Injectable hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable hydrogel and a preparation method and application thereof.The preparation method comprises the following steps that a NaIO4 solution and a hyaluronic acid solution are mixed, a reaction is conducted in a dark place for set time, and then dialysis purification and drying are conducted to obtain OHA; the preparation method comprises the following steps: adding OHA and EDS into a buffer solution in proportion, stirring for a set time under a weak acid condition, then adding NHS, and continuously stirring for a set time under the weak acid condition; after the reaction is completed, dopamine hydrochloride is added into the mixture, after the reaction is completed, dialysis is performed, and a product is dried to obtain OHD; the preparation method comprises the following steps: adding a mixed solution of SBMA, MBAA, ammonium persulfate and TI into a mixed solution of Tween 80 and Span 80, treating for a set time by using an ultrasonic wall breaking machine, heating, and reacting for a set time in an inert atmosphere; after the reaction is finished, cooling to completely crush and precipitate the microemulsion, washing, dialyzing and drying to obtain TI-Vpsb; the preparation method comprises the following steps: mixing TI-Vpsb, OHD, TA, GD, PNIPAM and water according to a ratio, so as to prepare the drug-loaded microsphere hydrogel TI-Vpsb-coated PGOT.

Description

Technical Field

[0001] The 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 first natural barrier of the human body, the skin plays an important role in preventing dehydration and protecting internal organs from damage by the external environment. However, the skin loses its protective function after severe damage and leads to wound formation. Wound healing is a complex and coordinated process that usually involves the following four stages: hemostasis, inflammation, new tissue formation, and final tissue remodeling and regression. When these stages progress abnormally, dysfunctional and disfiguring scars will result. Scars can cause various complications, such as destruction of appearance and functional activity disorders, which bring great physical and mental pain to patients. Many studies have promoted wound healing in various ways, such as promoting angiogenesis, reducing inflammatory responses, and inhibiting bacterial growth, but most of them have not considered the importance of scars in wound healing. In recent years, studies on promoting scarless wound healing have also been reported, but they only consider one of the four stages of wound healing, such as anti-inflammatory and antibacterial, and promoting tissue remodeling through various pathways. Considering that wound healing is a continuous four-stage process, it is necessary to promote the smooth progress of each stage as much as possible and inhibit scar formation. Summary of the invention

[0004] In view of the deficiencies in 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, and after stirring for a set time, dopamine hydrochloride is added thereto, and the reaction is stirred at room temperature in the dark under inert atmosphere protection, and maintained at weak acidity, and the product is desalted and dried to obtain GD;

[0008] Mix the NaIO4 solution and 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 and purification, and dry to obtain OHA;

[0009] OHA and EDS are added to a buffer solution in proportion, stirred for a set time under weak acid conditions, and then NHS is added thereto, and stirring is continued for a set time under weak acid conditions; after the reaction is completed, dopamine hydrochloride is added thereto, and stirred for a set time under weak acid conditions, 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 performed under ultraviolet light for a set time. The gel is dialyzed and dried to obtain PNIPAM;

[0011] The mixed solution of SBMA, MBAA, ammonium persulfate and TI and the mixed solution of Tween 80 and Span 80 are 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, the mixture is 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;

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

[0013] In the process of preparing GD, EDC and NHS are used to catalyze the carboxyl group of gelatin and the amino group of dopamine to form an amide bond to obtain dopamine-modified gelatin (GD). Inert gas protection prevents the oxidation of the phenolic hydroxyl group of dopamine. Dopamine hydrochloride contains phenolic hydroxyl groups, which are easily decomposed by light, so the reaction is avoided from light. EDC and NHS have the best activity in activating carboxyl groups in weakly acidic buffer solutions.

[0014] In the process of preparing OHA, sodium periodate is used to oxidize the hydroxyl groups of hyaluronic acid to obtain aldehyde-modified hyaluronic acid. Light energy excites the electrons of the vicinal diol molecules, making them in a high-energy state, which can react with sodium periodate, thereby producing an oxidation reaction. Therefore, reacting in a dark environment can avoid light interference and ensure the accuracy and stability of the reaction. Ethylene glycol contains hydroxyl groups, which can react with excess sodium periodate.

[0015] During the preparation of OHD, the carboxyl group of OHA can react with the amino group of dopamine under the catalysis of EDC and NHS to form an amide bond. At the same time, the aldehyde group of OHA can react with the amino group of dopamine to form a Schiff base reaction, further increasing the grafting rate of dopamine to obtain dopamine-modified oxidized hyaluronic acid. EDC and NHS have the best activity in activating carboxyl groups in a weakly acidic buffer solution, so weakly acidic conditions are required.

[0016] In the process of preparing TI-Vpsb, the role of ultrasonic wall breaking machine treatment is to mix the oil phase and the water phase evenly to obtain an oil-in-water colloidal dispersion system. The microspheres were prepared by the reverse microemulsion method to obtain microspheres encapsulating TI. Under the stabilizing effect of the emulsifier, the SBMA monomer aqueous solution was highly dispersed in the oil in the form of nano-sized micro-droplets to form a W / O colloidal dispersion system with a clear and transparent appearance. The orderly arranged monolayer of emulsifier on the oil-water interface surrounded the micro-droplets, and a "microreactor" was formed. Then the SBMA monomer in the "microreactor" polymerized under the action of thermal initiation, and the entire micro-droplet polymerized into microspheres. After demulsification, filtration, washing, vacuum drying and other operations, the microspheres were obtained.

[0017] During the preparation of TI-Vpsb@PGOT, the macromonomers OHD, PNIPAM, and GD interacted with each other through hydrogen bonds, Schiff base bonds, and covalent interactions to form hydrogels. TA and TI-Vpsb microspheres can further extend the retention time of the hydrogels 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, 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: generates NHS esters with activated intermediates; 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 the 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 a hemostatic effect in the early stage, anti-oxidation and antibacterial effect in the middle stage, and anti-fibrosis effect in the late stage, and is used to promote rapid healing of wounds without scars.

[0034] Dopamine-modified hyaluronic acid and gelatin are used as the skeleton of the hydrogel, which makes it have 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, which has a similar structure to dopamine. It can be used to enhance the adhesion properties of hydrogels and 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 and achieve long-lasting anti-inflammatory and antibacterial effects.

[0037] The present invention prepares poly(methacrylic acid 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 in the rat infected skin defect model, the 4D hydrogel dressing can be anti-inflammatory and antibacterial, accelerate skin wound closure, and inhibit scar formation. In short, TI-Vpsb@PGOT has great potential in clinical hemostasis, anti-inflammatory, antibacterial and anti-scar applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings in the specification, 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 of 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, 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 the 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 It is an image of the bleeding of the liver over time after treatment or no treatment in Example 1 of the present invention, wherein 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); 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 It is the H&E staining picture of each organ 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 descriptions are illustrative and 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 meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0049] The present invention will be further described below in conjunction with 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 (PBS) solution; 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 certain period of time, 1.0 g of dopamine hydrochloride (DOPA·HCl) was added to the reaction system, and the mixture was stirred at 37°C in the dark for 24 h under nitrogen protection, and the pH was maintained at 5.0-6.0. The mixture was dialyzed with deionized water for 48 h to remove salt, and then freeze-dried 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 at room temperature in the dark 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 in deionized water for 2 days and then lyophilized to obtain OHA with a yield of about 85%.

[0058] (3) Synthesis of OHD

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

[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 in 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 samples were characterized by H-NMR spectroscopy (400 Hz).

[0063] (4) Synthesis of PNIPAM

[0064] 1.0 g N-isopropylacrylamide (NIPAM) was mixed with 90 μL 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 an equilibrium state. Afterwards, the hydrogel was freeze-dried using a freeze dryer, and the 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 machine for 10 min, and then the solution was heated to 70°C, N2 was passed for protection, and the reaction was carried out for 4 hours.

[0071] After cooling to room temperature, 15 mL of tetrahydrofuran (THF) was added to completely break up the microemulsion and precipitate it, which was washed three times with THF to remove unreacted monomers, and 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 by dialysis and UV. TI-Vpsb@PGOT was transferred to a dialysis bag with PBS buffer on the outside, and placed on an oscillator for constant temperature culture at 37°C for 13 days.

[0076] PGOT was prepared in the same way. The specific preparation method was as follows: 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. The specific preparation method was as follows: 10 mg of freeze-dried TI-Vpsb was added to 10 mL of PBS to prepare a 1 mg / mL solution.

[0078] 1 mL of release medium was taken at 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, 240h, 264h, 288h, and 312h, and replaced with an equal volume of PBS. The content of TI in TI-Vpsb@PGOT and TI-Vpsb was determined by UV, and the content of TA in PGOT was determined by UV. Each group of samples was repeated 3 times, and the average value was taken to obtain the cumulative release rate, thereby obtaining the cumulative release curve.

[0079] Animal Experiments

[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 formed and a Staphylococcus aureus suspension (50 μL, 1 × 10 8 CFU / mL) were immediately dripped 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 wound with the corresponding hydrogels.

[0083] Then, the wound healing progress of mice was monitored and body weight was recorded using smartphones on days 0, 3, 7, 10, and 14. The wound area was calculated using ImageJ software. After 7 days of treatment, four rats in each group were killed and the wound skin tissue of the rats was harvested. After 14 days of treatment, the remaining rats were killed and the wound skin tissue and major organs (heart, liver, spleen, kidney, and lung) of the rats were harvested.

[0084] Results section:

[0085] (1) Characterization of polymers and TI-Vpsb@Gel hydrogels

[0086] Gel-DA was synthesized by condensation reaction between the carboxyl group of Gel and the amino group of dopamine. 1 H-NMR was used to characterize the synthesized structures, such as Figure 2 As shown, GD 1 In the HNMR spectrum, the peak at 2.75 ppm is attributed to the methylene protons close to 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 about 6.8 ppm in HNMR indicated the presence of aromatic protons, confirming the successful binding of OHA to DA.

[0087] SEM images of PGOT hydrogels are 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, 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 It can be seen that TA in PGOT is released rapidly and reaches a plateau on the 5th day, which is attributed to the mechanical contraction properties of PNIPAM, which can promote the rapid release of TA. TI in TI-Vpsb@PGOT begins to release on the fourth day and is almost completely released on the 13th day, with a release rate of 94.89%. Therefore, under the action of PNIPAM and TI-Vpsb, the sequential release of TA and TI in TI-Vpsb@PGOT basically 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. However, the liver bleeding time of rats in the TI-Vpsb@PGOT group was 144.57 seconds, and the blood loss was significantly reduced to 0.0336 g, which indicates 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 point of view, 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 show 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] Compared with the control group, no obvious abnormalities were found in the histological examination of the heart, liver, spleen, lung, and kidney of the treatment group (TI-Vpsb@PGOT 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 hemostasis in the early stage, antibacterial and antioxidant effects in the middle stage, and anti-fibrosis in the late stage, 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 (PBS) solution; 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 min, 2.0 g of dopamine hydrochloride (DOPA·HCl) was added to the reaction system, and the mixture was stirred at 37°C in the dark for 24 h under nitrogen protection, and the pH was maintained at 5.0-6.0. The mixture was dialyzed with deionized water for 48 h to remove salts, and then freeze-dried 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 at room temperature in the dark 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 in 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. Then 0.3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC was added to the solution and stirred for 15 min at pH 5.0-6.0. 0.3 g of N-hydroxysuccinimide NHS was added to the reaction solution and stirred for another 40 min at pH 5.5-6.0.

[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 in deionized water for 2 days and then lyophilized to obtain OHD.

[0109] (4) Synthesis of PNIPAM

[0110] 1.0 g N-isopropylacrylamide (NIPAM) was mixed with 100 μL 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 solution was treated with an ultrasonic wall breaking machine for 5 min, and then the solution was heated to 60°C, N2 was passed 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 it, which was washed three times with THF to remove unreacted monomers, and 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 (PBS) solution; 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 min, 1.5 g of dopamine hydrochloride (DOPA·HCl) was added to the reaction system, and the mixture was stirred at 37°C in the dark for 24 h under nitrogen protection, with the pH maintained at 5.0-6.0. The mixture was dialyzed with deionized water for 48 h to remove salts, and then freeze-dried 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 at room temperature in the dark 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 in 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. Then 0.4 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC was added to the solution and stirred for 10 min at pH 5.5-6.0. 0.35 g of N-hydroxysuccinimide NHS was added to the reaction solution and stirred for another 45 min at pH 5.5-6.0.

[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 in deionized water for 2 days and then lyophilized to obtain OHD.

[0130] (4) Synthesis of PNIPAM

[0131] 1.0 g N-isopropylacrylamide (NIPAM) was mixed with 80 μL 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 machine for 10 min, and then the solution was heated to 80°C, N2 was passed for protection, and the reaction was carried out for 4 hours.

[0136] After cooling to room temperature, 15 mL of tetrahydrofuran (THF) was added to completely break up the microemulsion and precipitate it, which was washed three times with THF to remove unreacted monomers, and 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 above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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, and after stirring for a set time, dopamine hydrochloride is added thereto, and the reaction is stirred at room temperature in the dark under inert atmosphere protection, and maintained at weak acidity, and the product is desalted and dried to obtain GD; Mix the NaIO4 solution and 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 and purification, and dry to obtain OHA; OHA and EDS are added to a buffer solution in proportion, stirred for a set time under weak acid conditions, and then NHS is added thereto, and stirring is continued for a set time under weak acid conditions; after the reaction is completed, dopamine hydrochloride is added thereto, and stirred for a set time under weak acid conditions, 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 performed under ultraviolet light for a set time. The gel is dialyzed and dried to obtain PNIPAM; The mixed solution of SBMA, MBAA, ammonium persulfate and TI and the mixed solution of Tween 80 and Span 80 are 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, the mixture is 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, 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, characterized in that: 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; Preferably, after adding dopamine hydrochloride, the reaction is stirred at room temperature in the dark for 10-30 hours.

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

5.

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

5. The method for preparing the injectable hydrogel according to claim 1, characterized in that: 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.

6. The method for preparing the injectable hydrogel according to claim 1, characterized in that: 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.

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

8. The method for preparing the injectable hydrogel according to claim 1, characterized in that: 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.

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

10. Use of the injectable hydrogel according to claim 9 in the preparation of a drug for promoting scar-free wound healing.

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