Injectable wound repair hydrogel and method of making same

Abstract

This invention discloses a method for preparing an injectable wound repair hydrogel. Polyethylene glycol diacrylate and diaminoguanidine hydrochloride are dissolved in dimethyl sulfoxide until fully dissolved. A catalyst, 4-dimethylaminopyridine, is added to activate the double bonds. The reaction is carried out at room temperature with stirring for 24–48 hours. The acrylate end groups of PEGDA and the amino groups of DAG undergo stepwise polymerization via Michael addition. Purification is performed to obtain hyperbranched PBAG macromolecules containing guanidine side groups. The obtained hyperbranched PBAG macromolecules are dissolved in deionized water or PBS buffer to prepare a solution A with a concentration of 5 wt%–15 wt%. Thiol-terminated polyethylene glycol (PEG-SH) is dissolved in the same solvent to prepare a solution B with a concentration of 5 wt%–15 wt%. Solutions A and B are mixed uniformly, and rapid gelation is achieved within 30 seconds at 37°C or with the addition of a very small amount of DMAP to the system, resulting in a PBAG-S-PEG crosslinked hydrogel with a certain degree of hardness.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to an injectable wound repair hydrogel and its preparation method. Background Technology

[0002] Wound repair is a common clinical problem, especially for difficult-to-heal wounds such as large-area skin defects, diabetic ulcers, and burns, causing severe pain and a heavy medical burden for patients. Traditional wound management methods (such as gauze dressings and autologous skin grafts) have drawbacks, including repeated dressing changes causing pain, donor site damage, and a high risk of infection. Injectable hydrogels have gained widespread attention in the field of wound repair in recent years due to their advantages such as minimally invasive operation, ability to fill irregular cavities, and provision of a moist healing environment.

[0003] However, injectable hydrogels still face multiple challenges in wound repair applications. On the one hand, the degradation rate of hydrogels is difficult to precisely control—degradation that is too rapid cannot support the growth of new tissue, while degradation that is too slow may hinder tissue remodeling and the normal healing process. Therefore, controlling the hydrogel degradation rate to match the tissue integration process is key to maximizing its advantages. On the other hand, the gelation time of traditional hydrogels is relatively long (usually several minutes to tens of minutes), which may affect the repair effect due to the loss of precursor solutions during clinical operations. There is an urgent need for material systems that can achieve rapid in-situ gelation. Furthermore, many hydrogel systems require initiation conditions such as ultraviolet light, chemical catalysts, or high temperatures, posing potential problems such as cytotoxicity and operational inconvenience.

[0004] Poly(β-amino esters) (PBAEs) are a class of cationic polymers synthesized via the Michael addition reaction of diacrylates with amine monomers. They are widely used in gene delivery, controlled drug release, and tissue engineering due to their biodegradability, ease of synthesis, and low cost. However, the uncontrollable degradation rate of traditional linear PBAE macromolecules limits their practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide an injectable PBAG-S-PEG hydrogel and its preparation method. It is formed by rapidly crosslinking hyperbranched PBAG macromolecules with linear PEG-SH through Michael addition reaction and guanidino-thiol synergistic effect. It has the characteristics of rapid crosslinking, adjustable degradation rate, and wound adhesion and repair, which solves the problems of long gelation time, mismatched degradation rate, cumbersome operation and need for toxic catalysts in existing wound repair hydrogels.

[0006] To achieve the above objectives, the following technical solution is adopted: A method for preparing an injectable wound repair hydrogel includes the following steps: (1) Polyethylene glycol diacrylate (PEGDA) and diaminoguanidine hydrochloride (DAG) were dissolved in dimethyl sulfoxide (DMSO) until fully dissolved; 4-dimethylaminopyridine (DMAP) catalyst was added to activate the double bond, and the reaction was stirred at room temperature for 24-48 hours. The acrylate end group of PEGDA and the amino group of DAG were polymerized stepwise by Michael addition. After purification, PBAG hyperbranched macromolecules containing guanidine side groups were obtained. (2) Dissolve the obtained hyperbranched PBAG macromolecules in deionized water or PBS buffer to prepare a solution A with a concentration of 5wt% to 15wt%; dissolve the mercapto-terminated polyethylene glycol (PEG-SH) in the same solvent to prepare a solution B with a concentration of 5wt% to 15wt%. (3) Mix solution A and solution B evenly, and achieve rapid gelation within 30 seconds under 37°C or with a very small amount of DMAP added to the system to obtain PBAG-S-PEG crosslinked hydrogel with a certain hardness.

[0007] In the hyperbranched PBAG macromolecule, the unreacted terminal acrylate double bonds chemically crosslink with the thiol groups of PEG-SH via Michael addition. Simultaneously, the guanidinium groups on the PBAG side chains can synergistically accelerate the crosslinking reaction and enhance network stability through hydrogen bonding and ionic interactions with the thiol groups. DMAP, as a nucleophilic catalyst, can further activate the double bonds, achieving gelation within seconds.

[0008] According to the above scheme, the molar ratio of PEGDA to DAG in step (1) is (2-5):1, preferably (3-4):1. This ensures that sufficient terminal acrylate double bonds are retained in the hyperbranched structure for subsequent crosslinking.

[0009] According to the above scheme, the purification process in step (1) includes the following steps: The reaction product was purified by dialysis using a dialysis bag with a molecular weight cutoff of 3500 Da for 1–2 days, with deionized water replaced every 2 hours to remove unreacted monomers, DMSO, and catalyst. The purified product solution was then freeze-dried to obtain a light brown, viscous, hyperbranched PBAG macromolecule.

[0010] In the preferred embodiment, the concentration of solution A in step (2) is 10 wt%.

[0011] In the preferred embodiment, the concentration of solution B in step (2) is 10 wt%.

[0012] According to the above scheme, the molecular weight of the thiol-terminated polyethylene glycol (PEG-SH) in step (2) is 2k to 10k, and the thiol functionality is ≥2.

[0013] According to the above scheme, in step (3), the degradation rate of the hydrogel is adjusted within the range of 6h to 48h by changing the molar ratio of PBAG to PEG-SH. The higher the PBAG ratio, the lower the crosslinking density and the faster the degradation; the higher the PEG-SH ratio, the higher the crosslinking density and the slower the degradation.

[0014] According to the above scheme, in step (3), solution A and solution B are mixed evenly at a volume ratio of (1-2):(2-1).

[0015] In the preferred embodiment, in step (3), solution A and solution B are mixed evenly at a volume ratio of 1:1.

[0016] According to the above scheme, the amount of 4-dimethylaminopyridine DMAP added in step (3) is 0.05-0.1 wt%.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is based on the design concept of PBAE hyperbranched macromolecules. Using polyethylene glycol diacrylate (PEGDA) and diaminoguanidine hydrochloride (DAG) as monomers, a novel PBAG hyperbranched macromolecular material is synthesized at room temperature via Michael addition reaction. The introduction of DAG not only provides the carbon-nitrogen double bond and secondary amine group of the guanidine group as active chemical modification sites, but its unique guanidine structure also synergizes with PEG-SH, endowing the hydrogel with rapid cross-linking ability. Gelation can be completed within 30 seconds at 37°C or under minimal DMAP catalysis. Furthermore, by adjusting the ratio of PBAG to PEG-SH, the degradation rate of the hydrogel can be adjusted from 6 hours to over 48 hours to match the tissue regeneration needs of different wound types.

[0018] The ester bonds in the PBAG backbone can be slowly hydrolyzed under physiological conditions, with degradation products including low-molecular-weight PEG segments, amino acid analogs (DAG derivatives), and diols, which are non-cytotoxic. The hyperbranched structure provides a three-dimensional porous network, offering space for cell migration and nutrient exchange. The guanidino group endows PBAG with a positive charge, allowing it to adhere to negatively charged cell membranes and tissue surfaces via electrostatic interactions, achieving wound adhesion and physical barrier functions.

[0019] Controllable gelation: Under mild conditions of 37°C or micro-dMAP catalysis, the gelation time can be shortened to less than 30 seconds, far exceeding that of traditional hydrogel systems (which usually take several minutes to tens of minutes), making it convenient for immediate clinical use and effectively preventing the loss of precursor solutions on the wound surface.

[0020] Adjustable degradation rate: By simply adjusting the ratio of PBAG to PEG-SH, the degradation rate can be continuously adjusted within the range of 6 to 48 hours or more, which can match the repair time window of wounds of different degrees.

[0021] Wound adhesion: Guanidinium groups allow the hydrogel to adhere directly to the wound tissue through electrostatic and hydrogen bonding interactions, eliminating the need for additional sutures or fixation and providing a moist healing environment.

[0022] Biocompatibility and biodegradability: The degradation products are non-toxic, and PEG is an FDA-approved biomaterial component; the hyperbranched structure improves the water solubility of macromolecules, avoiding the problem of poor solubility of traditional linear PBAE. Attached Figure Description

[0023] Figure 1 Synthetic route of PBAG hyperbranched macromolecules and schematic diagram of PBAG-S-PEG hydrogel crosslinking.

[0024] Figure 2 : 1H NMR spectrum of PBAG.

[0025] Figure 3 Visual images of color changes and UV-Vis absorption spectra for detecting free thiol groups using the DTNB method.

[0026] Figure 4 Digital photograph of PBAG-S-PEG hydrogel.

[0027] Figure 5 Comparison of gelation time under different ratios and conditions.

[0028] Figure 6 Photographs and tissue staining of the healing process of animal wound models. Detailed Implementation

[0029] The following embodiments further illustrate the technical solution of the present invention, but are not intended to limit the scope of protection of the present invention.

[0030] A specific embodiment provides a method for preparing an injectable wound repair hydrogel, as shown in the attached figure. Figure 1 As shown: (1) Polyethylene glycol diacrylate (PEGDA) and diaminoguanidine hydrochloride (DAG) were dissolved in dimethyl sulfoxide (DMSO) until fully dissolved; 4-dimethylaminopyridine (DMAP) catalyst was added to activate the double bond, and the reaction was stirred at room temperature for 24-48 hours. The acrylate end group of PEGDA and the amino group of DAG were polymerized stepwise by Michael addition. After purification, PBAG hyperbranched macromolecules containing guanidine side groups were obtained. (2) Dissolve the obtained hyperbranched PBAG macromolecules in deionized water or PBS buffer to prepare a solution A with a concentration of 5wt% to 15wt%; dissolve the mercapto-terminated polyethylene glycol (PEG-SH) in the same solvent to prepare a solution B with a concentration of 5wt% to 15wt%. (3) Mix solution A and solution B evenly, and achieve rapid gelation within 30 seconds at 37°C or with a very small amount of DMAP added to the system to obtain a PBAG-S-PEG crosslinked hydrogel with a certain hardness. The unreacted terminal acrylate double bonds in the hyperbranched PBAG macromolecules undergo chemical crosslinking with the thiol groups of PEG-SH through Michael addition; at the same time, the guanidinium groups of the PBAG side chains can accelerate the crosslinking reaction and enhance the network stability through hydrogen bonding, ionic interactions and synergistic effects with the thiol groups. DMAP, as a nucleophilic catalyst, can further activate the double bonds to achieve gelation in seconds.

[0031] Example 1 Synthesis of hyperbranched PBAG macromolecules: Weigh 3.5 g of PEGDA (Mn~700) and 0.25 g of DAG, and add them to 10–20 mL of DMSO. Sonicate for 5 minutes to completely dissolve the powder, and remove dissolved oxygen by venting for 15 minutes. Add 0.5 mM DMAP to initiate polymerization, and stir the reaction at room temperature (25°C) for 24–48 hours. After the reaction is complete, dialyze the mixture in deionized water for 1–2 days using a dialysis bag with a molecular weight cutoff of 3500 Da (changing the water every 2 hours). Freeze-dry for 48 hours to obtain light brown, viscous hyperbranched PBAG macromolecules. Yield: approximately 70%–85%.

[0032] Appendix Figure 2 The ¹H NMR spectrum of PBAG obtained in this embodiment confirms the presence of characteristic peaks of PEGDA ester bond and DAG guanidinium group, thus confirming successful synthesis.

[0033] Example 2 Preparation and characterization of PBAG-S-PEG hydrogel: The lyophilized PBAG macromolecules obtained in Example 1 were dissolved in deionized water to prepare a 10% wt solution (solution A). PEG-SH (molecular weight e.g., 3400 Da, thiol functionality ≥2) was dissolved in deionized water to prepare a 10% wt solution (solution B). 9 μL of solution A and 9 μL of solution B were mixed in a microcentrifuge tube, and 2 μL of 1% wt DMAP solution was added (or the tube was placed directly in a 37°C constant temperature environment), and the mixture was gently blown to mix. The gelation time was recorded (the gelation endpoint was defined as no liquid flow after inverting the centrifuge tube). Experimental results showed that complete gelation occurred within 30 seconds at room temperature after the addition of DMAP; the gelation time at 37°C without DMAP was approximately 1–3 minutes. The resulting hydrogel was light brown and transparent, with a storage modulus of 20–200 kPa.

[0034] The hydrogel obtained in this embodiment was analyzed using the DTNB method, and the resulting images show the visual color change of free thiol groups, along with its UV-Vis absorption spectrum. (See attached image.) Figure 3As shown, the PEG-SH solution turned yellow immediately upon the addition of DTNB, and an absorption peak appeared at 412 nm in the UV spectrum. When PBAG reacted with PEG-SH to form a gel, the addition of DTNB showed no color change and no absorption peak at 412 nm, confirming that the thiol groups of PEG-SH were fully involved in the cross-linking reaction. Scanning electron microscopy revealed that the hydrogel had a three-dimensional porous structure with a pore size range of approximately 10–50 μm, suitable for cell migration and nutrient exchange.

[0035] A digital photograph of the PBAG-S-PEG hydrogel obtained in this embodiment is attached. Figure 4 As shown.

[0036] Example 3 Degradation rate was controlled by different PBAG / PEG-SH ratios: The concentrations of solution A (PBAG) and solution B (PEG-SH) were fixed at 10% wt, and the mixing volume ratios were adjusted to 2:1, 1:1, and 1:2 (PBAG:PEG-SH). Hydrogels were prepared according to the method in Example 2 for each ratio, and then immersed in PBS buffer (pH 7.4, 37°C). The weight was recorded at regular intervals to determine the complete degradation time. Expected results: Degradation time for the 2:1 ratio was approximately 6–12 hours (high PBAG content, lower crosslinking density); degradation time for the 1:1 ratio was approximately 24–36 hours; degradation time for the 1:2 ratio was approximately 36–48 hours or more (high PEG-SH content, higher crosslinking density).

[0037] The comparison of gelation time under different formulations and conditions in this embodiment is shown in the appendix. Figure 5 As shown. The appropriate ratio can be selected according to the type of wound. For acute clean wounds, a fast-degrading formula (2:1) can be selected, while for chronic or large-area defect wounds, a durable formula (1:2) can be selected.

[0038] Example 4 Efficacy evaluation of an animal wound repair model: SD rats (6-8 weeks old) were used as the animal model. After hair removal from the back, a circular full-thickness skin defect of approximately 1 cm in diameter was surgically created. Groups: 8-10 rats per group. (a) Blank control group: the wound was covered only with medical gauze; (b) Pure PEG hydrogel group; (c) PBAG-S-PEG hydrogel treatment group (1:1 ratio, 10% concentration, 100 μL / wound). Postoperatively, the wound area was photographed and recorded periodically (on days 0, 3, 7, 10, and 14), and the healing rate was calculated. Rats were sacrificed on day 14, and the wound and surrounding tissue were harvested for H&E staining histological evaluation (epithelial reformation, collagen deposition, and inflammatory infiltration).

[0039] The attached images show the healing process of the animal wound model and tissue staining in this embodiment. Figure 6As shown, wounds treated with PBAG hydrogel healed significantly faster, with the wound area shrinking by more than 50% by day 7 and essentially closing by day 14; while the control group still had significant wound defects. PBAG hydrogel provides a suitable moist healing environment for the wound and a suitable microenvironment for accelerating tissue regeneration.

[0040] Example 5 Injectability and Clinical Simulation: PBAG precursor solution (Solution A) and PEG-SH solution (Solution B) were separately filled into the two chambers of a dual-syringe, mixed using a static mixing head, and then injected subcutaneously into the back of a rat or into a wound model via a 26G needle. The smoothness of injection and in-situ gelation of the precursor solution were observed. Results: The precursor solution was injected smoothly and without resistance through the 26G fine needle; at a body temperature of 37°C, a gel formed within 30 seconds to 2 minutes after injection, adhering tightly to the original site without flowing or detaching. Simulation results indicate that this system has good clinical operability.

Claims

1. A method for preparing an injectable wound repair hydrogel, characterized in that... Includes the following steps: (1) Polyethylene glycol diacrylate (PEGDA) and diaminoguanidine hydrochloride (DAG) were dissolved in dimethyl sulfoxide (DMSO) until fully dissolved; 4-dimethylaminopyridine (DMAP) catalyst was added to activate the double bond, and the reaction was stirred at room temperature for 24-48 hours. The acrylate end group of PEGDA and the amino group of DAG were polymerized stepwise by Michael addition. After purification, PBAG hyperbranched macromolecules containing guanidine side groups were obtained. (2) Dissolve the obtained hyperbranched PBAG macromolecules in deionized water or PBS buffer to prepare a solution A with a concentration of 5wt% to 15wt%; dissolve the mercapto-terminated polyethylene glycol (PEG-SH) in the same solvent to prepare a solution B with a concentration of 5wt% to 15wt%. (3) Mix solution A and solution B evenly, and achieve rapid gelation within 30 seconds under 37°C or with a very small amount of DMAP added to the system to obtain PBAG-S-PEG crosslinked hydrogel with a certain hardness.

2. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... In step (1), the molar ratio of PEGDA to DAG is (2-5):

1.

3. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... The purification process in step (1) includes the following steps: The reaction product was purified by dialysis using a dialysis bag with a molecular weight cutoff of 3500 Da for 1–2 days, with deionized water replaced every 2 hours to remove unreacted monomers, DMSO, and catalyst. The purified product solution was then freeze-dried to obtain a light brown, viscous, hyperbranched PBAG macromolecule.

4. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... In step (2), the concentration of solution A is 10 wt%.

5. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... In step (2), the concentration of solution B is 10 wt%.

6. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... The thiol-terminated polyethylene glycol (PEG-SH) mentioned in step (2) has a molecular weight of 2k to 10k and a thiol functionality ≥2.

7. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... In step (3), the degradation rate of the hydrogel is adjusted within the range of 6h to 48h by changing the molar ratio of PBAG to PEG-SH.

8. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... In step (3), solution A and solution B are mixed evenly at a volume ratio of (1-2):(2-1).

9. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... In step (3), solution A and solution B are mixed evenly at a volume ratio of 1:

1.

10. The method for preparing the injectable wound repair hydrogel as described in claim 1, characterized in that... In step (3), the amount of 4-dimethylaminopyridine DMAP added is 0.05-0.1 wt%.

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