Preparation method and application of injectable thermosensitive composite hydrogel for promoting wound repair

Abstract

This invention belongs to the field of biomedical polymer materials technology, and relates to a method for preparing an injectable thermosensitive composite hydrogel that promotes wound healing, and its application. Polyethylene glycol monomethyl ether is used as a macromolecular initiator, and stannous octoate is used as a catalyst to initiate the bulk polymerization of six-membered cyclic carbonate monomers and their derivatives. After polymerization, coupling is performed via hexamethylene diisocyanate to obtain an amphiphilic block polymer with hydrophilic ends and an oleophilic middle. The block polymer is then dissolved in PBS at low temperature, and a certain proportion of wound-healing nanoparticles are uniformly dispersed in the polymer aqueous solution to obtain the injectable thermosensitive composite hydrogel. This composite hydrogel is a viscous liquid at room temperature and has a viscosity of 34°C. o It rapidly gels in environments above °C, meaning that after injection into the body, it can gel quickly and release nanoparticles in situ to promote wound repair. It also possesses excellent wound-healing capabilities.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials technology, and particularly relates to a method for preparing an injectable thermosensitive composite hydrogel that promotes wound repair and its application. Technical Background

[0002] As the largest organ in the human body, the skin can be divided into three main layers: the epidermis, dermis, and subcutaneous tissue. It functions to sense external stimuli, regulate body temperature, and protect the body from external damage. However, due to its direct contact with the external environment, the skin is highly susceptible to accidental injury, losing its protective function and potentially leading to serious illness or even death, making it one of the most vulnerable tissues. The human body has the ability to repair the integrity of the skin through a complex process after injury. The wound healing process can be divided into several continuous and coordinated processes, including coagulation, inflammation control, angiogenesis, cell proliferation, tissue remodeling, and scar formation. In the early stages of wound repair, the proliferation of newly generated capillaries and fibroblasts greatly promotes the formation of granulation tissue. Angiogenesis is a key component of tissue regeneration, and one of the key problems after wound injury is impaired angiogenesis. Therefore, promoting angiogenesis at the affected area and restoring blood supply to damaged tissue to provide the oxygen and nutrients needed for cell growth and function is crucial. Therefore, to achieve the above objectives, methods for tissue regeneration using scaffold materials related to tissue engineering have been developed.

[0003] Hydrogels, as hydrophilic polymer materials, possess a slightly cross-linked three-dimensional network structure. They are well-known for their ability to absorb and retain large amounts of water while remaining water-insoluble in their structure. Due to their superior properties, including excellent water absorption and retention, good biocompatibility, and high drug loading capacity without loss of activity, hydrogels are widely used in tissue engineering scaffold materials, particularly in wound healing. In promoting wound healing, hydrogels provide a moist environment, absorb exudate, and clean the local environment to accelerate the healing process without causing toxicity, making them a highly competitive candidate material for wound repair. However, most hydrogel wound dressings are pre-formed before application, which cannot well adapt to the shape of irregular wounds, greatly limiting their application. Compared to pre-formed hydrogels, injectable hydrogels can adapt well to irregularly shaped wounds, making them highly suitable for use in tissue engineering. Summary of the Invention

[0004] To address the aforementioned problems with hydrogels, this invention provides a method for preparing an injectable thermosensitive composite hydrogel that promotes wound healing, as well as its application. This composite hydrogel possesses advantages such as good biocompatibility, low cytotoxicity, and the ability to promote cell proliferation and migration, as well as vascularization.

[0005] The first objective of this invention is to provide a method for preparing an injectable thermosensitive composite hydrogel that promotes wound healing. This method is simple, and the resulting hydrogel dressing has good biocompatibility and the ability to release nanoparticles in situ.

[0006] The second objective of this invention is that the injectable thermosensitive composite hydrogel loaded with nanoparticles plays an important role in wound repair and tissue regeneration.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A method for preparing an injectable thermosensitive composite hydrogel that promotes wound healing includes the following steps:

[0009] Step 1: Using polyethylene glycol monomethyl ether (mPEG) as an initiator and stannous octoate as a catalyst, the six-membered cyclic carbonate monomer and its derivatives are subjected to ring-opening polymerization via bulk polymerization to obtain mPEG-FPTMC.

[0010] Step 2: After coupling the prepared mPEG-FPTMC with hexamethylene diisocyanate (HDI), the product is precipitated and dried to obtain the target polymer mPEG-FPTMC-mPEG.

[0011] Step 3: Dissolve the polymer mPEG-FPTMC-mPEG in PBS buffer. After complete dissolution, add nanoparticles that can promote wound repair to form an injectable thermosensitive composite hydrogel that promotes wound repair.

[0012] Further, in step 1, the molecular weight of mPEG is between 300 and 2000, the molar ratio of the six-membered cyclic carbonate monomer and its derivatives to mPEG is between 5 and 100, and the molar ratio of the six-membered cyclic carbonate monomer and its derivatives to stannous octoate is between 100 and 10000; the reaction temperature is 90 to 200°C. o C, the reaction time is 12~72 hours.

[0013] Furthermore, in step 2, the ratio of HDI to mPEG-FPTMC is 1:2 to 1:20, and the reaction temperature is 50 to 100°C. o C, the reaction time is 4~24 hours.

[0014] Furthermore, in step 3, the nanoparticles that can promote wound repair are amorphous zinc phosphate, polydopamine, graphene, cerium oxide, europium hydroxide, or Mxene (including: single or multiple layers of titanium carbide, single or multiple layers of niobium carbide, single or multiple layers of vanadium carbide).

[0015] Furthermore, the polymer content of the injectable thermosensitive composite hydrogel that promotes wound repair is between 10% and 60% (w / w).

[0016] Furthermore, the injectable thermosensitive composite hydrogel that promotes wound repair contains 0.01% to 5% (w / w) of nanoparticles that promote wound repair.

[0017] The aforementioned injectable thermosensitive composite hydrogel loaded with nanoparticles is used as a product to promote wound healing.

[0018] Compared with the prior art, the present invention has the following beneficial effects.

[0019] 1. The injectable temperature-sensitive composite hydrogel of the present invention is at 34°C. o Rapid gelation occurs in environments above C.

[0020] 2. The injectable thermosensitive composite hydrogel of the present invention is very convenient to operate during application.

[0021] 3. The injectable thermosensitive composite hydrogel of the present invention can release nanoparticles in situ.

[0022] 4. The injectable thermosensitive composite hydrogel of the present invention has the advantages of good biocompatibility and low cytotoxicity.

[0023] 5. The injectable thermosensitive composite hydrogel of the present invention has the effect of promoting the proliferation and migration of vascular endothelial cells.

[0024] 6. The injectable thermosensitive composite hydrogel of the present invention has the effect of promoting angiogenesis around the wound.

[0025] 7. The water-injectable thermosensitive composite hydrogel of the present invention has the effect of promoting wound repair without producing scarring. Attached Figure Description

[0026] Figure 1 The structural formula, infrared spectrum, and nuclear magnetic resonance spectrum of the polymer in the hydrogel provided in Example 1 of the present invention are shown.

[0027] Figure 2 This is a diagram showing the gelation effect of the hydrogel provided in Example 1 of the present invention.

[0028] Figure 3 This is a rheological test diagram of the hydrogel provided in Example 1 of the present invention.

[0029] Figure 4 This is a diagram of the in vitro biocompatibility experiment of the hydrogel provided in Example 1 of the present invention.

[0030] Figure 5 This is a cell scratch experiment diagram of the hydrogel provided in Example 1 of the present invention.

[0031] Figure 6 This is a diagram of the in vivo wound healing experiment of the hydrogel provided in Example 1 of the present invention in mice.

[0032] Figure 7 HE staining and Masson staining images of tissue sections of the hydrogel provided in Embodiment 1 of the present invention.

[0033] Figure 8 This is an immunohistochemical image of the hydrogel provided in Example 1 of the present invention.

[0034] Figure 9 This is a q-PCR diagram of the hydrogel provided in Example 1 of the present invention. Detailed Implementation

[0035] To make the technical problems to be solved and the beneficial effects of this application clearer, the technical solutions of the embodiments of the present invention will be described completely and clearly below with reference to the accompanying drawings. It is obvious that the implementation examples described herein are merely for explaining this application and are not limited to this application. All reagents and materials used in the following experiments were purchased from conventional biochemical reagent companies. In the quantitative experiments in the following examples, three replicate experiments were set up, and the results were averaged.

[0036] A method for preparing an injectable thermosensitive composite hydrogel that promotes wound healing includes the following steps:

[0037] Step 1: Using polyethylene glycol monomethyl ether (mPEG) as an initiator and stannous octoate as a catalyst, the six-membered cyclic carbonate monomer and its derivatives are subjected to ring-opening polymerization via bulk polymerization to obtain mPEG-FPTMC.

[0038] Step 2: After coupling the prepared mPEG-FPTMC with hexamethylene diisocyanate (HDI), the product is precipitated and dried to obtain the target polymer mPEG-FPTMC-mPEG.

[0039] Step 3: Dissolve the polymer mPEG-FPTMC-mPEG in PBS buffer. After complete dissolution, add nanoparticles that can promote wound repair to form an injectable thermosensitive composite hydrogel that promotes wound repair.

[0040] Further, in step 1, the molecular weight of mPEG is between 300 and 2000, the molar ratio of the six-membered cyclic carbonate monomer and its derivatives to mPEG is between 5 and 100, and the molar ratio of the six-membered cyclic carbonate monomer and its derivatives to stannous octoate is between 100 and 10000; the reaction temperature is 90 to 200°C. o C, the reaction time is 12~72 hours.

[0041] Furthermore, in step 2, the ratio of HDI to mPEG-FPTMC is 1:2 to 1:20, and the reaction temperature is 50 to 100°C. o C, the reaction time is 4~24 hours.

[0042] Furthermore, in step 3, the nanoparticles that can promote wound repair are amorphous zinc phosphate, polydopamine, graphene, cerium oxide, europium hydroxide, or Mxene (including: single or multiple layers of titanium carbide, single or multiple layers of niobium carbide, single or multiple layers of vanadium carbide).

[0043] Furthermore, the polymer content of the injectable thermosensitive composite hydrogel that promotes wound repair is between 10% and 60% (w / w).

[0044] Furthermore, the injectable thermosensitive composite hydrogel that promotes wound repair contains 0.01% to 5% (w / w) of nanoparticles that promote wound repair.

[0045] The aforementioned injectable thermosensitive composite hydrogel loaded with nanoparticles is used as a product to promote wound healing.

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0047] Example 1.

[0048] This invention relates to an injectable thermosensitive polycarbonate composite hydrogel loaded with amorphous zinc phosphate (AZP) nanoclusters, the preparation method of which includes the following steps:

[0049] The ring-opening polymerization of trimethylene carbonate (TMC) was initiated via bulk polymerization using mPEG as the initiator and stannous octoate as the catalyst. The molecular weight of mPEG was 600, the molar ratio of TMC to mPEG was 25:1, the molar ratio of TMC to stannous octoate was 500:1, and the reaction temperature was 150°C. o C, after a reaction time of 48 hours, mPEG-PTMC was obtained; mPEG-PTMC and HDI were reacted at a molar ratio of 2:1 at 60 °C. o After 8 hours of coupling reaction, the product was precipitated and dried to obtain the target polymer mPEG-PTMC-mPEG. mPEG-PTMC-mPEG was dissolved in PBS buffer to prepare a 30% solution. 1% amorphous zinc phosphate (AZP) nanoclusters were added to obtain an injectable thermosensitive composite hydrogel that promotes wound repair.

[0050] Example 2.

[0051] The difference from Example 1 is that Example 2 adds 3% polydopamine nanoparticles to obtain an injectable thermosensitive composite hydrogel that promotes wound repair.

[0052] Example 3.

[0053] The difference from Example 1 is that Example 3 adds 2% graphene nanoparticles to obtain an injectable thermosensitive composite hydrogel that promotes wound repair.

[0054] Example 4.

[0055] The difference from Example 1 is that Example 4 adds 1.5% of monolayer titanium carbide MXene nanoparticles to obtain an injectable thermosensitive composite hydrogel that promotes wound repair.

[0056] Example 5

[0057] The difference from Example 1 is that Example 5 adds 0.5% of multilayer niobium carbide MXene nanoparticles to obtain an injectable thermosensitive composite hydrogel that promotes wound repair.

[0058] Example 6

[0059] The ring-opening polymerization of trimethylene carbonate (TMC) was initiated via bulk polymerization using mPEG as the initiator and stannous octoate as the catalyst. The molecular weight of mPEG was 750, the molar ratio of TMC to mPEG was 30:1, the molar ratio of TMC to stannous octoate was 1000:1, and the reaction temperature was 130°C. o C, after a reaction time of 24 hours, mPEG-FPTMC was obtained; mPEG-FPTMC and HDI were reacted at a molar ratio of 3:1 at 80 °C. o After C undergoes a coupling reaction for 12 hours, the product is precipitated and dried to obtain the target polymer mPEG-FPTMC-mPEG. mPEG-FPTMC-mPEG is dissolved in PBS buffer to prepare a 40% solution, and 1.5% amorphous zinc phosphate nanoparticles (AZP) clusters are added to obtain an injectable thermosensitive composite hydrogel that promotes wound repair.

[0060] The structure and performance of Example 1 are studied.

[0061] Figure 1 The infrared and nuclear magnetic resonance spectra of the polymer in the resulting injectable thermosensitive composite hydrogel that promotes wound healing are shown. In the infrared spectrum, 1184 and 1101 cm⁻¹ are particularly prominent. -1 The absorption peak at 2979 cm⁻¹ corresponds to the COC stretching vibration in mPEG. -1 The absorption peak at 1749 cm⁻¹ corresponds to the CH stretching vibration of CH₂ in the polymer. Additionally, the absorption peak at 1749 cm⁻¹...-1 The absorption peak at 1640 cm⁻¹ is a characteristic C=O stretching vibration of polycarbonate segments, while the peak at 1640 cm⁻¹ is a characteristic peak of polycarbonate segments. -1 The absorption peak at 1404 cm⁻¹ corresponds to the characteristic C=O stretching vibration of the amide bond. -1 The absorption peak at this point corresponds to the CN stretching vibration of the amide bond. These findings indicate that the polymerization of TMC is initiated by the terminal hydroxyl groups of mPEG and confirm the mPEG-b-PTMC... n The polymer successfully coupled with HDI to form an amide bond. In the NMR spectrum, the signal at 3.60 ppm represents -CH2 in mPEG, while -CH2 in PTMC is represented by signals at 2.00 and 4.10 ppm. The peaks observed at 1.28 and 3.21 ppm represent the two types of -CH2 in HDI. The characteristic peaks obtained in the IR spectrum and the analysis of the NMR spectrum confirm that the resulting polymer conforms to the molecular design.

[0062] Figure 2 The image shown is a diagram illustrating the gelation effect of the injectable thermosensitive composite hydrogel. 4 o C-type hydrogels exhibit fluidity, while 37-type hydrogels exhibit fluidity. o The hydrogel loses its fluidity at temperature C.

[0063] like Figure 3 As shown, injectable thermosensitive composite hydrogel in 4 o At C, it is a fluid, and at 37... o It transforms into a gel state at temperature C, and rheological tests have confirmed that the transition temperature of the injectable temperature-sensitive hydrogel is 34°C. o C is close to the actual transition temperature.

[0064] Figure 4 As shown, this invention uses human umbilical vein endothelial cells and L929 mouse fibroblasts to study the biocompatibility of injectable thermosensitive hydrogels and hydrogels loaded with AZP nanoparticles using an extract toxicity test according to national standard recommended methods. The biocompatibility test demonstrated that the injectable thermosensitive hydrogel is non-toxic and, after being loaded with AZP nanoparticles, significantly promotes the proliferation of human umbilical vein endothelial cells, while simultaneously not significantly promoting the proliferation of L929 mouse fibroblasts.

[0065] Depend on Figure 5 As shown, the effect of the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles provided in this invention on cell migration was investigated through a scratch healing experiment. The results showed that the hydrogel can significantly promote the migration of human umbilical vein endothelial cells.

[0066] Using SD rats as a wound injury model, in vivo animal experiments were conducted to further discuss the repair effects of the injectable thermosensitive hydrogel and the injectable thermosensitive composite hydrogel loaded with amorphous zinc phosphate (AZP) nanoparticles provided in this invention on wound defects. Figure 6 As shown, on day 6 of the experiment, compared with the control group (healing rate 44.94±4.4%) and the blank hydrogel group (healing rate 43.35±1.9%), the AZP nanoparticle group (healing rate 67.48±3.02%) and the injectable thermosensitive composite hydrogel group loaded with AZP nanoparticles (healing rate 74.22±2.92%) showed more significant repair effects on full-thickness skin defects. On day 15 of the experiment, the wound healing rate of the injectable thermosensitive composite hydrogel group loaded with AZP nanoparticles was 98.64±0.48%, which was significantly higher than that of the control group (healing rate 83.32±2.39%). Compared with the control group, the blank hydrogel (healing rate 90.1±1.26%) also showed a certain repair effect, which is due to the moisturizing properties of the hydrogel promoting wound healing. Similarly, compared with the control group, the AZP nanoparticle group (healing rate 95.47±1.53%) showed better repair effect due to its pro-angiogenic effect. More importantly, compared with the AZP nanoparticle group, the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles showed better repair effect. This is because the residence time and quantity of AZP nanoparticles at the wound site are both challenging, and the sustained-release effect of the hydrogel allows it to stay at the wound site for a longer time, providing a more stable release of zinc ions. Therefore, the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles is more conducive to the repair and healing of wound defects.

[0067] Depend on Figure 7The images show H&E and Masson staining of tissue sections near rat wounds. H&E staining results indicated that on day 9, compared to the control group, the number of neutrophils and lymphocytes near the wound in the injectable thermosensitive composite hydrogel group loaded with AZP nanoparticles was significantly reduced. By day 15, the injectable thermosensitive composite hydrogel group regenerated new epidermis, while the control group still showed varying degrees of inflammatory cell infiltration. This suggests that the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles helps reduce inflammation and promote epidermal remodeling. Compared to other groups, the wound epidermal thickness in the injectable thermosensitive composite hydrogel group loaded with AZP nanoparticles was close to the thickness of the surrounding undamaged epidermis, further confirming the role of the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles in reducing wound scar formation. Furthermore, a small number of glands were observed in the injectable thermosensitive composite hydrogel group loaded with AZP nanoparticles, indicating functional skin regeneration. Masson staining results showed that on day 9, the collagen deposition in the injectable thermosensitive composite hydrogel group loaded with AZP nanoparticles was higher than that in other groups, with a collagen deposition rate of 55.36±2.7%, nearly 1.9 times that of the control group. By day 15, the collagen deposition in the injectable thermosensitive composite hydrogel group loaded with AZP nanoparticles reached 59.26%±2.63%, 1.31 times that of the control group. Compared with the control group, the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles accelerated the formation of wound epithelium and collagen deposition, indicating that the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles has the potential to accelerate wound healing by promoting collagen deposition.

[0068] Depend on Figure 8 The image shows CD31 immunohistochemical staining of tissue sections near the wound in rats. Analysis showed that after treatment with the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles, CD31 expression was significantly higher in the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles compared to the control group. This further indicates that the injectable thermosensitive composite hydrogel loaded with AZP nanoparticles can promote CD3 expression to promote angiogenesis, growth, and recovery of tissues near the wound.

[0069] Depend on Figure 9The images show the Western blot and q-PCR characterization of the hydrogel. q-PCR analysis revealed that the α-SMA gene expression in the AZP-loaded injectable thermosensitive composite hydrogel group was significantly higher than that in the control group on day 9. Furthermore, the α-SMA gene expression in the AZP-loaded injectable thermosensitive composite hydrogel group was significantly lower on day 15 than on day 9. This indicates that after promoting wound vascularization in the early stages, the AZP-loaded injectable thermosensitive composite hydrogel does not lead to scarring in the later stages of wound healing. Therefore, the AZP-loaded injectable thermosensitive composite hydrogel can accelerate wound healing and reduce scarring by regulating the levels of α-SMA and CD31.

[0070] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and does not limit the implementation methods and scope of protection of the present invention. Those skilled in the art should recognize that any equivalent substitutions and obvious changes made based on the description and figures of this invention should be included within the scope of protection of this invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should also be included within the scope of protection of this invention.

Claims

1. A method for preparing an injectable thermosensitive composite hydrogel that promotes wound healing, characterized in that, Includes the following steps: Step 1: Using polyethylene glycol monomethyl ether (mPEG) as an initiator and stannous octoate as a catalyst, the six-membered cyclic carbonate monomer and its derivatives are subjected to ring-opening polymerization via bulk polymerization to obtain mPEG-PTMC. Step 2: After coupling the prepared mPEG-PTMC with hexamethylene diisocyanate (HDI), the product was precipitated and dried to obtain the target polymer mPEG-PTMC-mPEG. Step 3: Dissolve the polymer mPEG-PTMC-mPEG in PBS buffer. After complete dissolution, add nanoparticles that can promote wound repair to form an injectable thermosensitive composite hydrogel that promotes wound repair. In step 1, the molecular weight of mPEG is between 300 and 2000, the molar ratio of six-membered cyclic carbonate monomers and their derivatives to mPEG is between 5 and 100, and the molar ratio of six-membered cyclic carbonate monomers and their derivatives to stannous octoate is between 100 and 10000. In step 3, the nanoparticles that can promote wound repair are one or more combinations of amorphous zinc phosphate, polydopamine, graphene, cerium oxide, europium hydroxide, or Mxene.

2. The method for preparing the injectable thermosensitive composite hydrogel for promoting wound repair according to claim 1, characterized in that, In step 1, the reaction temperature of the ring-opening polymerization reaction is 90~200°C. o C, the reaction time is 12~72 hours.

3. The method for preparing the injectable thermosensitive composite hydrogel for promoting wound repair according to claim 1, characterized in that, In step 2, the ratio of HDI to mPEG-PTMC is 1:2 to 1:

20.

4. The method for preparing the injectable thermosensitive composite hydrogel for promoting wound repair according to claim 1, characterized in that, In step 2, the reaction temperature is 50~100℃. o C, the reaction time is 4~24 hours.

5. The method for preparing the injectable thermosensitive composite hydrogel for promoting wound repair according to claim 1, characterized in that, The polymer content of injectable thermosensitive composite hydrogels that promote wound healing is between 10% and 60% (w / w).

6. The method for preparing the injectable thermosensitive composite hydrogel for promoting wound repair according to claim 1, characterized in that, The injectable thermosensitive composite hydrogel that promotes wound healing contains 0.01% to 5% (w / w) of nanoparticles that promote wound healing.

7. The method for preparing the injectable thermosensitive composite hydrogel for promoting wound repair according to any one of claims 1-6, characterized in that, Application of the prepared injectable thermosensitive composite hydrogel that promotes wound healing in products that promote wound healing.

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