Description:FORM 2
THE PATENT ACT, 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. Title of the invention: “BIOPOLYMER GEL COATING ON NONWOVEN FABRIC AS HAEMOSTATIC AGENT”
2. Applicant:
NAME NATIONALITY ADDRESS
SWAKIT BIOTECH PRIVATE LIMITED INDIA

39, S L V PLAZA, BULL TEMPLE ROAD, BASAVANGUDI, BANGALORE 560004
Email:
shreedattalawconsultancy@gmail.com
Chothani18preeti@gmail.com
info@swakitbiotech.com
(M) +91 9448532321
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of the Invention:
The present invention relates to a medical bio-polymer gel coatings for the wound dressings based on the chitosan as the haemostatic agent during the arterial, venous and capillary bleeding in humans and animals especially during surgical interventions including dental procedure.
Background of the Invention:

The term “Haemostasis” is the stoppage of bleeding, haemorrhage or blood flow through a blood vessel or body part – human or animal. This is a new method & means for haemorrhage control to reduce future haemorrhage morbidity and mortality. The excessive and uncontrolled blood loss (Haemostasis) due to the injuries, accidents and operations may lead to the severe trauma including arterial, venous & capillary bleeding which may further lead to the shock, inflammation, organ failure and even death. Overall, around 35% of the pre-hospital deaths are reported due to the traumatic haemorrhage and these deaths could be reduced if efficiently haemorrhage control is provided at the time of emergency.
The present invention is a new method and means for haemorrhage control to reduce future haemorrhage morbidity and mortality. Various haemostasis materials such as collagen-based powders, sponges, or cloths are of a particulate nature are widely used to control the haemorrhage condition at the time of emergency. Moreover, various fibres such as polysaccharide fibres based on the alginate or carboxymethyl cellulose (CMC) are also being utilized as an absorbent dressing being capable of adapting to the conformation of the individual wound using fibre dressings or hydrogels. The use of fibres often suffers from the drawback of a limited absorption and/or lack of cohesion, leading to difficulties with respect to removing the fibre product from the wound as the product does not constitute a cohesive part. Therefore, there is a need to develop the haemostasis agent with good absorption and cohesion properties.
In this regards, chitosan is emerged as a novel bio-polymeric agent to be used as an efficient haemostasis agent due to its biocompatibility and physico-chemical properties.
Basically, Chitosan (Chitosan) is the product after the partly or entirely de-acyl group of chitin (Chitin). Chitosan is cationic polymer, positively charged, and charge density is high, can form water-fast composite, have very large and potential application in technical field of biological material with a large amount of anionic polymer. At low pH values (lower than 6), the amino of chitosan, by protonated, makes chitosan positively charged. At a high pH (higher than 6.5), chitosan will go matter, and chemical property is enlivened. And at a high pH, the copolymerization of chitosan energy forms fibre or network structure (as film or gel). Furthermore, chitosan has excellent biocompatibility, have the biological function of cellulose in collagen and higher plant tissue in higher mammal tissue concurrently, to animal and plant, all there is good adaptability, be embodied on cellular level with the affine performance of organism, the probability producing antigen is very little, chitosan contributes to repair in trauma, nontoxic, implant and almost there is no rejection, and angiogenesis.
Recently, chitosan is mainly used as the haemostasis material by many researchers, because chitosan has lot of advantages for wound healing which accelerate tissue regeneration and stimulate fibroblast synthesis collagen and have been disclosed in the various prior arts as mentioned below.
CN104208741A discloses the chitosan based adhesive bandage composed of chitosan, oligochitosan, and enhancing polymer, wherein the chitosan and oligochitosan accounts for 85 to 95% of the total weight of the adhesive bandage, and the enhancing polymer is one or more of sodium alginate, glucan, hyaluronic acid polymer, and collagen. The provided adhesive bandage can rapidly and high efficiently control the severe bleeding, reduces the wound treatment time, can be used in wild rescue and operation for stopping the wound bleeding, and thus a novel method is provided for local haemostasis. Moreover, the adhesive bandage has a very good antiseptic performance and has a prominent inhibiting effect on most of the bacterias.
IN201921039392 discloses the chitosan-based dressing for rapid hemostasis which includes silica nanoparticles (SiNPs) synthesized by sol-gel method are incorporated into a chitosan gauze for improving blood clotting efficiency of said hemostatic dressing.
US11160901B2 discloses the bio-adhesive chitosan gel for controlling bleeding and for promoting healing with scar reduction without obscuring or interfering with access to a surgical field with the composition of 3% (w/w) of chitosan; 0.5% to about 7% (w/w) of mono-functional acid, 5% (w/w) of di-functional or tri-functional organic acid, 3% (w/w) of polyfunctional organic acid, about 3% (w/w) of inorganic phosphoric, triphosphoric, or polyphosphoric acid for the development of chitosan gel.
CN109999214A discloses the preparation method of bacteriostatic medical bandage which includes antimicrobial composites of nano-montmorillonite and a Ti(SO4)2 solution along with the chitosan oligosaccharide. the bandage base material was subjected to high temperature steaming treatment in a steam chamber, then taken out while the base material was hot, treated in antibacterial liquid and taken out for drying to get the product with good strength, degradable, high in biocompatibility and durable in antimicrobial property and had certain self-stickiness.
Thus, there is still a need for a wound care haemostatic agent comprising a material being capable of absorbing large amount of liquid by gelling, having a three-dimensional structure in the form of fibres, and having a high degree of cohesion after absorption which can be fulfilled by the present invention.
The present invention discloses the biopolymer gel coating on nonwoven cotton fabric as haemostatic agent using chitosan which accelerated the clotting time. The medicated gauze is known to have haemostasis properties such as promotion of platelet adhesion, platelet aggregation, and platelet activation. Additionally it absorb exudates, cushion the wound, hide the wound from view, and provide a barrier to contamination. This product can save many lives & it can used being widely used to stop bleeding due to arterial, venous & capillary bleeding.
Object of the Invention:
Main objective of present invention is to provide a kind of chitosan-based gel coating nonwoven cotton fabric as an antibacterial agent which can be used for field trauma and to stop bleeding in time rescue, surgical haemostasis.

Another objective of the present invention is to provide chitosan based gel coating non-woven cotton fabric with specific composition of various GSM and thickness of the cotton gauze.

Yet another objective of the present invention is to accelerate the clotting time up to 435 sec against the haemostasis.

Yet another objective of the present invention is to provide gamma sterile chitosan gel coated non-woven cotton fabric with the shelf life of up to 4 years.

Summary of the Invention:

The present invention is related to biopolymer gel coating on non-woven cotton fabric as haemostatic agent which can be used for the bleed management from injuries in humans and animals. The biopolymer is prepared from chitosan and its oligosaccharides with chelating agent. The biopolymer gel coated non-woven cotton fabric can be useful for dressing in wound care with the ability to accelerate clotting time with the excellent properties like platelet adhesion, platelet aggregation, and platelet activation. Moreover, biopolymer gel coated nonwoven cotton fabric absorb exudates, cushion the wound, hide the wound from view, and provide a barrier to contamination. The present invention is Gamma sterile with 4 years shelf life and further can be used to stop bleeding due to arterial, venous & capillary bleeding. The composition of the present invention comprises of Chitosan - 1 -2% [Crab origin], Lactic acid 0.5 - 2%, Calcium chloride - 0.3 - 1.2% and made in Demineralised water.

Detailed Description of the Invention:
The following description relates to a particular manifestation of the present invention. The present invention relates to biopolymer gel coating on nonwoven cotton fabric as haemostatic agent includes the process of the coating woven & nonwoven absorbent cotton fabrics. Moreover, the present invention is related to the application of biocompatible, biodegradable natural polymer with additional properties of swelling, absorbency and haemostatic. The biopolymer gel coating has many advantages over exiting coating such as:
- It is applicable to both human and animals.
- It can be applied to the trauma inducing arterial, venous & capillary bleeding.
- It can be used in all kind of surgical interventions including dental procedures.

The normal absorbent cotton gauze can absorb blood or body exodus only. It cannot stop bleeding at the time of injury. To overcome this, the coated gauze is the best option to prevent blood loss due to clotting action by the medicated gauze. Then the injured person/animal can be taken to hospital for further treatment. Thus, the natural biopolymer is considered as the best alternative to overcome the above disadvantages of ordinary medicated gauze.

Recently, absorbent cotton (woven and nonwoven) coated with chitosan are intended for approval for medical uses due to their advantages superior to pure absorbent cotton gauze. Coated gauze has capability to clot blood with few minutes of application on wounded skin surface which prevents further blood loss from the patient before appropriate treatment whereas non-woven coated gauze has nearly 10 times more absorption capacity to its weight. In this connection, chitosan coating has been found to be most promising biopolymer as coating due to its cost effectiveness and very high efficacy.

Chitosan based haemostatic agents have been already marketed in various countries like United States, Korea and United Kingdom as chitosan based haemostatic agents function independently on platelets rather than following the normal clotting mechanism.
Unique properties of natural polymer on chemical cross linking which determines features of hydrogel. Other than antimicrobial properties, swelling action leads to absorption of body fluids. Chitosan (CS) is an amino-polysaccharide molecule with a strong positive electric charge, which strongly attracts and bonds to negatively charged molecules. On the surface of the RBC cell membrane, there are various proteins and glycol-lipids that are negatively charged. HARMOSTAT is coated with hydro-gel which makes blood to clot.

In addition, it is generally known that chitosan has excellent molding processability when it is used in a small amount as an additive in the existing main component, and when the chitosan is used as the main component, the fiber, membrane, and sheet-like structures have a soft property. Therefore, there was a problem that the utilization of gauze, bandages, mask packs, etc. is limited.

The present invention addresses the limitations associated with traditional chitosan packs or films by creating a nonwoven fabric comprising chitosan fibers and cotton fibers. Subsequently, this fabric undergoes an acid treatment to solidify the chitosan within it, facilitating direct interaction between the solidified chitosan and the skin.

Furthermore, the chitosan within the nonwoven fabric is transformed into a gel state, allowing the fabric to maintain its soft texture even with higher chitosan concentrations. As a result, it can serve various non-pharmaceutical purposes such as gauze, bandages, wound treatment, and cosmetic mask packs, where the chitosan is gelled through the utilization of lactic acid.

The present invention is made up of components/ingredients as mentioned below:
1. Natural bio-polymer such as chitosan and its oligosaccharide with chelating agent
2. Cotton nonwoven fabric of various GSM and thickness
3. Packed double wall paper and aluminium pouches.
4. Gamma sterile upto 4 years shelf life.

The dried coated nonwoven fabric is cut to various sizes and packed in heat sealed double layer pouches. It is further gamma sterilized.

Solubility data
Generally, batch-to-batch variability, non-precise characterization and randomly distributed acetyl groups of chitin structure eventually results in a bad reproducibility of chitin solubility. Therefore, the choice of an appropriate solvent for chitin and chitosan solubilization is important and primary issue for lab scale research and scaling up for industrial practice. Moreover, the acetyl groups in chitin can be removed by deacetylation to convert insoluble chitin into a more soluble compound, namely chitosan (this name is given to chitin with at least 50% degree of deacetylation, DD). Therefore, solubility study of chitosan based haemostatic agent was carried out to get the information of chitosan structure at molecular level and correlate solubility with chitosan structure to broaden the scope and use of the developed haemostatic agent for medical purpose.

Factors affecting solubility of chitosan

1. Effect of N-acetyl-d-glucosamine units
There are two monomer units present in the chitin structure in different fraction:
(i) 2-acetamino-2-deoxy-d-glucopyronase (N-acetyl-d-glucosamine) and (ii) 2-amino-2-deoxy-d-glucopyronase (N-amino-d-glucosamine). The first one, 2-acetamino-2-deoxy-d-glucopyronase, displays insolubility due to the strong hydrogen bonds between the acetyl groups of the same or adjacent chitin chains. Hydrogen bonds network builds a three-dimensional crystalline matrix by sequencing the following bonds -NH?O-C and -OH?O-C. The other unit, N-amino-d-glucosamine shows a distinct property such as hydrophilic nature and positively charged in acidic solution. The domination of the hydrophilic character with a high amount of N-amino-d-glucosamine unit in the chitin backbone can be determined by degrees of deacetylation (DD). The DD is determined from the ratio of N-amino-d-glucosamine to N-acetyl-d-glucosamine while the degree of acetylation (DA) represents the deduction from 100 (i.e. 100—DD). When DD is between 60 and 90% a new chemical entity “Chitosan” is baptized which is soluble in organic acids such as acetic acid.

2. Effect of solution pH
The aggregation behaviour of chitosan is strongly influenced by the pH of the solvent medium. In general, chitosan molecules are more or less ionized up to pH 6.0, and the ionization increases as the pH moves to low values. Therefore, the amino groups of chitin chains (low DA) at a particular pH (<6.0) capture H+ solution ions and exhibited positive surface charge which can be determined by zeta potential value. The charged amino groups resist the aggregation of chitosan in the solution, but when the DA value increases from zero to higher value the aggregation starts to dominate over the coulombic repulsion forces of the charged groups. The pH at which the net charge of a chitosan solution prevents aggregation is called critical pH.

3. Effect of molecular weight
Apart from the degree of deacetylation and pH, molecular weight influences the conformational changes and solubility of chitosan. Chitosan solubility increases with the decrease of molecular weight. The solubilization process of chitosan, as it happens for functionalized polymers, involves different types of chemical and physical interactions such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.

4. Salt effect/ionic strength and temperature
Ionic strength is a measure of the total concentration of ions present in a solution. Chitosan acts as polyelectrolyte, when it is dissolved in a solvent. The polycationic solutions develops electrostatic repulsive interactions while masking other possible interactions. The addition of a salt or the increase of ionic strength of the chitosan solution results in an inversion from repulsive to attractive interactions. The attraction inspired by the screening of amino-charged chitosan chains with the anions such as CH3COO- and Cl- increases the tendency of flocculation or precipitation of chitosan. Therefore, the increase of ionic strength enhances the aggregation by chitosan-chitosan attraction over the chitosan-solvent interaction and influences chitosan solubility. Chitosan in acidic medium shows an expanded conformation structure since the amino groups exert repulsive force with each other, but the addition of salt or increase of ionic strength shrink the structure by increasing chain flexibility. As a result, the occupied volume of chitosan chains in solution is reduced by increasing the ionic strength and a decrease of intrinsic viscosity of chitosan solution is observed.

Solvability of different solvents
Various kind of solvents such as formic acid, inorganic solvents and ionic liquids have been widely used for solubility of chitin/chitosan. The following Table 1 shows the various kind of solvents used for the solubility of chitosan.

Table 1: Various solvents used for the in the modification of chitin and chitosan to enhance the solubility
Chitin/chitosan
Solvent
Acetylated chitin Dimethyl sulfoxide (DMSO)
Chitin-graft-polystyrene Dimethyl sulfoxide (DMSO)
Monomethyl-modified chitosan Water
O-alkylated chitosan Chloroform, ethanol, water and acetic acid
Chitosan-graft-polycaprolactone Dimethylformamide (DMF), DMSO, ethanol and toluene

Enzymatic hydrolysis
Enzymatic hydrolysis is a green process to achieve chitosan excellent solubility in water by producing chitooligosaccharides (COS). The process does not require extreme conditions (very low pH or high concentration of acids) and the tuning of molecular weight, and DD of final product can be achieved by avoiding any unwanted yield. Chitinase, chitosanase are specific enzymes and many other nonspecific enzymes such as glycanases, proteases, lipase are isolated from many biological sources. Unlike acid hydrolysis, enzymatic hydrolysis affects both the depolymerization and deacetylation of chitin or chitosan through the catalytic activities, which mainly depends on the molecular structure of chitinase (enzyme).

The following Table 2 provides the other enzymes used for chitosan enzymatic treatments and the operation conditions.

Table 2: List of enzymes for the hydrolysis of chitin/chitosan
Enzyme Water soluble modified chitosan
Lysozyme Chitooligosaccharides (COS)
Papain
Cellulase
O-glycoside hydrolase Low MW chitosan (3–6 kDa)
Chitosanase and ß-d-glucosaminidase d-glucosamine
Chitin deacetylases Chitin and chitosan oligomers
Carbohydrases from Myceliophthora sp Low MW chitosan (4–28 kDa with 85% DD)

The following table summarizes the solubility of different formulation used in present invention to get the suitable solubility behaviour of chitosan. The adequate viscosity helped to desired product which is more absorbance of water and decrease the bleeding time. The following Table 3 shows the different formulation based on their viscosity for the chitosan-based haemostat preparation.

Table 3: Viscosity parameter of different formulation of chitosan
Parameter Formulation 1% Formulation 2% Formulation 3% Formulation 4% Formulation 5%
Qty
Chitosan 1 gm in 100 mL 2 gm in 100 mL 3 gm in 100 mL 4 gm in 100 mL 5 gm in 100 mL
Master Mixture 0.5 mL/1 gm 0.5 mL/1 gm 0.5 mL/1 gm 0.5 mL/1 gm 0.5 mL/1 gm
RPM 300 RPM 350 RPM 400 RPM 500 RPM 800 RPM
Type impeller Anchor Anchor Anchor Anchor Anchor
Time duration 3.0 Hrs 3.30 Hrs 4.30 Hrs 5.0 Hrs 6.0 Hrs
Viscosity 820 Cps 1800 Cps 3900 Cps 5200 Cps 7800 Cps
Solubility 100 % 100 % 100 % 99% 99%
Colour Colourless Colourless Colourless Colourless Colourless
Cotton Roll 300 GSM 300 GSM 300 GSM 300 GSM 300 GSM
Loading gel 3 GM 3 GM 3 GM 3 GM 3 GM
Size 10 cm x10 cm 10 cm x10 cm 10 cm x10 cm 10 cm x10 cm 10 cm x10 cm
Recover after compression 1.8 gm 1.2 gm 0.8 gm 0.0 gm 0.0 gm
Heat 42?C 42?C 42?C 42?C 42?C
Dressing Abs
3 MM (1 Ply) 20 22 51 US US
6 MM (2 Ply) 40 46 63 US US
9 MM (6 Ply) 60 65 56 US US
Haemostat 159 159 140 US US
US – Under study
Based on the above observations and solubility data the developed gauze is better than the other marketed gauze and developed gauze induced quick haemostatic action.
The composition of the present invention comprises of Chitosan - 1 -2% [Crab origin], Lactic acid 0.5 - 2%, Calcium chloride - 0.3 - 1.2% (Master mix) and made in Demineralised water. The present invention is Gamma sterile upto 4 years shelf life and further can be used to stop bleeding due to arterial, venous & capillary bleeding.

Coating procedure of chitosan on cotton gauze:
The woven gauzes may be lino weave, normal weave types of cotton gauzes or needle punched nonwoven fabrics coated with a biopolymer chitosan. The required viscosity of chitosan is achieved by a process of de-polymerization. The desired concentration of chitosan is achieved by addition of weighed quantity in organic acids diluted with demineralized water. The chitosan solution is then applied to the cotton fabrics by padding process. The fibrous structure of the chitosan according to the present invention may provide an essential coherence for use in a wound dressing. The wound contacting fabric is the absorbent material has coherent rendering property to remove the wound dressing in one piece from the wound.

Furthermore, the chitosan coating would provide improved platelets and iron metal complexes by tissue displacement with high efficacy for haemostatic properties, eliminating the side effects of existing vasoconstrictor and haemostatic agents, preventing further bleeding. The chitosan also includes the property of swelling so when the coated gauze is applied on bleeding surface it clots blot within short time.

The below mentioned various formulation were used for the preparation of chitosan-based gauze.

1. Concentration:
Molecules: CH750C (Chitosan high molecular weight)
Table 4: Different formulations prepared from CH750C
Sr. No. Formulation F1 F2 F3
1. Chitosan 1% 2% 3%
2. Master Mix 10 mL Not added 30 mL
3. Demineralized Water QS QS QS
* QS: Quantity Sufficient

Molecules: CH200C (Chitosan low molecular weight)
Table 5: Different formulations prepared from CH200C
Sr. No. Formulation F1’ F2’ F3’
1. Chitosan 1% 2% 3%
2. Master Mix 20 mL Not added 60 mL
3. Demineralized Water QS QS QS

2. In process test result (IPT) Applicable before Gel state and sterilization

Molecules: CH750C
Table 6: Test result of the formulations prepared from the CH750C
Sr. No. Parameter Acceptance criteria Results/Observations
F1 F2 F3
1. Formulation viscosity
(Upper and lower) Not less than 800Cps 820 cps 1800 cps 3900 cps
2. Formulation pH 5.0 to 7.0 5.6 6.0 6.2
3. Appearance Creamy white Creamy white Creamy white Creamy white
4. Moisture content Not more than 10% NA NA NA
5. Absorbency
(If dressing) Non absorbable chitosan / chitosan dressing absorbs NLT 20-time water NA NA NA
6. Final pH testing 5.5 to 7.0 5.6 6.2 6.3

Molecules: CH200C
Table 7: Test result of the formulations prepared from the CH200C
Sr. No. Parameter Acceptance criteria Results/Observations
F1’ F2’ F3’
1. Formulation viscosity
(Upper and lower) Not less than 300 Cps 300 cps 710 cps 1000 cps
2. Formulation pH 5.0 to 7.0 5.7 5.9 6.5
3. Appearance Creamy white Creamy white Creamy white Creamy white
4. Moisture content Not more than 10% NA NA NA
5. Absorbency (If dressing) Non absorbable chitosan / chitosan dressing absorbs NLT 20-time water NA NA NA
6. Final pH testing 5.5 to 7.0 5.6 6.2 6.3

3. Test result for before sterilization product
Molecules: CH750C
Table 8: Test result of the formulation prepared from the CH750C before sterilization
Sr. No. Parameter Acceptance criteria Results/Observations
F1 F2 F3
1. Appearance Yellow to white in colour Flexible No wet patches White White White
2. Moisture Content Not More than 10% of its total weight NA NA NA
3. Absorbency Non-Absorbable Chitosan wound healing dressing absorbs not less than 20 times its weight of water Mm Time
Abs Mm Time
Abs Mm Time
Abs
3 20 3 22 3 51.26
6 40 6 46 6 63.57
10 60 0 65 10 56.23
4. PH testing 6.0 to 7.0 5.6 6.2 6.3
5. Sterility Product should be sterile NA NA NA
6. Bacteria Endotoxin test ? 0.25 EU/ML of product wash with endotoxin free water NA NA NA
7. Seal Strength (Wet Condition) Not Less than 1.2N/15mm 1.5 N 1.9 N 2.1 N
8. Seal Strength (Dry Condition) Not Less than 1.5N/15mm 1.3 N 1.6 N 1.9 N
9. Brust test (For Blister Packing) Not Less than 0.25kg/cm2 NA NA NA
10. Tensile Strengths (Wet Condition) Not Less than 5n/25mm NA NA NA
11. Tensile Strengths (Dry Condition) Not Less than 7n/25mm NA NA NA
12. Dye penetration test No penetration of dye into the sealed pouch NA NA NA
13. Micro biology test (S. Aureus) Media peptone broth Zone of inhibition In diameter Between range of Diameter (mm) 10 mm to 15 mm 11 mm 11.5 mm 11.2 mm
14. Hemostat 180 sec 159 sec 159 sec 14 sec
15. MIC Microtiter plate Study on going S-100 ?g/ml E-108 ?g/ml SM-10 ?g/ml S-106 ?g/ml E-109 ?g/ml SM-11 ?g/ml S-100 ?g/ml E-108 ?g/ml SM-10 ?g/ml

Main embodiment of the present invention, a biopolymer gel coating on nonwoven fabric as haemostatic agent for dressing comprising of:
a) Chitosan;
b) Lactic acid; and
c) Calcium Chloride; and
d) Demineralize water;
wherein said gamma sterile chitosan gel coated non-woven cotton fabric with the shelf life of up to 4 years and has clotting time up to 435 sec against the haemostasis.

Another embodiment of the present invention is the said biopolymer gel comprising of 1-2 % of chitosan originated from crab, 0.5 -2% of lactic acid and 0.3 -1.2 % of calcium chloride in demineralized water.

Another embodiment of the present invention is the method of preparation of biopolymer gel coating on nonwoven fabric comprising the steps of:
a. Selection of a fabric such as lino weave cotton gauze, normal weave cotton gauze, and needle-punched nonwoven fabrics for coating;
b. Preparation of chitosan solution by addition of chitosan to organic acids diluted with demineralized water having the viscosity of through a de-polymerization process;
c. The prepared chitosan solution is applied to the fabric using padding process; and
d. The chitosan coated fabric is dried at the temperature to obtain the chitosan coated wound dressing.

Another embodiment of the present invention is chitosan-coated fabric is absorbent and possesses a coherent rendering property, allowing for the easy removal of the wound dressing in one piece from the wound.

Another embodiment of the present invention is chitosan-coated fabric exhibits high efficacy in promoting haemostatic properties by improving platelet and iron metal complexes through tissue displacement.

Another embodiment of the present invention is chitosan-coated fabric has the property of swelling which further promotes rapid clot formation when applied to a bleeding surface without any side effects.
Therefore, the present invention is having the following advantage over other haemostatic agents available in the market.
• It is gamma sterile
• Pain less application
• Effective in bleeding
• Versatile due to immediate blood clotting
• Antibacterial hence no infection to wound
• Bioactive adhesive
• Muco-adhesive hence covers injured tissue
• Remains on bleeding site

Furthermore, the present invention has novel features as mentioned below:
• Application of natural bio-polymer
• It is biocompatible, with swelling & haemostatic properties
• Nonwoven cotton or viscos fabric
• Unique coating process on nonwoven cotton fabric
• Packed in double layer pouches which is Gamma sterile
• Available in various sizes including rolls or ‘Z’ folded

The present invention discloses the following various chitosan based haemostatic agent for the different applications as per the requirement.

Table 9: Various chitosan based haemostatic agent
Model Name Pouch Size Contents Application
HRMST-A-10*10 MM, 150 W x 130 H mm 30 pcs Dental
HRMST-B- 50*50 MM, 150 W x 130 H mm 04 pcs Domestic
HRMST-C -50*80 MM, 150 W x 130 H mm 04 pcs BSF, Police
HRMST-D-Z-75*200 MM, 150 W x 130 H mm 1 pcs Travelers
HRMST-E-150*150 MM, 105 W x 170 H mm 1 pcs Severe bleeding
HMRST-F-12.5KR-125*1500MM Roll 120 W x 210 H mm 1 pcs Defense forces, Trekking

Test for in vitro cytotoxicity of chitosan based haemostatic agent: Direct contact method
The direct contact method is used to evaluate whether the test item Root Canal Irrigant induces in vitro cytotoxicity in Balb/c 3T3.
The test item Root Canal Irrigant and Reference control (EDTA) was stored at room temperature (20 – 30 °C). Root Canal Irrigant is modified chitosan which is non-sterile clear liquid. It is used prior to tooth root canal treatment as irrigant, to assist root canal bridging efficient. It is a device which comes in contact with mucous membrane, tissue/bone and dentin communicating. The duration of contact is maximum of 10 minutes.
Preparation of the test item
Test item and reference control was used as such. 200 µL of the Test item and reference control was loaded on to the respective filter paper of size 1cm x 2.5 cm and used.
Test Procedure
Confluent culture flask was trypsinised and reseeded into culture flasks for the cytotoxicity assay at a concentration of approximately 1 x 105 cells per mL. Triplicate cultures were set up for negative control, test item, reference control and positive control. Prior to the test item addition the cell confluency and morphology were confirmed by microscopy and found to be 80% confluent. The culture media was carefully removed and replaced with fresh media. Test item and the reference control were placed in the center of each culture flask. The cell culture medium serves as negative control and natural rubber latex gloves of size 2.5 cm2 (1 cm x 2.5 cm) serves as positive control. Cultures flasks were incubated at 37 °C for 24 h in an atmosphere of 5% CO2.
After 24 h incubation, the cultures were analysed for microscopic evidence of cytotoxicity under microscope. Cell confluency and morphology were specifically looked into using a grading scheme as described in Table 10.
Quantitative evaluation using the Neutral red uptake assay and the absorbance was measured at 550 nm using colorimeter described in Table 12.
Data evaluation
Qualitative evaluation was assessed by following the grading system given in
Table 10. If the numerical grade obtained is greater than 2 the test item was considered as cytotoxic. Quantitative evaluation: Test item will be considered non-cytotoxic if the viability as measured by neutral red uptake is equal to or greater than 70% that of the untreated control. Viability of less than 70% indicated cytotoxicity. The assay was considered valid, since the positive control treated cultures gave a clear increase in cytotoxicity compared to that observed in negative control cultures. Good scientific judgment was used in interpreting the data.
Acceptance criteria
1. The assay was considered valid since all the following criteria were met:
2. Before inoculation, cells had a confluency of greater than 70%.
3. Mean absorbance value of negative control is = 0.3.
4. The positive controls showed a strong positive cytotoxic response >30%.
Results
Qualitative evaluation: The cells treated with the positive control showed a complete destruction of cell layer compared with negative control. Quantitative evaluation: The cells treated with the negative control did not induce any cytotoxicity and the positive control induced 88.30 % cytotoxicity. Based on qualitative and quantitative evaluations, the assay was considered as valid.
The determination of cytotoxicity of the test item and the reference control was performed by qualitative evaluation by examining the cells under the microscope to assess the general morphology of the cell and graded as 0 and results are given in Table 11. Quantitative evaluation using neutral red uptake assay for the test item Root Canal Irrigant showed a viability of 88.29% and reference control shows 84.04% viability, when compared with the negative control (Refer Table 12).

Conclusion
Based on the results obtained in the study, it is concluded that the given test item Root Canal Irrigant and reference control EDTA is considered non - cytotoxic under the conditions of the present test carried out using Balb/c 3T3 cell line.
Table 10: Grading scheme for cytotoxicity
Grade Reactivity Description of reactivity zone
0 None No detectable zone around or under specimen
1 Slight Some malformed or degenerated cells under specimen
2 Mild Zone limited to area under specimen
3 Moderate Zone extending specimen size up to 1.0 cm
4 Severe Zone extending farther than 1.0 cm beyond specimen

Table 11: Qualitative evaluation
Sample Culture Reactivity Grade
Negative Control 1 None 0
2 None 0
3 None 0
Test item 1 None 0
2 None 0
3 None 0
Reference control 1 None 0
2 None 0
3 None 0
Positive control 1 Severe 4
2 Severe 4
3 Severe 4

Table 12: Quantitative evaluation (absorbance at 550 nm)
Sample Replicate 1 Replicate 2 Replicate 3 Mean Viability (%) Cytotoxicity (%)
Negative control 0.95 0.93 0.94 0.94 100.00 0.00
Test item 0.80 0.87 0.81 0.83 88.29 11.71
Reference control 0.79 0.76 0.81 0.79 84.04 15.96
Positive control 0.10 0.09 0.13 0.11 11.70 88.30

Acute systemic toxicity study of chitosan based haemostatic agent in Swiss albino mice
Acute systemic toxicity of the test item (Root Canal Irrigant), and reference control item (17% EDTA), was evaluated in Swiss albino mice. The test and reference control items were administered at the recommended dose volume of 50 mL/Kg b.w. Two groups of mice, each comprising of five males and five females were treated intraperitoneally. The animals, survived, were observed for morbidity, mortality, and abnormal clinical signs and symptoms following administration. The results indicated that the animals treated with test item showed reversible clinical signs. However, the reference control treated animals showed severe signs of multi-organ failure as indicated by gross lesions such as haemorrhages in kidney, liver and lungs; and ultimately death. Based on the results, it is concluded that the test item, Root Canal Irrigant, showed less acute systemic toxicity compared to similar doses of reference control.
The aim of the present invention is to determine the acute systemic toxicity potential of the test item in Swiss albino mice.
The test item for this study was Root Canal Irrigant which is modified chitosan non sterile clear liquid. It was received and stored at room temperature (20 - 30 °C). Root Canal Irrigant is used prior to tooth root canal treatment as irrigant to assist root canal bridging efficient. This device comes in contact with mucous membrane, tissue/bone and dentin communicating. The duration of contact is maximum 10 minutes.
Test Method
The test item and the reference control were administered as such to the animals without any dilutions.
Dosing Procedure
Justification for dose selection
Specified in ISO 10993, Part-11 standard, intraperitoneal route is the appropriate route for test item administration to evaluate the acute systemic toxicity of the medical devices.
The test item and reference control were administered to mice via intraperitoneal (IP) route using sterile syringes and needles as given in the table below:
Table 13: The administration details of test item and reference control via IP route
Group No. No. of animals Sample Route of administration Dose Volume*
G1 5M + 5F Test Item Intraperitoneal 50 mL/Kg b.w.
G2 5M + 5F Reference Control Intraperitoneal 50 mL/Kg b.w

Observations
Mortality & Morbidity
All the animals were observed daily for mortality and morbidity for a total of three days, following test item administration.
Body Weight Recording
Body weights of each animal were recorded at start of the experiment and at 24, 48 and 72 h following test item administration.
Clinical Observation
All the animals were observed at the time of test item administration (0 h), then within 30 minutes and at 4, 24, 48 and 72 h following the test item and reference control administration for any clinical signs of toxicity.
Gross Pathology, Clinical Pathology and Histopathology
Gross pathology, clinical pathology and histopathology were to be conducted as abnormal clinical signs and symptoms were observed.
Data evaluation
The findings of the acute systemic toxicity study shall be evaluated in conjunction with the findings of the toxic effects and the gross pathology examination, if performed. The evaluation shall include the presence or absence and the incidence and severity of abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, effects on mortality and any other general or specific effects. If during the observation period, none of the mice treated with the individual test exhibited a significantly greater reaction than the corresponding control mice, then the test item meets the requirements of ISO 10993-Part 11:2006. If two or more mice die, or if abnormal behaviour such as convulsions or prostration occurred in two or more mice, or if body weight loss is greater than 2 grams occurred in three or more mice, then the test item does not meet the test requirements.
Results
Mortality & Morbidity
Mortality and morbidity were observed in reference control group of the animals used in this study. All the animals in reference control group were dead within 2 minutes after administration. No mortality was observed in test item treated animals.
Body Weight Recording
The survival animals showed increase in body weight at the end of the experiment. Individual body weights of the animals are presented in Table 15.
Clinical Observation
A sign of ill health or overt toxicity was observed in both, test item and reference control groups which are presented in Table16.
Necropsy
All the survival animals were euthanized by Carbon dioxide exposure at the end of the experiment.
Gross Pathology
Gross pathology was conducted for all, the test item and reference control treated animals. The results are in Table17.
Discussion and conclusion
The test item showed acute systemic toxicity in mice following intraperitoneal administration. The clinical signs were reversible. However, the reference control treated animals showed severe signs of multi-organ failure as indicated by haemorrhages in kidney, liver and lungs; and ultimately death. This is not unexpected as the LD50 of EDTA is in the range of 300 - 900 mg/Kg body weight. Based on the results, it is concluded that the Test Item, Root Canal Irrigant, showed less acute systemic toxicity compared to similar doses of reference control.
Table 14: Details of administration via intraperitoneal route
Group No. Animal No Sex Volume administered (mL) Duration of administration (seconds)
G1 1 M 1.0 19
2 M 1.0 19
3 M 1.1 21
4 M 1.0 20
5 M 0.9 18
G2 6 F 0.9 18
7 F 1.0 19
8 F 1.1 22
9 F 1.0 20
10 F 0.9 18

G3 11 M 1.1 20
12 M 1.0 20
13 M 1.0 19
14 M 1.0 20
15 M 0.9 19
G4 16 F 1.1 22
17 F 1.0 20
18 F 1.0 19
19 F 1.1 21
20 F 1.0 20
M-Male; F-Female
Table 15: Individual body weights
Group No. Animal No. Sex Weight (in grams)
Days
0 1 2 3
G1 1 M 19.88 19.91 19.99 20.06
2 M 19.75 19.80 19.88 19.95
3 M 21.97 22.03 22.11 22.18
4 M 20.91 20.97 21.06 21.12
5 M 18.84 18.88 18.99 19.05
G2 6 F 18.16 18.20 18.30 18.37
7 F 20.66 20.68 20.75 20.83
8 F 21.24 21.27 21.36 21.44
9 F 19.78 19.82 19.90 19.98
10 F 18.44 18.49 18.58 18.64
G3 11 M 21.50 Mortality Mortality Mortality
12 M 20.84 Mortality Mortality Mortality
13 M 20.26 Mortality Mortality Mortality
14 M 20.45 Mortality Mortality Mortality
15 M 18.91 Mortality Mortality Mortality
G4 16 F 22.16 Mortality Mortality Mortality
17 F 19.80 Mortality Mortality Mortality
18 F 19.36 Mortality Mortality Mortality
19 F 21.59 Mortality Mortality Mortality
20 F 20.42 Mortality Mortality Mortality
M-Male; F-Female

Table 16: Clinical Observations
Group No. Animal No. Sex Observation at
0 h 30 min 4 h 24 h 48 h 72 h
G1 1 M N A A N N N
2 M N A A N N N
3 M N A A N N N
4 M N A A N N N
5 M N A A N N N
G2 6 F N A A N N N
7 F N A A N N N
8 F N A A N N N
9 F N A A N N N
10 F N A A N N N
G3 11 M A Mortality Mortality Mortality Mortality Mortality
12 M A Mortality Mortality Mortality Mortality Mortality
13 M A Mortality Mortality Mortality Mortality Mortality
14 M A Mortality Mortality Mortality Mortality Mortality
15 M A Mortality Mortality Mortality Mortality Mortality
G4 16 F A Mortality Mortality Mortality Mortality Mortality
17 F A Mortality Mortality Mortality Mortality Mortality
18 F A Mortality Mortality Mortality Mortality Mortality
19 F A Mortality Mortality Mortality Mortality Mortality
20 F A Mortality Mortality Mortality Mortality Mortality

M- Male; F- Female; G- Group; h- hour; min- minutes; N- Normal; A- Abnormal (see Table 17)

Table 17: Gross Pathology and Clinical signs
Group Animal No. Sex Clinical Signs Mortality & Morbidity Gross Pathology
G1 1 M All animals showed dyspnoea, abdominal breathing, catatonia, somnolence, unusual locomotion, fasciculation, bradycardia, piloerection, and opisthotonus for initial few hours following test item administration. Nil
NAD
2 M NAD
3 M NAD
4 M NAD
5 M NAD
G2 6 F All animals showed dyspnoea, abdominal breathing, catatonia, somnolence, unusual locomotion, fasciculation, bradycardia, piloerection, and opisthotonus for initial few hours following test item administration. Nil NAD
7 F NAD
8 F NAD
9 F NAD
10 F NAD
G3 11 M All animals were in dyspnoea, abdominal breathing, catatonia, somnolence, unusual locomotion, fasciculation, bradycardia, piloerection, opisthotonus, tremors and ataxia following reference control administration. 5 Haemorrhages, severe congestion of liver, kidney and lungs
12 M Haemorrhages, severe congestion of liver, kidney and lungs
13 M Haemorrhages, severe congestion of liver, kidney and lungs
14 M Haemorrhages, severe congestion of liver, kidney and lungs
15 M Haemorrhages, severe congestion of liver, kidney and lungs
G4 16 F All animals were in dyspnoea, abdominal breathing, catatonia, somnolence, unusual locomotion, fasciculation, bradycardia, piloerection, opisthotonus, tremors and ataxia following reference control administration 5 Haemorrhages, severe congestion of liver, kidney and lungs
17 F Haemorrhages, severe congestion of liver, kidney and lungs
18 F Haemorrhages, severe congestion of liver, kidney and lungs
19 F Haemorrhages, severe congestion of liver, kidney and lungs
20 F Haemorrhages, severe congestion of liver, kidney and lungs
M- Male; F- Female; G- Group; NAD- No Abnormality Detected
1. IPA Iso- propyl alcohol
2. NMT Not more than
3. NLT Not less than
4. CH Chitosan
5. AA Ascorbic acid
6. LA Lactic acid
7. PPM Part per million , Claims:We claim,
1. A biopolymer gel coating on nonwoven fabric as haemostatic agent for dressing comprising of:
a) Chitosan;
b) Lactic acid; and
c) Calcium Chloride; and
d) Demineralize water;
wherein said gamma sterile chitosan gel coated non-woven cotton fabric with the shelf life of up to 4 years and has clotting time up to 435 sec against the haemostasis.

2. The biopolymer gel coating on nonwoven fabric as haemostatic agent for dressing as claimed in claim 1, wherein the said biopolymer gel comprising of 1-2 % of chitosan originated from crab, 0.5 -2% of lactic acid and 0.3 -1.2 % of calcium chloride in demineralized water.

3. The biopolymer gel coating on nonwoven fabric as haemostatic agent for dressing as claimed in claim 1, wherein the method of preparation of biopolymer gel coating on nonwoven fabric comprising the steps of:
a. Selection of a fabric such as lino weave cotton gauze, normal weave cotton gauze, and needle-punched nonwoven fabrics for coating;
b. Preparation of chitosan solution by addition of chitosan to organic acids diluted with demineralized water having the viscosity of through a de-polymerization process;
c. The prepared chitosan solution is applied to the fabric using padding process; and
d. The chitosan coated fabric is dried at the temperature to obtain the chitosan coated wound dressing.

4. The biopolymer gel coating on nonwoven fabric as haemostatic agent for dressing as claimed in claim 1, wherein chitosan-coated fabric is absorbent and possesses a coherent rendering property, allowing for the easy removal of the wound dressing in one piece from the wound.

5. The biopolymer gel coating on nonwoven fabric as haemostatic agent for dressing as claimed in claim 1, wherein chitosan-coated fabric exhibits high efficacy in promoting haemostatic properties by improving platelet and iron metal complexes through tissue displacement.

6. The biopolymer gel coating on nonwoven fabric as haemostatic agent for dressing as claimed in claim 1, wherein chitosan-coated fabric has the property of swelling which further promotes rapid clot formation when applied to a bleeding surface without any side effects.

Dated 10th Oct, 2023

Chothani Pritibahen Bipinbhai
Reg. No.: IN/PA-3148
For and on behalf of the applicant