Preparation method of photo-thermal-MMP dual-response matrix hydrogel and application of photo-thermal-MMP dual-response matrix hydrogel in diabetic wound repair

Abstract

The invention discloses a preparation method of photo-thermal-MMP dual-response matrix hydrogel and application of the photo-thermal-MMP dual-response matrix hydrogel in diabetic wound repair, and belongs to the technical field of biological medicine. According to the present invention, the Cur (at) ZIF-8 (at) MMPDA / dAMG composite hydrogel is composed of an amnion source photo-crosslinked matrix hydrogel, curcumin-loaded ZIF-8 nanoparticles and MMP2 peptide modified polydopamine, and the Cur (at) ZIF-8 (at) MMPDA / dAMG composite hydrogel is obtained; the ZIF-8 nanoparticles decline in an acid environment to release zinc ions, destroy bacterial cell membranes, enhance the permeability of a biological membrane and cooperatively kill drug-resistant bacteria in combination with a photothermal effect, Cur is loaded through ZIF-8 to form Cur-coated ZIF-8, then a Cur-coated ZIF-8-coated MMPDA / dAMG double-drug-loading system is constructed through an MMPDA medium, and a photothermal antibacterial, anti-inflammatory and regeneration-promoting multi-mode therapy is formed for a wound surface in the acid environment. Therefore, wound healing is accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing a photothermal-MMP dual-responsive matrix hydrogel and its application in diabetic wound repair. Background Art

[0002] Diabetes, a metabolic disorder caused by excessive blood glucose or insufficient insulin secretion, has become one of the most prominent global public health threats. Current treatment options for diabetic wounds include: 1) basic care, such as blood glucose control, exercise guidance, and medication care; and 2) restorative care, such as debridement, revascularization, local decompression, hyperbaric oxygen therapy, electrical stimulation, negative pressure wound therapy, antibiotic infection treatment, skin grafting, and wound dressings. All treatments aim to improve the wound's regenerative microenvironment, promote tissue regeneration, and achieve wound healing. Therefore, autologous and allogeneic skin grafts remain the primary clinical treatments for wound healing, but they face challenges such as limited access to resources, secondary injury, immune rejection, and storage difficulties. Based on the principles and technologies of regenerative medicine, wound repair materials (such as scaffolds, nanofibers, films, and hydrogels) prepared from modified natural or synthetic polymers can be endowed with biological functions such as promoting angiogenesis and tissue regeneration, anti-inflammatory, and antibacterial properties, thereby meeting diverse clinical needs and improving clinical efficacy. Therefore, in response to the clinical treatment needs of diabetic trauma, it is very important to construct new multifunctional wound repair materials to improve the treatment of diabetic trauma.

[0003] Growth factors and drugs are released in large quantities in a short period of time, which does not match the continuous and gradient signals required for wound repair. They lack physiological spatial targeting and cell specificity, making it difficult to maintain effective concentrations locally. This non-directional release pattern leads to low growth factor utilization and may cause off-target effects. How to achieve "on-demand release" of growth factors and drugs to maximize their therapeutic effects is one of the currently unresolved problems. Ordinary hydrogels as local drug sustained-release systems mainly rely on the diffusion of the drug itself and the degradation of the material to release the drug. This release lacks specificity and controllability, and cannot achieve "on-demand release".

[0004] Smart responsive hydrogels are a new type of hydrogel that can sense external environmental stimuli such as temperature, pH, light, electricity, and biological enzymes, and are one of the important directions for the development of modern high-tech materials. Smart bioresponsive hydrogels can be designed as responsive drug carriers that can "sense" the surrounding physiological environment and release the therapeutic drugs encapsulated therein to highly specific targets on demand. Exogenous stimulus-responsive hydrogels, such as magnetic responsiveness and photodynamic responsiveness, are characterized by the need for in vitro auxiliary magnetic fields or in vitro light sources, while endogenous stimulus-responsive hydrogels use internal body signals such as pH, enzymes, etc. as stimulus sources, do not require the aid of other medical instruments, and have feedback and balance systems similar to those of organisms. The characteristics of MMP-2 imbalance in diabetic wounds are: excessive activation in the early stage (inflammatory stage), and the high-sugar environment activates MMP-2 prematurely through AGEs-RAGE signaling and oxidative stress (24-48 hours earlier than normal wounds); continuous high expression during the proliferation stage, and polarized macrophages M1 (common in diabetes) continuously secrete IL-1β, stimulating fibroblasts to overproduce MMP-2; activity increases instead of decreases during the remodeling stage, TIMP-1 / 2 expression is insufficient (downregulated in diabetes), and MMP-2 continuously cuts type I collagen, leading to loose scars and re-rupture of the wound; therefore, it is necessary to develop a photothermal-MMP dual-responsive matrix hydrogel with hemostasis, antibacterial and anti-inflammatory, antioxidant properties, and accelerated wound healing. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a photothermal-MMP dual-responsive matrix hydrogel, which can be used as an injectable or applyable functional dressing, adapted to different wound morphologies, and photothermal therapy can be precisely controlled by an external near-infrared light source to reduce systemic toxic side effects; the second technical problem to be solved by the present invention is to provide a preparation method of a photothermal-MMP dual-responsive matrix hydrogel, which has a wide source of raw materials. The preparation method uses MMPDA as a bridge to connect Cur@ZIF-8 and dAMG to form a porous and dispersed three-dimensional network structure, which not only enhances the mechanical strength of the hydrogel, but also facilitates drug sustained release and tissue ingrowth; the third technical problem to be solved by the present invention is to provide the application of the hydrogel in diabetic wound repair.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A method for preparing a photothermal-MMP dual-responsive matrix hydrogel comprises the following steps:

[0008] 1) dHAM was stirred in a rabbit pepsin solution, acetic acid was added and stirred to precipitate and dissolve the ECM components, NaCl was added and centrifuged, and the solution was dialyzed. dHAM was dissolved in acetic acid, the pH was adjusted, methacrylic anhydride was added, and the solution was dialyzed and lyophilized to obtain dHAMMA;

[0009] 2) Cur and zinc nitrate hexahydrate were dissolved in water and mixed with stirring, and then 2-methylimidazole solution was added dropwise. After centrifugation, the mixture was washed and dried to obtain Cur@ZIF-8;

[0010] 3) MMP2 peptide and DA were dissolved in Tris buffer, stirred and polymerized, and then dialyzed and purified to obtain MMPDA;

[0011] 4) After ultrasonicating the Cur@ZIF-8 obtained in step 2), the MMPDA obtained in step 3) was added, and after stirring, the dHAMMA obtained in step 1) and the photoinitiator were added, and a cross-linking reaction was carried out under ultraviolet light to obtain the Cur@ZIF-8@MMPDA / dAMG composite hydrogel.

[0012] Furthermore, in step 1), the mass concentration ratio of dHAM to rabbit pepsin solution is 10:1.

[0013] Furthermore, in step 1), the concentration of acetic acid is 0.5M.

[0014] Furthermore, in the step 1), the pH is adjusted to 8-9.

[0015] Furthermore, in the step 2), the mass ratio of Cur to zinc nitrate hexahydrate is 1:30.

[0016] Furthermore, in step 3), the mass ratio of MMP2 peptide to DA is 1:2.

[0017] Furthermore, in the step 3), the stirring polymerization time is 24 hours.

[0018] Furthermore, the method for preparing the photothermal-MMP dual-responsive matrix hydrogel prepares the photothermal-MMP dual-responsive matrix hydrogel.

[0019] Furthermore, the photothermal-MMP dual-responsive matrix hydrogel is used in the repair of diabetic wounds.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) This invention achieves "on-demand release" of drugs through a dual response mechanism of photothermal and MMP2 enzymes. The photothermal effect can quickly heat up and sterilize the infected area, while the MMP2 enzyme response targets abnormal enzyme activity during the inflammatory phase of the wound, continuously releasing the drug to meet the dynamic needs of each stage of wound repair.

[0022] (2) The present invention releases zinc ions through the degradation of ZIF-8 nanoparticles in an acidic environment, destroys the bacterial cell membrane, and enhances the permeability of the biomembrane in combination with the photothermal effect, thereby synergistically killing drug-resistant bacteria; polydopamine (PDA) is used as a photothermal agent, which not only has the advantages of simple synthesis, good biocompatibility, and high photothermal conversion efficiency, but also the multifunctional "encapsulation" of PDA can achieve stable and in situ release of drugs.

[0023] (3) The present invention loads Cur on ZIF-8 to form Cur@ZIF-8, and then constructs a Cur@ZIF-8@MMPDA / dAMG dual drug delivery system through MMPDA medium, forming a photothermal antibacterial, anti-inflammatory, and pro-regenerative multimodal therapy for the acidic environment of the wound, thereby accelerating wound healing.

[0024] (4) The hydrogel provided by the present invention has a simple preparation process and readily available raw materials. It can be used as an injectable or applyable functional dressing, which is adaptable to different wound morphologies. In addition, photothermal therapy can be precisely controlled by an external near-infrared light source to reduce systemic toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Characterization of the raw materials of this application; wherein, Figure A is the XRD diffraction pattern of ZIF, Figure B is the SEM image of ZIF, Figure C is the SEM image of PDA, and Figure D is the TEM image of PDA;

[0026] Figure 2 SEM image of the composite hydrogel prepared in this application;

[0027] Figure 3 This is a diagram for evaluating the photothermal performance of the composite hydrogel prepared in this application;

[0028] Figure 4 Figure 1 is a cell compatibility graph of the composite hydrogel prepared in this application; Figure A is a cell viability detection graph, and Figure B is a cell migration graph;

[0029] Figure 5 Graph showing the antibacterial performance of the composite hydrogel prepared in this application; Graph A is a plate count graph; Graph B is a bacterial viability detection graph. DETAILED DESCRIPTION

[0030] The present invention will be further illustrated below with reference to specific examples. The examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0031] Example 1

[0032] A method for preparing a photothermal-MMP dual-responsive matrix hydrogel comprises the following steps:

[0033] (1) Preparation of dAMG hydrogel: 10 mg / mL decellularized amniotic membrane (dHAM) was stirred in a 1 mg / mL New Zealand rabbit pepsin solution, 0.5 M acetic acid was added, and the mixture was stirred at room temperature for 48 h to precipitate and dissolve the ECM components. 20% w / v NaCl was added and centrifuged at 3000 rpm for 15 min at 4°C, followed by dialysis for two days. 10 mg / mL dHAM was dissolved in 0.5 M acetic acid and the pH was adjusted to 8-9 with NaOH. After dissolution, 4 wt % methacrylic anhydride was added and reacted for 24 h. The mixture was then dialyzed in a 3500 molecular weight dialysis bag for two days and then freeze-dried to obtain dHAMMA.

[0034] (2) Preparation of Cur@ZIF-8: 5 mg of curcumin (Cur) and 150 mg of zinc nitrate hexahydrate were dissolved in 5 mL of water, and the mixture was stirred for 5 min. Then, 10 mL of 2-methylimidazole solution was gradually added to the mixture, and the mixture was stirred for another 15 min. After centrifugation, the mixture was washed three times with a mixture of H2O and methanol, and then dried under vacuum to obtain Cur@ZIF-8.

[0035] (3) Preparation of MMPDA: 5 mg of MMP2 peptide and 10 mg of DA were dissolved in Tris buffer and stirred for polymerization for 24 h. The peptide was embedded in PDA via Michael addition or Schiff base reaction and purified by dialysis (to remove free peptide) to obtain MMPDA.

[0036] (4) Preparation of Cur@ZIF-8@MMPDA / dAMG: Cur@ZIF-8 was ultrasonicated for 5 min, and then MMPDA was added. After stirring evenly, dHAMMA and PDA were added. The cross-linking reaction was carried out under ultraviolet light to obtain Cur@ZIF-8@MMPDA / dAMG composite hydrogel.

[0037] The composite hydrogel material prepared in Example 1 was characterized.

[0038] 1. Scanning electron microscope detection

[0039] Depend on Figure 1 The XRD diffraction pattern of ZIF shows that it has high crystallinity, and SEM shows that the particle size is about 1 μm; the SEM of PDA shows that it is uniform nano-scale spheres, and TEM shows that its size is about 250 nm.

[0040] Depend on Figure 2 From the SEM image of the composite hydrogel, it can be seen that loading ZIF has no effect on the morphology of the hydrogel. The hydrogel exhibits a porous and dispersed structure, which is conducive to material degradation and moisture transfer.

[0041] 2. Photothermal performance measurement

[0042] The prepared Cur@ZIF-8@MMPDA / dAMG composite hydrogel was placed in a centrifuge tube and irradiated with 808 nm NIR laser (laser power of 1.0 W / cm 2 ) for 10 min. At every 30 s interval, a near-infrared thermal imager (Fluke, Ti400) was used to record the temperature changes of solutions with different concentrations and to take near-infrared images. Sterile deionized water was used as the control group under the same conditions. Figure 3 As shown, it was found that the photothermal group had a better warming effect than the control group, thereby improving the bactericidal efficacy.

[0043] 3. Determination and evaluation of antibacterial activity

[0044] (1) Bacterial strains and cell culture

[0045] Gram-negative bacteria Escherichia coli (ATCC 25922, E. coli) and Gram-positive bacteria vancomycin-intermediate-resistant Staphylococcus aureus (Mu50) were selected for testing. A single monoclonal colony was picked from a solid agar plate and inoculated into the corresponding broth medium, then cultured in a shaking incubator (200 rpm) at 37°C until it entered the logarithmic phase, and the bacterial suspension was then centrifuged for 5 minutes. After washing three times with PBS, the bacterial suspension was resuspended in PBS and then diluted to 1.5×108 CFU / mL for further use. Cytotoxicity experiments were performed using human keratinocytes (HaCaT cells). HaCaT cells were cultured in Dulbecco's modified MEM medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0046] (2) In vitro antibacterial activity assay

[0047] Mu50 and E. coli were selected as representative Gram-positive and Gram-negative bacteria for the experiment. The bacterial concentration was diluted to 1.5 × 10 6 CFU / mL, added to 96-well plates. After the above-mentioned treatment, the bacterial mixture was cultured in a constant temperature incubator at 37°C for 2 hours. The NIR light group was irradiated with 808nm laser (1.0W / cm 2 ) for 10 minutes. After 2 hours of incubation, the bacterial mixture of each group was evenly spread on a solid agar plate and cultured in a constant temperature incubator at 37°C. After overnight incubation, the colonies growing on the agar plate were recorded with a camera, and the number of colonies visible to the naked eye was analyzed using Image J software. Figure 5 The photothermal effect of the plate counting surface of A reduced the number of colonies and had a significant antibacterial effect.

[0048] (3) Bacterial live / dead staining test

[0049] Bacterial viability kit ( Bac Light™ was used to perform bacterial live / dead staining experiments to detect the activity of bacteria after nanomaterial treatment. 8 CFU / mL) were further incubated at 37°C for 2 h, and the illumination group was irradiated with 808 nm NIR (1.0 W / cm 2 ) for 10 minutes. Each group of bacterial mixtures was stained with propidium iodide (PI) and SYTO9 for 30 minutes under dark conditions. The SYTO9 in the kit can label all bacterial cells, including living cells and dead cells, however, PI only labels dead cells. After this, the bacterial solution was washed three times with PBS to wash away excess dye. The fluorescence images of the bacterial samples were recorded using Zeiss CLSM (Zeiss, LSM880). Figure 5 The bacterial activity test of B showed that the fluorescence intensity of bacteria in the photothermal group was lower, and the photothermal effect reduced the bacterial activity.

[0050] 4. Evaluation of biocompatibility indicators of hydrogels

[0051] Fibroblasts in the logarithmic growth phase were randomly divided into three groups. 4 The cells were seeded at a density of 100 μg / L on each hydrogel with a diameter of 15 mm, and the number of cells was characterized by CCK-8 method on the 1st, 3rd, 6th and 9th day respectively to study the biocompatibility of Cur@ZIF-8@MMPDA / dAMG composite hydrogel. Figure 4 As shown in Figure 4, the green fluorescence intensity of cells in each hydrogel group is high, and there is no red fluorescence, indicating that the material has good cell compatibility. Figure 4 B cell migration assays showed that the hydrogels promoted cell migration, and Cur@PDA / dAMG enhanced this effect.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a photothermal-MMP dual-responsive matrix hydrogel, characterized by: The following steps are involved: 1) dHAM was stirred in a rabbit pepsin solution, acetic acid was added and stirred to precipitate and dissolve the ECM components, NaCl was added and centrifuged, and the solution was dialyzed. dHAM was dissolved in acetic acid, the pH was adjusted, methacrylic anhydride was added, and the solution was dialyzed and lyophilized to obtain dHAMMA; 2) Cur and zinc nitrate hexahydrate were dissolved in water and mixed with stirring, and then 2-methylimidazole solution was added dropwise. After centrifugation, the mixture was washed and dried to obtain Cur@ZIF-8; 3) MMP2 peptide and DA were dissolved in Tris buffer, stirred and polymerized, and then dialyzed and purified to obtain MMPDA; 4) After ultrasonicating the Cur@ZIF-8 obtained in step 2), the MMPDA obtained in step 3) was added, and after stirring, the dHAMMA obtained in step 1) and the photoinitiator were added, and a cross-linking reaction was carried out under ultraviolet light to obtain the Cur@ZIF-8@MMPDA / dAMG composite hydrogel.

2. The method for preparing the photothermal-MMP dual-responsive matrix hydrogel according to claim 1, characterized in that: In the step 1), the mass concentration ratio of dHAM to rabbit pepsin solution is 10:

1.

3. The method for preparing the photothermal-MMP dual-responsive matrix hydrogel according to claim 2, characterized in that: In the step 1), the concentration of acetic acid is 0.5M.

4. The method for preparing the photothermal-MMP dual-responsive matrix hydrogel according to claim 1, characterized in that: In the step 1), the pH is adjusted to 8-9.

5. The method for preparing the photothermal-MMP dual-responsive matrix hydrogel according to claim 1, characterized in that: In the step 2), the mass ratio of Cur to zinc nitrate hexahydrate is 1:

30.

6. The method for preparing the photothermal-MMP dual-responsive matrix hydrogel according to claim 1, characterized in that: In the step 3), the mass ratio of MMP2 peptide to DA is 1:

2.

7. The method for preparing the photothermal-MMP dual-responsive matrix hydrogel according to claim 1, characterized in that: In the step 3), the stirring polymerization time is 24 hours.

8. The method for preparing the photothermal-MMP dual-responsive matrix hydrogel according to any one of claims 1 to 7, wherein the photothermal-MMP dual-responsive matrix hydrogel is prepared.

9. Use of the photothermal-MMP dual-responsive matrix hydrogel according to claim 8 in diabetic wound repair.

Images