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

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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....................................................................................................... 1 CHAPTER 1: OVERVIEW ........................................................................................ 3 1.1. Cutaneous wound and wound healing process ................................................ 3 1.1.1. Cutaneous wound ...................................................................................... 3 1.1.2. The normal wound healing process .......................................................... 3 1.1.3. The two therapeutic targets in wound treatment ....................................... 5 1.2. AgNPs – an outstanding antimicrobial and anti-inflammatory agent in the inflammation phase ................................................................................................. 7 1.2.1. AgNPs as a topical antimicrobial agent .................................................... 7 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. ..........................................................................4 Figure 1.2. The types of wound treatment applied for different wound categories. ...6 Figure 1.3. Mechanism of antimicrobial action of AgNPs. ........................................9 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 4 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 4 media on the migration of fibroblast cells. ...........................45 Figure 3.10. The migration rate of fibroblast treated with 4 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; 1 (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. 2 CHAPTER 1: OVERVIEW 1.1. Cutaneous wound and wound healing process 1.1.1. Cutaneous wound Wounds are considered a growing challenge for the healthcare system. There are variety of reasons that can lead to injury, from extrinsic factors, such as shear, thermal, pressure to underlying causes such as diabetes, stress [61]. The injuries not only cause burden to patients, family, and healthcare system but also resulted in significant economic costs. A retrospective analysis of Medicare beneficiaries (2018) reported that approximately 8.2 million people who suffered from at least one type of wounds with or without infection. The cost of wound care ranged from $28.1 billion to $96.8 billion involving costs for chronic and acute wounds [52]. Wound injuries are often classified into acute wounds including surgical wounds, traumatic, abrasions, or superficial burn, and chronic wounds, such as ulcers, diabetic foot ulcers (DFUs). Risk of chronic wounds is developed from an increase of age, the complication of diabetes, vascular diseases, obesity, etc. The market for advanced wound care for chronic and surgical wounds is expected to $22 billion by 2024 [61]. On the other hand, acute wounds are at risk of wound infection, particularly in postsurgery [61]. Another challenge for acute wounds is that prolonged healing can lead the wounds to enter a chronic state (non-healing) [16]. Therefore, novel concepts to prevent infection and promote the healing process are vital to managing wounds. 1.1.2. The normal wound healing process Wound healing is a dynamic process involving 4 phases – hemostasis, inflammation, proliferation, and remodeling, that overlap in time This process is regulated by a complex system of mediators, which are responsible for cell-cell communication, involving various cytokine, growth factors, and chemokines [9], [13], [53] (Figure 1.1). 3 Figure 1.1. Phases in wound healing [43]. Hemostasis begins immediately after an injury created, platelets form a plug and release several mediators, for example, platelet-derived growth factor (PDGF), which subsequently recruit leukocytes to the wound site. In the inflammatory phase, neutrophils start to cleanse the injury area from microorganisms and foreign contaminants, and then phagocytosed by macrophages or formed the eschars. Chemokines, transforming growth factor β (TGF-β), and monocyte chemoattractant protein 1 (MCP-1) are released that lead to the infiltration of monocytes to the injury site that later transformed to macrophages. The monocyte and macrophages play a crucial role in inflammatory phase by releasing various cytokine such as vascular endothelial growth factor (VEGF), colony-stimulating factor 1 (CSF-1), PDGF, transforming growth factor α (TGF-α), TGF-β, interleukin-1 (IL-1), etc. that initiate the formation of granulation tissue. The proliferation phase started with re-epithelialization, in which several growth factors including TGF-α, epidermal growth factor (EGF) and keratinocyte growth factor (KGF) were released to stimulate the proliferation of epidermal cells at the margin. Granulation tissue, which is the new stroma, forms in the wound site. The 4 concert of extracellular matrix (ECM) molecules and growth factors, PDGF, TGF-β induce fibroblasts around the wound to proliferate and migrate into the wound area. The structural molecules of new ECM involving fibrin, fibronectin, hyaluronic, providing a scaffold for cell migration and the formation of granulation tissue. The fibroblasts play an important role in synthesis, deposition, and remodeling of the ECM. Besides, the formation of new blood vessels, called angiogenesis, initiates with the angiogenic molecules such as acidic or basic fibroblast growth factor (aFGF, b-FGF), VEGF, TGF-β, angiogenin. After proliferation, the final phase is remodeling including the contraction and reorganization of ECM. Fibroblast and macrophage release several proteolytic enzymes called matrix metalloproteinases that degrade collagen type III of the granulation tissues. Collagen type I was replaced and aligned into paralleled fibrils, resulting in the formation of a scar. In summary, cytokines and growth factors are protein molecules that coordinate cellular processes. These act to regulate a wide range of functions involving cell proliferation, cell differentiation, angiogenesis, wound healing, tissue modeling, immune cell activity through autocrine, paracrine, juxtacrine, or endocrine mechanisms [13]. Hence, modulation of cytokines and growth factors can enhance the healing process. 1.1.3. The two therapeutic targets in wound treatment Wound care depends on the wound type, along with the purpose of treatment. Superficial burns are normally needed primary care, which is cleaning the wound and applying antibiotics. For deep partial-thickness and full-thickness burn, common treatment are topical antimicrobial agents, and skin grafts in case of a large-area wound. For non-healing wounds, the purpose of care is more complex including wound debridement, preventing injection, and avoiding the pressure at the wound site. The treatment approach for improving wound healing, was listed in Figure 1.2 [54]. 5 Figure 1.2. The types of wound treatment applied for different wound categories (adopted from [54]). For active wound care, several therapeutic strategies have been studying including using growth factor, chemokine, cell therapies, microRNA, and non-endogenous molecules such as silver-based agents, synthetic chemical pirfenidone (PFD), etc. [54]. For instance, a gel containing recombinant PDGF, which is a vital growth factor throughout the healing process, called Becaplerim showed to enhance wound healing in DFUs, was approved by Food and Drug Administration (FDA) for nonhealing wound treatment [55]. These approaches target different cellular processes 6 in wound healing, but one of the major targets is to regulate the cytokines, growth factors, chemokines involved in wound healing. Therefore, two targets that may be intervened to promote the healing process are (1) prevent external infections, and (2) administration of internal cytokines, growth factors, chemokines. In a recent study, the topical antimicrobial agent (AgNPs) was aimed to prevent the infiltration of microorganisms that enhance the inflammatory phase. Simultaneously, a product derived from MSCs, herein, conditioned medium (CM) display healing ability through regulating mediators (cytokines, growth factors, chemokines). The combination of the two strategies, which use the basic wound care to support the active wound care, is expected to create a synergistic effect to accelerate the healing process. 1.2. AgNPs – an outstanding antimicrobial and anti-inflammatory agent in the inflammation phase 1.2.1. AgNPs as a topical antimicrobial agent Preventing infection is the crucial target of effective wound management [56], [61]. Recently, the use of topical antibiotics and antiseptics are markedly increased. Topical antibiotic shows several benefits over the systemic use, such as the reduction in systemic toxicity, but can lead to rising bacterial resistance [1], [56]. Antiseptics, on the other hand, prefer more useful in the reduction of bacteria but more toxic than antibiotics [58]. Current representatives of topical antimicrobial agents, in which their benefits and drawbacks were listed in Table 1.1. Table 1.1: Topical antimicrobial agents for wound healing. Type Antiseptics Description Ref. Advantages: Broad spectrum of antimicrobial activity [58] Disadvantages: toxic to host cells Hydrogen Use for wound irrigation and remove necrotic tissues. [21] peroxide H2O2 was detected in normal healing, rapidly 7 (H2O2) decomposed to water and oxygen. High concentration cause cell damage through corrosion, formation of oxygen gas, and lipid peroxidation Povidone- A solution of 0.1 – 0.2% minimize cytotoxicity and rise [56] iodine the release of free iodine for antibacterial activity. Disadvantages: irritation, allergy, cytotoxic to vulnerable people (pregnant women, newborns, etc.) Chlohexidine Chlohexidine showed long-lasting residual ability, and [1] active activity against Gram-positive and Gramnegative bacteria but poor activity against nonenveloped viruses and bacterial spores. The mechanism of its action is disrupting the cytoplasmic membrane Alcohol Bactericidal action of an aqueous solution of 70% - [56] 92% alcohol is rapid, but short-time action, and can cause irritation and dryness. Nanoparticles: silver, NPs exhibit the bactericidal effect with wide-spectrum [49] gold, by the release of metal ions or generation of Reactive zinc (NPs) Oxygen Species (ROS). The toxicity can be governed by modulating several factors such as size, shape, concentration. Antibiotics: Advantages: the cytotoxic to host cells is less than [58] Bacitracin, antiseptic. Mupirocin, Bacitracin, Silver Disadvantages: Antibacterial spectrum is narrower, and resistance to antibiotics is more frequent than that of antiseptics. sulfadiazine Taking the advantages and disadvantages of these topical antimicrobial agents, nanoparticles turn out one of the most promising candidates. Together with the 8 development of nanotechnology, decreasing the size of the material to the nanoscale, for example, increases its surface-to-volume ratios, leading numerous advantages for applications [42]. The use of silver for preventing microorganisms and treating burns have seen for hundreds of years [63]. In the 18th -20th century, silver nitrate was widely used for treating burns and then a commercial product of silver sulfadiazine has been commonly used as topical antibiotics [75]. Over the past few decades, silver nanoparticles (AgNPs) attract great interest due to their well-known antimicrobial activity. The mechanism of this action is not fully understood yet, but it is suggested that this action is in relation with (1) AgNPs anchor to the cell wall of bacteria then penetrate it, altering the permeability of cell membrane, (2) AgNPs penetration damage the bacterial organelles including mitochondria, vacuoles, ribosomes, and denature protein, as well as DNA, (3) The formation of free radicals and generation of ROS, (4) AgNPs can modulate the signal transduction, dephosphorylate of peptides substrate on tyrosine residues, resulting in inhibition of cell growth [14], [57], [76] (Figure 1.3). Figure 1.3. Mechanism of antimicrobial action of AgNPs [14]. 9 Besides, AgNPs are easily incorporated into the cotton fabric and dressing and able to synthesize by simple and safe approaches [49]. The anti-inflammatory activity is another advantage of AgNPs in wound healing, which then is discussed further. 1.2.2. AgNPs as an anti-inflammatory agent The inflammatory phase is a range of early immunological response against bacteria and other foreign particles by the production of pro-inflammatory cytokines. In normal healing, both the pro-inflammatory and anti-inflammatory are present. However, if these responses take place inappropriately, a prolonged inflammatory phase can lead to non-healing wounds [16]. Scientific studies suggest that AgNPs possess anti-inflammatory activity besides the well-known antimicrobial activity. Tian et al. (2007), who first found evidence in the anti-inflammatory activity of AgNPs, compared the healing process between the mice treated with AgNPs and those treated with amoxicillin and metronidazole, two widely used antibiotics. The result showed that the AgNPs-treated group healed faster than the antibiotic-treated group, suggesting other influence of AgNPs besides antimicrobial action. The findings showed the expression level of IL-6 (pro-inflammatory cytokines) was lower in the AgNPs-treated group whereas IL-10 (anti-inflammatory cytokines), VEGF (angiogenic cytokine), TFN-γ (cytokines in remodeling phase) were stayed higher than the control group. The overall reduction in inflammation might be predicted, and the decrease of neutrophils in the wound area confirms it [67]. The efficiency of AgNPs in inflammatory reduction without toxic effects was found in a postoperative peritoneal adhesion model [77]. On another paper, this team reported AgNPs accelerated the rate of wound closure through the migration and proliferation of keratinocytes in the stage of reepithelialization. AgNPs, at the same time, inhibited the fibroblast proliferation, not due to their toxic effect. The enhanced expression of α-SMA (myofibroblast’s marker), suggested AgNPs stimulate the differentiation of fibroblast into myofibroblast in the healing process [40]. Other finding showed nanocrystalline silver solution exhibited the anti-inflammatory activity with a pH of 9 [48]. 10 Besides, AgNPs showed an influence on improving tensile properties of healed wounds, modulating collagen deposition in the wound healing process [36]. Taken together, not only do AgNPs display broad antimicrobial spectrum, but also exhibit anti-inflammatory properties through cytokine regulation at the inflammatory phase. This reinforces the potential of using AgNPs for wound treatment. 1.2.3. Concerned factors for using AgNPs in wound treatment AgNPs differ from size, shape, surface electric charge, and other physicochemical properties. AgNPs are aimed to use for wound repair, hence, the cytotoxicity (ability to destroy cells), genotoxicity (property that damage genomic information) of AgNPs are needed to take into account apart from antimicrobial activity. Size, range of concentration, and agglomeration are vital factors impacting their cytotoxicity [3] (Figure 1.4). Figure 1.4. Factors impacted their cytotoxicity. 11 1.2.3.1. Effect of particle size Size is considered to be the most relevant factor to the cytotoxicity of AgNPs. AgNPs revealed the effect on cell viability, ROS generation, lactate dehydrogenase (LDH) activity relying on sizes as well as testing cell lines (Table 1.2). Table 1.2: Effect of AgNPs size on cytotoxicity. Synthesized Size method (nm) Chemical Cell type Findings 5, 20, Human cells: Smaller particles Ref. were [39] reduction method 50 A549, easier to enter cells than (PVP coated) HepG2, SGC- larger ones, causing higher 7901, MCF-7 toxicity Unknown method 10, 50, Human liver Size-dependent (PVP-coated) 100 cells HepG2 through toxicity [45] autophagy activation Unknown method 15, 30, Alveolar Increase of ROS level when [11] (Hydrocarbon- cells treated with AgNPs 15 55 macrophages coated) nm suggested cytotoxicity through oxidative stress. Unknown method 20, Citrate-coated AgNPs 20 [73] (citrate-coated) nm generates neutrophilic 110 inflammation in lung compared to citrate-coated 110 nm In general, smaller particles display higher toxic than larger counterparts since they internalize more easily into cells, leading to DNA damage, and ROS production. 12
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