Ansar EB

235 Pages·2016·4.96 MB·English

SURFACE MODIFIED SUPERPARAMAGNETIC
NANOPARTICLES FOR FUNCTIONAL BIOMEDICAL
APPLICATIONS

Ansar E B

Ph.D. THESIS
2016

SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL
SCIENCES AND TECHNOLOGY
THIRUVANANTHAPURAM
INDIA
2016
DECLARATION

I, Ansar E B, hereby certify that I had personally carried out the work
depicted in the thesis entitled, “Surface Modified Superparamagnetic
Nanoparticles for Functional Biomedical Applications”, except where
due acknowledgment has been made in the text. No part of the thesis has
been submitted for the award of any other degree or diploma prior to this
date.

Ansar E B

Reg.No: PhD/2011/03
Trivandrum
12-08-2016

ii
SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL
SCIENCES & TECHNOLOGY
Thiruvananthapuram – 695011, INDIA
(An Institute of National Importance under Govt. of India)
Phone: [phone] Fax: [phone]
Email: [email protected] Web site – www.sctimst.ac.in

CERTIFICATE

This is to certify that Mr. Ansar E B, in the Bioceramics laboratory of this institute
has fulfilled the requirements prescribed for the Ph. D. degree of the Sree Chitra
Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram. The
thesis entitled, “Surface Modified Superparamagnetic Nanoparticles for
Functional Biomedical Applications” was carried out under my direct supervision.
No part of the thesis was submitted for the award of any degree or diploma prior to
this date.

Dr. P.R. Harikrishna Varma
(Research Supervisor)
Trivandrum
12-08-2016

iii
Office Seal
The thesis entitled

Surface Modified Superparamagnetic Nanoparticles for Functional
Biomedical Applications

Submitted by

Ansar E B

for the degree of
Doctor of Philosophy
Of

SREE CHITRA TIRUNAL INSTITUTE
FOR MEDICAL SCIENCES AND TECHNOLOGY,
THIRUVANANTHAPURAM - 695011

is evaluated and approved by

…………………………………… …………………………………..
Dr. P.R. Harikrishna Varma Examiner
(Research Supervisor)

iv
DEDICATED TO
MY FAMILY & TEACHERS

v
ACKNOWLEDGMENT

I take this opportunity to express immeasurable gratitude to many people for
their support during the PhD work which made this thesis possible.
First of all, I would like to express my heartfelt gratitude and respect to my
supervisor Dr. P.R. Harikrishna Varma, scientist-F, BCL, SCTIMST is beyond
description. Dr. Harikrishna Varma has offered constant support, strong
encouragement and motivation throughout the course of this study. I thank him for
the systematic guidance, and for lighting the path of my budding research career via
preparation of this thesis.
The Director, SCTIMST and The Head, BMT Wing is greatly acknowledged
for the facilities provided throughout the doctoral programme. I am very much
indebted toThe Dean, The Registrar and The Deputy Registrar for all the academic
assistance in this venture.
I thank the members of my doctoral advisory committee, Dr. K. Sreenivasan,
scientist G, Polymer Analysis Lab, Dr. Mohanan P. V, scientist F, Toxicology
division and Dr. R. S. Jayasree, Scientist E, Biophotonics and Imaging Laboratory or
their timely suggestions, support and critical comments.
I thank Department of science and technology, Govt. Of India for the
fellowship provided during the doctoral programme and Department of
biotechnology Govt. of India for funding for the international conferences which I
attended in China.
This work was completed only because of the help and support given by
Dr.Manoj Komath, Mr. Vijayan S, Dr. Sureshbabu S, and Mr. Nishad KV. I also
thank them for the training given in material synthesis, analysis by XRD, FTIR, ICP,
SEM etc. during the work. I am grateful to Mr. Sreekanth PJ, Dr. Padmaja P, Dr.
Rajesh P, Ms Nimmy Mohan, Ms. Sandhya, for all the support during my work.
It is great pleasure for me to thank to Dr. Annie John, Dr. Francis BF, and
all members of TEM for their help and support in cell culture studies. My sincere
thank to Dr. Jayasree and Dr. Shaiju S Nazeer of BPL Lab for help, support and
conducting the MRI analysis. I thank Dr. Lissy K. Krishnan and Mr. Ranjith Katha of
TRU Lab for Hemolysis studies and FACS analysis. I thank to Dr. Sabareeswaran
and all members of Histopathology Lab and Dr. TV Kumary, Dr. Anil Kumar P. R
and all members of Tissue culture Lab for various aspects of cell study. I thank to
Dr. C.Radhakumary of Polymer analysis Lab and Mr. Willi Paul of Central
Analytical Facility for help thermal analysis and DLS studies.
I am very much indebted to Dr. Anil Sukumaran of King Saud University,
Prof. Yokogawa of Osaka City University and Dr. Wilfried Wunderlich of Tokai
University for providing various aspects of experimental study, instrumental
facilities and for fruitful discussions. I gratefully acknowledge the support and help
given by my friends, staff and students of BMT wing of SCTIMST.
I have no wards to express gratitude to my family members who provided the
most precious support. I am indebted to my parents, wife, brothers and sister for
their endless support, encouragement, love and prayers.
Ansar E B
vi
TABLE OF CONTENTS

DECLARATION OF AUTHORSHIP ......................................................................................ii
CERTIFICATE BY THE RESEARCH GUIDE ..................................................................... iii
APPROVAL OF THESIS ........................................................................................................ iv
ACKNOWLEDGMENTS ....................................................................................................... vi
TABLE OF CONTENTS ........................................................................................................ vii
LIST OF FIGURES ............................................................................................................... xiv
LIST OF TABLES ................................................................................................................. xxi
ABBREVIATION................................................................................................................. xxii
SYNOPSIS ........................................................................................................................... xxiii
Chapter 1 .................................................................................................................................. 1
INTRODUCTION ................................................................................................................... 1
1.1 Nanotechnology ............................................................................................................. 1
1.2 Nanoparticles ................................................................................................................. 2
1.3 Importance of Nanoparticles in the Biomedical Field ................................................... 4
1.4 Magnetism and Superparamagnetic Nanoparticles ........................................................ 6
1.5 Potential Application of Superparamagnetic Nanoparticles in the Biomedical Field .... 7
1.5.1 Targeted Cell Therapy ............................................................................................ 7
1.5.2 Diagnostic Tool- Magnetic Resonance Imaging (MRI) Contrast Agent ................ 8
1.5.3 Therapeutic Agent – Magnetic Hyperthermia Cancer Treatment ........................... 9
1.6 Limitation of the Current Approaches ........................................................................... 9
Hypothesis ............................................................................................................................. 12
Objectives of the Study .......................................................................................................... 12
Chapter 2 ................................................................................................................................ 14
LITERATURE REVIEW ...................................................................................................... 14
2. 1 Superparamagnetism and Superparamagnetic Iron Oxide Nanoparticles [SPION] .... 14
2.2 Different Method of SPION Synthesis and Importance of Co-precipitation ............... 15
2.3 Versatile Applications of SPION and Importance in Potential Biomedical Field ....... 16
2.3.1 Targeted Delivery and Therapy ............................................................................ 16
2.3.1.1 Three Dimensional Cell Culturing and Magnetic Microspheres ................... 18
vii
2.3.2 MRI Contrast Agent .............................................................................................. 20
2.3.2.1 Magnetic Resonance Imaging ........................................................................ 20
2.3.2.2 Magnetic Resonance Imaging- Importance of Contrast Agent ...................... 21
2.3.2.3 Spinel Crystal Structure and its Modification ................................................ 22
2.3.2.4 Synthesis of Spinel Ferrite ............................................................................. 23
2.3.3 Hyperthermia Cancer Therapy – Importance of Magnetic Field .......................... 23
2.3.3.1 Temperature Sensitivity of Cancer Cells ....................................................... 24
2.3.3.2 Hyperthermia Heating Mechanism of Magnetic Nanoparticles ..................... 25
2.3.4 Theranostic Application of Magnetic Nanoparticles ............................................ 27
2.4 Problems Associated with Bare SPION Particles in Biomedical Applications ........... 27
2.4.1 Importance of Surface Modification ..................................................................... 29
2.4.2 Inorganic Molecules Used as Surface Coating Agent – Hydroxyapatite Crystals 30
2.4.3 Surface modification – Trisodium citrate (TC) Molecules ................................... 32
Chapter 3 ................................................................................................................................ 34
MATERIALS AND METHODS ........................................................................................... 34
3.1 Developemtn of Superparamagnetic Iron Oxide Embedded Hydroxyapatite
Nanocomposite .................................................................................................................. 34
3.1.1 Materials ............................................................................................................... 34
3.1.2 Synthesis of Nano Iron Oxide Embedded Hydroxyapatite Composites (HAIO) .. 34
3.1.3 Physicochemical Characterizations HAIOs and SPION ....................................... 35
3.1.3.1 High Resolution Ttransmission Electron Microscopy (HRTEM) and Energy
Dispersive X-ray Spectra (EDS) ................................................................................ 35
3.1.3.2 Environmental Scanning Electron Microscopy (ESEM) and Energy
Dispersive X-ray Spectra (EDS) ................................................................................ 36
3.1.3.3 X-ray Diffraction Analysis (XRD)................................................................. 36
3.1.3.4 Dynamic Light Scattering (DLS) and Zeta Potential Measurements ............. 36
3.1.3.5 Fourier Transform Infrared Spectra (FTIR) ................................................... 37
3.1.3.6 Vibrating Sample Magnetometry (VSM) ....................................................... 37
3.1.4 Biological Evaluation of HAIOs ........................................................................... 37
3.1.4.1 In vitro Biocompatibility- cell Culture ........................................................... 37
3.1.4.2 Cell viability MTT Assay .............................................................................. 38
3.1.4.3 Cell viability Alamar blue Assay ................................................................... 38
3.1.4.4 In vitro Hemocompatibility ............................................................................ 39
viii
3.1.4.5 Cellular Uptake: Prussian blue Staining and Flow Cytometry Evaluations ... 40
3.2 HAIO50 Assisted Cell Separation, Manipulation and Culturing using External
Magnetic field for Introducing Targeted Cell Delivery and Therapy ................................ 40
3.2.1 Cell separation ...................................................................................................... 40
3.2.2 Morphological Study: Cell Separation .............................................................. 41
3.2.2 Cell Culture of Magnetically Separated Cells ....................................................... 41
3.2.2.1 Cytoskeleton, Morphology Evaluations by Confocal Laser Scanning
Microscopy (cLSM) ................................................................................................... 41
3.2.3 HAIO50 Aided Three Dimensional Cell Culture .................................................. 42
3.2.3.1 Morphological Evaluation-ESEM Technique ................................................ 42
3.2.3.2 DAPI Nuclear Staining and Phase Contrast Imaging.................................... 43
3.2.4 Magnetic Microsphere Synthesis .......................................................................... 43
3.2.5 Physicochemical Characterizations ....................................................................... 44
3.2.5.1 ESEM and EDS Analysis ............................................................................... 44
3.2.5.2 XRD and FTIR Analysis ................................................................................ 44
3.2.6 Biological Characterizations ................................................................................. 44
3.2.6.1Cell Culture ..................................................................................................... 44
3.2.6.2 Cytotoxicity - Alamar Blue Assay and Light Microscopic Technique .......... 44
3.2.6.3 Hemolysis and RBC Morphology Analysis ................................................... 45
3.2.7 Three Dimensional Cell Culture using Magnetic Microsphere ............................ 46
3.2.7.1 ESEM Analysis .............................................................................................. 46
3.2.7.2 Live- Dead Staining and DAPI Nuclear Staining Evaluation ........................ 46
3.3 Theranostic Efficiency Evaluation of HAIO50 (Hyperthermia Therapy and MRI
Contrast Agent) .................................................................................................................. 47
3.3.1 Magnetic Hyperthermia Evaluation of HAIO50 and SLP Calculation ................. 47
3.3.2 HAIO50 in vitro Hyperthermia Evaluation ........................................................... 48
3.3.2.1 Quantitative Estimation of Dead Cell Population – FACS Analysis ............. 48
3.3.2.2 Quantitative Estimation of Cell Death Mechanism - FACS Analysis ........... 49
3.3.2.3 Hyperthermia Treated Cells Morphology Evaluation – ESEM Technique ... 49
3.3.3 Magnetic Resonance Imaging Contrast Efficiency of HAIO50 ............................ 49
3.3.3.1 In vitro MRI Analysis .................................................................................... 50
3.4 Improve the Theranostic Efficiency of Superpramagnetic Nanoparticles Through
Crystal Modification .......................................................................................................... 51
ix
3.4.1 Development of Manganese Substituted SPION (MnIO) Nanocrystal via an
Aqueous Co-precipitation .............................................................................................. 51
3.4.1.1Materials: ........................................................................................................ 51
3.4.1.2 Synthesis of MnIO ......................................................................................... 51
3.4.1.3 Development of Various Concentration of Mn2+ Substituted SPION ............ 52
3.4.1.4 Physicochemical Characterizations of MnIOs ............................................... 52
3.4.1.4.1 TEM and HRTEM analysis ..................................................................... 52
3.4.1.4.2 Powder X-ray Diffraction ....................................................................... 53
3.4.1.4.3 Fourier Transform Infrared Spectra (FTIR) ............................................ 53
3.4.1.4.4 Thermogravimetric Analysis ................................................................... 53
3.4.1.4.4 Inductively Coupled plasma-Optical Emission Spectroscopy ................ 53
3.4.1.4.5 ESEM and EDS spectrum ....................................................................... 54
3.4.1.4.5 Magnetic Property Measurement of MnIOs ........................................... 54
3.4.1.4 Biological Evaluations of MnIOs................................................................... 54
3.4.1.4.1 Cell Culture ............................................................................................. 54
3.4.1.4.2 Cytotoxicity - Alamar Blue Assay and Light Microscopy ...................... 54
3.4.1.4.3 Hemolysis Assay ..................................................................................... 55
3.4.1.4.4 Clotting Time .......................................................................................... 55
3.4.1.4.5 RBC Aggregation .................................................................................... 56
3.4.1.4.6 WBC Aggregation................................................................................... 56
3.4.1.4.7 Platelet Aggregation ................................................................................ 56
3.4.1.4.8 Cell Uptake ............................................................................................. 57
3.4.1.5 MnIOs Contrast Effect in Magnetic Resonance Imaging .............................. 57
3.4.2 Development of Surface Modified Manganese Substituted SPION ..................... 58
3.4.2.1 Materials ........................................................................................................ 58
3.4.2.2 Synthesis of Surface Modified MnIO Nanoparticles (MnIOTCs) ................. 58
3.4.2.3 Physicochemical Characterizations ................................................................ 59
3.4.2.3.1 Dynamic Light Scattering ....................................................................... 59
3.4.2.3.2 X-ray Diffraction Technique ................................................................... 59
3.4.2.3.3 Thermogravimetric Analysis ................................................................... 59
3.4.2.3.4 Transmission Electron Microscopic Analysis ......................................... 59
3.4.2.3.5 Fourier Transform Infrared Spectra ........................................................ 60
3.4.2.3.6 Vibrating Sample Magnetometry analysis .............................................. 60
x