Study on synthesis of combination of silver nanoparticles and mesenchymal stem cell products for wound healing

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY NGUYEN THI THANH HOAI STUDY ON SYNTHESIS OF COMBINATION OF SILVER NANOPARTICLES AND MESENCHYMAL STEM CELL PRODUCTS FOR WOUND HEALING MASTER'S THESIS VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY NGUYEN THI THANH HOAI STUDY ON SYNTHESIS OF COMBINATION OF SILVER NANOPARTICLES AND MESENCHYMAL STEM CELL PRODUCTS FOR WOUND HEALING MAJOR: NANOTECHNOLOGY CODE: 8440140.11QTD RESEARCH SUPERVISORS: Prof Dr Sc NGUYEN HOANG LUONG Associate Prof HOANG THI MY NHUNG Hanoi, 2020 ACKNOWLEDGMENTS First of all, I would like to express my deepest gratitude to my supervisors Prof Dr Sc Nguyen Hoang Luong and Assoc Prof Hoang Thi My Nhung for their enthusiastic guidance and inspiration throughout the implementation of the thesis I also wish to thank Assoc Prof Nguyen Hoang Nam, Dr Luu Manh Quynh (Center for Materials Science, VNU University of Science), Dr Le Tra My, MSc Bui Thi Van Khanh (Department of Cell Biology, VNU University of Science) for the wholehearted instruction and useful suggestion Besides, I am extremely grateful to Dr Than Thi Trang Uyen (Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Health Care System) for all her support My sincere thanks to lecturers in the Nanotechnology program for their helpful instruction when I have learned at Vietnam Japan University I am truly thankful for all the encouragement from my family and my friends My thesis would not be done without their support Finally, I would like to thank my classmates and my friends from Vietnam Japan University, VNU University of Science, Vinmec Research Institute of Stem Cell and Gene Technology who help me accomplish this thesis Hanoi, July 2020 Student Nguyen Thi Thanh Hoai i TABLE OF CONTENTS Page ACKNOWLEDGMENTS i TABLE OF CONTENTS ii LIST OF FIGURES iv LIST OF TABLES v LIST OF ABBREVIATIONS vi INTRODUCTION CHAPTER 1: OVERVIEW .3 1.1 Cutaneous wound and wound healing process .3 1.1.1 Cutaneous wound 1.1.2 The normal wound healing process .3 1.1.3 The two therapeutic targets in wound treatment 1.2 AgNPs – an outstanding antimicrobial and anti-inflammatory agent in the inflammation phase .7 1.2.1 AgNPs as a topical antimicrobial agent 1.2.2 AgNPs as an anti-inflammatory agent 10 1.2.3 Concerned factors for using AgNPs in wound treatment .11 1.2.3.1 Effect of particle size 12 1.2.3.2 Effect of capping agents 12 1.3 Products derived from MSC - cytokines and growth factors-modulated agent in wound healing 14 1.3.1 Stem cells and mesenchymal stem cells 14 1.3.1.1 What are stem cells (SCs)? 14 1.3.1.2 Mesenchymal stem cells (MSCs) 14 1.3.1.3 Products derived from MSCs 15 1.3.2 MSC-derived conditioned medium (CM) in wound healing 16 1.4 Combined using of silver nanoparticles and bio-factors for wound healing 17 CHAPTER 2: MATERIALS AND METHODS 19 2.1 Overview of experimental design 19 2.2 Preparation of AgNPs 20 2.2.1 Synthesis of AgNPs 20 2.2.2 Characterization of AgNPs 21 2.2.2.1 Physicochemical properties 21 2.2.2.2 Evaluation of the antimicrobial activity of AgNPs 22 2.2.2.3 Determination of the cytotoxic effect of AgNPs on NIH 3T3 cell 24 2.3 Preparation of CM and effect of CM on NIH 3T3 migration in vitro 26 2.3.1 Preparation of CM 26 2.3.2 Effect of CM on NIH 3T3 migration - Scratch assay in vitro 26 2.4 Skin wound model in vivo 29 ii 2.4.1 Deep partial-thickness burn wound model 29 2.4.2 Excisional wound model 31 2.4.3 Wound analysis 31 2.5 Statistical analysis 33 CHAPTER 3: RESULTS AND DISCUSSION 34 3.1 Characterization of AgNPs 34 3.1.1 Physicochemical properties 34 3.1.1.1 XRD pattern 34 3.1.1.2 TEM image 35 3.1.1.3 UV-Vis spectra 36 3.1.2 Evaluation of the antimicrobial activity of AgNPs 37 3.1.2.1 Sterility of AgNPs 37 3.1.2.2 Antimicrobial effect of AgNPs 38 3.1.3 Cytotoxic effect of AgNPs solution on NIH 3T3 cells in vitro 39 3.2 Effect of CM on NIH 3T3 migration - Scratch assay in vitro .44 3.3 Skin wound model in vivo 48 3.3.1 Deep second-degree burn model 48 3.3.2 Excisional model 51 CONCLUSIONS AND PERSPECTIVES 59 CONCLUSIONS 59 PERSPECTIVES 59 REFERENCES 60 iii LIST OF FIGURES Page Figure 1.1 Phases in wound healing Figure 1.2 The types of wound treatment applied for different wound categories Figure 1.3 Mechanism of antimicrobial action of AgNPs Figure 1.4 Factors impacted their cytotoxicity 11 Figure 1.5 MSC capacity of differentiation 15 Figure 2.1 Overall experimental design of the study 19 Figure 2.2 Schematic procedure of AgNPs synthesis 21 Figure 2.3 Examination of media on NIH 3T3 cells migration 28 Figure 2.4 Analysis of wound images by Image-J 29 Figure 2.5 Analysis of wound area by Image-J 32 Figure 2.6 Determination of wound area based on the stage of healing process 32 Figure 3.1 XRD pattern of synthesized AgNPs 35 Figure 3.2 TEM image shows the morphology of AgNPs and sizes of particles ranged from 10 to 45 nm 35 Figure 3.3 UV-Vis spectra of synthesized AgNPs 37 Figure 3.4 Agar plate without detection of microbial colony .38 Figure 3.5 AgNPs plates with less of microorganisms than the Control (-) plates 39 Figure 3.6 Morphology of NIH 3T3 cells 40 Figure 3.7 Image of 96-well plate after SRB staining 42 Figure 3.8 Cell viability measured by SRB assay on NIH 3T3 cells 43 Figure 3.9 Effect of media on the migration of fibroblast cells 45 Figure 3.10 The migration rate of fibroblast treated with media .46 Figure 3.11 The healing process of burn wounds in mice 48 Figure 3.12 Statistical analysis of healing rate of burn wounds at day 23 and day 30 after creating burns Values are represented as mean ± SD 49 Figure 3.13 Uneven healing rate in the MSC group 51 Figure 3.14 Statistical analysis of healing rate of excisional wounds with different treatments 52 Figure 3.15 The healing rate of excisional wounds 53 iv LIST OF TABLES Page Table 1.1: Topical antimicrobial agents for wound healing .7 Table 1.2: Effect of AgNPs size on cytotoxicity 12 Table 3.1 Number and size of microbial colonies in each group 38 Table 3.2 Descriptive qualitative assessment for the healing process in the burn model 50 v LIST OF ABBREVIATIONS AgNPs CM DFUs DMEM EGF ECM EVs FBS fcc FDA hUCB CM hUCB MSCs IL-1, IL-6, IL-8 IGF KGF MSCs OD PDGF ROS SDF SRB TEM TGF-α, TGF-β TSC UV-Vis XRD Silver nanoparticles Conditioned medium Diabetic foot ulcers Dulbecco’s modified Eagle’s medium Epidermal growth factor Extracellular matrix Extracellular vesicles Fetal bovine serum Face centered cubic Food and Drug Administration Human umbilical cord blood-derived mesenchymal stem cell conditioned medium Human umbilical cord blood-derived mesenchymal stem cells Interleukin-1, Interleukin-6, Interleukin-8 Insulin-like growth factor Keratinocyte growth factor Mesenchymal stem cells Optical density Platelet-derived growth factor Reactive oxygen species Stromal cell-derived factor Sulforhodamine B Transmission electron microscopy Transforming growth factor α, transforming growth factor β Trisodium citrate Ultraviolet visible spectroscopy X-ray diffraction vi INTRODUCTION Wounds are a silent burden on the healthcare system In 2018, Medicare beneficiaries analyzed that around 8.2 million people who have at least one type of wounds Wounds often classified into acute (traumatic, abrasions, surgical) and chronic wounds (diabetic foot ulcers (DFUs), leg ulcers, and pressure ulcers) based on healing time The challenges of healing wounds are the increase of infection, age and pathological background of the patient Hence, we need to come up with novel strategies to solve these problems Over the past few decades, silver nanoparticles (AgNPs) attract rapt attention in wound treatment due to various featured natures such as the history of using silver, simple and effective synthesized methods, and above all the outstanding antimicrobial activity These make AgNPs become one of the most widely used agents for preventing infection On the other hand, mesenchymal stem cells (MSCs) and products derived from MSCs, which appear as advanced therapies, have recently been studied and applied in the field of medicine In terms of wound healing, many studies suggest that paracrine signaling of MSCs rather than tissue differentiation and engraftment is a pivotal element for promoting wound healing That indicates the capacity to use conditioned medium (CM), which is one of the products derived from MSCs for wound treatment CM contains a variety of cytokines, growth factors, chemokines that modulate the healing process through induction of re-epithelialization, angiogenesis, and remodeling Therefore, we assume the synergistic effect of the combined use of AgNPs and CM, in which AgNPs with antibacterial, anti-inflammatory activities support CM to promote wound healing Our target is chronic wounds that require advanced therapies for treatment At the beginning of the research process, we aim to examine the healing effect of the combined use of AgNPs and CM on an acute wound, then perform it on a chronic wound model at a later stage This thesis is the first step of research, so in this study, three objectives need to be fulfilled (1) Synthesize and characterize properties of silver nanoparticles (AgNPs) including physicochemical properties, sterility, antimicrobial activity and cytotoxicity; (2) Evaluate the healing potential of conditioned medium (CM) by scratch assay in vitro; (3) Initially evaluate the therapeutic effect of each treatment: AgNPs and CM and the combined use of AgNPs and CM on the wound models in vivo 59 REFERENCES [1] A Williamson, Glen P Carter, Benjamin P Howden (2017) Current and emerging topical antibacterials and antiseptics: Agents, Action, and Resistance Patterns American society for microbiology 30(3), 827–860 [2] Agnihotri, N., Gupta, V., & Joshi, R M (2004) Aerobic bacterial isolates from burn wound infections and their antibiograms - A five-year study Burns, 30(3), 241–243 https://doi.org/10.1016/j.burns.2003.11.010 [3] Akter, M., Sikder, M T., Rahman, M M., Ullah, A K M 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Stem cells... using of silver nanoparticles and bio-factors for wound healing AgNPs have been used in combination with other materials for wound treatment The common strategy is to use the combination of AgNPs-antibiotic