Preparation and application of double-layer hydrogel microneedle patch with ROS response

Abstract

The invention provides an anti-tissue adhesion hydrogel microneedle patch with ROS response as well as a preparation method and application thereof, and belongs to the field of myocardial repair materials. According to the inner-layer hydrogel disclosed by the invention, polycyclodextrin (p beta-CD) is prepared and is subjected to primary crosslinking with gelatin-adamantane (G-Ad) through subject-object recognition, and then a thioketal bond bridge crosslinking agent (TK-NHS) is synthesized and is subjected to secondary crosslinking with residual amino groups on the G-Ad, so that the hydrogel is formed. Succinimide (NHS) at the two ends of the synthesized cross-linking agent is not only cross-linked with amino groups on gelatin-adamantane, but also can be grafted with protein cargoes containing free amino groups, and responds to ROS to release drugs, when hydrogel is prepared into microneedles, hydrophilic groups exposed by dehydration can form a large number of hydrogen bonds with active groups on myocardium, and the hydrogel can be used for preparing the microneedles. Therefore, the adhesion effect is achieved. In actual in-vivo application, as the outer layer of the microneedle absorbs chest body fluid, hydrogen bonds are formed in the hydrogel, and unnecessary inflammation caused by adhesion with chest tissues is avoided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of myocardial repair materials, and particularly relates to preparation and application of a ROS-responsive tissue adhesion-preventing hydrogel microneedle patch. BACKGROUND

[0002] Cardiovascular diseases, especially MI, are the leading cause of death worldwide. When myocardial infarction occurs, the hypoxia of myocardium leads to mitochondrial dysfunction. In the hypoxic environment, the respiratory chain of mitochondria is inhibited, resulting in that oxygen cannot be completely reduced to generate ATP, and a large amount of ROS is generated. ROS exacerbates the damage of myocardial cells by inducing lipid peroxidation, protein oxidation, DNA damage and the like. Moreover, ROS is a key mediator of inflammatory response, and can activate various signaling pathways and induce the release of inflammatory factors. Such inflammatory response will lead to more cell necrosis after myocardial infarction, and inhibit tissue repair. In addition, long-term accumulation of ROS will affect the structure and function of the heart, leading to ventricular dilation, thin wall and other cardiac remodeling phenomena, thereby increasing the risk of heart failure.

[0003] The thioacetone bond can react with various active oxygen (ROS), such as hydroxyl radicals (·OH), hydrogen peroxide (H2O2) and the like. By directly reacting with ROS, ROS is effectively removed, and oxidative stress is reduced, so as to achieve effective treatment purposes. In addition, the hydrogel patch will be undesirably adhered to other tissues due to its adhesion, and additional inflammation will be generated. Therefore, a ROS-responsive hydrogel microneedle patch is proposed, so that the ROS-responsive hydrogel microneedle patch responds to ROS and removes excess ROS, and a treatment mode of resisting tissue adhesion becomes a problem to be solved. SUMMARY

[0004] In view of the above defects of the prior art, the purpose of the present application is to provide a preparation and application of a ROS-responsive tissue adhesion-preventing hydrogel.

[0005] A ROS-responsive hydrogel formed by a first crosslinking agent and a second crosslinking agent and a crosslinking product, and the crosslinking structure formula of the ROS-responsive hydrogel is: ; A preparation method of a ROS-responsive hydrogel, comprising the following preparation steps: S01, dissolving 1-adamantane acetic acid, EDC and NHS in a mixed solution of pure water and DMSO, activating for 6 hours, and reserving; S02, dissolving gelatin, EDC and NHS in a mixed solution of pure water and DMSO, adding ethylenediamine, reacting for 24 hours, then adding the mixed solution of S01 dropwise, reacting for 45 hours, dialyzing for at least 3 days, and then freeze-drying to obtain a crosslinking product; S03, 3-mercaptopropionic acid, acetone were mixed in a flask, a certain amount of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 3 h. After washing with n-hexane and water for several times and vacuum drying, a crystalline powder was obtained. The powder was dissolved with EDC and NHS in DMSO, and the mixed solution was poured into pure water after overnight reaction, filtered and washed with pure water for several times, and then vacuum dried to obtain the first crosslinking agent; S04, β-cyclodextrin was dissolved in sodium hydroxide solution, and epichlorohydrin was added. After reaction for 3 h, the reaction was terminated by adding acetone, and the oil was obtained after drying under reduced pressure. The pH of the product was adjusted to 10, and then dialysis was carried out for at least 3 days, followed by freeze-drying to obtain the second crosslinking agent.

[0006] S05, the crosslinking product and the second crosslinking agent were dissolved in PBS solution. The first crosslinking agent was dissolved in DMSO. After mixing the three solutions, the solution was poured into a microneedle mold, vacuum defoaming for several times, and then placed in an oven for drying and dehydration. The precursor solution was supplemented and dried for 6 h to obtain a microneedle patch.

[0007] Preferably, the ratio of 1-adamantane acetic acid, EDC and NHS in S01 is 0.48 g 1-adamantane acetic acid: 3 mmol EDC: 3 mmol NHS.

[0008] Preferably, the ratio of gelatin, EDC, NHS and ethylenediamine in S02 is 1 g gelatin: 3 mmol EDC: 3 mmol NHS: 3 mmol ethylenediamine.

[0009] Preferably, the ratio of 3-mercaptopropionic acid, acetone and trifluoroacetic acid in S03 is 1.14 g 3-mercaptopropionic acid: 0.29 g acetone: 20 μL trifluoroacetic acid.

[0010] Preferably, the ratio of the crystalline powder, NHS and EDC in S03 is 300 mg crystalline powder: 330 mg NHS: 687 mg EDC Preferably, the concentration of sodium hydroxide in S04 is 30%.

[0011] Preferably, the ratio of β-cyclodextrin, sodium hydroxide solution, epichlorohydrin and acetone in S04 is 5 g β-cyclodextrin: 15 mL sodium hydroxide solution: 3.5 mL epichlorohydrin: 25 mL acetone.

[0012] Preferably, the amount of the first crosslinking product in S05 is 300 μL of a 6.25% (w / w) solution, the amount of the first crosslinking agent is 100 μL of a 12.5% (w / w) solution, and the amount of the second crosslinking agent is 10 μL of a 10% (w / w) solution.

[0013] Preferably, the oven temperature in S05 is 37°C.

[0014] Application of a ROS-responsive hydrogel in the preparation of ROS-responsive myocardial repair compositions.

[0015] Preferably, it is applied in the field of myocardial repair.

[0016] The beneficial effects of this invention are: The hydrogel prepared by this method has a thioketone bond in its structure that can directly react with ROS, thus making the hydrogel prepared by this method ROS responsive, able to remove excess ROS, and inhibit the polarization of M0 macrophages to M1 macrophages, achieving anti-inflammatory and antioxidant effects and being used for myocardial repair.

[0017] 2. The hydrogel system developed using this method can prevent adhesion to other pleural tissues when used in vivo. While maintaining tight adhesion to the myocardium, it also prevents tissue adhesion and avoids further unwanted inflammation. Attached Figure Description

[0018] Figure 1 The synthetic route for this hydrogel is shown.

[0019] Figure 2 This is a partial characterization of the hydrogel.

[0020] A) Infrared characterization of each product.

[0021] B) 1H NMR of each product.

[0022] C) Three-dimensional mesh electron microscope image of the hydrogel using SEM. Scale bar: 20 μm.

[0023] D, E) Time scan and frequency scan of the hybrid hydrogel.

[0024] F) SEM images of the microneedle morphology. Scale bar: 200 μm.

[0025] Figure 3 The self-healing and adhesion properties of the hydrogel, as well as the performance of the microneedles, were tested.

[0026] A) Schematic diagram of microneedle fabrication.

[0027] B) Images demonstrating the tensile properties of microneedles.

[0028] C) Microneedles can fit closely to the heart and leave a uniform mark.

[0029] D. Images of hydrogels with different TK-NHS contents dissolving at 37°C.

[0030] Figure 4 This study aimed to test the ROS responsiveness of the hydrogel, its ROS scavenging ability, and its effect on macrophage polarization.

[0031] A) The hydrogel's ability to scavenge 2,2-diphenyl-1-picrylhydrazine (DPPH) and H2O2.

[0032] B, C) Representative fluorescence images and quantitative relationships of DHE and DCFH-DA in cardiomyocytes after hydrogel treatment. Scale bar: 150 μm. (n = 3, ***P < 0.001).

[0033] D) Macrophage polarization flow cytometry analysis after hydrogel treatment (n = 3, ***P < 0.001).

[0034] Figure 5 This is a graph showing the ROS scavenging performance of the hydrogel. Detailed Implementation

[0035] The embodiments of the present invention will be described in detail below. The embodiments described below are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the embodiments described below.

[0036] Preparation of hydrogels 1. Preparation of the first crosslinking product 1.1 Dissolve 0.48 g of 1-adamantaneacetic acid, 3 mmol of EDC, and 3 mmol of NHS in a fusion solution of pure water and DMSO (1:3) and activate for 6 h; 1.2 After dissolving gelatin (1 g), EDC (3 mmol), and NHS (3 mmol) in a fusion solution of pure water:DMSO (1:3), ethylenediamine (3 mmol) was added. After reacting for 24 hours, the solution obtained in 1.1 was added dropwise, and the reaction was carried out for 45 hours. The mixture was dialyzed for at least 3 days and then freeze-dried to obtain the first crosslinking product. 1.3 Mix 3-mercaptopropionic acid (1 g) and acetone (1.4 g) in a flask, add trifluoroacetic acid (20 μL), react at room temperature for 3 h, wash several times with n-hexane and water, and then vacuum dry to obtain crystalline powder. Dissolve the powder (300 mg) with EDC (687 mg) and NHS (330 mg) in 10 mL DMSO, react overnight, pour the mixed solution into pure water to precipitate, filter and wash several times with pure water, and then vacuum dry to obtain the first crosslinking agent; 1.4 Dissolve β-cyclodextrin (5 g) in 15 mL of 30% sodium hydroxide solution, add 3.5 mL of epichlorohydrin, react for 3 h, add 25 mL of acetone to terminate the reaction, dry under reduced pressure to obtain an oily substance, adjust the pH of the product to 10 with 1 mol / L dilute hydrochloric acid, dialyze for at least 3 days, and then freeze dry to obtain the second crosslinking agent.

[0037] 1.5. Prepare 10 μL of 10% (w / w) second crosslinking agent using DMSO, 300 μL of 12.5% ​​(w / w) first crosslinking product using PBS solution, and mix 100 μL of 12.5% ​​(w / w) first crosslinking agent. Pour the resulting precursor solution into a microneedle mold, degas it under vacuum three times, and then dry it in a 37°C oven for dehydration. Add the precursor solution again and continue drying for 6 hours to obtain ROS-responsive microneedle patches.

[0038] Following the steps described in the embodiments of this disclosure, the final product is: .

[0039] II. Characterization and Performance Determination of the Target Hydrogel (1) Rheological testing: Hydrogel samples with a diameter of 1 cm and a height of 1 cm were prepared. The storage modulus (G') and loss modulus (G") of the hydrogel samples were obtained by time scanning (1-200 s) and oscillation frequency scanning (0.1-10 Hz) using a rotational rheometer. The measurement temperature was fixed at 25℃, and the strain value was 1%. In time scanning mode, the G' and G" of the three proportion hydrogel samples were measured within 200 s. With the addition of TK-NHS, the modulus increased accordingly, indicating that the mechanical properties of the hydrogel gradually improved. Figure 2 (D). In frequency scanning mode, G' is consistently significantly higher than G" at frequencies from 0.1 to 10 Hz, indicating the formation of a covalently cross-linked network in the hydrogel, possessing a structure similar to that of an elastic solid. Figure 2 (E).

[0040] (3) SEM morphology detection: Cylindrical hydrogel samples were prepared, lyophilized, sputter-coated with gold, and the three-dimensional network structure of the hydrogel was captured by SEM. Pore structures with pore sizes of tens of micrometers could be observed in the lyophilized hydrogel samples, which is suitable for drug embedding, delivery, and cell growth. Figure 2 (C). After preparing microneedles from hydrogel, gold was sputtered onto the microneedles again, and SEM images were taken of the microneedle surface, revealing a uniform needle-like structure. Figure 2 (F).

[0041] 2. Self-healing property test of hydrogel The use of cyclodextrin and adamantane in the system endows the hydrogel with certain self-healing properties. These properties were assessed through shearing and rhodamine staining. Simply put, the hydrogel was cut into two pieces, one of which was stained with rhodamine for easy observation. The cut surfaces of the two pieces were then brought into contact to observe their self-healing properties. In this study, the self-healing property of the hydrogel was tested by mutual adhesion experiments between two rhodamine-stained and unstained gels. The results showed that one side could be lifted with tweezers within a very short time, causing the other side to move along with it. Figure 3 This is mainly because the hydrogel contains a large number of hydroxyl, amino, and NHS active ester groups, which can quickly cross-link with each other and restore part of the gel network.

[0042] 3. Adhesion test of hydrogel During the microneedle fabrication process, water evaporation exposes numerous hydrogen bonds within the hydrogel, which exhibit suitable adhesion strength to the heart. To elucidate the adhesiveness of this hydrogel, we conducted a series of in vitro and in vivo tests. The hydrogen bond interactions between the gel and the hydroxyl, amino, and carboxyl groups present in cardiac tissue demonstrate good adhesion to the heart. Figure 3 (E). Due to the infiltration of intrapleural fluids into the outer surface of the microneedles, the outer hydration layer exhibits an anti-tissue adhesion effect and will not adhere to surrounding tissues. Figure 3 (Middle F).

[0043] 6. ROS scavenging test of hydrogel The thioketal bonds in this hydrogel break under high ROS conditions, consuming ROS. UV characterization showed that both DPPH and H₂O₂ effectively scavenged (ROS). Figure 5 (A) 7. Biocompatibility testing of hydrogels The viability of cardiomyocytes in hydrogels was further assessed using a live / dead cell staining kit. Calcein AM can cross intact cell membranes and bind to nuclear DNA, producing green fluorescence, while PI can only enter cells with damaged membranes, emitting strong red fluorescence. The combination of Calcein AM and PI can roughly distinguish between live and dead cells. Figure 4 Figure A shows that cardiomyocytes showed very few dead cells within 7 days in our selected hydrogel formulation, indicating that the hydrogel has excellent biocompatibility.

[0044] ROS removal test of hydrogel ROS expression levels in cardiomyocytes were detected using a DHE and DCFH-DA kit. DHE can penetrate the cell membrane and is converted into superoxide anions inside the cell. Oxidation produces fluorescence, which is often used to directly detect superoxide anion levels. After entering the cell, DCFH-DA is hydrolyzed to DCFH by esterases, and then oxidized by intracellular reactive oxygen species (ROS) to generate DCF. The fluorescence intensity of DCF reflects the overall ROS level. Results B and C in Figure 5 show that ROS expression levels were low in cardiomyocytes after hydrogel treatment, indicating that the hydrogel we selected has a ROS scavenging effect.

[0045] Macrophage polarization flow cytometry experiment CD80 is primarily expressed on M1 macrophages and is a marker of immune activation. They possess strong antibacterial and antitumor capabilities and play a crucial role in inflammatory responses. CD206 is primarily expressed on M2 macrophages and is a marker of immunosuppression. It is associated with tissue repair, immune tolerance, and suppression of inflammatory responses. M2 macrophages play an important role in anti-inflammatory responses and tissue repair. Figure 4 B and C flow cytometry analysis and quantitative results showed that the expression of CD80 in macrophages decreased after hydrogel treatment, indicating that hydrogel has the effect of inhibiting macrophage M1 polarization.

[0046] Based on the above analysis and measurement results, the hydrogel of the present invention, composed of G-Ad, TK-NHS, and pβ-CD, exhibits the following characteristics: The thioketone bonds in this hydrogel can respond to ROS cleavage and can scavenge excess ROS, inhibiting M1 macrophage polarization and achieving anti-inflammatory and antioxidant effects for myocardial repair. Furthermore, the synthesized TK-NHS contains NHS active ester groups at both ends, possessing the potential to crosslink loaded protein intermediates and responding to the release of its loaded intermediates in the high ROS environment of infarction. The cyclodextrin cavity can also load suitable hydrophobic drugs, preventing adverse consequences from direct drug release. The hydrogel system prepared by this method can prevent adhesion to other pleural tissues during in vivo application. While maintaining tight adhesion to the myocardium, it also prevents tissue adhesion, avoiding further undesirable inflammation. Therefore, this hydrogel has multiple loading scenarios and possesses advantages such as adhesion, anti-tissue adhesion, excellent biocompatibility, and simple structural composition, promoting inflammation resolution and myocardial repair, and has broad potential clinical application prospects.

[0047] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A ROS-responsive hydrogel patch for preventing tissue adhesion, characterized in that, The cross-linking structure of the hydrogel patch is as follows: ； In this process, G-Ad is the cross-linking product, TK-NHS is the first cross-linking agent, and pβ-CD is the second cross-linking agent; the NHS ends at both ends of TK-NHS are used to perform self-cross-linking with the amino groups on G-Ad.

2. A method for preparing a ROS-responsive hydrogel patch for preventing tissue adhesion, characterized in that, The preparation steps include the following: S01. Dissolve 1-adamantaneacetic acid, EDC, and NHS in a fusion solution of pure water and DMSO, and activate for at least 6 hours, then set aside. S02: After dissolving gelatin, EDC, and NHS in a fusion solution of pure water and DMSO, add ethylenediamine and react for 24 hours. Then add the product of S01 dropwise and react for no less than 45 hours. Dialyze for no less than 3 days and then freeze dry to obtain the crosslinked product G-Ad. S03. Mix 3-mercaptopropionic acid and acetone, then add trifluoroacetic acid, react at room temperature for no less than 3 hours, then wash with n-hexane and water several times and vacuum dry to obtain crystalline powder. Dissolve the crystalline powder with EDC and NHS in DMSO, react overnight to obtain a mixed solution, pour the mixed solution into pure water, filter and wash with pure water several times, then vacuum dry to obtain the first crosslinking agent TK-NHS. S04. After dissolving β-cyclodextrin in sodium hydroxide solution, epichlorohydrin was added and the reaction was carried out for no less than 3 hours. Then, acetone was added to terminate the reaction. After drying under reduced pressure, an oily substance was obtained. The pH of the oily substance was adjusted to 10 and dialyzed for no less than 3 days. Then, it was freeze-dried to obtain the second crosslinking agent pβ-CD. S05. The first crosslinking product G-Ad and the first crosslinking agent TK-NHS are dissolved in PBS solution to obtain the first solution. The second crosslinking agent pβ-CD is dissolved in DMSO to obtain the second solution. The first solution and the second solution are mixed to obtain the precursor solution. The precursor solution is poured into a microneedle mold, vacuum defoamed multiple times, placed in an oven to dry and dehydrate, and then the precursor solution is added again and the product is placed in an oven to dry for no less than 6 hours to obtain the ROS-responsive anti-tissue adhesion hydrogel patch.

3. The method for preparing the ROS-responsive anti-tissue adhesion hydrogel patch according to claim 1, characterized in that, The mass-to-volume ratio of 1-adamantaneacetic acid, EDC, and NHS in step S01 is 0.24 g: 1.5 mmol: 1.5 mmol; the volume ratio of pure water to DMSO in the fusion solution of pure water and DMSO is 1:

3.

4. The method for preparing a ROS-responsive anti-tissue adhesion hydrogel patch according to claim 1, characterized in that, The mass-volume ratio of gelatin, EDC, NHS, and ethylenediamine in step S02 is 1 g : 3 mmol : 3 mmol : 3 mmol; wherein the mass ratio of gelatin to 1-adamantaneacetic acid in step S01 is 1 : 0.

24.

5. The method for preparing a ROS-responsive anti-tissue adhesion hydrogel patch according to claim 1, characterized in that, In step S03, the mass-to-volume ratio of 3-mercaptopropionic acid, acetone, and trifluoroacetic acid is 1 g: 1.4 g: 20 μL; the mass ratio of 3-mercaptopropionic acid to 1-adamantaneacetic acid in step S01 is 1:

1.

6. The method for preparing a ROS-responsive anti-tissue adhesion hydrogel patch according to claim 1, characterized in that, The sodium hydroxide concentration in step S04 is 30%.

7. The method for preparing a ROS-responsive anti-tissue adhesion hydrogel patch according to claim 1, characterized in that, The mass-to-volume ratio of β-cyclodextrin, sodium hydroxide solution, epichlorohydrin, and acetone in step S04 is 5 g : 15 mL : 3.5 mL : 25 mL, wherein the mass ratio of β-cyclodextrin to 1-adamantaneacetic acid in step S01 is 5 :

1.

8. The method for preparing a ROS-responsive anti-tissue adhesion hydrogel patch according to claim 1, characterized in that, In step S05, the first crosslinking product is a 6.25% mass solution, the first crosslinking agent is a 12.5% ​​mass solution, and the second crosslinking agent is a 10% mass solution; the volume ratio of the first crosslinking product, the first crosslinking agent, and the second crosslinking agent is 300:100:10 μL.

9. The method for preparing a ROS-responsive anti-tissue adhesion hydrogel patch according to claim 1, characterized in that, The oven temperature in step S05 is 37°C.

10. The application of a ROS-responsive anti-tissue adhesion hydrogel patch according to any one of claims 1-9, characterized in that, The ROS-responsive anti-tissue-adhesion hydrogel patch is used as a raw material for preparing myocardial repair compositions.

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