Chitosan-based aqueous suspensions and powders, and uses thereof for blood clotting

Abstract

Described are chitosan-based aqueous suspensions and powders useful for blood clotting. One embodiment concerns an hemostatic composition comprising an aqueous chitosan suspension in the form of a paste, an ointment, a cream, a lotion, a gel, a milk, or a foam, the hemostatic composition displaying blood clotting properties. Another embodiment concerns a hemostatic powder composition comprising dried ground chitosan particles possessing a micro or a nano fibrous structure, the hemostatic powder composition displaying blood clotting properties. Also described are materials for treating or preventing bleeding, the materials comprising a substrate impregnated or coated with a hemostatic composition and / or with a hemostatic powder composition as defined herein. Methods of treating or preventing bleeding at a tissue site are also described.

Description

CHITOSAN-BASED AQUEOUS SUSPENSIONS AND POWDERS, AND USES THEREOF FOR BLOOD CLOTTINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to provisional patent applications U.S. 63 / 728,742 (filed on Dec. 06, 2024), U.S. 63 / 783,454 (filed on April 04, 2025), U.S. 63 / 868.035 (filed on August 21 , 2025) and US 63 / 898,924 (filed on October 14, 2025), the content of which is incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The invention relates to the field of medicine, and more particularly to hemostasis or blood clotting.BACKGROUND OF THE INVENTION

[0003] Hemostasis is the mechanism that leads to cessation of bleeding from a blood vessel. It is a process that involves multiple interlinked steps. This cascade culminates into the formation of a “plug” or blood clot that closes up the damaged site of the blood vessel controlling the bleeding.

[0004] Chitosan is a biopolymer primarily derived from chitin, which is found in various natural sources, including the exoskeletons of crustaceans like shrimp, lobsters, and crabs as well as from mushrooms. Chitosan has been studied for various medical applications due to its biocompatibility, biodegradability, and non-toxic nature. Chitosan has been shown to accelerate hemostasis, which is the process that causes bleeding to stop. Because chitosan can promote blood clotting, chitosan has been used to develop various hemostatic products beneficial in managing bleeding, such as dressings, bandages, and other wound-care materials. International PCT publications WO 2002 / 102276, WO 2007 / 0104769, WO 2016 / 176186, WO 2023 / 225752 and US patent publications US 2007 / 0237811 , US 2007 / 0346239, US 2011 / 0052665, US 2008 / 0145455, US 2013 / 0096082 US 2016 / 0346239, US 2019 / 0240249, US 2022 / 01931115, provide examples of chitosan-based devices, bioabsorbable hemostatic gauzes, wounddressings, hemostatic compositions, materials, sponges, nanofibers, foamed gels, sprays, bioadhesives, etc.

[0005] Nevertheless, existing products, materials and compositions comprising chitosan are still not optimal for stopping bleeding. As such, there is still a need for better hemostatic products that can be used in emergency situations, military settings, or in surgery to control bleeding quickly.

[0006] There is particularly a need for products, materials, compositions and methods for that can reduce blood clot initiation time, increase speed or rate of blood coagulation, increase blood clot stiffness, accelerate coagulation in presence of anticoagulant(s), and / or promote plasma coagulation via both extrinsic tissue factor and intrinsic tissue factor.

[0007] The present invention addresses these needs and other needs as it will be apparent from the review of the disclosure and description of the features of the invention hereinafter.BRIEF SUMMARY OF THE INVENTION

[0008] According to one aspect, the invention relates to a hemostatic composition comprising an aqueous chitosan suspension in the form of a paste, an ointment, a cream, a lotion, a gel, a milk, or a foam, wherein said hemostatic composition displays blood clotting properties.

[0009] According to another aspect, the invention relates to a hemostatic powder composition, the composition comprising dried ground chitosan particles possessing a micro or a nano fibrous structure, wherein said hemostatic powder composition displays blood clotting properties.

[0010] According to another aspect, the invention relates to a hemostatic powder composition, the composition comprising dried ground chitosan particles possessing a specific surface are (SSA) of at least 2 m2 / g, wherein said hemostatic powder composition displays blood clotting properties.

[0011] According to another aspect, the invention relates to a hemostatic powder composition, the composition comprising dried ground chitosan particles possessing a pore volume of at least 0.016 cm3 / g, wherein said hemostatic powder composition displays blood clotting properties.

[0012] Preferably, the hemostatic composition defined herein provides for at least one of the following benefits when applied on a wound and / or bleeding site:- reduces blood clot initiation time;- increases speed or rate of blood coagulation;- increase blood clot stiffness;- accelerates coagulation in presence of anticoagulant(s);- promotes aggregation of red blood cells;- forms a gel-like structure with red blood cells;- enhances platelet adhesion and / or activation;- promotes plasma coagulation via both extrinsic tissue factor and intrinsic tissue factor;- triggers the blood clotting cascade;- clots citrated blood;- initiate clotting in calcium-depleted environments; and- forms clots exhibiting greater stiffness and strength than clots formed with Celox™ or raw chitosan.

[0013] According to another aspect, the invention relates to a material for treating or preventing bleeding, the material comprising a substrate impregnated or coated (i) with a hemostatic composition as defined herein, and / or (ii) with a hemostatic powder composition as defined herein.

[0014] According to another aspect, the invention relates to a method of treating or preventing bleeding at a tissue site comprising: applying to the tissue site a hemostatic composition as defined herein, a hemostatic powder composition as defined herein, and / or a material as defined herein.

[0015] According to another aspect, the invention relates to a method of making a hemostatic composition, the method comprising: mechanically processing chitosan inpresence of an aqueous solvent to obtain an aqueous chitosan suspension; wherein the mechanical processing is configured to produce said aqueous chitosan suspension in the form of a paste, an ointment, a cream, a lotion, a gel, a milk or a foam that: (i) displays blood clotting properties; and (ii) is compatible for application on a wound, a hemorrhage, a damaged tissue and / or a bleeding tissue.

[0016] According to another aspect, the invention relates to a method of making a hemostatic composition, the method comprising mechanically processing chitosan in presence of an aqueous solvent to obtain an aqueous chitosan suspension; wherein the mechanical processing is configured to produce chitosan particles possessing a micro or a nano fibrous structure, and wherein the chitosan particles possessing a micro or a nano fibrous structure display enhanced hemostatic properties compared to a raw chitosan powder.

[0017] According to another aspect, the invention relates to a wound dressing suitable for direct application to a wound, the wound dressing comprising a substrate layer impregnated or having layered thereon (i) a hemostatic composition as defined herein, and / or (ii) a hemostatic powder composition as defined herein.

[0018] According to another aspect, the invention relates to a kit for treating a bleeding wound of a human or an animal, the kit comprising: at least one of: (i) a container comprising a hemostatic composition as defined herein; (ii) a container comprising a hemostatic powder composition as herein; and (iii) a material as defined herein; and at least one of a patch, a bandage, a wound dressing, a gauze, a sponge, one or more blood clotting agent including, a disinfectant, an antimicrobial, an antiseptic, and a pamphlet with instructions.

[0019] According to another aspect, the invention relates to the use of at least one of (i) a hemostatic composition as defined herein, (ii) a hemostatic powder composition as defined herein; and (iii) a material as defined herein, for treating or preventing bleeding.

[0020] Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description ofpreferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0021] For the invention to be readily understood, embodiments of the invention are illustrated by way of example in the accompanying figures.

[0022] Figures 1A-1C are graphs showing the result of clotting experiments in accordance with Experiment 1. Figure 1A: Clotting profiles (representative G'-time graphs) of various systems of recalcified blood and mixed with different dosages of hemostatic agents and CTR (control: recalcified blood without any treatment or hemostatic agent). Figure 1 B: Clot initiation time of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents. Figure 1 C: Clotting rate of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents (n = 3), each bar is labeled with its average value (the error bars are standard deviation). Statistically significant compared to control (*p < 0.05) and compared to Celox™ (xp < 0.05).

[0023] Figure 2 is a graph displaying apparent stiffness (G') of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents at the end of their 30 min tests at 37 °C (n = 3), in accordance with Experiment 1. Each bar is labeled with its average value (the error bars are standard deviation). Statistically significant compared to control (*p < 0.05).

[0024] Figures 3A-3C are graphs showing the result of clotting experiments in accordance with Experiment 2. Figure 3A: Clotting profiles (representative G'-time graphs) of various systems of recalcified blood and mixed with different dosages of hemostatic agents and CTR (control: recalcified blood without any treatment or hemostatic agent). Figure 3B: Clot initiation time of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents. Figure 3C: Clotting rate of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents (n = 3), each bar is labeled with its average value (the error bars are standard deviation).Statistically significant compared to control (*p < 0.05) and compared to Celox™ (xp < 0.05).

[0025] Figure 4 is a graph displaying apparent stiffness (G') of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents at the end of their 30 min tests at 37 °C (n = 3), in accordance with Experiment 2. Each bar is labeled with its average value (the error bars are standard deviation). Statistically significant compared to control (*p < 0.05).

[0026] Figures 5A-5C are graphs showing the result of clotting experiments in accordance with Experiment 3. Figure 5A: Clotting profiles (representative G'-time graphs) of various systems of recalcified blood and mixed with different dosages of hemostatic agents and CTR (control: recalcified blood without any treatment or hemostatic agent). Figure 5B: Clot initiation time of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents. Figure 5C: Clotting rate of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents (n = 3), each bar is labeled with its average value (the error bars are standard deviation). Statistically significant compared to control (*p < 0.05) and compared to Celox™ (xp < 0.05).

[0027] Figure 6 is a graph displaying apparent stiffness (G') of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents at the end of their 30 min tests at 37 °C (n = 3), in accordance with Experiment 3. Each bar is labeled with its average value (the error bars are standard deviation). Statistically significant compared to control (*p < 0.05).

[0028] Figures 7A-7F are pictures showing the blood clots from various systems obtained at the end of the test, in accordance with Experiment 3. The pictures were captured at the end of the 30 min tests at 37°C. The resulting systems were cut open to better show their consistency, liquid residue and the final clot height. Figure 7A: Control clot formed by recalcified blood with no hemostatic agent. Figure 7B: Clot formed by recalcified blood upon addition of dried, ground suspension (P1). Figure 7C: Clot formed by recalcified blood upon addition of suspension (S10). Figure 7D: Clot formed byrecalcified blood upon addition of Celox™. Figure 7E: Clot formed by recalcified blood upon addition of Curad™. Figure 7F: Comparison of clot thickness (height) across control, P1 , and S10 formulations using equal blood volumes.

[0029] Figures 8A-8C are graphs showing the result of clotting experiments in accordance with Experiment 4. Figure 8A: Clotting profiles (representative G'-time graphs) of various systems of citrated blood and mixed with different dosages of hemostatic agents and CTR (control: citrated blood without any treatment or hemostatic agent). Figure 8B: Clot initiation time of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents. Figure 8C: Clotting rate of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents (n = 2 or 3), each bar is labeled with its average value (the error bars are standard deviation). Note that the clot initiation was considered as within the testing time(s) defined here for those systems of citrated blood that did not coagulate within the testing time frame.

[0030] Figures 9A-9H are pictures showing the blood clots from various systems obtained in accordance with Experiment 4. The pictures were captured at the end of the 480 min tests at 37°C. Figure 9A: Clot formed by citrated blood upon addition of suspension (S10). Figure 9B: Clot formed by citrated blood upon addition of suspension (S11). Figure 9C: Clot formed by citrated blood upon addition of suspension (S12). Figure 9D: Clot formed by citrated blood upon addition of Celox™. Figure 9E: Clot formed by citrated blood upon addition of dried, ground suspension (P1). Figure 9F: Clot formed by citrated blood upon addition of dried, ground suspension (P2). Figure 9G: Clot formed by citrated blood upon addition of dried, ground suspension (P3). Figure 9H: Clot formed by citrated blood upon addition of Curad™.

[0031] Figure 10 is a line graph displaying turbidity profiles of various systems of plasma and mixed with different dosage of hemostatic agents and control, is accordance with Example 2 (n = 1 or 2).

[0032] Figures 11A-11 H are Scanning Electron Microscopy (SEM) images of hemostatic agents and granules tested in accordance with Example 3. Fig. 11 A: S10, Fig.11 B: S11 , Fig. 11C: S12, Fig. 11 D: P1 , Fig. 11 E: P2, Fig. 11 F: P3, Fig. 11G: raw chitosan powder and Fig. 11 H: Celox™ granules. Scale bar: 1 pm.

[0033] Figures 12A-12H are SEM images of hemostatic agents and granules corresponding to Figs, 11A-11 H, but with a scale bar of 5 pm.

[0034] Figures 13A-13D are graphs showing the result of clotting experiments in accordance with Example 5. Figure 13A: Clotting profiles (representative G'-time graphs) of various systems of citrated blood and mixed with different dosages of hemostatic agents and control (CTR: citrated blood without any treatment or hemostatic agent). Figure 13B: Clotting profiles (representative G'-time graphs) of various systems of recalcified blood and mixed with different dosages of hemostatic agents and control (CTR: citrated blood without any treatment or hemostatic agent). Figure 13C: Clot initiation time of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents. Figure 13D: Clotting rate of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents (n = 3), each bar is labeled with its average value.

[0035] Figures 14A and 14B are bar graphs showing the result of clotting experiments in accordance with Example 5. Figure 14A: Strength (i.e. , maximum stress before failure) of various systems of pure blood (controls) both citrated and recalcified and mixed with different dosages of hemostatic agents at the end of their 55 min tests at 37 °C (n = 3). Each bar is labeled with its average value. Figure 14B: Stiffness (i.e., the tangent modulus of the blood clot between 10-20% strain) of various systems of pure blood (controls) both citrated and recalcified and mixed with different dosages of hemostatic agents at the end of their 55 min tests at 37 °C (n = 3). Each bar is labeled with its average value.

[0036] Figures 15A-15C are pictures showing blood clots from various systems obtained at the end of the test, in accordance with Example 5. The pictures were captured at the end of the 30 min tests at 37°C. Fig. 15A: raw chitosan; Fig. 15B and 15C: dried chitosan formulation P1 (images taken from different replicates and on different days). Figure 16 are graphs showing the result of clotting experiments in accordance with Example 5. Figure 16A: Clotting profiles (representative G'-time graphs) of various systems of recalcified blood and mixed with different dosages of hemostatic agents andCTR (control: recalcified blood without any treatment or hemostatic agent). Figure 16B:Clot initiation time of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents. Figure 16C: Clotting rate of various systems of pure blood (controls) and mixed with different dosages of hemostatic agents (n = 3), each bar is labeled with its average value.

[0037] Figure 17 is a bar graph showing clot initiation time of five independent blood batches, normalized to their respective control values, in accordance with Example 5.

[0038] Figure 18 is a bar graph showing clotting rate of five independent blood batches, normalized to their respective control values, in accordance with Example 5.

[0039] Figure 19 is a bar graph showing clotting strength of five independent blood batches, normalized to their respective control values, in accordance with Example 5.

[0040] Figure 19 is a bar graph showing clot stiffness of five independent blood batches, normalized to their respective control values, in accordance with Example 5.

[0041] Further details of the invention and its advantages will be apparent from the detailed description included below.DETAILED DESCRIPTION OF EMBODIMENTS

[0042] In the following description of the embodiments, references to the accompanying figures are illustrations of an example by which the invention may be practised. It will be understood that other embodiments may be made without departing from the scope of the invention disclosed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.General Overview

[0043] In PCT publication WO 2022 / 137184 entitled “HOMOGENEOUS BIOPOLYMER SUSPENSIONS, PROCESSES FOR MAKING SAME AND USES THEREOF” (the content of which is incorporated herein by reference in its entirety)Applicant has described the preparation of stable homogeneous suspensions of insoluble and / or semi-soluble biopolymers. Among described insoluble biopolymers there are chitin, chitosan, cellulose, hemicellulose, lignin, amylose, actin, fibrin, collagen, silk, fibroin, keratin, wool, and mixtures thereof.

[0044] The present inventor(s) have now shown that chitosan-based suspensions and powders as described herein both display unexpected beneficial properties for blood clotting.

[0045] Accordingly, the present invention generally relates to the uses of hemostatic composition in the form of aqueous chitosan suspensions or powders for blood clotting. The term “hemostasis”, “hemostatic”, and “blood clotting” are used herein interchangeably to generally refer to the mechanism that leads to cessation of bleeding and / or hemorrhaging. It is a process that involves multiple interlinked steps that culminates into the formation of a blood clot or “plug”, thereby stopping bleeding.

[0046] Hemostatic compositions

[0047] Hemostatic compositions in accordance with the present invention comprises chitosan, and these compositions may take the form of a suspension (e.g., aqueous suspensions) or a solid form (e.g., powder or granules).

[0048] In embodiments, the hemostatic composition consists of an aqueous chitosan suspension in the form of a paste, an ointment, a cream, a lotion, a gel, a milk or a foam.

[0049] In embodiments, the hemostatic composition consists of a powder or granules obtained subsequently to drying of an aqueous chitosan suspension as defined herein.

[0050] As is known, chitosan is a biopolymer primarily derived from chitin which is found in various natural sources. The present invention is not limited to particular sources of chitin or chitosan. For instance, suitable sources of chitin may include, but are not limited to, green plants, algae, and fungi. Suitable sources of chitin and chitosan may include, but are limited to, fungi, crustaceans (e.g. crabs, shrimps, lobsters) and insects. In embodiments the chitosan is obtained from fungi and mushrooms. In embodiments, the chitosan is a fungal chitosan produced by Chibio™ (Qingdao City, China). Inembodiments, the chitosan is a fungal chitosan produced by KitoZyme™ (Herstal, Belgium). In embodiments, the chitosan is produced by mushrooms such as oyster mushroom.

[0051] Preferably, the hemostatic compositions in accordance with the present invention are substantially free from chemical residues, meaning that any of such chemical residue is totally absent or present in undetectable or trace amounts. As used herein, “substantially free from chemical residues” means that chemical compounds, such as acids, bases, reactive chemicals, organic salts and / or inorganic salts, surfactants, dispersing agents (e.g., Tween 80™), a silanizing reagent, acrylamide, etc. are totally absent or merely present in undetectable or trace amounts in the final composition (final suspension or powder). In embodiments, chitosan will constitute at least 98%, or at least 99% or at least 99.9% or at least 99.99% by weight of the organic materials in the hemostatic composition, i.e., the hemostatic composition will contain less than 2%, or less than 1%, less than 0.1%, or less than 0.01 %, or less than 0.001 % by weight of organic components other than the chitosan or degradation product(s).

[0052] In embodiments where the final suspension or powder further comprises a preservative (e.g., about 1% w / w to about 5% w / w) and the ratio w / w of chitosan : other organic components will be of at least 2:1 , or at least 3:1 , at least 4:1 , at least 5:1 , at least 6:1 , at least 7:1 , at least 8:1 , at least 9:1 , at least 10:1 or more. In other embodiments wherein the final suspension or powder further comprises a preservative, the weight ratio of the preservative: chitosan in the suspension or powderwill be less than 1 :2, or less than 1 :3, or less than 1 :4, or less than 1 :5, or less than 1 :6, or less than 1 :7, or less than 1 :8, or less than 1 :9, or less than 1 :10. In other embodiments wherein the final suspension or powder further comprises a preservative, the chitosan represent at least 50% w / w, or at least 60% w / w, or at least 65% w / w, or at least 70% w / w, or at least 75% w / w, or at least 80% w / w, or at least 85% w / w, or at least 95% w / w, or at least 99% w / w of the organic material(s).

[0053] In embodiments, the hemostatic composition in accordance with the present invention may further comprise one or more of the following biopolymers: chitin, alginic acid, cellulose, and silk. Therefore, in such embodiments, the above percentage valuesmay refer not to chitosan alone, but to a mixture comprising chitosan and one or more additional biopolymer(s).

[0054] In embodiments, aqueous chitosan suspensions in accordance with the present invention comprise at least 0.1% w / w, or at least 0.5% w / w, or at least 1 % w / w, or at least 2% w / w, or at least 2.5% w / w, or at least 3% w / w, or at least 4% w / w, or at least 5% w / w, or at least 6% w / w, or at least 7% w / w, or at least 8% w / w, or at least 9% w / w, or at least 10% w / w or more, based on total weigh of the composition. In embodiments, aqueous chitosan suspensions in accordance with the present invention comprise about 0.5% w / w to about 10% w / w chitosan, or about 1 % w / w to about 9% w / w, or about 2% w / w to about 8% w / w, or about 3% w / w to about 7% w / w chitosan, or about 4% w / w to about 6% w / w chitosan.

[0055] In embodiments, dried chitosan powders in accordance with the present invention comprise at least 80% w / w chitosan, or at least 85% w / w chitosan, or at least 90% w / w chitosan, or at least 95% w / w chitosan, or at least 96% w / w chitosan, or at least 97% w / w chitosan, or at least 98% w / w chitosan, or at least 99% w / w chitosan, or at least 99.5% w / w chitosan, or at least 99.9% w / w chitosan, based on total weigh of the powder. In embodiments, dried chitosan powders in accordance with the present invention comprises pure chitosan (e.g., 100% w / w chitosan).

[0056] In embodiments, the hemostatic composition in accordance with the present invention may further comprise one or more additive including, but not limited to, preservatives, buffer, pH adjuster, and dyes. Examples of additives include, but are not limited to, Dermosoft™, Hydrolit™6 I benzyl alcohol, Geogard® ECT, Glucono Delta- Lactone (GDL) and silver citrate.

[0057] The hemostatic compositions in accordance with the present invention may further comprise one or more local (topical) or systemic blood clotting agent. Particularly the hemostatic compositions in accordance with the present invention may further comprise one or more blood clotting agent (or “hemostatic booster”) including, but not limited to fibrin sealants (e.g., Tisseel™, Evicel™), thrombin (e.g., Thrombin-JMI®), oxidized cellulose (e.g., Surgicel™), collagen (e.g., Helistat™, Instat™), kaolin (e.g., QuikClot™), tranexamic acid (TXA), fibrinogen concentrate (FC), and the like.

[0058] In embodiments, the compositions of the invention are formulated for a specific application to at least one of a wound, a hemorrhage, a damaged tissue and a bleeding tissue.Physical properties

[0059] In embodiments, the aqueous chitosan suspension in accordance with the present invention a colloidal homogeneous chitosan suspension. In embodiments, the colloidal chitosan suspension comprises colloids having a range from about 1 nm to about 100 pm, or about 5 nm to about 50 pm, or about 10 nm to about 10 pm, or about 100 nm to about 1 pm.

[0060] In embodiments, the chitosan suspension in accordance with the present invention comprises chitosan fibers. In embodiments the chitosan fibers have of a width of about 1 nm to about 5 pm, or about 5 nm to about 5 pm, about 7 nm to about 5 pm, or about 10 nm to about 5 pm, or about 20 nm to about 5 pm, or about 25 nm to about 5 pm, or about 30 nm to about 5 pm, or about 35 nm to about 5 pm, or about 35 nm to about 3 pm. In embodiments the fibers having of a width of at least 1 nm, or at least 5 nm, or at least 10 nm, or at least 20 nm, or at least 30 nm, or at least 40 nm, or at least 50 nm, or at least 75 nm, or at least 100 nm, or at least 250 nm, or at least 500 nm, or at least 750 nm, or at least 1 pm, or at least 2 pm, or at least 3 pm, or at least 4 pm, or at least 5 pm, or at least 6 pm, or at least 7 pm, or at least 8 pm, or at least 9 pm, or at least 10 pm, or wider.

[0061] In embodiments the chitosan suspension in accordance with the present invention comprises chitosan fibers having a length of about 1 nm to about 200 pm, of about 10 nm to about 100 pm, or about 50 nm to about 10 pm, or about 100 nm to about 10 pm, or about 500 nm to about 10 pm, or about 750 nm to about 10 pm, or about 800 nm to about 10 pm, or about 900 nm to about 5 pm, or about 1 pm to about 10 pm, or about 1 pm to about 5 pm, or about 1 pm to about 3 pm. In embodiments the fibers have of a length of at least 1 nm, or at least 10 nm, at least 50 nm, or at least 100 nm, or at least 250 nm or at least 500 nm, or at least 750 nm, or at least 800 nm, or at least about 900 nm, or at least 1 pm, or at least 2 pm, or at least 3 pm, or at least 4 pm, or at least 5 pm, or at least 6 pm, or at least 7 pm, or at least 8 pm, or at least 9 pm, or at least 10 pm,or at least 25 pm, or at least 50 pm, or at least 75 pm, or at least 100 pm, or at least 150 pm, or at least 200 pm or longer. In embodiments, a dry particle size range may be between about 1 nm to about 1 pm, or up to 10 pm, and a wet particle size range may be between about 200 nm to about 20 pm, or up to 200 pm.

[0062] In embodiments the chitosan suspension in accordance with the present invention comprises chitosan fibers having both: (i) a width greater than 20 nm (e.g., at least 25 nm, or at least 40 nm, or at least 50 nm,) and a length greater than 50 nm (e.g., at least 100 nm, or at least 500 nm, or at least 1 pm, or at least 2 pm); or (ii) a width greater than 32 nm (e.g., at least 35 nm, or at least 40 nm, or least 50 nm) and a length of than 50 nm (e.g., at least 100 nm, or at least 500 nm, or at least 1 pm, or at least 2 pm); or (iii) a width greater than 20 nm (e.g., at least 25 nm, or at least 40 nm, or least 50 nm)and a length of than 500 nm (e.g., at least 600 nm, or at least 750 nm, or at least 1 pm, or at least 2 pm); or (iv) a width greater than 30 nm (e.g., at least 35 nm, or at least 40 nm, or least 50 nm)and a length of than 800 nm (e.g., at least 900 nm, or at least 1 pm, or at least 2 pm); or (v) a width greater than 8 nm (e.g., at least 10 nm, at least 25 nm, or at least 35 nm, or at least 40 nm, or least 50 nm) and a length of than 340 nm (e.g., at least 350 nm, or at least 500 nm, at least 750 nm, or at least 900 nm, or at least 1 pm, or at least 2 pm); or (vi) a width greater than 11 nm (e.g., at least 15 nm, at least 25 nm, or at least 35 nm, or at least 40 nm, or least 50 nm) and a length of than 166 nm (e.g., at least 200 nm, or at least 350 nm, or at least 500 nm, at least 750 nm, or at least 900 nm, or at least 1 pm, or at least 2 pm); or (viii) a width greater than 32 nm (e.g., at least 35 nm, or at least 40 nm, or least 50 nm) and a length greater than 800 nm (e.g., at least 900 nm, or at least 1 pm, or at least 2 pm, or at least 3 pm, or at least 4 pm, or at least 5 pm, or at least 6 pm, or at least 7 pm, or at least 8 pm, or at least 9 pm, or at least 10 pm, or at least 25 pm, or at least 50 pm, or at least 75 pm, or at least 100 pm, or at least 150 pm, or at least 200 pm or longer).

[0063] In embodiments the chitosan suspension in accordance with the present invention comprises chitosan fibers wherein the average width and average length of the fibers in the suspension are as defined hereinabove, e.g. an average width greater than 20 nm (e.g., at least 25 nm, or at least 40 nm, or at least 50 nm) and an average length greater than 50 nm (e.g., at least 60 nm, at least 75 nm, or at least 100 nm, or at least 500nm, at least 750 nm, or at least 1 pm, or at least 2 pm, or at least 3 pm, or at least 4 pm, or at least 5 pm, or at least 6 pm, or at least 7 pm, or at least 8 pm, or at least 9 pm, or at least 10 pm, or at least 25 pm, or at least 50 pm, or at least 75 pm, or at least 100 pm, or at least 150 pm, or at least 200 pm or wider).

[0064] In embodiments the chitosan suspension in accordance with an average molecular weight of about 50 kDa to about 100 kDa (e.g., or about 50 kDa, or a about 75 kDa, or about 100 kDa, or about or about 125 kDa, or about 150 kDa).

[0065] In embodiments the chitosan suspension in accordance with the present invention has a pH between about 6.5 and about 9. In particular embodiments the pH is between 7.5 and about 9, or about 8 and about 9.

[0066] In embodiments the chitosan suspension in accordance with the present invention comprises chitosan fibers having both a crystalline region and an amorphous region. In embodiments the chitosan suspension in accordance with the present invention comprises chitosan fibers having a globular shape. In embodiments the chitosan suspension in accordance with the present invention is comprised of mainly, or only, of suspended chitosan nanofibrils.

[0067] Those skilled in the art are aware that particle size measurements may vary according to the measurement method and the state of the particles (e.g., particles in a wet state are typically larger than the same particles in a dry state). Typically, the particles will be in a wet or suspended stage when measured by dynamic light scattering (DLS) and in a dry stage when measured by scanning electron microscopy (SEM).

[0068] In embodiments the hemostatic composition in accordance with the present invention comprises agglomerated spheres of chitosan having an average size of about 75 nm to about 120 nm, or about 80 nm to about 115 nm, or about 85 nm to about 110 nm, as measured by scanning electron microscopy (SEM). In embodiments the chitosan suspension comprises agglomerated spheres of chitosan having a median size of about 70 nm to about 100 nm or about 75 nm to about 95 nm, as measured by scanning electron microscopy (SEM). In embodiments, the hemostatic composition in accordance with the present invention comprises agglomerated spheres of chitosan having a size from about1 nm to about 100 pm, or about 5 nm to about 50 pm, or about 10 nm to about 10 pm, or about 100 nm to about 1 pm.

[0069] In embodiments, the hemostatic compositions in accordance with the present invention comprise a particle size distribution spanning from about 1 pm to about 200 pm, or about 5 pm to about 175 pm, or about 5 pm to about 160 pm, or about 10 pm to about 100 pm.

[0070] In embodiments, the hemostatic composition in accordance with the present invention comprises a median particle size (D50) of about 5 to about 75 pm, or about 10 to about 50 pm, or about 15 to about 35 pm. In one particular embodiment 95% of the particles are below 160 pm (i.e., D95 value analysis ranging from 90 to 160 pm).

[0071] In embodiments the hemostatic composition in accordance with the present invention comprises particles of chitosan wherein the range of particle sizes, as measured by SEM is as defined in the tables and figures of WO 2022 / 137184.

[0072] In embodiments the hemostatic composition in accordance with the present invention and / or chitosan therein is(are) characterized by visual properties like those depicted in the SEM images shown in the figures of WO 2022 / 137184.

[0073] In embodiments the hemostatic composition in accordance with the present invention and / or the chitosan therein is(are) characterized by a Fourier Transform Infrared Spectroscopy (FTIR) spectrum as depicted in the figures of WO 2022 / 137184.

[0074] In embodiments the hemostatic composition in accordance with the present invention and / or the chitosan therein is(are) characterized by Solid-State Nuclear Magnetic Resonance characterization (SSNMR) as depicted in the figures of WO 2022 / 137184.

[0075] In embodiments the hemostatic composition in accordance with the present invention and / or the chitosan therein is(are) characterized by Power X-Ray Diffraction (PXRD) pattern(s) as depicted in the figures of WO 2022 / 137184.

[0076] In embodiments the hemostatic composition in accordance with the present invention and / or the chitosan therein is(are) characterized by Dynamic Light Scattering (DLS) measurements like those reported in WO 2022 / 137184.

[0077] In embodiments the hemostatic composition in accordance with the present invention and / or the chitosan therein is(are) characterized by a transmittance spectrum as shown in the figures of WO 2022 / 137184.

[0078] In embodiments the hemostatic composition in accordance with the present invention and / or the chitosan therein is(are) characterized by a sweep suspension test as reported in WO 2022 / 137184.

[0079] In embodiments the hemostatic composition in accordance with the present invention and / or the chitosan therein is(are) characterized by a rheological behaviour as depicted in the figure(s)s WO 2022 / 137184.Methods of preparation

[0080] In embodiments, the hemostatic composition comprises chitosan molecules that have been mechanically processed into a stable homogeneous aqueous chitosan suspension. As used herein the term “homogeneous” generally refers to the appearance of the suspension under the naked eye (e.g., uniform color, uniform texture, etc.). Homogenous as used herein does not exclude the possibility that the suspension is “heterogenous” at the molecular level (e.g., various particles size, presence of aggregates, etc.). As used herein, the terms “stable homogeneous aqueous chitosan suspension”, or similar terms that may be used herein interchangeably such as “homogenous chitosan suspension” or “stable chitosan suspension” or simply “chitosan suspension”, all refer to a suspension of insoluble chitosan particles that have been stably dispersed within a polar solvent. The polar solvent may be a polar protic solvent or a polar aprotic solvent. The polar solvent may be an aqueous solvent. The chitosan particles that are present in the hemostatic compositions (suspensions or powders) may be shaped like fibers and / or like agglomerated spheres or agglomerated bodies (e.g., so- called mophead). Stability of chitosan suspensions may be assessed by any suitable means. In preferred embodiments, stability is measured or observed by a lack ofseparation, i.e., one single phase instead of two separate phases in an aqueous mixture, for instance absence of chitosan deposits / sediments at a bottom or floating at the top of the aqueous mixture. Preferably, chitosan suspensions in accordance with the present invention are stable (e.g. absence of separation) for at least 1 day, or at least 1 week, or at least one month, or at least one year or more.

[0081] In embodiments, the chitosan molecules or particles comprised in the hemostatic compositions of the invention have been mechanically processed into a stable homogeneous aqueous chitosan suspension. In embodiments the mechanical processing involves high-shearing conditions and / or high mechanical energy. In embodiments the high-shearing conditions and / or high mechanical energy is obtained by a process including, but not limited to mechanical shearing, sheer thinning, planetary ball milling, rolling mill, vibrating ball mill, tumbling stirred ball mill, horizontal media mill, colloid milling. As indicated hereinafter, the high-shearing conditions and / or high mechanical energy can be carried out for a duration, under parameters, under suitable conditions, etc. until a desirable change of state is obtained, e.g., change of color, a change in viscosity, a change from a slurry to a paste, ointment, cream, lotion, gel, milk, or foam, etc.

[0082] Without wishing to be bound by theory, submitting the chitosan to high- shearing conditions and / or high mechanical energy improves chitosan’s performance to levels not seen using conventional processes, including improved rheological properties such as viscosity, shear thinning properties, high yield value and also improved blood clotting properties. Chitosan dispersed using high shear processes may contain lamellar crystalline gel networks (LGN) that may synergistically increase the viscosity of the chitosan dispersions. Oil + water chitosan dispersions in accordance with the present invention may also contain Pickering emulsion wherein the water-in-oil or oil-in-water emulsion is stabilized by the chitosan.

[0083] Chitosan dispersed using high shear processes in accordance with the present invention may also comprises positive surface charges enables the chitosan molecule to bind with negatively charged blood components, thereby promoting platelet activation and the agglutination of blood proteins, facilitating fibrin clot formation, while also forming a strong physical barrier that adheres to wet tissues and seals wounds.

[0084] In embodiments the high-shearing conditions and / or high mechanical energy requires using a suitable device or apparatus including, but not limited to, ball miller (e.g., planetary ball miller, rolling miller, vibrating ball miller, tumbling stirred ball miller, horizontal media mill, colloid miller, a magnetic miller, shaking miller), a twin-screw extruder, a high-pressure homogenizer, a blade homogenizer, a stirring homogenizer, a disperser, a rotor-stator homogenizer, a high-shear mixer, a plowshare mixer, a dynamic mixer, a plough mixer, a turbine mixer, a speed mixer, an attrition miller, a sonicator (e.g. high shear ultrasonic processes), a tissue tearor, a cell lysor, a polytron, a ribbon agitator, a microfluidizer, a high pressure homogenizer, and combinations thereof. In embodiments, the present invention utilizes ball milling under wet conditions. Particular examples of ball miller include, but are not limited to, vertical planetary mill (e.g., Tencan XQM-2A™) with 100 mL capacity zirconia jars and 10 mm diameter zirconia balls, Flacktek™ speed mixer (DAC 330-11 SE) with 40 mL zirconia jar with 5 mm diameter zirconia balls or zirconia rings, 1.5L Supermill Plus™ with 1.4-1.7 mm zirconia beads and Zeta™10 Mill (NETZSCH-Feinmahltechnik GmbH). In particular embodiments, the present invention utilizes bead shaking milling under wet conditions. On particular example of a suitable agitator bead mill includes, but is not limited to, laboratory agitator Bead Mill LabStar™ (NETZSCH-Feinmahltechnik GmbH) with 0.6-0.8 mm beads or 1 .4-1 .7 mm beads.

[0085] According to one particular embodiment, the hemostatic composition of the invention comprises chitosan molecules that have been mechanically processed using a Zeta™10 Mill (NETZSCH-Feinmahltechnik GmbH), or using a similar apparatus. As is known, the Zeta™ 10 Mill operates as a high-speed circulation mill engineered specifically for wet grinding and dispersing applications, utilizing a unique peg grinding system. This system features high-intensity grinding pegs mounted on a rotor, enabling efficient energy input and allowing for the fine grinding of particles down to the nanometer scale. The mill supports both circulation and multi-pass operations, providing flexibility to handle a range of viscosities and product types while ensuring consistent product quality. An advanced centrifugal separation system is integrated to retain grinding media within the grinding zone, thereby ensuring uniform particle size distribution and preventing pressure buildup. To manage the heat generated during the grinding process, the Zeta™10 Mill is equipped with an intensive cooling system for the grinding tank and agitator shaft, maintaining lowproduct temperatures to prevent overheating. Furthermore, the mill accommodates various grinding media diameters, from 0.3 mm to 3 mm, allowing for precise control over the grinding process and adaptability to different material types.

[0086] Accordingly, the present invention encompasses obtaining suitable chitosan- based hemostatic compositions and suspensions in accordance with the present invention with an apparatus providing one or more of the features and / or functionalities provided by the Zeta™10 Mill including, but not limited to, wet grinding and dispersing, high-speed circulation milling, peg grinding, circulation and multi-pass milling operations, centrifugal separation to retain grinding media within a grinding zone intensive cooling system for the grinding tank and / or agitator shaft.

[0087] In particular embodiments, chitosan-based hemostatic compositions and suspensions in accordance with the present invention are obtained using a particular protocol referred herein as the “10+1 Alt method”. This method comprises milling of the chitosan for a certain period of time (e.g., 10 min) followed by a short pause (e.g., 1 min) then milling in the opposite direction for a certain period of time (e.g., 10 min) for a total of 1 hour, or 2 hours, or 3 hours, or 5 hours, 10 hours, or 12 hours.

[0088] In particular embodiments, chitosan-based hemostatic compositions and suspensions in accordance with the present invention are obtained using a particular protocol wherein the solvent (preferably water) is mixed for 1-2 minutes in shaker mill (e.g., Bead Mill LabStar™), followed by the gradual addition of chitosan powder over 6-8 minutes to prevent clumping and pump blockage. After 90 minutes, one or more additional facultative components (e.g., preservative(s)) are added gradually, allowing the mixture to return to its original viscosity between additions. Milling is continued (e.g., up to 120 min) until a desired consistency is achieved, e.g., resembling a thick cream and off-white in color.

[0089] Advantageously, the viscosity of the compositions / suspensions can be altered by varying the high-shearing conditions and / or mechanical energy to which the chitosan molecules are submitted. These conditions can be adjusted to obtain a stable homogeneous suspension (e.g., a stable colloidal homogeneous suspension) having a desired viscosity. Typically, providing more mechanical energy will increase the shearingand will reduce accordingly the viscosity of the end product (e.g., from a paste to a milk). The final hemostatic compositions in accordance with the present invention may be formulated as a paste, an ointment, a cream, a lotion, a gel, a milk or a foam.

[0090] According to particular aspects, the hemostatic composition in accordance with the present invention consists of a hemostatic powder composition. In embodiments, the hemostatic composition consists of a dry composition comprising dried ground chitosan particles. In embodiments, the hemostatic composition comprises a chitosan powder or chitosan granules obtained subsequently to drying (e.g., air dry, oven, etc.) of an aqueous chitosan suspension as described hereinbefore, followed by grinding of dried chitosan. In one particular embodiment, dried chitosan is obtained by placing a known weight of chitosan suspension in a glass container and drying in an oven at 40°C for 24 hours to obtain dried chitosan films which are next ground using a mortar and pestle. In embodiments the dried ground chitosan particles have a shape of agglomerated micro / nano particles and fibers. In embodiments the dried ground chitosan particles have a size from about 1 nm to about 100 pm, or about 5 nm to about 50 pm, or about 10 nm to about 10 pm, or about 100 nm to about 1 pm.

[0091] In embodiments, the hemostatic powder composition comprises dried ground chitosan particles possessing a specific surface area(SSA)of at least 1 m2 / g, or at least 1 .5 m2 / g, or at least 2 m2 / g, or at least 2.5 m2 / g, or at least 3 m2 / g, or at least 3.5 m2 / g, or at least 3.5 m2 / g, or at least 4 m2 / g, or at least 5 m2 / g. In embodiments the SSA is about 1 m2 / g to about 5 m2 / g, or about 1.5 m2 / g to about 4 m2 / g, or about 2 m2 / g to about 3.5 m2 / g.

[0092] In embodiments, the hemostatic powder composition comprises dried ground chitosan particles possessing a pore volume of at least 0.010 cm3 / , or at least 0.015 cm3 / , or at least 0.02 cm3 / , or at least 0.025 cm3 / , or at least 0.03 cm3 / , or at least 0.035 cm3 / , or at least 0.04 cm3 / , or at least 0.05 cm3 / , or higher. In embodiments the pore volume is aboutO.010 cm3 / to about 0.05 cm3 / , or about0.015 cm3 / to about 0.04 cm3 / , about0.02 cm3 / to about 0.03 cm3 / .

[0093] In embodiments, the hemostatic powder composition comprises dried ground chitosan particles possessing a pore size of at least 200 A, or at least 250 A, or at least300 A, or at least 350 A, or at least 450 A, or at least 450 A, or at least 500 A, or at least 600 A, or higher. In embodiments, the pore size is about 200 A to about 500 A, or about 300 A to about 450 A.Impregnated Materials

[0094] An additional aspect of the invention concerns materials for treating or preventing bleeding. In embodiments the material comprises a substrate layer impregnated or having layered thereon a hemostatic composition as defined herein. For instance, the material may consist of a patch, a bandage, a wound dressing, a gauze, a sponge, etc. suitable for direct application to a wound (e.g., a bleeding wound).

[0095] The substrate layer is configured for receiving and releasing a hemostatic chitosan-based composition (aqueous or powder form) as defined herein. The substrate layer comprises a lower face for contacting a bleeding wound and releasing chitosan to the wound. Additionally, the substrate layer may advantageously possess properties such as flexibility for conforming to wound contours, durability to maintain structural integrity during application, bioadhesiveness for secure placement on moist tissue, and permeability to facilitate fluid absorption and promote wound healing.

[0096] In embodiments, the substrate layer is composed of a polymeric material. Envisioned material for the substrate layer includes low-modulus meshes and / or films and / or weaves of synthetic and naturally occurring polymers.

[0097] The substrate layer may also be composed of a porous material such as a sponge.

[0098] The material may comprise multiple layers including for instance, in addition to the substrate layer, a backing protective layer position deposited over the substrate layer. Additional layers may include an adhesive layer for secure application, a moisture control layer to manage exudate, and an antimicrobial layer to reduce infection risk.

[0099] Multiple layers can be attached, bonded by direct adhesion or held together with any suitable adhesive. One or more of the layers may be biodegradable. The backing protective layer may advantageously be substantially blood impermeable.[000100] In embodiments, the material which forms the support layer is a layer of biocompatible polymer, such as polylactic acid (PLA), designed to provide structural integrity. Alternatively, the support layer could be a woven or non-woven fabric that offers flexibility and durability.Methods of Uses[000101] Additional aspect of the invention concerns uses of the hemostatic compositions as defined herein (i.e., aqueous chitosan suspensions and / or corresponding powder compositions) and uses of a material as defined herein, for medical applications for humans and animals. These medical applications may include treating or preventing bleeding at a tissue site, treating an injury, promoting blood clotting, treating a bleeding wound.[000102] Typically, desirable medical benefits are obtained by contacting a wound, a hemorrhage, a damaged tissue, a bleeding tissue or the like, with the composition(s) and / or the materials of the invention. Multiple applications may be required over a period of a few minutes, a few hours or days. Pressure may also be required on the bleeding site or wound. Treatment may also further require using a blood clotting agent as those defined herein before.Kits[000103] Another aspect of the invention concerns kits for treating a bleeding wound of a human or an animal. In embodiments the kit comprises at least one of: (i) a container comprising a hemostatic composition comprising an aqueous chitosan suspension as defined herein; (ii) a container comprising a hemostatic powder composition as defined herein, and (iii) a material as defined herein; and at least one of a patch, a bandage, a wound dressing, a gauze, a sponge, one or more blood clotting agent including, a disinfectant, an antimicrobial, an antiseptic, and a pamphlet with instructions.[000104] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are consideredto be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following example(s)which should not be construed as further or specifically limiting.EXAMPLESExample 1 : Comparing blood clotting efficacy of the compositions of the invention with commercial benchmark products in bovine whole blood[000105] Experiments were carried out to investigate the effect of hemostatic compositions in accordance with the invention on the clotting time of bovine whole blood and plasma.Materials[000106] The compositions tested included chitosan suspensions in accordance with the present invention (with and without the presence of preservative(s) (e.g., Dermosoft™ and Hydrolit™6 / benzyl alcohol), their dried ground suspension equivalents) and two commercial benchmark products, namely Curad® BloodStop Hemostatic Gauze (a biodegradable dressing made of natural fiber cellulose that turns into a gel to seal a wound), and Celox™ hemostatic granules (a proprietary blend of materials that contains chitosan). Both commercial products were applied in accordance with the manufacturer's Instructions for Use (IFU). Table 1 below lists the hemostatic agents used in this study.Table 1 : Hemostatic agents used in this study[000107] Compositions in accordance with the present invention (i.e., DEF samples listed in Table 1) were manufactured as follows. Briefly, all required materials were accurately weighted, including chitosan powder and the preservatives (Dermosoft™ or premix of Hydrolite™6 + benzyl alcohol). Pre-weighed water was poured into the tank of a laboratory agitator Bead Mill LabStar™ (NETZSCH-Feinmahltechnik GmbH) and mixed for 1-2 minutes, followed by the gradual addition of chitosan powder over 6-8 minutes to prevent clumping and pump blockage. After 90 minutes, preservative(s) was added, allowing the formula to return to its original viscosity between additions. Milling was continued until the desired consistency was achieved, resembling thick cream and off- white in color (up to 120 min). Then the formula is allowed to rest overnight to reach room temperature. The mixing parameters were as followed: Pump Speed 300 rpm; Agitator Speed 2500 rpm; Bead size 1.7 mm; Screen size 0.7 mm; Flow rate: 19.24 ml I sec; Pressure on screen 8.0 - 8.2 PSI, Temperature of product: 87 - 90 Fahrenheit[000108] Dried ground suspension equivalents of these 3 suspensions were obtained. A known weight of each chitosan suspension was placed in a glass container and dried in an oven at 40°C for 24 hours. The dried chitosan films were then ground using a mortar and pestle.[000109] Three different media were utilized for the tests: as-received citrated blood, recalcified blood, and bovine plasma with citrate. Recalcified and citrated blood were chosen to model healthy and impaired clotting cascades, respectively, while bovine plasma was selected to represent the intrinsic pathway of clotting.[000110] Recalcified blood (7 mL) was prepared by mixing 0.5 mL of 0.06 M CaCI2solution (dissolved in deionized water) with 6.5 mL of citrated blood at 37°C. Citrated bloodwas incubated for at least 30 minutes to reach 37°C before recalcification or G' measurements. Three separate batches of citrated whole blood were used, sealed upon receipt, stored at 4°C, and utilized within seven days.[000111] Both commercial products were used as is and applied in accordance with the manufacturer's instructions for fse (IFU). The Celox™ granules were tested as received. The Cured™ gauze was cut into smaller pieces and weighed before being introduced into the media.[000112] The viscoelastic properties of the blood sample including G' properties of the systems were measured using ElastoSens™ Bio2 (Rheolution Inc., QC, Canada). The device was calibrated at 37°C with empty sample holders to record baseline readings before introducing the hemostatic agents.[000113] To prevent evaporation and artificial hardening of the blood samples, an antievaporation oil was added on top of all test specimens. The oil also helped eliminate surface bubbles. Real-time changes in shear storage modulus (G') were monitored to assess coagulation in both citrated (n=2 or 3) and recalcified (n=3) blood. Measurements were taken over 30 minutes for recalcified blood systems and 480 minutes for citrated blood systems, with intervals of 5 seconds and 60 seconds between readings, respectively.[000114] The G'-time curves were analyzed to determine the coagulation characteristics of each system, including clot initiation time, clotting rate, and final stiffness of the clots. Clot initiation time was defined as the point where G' increased by more than 5% from its initial value (Go') and continued to rise. Clotting rate was calculated as the slope of the linear section (R2> 0.95) of the G'-time graph where G' rapidly increased. The final stiffness of the clots was taken as the G' value at the end of the analysis, which was 30 minutes for all systems. After the analysis, all samples were removed from the holders and photographed with a high-definition digital camera.[000115] Activated Partial Thromboplastin Time (aPTT) evaluates the intrinsic pathway by measuring the time it takes for plasma the clot after adding an activator such as kaolin or ellagic acid, along with calcium ions. Typically, aPTT is used to monitor patients onheparin therapy, evaluate for inherited or acquired coagulation disorders, and assess bleeding tendencies. APTT-XL reagent (Thermo Fisher Scientific) is useful as a screening tool, and as a quantitative test for the intrinsic coagulation factors. It is a simple and versatile test which is sensitive to deficiencies of all plasma clotting factors except Factor VII. However, it is mainly used to detect deficiencies in Factors VIII, IX, XI, XII, and prekallikrein.[000116] Turbidimeter TURBIDI.T™ (Rheolution Inc., QC, Canada) was used to assess coagulation factors in the intrinsic and common pathways, as well as to evaluate the efficacy of various hemostatic agents in promoting plasma coagulation.[000117] In this procedure, 1.3 mL of plasma was mixed with 1.3 mL of APTT reagent (Thermo Fisher Scientific) and incubated for 3 minutes at 37°C. Then hemostatic agent and 1.3 mL calcium chloride (0.02M) was added to mix. Real-time changes in Formazin Turbidity Units (FTU) at a wavelength of 850 nm were monitored to assess plasma coagulation through the intrinsic pathway. Measurements were taken at intervals of 7 seconds over a total duration of 3 minutes.[000118] The FTU-time curves were analyzed to determine the clot initiation time. Clot initiation time was defined as the point where FTU increased by more than 5% from its initial value and continued to rise.[000119] Data for each time point were analyzed for statistical significance between conditions using Student t-test at a significance level of p<0.05.Experiment 1: One (1) gram of suspension or 0.1 gram of powder / qranules / qauze[000120] The main objective of the experiment was to assess the hemostatic efficacy of chitosan suspensions, with or without preservative(s).[000121] As shown in Figures 1A-1C, the chitosan suspensions in accordance with the present invention (i.e. , S10 and S11) exhibited faster coagulation and shorter clot initiation times compared to Curad™ gauze and the control. However, recalcified blood treated with Celox™ showed the fastest coagulation and the shortest clot initiation time overall. The clotting rate of samples treated with the chitosan suspension and granules in accordancewith the present invention (i.e., S10 and S11 , respectively) was significantly faster than those treated with Curad™ and Control and fell within the same range as samples treated with Celox™ granules (Fig. 1C).[000122] As shown in Figure 2, clots formed using the chitosan suspension in accordance with the present invention (i.e., S10 and S11 , respectively) were significantly stronger than those in the control and Curad™ systems, with stiffness values comparable to clots formed by Celox™ granules.Experiment 2: 1.5 gram of suspension or 0.15 gram of powder / qranules / qauze[000123] The main objective of the experiment was to assess the hemostatic efficacy of chitosan suspensions, with or without preservative(s), and their dried, ground equivalents.[000124] As shown in Figures 3A-3C, the chitosan suspensions in accordance with the present invention (i.e., S10 and S11) exhibited faster coagulation and shorter clot initiation times compared to Curad™ gauze and the Control. Recalcified blood treated with Celox™ demonstrated the fastest coagulation and the shortest clot initiation time overall.[000125] The clotting rate of samples treated with the chitosan suspensions in accordance with the present invention (i.e., S10 and S11) and their dried ground versions (i.e., P1 and P2) was significantly faster than those treated with Curad™ and the Control, and comparable to samples treated with Celox™ granules. Notably, the S11 suspension (containing Dermosoft™ preservative) showed a significantly slower clotting rate than Celox™, while the S10 suspension (without preservative) had a significantly higher clotting rate compared to Celox™.[000126] As shown in Figure 4, blood clots formed using the chitosan suspension in accordance with the present invention (i.e., S10 and S11) were significantly stronger than those in the Control, Celox™, and Curad™ systems. The clot stiffness of samples treated with the dried, ground versions (i.e., P1 and P2) was comparable to that of clots formed by Celox™ granules.Experiment 3: 1.5 gram of threes suspensions or 0.15 gram of powder / granules / gauze[000127] The main objective of the experiment was to assess the hemostatic efficacy of chitosan suspensions with or without two different combinations of preservatives, namely (i) Dermosoft™ alone, or (ii) Dermosoft™ + Hydrolite™ 6 + benzyl alcohol, and their dried, ground equivalents.[000128] As shown in Figures 5A-5C, the chitosan suspensions in accordance with the present invention (i.e., S10, S11 and S12) and their dried, ground equivalents (i.e. , P1 , P2 and P3) exhibited faster coagulation and shorter clot initiation times compared to Cured™ gauze and the Control. However, recalcified blood treated with Celox™ demonstrated the fastest coagulation and the shortest clot initiation time overall. There was no significant difference between chitosan suspensions with or without preservatives, or between their powder equivalents, as all showed a similar range of clot initiation times. The clotting rate of samples treated with the chitosan suspensions in accordance with the present invention and their corresponding dried powder versions was significantly faster than those treated with Cured™ and the Control, and comparable to samples treated with Celox™ granules. The S10 suspension without preservative, showed a significantly higher clotting rate than Celox™.[000129] As shown in Figure 6, clots formed using the three chitosan suspension in accordance with the present invention (i.e., S10, S11 and S12) were significantly stronger than those in the Control, Celox™, and Cured™ systems. The clot stiffness of samples treated with the corresponding dried, ground versions (i.e., P1 , P2 and P3) was comparable to that of clots formed by Celox™ granules.[000130] In Figure 7, final clots formed using various hemostatic systems and the control were compared. The clots were cut open to better show their consistency, liquid residue, and final clot height. It can be observed that the control clot had a significant amount of liquid residue and a notably smaller height — approximately 50% less than clots from samples treated with the three chitosan suspension in accordance with the present invention and their corresponding dried, ground versions. The clots formed by Celox™ (Fig. 7D) and Cured™ (Fig. 7E) were not coherent and lacked consistency: they collapsed and became disrupted when attempts were made to cut them open. On the other hand,the clot formed by the S10 suspension demonstrated the greatest thickness at 18 mm (Fig. 7C), followed by the clot formed by P1 at 15 mm (Fig. 7B), and the control clot at 8 mm (Fig. 7A). These results clearly demonstrate the superior quality of clots formed using any of the chitosan-based suspensions and powders of the present invention.Experiment 4: 1.5 gram of suspensions or 0.15 gram of powder / qranules / qauze of citrated blood[000131] The main objective of the experiment was to assess the hemostatic efficacy of chitosan suspensions, with or without preservative(s), and their dried, ground equivalents in promoting coagulation in citrated blood.[000132] Sodium citrate is an anticoagulant commonly used in blood product collection and storage. Citrate binds to free calcium, preventing it from interacting with the coagulation system. Citrate effectively prevents blood products from clotting. To enable citrated blood to coagulate again, it needs to be provided with a calcium source for recalcification. Having a hemostatic product that can accelerate coagulation in the presence of anticoagulants is advantageous. If a hemostatic agent can clot citrated blood that has not been decalcified, it suggests that the agent can bypass or overcome the inhibitory effects of citrate on clotting. This could be achieved by directly activating clotting factors that are downstream of the calcium-dependent pathways.[000133] As shown in Figure 8, Control, Celox™ and Curad™ citrated blood systems were not coagulated up to 1 day. On the other hand, citrated blood systems comprising the chitosan suspensions in accordance with the present invention (i.e., S10, S11 and S12) were coagulated and formed a well-formed clot in less than 4 hours, while their dried, ground equivalents (i.e., P1 , P2 and P3) exhibited significantly faster coagulation and shorter clot initiation times (between 3 to 7 minutes). The P1 (i.e., the dried ground powder without preservative) showed a significantly higher clotting rate.[000134] In Figure 9, visually, a solid mass (clot) can be observed when the chitosan suspensions in accordance with the present invention and their dried ground equivalents are applied to citrated blood (Figs. 9A, 9B, 9C, 9E, 9F and 9G). However, with Celox™and Curad™, the blood remains liquid, with no clot (solid mass) formation (See: Fig. 9D) and Fig. 9H). This observation aligns with the graph profiles shown in Figure 8.[000135] Example 2: Intrinsic vs extrinsic pathway of bovine plasma[000136] As is known, there are two main pathways involved in the body coagulation process: the intrinsic pathway and the extrinsic pathway. The extrinsic pathway (tissue factor-mediated) is primarily triggered by tissue damage or external trauma that exposes tissue factor (TF), also known as thromboplastin. On the other hand, the intrinsic pathway (contact system-initiated) is activated by factors present within the blood vessel walls. It begins with the activation of factor XI I (FXII) by contact with exposed collagen or negatively charged surfaces.[000137] Experiments were carried out to evaluate the coagulation efficacy of the compositions of the invention and to assess their functionality of both intrinsic and extrinsic pathways of coagulation.Materials and methods[000138] Compositions in accordance with the present invention (listed in Table 1 (see above) or in Table 2 (see below) were manufactured as described above for Example 1.[000139] The following methods were used to evaluate the coagulation efficacy of the compositions of the invention and to assess their functionality of both intrinsic and extrinsic pathways of coagulation. (1) Prothrombin Time (PT): PT measures the extrinsic pathway's functionality by assessing the time taken for plasma to clot after adding calcium and tissue factor. (2) Activated Partial Thromboplastin Time (aPTT): aPTT evaluates the intrinsic pathway by measuring the time it takes for plasma to clot after adding an activator such as kaolin or ellagic acid, along with calcium ions.Table 2: Hemostatic agents used in this studyExperimental Methodology:[000140] Prothrombin Time (PT), Extrinsic Pathway:A. Prewarm Thromboplastin-DS to 37°C.B. Add 0.5 mL test plasma to cuvette and prewarm to 37°C.C. Forcibly add 1 mL warmed Thromboplastin-DS to the test plasma and time clot formation.[000141] Activated Partial Thromboplastin Time (aPTT), Intrinsic Pathway:A. Prewarm Calcium Chloride (0.02M) to 37°C.B. Add 0.5 mL test plasma to cuvette and prewarm to 37°C.C. Add 0.5 mL APTT-XL to the test plasma. Mix.D. Incubate the plasma-reagent mixture at 37°C for 3 minutes (activation time).E. Forcibly add 0.5 mL prewarmed Calcium Chloride and time clot formation.[000142] Turbidity profiles: TURBIDI.T™ (Rheolution Inc., QC, Canada) was used to assess coagulation factors in the intrinsic and common pathways, as well as to evaluate the efficacy of various hemostatic agents in promoting plasma coagulation. In this procedure, 1.3 mL of plasma was mixed with 1.3 mL of APTT reagent and incubated for 3 minutes at 37°C. Then hemostatic agent and 1.3 mL calcium chloride (0.02M) was added to mix. Realtime changes in Formazin Turbidity Units (FTU) at a wavelength of 850 nm were monitored to assess plasma coagulation through the intrinsic pathway. Measurements were taken at intervals of 7 seconds over a total duration of 3 minutes. The FTU-time curves were analyzed to determine the clot initiation time. Clot initiation timewas defined as the point where FTU increased by more than 5% from its initial value and continued to rise.Results:1) Prothrombin Time (PT), Extrinsic Pathway.[000143] The one-stage PT assay measures plasma clotting time after introducing tissue factor(thromboplastin). The addition of tissue factor leads to the activation of Factor Xa (F.Xa) via plasma recalcification. F.Xa then activates Prothrombin, converting fibrinogen into an insoluble fibrin clot.[000144] Although not shown, it was found that chitosan suspensions in accordance with the present invention with or without cellulose (i.e. S6: NTZ2402701 and S4: NTZ230913 showed faster plasma coagulation compared to Curad™ gauze and the control. Plasma treated with Celox™ granules coagulated first and at a quicker rate. Also, it should be noted that the Curad™ gauze was only effective within the immediate vicinity of the gauze.[000145] However, clots formed in the presence of chitosan suspensions in accordance with the present invention were notably stronger and more resistance to rupture (data not shown). This strength is advantageous for hemostatic performance as it efficiently stops bleeding, reduces the risk of rebleeding, accelerates wound healing, minimizes infection risk, and enhances overall patient outcomes.2) Activated Partial Thromboplastin Time (aPTT), Intrinsic Pathway:[000146] Although not shown, it was found that the two chitosan suspensions in accordance with the present invention, regardless of the presence of cellulose, (i.e. S6: NTZ2402701 and S4: NTZ230913), exhibited accelerated plasma coagulation compared to other treatments.[000147] It was also found that plasma treated with Curad™ gauze displayed coagulation primarily near the gauze application site. Control plasma showed the slowest coagulation response. Interestingly, plasma treated with Celox™ granules did not demonstrate any visible signs of coagulation. Furthermore, clots formed in the presenceof the chitosan suspensions of the invention were notably stronger and more resistant to rupture compared to the control and other treatments.[000148] 3) Turbidity-APTT[000149] As shown in Figure 10, APTT plasma testing of the hemostatic agents demonstrated that Celox™ took significantly longer to initiate coagulation in the intrinsic pathway compared to the chitosan suspensions in accordance with the present invention and their dried, ground equivalents and their dried, ground equivalents.3) Conclusions[000150] Celox™ granules demonstrated the fastest plasma coagulation in the presence of extrinsic tissue factor (PT-DS). However, plasma treated with Celox™ granules did not exhibit visible signs of coagulation in the presence of intrinsic tissue factor (APTTXL).[000151] As observed during the study, chitosan suspensions in accordance with the present invention, with or without cellulose (i.e., S6: NTZ2402701 and S4: NTZ230913), showed significant effectiveness in promoting plasma coagulation via both extrinsic tissue factor (PT-DS) and intrinsic tissue factor (APTTXL) compared to benchmark products Celox™ and Curad™ gauze, and the control group. Their effect was notably more significant in the presence of intrinsic tissue factor (APTTXL).[000152] Without being bound by theory, the present results suggest that the hemostatic compositions in accordance with the present invention leads to the cessation of bleeding “mostly” via the intrinsic pathway, a pathway not triggered by Celox™ since Celox™ seems to work only the extrinsic pathway which contains Factor 7. The present results further suggest that the hemostatic compositions in accordance with the present invention initiate secondary hemostasis. The main difference between primary and secondary hemostasis is that primary hemostasis is defined by the formation of the primary platelet plug whereas the secondary hemostasis or coagulation is defined by the formation of insoluble, cross-linked fibrin. Furthermore, activated platelets are responsible for primary hemostasis while activated coagulation factors are responsible for secondary hemostasis. The present invention thus provides these additional benefits.[000153] Clots formed in the presence of both chitosan suspensions in accordance with the present invention were notably stronger and more resistant to rupture than the two benchmark products (i.e., Celox™ and Curad™) and the control group. This increased strength is advantageous for hemostatic performance as it efficiently stops bleeding, reduces the risk of rebleeding, accelerates wound healing, minimizes infection risk, and enhances overall patient outcomes.[000154] Therefore, the chitosan suspensions in accordance with the present invention provides numerous benefits. Indeed, a stronger clot is generally considered better in terms of hemostatic performance for several reasons, including:1 . Efficiency in Stopping Bleeding-. A strong clot forms a more effective barrier to halt bleeding from a wound or injury. It helps prevent excessive blood loss, which is crucial for the body to maintain stable blood pressure and ensure proper oxygen delivery to tissues.2. Reduced Risk of Rebleeding-. A stronger clot is less likely to break down or dislodge prematurely, reducing the risk of rebleeding. This stability is important for the healing process and prevents complications that can arise from recurrent bleeding.3. Enhanced Wound Healing-. A robust clot provides a scaffold for the migration of cells involved in the healing process, such as platelets, immune cells, and fibroblasts. This facilitates tissue repair and regeneration, leading to faster and more efficient wound healing.4. Lower Chance of Infection-. A strong clot can help seal off the wound site, reducing the risk of pathogens entering the body and causing infections. It acts as a physical barrier that limits microbial infiltration and promotes a sterile environment for healing.5. Improved Patient Outcomes-. Overall, a stronger clot contributes to better patient outcomes by minimizing complications associated with prolonged bleeding, rebleeding, delayed healing, and infections. It supports the body's natural hemostatic mechanisms and promotes a favorable environment for recovery.Example 3: SEM characterization of blood-clotting composition[000155] Purpose: Scanning Electron Microscopy (SEM) is a powerful tool for analyzing hemostatic agents. SEM analysis reveals detailed microstructural characteristics which are critical for understanding how a homeostatic agent performs. A SEM analysis was thus conducted to further understand the performance and morphological differences of the chitosan suspensions.[000156] Methods: Sputter coating and SEM samples were sputter-coated with a 4 nm layer of Pt (EM ACE600™, Leica Microsystems, Buffalo Grove, IL, USA). A FEI Quanta™ 450 ESEM (FEI Corporation, Hillsboro, OR, USA) was used with an acceleration voltage of 5 kV to image the topography and morphology of hemostatic particles.Materials:[000157] Table 3 below lists the hemostatic agents used in this study. The raw chitosan is a fungal chitosan produced by Chibio™ (Qingdao City, China). The remainder hemostatic agents were manufactured as described above for Example 1.Table 3: Hemostatic particles / granules used in this studyResults and Conclusions:[000158] The SEM images of the hemostatic agents and granules tested are displayed in Figures 11A-11 H and Figures 12A-12H. The images reveal a micro / nano fibrous structure with micro / nano particle agglomeration within the fibrous matrix for both, the chitosan suspensions in accordance with the invention (i.e., S10, S11 and S12) and their dried, ground equivalents (i.e., P1 , P2 and P3) (Figs. 11A-11 F; Figs. 12A-12F). In contrast, the raw chitosan powder (Fig. 11G, Fig. 12G) and Celox™ granules (Fig. 11 H, Fig. 12H) exhibit a dense, non-porous structure.[000159] While the Celox™ granules and raw chitosan powder both appear as densely packed and non-porous particles agglomerated microparticles, the chitosan suspensions in accordance with the invention and their dried, ground equivalents exhibit nano / microporous structures comprising agglomerated chitosan spheres and microfibers. Without being bound by theory, it is expected that in such suspensions, microfibers can self-assemble due to chitosan molecules’ tendency to interact through hydrogen bonding and other intermolecular forces, especially in neutral-pH water. These interactions can thus lead to microfibrillar structures that remain uniformly dispersed, leading to a matrix in an aqueous solution.[000160] Accordingly, it is envisioned that the enhanced hemostatic properties possessed by the chitosan suspensions in accordance with the invention and their dried, ground equivalents, compared to raw chitosan powder, can be explained (at least partially) by their particular microfibrous structure that is absent in raw chitosan powders and Celox™. This assumption makes perfect sense considering the following properties of microfibrous structures:1 . Increased surface area: Microfibers have a significantly higher surface area compared to raw powder. (See. Figs. 11A-11 F; Figs. 12A-12F). This larger surface area allows for better contact with blood and tissue, which is critical for the hemostatic process. More surface area can improve the rate at which chitosan interacts with blood, promoting faster clotting.2. Higher porosity: The microfibrous structure is more porous, which can help in absorbing blood and fluids more efficiently. This can lead to faster aggregation of blood cells and platelets, accelerating the clotting process and improving hemostasis.3. Mechanical support for clot formation: Microfibers can provide a scaffoldlike structure that physically supports the formation of a clot. The microfibers can trap red blood cells and platelets, aiding in the formation of a stable clot at the site of injury.4. Enhanced chitosan action: Chitosan is known for its ability to interact with blood components, especially through its positive charge, which attracts negatively charged elements such as red blood cells and platelets. When in microfibrillar form, these interactions can be more effective, leading to improved hemostatic activity.[000161] In contrast, raw chitosan powders might not provide the same level of interaction because the particles may not form a consistent or supportive matrix for the clotting process. While raw chitosan still has hemostatic properties, it may be less efficient in promoting rapid and stable clot formation compared to a fibrous form that mimics natural tissue structures more closely.[000162] Therefore, microfibrous chitosan structures in accordance with the present invention offer distinct advantages for hemostatic applications due to their increased surface area, enhanced porosity, and ability to support stable clot formation. These features make them superior to raw chitosan powder and Celox™ granules for promoting rapid and efficient hemostasis, highlighting their potential for advanced medical applications.Example 4: Increased specific surface area of the blood-clotting composition[000163] Purpose: There is a known correlation between surface area and effectiveness of hemostatic agents. Indeed, hemostatic effectiveness is significantly influenced by the material's specific surface area (SSA), pore volume, and pore size. These parameters affect the material's ability to rapidly interact with blood components, absorb fluids, and initiate clotting. The present study was carried out to compare specific surface area, pore volume and pore size of chitosan suspensions in accordance with the present invention with those of raw chitosan powder and Celox™ granules.Materials and methods:[000164] Samples P2: DEF24072902, P3: DEF24082802, Celox™ and raw chitosan were tested. These samples correspond to those described hereinbefore (see Table 3 and also Table 4 below). The specific surface area (SSA) of each sample was measured using nitrogen gas adsorption and desorption isotherms collected with a Micromeritics TriStar™ 3000 (Micromeritics Instrument Corporation, USA) gas sorption system. SSA values were determined using the Brunauer-Emmett-Teller (BET) method, while the Barrett-Joyner-Halenda (BJH) method, utilizing the desorption isotherms, provided the average pore width and pore volume.Results:[000165] Table 4 below provides the Brunauer-Emmett-Teller (BET) surface area, pore volume and pore size information of the tested hemostatic agents.Table 4: BET surface area, pore volume and pore size information of tested hemostatic agents[000166] Interestingly, chitosan suspensions in accordance with the present invention (i.e. samples P2: DEF24072902 and P3: DEF24082802) exhibited the greatest SSA (2.12-3.20 m2 / g) and the greatest pore volume (0.016-0.020 cm3 / g). These SSA values exceed by 10 times that of the Celox™ granules, thereby advantageously providing a substantially larger surface area for interaction with blood. This structural characteristic ispresumably an important advantage to promote a rapid absorption of plasma and an efficient platelet adhesion, which are both critical for initiating the clotting cascade.[000167] The chitosan suspensions in accordance with the present invention also had a much greater pore volume compared to Celox™ and raw chitosan. A higher pore volume may enhance fluid absorption, which is vital for forming a stable clot. The pore size was similar to Celox™, which suggests a porous structure that balances fluid uptake with structural integrity.[000168] All these attributes make the dried ground chitosan suspensions in accordance with the present invention particularly suitable for applications requiring high surface interaction, such as rapid bleeding control in surgical or trauma settings.[000169] On the other hand, the Celox™ granules had a significantly lower SSA (0.271 m2 / g) and lower pore volume (0.002 cm3 / g), compared to the chitosan suspensions of the present invention, suggesting a more compact and less porous material. While the larger average pore size (513.67 A) may facilitate initial fluid absorption, the limited SSA may restrict the material's interaction with blood components, reducing its overall hemostatic efficiency compared to the dried suspensions of the invention. These properties suggests that Celox™ granules may be less effective in scenarios requiring rapid clot formation, though their compact nature could still offer structural stability in specific applications.[000170] The raw chitosan sample provided a baseline for comparison, with moderate SSA (0.9775 m2 / g) and low pore volume (0.005 cm3 / g). Its porosity and surface characteristics were less enhanced than the dried ground chitosan suspensions of the present invention, but more pronounced than Celox™ granules, suggesting intermediate performance as a hemostatic agent.Conclusions:[000171] Higher SSA and pore volume: The dried ground dried ground chitosan suspensions in accordance with the present invention possess advantageous structural characteristics for hemostatic applications due to their enhanced ability to absorb fluidsand interact with blood components. These properties support a faster clot formation and a more efficient hemostasis.[000172] Compact Materials: The lower SSA and lower pore volume of Celox™ granules, compared to those of the chitosan suspensions in accordance with the present invention, likely limit the interaction of these granules with blood. This could reduce their effectiveness in high-demand situations.[000173] Material engineering opportunities: The present comparison study underscores the importance of optimizing porosity and surface area in developing effective hemostatic agents. The present study further highlights the critical role of material surface characteristics in the design and selection of hemostatic agents for various medical applications and confirm the beneficial features of the chitosan suspensions in accordance with the present invention.Example 5: Clot initiation time, clotting rate, mechanical strength, and stiffness of hemostatic chitosan compositions in accordance with the present invention[000174] Purpose: This study investigates how hemostatic chitosan aqueous suspensions in accordance with the present invention (and their corresponding dried powder versions) affect key clotting efficacy factors, specifically clot initiation time, clotting rate, mechanical strength of clots, and clot stiffness, when used in bovine whole blood. The tested formulations varied in pH, in chitosan concentration, in chitosan sources, and preservatives. A dried and ground equivalent of a preservative-free powder (P1) was also tested and compared to a well-known benchmark product. Additionally, the ability of the chitosan compositions in accordance with the present invention to initiate clotting in citrated (non-recalcified) blood was evaluated, highlighting the potential for use in trauma and emergency scenarios where coagulation pathways may be compromised.Materials and methods:[000175] The followings were prepared. Unless specified otherwise, these were prepared in accordance with the method(s) of Example 1 .a. Chitosan Suspension Bases: i. S11 : DEF25041501 : Chibio Chitosan 10% I Dermosoft Pentiol ECO 5% (pH 8.8) ii. S13: DEF25042202: Chibio Chitosan 5.8% I Dermosoft Pentiol ECO 5% 1 1 ,5%GDL I 0.32% Xanthan Gum (pH 6.4) iii. S12: DEF25042402: Chibio Chitosan 10% I Hydrolite 6 + Benzyl Alcohol 2% (pH 8.1) iv. S14: NTZ24110401 : Oyster Mushroom Chitosan 5% I Geogard ECT 1.25% (pH 7.3) b. Dried, Ground Suspension Equivalents: i. P1 : DEF25042403: Dried powder derived from an aqueous suspension comprising water and 10% Chibio Chitosan, in accordance with the present invention. Water was removed by controlled drying, yielding a final effective chitosan concentration of 91-93% with residual moisture (7-8%) and ash content (0.3-0.8%). c. Benchmark Product: i. Celox™ granules: Proprietary blend of materials that contains chitosan. d. Bovine whole blood sterile sodium citrate e. Calcium chloride f. Raw chitosan powder[000176] Table 5 below lists the hemostatic agents used in this study. The raw chitosan is a fungal chitosan produced by Chibio™ (Qingdao City, China). The remainder hemostatic agents were manufactured as described above for Example 1.Table 5: Hemostatic particles / granules used in this studyDEF25042402 (S 12) is a new batch corresponding to DEF24082802; P1-DEF25042403 (P1) is a new batch corresponding to DEF24072901 (P1).** DM - Dermosoft™ Pentiol ECO 5% (pH 8.8; H6 = Hydrolite™ 6 ; BA = benzyl alcohol; GDL = Glucono Delta-Lactone; GeoG = Geogard™ ECT.Methodology[000177] The topical hemostatic agents used in this study were categorized by texture into two types: granular (powder) and suspension-like (liquid slurry) as described in Table 5. Each agent was applied in accordance with the manufacturer's Instructions for Use (IFU). Prior to testing, all agents were stored in sealed containers, protected from air and moisture. Two different media were utilized: as-received citrated blood and recalcified blood. Recalcified and citrated blood were chosen to model healthy and impaired clotting cascades, respectively.[000178] Recalcified blood (7 mL) was prepared by mixing 0.5 mL of 0.07M CaCI2solution (dissolved in deionized water) with 6.5 mL of citrated blood at 37°C. Citrated bloodwas incubated for at least 30 minutes to reach 37°C before recalcification or G' measurements. The citrated whole blood was stored at 4°C and utilized within four (4) days.[000179] The viscoelastic properties of the sample, including the G' properties of the systems, were measured using the ElastoSens™ Bio2 (Rheolution Inc., QC, Canada). The device was calibrated at 37°C with empty sample holders to record baseline readings before introducing the hemostatic agents.[000180] To prevent evaporation and artificial hardening of the samples, an antievaporation oil was added on top of all test specimens, which also helped eliminate surface bubbles. Real-time changes in shear storage modulus (G') were monitored to assess coagulation in both citrated (n=3) and recalcified (n=3) blood. Measurements were taken over 55 minutes for recalcified blood systems and 300 minutes for citrated blood systems, with intervals of 5 seconds and 60 seconds between readings, respectively.[000181] The G'-time curves were analyzed to determine the coagulation characteristics of each system, including clot initiation time, clotting rate of the clots. Clot initiation time was defined as the point where G' increased by more than 5% from its initial value (Go') and continued to rise. Clotting rate was calculated as the slope of the linear section (R2> 0.95) of the G'-time graph where G' rapidly increased.[000182] The storage modulus (G') measured over time provides a clear, real-time assessment of blood clot formation. At the start, whole blood has a low G', indicating a fluid state. As clotting begins, fibrin fibers form a solid network that increases G', reflecting the developing clot’s stiffness. The point where G' first rises defines how quickly clotting is initiated, while the slope of the rising curve shows the clotting rate, a steeper slope means the clot forms faster and strengthens more rapidly. Together, G' over time quantifies both the speed and the quality of clot development.[000183] After the analysis, all samples were removed from the holders for mechanical testing. Compression testing of blood clots was performed using the UniVert mechanical testing system (CellScale™, Canada). Cylindrical clot samples were carefully prepared and placed between the compression platens of the instrument. The test was conductedat room temperature under unconfined conditions. A constant displacement rate was applied until significant deformation was observed or the sample failed. Force and displacement data were continuously recorded and used to calculate stress-strain curves, providing quantitative information on the mechanical properties, such as stiffness and compressive modulus, of the blood clots.[000184] Stiffness: The tangent modulus of the blood clot was calculated between 10- 20% strain to represent the effective stiffness during physiological deformation, avoiding preconditioning artifacts at very low strains.[000185] Strength: The maximum stress recorded before failure.Statistical analysis:[000186] Data for each time point were analyzed for statistical significance between conditions using the Student t-test at a significance level of p<0.05.Results:[000187] The present study evaluated the hemostatic efficacy of many different the chitosan suspensions in accordance with the present invention. These were formulated with three different preservative systems, namely Dermosoft™ Pentiol ECO, Hydrolite™ 6 / benzyl alcohol, and Geogard™ ECT, as well as two different chitosan sources, namely mushroom or oyster mushroom. In addition, a dried and ground equivalent of a 10% chitosan suspension without any preservatives was tested ( this formulation containing a final effective chitosan concentration of 91-93%).[000188] Overall, the chitosan suspension formulations of the invention exhibited faster coagulation rates and shorter clot initiation times compared to Celox™, the benchmark control (see S11 , S13, S12 and S14 in Figures 13D and 13C).[000189] Notably, treatment of citrated (non-recalcified) blood with dried formulation P1 resulted in clot initiation time and a clotting rate comparable to those observed in reactivated and recalcified control blood (see Figures 13A, 13C and 13D). This finding suggests that certain chitosan formulations of the invention may be capable of initiatingclot formation even in the absence of calcium, which has significant implications for emergency and battlefield settings where calcium replenishment may not be immediately possible.[000190] Furthermore, the S11 and S12 suspensions manufactured in accordance with the present invention to containing 10% chitosan, demonstrated significantly higher clotting rates and faster clot initiation times compared to S13 and S14, which contained approximately 5% treated chitosan in accordance to the present invention (see Figures 13C and 13D). These results highlight a clear dose-dependent relationship between chitosan concentration and hemostatic performance, emphasizing the importance of chitosan content in optimizing clotting efficiency. This information is critical for tailoring treated chitosan formulations for different clinical and field applications, especially where rapid hemostasis is essential.[000191] As seen in Figures 15A-15C, visual inspection of the clots formed after treatment with raw chitosan and dried formulation P1 , the dried ground powder in accordance to the present invention (P1) reveals distinct differences in powder-blood interaction. The clot resulting from raw chitosan treatment (Fig. 15A) shows a localized agglomeration of chitosan particles, indicating poor dispersion and limited interaction with the surrounding blood. This agglomerated mass appears to act as a physical barrier, preventing the clot from penetrating or forming uniformly through the chitosan material, consequently suggesting inefficient hemostatic integration. In contrast, the clots formed in the presence of P1 (Figs. 15B and 15C) demonstrated a homogenous distribution of the chitosan material throughout the clot structure. The chitosan powder P1 in accordance with the present invention appears to be uniformly dispersed and effectively absorbs blood, facilitating comprehensive contact between the chitosan and blood components. This even distribution likely promotes consistent platelet activation and red blood cell adhesion across the clot volume, leading to a more cohesive and structurally integrated clot. Accordingly, composition P1 played a key role in promoting uniform blood absorption and clot integration, which are critical for effective and reliable hemostasis.[000192] These results highlight the benefits of the present chitosan formulations in enhancing the functional performance of known chitosan-based hemostatic agents. It ishypothesized that present invention provides chitosan molecules having different physical, chemical and / or structural characteristics when compared to raw chitosan or commercial product such as Celox™. Without being bound by any theory, the present invention may be providing chitosan particles having a different size and / or different surface characteristics.[000193] Example 6: Testing chitosan systems with a hemostatic booster.[000194] Purpose: This study evaluated chitosan suspensions with and without kaolin as a hemostatic booster.Materials and methods:[000195] The followings were prepared. Unless specified otherwise, these were prepared in accordance with the method(s) of Example 1 . a. Chitosan Suspension Bases: i. S11 : DEF25041501 : Chibio Chitosan 10% I Dermosoft Pentiol ECO 5% (pH 8.8). See previous samples. b. Dried, Ground Suspension: i. P4: BLD25091101 : 70% P1 (DEF25042403) +30% Kaolin (70% effective dry chitosan) ii. P5: DEF25090401 : 7% Chibio™ Chitosan + 5% Kaolin + 5% Dermosoft Pentiol ECO 5% (41 % effective dry chitosan after evaporation of water) c. Benchmark Product: i. Celox™ granules: Proprietary blend of materials that contains chitosan.[000196] Table 6 hereinafter lists the hemostatic agents used in this study.Table 6: Hemostatic particles / granules used in this studyMethodology[000197] The hemostatic agents used in this study were tested in accordance with the methodology of previous examples (e.g. Example 5).[000198] Both fresh suspensions and their dried / ground equivalents were tested and compared with a benchmark product. Their effects on clotting time, clotting rate, and the mechanical properties of the resulting clots were analyzed to identify synergistic effects.[000199] The efficacy of each formulation was first evaluated within its respective batch, relative to the control tested under identical conditions, to ensure precise and internally consistent interpretation.[000200] As a final exercise, all data from previous examples, covering a total of five independent blood batches, were normalized to their respective control values. This normalization enabled cross-stage analysis of clotting initiation time, clotting rate, strength and stiffness under variable biological conditions. Similar to the approach used in clinical trials, this method provides a more realistic representation of product efficacy across samples with inherent physiological variability. The normalized dataset thus serves as a reliable indicator of each formulation’s overall performance and reproducibility under diverse conditions.[000201] Results:[000202] As shown in Figure 16A, the G'-time curves show that all tested hemostatic agents enhanced clot stiffness compared to the untreated control (CTR). Among all samples, P4 produced the most rapid and highest increase in shear storage modulus, reaching ~220 Pa within ~10 minutes, indicating very fast and strong clot formation. S11 also demonstrated robust performance, with faster G' development and higher final modulus compared to Celox™. P5 showed moderate improvements, producing intermediate G' values. In contrast, the control and Celox™ exhibited the lowest and slowest increases, confirming weaker clot formation relative to the optimized formulations.[000203] As shown in Figure 16B, clot initiation time was significantly reduced for all formulations relative to the control. S11 and P4 showed the fastest clot initiation, reflecting rapid onset of coagulation. P5 also improved clot initiation compared to CTR but were slower than the top-performing samples. Celox™ showed only a modest reduction relative to the control and was slower than most of the engineered formulations.[000204] As shown in Figure 16C, clotting rate followed the same trend observed in the G' profiles. P4 exhibited by far the highest clotting rate, exceeding 250 Pa / min, confirming its ability to accelerate clot formation more effectively than all other samples. S11 also demonstrated strong clotting rates, outperforming Celox™ and the control. P5 showed moderate improvement. Celox™ again showed only limited enhancement relative to CTR and was outperformed by nearly all other formulations tested.[000205] Based on the normalized results across all stages and batches (see Figures 17 to 20, the 10% chitosan suspension in accordance to the present invention (e.g., S10, S11 and S12), along with the chitosan-kaolin powder formulation (P4, P5), demonstrated the best overall performance in terms of clot initiation time, clotting rate, and final clot stiffness. These formulations consistently induced faster and stronger clot formation across different blood batches, confirming their superior hemostatic potential and robustness across biological variability.[000206] Referring particularly to Figure 17, across all batches, formulations in accordance to the present invention exhibited shorter initiation times than control. The10% chitosan suspension (S11) and chitosan-kaolin powder (P4) were the most consistent performers, maintaining initiation times between 1.1-3.5 (normalized units) across all blood batches, compared to Celox™ (~10.7) and Curad™ (~1.0 baseline). Although the normalized data suggest a faster apparent initiation time for Celox™, this reflects its tendency to rapidly form a superficial gel layer upon contact with blood, rather than initiating a true fibrin-based clot. This distinction highlights the mechanistic difference between Celox™’s ionic gelation, and the physiological coagulation cascade enhanced by the formulations in accordance to the present invention.[000207] A similar trend was observed in clotting rate (Figure 18), where the chitosan- kaolin powder (P4), 10% chitosan suspensions (S10 and S11) and their dried equivalents (P1 and P2) exhibited the highest normalized values, surpassing Celox. These results indicate faster fibrin propagation and the formation of a denser, more cohesive gel network in the treated samples in accordance to the present invention, reflecting enhanced overall hemostatic performance.[000208] Referring particularly to Figure 19, Celox™ exhibited the lowest clot strength, performing below both the control and all formulations in accordance to the present invention. In contrast, all treated samples in accordance to the present invention demonstrated final clot strength comparable to or exceeding the control, remaining consistently within the same performance range.[000209] Referring particularly to Figure 20, mechanical evaluation revealed that all formulations in accordance to the present invention generated clots with higher normalized stiffness (1 .3-1.9) compared to Celox™ (0.5). The chitosan-kaolin powders (P4, P5) and 10% chitosan suspensions (S11 , S12) showed the greatest improvement confirming the significance of cationic polymer-plasma interactions in clot stabilization.Conclusions[000210] The present examples demonstrate that the chitosan-based formulations in accordance with the present invention significantly enhanced both the speed of clot formation and the mechanical quality of the clots in bovine whole blood, compared to conventional hemostatic agents such as Celox™ and raw chitosan.[000211] Convincing positive results were obtain using various chitosan suspensions, two physical states (e.g., suspension vs. dried powder), different concentrations of chitosan, various sources of chitosan, in presence of different preservatives.[000212] Among the tested formulations, suspensions S11 and S12 containing 10% chitosan consistently demonstrated superior hemostatic performance, as evidenced by significantly shorter clot initiation times and higher clotting rates.[000213] The suspension S13 having a lower chitosan content (5.8%) and lower pH (6.4), which also included xanthan gum and GDL (Glucono Delta-Lactone, a pH regulator / acidifier), still exhibited enhanced clotting properties compared to Celox™ and the control.[000214] Additionally, the S14 suspension comprising chitosan derived from oyster shell also demonstrated better hemostatic efficacy than Celox™ and the control. This indicates that the present invention may provide effective hemostatic products using chitosan from various sources.[000215] Notably, P1 , the dried version derived from a preservative-free aqueous 10% chitosan suspension, retained the functional advantages of its aqueous suspension precursor. Indeed P1 achieved homogenous clot integration and outperforming raw chitosan powders in both distribution and efficacy.[000216] The mechanical analysis of the formed clots revealed that clots obtained from suspensions or dried ground powder in accordance with the present invention, exhibited greater stiffness and strength than those formed with Celox™ or raw chitosan. These findings suggest that hemostatic chitosan compositions in accordance with the present invention promote denser, more robust fibrin networks, which is essential for ensuring mechanical stability in dynamic and high-stress environments. This mechanical resilience is particularly relevant in military and emergency settings where wound sites are exposed to movement and mechanical load.[000217] Importantly, the study also evaluated the ability of hemostatic chitosan compositions in accordance with the present invention to initiate clotting in citrated (nonrecalcified) blood, a model for anticoagulated conditions. The positive clotting responseobserved, particularly with the P1 formulation, underscores the potential of these materials to function in calcium-depleted environments, further supporting their utility in trauma care where coagulopathy is a concern. This is especially significant because citrated blood simulates clinical scenarios involving anticoagulated patients or severe hemorrhage, where natural coagulation is impaired due to calcium chelation. The ability of the hemostatic compositions of the invention to trigger clotting independently of the calcium- mediated coagulation cascade presents a substantial advantage, enabling rapid hemostasis when conventional clotting mechanisms are suppressed. Furthermore, the dose-dependent efficacy of P1 , demonstrated across a range from 0.15 g to 0.3 g, confirmed enhanced clotting rates and overall performance, reinforcing its therapeutic potential in high-risk bleeding situations.[000218] The studies further confirmed that higher chitosan concentration (10%) were key to rapid and robust hemostasis. Formulations in accordance to the present invention incorporating chitosan performed strongly. The chitosan-kaolin composite (P4) demonstrated the fastest and strongest response, with nearly ten-fold higher clotting rate than Celox™ and the greatest shear modulus (=220 Pa), indicating both rapid fibrin formation and enhanced mechanical resilience. Mechanical testing revealed that clots formed with the formulations in accordance to the present invention exhibited higher stiffness and strength, signifying a denser fibrin network capable of withstanding physiological stress.[000219] Collectively, these results confirm that biopolymer formulations in accordance to the present invention, particularly chitosan-based and chitosan-kaolin systems, combine rapid clot initiation, mechanical robustness, and functionality in anticoagulated blood, making them strong candidates for emergency, surgical, and field-deployable hemostatic applications.[000220] The present invention encompasses all novel compounds, compositions, processes, methods, devices, systems and uses substantially as hereinbefore described with particular references to the Examples and the Figures.[000221] Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein, and these concepts may have applicability in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.[000222] The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes one or more of such compounds and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.[000223] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentrations, properties, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors resulting from variations in experiments, testing measurements, statistical analyses, and such.[000224] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.

Claims

CLAIMS:1 . A hemostatic composition comprising: an aqueous chitosan suspension in the form of a paste, an ointment, a cream, a lotion, a gel, a milk, or a foam; wherein said hemostatic composition displays blood clotting properties.

2. The hemostatic composition of claim 1 , wherein said aqueous chitosan suspension comprises chitosan molecules that have been mechanically processed.

3. The hemostatic composition of claim 1 or 2, wherein said aqueous chitosan suspension comprises chitosan molecules possessing a micro or a nano fibrous structure.

4. The hemostatic composition of any one of claims 1 to 3, wherein said aqueous chitosan suspension comprises chitosan fibers having a width of about 1 nm to about 10 pm.

5. The hemostatic composition of any one of claims 1 to 4, wherein said aqueous chitosan suspension comprises chitosan fibers having a length of about 1 nm to about 200 pm.

6. The hemostatic composition of any one of claims 1 to 5, wherein said aqueous chitosan suspension is stable and homogeneous.

7. The hemostatic composition of any one of claims 1 to 6, wherein said hemostatic composition is formulated for a specific application to at least one of a wound, a hemorrhage, a damaged tissue and a bleeding tissue.

8. The hemostatic composition of any one of claims 1 to 7, wherein said hemostatic composition further comprises one or more preservative.

9. The hemostatic composition of any one of claims 1 to 8, further comprising a blood clotting agent selected from the group consisting of fibrin sealants, thrombin, oxidized cellulose, collagen, kaolin and tranexamic acid.

10. The hemostatic composition of any one of claims 1 to 9, further comprising a biopolymer selected from the group consisting of chitin, alginic acid, cellulose, and silk.11 . The hemostatic composition of any one of claims 1 to 10, wherein said hemostatic composition provides for formation of stronger blood clots compared to Celox™ granules and Curad™ gauze.

12. The hemostatic composition of any one of claims 1 to 11 , wherein said hemostatic composition promotes plasma coagulation via both intrinsic and extrinsic pathways of coagulation.

13. The hemostatic composition of any one of claims 1 to 12, wherein said composition provides for at least one of the following benefits when applied on a wound and / or bleeding site: reduces blood clot initiation time; increases speed or rate of blood coagulation; increase blood clot stiffness; accelerates coagulation in presence of anticoagulant(s); promotes aggregation of red blood cells; forms a gel-like structure with red blood cells; enhances platelet adhesion and / or activation; promotes plasma coagulation via both extrinsic tissue factor and intrinsic tissue factor; triggers the blood clotting cascade; clots citrated blood; initiate clotting in calcium-depleted environments; and forms clots exhibiting greater stiffness and strength than clots formed with Celox™ or raw chitosan.

14. A hemostatic powder composition, said composition comprising dried ground chitosan particles possessing a micro or a nano fibrous structure, wherein said hemostatic powder composition displays blood clotting properties.

15. The hemostatic powder composition of claim 14, wherein said aqueous chitosan suspension comprises chitosan fibers having a width of about 1 nm to about 10 pm.

16. The hemostatic powder composition of claim 14 or 15, wherein said aqueous chitosan suspension comprises chitosan fibers having a length of about 1 nm to about 200 pm.

17. The hemostatic powder composition of any one of claims 14 to 18, wherein said aqueous chitosan suspension comprises chitosan molecules that have been mechanically processed.

18. A hemostatic powder composition, said composition comprising dried ground chitosan particles possessing a specific surface area (SSA) of at least 2 m2 / g, wherein said hemostatic powder composition displays blood clotting properties.

19. A hemostatic powder composition, said composition comprising dried ground chitosan particles possessing a pore volume of at least 0.016 cm3 / g, wherein said hemostatic powder composition displays blood clotting properties.

20. The hemostatic powder composition of claim 18 or 19, further comprising a pore size of at least 300 A.21 . A material for treating or preventing bleeding, said material comprising a substrate impregnated or coated (i) with the hemostatic composition as defined in any one of claims 1 to 13, and / or (ii) with the hemostatic powder composition as defined in any one of claims 14 to 17.

22. The material of claim 21 , wherein said material is selected from the group consisting of a patch, a bandage, a wound dressing, a gauze, and a sponge.

23. A method of treating or preventing bleeding at a tissue site comprising: applying to the tissue site a hemostatic composition as defined in any one of claims 1 to 13, a hemostatic powder composition as defined in any one of claims 14 to 20, and / or a material as defined in claim 21 or 22.

24. The method of claim 23, wherein said tissue site is selected from the group consisting of a wound, a hemorrhage, a damaged tissue and a bleeding tissue.

25. The method of claim 23 or 24, wherein said applying promote blood clotting at the tissue site.

26. A method of making a hemostatic composition, the method comprising: mechanically processing chitosan in presence of an aqueous solvent to obtain an aqueous chitosan suspension; wherein said mechanical processing is configured to produce said aqueous chitosan suspension in the form of a paste, an ointment, a cream, a lotion, a gel, a milk or a foam that:(i) displays blood clotting properties; and(ii) is compatible for application on a wound, a hemorrhage, a damaged tissue and / or a bleeding tissue.

27. A method of making a hemostatic composition, the method comprising: mechanically processing chitosan in presence of an aqueous solvent to obtain an aqueous chitosan suspension; wherein said mechanical processing is configured to produce chitosan particles possessing a micro or a nano fibrous structure, and wherein said chitosan particles possessing a micro or a nano fibrous structure display enhanced hemostatic properties compared to a raw chitosan powder.

28. The method of claim 26 or 27, wherein the aqueous chitosan suspension obtained provides for at least one of the following benefits when applied on a wound and / or bleeding site: reduces blood clot initiation time; increases speed or rate of blood coagulation; increase blood clot stiffness; accelerates coagulation in presence of anticoagulant(s); promotes aggregation of red blood cells; forms a gel-like structure with red blood cells;enhances platelet adhesion and / or activation; promotes plasma coagulation via both extrinsic tissue factor and intrinsic tissue factor; triggers the blood clotting cascade; clots citrated blood; initiate clotting in calcium-depleted environments; and forms clots exhibiting greater stiffness and strength that clots formed with Celox™ or raw chitosan.

29. A wound dressing suitable for direct application to a wound, said wound dressing comprising a substrate layer impregnated or having layered thereon (i) a hemostatic composition as defined in any one of claims 1 to 13, and / or (ii) a hemostatic powder composition as defined in any one of claims 14 to 20.

30. A kit for treating a bleeding wound of a human or an animal, the kit comprising: at least one of: (i) a container comprising a hemostatic composition as defined in any one of claims 1 to 13; (ii) a container comprising a hemostatic powder composition as defined in any one of claims 14 to 20; and (iii) a material as defined in claim 21 or 22; and at least one of a patch, a bandage, a wound dressing, a gauze, a sponge, one or more blood clotting agent including, a disinfectant, an antimicrobial, an antiseptic, and a pamphlet with instructions.31 . Use of at least one of (i) a hemostatic composition as defined in any one of claims 1 to 13, (ii) a hemostatic powder composition as defined in any one of claims 14 to 20; and (iii) a material as defined in claim 21 or 22, for treating or preventing bleeding.

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