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.
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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.
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