LUẬN văn THẠC sĩ 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 1.1 Cutaneous wound and wound healing process 1.1.1 Cutaneous wound 1.1.2 The normal wound healing process 1.1.3 The two therapeutic targets in wound treatment 1.2 AgNPs – an outstanding antimicrobial and anti-inflammatory agent in the inflammation phase 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com LIST OF FIGURES Page Figure 1.1 Phases in wound healing Figure 1.2 The types of wound treatment applied for different wound categories .6 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com LIST OF TABLES Page Table 1.1: Topical antimicrobial agents for wound healing 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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; LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com (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 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Figure 3.15 The healing rate of excisional wounds All values are expressed as the mean ± SD p ≤ 0.05 compared with groups On day 3, the wounds in the AgNPs-treated groups were significantly closed, with approximately 47% Followed by combined use group and control group, with around 41% and 40%, respectively On day 7, the healing rate of AgNPs and combined use groups were nearly equal, with approximately 80%, and the control group with 77% Meanwhile, CM and MSCs displayed similar healing ability and were slower than the other groups in early-stage and subsequently accelerated in the later stage of the healing process The wound closure rates in both groups were about 35% and 65% on day and day 7, whereas completed 96% wound area in day 10 (Figure 3.15) Discussion a) AgNPs Burn wound supplied a suitable environment for bacterial multiplication and was the predominant reason for infections normally due to the large area impacted [2] 53 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com In many cases, the level of bacteria that suppressed the healing process but did not express the standard clinical sign of infection has been called “critical colonization” At this level, burn wounds have become infected and virtually hard to heal [46] On the other hand, removing skin in the excisional wounds (open injuries) facilitated bacteria to penetrate the wound The risk of wound infection after trauma ranges from to 17.5% [16], [19] In this study, AgNPs displayed the most remarkable healing effect among groups in both wound models In the burn wound model, the presence of pus, redness in the wound site, which was the normal inflammatory response seen by the naked eyes, was not observed due to the covered burnt skin However, it could be seen the eschar of wounds in the AgNPs group had completely peeled off after 15 days This demonstrated that epidermal cells at the margin of the wound had proliferated and fibroblast cells around the wound site had migrated and proliferated into the wound area That means the inflammatory phase was promoted and the re-epithelialization phase had been started earlier in the AgNPs than other groups Similar observation was seen on the excisional wound model, re-epithelialization phase demonstrated the significant healing rate in the early stage (i.e day and day 7) The key factor to enhance the inflammatory phase is the prevention of bacteria It was clear that AgNPs have played excellent roles in preventing infection in both experiments AgNPs exhibited the antimicrobial activity through the ability to adhere to the membrane of bacteria, lead to altering the permeability of the membrane, penetrate the cells and interact with organelles and biomolecules, result in changing mitochondria function, denaturing proteins, and DNA [14], [24], [76] These results are matched to other studies on the treatment of burn and excisional wounds On the study on the burn injuries which were created by contacting mouse skin with a heated metal plate (same approach described in part 2.4.1), and subsequently examined the antimicrobial effect of AgNPs against Staphylococcus aureus and Escherichia coli detected in the burn wound In the control and commercial silver sulfadiazine (SSD) group, S aureus count rose and reached the 54 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com maximum from day to day 17, whereas that of the AgNPs-treated group was reached a peak on day 12 and then significantly decreased Surprisingly, E coli was still detected in the control and SSD-treated group and was not markedly found in the AgNPs-treated group throughout the healing process [74] Also, the enhanced and excessive production of pro-inflammatory cytokines could tend to prolong the inflammatory phase [16] It is suggested that apart from preventing infection, AgNPs might display anti-inflammatory activity by regulating the expression of pro-inflammatory cytokines and anti-inflammatory cytokines in the inflammatory phase Tian el at., 2007 reported that during the process of healing, level of cytokines including IL-6, TGF-β1, IL-10, VEGF, and IFN-γ in the AgNPstreated group were significantly different compared to the control group They found that level of IL-6 was maintained lower during the process IL-6 was proinflammatory cytokine, which considered as a pioneer of inflammation events after injury IL-6 enhanced inflammatory response through the activation of monocyte and macrophage Decreased level of IL-6 might result in fewer immune cells (neutrophils and macrophages) recruited to the wound site, then lower paracrine signal for cellular migration and proliferation, and synthesis of ECM Therefore, their findings confirmed AgNPs induced IL-6 reduction, leading to an accelerating inflammatory phase and overall healing process [7], [67] Additionally, AgNPs regulated pro-inflammatory and anti-inflammatory cytokines in the early stage of the healing process AgNPs suppressed IL-6 expression significantly at day and day 3, resulting in the reduction of prolonged inflammation, hence promoting wound healing [81] b) CM, MSC and Culture medium In the burn model, the eschar of wounds in the CM group had also completely peeled off after 15 days, suggesting the proliferation of epidermal cells and fibroblast cells at the wound site That means CM enhanced the re-epithelialization phase in the healing process However, in the excisional model, the CM group exhibit slower closure than the other groups in early-stage and then accelerated in 55 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com the later stage of the process, by 35% and 65% on day and day 7, and completed 96% wound area in day 10 Fibroblasts located surround the wound migrate and proliferate to the wound area is one of the early events in the proliferation phase In this study, the effect of CM on the migration of fibroblasts has been proved in the scratch assay in vitro Therefore, it was likely that in this burn model, CM promoted the migration of fibroblasts, thus accelerated wound healing Besides, wounds of the CM groups nearly healed after 23 days, with approximately 90%, and completed after 30 days with normal scars, suggesting CM affected wound contraction and remodeling Several studies reported a variety of cytokines and growth factors found in CM that involved these stages Umbilical cord CM (UC CM) increased fibroblast migration and proliferation and changed the phenotypic characteristics of fibroblast, including a decreased ratio of TGF-β1/TGF-β3 and an increased ratio of MMP/TIMP [38] Human bone marrow-derived MSC CM (BM CM) induces re-epithelialization, tissue formation, angiogenesis in a deep second-degree burn model in rat [6] Meanwhile, many crucial anti-aging cytokines and growth factors in hUCB MSC CM such as EGF, bFGF, TGF-β, PDGF, HGF in which these cytokines mostly acted to induce fibroblast migration and proliferation, forming of new blood vessels and aligning ECM They also reported growth differentiation factor (GDF-11) induced growth and production of ECM protein involving Collagen type I, III, elastin, and fibronectin in human dermal fibroblast [34] In the case of MSCs, the MSCs-treated groups showed almost the same healing rate as the CM-treated group This result coincided with recent studies on its healing mechanisms that paracrine signaling is the main mechanism for the healing effects of MSCs [10], [27], [41] That means CM acts relatively similar mechanisms as MSCs on wound healing, primarily through autocrine and paracrine effects of cytokines and growth factors in the healing process Compared between Culture medium and CM groups, there was a conflict between results of the scratch assay in vitro and wound burn model in mice in vivo In the 56 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com scratch assay, the Culture medium group accelerated artificial “wound” closure faster than the CM group However, in the burn wound model, CM revealed better healing properties than the Culture medium group This could be explained by secretome, which means trophic factors presented in these media Culture medium was serum-free optimized for cell expansion in vitro, including manipulated recombinant factors Therefore, it was understandable that the Culture medium group healed faster than other groups in vitro On the other hand, CM involved in various factors released from MSC, these are key elements that contributed to wound repair This proved that trophic factors secreted from MSCs were more natural and exhibited better wound healing properties than recombinant factors in Culture medium c) The combined use of AgNPs and CM The result from the burn model suggested the potential of using CM derived from hUCB MSCs for burn wound treatment besides MSCs-based therapy Both AgNPs and CM exhibited healing capacity for burn treatment Therefore, it was expected the synergic effect of the combined use of AgNPs and CM for wound repair Interestingly, in cases of combined use and CM only groups, the combined-use group was seen accelerated healing rate, by 41% compared with 35% of the CM group in day The difference between healing rate of the combined use and CM group was more noticeable with 80% and 65% in day 7, respectively Due to the noticeable difference in the early stage, it is suggested that AgNPs promoted the inflammatory phase of the combined group via antimicrobial and anti-inflammatory activity, thereby enhance the proliferation, re-epithelialization and contraction phases For this reason, it might be the first signs of the synergic effects of using both treatments, although the mechanism of action (i.e which cytokines, growth factor are contained in CM, and the action of AgNPs and CM in each phases) needs to be studied further It could be found that the excisional model (acute wounds) is not a suitable model for examining the healing effect of treatments because it only can be seen the 57 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com difference among the groups at the beginning stage and it is difficult to see the distinction at the later stage However, the combined use of AgNPs and CM displays potential for acute treatment, so we can expect its effectiveness in other mouse wounds such as chronic wounds Otherwise, it is needed to consider a more effective method to combine them and investigate the underlying mechanisms of this combination 58 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com CONCLUSIONS AND PERSPECTIVES CONCLUSIONS Citrated-coated AgNPs were synthesized by the chemical reduction method AgNPs exhibited the spherical shape with the size ranged from 17.5 to 42.5 nm AgNPs possess sterility, antimicrobial activity and mild cytotoxicity on mouse fibroblasts with the values of IC50 from 26.02 – 30 µg/ml CM derived from human umbilical cord blood MSCs exhibited the effect on the migration of NIH 3T3 fibroblast cells in vitro Both AgNPs and CM displayed the healing nature for wound treatment, in which AgNPs showed the most prominent influence among treatments in both two models Besides, the combined use of AgNPs and CM revealed as the promising candidate for wound repair on the excisional wound model PERSPECTIVES In the future, we look forward to researching further on a more effective approach to combine AgNPs and CM as well as mechanisms of this combination 59 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 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., 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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