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\begin{document}

\title{Preparation of Chitosan Embedded Recombinant Human Epidermal Growth
Factor Nanoparticles as Accelerating Compounds for Skin Remodeling in
Chronic Lesions}



\author[1]{saadeh hashemi}%
\author[1]{Elnaz Mihandoost,}%
\author[2]{Sepideh Khaleghi}%
\affil[1]{Islamic Azad University Tehran Medical Sciences}%
\affil[2]{Islamic Azad University}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Chronic lesion has become a major biological burden for individual
patients and health organizations. Using nanoparticles as drug delivery
systems is remarkable nowadays. The unique properties of chitosan
without any toxicity for living creations make it a suitable option for
drug delivery. Epidermal growth factor (EGF) is one of the important
agents for wound healing, cellular proliferation, extracellular matrix
formation, and skin remodeling. A combination of these properties can
accelerate the wound healing process. In this study, rh-EGF is embedded
into the chitosan nanoparticles by the Ion-gelation method.
Nanoparticles are characterized by TEM microscopy and the DLS method and
conjugation efficacy is measured by FT-IR radiation. The antibacterial
effect of manipulated nanoparticles was estimated by MIC/MBC methods.
The cytotoxicity and proliferation were measured by MTT assay on the
HFF-1 human fibroblast cell line. Migration assay was accomplished by in
vitro scratch model and the gene expression analysis for TGF-\selectlanguage{greek}β, \selectlanguage{english}VEGF,
and PDGF were manipulated by the real time-PCR method. The obtained
results were considered statistically significant with P \textless{}
0.05. Obtained results illustrated no toxic effect on the HFF-1 cell
line treated with Chitosan-EGF (CS-EGF). In cellular proliferation and
migration assays, CS-EGF nanoparticles demonstrated a better effect than
free rh-EGF. For the duration of 72h of the experiment, the whole
scratch was covered by fibroblasts. The real time-PCR analysis also
showed upregulation of all TGF-\selectlanguage{greek}β, \selectlanguage{english}VEGF, and PDGF genes. As CS-EGF
nanoparticles in the acceleration of the skin remodeling process showed
promising results, subsequent studies might be useful.%
\end{abstract}%



\sloppy


\textbf{Preparation of Chitosan Embedded Recombinant Human Epidermal
Growth Factor Nanoparticles as Accelerating Compounds for Skin
Remodeling in Chronic Lesions}

Saadeh Hashemi\textsuperscript{\#1}, Elnaz
Mihandoost\textsuperscript{1\#}, Sepideh Khaleghi\textsuperscript{1*}

\emph{\textsuperscript{1} Department of Biotechnology, Faculty of
Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad
University, Tehran, Iran.}

\textsuperscript{\#} Co-first author: Saadeh Hashemi, Elnaz Mihandoost

\textsuperscript{*} Corresponding Author: Sepideh Khaleghi, Ph.D.,
s.khaleghi@iautmu.ac.ir

\textbf{Acknowledgments}

We are grateful to Tehran Medical Sciences, Islamic Azad University for
their general support. There is no financial support in this study.

\textbf{Abstract}

Chronic lesion has become a major biological burden for individual
patients and health organizations. Using nanoparticles as drug delivery
systems is remarkable nowadays. The unique properties of chitosan
without any toxicity for living creations make it a suitable option for
drug delivery. Epidermal growth factor (EGF) is one of the important
agents for wound healing, cellular proliferation, extracellular matrix
formation, and skin remodeling. A combination of these properties can
accelerate the wound healing process.

In this study, rh-EGF is embedded into the chitosan nanoparticles by the
Ion-gelation method. Nanoparticles are characterized by TEM microscopy
and the DLS method and conjugation efficacy is measured by FT-IR
radiation. The antibacterial effect of manipulated nanoparticles was
estimated by MIC/MBC methods. The cytotoxicity and proliferation were
measured by MTT assay on the HFF-1 human fibroblast cell line. Migration
assay was accomplished by\emph{in vitro} scratch model and the gene
expression analysis for TGF-\selectlanguage{greek}β, \selectlanguage{english}VEGF, and PDGF were manipulated by the
real time-PCR method. The obtained results were considered statistically
significant with P \textless{} 0.05.

Obtained results illustrated no toxic effect on the HFF-1 cell line
treated with Chitosan-EGF (CS-EGF). In cellular proliferation and
migration assays, CS-EGF nanoparticles demonstrated a better effect than
free rh-EGF. For the duration of 72h of the experiment, the whole
scratch was covered by fibroblasts. The real time-PCR analysis also
showed upregulation of all TGF-\selectlanguage{greek}β, \selectlanguage{english}VEGF, and PDGF genes. As CS-EGF
nanoparticles in the acceleration of the skin remodeling process showed
promising results, subsequent studies might be useful.

\textbf{Keywords:} Chitosan nanoparticles; Epidermal growth factor; Skin
lesion; \emph{In vitro} model\emph{.}

\textbf{Introduction} :

Wound healing is a complex physiological process conducted by a variety
of cellular and molecular interactions involving fibroblasts,
endothelial cells, keratinocytes, and immune cells which are mediated by
essential factors such as growth factors and cytokines. There are four
levels of wound healing: hemostasis, inflammation, proliferation, and
remodeling. In the initial phase, the onset of hemorrhage in the wound
site triggers the platelets. Their degranulation, then, releases alpha
granules which cause the abandonment of growth factors such as epidermal
growth factor (EGF), platelets-derived growth factor (PDGF), and
transforming growth factor-beta (TGF-\selectlanguage{greek}β\selectlanguage{english}). The outcome of this releasing
cascade is an influx of inflammatory cells, like neutrophils and
macrophages, into the wound site and starting the next phase:
inflammation. Within the next 24h neutrophils, macrophages, and
lymphocytes influx at the site of injury. They begin to clear debris and
bacteria from the wound site to facilitate the next phases of wound
healing. In this process, macrophages are the key cells to intricate
re-epithelialization, granulation, angiogenesis, and wound contracture.

Among the aforementioned phases, inflammation is an important one in the
process of wound healing. Disruptions in this phase, such as infection
and poor cellular response, can lead to poor reformation, scar
formation, and eventually chronic wounds. The proliferative phase
involves capillary budding, extracellular matrix formation, and
proliferation and influx of keratinocytes by the migration of the hair
follicle stem cells near the leading edge of the wound. Then
angiogenesis begins by the vascular endothelial growth factor (VEGF) and
some of the fibroblasts differentiate to myofibroblasts to close the
wound using their contractile function. Moreover, immature collagen
(type III) will also be generated, which has the main role in the
perfect appearance of the skin. In the next phase, the type III collagen
replaces the type I and the apoptosis of the remaining cells of the
previous phase happens (1, 2).

Flagging at the inflammation phase may cause a chronic wound. This
problem occurs by systemic and local reasons such as age, obesity,
vascular disease, and infection. The main reason for the infection is
bacterial colonization in the wound site. Other factors such as diabetes
and immunosuppressive disease can turn an acute wound into a chronic
ulcer, as well. Arterial and venous leg ulcers, diabetic foot ulcers,
pressure ulcers, rheumatologic ulcers and burn ulcers are the most
important chronic wound types (3, 4).

Epidermal growth factor (EGF) is a 6-KDa protein that is secreted
primarily by platelets, macrophages, and fibroblasts and has a paracrine
effect on keratinocytes. It has been confirmed that EGF accelerates the
migration of fibroblasts to promote the proliferation phase. EGF
expression is upregulated in the initial phases of wound healing.
Epidermal Growth Factor Receptors (EGFR) turn on a signaling cascade to
promote cell motility, cellular differentiation, protein secretion,
mutagenesis, and apoptosis. Inordinate infiltration of neutrophils
causes over-production of ROS, which damages ECM and cell membrane.
Moreover, the release of serine protease from neutrophils degrades
essential growth factors such as EGF and PDGF. On the other hand, lack
of sensation in newborn cells leads to bad localization and
down-regulating in EGF receptors. These incidents suggest that although
EGF family production is increased, their bio-availability is decreased.
Hence, the process of wound healing stops at the inflammatory phase,
making the wound a chronic ulcer (1, 5, 6). Certain types of wounds
require long-term hospital care where all kinds of nosocomial infections
happen. From a molecular point of view, bacterial infection is one of
the important reasons for turning an acute wound into a chronic one.
Bacterial colonization in the epidermis layer, the presence of matrix
metalloproteins in the dermis layer, and abnormal filtration of
neutrophils and macrophages, as a result of bacterial infection
response, impede the wound healing process in the inflammation phase.
Underlying disease and obesity will also make the repair process more
difficult (4). In the progression of a chronic wound, bacterial presence
in the wound site can lead to an abnormal neutrophil secretion which
aims to remove bacterial growth; however, the abandonment of serine
proteinase and over-producing reactive oxygen species (ROS) may degrade
both ECM and the growth factors. The use of nanomedicine can inhibit
bacterial infection while improving the absorption and stability of
biological drugs.

Chitosan is a natural linear polysaccharide that is obtained from
crustacean shell wastes. The composition of two monomeric units, namely
N-acetyl-2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-glucose, make
it soluble in acidic solvents, and the \selectlanguage{greek}β\selectlanguage{english}1,4-linkage results in the
unbranched structure of this biopolymer (7). \emph{In vivo} and \emph{in
vitro} studies on chitosan show no toxic effects on human and animal
models (8-10). Its antibacterial effects on gram-negative and
gram-positive bacteria (8, 11, 12), hemostatic function (13), and
biodegradable properties (14) also make chitosan a suitable choice for
dressings and drug delivery. In drug delivery systems based on chitosan
nanoparticles, many methods such as emulsion cross-linking,
Coacervation-precipitation, spray drying, Emulsion-droplet coalescence,
Ionic gelation, Reverse micellar, and Sieving has been developed (8,
15).

Chitosan nanoparticles can accelerate the remodeling phase of the wound
healing process by stimulating interleukin 8 (IL-8) secretion from
fibroblasts, which results in angiogenesis and migration of neutrophils
to the wound site (16). Chitosan also promotes granulation by the
proliferation of fibroblasts and enhances neutrophil migration (17, 18).
It can decrease scar formation in the re-epithelialization phase as well
(17). The ability of chitosan to increase the expression of TGF-\selectlanguage{greek}β
\selectlanguage{english}accompanied by collagen production in the early post-injury phase and
decreasing TGF-\selectlanguage{greek}β \selectlanguage{english}expression in the last injury phase prevents scar
formation and causes better re-epithelialization in the process of wound
healing (7, 19).

Time-controlled delivery of drugs is another advantage of chitosan gels,
which enhances the treatment process in wound dressing (8, 20). Chitosan
can also pass through cellular junctions, subsequently affecting claudin
and occludin expression depending on the cell lines and this, in turn,
facilitates drug delivery (21, 22). The combination of the growth factor
family and chitosan nanoparticles increases the GF family half-life,
shows no toxicity and does not affect the expression of interleukin 6
(IL-6) and TNF-\selectlanguage{greek}α \selectlanguage{english}(23).

In the present study, rh-EGF nanoparticles were manipulated by embedding
rh-EGF into the chitosan nanoparticles to accelerate the wound healing
process, by omitting the infection from the lesion site, and fibroblasts
proliferation as migratory and productive cells for rapid treatment.
Although chitosan nanoparticles as carriers might prevent scar formation
at the end of the remodeling process, using a combination of
antibacterial agents with a growth factor from upstream of a healing
process cascade makes a proper dressing to avoid chronic lesions.

\textbf{Materials and Methods}

Materials:

Chitosan (CS) (Low molecular weight), Human Recombinant Epidermal Growth
Factor (rh-EGF), sodium tripolyphosphate (TPP), and sodium hydroxide
(NaOH) were purchased from Sigma Aldrich. Human normal fibroblast cell
line (HFF1) for cytotoxicity assay and \emph{Staphylococcus aureus (S.
aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E.
coli),}and \emph{Pseudomonas aeruginosa (P. aeruginosa)} for
antibacterial studies were obtained from Pasture Institute of Iran.

Preparation of CS Nanoparticles:

CS nanoparticles were produced as the result of ionic cross-linking of
CS with TPP (24-27). About 1\% CS solution was prepared in 20ml of 1\%
acetic acid followed by adding 2ml of TPP 1\% to the aqueous solution
while sonicating. Then the solution was stirred for half-hour at room
temperature. The final product was obtained by dialyzing in PBS buffer
overnight at 4\selectlanguage{ngerman}°c temperature.

Preparation of CS-EGF Nanoparticles

About 1\% CS become solved in 20ml of 1\% acetic acid. 1 mg/ml rh-EGF
was added to the solution while stirring at room temperature for 1h,
then 2ml of TPP 1\% was added to the aqueous solution while sonicating.
The dissolved nanoparticles were stirred for 30 minutes at room
temperature. The final product was obtained by dialyzing in PBS buffer
overnight at 4°c temperature.

Physicochemical Characterization of Nanoparticles

The size and dispersion of nanoparticles were measured by dynamic light
scattering (DLS) (28) at 623nm (Scatterscope I, Qudix company, south
Korea). The size of particles was further measured by Transmission
Electron Microscopy (TEM) and FT-IR spectra of the corresponding
nanoparticles were recorded to measure the protein-nanoparticle
conjugation (29) (8400S, Shimadzu, Japan).

Determination of Conjugation Efficiency

To purify rh-EGF non-covalent conjugated nanoparticles from free rh-EGF,
the ensuing nanoparticles were centrifugated at 14000 rpm for thirty
minutes, and then, the absorbent of supernatant was measured at 280nm
wavelength to assess the concentration of free rh-EGF. After that, the
efficiency of conjugation was calculated by the following equation:

\begin{equation}
\text{The\ efficiency\ of\ conjugation}=\frac{The\ concentration\ of\ total\ protein-\ Concentration\ of\ free\ protein}{\text{The\ concentration\ of\ total\ protein}}\ \times 100\nonumber \\
 \end{equation}

\emph{In vitro} Release Studies of CS-EGF Nanoparticles

In this phase, 1mL of CS-EGF nanoparticles with a concentration of 1
mg/ml was placed in a dialysis sac, with a pore size of 12 KDa,
containing PBS buffer (pH 7.4) at 37\selectlanguage{ngerman}°c temperature. At the time of the
experiment, the dialysis tube was placed in a beaker containing 100 mL
of the release medium, maintained at 37ºC, and agitated at 80 rpm in a
water bath. The samples were then analyzed using a spectrofluorometer
and the release profile was observed (30). At different time intervals,
30\selectlanguage{greek}μ\selectlanguage{english}L of the medium was removed and replaced with fresh medium. The
excitation wavelengths were 342nm with an emission wavelength of 260nm.

Antibacterial Activity Measurement

Antibacterial activity of CS-EGF NPs and CS NPs was assessed against 4
strains, including \emph{Staphylococcus aureus (S. aureus)}
and\emph{Bacillus subtilis (B. subtilis)} as gram-positive bacteria,
and\emph{Escherichia coli (E. coli)} and \emph{Pseudomonas aeruginosa
(P. aeruginosa)} as gram-negative ones, by determining the MIC and MBC
concentrations. The prepared nanoparticle, CS and CS-EGF, were added to
all 4 groups of bacterial culture medium with 1 mg/ml concentration and
the amount of each nanoparticle increased within hours. The results were
measured using broth microdilution. The data were obtained by analyzing
minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC). In each test, an antibiotic was used as a positive
control for monitoring the influence of reagent on the growth and
mortality of bacteria. In this test, Vancomycin was used as a positive
control.

Cell culture

The human HFF-1 normal cell line was purchased from the Pasture
Institute of Iran. This cell line was separately cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10\% FBS, 2 mM
glutamine, 100 \selectlanguage{greek}μ\selectlanguage{english}gmL-1 streptomycins, and 100 IU mL-1 penicillin for
cellular assessment at 37 \selectlanguage{ngerman}°C in a humidi\selectlanguage{english}fied atmosphere of 5\% of CO2.

Cell viability and proliferation assay by MTT assessment

Cells were grown in a 96-well culture plate (Corning, NY) containing
15x10\textsuperscript{3} cells in 200 \selectlanguage{greek}μ\selectlanguage{english}L of DMEM medium. The
cytotoxicity measurement was accomplished with 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (MTT)
reagent, as a colorimetric assay, by transforming yellow Tetrazolium to
purple. For measuring NPs cytotoxicity, free rh-EGF, CS, and CS-EGF were
added to each sample at three frequencies at the concentration of 10\selectlanguage{greek}μ\selectlanguage{english}M
followed by performing an MTT assay after 24, 48, and 72h. Optical
density was measured at 570nm wavelength. Three parallel measurements
were carried out for each sample.

Determination of Cellular Migration

To determine cellular migration, the HFF-1 cell line was cultured in
6-well culture plates. Cells were cultured overnight to cover the
surface completely. Then, a scratch was made in exact dimensions to make
a break between cells. Subsequently, the culture media was limited to
the minimum concentration without FBS to prevent the effect of
proliferation on the closure of the scratch area. Afterward, free
rh-EGF, CS, and CS-EGF samples were added to three different wells at
the concentration of 10\selectlanguage{greek}μ\selectlanguage{english}M and three wells remained intact as control.
After 24h cell migration was observed by invert microscopy of the cell
culture.

Gene Expression Studies

Three main genes in the wound healing process, including TGF\selectlanguage{greek}β, \selectlanguage{english}VEGF, and
PDGF, were studied by the Real time-PCR method. After cellular treatment
by free rh-EGF, CS, and CS-EGF, the maximum response dose was chosen for
determining gene expression (10\selectlanguage{greek}μ\selectlanguage{english}M). To extract RNA, Trizol reagent was
used (31), with the minimum cell number of one million in each flask. To
ensure the purity and quality of the extracted RNA, the wavelength of
260nm and the ratio of 260/280nm were measured using a UV
visible-spectrophotometer (BIOTEK). Moreover, the quality assessment of
RNA extraction was determined using electrophoresis in agarose gel (2\%)
and three bands of 28s, 18s, and 5s rRNAs were observed (data not
shown). Complementary DNA synthesis was conducted based on the protocols
proposed in the BIOFACT kit (BioFACT, Korea). In this method, the
reverse transcriptase (RT) enzyme and a mix of oligo (dt) and random
hexamer primers were used. Then cDNAs obtained from the treated cell
lines were amplified with particular primers of the wound healing genes
and the internal control gene (GAPDH). The information related to the
sequence and length of the primers and the accession numbers is shown in
Table 1. To analyze the data and quantitatively assess gene expression,
the Pfaffl method (32) is used to calculate relative gene expression
data while accounting for differences in primer efficiencies.

\(Gene\ expression\ ratio=\frac{{(E_{\text{GOI}})}^{Ct\ GOI}}{{(E_{\text{HKG}})}^{Ct\ HKG}}\)

Statistical Analysis:

Statistical analysis was accomplished by IBM SPSS V.22.0 (IBM Inc, USA)
using a ONE-WAY ANOVA sample and T-test. Values are considered
statistically significant with P \textless{} 0.05.

\textbf{Results}

3.1. Physicochemical Characterization of Nanoparticles:

Nanoparticles of CS and CS-EGF were synthesized by chemical
cross-linking with TPP. CS nanoparticles were obtained as a result of
the ionic cross-linking between the positively charged protonated amine
of chitosan and negatively charged phosphate groups of TPP. By adjusting
the concentration of the precursors and stirring speed, the degree of
cross-linking and, thus, the particle size will be controlled. CS-EGF
nanoparticles were formed using the same mechanism; the positively
charged amino groups of chitosan were absorbed into the negatively
charged carboxyl groups of EGF-chitosan.

3.1.1. TEM Analysis:

The size and morphology of the prepared nanoparticles were determined
using TEM. The absolute diameters of chitosan and EGF-Chitosan
nanoparticles were approximately 100\selectlanguage{ngerman}±10nm and 100±50nm respectively
(Fig. 1A, B).

3.1.2. Particle-size Analysis:

Using dynamic light scattering (DLS) measurements, the size distribution
of the 2 types of nanoparticles were obtained. The average hydrodynamic
diameter of chitosan and EGF-Chitosan nanoparticles were 150±10nm and
150±50nm respectively (Fig. 1C).

3.2. FT-IR Studies:

Fig. 2 shows the FT-IR spectrum of chitosan and EGF-Chitosan
nanoparticles. A characteristic peak at 344.98
cm\textsuperscript{-1}appeared in the infrared spectrum of chitosan,
which can be attributed to the --OH groups stretching vibration, and a
peek at 1600.97 cm\textsuperscript{-1} for the amide I. Moreover, the
spectrum of polysaccharide conjugation of C-O and C-O-C bands is visible
at 993.37-1157.33 cm\textsuperscript{-1}. In the EGF-Chitosan spectrum,
a peak at 2924.18 cm-\textsuperscript{1} appeared that is related to the
conjugation between amino groups of chitosan and phosphates groups of
TPP. Also, the 1635.69 cm-\textsuperscript{1} spectrum shows the
presence of non-covalent bonds between carboxyl groups of protein and
amino groups of chitosan, which indicates chitosan and rh-EGF
absorption.

3.3. Conjugation Efficiency Studies:

The primary rh-EGF concentration was 1mg. The protein absorbance was
0.712 at 280nm wavelength associated free protein concentration was
0.324mg with an absorbance of 0.231 at 280nm wavelength. the ultimate
conjugation effectivity was calculated at regarding 70\% using the
subsequent equation:

\begin{equation}
Conjugation\ efficiency=\frac{1-0.231}{1}\times 100=67\%\simeq 70\%\nonumber \\
 \end{equation}

3.4\emph{. In vitro} Release of Embedded EGF CS NPs:

The release profile of EGF-CS NPs in phosphate buffer saline (PBS) under
the physiological conditions (pH 7.4 and 37\selectlanguage{ngerman}°c temperature) had a
releasing rate of 89.3\% during the first 24h of the experiment. The
results confirmed that the drug release of chitosan nanoparticles
follows a time-dependent manner, which is greater in the first 8 hours
of the experiment (the main release within 4h was 62.8\%, 76.2\% in 8h,
85.1\% in 16h, and 89.3\% in the next 8h respectively).

3.5. Antibacterial Studies:

The antibacterial activity of chitosan and EGF-Chitosan nanoparticles
against four different strains of the bacteria was assessed by
determining the MIC and MBC values (Table 2). The numbers show the
concentration of NPs which had inhibitory and mortality effects on the
bacteria during experiments. The zeta potential of CS and EGF-CS NPs
were 41±0.25 and 26±0.15 mV respectively. As shown in Table 1, chitosan
NPs only possess bactericidal effect on gram-negative and also
gram-positive bacteria. Additionally, a higher concentration of EGF-CS
NPs had the same effect as the rh-EGF promotes cellular growth further.
Obtained results show that EGF-CS NPs have lower inhibitory and
bactericidal effects on both gram-negative and gram-positive bacteria.
As can be seen, \emph{B. subtilis} from the gram-positive group
and\emph{P. aeruginosa} from the gram-negative ones showed more
sensitivity against treated NPs while the most resistant spices were
\emph{E. coli.}These bacteria also had different behavior toward
exposure to EGF-CS and CS NPs regarding mortality, with the MIC level of
24 against 27.4. Principally the MIC range of EGF-CS NP is between 2.53
for \emph{B. subtilis} and 8.5 for \emph{S. aureus} while the MBC range
is between 4.75 to 24 \selectlanguage{greek}µ\selectlanguage{english}g/ml. CS NP has a MIC range between 1.25 to 7.5
and an MBC range of 3.42 to 27.4. While Vancomycin was used as a
positive control, the control did not exhibit any additional harmful
effects on bacterial turbidity at the concentration of 0.5\% acetic
acid.

3.6. Cell Viability and Proliferation Experiments:

Cell viability and proliferation experiments were conducted 24, 48, and
72h after treatment by the concentration of 5, 10, and 15\selectlanguage{greek}μ\selectlanguage{english}M of rh-EGF
and chitosan NPs in the same concentration of EGF-CS NPs. The results
presented 100\% viability of controls within 3 days of the experiment.
The chitosan NPs enhance cell viability by 90\% in the first 24h of
incubation. A decrease in cell number after 48 and 72h incubation was
observed. There was major growth in cell number in the first 24h after
treatment with EGF-Chitosan NPs, which confirm the cellular
proliferation duration of the experiment. There is a direct relation
between cellular population growth and the increase in the concentration
of EGF-CS NPs. The cellular population grew by 144, 162, and 170\%
within the first 24h of the experiment by the nanoparticle concentration
of 5, 10, and 15\selectlanguage{greek}μ\selectlanguage{english}M respectively. Within the next 24h of experiments, the
rate of cellular growth decreased to 118\% for 5 \selectlanguage{greek}μ\selectlanguage{english}M, 125\% for 10\selectlanguage{greek}μ\selectlanguage{english}M, and
142\% for 15\selectlanguage{greek}μ\selectlanguage{english}M of NP concentrations. In the last 24h of the exposure
time (after 72h from the beginning), 145, 162, and 160\% of cellular
growth rates were observed after being treated with 5, 10, and 15\selectlanguage{greek}μ\selectlanguage{english}M of
NPs. This outcome may be the result of culture medium shortage as a
result of cellular uptake. The last experiment was done by cellular
treatment using free rh-EGF, which resulted in 118, 122, and 140\%
increase in cell number in the first 24h (5, 10, and 15\selectlanguage{greek}μ\selectlanguage{english}M of NP
exposure). In the next 48h, unusual behavior in the cell number growth
was observed, which was 75\%, 32\%, and 50\% decreasing in 5,10 and 15\selectlanguage{greek}μ\selectlanguage{english}M
concentrations (from 115 to 90\%, 118 to 50\%, and 120 to 70\%), which
indicates the short half-life of rh-EGF and the active presence of
protease (Fig. 3).

3.7. Cellular Migration Experiments:

Cellular migration is determined by treating the HFF-1 cell line with
each of the 3 provided samples (rh-EGF, CS-EGF, and CS). HFF-1 is a
human normal fibroblasts cell line that originated from the human
foreskin. The treatment solution had a concentration of 10\selectlanguage{greek}μ\selectlanguage{english}M of rh-EGF
and chitosan NPs, the same concentration of EGF-CS NPs, for 24h. The
test was conducted on 12 wells plates and 3 wells remained intact as
control. As shown in Fig. 4, there was 50\% cellular migration in the
first 24h for the sample which was treated by free rh-EGF. The result of
treatment using CS NPs showed 20\% cellular migration, which is
negligible compared to 90\selectlanguage{ngerman}±10\% cell migration in 24h after being treated
using CS-EGF NPs. This significant migration resulted in complete
scratch rehabilitation. (Fig. 4).

3.8. Gene Expression Assessments

As discussed above, the best result of cell viability and proliferation
experiments was used for real-time PCR and gene expression assessments.
The candidate genes were Platelet-derived Growth Factor (PDGF), which
regulate cell growth and division, Vascular Endothelial Growth Factor
(VEGF), which is a cell signal protein that stimulates the formation of
blood vessels, and Transforming Growth Factor beta (TGF-\selectlanguage{greek}β\selectlanguage{english}), which is
produced mostly by white blood cells and acts as a multifunctional
cytokine. The expression amount of these three genes is really important
in the wound healing process and efficient skin remodeling. To assess
the influence of manipulated nanoparticles on the gene expression
profile of the wound healing process, the best results of cell viability
experiments were chosen. The selected concentration was 10\selectlanguage{greek}μ\selectlanguage{english}M for free
rh-EGF and CS-EGF NPs and 5\selectlanguage{greek}μ\selectlanguage{english}M for free chitosan, the same as CS-EGF.
Also, the selected duration of the experiment was 24h. According to
real-time PCR analysis, there were no significant differences between
PDGF, VEGF, and TGF-\selectlanguage{greek}β \selectlanguage{english}gene expression in the HFF-1 cell line after and
before exposure to chitosan nanoparticles and the results were very
close to the control group. On the other hand, in comparison to the
control gene (GAPDH), remarkable elevation was observed in PDGF gene
expression of the samples treated with free rh-EGF (Fig. 5A). As shown
in Figure 5, as a result of treatment with rh-EGF, PDGF gene expression
increased by about 4.2-fold compared to the control group while the
expression of PDGF is 7-fold in cells treated with CS-EGF and also
1.5-fold from free rh-EGF treatments. Moreover, the expression of the
VEGF gene treated with CS-EGF and rh-EGF reached about 4.2 and 2.8-fold
in comparison to the control group, respectively (Fig. 5B). The results
also illustrated a 4.5-fold increase in TGF-\selectlanguage{greek}β \selectlanguage{english}gene expression in
comparison to the control group and the same difference with chitosan
NPs. TGF-\selectlanguage{greek}β \selectlanguage{english}gene expression in cells treated with CS-EGF and rh-EGF is 7
and 3.3-fold relative to the control group, respectively (Fig. 5C). As
can be seen in figure 5, EGF-CS NPs are more effective than free rh-EGF
of chitosan nanoparticles among all of the treated cells (Fig. 5).

\textbf{Discussion}

Nowadays, the treatment of a lesion has become a major problem in the
healthcare system. Many kinds of diseases such as diabetes, and
immunodeficiencies, along with many accidents cause one or more dermal
lesions which need treatment. Generally, poor responses to other growth
factors, bacterial colonization, poor immune response, collagen
degradation, and cellular miss-sensation promote scar formation (1, 2,
5, 6). The role of the Epidermal Growth Factor (EGF) in the treatment of
many wound types, involving chronic ones, has already been proven in
human and animal studies (33, 34). A systematic review and meta-analysis
studies, which were confirmed by clinical trials, demonstrated that the
usage of rh-EGF in the cure of chronic ulcers, namely diabetic foot
ulcers and burn wounds, was effective (35-37). On the other hand, many
studies show the success of using chitosan NPs for wound dressing due to
time-controlled drug delivery applications (38), antibacterial effect
against gram-positive and negative bacteria (10, 12), and
biodegradability without any kind of toxicity to human and animals (19).

In this study, chitosan NPs were manipulated using the Ion-gelation
method. Based on previous studies, the Ion-gelation method has been more
effective in protein-based drugs for smart drug delivery. A higher
concentration of loaded protein encompasses, drug-releasing with a
steady speed, weight balancing of the selected drug to the cellular
entrance and easy preparation were the reasons for choosing this method
instead of the other ones (26). Chitosan chemical reagent provides a
high positive surface charge for manipulated nanoparticles across a wide
range of pH, which may enhance nanoparticle stability in the acidic
wound site. Moreover, preserving the typical shape of polyhedrons as a
result of using the NPs is another benefit of TPP (24, 25, 39). TEM and
DLS were performed to determine the size range of NPs and the obtained
results reported the size of NPs around 150\selectlanguage{ngerman}±50nm, as confirmed in past
studies on drug delivery uses (40). It is notable that, as discussed
above, EGF has a positive charge while it is coated with the negative
charges of chitosan. As the result of biochemical cross-linkage of these
two different types of particles, the CS-EGF nanoparticle becomes either
smaller in size or the same in comparison to free chitosan. The protein
release of about 90\% on the first day of the experiment in
physiological conditions suggests successful nanoparticle manipulation
and a steady rate of drug release, in line with previous studies.

Results validate EGF-Chitosan conjugations and the obtained conjugation
efficacy was around 70\%. Regarding the antibacterial effect of the
manipulated nanoparticles, the obtained result demonstrated the lethal
effect on both kinds of gram-positive and gram-negative bacteria. As
reported in clinical studies, the selected bacterial strain may cause
different kinds of bacterial infections and turn an acute wound into a
chronic one (6). To overcome bacterial colonization in the wound site,
MBC results are more important to cure a chronic wound. However, both CS
and CS-EGF NPs showed a greater bactericidal effect on gram-positive
species, the CS NPs influence diminished after adding rh-EGF due to a
decrease in free amine groups. Although for all the bacteria species
involved in the experiments more volume of EGF-CS NPs must be used to
overcome bacterial colonization, the \emph{E. coli} strain, which was
treated with EGF-CS NPs, may cause a better response for bacterial
clearance in this case. However, being exposed to 1 mg/ml of both CS and
CS-EGF NPs, the most durable strain was common \emph{E. coli} and the
most sensitive one was \emph{B. subtilis} . As shown in the result, it
seems that using about 30 \selectlanguage{greek}µ\selectlanguage{english}g/ml of each nanoparticle can lead to the
demise of all colonies. This hypothesis was approved by the past studies
which examined chitosan nanoparticles against \emph{S. aureus}
and\emph{E. coli} species. The present study suggests that CS NPs can
overcome a wide range of bacteria and also have a wider bactericidal
effect (10, 12). As can be seen in cytotoxicity assay results, not only
no cytotoxicity was observed against any of the treated nanoparticles on
the HFF-1 human fibroblast cell line, but also the proliferation effects
were impressive. Moreover, chitosan can increase rh-EGF stability as a
protective cover and improve its efficiency during exposure time.
Migration assay consequences showed a parallel effect on proliferation.
As illustrated in the results section, the wound model treated with
manipulated nanoparticles was completely renewed with fibroblasts when
treated with EGF-CS NPs. Free chitosan NPs did not show significant
success in wound healing during the duration of the experiment. However,
the cellular migration was good in the first 24h of free rh-EGF
treatment. Although in previous studies rh-EGF protein exposure has
resulted in better wound healing, the short half-life of this protein
leads to the administration of continuous and higher doses of the drug
(36). Improvement of rh-EGF half-life by chitosan NPs might be a better
alternative in comparison with free rh-EGF exposure. The ability of
chitosan to coat and protect of Epidermal Growth Factor enhances the
rh-EGF stability in 3D structure and preserves it from environmental
proteases such as Matrix Metalloprotein 9 (MMP9), which increases during
the inflammation phase of the wound healing process. Principally the
results showed that chitosan nanoparticle has a negligible effect on
cellular migration, which can be considered an advantage as uncontrolled
cellular migration may cause cellular invasion and lead to poor
regeneration and neoplasms in higher concentrations. At the same time,
the protective effect of the NP for rh-EGF structure and half-life
enhancement make the CS-EGF nanoparticle a suitable choice for
accelerating the lesion remodeling process without any side effects. A
high level of available rh-EGF can also lead to scar formation, which is
a detrimental process in chronic wound healing (1). This can be
controlled by the steady speed of chitosan nanoparticles drug release.

On the other hand, there are many studies using lipoid-based
nanoparticles as an rh-EGF carriers \emph{in vitro} and \emph{in
vivo}for improving the wound healing process (41, 42). There are several
studies using hydrogel chitosan dressing to cure diabetic and burn
wounds, which confirm the results of this study (43, 44). All of these
outcomes advocate the idea that chitosan can enhance the epidermal
growth factor effect in the wound dressing process, especially in
chronic wounds because of its antibacterial effects. Eventually, the
effectiveness of EGF-CS NPs in 72h, shows that it can be used in fewer
dosages or even a single dose to enhance the cellular remodeling process
in many types of wounds. Other benefits of this treatment method include
cost-effectiveness and side effect reduction.

An increase in the growth factors and cytokines leads to better cellular
regeneration. In the meantime, the master key of the secretion cascades
is EGF, and the main response growth factors are VEGF, PDGF, and TGF-\selectlanguage{greek}β
\selectlanguage{english}subsequently. On the other hand, in the case of chronic wounds, a
decrease in the level of the aforementioned factors was observed (1). As
can be seen in the results, increasing the level of EGF in the wound
site may cause overexpression of forgoing genes. In a step-by-step
review of the wound healing process in the hemostasis phase, an increase
in the amount of EGF, and subsequently PDGF, triggers peripheral cells
to divide. Then overexpression of VEGF causes immune cell filtration and
TGF-\selectlanguage{greek}β \selectlanguage{english}causes the wound site to enter the inflammatory phase. In the next
phase, immune cells, especially macrophages, neutrophils, and
lymphocytes, upregulate the expression of TGF-\selectlanguage{greek}β, \selectlanguage{english}which causes the
release of the cytokine. PDGF also manages cellular proliferation. Up to
this point, there have been no differences in the expression of the
desired genes concerning chronic and acute wounds, however, as time goes
on, cytokine overload and bacterial existence in the wound site destroy
secreted EGF. Therefore, the proliferation phase becomes abnormal. VEGF,
which is secreted from fibroblasts, leads to angiogenesis and PDGF
consequently causes tissue remodeling. Excessive expression of TGF-\selectlanguage{greek}β
\selectlanguage{english}still calls for cytokines that may lead to over-destruction of EGF.
After that, the remodeling phase starts with fibroblasts and smooth
muscle cells, which secret EGF, PDGF, and TGF-\selectlanguage{greek}β. \selectlanguage{english}In this phase, the
existence of EGF leads to damaging the cell apoptosis process and
remodeling. A high level of PDGF and TGF-\selectlanguage{greek}β \selectlanguage{english}expression may also lead to
cellular mitosis and then scar formation. This can cause an untreated
wound, long-term infection, and subsequent problems (5, 6). Since EGF
plays an important role in all stages of hemostasis, proliferation, and
remodeling, any changes in its concentration lead to poor regeneration.
Cytokine degeneration is the reason why they become less bio-available,
which can be overcome by adding an exogenic rh-EGF. This enhances the
gene expression and results in the prevention of scar formation. Gene
expression studies also confirm this hypothesis. Nevertheless, bacterial
colonization still possesses a threat in the wound site and can be can
overcome by the bactericidal property of chitosan. Adding rh-EGF to
wound dressing should also occur in short intervals and continuously due
to the short half-life of this protein. Chitosan can overcome this by
enhancing rh-EGF efficiency and its stability. Compared with free
rh-EGF, the results of chitosan embedded rh-EGF treatment show a more
efficient effect on gene expression. Previous studies reported that
chitosan also decreases the level of TGF-\selectlanguage{greek}β \selectlanguage{english}in long-term administration
and causes ECM formation by accelerating collagen formation, which may
reduce the chances of scar generation and speed up recovery (18, 19).
Real-time PCR analysis illustrated that by long-term rh-EGF availability
the expression of TGF-\selectlanguage{greek}β, \selectlanguage{english}VEGF, and PDGF remains higher than normal,
increasing the efficiency of the wound healing process in different
stages.

To summarize, the dual function of CS-EGF NPs can be concluded as the
antibacterial effect of chitosan can inhibit bacterial colony formation
in the inflammation phase (12) and the effect of rh-EGF on gene
expression upregulates the epidermal growth factor receptors (2). As
chitosan can pass through the squamosal layer, rh-EGF can be more
reachable (45). Moreover, the secretion of vascular endothelial growth
factor may be increased by chitosan, which can improve angiogenesis.
Subsequently, cell filtration in the wound area may accelerate the
hemostasis phase, reducing the time for possible pathogens infection
(20). In the next phase, chitosan's existence, as a collagen forming
enhancer, and its role in the downregulation of TGF-\selectlanguage{greek}β \selectlanguage{english}in the wound
healing process can even completely reduce the chances of scar formation
(9). On the other hand, capillary budding, extracellular matrix
formation, and keratinocyte migration are considered subsequent effects
of rh-EGF. Rh-EGF existence in a wound site can also accelerate degraded
cell apoptosis (2). The studies on the effectiveness of rh-EGF as a
wound dressing have reached a point that its use in clinical trials is
becoming prevalent (37). Although systematic review and meta-analysis
proved the rh-EGF effectiveness, elimination of nosocomial infections
remains a problem (35, 36). Dextrin-rhEGF also shows a better result
than free rh-EGF in diabetic wound cures (33). The histological
evaluation in a mouse model of wound healing, which was treated by
hydrogel embedded polymer-growth factor conjugated nanoparticles,
confirmed the obtained results of the current study regarding cell
viability and proliferation in the wound sites (34), except not
considering the genes involved in this process.

\begin{enumerate}
\tightlist
\item
  \textbf{Conclusions}This study confirmed the fact that using chitosan
  nanoparticles as a carrier for epidermal growth factors in an \emph{in
  vitro} model for dressing a chronic wound can accelerate the cellular
  remodeling and eradicate the concern of bacterial infection in the
  process of treatment. The superiority of the manipulated nanoparticle
  in this study is the slow release of the epidermal growth factor,
  bactericidal effect, collagen formation, passing through the cell
  layer, thermo-sensitivity, biodegradability, and harmlessness for
  organisms. The improvement in rh-EGF half-life makes these CS-EGF NPs
  a suitable choice for chronic wound dressing.
\item
  \textbf{Conflict of Interest}The authors declare no conflict of
  interest.
\item
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\end{enumerate}

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\textbf{Tables}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
gene & Forward/Reverse & Sequence & Sequence & Product length
(bp)\tabularnewline
\midrule
\endhead
\emph{PDGF} & F & 5\selectlanguage{ngerman}´CGTGCGTGACATTAAAGAGAA3´ & 5\selectlanguage{ngerman}´CGTGCGTGACATTAAAGAGAA3´
& 135\tabularnewline
& R & 5\selectlanguage{ngerman}´CGCTCATTGCCGATAGTGAT3´ & 5\selectlanguage{ngerman}´CGCTCATTGCCGATAGTGAT3´
&\tabularnewline
\selectlanguage{ngerman}\emph{TGF-ß} & F & 5\selectlanguage{ngerman}´AAGGCCTGGTCTGGGAGTAT3´ & 5\selectlanguage{ngerman}´AAGGCCTGGTCTGGGAGTAT3´ &
149\tabularnewline
& R & 5\selectlanguage{ngerman}´ GCATCCACTCGCTCACTACA3´ & 5\selectlanguage{ngerman}´ GCATCCACTCGCTCACTACA3´
&\tabularnewline
VEGF & F & F & 5' CTGGAGCGTGTACGTTGGT 3' & 179\tabularnewline
& R & R & 5' CGTTTAACTCAAGCTGCCTCG 3' &\tabularnewline
\emph{GAPDH} & F & 5\selectlanguage{ngerman}´ CGTAAGGATTGCATCGGACT3´ & 5\selectlanguage{ngerman}´ CGTAAGGATTGCATCGGACT3´
& 138\tabularnewline
& R & 5\selectlanguage{ngerman}´ AGGGGGACTAAGGAGGTGAA3´ & 5\selectlanguage{ngerman}´ AGGGGGACTAAGGAGGTGAA3´
&\tabularnewline
\bottomrule
\end{longtable}

Table 1. Forward and reverse primers sequences of PDGF, \selectlanguage{ngerman}\emph{TGF-ß} and
VEGF genes for gene expression analysis by real-time PCR
assay.\emph{GAPDH} gene was used as control.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Treatments\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Particle size (nm)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Zeta Potential (mV)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Concentration (mg/ml)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
MIC/MBC (\selectlanguage{greek}µ\selectlanguage{english}g/ml)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
MIC/MBC (\selectlanguage{greek}µ\selectlanguage{english}g/ml)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
MIC/MBC (\selectlanguage{greek}µ\selectlanguage{english}g/ml)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
MIC/MBC (\selectlanguage{greek}µ\selectlanguage{english}g/ml)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Gram-Positive bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Gram-Positive bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Gram Negative bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Gram Negative bacteria\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
S. aureus\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
B. subtilis\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
E. coli\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
P. aeruginosa\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
EGF-C-TPP\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
147\selectlanguage{ngerman}±10\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
26\selectlanguage{ngerman}±0.15\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1 mg/ml\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8.5/15.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2.53/4.75\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
11.4/24\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
3.28/7.10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
C-TPP\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
126\selectlanguage{ngerman}±10\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
41\selectlanguage{ngerman}±0.25\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1 mg/ml\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.5/13.6\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.25/3.42\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.5/27.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
1.74/6.5\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Table 2. The antibacterial activity of CS-EGF and CS was assessed
against four different strains by determining the MIC and MBC values.
While Vancomycin was used as a positive control, the control did not
exhibit any additional harmful effects on bacterial turbidity at the
concentration of 0.5\% acetic acid.

\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 1. Topological characterization of nanoparticles. A) Transmission
electron microscopy results of chitosan nanoparticles. The absolute
diameters of chitosan nanoparticles were around 100\selectlanguage{ngerman}±10nm. B) TEM
microscopy of rh-EGF chitosan nanoparticles had particle diameters of
around 100±50nm. C) \textsubscript{}The result of measuring the size of
the conjugated nanoparticles with rh-EGF protein using the DLS method
and its analysis.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 2. Physicochemical characterization and drug release of
manipulated nanoparticles. A) The FTIR spectrum of free chitosan
nanoparticles and B) Chitosan nanoparticles conjugated with rh-EGF
protein. The 1635.69 cm-\textsuperscript{1} spectrum shows the
non-covalent bonds between carboxyl groups of the protein and amino
groups of chitosan, which indicates the difference between two groups of
nanoparticles, showing the connection between chitosan and rh-EGF in
CS-EGF nanoparticles. C) CS-EGF NPs drug release profile, measured at 0,
4, 8, 16, 24, and 30h of the experiment and T=37\selectlanguage{ngerman}±1 \selectlanguage{english}*C, pH 7.4.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 3. The diagram of cell viability assay at 24, 48, and 72 hours by
MTT method. Empty chitosan samples did not show a significant difference
after 24, 48, and 72 hours. This increase can be seen after 48 and 72
hours, however, after 72 hours rh-EGF protein has decreased
significantly, which is due to protein instability after 3 days.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 4. A) The evaluation of cell proliferation after 24, 48, and 72
hours at different concentrations of 5, 10, and 15\selectlanguage{greek}μ\selectlanguage{english}M for rh-EGF protein,
60, 120, and 180 \selectlanguage{greek}μ\selectlanguage{english}l for non-conjugated chitosan nanoparticles, and 5,
10, and 15\selectlanguage{greek}μ\selectlanguage{english}M for nanoparticles conjugated with rh-EGF protein with an
optical microscope. B) Cell migration rate diagram which illustrates a
5-time increase against the control group for the cells that were
treated with CS-EGF.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 5. Real-time PCR analysis results showed that treatment with
rh-EGF protein results in an over-expression of PDGF, VEGF, and TGF-\selectlanguage{greek}β
\selectlanguage{english}genes. A, B, and C diagrams are related to PDGF, VEGF, and TGF-\selectlanguage{greek}β \selectlanguage{english}gene
expressions respectively. The results show no significant changes in the
expression level of the aforementioned gene treated with free chitosan.
When treated with EGF-CS NPs, a higher expression level of the three
intended genes has been observed.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/EGF-wound-healing/EGF-wound-healing}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/DLS/DLS}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/FTIR-and-drug-release/FTIR-and-drug-release}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/MTT/MTT}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/migration-new/migration-new}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/realtime/realtime}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`table 1.docx`  available at \url{https://authorea.com/users/505891/articles/584826-preparation-of-chitosan-embedded-recombinant-human-epidermal-growth-factor-nanoparticles-as-accelerating-compounds-for-skin-remodeling-in-chronic-lesions}

\textbf{Hosted file}

\verb`table 2.docx`  available at \url{https://authorea.com/users/505891/articles/584826-preparation-of-chitosan-embedded-recombinant-human-epidermal-growth-factor-nanoparticles-as-accelerating-compounds-for-skin-remodeling-in-chronic-lesions}

\selectlanguage{english}
\FloatBarrier
\end{document}

