Biomedical hydrogels for preventing postoperative tissue adhesions, their preparation methods and applications

Abstract

This invention provides a biomedical hydrogel that can prevent postoperative tissue adhesion, its preparation method, and its application. The method involves dissolving polyamide-amine dendritic macromolecules in a solvent, with the dissolved dendritic macromolecules having a mass fraction of 10%-40%; taking a certain amount of the dendritic macromolecule solution and adding an anti-inflammatory drug to it; dissolving γ-polyglutamic acid in a solvent and stirring, with the dissolved γ-polyglutamic acid having a mass fraction of 1%-10%; mixing the two solutions thoroughly; and then adding a carboxyl activator to the mixture, wherein the activator is any one or two of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. After the reaction is complete, the biomedical hydrogel of this invention is obtained. The biomedical hydrogel prepared by this invention has good biocompatibility and exhibits excellent anti-adhesion effects in postoperative tissue adhesion applications. It can also be used for safe and efficient drug delivery and sustained drug release.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a gel complex, specifically a biomedical hydrogel suitable for preventing postoperative tissue adhesion, its preparation method, and its application. Background Technology

[0002] Polyamide-amine dendrimers (PAMAMs) are monodisperse and highly branched units with well-defined structures. Their surface cationic primary amine groups allow them to bind with other chemical entities. PAMAM dendrimers possess excellent water solubility and, due to their unique structure, are frequently used as carriers for various hydrophilic and hydrophobic drugs and genes. Furthermore, PAMAM dendrimers are a popular choice due to their passive targeting, high drug loading, solubilization, spherical shape, monodispersity, and ability to increase drug half-life, purity, and stability. Therefore, PAMAM dendrimers have been widely applied in biomedicine, drug delivery, tissue engineering, and other fields, showing promising application prospects.

[0003] Polyglutamic acid (γ-PGA) is a biodegradable polymer with excellent biocompatibility. It is polymerized from the natural amino acid glutamic acid and can be degraded by microorganisms into carbon dioxide and water. The physicochemical properties of polyglutamic acid can be controlled by altering 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] Postoperative abdominal adhesions are a very common problem. The presence of adhesions from a previous surgery not only increases the difficulty and risk of subsequent surgeries but also leads to a range of complications, including bowel obstruction, chronic pelvic pain, female infertility, and even death. Currently, clinically, adhesion formation can be reduced through delicate and minimally invasive surgeries, such as laparoscopic surgery. Aside from surgery, the main anti-adhesion strategies focus on physical barriers. However, these methods cannot completely eliminate adhesion formation, often resulting in poor outcomes and low efficiency. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a biomedical hydrogel suitable for preventing postoperative tissue adhesion, its preparation method, and its application. This biomedical hydrogel aims to solve many problems associated with existing postoperative adhesion materials, such as unsatisfactory results and the need for secondary surgery.

[0006] This invention provides a method for preparing a biomedical hydrogel suitable for preventing postoperative tissue adhesion. The method involves dissolving polyamide-amine dendritic macromolecules in a first solvent until completely dissolved, resulting in a dendritic macromolecule mass fraction of 10%-40%. A certain amount of the dissolved dendritic macromolecule solution is taken, and an anti-inflammatory drug is added to it, with the dispersed antibacterial drug concentration being 0.5-10 mg / mL, yielding solution A. γ-polyglutamic acid is dissolved in a second solvent and stirred until completely dissolved, resulting in a γ-polyglutamic acid mass fraction of 1%-10%, yielding solution B. Solution A and solution B are mixed thoroughly to obtain a mixed solution. A carboxyl activator is then added to the mixed solution; the activator is any one or both of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. After the reaction is complete, the biomedical hydrogel is obtained.

[0007] Further, the first solvent is methanol, ethanol, water, DMSO, distilled water, phosphate buffer solution, or physiological saline, and the second solvent is any one of distilled water, phosphate buffer solution (pH = 7.4), and physiological saline (w / v = 0.9%).

[0008] Furthermore, the polyamide-amine dendritic macromolecule can be any one of the third, fourth, fifth, sixth, seventh, or eighth generation.

[0009] Furthermore, in the preparation method of biomedical hydrogels, the mass concentration percentage of dendritic macromolecules and γ-polyglutamic acid is 10%-40%.

[0010] Furthermore, in the preparation method of biomedical hydrogels, the dissolution temperature of dendritic macromolecules and γ-polyglutamic acid is 20-80°C. o C.

[0011] Furthermore, in the preparation method of biomedical hydrogels, the mass fraction of carboxyl activator is 1%-30%.

[0012] Furthermore, in the preparation method of biomedical hydrogels, the mass fraction of the carboxyl activator added to the final reaction solution is 0.5% - 30%.

[0013] Furthermore, the method for preparing biomedical hydrogels is characterized by: a reaction temperature of 10-30°C. o C.

[0014] In the method of this invention, a carboxyl activator can activate the carboxyl groups in γ-polyglutamic acid, enabling γ-polyglutamic acid to undergo a cross-linking reaction with the amino groups in polyamide-amine dendritic macromolecules. This invention provides a method for preparing a gel by solubilizing meloxicam with polyamide-amine dendritic macromolecules and cross-linking it with γ-polyglutamic acid under the activation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS).

[0015] The present invention also provides a biomedical hydrogel obtained by the above method, and the application of such a biomedical hydrogel in the preparation of anti-inflammatory biomaterials that have drug delivery and sustained-release functions and prevent postoperative tissue adhesion.

[0016] This invention features a simple process and rapid product preparation; the two solutions rapidly gel within approximately 30 seconds of mixing. The resulting product exhibits excellent biocompatibility in both in vitro and in vivo simulation experiments. This invention selects polyamide-amine dendritic macromolecules and γ-polyglutamic acid as the matrix, then adds an anti-inflammatory drug. Simultaneously, EDC-NHS is used to activate the carboxyl groups in γ-polyglutamic acid, enabling them to crosslink with the numerous amino groups in the polyamide-amine dendritic macromolecules, thereby preparing a biomedical hydrogel. Experiments show that the hydrogel prepared by this method possesses excellent flexibility and elasticity, as well as good water swelling and degradation properties. After loading the drug, this medical hydrogel can gradually release the drug after entering the body, increasing the drug's stability and persistence while reducing side effects. Furthermore, due to the excellent in vivo degradation performance of this invention, no secondary surgery is required after surgery, reducing the harm caused to patients by secondary surgery. Such a material not only improves drug bioavailability but also reduces damage and impact on normal tissues and organs.

[0017] Compared with existing technologies, the present invention represents a significant technological advancement. The biomedical hydrogel prepared by this invention is simple to process and the product is readily available. Furthermore, the product possesses properties such as softness and injectability, allowing it to adapt to various shapes and visceral surfaces. The drug-loaded hydrogel holds promise for applications in drug delivery and sustained-release, demonstrating considerable clinical value. Through establishing a mouse cecal-abdominal wall adhesion model, our experiments ultimately proved that the designed biomedical hydrogel can significantly reduce postoperative peritoneal adhesions in the mouse model, exhibiting a good anti-adhesion effect and demonstrating its promising clinical application prospects. Attached Figure Description

[0018] Figure 1 a is a FESEM image of the biomedical hydrogel prepared in Example 1. Figure 1 b is an FESEM image of the biomedical hydrogel prepared in Example 2; Figure 1c is an FESEM image of the biomedical hydrogel prepared in Example 3; Figure 1 Image d is a magnified FESEM image of the biomedical hydrogel from Example 2.

[0019] Figure 2 Dynamic time-scan rheological analysis of the biomedical hydrogel prepared in Example 1; Figure 2 b. Dynamic time-scan rheological analysis of the biomedical hydrogel prepared in Example 2; Figure 2 c. Dynamic time-scan rheological analysis diagram of the biomedical hydrogel prepared in Example 3.

[0020] Figure 3 a represents the compressive strain-stress curve test results of the biomedical hydrogels prepared in Examples 1, 2, and 3; Figure 3 b represents the average compressive stress test results of the biomedical hydrogels prepared in Examples 1, 2, and 3.

[0021] Figure 4 a represents the survival rate of L929 cells after co-culturing with the biomedical hydrogel prepared in Example 2; Figure 4 b shows the DEAD / LIVE staining results without material treatment; Figure 4 c to Figure 4 f represents the DEAD / LIVE staining results after treatment with the biomedical hydrogel prepared in Example 2, wherein... Figure 4 The concentration of the biomedical hydrogel in component c is 5 mg / mL. Figure 4 The concentration of the biomedical hydrogel in d is 10 mg / mL; Figure 4 The concentration of the biomedical hydrogel in e is 25 mg / mL; Figure 4 The concentration of the biomedical hydrogel in f is 50 mg / mL.

[0022] Figure 5 a to Figure 5 f represents the relevant test results of the biomedical hydrogels prepared in Examples 1 to 3, wherein... Figure 5 a represents the hemolysis test results of the biomedical hydrogel prepared in Example 2; Figure 5 b represents the BSA protein adsorption result; Figure 5 c represents the swelling kinetics curve; Figure 5 d represents the swelling ratio; Figure 5 e represents the in vitro degradation curve of the biomedical hydrogel in phosphate buffer solution; Figure 5 f is the degradation curve of the biomedical hydrogel prepared in Example 2 in mice.

[0023] Figure 6a represents the in vitro release analysis results of meloxicam loaded onto the biomedical hydrogels prepared in Examples 1 to 3; Figure 6 b represents the standard curve for meloxicam.

[0024] Figure 7 a to Figure 7 d represents the in vitro antibacterial test results of the biomedical hydrogel prepared in Example 2: Figure 7 a represents the antibacterial rate of the biomedical hydrogel against Escherichia coli; Figure 7 b is an image showing the antibacterial results of Escherichia coli; Figure 7 c represents the antibacterial rate of the biomedical hydrogel against Staphylococcus aureus; Figure 7 Image d shows the antibacterial results of Staphylococcus aureus.

[0025] Figure 8 a and Figure 8 b represents the in vivo degradation results of the biomedical hydrogel: Figure 8 a is a photograph of the skin around the dorsal incision of the hydrogel. Figure 8 b shows images of the hydrogel size at different time points.

[0026] Figure 9 Tissue and organ safety results of biomedical hydrogels: H&E staining images of mouse heart, liver, spleen, lung, and kidney at different time points.

[0027] Figure 10 a to Figure 10 c represents the experimental results of the biomedical hydrogel in preventing postoperative tissue adhesion: Figure 10 Figure a shows the rat model of lateral wall defect-cecal abrasion. Figure 10 b. Adhesion score for adhesion formation; Figure 10 c represents the representative observation results of postoperative adhesion formation on days 7 and 14.

[0028] Figure 11 The results of t-PA and DAPI immunostaining of biomedical hydrogels.

[0029] Figure 12 a to Figure 12 c represents the real-time quantitative PCR result of the biomedical hydrogel: Figure 12 a represents the TNF-α mRNA expression level; Figure 12 b represents the t-PA mRNA expression level; Figure 12 c represents the PAL-1 mRNA expression level. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0031] This invention involves adding an anti-inflammatory drug and a carboxyl activator to a solution of γ-polyglutamic acid and polyamidoamine dendritic macromolecules. The anti-inflammatory drug chosen is the non-steroidal anti-inflammatory drug meloxicam, and the carboxyl activator is EDC-NHS. Three different proportions of γ-polyglutamic acid gel were selected for preparation (20% in Example 1, 30% in Example 2, and 40% in Example 3). Specifically, meloxicam is first mixed with the dendritic macromolecule solution and stirred until homogeneous. Then, it is mixed with different proportions of γ-polyglutamic acid solution and stirred again. Finally, EDC-NHS is added to form a gel. All three methods can yield biomedical hydrogels with a certain network structure.

[0032] Example 1

[0033] The third-generation polyamide-amine dendritic macromolecule (G3) was dissolved in methanol at a concentration of 200 mg / mL. 100 μL of the G3 methanol solution and 5 mg of meloxicam (MX) were dissolved in 1 mL of deionized water and 100 μL of DMSO solution, respectively. These solutions were then mixed to obtain a clear, transparent solution. Next, 0.1 g of γ-polyglutamic acid was dissolved in 4 mL of deionized water and stirred until homogeneous at room temperature. This solution was then mixed with the above solution to obtain a dendritic macromolecule-meloxicam-γ-polyglutamic acid mixed solution. Then, approximately 80 mg of EDC and 80 mg of NHS were added to 1 mL of deionized water to prepare an EDC-NHS mixed solution. Finally, the EDC and NHS mixed solution was added to the dendritic macromolecule-meloxicam-γ-polyglutamic acid mixed solution and stirred until homogeneous to obtain the biomedical hydrogel.

[0034] Example 2

[0035] The third-generation polyamide-amine dendritic macromolecule (G3) was dissolved in methanol at a concentration of 200 mg / mL. 100 μL of the G3 methanol solution and 5 mg of meloxicam (MX) were dissolved in 1 mL of deionized water and 100 μL of DMSO solution, respectively. These solutions were then mixed to obtain a clear, transparent solution. Next, 0.15 g of γ-polyglutamic acid was dissolved in 4 mL of deionized water and stirred at room temperature until homogeneous. This solution was then mixed with the above solution to obtain a dendritic macromolecule-meloxicam-γ-polyglutamic acid mixed solution. Then, approximately 80 mg of EDC and 80 mg of NHS were added to 1 mL of deionized water to prepare an EDC-NHS mixed solution. Finally, the EDC and NHS mixed solution was added to the dendritic macromolecule-meloxicam-γ-polyglutamic acid mixed solution and stirred until homogeneous to obtain the biomedical hydrogel.

[0036] Example 3

[0037] The third-generation polyamide-amine dendritic macromolecule (G3) was dissolved in methanol at a concentration of 200 mg / mL. 100 μL of the G3 methanol solution and 5 mg of meloxicam (MX) were dissolved in 1 mL of deionized water and 100 μL of DMSO solution, respectively. These solutions were then mixed to obtain a clear, transparent solution. Next, 0.2 g of γ-polyglutamic acid (γ-PGA) was dissolved in 4 mL of deionized water and stirred at room temperature until homogeneous. This solution was then mixed with the above solution to obtain a dendritic macromolecule-meloxicam-γ-polyglutamic acid mixed solution. Then, approximately 80 mg of EDC and 80 mg of NHS were added to 1 mL of deionized water to prepare an EDC-NHS mixed solution. Finally, the EDC and NHS mixed solution was added to the dendritic macromolecule-meloxicam-γ-polyglutamic acid mixed solution and stirred until homogeneous to obtain the biomedical hydrogel.

[0038] Example 4

[0039] To analyze the morphology of biomedical hydrogels, samples with 20% PGA (Example 1), 30% PGA (Example 2), and 40% PGA (Example 3) were analyzed using a FEI Magellan 400 field emission scanning electron microscope.

[0040] As can be seen from the electron microscopy images, the biomedical hydrogels all exhibit a three-dimensional porous structure. Figure 1 ad). Figure 1 a, Figure 1 b、 Figure 1 c represents scanning electron microscope (SEM) images of Examples 1, 2, and 3 at different angles at 100 μm. To further observe the internal structure of the hydrogel, the hydrogel prepared in Example 2 (… Figure 1 b) Perform local magnification and take scanning electron microscope images ( Figure 1 d).

[0041] Example 5

[0042] The gelation process of the hydrogel was investigated by observing the final storage modulus (G′) and final loss modulus (G′′) in dynamic time-scan rheological experiments. Dynamic rheological studies were conducted using a rotational rheometer (MARS III HAAKE) with a flat plate (P20 TiL, 20 mm in diameter). The hydrogels in Examples 1, 2, and 3 were subjected to time-scan oscillation experiments at a frequency of 1 Hz, a gap of 1 mm, and a strain of 1%. The corresponding hydrogel precursor solutions were injected onto the plate, with the gap adjusted to 1 mm. The frequency scan measurements of the hydrogel are expressed as G′ and G′′. Due to intermolecular crosslinking, the G′ of the hydrogel increased rapidly, indicating a high hydrogel formation efficiency. Figure 2 (a, 2b, 2c)

[0043] Example 6

[0044] Mechanical evaluation was performed using a 2.5 kN sensor on a Zwick Roell Z2.5 TH universal testing machine. The compressive properties of the biomedical hydrogel were investigated using a modified American Society for Testing and Materials (ASTM) method. In the compression test, the hydrogel was prepared in a cylindrical model with a diameter of 8 mm and a thickness of 4 mm, and the compression strain rate was 0.5 mm / min. The compressive modulus was recorded by linear fitting of the stress-strain curve within a strain range of 10–20%. Figure 3 a). The maximum compressive stress of the hydrogel prepared in Example 1 was 19.467 kPa, the maximum compressive stress of the hydrogel prepared in Example 2 was 75.297 kPa, and the maximum compressive stress of the hydrogel prepared in Example 3 was 157.648 kPa. Figure 3 (a, 3b). Experimental results show that the hydrogel prepared in Example 2 has a relatively moderate degree of softness, neither too soft to withstand pressure nor too hard to conform well to tissues in vivo.

[0045] Example 7

[0046] The hydrogel prepared in Example 2 was co-incubated with L929 cells for 3 days. The cytotoxicity of the hydrogel was quantitatively and qualitatively detected using the CCK-8 and LIVE / DEAD cell viability assay kits on days 1, 2, and 3. Different concentrations of hydrogel extract (5, 10, 25, and 50 mg / mL) were added to the cell culture medium, while the control group received only 100 μL of cell culture medium (viability set at 100%). After 1 day of culture, the cell viability rates were 98.88±4.67%, 99.34±3.48%, 98.56±4.23%, and 99.32±10.51%, respectively. After 2 days, the cell viability rates were 99.98±7.35%, 99.81±6.95%, 98.77±2.94%, and 97.23±5.72%, respectively. After 3 days of culture, the cell viability rates were 99.31±2.17%, 98.27±0.51%, 99.95±1.89%, and 98.78±0.62%, respectively. The results indicate that the cell viability rates were all above 95%, and the biomedical hydrogel prepared in Example 2 showed no significant toxicity to L929 cells. Figure 4 a). LIVE / DEAD cell viability assay results showed similarity to the control group ( Figure 4 (b) All cells treated with the hydrogel were stained green by the LIVE / DEAD reagent (living cells were stained green), and almost no cells were stained red (dead cells were stained red). Figure 4 c, 4d, 4e, and 4f). CCK-8 and LIVE / DEAD cell staining results indicate that the prepared biomedical hydrogel has good cell compatibility.

[0047] Example 8

[0048] Example 7 investigated the blood compatibility of the biomedical hydrogel. Two mL of fresh mouse whole blood was centrifuged (3000 rpm, 5 min) to collect plasma, which was then washed three times with phosphate buffer solution to collect red blood cells. The resulting red blood cells were dissolved in 50 mL of phosphate buffer solution for further use.

[0049] In the hemolysis experiment, 2.4 mL of phosphate buffer solution and 600 μL of red blood cell suspension were added to a 5 mL centrifuge tube. Hydrogels of different masses prepared in Example 2 were placed in DMEM cell culture medium (50 mg hydrogel, 1 mL culture medium) and incubated overnight at 37°C to prepare the extract. The extract concentrations were set at 5 mg / mL, 10 mg / mL, 25 mg / mL, and 50 mg / mL. Two other groups served as control groups, treating red blood cell suspensions with phosphate buffer solution (negative control) and deionized water (positive control), respectively. The above mixed solutions were incubated at 37°C for 2 h, then centrifuged at 5000 rpm for 3 min. The absorbance of the supernatant at 541 nm was collected (using a Shimadzu UV-3600 UV-Vis-NIR spectrometer), and the hemolysis ratio of red blood cells was calculated. Figure 5 As shown in figure a, the calculated hemolysis rates of the hydrogels were all less than 5%. As can be seen from the photographs, the red blood cell supernatant incubated with the hydrogel and phosphate buffer solution was transparent. However, the blood treated with deionized water appeared distinctly red due to positive hemolysis. These results indicate that the biomedical hydrogel has good blood compatibility.

[0050] Example 9

[0051] The antifouling ability of the biomedical hydrogel was assessed by measuring its resistance to nonspecific protein adsorption and cell adhesion. Nonspecific protein adsorption was measured using a BCA protein assay kit. The hydrogels prepared in Examples 1, 2, and 3 were placed in 24-well plates, and a bovine serum albumin (BSA) solution at a concentration of 2 mg / mL was added. After incubation at 37°C for 2 h, the hydrogels were removed from the protein solution and rinsed three times with phosphate buffer to remove loosely adsorbed proteins. Then, the hydrogels were immersed in 1 mL of fresh phosphate buffer and sonicated for 10 minutes to remove proteins firmly adsorbed on the hydrogel. The protein concentration in the solution was obtained using a micro-BCA protein assay kit based on absorbance measured at 562 nm using a microplate reader (Bio-Tek, ELx808, USA). The results showed that the hydrogels prepared in Examples 1, 2, and 3 adsorbed 14.42, 16.39, and 17.05 μg / cm³ of BSA protein, respectively. −2 ( Figure 5 b). This indicates that the hydrogel has good resistance to non-specific protein adsorption.

[0052] Example 10

[0053] To investigate the swelling ability of this biomedical hydrogel in phosphate buffer solution, the hydrogels obtained in Examples 1, 2, and 3 were freeze-dried, weighed to determine their initial mass, and then added to phosphate buffer solution. They were soaked at 37°C for a total of 24 hours. At different time points, the hydrogels were removed, gently wiped to remove surface moisture, and then weighed. Finally, the swelling ratio was calculated by comparing the obtained mass with the initial mass. Figure 5 d). Furthermore, the swelling kinetics of the hydrogel in deionized water were investigated. Figure 5 c) demonstrates that the hydrogel absorbs fluids rapidly, reaching 16.34 g / g, 27.52 g / g, and 30.25 g / g, respectively, after 6 to 8 hours of storage in phosphate buffer solution.

[0054] Example 11

[0055] The freeze-dried biomedical hydrogels prepared in Examples 1, 2, and 3 were weighed and incubated with phosphate buffer solution. The degradation rate of the hydrogels was continuously measured over 28 days at a constant temperature of 37°C. The phosphate buffer solution was replaced every other day. At each time point, the biomedical hydrogels were removed from the culture medium, gently rinsed with phosphate buffer solution, and then freeze-dried. The mass of the freeze-dried biomedical hydrogels was weighed to calculate the degradation rate. The experimental results showed that the hydrogels were almost completely degraded in phosphate buffer solution within approximately 28 days, demonstrating the degradability of the hydrogels. Figure 5 e).

[0056] Example 12

[0057] The in vivo degradability of the biomedical hydrogel was assessed by monitoring its degradation in mice. Kunming (KM) mice (approximately 25 g, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were anesthetized with 4% chloral hydrate and randomly divided into five groups at 2, 4, 8, 12, and 24 h. Each group had three replicates. A dorsal incision of approximately 15 mm was made along the mid-dorsal skin of the KM mice using surgical scissors. A meloxicam-containing hydrogel sheet (GP@MX) prepared in Example 2, sterilized with alcohol, was implanted under the incision. Mouse weight was recorded at the corresponding time points, and the mice were euthanized. The size of the hydrogel removed from the body and the area of ​​skin around the dorsal incision were then measured and recorded using a camera. Figure 8 a, b). After approximately 24 hours, the hydrogel prepared in Example 2 was completely degraded ( Figure 5f). Notably, the hydrogel mass increased at 2 h, possibly due to the absorption of some tissue fluid. In vivo degradation studies demonstrate that the prepared biomedical hydrogel is biodegradable and does not produce any toxicity to the organism, ensuring its biocompatibility in vivo.

[0058] Example 13

[0059] The absorbance of meloxicam solutions with different concentration gradients at a wavelength of 272 nm was measured using a UV-Vis spectrophotometer to obtain a standard curve. Figure 6 (b) To assess the drug release, the biomedical hydrogel prepared in Example 2 was placed into a dialysis bag with a cutoff molecular weight of 1000 kDa, and then the dialysis bag was placed into a 50 mL centrifuge tube containing 15 mL of deionized water. The centrifuge tube was then incubated in a 37°C steam bath shaker. At each specific time point, 1 mL of deionized water was taken, and 1 mL of fresh deionized water was added. Finally, the concentration of meloxicam released was determined using a UV-Vis spectrophotometer. Figure 6 As shown in Figure a, meloxicam in the hydrogel exhibits a significant sustained release in phosphate buffer solution. Subsequently, the release tends to stabilize, indicating that the prepared biomedical hydrogel has good application potential in drug sustained release.

[0060] Example 14

[0061] Using *Escherichia coli* (Gram-negative) and *Staphylococcus aureus* (Gram-positive) as bacterial models, the liquid antibacterial activity of this biomedical hydrogel was studied. The hydrogel prepared in Example 2 was used as the experimental group for antibacterial testing. All bacteria were cultured in Luria-Bertani broth (LB) medium. Simultaneously, pure LB medium without bacterial suspension served as the blank group, and the hydrogel group without meloxicam (GP) served as the control group. Different concentrations of hydrogel were added to medium containing bacterial suspension (100 mg / mL, 200 mg / mL, 300 mg / mL) as experimental groups, and incubated at 37°C for 1 day. Each group had three parallel experiments. Finally, the bacterial suspensions after different treatments were collected, and their absorbance at 625 nm was measured. Figure 7 As shown in a and 7c, after 24 hours of incubation, the final inhibition rates of the 300 mg / mL hydrogel against *Escherichia coli* and *Staphylococcus aureus* were 81.77% and 76.16%, respectively. Macroscopic images of the bacterial suspensions after 24 hours of hydrogel culture show that the clearer the bacterial suspension, the less bacterial proliferation, with the drug-containing hydrogel exhibiting a particularly prominent antibacterial effect. Figure 7 b, 7d).

[0062] Example 15

[0063] To further investigate whether biomedical hydrogels cause organ damage, Kunming (KM) mice (approximately 25 g in weight, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were anesthetized with 4% chloral hydrate. GP@MX hydrogel sheets (11 mm in diameter, 2 mm thick) prepared in Example 2, sterilized with alcohol, were subcutaneously implanted into the backs of KM mice. Healthy mice not injected with the hydrogel served as the control group, with three replicates per group. Euthanasia was performed on days 7, 14, and 30, and blood samples were collected for routine and biochemical examinations. Hematoxylin and eosin (H&E) staining of major mouse organs was used to evaluate the safety in mice. H&E staining was performed on mouse heart, liver, spleen, lungs, and kidneys. Figure 9 After 7, 14, and 30 days of treatment, there were no significant differences in the major organs compared to healthy mice, confirming that the GP@MX hydrogel prepared in Example 2 did not cause toxicity to mouse organs.

[0064] Example 16

[0065] To investigate the role of the GP@MX hydrogel prepared in Example 2 in preventing postoperative tissue adhesions, we established a rat model of lateral wall defect-cecal abrasion. After anesthetizing the rats with chloral hydrate, the skin was shaved and disinfected using a razor and 75% alcohol, respectively. A 5 cm midline incision was then made along the linea alba on the rat's abdominal wall, and the abdomen was dissected layer by layer to expose the normal abdominal wall and cecum. The cecum surface was carefully scraped with sterile surgical gauze, and the corresponding abdominal wall was scraped with a scalpel. The area of ​​damage to the abdominal wall and cecum in each rat was approximately 1 × 2 cm. 2 The group treated with only 1 mL of physiological saline was designated as the PBS control group. In the hydrogel experimental groups with different compositions, 1 mL of hydrogel was placed to cover the entire surface of the damaged cecum and the abdominal wall defect. Figure 10 a) Three parallel groups were formed. Finally, the peritoneal cavity was sutured to the abdominal wall using 4-0 sutures, layer by layer. On postoperative days 7 and 14, the rats were euthanized, and the adhesions between the abdominal wall and cecum were examined. The adhesion fraction and representative gross observations are as follows: Figure 10 b and Figure 10 As shown in Figure c, the PBS control group showed strong adhesions between the peritoneum and cecum in rats. The commercial HA hydrogel and drug-free GP hydrogel groups exhibited mild bridging around the cecum and peritoneum due to the barrier effect of the hydrogel, with moderate adhesions. However, most rats treated with GP@MX hydrogel showed no tissue adhesions, and cecal damage and abdominal wall defects partially healed. This demonstrates that the GP@MX hydrogel prepared in Example 2 has a certain effect in preventing tissue adhesions.

[0066] Example 17

[0067] To investigate the expression of t-PA in adhesion-related tissues after different treatment groups, we performed immunostaining on t-PA. Paraffin-embedded (5 μm) tissue sections were dewaxed, rehydrated, blocked with goat serum, and incubated with primary antibody. The tissue sections were then incubated with secondary antibodies labeled with two different fluorescent dyes, and finally, reverse stained with DAPI for 10 minutes. Images were acquired using a fluorescence microscope (Nikon Eclipse Ti-SR, Nikon, Japan). Figure 11 In the control group, surgical trauma led to low t-PA expression (red fluorescence), and the excessively low t-PA activity resulted in postoperative adhesions. After treatment with HA hydrogel, this expression only increased slightly, which may be the main reason for the poor therapeutic effect of HA hydrogel. Conversely, the GP@MX hydrogel group prepared in Example 2 showed more red fluorescence (t-PA), indicating that GP@MX hydrogel can significantly improve t-PA expression levels, demonstrating that GP@MX hydrogel has a better therapeutic effect in postoperative anti-adhesion.

[0068] Example 18

[0069] The expression levels of TNF-α, PAL-1, and t-PA mRNA in locally injured rat tissue were detected using real-time quantitative PCR (qPCR). Rats in each group were euthanized on days 7 and 14 post-surgery, and approximately 80–100 mg of abdominal wall tissue or adhesion tissue from the adhesion sites was collected. Total RNA was extracted using Trizol reagent. The purified RNA was reverse transcribed into cDNA using a reverse transcription kit. A real-time quantitative PCR instrument (MX3005P, Agilent, USA) was used to detect the reaction system containing cDNA, 10 μM gene-specific primers, 1×SYBR™, and ROX™. Gene (mRNA) expression levels were normalized to GAPDH expression levels and analyzed using 2... -ΔΔCT Method for calculating relative expression levels ( Figure 12 TNF-α plays a crucial role in adhesion formation, not only promoting inflammation and coagulation but also reducing fibrinolysis by stimulating the release of plasminogen activator inhibitors (PAIs) and inhibiting the production of PAIs in the peritoneal cavity. In the control group, the relative expression level of TNF-α mRNA in the injured tissue was significantly increased on postoperative days 7 and 14. Figure 12 a). After hydrogel treatment, TNF-α levels were significantly reduced. In contrast, the GP@MX hydrogel prepared in Example 2 showed a more significant inhibitory effect on TNF-α expression. Furthermore, the expression of t-PA and PAL-1 is also closely related to the formation of postoperative adhesions; high PAL-1 expression can inhibit t-PA activity to some extent, leading to postoperative adhesions. GP@MX hydrogel can increase t-PA expression ( Figure 12b) and attenuation of PAL-1 expression ( Figure 12 c). This indicates that PNAAA hydrogel has a significant inhibitory effect on the inflammatory response caused by peritoneal injury.

Claims

1. A method for preparing a biomedical hydrogel, characterized in that, Polyamide-amine dendritic macromolecules are dissolved in a first solvent until completely dissolved, and the mass fraction of the dissolved polyamide-amine dendritic macromolecules is 10%-40%. An anti-inflammatory drug is added to the dissolved dendritic macromolecule solution to obtain solution A, wherein the concentration of the anti-inflammatory drug is 0.5-10 mg / mL, and the anti-inflammatory drug is meloxicam. γ-polyglutamic acid was dissolved in a second solvent and stirred until completely dissolved. The mass fraction of the dissolved γ-polyglutamic acid was 1% - 10%, which yielded solution B. Solution A and solution B are mixed evenly to obtain a mixed solution. Then, a carboxyl activator is added to the mixed solution. The carboxyl activator is any one or two of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. After the reaction is complete, the biomedical hydrogel is obtained.

2. The method for preparing the biomedical hydrogel according to claim 1, characterized in that, The first solvent is methanol, ethanol, water, DMSO, distilled water, phosphate buffer solution, or physiological saline, and the second solvent 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 is 0.9% w / v.

3. The method for preparing the biomedical hydrogel according to claim 1, characterized in that, The polyamide-amine dendritic macromolecule is any one of the third, fourth, fifth, sixth, seventh, or eighth generation.

4. The method for preparing the biomedical hydrogel according to claim 1, characterized in that, The dissolution temperatures of both the γ-polyglutamic acid and the polyamide-amine dendritic macromolecules are 20-80°C. o C.

5. The method for preparing the biomedical hydrogel according to claim 1, characterized in that, The mass fraction of the carboxyl activator is 1% - 30%.

6. The method for preparing the biomedical hydrogel according to claim 1, characterized in that, The carboxyl activator added to the final reaction solution has a mass fraction of 0.5% - 30%.

7. The method for preparing the biomedical hydrogel according to claim 1, characterized in that, The reaction temperature of the mixed solution with the carboxyl activator is 10-30°C. o C.

8. The biomedical hydrogel for preventing postoperative tissue adhesion obtained by the method of any one of claims 1 to 7.

9. The application of the biomedical hydrogel as described in claim 8 in the preparation of anti-inflammatory biomaterials that have drug delivery and sustained-release functions and prevent postoperative tissue adhesion.

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