Purified hemostatic sponge based on heat-assisted cross-linked pullulan

Abstract

This invention discloses a pullulan polysaccharide composite hemostatic sponge based on heat-assisted citric acid crosslinking and its preparation method, belonging to the field of biomedical materials technology. This invention uses pullulan polysaccharide as the base material, citric acid as the biocompatible crosslinking agent, and tilapia skin collagen as the functional enhancing component. A sponge precursor is prepared by collagen solution preparation, mixing and dissolving, centrifugation to remove bubbles, and gradient freeze-drying. This precursor is then subjected to solvent-free heat-assisted esterification crosslinking at 130℃ and water washing purification to obtain a pullulan polysaccharide-collagen composite sponge matrix. Finally, the target composite hemostatic sponge is obtained by co-incubating with tissue factor (TF)-liposomes. The preparation process of this invention is mild and simple, leaves no toxic residues, and is easy to scale up for production. The prepared composite sponge has a porosity ≥80%, a water absorption rate of 13.5-28.7 times, a blood absorption rate of 17.4-37.0 times, high mechanical strength and good shape recovery, a hemolysis rate <2%, cell viability ≥85%, and excellent biocompatibility. This sponge integrates the synergistic effects of pullulan, collagen, and TF-liposomes, which can quickly initiate the coagulation cascade to achieve efficient hemostasis, while promoting wound tissue repair. It is suitable for the care of various bleeding wounds such as military trauma, surgical procedures, and accidental injuries, and has good clinical application prospects.

Description

Technical Field

[0001] This invention relates to a pullulan polysaccharide composite hemostatic sponge loaded with tissue factor-liposomes and collagen, prepared by heat-assisted citric acid crosslinking, which is mainly used for wound hemostasis and promoting tissue healing. Background Technology

[0002] Uncontrolled bleeding is a leading cause of death in military trauma, surgical procedures, and accidental injuries. Uncontrolled bleeding is often accompanied by hypothermia, acidosis, and coagulation disorders, resulting in extremely high mortality and disability rates. Currently, commonly used hemostatic materials in clinical practice include hemostatic gauze, collagen sponges, chitosan dressings / sponges, oxidized regenerated cellulose (ORC), and fibrin glue. While these materials can achieve hemostasis to some extent, they generally suffer from limitations such as limited functionality, insufficient bioactivity, weak mechanical strength, poor biocompatibility, or high manufacturing costs.

[0003] Biopolymer porous sponges, with their core advantages such as high porosity, high liquid absorption capacity, compressive hemostasis, and ability to closely adhere to irregular wound surfaces, have shown great application potential in wound hemostasis and tissue repair, making them a current research hotspot in biomedical materials. These porous materials can rapidly absorb wound exudate, providing a three-dimensional scaffold for coagulation factor accumulation and platelet adhesion, while also possessing good mechanical adaptability and ease of handling, effectively addressing various complex wound bleeding problems in clinical practice. Among numerous biopolymer substrates, polysaccharide sponges, with their wide availability of raw materials, excellent biocompatibility, complete biodegradability, and no risk of in vivo residue, have become the preferred system for preparing hemostatic and repair materials, and also provide an important direction for developing safe and efficient novel hemostatic dressings. Among these, pullulan, as a high-performance natural microbial polysaccharide, has significant research and application value in polysaccharide-based hemostatic sponge substrates.

[0004] Despite the numerous advantages of polysaccharide-based hemostatic sponges, currently reported and applied polysaccharide-based materials still suffer from several common and insurmountable problems, severely limiting their effectiveness in emergency massive bleeding and refractory wounds. Most natural polysaccharides are highly water-soluble and easily swell and collapse in blood and exudate environments, resulting in insufficient structural stability and mechanical strength, failing to maintain long-term hemostatic support. To improve structural strength, toxic chemical cross-linking agents such as glutaraldehyde and epoxy compounds are often used, which can easily lead to cytotoxicity and safety hazards. More importantly, traditional polysaccharide sponges rely solely on physical liquid absorption and passive packing to achieve hemostasis, lacking the efficient procoagulant function to actively initiate coagulation and rapidly accelerate thrombus formation. They also lack the ability to actively promote cell proliferation, tissue remodeling, and wound healing, resulting in slow hemostasis, limited hemostatic effect, and long wound repair cycles, failing to meet the modern clinical demand for integrated rapid blood control and wound repair. Therefore, there is an urgent need to develop safe cross-linking modification technology and introduce functional components for compounding, so as to endow polysaccharide sponges with the dual functions of active coagulation and hemostasis and efficient healing promotion, thereby breaking through the technical bottleneck of existing hemostatic materials.

[0005] Pullulan is a natural microbial polysaccharide produced by the fermentation of *Brachystomata buddingae*. It possesses excellent biocompatibility and biodegradability, and is easy to process and inexpensive, making it ideal as a substrate for hemostatic sponges. However, pullulan molecules are rich in hydroxyl groups and are highly water-soluble, making them unsuitable for direct application to bleeding wounds. Cross-linking modification is necessary to improve structural stability. Currently, pullulan sponges often use chemical cross-linking agents such as glutaraldehyde, EDC / NHS, and epoxy resins. While these achieve cross-linking effects, they present issues such as cytotoxicity, residual risks, and insufficient biocompatibility. Furthermore, single pullulan sponges only provide passive hemostasis through fluid absorption, lacking active coagulation and healing-promoting capabilities, resulting in low hemostatic efficiency and slow wound repair, failing to meet the dual requirements of efficient hemostasis and rapid healing.

[0006] Tissue factor (TF) is the strongest natural coagulation initiator in the human body. When exposed to blood, it rapidly activates the extrinsic coagulation cascade, significantly accelerating clot formation and possessing irreplaceable advantages in controlling acute massive bleeding. However, TF has poor stability, is easily inactivated when used directly, and poses a risk of systemic thrombosis. Therefore, it requires a carrier to achieve stable loading and controlled release. Collagen is a natural structural protein that can activate platelets, initiate the intrinsic coagulation pathway, and provide a scaffold for cell adhesion and proliferation, possessing both hemostatic and healing-promoting functions.

[0007] Among the existing publicly available technologies, patent CN117357687A discloses a method for preparing pullulan polysaccharide hemostatic sponges by constructing a double network structure through sodium periodate oxidation self-crosslinking, gallium ion ion crosslinking, and borax borate ester bond crosslinking. Although this method effectively improves the mechanical and water absorption properties of pullulan polysaccharide sponges, it lacks active coagulation and efficient healing promotion functions. Patent CN116983461A discloses a hemostatic material based on alginate and composite liposomes. Although it can achieve filling hemostasis for irregular wounds, it has defects such as low mechanical strength, limited liquid absorption ratio, and single application dosage form. Patent CN114805628A discloses a method for crosslinking polysaccharides and collagen using epoxy crosslinking agents. Although it has a certain hemostatic effect, the crosslinking agents have potential cytotoxicity and residue risks, resulting in insufficient biosafety. Based on this, the present invention provides a pullulan polysaccharide composite sponge with heat-assisted citric acid crosslinking. Citric acid is used as a biocompatible crosslinking agent to integrate TF-liposomes and collagen, addressing the core shortcomings of existing dressings such as poor safety, limited functionality, low hemostatic efficiency, and poor wound healing effects. The combination of pullulan polysaccharide, collagen, and tissue factor achieves a synergistic improvement in porous structure construction, enhanced mechanical properties, active coagulation initiation, and wound repair promotion. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing pullulan polysaccharide composite sponges with heat-assisted citric acid crosslinking. This method uses citric acid as a biocompatible crosslinking agent and employs solvent-free heat-assisted esterification crosslinking. The process is mild, leaves no toxic residues, and the resulting composite sponge possesses high porosity, high liquid absorbency, excellent mechanical properties, efficient hemostatic ability, and good healing-promoting properties, while exhibiting high biosafety.

[0009] To achieve the above-mentioned technical effects, the present invention relates to a method for preparing pullulan polysaccharide hemostatic sponge based on heat-assisted crosslinking, specifically including the following steps:

[0010] (1) Dissolve collagen in water to prepare collagen aqueous solutions with concentrations of 7.5 mg / mL, 15 mg / mL or 30 mg / mL, and stir until completely dissolved;

[0011] (2) Add 0.6 g pullulan and 0.4 g citric acid to the collagen aqueous solution obtained in step (1), and continue stirring until completely dissolved to obtain a mixture;

[0012] (3) Transfer the mixture obtained in step (2) into a mold, centrifuge to remove air bubbles, freeze at -20°C for 12 hours, then transfer to -80°C for further freezing, and then freeze-dry with pulluland to obtain the sponge precursor;

[0013] (4) The sponge precursor obtained in step (3) is heated in an oven at 130°C for 3 hours to carry out esterification and crosslinking reaction. After cooling, it is rinsed with deionized water to remove unreacted components and obtain collagen composite sponge (PC@COL).

[0014] (5) The composite sponge obtained in step (4) and TF-liposome stock solution were incubated together at 37°C for 0.5 hours. The volume ratio of liposome solution to sponge was 3:1. After incubation, the above composite sponge was obtained.

[0015] The collagen involved in this invention has a molecular weight of 380 kDa and is derived from fresh tilapia skin, but is not limited to this type.

[0016] The TF-liposomes involved in this invention are recombinantly expressed in Escherichia coli with a molecular weight of approximately 27 kDa, purified to approximately 95% purity, and reconstructed into DOPC / DOPS liposomes. However, this is not the only method.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) Citric acid is used as a biocompatible crosslinking agent, and thermal assisted esterification crosslinking is used to avoid toxic chemical crosslinking agents, resulting in high biosafety; (2) The preparation process is solvent-free, simple in steps, mild in conditions, and easy to scale up production; (3) The composite sponge has a porosity of ≥80%, a water absorption rate of 13.5-28.7 times, a blood absorption rate of 17.4-37.0 times, high mechanical strength, no cracking under compression, and good shape recovery; (4) It integrates pullulan polysaccharide, collagen, and tissue factor to achieve rapid hemostasis and efficient healing promotion; (5) Tissue factor is encapsulated by liposomes, has high stability, and is released slowly, achieving local strong hemostasis and avoiding the risk of systemic thrombosis; (6) The hemolysis rate is <2%, and the cell viability is ≥85%, which fully meets the clinical biosafety requirements. Attached Figure Description

[0018] Appendix Figure 1 An image showing the shape recovery of the PC@COL sponge after absorbing water, which is part of the invention described in this paper.

[0019] Appendix Figure 2 The microstructure morphology of PC and PC@COL sponges in Embodiment 1 of the present invention;

[0020] Appendix Figure 3 The FTIR spectra of pullulan, PC, and PC@COL sponges with different collagen contents in Example 1 of this invention are shown below.

[0021] Appendix Figure 4 The porosity of PC@COL sponge in Example 1 of this invention;

[0022] Appendix Figure 5The water absorption rate of PC@COL sponge in Example 1 of this invention;

[0023] Appendix Figure 6 The blood absorption rate of PC@COL sponge in Example 1 of this invention;

[0024] Appendix Figure 7 The stress-strain curve of PC@COL sponge under 80% compressive stress in Example 1 of the present invention;

[0025] Appendix Figure 8 The accompanying drawings are for the abstract of this invention.

[0026] Detailed Implementation of the Invention

[0027] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0028] Example 1:

[0029] (1) Preparation of composite sponge

[0030] Collagen extracted from tilapia skin was dissolved in water and stirred until completely dissolved to obtain a collagen aqueous solution. Pullulan powder and citric acid were added to the aqueous solution at a mass ratio of pullulan to citric acid of 3:2, and stirring continued until all components were completely dissolved to obtain a mixture. The mixture was transferred to a mold, centrifuged to remove air bubbles, and then frozen at -20°C for 12 hours, followed by further freezing at -80°C and then freeze-drying to obtain a sponge precursor. The sponge precursor was heated in an oven at 130°C for 3 hours to carry out an esterification and cross-linking reaction. After cooling, it was rinsed with deionized water to remove unreacted components to obtain a pullulan-collagen composite sponge. The composite sponge was co-incubated with tissue factor TF-liposome stock solution at 37°C for 0.5 hours at a volume ratio of liposome solution to sponge of 3:1. After incubation, a TF-loaded composite sponge was obtained.

[0031] (2) Microscopic morphological characterization

[0032] The composite sponge and TF-liposome stock solution were co-incubated at 37°C for 0.5 hours, with a volume ratio of liposome solution to sponge of 3:1. After incubation, TF-loaded composite sponges were obtained. The microstructure of the composite sponge was observed using scanning electron microscopy with an accelerating voltage of 3.0 kV. The samples were tested after gold sputtering treatment.

[0033] (3) Chemical structure characterization

[0034] The composite sponge was tested using Fourier transform infrared spectroscopy (FTIR) with the KBr pellet method, and the scanning range was 400-4000 cm⁻¹. -1The C1s high-resolution spectrum of the sample was measured using an X-ray photoelectron spectrometer, with 128 scans performed.

[0035] (4) Porosity test

[0036] The porosity of the composite sponge was determined by the ethanol displacement method. The mass of the dry sponge and the mass after ethanol saturation were recorded, and the porosity was calculated according to formula (1). Wherein, ρ is the density of anhydrous ethanol (0.789 g / cm³), and V is the volume of the sponge.

[0037] Porosity = ×100% formula (1)

[0038] (5) Water absorption rate and blood absorption rate test

[0039] The water absorption rate and blood absorption rate of the composite sponge were determined by gravimetric method. The dry sponge was immersed in deionized water and rabbit whole blood for 1 hour, respectively. The surface fluid was wiped off and the sponge was weighed. The water absorption rate and blood absorption rate were calculated by formula (2).

[0040] Liquid absorption rate = ×100% formula (2)

[0041] (6) Mechanical property testing

[0042] A cylindrical sponge sample with a diameter of 20 mm and a height of 20 mm was subjected to compression testing using a universal testing machine at a compression rate of 10 mm / min until the strain reached 80%. The stress-strain curve was then recorded.

[0043] Example 2:

[0044] This embodiment is the same as Example 1 except that the concentration of collagen aqueous solution in step (1) is set to 15 mg / mL. The other preparation steps, raw material ratios and characterization test methods are the same as those in Example 1.

[0045] Example 3:

[0046] This embodiment is the same as Example 1 except that the concentration of collagen aqueous solution in step (1) is set to 30 mg / mL. The other preparation steps, raw material ratios and characterization test methods are the same as those in Example 1.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still make modifications or equivalent substitutions to the foregoing solutions, and these modifications or substitutions do not cause the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing pullulan polysaccharide hemostatic sponge based on heat-assisted crosslinking, characterized in that, Specifically, the following steps are included: (1) Dissolve collagen in water to prepare collagen aqueous solutions with concentrations of 7.5 mg / mL, 15 mg / mL or 30 mg / mL, and stir until completely dissolved; (2) Add pullulan powder and citric acid to the collagen aqueous solution obtained in step (1), and continue stirring until all components are completely dissolved to obtain a mixture. The mass ratio of pullulan to citric acid is 3:

2. (3) Transfer the mixture obtained in step (2) into a mold, centrifuge to remove air bubbles, freeze at -20°C for 12 hours, then transfer to -80°C for further freezing, and then freeze dry to obtain the sponge precursor; (4) The sponge matrix obtained in step (3) is heated in an oven at 130°C for 3 hours to carry out esterification and crosslinking reaction. After cooling, it is rinsed with deionized water to remove unreacted components and obtain plutol-collagen composite sponge (PC@COL). (5) The composite sponge obtained in step (4) and tissue factor (TF)-liposome stock solution were incubated together at 37°C for 0.5 hours. The volume ratio of liposome solution to sponge was 3:

1. After incubation, the composite sponge was obtained.

2. The method for preparing pullulan polysaccharide composite sponge crosslinked with citric acid according to claim 1, characterized in that, The collagen, with a molecular weight of 380 kDa, was derived from fresh tilapia skin. The TF-liposomes were recombinantly expressed from tissue factor with a molecular weight of approximately 27 kDa in E. coli, purified to approximately 95% purity, and reconstructed into DOPC / DOPS liposomes.

3. The application of the composite sponge as described in claims 1-2 in the preparation of hemostatic wound care materials.

4. The application according to claim 3, characterized in that, The hemostatic wound care materials include military wound hemostatic dressings, surgical wound hemostatic dressings, and accidental wound hemostatic dressings.

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