Multifunctional nasal cavity hemostatic sponge as well as preparation method and application thereof

Abstract

The invention discloses a preparation method and application of a multifunctional nasal cavity hemostatic sponge. The preparation method comprises the following steps: dissolving gamma-polyglutamic acid in a solvent, and stirring until the gamma-polyglutamic acid is completely dissolved; the preparation method comprises the following steps: dissolving quaternized carboxymethyl chitosan in a solvent, and stirring until the quaternized carboxymethyl chitosan is completely dissolved; uniformly mixing the two obtained solutions to obtain a mixed solution; adding a high polymer into the mixed solution, then adding an activating agent, and reacting completely to obtain the multifunctional nasal cavity hemostatic hydrogel. Or adding a medicine, a high polymer and an activating agent, and reacting completely to obtain the multifunctional nasal cavity hemostasis hydrogel. And then freeze-drying the obtained hydrogel to obtain the multifunctional nasal cavity hemostatic sponge. The prepared multifunctional nasal cavity hemostatic sponge has good biocompatibility and can be applied to safe and efficient postoperative hemostasis, wound healing and nasosinusitis treatment.

Description

Technical Field

[0001] The invention relates to a gel composite, in particular to a multifunctional nasal hemostatic sponge and a preparation method and application thereof, belonging to the technical field of bionanomaterials. Background Art

[0002] Carboxymethyl chitosan (CMCS) is a natural polymer obtained by carboxymethylation of chitosan. Its chemical structure contains carboxymethyl functional groups, resulting in excellent water solubility and biocompatibility. Furthermore, CMCS can be further functionalized through chemical modification and self-assembly to achieve diverse physicochemical properties and functions to meet the needs of diverse applications. Consequently, CMCS has been widely used in areas such as drug sustained release, tissue engineering, and food preservation, and shows promising application prospects.

[0003] Polyglutamic acid (γ-PGA) is a biodegradable polymer with excellent biocompatibility. It is formed by polymerizing the natural amino acid glutamic acid and can be degraded into carbon dioxide and water by microorganisms. The physicochemical properties of polyglutamic acid can be manipulated by adjusting the degree of polymerization, substituents, and cross-linking, thus offering broad application prospects. Currently, polyglutamic acid has been applied in medicine, food, agriculture, and other fields, becoming an important biodegradable polymer material.

[0004] Polyvinyl alcohol (PVA) is a synthetic linear semi-crystalline polymer with a carbon chain as the main chain and hydroxyl groups as functional groups. It is an important and widely used non-ionic hydrophilic polymer. Polyvinyl alcohol has the characteristics of low cost, biodegradability, biocompatibility, non-toxicity, water solubility, excellent film-forming ability, thermal stability and adhesion, making it widely used in medicine, cosmetics, food and other fields. Cross-linking is the most attractive and widely used method to change the properties of polyvinyl alcohol to make it a more valuable material. Polyvinyl alcohol can be cross-linked by two different methods: physical methods or chemical methods. Among them, physical methods do not increase cytotoxic cross-linking, thereby avoiding adverse effects in biomedical applications, and have attracted widespread attention in the field of hydrogels. With the continuous deepening of research on the functionalization of polyvinyl alcohol, its application prospects in various fields will be broader.

[0005] Hydrogels are materials composed of polymers with a three-dimensional network structure that can form a gel in water. Hydrogels possess excellent biocompatibility and biodegradability, making them suitable for applications in tissue engineering, drug delivery, and biosensing. The physicochemical properties of hydrogels can be manipulated by adjusting the type of polymer, degree of cross-linking, and pore size, thereby controlling the gel's mechanical properties, water absorption, and drug release. Furthermore, hydrogels can be prepared using a variety of methods, such as self-assembly, template generation, and microfluidics, to meet the needs of diverse applications. Due to their unique structure and excellent properties, hydrogels have become an important biomaterial with broad application prospects in the biomedical field. Further processing hydrogels into a sponge form can significantly enhance their physical properties and application potential. This hydrogel-like sponge not only retains the inherent biocompatibility and high fluid absorption capacity of hydrogels, but also exhibits a larger specific surface area, faster fluid absorption rate, and excellent compressible resilience. These properties make it particularly suitable as a physical packing hemostatic material, which can quickly absorb blood and apply local pressure. At the same time, it is easier to operate and fit the wound surface in complex cavities such as the nasal cavity. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing nasal hemostatic materials have a single effect and cannot promote wound healing.

[0007] In order to solve the above problems, the present invention provides a method for preparing a multifunctional nasal hemostatic sponge, which is characterized by comprising the following steps:

[0008] Step A: dissolving carboxymethyl chitosan in a solvent and stirring to completely dissolve it;

[0009] Step B: grafting a quaternary ammonium salt into a carboxymethyl chitosan solution to react and obtain quaternized carboxymethyl chitosan;

[0010] Step C: dissolving γ-polyglutamic acid in a solvent and stirring to completely dissolve it;

[0011] Step D: dissolving the quaternized carboxymethyl chitosan in a solvent and stirring to completely dissolve it;

[0012] Step E: The two solutions obtained in Steps C and D are mixed uniformly to obtain a mixed solution; a polymer is then added to the mixed solution, followed by an activator, and the reaction is complete to obtain a multifunctional nasal hemostatic hydrogel. Alternatively, a drug, a polymer, and an activator are added, and the reaction is complete to obtain a multifunctional nasal hemostatic hydrogel.

[0013] Step F: freeze-drying the hydrogel obtained in step E to obtain a multifunctional nasal hemostatic sponge.

[0014] Preferably, the solvent in step A is any one of distilled water, phosphate buffer solution (pH=7.4), and physiological saline (w / v=0.9%); the mass concentration of the dissolved carboxymethyl chitosan is 0.5-15%; and the dissolution temperature of the carboxymethyl chitosan is 30-80°C.

[0015] Preferably, the quaternary ammonium salt in step B is any one of glycerol trimethylammonium chloride and 2,3-epoxypropyltrimethylammonium chloride; the mass concentration of the quaternary ammonium salt is 1-10%; the reaction temperature is 30-100° C.; the reaction time is 5-72 hours; and the mass ratio of the quaternary ammonium salt to carboxymethyl chitosan is 0.3-1.5:1.

[0016] Preferably, the solvent in step C is any one of distilled water, phosphate buffer solution (pH=7.4), and normal saline (w / v=0.9%); the mass concentration of the dissolved γ-polyglutamic acid is 0.5-15%; and the dissolution temperature of the γ-polyglutamic acid is 30-80°C.

[0017] Preferably, the solvent in step D is any one of distilled water, phosphate buffer solution (pH=7.4), and normal saline (w / v=0.9%); the mass concentration of the dissolved quaternized carboxymethyl chitosan is 0.5-15%; and the dissolution temperature of the quaternized carboxymethyl chitosan is 30-80°C.

[0018] Preferably, the activator in step E is any one or both of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; the mass concentration of the activator is 1 to 30%; the mass concentration of the activator (any one or both of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide) in the mixed solution is 0.5 to 50%; the mass ratio of the γ-polyglutamic acid to the quaternized carboxymethyl chitosan is 0.5 to 2:1.

[0019] Preferably, the reaction temperature in step E is 10-30°C.

[0020] Preferably, the reaction time in step E is 0.01 to 1 h.

[0021] Preferably, the polymer in step E is any one of polyvinyl alcohol and polyethylene glycol; the mass concentration of the polymer is 5-30%; the mass concentration of the polymer (any one of polyvinyl alcohol and polyethylene glycol) in the mixed solution is 5-50%.

[0022] Preferably, the drug in step E is various hormone drugs, such as mometasone furoate or budesonide, etc. The concentration of the drug in the mixed solution is 0.1-10 mg / mL.

[0023] Preferably, the freezing time in step F is 1 to 72 hours, and the freezing temperature is -10 to -40°C.

[0024] The present invention also provides an application of the multifunctional sponge prepared by the above preparation method in nasal antibacterial hemostasis and drug delivery or drug sustained-release biomaterials.

[0025] The present invention adds a co-crosslinked polymer and an activator to a solution of γ-polyglutamic acid and quaternized carboxymethyl chitosan. Taking mometasone furoate as a hormonal drug, polyvinyl alcohol (Type 1799) as a co-crosslinked polymer, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS) as an activator as an example, two sponge preparation methods are provided: ① Dissolve γ-polyglutamic acid and quaternized carboxymethyl chitosan in a solvent, stir evenly, add the polyvinyl alcohol solution, and then add EDC-NHS to form a hydrogel. This is followed by freeze-drying to form a sponge; ② Dissolve γ-polyglutamic acid and quaternized carboxymethyl chitosan in a solvent, stir evenly, add the mometasone furoate and polyvinyl alcohol solution, and then add EDC-NHS to form a hydrogel. This is followed by freeze-drying to form a sponge. Both methods can produce a multifunctional nasal hemostatic sponge with a specific network structure.

[0026] The invention utilizes the property of an activator that can activate the carboxyl groups in gamma-polyglutamic acid so that the activator can undergo a cross-linking reaction with quaternized carboxymethyl chitosan and polyvinyl alcohol, and provides a preparation method for cross-linking gamma-polyglutamic acid, quaternized carboxymethyl chitosan and polyvinyl alcohol to form a gel under the activation effect of EDC-NHS.

[0027] The process of the present invention is simple, the product preparation time is short, and the obtained product has demonstrated good biocompatibility in both in vivo and in vitro simulation experiments. The present invention selects γ-polyglutamic acid and quaternized carboxymethyl chitosan as matrices, polyvinyl alcohol as a co-crosslinked polymer, and utilizes EDC-NHS to activate the carboxyl groups in γ-polyglutamic acid, so that it can be cross-linked with quaternized carboxymethyl chitosan and can also undergo cross-linking reaction with polyvinyl alcohol to prepare a multifunctional nasal hemostatic sponge. Experiments show that the sponge prepared by the present invention has good flexibility and elasticity. The sponge can enter the nasal cavity through a packing route, quickly stop bleeding at the bleeding site, and has a sustained antibacterial effect, which can effectively reduce nasal damage and infection.

[0028] Compared with existing technologies, the present invention represents a significant technological advancement. The sponge produced by the present invention is simple to manufacture, readily available, and exhibits excellent flexibility and elasticity, adapting to various shapes and surfaces. This approach addresses the shortcomings of existing nasal hemostatic materials, such as their limited effectiveness and inability to promote wound healing. The present invention is expected to be applied to hemostasis and wound healing in patients undergoing nasal surgery, demonstrating significant clinical application value.

[0029] The present invention also provides the use of the sponge as a safe and efficient antibacterial biomaterial. The multifunctional nasal hemostatic sponge prepared by the present invention has excellent flexibility and elasticity, good biocompatibility, and can be used for safe and efficient postoperative hemostasis and wound healing.

[0030] The present invention also provides the use of the sponge as a biomaterial for safe and efficient drug delivery and sustained drug release, which can be used for safe and efficient drug delivery and sustained drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 a is a FESEM image of the multifunctional nasal hemostatic sponge in Example 7, with a scale of 200 μm;

[0032] Figure 1 b is a FESEM image of the multifunctional nasal hemostatic sponge in Example 7, with a scale of 100 μm;

[0033] Figure 2 This is a dynamic time-scan rheological analysis of the multifunctional nasal hemostatic sponge in Example 8;

[0034] Figure 3 a is the compression strain-stress curve of the mechanical properties of the multifunctional nasal hemostatic sponge in Example 9;

[0035] Figure 3 b is the average compressive stress and compression modulus test results of the mechanical properties of the multifunctional nasal hemostatic sponge in Example 9;

[0036] Figure 4 a is the in vitro hemolysis test result of the multifunctional nasal hemostatic sponge prepared in Example 6;

[0037] Figure 4 b is the survival rate of fibroblasts after co-culture with the multifunctional nasal hemostatic sponge prepared in Example 6,

[0038] Figure 4 c is the DEAD / LIVE staining result of fibroblasts without material treatment;

[0039] Figure 4 d DEAD / LIVE staining results of fibroblasts treated with 2.5 mg / mL of the multifunctional nasal hemostatic sponge prepared in Example 6;

[0040] Figure 4 e DEAD / LIVE staining results of fibroblasts treated with 5 mg / mL of the multifunctional nasal hemostatic sponge prepared in Example 6;

[0041] Figure 4 f DEAD / LIVE staining results of fibroblasts treated with 10 mg / mL of the multifunctional nasal hemostatic sponge prepared in Example 6;

[0042] Figure 5 a is the swelling ratio of the multifunctional nasal hemostatic sponge in deionized water in Examples 12 and 13;

[0043] Figure 5 b is the swelling kinetics curve of the multifunctional nasal hemostatic sponge in deionized water in Examples 12 and 13;

[0044] Figure 5 c is the swelling rate of the multifunctional nasal hemostatic sponge in Examples 12 and 13 in simulated body fluid;

[0045] Figure 5 d is the swelling kinetics curve of the multifunctional nasal hemostatic sponge in Examples 12 and 13 in simulated body fluid;

[0046] Figure 5 e is the in vitro degradation curve of the multifunctional nasal hemostatic sponge in deionized water in Examples 12 and 13;

[0047] Figure 5 f is the in vitro degradation curve of the multifunctional nasal hemostatic sponge in Examples 12 and 13 in simulated body fluid;

[0048] Figure 6 a is the in vitro antibacterial test results of the multifunctional nasal hemostatic sponge in Examples 14 and 15, showing the proliferation of Escherichia coli colonies on an agar plate;

[0049] Figure 6 b is the in vitro antibacterial test results of the multifunctional nasal hemostatic sponge in Examples 14 and 15, and the antibacterial rate of the multifunctional nasal hemostatic sponge against Escherichia coli;

[0050] Figure 6 c is the in vitro antibacterial test results of the multifunctional nasal hemostatic sponge in Examples 14 and 15, showing the proliferation of Staphylococcus aureus colonies on an agar plate;

[0051] Figure 6 d is the in vitro antibacterial test result of the multifunctional nasal hemostatic sponge in Examples 14 and 15, and the inhibition rate of the multifunctional nasal hemostatic sponge against Staphylococcus aureus;

[0052] Figure 7The results of routine blood tests and blood biochemistry tests for the in vivo biological safety of the multifunctional nasal hemostatic sponge prepared in Example 6 are shown, where a, b, c, and d represent routine blood tests, and e and f represent blood biochemistry tests;

[0053] Figure 8 The H&E staining results of the in vivo biosafety of the multifunctional nasal hemostatic sponge prepared in Example 6 are as follows;

[0054] Figure 9 a is a photo of the in vitro coagulation of the multifunctional nasal hemostatic sponge prepared in Example 4;

[0055] Figure 9 b is the in vitro coagulation index of the multifunctional nasal hemostatic sponge prepared in Example 4;

[0056] Figure 10 a is a photograph of the in vivo nasal hemostasis process of the multifunctional nasal hemostatic sponge prepared in Example 4;

[0057] Figure 10 b is the in vivo nasal hemostasis time of the multifunctional nasal hemostatic sponge prepared in Example 4;

[0058] Figure 10 c is the amount of bleeding in vivo after nasal hemostasis using the multifunctional nasal hemostatic sponge prepared in Example 4;

[0059] Figure 11 This is the in vivo treatment result of sinusitis using the multifunctional nasal hemostatic sponge prepared in Example 6 (PAS staining image of the nasal septum). DETAILED DESCRIPTION

[0060] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0061] Example 1

[0062] 2.0 g of carboxymethyl chitosan was dissolved in 80 mL of deionized water, and then 3.6 g of 2,3-epoxypropyltrimethylammonium chloride was dispersed in the above solution. After reacting at 80°C for 36 h, the solution was dialyzed in deionized water for 72 h and finally freeze-dried using a freeze dryer to obtain quaternized carboxymethyl chitosan.

[0063] Example 2

[0064] 2.0 g of carboxymethyl chitosan was dissolved in 80 mL of deionized water, and then 3.0 g of glycerol trimethylammonium chloride was dispersed in the above solution. After reacting at 80° C. for 24 h, the solution was dialyzed in deionized water for 72 h and finally freeze-dried using a freeze dryer to obtain quaternized carboxymethyl chitosan.

[0065] Example 3

[0066] Take 0.5g of quaternized carboxymethyl chitosan and 0.25g of γ-polyglutamic acid, dissolve them in 10mL of deionized water, and stir them evenly at 60°C to obtain a carboxymethyl chitosan-γ-polyglutamic acid mixed solution. Take 0.1g of polyvinyl alcohol and dissolve it in 10mL of deionized water to obtain a polyvinyl alcohol solution. Then, take 1mL of the carboxymethyl chitosan-γ-polyglutamic acid mixed solution and 500μL of polyvinyl alcohol solution in a glass bottle, add 200μL of EDC-NHS mixed solution (concentration 0.1g / mL) to the glass bottle, stir evenly, and obtain a multifunctional nasal hemostatic hydrogel. Subsequently, freeze it at -20°C for 12h and dry it to obtain a multifunctional nasal hemostatic sponge.

[0067] Example 4

[0068] Take 0.5g of quaternized carboxymethyl chitosan and 0.5g of γ-polyglutamic acid, dissolve them in 10mL of deionized water, and stir them evenly at 60°C to obtain a carboxymethyl chitosan-γ-polyglutamic acid mixed solution. Take 0.1g of polyvinyl alcohol and dissolve it in 10mL of deionized water to obtain a polyvinyl alcohol solution. Then, take 1mL of the carboxymethyl chitosan-γ-polyglutamic acid mixed solution and 500μL of polyvinyl alcohol solution in a glass bottle, add 200μL of EDC-NHS mixed solution (concentration 0.1g / mL) to the glass bottle, stir evenly, and obtain a multifunctional nasal hemostatic hydrogel. Subsequently, freeze it at -20°C for 12h and dry it to obtain a multifunctional nasal hemostatic sponge.

[0069] Example 5

[0070] Take 0.5g of quaternized carboxymethyl chitosan and 0.5g of γ-polyglutamic acid, dissolve them in 10mL of deionized water, and stir them evenly at 60°C to obtain a carboxymethyl chitosan-γ-polyglutamic acid mixed solution. Take 0.1g of polyvinyl alcohol and dissolve it in 10mL of deionized water to obtain a polyvinyl alcohol solution. Then, take 1mL of the carboxymethyl chitosan-γ-polyglutamic acid mixed solution and 300μL of polyvinyl alcohol solution in a glass bottle, add 200μL of EDC-NHS mixed solution (concentration 0.1g / mL) to the glass bottle, stir evenly, and obtain a multifunctional nasal hemostatic hydrogel. Subsequently, freeze it at -20°C for 12h and dry it to obtain a multifunctional nasal hemostatic sponge.

[0071] Example 6

[0072] 0.5 g of quaternized carboxymethyl chitosan and 0.5 g of γ-polyglutamic acid were dissolved in 10 mL of deionized water and stirred at 60°C to obtain a carboxymethyl chitosan-γ-polyglutamic acid mixed solution. Subsequently, 2 mg of mometasone furoate was dissolved in 1 mL of the carboxymethyl chitosan-γ-polyglutamic acid mixed solution. 0.1 g of polyvinyl alcohol was dissolved in 10 mL of deionized water to obtain a polyvinyl alcohol solution. Then, 1 mL of the carboxymethyl chitosan-γ-polyglutamic acid mixed solution and 500 μL of the polyvinyl alcohol solution were placed in a glass bottle. 200 μL of the EDC-NHS mixed solution (concentration 0.1 g / mL) was added to the glass bottle and stirred to obtain a multifunctional nasal hemostatic hydrogel. Subsequently, the solution was frozen at -20°C for 12 hours and dried to obtain a multifunctional nasal hemostatic sponge.

[0073] Example 7

[0074] The morphology of the multifunctional nasal hemostatic sponge in Example 4 was analyzed. The sample was analyzed on a FEI Magellan 400 field emission scanning electron microscope. As can be seen from the electron microscope scanning image, the multifunctional nasal hemostatic sponge exhibits a three-dimensional pore structure ( Figure 1 ), and the pores are densely distributed and the pore size is small.

[0075] Example 8

[0076] The gelation process of the multifunctional nasal hemostatic sponge was studied by observing the final storage modulus (G′) and the final loss modulus (G″) in the dynamic time sweep rheological test. The dynamic rheological study was performed using a rotational rheometer (MARSⅢHAAKE) with a flat plate (P20TiL, 20 mm in diameter) geometry. The multifunctional nasal hemostatic sponge in Example 4 was subjected to a time sweep oscillation test at a frequency of 1 Hz, a gap of 1 mm, and a strain of 10%. The corresponding sponge precursor solution was injected into the flat plate, and the gap was adjusted to 1 mm. The frequency sweep measurement values of the hydrogel are expressed as G′ and G″. When G′ exceeds G″, the gel point is determined. Due to intermolecular cross-linking, the G′ of the sponge increases rapidly, indicating that the sponge formation efficiency is very high ( Figure 2 ).

[0077] Example 9

[0078] The multifunctional nasal hemostatic sponge prepared in Example 4 was mechanically evaluated on a Zwick Roell Z2.5 TH universal material testing machine using a 2.5 kN sensor. The compression properties of the multifunctional nasal hemostatic sponge were studied using a modified American Society for Testing and Materials method. In the compression test, the multifunctional nasal hemostatic sponge was formed into a cylinder with a diameter of 10 mm and a thickness of 3 mm, and the compression strain rate was 1 mm / min ( Figure 3a). In the strain range of 30-40%, the compression modulus was recorded by the linear fitting value of the stress-strain curve. The maximum compressive stress and compression modulus of the multifunctional nasal sponge prepared in Example 4 were 342.12 kPa and 6.77 kPa ( Figure 3 b).

[0079] Example 10

[0080] The blood compatibility of the multifunctional nasal hemostatic sponge was studied (Example 6). 2 mL of whole blood was centrifuged (5000 rpm, 5 minutes) and rinsed three times with phosphate buffer solution to obtain red blood cells. The obtained red blood cells were stored in 50 mL of phosphate buffer solution for further use. In the hemolysis test, 0.5 mL of the above mouse red blood cells were placed in a 5.0 mL centrifuge tube and incubated with (1) 2.5 mL of phosphate buffer solution (negative control), (2) 2.5 mL of deionized water (positive control) and (3) different masses of the multifunctional hemostatic sponge prepared in Example 6 (2.5, 5, 10, 20 mg / mL, in 2.5 mL of phosphate buffer solution) in a 37°C water bath for 2 hours. The absorbance value of the supernatant at 541 nm was collected (Shimadzu UV-3600 ultraviolet-visible near-infrared spectrometer, Japan), and the hemolysis rate of the red blood cells was calculated. As Figure 4 As shown in Figure a, the calculated hemolysis rates of the sponges were all less than 5%. As can be seen from the supernatant photograph, the supernatant of red blood cells incubated with the multifunctional sponge and phosphate buffer solution is transparent. However, the blood treated with deionized water exhibits a distinct red color due to positive hemolysis. These results demonstrate that the multifunctional nasal hemostatic sponge has excellent hemocompatibility.

[0081] Example 11

[0082] Fibroblasts were seeded in a 96-well plate and 100 μL of cell culture medium was added and cultured overnight. The above culture medium was discarded, and different weights of the multifunctional nasal hemostatic sponge obtained in Example 6 (2.5, 5, 10 mg / mL) and 100 μL of new cell culture medium were added. The control group only added 100 μL of cell culture medium (the survival rate was set to 100%). The above cells were placed in a CO2 constant temperature incubator and incubated for 24 hours. The cell survival was quantitatively and qualitatively detected using CCK-8 and LIVE / DEAD cell activity detection kits. Figure 4 As shown in middle b, the multifunctional nasal hemostatic sponge did not affect the survival of cells. Similar to the control group ( Figure 4 c), cells treated with multifunctional nasal hemostatic sponge can be stained green by LIVE / DEAD reagent (live cells are stained green), and almost no cells are stained red (dead cells are stained red) ( Figure 4 d. Figure 4 e and Figure 4f). The results of CCK-8 and LIVE / DEAD cell staining showed that the prepared multifunctional nasal hemostatic sponge had good cell compatibility.

[0083] Example 12

[0084] In order to investigate the liquid absorption capacity of the sponge, the multifunctional nasal hemostatic sponge obtained in Example 4 was added to deionized water and soaked at 37°C for 24 hours. The sponge was then gently wiped to remove surface moisture and weighed. Finally, the sponge was freeze-dried and the initial mass was weighed to determine the expansion ratio ( Figure 5 a). In addition, the swelling kinetics of the sponge in deionized water were studied ( Figure 5 b) demonstrates that the sponge absorbs fluid quickly, with a swelling rate of 84.99 g / g after 20 minutes of storage in deionized water. The swelling of the multifunctional nasal hemostatic sponge in simulated body fluid was performed using the same experimental procedures as described above for deionized water. After 20 minutes of storage in simulated body fluid, the sponge's swelling rate reached 37.80 g / g. Figure 5 c and Figure 5 d)

[0085] Example 13

[0086] Then the multifunctional nasal hemostatic sponge obtained in Example 4 was weighed and mixed with 10 mL of lysozyme (1×10 4 U / mL) of deionized water or simulated body fluid and incubated at 37°C with continuous stirring for 16 days. The culture medium was changed every other day. At each time point, the multifunctional nasal hemostatic sponge was removed from the culture medium, gently rinsed with deionized water or simulated body fluid, and freeze-dried. The mass of the freeze-dried sponge was weighed to calculate the degradation rate. The study found that after the sponge was degraded in deionized water containing lysozyme for 16 days, the degradation rate was similar, and about 26.91% of the original mass ( Figure 5 e). In contrast, the sponge degraded slightly faster in simulated body fluids, retaining approximately 21.52% of its original mass after 16 days of degradation ( Figure 5 f). The sponge completely degraded after 21 days, thus ensuring the long-term biocompatibility of the sponge.

[0087] Example 14

[0088] Escherichia coli was activated in liquid culture medium and cultured in a three-gas incubator at 37°C for 24 hours at a speed of 120 rpm. Then they were added to sterile liquid culture medium respectively, and the OD value of the bacterial suspension at 600nm was adjusted to about 0.1. The multifunctional nasal hemostatic sponge (prepared in Example 4) was placed in a test tube containing 5mL of bacterial suspension and incubated at 37°C in a three-gas incubator for 24 hours. Under the same conditions, a pure bacterial suspension was taken as a positive control. Subsequently, the above-mentioned resuscitated bacterial suspension (10μL) was diluted and spread on an agar plate, and cultured at 37°C in a three-gas incubator for 24 hours. The number of bacteria grown on the agar plate was counted. As Figure 6 As shown in a, after 24 hours of incubation, the growth of Escherichia coli colonies proved that the multifunctional nasal hemostatic sponge had a certain antibacterial effect on Gram-negative bacteria, with an antibacterial rate of 97.70% ( Figure 6 b).

[0089] Example 15

[0090] Staphylococcus aureus was activated in a liquid culture medium and cultured in a three-gas incubator at 37°C for 24 hours at a speed of 120 rpm. They were then added to a sterile liquid culture medium, and the OD value of the bacterial suspension at 600nm was adjusted to about 0.1. The multifunctional nasal hemostatic sponge (prepared in Example 4) was placed in a test tube containing 5mL of bacterial suspension and incubated in a three-gas incubator at 37°C for 24 hours. Under the same conditions, a pure bacterial suspension was taken as a positive control. Subsequently, the above-mentioned revived bacterial suspension (10 μL) was diluted and spread on an agar plate, and cultured in a three-gas incubator at 37°C for 24 hours. The number of bacteria grown on the agar plate was counted. As Figure 6 As shown in Figure c, after 24 hours of incubation, the colony growth of Staphylococcus aureus proved that the multifunctional nasal hemostatic sponge had a certain antibacterial effect on Gram-positive bacteria, with an antibacterial rate of 95.03% ( Figure 6 d).

[0091] Example 16

[0092] The multifunctional nasal hemostatic sponge obtained in Example 6 was implanted subcutaneously into Kunming mice. Blood routine and blood biochemistry tests were performed on Kunming mice on days 1, 3, 7, and 14. The results showed that the blood routine ( Figure 7 a- Figure 7 d) and blood biochemistry ( Figure 7 e and Figure 7 f) All indicators were within the standard range. The main organs of the mice (heart, liver, spleen, lung, and kidney) were then sectioned and stained with H&E for further histological study. Compared with the control group, the multifunctional nasal hemostatic sponge had no adverse effects on the important organs of the mice ( Figure 8), indicating that the multifunctional nasal hemostatic sponge has good in vivo safety. (WBC: white blood cell count; RBC: red blood cell count; RDW: red blood cell volume distribution width; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; HGB: hemoglobin; MCHC: mean corpuscular hemoglobin concentration; PLT: platelet count; TB: total bilirubin; Urea: urea; Crea: creatinine; ALT: alanine aminotransferase; AST: aspartate aminotransferase.)

[0093] Example 17

[0094] The in vitro coagulation ability of the multifunctional nasal hemostatic sponge was studied. The anticoagulant sodium citrate was mixed with mouse blood and set aside. 50 μL of whole blood and 10 μL of 0.1M CaCl2 aqueous solution were mixed and dripped onto the surface of the multifunctional nasal hemostatic sponge (prepared in Example 4). In addition, 50 μL of whole blood and 10 μL of 0.1M CaCl2 aqueous solution were mixed and dripped onto the surface of gauze or allowed to coagulate naturally in the air. All samples were stored at 37°C for 60, 90, and 120 seconds. Finally, 10 mL of deionized water was added to release the hemoglobin of uncoagulated mouse red blood cells. The absorbance value of the supernatant at 545 nm was collected using a UV-Vis-NIR spectrophotometer (Japan Shimadzu UV-3600 UV-visible near-infrared spectrometer), and the coagulation index was calculated. Figure 9 As shown in a, the supernatant of the gauze group and the natural coagulation group was red, while the supernatant of the blood treated with the multifunctional nasal hemostatic sponge group was lighter in color and almost transparent, indicating that the blood treated with the multifunctional nasal hemostatic sponge had almost completely coagulated. The dynamic coagulation index was calculated based on the absorbance value of the collected supernatant. At each culture time point, the dynamic coagulation index of the multifunctional nasal hemostatic sponge group was lower than that of the natural coagulation group and the gauze group ( Figure 9 b).

[0095] Example 18

[0096] The in vivo hemostatic ability of the multifunctional nasal hemostatic sponge was studied. Six healthy SD rats (300±20g) were selected and randomly divided into an experimental group and a control group (n=3). A nasal bleeding model was established: after anesthesia, a standard wound with a diameter of 2 mm was made in the sinus area using a microsyringe. The experimental group was immediately filled with the multifunctional nasal hemostatic sponge (diameter: 2 mm, height: 3 mm) prepared in Example 4, while the control group was only exposed to the wound without hemostatic intervention ( Figure 10 a). The results showed that the hemostasis time of the experimental group was 34.67s, which was significantly shorter than that of the control group (272.33s). Figure 10 b), the amount of bleeding in the experimental group (2.5 mg) was significantly less than that in the control group (118.7 mg) ( Figure 10c) It shows that the multifunctional nasal hemostatic sponge prepared by the present invention has excellent in vivo hemostatic performance.

[0097] Example 19

[0098] The efficacy of multifunctional nasal hemostatic sponge loaded with drugs in treating sinusitis was studied. SPF-grade C57BL / 6 mice were used as experimental subjects, and a sinusitis model was established by nasal inoculation: the concentration of Staphylococcus aureus suspension was adjusted to 1×10 8 CFU / mL, use a micropipette to drip into the bilateral nasal cavity of the mouse (10 μL / time on each side), once a day, for 28 consecutive days to establish an acute inflammation model. After the model was successfully established, the mice were divided into an experimental group and a positive control group (n=3) using a random number table method. Mice that were not infected with inflammation served as the negative control group. The negative control group and the positive control group did not receive any intervention treatment. Starting from the 30th day of modeling, the experimental group was filled with the multifunctional nasal hemostatic sponge (2 mm in diameter, 3 mm in height) prepared in Example 6 in both nasal cavities. Euthanasia was performed after 7 days of intervention, and sinus tissue was taken for Periodic Acid-Schiff (PAS) staining. Histopathological analysis showed that ( Figure 11 ), while the positive control group showed significant goblet cell proliferation in the sinus mucosa. The experimental group showed significant improvement in sinus tissue structure, with a significantly reduced number of goblet cells compared with the positive control group and similar to that of the negative control group. This indicates that the multifunctional nasal hemostatic sponge prepared by the present invention can effectively inhibit inflammatory responses in the sinuses.

[0099] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional nasal hemostatic sponge, characterized in that: The following steps are involved: Step A: dissolving carboxymethyl chitosan in a solvent and stirring to completely dissolve it; Step B: grafting a quaternary ammonium salt into a carboxymethyl chitosan solution to react and obtain quaternized carboxymethyl chitosan; Step C: dissolving γ-polyglutamic acid in a solvent and stirring to completely dissolve it; Step D: dissolving the quaternized carboxymethyl chitosan in a solvent and stirring to completely dissolve it; Step E: The two solutions obtained in Steps C and D are mixed to obtain a mixed solution; a polymer is then added to the mixed solution, followed by an activator, and the reaction is complete to obtain a multifunctional nasal hemostatic hydrogel. Alternatively, a drug, a polymer, and an activator are added to the mixed solution, and the reaction is complete to obtain a multifunctional nasal hemostatic hydrogel. Step F: freeze-drying the hydrogel obtained in step E to obtain a multifunctional nasal hemostatic sponge; The polymer in step E is any one of polyvinyl alcohol and polyethylene glycol; the mass concentration of the polymer is 5-30%; the mass concentration of the polymer in the mixed solution is 5-50%.

2. The method for preparing the multifunctional nasal hemostatic sponge according to claim 1, wherein: The solvent in step A is any one of distilled water, phosphate buffer solution, and physiological saline, the pH of the phosphate buffer solution is 7.4, and the concentration of the physiological saline w / v is 0.9%; the mass concentration of the dissolved carboxymethyl chitosan is 0.5-15%; and the dissolution temperature of the carboxymethyl chitosan is 30-80°C.

3. The method for preparing the multifunctional nasal hemostatic sponge according to claim 1, wherein: The quaternary ammonium salt in step B is any one of glycerol trimethylammonium chloride and 2,3-epoxypropyltrimethylammonium chloride; the mass concentration of the quaternary ammonium salt is 1-10%; the reaction temperature is 30-100° C.; the reaction time is 5-72 hours; and the mass ratio of the quaternary ammonium salt to carboxymethyl chitosan is 0.3-1.5:

1.

4. The method for preparing the multifunctional nasal hemostatic sponge according to claim 1, wherein: The solvent in step C is any one of distilled water, phosphate buffer solution, and physiological saline, the pH of the phosphate buffer solution is 7.4, and the concentration of the physiological saline w / v is 0.9%; the mass concentration of the dissolved γ-polyglutamic acid is 0.5-15%; and the dissolution temperature of the γ-polyglutamic acid is 30-80°C.

5. The method for preparing the multifunctional nasal hemostatic sponge according to claim 1, wherein: The solvent in step D is any one of distilled water, phosphate buffer solution, and normal saline, the pH of the phosphate buffer solution is 7.4, and the concentration of the normal saline w / v is 0.9%; the mass concentration of the dissolved quaternized carboxymethyl chitosan is 0.5-15%; and the dissolution temperature of the quaternized carboxymethyl chitosan is 30-80°C.

6. The method for preparing the multifunctional nasal hemostatic sponge according to claim 1, wherein: The activator in step E is any one or both of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; the mass concentration of the activator is 1 to 30%; the mass concentration of the activator (any one or both of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide) in the mixed solution is 0.5 to 50%; the mass ratio of the γ-polyglutamic acid to the quaternized carboxymethyl chitosan is 0.5 to 2:

1.

7. The method for preparing the multifunctional nasal hemostatic sponge according to claim 1, wherein: The reaction temperature in step E is 10-30° C.; the reaction time in step E is 0.01-1 h; the hormone drug in step E is mometasone furoate or budesonide; and the concentration of the drug in the mixed solution is 0.1-10 mg / mL.

8. The method for preparing the multifunctional nasal hemostatic sponge according to claim 1, wherein: The freezing time in step F is 1 to 72 hours, and the freezing temperature is -10 to -40°C.

9. The multifunctional nasal hemostatic sponge obtained by the method according to any one of claims 1 to 8.

10. Use of the multifunctional nasal hemostatic sponge according to claim 9 in preparing biomaterials for postoperative hemostasis, wound healing and sinusitis treatment.

Images