Title of Invention	A BIOPOLYMER MATRIX
Abstract	A BIOPOLYMER MATRIX This invention relates to a biopolymer matrix comprising of gelatin crosslinked with alginate dialdehyde.

Full Text

FIELD OF THE INVENTION This invention relates to alginate dialdehyde (ADA) crosslinked gelatin and a process for the preparation thereof. This invention further relates to alginate dialdehyde crosslinked gelatin as a wound dressing material BACKGROUND OF THE INVENTION Large scale burn wounds and other open wounds with skin loss often require treatment with temporary dressings. Their main functions are : 1. Reduce the evaporation of water from the wound bed and associated energy loss; 2. Prevention or minimisation of microbial invasion at the wound site; 3. Encouragement of vascularisation and tissue regeneration at the interface between the dressing and the wound surface. Numerous wound dressing materials are available and are also being investigated. Dressings help to achieve haemostasia and thus control blood loss and protects the wound from microbial contamination by shielding the wound from the environment. Some dressings are designed to prevent evaporative fluid loss and thereby keep the wound bed in a moist condition which is beneficial for healing. Some wound dressings contain components which directly promote cell growth or migration or which attract or activate cells from the immune system which secrete growth-promoting substances. Dressings may also contain antimicrobial drugs, which help control infection of the wound. Paraffin-em bedded cotton gauze is widely used as wound dressing but has many limitations. 'Hydrogel' dressings, which are non-occlusive dressings made of hydrophilic polymers such as gelatin, polysaccharides, polyacryl-amide, polyhydroxyethyl meth acry late, polyvinyl pyrrolidone, etc. are another type of dressing with a high capacity for the absorption of exudates. These dressings swell upon contact with wound fluid and can absorb several times their own weight of exudate. Commercially available hydrogel dressings include intrasite gel (Smith and Nephew, UK) and Vigilon (CR Bard, USA). A special type of hydrogels are the alginates, which are hydrophilic polysaccharides extracted from seaweed. They are produced as thin non-woven tissues or as fibres. Upon contact with the wound fluid, they turn into a gel which has a high absorptive capacity for wound fluid. Examples include Kaltostat (Br it-C air, UK) and Sorbsan (Steriseal, UK). Occlusive or semi-ocelusive dressings prevent or retard the evaporation of water from the wound bed thereby keeping it moist. They are usually polymer films such as that fabricated from flexible polyurethane and contain a self-adhesive coating. Examples of such dressings are Opsite (Smith and Nephew, UK) and Tegaderm (3M, USA). Examples of semi-occlusive dressings are Omiderm (Latro Medical Systems, UK) and Ex kin (Konin- klijke Utermohien, The Netherlands). A more complex type of occlusive dressings is the hydrocolloid (HCD) dressings consisting of hydrocollo id particles (eg. gelatin, pectin, etc.) embedded in a hydrophobic matrix (eg. polyisobutylene).HCD dressings have a high absorptive capacity, and are very useful for the treatment of wounds producing high amounts of exudate. Commercially available examples of these dressings include Duo derm degree. (Convatec, UK) and Tegasorb TM. (3M, USA). Wound dressings is an area which is undergoing sweeping changes in its technology with the advent of genetic engineering and the emergence of many protein- and peptide-based therapeutic substances and cell culture technologies. The major technological developments in the area of wound dressings have principally sought to obtain simple or composite materials able to match most of the characteristics of an ideal skin replacement. Among dressings composed of natural polymers, alginates have found wide acceptability (vide infra). Non-woven calcium alginate dressings have been increasingly used in the management of partial and full thickness wounds. Calcium alginate is converted into a hydrophilic gel at the wound surface by an ion exchange reaction between the calcium in the alginate and sodium in the blood and wound exudates. The hydrophilic gel provides a moist environment which promotes healing and epidermal regeneration. Calcium alginate has also a role in blood coagulation. The calcium ions released in exchange for sodium ions promote hemostasis while the sodium alginate serves as a matrix for the aggregation of platelets and erythrocytes. Gelatin is obtained by the controlled hydrolysis of the fibrous insoluble protein collagen which is the major constituent of skin, bones and connective tissue. Gelatin contains a high concentration of amino acids such as glycine, proline and hydroxyproline. Gelatin possess a high hemostatic effect. Gelatin-based wound dressings have been conventionally prepared by crosslinking gelatin using formaldehyde glutaraldehyde, carbodiimidc, diisocyanate, etc. and recently a wound dressing based on gelatin crosslinked with dextran dialdehyde has been reported. (US Pat. No.6132759). Although both alginate and gelatin have been investigated individually for many biomedical applications including wound dressings, published reports or patents on combining the two and thus achieving synergic beneficial effects of both for medical use has been rare. In the single known instance of gelatin-alginate composite as a possible wound dressing material A mixture of gelatin and sodium alginate in water is cast as a film and crosslinked using carbodihnide in 90% acetone-water mixture. However, the method involves crosslinking bom gelatin and alginate using a toxic compound such ascarbodiimide. Reports on the toxicity of alginate dressings prepared using calcium cross-linking method have caused concern on the use of these dressings. Calcium sodium alginate dressing (Kaltostat) is found to be cytotoxic to fibroblasts and keratinocytes in culture and high calcium concentration is known to inhibit the growth of cells in culture. OBJECTS OF THE INVENTION It is therefore an object of this invention to propose an alginate dialdehyde crosslinked gelatin whose preparation does not involve any toxic compounds. It is a further object of this invention to propose an alginate dialdehyde crosslinked gelatin which is prepared by a rapid process. Another object of this invention is to propose an ADA crosslinked gelatin which helps to prevent or check wound infection, and helps in healing and epithelialization of the wound. These and other objects of the invention will be apparent from the ensuing description. BRIEF DESCRIPTION OF THE INVENTION Thus, according to this invention is provided an alginate dialdehyde cross-linked gelatin as a wound dressing material. According to the invention is further provided a porous for the preparation of an alginate dialdehyde crosslinked gelatin. In accordance with this invention, a polysaccharide is subjected to period ate oxidation to produce the dialdehyde. Period ate oxidation is a well-known reaction. Oxidation can be accomplished on any polysaccharide having geminal hydroxy I or one hydroxy I and one amino group in adjacent positions using reagents such as sodium or potassium period ate in aqueous medium. The reaction produces dialdehyde residues in the polysaccharide. The extent of oxidation depends on the concentration of the reagents,substrate (alginate), time and temperature of the reaction and the molecular weight of the substrate. Although sodium or potassium periodate is the preferred reagent for such reactions, those skilled in the art would know that reagents such as periodic acid or lead tetra acetate in an organic solvent such as dimethyl sulphoxide may also be used. After oxidation, the dialdehyde derivative of alginate can be conveniently purified and separated from low molecular weight reaction components by using dialysis membranes, precipitation, ultrafiltration or gel permeation chromatography, followed by ryophilization. The oxidation reaction also can be conducted in ethanol/water mixture, with the advantage that more amount of alginate can be used for the oxidation in comparison with the aqueous solution thereby yielding siginificantly larger quantity of ADA in a single reaction. Alginates cannot be dissolved in water to prepare solutions of high concentration since their viscosity is very high which result in gels rather than solutions making the handling of the same difficult. In ethanol/water mixture, it is demonstrated that a dispersion of alginate can undergo oxidation by periodate. Periodates are known not to oxidise ethanol, making the process simple and advantageous for the preparation of large quantities of ADA. Cross-linking between gelatin and ADA is achieved by the formation of Schiff base linkages between free amino groups present on the gelatin and the dialdehyde residues on ADA. This reaction is performed in aqueous medium and the speed and degree of cross-linking depend on a variety of parameters, such as the type of gelatin and its concentration, degree of dialdehyde substitution and molecular weight of the ADA, pH , buffer type and the presence of electrolytes in the reaction medium, etc.. The ADA is crosslinked with gelatin in the presence of a reagent selected from borax solution, phosphate buffer and phosphate buffered saline. Two different grades of sodium alginate are employed in the embodiment of the invention. High (viscosity of 2% solution 14,000 cps at 25°C) and medium viscosity (viscosity of 2% solution 3,500 cps at 25°C) sodium alginate from the brown algae Macro cyst is pyrifera, are obtained from Sigma Chemical Co., St. Louis, MO, USA. However, those skilled in the art would understand that such viscosities are not a limiting factor for the invention, other viscosities could also be employed. This is particularly the case with biopolymers which are natural products and the viscosities of the material often depend on the biological source from which it is derived and can never be the same or identical. Gelatin employed in the invention is type A (Bloom 300) also obtained from Sigma, USA. Several types of gelatin exist, depending on the source of collagen used, and on the extraction and production process employed. Type A is prepared by acid hydrolysis and type B is prepared by basic hydrolysis of the collagen. Both types of gelatin can be used for the preparation of the composite matrix, but type A is preferred since it is known to contain more amino groups and therefore is more efficient in the crosslink in g reaction. Gelatins with a variety of Bloom numbers exist, the Bloom number being a measure of the rigidity of the gel formed from the material. Although the present embodiment is using Bloom 300, other Bloom numbers are also suitable, but those skilled in the art would know mat the lower the Bloom number, the weaker will be the mechanical strength of the films formed. Again, type A gelatins have higher Bloom numbers and therefore, this type is preferred in the invention. The composite matrix according to the invention may be prepared as membranes, sheets or foams: Foams have the advantage that they will be able to absorb large amount of the wound exudates because of the macro-porous nature. The fabrication of such foams is simple and usually involves passing air, nitrogen or an inert gas such as helium through the gelatin solution under agitation and then adding the ADA to crosslink the same. Foams thus produced can be shaped as sheets, rods, plugs, pads, etc. and can be used in the hydrated, semi-hydrated or dry form suitable for application in the wound site. In accordance with an embodiment, the composite matrix may be prepared in situ in the would bed itself. The in situ application will have several conform ability of the dressing on wounds without the dressing wrinkling or fluting in the area covering the wound. Most commercially available dressings in the form of membranes and sheets are problematic as far as the conform ability is concerned and the in situ formation of the dressings will therefore be superior to such dressings. The composite matrix may also be fabricated into microparticles or microspheres which could find applications as a controlled drug delivery system. The fabrication of such geometries of the composite matrix involves dispersing the gelatin solution as fine droplets in a suitable dispersion medium such as vegetable oil or other hydroxycarbon oils such as paraffin stabilised by a suitable surfactant and crosslinking the particles by the introduction of ADA. Various techniques could be employed for generating the dispersion such as mechanical agitation, ultrasonic at ton, vortex mixing, etc. depending on the size of the particles desired. Many drugs could be incorporated into the matrix during the fabrication of such particles. Such drug-loaded particles could be used for injection intramuscularly or subcutaneousiy for their slow release in the human body. They could also be used for covering the wound bed since the large surface area of the particles would be helpful in absorbing wound exudates especially in chronic ulcers. Proteins and polypeptides also could be incorporated into the matrix for their slow release up on implantation. The alginate dialdehyde crosslinked gelatin may be provided with antibacterial agents such as silver sulphadiazine or gentamycin or other antibacterials which are beneficial in preventing or checking wound infection. Such antibacterials could be incorporated into the composite matrix either during the preparation of the matrix itself or could be incorporated later after the formation of the matrix by sorption methods which usually involves socking the matrix in a solution of the antibiotic (antibacterial) compound and allowing its sorption into the matrix. Such antibiotics would be released slowly from the matrix into the wound bed thereby help checking the bacterial colonization of the wound. The compounds are not limited to antibiotics or antibacterial and those skilled in the art would know that there are many other compounds that helps wound healing and epithelialization such as many Growth Factors currently available. Growth factors such as those belonging to the class of the EGF, FGF, PDGF, TGF, VEGF, PD-ECGF or IGF families are suitable for the said purpose. The invention will now be explained in greater detail with the help of the following non-limiting examples: Example 1 Preparation of Alginate Dialdehyde (ADA): Method A Medium viscosity sodium alginate, 20 gm or high viscosity sodium alginate 5 g was dissolved in 500 mL and 450 mL distilled water respectively by magnetic stirring in a beaker at Table 3. Gelling time of gelatin using ADA of different percentage oxidation The gelling time was also determined by varying the concentration of gelatin, ADA and Borax for the 84% oxidized ADA and the representative data are shown in the following tables. Table 4. Variation of the concentration of ADA (84% oxidized-Method B) in 0.1 M Borax on the gelling time of 20% solution of gelatin (Bloom 300, type A). Table 5. Variation of the concentration of gelatm (Bloom 300, type A) on the gelling time (ADA 84% oxidized-Method B, 20% solution in 0.1 M Borax): Table 6. Variation of the concentration of Borax on gelling time of 20% solution of gelatin (Bloom 300, type A) with 20% solution of ADA (84% oxidized-Method B): EXAMPLE 3 Rate of Swelling of the Gels in Phosphate Buffered Saline One half mL 10% and 20% ADA from medium viscosity sodium alginate 0.1M Borax (percent oxidation 84,55 and 20-Method B) and one half mL gelatin solution (15% solution of Bloom 300, Type A in water) were mixed using a vortex mixer in a glass vial of 15 mL capacity and allowed to form gel of approximately 26 mm diameter and 20 mm thickness. It was then kept for 10 nrin at 37°C and 5mL of phosphate buffered saline (0.1 M, pH 7.4) was added to the gel and incubated the same at 37°C. At regular intervals of time, the weight of the gel was noted after removing PBS using Pasteur pipette. The percentage swelling was calculated based on the initial weight of the gel and its swollen weight as: Table 7. Rate of swelling of gel in phosphate buffered saline with respect to time: The gels tons prepared were then subjected to various experiments to study the rate of evaporation of water from the gels and tfae release pattern of proteins from toe gels, as h ighlighted below Rate of Evaporation of Water from the Gels: Gels for the experiment were prepared as before by combining 0.5 mL 10% and 20% ADA from medium viscosity sodium alginate in 0.1M Borax (percent oxidation 84, 55 and 20) and 0.5 mL gelatin solution (15% solution of Bloom 300, Type A in water) using a vortex mixer in a glass vial It was then kept open at 37°C in an incubator at 35 ± 2% relative humidity and periodically weighed in an analytical balance The percentage weight loss was calculated based on the initial weight of the gel. This is illustrated in the example shown in Table 8. Gelling experiments in the absence of borax, ADA (55% oxidized - Medium viscosity) prepared by method B was tried to crosslink with gelatin (Type A, Bloom 300) in the absence of Borax. Gelling time was noted in the presence of buffers Eke phosphate buffered saline (0.1M, pH7.4) and phosphate buffer (0.1 M, pH 7.4). Representative data for the gelling time is illustrated in the example shown in Table 9. Table 9. Variation of type of buffer on gelling time of 20% solution of gelatin (Bloom 300, type A) with 20% solution of ADA (55% oxidized-method B): In all other buffers, gelling time was prolonged (more than 2 hours). ADA (60% oxidized) prepared by method A was also used to crosslink with gelatin. Gelling time obtained was more than 15 minutes. The reason for this high gelling time was examined and it was found that there is decrease in molecular weight for ADA prepared by method A Molecular weights of ADA determined at different degree of oxidation by the two methods are shown in Table 10. Table 10. Molecular weight of ADA having different percentage oxidation (Medium viscosity) Controlled Release of FTTC Albumin from the Gel To study the release profile of high molecular weight proteins and how the method of preparation, viscosity and percentage oxidation affect the release profile, different types of gels were loaded with FITC-Iabeled bovine serum albumin (1% loading) and cumulative release was followed. ADA prepared from medium viscosity (BM65, AM55) and high viscosity (BH61, AH77) by both methods was used for this experiment. A and B denote alginate dialdehyde prepared by method A and B respectively and M and H stand for medium and high viscosity alginate and number denotes percentage oxidation. Briefly, 0.15 mL of ADA (20% solution in 0.1M borax) was taken in a capped test tube, to which was added 0.15 mL of gelatin(15% solution) containing FITC- albumin (1% loading) and allowed to form geL It was then kept at 37°C for 10 minutes. 10 mL of PBS (pH 7.4) was introduced to it and incubated at 37°C At regular intervals, lmL aliquots were withdrawn and absorbance of released FITC-albumin was read at 496 nmina LTV-Visible spectrophotometer. Cumulative release was then calculated. All the experiments were done in triplicate. Representative data are illustrated in the example shown in Table 11. Controlled Release of Silver sulphadiazine from Gels. About 0.15 mL of (10 and 20% solution)ADA (medium viscosity) prepared by method B (percentage oxidation 84, 50 and 20%) was taken in capped test tube. Gelatin (0.15 ml of 15 % solution, Type A, Bloom 300) containing silver sulphadiazine (USP) was added to it and allowed to form gel. The gel was kept for 10 minutes at 37°C. 10 mL of PBS (pH 7.4) was introduced to this and incubated at 37°C. At regular intervals, one mL aliquot solution was withdrawn and absorbance of the released silver sulphadiazine was read at 256nm in a UV- Visible spectrophotometer. Cumulative release was then calculated. All the experiments were done in triplicate. Data are illustrated in the example shown in Table 12. WE CLAIM: 1. A composite biopolymer matrix comprising of gelatin crosslinked with alginate dialdehyde. 2. The biopolymer matrix of claim 1 wherein said alginate dialdehyde is obtained by the perlodate oxidation of an aqueous solution of sodium alginate. 3. The biopolymer matrix of claim 1 wherein said alginate dialdehyde Is obtained by the perlodate oxidation of a dispersion of sodium alginate In ethanol/water mixture. 4. The biopolymer matrix of claim 1 wherein the matrix is formed by crosslinking of gelatin with alginate dialdehyde in the presence of an agent such as phosphate buffer, or phosphate buffered saline, and Borax. 5. The biopolymer matrix of claim 1 where In the matrix is formed in situ in the wound bed by the crosslinking of gelatin with alginate dialdehyde in the presence of Borax or In the presence of phosphate buffer or phosphate buffered saline. 6. The biopolymer matrix of claim 1 wherein the matrix is used for wound and burn dressing. 7. The biopolymer matrix of claim 1 wherein the matrix is prefabricated in the form of films, sheets or foams In their dry or wet forms and applied as a wound or burn dressing. 8. The blopofymer matrix or claim 1 wherein the matrix is prefabricated in the form of microparticles or microspheres and then applied on the wound bed. 9. The biopolymer matrix of claim 1 wherein the matrix is loaded with antiseptics, antibiotics, antibacterial drugs or growth factors. 10. The biopolymer matrix of claim 1 wherein said antibacterial agent Is such as silver sulphadiazine, gentamycin. 11. The biopolymer matrix as claimed in claim 1, wherein the matrix is used as an injectable system. 12. The biopolymer matrix of claim 1 wherein the matrix is used for the controlled or slow delivery of drugs. Dated this 1st day of JULY, 2005.

Documents:

0847-che-2005-claims.pdf

0847-che-2005-correspondnece-others.pdf

0847-che-2005-description(complete).pdf

0847-che-2005-form 1.pdf

0847-che-2005-form 26.pdf

847-CHE-2005 ABSTRACT.pdf

847-CHE-2005 CLAIMS.pdf

847-CHE-2005 CORRESPONDENCE OTHERS.pdf

847-CHE-2005 CORRESPONDENCE PO.pdf

847-CHE-2005 DESCRIPTION (COMPLETE).pdf

847-CHE-2005 FORM 1.pdf

847-CHE-2005 FORM 18.pdf

847-CHE-2005 FORM 3.pdf

847-CHE-2005 PETITION.pdf

847-che-2005.tif

PDF.PDF