheet, the overall diamagnetic anisotropy can be much stronger [61. Therefore, although most proteins have very weak diamagnetic anisotropy, their response to the magnetic field should be able to be amplified when they are constrained in membrane sheets, in liquid crystal phase, or in highly ordered polymers in which the additive diamagnetic anisotropy can be significant. However, people found that there are enormous reports showing completely different results, such as cell growth promotion vs. inhibition. In addition, magnetic field associated therapies were also shown to be less effective than expected, except for pain relief. Therefore, the enthusiasms about the biological effects of magnetic fields eventually fade away. Luckily, although this flourishing era has passed, there were still some people did not give up. In the past few decades, researchers are still doing magnetic field related biological studies worldwide, although mostly sporadically. Now we are clear that the seemingly inconsistent results from independent studies are caused by multiple

Chapter 2

Progress of Static Magnetic Fields and Cancer Research in China

33

reasons, such as the magnetic field types, intensities, cell types, etc. Moreover, with the help of more advanced techniques, we nowadays are able to reexamine these "old questions" again and try to get a clearer understanding for this nature power on our human bodies.

Progress of static magnetic fields and cancer research in China Although overall the studies about biological effects of SMFs are still at preliminary stage, many progresses have been made in the field of SMF and cancer research in the past few years. Experimental evidences from multiple studies have indicated that SMFs could in fact inhibit the growth of some cancer cells, while have minimum effect on non-transformed cells. In addition, efforts have also been made toward mechanistic studies, which showed that several aspects of cellular activities are involved in SMF-induced cancer growth inhibition, such as cell division, EGFR (epidermal growth factor receptor) and cell membrane. In this chapter, I mainly outline recent advances reported by the Chinese researchers about how SMFs affect biological objects in the field of cancer biology in the past decades, including research of our own group

[8- 141,

and also discuss the possible relevance of these studies to

their potential clinical applications. Based on their magnetic field intensities, the classification of SMFs and timevarying magnetic fields can be different in various research areas. In general, they can be classified as weak (

Js.o

{b)

7.5 7.0

----=~

- ·---- ---- -· .•

-8-4

-a o

:~

4

6

~ dlM:tion/JDm (c)

Figure 6.3 SMF exposure system. (a) Physical Properties MeaBurement System (PPM). (b) Schematic diagram of the i.oside structure of PPMS. (c) Field lllliformity at 9T in axial/diametrical direction. The figure was adapted from Ref [38].

6.3

SMFs on the lifespan and development process of C. elegans

The embryo development is a highly sensitive ewnt in the life cycle, which are wry susceptible to environmental stress. The Food and Drug Administration (FDA)

has considered magnetic resonance diagnostic devices significant risk when adults, children, and infants aged

>1 month expose to the main SMF greater than 8T and

neonates expose to the main SMF greater than 4T1391. The whole life cycle of C.

elegans includes the embryonic stage, four larval stages {Ll,.,L4), and adulthood£401. It takes approximate 3.5 days for 0. elegans developing from Lllarvae to egg-laying adults at 2(TC under normal laboratory conditions. The length of the life cycle vary with temperature for the growth rate is increased by the temperature. The average

Chapter 6 High Static Magnetic Fields (SMFs) on Reproduction...

150

lifespan of wild-type worm used here was ,..,15 days. S::MFs at 8.5T had slight effect on the lifespan of a. elegans adults. Comp&red to adults, e~ were more sensitive to S::MFs. The lifespan was reduced by 15% in

a. elegans hatching from e~ exposed

to 8.5 T SMF for 5 hours (Figure 6.4(a)). In contrast, 5T SMF had no such influence on the lifespan no matter adults or eggs exposed. The development time for

a. elegam from eggs toLl, L2, L3, L4 and young adult

is generally 12, 26, 34.5, 43.5 and 60 hours, respectively. The body length for L1, L2, L3, L4 and young adult is 250, 360,.,.380, 490,.,.510, 620,.,.650, and 1060,..,1150JUD., respectively. The hatchability of eggs generated from wild type worms is 95%""100% in the present study. An exposure-time dependent decrease of hatchability was observed in

a. elegans exposed to 8.5T for 1, 3, 5 hours, respectively (Figure 6.4(b)).

Moreover, the development time of all stages was slightly extended by 8.5T S::MF. Although the magnetic fields up to 0.16T had no effect on fresh insect eggs (Heli.othis 120 ~

'0

!P~

';;- 100

so

1}60 ~ ; 40 8.5T SMFs

~~

(/.)

-- Oh _.,_ lh

20 0

80

l

60

'a t:l

40

~

- - 3h -- 5h

o

~

..CI

0

30

5

20 Oh

lh 3h S.MF ex;poBUI'e periods (b)

5h

40

daf-2(e1370) 20 G Control --8.6T,6h 0

o

10

20 30 Time/days

50

(c)

Figure 6.4 Effects of SMFs on C. elegans. (a) LifespaDB of C. elegans eggs exposed to

SMF. (b) SMF on the development stages. (c) Lifespan of d4/-B mutants exposed to SMF. The figure was adapted &om Ref [38J.

SMF on brood size and germline apoptosis in C. elegans

6.4

151

virescens or tobacco bugworm), significant effects on the hatching was observed in eggs exposed to magnetic field of 7T[411. The hatching time of mosquito eggs exposed to 9.4T and 14.1Twas delayed 32h and 71h, respectively[421. The insulin/IGF-1 signaling (liS) pathway is evolutionarily conserved pathway that modulates dauer formation, stress response, and metabolism in C. elegans[431. The C. elegans liS pathway consists of an insulin/IGF-1 receptor (DAF-2), a PI 3kinase (AGE-l/ AAP-1) serine/threonine kinases (PDK-1, AKT-1, and AKT-2), and aForkhead Box 0 (FOXO) transcription factor (DAF-16). Inhibition ofinsulin/IGF1 signaling can stimulate DAF-16/FOXO, which extends the lifespan by up- or downregulation of a variety of genes. Genetic disruption of insulin-like signaling extends remarkable longevity in C. elegans[441. Compared to wild type worms, the lifespan of daf-2 mutants was extended by 9.62% in the presence of 8.5T SMF (Figure 6.4(c)); in contrast, SMF has little effect on daf-16 mutants. The mRNA level of daf-2 was greatly increased in daf-16 mutants while decreased in wild-type C. elegans

under the magnetic field. SMF had no effect on the mRNA level of daf-16 in exposed daf-2 mutants.

6.4

SMF on brood size and germline apoptosis in C.elegans

C. elegans exists as either self-fertilizing hermaphrodite or males[45l. In general,

the majority of wild type worms are hermaphrodite and less than 0.2% is male. The male worm only produces sperms, which have to mate with hermaphrodites to generate progeny. The hermaphrodite itself can generate approximately 300 progenies, while the mated hermaphrodite can produce more than 1000 progeny. In our experiments, C. elegans are generated by self-fertilization, which the average offspring of one hermaphrodite are 300 produced within three days of peak fecundity. In the present study exposure to 8.5T SMF had no effect on the brood size of wild type worms (Figure 6.5). There is a close relationship between aging and reproduction in C. elegans. Germline cells including perm and oocytes influence the lifespan of the worms, which the lifespan is extended by 40%·... 60% when the germline precursor cells are removed[461. In adult hermaphrodite, the germline is a unique tissue containing two symmetrical U-shaped tubes which are connected to the uterus in the middle of body[471. The distal part of each gonadal tube is composed of germ cells

Chapter 6 High Static Magnetic Fields (SMFs) on Reproduction...

152

120 100 80 ~

l 1 40

60

~

20

0

Oh

lh

Sh

5h

SMF e:xpo!JIIJe time

Figure 6.5 Efrects of SMF on brood size of C. eiqJanB. The figure was adapted &om Ref [38].

with stem cell potential, which transition into the stages of meiosis I as they move proximally towards the uterus. The sperm is produced during the .last (L4) larval stage and stored in the sperm.atheca. Then, the hermaphrodites permanently switch over to the production of oocytes, which form in the loop region (Figure 6.6)1481. Apoptosis is a common event of the reproductive system in maintaining appropriate germ cell to sertoli cell ratio, removing defective germ cells, and controlling the sperm production1491. During normal oogenesis, approximate 50% germ cells undergo physiological apoptosis; however, the germ cell apoptosis can be triggered by various environmental stresses including starvation, heat shock, radiation, chemical pollutants, pathogenic bacterial, and oxidative stressl50•511. Several methods have been established to determine germ cell apoptosis including TdT-mediated dUTP Nick End Labeling (TUNEL) assay, acridine orange (AO) and SYT012 staing, and distinct morphology under DIC microscopyl521. In the present study, living worms were stained with AO detecting nucleic acids in the apoptotic cells. The germ cell apoptosis in the meiotic zone of the gonad was significantly increased by exposure to 8.5T SMF, which WBSin a ti~depen.den.t manner (Figure 6.7). This observation

6.4

S:MF on brood size and germl.ine apoptosis in C. elegtlns

153

was in consistent with the increase of apoptosis in immune cells exposed to lOT SMFI531. SMF of 6mT induced the apoptosis and alter the cell cycle in p53 mutant

Jurkat cells.

(a)

(b)

(c)

Figure 6.6 Hermaphrodite C. elegans and their germ]ine. (a) An adult wild-type (N2)

hermaphrodite stained with DAPI {4',~diamidin~2-phenylindole). (b) A dissected adult hermaphrodite germline stained with DAPI. (c) Schematic of an adult C. elegtlns hermaphrodite gonad. Somatic DTC is located at the distal end. Cells at the distal end of the germlioe, including germline stem cells, divide mitotically (yellow). As cells move proximally, they enter meiosis (green) and differentiate into either aperm (blue) or oocytee (pink). This figure was adapted from Ref [48].

154

Chapter 6 High Static Magnetic Fields (SMFs) on Reproduction...

Recovery time/hour (b)

Rerovmy time/hour

(a)

~ Control

-

6T S.MF,6h(24h recovery)

lh Recowryti:mefhour

(c)

3h 5h Exposure time/hour (d)

Figure 6.7 Germline apoptoeia induced by SMF of 8.5T in C. ek4ans. (a) Germline apoptosis in C. elegans exposed to SMF of 8.5T for lb. (b) Germlliae apoptosis .in C. elegaos exposed to SMF of 8.5T for 3h. (c) Germl.ine apoptosis in C. ek4ans exposed to SMF of 8.5T for 5h. (d) Germl.ine apoptosis induced by SMF of 5T in C. ek4ans. The figure was adapted from Ref [38).

The core apoptosis signaling pathway regulating the germ cell apoptosis is highly conserved in eukaryotes and consists of the BH3-only protein EGL-1, the antiapoptotic Bcl2 orthologue CED-9, the apoptotic protease activation factor 1 (Apaf1) orthologue CED-4, and the caapaae CED-3 inC elegans (Figure 6.8)[641. StreiiSinduced apoptosis is dependent on ced-9 and ced-,4, but is negatively mediated by

ced-9. The germ cell corpses induced by 8.5T SMF were greatly suppressed in ced-9 (n717) and ced-4 (n1162) mutants, respectively, indicating that SMF-induced germ cell death was regulated by the key apoptosis signaHng pathway in C. elegans (Figure

6.9).

6.4

155

S:MF on brood size and germl.ine apoptosis in C. elegtlns

C.elega'M Apoptotic signals

Droaophil4 Apoptotic signals

t

+

e

Mammals Apoptotic signals

t

Mitochondria

1

Apoptoeis

t

Apoptoeis

Apoptoais

Figure 6.8 Conserved core caap88e-8ignaliog cascades. (a) In C. elegans. (b) In Drosophila.

(c) In mammals. Functional homologous proteins across species are similarly represented

by the color and shape. The figure was adapted from Ref [54].

Chapter 6 High Static Magnetic Fields (SMFs) oo Reproduction...

156

5

Control 8.5T,5h

0 Wild Type

Figure 6.9

6.5

Ced-9(n117)

Ced-4(n1162}

Germlioe apoptosis induced by SMF. The figure was adapted &om Ref [38].

SMF on mitochondrial damage and oxidative stress in

C.elegans Free radicals are a group of chemical species that are paramagnetic, due to their possession of at least one Wlpaired electron. SMF can influence singlet-triplet transitioDB in unpaired electroDB and the .increased lifetime of free radicals interact directly or indirectly with biomolecules, leading to oxidative damage[551. Reactive oxygen/nitrogen species (ROS/RNS) including superoxide anion (02-), hydroxyl radical (OH·), singlet oxygen(10 2 ), and nitric oxide (NO) have been indicated as one of the key determinants in SMF-induced biological responses. The increased levels of metallothionein and lipid peroxida.tion was monitored in the liver of mice exposed to S:MF of 4.7T[56]. Our previous study showed that the generation of ROS was dramatically increased in human-hamster cells (Figure 6.10)1511. Mitochondria is the most important cellular source of free radicals and the main target for free radical regulatory and toxic actioDB.

me~1

encodes the C. ele-

gans ortholog of the succinate dehydrogenase cytochrome b560 subunit, an integral

membrane protein that is a subunit of mitochondrial respiratory chain complex II (ubiquinol-cytochrome c reductase)l58l. 8.5T SMF reduced the lifespan by 19% in wild type worms; in contrast, mev-1 (knl) mutants were more sensitive to S:MF, which the lifespan W89 reduced by 21.39%. Moreover, the germ cell corpses induced

6.5

SMF on mitochondrial damage and oxidative stress in C. elegans

157

by 8.5T S:MF in mev-1 (kn-1) mutant were signifiC&llt higher than that wild type

worms, indicating that the mev-1 (kn-1) mutant a.re hypersensitive to high S:MF exposure (Figure 6.ll(a)). Dimethyl sulfoxide (DMSO) is an efficient free radical scavenger. The presence of DMSO significantly extended the lifespan by 12% in S:MF-exposed groups, while deduced the number of germ cell corpse to near background in S:MF-exposed groups (Figure 6.11(b)). These data suggested that the S:MF-induced lifecycle abnormal and reproductive toxicity were mediated by mitochondrial dysfunction and oxidative stress in C. elegans.

(a)

blOO ..... X

i: I 40

Incubation Incubation Incubation Oh 12h. 24h

(c)

·~

20

~

0

CON

DMSO (0.2%)

8.5T 8.5T+DMO

(d)

Figure 6.10 Cellar ATP content was decreased by SMF of 8.5T, which was mediated by ROS. (a) Effect of SMF on ATP content with various exposure periods in wild type cells.

(b) Post-exposure effects of SMF on ATP content in wild type cells. (c) Effect of SMF on ATP content in mitochonchia deficient (p0 ) cells. (d) Protective effect of free radical quencher, DMSO, on the content of ATP in SMF exposed cells. This figure was adapted from Ref [57). The biological effects mediated by free radical has been proposed by the disruption of antioxida.nt defeDSe system via suppression the expression of antioxidant

Chapter 6 High Static Magnetic Fields (SMFs) on Reproduction...

158

enzymes1591. Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. In C. elt!Jcns, SOD is encoded by five different genes: sod-1 and sod-5 encode cytosolic CuZnSODs; JJod-4 expresses two splice variants, one membrane bound and one se-

creted; sod-2 and sod-9 encode mitochondrial MnSODsl601. With CF1553 (muls84) strain containing sod-9p::gfp reporter gene, we found that the fluorescence intensity in CF1553 strain was greatly increased by exposure to 8.5T SMF as compared to unexposed groups (Figure 6.11(c)). Moreover, the mRNA level of sod-3 was increased greatly in 8.5T SMF exposed worms. In male volunteers, SMF of 1.5T significantly increased the total antioxidant capacity. The protective effects of vitamins C and E on germ cells was reported in rats exposed to SMF of 1.5TI611.

(b)

Figure 6.11

(c)

Mitochondrial damage and oxidative stress mediated the germline apoptoeis

induced by SMF. (a) Germline apoptosis in wild type and mw-1 mutants exposed to SMF.

(b) Effect of DMSO on the germline apoptosis induced by SMF. (c) Expression of SOD-3 in C. elf'4GnB exposed to SMF. The figure was adapted from Ref 138].

Conclusion The present study showed that 8.5T SMF induced developmental and reproductive abnormalities during the lifecycle in C. elf>4ans. Exposure of eggs to 8.5T SMF

Reference

159

led to a time-dependent decrease of lifespan, while 5T had slight influence on the lifespan of exposed eggs. Although brood size was not altered, 8.5T SMF modified development rate and stages in the lifecycle, which was regulated by insulin/IGF-1 (insulin-like growth factors-1) signaling pathways. In adult worms, 8.5T SMF dramatically increased the germ cell apoptosis in an exposure time dependent manner. Moreover, our findings suggested that developmental and reproductive abnormality under 8.5T SMF was mediated by mitochondrial dysfunction and oxidative stress, which might help better understanding the effects of high SMFs in living organisms. Acknowledgements Nematode strains used in this work were provided by the Caenorhabditis Genetics Center. This investigation was financially supported by National Natural Science Foundation of China grants U1232144 and the Major/Innovative program of Development Foundation of Hefei Center for Physical Science and Technology 2017FXZY005.

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Non-ionizing Radiation, Part 1: Static and extremely low-frequency

(ELF) electric and magnetic fields. Health Physics, 2002. 83(6): p. 920. [7] P.W.Neurath, High gradient magnetic field inhibits embryonic development of frogs. Nature, 1968. 219{5161): p. 1358-1359. [8] J.M.Denegre, J.M. Valles, K. Lin, W. Jordan, and K.L. Mowry, Cleavage planes in frog eggs are altered by strong magnetic fields. Proceedings of the National Academy of Sciences, 1998. 95(25): p. 14729-14732.

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Chapter 7 Low Frequency Magnetic Field Regulates Immunity and Inhibits Cancer Tingting Wang 1•2 •*, Jing Ren 1•2 , Yayi Hou1•2 1

The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing, 210093, China; 2

Jiangsu Key Laboratory of Molecular Medicine, Nanjing, 210093, China. *Correspondence should be sent to: [email protected].

The treatment of low frequency magnetic fields (LF-MF) is thought to be non-invasive and non-ionizing and even possess non-thermal effects on cells and tissues. Thus the possible effects ofLF-MF on human health have been attractively explored. In this chapter, using different cells and mice models, we summarize our recent researches on LF-MF, including its potential roles in cancer treatment and immunity regulation. We also update recent progress and application of LF-MF in China.

7.1

Low frequency magnetic field (LF-MF) exposure system

With the development of science and technology, more and more electromagnetic appliances are used in the environment and at the workplace. Scientists began to focus on various magnetic field (MF) effects on the human body, which has gained worldwide attention. We had established a revolving LF-MF system. AI3 shown in Figure 7.1 (a), two pairs of fan-shaped N dFeB permanent magnets were embedded into a circular iron plate and arranged to establish LF-MF. The bottom two magnets rotated at a certain frequency driven by a step motor, which was controlled by using a functional signal generator. Due to the strong magnetic interaction, the

7.1

Low frequency magnetic field (LF-MF) exposure system

165

Step motor

(a.}

For cell

(b)

Figure 7.1

Magnetic field exposure system. (a) lostrument of magnetic field exposure system. Two pairs of fan-ahaped NdFeB permanent magnets were arranged to establish magnetic fields. LF-MF at the target site is alternative pu1ses with a maximum flux density of about 0.4T. (b)The smaller instrument with similar structure was instaU.ed in Thermo Scientific Forma Series II 3120 Water-Jacketed C02 Incubators. (Figure was adapted with permission from Ref [5], copyright@ 2017, Nature Publishing Group)

top two magnets rotated synchronously. The flux density and frequency of LF-

MF at the target site were alternative and the magnetic flux density was measured by a gauss meter. Fbr in vivo experiment, mice were placed in the middle of the magnets in a lucent and breathable box and could move freely in the box. There

166

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Low Frequency Magnetic Field Regulates Immunity...

was an internal column in the center of the box, and mice were put between ex-

ternal column (D=20cm) and internal column (D=Bcm). Control mice were placed in a. similar apparatus except that there were two rotating iron plates instead of

magnets, thus lacking a LF-:MF. The entire magnetic apparatus was loca.ted in a hood with hU.IDidity and temperature controller. For cellular experiments, a swa.ller instrument with similar structure was installed in Thermo Scientific Forma Series Water-Jacketed C02 Incubators (Figure 7.l(b)). Control cells were placed in a. similar apparatus except that there were two rotating iron plates instead of magnets

(Sham :MF). The instrument was fabricated by the National Laboratory of Solid Microstructures, Nanjing University.

7.2

Screening for suitable tumor parameters of LF-MF

In our LF-:MF system, the maximum magnetic flux density was 0.4T and the frequency of LF-MF was

a. . . 15Hz. Using hUIIl3Il gastric cancer cells BGC-823 cells,

we found that the inhibitory effect of :MFs on cell growth was enhanced by increasing the magnetic flux density from 0.05 to 0.4T (Figure 7.2(a.)) B.II.d increasing the 25

~20

~...

15

jiO

:a 5 ~ 00~....,........,,.....,.......,,......,_~~...,..,. Magnetic frequency /Hz.

(b) ~

25

-.... 20

~... 15

.i

~

10

5 ~~1--2~~3~~4~5~~6

Exposure time/day

Figure 7.2

(c) Inhibitory effect of LF-MF exposure on BGC-823 cells with different stimu-

lation conditions. BGC-823 cells were treated with (a) different magnetic flux densities, (b) different magnetic frequencies and (c) different exposure time. Data points are mean values ±standard deviations of three separate experiments. (Figure was adapted with permission from Ref [1), copyright @ 2011 Wiley-Lias, Inc.)

7.3

Thmor inhibition effect of LF-MF

167

frequency from 0 to 7.5Hz. However, the inhibitory effect was similar from7.5 to 15Hz (Figure 7.2(b)). Additionally, the inhibitory effect of this LF-MF on BGC-823 cells was also time dependent (Figure 7.2(c)). After exposure to LF-MFs for 2h/day and intermittently for 0.5, 1 and 2 days, no inhibition was observed in BGC-823 cell growth. Even though the inhibitory effect of LF-MFs was observed at4, 5, and 6 days, no significant change was found. Therefore, we selected a OAT, 7.5Hz LF-MF for further analysis, with cells intermittently exposed for 4 days (2h/day).

7.3 7.3.1

Tumor inhibition effect of LF-MF Inhibitory effect of LF-MF on different cancer cells

7.3.1.1 Effects of LF-MF on proliferation in different cancer cells The inhibitory effect of the OAT, 7.5Hz LF-MF on gastric carcinoma cells (BGC823, MKN-28 and MKN-45), lung cancer cells(SPC-Al, A549, H460 and LLC), colorectal cancer cells(LOVO), hepatocellular Carcinoma (H22) and melanoma (B16F10) cell lines were evaluatedl1- 5l. We found that LF-MFs could significantly inhibit the proliferation of BGC-823, MKN28, A549, H460 LLC, B16-F10, LOVO and H22 cells, while no significant effect was detected on MKN-45 and SPC-Al cells. These results suggest that LF-MFs may have a selective inhibitory effect on specific cancer cells. In gastric carcinoma cells, we found ultrastructural changes of BGC-823 cells. Compared to control cells, the cytoplasm of treated cells was characterized by moderate edema with mitochondrial disorganization, accompanied by degeneration or hypercondensation of cristae, expansion of the endoplasmic reticulum (ER), villous changes on cytoplasmic surface, appearance of myelin-like corpuscles, and decreased electron density of nuclear chromatin (Figure 7.3(a)). These data indicate that LF-MFs can directly alter the ultrastructure of BGC-823 cells, which suggests that the inhibitory effect of LF-MFs on cancer cells was associated with their intact ultrastructuresl 11. 7.3.1.2 Effects of LF-MF on cells cycle arrest and senescence in different cancer cells Cell cycle progression is an essential process by which cell monitors its growth and differentiation. We then examined the effect of this OAT, 7.5Hz LF-MF on cell cycle. Exposure to LF-MFs led to a significant increase in the cell population in the

Chapter 7

168

Low Frequency Magnetic Field Regulates lmmUDity...

S phase from 40.1% to 47.5% in BGCr823 cells, and41.3% to 47.0% in BGCr3.1 cells. However, no significant change in the cell cycle was found in BGC-:MK cells (Figure 7.3(b) and (c)). During the S phase of the cell cycle, DNA replication occurs and the amount of DNA in the cell has effectively doubled. These results suggest that the Sph.a.se arrest effect of LF-MFs W89 eliminated in BGCrMK cellsl11. However, in lung cancer cells, AS49 cells exposed to LF-MF displayed strikingly decreased number of

cells in the Sand G2/M phases and increased arrest in GO/Gl phase from 53.88% to 77.90%compared to Sham LF-MF (Figure 7.3(d)). A simil.a.r phenomenon was observed in LLC cells exposed to LF-MF for 6 days161. Furthermore, after exposure to LF-MF for 6 days, the morphology of cells partially changed and became hypertrophic, suggesting the appearance of senescence. Staining of senescence-associated ~galactosidase (SA-~gal)in lung cancer cells showed that LF-MF treatment up-

regulate frequency of ~ga).actosidase (~gal)-positive cells(Figure 7.3(e) and (f))l151. 7.3.1.3 LF-MF inhibits iron metabolismin lung cancer cells. Iron (Fe) is an essential element for all living organisms. It is involved in several fundamental biological processes161. Accumulation of iron in tissues increases the risk of ca.ncer and TfR (tr811Sferrin receptor) is frequently expressed in multiple carcinoma celllines11l. The iron deficiency results in cell proliferation reduction and Gl/S arrest of tumor cell. Depriving essential nutrient iron of cells by chelators has been used as an approach for cancer treatmentl81. A significant change of iron

and copper concentration in the liver and kidneys of fertilized rats exposed to static and LF-MFs has been observedl91. Cellular iron metabolism is critical for many cellular processes, including oxygen tra.nsport, respiration and DNA synthesisl101.

(a)

(b)

169

'1\mwr iohibition efFect of LF-MF

7.3

-BGC-823 ID BGC-823(MP) EIBGC-3.1 IDD BGC-:U(MF) CBGC-MK C BGC-MK{MF)

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