Postoperative anti-adhesion sponge patch as well as preparation method and application thereof

Abstract

The invention discloses a postoperative anti-adhesion sponge patch as well as a preparation method and application thereof, and particularly relates to the technical field of biomedical materials. The sponge patch comprises an HAMA sponge of an anti-adhesion layer and a CS-CA sponge of an adhesion layer; wherein the HAMA layer and the CS-CA layer form polymer network interpenetrating interaction (IPN) and physical interaction through permeation interlocking, and the HAMA layer and the CS-CA layer are tightly combined together after being freeze-dried. The HAMA-CS-CA sponge patch disclosed by the invention has the advantages that interface water is efficiently removed, wet tissue adhesion is enhanced, and cationic charges in catechol and chitosan synergistically enhance hemostatic performance; meanwhile, according to the HAMA-CS-CA sponge patch disclosed by the invention, the HAMA contains anionic charges, so that proteins and cells with negative charges on the surfaces can be effectively dispersed, and the functions of preventing protein adsorption and cell adhesion are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a postoperative anti-adhesion sponge patch and a preparation method and application thereof. Background Art

[0002] Postoperative adhesions are fibrous scar complications caused by peritoneal injury after abdominal surgery. Clinical data show that approximately 54% of patients undergoing abdominal surgery develop postoperative adhesions, which can lead to secondary conditions such as chronic abdominal pain and infertility, placing a heavy health burden on patients. The primary pathogenesis is the abnormal accumulation of macrophages and fibroblasts at the site of peritoneal injury, which in turn triggers a cascade of inflammatory responses and fibrosis, ultimately leading to the formation of postoperative adhesions. Therefore, the development of a postoperative protective patch that combines wound closure with anti-adhesion capabilities is of great significance. An ideal anti-adhesion barrier should possess the following characteristics: forming a stable physical isolation layer; dispersing macrophages to regulate local inflammatory responses; and inhibiting excessive fibrin deposition.

[0003] Hyaluronic acid (HA), a natural anionic polysaccharide, has been used clinically as an anti-adhesion barrier due to its excellent lubricity, biocompatibility, and biodegradability. In addition, high molecular weight HA can not only form a physical barrier, but also disperse negatively charged fibroblasts and macrophages on the surface through charge repulsion, and regulate local inflammatory responses. Currently, there are three main application forms of hyaluronic acid-based anti-adhesion materials: (1) in situ injection of pure HA solution, which has the disadvantage of short retention time in the body; (2) photocrosslinked HA hydrogel, which requires in situ gelation induced by ultraviolet light and has high operational complexity; (3) thermal polymerization gel patch, such as Chinese patents CN102908626A and CN115282339A, which are both hyaluronic acid and its derivatives and can achieve physical isolation, but the preparation cycle is long and there is a risk of dehydration failure. In comparison, sponge patches can be gelled by in situ absorption of exudate, and have the advantages of simple operation, long retention time, and easy storage.

[0004] While preventing adhesion, the new anti-adhesion barrier should also have hemostatic and tissue adhesion properties to achieve wound closure, replacing surgical sutures. The dry cross-linking mechanism is an effective strategy for wet tissue adhesion, achieving immediate adhesion by quickly removing interfacial water, and its core lies in the effective removal of interfacial water. Chinese patent CN104874029A, the material includes an anti-adhesion layer with a dense structure and a hemostatic layer with a loose structure, wherein the anti-adhesion layer includes at least two layers of anti-adhesion sublayers wrapped layer by layer, and the pore size of the pores of each anti-adhesion sublayer is a gradient structure that gradually decreases from the outer layer to the inner layer, and the hemostatic layer also has sponge-like pores. The method adopts drying and freeze-drying as well as activation and complexation to prepare an anti-adhesion layer with a dense structure and a hemostatic layer with a loose structure, which significantly improves the hemostatic and anti-adhesion effects of the material and is convenient for clinical application. However, the patent uses simple hyaluronic acid HA and chitosan, but its water absorption capacity is limited, which limits its further application. The three-dimensional porous sponge structure can significantly improve its continuous liquid absorption capacity due to its high specific surface area and capillary force, providing new ideas for the development of high-performance adhesive patches. Summary of the Invention

[0005] To this end, the present invention provides a postoperative anti-adhesion sponge patch and a preparation method and application thereof to solve the problems in the prior art.

[0006] The present invention designs a sponge patch (HAMA-CS-CA) with Janus adhesion / anti-adhesion dual functions. As a sponge, the HAMA-CS-CA patch can efficiently remove interfacial water and enhance wet tissue adhesion. It also has good tissue compliance and is easy to store for a long time. The CS-CA layer uses catechol and cationic charges to synergistically quickly absorb fluid to stop bleeding and enhance adhesion. The HAMA layer gels through rapid fluid absorption without the need for light initiation, and has better retention than pure HA solution.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] According to the first aspect of the present invention, a postoperative anti-adhesion sponge patch is provided, which includes a HAMA sponge as an anti-adhesion layer and a CS-CA sponge as an adhesion layer; wherein the HAMA layer and the CS-CA layer form an interpenetrating polymer network (IPN) and physical interaction through permeation interlocking, and are tightly combined together after freeze-drying.

[0009] According to the second aspect of the present invention, a method for preparing the postoperative anti-adhesion sponge patch as described above is provided, the method comprising: first preparing a HAMA hydrogel by ultraviolet light curing, then depositing a CS-CA solution on the surface of the HAMA gel, and finally directly obtaining a HAMA-CS-CA double-layer sponge patch by a one-step freeze-drying method.

[0010] As an example, the preparation steps of the postoperative anti-adhesion sponge patch (HAMA-CS-CA sponge patch) are as follows:

[0011] First, prepare a 0.5-5% (w / v) HAMA solution by dissolving 1-100 mg of HAMA in 0.5-20 mL of ultrapure water. Then, add 0.005-10% (relative to HAMA) of I1173 to each 0.5-20 mL of the 0.005-20% HAMA solution and mix thoroughly to obtain a precursor solution. This precursor solution is then poured into a circular mold and irradiated with UV light (5-30 W) for 0.5-15 minutes in an ice bath to produce a HAMA hydrogel. Dissolve 1-200 mg of CS-CA in 0.5-10 mL of ultrapure water to prepare a 0.001-40% CS-CA solution. An equal volume of CS-CA solution is added dropwise to the surface of the HAMA gel and placed in a refrigerator at -20 to -5°C for 5-48 hours. Due to the water absorption, swelling, and mechanical interlocking of the HAMA gel, the CS-CA layer will be tightly combined with the HAMA layer. After freeze-drying for 5-48 hours, a HAMA-CS-CA double-layer sponge patch can be obtained.

[0012] Furthermore, the method for preparing HAMA hydrogel includes: first dissolving HAMA in deionized water to prepare a HAMA solution, then adding a photoinitiator to the solution, and obtaining a HAMA precursor solution after thorough mixing, and finally adding the precursor solution to a circular mold, and irradiating the mold with ultraviolet light in an ice bath to obtain the HAMA hydrogel.

[0013] Furthermore, the mass ratio of HAMA:photoinitiator:deionized water in the HAMA precursor solution is 1:0.005-1:20-200.

[0014] As an example, the photoinitiator is preferably I1173.

[0015] Furthermore, the conditions for ultraviolet irradiation in the ice bath are: ice bath temperature 1-9° C., ultraviolet irradiation time 0.5-10 min.

[0016] Furthermore, the HAMA is methacryloyl hyaluronic acid HAMA, which is synthesized by acylation reaction between hyaluronic acid and methacrylic anhydride in a system of pH=7-10.

[0017] Specifically: The synthesis route of methacryloyl hyaluronic acid (HAMA) is shown below.

[0018]

[0019] The specific experimental steps are as follows: Accurately weigh 0.5-5g of hyaluronic acid (molecular weight 100-150w) into a 500mL three-necked flask, add 50-250mL of ultrapure water and stir until completely dissolved. Add 0.5-15mL of methacrylic anhydride (MA), then slowly add 1-8M NaOH solution dropwise to adjust the pH of the system to between 7-10. The entire reaction is carried out in a low-temperature constant temperature reactor (5±4°C), with magnetic stirring (100-1000rpm), and nitrogen protection is adopted. The reaction is kept away from light for 5-48 hours to obtain methacryloyl hyaluronic acid (HAMA).

[0020] Prepare a 0.05-2% (w / v) HAMA solution by dissolving 1-10 mg of HAMA in 0.5-2 mL of ultrapure water. Then, add 0.005-10% (relative to HAMA) of I1173 to each 0.5-10 mL of the 0.05-2% HAMA solution and mix thoroughly to obtain a precursor solution. This precursor solution is then poured into a circular mold and irradiated with UV light (5-30 W) for 0.5-15 minutes in an ice bath (1-9°C) to produce a HAMA hydrogel. Freeze-dry for 5-48 hours to obtain a HAMA sponge.

[0021] Furthermore, the method for directly obtaining the HAMA-CS-CA double-layer sponge patch by the one-step freeze-drying method includes: dripping the CS-CA solution onto the surface of the HAMA gel, and freeze-drying to obtain the HAMA-CS-CA double-layer sponge patch.

[0022] Furthermore, the mass concentration of the CS-CA solution is 0.5-5%.

[0023] Furthermore, the CS-CA is prepared by esterifying chitosan and catechol in a pH system of 2-7 to synthesize catechol-modified chitosan CS-CA.

[0024] The preparation of catechol-modified chitosan (CS-CA) is based on the EDC / NHS-mediated carboxyl-amino coupling method. EDC / NHS is used to activate the carboxyl groups in catechol to generate an active ester intermediate. The active ester reacts with the amino groups on chitosan under weak acidic conditions (pH 2-7) to form a stable amide bond, achieving covalent grafting of the catechol group. The synthetic route is shown below.

[0025]

[0026] The specific experimental steps are as follows: 0.5-5.0g chitosan (degree of deacetylation ≥95-99%) is dissolved in 50-300mL of HCl solution at pH 2-6. Subsequently, 0.5-10g of caffeic acid (HCA) and 0.5-10g of EDC are dissolved in 20-200mL of ultrapure water. 10-100mL of ethanol is added to this solution to prevent precipitation during the EDC coupling reaction. The HCA / EDC mixture is then added dropwise to the chitosan solution. The reaction is maintained at pH 2-6 and in the dark at room temperature for 5-48 hours.

[0027] The present invention synthesizes a modified natural polysaccharide material, catechol-modified chitosan (CS-CA), with strong wet tissue adhesion and hemostasis. The CS-CA solution is deposited on the surface of the HAMA gel, which facilitates the formation of an interpenetrating polymer network (IPN) and physical interaction between the HAMA and CS-CA layers. The HAMA-CS-CA sponge patch is then directly obtained through a one-step freeze-drying process. Upon contact with a wet tissue surface, the patch rapidly absorbs interfacial water and gels, eliminating the effects of the hydration layer and significantly enhancing adhesion strength. The synergistic procoagulant effect of the catechol and cationic groups simultaneously effectively stops bleeding and maintains long-lasting adhesion.

[0028] According to the third aspect of the present invention, a postoperative anti-adhesion sponge patch is provided for use in preparing postoperative wound repair materials.

[0029] The present invention enhances the adhesion between the two layers by depositing a CS-CA precursor solution on the HAMA gel layer, which helps to form a polymer network interpenetration (IPN) and physical interaction between the HAMA layer and the CS-CA layer. Its adhesion mechanism is mainly based on a dry cross-linking mechanism. When HAMA-CS-CA contacts the wet tissue surface, it quickly absorbs interfacial water and gels, eliminating the influence of the hydration layer and greatly enhancing the adhesion strength. At the same time, the synergistic procoagulant effect of catechol and cationic groups can effectively stop bleeding and maintain long-term adhesion. Its anti-adhesion mechanism is mainly through the repulsive and dispersive effect of the anionic charge on HA on macrophages, effectively avoiding the aggregation of macrophages and fibroblasts. In addition, according to relevant literature reports, high molecular weight HA has a regulatory effect on inflammation. Therefore, the present invention uses hyaluronic acid with a molecular weight of 100-150w to prepare HAMA. In summary, the HAMA-CS-CA patch, as a sponge, can efficiently remove interfacial water, enhance wet tissue adhesion, and has good tissue compliance and is easy to preserve for a long time. The CS-CA layer rapidly absorbs fluid to stop bleeding and enhances adhesion based on the synergistic effect of catechol and cationic charge. The HAMA layer gels through rapid fluid absorption without the need for photoinitiation, and has better retention than pure HA solution.

[0030] The present invention has the following advantages:

[0031] The HAMA-CS-CA patch, in the form of a sponge, can efficiently remove interfacial water, eliminate the influence of the hydration layer between the patch and tissue, and enhance wet tissue adhesion.

[0032] The HAMA sponge layer in the HAMA-CS-CA sponge patch of the present invention gels by rapid liquid absorption without the need for light initiation. Compared with the fluid pure HA solution, it has better retention and is easier to store for a long time.

[0033] In the HAMA-CS-CA sponge patch of the present invention, the CS-CA layer introduces catechol into CS. Based on the catechol and cationic charge, it can synergistically enhance hemostasis, accelerate hemostasis time, and improve coagulation performance. In addition, the introduction of catechol groups enhances the hydrophilicity of CS, thereby further improving the ability of instantaneous liquid absorption and removal of interfacial water. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0035] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.

[0036] Figure 1 This is a H NMR spectrum of methacryloyl hyaluronic acid (HAMA) provided in Example 1 of the present invention;

[0037] Figure 2 This is the H NMR spectrum of catechol-modified chitosan (CS-CA) provided in Example 2 of the present invention;

[0038] Figure 3 A physical image of the HAMA-CS-CA double-layer sponge patch provided in Example 3 of the present invention; wherein a- is a cross-sectional view of the double-layer sponge patch; b is a physical image of the double-layer patch in a torsion state;

[0039] Figure 4 This is a graph showing the time-strength results of the pigskin adhesion performance of the HAMA-CS-CA sponge patch obtained in Example 3 provided in Experimental Example 1 of the present invention;

[0040] Figure 5 The linearity and adsorption capacity diagram of the HAMA-CS-CA sponge patch obtained in Experimental Example 3 provided in Experimental Example 2 of the present invention; wherein, a-standard curve; b-adsorption capacity diagram;

[0041] Figure 6 This is a graph showing the anti-cell adhesion performance of the HAMA-CS-CA sponge patch obtained in Experimental Example 3 of the present invention; wherein, a is a fluorescent staining image of cells adhered to the surface of each material; b is a statistical count of the number of cells adhered to the surface of each material;

[0042] Figure 7 This is a diagram of the cell compatibility of the double-layer sponge patch HAMA-CS-CA provided in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0043] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. It is apparent that the described embodiments are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0044] The present invention synthesizes methacryloylated hyaluronic acid (HAMA) and catechol-modified chitosan (CS-CA); prepares a HAMA hydrogel by ultraviolet curing, and then deposits a CS-CA solution on the surface of the HAMA gel, which helps to form a polymer network interpenetration (IPN) and physical interaction between the HAMA layer and the CS-CA layer, thereby enhancing the adhesion between the two layers; directly obtains a HAMA-CS-CA sponge patch by a one-step freeze-drying method; when CS-CA contacts a wet tissue surface, it rapidly absorbs interfacial water and gels, eliminating the influence of the hydration layer and greatly enhancing the adhesion strength; at the same time, the synergistic pro-coagulant effect of catechol and cationic groups can effectively stop bleeding and maintain long-lasting adhesion; the HAMA layer gels by rapid liquid absorption without the need for light initiation, and has better retention than a pure HA solution; the anionic charge on the HA repels and disperses macrophages, effectively preventing the aggregation of macrophages and fibroblasts.

[0045] According to literature reports, high molecular weight HA (molecular weight of 100-150w) has a regulatory effect on inflammation. The small-scale experiment of the present invention has verified that the HAMA synthesized by modifying high molecular weight HA also has the effect of regulating local inflammation. 120w The effect is stronger than HA 20w Effect of MA-modified HAMA 120w The effect is stronger than HAMA 20wIt is known in the art that the so-called 120w molecular weight of HA is actually a fluctuation of 120±20w, so the molecular weight of hyaluronic acid used in the present invention is 100-150w.

[0046] Example 1

[0047] This example provides a method for synthesizing high molecular weight methacryloyl hyaluronic acid (HAMA) 120W The specific experimental steps are as follows:

[0048] Accurately weigh 2.0g of hyaluronic acid (HA, molecular weight of 120w) in a 500mL three-necked flask, add 200mL of ultrapure water and stir until completely dissolved. Add 5mL of methacrylic anhydride (MA), and then slowly add 5M NaOH solution to adjust the pH of the system to between 8-9. The entire reaction was carried out in a low-temperature constant temperature reactor (4±0.5℃), magnetically stirred (600rpm), and protected by nitrogen, and the reaction was carried out in the dark for 24h. After the reaction, the mixed solution was transferred to a 3500Da dialysis bag and dialyzed with ultrapure water in the dark for 72h (dialysis water was replaced every 8h) to remove small molecules. After freeze-drying, a white flocculent HAMA product was obtained. Nuclear magnetic resonance hydrogen spectroscopy ( 1 H NMR) characterization of double bond grafting rate: Taking the methyl proton peak on HA (δ = 1.95 ppm) as a reference, the grafting rate was calculated by integrating the peak area of hydrogen atoms on the methacrylic acid double bond (5.75 ppm and 6.19 ppm), as shown in Figure 5. Figure 1 As shown, H NMR spectrum showed that HAMA was successfully synthesized.

[0049] Example 2

[0050] This example provides the following specific experimental steps for synthesizing catechol-modified chitosan (CS-CA):

[0051] 3.0g chitosan (deacetylation degree ≥95%) was dissolved in 100mL of HCl solution at pH 5.0. Then 2.37g of caffeic acid (HCA) and 2.02g of EDC were weighed and dissolved in 50mL of ultrapure water, and 50mL of ethanol was added to the solution to dissolve them together to avoid precipitation during the EDC coupling reaction. The HCA / EDC mixture was then added dropwise to the chitosan solution, maintaining the pH of the reaction system at 5.5±0.2, and reacted at room temperature in the dark for 12h. After the reaction was completed, the mixed solution was transferred to a 3500Da dialysis bag and dialyzed with a NaCl solution at pH 4.0 for 72h to remove small molecules. The light yellow flocs obtained by freeze drying were CS-CA. 1Quantitative analysis of CA grafting rate by H NMR: The grafting rate was calculated by integrating the area of the proton peak on catechol (δ = 6.67 ppm) with the chitosan acetyl proton peak (δ = 2.05 ppm) as the reference. Figure 2 As shown, H NMR spectrum showed that CS-CA was successfully synthesized.

[0052] Example 3

[0053] This example provides the following specific experimental steps for the HAMA-CS-CA sponge patch:

[0054] First, a 1% (w / v) HAMA solution from Example 1 was prepared by dissolving 5 mg of HAMA in 1 mL of ultrapure water. Then, 1% (relative to HAMA) of I1173 was added to 2 mL of the 1% HAMA solution and mixed thoroughly to obtain a precursor solution. This precursor solution was then poured into a circular mold and irradiated with UV light (10 W) for 2 minutes in an ice bath to produce a HAMA hydrogel.

[0055] 20 mg of CS-CA obtained in Example 2 was dissolved in 1 mL of ultrapure water to prepare a 2% CS-CA solution.

[0056] Finally, an equal volume of CS-CA solution was added dropwise to the surface of the HAMA gel and placed in a -20°C refrigerator for 24 hours. Due to the water absorption and swelling of the HAMA gel and the mechanical interlocking effect, the CS-CA layer was tightly combined with the HAMA layer. After freeze-drying, a HAMA-CS-CA double-layer sponge patch was obtained. Figure 3 As shown, the HAMA-CS-CA sponge patch was successfully prepared. Figure 3 a is a cross-sectional view of the double-layer sponge patch, where the tightly bound double layers can be observed; Figure 3 b is a real-life image of the double-layer patch being twisted, showing its flexibility and ability to fit onto the uneven dynamic tissue surface.

[0057] Example 4

[0058] This example provides the specific experimental steps of the HAMA-CS-CA sponge patch:

[0059] In this embodiment, the mass ratio of HAMA:photoinitiator:deionized water in the HAMA precursor solution is 1:0.005:20, and the other conditions are the same as those in Example 3.

[0060] Example 5

[0061] This example provides the specific experimental steps of the HAMA-CS-CA sponge patch:

[0062] In this embodiment, the mass ratio of HAMA:photoinitiator:deionized water in the HAMA precursor solution is 1:0.5:100, and the other conditions are the same as those in Example 3.

[0063] Example 6

[0064] This example provides the specific experimental steps of the HAMA-CS-CA sponge patch:

[0065] In this embodiment, the mass ratio of HAMA:photoinitiator:deionized water in the HAMA precursor solution is 1:1:200, and the other conditions are the same as those in Example 3.

[0066] Comparative Example 1

[0067] This comparative example provides the following specific experimental steps for synthesizing methacrylated hyaluronic acid HAEA:

[0068] Accurately weigh 2.0g of hyaluronic acid (HA, molecular weight 120w) into a 500mL three-necked flask, add 200mL of ultrapure water and stir until completely dissolved. Dissolve 0.6g of NHS and 0.9g of EDC in 50mL of ultrapure water and add them dropwise to the reaction solution. After reacting for 12h, add 6.55g of 2-aminoethyl methacrylate hydrochloride (AEMA) and continue until homogeneous. Adjust the pH to 4.75±0.05, maintain a water bath temperature of 25.0℃, and stir magnetically (600rpm) for 12h. After the reaction is completed, transfer the solution to a dialysis bag (3500Da) and dialyze with ultrapure water for 72h to remove small molecular impurities. The white flocs obtained after freeze-drying are HAEA.

[0069] Comparative Example 2

[0070] This comparative example provides the following specific experimental steps for synthesizing ferulic acid-modified chitosan (CS-FA):

[0071] In this comparative example, an equal amount of ferulic acid (FA) was used to replace caffeic acid (HCA), and the other steps were the same as those in Example 2 to obtain CS-FA.

[0072] Comparative Example 3

[0073] This comparative example provides the following specific experimental steps for the HA-CS-CA sponge patch:

[0074] This comparative example uses an equal amount of HA to replace HAMA, and the rest is exactly the same as in Example 3 to obtain a HA-CS-CA sponge patch.

[0075] Comparative Example 4

[0076] This comparative example provides the following specific experimental steps for the HA-CS sponge patch:

[0077] In this comparative example, an equal amount of HA was used to replace HAMA, and an equal amount of CS was used to replace CS-CA. Other modifications were the same as in Example 3, to obtain an HA-CS sponge patch.

[0078] Comparative Example 5

[0079] This comparative example provides the following specific experimental steps for the HAMA-CS sponge patch:

[0080] In this comparative example, an equal amount of CS was used to replace CS-CA, and the other aspects were completely consistent with Example 3 to obtain a HAMA-CS sponge patch.

[0081] Comparative Example 6

[0082] This comparative example provides the following specific experimental steps for the HAMA-CS-FA sponge patch:

[0083] In this comparative example, an equal amount of CS-FA was used to replace CS-CA, and other aspects were the same as those in Example 3 to obtain a HAMA-CS-FA sponge patch.

[0084] Comparative Example 7

[0085] This comparative example provides the following specific experimental steps for the HAEA-CS-CA sponge patch:

[0086] This comparative example uses an equal amount of HAEA instead of HAMA, and the rest is exactly the same as in Example 3 to obtain a HAEA-CS-CA sponge patch.

[0087] Experimental Example 1

[0088] This experimental example provides the following experimental steps for testing the adhesion performance of a sponge patch to pig skin:

[0089] The lap shear test was used to evaluate the pigskin adhesion performance of the HAMA-CS-CA sponge patch. Referring to the ASTM F2255 standard, fresh pigskin was pretreated and cut into strips of 2.5 cm × 1 cm in size. Then, the sponge patches of Example 3 and Comparative Examples 3-7 of equal size were placed on the pigskin surface coated with 70 μL of fresh rat blood and pressed for 1 minute to ensure interfacial adhesion. A uniaxial tensile universal testing machine (50N load cell) was used to perform tensile tests at a constant rate of 5 mm / min. In order to eliminate the interference of soft tissue deformation, polymethyl methacrylate (PMMA) film was used as a rigid support. The adhesion strength was measured by comparing the maximum breaking load with the adhesion area (2.5 cm 2 The results of 6h and 24h are shown in Table 1. The time-intensity results of Example 3 are shown in Table 1. Figure 4 As shown, the results showed that the HAMA-CS-CA sponge patch had good adhesion properties.

[0090] Table 1

[0091] Example 3 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 6h / KPa 20 19.6 5.3 5.1 12.4 19.8 24h / KPa 18.2 18.4 2.0 1.8 6.7 18.2

[0092] Table 1 shows that catechol-modified chitosan (CS-CA) exhibits superior adhesion properties. The long-term adhesion performance of CS-CA was tested over different time periods (0, 2, 6, 12, and 24 hours), and its adhesion strength remained within the 17-20 kPa range for 24 hours, showing no significant attenuation. Unmodified chitosan, lacking the corresponding adhesive functional groups, exhibited only approximately 5 kPa adhesion strength, and after 24 hours, its strength dropped to 2 kPa, nearly losing its adhesive ability. Ferulic acid-modified chitosan (CS-FA), due to its lack of a phenolic hydroxyl group compared to CS-CA, exhibited a weaker adhesion strength of only 12 kPa. Furthermore, its adhesion strength decreased significantly over time, reaching only 6.7 kPa after 24 hours. Therefore, catechol-modified chitosan significantly enhances its adhesion.

[0093] Experimental Example 2

[0094] This experimental example provides the anti-protein adhesion performance of the sponge patch. The specific experimental steps are as follows:

[0095] The anti-protein adsorption performance of the HAMA-CS-CA sponge patch was evaluated using a bovine serum albumin (BSA) model. First, 1 cm × 1 cm sponge patches of Example 3 and Comparative Examples 3-7 were immersed in PBS buffer for 1 hour to allow them to swell to equilibrium. They were then transferred to 5 mL of BSA solution (5 mg / mL) and incubated at 37°C for 4 h. The samples were removed and rinsed three times with PBS before being immersed in a 1% (w / v) sodium dodecyl sulfate (SDS) solution, shaken at 37°C for 2 h, and supplemented with ultrasonic treatment (10 min) to fully release the adsorbed proteins.

[0096] The protein concentration was quantified using a BCA kit: 200 μL of the desorbed solution was mixed with an equal volume of BCA working solution (reagent A:B = 50:1, v / v), incubated at 37°C for 30 min, and the absorbance at OD562 was measured. The standard gradient dilution in the BCA kit was used as the marker (the standard was 2 mg / mL, and the dilutions were 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL, respectively). Each set of experiments was measured in triplicate. The results of protein adsorption are shown in Table 2. The linearity and adsorption capacity of Example 3 are shown in Table 2. Figure 5 As shown, the results indicate that the HAMA-CS-CA sponge patch of Example 3 has good anti-protein adsorption performance.

[0097] Table 2

[0098]

[0099] As shown in Table 2, HAMA has good anti-protein adsorption performance. Bovine serum albumin (BSA) was used to evaluate the anti-protein adhesion effect of different materials. According to the BCA standard curve ( Figure 5 a) Calculate the protein adsorption capacity of each group. HAMA showed the best anti-protein adsorption effect, with an adsorption capacity of 1.41 μg / cm for BSA. 2 , compared with the HA group (6.73 μg / cm 2 ), the blank glass group (25.93μg / cm2), and the adsorption of BSA decreased by 78.3% and 94.3%, respectively. , and was superior to the HAEA obtained by another modification method (6.73μg / cm 2 ). Therefore, HAMA has the most ideal anti-protein adsorption ability.

[0100] Experimental Example 3

[0101] This experimental example provides the anti-cell adhesion performance of the sponge patch. The specific experimental steps are as follows:

[0102] The anti-cell adsorption performance of the HAMA-CS-CA sponge patch was evaluated using L929 mouse fibroblasts as a model (simulating the main effector cells of postoperative adhesion). First, the 1 cm × 1 cm sponge patches of Example 3 and Comparative Examples 3-7 were immersed in PBS buffer for 1 hour to allow them to swell to equilibrium. A glass slide was used as a blank control group. Each group of materials was placed in a 24-well plate, and after L929 cells were revived and passaged, they were (3 × 10 5 / cm 2 ) were inoculated on the surface of each material (n = 4 per group) and incubated for 24 hours to allow cells to fully adhere. After that, the culture medium was removed and the cells were washed three times with PBS. The cells were stained with a calcein AM / propidium iodide (AM / PI) double staining kit for 25 minutes, and the surface cell adhesion was observed under a laser confocal microscope. The number of cells attached to the sample surface was calculated using Image J. The results are shown in Table 3. The results of the blank group and Example 3 are shown in Table 3. Figure 6 As shown, the results showed that the HAMA-CS-CA sponge patch had good anti-cell adhesion properties.

[0103] Table 3

[0104]

[0105] As shown in Table 3, HAMA has good anti-cell adhesion performance. L929 mouse fibroblasts were used for evaluation. Glass slides were used as the control group. After L929 cells were co-cultured with each group of materials for a period of time, the cell density on the surface of the materials was recorded by laser confocal microscopy ( Figure 6), and conduct quantitative statistical analysis, such as Figure 6 As shown: the amount of cell adhesion on the HAMA surface was reduced by 78.3% compared with the control group, indicating that based on the strong negative charge of HAMA, the negatively charged fibroblasts could not effectively stay on the HAMA surface through electrostatic repulsion, which greatly weakened the cell adhesion effect and effectively prevented the aggregation of fibroblasts. It also has a stronger effect in preventing cell adhesion than HA and HAEA.

[0106] Experimental Example 4

[0107] This experimental example provides the cell compatibility of the sponge patch. The specific experimental steps are as follows:

[0108] The biocompatibility of the HAMA-CS-CA sponge patch was evaluated using the L929 mouse fibroblast cell line. The evaluation was performed by MTT cytotoxicity assay: HAMA, CS-CA, the HAMA-CS-CA sponge patch of Example 3, and the sponge samples of Comparative Examples 3-7 were first sterilized by UV (30 min), then converted into a gel state with PBS, and incubated with conditioned medium (containing 10% FBS) at a ratio of 1:5 (w / v) at 37°C for 24 h. The extract was collected and mixed with equal volumes of fresh 1640 medium (containing 20% FBS + 2% P / S), and sterilized with a 0.22 μm sterile needle filter for later use. The cell suspension (density 1×10 4 Cells / well) were seeded in a 96-well plate. 100 μL of extract medium was added to the experimental group, and a negative control (sterile water) and a positive control (conditioned medium) were set up in the control group. After incubation in a cell incubator for 24 hours, fresh medium containing 0.5 mg / mL MTT was replaced. After further incubation for 4 hours, the culture medium was removed and 100 μL of DMSO was added to dissolve the crystals. After micro-oscillation for 10 minutes, the absorbance (OD) was measured at 490 nm on a microplate reader to calculate the cytotoxicity. The results are shown in Table 4. Figure 7 As shown, the results showed that the HAMA-CS-CA sponge patch had good cell compatibility.

[0109] Table 4

[0110]

[0111] As shown in Table 4, the HAMA-CS-CA sponge patch has good cell compatibility. The cytotoxicity of HAMA-CS-CA and its various layers to L929 fibroblasts was evaluated by MTT assay. Figure 7As shown, after 24 hours of co-culture of cells with extracts from each material group, the relative cell viability remained above 80% (vs. the negative control group). Furthermore, the relative cell viability of the HAMA-C and HAMA groups exceeded 100%, likely due to the proliferation-promoting effect of HA in HAMA on L929 cells. The other control groups, all made of HA and CS derivatives, exhibited excellent biocompatibility. These results confirm that HAMA-C has no significant cytotoxicity and meets biosafety standards for biomaterials.

[0112] Experimental Example 5

[0113] This experimental example provides the instantaneous liquid absorption rate of the sponge patch of Example 3 and Comparative Examples 3-7:

[0114] Take the same mass of sponge material, record the initial weight, then place the sponge in a culture dish filled with PBS buffer. After a predetermined time (10 seconds, 30 minutes, 24 hours), gently lift a corner with tweezers, let it stand in the air, gently wipe off the unabsorbed droplets, and weigh it again. Finally, convert it into water absorption rate. The calculation formula is as follows:

[0115]

[0116] Wherein, W0 is the weight of the sponge at 0 min, and WS is the weight after absorbing water.

[0117] The results are shown in Table 5.

[0118] Table 5

[0119] 10s 30min 24h Example 3 28 43 47 Comparative Example 3 35 45 18 Comparative Example 4 17 22 25 Comparative Example 5 12 19 23 Comparative Example 6 20 26 28 Comparative Example 7 27 41 45 Single HAMA 22 33 35 Single CS-CA 37 61 65

[0120] As shown in Table 5, the HAMA-CS-CA sponge patch obtained in Example 3 of the present invention can absorb 43 times the liquid in 30min. In the prepared double-layer structure, the CS-CA of the adhesive layer introduces catechol into CS, so that the introduction of polyphenols brings more hydrophilic groups, so that the CS-CA sponge shows a stronger liquid absorption capacity in a short time, reaching 37 times in 10s. Compared with the separate HAMA sponge layer, the CS-CA sponge layer shows a stronger instantaneous water absorption capacity. After HAMA and CS-CA are compounded, the instantaneous water absorption rate of Example 3 is 28 times, and compared with the HA-CA-CS in Comparative Example 3, different water absorption capacities are also shown, among which the HA-CS-CA sponge shows a better instantaneous water absorption capacity, with a water absorption capacity of 35 times. This is because the cross-linked structure of HAMA is denser, thereby blocking the penetration of water molecules to a certain extent, but as time goes on, the two groups of water absorption gradually approach each other. Compared to HAMA-CS in Comparative Example 5, the instantaneous water absorption of Comparative Example 5 was only 12 times due to the fewer hydrophilic groups in CS. Compared to Example 3, CS-FA, due to the presence of only one phenolic hydroxyl group, was less hydrophilic than CS-CA, with an instantaneous water absorption of 20 times. Compared to HAEA-CS-CA in the Comparative Example, the water absorption of HAEA and HAMA was similar, as HAEA's cross-linking mechanism was similar.

[0121] Experimental Example 6

[0122] This example provides the in vitro hemostatic performance (whole blood coagulation index BCI) of the sponge patches of Example 3 and Comparative Examples 3-7:

[0123] First, 10 mg of the sample of Example 3 and Comparative Examples 3-7 was weighed in a 1.5 mL centrifuge tube, and then re-coagulated blood (blood with restored coagulation ability) was prepared: 10 μL of 0.2 M CaCl2 aqueous solution was added to a 0.5 mL centrifuge tube, followed by addition of 100 μL of sodium citrate anticoagulated whole blood, and the re-coagulated blood was obtained after mixing. The re-coagulated blood was quickly added dropwise to the sample surface and incubated in a 37 ° C water bath for 70 s. After the incubation, 10 mL of ultrapure water preheated to 37 ° C was added to continue lysing the uncoagulated blood cells for 3 min. 100 μL of liquid was taken in a 96-well plate, and the absorbance at a wavelength of 545 nm was measured using an enzyme marker. Chitosan sponge (CS) was used as the control group, and the blank control group was directly added to 10 mL of ultrapure water (without adding CaCl2 and without contact with the material), and the same 3 min standard lysis treatment was performed. The whole blood coagulation index (BCI) of each group was calculated according to the formula:

[0124]

[0125] Where A S is the absorbance of the material group; A0 is the absorbance of the blank group.

[0126] The results are shown in Table 6.

[0127] Table 6

[0128] BCI% Example 3 33 Comparative Example 3 35 Comparative Example 4 63 Comparative Example 5 62 Comparative Example 6 45 Comparative Example 7 34 Single CS-CA 32 Single CS 61

[0129] As shown in Table 6, the BCI value of the bare CS-CA group was significantly enhanced compared to the bare CS group, indicating that the introduction of catechol effectively enhanced the hemostatic properties of chitosan. The effect of CS-CA combined with HAMA on the BCI index was minimal. Comparative Examples 3 and 7 show that hemostatic performance is primarily dependent on the CS-CA layer; combination with HA and HAEA, respectively, has no effect. Compared to the CS-FA in Comparative Example 6, due to its single phenolic hydroxyl group, the synergistic hemostatic performance was significantly reduced, but slightly superior to that of the unmodified CS group.

[0130] Experimental Example 7

[0131] This example provides the in vivo coagulation performance of the sponge patches of Example 3 and Comparative Examples 3-7:

[0132] SD rats weighing 180-220 g were selected to establish a rat femoral artery hemostasis experimental model. The rats were anesthetized with 10% (w / v) chloral hydrate by intraperitoneal injection. After fixing the rat's limbs, the skin on the rat's left thigh was cut to expose the femoral artery, and the fascia around the femoral artery was gently removed. The femoral artery of the rat was cut with a scalpel, and the timing was started at the same time. It was allowed to bleed freely for 10 seconds. The amount of blood loss during this period was collected with gauze and recorded as the initial blood loss. In order to eliminate the influence of individual differences in rats, samples with an initial blood loss of 100-400 mg were selected, and samples with blood loss outside this range were excluded. Subsequently, a 2cm×2cm sponge patch of Example 3 and Comparative Examples 3-7 was covered on the bleeding point, and a 100g weight was pressed on the material. After 3 minutes, the weight was removed for the first time, the material was uncovered, and the bleeding at the wound was observed. If bleeding continued, the material was covered again and the weight was pressed. Thereafter, the material was removed and observed once every 1 minute until the wound stopped bleeding, which was considered a successful hemostasis. The amount of blood loss during this period was collected with filter paper and recorded as the post-bleeding amount. The amount of blood loss before and after was calculated as follows: amount of blood loss before = m4-m1; amount of blood loss after = m5-m3-m2. The results are shown in Table 7.

[0133] Where m1, m2, and m3 are the weights of the pre-weighed ziplock bags containing filter paper, gauze, and materials; m4 is the weight of the gauze stained with blood; and m5 is the total weight of the ziplock bags containing filter paper and materials stained with blood.

[0134] Table 7

[0135]

[0136]

[0137] As shown in Table 7, while there was no significant difference in the initial blood loss, the subsequent blood loss decreased from 394 mg in the CS group to 147 mg, a decrease of 62.7%, and the hemostasis time was also significantly shortened. In vivo hemostasis experiments showed that CS-CA has better hemostatic properties than CS, mainly due to the introduction of catechol groups, which synergistically enhance the hemostatic effect. After compounding CS-CA with HAMA, there was little effect on various indicators. Comparative Examples 3 and 7 show that the hemostatic performance mainly depends on the CS-CA layer. After compounding with HA and HAEA, respectively, there is no effect on the hemostatic performance. Compared with CS-FA in Comparative Example 6, due to its only phenolic hydroxyl group, the synergistic hemostatic performance is significantly reduced, but it is slightly better than the unmodified CS group.

[0138] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A postoperative anti-adhesion sponge patch, characterized in that: The sponge patch includes a HAMA sponge as an anti-adhesion layer and a CS-CA sponge as an adhesion layer; wherein the HAMA layer and the CS-CA layer form an interpenetrating polymer network (IPN) and physical interaction through permeation interlocking, and are tightly combined together after freeze-drying.

2. A method for preparing the postoperative anti-adhesion sponge patch according to claim 1, characterized in that: The method comprises: first preparing a HAMA hydrogel by ultraviolet curing, then depositing a CS-CA solution on the surface of the HAMA gel, and finally directly obtaining a HAMA-CS-CA double-layer sponge patch by a one-step freeze-drying method.

3. The preparation method of the postoperative anti-adhesion sponge patch according to claim 2, wherein The method for preparing the HAMA hydrogel includes: first dissolving HAMA in deionized water to prepare a HAMA solution, then adding a photoinitiator to the solution, and thoroughly mixing to obtain a HAMA precursor solution, and finally adding the precursor solution to a circular mold, and irradiating the mold with ultraviolet light in an ice bath to obtain the HAMA hydrogel.

4. The method for preparing the postoperative anti-adhesion sponge patch according to claim 3, wherein: The mass ratio of HAMA: photoinitiator: deionized water in the HAMA precursor solution is 1:0.005-1:20-200.

5. The method for preparing the postoperative anti-adhesion sponge patch according to claim 3, wherein: The conditions for ultraviolet irradiation in the ice bath are: ice bath temperature 1-9° C., ultraviolet irradiation time 0.5-10 min.

6. The method for preparing the postoperative anti-adhesion sponge patch according to claim 3, wherein: The HAMA is methacryloyl hyaluronic acid HAMA, which is synthesized by acylation reaction between hyaluronic acid and methacrylic anhydride in a system of pH=7-10.

7. The method for preparing the postoperative anti-adhesion sponge patch according to claim 2, wherein: The one-step freeze-drying method for directly obtaining the HAMA-CS-CA double-layer sponge patch comprises: dripping the CS-CA solution onto the surface of the HAMA gel, and freeze-drying to obtain the HAMA-CS-CA double-layer sponge patch.

8. The method for preparing the postoperative anti-adhesion sponge patch according to claim 7, wherein: The mass concentration of the CS-CA solution is 0.5-5%.

9. The method for preparing the postoperative anti-adhesion sponge patch according to claim 8, wherein: The CS-CA is prepared by esterification reaction between chitosan and catechol in a system of pH=2-7 to synthesize catechol-modified chitosan CS-CA.

10. Application of a postoperative anti-adhesion sponge patch in the preparation of postoperative wound repair materials.

Images