This invention relates to a wound care product'
wound healing is a dynamic and orchestrated process which involves
cellular and matrix components acting together to re-establish the lost
tissue.Generally,a.rleffectivewounddressinghasthefollowing
properties: (i) a suitable water vapor transmission rate (wvTR) that
producesamoistenvironmentonthewoundbedswithoutrisking
dehydration or e:mdates accumulation; (ii) sufficient gas permeability for
oxygen required for tissue reparative processes; (iii) a high level of fluid
absorption capability to remove excessive exudates that contain
nutrients for bacteria from the wound beds; (iv) a good barrier against
the penetration of infection causing microorganisms; (v) antimicrobial
activity to suppress microbial growth beneath the dressing; and (vi) the
absence of any rytotoxic effects in case of secondary damage to the
neonatal tissues.
The management of wounds began in ESrptian times with grease-soaked
gauzebandages - with little thought to wound management' wound care
in ancient followed by treatment with astringents' herbs ald honey' After
the usage of bandage as a dressing, astringents, herbs and honey were
used in the treatment of wounds'
Though sterile gauze dressings can provide some bacterial protection'
this is lost when the outer surface of the dressing becomes moistened
either by wound e:nrdate or external fluids' The problem associated with
these dressings are adherence which causes pain to patients and later
leads to trauma.
3
winter introduced concept of moist wound heating in L962 after that
woundcaretookanewdirection.Afterthatsomearrtibacterialagents
like silver, povidone-iodine were incorporated into dressings to prevent
infection from dressings. In a modern concept brief description of
available dressings up to date are Semi-permeable film dressings' nonadherent
contact layer dressings, hydrocolloid dressings, alginate
dressings, hydrofrbre dressings, foam dressings, hydrogel dressings, and
antimicrobial dressings.
semipermeable dressings transmit moisture vapour but do not absorb
sufficient exudates. opsiteffi (Smith and Nephew, Hull, uK), CutifilmrM
(B.D.F. Medical, Milton KeSmes, UK), BiooclusiverM (Johnson & Johnson)
and Tegaderm (3M Healthcare) are the examples of semipermeable
dressings available in the market'
Non-adherent contact Layer Dressings are applied directly onto the
wound to provide an interface with the secondary dressing or pad' Some
non-adherent dressings are impregnated with paraffrn or silicone to
improve non-adherence.
Foam dressings are mostly made of polyurethane or silicone which
enables them to handle large volumes of wound fluid' Examples of foam
dressing include: Lyofoamo (conva Tec) and Allevyno(Smith and Nephew)'
Hydrogel dressings are made up of insoluble polymers with a high degree
of water content which makes them ideat dressings to facilitate autolytic
debridement of necrosis and slough. Some hydrogel dressings are Nugeltrvr
(Johnson & Johnson, Ascot, UK) and PurilonrM (Coloplast)'
Antimicrobial dressings are used to minimize growth of microorganisms
on acute and chronic wounds. silver and Iodine are two main types of
antimicrobial dressings which is mostly used'
4
Chitosan dressing has been deVeloped by inCOrpOrating a prOCOagulant
(pOlyphOSphate)and an anti-lnicrobial(SilVerl. silVer-10aded ChitOSan―
pOlyphosphate(ChiPP― Agl exhibited higher bactericidal aCtiVity than
achie宙ng a cOmplete kil1 0f
Chitosan― polyphoSphate in υitro,
Pscudomonas aerllginosa and a>99。99%kil1 0f Staphy10COCCus aureus
cOnsistently.On 14th day demis shOWed abundant c01lagen flbers that
were thiCker and inOre organized,abundant ibroblasts in the dermiS and
good keratinOCyte maturation in the epidemis With ChiPP―
Ag treated
wounds。
Another dresSing waS prepared wOund dressing composed Of nanO―
SilVer
and ChitOSano They fOund negatiVe reSult With Sterility and pyrogen teStS
宙th silVer nanocryStalline chitOSan dressing.The wound healing rates
were 98。98± 6.090/0,81.67± 6.300/o and healing time waS13.51± 4.56
days and 17.45± 6.23 days fOr silver nanocrystalline chitOSan dresSing
dressmg treated grOup and SilVer sulfadi〔Mttne dressing treated COntr01
groups respectiVely.
Silver nanoparticles added cOmposite dreSSmg Was prepared With
chitosan,sago starch and With and withOut antibiOtic gentamicin(G}by
solvent casting methOd.They found that addition of 2 mL of ethylene
glycol as plaStiCizer increased tenSne strengho cOmplete healing time fOr
control, ChitOSan― SagO― Silver nar10particles and ChitOsan― sago― silVer
nanoparticles with gentamlcin were 24, 18 and 16 days reSpectively。
Same proved by histOpatholo,Cal study.InCreased tenSile StrengJ■
thus
increase in collagen matr破in ChitOsan― sago― silver nanoparddes With
gentamicin treated wounds renects faster healingo They conCluded that
this prepared dresSing may be uSed as WOund healing material.
In a very recent study a flbrous mats uSing pOly(Vinyl alCOholl{PVAl,
chitosan oligosacchandes(COS)and SilVer nanoparticles lAgNPs)by
5
wound healing aPPlications'
nanofibrous membrane could
They concluded that
be used for wound
electrosPun for
PVA/COS-AgNP
healing.
In a study copper oxide nano sheets synthesized in PVP was tested as
antifungalarrdantibacterialagents.Theyfoundgoodantimicrobial
activity with these nanosheets. A copper oxide load'ed film was prepared
with chitosan and they concluded that in corporation of nano copper in
the chitosan forms an appropriate combination, which efficiently promote
different phases of cutaneous wound healing by systematic modulation
of different cytokines and growth factors on different days'
A Zinc oxide loaded composite bandage was prepared by using chitosan
for wound healing applications. They concluded that these advanced cart
beusedforburn,chronicanddiabeticwoundinfections.
Nano-Tioz-chitosanwit}rcollagenartjficiatskin(NTCAS)wasprepared
for wound healing applications. Animals used in in uiuo experiment were
Sprague-Dawley rats and results were compared between uncovered
group(control),Duod,erm(positivecontrol)andNTCAS.Senrmlevelsof
hrmor necrosis factor alpha (TNF-a) and (interleukin 1) lL-6 were
measured to evaluate inflammation status. Low levels of IL-6 and TNF-a
in rat serum, better healing (photographic view) in NTCAS-treated group
when compared to other control groups'
In one study titanium dioxide nano particle loaded chitosan-pectin
dressing was prepared to investigate wound healing efficacy' The
incorporation of nano particle increased the mechanical strength of
dressing. In another study chitosan-PVP-titanium dioxide
nanocomposites prepared, for wound healing applications' The dressing
6
contain antibacterial activity, hemocompatible nature and nontoxic
nature.
The need exists, to provide wound dressings which can overcome all the
disadvantages of the Prior art'
OBJECTS OF THE INIIENTION:
It is therefore an object of this invention to propose a wound care
product which is easY to PrePare'
It is a further object of this invention to propose a wound care product'
which is cost-effective and can be made available at a lower price'
Another object of this invention
which has good mechanical and
wound dressing material.
is to propose a wound care Product,
biological properties for use as a ideal
yet another of this invention is to propose a wound care product, which
is biocompatible, biodegfadable, has haemostatic activity, arrests
infections and accelerates wound healing'
These and other objects of the invention will be apparent from the
ensuing descriPtion.
Thus according to this invention is provided a wound care product'
In accordance with this invention, a wound care product is provided
which is a film based antimicrobial dressing comprising chitosan'
polyvinyl pyrrolidone (PVP) and metal oxide nano particles, preferably
nanoparticles of coPPer oxide'
7
A mi:rfure of chitosan and polyvinyl pyrrolidone (PVP) is prepared and
metal nano particles is added to the mixture and the resulting solution is
cast on a ceramic plate in a dust free environment and dried in air at
room temperature. This is followed by dryrng in vacuum to obtain the
wound care Product.
ThechitosanandPVPareusedinaratioofl:1to2:lvlv'
Metat oxide nanoparticles is used in a proportion of 0'01 to 0'100 mg per
100 ml of the chitosan-PVP mixture'
The metal oxide nanoparticles is preferably copper oxide nanoparticles'
The invention will now be explained in greater detail wittr tl.e help of tl.e
following non limiting examPle'
Examples:
A mixture of 0.5 gms/50 mI of chitosan and 0.5 gms/50 ml of PVP was
prepared and 0.001 mg of copper oxide nano particles was added to it'
The resulting solution was cast on a ceramic plate in a dust free
environment and left to complete dried in air at room temperature for48
h. Then it was kept in vacuum to complete dry and kept the film for
further usage.
A mixture of 0.5 gms/So ml of chitosan and 1 gm/50 ml of PVP was
prepared and 0.010 mg of copper oxide nano particles was added to it'
The resulting solution was cast on a ceramic plate in a dust free
environment ald left to complete dried in air at room temperature for48
h. Then it was kept in vacuum to complete dry and kept the film for
further usage.
8
Amixtureoflgms/50mlofchitosarrarrd0.5gms/50mlofPVPwas
preparedando.l.o0mgofcopperoxidenalroparticleswasaddedtoit.
Theresultingsolutionwascastonaceramicplateinadustfree
environment and left to complete dried in air at room temperature for48
h.Thenitwaskeptinvacuumtocompletedryandkeptthefilmfor
further usage.
The prepared composite films were characterved using FTIR, XRD' TGA'
SEM,TEM,swellingtest,WVTR,watercontactinEarrEle,mechanical
testing, antimicrobial assessment, hemolysis assay, whole blood clotting'
cytotoxicity test, in uiuowound healing and histopathological analysis'
Evaluatlon Processes
Swelling Test
Swelling medium was buffer solutions of pH 3-L2 and phosphate
buffered saline (PBS) solution. The standard PBS solution (pH=7 '4' O'1M)
was prepared by dissolving L7.g7 g of di-sodium hydrogen phosphate'
5.73 g of monosodium hydrogen phosphate, and 9 g of sodium chloride
in 1 L distilled water. The pH values were precisely checked by a pHmeter
(Decibel DP51O) previously standardized with buffer solutions of
pH4andg.2.Theswellingratio,wasmeasuredbyimmersingapre
weighed dry sample in appropriate swelling medium at a pre-determined
time interval. Excess surface water was blotted out with filter paper
before weighing. Highly swollen samples were placed like sand witch
between two sieves and then blotted with frlter paper. Percentage swelling
of the prepared dressings at equilibrium was calculated from the
following formula
DS = l*"-*olrroo
LWo J
Where, Ww and Wa are weights
respectivelY.
Water VaPor TYansmission Rate
9
of wet and dry dressing material
water absorption studies are of great importance for a biodegradable
material. water vapor transmission (!vw) of dressing materials was
determined gravimetrically at 25 "C to find the moisture barrier
properties of free dressing materials at room temperature and also their
tightness and homogeneity. Free dressing materials with appropriate
dimensions were sealed to WVT cups containing 10 mL of distilled water'
The cups were accurately weighed and placed in a desiccator containing
silica gel and appropriate amounts of calcium chloride to create a climate
of low relative humidity (approximately o%1. Then, the cups were
reweighed at determined. intervals (24, 48, 72,96 and 120 h) and the
profile of mass change versus time was plotted for each free dressing
material. wvT was calculated using following equation:
f t s x24\1
WVTR (grn-2 dar') = L-04-l
where g represents mass loss, t is time (measured in hours during which
the weight loss occurred), and A is ttre exposed area of the dressing
material.
Mechanical ProPerties
The mechanical properties of the prepared composite films were
determined using a universal testing machine (Model 1185, Instron' USA)
witlr a cross-head speed of 5 mm/min under Lo Hz at 23 "C. The film
material thickness (mm) was individually determined for six dressing
materials averaging the measurements taken at five points for each
l0
dressing material using a screw gauge and simultaneously' verified with
vernier caliPer.
Antlmicrobtal Assessment
Microbiological efficiency of antimicrobial chitosan frlms and their
solutions were evaluated by using agar diffusion method' Nutrient agar
was placed in petri dish and a loopful of each bacterial strain was spread
on nutrient agar and incubated at 37"C for 24 h to give the sing[e
colonies. A representative bacteria colony was picked off with a wire loop'
placed in pre-sterilized nutrient broth and then incubated overnigfut at
3T"cforL2h.Byappropriatelydilutingwithsteriledistilledwaterarrd
nutrient broth, the cultures of bacteria containin8 -10e CFU/mL were
prepared. Then the prepared bacteria medium was dispensed on to agar
plate and the dressing material or solution was placed' The incubation
was continued for l2:- at gr"c. Finally, the inhibition ?,orre was
measured. For antifungal activity sabouraud dextrose agar and broth
was used and activity was checked against A. niger as described above'
The activity was determined after 48 h incubation at28"C'
Blo o d ComP atlbllltg As sclg
Hemolysis AssaY
Blood testing solution was prepared by diluting 4 mL fresh ACD human
blood with 5 mL O.9o/o saline. Dressing material was cut into small pieces
(approximately t cm x 1 cm) and equilibrated in 4 mL saline for 3o min
at 37 0 c. Diluted blood lo.2 mL) was added to each sample and
incubated for 60 min at ST "C. Positive or negative controls, which did
not contain dressing materials, were performed by adding O'2 mL of
human blood to 4 mL of distilled water (1OO% hemolysis) or saline
solution (0% hemolysis), respectively. Al1 solutions were centrifuged at
1OO0 rpm for 5 min and absorbance of the supernatant was measured at
It
545 nm. Hemolysis was calculated as described by Dey and Ray [4] as
following:
| (oo,* - oodl*roo
Hemolysis (%) =
lloo*,_oo_d)"
where oDneg control is the adsorption of 0.2 mL human blood dissolved
in 4 mL distilled saline solution; ODpos control is the adsorption of 0'2
mL huma' blood dissolved in 4 mL water; oDtest is the absorption of
sample. All the hemolysis experiments were performed in triplicate'
CytotoxicitY Test
TWo cell lines NIH3T3 and, Lg2g (mouse Iibroblast cell line, NCCS Pune),
NIH3T3 (Mouse embryonic fibroblast cell line, NCCS Plrne) were used in
our present study. For the cytotoxicity test, NIH3T3 and L929 fibroblast
cells were cultured. in minimum essential medium (MEM) supplemented
with 10% fetal bovine Serum (FBS), 50 ru ml-l penicillin, 50 F8 ml-t
streptomycin (Invitrogen, cA, usA). NIH3T3 and, L929 cells were seeded
into 96 well plates at a seeding density 1 x 10+ cells/well and incubated
for overnigfrt.
cell viability of the prepared dressing was evaluated by indirect
cytotoxicity test using Alamar blue. The cytotoxicity test of samples was
done according to ISO 10993-5 protocol. The cytotoxic effect of samples
was evaluated on NIH3T3 and, L929 cells. samples were sterilized by
ethyleneoxidegastreatmentandextractionratiowas50mglmL
(sample/medium).Triplicatesofeachsampleweretakenandincubated
inserumcontainingmediafor24hat3T.C.lo0pLoft}remediafrom
each sample was taken and transferred into each well' The cells were
then incubated for 1 , 3, 7 days and Alamar blue assay (Invitrogen', USA)
wasperformed.Theopticaldensitywasmeasuredat5Tonmwith620
t2
nm set as the reference wavelength using a microplate
spectrophotometer (Biotek PowerWave XS, USA)'
In vivo Test
To perform wound healing test, 2 cm x 2 cm (400 mm2) open excision
type wound was created to the depth of loose subcutaneous tissue' The
animal study was carried out abiding by the national regulations related
to conduct the experiments. Adult male albino rats (140-180 g) were
used in the present study. The animals were anesthetized with €rn
intraperitoneal (i.p.) injection of pentobarbitone sodium (5o mg/kg)'
Animals after recovery from anaesthesia were housed individually in
properly disinfected cages and rats were divided into three different
groups consisting of six animals each. out of them, first group was
treated with gauze, the second group with chitosan and tl.e third goup
treated with nano dressing. The rats were kept at room temperature of
25 *.2.C. During this period, the animals were repeatedly and gently
handled to minimizn ttre stress and to get them acclimatized to the
laboratory environment. On Oth, 7th, 1lth and 16th postoperative days,
the d.ressings were removed and the appearance of the wound was
photographed. The rate of wound closure rate was determined by the
f .n,1
following equation: Wound closure rate = L;lx 100
Where At and Ao ue the wound areas on the specified day and the day of
operation, respectivelY.
Histological Obsenration
For histological analysis, the harvested samples were frxed in 4o/o
formaldehyde/PBS solution at 4 "C, dehydrated with a graded series of
etl-anol and embedded into paraffin. The sectioned samples with a
13
thickness of 5 pm were then stained by haemato>rylin eosin (H&E) and
visualized by an optical microscope.
Statlstical AnalYsis
All experimental values are presented as means t s.D. The data were
anaTyzed. using ANOVA, and then T\rkey's test was used to artaTyze
multiple differences between the groups by using Origin Pro software'
version g. p varue at less than 0.05 was considered statistically
significant.

WE CLAIM:
1. A wound care product comprising chitosan, polyvinylpyrrolidone (PVP)
and metal oxide nanoparticles.
2. The wound care product as claimed in claim 1, wherein said metal
oxide nanoparticles is copper oxide nanoparticles.
3. The wound care product as claimed in claim 1, wherein said chitosan
and PVP are present in 1:1 to 2:l vlv.
4. A process for the preparation of a wound care product, comprising
mixing chitosan and PVP to obtain a mixture, adding metal oxide
nanoparticles to said mixture and mixing to obtain a solution,
casting said solution on a plate followed by drying the same to obtain the
wound care product.