Oxygen-producing hydrogel for myocardial repair and preparation method thereof

Abstract

This invention belongs to the technical field of hydrogel materials for myocardial repair, specifically relating to an oxygen-generating hydrogel for myocardial repair and its preparation method. The invention involves modifying GelMA with KH570 in PBS buffer to prepare KH570-GelMA, and oxidizing cobalt chloride, strontium chloride, sodium hydroxide, and sodium hypochlorite in aqueous solution to prepare CoOOH-Sr nanosheets. The CoOOH-Sr nanosheets are then mixed with the KH570-GelMA solution and a photoinitiator is introduced for in-situ curing to prepare the oxygen-generating hydrogel. By modifying GelMA with KH570, the crosslinking density is controlled, increasing the hydrogel porosity. The introduction of CoOOH-Sr functional molecules enables controlled oxygen production, alleviating local hypoxia, improving the cell compatibility and bioactivity of the hydrogel, promoting cardiomyocyte proliferation and tissue regeneration, and allowing for the construction of injectable or patch-like hydrogel patches suitable for myocardial infarction models.

Description

Technical Field

[0001] This invention belongs to the technical field of hydrogel materials for myocardial repair, specifically relating to an oxygen-generating hydrogel for myocardial repair and its preparation method. Background Technology

[0002] Currently, hydrogel materials used for myocardial repair mainly include natural polymers (such as gelatin, hyaluronic acid, and chitosan) and their derivatives (such as gelatin methacryloyl (GelMA)). These materials have good biocompatibility, but the following problems exist in practical applications:

[0003] First, the cross-linking methods of traditional GelMA hydrogels (such as UV cross-linking) are not properly controlled, resulting in high cross-linking density and the formation of a dense network with a dense pore structure. This leads to insufficient porosity, which is not conducive to cell migration, nutrient transport, and metabolic waste removal. Second, conventional hydrogel materials lack the function of actively promoting cell proliferation and tissue repair, have limited biological activity, and cannot effectively stimulate myocardial cell regeneration. In addition, the blood supply in the myocardial infarction area is poor, and local hypoxia is severe. Traditional hydrogels lack functional modifications and cannot introduce oxygen-producing components. Furthermore, the material itself has the function of regulating oxygen release, which cannot improve the local microenvironment, resulting in hypoxia and limiting cell survival and functional recovery.

[0004] Therefore, it is necessary to design a hydrogel with high porosity, self-oxygenation, and high bioactivity for myocardial infarction models. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an oxygen-generating hydrogel for myocardial repair and its preparation method. Specifically, the oxygen-generating hydrogel is a γ-methacryloyloxypropyltrimethoxysilane (KH570) modified GelMA hydrogel, in which a cobalt oxide hydroxide-strontium complex (CoOOH-Sr) functional molecule with oxygen-generating function is introduced to improve the porosity and bioactivity of the hydrogel. This hydrogel can be used as a myocardial repair patch for tissue repair and regeneration therapy in myocardial infarction (MI) models. This invention modifies GelMA with KH570 to regulate crosslinking density and increase hydrogel porosity; by introducing the CoOOH-Sr functional molecule, controlled oxygen production is achieved, alleviating local hypoxia, improving the cell compatibility and bioactivity of the hydrogel, and promoting cardiomyocyte proliferation and tissue regeneration; and an injectable or patch-like hydrogel patch can be constructed to suit myocardial infarction models.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a method for preparing an oxygen-generating hydrogel for myocardial repair, comprising the following steps:

[0008] Step S1: Add gelatin methacryloyl groups to preheated PBS buffer, stir in the dark, then add γ-methacryloyloxypropyltrimethoxysilane, react under water bath stirring, dialyze, freeze dry to obtain KH570-GelMA flocculent sponge, store at room temperature in the dark;

[0009] Step S2: Add sodium hydroxide aqueous solution to the mixed aqueous solution of cobalt chloride and strontium chloride, sonicate in a water bath at room temperature, then add sodium hypochlorite solution as an oxidant, sonicate in a water bath to cause co-precipitation and oxidation reaction, and obtain CoOOH-Sr nanosheet suspension. Centrifuge, dry, grind to obtain CoOOH-Sr nanosheet powder.

[0010] Step S3: Prepare a KH570-GelMA solution by mixing KH570-GelMA flocculent sponge with PBS buffer. Add CoOOH-Sr nanosheet powder to the KH570-GelMA solution, sonicate, add photoinitiator under light-protected conditions, shake or stir to obtain a mixture, and then solidify in situ under ultraviolet light to form a porous oxygen-generating hydrogel.

[0011] Further, in step S1, the preheating temperature is 40-50℃, the stirring time in the dark is 30-60 min, the water bath stirring temperature is 40-50℃, and the reaction time is 2-4 h.

[0012] Further, in step S1, the ratio of gelatin methacryloyl group to γ-methacryloyloxypropyltrimethoxysilane is 3-5 g / ml.

[0013] Furthermore, in step S1, the molecular weight cutoff for dialysis is 8000-14000 Da, and the dialysis time is 3-5 days.

[0014] Further, in step S2, the molar ratio of cobalt chloride, strontium chloride, sodium hydroxide and sodium hypochlorite is 95-100:1-5:280-300:450-500.

[0015] Furthermore, in step S2, the time for room temperature water bath ultrasound is 1-2 min, and the time for water bath ultrasound is 5-10 min.

[0016] Further, in step S3, the ultrasound time is 10-20 min, and the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP).

[0017] Further, in step S3, the mass ratio of the CoOOH-Sr nanosheet powder to the initiator is 1-2:3-5, and the amount of CoOOH-Sr nanosheet powder and KH570-GelMA is controlled such that the mass ratio of the CoOOH-Sr nanosheet powder to the gelatin methacryloyl group is 1-10:50-100.

[0018] Further, in step S3, the in-situ curing method involves injecting the mixture into a mold or directly dripping / injecting it into the myocardial defect model area. The ultraviolet light has a wavelength of 405 nm and an intensity of 10-30 mW / cm². 2 The irradiation time is 15-60 seconds.

[0019] A second aspect of the present invention provides an oxygen-generating hydrogel for myocardial repair prepared by the above-described preparation method.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) This invention modifies the cross-linking structure of the hydrogel by modifying it with KH570 in GelMA, thereby increasing the porosity and facilitating cell migration and material exchange.

[0022] (2) The present invention introduces CoOOH-Sr functional molecules into KH570-GelMA to enable the hydrogel to produce oxygen and improve the hypoxic microenvironment of the myocardial infarction model.

[0023] (3) This invention improves the activity of biomaterials by releasing strontium ions from the introduced CoOOH-Sr functional molecule in synergy with high porosity and oxygen production function, thereby promoting cell proliferation and tissue regeneration.

[0024] (4) The hydrogel material of the present invention has good plasticity and controllable cross-linking, and can be used to make myocardial repair patches by injection or patch, which is suitable for myocardial infarction models. Attached Figure Description

[0025] Figure 1 SEM images of GelMA hydrogel and modified KH570-GelMA hydrogel.

[0026] Figure 2 In the image, A represents the transmission electron microscope (TEM) morphology of the CoOOH-Sr nanosheet powder. Figure 2 In the image, B represents the high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image and its corresponding EDS elemental mapping.

[0027] Figure 3 Verification of the oxygen generation capacity of CoOOH-Sr nanosheet powder.

[0028] Figure 4 This is a scanning electron microscope (SEM) image of the oxygen-producing hydrogel after lyophilization (scale bar: 100 μm).

[0029] Figure 5 Comparison of reactive oxygen species (ROS) in cells after oxygen-generating hydrogel treatment, GelMA hydrogel treatment, and no treatment following reoxygenation.

[0030] Figure 6 This figure shows the tissue repair evaluation results of an in vivo rat model of myocardial infarction (MI). Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0033] Example 1: Preparation and characterization of oxygen-generating hydrogels

[0034] Step S1: Slowly add 0.5 g of GelMA to 10 mL of PBS buffer (pH=7.4) preheated to 50°C. Stir magnetically for 45 minutes in the dark until the GelMA is completely dissolved, forming a homogeneous and transparent solution. Slowly add 100 μL of KH570 to the GelMA solution using a pipette. Place the reaction system in a 50°C water bath and react continuously with magnetic stirring (approximately 500 rpm) for 4 hours. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 11000 Da and dialyze in deionized water for 4 days to remove unreacted KH570. Then, freeze-dry to obtain KH570-GelMA flocculent sponge, and store at room temperature in the dark for later use. Prepare a 5% KH570-GelMA solution using PBS buffer (pH=7.4). The SEM image of the KH570-GelMA flocculent sponge compared to the unmodified GelMA hydrogel is shown below. Figure 1 As shown in the figure, the modified hydrogel has larger pores.

[0035] Step S2: Accurately measure 9.7 mL of cobalt chloride aqueous solution (CoCl2, 10 mM) and 0.3 mL of strontium chloride aqueous solution (SrCl2, 10 mM) and mix them in a reaction vessel. Then, add 300 μL of sodium hydroxide aqueous solution (NaOH, 1 M) to this mixture and sonicate in a water bath for 2 minutes at room temperature to ensure thorough mixing. Quickly inject 500 μL of sodium hypochlorite solution (NaClO, 0.9 M) as an oxidant into the homogenized precursor solution. After addition, keep the reaction system undisturbed and continue sonication in a water bath for 5 minutes. During this time, co-precipitation and oxidation reactions occur in the solution, generating a CoOOH-Sr nanosheet suspension. After the reaction is complete, transfer the reaction solution to a centrifuge tube and centrifuge at 10,000 rpm for 10 minutes. Discard the supernatant and resuspend the precipitate at the bottom in deionized water. Repeat the "centrifugation-resuspending and washing" step three times to thoroughly remove unreacted ions and free impurities. Finally, place the collected purified precipitate in a 60°C vacuum drying oven overnight (or freeze-dry it), and grind it to obtain pure CoOOH-Sr nanosheet powder, which appears as follows under an electron microscope: Figure 2 As shown, (a) is a transmission electron microscope (TEM) image, with the left side showing a low-magnification image (scale bar 200 nm) and the right side showing a high-magnification image (scale bar 50 nm) within the dashed box, visually demonstrating the nanosheet-like microstructure and agglomeration of the powder. (b) The first image on the left is a HAADF (high-angle annular dark field) image, showing the dark field morphology of a single nanosheet cluster; the images on the right are the corresponding energy-dispersive X-ray spectroscopy (EDS) elemental mapping maps, clearly demonstrating the uniform distribution of cobalt (Co, red), strontium (Sr, cyan), oxygen (O, yellow), and carbon (C, blue) elements in the nanosheets (Merge figure shows the multi-element overlap effect).

[0036] The oxygen generation capacity verification diagram of CoOOH-Sr nanosheet powder is shown below. Figure 3 As shown, the left tube contained only a certain concentration and volume of hydrogen peroxide (H2O2) solution. The right tube contained an equal volume of H2O2 solution, followed by a measured amount of CoOOH-Sr nanosheet powder. After allowing the reaction to proceed at room temperature for a period of time, macroscopic images of both tubes were taken (with local magnification to clearly observe the bubble state). The solution on the left was very calm, with almost no bubbles produced; while the right tube containing CoOOH-Sr exhibited vigorous bubble turbulence, forming a thick foam layer. The CoOOH-Sr nanosheets here demonstrated strong catalase-like activity. This image strongly demonstrates the excellent catalytic decomposition and oxygen production capabilities of the CoOOH-Sr nanosheets.

[0037] Step S3: Prepare a 5% KH570-GelMA solution using PBS buffer (pH=7.4) with the KH570-GelMA flocculent sponge obtained in Step S1. Weigh 10 mg of CoOOH-Sr nanosheet powder and add it to the KH570-GelMA solution. Sonicate the solution for 20 minutes to ensure uniform dispersion, obtaining the composite precursor solution. Under light-protected conditions, add 25 mg of photoinitiator LAP to the mixture and gently shake or stir for 5 minutes to ensure complete dissolution. Inject the mixture into a mold or directly drop / inject it into the myocardial defect model area. Use a 405 nm ultraviolet light source at 30 mW / cm². 2 When exposed to light intensity for 40 seconds, the solution rapidly undergoes a free radical polymerization reaction, cross-linking and solidifying to form a porous hydrogel, such as... Figure 4 As shown in the image, the color plot on the right is an energy-dispersive X-ray spectroscopy (EDS) elemental mapping based on this field of view, displaying the spatial distribution of six elements: C (carbon), N (nitrogen), O (oxygen), Sr (strontium), Si (silicon), and Co (cobalt). The SEM image reveals a typical, interconnected three-dimensional porous network structure within the hydrogel. This porous structure is highly conducive to water retention, nutrient exchange, cell ingrowth, and the outward diffusion and release of generated oxygen. The EDS elemental mapping shows a dense distribution of conventional elements representing the hydrogel matrix (C, N, O, etc.); more importantly, the unique elements Co (cobalt) and Sr (strontium), representing the functional nanosheets, exhibit a uniform scattered distribution throughout the gel framework. This demonstrates that CoOOH-Sr nanosheets have been successfully and uniformly dispersed within the hydrogel matrix, and that the addition of the nanomaterials has not disrupted the hydrogel's original excellent porous network structure.

[0038] Test Example 1: In vitro test to alleviate oxidative stress and promote cell viability

[0039] (1) Experimental materials

[0040] 1) Cells: Neonatal rat cardiomyocytes were isolated from SD rats aged 1 to 3 days. Hearts were rapidly harvested, rinsed in ice-cold PBS, and minced into 1 mm pieces. 3Tissue blocks were collected. The tissue blocks were placed in a solution containing 0.12 mg / ml trypsin and 0.08 mg / ml collagenase and subjected to continuous enzymatic digestion at 37°C (6 minutes per cycle). After each cycle, the supernatant was collected into 50 ml conical centrifuge tubes and centrifuged at 1100 rpm for 5 minutes. The cell pellet was then resuspended in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The digestion process was repeated for 6 cycles. The combined cell suspension was then filtered through a 120 μm sterile filter, seeded into 10 cm culture dishes, and incubated at 37°C and 5% CO2 for 2 hours to promote fibroblast adhesion using differential adhesion. Finally, unattached cardiomyocytes were collected, counted, and seeded at the desired density into gelatin-coated culture plates and maintained in complete DMEM medium.

[0041] 2) Samples: The oxygen-producing hydrogel and the cells after hypoxia-reoxygenation were co-cultured in a chamber.

[0042] (2) Experimental methods

[0043] 1) Group processing

[0044] Cells were subjected to hypoxia treatment (1% O2, 5% CO2, 94% N2) for 6 hours, followed by reoxygenation for 24 hours. Cells were randomly divided into three groups: a control group (H / R), a GelMA group, and a GelMA / KH570 / CoOOH-Sr group. At the start of treatment, the GelMA group and the GelMA / KH570 / CoOOH-Sr group were co-cultured with hydrogels. After 24 hours of culture, analysis was performed.

[0045] (3) Experimental results

[0046] Analysis results as follows Figure 5 As shown, the H / R group exhibited very strong green fluorescence, indicating that the ischemic-reoxygenated environment led to the production of a large amount of reactive oxygen species (ROS), causing severe oxidative stress. The GelMA group still showed significant green fluorescence, indicating that the GelMA hydrogel framework alone does not have the ability to scavenge ROS. In the GelMA / KH570 / CoOOH-Sr group, the green fluorescence was significantly weakened, almost disappearing. This demonstrates that the oxygen-generating composite hydrogel loaded with CoOOH-Sr can significantly scavenge intracellular reactive oxygen species (ROS), significantly alleviating cellular oxidative stress damage caused by hypoxia / reoxygenation (H / R). Combined with the previous oxygen production diagram, it can be inferred that the material not only generates oxygen to alleviate hypoxia but also consumes / removes harmful peroxides (ROS), playing a dual role in protecting cells.

[0047] Test Example 2: Evaluation of tissue repair in an in vivo rat model of myocardial infarction (MI)

[0048] (1) Experimental methods (brief description)

[0049] A rat model of myocardial infarction (MI) was established, and rats were randomly divided into five groups: sham operation group (Sham), myocardial infarction group (MI), simple hydrogel group (GelMA), modified hydrogel group (GelMA / KH570), and oxygen-generating composite hydrogel group (GelMA / KH570 / CoOOH-Sr). After a period of treatment, heart tissue from each group of rats was sectioned and stained with H&E, Masson's trichrome, and Sirius red to assess morphological changes and the degree of fibrosis in the myocardial tissue.

[0050] (2) Experimental results and analysis

[0051] To verify the promoting effect of oxygen-producing hydrogel on tissue repair after myocardial infarction, histological evaluation was performed on heart sections from each group.

[0052] Tissue morphology and ischemic area assessment (H&E staining): For example Figure 6 H&E staining results showed that the left ventricular wall thickness was normal and the cardiomyocytes were neatly arranged in the Sham group rats; while the MI group showed severe left ventricular wall thinning and extensive tissue necrosis. Although the GelMA and GelMA / KH570 groups alleviated ventricular wall thinning to some extent, the effect was limited. In contrast, the GelMA / KH570 / CoOOH-Sr oxygen-generating hydrogel group significantly preserved the thickness of the left ventricular wall. Quantitative analysis further confirmed that the ischemic area in the oxygen-generating hydrogel group was significantly lower than that in the MI group and other hydrogel control groups, indicating its excellent ability to reduce myocardial injury.

[0053] Assessment of Collagen Deposition and Fibrosis (Masson and Sirius Red Staining): Extensive proliferation of fibroblasts after myocardial infarction leads to irreversible tissue fibrosis. Masson staining (staining collagen blue) and Sirius red staining (staining collagen fibers reddish-yellow) results showed abundant collagen deposition in cardiac sections from the MI group, forming significant scar tissue. The degree of fibrosis was slightly reduced in the GelMA and GelMA / KH570 groups, but large areas of scarring remained. In the GelMA / KH570 / CoOOH-Sr treatment group, the blue and reddish-yellow stained areas were significantly reduced. Correspondingly, quantitative statistics showed that the collagen volume and fibrotic area in this oxygen-producing hydrogel group were reduced to extremely low levels, with statistically significant differences compared to other intervention groups (p < 0.05 or higher).

[0054] (3) Experimental results

[0055] The above histological evaluation results are as follows Figure 6 As shown, it is fully demonstrated that the GelMA / KH570 / CoOOH-Sr oxygen-generating hydrogel can effectively and continuously supply oxygen, rescue ischemic cardiomyocytes, significantly reduce the infarct area, and strongly inhibit ventricular remodeling and tissue fibrosis after myocardial infarction, exhibiting extremely excellent in vivo tissue repair capabilities.

[0056] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing an oxygen-generating hydrogel for myocardial repair, characterized in that, Includes the following steps: Step S1: Add gelatin methacryloyl groups to preheated PBS buffer, stir in the dark, then add γ-methacryloyloxypropyltrimethoxysilane, react under water bath stirring, dialyze, freeze dry to obtain KH570-GelMA flocculent sponge, store at room temperature in the dark; Step S2: Add sodium hydroxide aqueous solution to the mixed aqueous solution of cobalt chloride and strontium chloride, sonicate in a water bath at room temperature, then add sodium hypochlorite solution as an oxidant, sonicate in a water bath to cause co-precipitation and oxidation reaction, and obtain CoOOH-Sr nanosheet suspension. Centrifuge, dry, grind to obtain CoOOH-Sr nanosheet powder. Step S3: Prepare a KH570-GelMA solution by mixing KH570-GelMA flocculent sponge with PBS buffer, add CoOOH-Sr nanosheet powder to the KH570-GelMA solution, sonicate, add photoinitiator under light-protected conditions, shake or stir to obtain a mixture, and then solidify in situ under ultraviolet light to form a porous oxygen-generating hydrogel. In step S1, the preheating temperature is 40-50℃, the stirring time in the dark is 30-60 min, the water bath stirring temperature is 40-50℃, and the reaction time is 2-4 h; In step S1, the ratio of gelatin methacryloyl group to γ-methacryloyloxypropyltrimethoxysilane is 3-5 g / ml.

2. The method for preparing an oxygen-generating hydrogel for myocardial repair according to claim 1, characterized in that, In step S1, the molecular weight cutoff for dialysis is 8000-14000 Da, and the dialysis time is 3-5 days.

3. The method for preparing an oxygen-generating hydrogel for myocardial repair according to claim 1, characterized in that, In step S2, the molar ratio of cobalt chloride, strontium chloride, sodium hydroxide and sodium hypochlorite is 95-100:1-5:280-300:450-500.

4. The method for preparing an oxygen-generating hydrogel for myocardial repair according to claim 1, characterized in that, In step S2, the time for ultrasonication in a room temperature water bath is 5-10 minutes.

5. The method for preparing an oxygen-generating hydrogel for myocardial repair according to claim 1, characterized in that, In step S3, the ultrasound duration is 10-20 min, and the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

6. The method for preparing an oxygen-generating hydrogel for myocardial repair according to claim 1, characterized in that, In step S3, the mass ratio of the CoOOH-Sr nanosheet powder to the photoinitiator is 1-2:3-5.

7. The method for preparing an oxygen-generating hydrogel for myocardial repair according to claim 1, characterized in that, In step S3, the in-situ curing method involves injecting the mixture into a mold or directly dripping / injecting it into the myocardial defect model area. The ultraviolet light has a wavelength of 405 nm and an intensity of 10-30 mW / cm². 2 The irradiation time is 15-60 seconds.

8. An oxygen-producing hydrogel for myocardial repair prepared by the preparation method according to any one of claims 1-7.

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