CN117100918A

CN117100918A - Biomedical hydrogel for preventing postoperative tissue adhesion and preparation method and application thereof

Info

Publication number: CN117100918A
Application number: CN202311328461.7A
Authority: CN
Inventors: 王世革; 蔡洁; 安潇
Original assignee: Shanghai First Peoples Hospital; University of Shanghai for Science and Technology
Current assignee: Shanghai First Peoples Hospital; University of Shanghai for Science and Technology
Priority date: 2023-10-13
Filing date: 2023-10-13
Publication date: 2023-11-24

Abstract

The application provides biomedical hydrogel capable of preventing postoperative tissue adhesion, and a preparation method and application thereof. According to the method, polyamide-amine dendritic macromolecules are dissolved in a solvent, and the mass fraction of the dissolved dendritic macromolecules is 10% -40%; taking a certain amount of dendritic macromolecule solution, and adding an anti-inflammatory drug into the dendritic macromolecule solution; dissolving gamma-polyglutamic acid in a solvent, and stirring, wherein the mass fraction of the dissolved gamma-polyglutamic acid is 1% -10%; mixing the two solutions uniformly, adding a carboxyl activating agent into the mixed solution, wherein the activating agent is any one or two of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and obtaining the biomedical hydrogel after the reaction is completed. The biomedical hydrogel prepared by the application has good biocompatibility, shows excellent anti-adhesion effect in postoperative tissue adhesion application, and can be applied to safe and efficient drug delivery and drug slow release.

Description

Biomedical hydrogel for preventing postoperative tissue adhesion and preparation method and application thereof

Technical Field

The application belongs to the field of biomedical materials, and relates to a gel compound, in particular to biomedical hydrogel suitable for preventing postoperative tissue adhesion, and a preparation method and application thereof.

Background

Polyamide-amine dendrimers (PAMAM), which are units that are monodisperse and highly branched, have a defined structure. The cationic primary amine groups on their surface allow them to bind to other chemical entities. PAMAM dendrimers have good water solubility themselves and are often used as carriers for various hydrophilic and hydrophobic drugs and genes due to their special structure. In addition, PAMAM dendrimers are a popular choice due to their passive targeting, high drug loading, solubilization, sphericity, monodispersity, ability to increase drug half-life, purity and stability, etc. Therefore, PAMAM dendrimers have been widely used in various fields such as biological medicine, drug carriers, tissue engineering, etc., and show good application prospects.

Polyglutamic acid (gamma-PGA) is a biodegradable polymer material with good biocompatibility. The natural amino acid glutamic acid is polymerized to form the natural amino acid glutamic acid which can be degraded and metabolized into carbon dioxide and water through microorganisms. The physicochemical properties of the polyglutamic acid can be regulated and controlled by changing the polymerization degree, substituent groups, crosslinking and the like, so that the polyglutamic acid has wide application prospect. Currently, polyglutamic acid is applied to the fields of medicine, food, agriculture and the like, and becomes an important biodegradable polymer material.

The abdominal adhesions that occur after surgery are a very common problem, as the presence of adhesions from the previous surgery not only increases the difficulty and risk of the next surgery, but also causes a series of complications including ileus, chronic pelvic pain, female infertility, and even death. Currently, the clinical approach to reducing adhesion formation through delicate and minimally invasive procedures, such as laparoscopic procedures, has focused on physical barriers to the primary anti-adhesion strategy, but these approaches have resulted in incomplete elimination of adhesion formation, often resulting in poor and inefficient results.

Disclosure of Invention

Aiming at the problems existing in the prior art, the application provides biomedical hydrogel suitable for preventing postoperative tissue adhesion, and a preparation method and application thereof. The biomedical hydrogel aims to solve the problems that the existing postoperative adhesion material is not ideal in effect, secondary operation is needed and the like.

The application provides a preparation method of biomedical hydrogel suitable for preventing postoperative tissue adhesion, which comprises the steps of dissolving polyamide-amine dendritic macromolecules in a first solvent to completely dissolve the polyamide-amine dendritic macromolecules, wherein the mass fraction of the dissolved dendritic macromolecules is 10% -40%; taking a certain amount of dissolved dendritic macromolecule solution, adding an anti-inflammatory drug into the solution, wherein the concentration of the dispersed antibacterial drug is 0.5-10mg/mL, and obtaining solution A. Dissolving gamma-polyglutamic acid in a second solvent, stirring to completely dissolve the gamma-polyglutamic acid, wherein the mass fraction of the dissolved gamma-polyglutamic acid is 1-10%, so as to obtain a solution B; and uniformly mixing the solution A and the solution B to obtain a mixed solution. And adding a carboxyl activating agent into the mixed solution, wherein the activating agent is one or two of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and obtaining the biomedical hydrogel after complete reaction.

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

Further, the polyamide-amine dendrimer is any one of the third generation, fourth generation, fifth generation, sixth generation, seventh generation and eighth generation.

Further, the mass concentration percentage of the dendritic macromolecule and the gamma-polyglutamic acid is 10-40% in the preparation method of the biomedical hydrogel.

Further, the dissolution temperature of the dendrimer and the gamma-polyglutamic acid is 20-80 ℃ in the preparation method of the biomedical hydrogel.

Further, the mass fraction of the carboxyl activating agent in the preparation method of the biomedical hydrogel is 1% -30%.

Further, the mass fraction of the carboxyl activating agent added into the final reaction solution is 0.5-30% in the preparation method of the biomedical hydrogel.

Further, the preparation method of the biomedical hydrogel is characterized by comprising the following steps: the reaction temperature is 10-30 ℃.

According to the method, the carboxyl activating agent can activate carboxyl groups in the gamma-polyglutamic acid, so that the gamma-polyglutamic acid can carry out a crosslinking reaction with amino groups in the polyamide-amine dendrimer. The application provides a preparation method for realizing the solubilization of meloxicam drugs and gamma-polyglutamic acid crosslinking gel by adopting polyamide-amine dendritic macromolecules under the activation action of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride-N-hydroxysuccinimide (EDC-NHS).

The application also provides the biomedical hydrogel obtained by the method and the application of the biomedical hydrogel in preparing anti-inflammatory biological materials with drug delivery and drug slow release effects for preventing postoperative tissue adhesion.

The application has simple process, the product preparation is very rapid, and the two solutions are mixed to form glue rapidly within about 30 seconds. The obtained product shows good biocompatibility in-vitro and in-vivo external mold experiments. According to the application, polyamide-amine dendritic macromolecules and gamma-polyglutamic acid are selected as matrixes, and anti-inflammatory drugs are added into the matrixes, and meanwhile, EDC-NHS is utilized to activate carboxyl groups in the gamma-polyglutamic acid, so that the carboxyl groups can be crosslinked with a large number of amino groups in the polyamide-amine dendritic macromolecules, and the biomedical hydrogel is prepared. Experiments show that the hydrogel prepared by the method has the characteristics of excellent flexibility and elasticity, good water swelling, degradation and the like. After the medicine is loaded, the medical hydrogel can gradually release the medicine after entering the body, so that the stability and the persistence of the medicine are improved, and meanwhile, the side effect of the medicine is reduced. Meanwhile, the application has good in vivo degradation performance, so that secondary operation removal is not needed after operation, and the damage to patients caused by the secondary operation is reduced. The material not only improves the bioavailability of the medicine, but also reduces the damage and influence on normal tissues and organs.

Compared with the prior art, the application has obvious technical progress. The biomedical hydrogel prepared by the application has simple process and easily obtained product. Furthermore, the product has soft, injectable and other properties that allow it to adapt to different shapes and visceral surfaces. The hydrogel loaded with the medicine is expected to be applied to the fields of medicine delivery, medicine slow release and the like, and has a certain clinical application value. By establishing a mouse cecum-abdominal wall adhesion model, experiments finally prove that the designed biomedical hydrogel can remarkably reduce the formation of postoperative abdominal adhesion in the mouse model, plays a good role in resisting adhesion, and proves the clinical application prospect.

Drawings

FIG. 1a is a FESEM photograph of the biomedical hydrogel prepared in example 1, and FIG. 1b is a FESEM photograph of the biomedical hydrogel prepared in example 2; FIG. 1c is a FESEM image of biomedical hydrogels prepared in example 3; fig. 1d is a FESEM picture of the biomedical hydrogel of example 2, partially enlarged.

FIG. 2a is a graph of dynamic time-scanning rheological analysis of biomedical hydrogels prepared in example 1; FIG. 2b is a graph of dynamic time-scanning rheological analysis of biomedical hydrogels prepared in example 2; FIG. 2c is a graph of dynamic time-scanning rheological analysis of biomedical hydrogels prepared in example 3.

FIG. 3a is a compression strain-stress curve test result of biomedical hydrogels prepared in examples 1, 2 and 3; FIG. 3b shows the results of the average compressive stress test of biomedical hydrogels prepared in examples 1, 2 and 3.

FIG. 4a shows the viability of L929 cells after co-culture with the biomedical hydrogels prepared in example 2; FIG. 4b shows the DEAD/LIVE staining results without material treatment; FIGS. 4c to 4f are the results of DEAD/LIVE staining of biomedical hydrogels prepared in example 2, wherein the biomedical hydrogels in FIG. 4c have a concentration of 5mg/mL and the biomedical hydrogels in FIG. 4d have a concentration of 10mg/mL; the biomedical hydrogel in FIG. 4e has a concentration of 25mg/mL; the biomedical hydrogel in FIG. 4f was 50mg/mL.

FIGS. 5a to 5f are relevant test results of biomedical hydrogels prepared in examples 1 to 3, wherein FIG. 5a is a hemolysis test result of biomedical hydrogels prepared in example 2; FIG. 5b shows the BSA protein adsorption results; FIG. 5c is a swelling kinetics plot; FIG. 5d shows the swelling ratio results; FIG. 5e is an in vitro degradation curve of biomedical hydrogels in phosphate buffered saline; FIG. 5f is a degradation curve of the biomedical hydrogel prepared in example 2 in mice.

FIG. 6a shows the results of in vitro release analysis of the biomedical hydrogels prepared in examples 1 to 3, loaded with meloxicam; figure 6b is a standard curve of meloxicam.

Fig. 7a to 7d are in vitro antibacterial test results of biomedical hydrogels prepared in example 2: FIG. 7a shows the antibacterial rate of biomedical hydrogels against E.coli; FIG. 7b is a graph showing the antibacterial effect of E.coli; FIG. 7c shows the antibacterial rate of biomedical hydrogels against Staphylococcus aureus; fig. 7d is a graph of the bacteriostatic results of staphylococcus aureus.

Fig. 8a and 8b show in vivo degradation experimental results of biomedical hydrogels: FIG. 8a is a photograph taken with a camera of the skin around a back incision in a hydrogel; fig. 8b is a photograph of hydrogel size for various time periods.

Fig. 9 tissue organ safety results for biomedical hydrogels: h & E staining patterns of mice heart, liver, spleen, lung, kidney at different time points.

Fig. 10a to 10c show experimental results of biomedical hydrogels for preventing postoperative tissue adhesion: FIG. 10a is a graph of a model of rat sidewall defect-cecal abrasion; figure 10b adhesion score for adhesion formation; fig. 10c is a representative observation of adhesion formation after day 7, day 14.

FIG. 11 shows the results of t-PA and DAPI immunostaining of biomedical hydrogels.

Fig. 12a to 12c show real-time fluorescent quantitative PCR results for biomedical hydrogels: FIG. 12a is TNF- α mRNA expression levels; FIG. 12b is t-PA mRNA expression levels; FIG. 12c shows the PAL-1mRNA expression level.

Detailed Description

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

According to the application, an anti-inflammatory drug and a carboxyl activating agent are added into a gamma-polyglutamic acid and polyamide-amine dendritic macromolecule solution, the anti-inflammatory drug is non-steroidal meloxicam, EDC-NHS is taken as the carboxyl activating agent, three preparation methods of gamma-polyglutamic acid gel (20% in example 1, 30% in example 2 and 40% in example 3) with different proportions are selected, specifically, meloxicam and the dendritic macromolecule solution are firstly mixed and stirred to be uniform, then the mixture is stirred and mixed with the gamma-polyglutamic acid solution with different proportions, and EDC-NHS is added to enable the solution system to be gel. All three methods can obtain biomedical hydrogel with a certain network structure.

Example 1

The third generation polyamide-amine dendrimer (G3) was dissolved in methanol at a concentration of 200mg/mL, 100. Mu. L G3 in methanol and 5mg Meloxicam (MX) were dissolved in 1mL deionized water and 100. Mu.L DMSO solution, respectively, and then the two were mixed to give a clear and transparent solution. And then 0.1g of gamma-polyglutamic acid is dissolved in 4mL of deionized water, stirred uniformly at room temperature, and then mixed with the solution to obtain the dendritic macromolecule-meloxicam-gamma-polyglutamic acid mixed solution. Then, about 80mg of EDC and 80mg of NHS were added to 1mL of deionized water, respectively, to prepare EDC-NHS mixed solutions. And finally, adding the mixed solution of EDC and NHS into the mixed solution of dendritic macromolecules-meloxicam-gamma-polyglutamic acid, and uniformly stirring to obtain the biomedical hydrogel.

Example 2

The third generation polyamide-amine dendrimer (G3) was dissolved in methanol at a concentration of 200mg/mL, 100. Mu. L G3 in methanol and 5mg Meloxicam (MX) were dissolved in 1mL deionized water and 100. Mu.L DMSO solution, respectively, and then the two were mixed to give a clear and transparent solution. And then 0.15g of gamma-polyglutamic acid is dissolved in 4mL of deionized water, stirred uniformly at room temperature, and then mixed with the solution to obtain the dendritic macromolecule-meloxicam-gamma-polyglutamic acid mixed solution. Then, about 80mg of EDC and 80mg of NHS were added to 1mL of deionized water, respectively, to prepare EDC-NHS mixed solutions. And finally, adding the mixed solution of EDC and NHS into the mixed solution of dendritic macromolecules-meloxicam-gamma-polyglutamic acid, and uniformly stirring to obtain the biomedical hydrogel.

Example 3

The third generation polyamide-amine dendrimer (G3) was dissolved in methanol at a concentration of 200mg/mL, 100. Mu. L G3 in methanol and 5mg Meloxicam (MX) were dissolved in 1mL deionized water and 100. Mu.L DMSO solution, respectively, and then the two were mixed to give a clear and transparent solution. Then 0.2g of gamma-polyglutamic acid (gamma-PGA) is dissolved in 4mL of deionized water, stirred evenly at room temperature, and then mixed with the solution to obtain the dendrimer-meloxicam-gamma-polyglutamic acid mixed solution. Then, about 80mg of EDC and 80mg of NHS were added to 1mL of deionized water, respectively, to prepare EDC-NHS mixed solutions. And finally, adding the mixed solution of EDC and NHS into the mixed solution of dendritic macromolecules-meloxicam-gamma-polyglutamic acid, and uniformly stirring to obtain the biomedical hydrogel.

Example 4

To analyze the morphology of biomedical hydrogels, samples of 20% (example 1), 30% (example 2) and 40% (example 3) PGA were analyzed on a FEI Magellan 400-type field emission scanning electron microscope.

As can be seen from the electron microscopy images, the biomedical hydrogels all exhibit three-dimensional pore structures (fig. 1 a-d). Fig. 1a, 1b and 1c are scanning electron microscope pictures of example 1, example 2 and example 3 at different angles of 100 μm, respectively. To further observe the intrinsic structure of the hydrogel, the hydrogel prepared in example 2 (fig. 1 b) was partially enlarged and a scanning electron microscope picture was taken (fig. 1 d).

Example 5

The gelation process of the hydrogels was studied by observing the final storage modulus (G') and the final loss modulus (G ") in a dynamic time-sweep rheological test. The rotational rheometer (MARS III HAAKE) was studied dynamically using the geometry of a flat plate (P20 TiL, diameter 20 mm). The hydrogels of example 1, example 2 and example 3 were each subjected to time-sweep oscillation tests at a frequency of 1Hz, a gap of 1mm and a strain of 1%. The corresponding hydrogel precursor solution was poured onto the plate and the gap was adjusted to 1mm. Frequency sweep measurements of hydrogels are denoted as G' and G ". The G' of the hydrogels increased rapidly due to intermolecular crosslinking, indicating that the hydrogel formation efficiency was very high (FIGS. 2a,2b,2 c).

Example 6

Mechanical evaluation was performed on a Zwick Roell Z2.5 TH universal material tester using a 2.5kN sensor. Compression properties of biomedical hydrogels were studied using the modified American society for Material testing and materials method. In the compression test, hydrogels were prepared in a cylindrical model with a diameter of 8mm and a thickness of 4mm, with a compressive strain rate of 0.5mm/min. The compressive modulus was recorded by a linear fit of the stress-strain curve over a strain range of 10-20% (fig. 3 a). The maximum compressive stress for the hydrogel prepared in example 1 was 19.467kPa, the maximum compressive stress for the hydrogel prepared in example 2 was 75.297kPa, and the maximum compressive stress for the hydrogel prepared in example 3 was 157.648kPa (FIGS. 3a,3 b). The experimental results show that the hydrogels prepared in example 2 have a relatively moderate degree of softness, are neither too soft to resist compression nor too hard to fit well with tissue in vivo.

Example 7

The hydrogels prepared in example 2 were incubated with L929 cells for 3 days, and cytotoxicity of the hydrogels was quantitatively and qualitatively detected using CCK-8 and LIVE/DEAD cell activity detection kits at days 1, 2, and 3, respectively. Hydrogel leachates (5, 10, 25 and 50 mg/mL) with different concentrations are respectively added into the cell culture media, only 100 mu L of the cell culture media (the survival rate is set to be 100%) is added into the control group, and the cell survival rates after 1 day of culture are 98.88+/-4.67%, 99.34+/-3.48%, 98.56+/-4.23% and 99.32+/-10.51% respectively. After 2 days, the cell viability was 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 was 99.31.+ -. 2.17%, 98.27.+ -. 0.51%, 99.95.+ -. 1.89% and 98.78.+ -. 0.62%, respectively. The results showed that the cell viability was above 95% and that the biomedical hydrogels prepared in example 2 had no significant toxic effect on L929 cells (fig. 4 a). The LIVE/DEAD cell activity assay showed that, similar to the control (fig. 4 b), all hydrogel-treated cells could be stained green with LIVE/DEAD reagent (LIVE cells were stained green) and almost no cells were stained red (DEAD cells were stained red) (fig. 4c,4d,4e and 4 f). The result of CCK-8 and LIVE/DEAD cell staining shows that the prepared biomedical hydrogel has good cell compatibility.

Example 8

Example 7 the blood compatibility of biomedical hydrogels was investigated. Fresh whole blood from mice was centrifuged (3000 rpm,5 min), plasma was taken and washed 3 times with phosphate buffer solution to collect red blood cells. The resulting red blood cells were dissolved in 50mL of phosphate buffer solution for further use.

Hemolysis experiments 2.4mL of phosphate buffer solution and red blood cell suspension (600 μl) were added to a 5mL centrifuge tube. The hydrogels prepared in example 2 were placed in DMEM cell culture medium (50 mg hydrogel, 1mL medium) with different mass, and incubated overnight at 37 ℃ to prepare a leachate. The concentration of the leachate was set to 5mg/mL, 10mg/mL, 25mg/mL, and 50mg/mL. The other two groups were designated as control groups, and the red blood cell suspensions were treated with phosphate buffer (negative control group) and deionized water (positive control group), respectively. The above mixed solution was incubated at 37℃for 2 hours, and then centrifuged at 5000rpm for 3 minutes, and the absorbance of the supernatant at 541nm (Shimadzu UV-3600 ultraviolet visible near infrared spectrometer) was collected, and the hemolysis ratio of erythrocytes was calculated. As shown in FIG. 5a, the calculated hemolysis rates of the hydrogels were all less than 5%. As can be seen from the photographs on the figures, the red cell supernatant incubated with the hydrogel and phosphate buffer solution was transparent. However, blood treated with deionized water appears significantly red due to hemolysis positivity. The result shows that the biomedical hydrogel has good blood compatibility.

Example 9

The biomedical hydrogels were tested for their anti-fouling ability by measuring their resistance to nonspecific protein adsorption and cell attachment. Nonspecific protein adsorption assay was performed using BCA protein assay kit. The hydrogels prepared in example 1, example 2 and example 3 were placed in 24-well plates, respectively, and bovine serum albumin (BSA protein) solutions at a concentration of 2mg/mL were added, respectively. After incubation at 37 ℃ for 2h, the hydrogel was removed from the protein solution and rinsed 3 times with phosphate buffer solution to remove loosely adsorbed proteins from the surface. The hydrogel was then immersed in 1mL of fresh phosphate buffer solution and the ultrasonic cleaning was continued for 10 minutes to remove the proteins firmly adsorbed on the hydrogel. The protein concentration in the solution was obtained from the micro BCA protein assay kit from absorbance measured at 562nm wavelength using a microplate reader (Bio-Tek company, usa, ELx 808). The results showed that the hydrogels prepared in example 1, example 2, and example 3 adsorbed BSA protein in amounts of 14.42, 16.39, and 17.05. Mu.g cm, respectively ^-2 (FIG. 5 b). This indicates that the hydrogels have good resistance to non-specific protein adsorption.

Example 10

To investigate the swelling ability of the biomedical hydrogels in phosphate buffer solution, first, the hydrogels obtained in example 1, example 2 and example 3 were freeze-dried to weigh the initial mass, then added to phosphate buffer solution, soaked at 37 ℃ for 24 hours in total, taken out at different time points, gently rubbed to remove surface moisture, and then weighed. The expansion ratio is finally determined after calculation of the resulting mass and the initial mass (fig. 5 d). In addition, the swelling kinetics of the hydrogels in deionized water were studied (FIG. 5 c), demonstrating that the hydrogels absorbed fluid very rapidly, reaching 16.34g/g,27.52g/g and 30.25g/g, respectively, after 6 to 8 hours of storage in phosphate buffer solution.

Example 11

The biomedical hydrogels prepared in example 1, example 2 and example 3 after freeze-drying were weighed separately, incubated with phosphate buffer solution, and the degradation degree of the hydrogel was measured continuously for 28 days at a constant temperature of 37 ℃. Wherein the phosphate buffer solution is replaced every other day. At each time point, biomedical hydrogels were removed from the culture medium, gently rinsed with phosphate buffer solution, and then lyophilized. The mass of the lyophilized biomedical hydrogel was weighed to calculate the degradation rate. The experimental results showed that the hydrogel was substantially completely degraded in phosphate buffered saline, around 28 days, demonstrating the degradability of the hydrogel (fig. 5 e).

Example 12

The in vivo degradability of the hydrogel was evaluated by monitoring the degradation of the biomedical hydrogel in mice. Kunming (KM) mice (body weight about 25g, peking Wei Tongli Hua Experimental animal technologies Co., ltd.) were anesthetized with 4% chloral hydrate and randomly divided into five groups: 2.4, 8, 12 and 24 hours. Each group of three are parallel. Then, a dorsal incision of about 15mm was made along the medial dorsal skin of the KM mouse with surgical scissors. The alcohol-sterilized meloxicam drug-containing hydrogel (gp@mx) hydrogel sheet (diameter 11mm, thickness 2 mm) prepared in example 2 was implanted under the incision. The body weight of the mice was recorded at the corresponding time points and euthanized, and then the size of the hydrogel removed in the body and the skin around the incision in the back side of the mice were measured and recorded by a camera (fig. 8a, b). After about 24 hours, the hydrogel prepared from example 2 was completely degraded (fig. 5 f). Notably, at 2h, the hydrogel mass increased, probably due to absorption of a portion of the interstitial fluid. The research result of in vivo degradation shows that the prepared biomedical hydrogel has biodegradability, does not generate any toxicity to organisms, and ensures the biocompatibility of the hydrogel in vivo.

Example 13

The absorbance of meloxicam solutions with different concentration gradients at 272nm wavelength was measured using an ultraviolet-visible spectrophotometer (UV-vis) to give a standard curve (fig. 6 b). To evaluate the amount of drug released, the biomedical hydrogel prepared in example 2 was filled into a dialysis bag having a cut-off molecular weight of 1000kDa, and then the dialysis bag was placed into a 50mL centrifuge tube containing 15mL of deionized water. Subsequently, the centrifuge tube was incubated in a steam bath shaker at 37 ℃. At each particular time point, 1mL of deionized water was taken and 1mL of fresh deionized water was added. Finally, the released meloxicam concentration was determined using a UV-vis spectrophotometer. As shown in fig. 6a, meloxicam drug in the hydrogel showed a more pronounced sustained release in phosphate buffered solution. Then, the release tends to be in a stable state, which shows that the prepared biomedical hydrogel has good application potential in the aspect of drug slow release.

Example 14

The liquid antibacterial activity of the biomedical hydrogel was studied using escherichia coli (gram negative) and staphylococcus aureus (gram positive) as bacterial models. The hydrogels prepared in example 2 were used as experimental groups for bacteriostasis tests, and all bacteria were cultivated in Luria-Bertani broth (LB) medium. Meanwhile, LB pure culture medium without bacterial liquid is taken as a blank group, hydrogel Group (GP) without meloxicam drug is taken as a control group, hydrogels with different concentrations are respectively added into culture medium (100 mg/mL,200mg/mL,300 mg/mL) with bacterial liquid as an experimental group, and incubated for 1d in an incubator at 37 ℃. Each set was set up with 3 replicates. Finally, the various treated bacterial suspensions were collected and their absorbance at 625nm was measured. As shown in FIGS. 7a,7c, after 24 hours incubation, the final 300mg/mL hydrogel had bacteriostasis rates of 81.77% and 76.16% for E.coli and Staphylococcus aureus, respectively. The macroscopic image of the bacterial suspension after 24 hours of incubation of the hydrogel shows that the clearer the bacterial suspension, the less bacteria proliferate, and the antibacterial effect of the hydrogel containing the drug is particularly remarkable (figures 7b,7 d).

Example 15

To further investigate whether biomedical hydrogels caused damage to viscera, kunming (KM) mice (weighing about 25g, beijing Wei Tongli Hua Experimental animal technologies Co., ltd.) were anesthetized with 4% chloral hydrate. The back of KM mice was subcutaneously implanted with GP@MX hydrogel sheets (diameter 11mm, thickness 2 mm) prepared in example 2 after alcohol sterilization. Healthy mice without hydrogel injection were used as control groups, three groups were euthanized on days 7, 14, and 30, respectively, and blood was collected for routine and biochemical examination. The safety of the main organ sections of mice in the mice was evaluated by hematoxylin-eosin staining (H & E). Mice were heart, liver, spleen, lung, kidney were H & E stained (fig. 9). After 7, 14 and 30 days of treatment, the main organs are not significantly different from healthy mice, and the gp@mx hydrogel prepared in example 2 is proved to be non-toxic to the organs of the mice.

Example 16

To investigate the role of the gp@mx hydrogel prepared in example 2 in preventing post-operative tissue adhesions, we established a rat lateral wall defect-cecal abrasion model. After anesthetizing the rats with chloral, the skin of the rats was shaved and disinfected with a razor and 75% alcohol, respectively. Then, a 5cm median incision was made along the white line on the abdominal wall of the rat, and the abdomen of the rat was incised layer by layer to expose the normal abdominal wall and cecum. The cecal surface was carefully scraped with sterile surgical gauze, the corresponding abdominal wall was scraped with a scalpel, and the area of abdominal wall and cecal lesions was about 1X 2cm per rat ² . A1 mL rinse with saline alone was set as the PBS control group. Different composition hydrogel experimental groups 1mL of hydrogel was placed to cover the entire damaged cecal surface and abdominal wall defect (fig. 10 a). Each group of three are parallel. Finally, the peritoneal cavity is sutured on the abdominal wall layer by layer with a 4-0 needle. After euthanizing the rats on postoperative days 7 and 14, the rats were examined for adhesion between the abdominal wall and the cecum. The adhesion score and representative general observations of adhesion formation are shown in fig. 10b and 10 c. The peritoneum and cecum adhesion of the rats in the PBS control group were stronger. Commercial HA hydrogels and drug-free GP hydrogel groups exhibited slight bridging around the cecum and peritoneum due to the barrier effect of the hydrogels, with moderate adhesion. Whereas most rats treated with gp@mx hydrogel had no tissue adhesion and recovery of cecal lesions and abdominal wall defects. The GP@MX hydrogel prepared in the example 2 has a certain tissue adhesion preventing effect.

Example 17

To study t-PA expression in adhesion-associated tissues treated with different groups, we immunostained t-PA. Paraffin-embedded (5 μm) tissue sections were deparaffinized, rehydrated, goat serum blocked, primary anti-incubated. The tissue sections were then incubated with two different fluorescent dye labeled secondary antibodies and finally counterstained with DAPI for 10 minutes. Images were acquired using a fluorescence microscope (Nikon Eclipse Ti-SR, nikon, japan) (FIG. 11). Surgical trauma in the control group resulted in low t-PA expression (red fluorescence), and too low t-PA activity resulted in post-operative adhesions. This expression increases only slightly after HA hydrogel treatment, which may be the main reason for the poor therapeutic effect of HA hydrogels. In contrast, the group of gp@mx hydrogels prepared in example 2 detected more red fluorescence (t-PA), which may indicate that the gp@mx hydrogels may significantly increase the expression level of t-PA, demonstrating that the gp@mx hydrogels have better therapeutic effect in terms of post-operative anti-adhesion.

Example 18

The expression level of TNF-alpha, PAL-1 and t-PA mRNA in the local damaged tissue of the rat is detected by adopting real-time fluorescence quantitative PCR (qPCR). On the 7 th and 14 th days after the operation, each group of rats was euthanized, and about 80-100 mg of the abdominal wall tissue or the adhesion tissue at the adhesion site was taken. Total RNA was extracted with Trizol reagent. The isolated and purified RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction system containing cDNA, 10. Mu.M gene-specific primers, 1 XSYBRTM and ROXTM was detected using a real-time fluorescent quantitative PCR apparatus (MX 3005P, agilent, USA). Normalizing the expression level of a gene (mRNA) to the expression level of GAPDH by 2 ^-ΔΔCT The relative expression level was calculated by the method (FIG. 12). Of these, TNF- α is important for adhesion formation, and it not only promotes inflammation and coagulation, but also reduces fibrinolysis by stimulating release of a plasminogen activator inhibitor and inhibiting production of a plasminogen activator in the abdominal cavity. In the control group, the relative expression of TNF- α mRNA in the injured tissue was significantly increased on both postoperative day 7 and day 14 (fig. 12 a). After hydrogel treatment, TNF- α levels were significantly reduced. In contrast, the GP@MX hydrogel prepared in example 2 had a more pronounced inhibition of TNF- α expression. In addition, the expression of t-PA and PAL-1 is also closely related to the formation of postoperative adhesion, and the high expression of PAL-1 can inhibit the activity of t-PA to a certain extent so that the postoperative adhesion is generated. GP@MX hydrogels increased expression of t-PA (FIG. 12 b) and decreased expression of PAL-1 (FIG. 12 c). Elucidation of inflammation caused by PNAAA hydrogel on peritoneal injuryThe symptom response has obvious inhibition effect.

Claims

1. A preparation method of biomedical hydrogel is characterized in that polyamide-amine dendritic macromolecules are dissolved in a first solvent to be completely dissolved, and the mass fraction of the dissolved polyamide-amine dendritic macromolecules is 10% -40%; adding the anti-inflammatory drug into the dissolved dendrimer solution to obtain solution A;

dissolving gamma-polyglutamic acid in a second solvent, stirring to completely dissolve the gamma-polyglutamic acid, wherein the mass fraction of the dissolved gamma-polyglutamic acid is 1-10%, so as to obtain a solution B;

uniformly mixing the solution A and the solution B to obtain a mixed solution, and adding a carboxyl activating agent into the mixed solution, wherein the carboxyl activating agent is any one or two of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the biomedical hydrogel is obtained after the reaction is completed.

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

3. The biomedical hydrogel according to claim 1, wherein the polyamidoamine dendrimer is any one of the third, fourth, fifth, sixth, seventh and eighth generations.

4. The method for preparing biomedical hydrogels according to claim 1, wherein the dissolution temperature of said gamma-polyglutamic acid and polyamidoamine dendrimer is 20-80 ℃.

5. The method for preparing biomedical hydrogels according to claim 1, wherein the mass fraction of the carboxy activator is 1% -30%.

6. The method for preparing biomedical hydrogels according to claim 1, wherein the mass fraction of the carboxyl activator added to the final reaction solution is 0.5% -30%.

7. The method for preparing biomedical hydrogels according to claim 1, wherein the reaction temperature of the mixed solution and the carboxyl activating agent is 10-30 ℃.

8. The method for preparing biomedical hydrogel according to claim 1, wherein the concentration of the anti-inflammatory drug is 0.5-10mg/mL, and the anti-inflammatory drug is meloxicam.

9. Biomedical hydrogels obtained by the method according to any one of claims 1 to 8 for preventing postoperative tissue adhesion.

10. The use of the biomedical hydrogel according to claim 9 for preparing an anti-inflammatory biomaterial with drug delivery and drug sustained release effects for preventing postoperative tissue adhesion.