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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: (91)0471-2520271 Fax: (91)0471-2341814 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

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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 1.4 Magnetism and Superparamagnetic Nanoparticles . 5.2.3 Three Dimensional Cell Culture using Magnetic Levitation Technique 138. 5.2.4 Magnetic
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