A method for preparing an immunomodulatory nanocomposite hydrogel

Abstract

An immunomodulatory nanocomposite hydrogel and its preparation method are disclosed. The preparation process includes: firstly, preparing polydopamine-coated manganese dioxide nanoparticles loaded with ginsenoside Rk3; then dispersing these nanoparticles in a solution of polyethyleneimine-grafted γ-polyglutamic acid; mixing this with a methacrylated oxidized hyaluronic acid solution; and finally preparing the immunomodulatory nanocomposite hydrogel via dynamic Schiff base bonding and free radical polymerization. This hydrogel effectively responds to pH and ROS, exhibiting oxygen-generating, photothermal antibacterial, and anti-inflammatory activities. It specifically addresses the immune dysregulation problem during the healing process of deep second-degree burns, promoting the transformation of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype, and effectively shortening the healing cycle of deep second-degree burns.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing and applying an immunomodulatory nanocomposite hydrogel. Background Technology

[0002] The skin, the largest organ in the human body, acts as a physical barrier, providing primary protection against various pathogens. Burns are injuries to the skin or other organic tissues. The severe inflammatory infiltration caused by the post-burn inflammatory response, the pain, the immediate loss of blood flow to the injured site, and the altered blood flow to surrounding areas all negatively impact wound healing and can lead to scarring. Accelerating wound healing and minimizing scarring is a significant challenge in post-burn surgery. In recent years, multifunctional hydrogel wound dressings have attracted increasing interest in addressing this issue.

[0003] Rare ginsenoside Rk3 exhibits effects such as inhibiting the production of oxidative molecules, increasing the production of antioxidant molecules, reducing the infiltration of inflammatory cells, and decreasing the production of pro-inflammatory factors, making it highly applicable in burn wound healing. However, rare ginsenosides are lipid-soluble drugs with low water solubility and short half-lives, resulting in poor bioavailability and limiting their medical applications. Nanostructured integrated hydrogels, acting as bioactive reservoirs, not only improve the efficiency and safety of biodistribution, bioavailability, and delivery processes, but also increase the retention time of loaded drugs compared to conventional hydrogels. More importantly, these hybrid networks of hydrogels can impart responsiveness to controlled drug release. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for preparing an immunomodulatory nanocomposite hydrogel, addressing the shortcomings of the prior art. The method of this invention uses γ-polyglutamic acid grafted with polyethyleneimine, PM@Rk3 nanoparticles, and oxidized methacrylated hyaluronic acid as main raw materials. After mixing and stirring, and then irradiating with ultraviolet light, a composite hydrogel of PM@Rk3 nanoparticles is obtained. This composite hydrogel can effectively respond to pH and ROS, exhibiting oxygen-generating, photothermal antibacterial, and anti-inflammatory activities, and can be applied to the healing of burn wounds.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] An immunomodulatory nanocomposite hydrogel is composed of a hydrogel colloid and drug-loaded nanoparticles loaded within the hydrogel colloid. The hydrogel colloid is formed by Schiff base bonding and free radical polymerization of methacrylated oxidized hyaluronic acid and γ-polyglutamic acid grafted with polyethyleneimine. The drug-loaded nanoparticles are polydopamine-coated manganese dioxide nanoparticles loaded with ginsenoside Rk3. The concentration of the methacrylated oxidized hyaluronic acid is 3% (w / v), the concentration of the γ-polyglutamic acid grafted with polyethyleneimine is 5% (w / v), the concentration of the phenyl-2,4,6-trimethylbenzoyl phosphite lithium photoinitiator is 0.2% (w / v), and the concentration of the polydopamine-coated drug-loaded nanoparticles is 100 μg / mL. The volume ratio of the methacrylated oxidized hyaluronic acid to the γ-polyglutamic acid grafted with polyethyleneimine is 2:1.

[0007] The hydrogel is an injectable hydrogel suitable for wounds of various shapes. It gels in 30 seconds, exhibiting rapid gelation and preventing rapid diffusion at the wound site. Due to the high water absorption capacity of hyaluronic acid and γ-polyglutamic acid, the hydrogel displays a high swelling level, which is beneficial for cell migration, oxygen and nutrient diffusion, and wound healing. The hydrogel also has good compressibility, meeting the mechanical requirements of soft tissues and showing promising application prospects in soft tissue engineering.

[0008] A method for preparing an immunomodulatory nanocomposite hydrogel, comprising:

[0009] Provides honeycomb-shaped manganese dioxide nanoparticles;

[0010] Grafting amino groups onto manganese dioxide nanoparticles yields positively charged manganese dioxide nanoparticles.

[0011] Ginsenoside Rk3 was loaded onto positively charged manganese dioxide nanoparticles to obtain drug-loaded nanoparticles.

[0012] A layer of polydopamine was coated onto the outer layer of drug-loaded nanoparticles to obtain polydopamine-coated drug-loaded nanoparticles.

[0013] Provides γ-polyglutamic acid for grafting polyethyleneimine;

[0014] Provides methacrylated oxidized hyaluronic acid;

[0015] Drug-loaded nanoparticles coated with polydopamine were dispersed in a γ-polyglutamic acid solution grafted with polyethyleneimine. A methacrylated oxidized hyaluronic acid solution was mixed with the γ-polyglutamic acid solution grafted with polyethyleneimine containing drug-loaded nanoparticles coated with polydopamine to obtain an immunomodulatory nanocomposite hydrogel.

[0016] Provides honeycomb-shaped manganese dioxide nanoparticles, specifically including:

[0017] Potassium permanganate was dissolved in deionized water and the mixture was stirred rapidly for about 0.5 hours. Then total oleic acid was added to allow the resulting mixture to react for 48 hours. The crude brown-black product was collected by centrifugation and washed several times with deionized water and ethanol to remove any possible residual reactants. The purified manganese dioxide nanoparticles were dispersed in anhydrous ethanol.

[0018] Furthermore, the concentration of potassium permanganate was 3.16 mmol, the concentration of oleic acid was 15.75 mmol, the stirring speed was 900 r / min, the reaction speed was 500 r / min, the concentration of honeycomb manganese dioxide was 0.83 mg / mL, and the concentration of 3-aminopropyltriethoxysilane was 6.67 μg / mL.

[0019] Furthermore, amino groups are grafted onto manganese dioxide nanoparticles to obtain positively charged manganese dioxide nanoparticles, specifically including:

[0020] The prepared honeycomb manganese dioxide was poured into a round-bottom flask containing anhydrous ethanol and carried out under ultrasonic conditions to ensure uniform dispersion of the suspension. 3-aminopropyltriethoxysilane was added to the suspension at 40°C and stirred for 12 hours. The precipitate was collected by centrifugation and dried overnight.

[0021] Furthermore, ginsenoside Rk3 was loaded onto positively charged manganese dioxide nanoparticles to obtain drug-loaded nanoparticles, specifically including:

[0022] First, ginsenoside Rk3 was dissolved in ethanol. Then, positively charged manganese dioxide nanoparticles were dispersed in the ethanol solution containing ginsenoside Rk3 and ultrasonically mixed. After reacting for an appropriate time at an appropriate stirring speed, the precipitate was separated and collected by centrifugation at 10,000 rpm for 20 min and dried in a vacuum drying oven.

[0023] Furthermore, the concentration ratio of ginsenoside Rk3 to positively charged manganese dioxide nanoparticles was 10:1, the stirring speed was 300 r / min, the reaction time was 3 h, and the concentration of ginsenoside Rk3 was mg / mL, and the concentration of positively charged manganese dioxide nanoparticles was mg / mL.

[0024] Furthermore, a layer of polydopamine is coated onto the outer layer of the drug-loaded nanoparticles to obtain polydopamine-coated drug-loaded nanoparticles, specifically including:

[0025] First, prepare a 0.01M pH-8.5 Tris-HCl buffer solution by weighing 0.1211g of Tris and dissolving it in 100mL of water and stirring until the solid is completely dissolved. Then, use a pH meter and add HCl aqueous solution to bring the pH value to 8.5.

[0026] Drug-loaded nanoparticles were dispersed in Tris-HCl buffer, then dopamine hydrochloride was added and stirred slowly. The nanoparticles were washed with deionized water by centrifugation and centrifuged three times at 10,000 rpm to obtain polydopamine-coated drug-loaded nanoparticles, which were then vacuum dried and stored.

[0027] Furthermore, the concentration of the drug-loaded nanoparticles dispersed in Tris-HCl buffer was 0.2 mg / mL, the concentration of dopamine hydrochloride was 0.1 mg / mL, and the reaction time was 20 min.

[0028] Furthermore, the γ-polyglutamic acid grafted with polyethyleneimine specifically includes:

[0029] γ-Polyglutamic acid was completely dissolved in water; the pH of the γ-polyglutamic acid solution was adjusted to 5.5 with HCl, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added sequentially. After reacting for 60 minutes, the pH of the reaction solution was adjusted to 8 with NaOH, and polyethyleneimine was added. The system was allowed to continue reacting at room temperature for 24 hours. When the reaction was complete, the product was dialyzed (molecular weight cutoff (MWCO) 8000-14000 kDa) for 3 days, and the resulting solution was lyophilized to obtain polyethyleneimine-grafted γ-polyglutamic acid; the concentration of the γ-polyglutamic acid was 2% (w / v), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to γ-polyglutamic acid was 1:1, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1:1.

[0030] Furthermore, methacrylated hyaluronic acid is provided, specifically including:

[0031] Hyaluronic acid was dissolved in deionized water and stirred until completely dissolved; then sodium periodate solution was added dropwise and stirred in the dark at room temperature for 2 hours; then ethylene glycol was added, and unreacted periodate was soaked for hours; after thorough dialyzing with deionized water for 3 days (molecular weight cutoff (MWCO) 8000-14000 kDa), oxidized hyaluronic acid was obtained by freeze-drying; the oxidized hyaluronic acid was given double bonds by reacting with methyl methacrylate; in short, oxidized hyaluronic acid was dissolved in water and stirred until completely dissolved, then methyl methacrylate was added and the reaction was carried out at pH... The solution was kept at 8-8.5°C for 12 hours; the entire process, including dissolution and reaction, was carried out on ice; after the reaction, the solution was dialyzed for 2 days (molecular weight cutoff (MWCO) 8000-14000 kDa) and freeze-dried to obtain methacrylated oxidized hyaluronic acid; the concentration of the hyaluronic acid was 1% (w / v), the concentration of the oxidized hyaluronic acid was 1% (w / v), the molar ratio of MA:COOH was 3:1, and the molar ratio of -OH:NaIO3 was 1:1;

[0032] The concentration of methacrylated oxidized hyaluronic acid is 3% (w / v), the concentration of γ-polyglutamic acid grafted with polyethyleneimine is 5% (w / v), the concentration of phenyl-2,4,6-trimethylbenzoyl phosphite lithium photoinitiator is 0.2% (w / v), and the concentration of polydopamine-coated drug-loaded nanoparticles is 100 μg / mL.

[0033] The volume ratio of the methacrylated oxidized hyaluronic acid to the γ-polyglutamic acid grafted with polyethyleneimine is 2:1.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. The preparation method of the immunomodulatory nanocomposite hydrogel of the present invention includes preparing manganese dioxide nanoparticles and loading lipid-soluble ginsenoside Rk3 onto them, achieving a drug loading rate of 81%, thereby improving the bioavailability of ginsenoside Rk3 and enhancing the efficiency and safety of the delivery process. It can specifically address the immune dysregulation problem during the healing process of deep second-degree burns, promote the transformation of macrophages from a pro-inflammatory phenotype to an anti-inflammatory phenotype, improve the healing efficiency of deep second-degree burns, and effectively shorten the healing cycle of deep second-degree burns.

[0036] 2. The preparation method of the immunomodulatory nanocomposite hydrogel of the present invention involves using γ-polyglutamic acid grafted with polyethyleneimine, PM@Rk3 nanoparticles, and methacrylated oxidized hyaluronic acid as main raw materials. The mixture is stirred and then irradiated with ultraviolet light to obtain a composite hydrogel of PM@Rk3 nanoparticles. This composite hydrogel can be used for combined material and photothermal antibacterial therapy to kill bacteria at the burn wound site and improve the healing efficiency of deep second-degree burns.

[0037] 3. The immunomodulatory nanocomposite hydrogel of the present invention can effectively respond to pH and ROS, convert H2O2 in the burn wound site into O2, alleviate the hypoxic environment of the wound site, promote angiogenesis, and improve the healing efficiency of deep second-degree burn wounds.

[0038] 4. The nanocomposite hydrogels prepared by the method of the present invention have stable structures and simple preparation processes, which are conducive to their widespread application. Attached Figure Description

[0039] Figure 1 This is a SEM image of manganese dioxide nanoparticles from Example 1.

[0040] Figure 2 This is a TEM image of manganese dioxide nanoparticles from Example 1.

[0041] Figure 3 XPS image of manganese dioxide nanoparticles in Example 1.

[0042] Figure 4 The image shows the XRD pattern of manganese dioxide nanoparticles from Example 1.

[0043] Figure 5 The Fourier transform infrared absorption spectrum of γ-polyglutamic acid grafted polyethyleneimine in Example 2 is shown.

[0044] Figure 6 The Fourier transform infrared absorption spectrum of methacrylated oxidized hyaluronic acid from Example 3 is shown.

[0045] Figure 7 This is a SEM image of the nanocomposite hydrogel from Example 4.

[0046] Figure 8 The photothermal images of the nanocomposite hydrogel in Example 4 are shown at different powers.

[0047] Figure 9 This is a cyclic photothermal image of the nanocomposite hydrogel in Example 4.

[0048] Figure 10 This is a cell viability graph of the nanocomposite hydrogel in Example 4.

[0049] Figure 11 This is a graph showing the growth inhibition of the nanocomposite hydrogel in Example 4 on Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0050] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0051] like Figure 1-11As shown, Example 1

[0052] This embodiment provides a method for preparing PM@Rk3 nanoparticles, including the following steps:

[0053] Step 1: Provide manganese dioxide nanoparticles, specifically including

[0054] Step 101: Dissolve 3.16 mmol of potassium permanganate in 250 mL of deionized water and stir the mixture rapidly at 900 rpm for about 0.5 hours.

[0055] Step 102: Add 15.75 mmol of oleic acid, and react the resulting mixture at 500 rpm for 48 hours.

[0056] Step 103: Collect the crude brownish-black product by centrifugation, wash it several times with deionized water and ethanol to remove any possible residual reactants, and disperse the purified manganese dioxide nanoparticles in anhydrous ethanol.

[0057] Step 2: Provide positively charged manganese dioxide nanoparticles, specifically including:

[0058] Step 201: Pour 25 mg of the manganese dioxide nanoparticles into a round-bottom flask containing 30 mL of anhydrous ethanol, and carry out the process under ultrasonic conditions to ensure uniform dispersion of the suspension.

[0059] Step 202: Add 200 μL of 3-aminopropyltriethoxysilane to the above suspension at 40°C and stir for 12 h. Centrifuge to collect the precipitate and dry it overnight.

[0060] Step 3: Provide drug-loaded nanoparticles, specifically including:

[0061] Step 301: Dissolve 50 mg of ginsenoside Rk3 in 10 mL of ethanol.

[0062] Step 302: Disperse 2.5 mg of the positively charged manganese dioxide nanoparticles in an ethanol solution containing ginsenoside RK3 and sonicate for 1 h.

[0063] Step 303: After reacting at a stirring speed of 300 r / min for 3 h, the precipitate is separated and collected by centrifugation at 10000 rpm for 20 min, and then dried in a vacuum drying oven.

[0064] Step 4: Provide drug-loaded nanoparticles coated with polydopamine, specifically including:

[0065] Step 401: Prepare a 0.01M pH-8.5 Tris-HCl buffer solution. Weigh 0.6g of Tris and dissolve it in 500mL of water. Stir until the solid is completely dissolved. Use a pH meter to add HCl aqueous solution to bring the pH value to 8.5.

[0066] Step 402: Disperse 5 mg of the drug-loaded nanoparticles in 25 mL of Tris-HCl buffer, then add 2.5 mg of dopamine hydrochloride and stir slowly for 20 min.

[0067] Step 403: Wash the nanoparticles three times with deionized water by centrifugation at 10,000 r / min to obtain drug-loaded nanoparticles coated with polydopamine, and then dry them under vacuum at 40°C.

[0068] Example 2

[0069] This embodiment provides a γ-polyglutamic acid grafted with polyethyleneimine, comprising the following steps:

[0070] 1 g of γ-polyglutamic acid was completely dissolved in 50 mL of water. The pH of the γ-PGA solution was adjusted to 5.5 with HCl. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added sequentially. After reacting for 60 minutes, the pH of the reaction solution was adjusted to 8 with NaOH. 0.5 g of polyethyleneimine was then added, and the system was allowed to react for another 24 hours at room temperature. When the reaction was complete, the product was dialyzed (molecular weight cutoff (MWCO) 8000-14000 kDa) for 3 days. The resulting solution was then lyophilized to obtain polyethyleneimine-grafted γ-polyglutamic acid.

[0071] Example 3

[0072] This embodiment provides methacrylated oxidized hyaluronic acid, specifically including:

[0073] 1 g of hyaluronic acid was dissolved in 100 mL of deionized water, and then 5 mL of 0.5 M sodium periodate was added dropwise. The mixture was stirred at 250 rpm for 2 hours at room temperature in the dark. Then, 1 mL of ethylene glycol was added, and the unreacted periodate was soaked for 1 hour. After thorough dialyzing with deionized water for 3 days (molecular weight cutoff (MWCO) 8000-14000 kDa), oxidized hyaluronic acid was obtained by freeze-drying. Oxidized hyaluronic acid was given a double bond by reacting with methyl methacrylate. In short, 1 g of oxidized hyaluronic acid was dissolved in 100 mL of deionized water and stirred until completely dissolved. Then, 1 mL of methyl methacrylate was added, and the reaction was maintained at pH 8-8.5 for 12 hours. The entire process, including dissolution and reaction, was carried out on ice. After the reaction, the solution was dialyzed for 2 days (molecular weight cutoff (MWCO) 8000-14000 kDa) and freeze-dried to obtain methacrylated oxidized hyaluronic acid.

[0074] Example 4

[0075] This embodiment provides a method for preparing an immunomodulatory nanocomposite hydrogel, comprising the following steps:

[0076] Step 1: Place 0.5g of the γ-polyglutamic acid grafted with polyethyleneimine from Example 2 into 10mL of PBS and sonicate to dissolve it, thus obtaining system A;

[0077] Step 2: Add 1 mg of the polydopamine-coated drug-loaded nanoparticles from Example 1 to system A described in Step 1, and disperse them evenly by ultrasonication to obtain system B; the frequency of the ultrasonication is 40 Hz.

[0078] Step 3: Place 0.3g of the methacrylated oxidized hyaluronic acid from Example 3 into 10mL of deionized water and dissolve by sonication to obtain system C;

[0079] Step 4: Add 0.02g of phenyl-2,4,6-trimethylbenzoyl lithium phosphite photoinitiator to system C described in step 3 to obtain system D;

[0080] Step 5: Mix system B from step 2 and system C from step 4 at a volume ratio of 1:2 until homogeneous, and irradiate with ultraviolet light for 1 minute to obtain an immunomodulatory nanocomposite hydrogel.

[0081] Performance Evaluation

[0082] Figure 1 The SEM test method for Example 1 includes: ultrasonically dispersing the manganese dioxide nanoparticles in anhydrous ethanol to obtain a sample; dropping the sample onto a silicon wafer; and observing the morphology using a scanning electron microscope. The results are as follows: Figure 1 As shown, according to Figure 1 As can be seen, the nanoparticles exhibit a honeycomb structure.

[0083] Figure 2 For the TEM of Example 1, the testing method includes: ultrasonically dispersing the manganese dioxide nanoparticles in anhydrous ethanol to obtain a sample; dropping the sample onto a copper grid; and observing the morphology using a transmission electron microscope. The results are as follows: Figure 2 As shown, according to Figure 2 As can be seen, the nanoparticles exhibit a honeycomb structure.

[0084] Figure 3 The XPS spectrum for Example 1 was obtained by placing the manganese dioxide nanoparticles on an XPS testing platform and exporting the elemental distribution map. The results are as follows: Figure 3 As shown, according to Figure 3As can be seen, the XPS measurement results confirm that the constituent elements of MnO2 are Mn and O, and the characteristic peaks of 642.2 and 654.1 eV are attributed to Mn(IV)2p1 / 2 and Mn(IV)2p2 / 3.

[0085] Figure 4 The XRD pattern of Example 1 was obtained by placing the manganese dioxide nanoparticles on a glass slide, inserting it into an XRD analyzer, and obtaining its peak distribution map, as shown below. Figure 4 As shown, according to Figure 4 It can be seen that there are four distinct peaks at 2θ = 12.16, 24.25, 36.49 and 65.81°, which belong to the (001), (002), (100) and (110) crystal planes of naphthoic MnO2, respectively.

[0086] Figure 5 This is the Fourier transform infrared absorption spectrum analysis diagram of Example 2. (See attached image.) Figure 6 As shown, a CN vibration peak of amide III appeared at 1300 cm⁻¹, and a broader infrared absorption peak appeared in the range of 3000-3700 cm⁻¹. This is due to the introduction of a large number of amino groups after the grafting reaction, indicating that polyethyleneimine was successfully grafted onto γ-polyglutamic acid.

[0087] Figure 6 This is the Fourier transform infrared absorption spectrum analysis diagram of Example 3. Figure 5 As shown, the characteristic absorption peak at wavenumber 1734 cm⁻¹ belongs to the aldehyde group, and the characteristic absorption peak at 1700-1750 cm⁻¹ belongs to the ester bond, indicating that the modification of methacrylic acid oxidized hyaluronic acid was successful.

[0088] Figure 7 The image shown is a SEM image of Example 4. The testing method included: extracting the lyophilized hydrogel with liquid nitrogen and observing the cross-sectional morphology using SEM, as shown below. Figure 7 As shown, according to Figure 7 It is evident that the hydrogel exhibits a three-dimensional porous structure.

[0089] Figure 8 The figure shows the photothermal conversion capability at different powers in Example 4. The testing method includes: measuring the degree of thermal conversion of the hydrogel system containing the polydopamine-coated drug-loaded nanoparticles at different powers under an 808 nm exciter. The results are as follows. Figure 8 As shown, according to Figure 8 It is evident that as the power increases, the temperature also increases.

[0090] Figure 9The image shows the cyclic photothermal performance of Example 4 under cyclic laser irradiation. The test method included measuring the heating-cooling curve of Example 4 under an 808nm exciter. The results show that Example 4 can still reach a stable temperature after three cycles, indicating that Example 4 has stable photothermal performance.

[0091] Figure 10 The figure shown is a cell viability graph from Example 4. The effect of nanoparticles on the growth of L929 cells was detected using the CCK8 assay. The test method included: co-culturing L929 cells with hydrogel extract for 24 h, and measuring the absorbance at 450 nm using the CCK8 assay; the co-culture medium was RPMI-1640 medium; the culture conditions were 37℃; the blank group did not contain any additives from the example. The results showed that the cell viability was significantly increased, indicating that the hydrogel containing drug-loaded nanoparticles is conducive to cell proliferation.

[0092] Figure 11 To test the antibacterial growth ability of Example 7, the method included: adding 1g of the material from Example 4 to 10mL of *E. coli* or 10mL of *Staphylococcus aureus* bacterial suspension, irradiating with an 808nm exciter for 10 minutes, and then co-culturing for 24 hours. The mixture was then spread on plates and incubated for another 24 hours, and the number of colonies was counted. The co-culturing medium was LB medium at 37°C. The blank group consisted of 10mL of *E. coli* or 10mL of *Staphylococcus aureus* bacterial suspension without the addition of Example 4. The results showed that the antibacterial effect of the material containing Example 4 was better, and the antibacterial effect after irradiation with an 808nm exciter was the best.

[0093] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions conceived without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. An immunomodulatory nanocomposite hydrogel, characterized in that, The product consists of a hydrogel and drug-loaded nanoparticles mounted within it. The hydrogel is formed by Schiff base bonding and free radical polymerization of methacrylated oxidized hyaluronic acid and γ-polyglutamic acid grafted with polyethyleneimine. The drug-loaded nanoparticles are polydopamine-coated manganese dioxide nanoparticles loaded with ginsenoside Rk3. The concentration of the methacrylated oxidized hyaluronic acid is 3% (w / v), the concentration of the γ-polyglutamic acid grafted with polyethyleneimine is 5% (w / v), the concentration of the phenyl-2,4,6-trimethylbenzoyl phosphite lithium photoinitiator is 0.2% (w / v), and the concentration of the polydopamine-coated drug-loaded nanoparticles is 100 μg / mL. The volume ratio of the methacrylated oxidized hyaluronic acid to the γ-polyglutamic acid grafted with polyethyleneimine is 2:

1. The hydrogel is an injectable hydrogel with a gelation time of 30 seconds, exhibiting rapid gelation ability and preventing rapid diffusion of the hydrogel at the wound site. Due to the high water absorption capacity of hyaluronic acid and γ-polyglutamic acid, the hydrogel exhibits a high swelling level. This high swelling rate will facilitate cell migration, oxygen and nutrient diffusion, and promote wound healing. The hydrogel has good compressibility, meeting the mechanical requirements of soft tissue.

2. A method for preparing an immunomodulatory nanocomposite hydrogel, characterized in that, include: Provides honeycomb-shaped manganese dioxide nanoparticles; Grafting amino groups onto manganese dioxide nanoparticles yields positively charged manganese dioxide nanoparticles. Ginsenoside Rk3 was loaded onto positively charged manganese dioxide nanoparticles to obtain drug-loaded nanoparticles. A layer of polydopamine was coated onto the outer layer of drug-loaded nanoparticles to obtain polydopamine-coated drug-loaded nanoparticles. Provides γ-polyglutamic acid grafted with polyethyleneimine; Provides methacrylated oxidized hyaluronic acid; Drug-loaded nanoparticles coated with polydopamine were dispersed in a γ-polyglutamic acid solution grafted with polyethyleneimine. A methacrylated oxidized hyaluronic acid solution was mixed with the γ-polyglutamic acid solution grafted with polyethyleneimine containing drug-loaded nanoparticles coated with polydopamine to obtain an immunomodulatory nanocomposite hydrogel. Provides honeycomb-shaped manganese dioxide nanoparticles, specifically including: Dissolve potassium permanganate in deionized water and stir the mixture rapidly for 0.5 hours; Oleic acid was then added to allow the resulting mixture to react for 48 hours. The crude brown-black product was collected by centrifugation and washed several times with deionized water and ethanol to remove any possible residual reactants. The purified manganese dioxide nanoparticles were then dispersed in anhydrous ethanol.

3. The preparation method according to claim 2, characterized in that, The concentration of potassium permanganate was 3.16 mmol, the concentration of oleic acid was 15.75 mmol, the stirring speed was 900 r / min, and the reaction speed was 500 r / min; the concentration of honeycomb manganese dioxide was 0.83 mg / mL, and the concentration of 3-aminopropyltriethoxysilane was 6.67 μg / mL.

4. The preparation method according to claim 2, characterized in that, Grafting amino groups onto manganese dioxide nanoparticles yields positively charged manganese dioxide nanoparticles, specifically including: The prepared honeycomb manganese dioxide was poured into a round-bottom flask containing anhydrous ethanol and carried out under ultrasonic conditions to ensure uniform dispersion of the suspension. 3-aminopropyltriethoxysilane was added to the suspension at 40°C and stirred for 12 h. The precipitate was collected by centrifugation and dried overnight.

5. The preparation method according to claim 2, characterized in that, By loading ginsenoside Rk3 onto positively charged manganese dioxide nanoparticles, drug-loaded nanoparticles are obtained, specifically including: First, ginsenoside Rk3 was dissolved in ethanol. Then, positively charged manganese dioxide nanoparticles were dispersed in the ethanol solution containing ginsenoside Rk3 and ultrasonically mixed. After reacting for an appropriate time at an appropriate stirring speed, the precipitate was separated and collected by centrifugation at 10,000 rpm for 20 min and dried in a vacuum drying oven.

6. The preparation method according to claim 2, characterized in that, The concentration ratio of ginsenoside Rk3 to positively charged manganese dioxide nanoparticles was 10:1, the stirring speed was 300 r / min, and the reaction time was 3 h. The concentration of ginsenoside Rk3 was in mg / mL, and the concentration of positively charged manganese dioxide nanoparticles was in mg / mL.

7. The preparation method according to claim 2, characterized in that, Coating the outer layer of the drug-loaded nanoparticles with a layer of polydopamine to obtain polydopamine-coated drug-loaded nanoparticles, specifically including: First, prepare a 0.01M Tris-HCl buffer solution with pH 8.5 by weighing 0.1211 g of Tris and dissolving it in 100 mL of water and stirring until the solid is completely dissolved. Then, use a pH meter and add HCl aqueous solution to bring the pH value to 8.

5. Drug-loaded nanoparticles were dispersed in Tris-HCl buffer, then dopamine hydrochloride was added and stirred slowly. The nanoparticles were washed with deionized water by centrifugation and centrifuged three times at 10,000 r / min to obtain polydopamine-coated drug-loaded nanoparticles, which were then vacuum dried and stored.

8. The preparation method according to claim 2, characterized in that, The concentration of drug-loaded nanoparticles dispersed in Tris-HCl buffer was 0.2 mg / mL, the concentration of dopamine hydrochloride was 0.1 mg / mL, and the reaction time was 20 min.

9. The preparation method according to claim 2, characterized in that, Gamma-polyglutamic acid grafted with polyethyleneimine, specifically including: γ-Polyglutamic acid was completely dissolved in water. The pH of the γ-polyglutamic acid solution was adjusted to 5.5 with HCl, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added sequentially. After reacting for 60 minutes, the pH of the reaction solution was adjusted to 8 with NaOH, and polyethyleneimine was added. The system was allowed to continue reacting at room temperature for 24 h. When the reaction was complete, the product was dialyzed for 3 days, and the resulting solution was lyophilized to obtain polyethyleneimine-grafted γ-polyglutamic acid. The concentration of the γ-polyglutamic acid was 2% (w / v), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to γ-polyglutamic acid was 1:1, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 1:

1.

10. The preparation method according to claim 2, characterized in that, Provides methacrylated hyaluronic acid, specifically including: Hyaluronic acid was dissolved in deionized water and stirred until completely dissolved. Sodium periodate solution was then added dropwise and stirred in the dark at room temperature for 2 hours. Ethylene glycol was then added, and the unreacted periodate was soaked for 1 hour. After thorough dialyzing with deionized water for 3 days to remove a molecular weight cutoff of 8000-14000 kDa, oxidized hyaluronic acid was obtained by freeze-drying. The oxidized hyaluronic acid was then given double bonds through a reaction with methyl methacrylate. In short, oxidized hyaluronic acid was dissolved in water and stirred until completely dissolved. Then, methyl methacrylate was added and the reaction was maintained at pH 8-8.5 for 12 hours. The entire process, including dissolution and reaction, was carried out on ice. After the reaction, the solution was dialyzed for 2 days to remove a molecular weight cutoff of 8000-14000 kDa. kDa was freeze-dried to obtain methacrylated oxidized hyaluronic acid; the concentration of hyaluronic acid was 1% (w / v), the concentration of oxidized hyaluronic acid was 1% (w / v), the molar ratio of MA:COOH was 3:1, and the molar ratio of -OH:NaIO3 was 1:1; The concentration of methacrylated oxidized hyaluronic acid is 3% (w / v), the concentration of γ-polyglutamic acid grafted with polyethyleneimine is 5% (w / v), the concentration of phenyl-2,4,6-trimethylbenzoyl phosphite lithium photoinitiator is 0.2% (w / v), and the concentration of polydopamine-coated drug-loaded nanoparticles is 100 μg / mL. The volume ratio of the methacrylated oxidized hyaluronic acid to the γ-polyglutamic acid grafted with polyethyleneimine is 2:1.

Images