Cryogels and methods of use thereof

Abstract

Cryogels are provided as well as methods of use thereof and methods of making.

Description

[0001] CRYOGELS AND METHODS OF USE THEREOF

[0002] Jingwei Xie Syed Muntazir Andrabi Mark Carlson

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 553,396, filed February 14, 2024. The foregoing application is incorporated by reference herein.

[0004] This invention was made with government support under Grant Nos. W81XWH- 20-1-0207 and W81XWH-20- 1-0208 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] This application relates to the fields of cryogels. More specifically, this invention provides cryogels, methods of synthesizing, and methods of use thereof.

[0007] BACKGROUND OF THE INVENTION

[0008] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0009] Traumatic injuries leading to massive hemorrhage emerge as the foremost avoidable cause of fatalities within military combat, with the potential to prevent up to 90% of these deaths (Jamal, et al. (2021) Ann. Transl. Med., 9(14): 1192). A range of injuries contributes to this substantial hemorrhage, with the majority of the wound cases stemming from truncal (67.3 %) followed by junctional (19.2%) and extremity (13.5%) (Jamal, et al. (2021) Ann. Transl. Med., 9(14): 1192; Yang, et al. (2019) Am. J. Emerg. Med., 37(5):973-974; Gao, et al. (2020) Sci. Adv., 6(31):eaba0588). Within the civilian context, traumatic hemorrhage represents the second most prominent cause of mortality among trauma patients, as studies report rates ranging from 26% to 40%. In addition, the complete treatment of such injuries predominantly involves surgical procedures and requires transporting the patients to a point of care within a narrow time window of up to 3 hours (Jamal, et al. (2021) Ann. Transl. Med., 9(14): 1192; Yang, et al. (2023) Adv. Sci., 10(16):e2207347). Hence, this underscores the critical significance of having highly effective hemostatic materials to promptly control bleeding, providing a vital bridge until patients can access hospital care. Unfortunately, there remains a significant gap in the availability of satisfactory hemostatic materials for addressing marginally / non- compressible hemorrhages.

[0010] A variety of hemostatic materials have been developed with distinct designs and functionality to stop massive bleeding and several were clinically approved, such as Celox™, Axiostat®, QuikClot®, HemCon®, etc. (Andrabi, et al. (2023) Biomater. Adv., 150:213424; Fang, et al. (2020) Chem. Eng. J., 388: 124169; Liu, et al. (2019) Biomaterials 205:23-37). However, in the truncal or groin regions, high blood pressure and extensive bleeding pose a major challenge for their failure (Liu, et al. (2019) Biomaterials 205:23-37). XStat®, an FDA-approved product consisting of compressed cellulose-based sponges, functions as a hemostatic adjunct with a distinct focus on controlling bleeding emanating from junctional wounds in the groin or axilla. Although XStat® shows some efficacy, several limitations, including lack of clotting ability, prolonged recovery time, risk of damaging surrounding tissues due to its high mechanical stress, high cost, and non-biodegradability, are still of grave concern (Gao, et al. (2020) Sci. Adv., 6(31):eaba0588; Beaman, et al. (2022) Acta Biomater., 137: 112- 123; Li, et al. (2023) Chem. Eng. J., 469: 143758). Another FDA product for controlling bleeding in penetrating wounds is QuikClot® Combat Gauze (CG), but the hemostasis is ineffective with a long application time (Beaman, et al. (2022) Acta Biomater., 137: 112- 123; Hickman, et al. (2018) Adv. Mater., 30(4): 1700859). In addition, several types of materials with shape memory properties have been studied for controlling massive bleeding in deep irregular wounds, which were fabricated using multiple strategies such as 3D printing, freeze drying, gas foaming, and electrospinning in the form of hydrogels, cryogels, and nanofiber peanuts (Andrabi, et al. (2023) Biomater. Adv., 150:213424; Zhao, et al. (2018) Nat. Commun., 9(1):2784; Ai, et al. (2018) J. Mater. Chem. B 6(37):5940-5948; Du, et al. (2021) Nat. Commun., 12(1):4733; Chen, et al. (2018) Biomaterials 179:46-59). However, these hemostats are associated with several potential drawbacks, such as unsuitable mechanical properties, lack of vital blood coagulation factors, limited testing primarily in small animal or non-critical hemorrhage models, and non-optimal delivery methods for the intended purpose (Yang, et al. (2023) Adv. Sci., 10(16):e2207347; Chan, et al. (2015) Sci. Transl. Med., 7(277):277ra29-277ra29).

[0011] Zhao et al. developed injectable cryogels to cease fatal noncompressible and coagulopathic bleeding (Zhao, et al. (2021) Chem. Eng. J., 403: 126329). The degradable cryogels presented promising results when evaluated for hemostatic efficiency in rabbit liver and subclavian swine models, apart from promoting wound healing compared to the control groups. In another study, Yoo et al. prepared cryogels from quaternized chitosan and mesoporous bioactive glass to cope with massive bleeding (Yao, et al. (2022) Chem. Eng. J., 428: 131005). The hemostatic potential of these cryogels was demonstrated in a mouse liver model. Similarly, considering other ideal characteristics of cryogels, studies have substantiated the effectiveness of cryogels in various animal models of bleeding, but excluding majorly those related to lethal junctional hemorrhage (Andrabi, et al. (2023) Biomater. Adv., 150:213424; Li, et al. (2023) Chem. Eng. J., 469: 143758; Du, et al. (2021) Nat. Commun., 12(1):4733; Qi, et al. (2023) Adv. Funct. Mater., 33(16):2212231 ; Zhu, et al. (2023) Adv. Healthcare Mater., 12(5):e2202122). However, these cryogels typically exhibit low mechanical strength and various strategies, including the use of multiple polymers / components, composites, and cotton, have been explored to enhance mechanical strength. Nevertheless, these endeavors frequently result in heightened complexity, prolonged fabrication time, and increased costs (Fang, et al. (2020) Chem. Eng. J., 388: 124169; Zhao, et al. (2018) Nat. Commun., 9(1):2784).

[0012] The shortcomings of these cryogels show that improved cryogels are needed.

[0013] SUMMARY OF THE INVENTION

[0014] In accordance with the instant invention, cryogels are provided. The cryogels of the instant invention demonstrate superior absorptive properties and an ability to achieve rapid hemostasis. In certain embodiments, the cryogel comprises a polymer, particularly a hydrophilic polymer. In certain embodiments, the cryogel comprises polyacrylamide. In certain embodiments, the average diameter of the pores of the cryogel is about 10 pm to about 100 pm. The cryogels of the instant invention may comprise one or more therapeutic agents. In certain embodiments, the therapeutic agents are loaded or embedded within the cryogel. In certain embodiments, the therapeutic agent is a hemostatic or clotting agent, such as, without limitation thrombin, fibrinogen, tranexamic acid, Factors II, V, VIII, and IX, or other like agents. In certain embodiments, the cryogel comprises an antimicrobial such as antibiotic, an antimicrobial peptide, or metal ions (e.g., Ag+, Ga3+, Cu2+, Zn2+). Compositions comprising the cryogel of and a pharmaceutically acceptable carrier are also encompassed by the present invention.

[0015] In accordance with another aspect of the instant invention, methods of synthesizing a cryogel are provided. In certain embodiments, the method comprises preparing a solution (e.g., a precursor solution) comprising a monomer and a crosslinker and maintaining the solution at a temperature below freezing (e.g., at about -14°C or lower) to allow for cryo-polymerization. In certain embodiments, the method comprises warming the solution to above freezing temperatures (e.g., room temperature) after maintenance at sub-freezing temperatures. In certain embodiments, the concentration of the monomer in the solution is about 1% to about 5%. In certain embodiments, the monomer is acrylamide. In certain embodiments, the crosslinker is N’-N’-methylene- bis-acrylamide. In certain embodiments, the solution further comprises a catalyst and / or initiator. In certain embodiments, the solution further comprises a therapeutic agent. In certain embodiments, the therapeutic agent is a hemostatic agent or clotting agent, such as, without limitation: thrombin, fibrinogen, tranexamic acid, Factors II, V, VIII, and IX or other like agents. In certain embodiments, the solution comprises an antimicrobial such as antibiotic, an antimicrobial peptide, or metal ions (e.g., Ag+, Ga3+, Cu2+, Zn2+).

[0016] In accordance with another aspect of the instant invention, methods of treating or inhibiting a hemorrhage and / or reducing blood loss in a subject are provided. Methods of absorbing a fluid (e.g., blood, exudate, draining fluid, etc.) in a subject are provided. In certain embodiments, the methods comprise administering a cryogel of the instant invention to the subject (e.g., at the desired site). In certain embodiments, the cryogel are administered by direct injection.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1A-1C provide schematic illustrations of synthesizing cryogels, their shape recovery property and application in lethal junctional hemorrhage. Fig. 1 A: Schematic of the synthetic procedure of pAAm-based cryogels. Fig. IB: Schematic of the shape recovery property of cryogels. Fig. 1C: Schematic of the application of injectable and expandable cryogels for managing marginally compressible junctional hemorrhage in swine. Fig. ID provides the shape and surface morphology of the pAAm cryogels. Digital images show the irregular shapes of pAAm-1 and pAAm-2, while a regular cylindrical shape is retained in pAAm-3 and pAAm-4. SEM images show that large pores are visible in pAAm-1 and pAAm-2, compared to pAAm-3 and pAAm-4. Figures 1E-1G show the effect of acrylamide concentration on pore size. Fig. IE: Micro-CT image showing the morphological difference of pAAm cryogels. Fig. IF: Porosities of pAAm cryogels. Fig. 1G: Pore size of pAAm cryogels. Pore size in pAAm-1 and pAAm-2 was larger (~85 pm) than pAAm-3 and pAAm-4 (~50 pm). Figures 1H-1K show the water / blood uptake and absorption capacity of pAAm cryogels. Fig. 1H: Water uptake. Fig. II: Water absorption capacity. Fig. 1 J: Blood absorption. Fig. IK: Blood absorption capacity of pAAm cryogels. Figures IL- IN show the mechanical property of pAAm cryogels. Fig. IL: Digital images of the developed pAAm cryogels in wet form. Figs. IM and IN: Cyclic compression of pAAm-3 and pAAm-4 cryogels, respectively.

[0019] Figures 2A-2C provide the morphology and structure of pAAm-based cryogels (thrombin-loaded pAAm cryogels (AT cryogels) or non-loaded pAAm-3 cryogel (AAm)) and XStat®. Fig. 2A: Digital images, SEM images, and micro-CT images of pAAm-based cryogels (thrombin-loaded pAAm cryogels (AT cryogels) or non-loaded pAAm-3 cryogel (AAm)) and XStat®. Figs. 2B and 2C: The porosity and pore size of pAAm-based cryogels (thrombin-loaded pAAm cryogels (AT cryogels) or non-loaded pAAm-3 cryogel (AAm)) and XStat®.

[0020] Figures 3 A-3F show the absorption and swelling properties of the AT cryogels. Figs. 3A-3D: The absorption rate and capacity of the AAm (pAAm-3 not loaded with thrombin), AT cryogels and XStat® in water / blood. The cryogels showed a significantly higher water / blood absorption rate compared to XStat®. Figs. 3E and 3F: The swelling ratio of the AT cryogels in water and blood, respectively. Figs. 3G-3L show the mechanical and cyclic compression testing of the AT cryogels. Figs. 3G and 3H: The mechanical strength of the cryogels AAm, AT-1, AT -2, and AT-3 in a dry state. Figs. 31 - 3L: The cyclic compressive strength of the cryogels AAm, AT-1, AT -2, and AT-3 in a wet state, respectively. All the samples were compressed up to 80% in both tests. Cryogels (diameter = 8 mm; length = 2 cm). Hold time in compressed state = 5 seconds.

[0021] Figures 4A-4D show the shape fixing and recovery properties of pAAm-based cryogels. Fig. 4A: The digital images of the pAAm-based cryogels in the original, compressed, and shape recovery states triggered by water / blood. Fig. 4B: Digital images and scanning electron microscopy (SEM) images show the morphological properties of the cryogels in the original, compressed, and shape recovery states. Fig. 4C: The schematic representation shows the effect of applied force and shape recovery of the cryogel. Fig. 4D: Digital images of the cryogels before / after compression and subsequent complete shape recovery of all the samples. Figures 4E-4H show the shape recovery property of the developed cryogels. Figs. 4E-4F and 4G-4H show the timedependent recovery and recovery ratio of the cryogels and XStat® in water and blood, respectively. The results indicate a shorter recovery time attained by the developed cryogels in both water and blood compared to commercially available XStat®. Figures 5A-5E show the packaging of the developed cryogels. Fig. 5A: AT shape memory cryogels in expanded form. Fig. 5B: Cryogels were compressed and packed in a syringe. Figs. 5C-5E: Injecting compressed cryogels into the water at 0 second, 1 second, and 4 seconds, respectively. Figures 5F-5I show the hemocompatibility and cytotoxicity of the AT cryogels. Fig. 5F: Thrombin release from the cryogels over a period of 3 days. Fig. 5G: Hemolysis assay of the AAm and AT cryogels and XStat®. Figs. 5H and 51: Cell viability of HDFs and HUVECs when incubated with the cryogels, respectively.

[0022] Figures 6A-6I show the blood clotting and material-cell interaction of pAAm- based cryogels and XStat®. Figs. 6A and 6B: Blood clotting time and blood clotting index of pAAm-based cryogels and XStat®. Fig. 6C: Digital image shows the blood clotting ability of acrylamide-based cryogels and XStat®. Figs. 6D-6G: Digital images of the AT -2 cryogel and XStat® before and after blood absorption. Fig. 6H: SEM images show the enhanced adhesion of RBCs and platelets on thrombin-loaded cryogels (AT-1, AT-2 and AT-3) in contrast to groups AAm cryogel and XStat®. Fig. 61: Schematic illustration of blood clotting formation in the AT cryogels with interconnected pores.

[0023] Figures 7A-7D show the hemostatic performance of cryogels in managing lethal junctional hemorrhage in swine. Fig. 7A: Schematic illustrating the procedure for developing a swine lethal groin injury model and treatment with injectable and expandable cryogels. Fig. 7B: Digital images show hemostasis of the bleeding site and after treatment with the AAm and AT-2 cryogels, XStat®, and combat gauze (CG), while no hemostasis was observed in the control group (Ctr). Fig. 7C: The time taken to apply the treatment at the injury site. Fig. 7D: Total blood loss after 30 seconds of postinjury and after applying the treatment. Figure 7E shows the XStat® expansion posttreatment. The digital images of XStat® in the wound cavity and subsequent removal at the terminal point are provided (top images). The opened pouch displayed a very low expansion of XStat® pellets (bottom left) and their respective original thickness (white pellets) (bottom right).

[0024] Figures 8A-8D show the in vivo efficacy of the cryogels on survivability and rebleeding time. Fig. 8A: Post-treatment survival time for all the treatment groups. Fig. 8B: Survival rate for all the treatment groups. Fig. 8C: Rebleeding time for all the treatment groups. Fig. 8D: Schematic illustrating the treatment and hemostatic mechanism of the AT-2 cryogels for efficient hemostasis. DETAILED DESCRIPTION OF THE INVENTION

[0025] The indispensable role of interconnected pores for hemostatic application has received considerable attention (Zhao, et al. (2021) Chem. Eng. J., 403: 126329). In this context, cryogels possess an inherent property of forming interconnected porous structures that impart cryogels with characteristics of high blood absorption, mechanical flexibility, shape memory and injectable properties. Additionally, cryogels can be fabricated from a wide range of highly adaptable polymeric precursors, making them an ideal platform for integrating desired hemostatic agents (Zhao, et al. (2018) Nat. Commun, 9(1):2784; Shiekh, et al. (2021) Eur. Polym. J., 144: 110234).

[0026] It is desirable to fabricate economically feasible, shape memory and injectable cryogels with desired mechanical strength for lethal hemorrhages, especially junctional hemorrhage. pAAm was chosen owing to its highly hydrophilic nature, biocompatibility, and ease in tunability to achieve desired mechanical robustness, apart from its proven role in many biomedical applications, including bio-separation, tissue engineering, and drug delivery (Zhou, et al. (2011) J. Colloid Interface Sci., 353(1): 116- 123; Kang, et al. (2021) J. Mater. Chem. B 9(6): 1503-1520; Plieva, et al. (2005) Soft Matter l(4):303-309; Zheng, et al. (2021) Adv. Funct. Mater. 31 (34):2102599). pAAm- based materials exhibit quick and effective hemostatic applications but are still largely unexplored (Zheng, et al. (2021) Adv. Funct. Mater. 31(34):2102599; Fan, et al. (2020) Mater. Sci. Eng. C 109: 110649; Wang, et al. (2019) Colloids Surf. B 182: 110367). Moreover, incorporating bioactive / clotting factors elevates the procoagulant activity of hemostats. Thrombin, one of the central components of the clotting pathway, is crucial for fibrin generation besides promoting aggregation of platelets, necessary for endorsing rapid clotting not only in normal but also in coagulopathies (Cheng, et al. (2018) Macromol. Mater. Eng., 303(2): 1700395; Sekhon, et al. (2022) Sci. Transl. Med., 14(629):eabb8975; Baylis, et al. (2015) Sci. Adv., 1 (9):el 500379). In clinical settings, thrombin is directly employed as a freshly prepared solution to the targeted site, which, however, is associated with specific challenges such as risk of contamination, short halflife, and use of high doses (Hickman, et al. (2018) Adv. Mater., 30(4): 1700859; Cheng, et al. (2018) Macromol. Mater. Eng., 303(2): 1700395; Mahbub, et al. (2022) J. Biomater. Sci. Polym. Ed., 33(4):499-516; Shukla, et al. (2012) Adv. Mater., 24(4):492-496). Therefore, thrombin incorporation into the relevant material can reduce pre-application time, improve its storage shelf-life, and help overcome other limitations (Cheng, et al. (2018) Macromol. Mater. Eng., 303(2): 1700395; Mahbub, et al. (2022) J. Biomater. Sci. Polym. Ed., 33(4):499-516).

[0027] Herein, injectable and quickly responsive shape-memory pAAm-based cryogels with high blood absorption and excellent clotting abilities for effectively managing lethal junctional hemorrhage in swine were fabricated. Effective therapies are urgently needed to stabilize patients with marginally compressible junctional hemorrhage long enough to get them to the hospital alive. The pAAm cryogels were loaded with thrombin (AT cryogels) to enhance comprehensive hemostatic efficacy. The developed shape memory cryogels exhibited the desired small pore size and interconnected porous architecture apart from good mechanical strength and shorter recovery time in contrast to commercial products XStat® and CG. The cryogels exhibit rapid shape memory properties and prove to be resilient against fatigue. These cryogels also show high water / blood absorption capacity, fast blood clotting effect, and enhanced adhesion of red blood cells and platelets in vitro. The AT cryogels were further evaluated for physiochemical properties in vitro and the pre-clinical testing was carried out in a lethal swine junctional hemorrhage model. Next, the pre-clinical hemostatic efficacy of the developed thrombin-loaded pAAm (AT-2) cryogels and other commercial hemostats was measured in terms of blood loss, rebleeding time, post-treatment survival time, and survival rate. The results indicate that AT-2 cryogels achieve the least blood loss and the highest survival rate (100%) compared to currently employed products such as XStat® and combat gauze. The high hemostatic performance of the cryogels may be attributed to highly interconnected porous architecture with small pore size and the use of thrombin as a pro-coagulant agent. Collectively, injectable and rapidly expandable thrombindecorated polyacrylamide-based cryogels show significant ability as hemostatic material, offering effective management of marginally compressible junctional hemorrhages in prehospital settings.

[0028] In accordance with the instant invention, cryogels are provided. Methods of synthesizing the cryogels and methods of using the cryogels are also encompassed by the instant invention. As described herein, cryogels are macroporous polymeric structures formed from the cryogelation or cryo-polymerization of monomers or polymers in a solvent below freezing temperature. Cryogelation causes part of the solvent to crystallize and the polymer network to form around the formed ice crystals. Thawing of the mixture (e.g., to room temperature) melts the ice crystals, leaving behind the cryogel. As stated herein, cryogels have a highly interconnected porous architecture. In certain embodiments, the average pore size (e.g., diameter) of the cryogel is from about 1 pm to about 200 pm, from about 10 pm to about 100 pm, from about 20 pm to about 80 pm, from about 30 pm to about 70 pm, from about 40 pm to about 65 pm, or from about 45 pm to about 60 pm. In certain embodiments, the average pore size of the cryogel is at least from about 1 pm, at least from about 5 pm, at least from about 10 pm, at least from about 15 pm, at least from about 20 pm, at least from about 25 pm, at least from about 30 pm, at least from about 35 pm, at least from about 40 pm, or at least from about 45 pm. In certain embodiments, the average pore size of the cryogel is less than about 200 pm, less than about 150 pm, less than about 100 pm, less than about 90 pm, less than about 80 pm, less than about 75 pm, less than about 70 pm, less than about 65 pm, or less than about 60 pm.

[0029] The cryogels of the instant invention may comprise any polymer and may comprise more than one polymer. In certain embodiments, the polymer is biocompatible. In certain embodiments, the polymer is biodegradable. In certain embodiments, the polymer is non-biodegradable. The polymer may by hydrophobic, hydrophilic, or amphiphilic. In certain embodiments, the polymer is hydrophilic. The polymer may be, for example, a homopolymer, random copolymer, blended polymer, copolymer, or a block copolymer. Block copolymers are most simply defined as conjugates of at least two different polymer segments or blocks. The polymer may be, for example, linear, star-like, graft, branched, dendrimer based, or hyper-branched (e.g., at least two points of branching). The polymer of the invention may have from about 2 to about 10,000, about 2 to about 1000, about 2 to about 500, about 2 to about 250, or about 2 to about 100 repeating units or monomers. The polymers of the instant invention may comprise capping termini.

[0030] Examples of hydrophobic polymers include, without limitation: poly(N-isopropyl acrylamide), poly(lactic acid) (PLA (or PDLA)), poly(lactide-co-glycolide) (PLG), poly(lactic-co-glycolic acid) (PLGA), polyglycolide or polyglycolic acid (PGA), polycaprolactone (PCL), poly(aspartic acid), polyoxazolines (e.g., butyl, propyl, pentyl, nonyl, or phenyl poly(2-oxazolines)), polyoxypropylene, poly(glutamic acid), polypropylene fumarate) (PPF), poly(trimethylene carbonate), polycyanoacrylate, polyurethane, polyorthoesters (POE), polyanhydride, polyester, polypropylene oxide), poly(caprolactonefumarate), poly(l,2-butylene oxide), polyp-butylene oxide), poly(tetrahydrofurane), ethyl cellulose, polydipyrolle / dicabazole, starch, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polydioxanone (PDO), polyether poly(urethane urea) (PEUU), cellulose acetate, polypropylene (PP), polyethylene terephthalate (PET), nylon (e.g., nylon 6), polycaprolactam, PLA / PCL, poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), PCL / calcium carbonate, and / or poly (styrene).

[0031] Examples of hydrophilic polymers include, without limitation: polyacrylamide, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol) and poly(ethylene oxide) (PEO), poly(hydroxyethyl methacrylate), poly (ethyleneimine), chitosan, collagen, chondroitin sulfate, sodium alginate, gelatin, elastin, hyaluronic acid, silk fibroin, sodium alginate / PEO, silk / PEO, silk fibroin / chitosan, hyaluronic acid / gelatin, collagen / chitosan, chondroitin sulfate / collagen, and chitosan / PEO.

[0032] Amphiphilic copolymers or polymer composites may comprise a hydrophilic polymer (e.g., segment) and a hydrophobic polymer (e.g., segment) from those listed above (e.g., gelatin / polyvinyl alcohol (PVA), PCL / collagen, chitosan / PVA, gelatin / elastin / PLGA, PDO / elastin, PHBV / collagen, PLA / hyaluronic acid, PLGA / hyaluronic acid, PCL / hyaluronic acid, PCL / collagen / hyaluronic acid, gelatin / siloxane, PLLA / MWNTs / hyaluronic acid).

[0033] In certain embodiments, the polymer is selected from the group consisting of polymethacrylate, poly vinyl phenol, polyvinylchloride, cellulose, polyvinyl alcohol, polyacrylamide, acrylamide, N', N'- methylene-bis-acrylamide, poly(lactic-co-glycolic) acid (PLGA), collagen, polycaprolactone (PCL), polydioxanone (PDO), polyurethanes, polyvinyl fluoride, polyamide, silk, nylon, polybennzimidazole, polycarbonate, polyacrylonitrile, polyvinyl alcohol, polylactic acid, polyethylene-co-vinyl acetate, polyethylene oxide, polyaniline, polystyrene, polyvinylcarbazole, polyethylene terephthalate, polyacrylic acid-polypyrene methanol, poly(2 -hydroxyethyl methacrylate), polyether imide, polyethylene gricol, polyethylene glycol, poly(ethylene-co-vinyl alcohol), polyacrylnitrile, polyvinyl pyrrolidone, polymetha-phenylene isophthalamide, gelatin, alginate, chitosan, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starch-acrylonitrile co-polymers, bioactive glass, and combinations of two or more materials. Multiple materials may be mixed to form the cryogel. They may be mixed evenly or in various ratios depending on the desired properties of the cryogels. In certain embodiments, the polymer is synthesized by polymerizing a monomer with a crosslinker, optionally in the presence of a catalyst and / or initiator. In certain embodiments, the polymer is hydrophilic. In certain embodiments, the polymer is a polyacrylamide. In certain embodiments, the polyacrylamide is made from acrylamide and a crosslinker. In certain embodiments, the crosslinker is bis-acrylamide or N', N'- methylene-bis-acrylamide. The amount of crosslinker to monomer can be varied. In certain embodiments, the ratio (e.g., w / w) of the monomer to crosslinker is from about 1:2 to about 10: 1, about 1 : 1 to about 5: 1, or about 3: 1.

[0034] The concentration of the monomer or polymer in the solution for cryogelation can be varied. In certain embodiments, the monomer concentration is about 1% to about 10% (e.g., w / v). In certain embodiments, the monomer concentration is about 1% to about 7.5%. In certain embodiments, the monomer concentration is about 1% to about 5%. In certain embodiments, the acrylamide concentration is about 1% to about 10%. In certain embodiments, the acrylamide concentration is about 1% to about 7.5%. In certain embodiments, the acrylamide concentration is about 1% to about 5%. In certain embodiments, the acrylamide concentration is about 3% to about 7.5%. In certain embodiments, the acrylamide concentration is about 3% to about 5%. In certain embodiments, the acrylamide concentration is about 3% to about 4%. In certain embodiments, the acrylamide concentration is about 3.5%.

[0035] The cryogels may comprise (e.g., be loaded with or embedded with) one or more therapeutic agents such as drugs (e.g., small molecules), biologies, and combinations thereof. Generally, the cryogels comprise the therapeutic agents, but the instant invention also encompasses co-administering the therapeutic agent (e.g., before, after, and / or at the same time as the administration of the cryogel). In certain embodiments, the therapeutic agent is useful in applications such as hemostasis, surgical applications and / or for wounds to absorb blood, draining fluid, or exudate. In certain embodiments, the cryogel is conjugated or linked to the therapeutic agent. The therapeutic agent may be directly conjugated or conjugated via a linker (e.g., a cleavable linker). Biologies include, but are not limited to: proteins, peptides, antibodies, antibody fragments, growth factors, DNA, RNA, vitamins, and other known biologic substances that have therapeutic use. Specific examples of biologies include, but are not limited to: hemostatic or clotting agents including but not limited to thrombin, fibrinogen, prothrombin, tissue thromboplastin, ionized calcium, proaccelerin, and coagulation factors (e.g., Factors VII, IX, X, XI, V, XII, II, and / or von Willebrand factor). In certain embodiments, the biologic is an antimicrobial peptide. In certain embodiments, the therapeutic agent is an antibiotic. In certain embodiments, the therapeutic agent is an antimicrobial peptide. The antibiotic and / or antimicrobial peptide may prevent and / or inhibit bacterial growth and / or biofilm formation and / or promote wound healing. In certain embodiments, the antimicrobial peptide is selected from the antimicrobial peptide database (aps.unmc.edu). In certain embodiments, the antimicrobial peptide is a fragment of LL37 or an LL37 analog. In certain embodiments, the antimicrobial peptide comprises W379. In certain embodiments, the antimicrobial peptide has activity against Gram-positive and / or Gramnegative bacteria. In certain embodiments, the antimicrobial peptide has activity against biofilms. In certain embodiments, the antimicrobial is selected from the group consisting of W379, antibiotics, Aurein 1-2, Mellitin, Brevinin, Maculatins, Citropin, Buforin, Cathelicidins, LL37, W379, BMAP-27, 28, 34, Magainins, Cecropin, Protegrins, Bactenecin, Defensins, Tachyplesins, Polyphemusin, PR-39, TRitrpticin, Indolicidn, Crotalcidin, Histatins, Pexiganan, OP145, Omiganan, PAC 113, Iseganan, IMX942, Dalbavancin, Dalvance, PAC-113, P-113, Fuzeon, Baciim, Vancocin, Daptomycin, Telavancin, Colistin, Gramicidin, D2A21, PXL01, Omiganan, NISin, or combinations thereof. Examples of antimicrobial peptides are also disclosed in U.S. Patent No. 7,465,784, U.S. Patent No. 9,580,472, U.S. Patent No. 10,144,767, U.S. Patent Application Publication No. 20090156499, U.S. Patent Application Publication No. 20150259382, U.S. Patent Application Publication No. 20140303069, and PCT / US2019 / 039792, each incorporated by reference herein. In certain embodiments, the antimicrobial peptide has fewer than about 50 amino acids, fewer than about 25 amino acids, fewer than about 20 amino acids, fewer than about 17 amino acids, fewer than about 15 amino acids, fewer than 12 amino acids, fewer than 10 amino acids, or fewer than 9 amino acids. In certain embodiments, the antimicrobial peptide has more than about 6 amino acids, particularly more than about 7 amino acids.

[0036] In certain embodiments, the therapeutic agent is a drug. Drugs include, but are not limited to: anti-inflammatory drugs, antimicrobials (including but not limited to antibacterials, antivirals, and antifungals), pain medications, analgesics, and other drugs useful for treating specific diseases or disorders. Various concentrations of drugs, biologies, and other factors may be used to achieve the desired therapeutic effect.

[0037] In certain embodiments, the cryogel contains a clotting agent. In certain embodiments, the clotting agent is thrombin or fibrinogen. In certain embodiments, the cryogel contains thrombin. Concentrations of thrombin can vary in the cryogel. In certain embodiments, the cryogel comprises between about 1 international unit and about 500 international units of thrombin. In certain embodiments, the cryogel comprises between about 10 international unit and about 100 international units of thrombin. In certain embodiments, the cryogel comprises between about 50 international unit and about 100 international units of thrombin.

[0038] The cryogel of the present invention can be formed and manufactured into any shape, size, and / or thickness. In certain embodiments, a mold may be used (e.g., during the cryogelation process) to shape the cryogel. In certain embodiments, the cryogel is cut, trimmed or shaped into a desired shape after production. In certain embodiments, the nanofiber mat is cut, trimmed, or shaped under cryogenic or frozen conditions (e.g., in liquid nitrogen). The cryogel may have a three dimensional shape such as, without limitation: a capsule, cylinder, tube, cone, rectangle, dome, sphere (spherical), prolate spheroid, elongated sphere, or cube (cuboidal) or other like shapes. Generally, a capsular shape approximates the shape of a capsule (e.g., a geometric shape consisting of a cylinder with hemispherical ends). In certain embodiments, the cryogel is cuboidal, spherical, cylindrical, or capsular in shape. In certain embodiments, the cryogel has a cylindrical shape. The cryogel may be of any size, but desirably of a size to be injectable and / or to fit within a wound (e.g., in a compressed state). In certain embodiments, the cryogels are less than about 5 cm in length, less than about 4 cm in length, less than about 3 cm in length, less than about 2 cm in length, or less than about 1 cm in length. In certain embodiments, the cryogels are more than about 0.25 cm in length, more than about 0.5 cm in length, more than about 0.75 cm in length, or more than about 1 cm in length. In certain embodiments, the cryogels are less than about 3 cm in width (e.g., diameter), less than about 2 cm in width, less than about 1.5 cm in width, or less than about 1 cm in width. In certain embodiments, the cryogels are more than about 0.25 cm in width (e.g., diameter), more than about 0.5 cm in width, more than about 0.75 cm in width, or more than about 1 cm in width.

[0039] In accordance with the instant invention, methods of synthesizing a cryogel are also provided. In certain embodiments, the method comprises adding monomer and crosslinker to a solution and maintaining the solution at a temperature below freezing (e.g., below the freezing temperature for the solvent) such that cryogelation or cryopolymerization occurs. In certain embodiments, the solution is at a temperature below room temperature but above freezing (e.g., between about 1°C and about 10°C, particularly about 4°C) prior to cryogelation. In certain embodiments, the monomer is acrylamide. In certain embodiments, the crosslinker is bis-acrylamide or N’,N’- methylene-bis-acrylamide. In certain embodiments, the solution is maintained at 0°C or lower. In certain embodiments, the solution is maintained at -10°C or lower. In certain embodiments, the solution is maintained at -14°C or lower. The solution may be maintained at cold (e.g., below freezing) temperatures for at least one hour, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least one day, or more.

[0040] In certain embodiments, the solution further comprises a catalyst and / or initiator of the polymerization process. In certain embodiments, the catalyst and / or initiator is ammonium persulfate and / or tetramethylethylenediamine (TEMED).

[0041] In certain embodiments, the solution further comprises adding a therapeutic agent (e.g., to the solution prior to cryogelation). In certain embodiments, the therapeutic agent is a hemostatic agent or clotting agent such as thrombin.

[0042] The methods of the instant invention may further comprise washing and / or rinsing the cryogel. In certain embodiments, the cryogel is washed and / or rinsed in water and / or a desired carrier or buffer (e.g., a pharmaceutically or biologically acceptable carrier). In certain embodiments, the cryogel is washed and / or rinsed at least one time, at least times, at least three times, or more.

[0043] The methods of the instant invention may further comprise freeze-drying the cryogel (e.g., after washing). The methods of the instant invention may further comprise storing the cryogel in cold conditions (e.g., at 4°C, 0°C, -20°C, or -80°C). The methods of the instant invention may further comprise lyophilizing the cryogel. The methods of the instant invention may further comprise placing the cryogel in storage in cold solution (e.g., at 4°C, 0°C, -20°C, or -80°C).

[0044] The methods of the instant invention may further comprise sterilizing the cryogel. For example, the cryogel can be sterilized using various methods (e.g., by treating with ethylene oxide gas, gamma irradiation, or 70% ethanol). In certain embodiments, the cryogel are sterilized by treating with ethylene oxide.

[0045] In accordance with the instant invention, the cryogel may be used in inducing and / or improving / enhancing wound healing. Methods for inducing and / or improving / enhancing wound healing in a subject are also encompassed by the instant invention. Methods of inducing and / or improving / enhancing hemostasis in a subject are also encompassed by the instant invention. Methods of treating or inhibiting hemorrhaging are also encompassed by the instant invention. The cryogel of the present invention can be used for the treatment, inhibition, and / or prevention of any injury (e.g., traumatic injury) or wound and / or reducing, inhibiting, and / or preventing blood loss. In certain embodiments, the wound, injury or hemorrhage is truncal, junctional, or extremity. In certain embodiments, the wound, injury or hemorrhage is truncal or to the torso. In certain embodiments, the wound, injury or hemorrhage is junctional. In certain embodiments, the wound, injury or hemorrhage is to the groin area. In certain embodiments, the wound, injury or hemorrhage would be fatal to the subject (e.g., within an hour) within intervention with the methods of the instant invention. In certain embodiments, the wound, injury or hemorrhage is only marginally compressible. In certain embodiments, the wound, injury or hemorrhage is non-compressible.

[0046] In certain embodiments, the method comprises administering a cryogel as described herein to a subject in need thereof. In certain embodiments, the methods of the instant invention comprise administering or applying a cryogel of the instant invention to the subject (e.g., at or in a wound and / or place of hemorrhage and / or bleeding). In certain embodiments, the cryogel comprises a blood clotting factor (e.g., for accelerating blood clot formation and / or preventing blood loss). For example, the cryogel can be used to induce, improve, or enhance wound healing associated with traumatic injury such as cavitary wounds, cuts, punctures, stab wounds, gunshot wounds, and shrapnel wounds. In certain embodiments, the cryogel can be used in an operative setting (e.g., to treat or inhibit operative hemorrhage). The cryogels may be effective in the management of junctional hemorrhage, noncompressible torso hemorrhage, postpartum hemorrhage, dental bleeding, bleeding during surgery (e.g., non-elective or elective surgery), obstetrical hemorrhage, and in other scenarios involving hemorrhage that is difficult to control. In some embodiments, the cryogels are used in surgical applications and / or for wounds to absorb blood, draining fluid, or exudate.

[0047] The cryogel of the present invention can also be incorporated into delivery devices (e.g., a syringe) that allow for their injection / delivery directly into a desired location (e.g., a wound). The cryogel also may be delivered directly into a cavity (such as the peritoneal cavity) using a needle, syringe, or cannula (e.g., pressurized cannula). Due to their compressibility and flexibility, cryogels of the present invention can be incorporated into delivery devices such as syringes (automated and non-automated) that allow for their injection / delivery directly into a wound (such as a gunshot wound) or internally into a body cavity (such as the peritoneal cavity) to treat bleeding.

[0048] The cryogel of the present invention may be administered by any method. The cryogels of the instant invention may be administered dry and / or compressed. The cryogel described herein may be administered to a subject or a patient as a pharmaceutical composition. The compositions of the instant invention comprise a cryogel and a pharmaceutically acceptable carrier. The term “patient” as used herein refers to human or animal subjects. These cryogel may be employed therapeutically, under the guidance of a physician (e.g., in a hospital setting) and / or may be employed in to stabilize a subject in a prehospital setting.

[0049] The compositions of the instant invention may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). For example, the cryogel may be formulated with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the cryogel in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agents to be administered, its use in the pharmaceutical preparation is contemplated. Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the composition should be compatible with the mode of administration.

[0050] Cryogels of the instant invention may be administered by any method. For example, the cryogels of the instant invention can be administered, without limitation, parenterally, subcutaneously, orally, topically (e.g., wound surface), pulmonarily, rectally, vaginally, intravenously, intraabdominally, peri-arterially, peri-venously, intrathoracically, intraperitoneally, intrathecally, intracerebrally, epidurally, intramuscularly, intradermally, intratumoral, intracarotidly, intravascularly (e.g., embolization of a hemorrhage), or by direct or local injection (e.g., injection into a specific tissue or organ). In certain embodiments, the cryogel is administered by injection (e.g., directly to the desired site). In certain embodiments, the cryogel is implanted and / or inserted (e.g., manually). In certain embodiments, the cryogel is delivered directly to the wound site or site of bleeding.

[0051] In certain embodiments of the instant invention, methods for modulating (increasing) hemostasis; inhibiting blood loss; and / or treating hemorrhage are provided. In certain embodiments, the method comprises administering the cryogel to the wound or site of bleeding. In certain embodiments, the cryogel comprises a blood clotting factor such as thrombin. In certain embodiments, the cryogel is delivered directly into a cavity (such as the peritoneal cavity) (e.g., using a cannula, syringe, etc.).

[0052] The invention includes, but is not limited to, the embodiments of the following numbered paragraphs:

[0053] 1. A cryogel.

[0054] 2. The cryogel of paragraph 1, wherein the cryogel comprises polyacrylamide.

[0055] 3. The cryogel of paragraph 1 or 2, wherein the cryogel is loaded or embedded with a therapeutic agent.

[0056] 4. The cryogel of paragraph 3, wherein the therapeutic agent is a hemostatic or clotting agent.

[0057] 5. The cryogel of paragraph 4, wherein the hemostatic or clotting agent is thrombin.

[0058] 6. The cryogel of any one of paragraphs 1-5, wherein the cryogel further comprises an antimicrobial.

[0059] 7. The cryogel of paragraph 5, wherein the antimicrobial is an antimicrobial peptide.

[0060] 8. The cryogel of any one of paragraph 1-7, wherein the average diameter of the pores of the cryogel is about 10 pm to about 100 pm.

[0061] 9. A composition comprising the cryogel of any one of paragraphs 1-8 and a pharmaceutically acceptable carrier.

[0062] 10. A method of synthesizing a cryogel, the method comprising: a) preparing a solution comprising a monomer and a crosslinker; and b) maintaining the solution of a) at a temperature below freezing to allow for cryo-polymerization.

[0063] 11. The method of paragraph 10, wherein the monomer is acrylamide. 12. The method of paragraph 10 or 11, wherein the crosslinker is N’-N’-methylene- bis-acrylamide.

[0064] 13. The method of any one of paragraphs 10-12, wherein step b) comprises maintaining the solution at -14°C or lower.

[0065] 14. The method of any one of paragraphs 10-13, wherein the concentration of the monomer in the solution is about 1% to about 5%.

[0066] 15. The method of any one of paragraphs 10-14, further comprising adding a therapeutic agent to the solution.

[0067] 16. The method of paragraph 15, wherein the therapeutic agent is a clotting agent.

[0068] 17. The method of paragraph 16, wherein the clotting agent is thrombin.

[0069] 18. A method of treating or inhibiting a hemorrhage and / or reducing blood loss in a subject, the method comprising administering a cryogel of any one of paragraphs 1-8 to the subject.

[0070] 19. The method of paragraph 18, wherein the cryogel is injected directly to the site of hemorrhage and / or blood loss.

[0071] 20. A method of absorbing a fluid in a subject, the method comprising administering a cryogel of any one of paragraphs 1-8 to the subject.

[0072] 21. The method of paragraph 20, wherein the fluid is blood, draining fluid, or exudate.

[0073] Definitions

[0074] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0075] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., TrisHCl, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington.

[0076] As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind.

[0077] “Hydrophobic” designates a preference for apolar environments (e.g., a hydrophobic substance or moiety is more readily dissolved in or wetted by non-polar solvents, such as hydrocarbons, than by water). In certain embodiments, hydrophobic polymers may have aqueous solubility less than about 1% wt. at 37°C. In certain embodiments, polymers that at 1% solution in bi-distilled water have a cloud point below about 37°C, particularly below about 34°C, may be considered hydrophobic.

[0078] As used herein, the term “hydrophilic” means the ability to dissolve in water. In a particular embodiment, polymers that at 1% solution in bi-distilled water have a cloud point above about 37°C, particularly above about 40°C, may be considered hydrophilic.

[0079] As used herein, the term “amphiphilic” means the ability to dissolve in both water and lipids / apolar environments. Typically, an amphiphilic compound comprises a hydrophilic portion and a hydrophobic portion.

[0080] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0081] The term “crosslink” refers to a bond or chain of atoms attached between and linking two different molecules (e.g., polymer chains). The term “crosslinker” refers to a molecule capable of forming a covalent linkage between compounds. A “photocrosslinker” refers to a molecule capable of forming a covalent linkage between compounds after photoinduction (e.g., exposure to electromagnetic radiation in the visible and near-visible range). Crosslinkers are well known in the art (e.g., formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, etc.). The crosslinker may be a bifunctional, trifunctional, or multifunctional crosslinking reagent.

[0082] As used herein, a linker is generally a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches two compounds. The linker can be linked to any synthetically feasible position of the two compounds. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic aliphatic group, an alkyl group, or an optionally substituted aryl group. The linker may be a lower alkyl or aliphatic. The linker may also be a polypeptide (e.g., from about 1 to about 10 amino acids, particularly about 1 to about 5). The linker may be non-degradable and may be a covalent bond or any other chemical structure which cannot be substantially cleaved or cleaved at all under physiological environments or conditions.

[0083] The term “antimicrobials” as used herein indicates a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, viruses, or protozoans.

[0084] As used herein, the term “antiviral” refers to a substance that destroys a virus and / or suppresses replication (reproduction) of the virus. For example, an antiviral may inhibit and or prevent: production of viral particles, maturation of viral particles, viral attachment, viral uptake into cells, viral assembly, viral release / budding, viral integration, etc.

[0085] As used herein, the term “antibiotic” refers to antibacterial agents for use in mammalian, particularly human, therapy. Antibiotics include, without limitation, betalactams (e.g., penicillin, ampicillin, oxacillin, cioxacillin, methicillin, and cephalosporin), carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides (e.g., gentamycin, tobramycin), glycopeptides (e.g., vancomycin), quinolones (e.g., ciprofloxacin), moenomycin, tetracyclines, macrolides (e.g., erythromycin), fluoroquinolones, oxazolidinones (e.g., linezolid), lipopetides (e.g., daptomycin), aminocoumarin (e.g., novobiocin), co-trimoxazole (e.g., trimethoprim and sulfamethoxazole), lincosamides (e.g., clindamycin and lincomycin), polypeptides (e.g., colistin), and derivatives thereof. As used herein, the term “analgesic” refers to an agent that lessens, alleviates, reduces, relieves, or extinguishes pain in an area of a subject's body (i.e., an analgesic has the ability to reduce or eliminate pain and / or the perception of pain).

[0086] As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 2,000). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids.

[0087] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0088] As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition.

[0089] The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0090] The following example illustrates certain embodiments of the invention. It is not intended to limit the invention in any way.

[0091] EXAMPLE

[0092] Materials and Methods

[0093] Materials

[0094] Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were procured from Gibco (Thermo Fisher Scientific Inc., Waltham, MA). Human whole blood was purchased from Research Blood Components, LLC (Boston, MA). Thrombin (human source) was purchased from Sigma-Aldrich (St. Louis, MO). Thrombin ELISA kit was purchased from Abeam (Cambridge, MA). All other chemicals were acquired from Sigma-Aldrich (St. Louis, MO).

[0095] Synthesis of polyacrylamide and thrombin-loaded cryogels

[0096] The pAAm cryogels were prepared to free radial polymerization involving acrylamide (AA) as monomers units and N', N'- methylene-bisacrylamide (MBAAm) as crosslinker. A series of pAAm cryogels named pAAm-1, pAAm-2, pAAm-3 and pAAm-4 were prepared from different acrylamide concentrations, 1.5 %, 2.5 %, 3.5 % and 4.5 %, respectively. To prepare pAAm-1 cryogels, 150 mg of AA was added to 8 ml of degassed distilled water and dissolved completely using a magnetic stirrer. After complete dissolution, 50 mg of MBBAm in the ratio of 3: 1 (AA: MBAAm) was added to this solution and stirred further. Next, 10 mg of ammonium persulfate (APS) and 100 pl of tetramethylethylenediamine (TEMED) were separately dissolved in 1 ml of degassed distilled water. All the solutions were allowed to precool at 4°C for 30 minutes. The precooled AA and MBBAm solution was first added with APS with quick mixing, followed by the addition of TEMED. The solution was immediately mixed, poured into the desired molds, and transferred to a cryostat, maintained at -14°C for 16 hours.

[0097] Before pouring the solution into the molds, plastic syringes (2.5 ml), in this case, were completely sealed with parafilm at the bottom nozzle to avoid any leakage into or out of the mold. The cryogels were later freeze-dried for future use. Subsequently, all other pAAm cryogels, i.e., pAAm-2, pAAm-3, and pAAm-4, were fabricated by following the same procedure.

[0098] Similarly, for synthesizing the thrombin-loaded cryogels, 350 mg of AA (optimal concentration) was dissolved in degassed distilled water, followed by adding MBAAm in the same ratio. The solution was first precooled and added with varying concentrations of thrombin (T-l: 10 IU; T-2: 50 IU; T-3: 100 IU). The precursor solution was mixed with precooled APS and then TEMED solutions sequentially, and the rest of the steps were similar to those described above. The prepared AT cryogels (AT-1, AT -2, and AT- 3) were washed three times to remove loosely bound polymer / thrombin and dried and stored at -80°C for future use. The cryogel samples were evaluated to determine the presence of any residual acrylamide using liquid chromatography-mass spectrometry.

[0099] Determination of acrylamide by LC-MS (Liquid Chromatography- Mass spectrometry) An aliquot of the solid sample was extracted using 500 pL of 100 % acetonitrile.

[0100] The samples were centrifuged at 16,000 g, and the supernatant was collected into a new tube. The sample was dried down using a speed-vac and then resuspended in 500 pL of water. An aliquot of the liquid sample was diluted 200 times with water before transferring the sample from the solid and liquid samples into the HPLC vial. The samples were run using an LC-MS MRM (Multiple Reaction Monitoring) targeted assay. Briefly, LC separation is done on a Synergi™ 2.5 u Hydro-RP (100 x 2.0 mm, Phenomenex) flowing at 0.3 mL / minute. The gradient of the mobile phases A (0.1% formic acid) and B (0.1 % formic acid in 100 % acetonitrile). The Shimadzu LC system was interfaced with a Sciex QTRAP 6500+ mass spectrometer equipped with a Turbo Ion Spray (TIS) electrospray ion source. The instrument was set up to acquire in positive ion mode. Analyst software (version 1.6.3) was used to control sample acquisition and data analysis. The acrylamide was detected using MRM transition (72-55) optimized using a standard. For quantification, an external standard curve was prepared using a dilution series of known acrylamide concentrations.

[0101] Characterization of morphology, microstructure and porosity

[0102] The developed pAAm and AT cryogels were assessed for morphology, pore size and porosity using SEM and Micro-CT analysis. The samples of pAAm and AT cryogels and XStat® were assessed for surface morphology and macro / micro-structure using SEM (FEI Quanta 200, Hillsboro, OR) and Micro-CT analysis (Bruker SkyScan 1276, CMOS Edition, Kartuizersweg, Kontich, Belgium). The porosity was characterized by Micro-CT at a resolution of 4 pm followed by a 3D analysis of the reconstructed scaffolds using CTAn (Bruker). For pore size determination, SEM images of the desired samples were assessed to measure the size of each pore in multiple regions of interest (RO I) by using Image-J software. The ROI in SEM images were selected randomly, and the final average pore size was calculated.

[0103] Water / blood absorbability

[0104] The fluid absorption rate and swelling ratio of the developed cryogels were evaluated by immersing the samples in water and anti coagulated blood. Analysis of the fluid absorption and swelling ratio of AAm and AT cryogels and XStat® were analyzed by submerging a sample of each group into distilled water and anticoagulated human blood at 37°C for set time points. The samples used were cylindrical with height dimensions (1.5 cm) x diameter (8 mm). Before submergence, the dry weight (Wdry) of the lyophilized samples was recorded. The wet weight (Wwet) of the samples was assessed over specified intervals of 2.5 seconds and this process continued until equilibrium was reached (Weq). Finally, the water / blood absorption rate (W) was estimated using the formula:

[0105] W (%) = (Wwet-Wdry)AVeqx 100 Moreover, the water / blood absorption capacity (Wcap) of the sample having the same volume (V) was also evaluated according to the following equation: Wcap (g cm'3) = (Wwet - Wdry) / V

[0106] For the swelling ratio (SR) study, samples from all groups, pAAm, AT cryogels and XStat®, were placed separately in a closed distilled water system and anti coagulated blood for 5 minutes. After the pre-determined time, the wet weight of the samples was measured to determine their swollen weight (Wwet). The equation for quantification of the swelling ratio is given below:

[0107] %SR=(Wwet-Wdry) / Wdryx 100

[0108] Mechanical characterization

[0109] The mechanical strength of the cryogels was determined in both dry and wet states by cyclic compression testing. The mechanical study was conducted on both dry and wet samples of AT cryogels using CellScale at room temperature. The mechanical performance was determined in terms of compression testing for dry samples and cyclic compression testing for wet samples. All the cryogel samples (pAAm or AAm, AT-1, AT-2, and AT-3) having cylindrical shapes were finely cut 15 mm long (8 mm in diameter) using a digital vernier caliper. Using the load cell of 100 N and displacement of 1 mm / min, 80% compressive strain was applied to the dry samples. Finally, from the stress-strain curve, maximum compressive stress was determined. For the cyclic compressive strain test, the cryogel samples were subjected to wet form by submerging in distilled water for 5 minutes before the test started. A compressive strain of 80 % was employed on the wet samples, which were allowed to stay in a compressive state for 5 seconds before releasing the strain. These parameters were maintained constant for 3 repetitive cycles.

[0110] Shape memory property and injectability

[0111] The shape-memory capability of the AAm, AT-1, AT-2, and AT-3 cryogels was examined (Zhao, et al. (2021) Chem. Eng. J., 403: 126329). The AAm and AT cryogels were prepared in cylindrical form and were initially cut to 1 cm in length. The samples were submerged in water and squeezed to drain water to attain a shape fixed state. Next, the samples in the fixed form were exposed to water / blood to measure recovery time and shape recovery ratio. Further, compression testing was performed to accomplish the shape-fixed state of AAm and AT cryogels. The testing was conducted by applying an 80% compression strain to the cryogels for 5 minutes and then adding water while still under the same strain. Compression force was gradually released and the ability of the cryogels to revert to a normal state was observed and photographed. In addition, the change in surface morphology in original form, shape fixed state and recovery state was analyzed by SEM imaging.

[0112] The cryogel samples were prepared in cylindrical shapes (8 mm diameter) to validate their injectable property. Once produced, the water from the samples was impelled out to establish a shape-fixed form. Subsequently, the shape-fixed cryogels were packed into an applicator with a 20 mm diameter and injected into an enclosed water system. The ability of the cryogels to absorb the fluid and recover to their original form was recorded and photographed.

[0113] Release of thrombin from AT cryogels

[0114] The thrombin release from the AT cryogels was evaluated using an ELISA kit assay (Chen et al. (2018) Biomaterials 179:46-59; Zhao, et al. (2021) Chem. Eng. J., 403 : 126329). The thrombin-loaded cryogel samples (40 mg) in their dried form were used to determine the thrombin release. The cryogel samples were placed in a glass bottle and filled with 10 ml of phosphate buffer saline (PBS). The sample was kept on gentle stirring at 37°C. At desired time points, 100 pl of the sample was taken out and the same volume was replaced by adding fresh PBS. The amount of thrombin released was later quantified using a thrombin ELISA kit.

[0115] Hemocompatibility study

[0116] The compatibility of the developed cryogels was examined by a hemolysis assay using anticoagulated blood. The hemolytic assay of the AAm and AT cryogels was evaluated as reported previously with slight modifications (Andrabi, et al. (2023) Biomater. Adv., 150:213424). Briefly, the anti coagulated human blood was used for the erythrocyte separation. At 1000 rpm, the blood was centrifuged for 15 minutes to obtain a thick erythrocyte pallet. The supernatant was discarded, and the pallet was carefully washed with PBS. Next, the erythrocytes were diluted with PBS in a ratio of 1 :9 and subsequently, the cryogels samples (5 mm thick and 8 mm diameter) of each group were incubated individually for 1 hour at 37°C. This was followed by centrifugation and measuring absorbance (Abs) 540 nm. PBS was used as the negative control and 0.1 % of Triton X-100 was used as a positive control. The hemolysis was determined according to the given equation as below: Hemolysis (%) = (Abs of sample - Abs of negative control) / (Abs of positive control - Abs of negative control) x 100

[0117] Cell viability study

[0118] The toxicity of the AT cryogels was carried out in vitro using human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVEC). To determine cell toxicity, the cryogel samples, in their cylindrical form, were cut into thin disc sections and sterilized. The cytotoxicity of the AAm and AT cryogels was evaluated against HDFs and HUVEC. Before seeding cells, the cryogel scaffolds were placed in a non-treated 48-well plate and allowed to saturate with complete media (10% FBS and 1 % penicillin / streptomycin) for 4 hours. The wells containing scaffold were then separately seeded with HDF and HUVEC at a density of 1 x 104and 2 x 104. The cryogel samples with cells were allowed to culture for predetermined durations, 24 hours, 48 hours and 72 hours, by placing them in a CO2 incubator at 37°C. Next, at preset time points, using CCK-8 assay, cell viability was assessed. The quantification of viable cells was determined by measuring the absorbance at 540 nm using a plate reader (Biotek Synergy Hl).

[0119] Hemostatic efficacy in vitro

[0120] The whole blood clotting index (BCI) was employed to validate the clotting efficiency of the developed cryogels (Li, et al. (2023) Chem. Eng. J., 469: 143758). The cryogels and XStat® were precisely cut and transferred in the glass vials and prewarmed at 37°C. Each scaffold containing vial was added with 100 pl of anti coagulated human blood and incubated at 37°C. At pre-set time points ranging from 30 seconds to 10 minutes, 15 ml of deionized water was added to the vials without disturbing the clot. Adding water ensures that the loosely bound erythrocytes are released, which undergo hemolysis. Consequently, to quantify the released hemoglobin from lysed erythrocytes, absorbance at 542 nm was measured using a microplate reader. For reference, 100 pl of blood in 10 ml of deionized water was also measured to be an absorbance reference. The BCI was calculated using the formula as follows:

[0121] BCI (%) = (Is - Io) / (Ir - Io) x 100 where Is, Io and Ir are the absorbance of a scaffold containing group, PBS, and reference, respectively. Material-cell interaction: in vitro adhesion of platelets and red blood cells

[0122] The effect of material interface on blood cells was assessed by platelet and red blood cells (RBCs) adhesion test (Du, et al. (2021) Nat. Commun., 12( 1 ):4733 ; Zhao, et al. (2021) Chem. Eng. J., 403: 126329). The various groups examined in this study include AAm, AT-1, AT-2, AT-3 cryogel groups and commercial product XStat® as positive control. All samples were fabricated in cylindrical shape (8 mm diameter) and cut into discs of 3 mm thickness. The anti coagulated human blood was used to produce platelet-rich plasma (PRP), following centrifugation at 2500 rpm for 15 minutes. The platelet adhesion on the AT cryogel samples and other groups was tested by adding 100 pl of PRP on their surfaces. The PRP-added samples were incubated for 1 hour at 37°C. The platelet-adhered scaffolds were gently washed with PBS (three times) to wipe out the material surface from non-adherent platelets. Afterward, the samples underwent fixation with 2.5 % glutaraldehyde for a period of 4 hours. Following fixation, the samples were subjected to dehydration using varying ethanol gradients (50%, 70%, 90% and 100%), with each step lasting 10 minutes. Once the samples dried, the adhered platelets and their morphology were observed under SEM. RBC adhesion was determined using a similar method; however, separation of erythrocytes was done by centrifuging human blood at 1000 rpm for 15 minutes and then gently PBS washed. Next, 100 pl of RBCs were dropped on the surface of cryogels and XStat®. The samples were PBS washed post 3 minute incubation at 37°C and the subsequent steps closely followed those mentioned earlier.

[0123] In vivo hemostatic efficacy test in a lethal swine junctional hemorrhage model

[0124] The efficacy of hemostatic cryogels in addressing marginally compressible junctional hemorrhages was assessed through a swine model involving the complete transection of the femur artery and vein (Zhao, et al. (2021) Chem. Eng. J., 403: 126329; Kheirabadi, et al. (2009) J. Trauma 67(3):450-459). The in vivo studies were conducted in strict adherence to the protocol approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC) (Protocol No.: 22-051- 08-EP). The studies involved a lethal swine femur artery -venous complete transection hemorrhage model as a preclinical model. Swine were randomly distributed into 5 groups to have average weight; each group had 5 animals.

[0125] Sample preparation'. A total of 25 pigs were used for this lethal junctional hemorrhagic model, with each group having 5 animals. The various groups included

[0126] T1 were no treatment group as control (Ctr), treatment groups: AAm group (pAAm cryogels without thrombin), AT -2 group (pAAm cryogels containing thrombin) and commercial products, XStat® and QuikClot® Combat Gauze (CG). In treatment groups, Aam and AT-2 shape memory groups were compressed and packed in the syringes, while XStat® and CG were applied directly as received. The treatment administered to each swine of the respective groups involves 2 syringes of AAm cryogels (~36 g), 1 syringe of AT-2 cryogels (~15 g), 2 syringes of XStat®, and 1 pack of CG.

[0127] Swine preparation and surgical procedure'. Before the surgical procedure, the animals underwent a 12 hour fasting period, with unrestricted access to water and were pre-medicated on the morning of surgery. They were pre-anesthetized using an intramuscular injection of Telazol®-Ketamine-Xylazine and taken to the operating room, placed in the supine position with legs secured to an operating table, and intubated with 6-7 French endotracheal tube. The animals were anesthetized and maintained under isoflurane anesthesia with supplemental oxygen at a rate of 1-2 L / minute throughout the entire procedure until the point of either death or euthanasia. A peripheral ear intravenous (IV) line (20-22 G) was placed as needed for additional medications. A right neck cut down was carried down through subcutaneous tissue and platysma down to the sternocleidomastoid muscle (SCM) at which point dissection was carried laterally to expose the external jugular artery. The vein was canalized with 16G angiocath for fluid and medication administration, and the IV catheter was secured surrounding tissue. Dissection was then carried medial and deep to the SCM to expose the carotid artery, after which the anterior wall of the artery was canalized with a 20G angiocath, secured to surrounding tissue, and connected to an arterial line monitoring device.

[0128] To make the porcine hemorrhage model more closely resemble human physiology, a midline abdominal laparotomy followed by splenectomy was performed. This step aimed to reduce auto-transfusion by the contractile porcine spleen, following the recommendation from the US Army Institute of Surgical Research in San Antonio, TX. Following splenectomy, the free spleen was weighed, and warm lactated Ringers (LR) fluid was given 3 x the spleen weight to replace the reserved volume of blood in the spleen. Subsequently, a bladder cystostomy was done to prevent inadvertent compression of pelvic vascular structures from an over-distended bladder. The abdominal wall was then reapproximated with multiple penetrating clamps.

[0129] Finally, an 8 cm incision in the right groin was made to expose the underlying femoral vessels (artery and vein). A 2-0 silk vessel loop was used to encircle the femoral artery and vein proximally and a complete transaction was created to simulate junctional hemorrhage. Blood was freely allowed to flow for 30 seconds and suctioned to measure post-injury blood loss. After 30 seconds of free bleeding, in the case of treated groups, samples of each group fixed in the syringes were injected into the groin wound, and a slight manual pressure was applied for 3 minutes. Later, the pressure was removed, and blood was collected from the arterial line at 0, 15, 30, 60, 120, 180 minutes or preterminal point if the subject expired before 3 hours. In addition, vitals were recorded every minute for the first 10 minutes, then at 15, 30, 60, 120, 180 or pre till the terminal point. The hemostatic potential of the AT -2 cryogels and other groups was measured in terms of blood loss (post-injury + post-treatment), bleeding time and survivability. After 3 hours of observation, the abdomen was reentered, the diaphragm was incised to expose the IVC; weight-based fetal plus (1 ml / 10 kg) of fatal plus was injected into the IVC and after cessation of cardiac activity, the IVC was transected.

[0130] Statistical analysis

[0131] All the experiments were carried out at least in triplicates and the results are presented as the means ± SD. GraphPad Prism 10 software was used to analyze the data. The statistical difference was determined by applying the student’s t-test, one-way and two-way ANOVA test. For statistical significance, the value of p < 0.05 was considered. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = non-significant. To achieve a statistical power of 0.9 and maintain a significance level of 0.5, five rats were required for each treatment group.

[0132] Results

[0133] Fabrication of acrylamide-based cryogels without and with the incorporation of thrombin

[0134] Herein, a series of cryogels based on hydrophilic pAAm and thrombin were fabricated using cryogelation technology, followed by their characterization and application in a lethal swine hemorrhagic model (Fig. 1). The cryogels should fulfill the following design criteria: (i) having a uniformly interconnected macroporous structure; (ii) having high porosity and suitable mechanical strength; and (iii) having quickly responsive shape-recovery and fast blood clotting properties. Based on these criteria, pAAm-based cryogels were initially synthesized using four concentrations of AA (e.g., 1.5 %, 2.5 %, 3.5 %, and 4.5 %) and crosslinker MBAAm (AA: MBAAm = 3: 1) via free radical polymerization. APS / TEMED were incorporated into the polymer solution as free radical initiators / accelerators before subjecting it to subzero temperature (-14°C) for cryo-polymerization. During the cryogelation process at sub-zero temperatures, the homogenized polymeric solution undergoes phase separation, forming aqueous frozen and unfrozen polymeric phases. The ice crystals, later on thawing / drying create interconnected porous structures in the cryogels with high porosity (Fig. 1 A). Using LCMS, the cryogel samples showed a very minimal amount of residual acrylamide after the first wash (1.2 pg / g) and was clear after the third wash. The synthesized cryogels exhibit shape memory properties triggered by water / blood, rapidly expanding to fill the injured bleeding site for effective hemostasis (Figs. IB and 1C).

[0135] The developed pAAm cryogels were validated to identify the optimal concentration for the desired morphological architecture, mechanical stability, and clotting ability. The cryogels (dry state) showed uneven and irregular shapes at lower concentrations (pAAm-1 and pAAm-2), while uniform cylindrical shape was maintained in the cryogels prepared at higher concentrations (pAAm-3 and pAAm-4) (Fig. ID). SEM images revealed larger and non-uniform porous structures in pAAm-1 and pAAm- 2, while uniform porous structure was observed in pAAm-3 and pAAm-4 (Fig. ID). These variations were further confirmed by micro-CT analysis, showing more irregular morphology and larger pores (marked by arrows) for the cryogels, pAAm-1 and pAAm- 2, when compared to the cryogels, pAAm-3 and pAAm-4 (Fig. IE). The variations in morphology and porous architecture among the developed cryogels can be ascribed to differences in concentration (Plieva, et al. (2005) Soft Matter 1 (4): 303-309). In addition, all the pAAm cryogels displayed high porosity, while varying pore sizes were observed in the pAAm cryogels, specifically pAAm-1 and pAAm-2, exhibiting large pore sizes (Figs. IF and 1G).

[0136] Next, the developed cryogels were examined for water / blood absorption rate and swelling capacity to validate their potential application in hemostasis. The results showed that pAAm-3 and pAAm-4 cryogels absorbed water / blood more rapidly and exhibited higher absorption capacity than pAAm-1 and pAAm-2 (Figs. 1H-1K). Moreover, physical observation of the developed cryogels (wet state) revealed significant frailty of pAAm-1 followed by pAAm-2 in contrast to the pAAm-3 and pAAm-4 cryogels (Fig. IL). Due to the weak mechanical properties owing to large pore sizes, slow fluid uptake rate, and low fluid absorption capacity of pAAm-1 and pAAm-2, no detailed mechanical study was performed and these cryogels were unsuitable for the desired application. The cyclic compressive testing of pAAm-3 exhibited good mechanical strength and shape recovery properties. In the case of pAAm-4, the strength was higher, but this resulted in a loss of recovery (Figs. IM and IN). Hence, considering these results, pAAm-3 (now named AAm) was selected for further study.

[0137] During cryogelation, pAAm forms the essential backbone of the cryogel network for hemostatic applications by providing an interconnected porous network with suitable flexibility, mechanical and rapid shape recovery properties. In addition to possessing these properties ideal for hemostatic applications, the ability to swiftly staunch excessive bleeding is a prerequisite for practical use in managing lethal junctional hemorrhage. Therefore, thrombin, a potent coagulation factor, was incorporated into the AAm cryogels in three different concentrations (e.g., AT-1, AT-2, and AT-3) to enhance their hemostatic efficacy, which was comprehensively validated both in vitro and in vivo. Fig. 2A shows the digital images of the AAm cryogels with and without the incorporation of thrombin and XStat®, indicating their cylindrical shape. The strategy involving acrylamide as the material choice and the use of the cryogelation process provides a straightforward approach, facilitating the integration of hemostatic agents and enabling scale-up production for commercialization.

[0138] Characterizations of morphology, microstructure and porosity

[0139] The surface morphology and porous architecture of AAm and AT cryogels were examined by scanning electron microscopy (SEM) and micro-CT (Fig. 2). All the cryogels (AAm, AT-1, AT-2 and AT-3) exhibit uniform pore distributions and well- interconnected porous architectures, and thin, and smooth pore walls, while the commercially available hemostat XStat® display rough and thick pore walls, along with uneven pore size distributions (Fig. 2A). The results were further confirmed by the micro-CT analysis, which revealed a more uniform porous structure of cryogels as compared to XStat® (Fig. 2A). This is illustrated through 3D and cross-sectional images, respectively. A nonhierarchical porous surface with a substantial difference in pore size distribution was evident from the micro-CT images (longitudinal and cross- sectional) (Fig. 2A). Further micro-CT analysis demonstrated that all the cryogels exhibit high and similar porosity of approximately 85 ± 7 %, significantly higher than the porosity of XStat® (~75 ± 4 %) (Fig. 2B). In addition, pore size estimation revealed comparable results among the cryogel groups, with pore sizes ranging from 45 to 60 pm. In contrast, XStat® exhibited larger pore size in the range of 130-190 pm (Fig. 2C). These results also revealed that thrombin incorporation does not affect the morphology of AAm cryogels. The cryogels’ uniform and interconnected porous architecture, coupled with the desired small pore size, may contribute to enhancing the hemostatic effect. This enhancement is achieved by boosting the fluid absorption rate and facilitating the entrapment of clotting factors, thereby rapidly activating the coagulation cascade.

[0140] Water / blood absorbability

[0141] For an ideal hemostatic material, a fast liquid absorptive rate and high absorption capacity are the primary factors conducive for promoting hemostasis. The well- interconnected porous structure of cryogels essentially facilitates fluid absorption capacity and provides a high ability to amass blood cells / clotting factors, promoting rapid coagulation (Zhao, et al. (2018) Nat. Commun., 9(1):2784; Du, et al. (2021) Nat. Commun., 12(1) :4733). Fig. 3 shows the fluid absorption rate / capacity of the AAm, AT cryogels (AT-1, AT -2 and AT-3), and XStat®. All the cryogels, both with and without incorporation of thrombin, exhibited approximately 96 ± 7 % water absorption within just 5 seconds. In comparison, the absorption rate of XStat® was slower, demonstrating only about 75 ± 5 % water absorption in 5 seconds, reaching approximately 97 ± 4 % in 10 seconds (Fig. 3A). As expected, the cryogels initially displayed a higher water absorption capacity. However, once swelling equilibrium was reached, there was no difference in water absorption capacity between the cryogels and XStat®, but estimated at approximately 1.6 ± 0.1 g / cm3(Fig. 3B). A similar trend was observed for blood absorption, with cryogels showing a faster blood absorption rate initially compared to XStat®. The blood absorption rate in cryogels reached approximately 90 ± 5% within 5 seconds, while for XStat®, only around 60 ± 6 % blood uptake was observed after 5 seconds (Fig. 3C). The blood absorption of XStat® gradually increased over time, reaching approximately 98 ± 8% absorption in 15 seconds. In all cryogel groups and XStat®, the blood absorption capacity became comparable after reaching the swelling equilibrium point, demonstrating a capacity of approximately 1.52 g / cm3(Fig. 3D).

[0142] Figs. 3E and 3F shows the swelling ratio (%) of the cryogels and XStat®, indicating a high swelling ratio (%) of cryogel groups (AAm, AT-1, AT-2, and AT-3) and XStat® when submerged in water and blood. The cryogel groups showed a swelling ratio (%) of approximately 970 ± 130 in water and 1090 ± 110 in blood. The value of swelling ratio (%) of XStat® was slightly higher (1170 ± 165 in water and 1110 ± 175 in blood) compared to the cryogels groups but there was no statistically significant difference. Overall, the results revealed that the cryogel groups and XStat® exhibited a similar fluid absorption rate and swelling behavior. The results also indicated thrombin loading did not influence the absorption and swelling behavior of the AT cryogels. The difference in absorption rate between cryogels and XStat® could be attributed to the disparity in porosity, pore size and interconnected porous structure. The water / blood absorbability and swelling properties may make AT cryogels an excellent candidate for hemostatic applications, especially in the context of junctional hemorrhage.

[0143] Mechanical characterization

[0144] Mechanical strength and flexibility are primary requirements of hemostats during practical scenarios, and the AT cryogels, as a hemostatic material, should possess suitable mechanical properties. The mechanical property of the AAm and AT cryogels was tested in both dry and wet states (Figs. 3G-3K). All the samples were compressed by 80% with respect to the original length in a compressive strain test (Figs. 3G and 3H). The results indicated that the AT-1, AT-2 and AT-3 groups had compressive stress of 125 ± 24 kPa, 140 ± 30 kPa, and 105 ± 40 kPa, respectively, slightly lower than the AAm group (145 ± 35 kPa). Although there was a slight difference in the compressive stress between AAm and other AT groups, it was statistically non-significant. In addition, the subtle difference among AT cryogel groups might be accredited to thrombin loading, showing a marginal decrease in the mechanical strength with increasing thrombin amount. The compressive stress-strain curve of the samples revealed a nonlinear behavior, primarily observed in flexible and shape memory sponges. From the curve, the cryogels during compression, sequentially undergo elastic and plastic deformations. This usually occurs by expelling air from pores, followed by subsequent flexing, collapse, or fracture in sample walls. Finally, a quick increase in the slope leading to maximum stress point is attained due to cryogel compactness, observed at a pre-set strain of 80%. The compression test showed that all AT cryogels exhibit nonlinear behavior and excellent mechanical strength in a dry state, essential for maintaining structural integrity during handling and storage.

[0145] Further, the AAm and AT cryogels were evaluated for the effect of thrombin concentration on mechanical stability in a wet state using cyclic compression at an 80% compressive strain. The results showed that the cryogels maintained their original shape and were flexible enough to withstand compression (Figs. 3I-3L). As expected, the cryogels in the wet state exhibited lower compressive stress, in contrast to the dry state, having more stiffness (Fig. 31). The AAm group showed a compressive stress of ~1.7 kPa, similar to thrombin-loaded groups. The compressive stress in AT cryogel groups was determined in the range of 1.5 kPa-1.8 kPa (Figs. 3J-3L). The results demonstrated the sample’s suitable mechanical strength, essential for withstanding the strain during injection at the bleeding site and retaining structural integrity to restore the original shape. The excellent mechanical properties of the AAm and AT cryogels might be attributed to the highly porous and flexible network of cryogels.

[0146] Shape recovery and injectable properties

[0147] During practical applications, hemostatic materials may encounter diverse wounds, necessitating their adaptation to various configurations. For critical injuries at junctional extremities, it is strongly advised to pursue hemostats that possess injectable properties and robust shape-memory capability. Given their excellent mechanical properties, the water / blood-induced shape memory capability and injectability of the AT cryogels were investigated (Fig. 4). The dry / wet samples of AAm and AT cryogels underwent structural compactness upon 80% compression to attain a fixed state and rapidly recovered their original shape upon contacting water / blood (Fig. 4A). The shape recovery time observed in AAm and AT cryogels was remarkably shorter (water, 4.5 ± 1 seconds and blood, 6 ± 1 seconds), contrary to XStat® (water, 8 ± 1.5 seconds and blood, 13 ± 1 seconds) (Figs. 4E-4H). In addition, all cryogels and XStat® achieved a 100 % recovery ratio when exposed to water and blood.

[0148] All the samples were compressed to 80 % of the initial length for 5 minutes, and the digital images of respective shapes of the AAm and AT cryogels (AT-1, AT -2 and AT-3) before and after compression were shown in the first column of Fig. 4B. The morphological and structural changes in the cryogels in compressed and recovery states were further analyzed by SEM (Fig. 4B). The SEM images of the shape memory cryogels showed closed pores in a compressed state. In the shape-recovery state, the cryogels restored a uniform porous structure comparable to the original state. Fig. 4C shows a schematic illustrating the effects of applied forces in various directions and subsequent water / blood-triggered shape recovery. The AT cryogels exhibited rapid shape recovery from compressed states in the forms of discs, bent shapes, and flat sheets to their original shape once the applied force was released upon contact with water (Fig. 4D). The consistent shape and strength retention highlight a resilient and flexible cryogel network with robust fatigue resistance. These characteristics enable the compression of the cryogels into a syringe, followed by rapid water / blood absorption and shape recovery after injection. This effectively creates a sealing barrier at the injection site, filling the cavity. Fig. 5 illustrates the compressed AT-2 cryogels packed in an injecting device (a modified plastic syringe). The demonstration revealed the ease of injecting the compressed cryogels and their instant ability to re-expand to the initial state within 4 seconds (Figs. 5C and 5D). Additionally, XStat®, available in the form of compact discs, expands only in the longitudinal direction, exerting pressure primarily in that direction and may pose challenges in effectively compressing the arteries, particularly in cases of narrow and deep wounds (Fan, et al. (2020) Int. J. Biol. Macromol., 164:2769-2778). In the case of AT cryogels, the ability to recover shape from distinct types of compressed forms makes them desirable hemostats to fill narrow, irregular and deep wounds. The results elucidated the remarkable compressibility, resistance to fatigue, and swift shape recovery of the AT cryogels. This balance between mechanical strength and compressibility positions the AT cryogels as highly useful for contemporary hemostatic applications, particularly in the context of junctional hemorrhage.

[0149] Thrombin release from AT cryogels

[0150] To augment the hemostatic effectiveness of cryogels, thrombin, a clotting factor commonly used in clinical settings, was incorporated. Fig. 5F illustrates the release kinetics of thrombin from the AT-2 cryogel. The results revealed a slower release of thrombin, around -20% within the initial 4 hours, and increased up to 60% after 72 hours, which may be ascribed to the fabrication process and non-covalent intermolecular interaction. The released thrombin plays a central role directly in the final step of the blood coagulation process by facilitating the insoluble fibrin conversion from fibrinogen. Therefore, incorporating thrombin would notably enhance the hemostatic efficiency of the cryogel, particularly in cases involving coagulation disorders that are characterized by thrombin deficiency.

[0151] Hemocompatibility study

[0152] Hemocompatibility, a crucial factor in determining the translational application of the cryogels, was assessed by a hemolysis test using isolated human erythrocytes. This test signifies higher cell lysis at elevated absorbance of hemoglobin (Hb) and minimal to no lysis of erythrocytes at lower absorbance. Fig. 5G shows the hemolytic effect of the cryogels and other control groups. The AAm and AT cryogel (AT-1, AT-2 and AT-3) groups and XStat® exhibited negligible hemolysis (<2 ± 1%), showing a nonsignificant effect on erythrocytes. As expected, no hemolysis was observed in negative PBS control, in contrast to the Triton X-100 (positive control) group, which induced 100% hemolysis of RBCs. The results illustrated that the hemolytic effect of approximately 2% falls below the safe range of 5%, aligning with findings reported in the literature (Lv, et al. (2022) Compos. B Eng., 247: 110263). Therefore, the developed cryogels demonstrate good hemocompatibility and are deemed safe for use as hemostats.

[0153] Cell viability study

[0154] As an ideal hemostat and its direct contact with injured tissue, the material should be cytocompatible. The cell viability of cryogels, both with and without the incorporation of thrombin, was assessed in vitro using the CCK-8 assay over a three-day period. Figs. 5H and 51 show the effect of the cryogels with / out thrombin on HDFs and HUVEC, respectively. The cryogel group’s AAm, AT-1, AT-2 and AT-3 showed similar cell growth as observed in the 2D control group after 24 hours. On days 2 and 3, the cryogels showed a slight decrease in HDFs growth compared to the 2D group; however, the difference was statistically non-significant. Similarly, the cell viability of HUVEC cells in the cryogel-treated groups was comparable to that of the 2D group throughout the experiment. In both HDFs and HUVECs, cell proliferation generally exhibited an upward trend upon treatment with AAm, AT-1, AT-2, and AT-3 cryogel groups. In addition, the results showed that incorporating thrombin into the cryogels did not induce any cell toxicity. Overall, the results indicated that all cryogel groups displayed good cytocompatibility.

[0155] Hemostatic efficacy in vitro

[0156] The blood clotting ability of the cryogels was determined by assessing the BCI. The BCI determines the non-clotting RBCs by measuring the absorbance of Hb. The BCI value is inversely related to the clotting ability, where a low value indicates a higher clotting ability of the material (Wang, et al. (2019) ACS Appl. Mater. Interfaces 11 (38) :34595-34608). The results demonstrated the Ctr and XStat® groups had a clotting time of approximately 605 seconds, whereas AT-2 and AT-3 exhibited faster clotting (60 ± 5 seconds), followed by AT-1 (-200 seconds) and AAm (-380 seconds) groups (Fig. 6A). As expected, the BCI value was the highest in the Ctr group (100%) due to the absence of any coagulation ability (Fig. 6B). The BCI value in the AT -2 and AT-3 groups was -22% after 180 seconds which was significantly lower compared to the AT-1 group and other groups. However, the clotting ability of AT-1 gradually improved, and eventually, the difference in BCI values became negligible compared to that in the AT -2 and AT-3 groups. The BCI value in the AAm group (-40%) indicated lower clotting ability than AT cryogel groups but was higher compared to the ones in the XStat® (-65%) and control groups. Notably, the XStat® group showed a higher BCI value than all cryogel groups, indicating its poor clotting ability.

[0157] Fig. 6C shows the photos of blood in the vials after different treatments, clearly indicating better clotting ability of the AT cryogel groups than other control groups. Particularly, AT-2 and AT-3 groups showed rapid and stable clot formation, followed by AT-1 and AAm groups. The XStat® group exhibited slow coagulation with blood cells diffusing from a weak clot, and no clotting was observed in the Ctr group. In addition, for the intended application, AT-2 cryogel and XStat® were used in the compressed states to evaluate their clotting ability while quickly returning to their original shapes (Figs. 6D-6G). The remarkable clotting property of AT cryogel groups may be primarily endorsed to thrombin incorporation. The clotting effect in AT cryogel and AAm groups could be attributed to the small pore size and interconnected porous architecture. This property significantly aids in absorbing the blood quickly and amasses RBCs, platelets, and clotting factors inside the cryogels, leading to rapid clot formation. The poor clotting ability in XStat® may be ascribed to its large pore size, absence of an interconnected porous structure, and lack of clotting factors (Andrabi, et al. (2023) Biomater. Adv., 150:213424; Zhao, et al. (2021) Chem. Eng. J., 403: 126329; Yao, et al. (2022) Chem. Eng. J., 428: 131005).

[0158] Material-cell interaction: in vitro adhesion of platelets and RBCs

[0159] To better understand the effect of AT cryogels on blood coagulation and determine the clotting mechanism, the influence of material interface on RBCs / platelets was evaluated. The adhesion of RBCs and platelets interacting with the material surface and the subsequent changes in their shape and morphology are shown in Fig. 6H. SEM images showed the adhesion of RBCs in all cryogel and XStat® groups. The number of adhered RBCs varied substantially among the groups. A higher number of aggregated RBCs were observed in the AT -2 and AT-3 groups, followed by the AT-1 group, than in the AAm and XStat® groups. Interestingly, the AAm cryogels exhibited a larger number of adhered RBCs than XStat®. The platelet aggregation and activation play a central role during the initial steps of the coagulation cascade in forming a clot (Sekhon, et al. (2022) Sci. Transl. Med., 14(629):eabb8975). The results demonstrated a higher number of adherent platelets in AT -2 and AT-3 groups than in AT-1, AAm and XStat® groups. The AT-1 and AAm groups demonstrated a similar number of adherent platelets, which were significantly higher than those observed in the XStat® group. In the XStat® group, only a sparse number of platelets adhered, and no aggregation was observed. In addition, the SEM images of AT-1, AT -2 and AT-3 groups revealed an irregular shape and pseudopodia stretching, indicating a distinctive platelet morphology that endorses the activated forms of platelets. In the case of the AAm group, a certain number of platelets showed morphological changes, while the XStat® group failed to promote sufficient platelet adhesion and activation.

[0160] These results validated excellent adhesion and aggregation of RBCs / platelets and platelet activation in the AT cryogels, particularly AT-2 and AT-3 cryogels, aligning well with their rapid blood clotting ability. Moreover, the synergistic effect of thrombin incorporation, small pore size, and a highly interconnected porous architecture may contribute to the rapid blood clotting observed in injectable and shape-adaptable AT cryogels (Fig. 61).

[0161] Hemostatic efficacy in a lethal swine junctional hemorrhage model

[0162] The pre-clinical hemostatic efficacy of the injectable and rapid shape memory AT cryogels was examined in a lethal swine junctional hemorrhage model. A lethal swine model was established through the complete transection of the femoral artery and vein to replicate penetrating junctional injuries, encompassing scenarios related to the axillary and subclavian arteries (Fig. 7). These injuries are associated with considerable rates of mortality in both military and civilian environments (Zhao, et al. (2021) Chem. Eng. J., 403 : 126329). To more closely emulate human physiology, a midline abdominal laparotomy followed by splenectomy was conducted in this porcine hemorrhage model. The animals were randomly distributed into 5 groups, including the control group without treatment (Ctr), AAm, AT-2, XStat® and CG. Among the AT cryogel groups, the AT-2 group was selected and further validated for pre-clinical hemostasis because of its rapid clotting ability and enhanced adhesion and activation of RBCs / platelets. The XStat® and CG, used for junctional hemorrhage in prehospital and hospital scenarios, were used as positive control groups. Fig. 7A presents a schematic illustrating the surgical intervention and subsequent treatment steps followed until the end of the experiment, which lasted 180 minutes. Briefly, the swine femoral artery and vein were transected, followed by free blood blow for 30 seconds to mimic the injury. For the control group, no treatment was applied. For the treatment groups, the materials were applied through either injection (AAm, AT -2, XStat®) or packing (CG), and cotton gauze was placed on the top and compressed manually for 3 minutes. Then, the animals were monitored for 180 minutes.

[0163] Fig. 7B shows the representative images of the treated injured site immediately after releasing manual compression and at the end of the experiment, indicating the order of rapid and effective blood coagulation was AT -2, AAm, XStat®, and then other groups. The treatment with injectable and rapid shape-memory AAm and AT-2 cryogels showed rapid blood absorption and filling of the injury site instantaneously to stop massive bleeding. Further, the AAm and AT-2 groups exhibited high resilience properties, quickly re-expanding while maintaining their structural integrity. As expected, the control group showed a quick and massive blood loss after the injury and failed to attain any hemostasis on its own. It is worth mentioning that only a single applicator containing AT-2 cryogel samples (~14 g) was sufficient to stop the lethal junctional hemorrhage compared to the groups treated with two syringes of each AAm cryogel (~40 g) and XStat®. Hence, the time to inject the AT-2 cryogels into the injured site was significantly shorter (3 seconds) than Aam and XStat® treated groups (~8 seconds) (Fig. 7C). The time duration for applying the CG treatment was the longest (20 seconds) among all the treated groups. The hemostatic efficiency was further evaluated by quantifying the total blood loss post-treatment (Fig. 7D). As anticipated, due to continuous bleeding, the total blood loss in the control group was the highest (2200 ± 330 ml) among all the groups. Remarkably, the AT-2 treated group showed significantly less blood loss (110 ± 90 ml) compared to the AAm (476 ± 280 ml), XStat® (770 ± 140 ml), and CG (1540 ± 240 ml) groups. Obviously, the CG group had a large amount of blood loss, indicating its poor clotting ability. The blood loss in the AAm and XStat® groups varied slightly but was statistically non-significant. Surprisingly, some of the pouch-packed pellets in the XStat® product failed to expand, which could be attributed to the hindrance of the mesh pouch itself and potential quality issues of those pellets (Fig. 7E). Next, the effect of treatment groups on the survival time and survivability of the animals were examined (Fig. 8). The post-treatment results manifested an interesting outcome and revealed that all the subjects in the control group died within 15 ± 6 minutes, followed by the CG group with a survival time of 70 ± 50 minutes. Notably, in both the AAm and AT-2 groups, the animals survived until the end of the experiment, i.e., 180 minutes, demonstrating a 100% survival rate, unlike the XStat® and CG groups. In the case of the XStat® treated group, an average of 70% of the pigs survived until 140 ± 60 min, while the remaining 30% died after 30 minutes (Figs. 8A and 8B). In the CG group, 50% of the animals died within 1 hour, and approximately 70% succumbed around the 2-hour mark. The final mean artery pressure (MAP, mmHg) monitored in the AT-2 group was higher, at approximately 62 mmHg, compared to the group’s AAm cryogels, XStat® and CG, recorded at approximately 32 mmHg. Moreover, to achieve proficient hemostasis in junctional hemorrhage in clinical settings, hemostats should not only demonstrate initial clotting but also be effective in preventing post-treatment rebleeding. No rebleeding was observed in the AT-2 group except in one pig that only lasted for approximately 4 minutes. This was in contrast to the AAm groups, which also experienced rebleeding but for a shorter duration (~7 minutes), compared to the XStat® and CG groups, where rebleeding lasted for 15 minutes (Fig. 8C). Fig. 8D shows a schematic illustrating the procedure of AT-2 treatment and possible clotting mechanism induced by blood-triggered rapid expansion and accumulation / activation of blood cells and clotting factors. In summary, the AT-2 cryogels show significantly higher hemostatic efficacy than XStat® and CG in the lethal and marginally compressible swine junctional hemorrhage model.

[0164] The majority of bleeding casualties occur within the initial 30 minutes following a severe injury, such as junctional wounds, contributing to high patient mortality rates (Andrabi, et al. (2023) Biomater. Adv., 150:213424; van Oostendorp, et al. (2016) Scand. J. Trauma Resuscitation Emerg. Med., 24(1): 110). These fatalities can be mitigated by promptly employing effective hemostats. Despite considerable progress in material science that has yielded several promising hemostats, only a select few have successfully transitioned into clinical application. Therefore, effectively achieving hemostasis for severe bleeding still poses a significant challenge. Here, a straightforward, easy, and cost-effective method is provided to fabricate the injectable and rapidly expandable AT cryogels for use as a propitious hemostat, efficiently managing marginally compressible junctional hemorrhage.

[0165] The ability of any hemostat to stop excessive bleeding rapidly and effectively has been primarily shown to depend on various key parameters, including fabrication technology, pore size, porosity, and the incorporation of desired hemostatic agents (Zhao, et al. (2018) Nat. Commun., 9(1):2784; Yao, et al. (2022) Chem. Eng. J., 428: 131005; Yang, et al. (2019) Acta Biomater., 99:220-235). Herein, cryogelation technology was used to fabricate AT cryogels with a unique interconnected porous architecture, desired pore size, and rapid shape memory properties. During the optimization, it was observed that the pAAm cryogels with either smaller or larger pore sizes were less effective in blood clotting and also exhibited compromised mechanical strength. Subsequently, pAAm-3, which demonstrated the required ideal properties, was selected and used to prepare AT cryogels for further in vitro and in vivo validation. One of the essential properties of hemostats is their significant liquid absorptive capacity, which aids in speeding up the blood coagulation cascade (Udangawa, et al. (2019) ACS Appl. Mater. Interfaces 11(17): 15447-15456; Wang, et al. (2020) Acta Biomater., 114: 193-205). Because of high porosity as well as a uniform interconnected porous architecture, the AT cryogels showed a higher fluid absorption rate and capacity, like XStat®.

[0166] In prehospital settings, persistent cases of extensive bleeding from deep and irregular wound sites present significant challenges, especially junctional hemorrhage. To mitigate these complications, it is strongly recommended to employ hemostats possessing both injectable and rapid expandable properties. The AT cryogels, after being compressed into various shapes, showed remarkable water / blood-triggered shape memory and can rapidly restore their original shape in a few seconds. The mechanical study proved that the cryogels withstand high stress without experiencing mechanical fractures, even after 80% compression. The outstanding mechanical stability and shape memory capability of the cryogels can be attributed to its interconnected porous network, which allows for reversible pore deformation, ensuring the unimpeded penetration of water / blood (Bencherif, et al. (2012) Proc. Natl. Acad. Sci., 109(48): 19590-19595). Additionally, its high flexibility supports effective recovery, and its polarity facilitates water uptake (Andrabi, et al. (2023) Biomater. Adv., 150:213424; Zhao, et al. (2018) Nat. Commun., 9(1 ):2784). Overall, these properties indicate the applications of the cryogels in the injectable form for managing junctional hemorrhage and addressing noncompressible torso hemorrhage, a leading cause of potentially preventable trauma mortality (Jamal, et al. (2021) Ann. Transl. Med., 9(14): 1192).

[0167] Swift cessation of bleeding by forming a stable clot has been established as a critical factor in intervening effectively in cases of massive hemorrhages (Teuschl, et al. (2017) J. Biomed. Mater. Res., 105(3):687-696). An in vitro blood clotting assay was performed to investigate the impact of AT cryogels in promoting blood clotting. The incorporation of thrombin into cryogels, specifically AT -2, indicated better blood clotting capability in a shorter time than the XStat® and other groups. Interestingly, the clot formed in the AT-2 group was firm and stable. In contrast, XStat® showed poor clotting ability with continuous diffusion of erythrocytes from the unstable clot. Studies have reported the key role of RBCs in activating platelets (Zhao, et al. (2021) Chem. Eng. J., 403: 126329). Upon activation, platelets have the ability to transport numerous other clotting factors, aggregate on a wound surface to form a platelet plug and initiate the blood clotting cascade by facilitating a rapid generation of thrombin (Chan, et al. (2015) Sci. Transl. Med., 7(277):277ra29-277ra29; Zhao, et al. (2021) Chem. Eng. J., 403: 126329; Sekhon, et al. (2022) Sci. Transl. Med.,14(629):eabb8975; Yang, et al. (2019) Acta Biomater., 99:220-235; Guo, et al. (2021) Sci. Adv., 7(29):eabf9635). The improvement of this process mainly relies on the interaction between the material surface and blood cells. A sparse number of RBCs adhered to XStat®, which could be due to the lack of an interconnected porous network and the presence of larger pore size. On the contrary, AT-2 and AT-3 attracted abundant RBCs and platelets on their surfaces and favored their activation compared to other groups. This may be attributed to an interconnected porous architecture and the small pore size of cryogels that are crucial for high fluid absorption, thereby allowing cryogels to concentrate RBCs / platelets and plasma proteins (Wang, et al. (2019) ACS Appl. Mater. Interfaces l l(38):34595-34608; Li, et al. (2016) ACS Appl. Mater. Interfaces 8(51):35071-35080). Moreover, thrombin plays a vital role in accelerating the coagulation cascade and establishes a stable and firm clot by augmenting localized activation of platelets as well as amplifying the fibrin generation from fibrinogen (Hickman, et al. (2018) Adv. Mater., 30(4): 1700859; Chen, et al. (2018) Biomaterials 179:46-59; Sekhon, et al. (2022) Sci. Transl. Med., 14(629):eabb8975).

[0168] The AAm and AT-2 cryogels can be compressed and packed into an applicator (e.g., a syringe device) to deliver them into narrow and irregular wounds (e.g., junctional, truncal, and gunshot wounds). Next, the preclinical efficacy testing of the AT-2, AAm, XStat®, and CG was assessed in a lethal swine junctional hemorrhage model. Among all groups, AT-2 demonstrated a remarkable hemostatic effect in a shorter time with a 100 % survival rate. Although AAm and XStat® demonstrated comparable hemostatic effects, considering the obtained results, AAm outperformed XStat® in terms of the survival rate, which was 100% in the former and 70% for the latter. The CG, another commercial hemostat, exhibited poor hemostatic effectiveness with high blood loss, along with a significantly lower survival rate of 30%. The rapid blood clotting ability, contributing to the excellent hemostatic efficacy of AT-2 cryogels, can be attributed to a combination of cryogelation technology, a hydrophilic polymer, small pore size, a uniformly interconnected porous architecture, and the incorporation of thrombin as an active hemostatic agent.

[0169] While the AT-2 cryogels lack antibacterial property, these cryogels may only remain at the injury site for a short period of time until surgical intervention begins. If desired, antimicrobial agents can be incorporated into the cryogels. The cryogels were comprehensively assessed for physiochemical characteristics and evaluated in a lethal and marginally compressible swine junctional hemorrhage model. A swine noncompressible torso hemorrhagic model may also be used for further hemostatic efficacy testing. The storage and shelf life of the cryogels were examined for several weeks and are expected to be stable for years. Some of the major disadvantages of XStat® compared to the instant cryogels include, without limitation: less porosity with closed pores; larger pore size; more shape recovery time; increased clotting time; no clotting ability; low platelets and RBCs adhesion; lack of clotting factors; longer application time; high material quantity needed (two syringes); more blood loss in vivo, 30% deaths due to more blood loss; risk of secondary damage to adjacent tissue; and nonbiodegradable.

[0170] In conclusion, AAm and AT cryogels have been fabricated with injectable and rapidly expandable properties for managing lethal junctional hemorrhage in swine. In contrast to commercial hemostats XStat® and CG, the AT-2 cryogels showed significantly less blood loss and a higher survival rate (100%) in a lethal swine junctional hemorrhage model, indicating their rapid and efficient clotting ability to control massive bleeding. Overall, these results demonstrate the AT-2 cryogels as a hemostat for effectively managing marginally compressible junctional hemorrhage. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A cryogel comprising polyacrylamide.

2. The cryogel of claim 1, wherein said cryogel is loaded or embedded with a therapeutic agent.

3. The cryogel of claim 2, wherein said therapeutic agent is a hemostatic agent or clotting agent.

4. The cryogel of claim 3, wherein said clotting agent is thrombin.

5. The cryogel of any one of claims 1-4, wherein said cryogel further comprises an antimicrobial.

6. The cryogel of claim 5, wherein said antimicrobial is an antimicrobial peptide.

7. The cryogel of claim 1, wherein the average diameter of the pores of the cryogel is about 10 pm to about 100 pm.

8. A composition comprising the cryogel of claim 1 and a pharmaceutically acceptable carrier.

9. A method of synthesizing a cryogel, said method comprising: a) preparing a solution comprising a monomer and a crosslinker; and b) maintaining the solution of a) at a temperature below freezing to allow for cryo-polymerization.

10. The method of claim 9, wherein said monomer is acrylamide.

11. The method of claim 9, wherein said crosslinker is N’-N’-methylene-bis- acrylamide.

12. The method of claim 9, wherein step b) comprises maintaining the solution at - 14°C or lower.

13. The method of claim 9, wherein the concentration of the monomer in the solution is about 1% to about 5%.

14. The method of claim 9, further comprising adding a therapeutic agent to the solution.

15. The method of claim 14, wherein said therapeutic agent is a clotting agent.

16. The method of claim 15, wherein said clotting agent is thrombin.

17. A method of treating or inhibiting a hemorrhage and / or reducing blood loss in a subject, said method comprising administering a cryogel of claim 1 to the subject.

18. The method of claim 17, wherein said cryogel is injected directly to the site of hemorrhage and / or blood loss.

19. A method of absorbing a fluid in a subject, said method comprising administering a cryogel of claim 1 to the subject.

20. The method of claim 19, wherein said fluid is blood, draining fluid, or exudate.

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