WO2011004399A2 - Liquid filmogenic chitosan skin for wound healing and a method for preparation thereof - Google Patents

Abstract

This invention is related to chitosan filmogenic liquid for wound healing. It particularly relates to small to chronic wounds healing using filmogenic liquid applied on site which is made of pharmaceutical grade chitosan. The proposed bioactive wound healing chitosan filmogenic liquid heals even the traumatic wounds by promoting natural cell growth factors and resulting into pleasing wound healing. The filmogenic liquid chitosan and a method of obtaining the same are also disclosed. A method of obtaining pharmaceutical grade chitosan from crude chitosan is also proposed.

Description

Description

"LIQUID FILMOGENIC CHITOSAN SKIN FOR WOUND HEALING AND A METHOD FOR PREPARATION THEREOF"

FIELD OF INVENTION

This invention is related to chitosan filmogenic liquid for wound healing and a method for preparation thereof. It particularly relates to small to chronic wounds healing which are always irregular in size and depth using above filmogenic liquid applied on site which is made of pharmaceutical grade chitosan of (1.5-2% re-acetylated polyglucosamine). The proposed bioactive wound healing chitosan filmogenic liquid heals even the traumatic wounds by promoting natural cell growth factors and resulting into pleasing wound healing. This filmogenic liquid skin is prepared from high molecular weight pharmaceutical grade highly deacetylated (re-acetylated polyglucosamine 1.5- 2%) with lowest polydispersity index and allows natural healing process by promoting growth factors, scaffolds for growth of cells resulting in aesthetically pleasing wound healing. The filmogenic liquid chitosan and a method of obtaining the same are also disclosed. BACKGROUND OF THE INVENTION

Chronic non-healing wounds are major problems all over the world leading to pain, incapacitation, limb loss and death. Moreover, patients with diabetes ulcers often do not heal due to a coalescence of factors including neuropathy, macro vascular disease and Extra Cellular Matrix (ECM) proteins like collagen being glucosylated rather being glycosylated. In this situation, amputation is performed but it is not patient-friendly. A step ahead is dressings and skin grafting which do not always provide required results in healing and often lead to scarring, keloids formation etc. In case of recalcitrant wounds which are seen in certain diseases like diabetic and venous ulcers and burns, there is insufficient availability of oxygen by blood and occlusive dressings do not allow the wound to breathe.

Over the last few years, regenerative medicine approach has been taken to heal recalcitrant wounds. Unlike plain and interactive wound dressing, the regenerative medicine approach involves

"Bioactive dressing". Bioactive dressings not only function as Interactive wound dressings but also interact with cells, promote growth factors, stabilize growth factors and thus promote rapid and aesthetically pleasing wound healing.

In Bioactive dressings often proteins and carbohydrates of the extra cellular matrix (ECM), alone or in combinations are used to prepare dressing materials. For Example Integra™ is made from Bovine Collagen and Shark Chondroitin sulfate. This first artificial skin is a porous matrix which induces fibroblast migration and allows the formation of Dermis. Similarly hyaluronic acid based dressing materials have been available in International markets called as

Hyalografts etc. Apart from being very expensive, they may also require skin grafting. Moreover, it fails to provide protection against infection. Chitosan is derived from chitin, an Extra Cellular Matrix carbohydrate of invertebrates and is very abundant, renewable natural source. Chitosan structurally and biologically mimics hyaluronan and is expected to, like hyaluronan, heal adult wounds scar-free. Because of this and other literature a lot of animal studies on wound healing there have been a lot of expectation of chitosan becoming a good medical dressing. However, chitosan extracted from various sources differ significantly in terms of its molecular weight, degree of acetylation, polydispersity, endotoxin levels etc. The American Society of Testing of Materials (ASTM), in their Tissue Engineering Medical Devise has come up with standards and specifications for chitosan to be used in medical applications. Thus a few products made from pharmaceutical grade chitosan, especially Hemostat Bandages are now available and lots of research and development is being made in this specific field to improve patient compliance particularly in developing certain bioactive dressings utilizing chitin and chitosan. Chitin and chitosan have been considered as non-toxic and biodegradable renewable sources of polyglucosamine to attain bioactive dressing.

Chitosan are copolymers made out of two types of monomer units:

(a) Glucosamine (G) units, and

(b) N-Acetyl-Glucosamine (NAG) units.

Certain specific properties and quality of chitosan is decided by its molecular weight, polydispersity index, degree of deacetylation (DDA) etc. The ratio of G to (G + NAG) is called DDA along with pattern of acetylation (distribution of "G" and "NAG" units on the polymeric chain) which plays an important role to decide whether to allow or not to allow the chitosan to be soluble in acidic water. The higher DDA shows good bacteriostatic properties due to the cationicity of free amine groups. Hence, it is always desirable to have higher DDA of chitosan for topical applications/wound healing. But it is very challenging and difficult to produce high DDA with high Molecular weight Chitosan with low polydispersity index which is expected to produce excellent healing results.

The given molecular weight (Mw) of a chitosan is always represented as average of all the molecules in the population. The most common way to express the molecular weight (Mw) is the number average (Mn) and the Weight average (Mw) of chitosan. The coefficient Mw/Mn is referred to as the polydispersity index. In a polydispersity molecular population, the relation Mw > Mn is always valid. The coefficient Mw/Mn is referred to as the polydispersity index of that chitosan. It is always considered that there is good homogeneity of the molecules in the population when the polydispersity index is preferably below (<) 2%.

There are mainly two categories of bioactive dressing made from Chitosan:

(a) Haemostatic dressing e.g. haemostatic sponge developed by HemCon Ltd. to stop profuse bleeding and hemorrhage;

(b) Wound Healing dressing for healing wound; Generally, the haemostatic sponge used in haemostatic dressing is developed using solid form of chitosan and is preferably chitosan acetate (acetic acid) which is not naturally available in the human metabolic system. The origin of this type of sponge is alpha chitosan of crustacean exoskeleton. The alpha chitin contains heavy metals present in the sea which causes allergy. The problem with crustacean origin, is the incorporated toxic heavy metals (coming from the sea) to the exoskeleton during the process of calcification) and also the presence of astaxanthin associated to lipo-proteins, which also may cause allergy at trace level in the finish chitosan. So far, Wound Healing dressing developed has used chitin fiber and synthetic fibrous binder or cross linked chitosan with an adjuvant producing thermo reversible gel. So far, Wound Healing dressing developed has used chitin fiber and synthetic fibrous binder or cross linked chitosan with an adjuvant producing thermo reversible gel.

HemCon uses DDA ranging from 78 to 97%, most preferably greater than 85% but less than 95% which does not provide as good a bacterio-static and fungi-static properties as DDA above 98%. This type of sponge stops bleeding immediately and is used as an emergency remedy.

But no one has yet developed any wound healing dressing using high grade chitosan with consistent properties. Thus there is an urgent unmet need to come up with effective bio active dressing for wound healing

PRIOR ART

There are various literatures in the field of chitosan and in particular to wound healing and wound dressing.

WO/2005/062896 filled by HEMCON Co. discloses "Tissue Dressing Assemblies, Systems, And Methods Formed From Hydrophilic Polymer Sponge Structures Such As Chitosan" Related

Applications). This application is a continuation-in-part of U. S. Patent Application No. 10/743,052, filed on December 23, 2004, entitled "Wound Dressing and Method of Controlling Severe Life- Threatening Bleeding,", which is a continuation-in-part of U. S. Patent Application No. 10/480, 827, filed on December 15,2003, entitled "Wound Dressing and Method of Controlling Severe Life- Threatening Bleeding", which was a national stage filing under 37 C. F. R. § 371 of International Application No. PCT/US502/18757, filed on June 14, 2002, which claims the benefit of provisional patent application Serial No. 60/298,773 filed in June 14, 2001. This invention is granted a patent for its haemostatic sponge to stop profuse bleeding. It is continuation of various patents as stated. All these applications use alpha chitin which is chitosan extracted from exo-skeletons (prawn, shrimps lobsters, crabs etc) and its DDA ranging from 78 % to 97%, most preferably greater than 85% but less than 95%. These type of sponge stops bleeding immediately and are used as an emergency remedy. It claims for tissue dressings applied on a site of tissue injury, or tissue trauma, or tissue access to ameliorate bleeding, fluid seepage or weeping, or other forms of fluid loss, as well as provide a protective covering over the site and mainly suffer from the defects by not providing so good bacterio-static and fungi-static properties.

US 4651725 titled "Wound dressing " filed by UNIKITA (Japan) claims for wound dressing material made of chitin fiber non woven fabric plus naturals or synthetics fibrous binders. Another patent application filed by BIOSYNTECH (Canada) numbered as US 7098194 titled "Composition And Method To Homogeneously Modify Or Cross Link Chitosan Under Neutral Condition". In this patent, chitosan is cross linked by an adjuvant in order to produce a thermo reversible gel. The above mentioned wound healing dressing fails to be produced from pharma grade chitosan and hence healing of wound is associated with allergy or infection problems. Extra Cellular Matrix derived proteins and carbohydrate polymers such as collagen, elastin, hyaluronan (hyaluronic acid), chondroitin sulphate etc. have been used to make biological dressing materials for wound healing. Bovine collagen and shark chondroitin sulphate have been combined to make porous 3-D templates for deep wounds. The porous template allows fibroblasts to migrate into the holes and form dermis. This has been first artificial skin for medical applications. However, these templates can not be used on infected wounds and also management of infection with these dressings is difficult.

There are many inventions related to chitosan and chitin. But as stated earlier, haemostatic sponges using chitosan is for stopping profuse bleeding with traumatic wounds. And it can be used for healing the wound. Whereas, the wound healing dressings which is our prime concern and we have tried to solve the associated problems, developed and taught in the literatures so far have many disadvantages: DISADVANTAGES OF PRIOR ART

• The teachings of almost all prior art use chitosan extracted from exoskeletons (prawn, shrimps lobsters, crabs etc.) which contains a lot of heavy metal contamination (mercury, cadmium, etc from sea water).

• Many of the prior arts use chemically modified cross linked chitosan. However, none of the prior art has used natural form of chitosan.

• Most literatures prefer chitosan acetate which is not naturally available in the human metabolic system.

• These are available in solid, fibrous, sponge or thermo reversible gel form which is not as easy to apply as is liquid form.

• None have developed such dressing using pharmaceutical grade chitosan, which is base of the dressing using high grade chitosan with consistent properties.

• The prior art use low molecular weight chitosan i.e. very low viscosity chitosan and not able to meet all complications of a patent.

• Most of the these prior arts have no proper control on the Molecular weight and use chitosan with highly variable range of molecular weight ^" thus resulting into high molecular weight polydispersity. Many of them do not consider polydispersity index. Hence the end product and its result vary a lot.

• Almost all prior art using maximum 95% deacetylated chitosan.

No one has claimed the use of polyglucosamine i.e. more than 98% deacetylated chitosan. They do not contour to wound's irregularity in size, shape and depth.

• Their application creates an anaerobic (non availability of oxygen) condition in the wound and hence bacterial growth is increased.

• In recalcitrant wounds occlusive dressings do not allow the wound to breathe which delays the process of healing.

• The current dressings are not interactive (i.e. they do not induce growth factors in the wound bed).

• During peeling off these dressings by applying force around 30-

40% of the healed tissue also comes off which disturb the new cell growth in the wound.

• These dressing materials are not patient friendly and are not self dressable.

• Current dressings do not allow the patients to take a bath.

• None of these dressings are cost effective

• The dressing frequency in the prevailing method of dressing is 2- 3 times a week. Every time of dressing, it peels off 30-40% healed tissues comes off, resulting in delay wound healing.

• Growth factors used as ointments do not match with the growth factor requirements of wound healing during different stages of wound healing. • Wound debridement done to obtain healthy tissue does not normally remove all narcotized tissue, infection tendons, muscles, bones not removed and surgical debridement often damages nerves.

• Due to all above reasons, severe burns results in death, where as diabetic and venous ulcers often lead to limb amputation.

Therefore, there is an urgent unmet need to come up with effective bioactive dressing for healing the wound. But no one has yet developed any wound healing dressing using pharma grade chitosan with consistent quality of high MW and high DDA. Chitosan procured from various companies show lot of variations. Even if procured from the same company, it will show batch to batch variations. High Polydispersity, inconsistent performance, very high endotoxins levels, unsafe for medical applications etc.

To overcome the problems associated in the said technology currently in practice (bio-dressings), we have come out with an invention, a Liquid Filmogenic Chitosan Skin which provides very fast healing of wounds of any intensity. Chitosan of high purity is to be converted into pharma Grades products to achieve the objective set. A consistent method for purification of squid Chitosan and its further refinement to ultra pure pharma grade has been developed by us.

PRESENT DRESSING METHODS AND DISADVANTAGES ASSOCIATED WITH THE SAME

While debridement is done to obtain healthy non-infected tissue to aid healing process, it is not possible to remove all the necrotic tissues. The infected tendons, muscles and bones may not get removed. Also there are very high chances of damaging the nerves while the dressing is removed for the reapplication of a fresh dressing. The following are the disadvantages of the present dressing methods:

1. Dressing frequency: 2-3 times a week. Every time of dressing, peels off 30-40% healed tissue comes off, resulting in delayed wound healing

2. While external infection prevented in the wound they create anaerobic situation resulting in less supply of oxygen and anaerobic bacterial growth

3. Growth factors used as ointments do not match with the growth factor requirements of wound healing during different stages of wound healing

4. Wound debridement done to obtain healthy tissue does not normally remove all narcotize tissue, infection tendons, muscles, bones not removed and surgical debridement often damages nerves

5. Tissue Engineering based artificial and bioartificial skins are expensive can not used on infective tissue and are not always useful because there is always a sub-clinical infection even in good healthy looking wounds

6. Due to all above reasons, severe burns results in death, whereas diabetic and venous ulcers often lead to limb amputation

7. Most of wound covers available in market do not contour to irregular depths and size of wounds

8. In the present method of dressing patients are advised not to take a bath or put water over the dressing. This results in wound surrounding not being cleaned and thus raising chances of infection

OBJECTIVE OF THE INVENTION

The main object of the invention is to provide a Liquid Filmogenic Chitosan skin which contains natural form of Chitosan that promotes natural cell growth factors and results into fast wound healing even the traumatic wounds. Further object of the invention is to provide Liquid Filmogenic Chitosan Skin which is made from endoskeleton (squid), wherein the endoskeleton is not calcified and does not contain pigments like astaxanthin and does not have contact with the sea contaminant (like heavy metals) at the time of its synthesis.

Further more object of the invention is to provide Liquid Filmogenic Chitosan Skin which is in transparent liquid form and easy to apply. Further object of the invention is to provide Liquid Filmogenic

Chitosan Skin which uses chitosan lactate that is naturally available in the human metabolic system that promotes natural cell growth factors. Further object of the invention is to provide Liquid Filmogenic

Chitosan Skin which uses pharmaceutical grade chitosan, eliminating the risk of infections while healing.

Further object of the invention is to provide Liquid Filmogenic Chitosan Skin using high Molecular weight with better control over polydispersity index of the chitosan thus producing consistent end product. Further object of the invention is to provide Liquid Filmogenic Chitosan Skin which uses polyglucosamine i.e. more than 98% deacetylated chitosan. Further object of the invention is to provide Liquid Filmogenic

Chitosan Skin which is interactive inducing growth factors in the wound bed resulting into fast healing.

Further object of the invention is to provide Liquid Filmogenic Chitosan Skin which allows oxygen from the air to go in the wound bed again promoting fast healing.

Further object of the invention is to provide Liquid Filmogenic Chitosan Skin which does not require dressing hence the healed tissues are not disturbed.

Further object of the invention is to provide Liquid Filmogenic Chitosan Skin which is cost effective and patient friendly which is self dressable.

Further more objective of the invention is to provide a filmogenic liquid chitosan and a method of preparing the same. Further more objective of the invention is purification of chitosan and then its conversion pharma grade chitosan.

Further more objective of the invention is to provide liquid filmogenic chitosan skin with an increased film forming property due to the presence of glycerol that acts as plasticizer and having antibacterial preservative property.

Further more objective of the invention is is to encourage autolytic debridement of tissue rather surgical debridement method used

. currently that results in partial removal of necrotic tissue, often damages nerves, resulting in scar formation, keloids formation etc.

SUMMARY OF THE INVENTION

The summary of the invention is the development of a chitosan filmogenic liquid for wound healing of small to chronic type wherein the said filmogenic liquid is made of pharmaceutical grade chitosan. The bioactive wound healing chitosan filmogenic liquid heals even the traumatic wounds by promoting natural cell growth factors and resulting into pleasing wound healing. Such an invention involves the improvement and modification in production of chitin prepared from high molecular weight pharmaceutical grade highly deacetylated (re-acetylated polyglucosamine 1.5- 2%) with lowest polydispersity index and allows natural healing process by promoting growth factors, scaffolds for growth of cells resulting in aesthetically pleasing wound healing. The use of pharmaceutical grade chitosan for obtaining filmogenic liquid chitosan and a method of obtaining the same are also disclosed. A method of obtaining pharmaceutical grade chitosan from crude chitosan is also within the purview of the invention. A method of fast treatment of wounds of any intensity comprising applying a liquid pharmaceutical grade chitosan of high molecular weight, polydispersity index less than 2, high viscosity and higher degree of deacetylation (DDA) around the wound and spreading with an applicator is also disclosed.

SALIENT FEATURES • Chitosan of the present invention is made from endoskeleton

(squid), wherein the body organs have removed much of these metallic elements

• Chitosan of the present invention has high molecular weight In other words it has high viscosity

• Chitosan of the present invention is low polydispersity chitosan

(>2%).

• Chitosan of the present invention uses beta chitosan from squid pens as the starting material

• Chitosan of the present invention is chitosan lactate

• Chitosan of the present invention is kept as natural as possible DESCRIPTION OF THE INVENTION

The present invention is a liquid filmogenic skin mainly comprising of highly deacetylated beta chitosan (poly-glucosamine) derived from endoskeleton (squid). This beta chitosan polysaccharide is solubilized in an organic acid like formic, acetic, lactic, malic, maleic, malonic, propionic, adipic, succinic, oxalic, pyruvic, citric, tartaric, benzoic, salicylic, 4-Amino-Benzoic, glutamic, glycolic in equally w/w. Most preferably lactic acid , is used as the solubilizing acid as it is naturally present in the human body.

To this chitosan lactate solution as stated above, we add a plasticizer like ethylene glycol, polyetheleneglycol, propylene glycol or glycerol in equally w/w. The most preferred plasticizer is glycerol whose use provides a proven result and is patient compliance. The basic role of the plasticizer is to increase film forming property of the solution as well as it also acts as antibacterial preservative.

The obtained chitosan polymer essentially possesses the following properties:

It has High Molecular weight, (soft film forming and high moisturizing effect); lowest polydispersity; higher degree of deacetylation (DDA) or lower degree of acetylation; lowest endotoxins levels and pharma grade artificial skins. The said material is so prepared that polydispersity index of the substance is maintained as <2 preferably between 1.5 to 2. The range of DDA (degree of deacetylation) is kept around 80% to 99.5% preferably more than 98%. With higher value of DDA, higher degree of bacterio-static and fungi-static properties are achieved. Hence this is best suited for topical applications. The present optimized values are maintained to make the objective of the invention fruitful and this has happened as a result of the proven and consistent results during extensive trials.

The chitosan used in the present invention is a natural form and not extracted from exoskeleton of marine animals. Thus there is no heavy metal contamination. As this present invention uses beta chitosan that is derived from cephalopods endoskeleton, wherein no micro pollutant coming from the sea can be integrated during the morphogenesis, and absence of astaxanthin. Hence, the chances of allergy and infections are almost eliminated due to judicious selection of the chitosan possessing this specific property.

The present invention uses pharma grade beta chitosan in our invention which can be used as artificial skin on small to traumatic wound that enhances healing. Trials in this context have shown proven results with the use of our invented product in curing wound healing. A comparative and analytical data with respect to the prior art shows the ultra high standard of the invented product and its proven use in the field of the proposed problem.

Polyglucosamine standards Many prior art disclose and claim pharma grade chitosan and one such example is shown in table 1 below. However, most of these do not meet the standards established by American Standards of Testing Materials (ASTM), in their Tissue Engineering Medical Device Standards and specifications for chitosan to be used in medical applications. Most of them do not even test for endotoxins and have no control over polydispersity indexes as shown in Table 1. In contrast our specification is highly stringent as can be seen in Table 2. The specifications of chitosan as approved by US-FDA for making Haemostatic Bandages for HemCon as discussed in the prior art and our ultra-pure polyglucosamine is given below for reference.

TABLE 1

Specification of Pharmaceutical Grade Chitosan (Daewoo)

TABLE 2

Ultra-Pure Folyglucosamine Specifications:

The present invention made using all pharmaceutical grade chemicals contour to wound/wound bed and which induces growth factors in human cells.

In our inventive ingenuity in the present invention we have used chitosan lactate which is naturally available in the human metabolic system that promotes natural cell growth factors.

The present invention uses pharmaceutical grade chitosan, eliminating the risk of infections while healing. The final product (i.e. filmogenic liquid) from the raw material is the crux of the invention which has final implication in wound healing properties. The invented filmogenic liquid chitosan skin of our invention bears all the properties for wound healing.

The present invention uses high Molecular weight with control over polydispersity index of the chitosan thus producing consistent end product. The present invention uses polyglucosamine i.e. more than 98% deacetylated chitosan.

The present invention allows oxygen from the air to go in the wound bed again promoting fast healing.

The present invention is in viscous transparent liquid form hence it is easy to apply as well the healing can be easily visualized.

The present invention does not require dressing hence the healed tissues are not disturbed.

The present invention is cost effective, patient friendly and self dressable. The invented Liquid Filmogenic Chitosan Skin is viscous transparent liquid and has many advantages over the conventional products used for wound healing.

The process flow diagram for purification of polyglucosamine to Pharma grade and the Pharma equipment needed are given below.

PROCESS FLOW DIAGRAM FOR FORMULATION INTO LIQUID SKIN

Polyglucosamine is boiled in distilled water for lOmin at a temperature 100⁰C

The reaction mixture is cooled to room temperature

To this suspension, solution A most preferred lactic acid and solution B most preferred glycerol are added by weight in a ration of 1 : 1: 1

Enough distill water is added to the resultant mixture to make up to 2% polyglucosamine

All the contents are mixed for 24hours with constant stirring and maintaining the viscosity at 500 to 5000 Cps. After complete solubilization, filtration is carried out through 100 mesh nylon cloth and then subsequent filtration is done through 0.22 micron PTFE filters

The product thus obtained is bottled up.

Summarizing the above process flow chart:

(i) obtaining pharma grade chitosan as per the specificity devised in the invention; (ii) mixing with distilled water and boiling the content at 100⁰C for the required time; (iii) adding required amount of solution A i.e. organic acid to the content of step (ii) to make it solubilize; (iv) adding required amount of solution B i.e. a plasticizer to the content of step (iii) and stirring for 24 hours;

(v) filtering the contents through nylon cloth meshes and subsequently through PTFE filters;

(vi) bottling the above content for commercial use. The present invented product when taken for various testing which are as follows gives the following results which indicate the potential application of such a product in wound healing and safe use. In Vitro Toxicity tests

Human skin keratinocytes, melanocytes and fibroblasts were established in tissue culture in their respective medium. Chitosan at lmg/ml were added to the medium and the cells were grown for two population doubling. This continuous exposure to chitosan did not have any effect on the survival, growth rates and doubling time. Thus this grade of pharmaceutical chitosan did not have any toxic effect which is presented in Table 3.

TABLE 3

In vitro Toxicology tests result

BATCH TESTING

The batch testing studies have been carried out using films made of this polymer and the comparative results are depicted in Table 4. It shows the enhanced keratinocytes growth profile on 3rd to 5th days on the biopolymer sheets developed.

TABLE 4

BATCH TESTING OF HUMAN KERATINOCYTES CELL GROWTH

Growth factor expression studies

In addition to showing that the present product is not toxic to cell and that growth rates could be faster, growth factors involved in wound healing was also investigate. For this keratinocytes were grown on biopolymer sheets developed and their mRNA (messenger ribonucleic acid) was isolated and expression studies were done using appropriate primers and RT PCR (reverse transcriptase polymer chain reaction. The results are tabulated in Table 5. TABLE 5

Growth Factor Expression in keratinocytes cultured on biopolymer sheets

The above studies clearly indicate this liquid filmogenic preparation will be very useful to treat recalcitrant wounds where neuropathy and vasculopathy is a common factor underlying their non-healing states.

The clinical trial experiments further shows some proven results on the implication of the invented chitosan containing liquid film to serve as a skin. linical Trial Experience

One drop of liquid skin covers approximately 2.5 sq. cm. skin. Since all wounds are irregular in shape and depth, it is impossible to estimate how much liquid skin would be needed for each wound. Our experience has been that patients start with 30 ml bottles, take 30 ml bottles midway and always would want to keep 10 ml bottles at home, even after the wound heals. Similarly it is difficult to estimate the number of days it would take to heal. In a normal individual liquid skin wound take half of the time it takes to heal with routine dressings. In diabetic individuals fluctuating blood sugar levels takes its toll on the healing process. Liquid skin induces dermalization even when sugar levels are high. Even here, a rough estimate would indicate that the time taken to heal would be half of what it would take otherwise.

Above all, the following Advantages have been noticed during Clinical trials.

1. Patient friendly, do it yourself dressing.

2. Cost effective treatment modalities.

3. Reduced frequency of hospital visits.

4. Transparent washable dressing.

5. Growth factor Induction during wound healing.

6. Liquid skin contours to wounds irregular depth.

7. Fast and aesthetic healing.

8. Skin grafting not needed in most cases.

9. Amputation prevention is 60-70% of cases.

10. Possible Nerve Regeneration.

Claims

1. A liquid filmogenic pharmaceutical grade chitosan skin for wound healing comprising;

(i) Chitosan of high molecular weight, high viscosity and higher degree of deacetylation (DDA) with lowest polydispersity;

(ii) Effective amount of an organic acid for formation of salt of chitosan;

(iii) Effective amount of glycerol to serve as a plasticizer for enhancing film forming property as well as antibacterial preservative.

2. The liquid chitosan skin as claimed in claim 1 wherein the molecular weight of the chitosan is 300000 g/mole.

3. The liquid chitosan skin as claimed in claim 1 wherein the polydispersity index of chitosan is less than 2.

4. The liquid chitosan skin as claimed in claim 1 wherein the degree of deacetylation (DDA) is in the range of 98 to 99.5%.

5. The liquid chitosan skin as claimed in claim 1 wherein the chitosan is a beta chitosan obtained from squid internal bone.

6. The liquid chitosan skin as claimed in claim 1 wherein the organic acids selected for salt formation of chitosan are selected from acetic, lactic, malic, maleic, malonic, propionic, adipic, succinic, oxalic, pyruvic, citric, tartaric, benzoic, salicylic, 4-Amino-Benzoic, glutamic or glycolic acid.

7. The liquid chitosan skin as claimed in claim 6 wherein the organic acid is preferably lactic acid.

8. The liquid chitosan skin as claimed in claim 1 wherein the said skin is a transparent liquid.

9. The liquid chitosan skin as claimed in claim 1 wherein the viscosity of the liquid is between 500 to 5000 Cps.

10. The liquid chitosan skin as claimed in claim 4 wherein the degree of deacetylation (DDA) is preferably in the range of

98%.

11. A process for preparation of a liquid filmogenic pharmaceutical grade chitosan skin for wound healing comprising the steps of:

(i) obtaining pharma grade chitosan as per the specificity devised in the invention;

(ii) mixing with distilled water and boiling the content at

100⁰C for the required time;

(iii) adding required amount of solution A i.e. organic acid to the content of step (ii) to make it solubilize;

(iv) adding required amount of solution B i.e. a plasticizer to the content of step (iii) and stirring for 24 hours;

(v) filtering the contents through nylon cloth meshes and subsequently through PTFE filters;

(vi) bottling the above content for commercial use.

12. The process as claimed in claim 11 wherein solution A and B are mixed with pharma grade chitosan in the ratio of 1: 1:1 with water so as to make 2% polyglucosamine.