Journal of Drug Delivery Science and Technology 54 (2019) 101308

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Journal of Drug Delivery Science and Technology

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Therapeutic potential of green synthesized silver nanoparticles loaded PVA T

hydrogel patches for wound healing
Anam Ahsana,b,∗, Muhammad Asim Farooqc
a
College of Animal Science & Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
b
Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad, 38040, Pakistan
c
Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, 211198, PR China

ARTICLE INFO ABSTRACT

Keywords: Silver nanoparticles were synthesized from cabbage (Brassica oleracia capitate) extract following green chemistry
Green chemistry approach and characterized by UV–Vis spectroscopy, DLS, SEM and FT-IR. Furthermore, PVA hydrogel patches
Silver nanoparticles loaded with these green synthesized AgNPs and AgNP/clay/activated carbon biocomposites were designed and
PVA hydrogels developed by Freeze-thaw method and their characterization was done by FT-IR and SEM. Swelling studies for
Wound healing
these hydrogel patches were performed and overall thickness of these hydrogel patches lied between 0.34 and
Transdermal drug delivery system
0.36 mm. Antibacterial activity of hydrogel patches (10% aqueous soln.) was checked against two representative
bacterial strains (Staphylococcus aureus and Escherichia coli) using microdilution method. The minimum in-
hibitory concentration (MIC) of AgNP-HP and AgNP-Bc-HP for Staphylococcus aureus was 25 μg/mL and 12.5 μg/
mL respectively whereas for Escherichia coli it was 3.13 μg/mL and 6.3 μg/mL respectively. Wound healing study
was carried out using rabbits as experimental models after giving wound incision on the dorsal skin of these
rabbits. Wounds were grossly observed for wound healing over a period of 20 days after application of hydrogel
patches on regular basis. Wound healing score was recorded after every 5 days and skin biopsies were taken at
2nd and 3rd weeks of study for histopathological examination. These PVA hydrogel patches proved to be an
excellent candidate for wound healing.

1. Introduction
The core aim of drug delivery systems is to deliver drugs to the
desired area of body in a controlled and efficient manner to ensure
optimum drug delivery. There are several innovative methods of drug
delivery to the biological systems e.g. liposomes, nanoparticles, nio-
somes, microsphere, microencapsulation, transdermal drug delivery
systems, mucoadhesive and supramolecular drug delivery systems.
These methods are gaining importance day by day particularly in those
countries where health care system is still under development [1].
Conventional dosage forms i.e. tablets, capsules, creams, ointments,
nasal sprays, injectable solution, emulsion and suspensions, pose var-
ious limitations regarding dosage, drug delivery and clearance time [2].
Risk of missing dose, fluctuations in plasma concentration of drug and
poor patient compliance are drawbacks which heads towards switching
from conventional to controlled release dosage forms. After adminis-
tration of controlled release formulations, they exhibit uniform release
and adequate delivery of drug over a dosage regimen and hence over-
come the disadvantages and barriers of conventional dosage forms [3].
Among these delivery systems, novel drug delivery system (NDDS) has
gained much more importance because of its increased stability and
therapeutic value and low toxicity rate. There are various NDDS which
have significant advantages and minimum drawbacks, such as nano-
particles, liposomes, vesicles, micelles, dendritic polymers, liquid
crystals, hydrogels and implants etc [4,5]. Transdermal drug delivery
systems (TDDS) have more advantages over conventional dosage forms.
These advantages are reduced frequency of dose, bypassing first pass
metabolism, increased activity of drugs expressed by short half-life,
maintenance of stable drug delivery profiles, therapy can be im-
mediately stopped by removal of patch, suitable route for patients who
show low compliance by oral route [6,7]. TDDS usually include semi
solid emulsions, ointments and polymer patches [8].
Hydrogels are basically three-dimensional network structures which
are formulated from specific natural and synthetic polymers. They have
the ability to absorb and carry sufficient quantity of water in their
porous structure. The three-dimensional network structure of hydrogel
is obtained by the hydrophilic domains and groups present in a poly-
meric network when they are hydrated in an aqueous medium [9]. The

∗
Corresponding author. College of Animal Science & Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China.
E-mail address: anamahsan267@gmail.com (A. Ahsan).

https://doi.org/10.1016/j.jddst.2019.101308
Received 5 July 2019; Received in revised form 29 September 2019; Accepted 1 October 2019
Available online 04 October 2019
1773-2247/ © 2019 Elsevier B.V. All rights reserved.
A. Ahsan and M.A. Farooq
formulation of hydrogel-based patch has recently gained much im-
portance because of its unique properties. The quality of an ideal
dressing is that it retains the moist environment at the site of wound
and do not allow the leakage or spread of fluid to adjacent healthy
unaffected skin area [10]. Hydrogels which are mostly synthesized from
hydrophilic polymers have the capacity to hold substantial amount of
water ranging from 10 to 20% and may goes up to 1000's times of their
dry weight. This substantial amount of water in hydrogels plays an
important role in skin elasticity and moisturization making them sui-
table dosage form for topical use. These hydrogel patches can be made
from either natural or synthetic polymers [11]. Hydrogel-based patches
delivers the drug in controlled manner over a specified time period thus
making them more important and acceptable over conventional drug
delivery methods. These are also easy to package and transport and
provide more specific drug delivery at the desired site. Hydrogel can
retain large amount of water and also exhibit occlusive behavior. Be-
cause of such properties hydrogel can hydrate the stratum corneum
very well which facilitate the delivery of active ingredient to a greater
extant. Hydrogel patch due to its adhesive nature also plays an im-
portant role in removing dead skin when peeled off [12].
Sliver nanoparticles have advantage over other metallic nano-
particles because of their unique properties e.g. optical properties,
chemical, magnetic, antimicrobial properties and excellent electrical
conductivity [13]. Pertaining to broad spectrum activity of silver
against many Gram positive, Gram negative, aerobic, anaerobic and
multidrug resistant strains, it can be employed as a topical anti-
microbial agent in the form of silver containing drugs and dressings for
wound healing purpose [14]. Mortality rate in case of large and deep
wounds is very high so the primary and most important aim in treat-
ment protocol is the closure of wound [15]. AgNPs because of their
large surface area to volume ratio are suitable candidates for topical
antibacterial dressings. Recently silver nanoparticles have been in-
corporated in hydrogels, polymers, burn dressings and medical devices
[16].
Acticoat which is a silver based wound dressing was developed by
Burrell et al. This dressing accelerates the healing process and also helps
to remove scar which are formed in healing process [17]. Nanoparticles
speeds up the healing process by increasing the neutrophils-based
apoptosis and reducing the activity of local metalloproteinase (MMP) at
the site of wound. In burn patients it is also reported that when AgNPs
were applied, the pro inflammatory cytokines level was significantly
reduced [18]. Previously, Silver nanoparticles and curcumin containing
antibacterial hydrogel composites has been developed for wound
dressing [19]. Similarly, a hydrogel wound dressing containing silver
and graphene polymer was designed for antibacterial activity [20]. It
has been investigated previously that both activated carbon and clay
possess good antibacterial activity. It has also reported that clay can
proved to be an excellent medicinal agent for wound healing although
the exact mechanism of action is still not clear. It has been investigated
that the Kisameet bay clay deposit has eradicated 16 pathogens which
include gram-positive, gram-negative and multi-drug resistant strains
[21,22]. The wound healing ability of activated carbon has also been
reported. Activated carbon fibers containing silver has been evaluated
for in vitro and in vivo wound healing study. Similarly activated carbon
cloth dressing has been used for the reduction of chronic leg ulcers
[23,24]. So, we designed to make biocomposites of both clay and ac-
tivated carbon with green synthesized AgNPs to maximize the anti-
bacterial efficacy and wound healing process after incorporation into
hydrogel network.
The aim of this study was to develop an alternative silver delivery
system which would have better surface binding ability and less toxicity
while enhanced antibacterial potential when applied topically, main-
tain the moist environment for prolong time at the site of wound and
release the AgNPs and AgNP/clay/activated carbon biocomposite in a
sustained manner hence accelerating the wound healing process i.e. to
formulate and characterize hydrogel patch containing green
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Journal of Drug Delivery Science and Technology 54 (2019) 101308
synthesized silver nanoparticles and AgNPs/clay/activated carbon
biocomposites. Further, in vitro antibacterial activity and in vivo wound
healing activity of these hydrogel patches was assessed.
2. Experimental
2.1. Materials
Silver nanoparticles (AgNPs) synthesized from cabbage (Brassica
Oleracea Capitata) and silver nanoparticles/Clay/activated carbon bio
composites (AgNP-Bc) which were obtained by the courtesy of Miss.
Faiza Rafique from Department of Chemistry, University of Punjab,
Pakistan. Poly Vinyl Alcohol (PVA) which is a hydrophilic biodegrad-
able polymer was purchased from Sigma Aldrich (Germany). Distilled
water was prepared in laboratory of Pharmacy, Physiology and
Pharmacology, University of Agriculture, Faisalabad, Pakistan. All the
chemicals used in research work were of analytical grade.
2.2. Preparation of silver nanoparticles (AgNPs) by green chemistry
approach
Silver nanoparticles were synthesized by following green approach.
For the reduction of the metallic silver to nanoparticles the plant extract
of Cabbage (Brassica Oleracea Capitata) was used and AgNO3 solution
was employed as a precursor for silver ions. The conditions for the
optimum synthesis i.e. ratio of extracts: precursor solution, concentra-
tion of the extract, concentration of precursor solution, time and tem-
perature for the reaction were augmented. For the synthesis of nano-
particles of desired size, fresh juice of cabbage, the boiled extract of
cabbage in water and soxhlet in water of different concentrations were
used.
2.2.1. Characterization of AgNPs
Characterization of synthesized AgNPs was done by UV–Visible
spectroscopy, dynamic light scattering (DLS), scanning electron mi-
croscopy (SEM) and FT-IR. For UV–Vis analysis (Shimadzu UV–visible
spectrophotometer) absorbance in the range of 300–600 nm was ob-
served. The Berker FT-IR (Tensor 27 series, Germany) spectro-
photometer was used to record FT-IR spectra. For recording FT-IR
spectra for Silver nanoparticles wave number range was 4000-
500 cm−1. For measuring size distribution and average size of AgNPs
dynamic light scattering (DLS) was done by using Brookhaven instru-
ment. SEM (FEI QUANTA) was performed for measuring size and
morphology of the AgNPs.
2.3. Preparation of AgNPs/clay/activated carbon bio composites (AgNP-
Bc)
A composite of silver nanoparticles, along with activated carbon and
clay was developed by treating the mixture at high temperature and the
concentrations of the components, reaction time and temperature of the
system were optimized. The whole reaction was carried out in Muffel
furnace at higher temperatures.
2.4. Preparation of PVA hydrogel
For the preparation of PVA hydrogel, first of all 10% w/v aqueous
solution of PVA was prepared by slowly dissolving PVA granules in
water at 80–100 °C for 40 min on magnetic stirrer (1500 rpm).
Sonication was done to remove air bubbles and clear homogeneous
solution was obtained. After complete dissolution of PVA in water, this
homogeneously prepared 10% aqueous solution was poured under la-
minar flow hood into five sterile petri plates and properly covered with
aluminum foil. It was monitored that every plate shouldn't contain
more than 20 mL of this 10% homogeneous mixture of PVA. It is done
to ensure that thickness of each hydrogel shouldn't exceed than 3 mm.
A. Ahsan and M.A. Farooq
All these petri plates were subjected to repeated freeze-thawing
cycle (3 cycles) for 6 days for developing physical crosslinking of PVA
solution. In first cycle freezing temperature was −20 ± 20 °C for 16 h
while thawing temperature was 20 ± 20 °C for 2 h. In second and third
cycle the freezing time was kept same while thawing time was in-
creased up to 4 h in second cycle and 6 h in third cycle. The complete
three cycles of freeze-thawing were repeated only once. The gelling as a
result of crosslinking of PVA solution was observed throughout the
process. In first cycle the hydrogel appearance was almost clear but till
the completion of freeze-thawing process of 6 days, it appeared milky
white in color [25].
2.5. Loading of AgNPs into PVA hydrogel
Silver nanoparticles (AgNPs) solution of 9.3 μg/mL was made. 10%
w/v homogeneous aqueous solution of PVA/AgNPs was prepared by
dissolving 10 g of PVA in 100 mL of AgNPs solution (9.3 μg/mL). This
homogeneous aqueous solution of PVA/AgNPs was prepared by the
above-mentioned procedure and poured under laminar flow hood into
five sterile petri plates and properly covered with aluminum foil in
order to maintain sterile environment. Hydrogels were prepared by
repeated freeze-thawing cycle for 6 days as described above.
2.6. Loading of AgNP-Bc into PVA hydrogel
AgNP-Bc solution of 9 μg/mL was made. 10% w/v homogeneous
aqueous solution of PVA/AgNP-Bc was prepared similarly as for PVA/
AgNPs and the rest of procedure was carried out similar to AgNPs
loading into hydrogel.
2.7. Characterization of hydrogel patches
2.7.1. Swelling studies and gel content
Swelling studies were carried out in terms of equilibrium swelling
ratio and gel content. Swelling studies were performed in distilled
water, separately for both formulations. Lyophilized hydrogels were cut
into 8 × 2 mm sized discs and weighed. Then these hydrogel discs were
immersed in distilled water. Swollen discs were taken out at regular
intervals, extra surface water was removed with the help of filter paper,
weighed on an analytical balance (n = 3) and again immersed into the
same distilled water. Process was continued till the equilibrium was
achieved. Once the equilibrium was reached, the hydrogel discs were
dried in an oven at 38–40 °C till the constant weight was attained.
2.7.1.1. Equilibrium swelling ratio (% ESR). Swelling study was
continued till equilibrium was achieved. The swelling ratio is defined
as “The fractional increase in the hydrogel's weight due to water
captivation.”
Equilibrium swelling ratio (% ESR) was calculated using the fol-
lowing equation (Eq. (1))
% ESR = Wte-Wti/Wti × 100 (1)
Where,
Wte = weight of the hydrogels after swelling in distilled water
Wti = weight of lyophilized hydrogels before swelling
2.7.1.2. Gel content (%). Once after drying the constant weight of
hydrogel discs was achieved, the gel content was calculated using the
following equation (Eq. (2))
% Gel content = Wt/ Wd × 100 (2)
Where,
Wt = weight of dry hydrogel discs
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Journal of Drug Delivery Science and Technology 54 (2019) 101308
Wd = Initial weight of hydrogel discs
2.7.2. Uniformity of patch thickness
The thickness of hydrogel patches was measured by micrometer
screw gauge in mm. Thickness measurement was carried out at 3 dif-
ferent places on patch and then mean value was calculated for both
formulated hydrogel patches [26].
2.7.3. UV–vis spectroscopic analysis
The UV–vis spectroscopic analysis was performed by (UV–visible
spectrophotometer, Shimadzu, Japan) to confirm the nanoparticles
formation. The UV spectra of silver nanoparticles were recorded at
300–600 nm.
2.7.4. Dynamic light scattering (DLS)
The hydrodynamic size of sliver nanoparticles was measured by
particle size analyzer (Brookhaven, USA). Briefly, 5 mg nanoparticles
were dispersed in 5 mL of double distilled water and sonicated for 5 min
and then particle size and PDI were measured by DLS. All the experi-
ment was performed in triplicate.
2.7.5. Fourier transform infrared spectroscopy (FT-IR)
The compatibility between drug (AgNPs & AgNP-Bc) and excipient
(PVA) was checked by performing FT-IR analysis. FT-IR was also per-
formed to observe the difference between loaded and unloaded hy-
drogel patch formulation which would be observed by any peak shifts
or formation of new peaks in spectrums [27]. Similarly, the FT-IR
spectrum for individual active ingredient, polymer and hydrogel patch
formulations before and after drug loading were taken. The Berker FT-
IR (Tensor 27 series, Germany) spectrophotometer was used to record
FT-IR spectra. For recording FT-IR spectra for PVA wave number range
was around 3500-1000 cm−1. The FT-IR spectra for loaded (AgNP-HP
and AgNP-Bc-HP) and unloaded (Blank-HP) hydrogel patches were
obtained at wave number range of 4000-750 cm−1. Before taking
spectrum of any sample a blank background scan was performed with
empty cell plate.
2.7.6. Scanning electron microscope (SEM) imaging
For performing surface morphology studies of PVA hydrogel patch
(Blank-HP) and drug loaded hydrogel patches, lyophilized samples
were used. All the samples were freeze dried and were kept in vacuum
oven overnight to remove any retained moisture. Morphology and
diameter range of hydrogel patches was evaluated from three different
positions. SEM (FEI QUANTA) was used for SEM analysis.
2.8. In vitro antibacterial activity
Bacterial infection is known to be the major source of wound in-
fection. So before using the hydrogel patches for wound healing pur-
pose, they must be evaluated for antibacterial activity in vitro [28]. In
vitro antibacterial activity of the prepared hydrogel patch formulations
was checked against two representative bacterial strains i.e. one Gram
negative strain (E. coli) and one Gram positive strain (S. aureus).
2.8.1. Minimum inhibitory concentration (MIC)
MIC is defined as the lowest concentration of the formulation at
which the organism doesn't demonstrate visible growth. To asses MIC
microdilution method was performed. 10% aqueous dilutions of the
prepared hydrogel patches i.e. AgNP-HP and AgNP-Bc-HP were pre-
pared in a 96-well micro titration plate. 10 μL of indicator solution and
10 μL of Muller Hinton Broth were added to each well. 50 μg of bac-
terial suspension was added to each well. Plates were prepared in tri-
plicates. Then plates were placed in an incubator at 37 °C for 18–24 h.
After that color change was evaluated visually. MIC value was taken as
the lowest concentration at which the color change was occurred. The
growth of microorganisms was indicated by turbidity [29].
A. Ahsan and M.A. Farooq
Fig. 1. (a)Equilibrium swelling ratio (% ESR) (b) %Gel content (c) Thickness of Blank-HP, AgNP-HP and AgNP-Bc-HP Gel content (%) of all the hydrogel patch
formulations & variation among the gel content (%) of all the formulations is shown in Fig. 1(b). A general trend of decreasing the gel content is observed between
PVA-HP, AgNP-HP and AgNP-Bc-HP. Gel content (%) decreased in AgNP-HP and even more decreased in AgNP-Bc-HP while Blank-HP showed increased gel content
(%).
2.9. In-Vivo wound healing activity
Sixteen healthy male rabbits weighing 2–3 kg were purchased from
local market of Faisalabad, Pakistan and were kept in animal room of
institute of Pharmacy, Physiology and Pharmacology, University of
Agriculture, Faisalabad, Pakistan. Rabbits were housed in individual
cages and were kept there for one week at ambient temperature with
12/12 h light/dark period. Afterwards all 16 rabbits were randomized
into four groups. Rabbits were fed on regular routine feed. Feed was
given twice a day i.e. in morning and evening but water was given
throughout the day.
Rabbits were divided into four groups with each group comprised of
four rabbits. Rabbits were anesthetized by administering IM injection of
Lignocaine. After giving anesthesia, the dorsal skin region of the rabbits
was clipped to remove hair present on the skin surface. After that skin
was cleaned with sprit and incisions (4 cm2 area, 0.5 cm thickness) were
made by using sterilized and sharp surgical blade on the dorsal skin
region. Following that the wound healing studies were performed to
check the wound healing activity of unloaded and drug loaded hydrogel
patch formulations and to compare their activity with standard (mar-
keted) drug formulation.
For evaluating wound healing activity of prepared hydrogel patch
formulations, these formulations i.e. Blank-HP, AgNPs-HP and AgNP-
Bc-HP were applied on the incised wounds. The contraction in wound
size was observed and calculated over a period of 20 days as compared
to original wound area and wound healing score was noted for each
group. As animals were divided into four groups, to first group assigned
as negative control group, Blank-HP were applied on wounds. To rab-
bits in 2nd and 3rd groups (Treatment groups), AgNPs-HP and AgNP-
Bc-HP respectively were applied on incised wounds. The rabbits in 4th
group (Positive control) received marketed drug formulation
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Journal of Drug Delivery Science and Technology 54 (2019) 101308
(Neosporin) as a standard. Wounds were observed regularly after an
interval of 5 days till the complete healing in order to observe wound
healing potential of all the formulations.
2.10. Histological evaluation
For observing wound contraction and reepithelization, skin biopsies
were collected from intact wound lesions including adjacent area after
2nd and 3rd week of wound incisions and formulations application.
Tissues were fixed in 10% formaldehyde. Afterwards these skin tissues
were processed and embedded in paraffin. Sections of 2–4 μm were
stained with Hematoxylin & Eosin (H&E) for histological evaluation
[30].
2.11. Statistical analysis
All the experiments were carried out in triplicates. Experimental
data is represented with mean ± SE (Standard Error) and statistically
analyzed using GraphPad Prism and two-way ANOVA (Analysis of
variance) between groups. A value of P ˂ 0.05 was considered as sta-
tistically significant for all the results.
3. Results and discussion
3.1. Swelling studies and gel content
Variation among the equilibrium swelling ratio (%) of all the for-
mulations are shown in Fig. 1(a). A general trend of increasing the
equilibrium swelling ratio is observed between Blank-HP, AgNP-HP and
AgNP-Bc-HP. Equilibrium swelling ratio (%) increased in AgNP-HP and
in AgNP-Bc-HP % ESR was increased but less as compared to AgNP-HP
A. Ahsan and M.A. Farooq
Fig. 2. UV–Vis spectrum of AgNPs synthesized from Brassica Oleracea Capitata.
while Blank-HP showed decreased swelling ratio. It may be explained in
terms that swelling ratio goes on increasing with increased porosity of
hydrogel networks. It was also observed that maximum swelling was
reached within 8 h and equilibrium was attained after 24 h.
3.2. Thickness
Variations among the thickness of all the prepared hydrogel patches
is shown in Fig. 1(c). Thickness of Blank-HP was 0.350 ± 0.005 mm,
for AgNP-HP patch was 0.357 ± 0.009 mm and for AgNP-Bc-HP was
0.343 ± 0.009 mm. The overall thickness of all the hydrogel patches
lies between 0.34 and 0.36 mm which confirms the uniformity of
thickness among all patches.
3.3. UV–Vis spectroscopic analysis
UV–Vis spectroscopic image of the silver nanoparticles is shown in
Fig. 2. It confirms the formation of AgNPs from Cabbage (Brassica
Oleracea Capitata) extract which was used for the reduction of silver
ions to silver nanoparticles. The characteristic broad sharp peak
Fig. 3. DLS histogram of AgNPs synthesized by Brassica Oleracea Capitata leave extract.
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Journal of Drug Delivery Science and Technology 54 (2019) 101308
ranging from 412 to 422 nm in various experiments confirms the for-
mation of nano scale silver particles [31].
3.4. Dynamic light scattering (DLS)
Dynamic light scattering (DLS) histogram for AgNPs size distribu-
tion shows that the average size of these AgNPs is 74.58 nm and
polydispersity (PDI) 0.387, respectively. DLS pattern of AgNPs sus-
pension synthesized from Brassica Oleracea Capitata leave extract is
shown in Fig. 3.
3.5. Fourier transform infrared spectroscopy (FT-IR)
In present study, FT-IR was done to investigate the chemical
changes in polymer structure due to the loading of AgNPs in the hy-
drogel network. The FT-IR spectrum of both drug loaded and unloaded
hydrogel patches were obtained as well of pure building materials. Here
emphasis was given on –OH group evaluation because –OH group is
present in both the raw ingredients i.e. PVA and AgNPs. Hydroxyl group
is mainly responsible for water holding capacity in the polymeric hy-
drogel networks [32]. The FT-IR spectra of AgNPs shows characteristic
peaks at 3822 cm-1, 2921 cm-1 and 1403 cm-1(Fig. 4(a)). FT-IR spec-
trum of pure PVA (Fig. 4 b) shows a broad peak at 3286cm-1 which
indicate the presence of intramolecular H-bonded –OH groups in single
bond compounds. Peaks can also be observed in PVA spectra at
2941cm-1 which indicate C–H stretching and at 1417cm-1 which in-
dicate the C–H bond bending. These C–H stretching and bending de-
picts that hydrocarbon chromophores are present in PVA.
Blank-HP (Fig. 4 c) shows a sharp peak at 3273cm-1 while in AgNP-
HP and AgNP-Bc-HP (Fig. 4 d, e), sharp peaks were observed at
3282cm-1 and 3296cm-1 which indicated the presence of in-
tramolecular hydrogen bonded –OH groups. These free hydroxyl groups
are thought to be responsible for water holding capacity in hydrogel's
polymeric network. Hydrogel patches also indicate peaks at 2933cm-
1,1714cm-1(unloaded hydrogel patch), 2924cm-1, 1712cm-1(AgNP-
HP) and 292cm-1,1716cm-1(AgNP-Bc-HP) which indicates C–H bond
A. Ahsan and M.A. Farooq
Fig. 4. FT-IR spectra of (a) AgNPs (b) PVA (c) Blank-HP (d) AgNP-HP (e) AgNPs-Bc-HP.
stretching and bending respectively and also confirms the presence of
hydrocarbon chromophores.
These FT-IR spectrums of all the formulations confirm the physical
crosslinking in hydrogel networks when all the spectrums seen in
comparison. It is confirmed from the results that there is only slight
shift in peaks of unloaded and loaded hydrogel patches as well as no
new peak was observed which confirms that the crosslinking between
PVA and AgNPs and between PVA and AgNPs biocomposites is only
physical and no chemical interaction occurred between the ingredients
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Journal of Drug Delivery Science and Technology 54 (2019) 101308
[33].
3.6. Scanning electron microscope (SEM) analysis
SEM image of Silver nanoparticles (Fig. 5(a)) was taken at high
magnification in order to determine the particle size. The SEM micro-
graph clearly indicates the existence of sub micrometer size AgNPs.
These silver nanoparticles range in size between 20 and 45 nm. For SEM
experiment, dried sample of AgNPs was used so the observed true
A. Ahsan and M.A. Farooq
Fig. 5. SEM micrographs of (a) AgNPs (b) AgNP-HP(c) AgNP-BC-HP (d) AgNP-HP (e) AgNP-Bc-HP
Table 1
Minimum Inhibitory Concentration (MIC) of hydrogel patch formulations.
Formulations E.coli (μg/mL) S.aureus (μg/mL)
AgNP-HP 3.13 25
AgNP-Bc-HP 6.3 12.5
Standard drug(Ampicilin) 12.5 3.25
E.coli and S.aureus concentration used = 50 μg/mL.
diameter of nanoparticles is decreased as in DLS where AgNPs sus-
pension is used for measuring the particle size [27].
Porous surface is essential for the passage of oxygen through wound
dressings and 3D network structure is important for captivating and
retaining large amount of water in hydrogel formulations. So, for this
purpose, surface and cross-sectional morphologies of AgNP-HP and
AgNP-Bc-HP were examined by scanning electron microscope [34].
SEM images of AgNP-HP and AgNP-Bc-HP are shown in Fig. 5(d and
e). These micrographs indicate a micro porous and filamentous poly-
meric network at higher magnification. Surface morphology of these
hydrogel patches also confirms the homogeneous distribution of AgNPs
and AgNPs biocomposites in hydrogel network. SEM images of both the
hydrogel patches indicates a porous web like structure as also pre-
viously evaluated by a group of researchers [25].
3.7. In vitro antibacterial activity
The in vitro antibacterial activity of the prepared hydrogel patches
was checked against 1 g negative strain (E.coli) and 1-g positive strain
(S.aureus). For the evaluation of antibacterial activity microdilution
method was employed and results were recorded as Minimum
Inhibitory Concentration (MIC). For checking antibacterial activity 10%
aqueous dilutions of both the loaded hydrogel patches i.e. AgNP-
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Journal of Drug Delivery Science and Technology 54 (2019) 101308
hydrogel and AgNP-BC, were prepared.
The results of antibacterial activity of these hydrogel patch for-
mulations against E.coli and S. aureus in terms of MIC are mentioned in
Table 1.
Both the formulations showed excellent antibacterial activity
against both strains. It is also evident from results that prepared hy-
drogel patches exhibited significantly higher activity against E.coli than
S.aureus i.e. MIC values for AgNP-HP against E.coli and S.aureus were
3.13 μg/mL and 25 μg/mL respectively while MIC values for AgNP-Bc-
HP against E.coli and S.aureus were 6.3 μg/mL and 12.5 μg/mL re-
spectively. The antibacterial activity of AgNP-Bc-HP is higher against
E.coli as compared to AgNP-HP and vice versa against S.aureaus. So, we
can say that our prepared hydrogel patch formulations have more ac-
tivity against Gram negative bacteria than Gram positive bacteria. It is
also seen that against E.coli the antibacterial activity of prepared hy-
drogel patch formulations is even more than the standard drug,
Ampicillin (MIC of ampicillin was 12.5 μg/mL and 3.25 μg/mL against
E.coli and S.aureus respectively) which proves our formulations have
even more antibacterial potential than the marketed drug.
3.8. In vivo wound healing activity
Apart from in vitro antibacterial tests, the in vivo studies are also
essential for the assessment of actual wound healing potential of for-
mulated hydrogel patches [28]. In addition to antimicrobial activity,
AgNPs have the potential to accelerate the wound healing process and
diminutions the scar formation [25]. Kataria et al. evaluated the in vivo
wound healing activity of ciprofloxacin loaded PVA and sodium algi-
nate electrospun composite nanofibers for transdermal application in
rabbits. They also performed the biochemical estimation of wound
healing [35].
Wound healing contraction score over a period of 20 days is
A. Ahsan and M.A. Farooq Journal of Drug Delivery Science and Technology 54 (2019) 101308

Fig. 6. Variation and comparison of wound contraction area (cm) in control and treatment groups of rabbits after regular intervals of time (days), receiving hydrogel
formulations and standard marketed drug.

Fig. 7. A gross illustration of wound healing process in incised wound lesions on rabbit's dorsal surface in control and treatment groups over a period of 20 days (A)
negative control group (Blank-HP), (B) treatment group (AgNP-HP (C) treatment group (AgNP-Bc-HP) (D) positive control group (Neosporin).

illustrated in Fig. 6. All the animal groups were provided with treat-
ments on regular basis and wound area was observed and calculated
after every 5 days in order to determine the wound healing activity of
hydrogel patch formulations. At first day of experiment, all the animals
(rabbits) have a wound area of 4 cm2 which goes on decreasing with
passage of time(days). The rate of contraction of these wound areas was
different in all the groups. In our experiment negative control group
wounds closed after 26 days. In rabbits treated with AgNP-HP and
AgNP-Bc-HP, this wound closure occurs within 12–14 days whereas
wounds closed in almost 23 days for rabbits treated with marketed
drug. The control group showed poor wound healing activity as a
wound area of 1.55 ± 0.06 cm2 was observed even on the last day of
observation while the hydrogel patch treatment groups showed ex-
cellent wound healing activity which is evident from wound healing
score on the last day of observation i.e. 0.00 ± 0.00 cm2 and
0.00 ± 0.00 cm2 for AgNP-HP application and AgNP-Bc-HP applica-
tion respectively which indicates a complete wound healing and epi-
thelization. Positive control group rabbits, to which standard marketed
drug was applied on the wounds showed good wound healing activity
i.e. 0.30 ± 0.08 cm2 as compared to control group but less activity
than hydrogel patch treated groups.
Hence it is proved that treatment groups receiving formulated hy-
drogel patches have significantly higher wound healing activity than
negative control and positive control groups while positive control
group depicts significantly higher wound healing activity than negative
control group but lower than hydrogel patch receiving groups as evi-
dent from Fig. 6. Grossly wound healing activity can be observed in all
the groups from Fig. 7 which shows the wound healing progress after
every 5 days. These studies added to the findings that AgNPs accelerates
the wound healing process.

8
A. Ahsan and M.A. Farooq
Fig. 8. Histological evaluation of wound lesions at 2nd and 3rd week of treatment
At 2nd week: a) Negative control b) AgNP-HP c) AgNP-Bc-HP d) Positive control
At 3rd week: e) Negative control f) AgNP-HP g) AgNP-Bc-HP h) Positive control.
3.9. Histological evaluation
Wound contraction is the primary and foremost histological al-
teration in the wound bed after wounding. It is an important biological
and natural process which basically involves the phenomenon of
growth and regeneration of tissues. Activated fibroblast which are si-
tuated at the wound area provoke the wound contraction. One way to
monitor/examine the wound contraction rate is to quantify the %age of
wound bed area at different time intervals till the proper wound closure
[27]. In current research, hematoxylin and eosin (H&E) stained tissue
sections were used for the assessment of wound healing potential. As
evident from Fig. 8 (a) more inflammatory cells are present on the
wound area of control group treated with blank hydrogel patch while
the number of inflammatory cells is reduced after 3rd week and col-
lagen fibers are also seen. Few blood vessels can be observed. Thin but
incomplete layer of epidermis is also formed. Hair follicles are present.
At most places epithelium is intact. Acute inflammatory response is
observed which shows poor wound healing and increased wound size.
Glands are present and few fibroblasts can also be observed as shown in
Fig. 8(e). Fig. 8(b, f) shows the histological analysis of AgNP-HP treated
group.
Less number of inflammatory cells are present. Collagen fibers, fi-
broblasts and glandular cavity appeared after 2nd week and after 3rd
week mature glandular cells develop, higher number of micro vessels,
inflammatory cells disappeared and thick and complete layer of epi-
dermis is formed. As evident from Fig. 8(c, g) AgNP-Bc-HP treated
group shows gradual healing of wound after 2nd and 3rd week re-
spectively. Inflammatory cells disappeared. More collagen fibers can
also be observed after 3rd week. Gland cells are present in higher
amount. Parenchyma is intact at some places but at most places is not
intact at 2nd week. More hair follicles are present. Less number of blood
vessels after 2nd week but blood vessels are present abundantly after
3rd week. Thick epithelium is present and deep scar formation is evi-
dent after 3rd week. Epithelization is complete after 3rd week. A similar
trend like AgNP-HP treated group was observed in Neosporin treated
group but all the wound healing stages were at lower speed as evident
from Fig. 8(d, h). So, we can conclude that AgNP-Bc-HP showed max-
imum wound healing potential than other patch formulations.
4. Conclusion
In conclusion, Silver nanoparticles synthesized by green approach
9
Journal of Drug Delivery Science and Technology 54 (2019) 101308
using cabbage (Brassica Oleracea Capitata) plant extract showed ex-
cellent antibacterial activity when incorporated in hydrogel network.
AgNP-hydrogel and AgNP-Bc-hydrogel patches were successfully de-
signed, developed and characterized. These hydrogel patches exhibit
excellent wound healing activity as clearly evident from histopatholo-
gical analysis of wound lesions at regular intervals. AgNP-Bc-HP
showed maximum wound healing potential than other patch formula-
tion hence our aim to maximize the wound healing activity by forming
the biocomposites of AgNPs with clay and activated carbon proved
beneficial. It is also investigated that these hydrogel patches can release
drug (AgNPs, AgNP-Bc) in a sustained manner so can be used as a re-
servoir for silver nanoparticles. The controlled release of AgNPs from
hydrogel polymeric network will keep a sterile environment at the site
of wound and hence will speed up the healing process. It is proved that
hydrogel patches with their moisturizing properties avoids scaling and
dryness and has better patient compliance. Improved swelling property
i.e. Equilibrium swelling ratio (% ESR) and Gel content (%), uniform
thickness, easy pealability and high water content which will absorb the
exudates, makes these hydrogel patches a suitable candidate for wound
dressings.
Declaration of competing interest
Each named author has substantially contributed to conducting the
underlying research and drafting this manuscript. Each of the authors
also confirms that this manuscript has not been previously published
and is not currently under consideration by any other journal.
Additionally, all of the authors have approved the contents of this paper
and have agreed to the Drug delivery science and technology submis-
sion policies. Furthermore, to the best of our knowledge, the named
authors have no conflict of interest, financial or otherwise.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jddst.2019.101308.
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