DESC:DESCRIPTION
Technical Field of the Invention

The present invention relates to biopolymer-based wound care technologies, specifically to hemostatic dressings composed mainly of chitosan, optionally combined with aminocaproic acid, collagen, gelatin, and/or inorganic salts like aluminum chloride, aluminum sulfate, and iron (III) sulfate. The invention provides a method for manufacturing a porous, non-fabric, non-woven chitosan hemostatic dressing using lyophilization techniques.

Background of the Invention

Hemorrhage control remains a crucial aspect of medical interventions, particularly in emergencies and surgical procedures. The development of effective hemostatic materials is imperative to enhance patient outcomes and reduce complications. Despite the existence of several commercially available absorbable hemostats and sealants, their usage remains limited due to higher costs, restricted absorption capacity, and their inability to effectively manage extensive or continuous bleeding. As a result, surgical procedures often resort to using laparotomy sponges and gauze swabs to absorb fluids and isolate the surgical area. However, these traditional gauze dressings fail to promote hemostasis, leading to prolonged surgeries and increased time spent in operation theatres, thereby escalating the overall surgical costs for patients. A similar situation arises with conventional dressings intended for various types of wounds, such as deep cavity wounds, puncture wounds, post-partum haemorrhage, and internal bleeding. Hence, there is a demand for advanced hemostatic dressings that can efficiently absorb fluids while facilitating hemostasis. Composite dressings, which incorporate distinct components like a porous matrix or fabric support, offer multiple benefits such as high fluid absorption, robust strength in wet and dry conditions, bacterial barrier properties, flexibility, ease of handling, and adhesion. Typically, these composite dressings consist of multiple layers fused together using adhesive or stitching. Developing innovative hemostatic materials that effectively promote blood clotting while ensuring biocompatibility is a significant area of research.

Chitosan, a biopolymer derived from chitin, has garnered significant attention for its multifaceted properties, including biocompatibility, antimicrobial activity, and hemostatic potential. Chitosan is a positively charged, linear polysaccharide and its cationic nature enables interactions with negatively charged cell surfaces and extracellular matrices, rendering it an ideal candidate for promoting blood clotting, with the potential to be used as a topical haemostatic dressing. It has an important role in mucoadhesion through its electrostatic interactions with the anionic mucin chains. A designed chitosan formulation called chitosan haemostatic dressing can be utilised as a haemostatic control dressing. This treatment has haemostatic properties that make topical administration a practical technique for reducing bleeding brought on by wound debridement. The chitosan haemostatic dressing also improves blood absorption, decreases antithrombin generation, and considerably extends partial thromboplastin time without changing the prothrombin ratio. The therapeutic potential of chitosan haemostatic dressing is therefore quite high, and it can significantly increase the survival rate of bleeding patients.

Chitosan haemostatic dressing is a single-use hemostatic dressing that is sterile and designed to control moderate to severe bleeding. Additionally, it also serves as a bacterial barrier. Through blood clotting, cell regeneration stimulation, and anti-microbial activity, Chitosan hemostatic dressing also reduces inflammation and speeds up wound healing.

Achieving hemostasis is a crucial aspect of wound management and surgical procedures to control bleeding and promote tissue healing. Collagen and gelatin have emerged as promising hemostatic agents due to their biocompatibility, biodegradability, and hemostatic efficacy.
Collagen: Collagen is a fibrous protein that forms the structural framework of connective tissues in the body, including skin, tendons, and blood vessels. Topical collagen can help promote hemostasis by providing a scaffold for platelet adhesion and aggregation, which are critical steps in the formation of blood clots. When collagen comes into contact with blood, it activates platelets and initiates the clotting cascade, leading to the formation of a stable clot at the site of injury.

Gelatin: It is derived from collagen and shares similar hemostatic properties. When applied topically to a bleeding wound, gelatin forms a gel-like matrix that adheres to the wound surface and promotes platelet activation and aggregation. This accelerates the formation of a fibrin clot, which helps to staunch bleeding.

The mechanism of action of collagen and gelatin in hemostasis involves several steps that facilitate clot formation and stabilization:
• Platelet Activation and Adhesion: Upon contact with blood, collagen and gelatin matrices provide a surface for platelet adhesion and activation. Platelets adhere to the exposed collagen or gelatin fibers via specific receptors, such as glycoprotein (GP) Ib and GPVI, initiating a cascade of platelet activation events.
• Platelet Aggregation: Activated platelets undergo shape change and release granule contents, including ADP, serotonin, and thromboxane A2 (TXA2), which further activate nearby platelets. This process leads to platelet aggregation, forming a platelet plug at the site of injury, known as primary hemostasis. Coagulation Cascade
• Activation: Collagen and gelatin matrices also serve as a platform for the activation of the coagulation cascade. Surface-bound clotting factors, such as factor XII (Hageman factor), factor XI, and factor IX, become activated upon interaction with collagen or gelatin, initiating the intrinsic pathway of coagulation.
• Thrombin Generation: The activation of the coagulation cascade results in the generation of thrombin, a key enzyme in the clotting process. Thrombin converts fibrinogen, a soluble plasma protein, into insoluble fibrin strands, forming a meshwork that reinforces the platelet plug and stabilizes the clot.
• Fibrin Clot Formation: The polymerization of fibrin strands creates a stable fibrin clot, which traps blood cells and platelets, further consolidating the hemostatic plug. Collagen and gelatin matrices enhance fibrin polymerization and clot formation, promoting hemostasis.
• Wound Healing and Tissue Repair: Beyond their hemostatic effects, collagen and gelatin also support wound healing and tissue repair processes. The formation of a stable fibrin clot provides a scaffold for migrating fibroblasts, which deposit collagen and other extracellular matrix components, facilitating tissue regeneration.

In hemostatic dressings, Chitosan can be combined with Collagen and/or Gelatin to create an efficient dressing that not only promotes clotting but also provides an environment conducive to infection prevention. The combination of these compounds can enhance the overall effectiveness of the dressing in various bleeding scenarios.

Aluminium chloride (AlCl3), Aluminum sulfate (Al2(SO4)3), and Iron(III) sulfate (Fe2(SO4)3) are commonly utilized inorganic salts due to their hemostatic properties.

Aluminium chloride and Aluminum sulfate act as an astringent, exerting their hemostatic effect by causing vasoconstriction and tissue contraction. Upon topical application, these chemical agents interact with proteins and mucopolysaccharides in the tissues, leading to protein precipitation and tightening of the skin. Additionally, these stimulate the aggregation of platelets, enhancing the formation of a temporary plug at the site of bleeding. They also promote the activation of the coagulation cascade, further contributing to blood clot formation.
Iron(III) sulfate and related iron compounds exert their hemostatic effects by promoting blood clot formation through the activation of the coagulation cascade. Upon contact with blood, Fe2(SO4)3 accelerates the conversion of fibrinogen to fibrin, leading to the formation of a stable blood clot. Additionally, iron ions released from Fe2(SO4)3 interact with platelets and clotting factors, enhancing platelet aggregation and thrombin formation.

In hemostatic dressings, the above inorganic salts can be combined with Chitosan to produce an efficient dressing that promotes efficient blood clotting. The combination of these compounds can enhance the overall effectiveness of the dressing in various bleeding scenarios.
Aminocaproic acid (ACA) is a synthetic molecule that has a potential role in hemostatic dressings and can be designed to control bleeding and promote clotting in various medical situations. ACA is a hemostatic agent used to control bleeding. It works by inhibiting fibrinolysis, the process of breaking down blood clots, by blocking the action of plasmin, an enzyme responsible for clot dissolution. This helps stabilize blood clots, making it useful in surgical settings, treating bleeding disorders, and managing excessive bleeding due to trauma or medical conditions. It is generally well-tolerated and can be administered orally or intravenously under medical supervision. In hemostatic dressings, ACA can be impregnated into the dressing material to enhance its clot-promoting properties.
The role of Aminocaproic acid in hemostatic dressings includes:

• Inhibiting fibrinolysis: Aminocaproic acid exerts its hemostatic effect primarily by inhibiting fibrinolysis, the process by which blood clots are broken down. It does this by blocking the activity of plasmin, an enzyme responsible for dissolving fibrin, a key component of blood clots.
• Stabilization of Clots: By interfering with plasmin's action, aminocaproic acid helps to stabilize and maintain blood clots that have formed, preventing premature dissolution. This is particularly useful in situations where excessive bleeding needs to be controlled.
• Postoperative bleeding: In surgical contexts, it may be administered before and after an operation to minimize the likelihood of postoperative bleeding or oozing from surgical sites.

A few patents that discuss on chitosan and hemolytic dressings have been discussed below:
US Patent Publication No. US 2021/0252182 A describes a composite dressing comprising at least one layer of a porous matrix composed of biomaterial and at least one layer of fabric support, wherein the fabric support is at least partially embedded in the porous matrix. The disclosure also provides a method for the preparation of said composite dressing, wherein said method enables the production of the composite dressing having cohesive bonding between porous matrix and fabric support without the use of any adhesive, stitching or chemical crosslinking agent between the two layers.

Unlike US Patent Publication No. US 2021/0252182 A, which consists of two layers including a porous matrix and a fabric support, the current invention is a single-layer matrix sheet comprising Chitosan, and/or Aminocaproic acid, Gelatin, Collagen that helps in hemostasis. The matrix sheet does not include any fabric layer within or above its structure and may also include aminocaproic acid as a hemostatic agent for enhanced hemostatic efficiency of the dressing.

US Patent No. US 7981872 B2 describes hemostatic powder comprising a chitosan salt together with at least one medical Surfactant. The hemostatic powder of the present invention can be applied to the wound area followed by pressure.

The current invention is a single-layer porous matrix sheet, that consists of various chitosan and synthetic and/or inorganic clotting agents for hemostasis. It is different from the disclosure of US Patent No. US 7,981,872 B2, in terms of using chitosan and/or aminocaproic acid, Gelatin, Collagen. The current invention is a sheet-like matrix form, unlike the powder form of US Patent No. US 7,981,872 B2 and does not include any surfactant. The current disclosure is compact, portable, and convenient for easy application and removal.
US Patent Publication No. US20140046236A1 describes a layered chitosan scaffold wherein said layered scaffold comprises at least two fused layers, wherein at least one of the fused layers comprises a chitosan nanofiber membrane and the other fused layer comprises a porous chitosan support layer. It also provides a process for the preparation of the layered chitosan scaffold.

Unlike US Patent Publication No. US20140046236A1, the current invention comprises a single layer, composed of chitosan and may include a synthetic and/or inorganic hemostatic agent. The current invention is a single-layer porous sheet-like matrix which is non-fibrous in nature and is processed by Lyophilization with processing times of approximately 20 hours.

Our present invention aims to tackle the significant deficiencies in the current range of hemostatic products. This invention holds substantial importance as it directly addresses a critical healthcare issue, which is the need for efficient hemorrhage control. By advancing the field of hemostatic technology, our present invention has the potential to save lives, improve patient outcomes by enhancing healthcare readiness for traumatic incidents, and ultimately reduce healthcare expenditures.

Brief Summary of the Invention

OBJECTS OF THE INVENTION
The main objective of our present invention is to provide a formulation of chitosan-based dressings for hemostasis applications. These dressings are designed to prevent bleeding, ward off microbial infections, and provide protection for wounds.

Another objective of our present invention is to prepare dressings using various acids alone like acetic acid, lactic acid, hydrochloric acid or any specific combinations of these acids to create a dressings formula containing chitosan and additional hemostatic agents like Aminocaproic acid, Collagen, Gelatin, Aluminum chloride, Aluminum sulphate or iron(III) sulfate.

Another objective of this innovation is the process of manufacturing the chitosan-based dressing which includes pre-freezing the solution by exposing it to liquid nitrogen or supercooled surfaces before the sublimation process.
Another objective of this innovation is to maintain the structural integrity of the biomaterial by avoiding the use of heat and instead employing the lyophilization process.

SUMMARY OF THE INVENTION
The following summary is provided to facilitate a clear understanding of the new features in the disclosed embodiment and it is not intended to be a full, detailed description. A detailed description of all the aspects of the disclosed invention can be understood by reviewing the full specification, the drawing and the claims and the abstract, as a whole.

Our present invention is a method to formulate a chitosan based hemostatic dressing. Its unique formulation, combined with its multifunctional properties, positions it as a versatile solution for various medical settings. The dressing's ability to swiftly promote hemostasis while exhibiting antimicrobial activity and biocompatibility makes it a promising tool for enhancing patient care and outcomes in critical situations.

Combining Chitosan with Aminocaproic acid / Gelatin / Collagen in a hemostatic dressing achieves a synergistic effect that addresses multiple aspects of wound management. The dressing helps control bleeding through clot stabilization and platelet aggregation while supporting wound healing and infection prevention. Incorporating inorganic salts like Aluminum chloride, Aluminum sulfate, and Iron (III) sulfate into chitosan hemostatic dressings enhances their hemostatic properties and promotes rapid blood clotting.

These compounds act synergistically with chitosan to tighten tissues, constrict blood vessels, and accelerate the coagulation cascade, effectively controlling bleeding and facilitating wound healing. These combinations can be particularly useful when rapid and effective hemostasis is crucial, such as surgical procedures, trauma, or patients on anticoagulant medications. Chitosan-based dressings supplemented with Aminocaproic acid, Collagen, Gelatin, AlCl3, Al2(SO4)3, and Fe2(SO4)3 represent an innovative approach to hemostasis, offering clinicians a versatile and effective solution for managing bleeding in various medical and surgical scenarios.
Brief Description of the Drawings

The manner in which the present invention is formulated is given a more particular description below, briefly summarized above, may be had by reference to the components, some of which is illustrated in the appended drawing It is to be noted; however, that the appended drawing illustrates only typical embodiments of this invention and are therefore should not be considered limiting of its scope, for the system may admit to other equally effective embodiments.

Throughout the drawings, the same drawing reference numerals will be understood to refer to the same elements and features. The features and advantages of the present invention will become more apparent from the following detailed description a long with the accompanying figures, which forms a part of this application and in which:

Fig 1 is a diagram showing the Schematic process for the manufacture of Hemostatic dressing, in accordance with our present invention;

Fig 2 is a diagram showing the Normal images of Hemostatic dressing, in accordance with our present invention;

Fig 3 is a diagram showing the Scanning Electron Microscope image of Hemostatic dressing at 500 µm, 200 µm and 100 µm magnification respectively, in accordance with our present invention;

Fig 4 is a diagram showing the Images of Dry hemostatic dressing and Wet hemostatic dressing respectively, in accordance with our present invention;

REFERENCE NUMERALS
101 – Raw material dispensing
102 – Formulation
103 – Lyophilization
104 – Slitting
105 – Packing
106- Gamma irradiation

Detailed Description of the Invention
It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. In addition, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

The principles of operation, design configurations and evaluation values in these non-limiting examples can be varied and are merely cited to illustrate at least one embodiment of the invention, without limiting the scope thereof.

The embodiments disclosed herein can be expressed in different forms and should not be considered as limited to the listed embodiments in the disclosed invention. The various embodiments outlined in the subsequent sections are constructed such that it provides a complete and a thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.

Throughout this specification various indications have been given as to preferred and alternative embodiments of the invention. It should be understood that it is the appended claims, including all equivalents, which are intended to define the spirit and scope of this invention.

The present invention provides a chitosan-based hemostatic dressing and a method for preparing the dressing that capitalizes on chitosan's solubility in acidic environments. The dressing is formulated using specific combinations (or alone) of acids such as acetic acid, lactic acid, and hydrochloric acid in various concentrations. This allows for the controlled solubility of chitosan, enabling the preparation of an effective hemostatic dressing.

In the process of developing a chitosan-based hemostatic dressing, various concentrations of chitosan were solubilized in different concentrations of acetic acid to determine the optimal conditions for achieving effective solubility. This process is critical for ensuring the production of a high-quality hemostatic dressing that can effectively control bleeding. Additionally, lactic acid and hydrochloric acid also present themselves as another potential solvent for chitosan, which could be considered for further exploration in hemostatic dressing formulations Further work outlines the solubility testing of 2% chitosan in various acidic solutions, including acetic acid, lactic acid, hydrochloric acid (HCl), and combinations of acetic acid with lactic acid or HCl. These experiments were conducted to assess the suitability of different acid combinations for solubilizing chitosan, a crucial step in the development of a chitosan-based hemostatic dressing.
Different acid compositions required for solubilizing 2% chitosan:
Sl. No. Acetic acid% Lactic acid% Water% Solution pH
1 0.65 - Upto 100% 2.83
2 - 0.9 Upto 100% 2.92
3 0.05 0.7 Upto 100% 2.85
4 0.1 0.6 Upto 100% 2.82
5 0.15 0.5 Upto 100% 2.79
6 0.2 0.45 Upto 100% 2.81
7 0.25 0.35 Upto 100% 2.73

Sl. No. Acetic acid% 2N HCL% Water% Solution pH
1 0.65 - Upto 100% 2.83
2 - 6.1 Upto 100% 2.86
3 0.05 5 Upto 100% 2.85
4 0.1 4.2 Upto 100% 2.82
5 0.15 3.6 Upto 100% 2.79
6 0.2 3.2 Upto 100% 2.81
7 0.25 2.8 Upto 100% 2.73

In one embodiment of our present invention, the chitosan hemostatic dressing is formulated by using the following steps:
• Contacting chitosan with an organic acid (102), resulting in the creation of a chitosan solution.
• This solution is then subjected to a lyophilization (103) process, involving freezing and drying cycles.
o During the freezing cycle, the chitosan solution is frozen either by rapid exposure to liquid nitrogen or by gradual reduction of temperature.
o This is followed by primary drying, where the frozen solution is subjected to controlled vacuum and temperature conditions, allowing for the sublimation of ice crystals.
o Secondary drying further removes residual moisture, yielding a dry chitosan matrix or sheet.
Embodiment-1:
This embodiment of our current invention pertains broadly to hemostatic dressings. Specifically, it introduces a biomaterial-based hemostatic dressing and a method for its preparation, eliminating the need for adhesive within or on its structure. The composite dressing finds utility as a hemostatic dressing for diverse scenarios including deep cavity wounds, puncture wounds, post-partum haemorrhage, and internal bleeding. Notably, this hemostatic dressing's structure doesn't incorporate adhesive or chemical crosslinking agents.
The invention also introduces the hemostatic dressing for use as a medicinal product. Additionally, it presents the application of the aforementioned hemostatic dressing to manage bleeding, alongside a method for controlling or halting bleeding. This method involves placing the hemostatic dressing at the site of bleeding.

The present invention relates to a method of obtaining a hemostatic preparation, comprising acts of:
a) Bringing chitosan in contact with an organic acid to generate a chitosan solution.
b) Subjecting the chitosan solution to lyophilization, resulting in a dry chitosan hemostatic preparation.
c) Fine-tuning the parameters of the lyophilization process to produce the chitosan hemostatic dressing.

The lyophilization process includes freezing and drying cycles. In one preferred approach, freezing is followed by primary and secondary drying to yield a porous sheet of dry chitosan or chitosan matrix.

The current disclosure pertains to a procedure for acquiring a hemostatic preparation, in accordance to one embodiment of our present invention, involving the following steps for the Lyophilization process:
1. Freezing Cycle: The chitosan solution freezes by exposure to liquid nitrogen or supercooled surfaces, gradually reducing the solution temperature from around 25 °C to about -40°C (preferably -20°C) for approximately 1 to 4 hours (preferably 3 hours).
2. Primary Drying: The frozen solution is exposed to a controlled vacuum (approximately 75 to 400 mTorr, preferably 200 mTorr) at temperatures ranging from around -20°C to 25°C for approximately 10 to 17 hours.
3. Secondary Drying: This stage involves drying at temperatures from around 25°C to 35°C for about 1 to 2 hours (preferably 30°C), at a vauum of 50 to 150 mTorr or any suitable combination of these steps.

A method of preparation of the said hemostatic dressing in accordance to one embodiment of our present invention comprises the following steps, as shown in fig 1:
Step no. Details of the Manufacturing Procedure
Step 1 A weighed quantity of purified water was taken in an SS container and kept under stirring at 500 RPM
Step 2 A weighed quantity of Glacial acetic acid / Lactic acid / hydrochloric acid was added to above step-1 under stirring at 500 RPM for 10 min
Step 3 A weighed quantity of Chitosan (101) was added to above step-2 under stirring at 800 RPM for 240 min
Step 4 The obtained homogenous viscous solution (102) was passed through a clarifying filter to remove the fibrous aggregates or particulates, if any
Step 5 The filtered viscous solution was kept aside for 60 min to remove all the entrapped air bubbles.
Step 6 The required quantity of the above solution was transferred into individual trays for the Lyophilization process (103) to obtain the porous hemostatic dressing (104).
Step 7 The obtained dressing was packed (105) in 4 layered laminated pouches and vacuum sealed which are then terminally sterilized using Gamma irradiation dose (106) of not less than 15 kGy.

Composition – A1 Solution Dressing
S No. Ingredients Function Range (%w/w) Range (%w/w)
1 Chitosan API 0.5 - 8 Up to 100%
2 Glacial Acetic acid Solubilizer 0.05 - 3.0 -
3 Purified water Vehicle Qs -
Composition – A2
1 Chitosan API 0.5 - 8 Up to 100%
2 Lactic acid Solubilizer 0.05 - 4.0 -
3 Purified water Vehicle Qs -
Composition – A3
1 Chitosan API 0.5 - 8 Up to 100%
2 Hydrochloric acid Solubilizer 0.05 - 7.0 -
3 Purified water Vehicle Qs -

Glacial acetic acid / Lactic acid / Hydrochloric acid and Purified water gets eliminated from the product by evaporation during the process. Hence, their concentration in the final product is negligible.

Results for the Chitosan haemostatic dressings composed with different individual acids
Tests Composition – A1 Composition – A2 Composition – A3
Description Pale yellow colored flexible cake Yellow colored hard cake Yellow colored brittle cake
pH 5.12 5.08 4.98
Absorbency Testing 25 times dry weight 37 times dry weight 18 times dry weight
Tensile Strength
Wet 0.0731 N/mm2 0.0672 N/mm2 0.0312 N/mm2
Dry 0.1636 N/mm2 0.0943 N/mm2 0.0557 N/mm2

Composition – B1 Solution Dressing
S No. Ingredients Function Range (%w/w) Range (%w/w)
1 Chitosan API 0.5 - 8 Up to 100%
2 Glacial Acetic acid Solubilizer 0.05 - 3.0 -
3 Lactic acid Solubilizer 0.1 – 4.0 -
4 Purified water Vehicle Qs -
Composition – B2
1 Chitosan API 0.5 - 8 Up to 100%
2 Glacial Acetic acid Solubilizer 0.05 - 3.0 -
3 Hydrochloric acid Solubilizer 0.1 – 7.0 -
4 Purified water Vehicle Qs -
*Glacial acetic acid / Lactic acid / Hydrochloric acid and Purified water gets eliminated from the product by evaporation during the process. Hence, their concentration in the final product is negligible.
Results for the Chitosan haemostatic dressings composed with different combination of acids
Tests Composition – B1 Composition – B1
Description Pale yellow colored flexible cake Pale yellow colored flexible cake
pH 4.98 5.02
Absorbency Testing 63 times dry weight 47 times dry weight
Tensile Strength
Wet 0.0864 N/mm2 0.0624 N/mm2
Dry 0.1928 N/mm2 0.11787 N/mm2

Embodiment-2:
Role of Aminocaproic acid in Hemostasis:
In hemostatic dressings, both Chitosan and Aminocaproic acid can be combined to create an efficient dressing that not only promotes clotting but also provides an environment conducive to infection prevention. The combination of these compounds can enhance the overall effectiveness of the dressing in various bleeding scenarios.

This embodiment of our present invention relates to a method of obtaining a hemostatic preparation, comprising acts of:
a) Solubilizing chitosan in an organic acid to generate a chitosan solution.
b) Addition of a synthetic hemostatic agent namely, aminocaproic acid and solubilizing the same.
c) Subjecting the above solution to lyophilization, resulting in a dry hemostatic preparation.
d) Fine-tuning the parameters of the lyophilization process to produce the hemostatic dressing.

The lyophilization process includes freezing and drying cycles. In one preferred approach, freezing is followed by primary and secondary drying to yield a porous sheet of dry sheet or matrix.

The current embodiment in accordance to our present invention pertains to a procedure for acquiring a hemostatic preparation, involving the following steps for the Lyophilization process:
1. Freezing Cycle: The chitosan solution freezes by exposure to liquid nitrogen or supercooled surfaces, gradually reducing the solution temperature from around 25 °C to about -50 °C (preferably -30°C) for approximately 1 to 4 hours (preferably 3 hours).
2. Primary Drying: The frozen solution is exposed to a controlled vacuum (approximately 100 to 800 mTorr, preferably 400 mTorr) at temperatures ranging from around -30°C to 30°C for approximately 3 to 18 hours.
3. Secondary Drying: This stage involves drying at temperatures from around 30°C to 45°C for about 1 to 2 hours (preferably 35°C to 40°C), or any suitable combination of these steps.
Embodiment 2 Solution Dressing
S No. Ingredients Function Range (%w/w) Range (%w/w)
1 Chitosan API 0.5 – 8 80 - 100%
2 Aminocaproic acid API 0.01 - 2 0.1 - 20%
3 Organic acid Solubilizer 0.05 - 3.0 -
4 Purified water Vehicle Qs -

Another method of preparation of the said hemostatic and wound care dressing in accordance to one embodiment (embodiment-2) of our present invention comprises the following steps:

Step no. Details of the Manufacturing Procedure - Embodiment 2
Step 1 A weighed quantity of purified water was taken in an SS container and kept under stirring at 500 RPM
Step 2 A weighed quantity of Aminocaproic acid was added to above step-1 under stirring at 500 RPM for 20 min
Step 3 A weighed quantity of Organic acid was added to above step-2 under stirring at 500 RPM for 10 min
Step 4 A weighed quantity of Chitosan was added to above step-3 under stirring at 800 RPM for 240 min
Step 5 The obtained homogenous viscous solution was passed through a clarifying filter to remove the fibrous aggregates or particulates, if any
Step 6 The filtered viscous solution was kept aside for 60 min to remove all the entrapped air bubbles.
Step 7 The required quantity of the above solution was transferred into individual trays for the Lyophilization process to obtain the porous hemostatic dressing.
Step 8 The obtained dressing was packed in 4 layered laminated pouches and vacuum sealed which are then terminally sterilized using Gamma irradiation dose of not less than 15 kGy.

Embodiment-3:
This embodiment of our present invention relates to a method of obtaining a hemostatic preparation, comprising acts of:
a) Solubilizing chitosan in an organic acid to generate a chitosan solution.
b) Addition of collagen and/or gelatin and solubilizing the same.
c) Subjecting the above solution to lyophilization, resulting in a dry hemostatic preparation.
d) Fine-tuning the parameters of the lyophilization process to produce the hemostatic dressing.
The lyophilization process includes freezing and drying cycles. In one preferred approach, freezing is followed by primary and secondary drying to yield a porous sheet of dry sheet or matrix.

The current disclosure pertains to a procedure for acquiring a hemostatic preparation, involving the following steps for the Lyophilization process:
1. Freezing Cycle: The chitosan solution freezes by exposure to liquid nitrogen or supercooled surfaces, gradually reducing the solution temperature from around 25 °C to about -50 °C (preferably -30°C) for approximately 1 to 3 hours (preferably 2 hours).
2. Primary Drying: The frozen solution is exposed to a controlled vacuum (approximately 100 to 800 mTorr, preferably 400 mTorr) at temperatures ranging from around -30°C to 30°C for approximately 4 to 16 hours.
3. Secondary Drying: This stage involves drying at temperatures from around 30°C to 45°C for about 0.5 to 2 hours (preferably 35°C to 40°C), or any suitable combination of these steps.
Embodiment 3 Solution Dressing
S No. Ingredients Function Range (%w/w) Range (%w/w)
1 Chitosan API 0.5 – 7.5 75 - 100%
2 Collagen API 0.01 – 2.5 0.1 - 25%
3 Organic acid Solubilizer 0.05 - 3.0 -
4 Purified water Vehicle Qs -

A method of preparation of the said hemostatic and wound care dressing comprises the following steps, in accordance with another embodiment (embodiment-3) of our present invention :

Step no. Details of the Manufacturing Procedure - Embodiment 3
Step 1 A weighed quantity of purified water was taken in an SS container and kept under stirring at 500 RPM
Step 2 A weighed quantity of Collagen was added to above step-1 under stirring at 500 RPM for 20 min
Step 3 A weighed quantity of Organic acid was added to above step-2 under stirring at 500 RPM for 10 min
Step 4 A weighed quantity of Chitosan was added to above step-3 under stirring at 800 RPM for 240 min
Step 5 The obtained homogenous viscous solution was passed through a clarifying filter to remove the fibrous aggregates or particulates, if any
Step 6 The filtered viscous solution was kept aside for 60 min to remove all the entrapped air bubbles.
Step 7 The required quantity of the above solution was transferred into individual trays for the Lyophilization process to obtain the porous hemostatic dressing.
Step 8 The obtained dressing was packed in 4 layered laminated pouches and vacuum sealed which are then terminally sterilized using Gamma irradiation dose of not less than 15 kGy.

Embodiment-4:
Role of Inorganic salts in Hemostasis:
The present disclosure relates to a method of obtaining a hemostatic preparation, comprising acts of:
a) Solubilizing chitosan in an organic acid to generate a chitosan solution.
b) Addition of a inorganic salts namely Aluminum chloride (AlCl3) / Aluminum sulfate (Al2(SO4)3) / Iron(III) sulfate (Fe2(SO4)3).
c) Subjecting the above solution to lyophilization, resulting in a dry hemostatic preparation.
d) Fine-tuning the parameters of the lyophilization process to produce the hemostatic dressing.
The lyophilization process includes freezing and drying cycles. In one preferred approach, freezing is followed by primary and secondary drying to yield a porous sheet of dry sheet or matrix.
The current disclosure pertains to a procedure for acquiring a hemostatic preparation, involving the following steps for the Lyophilization process:
1. Freezing Cycle: The chitosan solution freezes by exposure to liquid nitrogen or supercooled surfaces, gradually reducing the solution temperature from around 25 °C to about -50 °C (preferably -30°C) for approximately 1 to 3 hours (preferably 2 hours).
2. Primary Drying: The frozen solution is exposed to a controlled vacuum (approximately 100 to 800 mTorr, preferably 300 mTorr) at temperatures ranging from around -30°C to 30°C for approximately 5 to 18 hours.
3. Secondary Drying: This stage involves drying at temperatures from around 30°C to 45°C for about 1 to 2 hours (preferably 35°C to 40°C), or any suitable combination of these steps.
Embodiment 4 Solution Dressing
S No. Ingredients Function Range (%w/w) Range (%w/w)
1 Chitosan API 0.5 – 7.5 75 - 100%
2 Aluminum chloride (AlCl3) API 0.1 – 2.5 1 - 25%
3 Organic acid Solubilizer 0.05 - 3.0 -
4 Purified water Vehicle Qs -

A method of preparation of the said hemostatic and wound care dressing, in accordance with another embodiment (embodiment-3) of our present invention, comprises the following steps:
Step no. Details of the Manufacturing Procedure - Embodiment 4
Step 1 A weighed quantity of purified water was taken in an SS container and kept under stirring at 500 RPM
Step 2 A weighed quantity of Aluminum chloride (AlCl3) was added to above step-1 under stirring at 500 RPM for 20 min
Step 3 A weighed quantity of Organic acid was added to above step-2 under stirring at 500 RPM for 10 min
Step 4 A weighed quantity of Chitosan was added to above step-3 under stirring at 800 RPM for 240 min
Step 5 The obtained homogenous viscous solution was passed through a clarifying filter to remove the fibrous aggregates or particulates, if any
Step 6 The filtered viscous solution was kept aside for 60 min to remove all the entrapped air bubbles.
Step 7 The required quantity of the above solution was transferred into individual trays for the Lyophilization process to obtain the porous hemostatic dressing.
Step 8 The obtained dressing was packed in 4 layered laminated pouches and vacuum sealed which are then terminally sterilized using Gamma irradiation dose of not less than 15 kGy.

Evaluation Of The Different Embodiments Of Our Present Invention
In order to evaluate the functionality of the prepared hemostatic dressings, following evaluation tests were performed.
1. Physical characterization tests
2. Skin irritation test
3. In vitro cytotoxicity tests
4. Hemostatic efficiency tests
1. Physical characterization tests
Tests Reference product Embodiment 1 Embodiment 2 Embodiment 3 Embodiment 4
Description Pale yellow colored solid cake
pH 5.12 5.08 4.87 5.19 4.14
Absorbency Testing (water) 25 times dry weight 54 times dry weight 44 times dry weight 58 times dry weight 47 times dry weight
Tensile Strength (N/mm2)
Wet 0.0031 0.0731 0.0912 0.0863 0.0712
Dry 0.0457 0.1636 0.1964 0.2174 0.1836

2. Skin Irritation test
Method of Analysis:
ISO 10993-10:2002/Amd:1 2006(E) – Biological Evaluation of Medical Devices, Part 10: Test for Irritation and Delayed-Type Hypersensitivity, Clause 6.3 (Animal Skin Irritation Test).

Process Summary:
The hemostatic dressing (test material) was moistened with physiological saline and applied to the upper left side of the animal. A sterile gauze pad moistened with physiological saline served as a control and was applied to the lower side. Both application sites were covered, and observations were made at 1, 24, 48, and 72 hours following patch removal to assess any tissue reactions. Erythema and oedema were graded at each time point. The primary irritation index was calculated by summing the scores for each animal and dividing by the total number of animals tested.
Rabbit
ID Sample type Sample Location Erythema Score
(0-4) Edema Score (0-4) Other Observations Total Score (0-8)
Rabbit
1 Control- Gauze Back – Upper Left 0 0 Nil 0
Reference product Back – Lower right 0 0 Nil 0
E1 Back – Upper Right 0 0 Nil 0
E1 Back – Lower left 0 0 Nil 0
Rabbit
2 Control-
Gauze Back – Upper Left 1 0 Nil 0
Reference product Back – Lower Right O 0 Nil 0
E2 Back – Upper Right O 0 Nil 0
E2 Back – Lower left 0 0 Nil 0
Rabbit
3 Control-
Gauze Back – Upper Left 0 0 Nil 1
Reference product Back – Lower Right 0 0 Nil 0
E3 Back – Upper Right 1 0 Nil 0
E3 Back – Lower left 0 0 Nil 0
Rabbit
4 Control-
Gauze Back – Upper Left 0 0 Nil 1
Reference product Back – Lower Right 0 0 Nil 0
E4 Back – Upper Right 1 0 Nil 0
E4 Back – Lower left 0 0 Nil 0
** All the values mentioned are averages with N=3
E1, E2, E3 & E4 are In-House samples of Embodiment 1, Embodiment 2, Embodiment 3 & Embodiment 4.
Grading Scores:
Scores Erythema (Redness) Edema (Swelling)
0 No erythema No edema
1 Very slight erythema (barely perceptible) Very slight edema (barely perceptible)
2 Well-defined erythema Slight edema (edges of the area well defined)
3 Moderate to severe erythema Moderate edema (raised about 1 mm)
4 Severe erythema (beet redness) Severe edema (raised more than 1 mm, extending beyond area of exposure)

Total Score:
The total score for each sample is calculated as the sum of the erythema and edema scores, ranging from 0 to 8.
Conclusion:
The test samples generally demonstrated minimal to no skin irritation. The very slight erythema observed in two (E3 & E4) samples was isolated and does not indicate significant irritation potential. All other samples (E1, E2 and Standard) including controls, showed no adverse reactions, supporting the conclusion that the test samples are largely non-irritating under the conditions of the study.
2. In Vitro Cytotoxicity Testing
Analytical Method: Extract testing based on ISO 10993-5, 1999 guidelines.
Process Summary:
The extract was prepared by incubating the test material in physiological saline at 37?±?2°C for 24–26 hours. The resulting extract was then diluted with a serum-containing medium to achieve an extraction ratio of 1.25 cm²/ml. This diluted extract was subsequently incubated with L-929 cell lines for approximately 24–26 hours, with periodic observations during the incubation. Phenol served as the positive control, while high-density polyethylene (HDPE) was used as the negative control.

Cytotoxicity Evaluation:
Sl. No. Sample ID Sample Cytotoxicity Scale Interpretation
1 E1 Negative control 0 Non-cytotoxic
2 Positive control 4 Severely cytotoxic
3 Hemostatic dressing 0 Non-cytotoxic

4 E2 Negative control 0 Non-cytotoxic
5 Positive control 4 Severely cytotoxic
6 Hemostatic dressing 0 Non-cytotoxic

7 E3 Negative control 0 Non-cytotoxic
8 Positive control 4 Severely cytotoxic
9 Hemostatic dressing 0 Non-cytotoxic

10 E4 Negative control 0 Non-cytotoxic
11 Positive control 4 Severely cytotoxic
12 Hemostatic dressing 1 Non-cytotoxic

Conclusion:
Negative controls (E1, E2, E3, E4) consistently showed a cytotoxicity scale of 0, confirming they were non-cytotoxic as expected. Positive controls in all experiments exhibited a cytotoxicity scale of 4, indicating severe cytotoxicity, as is typical for a positive control (phenol). For E1, E2, and E3, the hemostatic dressing showed a cytotoxicity scale of 0 (non-cytotoxic). For E4, the hemostatic dressing showed a cytotoxicity scale of 1, which is still interpreted as non-cytotoxic according to ISO 10993-5 standards, where scores 0–2 indicate non-cytotoxic behaviour.

The in vitro cytotoxicity testing based on ISO 10993-5, 1999 demonstrated that the hemostatic dressing extracts were non-cytotoxic across all tested samples (E1 to E4). Negative controls confirmed the absence of cytotoxicity (scale 0), while positive controls validated the test system by showing severe cytotoxicity (scale 4). Although one dressing sample (E4) exhibited a slight response (scale 1), it still falls within the non-cytotoxic range. Therefore, the hemostatic dressing is considered safe and non-cytotoxic under the tested conditions.

3. Hemostatic Efficiency Testing
Method of Analysis:
The hemostatic efficiency of the in-house hemostatic dressing was evaluated using albino rabbits as the animal model. A comparative study was conducted against a commercially available reference hemostatic bandage, as the control. A group of healthy adult rabbits (n) was selected for the study. Hematological parameters were assessed and monitored throughout the study.
Parameters Evaluated:
o Time for hemostasis
o Animal survival and body weight monitoring
o Incidence of re-bleeding
o Time to onset of re-bleeding
o Time to achieve complete hemostasis
o Absorbency potential

Summary of the Procedure:
Each rabbit, weighing between 2 to 3 kg, was anesthetized using a combination of ketamine and xylazine. Both ears of the animals were shaved and disinfected. A 1 cm longitudinal incision was made on the marginal ear artery of both ears to induce bleeding.
Right Ear: The wound was treated with a 2 × 2 cm piece of the in-house hemostatic dressing (initial weight: W1). Left Ear: The wound was treated with a 2 × 2 cm piece of the reference hemostatic bandage (initial weight: W2). Immediately after the appearance of arterial bleeding, the dressings were applied directly to the wound sites, followed by the application of direct pressure for 2 minutes. After removal, each dressing was weighed again to determine the amount of blood absorbed: W3: Final weight of in-house hemostatic dressing with blood and W4: Final weight of reference bandage with blood

Calculation of Absorbency Potential:
Absorbency Potential (%) = {[Final weight – Initial weight] / Initial weight} X 100

Parameter Control Group Standard Group Test Rabbit 1 Test Rabbit 2 Test Rabbit 3 Test Rabbit 4
Injury Type Arterial Incision Arterial Incision Arterial Incision Arterial Incision Arterial Incision Arterial Incision
Hemostatic Agent Used Gauze Reference product In-House
Product - E1 In-House
Product – E2 In-House
Product – E3 In-House
Product – E4
Time for Hemostasis (mins) 3 min
24 sec 1 min
50 sec 1 min
15 sec 1 min
1 sec 1 min
16 sec 1 min
34 sec
Re-Bleeding Observed Yes Yes No No No No
Time to Re- Bleeding (mins) 1 min
19 sec 3 min
25 sec No No No No
Complete Hemostasis Achieved Yes Yes Yes Yes Yes Yes
Time to Complete Hemostasis (mins) 12 min
27 sec 7 min
35 sec 2 min
59 sec 1 min
46 sec 2 min
55 sec 3 min
34 sec
General Health of Rabbit Stable Stable Stable Stable Stable Stable
** All the values mentioned are averages with N=3
Conclusion:
The comparative evaluation of hemostatic performance across the control, standard, and in-house test groups reveals a distinct advantage of the in-house products (E1–E4). Time to achieve initial hemostasis was significantly shorter for the in-house dressings, with times ranging from 1 min 1 sec to 1 min 34 sec, compared to 3 min 24 sec for the control and 1 min 50 sec for the standard product. Re-bleeding was observed in both the control and standard groups but completely absent in all test rabbits treated with the in-house products, underscoring their effective wound sealing and hemostatic strength. The time to complete hemostasis was also substantially reduced in the test group, with durations between 1 min 46 sec and 3 min 34 sec, as opposed to over 7 minutes in both the control and standard groups. All animals across groups maintained stable general health, confirming the safety and biocompatibility of the in-house dressing.
The in-house hemostatic dressings (E1–E4) demonstrated superior efficacy in achieving rapid hemostasis, preventing re-bleeding, and significantly reducing overall hemostatic time when compared to conventional gauze and a commercially available reference product.
Results for Absorbency Potential:
Sample Average Initial Weight (gms) Average Final Weight (gms) Weight Increase
(%)
Control-Gauze 0.270 0.318 17.78
Standard – Reference Product 0.258 0.352 36.43
Test Sample - E1 0.253 0.510 101.58
Test Sample – E2 0.240 0.490 104.17
Test Sample – E3 0.271 0.493 81.92
Test Sample – E4 0.250 0.480 92.00

All the values mentioned are averages with N=3
Conclusion:
The absorbency potential results reveal a significant performance advantage of the in-house test samples (E1–E4) over both the control gauze and the standard reference product. The control gauze showed the lowest absorbency, with a 17.78% increase in weight. The standard reference product demonstrated improved absorbency at 36.43%. All test samples (E1 to E4) exhibited substantially higher absorbency, with weight increases ranging from 81.92% to 104.17%, indicating their superior capacity to retain blood. When averaged, the test samples showed an absorbency potential that was approximately 5 to 6 times higher than the control and more than double that of the standard product. This consistency across all test samples (N=3) confirms the reproducibility and reliability of the in-house hemostatic dressing.

The in-house hemostatic dressing significantly outperforms both conventional gauze and a commercial reference bandage in terms of blood absorbency. This enhanced absorption capacity supports its suitability and effectiveness in managing bleeding, making it a promising candidate for clinical hemostatic applications.

Extensive optimization has been conducted to develop a robust and reproducible lyophilization cycle critical for the preparation of advanced hemostatic dressings. In the present process, the freezing step is precisely controlled at -30°C for 3 hours, allowing uniform nucleation and ice crystal formation, which is essential for generating a porous, highly interconnected matrix. The primary drying phase is carried out over 15 hours under reduced pressure, facilitating sublimation of ice directly from the solid phase to vapor phase, thereby avoiding any intermediate liquid phase that could compromise scaffold structure. The total lyophilization cycle time is minimized to 21 hours, which is a significant improvement over conventional protocols, while still ensuring optimal matrix integrity and performance.

This lyophilization approach avoids the use of thermal drying and leverages rapid freezing to preserve the microstructure of biopolymeric and composite systems. The formulation includes biomaterials such as chitosan, collagen, and gelatin, which contribute to biocompatibility and promote cellular interaction and tissue regeneration. Additionally, to enhance hemostatic efficiency, the matrix is fortified with synthetic antifibrinolytic agents like e-aminocaproic acid, and inorganic coagulation promoters such as aluminum chloride, aluminum sulfate, and ferric sulfate. These additives synergistically improve clot formation, reduce bleeding time, and ensure efficient wound management. These materials combined with the optimized lyophilization cycle, enables the large-scale production of highly reproducible, structurally consistent hemostatic dressings with superior performance characteristics compared to existing predicate devices.
,CLAIMS:5. CLAIMS
We Claim
1. A method of chitosan hemolytic dressing preparation process, wherein the dressing comprises of chitosan, water, and acetic acid, and wherein the lyophilization cycle (103) comprises the following steps:
a. Preparing a solution containing chitosan dissolved in a mixture of water and organic acid.
b. Loading the solution into molds or trays at a controlled temperature.
c. Freezing the loaded solution rapidly by reducing the temperature in a staged manner down to -30?°C ± 2?°C over a period of 3 hours, to allow optimal pore formation and scaffold stabilization.
d. Maintaining the frozen state to ensure uniform solidification and prevent collapse of the porous matrix.
e. Initiating the primary drying phase by applying a vacuum not exceeding 400 µbar and gradually increasing the shelf temperature in controlled increments up to 30?°C ± 2?°C over 17 hours.
f. Ensuring sublimation of ice directly from the solid phase to vapor phase, thereby avoiding any intermediate liquid phase transition to preserve the structural integrity of the chitosan dressing.
g. Completing the entire freeze-drying cycle within 21 hours.
h. Obtaining a dry, porous, uncompressed chitosan dressing with preserved morphology, suitable for hemostatic dressing applications (104, 105).
wherein the laminate pouch-packed hemostatic dressing is subjected to gamma irradiation (106) for terminal sterilization and the hemostatic dressing is in the form of an uncompressed, porous sponge.

2. The method of chitosan hemolytic dressing preparation process, as claimed in claim 1, wherein:
a. the solvent system comprises an organic acid selected from the group consisting of acetic acid, lactic acid, hydrochloric acid, or combinations thereof, at a concentration ranging from 0.01% to 7% (v/v)
b. one or more biocompatible biomaterials selected from the group consisting of collagen and gelatin, in a concentration ranging from 0.01% to 2.5% (w/v).
c. optionally includes inorganic salts selected from the group consisting of aluminum chloride (AlCl3), Iron (III) sulfate (Fe2(SO4)3), and aluminum sulfate (Al2(SO4)3), in a concentration effective to enhance hemostatic activity.
d. formulation optionally comprises a synthetic hemostatic agent selected from the group consisting of aminocaproic acid, in a concentration ranging from 0.01% to 2% (w/v).
3. The method of chitosan hemolytic dressing preparation process, as claimed in claim 2, wherein the concentration of chitosan ranges from 0.5% to 8% (w/v).

4. The method of chitosan hemolytic dressing preparation process, as claimed in claim 1, wherein the degree of deacetylation of the chitosan is greater than 80%.

5. The method of chitosan hemolytic dressing preparation process, as claimed in claim 1, wherein the absorption efficiency of hemostatic dressing ranges to 105%.

6. The method of chitosan hemolytic dressing preparation process, as claimed in claim 1, wherein the time taken for complete hemostasis by the hemostatic dressing is less than 220 seconds.

6. DATE AND SIGNATURE
Dated this 16th May, 2025
Signature

(Mr. Srinivas Maddipati)
IN/PA 3124
Agent for Applicant