STATEMENT OF INVENTION:
This invention relates to biopolymer-based biodegradable and biocompatible patches loaded with or without somatic or stem cells for wound dressing and tissue engineering applications. More specifically, the invention relates to poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) blends incorporated with crosslinker and plasticizers to develop a ready-to-use, dry-patch which forms a water-insoluble hydrogel-patch upon contact with body fluids and therefore, has application in closure of clinical wounds, post-operative incisions, coating tissue constructs such as vascular grafts and, wound dressing. The polymeric patch seeded with patient-derived somatic or stem cells has application in soft tissue engineering.
BACKGROUND OF INVENTION:
A wound is an injury to the external (e.g. skin) or internal (e.g. muscle, bone, heart or other tissue) tissue caused by variety of etiological factors such as trauma, burns, ischemia, ulcers or pressure. It may be a simple abrasion, laceration, deep puncture, burn or an intentional incision such as a surgical wound. They are generally treated with application of local wound dressing materials, which provide a physical barrier from external environment such as dust and dirt and inhibit the microbial growth. Some wound dressing materials also support or promote wound healing mechanisms by incorporating growth factors. The wound dressing materials are generally applied as cream or ointments and often require covering with bandages.
Wound dressings in the form of patchs made from biodegradable materials are desirable because they eliminate the need of external bandage and may reduce the trauma during removal of the wound dressing from a wound surface. Natural biomaterials such as "Fibrin glue" and collagen have been used for surgical wounds but have been discouraged due to concerns relating to transmission of diseases such as AIDS and

Hepatitis and possible immune reaction in patients sensitive to them. Biodegradable hydrogels derived from natural polysaccharides such as microbs (e.g. dextran, glucan, and alginate), animals (e.g. chitosan and gelatin), and plants (e.g. starch and cellulose), have also been tested as wound dressing material due to their inherent biocompatibility. However, they also pose limitations such as batch-to-batch variability, possibility of immunogenicity and zoonosis such as AIDS and Hepatisis. To overcome these limitations, several biocompatible and biodegradable synthetic biomaterials have been explored as wound dressing materials. The majority of these biomaterials are thermoplastic materials based upon glycolide. lactide, caprolactone or their copolymers such as polyglycolide (US Patent 3297033) and, poly(L-lactide-co-glycolide) (US Patent 4523591). A US Patent (No. 5410016) has disclosed biocompatible and biodegradable polymers which can polymerized to form hydrogels.
PVA is the oldest and one of the most frequently used synthetic polymer hydrogels that has been tested as wound dressing material due to its good biocompatibility. PVA-based hydrogels are disclosed in US Patents (No. US55083I7, US5932674, US5846214). However, these hydrogels are not degradable. US Patent (No. US6710I26) disclosed hvdrogel made of pre-polymers having a PVA backbone and pendant chains that include polymerizable OH group, which adds versatility in terms of various modifications such as attachment of active agents or another polymer. With a PVA hydrogel, the choice of a suitable polymer can yield non-swellable or minimally swellable hydrogel. However, PVA-based hydrogels possessed insufficient mechanical properties such as elasticity, stiffness, and has very limited hydrophilicity characteristics which restrict its use alone as a wound dressing polymeric material. Blends of PVA with natural polymers such as alginate, dextran, starch, hydroxyethyl starch, chitosan/chitosan derivatives, glucan and gelatin (CN10526801 5, CN 101502667, KR20040085646) or synthetic polymers such as PVP, poly (ethylene glycol) and poly(isopropylacrylamide) have also been developed

(US 2009297587, WO2005120462) using different crosslinking methods such as radiation crosslinking, chemical reaction with glyoxal, bi-functional reagents with glutaraldehyde, reaction with borates and freezing-thawing. Many of these crosslinked polymers have satisfied biocompatibility. However, they failed to provide desirable sufficient mechanical properties for use as dressing material for soft tissues. PVA composite biopolymers using clays and nano-inorganic particles as fillers have also been used to improve the mechanical properties of the hydrogels (CN104059234, CN104804343). However, their use is very restricted due to their non-biodegradability at tissue environment and their somewhat cytotoxicity nature at high concentration.
PVP is a popular water-soluble, biodegradable, biocompatible, and extremely low cytotoxicity synthetic polymers. It has been used previously on PVA hydrogels for skin regeneration and wound dressing applications. The pyrrolidone group in the PVP can covalently bind with the OH group of the PVA and alter the swelling, degradation and gelling property. Further, due to their hydrocarbon backbone they can be stored conveniently. PVA-PVP hydrogels prepared by radiation crosslinking were elastic, fixable, transparent, impermeable for bacteria, the attached cells on the obtained hydrogel membranes. However it was suitable only for healthy skin but not for wound dressing or suitable for wound dressing only in a tropical environment (Darwis et al., 1993; Himly et al., 1993). Singh and Ray (1994) developed a radiation crosslinking for PVA-PVP modified with sterculia gum polysaccharide hydrogel and reported increased swelling degree with an increase of amounts of PVP and sterculia but the swelling degree decreased with an increase of the radiation dose due to formation of long crosslinked chains and, the mechanical properties were still weak. Razzak et al. (2001) developed co-polymeric hydrogels of PVA-PVP by 60Co v-ray irradiation crosslinking process and reported improved physico-chemical properties of PVA hydrogels such as water content and water adsorption. Park and Chang (2003) developed two-layer hydrogels consisting

of a polyurethane membrane cover and a mixture of PVA-PVP-glycerin-chitosan crosslinked by r-irradiation or two steps of freeze-thawing followed by r-irradiation and reported improved mechanical strength reduced evaporation speed of water and permeation rate of PVA-PVP hydrogel due to covering by polyurethane membranes. However, crosslinking by chemical reaction (e.g. free-radical polymerization, chemical reaction of complementary groups, using high energy irradiation, or enzymatic reaction) not only produce toxic compounds but also can affect the nature of the substances (e.g. proteins, drugs, and cells). Thus, physical crosslinking (e.g. ionic interaction, crystallization of the polymeric chain, hydrogen bond between chains, protein interaction, or design of graft copolymers) are to be preferred.
Another disadvantage of the PVA-PVP hydrogels that have been developed so far is that their mechanical properties are not desirable as they have low elasticity, high modulus and are more brittle that make then unsuitable for use on soft tissues. For use as wound dressing patch, it is desirable that they have properties such as biodegradable, biocompatible, elastic and durable, minimally swellable and greater adhesive. These properties can be imparted with the optimal use of cross-linkers and plasticizers.
OBJECTS OF-THE INVENTION:
An object of this invention is to develop a cross-linked PVA-PVP polymer-based patch for wound dressing which is biodegradable, biocompatible and designed for selected properties of compliancy (i.e high elastic modulus and low elongation at rupture) and elasticity for different wound types including the surgical incision. Another object of this invention is to provide a non-cross-linked PVA-PVP polymer-based patch, which can. support the growth and proliferation of somatic and stem cells for use as soft-tissue construct or as a coating materials for implants. A further object of this invention is to

disclose a kit for treating a wound, the kit comprising sterile packaging containing the patch and printed instructions describing the use of the patch and kit for treating wounds.
SUMMARY OF INVENTION:
The present invention discloses a biodegradable and biocompatible wound dressing patch comprising PVA or its derivatives blended with PVP or its derivatives, wherein the patch is a dry-patch at room temperature and forms a water-insoluble hydrogel-patch upon contact with the body fluids to have a desirable surface pH of at least 5.5, folding endurance of at least 200, residence time of at least 1 day, tensile strength of 10 to 25 MPa, extension at break of at least 15 mm, loaded with or without an antimicrobial agent and seeded with or without somatic or stem cells at a seeding density of at least 30,000 cells/cm". The present invention also discloses a kit for treating a wound, the kit comprising sterile packaging containing the patch and printed instructions describing the use of the patch and kit for treating wounds.
DRAWINGS (if any)
Nil
DETAILED DESCRIPTION
The disclosed patch can be prepared by dissolving the initially solid ingredients (e.g., water-soluble PVA and ethanol soluble PVP) in distilled water (PVA) or ethanol (PVP) at room temperature (e.g. 20 - 25°C) when the solid ingredients become solubilized. The solubilized ingredients and then mixed together in desired molar ratio and cross-linked and plasticized by adding cross-linker and plasticizers and casted on non-coated glass surface by solvent casting method. The casted film is air-dried for 24-48 h followed by oven-drying at 60°C for overnight. PVA and PVP concentrations may be from 0.25 to 0.70 mM and 1.25 to 10.0 mM, respectively and their ratio may be from 1:1 to 18:1. PVA, PVP and their derivatives may be obtained from a variety of commercial sources including but not limited to sources such as Sigma-Aldrich (MO, USA), Hi-Media (Mumbai, India) and Loba Chemie (Mumbai, India). The polymers may have a variety

of number average molecular weights, e.g., about 89,000 to 98,000, 85,000 to 124,000, 110,000-130,000, 146,000 to 186,000, 31,000 to 50,000 and 9,000 to 10,000 for PVA and about 10,000 to 360,000 for PVP. The PVA and PVP in the number average molecular weight range of 30,000-40,000 are preferred for the disclosed invention. PVA or PVP derivatives may also be employed, for example, derivatives in which one or more hydroxyl or pyrrolidone groups have been modified for the purpose of altering the solubility or tissue adhesive characteristics of the derivative. Cross-linkers and plasticizers are included to help maintain consistency and tensile strength. Examples of crosslinkers include gluteraldehyde, formaldehyde genipin etc. wherein gluteraldehyde (0-3%; v:v) is preferred in the disclosed invention. Examples of plasticizers include sucrose, hydroxymethyl cellulose, glycerol, propylene glycol etc. wherein glycerol or propylene glycol (3-12% v:v) is preferred. Antimicrobial agents also may be employed in the disclosed patch and include but not limited to antibiotics such as metronidazole (0.3 to 1.5% w:v).
The disclosed patch is a dry-patch and turns into a transparent water insoluble hydrogel-patch, which makes it easy to visualize the underlying tissue when applied. The disclosed patch is provided in a ready-to-use, storage-stable form, requiring no preparation prior to use. Additionally, it can be seeded with the patient-derived or autologous somatic or stem cells at a seeding density of at least 30,000 cells/cm' to promote fast healing. The patch can be stored for extended time periods and does not require low temperature for storage. For example, the patch stored for more than 15 months, 18 months or up to 24 months did not show discoloration, physical distortion or change in surface pH. The patch is stable at temperatures ranging from about 4° C to about 42° C.
The disclosed patch is biocompatible, biodegradable and, when applied to a wet surface (e.g. wound), the patch absorbs a substantial amount of water to cause absorption of wound exudate without undue desiccating the wound site. The disclosed patch will provide physical cover to healthy or healable tissue in the wound by adhering to tissues (e.g., cartilage or bone) at the treatment site and resists detachment or other disruption until natural degradation or degradation initiated by hydrolysis occurs. Thus, the patch will be useful in wound dressing in a variety of surgical procedures, treating ulcers.

burns, cuts etc. The disclosed patch may be used alone or in combination with commonly used antiseptic creams and ointments. Due to presence of antibiotics, the patch significantly reduces or prevents bacterial proliferation and therefore, would improve healing by preventing bacterial recolonization or reinfection.
The disclosed patch also has desirable tissue adhesion. Upon wetting, the disclosed patch adhered the specific body tissue to which it is applied. The patch has a residence time in the applied tissues for at least I day, with or without irrigation. The patch may degrade naturally or by irrigation (e.g. saline solution). The disclosed patch is non-cytotoxic with cytotoxicity scores of less than I, as measured by guidelines of ISO Guideline.
The disclosed patch typically will be placed in suitable sealed packaging (e.g. pouch or a vial made of suitable materials, or a kit containing such packaging and optionally contains printed instructions) and subjected to sterilization before being further packaged if need be with printed instructions describing the proper use of the patch or kit in the treatment of wounds. Suitable sterilization methods include ionizing radiation (e.g. gamma radiation) or ethanol treatment.
The disclosed patch may be applied directly (e.g. for clean wounds) or as a part of a multi-step treatment regimen (e.g. for infected wound). The multi-step treatment regimen may include or be followed by a cleaning and disinfection followed by the application of the disclosed patch.
The present invention is further described in the following examples.
EXAMPLE 1
PVA solution was prepared by dissolving in water whereas PVP was prepared by dissolving in ethanol. The PVA and PVP solutions were mixed in different ratios with or without glyceraldehydes (GA; 3%, v:v) and mixed at room temperature to form a homogeneous solution. The solution was then casted on clean petriplates and patches were prepared by solvent evaporation method. All formulations formed films and remained transparent at room temperature. Tables I and 2 shows the percentage of the ingredients different composition and their physical and mechanical properties. Figures I and 2 shows the swelling behavior and degradability of the patches.

EXAMPLE 2
Glycerol (0.1 - 0.3 ml of 30% v:v solution) or propylene glycol (0.1 - 0.3 ml of 30% v:v solution) were mixed with the PVA-PVP solution and the films were prepared by solvent casting method. All formulations formed patches and remained transparent at room temperature. Tables 3 and 4 show the percentage of the ingredients different composition and their physical and mechanical properties. Figure 3 shows the swelling behavior of the PVA-PVP patches.



Figure 3. Swelling behaviour of PVA-PVP patch plasticized without (SI) with Glycerol
(S2-S3) or propylene glycol (S4-S5). SI: control; S2-S4: 0.1 ml of plasticizer were added
to 8 ml of PVA-PVP; S3-S5: 0.2 ml of plasticizer was added to 8 ml of PVA-PVP
EXAMPLE 3
Antibiotic (Metronidazole) loaded PVA-PVP patches were evaluated for rate of drug release (Figure 3) and their antimicrobial activity against four common bacterial strains (Streptococcus spp., Bacillus substilis, Lactobacillus spp, and Escherichia coli by the zone of inhibition screening technique. The four bacterial strains were grown on Muller Hinton agar plates and, under sterile conditions, punched patches (~3 mm diameter) of each formulation was directly placed on the agar plates. The agar plates were incubated at 37°C for 18 hours and evaluated for bacterial growth. The diameter of clear "zone of inhibition" around the patches where measured. The results shown in Table 5.

Figure 4. Cumulative percentage drug release of metronidazole loaded on PVA-PVP patchs without (S1) or with Glycerol (RX1) or Propylene Glycol (RX2) plasticizer.

EXAMPLE 4
The PVA-PVP patches were sterilized by ethanol treatment and were evaluated for potential cytotoxic effects following the guidelines of ISO 10993-5. The patches were extracted in distilled water at 37°C for 24 h and the extracts were mixed with complete DMEM cell culture medium. A negative control (high density polyethylene) and reagent control (e.g. distilled water) were similarly prepared. A positive control (powder-free latex gloves which include natural rubber latex, zinc oxide and titanium dioxide) was extracted in DMEM at 37°C for 24 h. Triplicates of a H9C2 monolayer cell culture were dosed with each extract and incubated at 37°C in the humidified atmosphere of 5% CO2 in air for 48 h. Following incubation, the monolayers were examined microscopically for cell morphology and cellular degeneration. The cell proliferation was assessed by MTT assay and the cell viability was estimated by FDA assay. All formulations showed non-cytotoxicity with a cytoxicity score of 1.

Figure 5: Cell culture of H9C2 cells on PVA-PVP film, PP7.5 non-coated (PP7.5_NC) and collagen-coated (PP7.5_C), PPG7.5 non-coated (PPG7.5_NC) and collagen-coated (PPG7.5_C). Cell viability and proliferation was assessed by MTT assay.

CLAIMS
We claim:
1. A ready-to-use, biocompatible, biodegradable wound dressing patch comprising PVA or its derivatives blended with PVP or its derivatives, wherein the patch is a dry-patch at room temperature and forms a water-insoluble hydrogel-patch upon contact with body fluids to have a surface pH of at least 5.5, folding endurance of at least 200, residence time of at least I day, tensile strength of 10 to 25 MPa, extension at break of at least 15 mm, loaded with or without an antimicrobial agent and seeded with or without somatic or stem cells at a seeding density of at least 30,000 cells/cm2.
2. The patch according to claim 1, wherein the concentration of PVA or its derivative and PVP or its derivative vary from 0.25 to 0.70 mM and 1.25 to 10.0 mM, respectively and their molar ratio vary from 1:1 to 18:1.
3. The patch according to claim I, wherein the water- soluble PVA has a number average molecular weight of about 9,000 to about 186,000 and ethanol-soluble PVP has a number average molecular weight of about 10,000 to about 360,000.
4. The patch according to claim 1 wherein cross-linking agent is glutaraldehyde at a concentration of 0-3% (v:v).
-5 The-patch-according_tO-claim_l,-wherein_plasticizer_is-glycerol_or_propy.lene_
glycol at a concentration of 3-12% (v:v).
6. The patch according to claim I, comprising an antibiotic as antimicrobial agent at a concentration of 0.3-1.5% (w:v).
7. The patch according to claim I and 6, wherein the patch is sterilized by ethanol treatment or gamma radiation.
8. The patch according to claim I and 6, wherein the patch is hemocompatible and non-cytotoxic with a cytotoxity score of I or less.
9. The patch according to claim 1 to 8, seeded with or without somatic cells or stem cells at a seeding density of at least 30,000/cm2

10. A kit for treating a wound, the kit comprising sterile packaging containing a patch
according to claim 1-8, and printed instructions describing the use of the patch
and kit for treating the wounds.