Injectable hydrogel and its preparation method and application

Abstract

The invention discloses an injectable hydrogel and a preparation method and application thereof. The preparation method comprises the following steps: mixing a NaIO4 solution with a hyaluronic acid solution, reacting in the dark for a set time, then dialyzing and purifying, and drying to obtain OHA; adding OHA and EDS to a buffer solution in proportion, stirring for a set time under weak acid conditions, then adding NHS thereto, and continuing stirring for a set time under weak acid conditions; after the reaction is completed, adding dopamine hydrochloride thereto, dialyzing, and drying the product to obtain OHD; adding a mixed solution of SBMA, MBAA, ammonium persulfate and TI to a mixed solution of Tween 80 and Span 80, treating with an ultrasonic wall breaking machine for a set time, heating, and reacting in an inert atmosphere for a set time; after the reaction is completed, cooling to completely crush and precipitate the microemulsion, washing, dialysis, and drying to obtain TI-Vpsb; and mixing TI-Vpsb, OHD, TA, GD, PNIPAM and water in proportion to prepare a drug-loaded microsphere hydrogel TI-Vpsb@PGOT.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and in particular relates to an injectable hydrogel and a preparation method and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] As the body's first natural barrier, the skin plays a vital role in preventing dehydration and protecting internal organs from external damage. However, after severe injury, the skin loses this protective function, leading to wound formation. Wound healing is a complex, coordinated process that typically involves four phases: hemostasis, inflammation, new tissue formation, and ultimately, tissue remodeling and regression. Abnormal progression of these phases can lead to dysfunctional and disfiguring scars. Scars can cause various complications, such as cosmetic loss and functional impairment, causing significant physical and emotional distress to patients. Numerous studies have promoted wound healing through various approaches, such as promoting angiogenesis, alleviating inflammation, and inhibiting bacterial growth. However, most have failed to consider the importance of scarring in wound healing. Recent studies have reported on promoting scarless wound healing, but these have only considered one of the four phases, such as anti-inflammatory and antibacterial measures and promoting tissue remodeling through various pathways. Given that wound healing is a continuous process, it is necessary to maximize the smooth progression of each phase and inhibit scar formation. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide an injectable hydrogel and a preparation method and application thereof.

[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing an injectable hydrogel, comprising the following steps:

[0007] After adding EDC and NHS to the buffer solution of gelatin, the pH value of the buffer solution is adjusted to weak acidity. After stirring for a set time, dopamine hydrochloride is added thereto. Under inert atmosphere protection, the reaction is stirred at room temperature in the dark and maintained at weak acidity. The product is desalted and dried to obtain GD.

[0008] Mix the NaIO4 solution with the hyaluronic acid solution, protect from light and react for a set time. After the reaction is completed, add ethylene glycol to neutralize the excess NaIO4, then dialysis purification, and dry to obtain OHA;

[0009] OHA and EDS are added to a buffer solution in proportion, stirred under weak acidic conditions for a set time, and then NHS is added thereto, and stirring is continued under weak acidic conditions for a set time; after the reaction is completed, dopamine hydrochloride is added thereto, and stirred under weak acidic conditions for a set time. After the reaction is completed, dialyzed, and the product is dried to obtain OHD;

[0010] N-isopropylacrylamide, a crosslinker, and a photoinitiator are dissolved in water, and the reaction is carried out under ultraviolet light for a set time. The gel is dialyzed and dried to obtain PNIPAM.

[0011] A mixture of SBMA, MBAA, ammonium persulfate, and TI is placed in a mixed solution of Tween 80 and Span 80, treated with an ultrasonic wall breaking machine for a set time, then heated and reacted in an inert atmosphere for a set time; after the reaction is completed, the mixture is cooled, and tetrahydrofuran is added to the reaction system to completely break up the microemulsion and precipitate, which is then washed, dialyzed, and dried to obtain TI-Vpsb;

[0012] TI-Vpsb, OHD, TA, GD, PNIPAM and water were mixed in proportion to prepare the drug-loaded microsphere hydrogel TI-Vpsb@PGOT.

[0013] During the preparation of GD, EDC and NHS catalyze the formation of amide bonds between the carboxyl groups of gelatin and the amino groups of dopamine, resulting in dopamine-modified gelatin (GD). An inert gas atmosphere prevents oxidation of the phenolic hydroxyl groups of dopamine. Dopamine hydrochloride contains phenolic hydroxyl groups, which are easily decomposed by light, so the reaction should be kept away from light. EDC and NHS are most active in activating the carboxyl groups in a weakly acidic buffer solution.

[0014] During the preparation of OHA, sodium periodate is used to oxidize the hydroxyl groups of hyaluronic acid to produce aldehyde-modified hyaluronic acid. Light excites the electrons of the vicinal diol molecules, placing them in a high-energy state and enabling them to react with sodium periodate, resulting in an oxidation reaction. Therefore, performing the reaction in the dark avoids light interference, ensuring the accuracy and stability of the reaction. Ethylene glycol contains hydroxyl groups, which react with excess sodium periodate.

[0015] During the preparation of OHD, the carboxyl groups of OHA react with the amino groups of dopamine under the catalysis of EDC and NHS to form an amide bond. Simultaneously, the aldehyde groups of OHA react with the amino groups of dopamine in a Schiff base reaction, further increasing the grafting rate of dopamine and producing dopamine-modified oxidized hyaluronic acid. EDC and NHS have optimal activity in activating carboxyl groups in a weakly acidic buffer solution, thus requiring weakly acidic conditions.

[0016] During the preparation of TI-Vpsb, ultrasonic wall breaking is used to homogenize the oil and water phases, resulting in a water-in-oil colloidal dispersion. Microspheres are prepared using a reverse microemulsion method, yielding TI-encapsulated microspheres. Stabilized by an emulsifier, the aqueous solution of SBMA monomer is highly dispersed in the oil as nanosized droplets, forming a clear and transparent W / O colloidal dispersion. An orderly arranged monolayer of emulsifier at the oil-water interface surrounds the droplets, forming a "microreactor." Thermal initiation then triggers the polymerization of the SBMA monomer within the microreactor, transforming the droplets into microspheres. Demulsification, filtration, washing, and vacuum drying are then performed to obtain the microspheres.

[0017] During the preparation of TI-Vpsb@PGOT, the macromonomers OHD, PNIPAM, and GD form a hydrogel through hydrogen bonding, Schiff base bonds, and covalent interactions. TA and TI-Vpsb microspheres can further extend the retention time of the hydrogel through hydrogen bonding interactions.

[0018] In some embodiments, when preparing GD, the molar ratio of gelatin, EDC, NHS and dopamine hydrochloride is 1.5-3:2-4:3-5:0.5-1.5.

[0019] Gelatin: provides carboxyl groups, EDC: activates carboxyl groups, NHS: forms NHS esters with intermediates, and dopamine hydrochloride: provides amino groups.

[0020] Preferably, when preparing GD, the pH value of the buffer solution is 5-6.

[0021] Preferably, after adding EDC and NHS to the gelatin buffer solution, the stirring time is 20-40 minutes.

[0022] Preferably, after adding dopamine hydrochloride, the reaction is stirred at room temperature in the dark for 10-30 hours, preferably 24 hours.

[0023] In some embodiments, when preparing OHA, the molar ratio of NaIO4 to hyaluronic acid is 1-3:5.

[0024] Preferably, the NaIO4 solution is mixed with the hyaluronic acid solution and the reaction time is 2-4 hours in the dark. HA: provides hydroxyl groups; sodium periodate: oxidizes the hydroxyl groups of HA to aldehyde groups.

[0025] In some embodiments, when preparing OHD, the molar ratio of OHA, EDS, NHS, and dopamine hydrochloride is 0.5-1.5:1-3:1-3:0.5-1.5. OHA provides aldehyde and carboxyl groups; EDC activates carboxyl groups; NHS forms an NHS ester with an activated intermediate; and dopamine hydrochloride provides amino groups.

[0026] In some embodiments, when preparing TI-Vpsb, the mass ratio of SBMA, MBAA, ammonium persulfate, TI, Tween 80 and Span 80 is 1500-2500:40-60:1-3:3-7:400-600:1500-2500.

[0027] Sbma: monomer for synthesizing microspheres; MBAA: cross-linking agent; ammonium persulfate: initiator; TI: TGF-β inhibitor; Tween 80, Span 80: emulsifiers.

[0028] Preferably, the reaction system is treated with an ultrasonic wall breaking machine for 5-15 minutes, then heated to 50-90° C., and reacted in an inert atmosphere for 3-5 hours.

[0029] In some embodiments, when preparing the drug-loaded microsphere hydrogel TI-Vpsb@PGOT, the mass ratio of TI-Vpsb, OHD, TA, GD, PNIPAM and water is 0.1-0.5:5-15:15-20:1-5:0.5-1:50-80.

[0030] In a second aspect, the present invention provides a four-dimensional injectable hydrogel prepared by the preparation method.

[0031] In a third aspect, the present invention provides use of the four-dimensional injectable hydrogel in the preparation of a drug for promoting scarless wound healing.

[0032] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0033] The present invention develops a 4D injectable hydrogel dressing (TI-Vpsb@PGOT), which has an early hemostatic effect, a mid-term antioxidant and antibacterial effect, and a late anti-fibrosis effect, and is used to promote rapid, scar-free wound healing.

[0034] Dopamine-modified hyaluronic acid and gelatin are used as the skeleton of the hydrogel, giving it high biocompatibility and adhesion and hemostatic properties.

[0035] Poly(N-isopropylacrylamide) (PNIPAM) was further introduced into the system. The introduction of the double network enhanced the toughness and retention time of the hydrogel. At the same time, the thermosensitive properties of PNIPAM enabled the gel to promote wound contraction.

[0036] Tannic acid (TA) is a polyphenolic substance containing a large amount of catechol / triphenol. It has a structure similar to dopamine and can be used to enhance the adhesion properties of hydrogels. It has better antioxidant and antibacterial functions. It can also be used as a cross-linking agent to further prolong the retention time of the gel, achieving long-lasting anti-inflammatory and antibacterial effects.

[0037] The present invention prepares poly(methacrylate sulfobetaine) (PSBMA) microspheres encapsulating TGF-β inhibitors, which have the characteristics of pulsed drug release, making TI-Vpsb@PGOT a 4D drug release platform.

[0038] The results showed that the 4D hydrogel dressing had anti-inflammatory and antibacterial effects in a rat infected skin defect model, accelerated skin wound closure, and inhibited scar formation. In summary, TI-Vpsb@PGOT has great potential for clinical applications in hemostasis, anti-inflammatory and antibacterial properties, and anti-scarring. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0040] Figure 1 These are photos of the full-thickness skin defect model in rats infected with Staphylococcus aureus established in the examples of the present invention, and the wound conditions after treatment or without treatment.

[0041] Figure 2 : are the NMR spectra of Gel-DA, OHA and OHD in the embodiments of the present invention, wherein a is the NMR spectra of the raw materials Gel, DA and the product Gel-DA); b is the NMR spectra of the raw materials HA, DA and the products OHA and OHD.

[0042] Figure 3 is a SEM image of the PGOT hydrogel in Example 1 of the present invention.

[0043] Figure 4 This is a before-and-after comparison of the physical contraction behavior of the PGOT hydrogel in an embodiment of the present invention after being placed at 37° C. for 0.5 h. Scale: 1 cm.

[0044] Figure 5 1 is the release curve of TA and TI in different preparations (PGOT, TI-Vpsb, TI-Vpsb@PGOT) in Example 1 of the present invention.

[0045] Figure 6 : These are images of liver bleeding changes over time after treatment or without treatment in Example 1 of the present invention, where a is (the bleeding image of the untreated group at time 0); b is (the bleeding image of the untreated group when bleeding stops naturally); c is (the bleeding image of the treated group at time 0); and d is (the bleeding image of the treated group when bleeding stops).

[0046] Figure 7 These are representative images of the wound healing process of different groups on days 0, 3, 7, 10, and 14 in Example 1 of the present invention. Scale bar: 1 cm.

[0047] Figure 8 These are H&E staining images of various organs after treatment with different preparations in Example 1 of the present invention. Scale bar: 100 μm. DETAILED DESCRIPTION

[0048] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0049] The present invention will be further described below with reference to the embodiments.

[0050] Example 1

[0051] A method for preparing an injectable hydrogel comprises the following steps:

[0052] (1) Synthesis of GD

[0053] 2.0 g of gelatin was completely dissolved in 100 mL of phosphate buffer saline (PBS); subsequently, 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.3 g of N-hydroxysuccinimide (NHS) were added to the solution, and the pH of the mixture was adjusted to 5.0 with 1 M HCL.

[0054] After stirring for a period of time, 1.0 g of dopamine hydrochloride (DOPA·HCl) was added to the reaction system. The mixture was stirred in the dark at 37°C under nitrogen for 24 hours, maintaining the pH between 5.0 and 6.0. The mixture was dialyzed against deionized water for 48 hours to remove salts, and then lyophilized to obtain GD.

[0055] (2) Synthesis of OHA

[0056] 1.0 g of hyaluronic acid (HA) was dissolved in 100 mL of deionized water to obtain a HA solution. 0.12 g of NaIO4 was dissolved in water, and the NaIO4 solution was added dropwise to the HA solution and stirred in the dark at room temperature for 3 hours.

[0057] 1 mL of ethylene glycol was added to the reaction solution to neutralize the excess sodium periodate. After 1 h, the reaction mixture was dialyzed against deionized water for 2 days and then lyophilized to obtain OHA with a yield of approximately 85%.

[0058] (3) Synthesis of OHD

[0059] The synthesized OHA was dispersed in PBS buffer at a concentration of 5 mg / ml. 0.3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was then added to the solution and stirred at pH 5.5-6.0 for 15 min. 0.3 g of N-hydroxysuccinimide (NHS) was added to the reaction solution and stirred at pH 5.5-6.0 for an additional 45 min.

[0060] After the reaction, 0.25 g of dopamine hydrochloride (DOPA·HCl) was added to the solution, and stirred at pH 5.0-5.5 for 24 h.

[0061] The reaction mixture was dialyzed against deionized water for 2 days and then lyophilized to obtain OHD.

[0062] The synthesized GD, OHA and OHD polymer powders were dissolved in deuterated water, TMS was used as an internal reference, and 1 The characteristics were determined by H-NMR spectroscopy (400 Hz).

[0063] (4) Synthesis of PNIPAM

[0064] 1.0 g of N-isopropylacrylamide (NIPAM) was mixed with 90 μL of 5 × 10 -3 M aqueous solution crosslinker 6,6'-bisamino-3,3'-methylenedibenzoic acid (MBAA) was dissolved in 9 mL of deionized water, 0.02 g of photoinitiator (Irgacure 2959) was added, and the mixture was cured under ultraviolet light for 2 h. The gel was then dialyzed and freeze-dried to obtain PNIPAM.

[0065] (5) Synthesis of PGOT hydrogel

[0066] To form the hydrogel, 0.34 g GD and 0.2 g PNIPAM macromonomers were dissolved in 1 mL of deionized water and mixed with equal volumes of OHD solution and TA solution. The concentration of OHD solution was 60 mg / mL and the concentration of TA solution was 14 mg / mL. A dark brown PGOT hydrogel was quickly formed.

[0067] (6) SEM observation

[0068] 1 g of the prepared PGOT hydrogel was placed in 3 mL of deionized water until it expanded to equilibrium. The hydrogel was then freeze-dried using a freeze dryer, and its internal morphology was studied using a scanning electron microscope (SEM, COXEM, EM-30Plus, Korea).

[0069] (7) Synthesis of TI-Vpsb

[0070] 0.14 g of Tween 80 and 0.43 g of Span 80 were dissolved in 25 mL of n-hexane to obtain a mixed solution; 2 mL of an aqueous solution containing 0.57 g of sulfobetaine methacrylate (SBMA), 0.0126 g of a crosslinker 6,6'-bisamino-3,3'-methylenedibenzoic acid (MBAA), 0.47 mg of ammonium persulfate (APS) and 1 mg of TI was added dropwise to the above mixed solution, and the mixture was treated with an ultrasonic wall breaking device for 10 minutes. After that, the solution was heated to 70°C, passed through N2 for protection, and reacted for 4 hours.

[0071] After cooling to room temperature, 15 mL of tetrahydrofuran (THF) was added to completely break up the microemulsion and precipitate. The microemulsion was washed three times with THF to remove unreacted monomers. The precipitate was dialyzed and freeze-dried to obtain TI-Vpsb. The chemical structure of Vpsb was analyzed by FTIR.

[0072] (7) Synthesis of TI-Vpsb@PGOT

[0073] The prepared TI-Vpsb, OHD, TA, GD, PNIPAM and water were mixed. In the mixed system, the concentration of TI-Vpsb was 1 mg / mL, the concentration of OHD was 30 mg / mL, the concentration of TA was 7 mg / mL, the concentration of GD was 170 mg / mL, and the concentration of PNIPAM was 100 mg / mL to prepare the drug-loaded microsphere hydrogel TI-Vpsb@PGOT.

[0074] (8) In vitro release

[0075] The in vitro release behavior of TI-Vpsb@PGOT was studied using dialysis and UV filtration. TI-Vpsb@PGOT was transferred to a dialysis bag filled with PBS buffer and incubated on a shaker at a constant temperature of 37°C for 13 days.

[0076] PGOT was prepared in the same manner as above: 0.34 g GD and 0.2 g PNIPAM macromonomer were dissolved in 1 mL deionized water and mixed with equal volumes of OHD solution and TA solution. The concentration of OHD solution was 60 mg / mL and the concentration of TA solution was 14 mg / mL.

[0077] TI-Vpsb of the same concentration was prepared as follows: 10 mg of freeze-dried TI-Vpsb was added to 10 mL of PBS to prepare a 1 mg / mL solution.

[0078] At 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, and 312 hours, 1 mL of release medium was replaced with an equal volume of PBS. The TI content in TI-Vpsb@PGOT and TI-Vpsb was determined by UV, while the TA content in PGOT was determined by UV. Three replicates were performed for each sample, and the average value was used to calculate the cumulative release rate, thus generating the cumulative release curve.

[0079] Animal Experiment Section:

[0080] The effect of TI-Vpsb@PGOT hydrogel on wound healing was evaluated using a full-thickness skin defect model in rats infected with Staphylococcus aureus.

[0081] SD rats (6-8 weeks old, male) were randomly divided into four groups, with 7 rats in each group: Control group, Commercial gel (Hydrosorb Gel) group, PGOT group and TI-Vpsb@PGOT group.

[0082] After anesthesia with isoflurane and removal of dorsal hair, a full-thickness circular wound with a diameter of 10 mm was made and a Staphylococcus aureus suspension (50 μL, 1 × 10 8 CFU / mL) were immediately dropped onto the wound to establish a full-thickness Staphylococcus aureus infected wound model. Figure 1 The control group received no treatment, while the other groups used syringes to completely cover the wounds with the corresponding hydrogels.

[0083] The mice's wound healing progress was then monitored and their body weights were recorded using smartphones on days 0, 3, 7, 10, and 14. Wound area was calculated using ImageJ software. After 7 days of treatment, four rats in each group were sacrificed, and skin tissue from the wounds was harvested. After 14 days of treatment, the remaining rats were sacrificed, and skin tissue and major organs (heart, liver, spleen, kidney, and lungs) from the wounds were harvested.

[0084] Results section:

[0085] (1) Characterization of polymer and TI-Vpsb@Gel hydrogel

[0086] Gel-DA was synthesized by condensation reaction between the carboxyl group of Gel and the amino group of dopamine. 1 The synthesized structures were characterized by H-NMR. Figure 2 As shown, GD 1 In the HNMR spectrum, the peak at 2.75 ppm is attributed to the methylene protons near the phenyl group in dopamine. 1The HNMR spectrum confirmed the presence of aldehyde groups in OHA, and the peak between 5.0 and 5.1 ppm was attributed to the hemiacetal protons formed by the aldehyde and the adjacent hydroxyl groups. 1 The peak at approximately 6.8 ppm in HNMR indicated the presence of aromatic protons, confirming the successful binding of OHA to DA.

[0087] The SEM image of PGOT hydrogel is shown in Figure 2. Figure 3 As shown, the hydrogel has a porous and interconnected internal structure, which creates conditions for drug loading.

[0088] After the prepared PGOT hydrogel was placed in a 37°C incubator for 0.5 h, the volume of the PGOT hydrogel shrank significantly, with a shrinkage rate of 48.16%. Figure 4 As shown in Figure 3, this is attributed to the excellent thermal response of PNIPAM, which makes it possible for PGOT to promote skin contraction.

[0089] TI-Vpsb was mixed with PGOT to prepare a time-dependent four-dimensional TI-Vpsb@PGOT, and the feasibility of this strategy was verified by studying the in vitro release behavior. Figure 5 TA from PGOT was rapidly released, reaching a plateau on day 5, which was attributed to the mechanical contraction of PNIPAM, which facilitated the rapid release of TA. TI from TI-Vpsb@PGOT began to be released on day 4 and was almost completely released by day 13, with a release rate of 94.89%. Therefore, under the action of PNIPAM and TI-Vpsb, the sequential release of TA and TI from TI-Vpsb@PGOT generally conforms to the time-dependent process of several stages of wound healing.

[0090] (2) Hemostatic properties of TI-Vpsb@Gel hydrogel

[0091] like Figure 6 As shown, this is a rat liver bleeding model. The bleeding in the control group lasted for 349.18 seconds, and the blood loss was 0.4055 g. In the TI-Vpsb@PGOT group, the liver bleeding time of rats was 144.57 seconds, and the blood loss was significantly reduced to 0.0336 g, indicating that TI-Vpsb@PGOT has excellent hemostatic ability.

[0092] (3) Evaluation of scar-free healing of infected wounds:

[0093] like Figure 7As shown, from a macroscopic perspective, due to the active contractility of PGOT gel, it can be clearly observed that the healing rate of TI-Vpsb@PGOT is faster than that of the Control group and the Commercial gel group. On the 14th day, the wounds of the rats in the TI-Vpsb@PGOT group were completely healed, and the skin was smooth with almost no scars. The wounds of the PGOT group were also completely healed, but there were obvious scars, while the wounds of the Control group and the Commercial gel group were still clearly visible. These results indicate that TI-Vpsb@PGOT can promote scarless healing of infected wounds, which may be because TI-Vpsb@PGOT can release TA and TI successively. TA has good antibacterial and anti-inflammatory properties, and TI plays an anti-fibrotic role in the later stage.

[0094] (4) Biosafety evaluation

[0095] The histological examination of the heart, liver, spleen, lung, and kidney of the treatment group (TI-Vpsb@PGOT group) showed no obvious abnormalities compared with the control group, indicating that the TI-Vpsb@PGOT hydrogel has good biocompatibility, such as Figure 8 shown.

[0096] The results showed that TI-Vpsb@PGOT hydrogel has early hemostasis, mid-term antibacterial and antioxidant effects, and late-stage anti-fibrosis effects, which can promote scarless wound healing and provide a promising strategy for scarless wound repair.

[0097] Example 2

[0098] A method for preparing an injectable hydrogel comprises the following steps:

[0099] (1) Synthesis of GD

[0100] 2.0 g of gelatin was completely dissolved in 100 mL of phosphate buffer saline (PBS); subsequently, 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.3 g of N-hydroxysuccinimide (NHS) were added to the solution, and the pH of the mixture was adjusted to 5.0 with 1 M HCL.

[0101] After stirring for 20 minutes, 2.0 g of dopamine hydrochloride (DOPA·HCl) was added to the reaction system. The mixture was stirred in the dark at 37°C under nitrogen for 24 hours, maintaining the pH between 5.0 and 6.0. The mixture was dialyzed against deionized water for 48 hours to remove salts and then lyophilized to obtain GD.

[0102] (2) Synthesis of OHA

[0103] 1.0 g of hyaluronic acid (HA) was dissolved in 100 mL of deionized water to obtain a HA solution. 0.23 g of NaIO4 was dissolved in water, and the NaIO4 solution was added dropwise to the HA solution and stirred in the dark at room temperature for 4 hours.

[0104] 1 mL of ethylene glycol was added to the reaction solution to neutralize the excess sodium periodate. After 1 h, the reaction mixture was dialyzed against deionized water for 2 days and then lyophilized to obtain OHA.

[0105] (3) Synthesis of OHD

[0106] The synthesized OHA was dispersed in PBS buffer at a concentration of 5 mg / ml. 0.3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was then added to the solution and stirred at pH 5.0-6.0 for 15 min. 0.3 g of N-hydroxysuccinimide (NHS) was added to the reaction solution and stirred at pH 5.5-6.0 for an additional 40 min.

[0107] After the reaction, 0.3 g of dopamine hydrochloride (DOPA·HCl) was added to the solution, and stirred at pH 5.0-5.5 for 24 h.

[0108] The reaction mixture was dialyzed against deionized water for 2 days and then lyophilized to obtain OHD.

[0109] (4) Synthesis of PNIPAM

[0110] 1.0 g of N-isopropylacrylamide (NIPAM) was mixed with 100 μL of 5 × 10 -3 M aqueous solution crosslinker 6,6'-bisamino-3,3'-methylenedibenzoic acid (MBAA) was dissolved in 9 mL of deionized water, 0.02 g of photoinitiator (Irgacure 2959) was added, and the mixture was cured under ultraviolet light for 2 h. The gel was then dialyzed and freeze-dried to obtain PNIPAM.

[0111] (5) Synthesis of PGOT hydrogel

[0112] To form the hydrogel, 0.34 g of GD and 0.2 g of PNIPAM macromonomer were dissolved in 1 mL of deionized water and mixed with equal volumes of OHD solution and TA solution. The concentration of OHD solution was 60 mg / mL and the concentration of TA solution was 14 mg / mL. A dark brown PGOT hydrogel was quickly formed.

[0113] (6) Synthesis of TI-Vpsb

[0114] 0.1 g of Tween 80 and 0.4 g of Span 80 were dissolved in 25 mL of n-hexane to obtain a mixed solution; 2 mL of an aqueous solution containing 0.5 g of sulfobetaine methacrylate (SBMA), 0.012 g of a crosslinker 6,6'-bisamino-3,3'-methylenedibenzoic acid (MBAA), 0.4 mg of ammonium persulfate (APS) and 1 mg of TI was added dropwise to the above mixed solution, and the mixture was treated with an ultrasonic wall breaking device for 5 minutes. After that, the solution was heated to 60°C, nitrogen was passed through the solution for protection, and the reaction was carried out for 4 hours.

[0115] After cooling to room temperature, 15 mL of tetrahydrofuran (THF) was added to completely break up the microemulsion and precipitate. The microemulsion was washed three times with THF to remove unreacted monomers. The precipitate was dialyzed and freeze-dried to obtain TI-Vpsb. The chemical structure of Vpsb was analyzed by FTIR.

[0116] (7) Synthesis of TI-Vpsb@PGOT

[0117] The prepared TI-Vpsb, OHD, TA, GD, PNIPAM and water were mixed. In the mixed system, the concentration of TI-Vpsb was 1 mg / mL, the concentration of OHD was 30 mg / mL, the concentration of TA was 7 mg / mL, the concentration of GD was 170 mg / mL, and the concentration of PNIPAM was 100 mg / mL to prepare the drug-loaded microsphere hydrogel TI-Vpsb@PGOT.

[0118] Example 3

[0119] A method for preparing an injectable hydrogel comprises the following steps:

[0120] (1) Synthesis of GD

[0121] 2.0 g of gelatin was completely dissolved in 100 mL of phosphate buffer saline (PBS); subsequently, 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.3 g of N-hydroxysuccinimide (NHS) were added to the solution, and the pH of the mixture was adjusted to 5.5 with 1 M HCL.

[0122] After stirring for 40 minutes, 1.5 g of dopamine hydrochloride (DOPA·HCl) was added to the reaction system. The mixture was stirred in the dark at 37°C under nitrogen for 24 hours, maintaining the pH between 5.0 and 6.0. The mixture was dialyzed against deionized water for 48 hours to remove salts and then lyophilized to obtain GD.

[0123] (2) Synthesis of OHA

[0124] 1.0 g of hyaluronic acid (HA) was dissolved in 100 mL of deionized water to obtain a HA solution. 0.34 g of NaIO4 was dissolved in water, and the NaIO4 solution was added dropwise to the HA solution and stirred in the dark at room temperature for 2 hours.

[0125] 1 mL of ethylene glycol was added to the reaction solution to neutralize the excess sodium periodate. After 1 h, the reaction mixture was dialyzed against deionized water for 2 days and then lyophilized to obtain OHA.

[0126] (3) Synthesis of OHD

[0127] The synthesized OHA was dispersed in PBS buffer at a concentration of 5 mg / ml. 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was then added to the solution and stirred at pH 5.5-6.0 for 10 min. 0.35 g of N-hydroxysuccinimide (NHS) was added to the reaction solution and stirred at pH 5.5-6.0 for an additional 45 min.

[0128] After the reaction, 0.3 g of dopamine hydrochloride (DOPA·HCl) was added to the solution, and stirred at pH 5.0-5.5 for 24 h.

[0129] The reaction mixture was dialyzed against deionized water for 2 days and then lyophilized to obtain OHD.

[0130] (4) Synthesis of PNIPAM

[0131] 1.0 g of N-isopropylacrylamide (NIPAM) was mixed with 80 μL of 5 × 10 -3 M aqueous solution crosslinker 6,6'-bisamino-3,3'-methylenedibenzoic acid (MBAA) was dissolved in 9 mL of deionized water, 0.02 g of photoinitiator (Irgacure 2959) was added, and the mixture was cured under ultraviolet light for 3 h. The gel was then dialyzed and freeze-dried to obtain PNIPAM.

[0132] (5) Synthesis of PGOT hydrogel

[0133] To form the hydrogel, 0.32 g of GD and 0.2 g of PNIPAM macromonomer were dissolved in 1 mL of deionized water and mixed with equal volumes of OHD solution and TA solution. The concentration of OHD solution was 80 mg / mL and the concentration of TA solution was 14 mg / mL. A dark brown PGOT hydrogel was quickly formed.

[0134] (6) Synthesis of TI-Vpsb

[0135] 0.14 g of Tween 80 and 0.48 g of Span 80 were dissolved in 25 mL of n-hexane to obtain a mixed solution; 2 mL of an aqueous solution containing 0.6 g of sulfobetaine methacrylate (SBMA), 0.013 g of a crosslinker 6,6'-bisamino-3,3'-methylenedibenzoic acid (MBAA), 0.51 mg of ammonium persulfate (APS) and 1 mg of TI was added dropwise to the above mixed solution, and the mixture was treated with an ultrasonic wall breaking device for 10 minutes. After that, the solution was heated to 80°C, passed through N2 for protection, and reacted for 4 hours.

[0136] After cooling to room temperature, 15 mL of tetrahydrofuran (THF) was added to completely break up the microemulsion and precipitate. The microemulsion was washed three times with THF to remove unreacted monomers. The precipitate was dialyzed and freeze-dried to obtain TI-Vpsb. The chemical structure of Vpsb was analyzed by FTIR.

[0137] (7) Synthesis of TI-Vpsb@PGOT

[0138] The prepared TI-Vpsb, OHD, TA, GD, PNIPAM and water were mixed. In the mixed system, the concentration of TI-Vpsb was 1 mg / mL, the concentration of OHD was 40 mg / mL, the concentration of TA was 7 mg / mL, the concentration of GD was 160 mg / mL, and the concentration of PNIPAM was 100 mg / mL to prepare the drug-loaded microsphere hydrogel TI-Vpsb@PGOT.

[0139] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing an injectable hydrogel, characterized in that: The steps include: After adding EDC and NHS to the buffer solution of gelatin, the pH value of the buffer solution is adjusted to weak acidity. After stirring for a set time, dopamine hydrochloride is added thereto. Under inert atmosphere protection, the reaction is stirred at room temperature in the dark and maintained at weak acidity. The product is desalted and dried to obtain GD. Mix the NaIO4 solution with the hyaluronic acid solution, protect from light and react for a set time. After the reaction is completed, add ethylene glycol to neutralize the excess NaIO4, then dialysis purification and drying to obtain OHA; OHA and EDS are added to a buffer solution in proportion, stirred under weak acidic conditions for a set time, and then NHS is added thereto, and stirring is continued under weak acidic conditions for a set time; after the reaction is completed, dopamine hydrochloride is added thereto, and stirred under weak acidic conditions for a set time. After the reaction is completed, dialyzed, and the product is dried to obtain OHD; N-isopropylacrylamide, a crosslinker, and a photoinitiator are dissolved in water, and the reaction is carried out under ultraviolet light for a set time. The gel is dialyzed and dried to obtain PNIPAM. A mixture of methyl methacrylate sulfobetaine SBMA, 6,6'-bisamino-3,3'-methylenedibenzoic acid MBAA, ammonium persulfate and TGF-β inhibitor TI is placed in a mixed solution of Tween 80 and Span 80, treated with an ultrasonic wall breaking machine for a set time, and then heated and reacted in an inert atmosphere for a set time; after the reaction is completed, cooled, tetrahydrofuran is added to the reaction system to completely break up the microemulsion and precipitate, and TI-Vpsb is obtained after washing, dialyzing and drying; TI-Vpsb, OHD, tannic acid TA, GD, PNIPAM and water were mixed in proportion to prepare the drug-loaded microsphere hydrogel TI-Vpsb@PGOT.

2. The method for preparing the injectable hydrogel according to claim 1, wherein: When preparing GD, the molar ratio of gelatin, EDC, NHS and dopamine hydrochloride is 1.5-3:2-4:3-5:0.5-1.

5.

3. The method for preparing the injectable hydrogel according to claim 2, wherein: After adding dopamine hydrochloride, the reaction is stirred at room temperature in the dark for 10-30 hours.

4. The method for preparing the injectable hydrogel according to claim 1, wherein: When preparing OHA, the molar ratio of NaIO4 to hyaluronic acid is 1-3:

5.

5. The method for preparing the injectable hydrogel according to claim 1, wherein: Mix the NaIO4 solution with the hyaluronic acid solution and allow to react for 2-4 hours in the dark.

6. The method for preparing the injectable hydrogel according to claim 1, wherein: When preparing OHD, the molar ratio of OHA, EDS, NHS and dopamine hydrochloride is 0.5-1.5:1-3:1-3:0.5-1.

5.

7. The method for preparing the injectable hydrogel according to claim 1, wherein: When preparing TI-Vpsb, the mass ratio of SBMA, MBAA, ammonium persulfate, TI, Tween 80 and Span 80 is 1500-2500:40-60:1-3:3-7:400-600:1500-2500.

8. The method for preparing the injectable hydrogel according to claim 1, wherein: The reaction system is treated with an ultrasonic wall breaking machine for 5-15 minutes, then heated to 50-90°C and reacted in an inert atmosphere for 3-5 hours.

9. The method for preparing the injectable hydrogel according to claim 1, wherein: When preparing drug-loaded microsphere hydrogel TI-Vpsb@PGOT, the mass ratio of TI-Vpsb, OHD, TA, GD, PNIPAM and water is 0.1-0.5:5-15:15-20:1-5:0.5-1:50-80.

10. An injectable hydrogel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

11. Use of the injectable hydrogel according to claim 10 in the preparation of a drug for promoting scarless wound healing.

Images