Freezing gel microneedle as well as preparation method and application thereof

Abstract

The invention discloses a frozen gel microneedle as well as a preparation method and application thereof, the preparation method comprises the following steps: (1) adding PBS (Phosphate Buffer Solution) into a brown bottle filled with an initiator LAP, and heating and stirring in a water bath at 40-50 DEG C for 15 minutes to obtain an initiator standard solution; (2) adding the initiator standard solution obtained in the step (1) into a brown glass bottle with HAMA, and stirring at room temperature in a dark place for 1 hour to obtain a photo-crosslinked HAMA hydrogel solution; (3) preparing a complete porous gel microneedle mold needle tip loaded with a gene therapy vector; (5) preparing a microneedle mold substrate; (6) freezing the microneedle mold with the formed needle tip and substrate to obtain a frozen gel microneedle array; and (7) demolding the frozen gel microneedle array to obtain the frozen gel microneedle. The obtained microneedle can be used for long-term collaborative treatment of chronic inflammatory diseases through percutaneous delivery of anti-inflammatory genes coded by CeNPs and rAAV2.

Description

Technical Field

[0001] The present invention belongs to the technical field of gel microneedles, and in particular relates to a cryogel microneedle and a preparation method and application thereof. Background Art

[0002] Chronic inflammatory diseases (such as autoimmune diseases, chronic skin inflammation, and fibrotic disorders) are pathological conditions caused by persistent, abnormal activation of the immune system. They are characterized by excessive production of proinflammatory cytokines, immune cell infiltration, and elevated oxidative stress in the local microenvironment. These diseases are often accompanied by persistent inflammation, impaired tissue repair, and progressive organ dysfunction, posing significant challenges to clinical treatment. While existing conventional treatments can manage symptoms to a certain extent, they remain significantly inadequate in terms of long-term efficacy and relapse prevention. In recent years, biomedical treatment strategies for these diseases have undergone a significant shift, from simply controlling symptoms to addressing the root causes of the disease. In this context, gene therapy offers unique advantages due to its precise targeting and long-lasting effects. However, clinical application of this technology faces a common challenge: the transfection efficiency of target genes is significantly reduced in the oxidative stress microenvironment characteristic of chronic inflammatory diseases. This phenomenon has been reported in various chronic inflammatory diseases and represents a key bottleneck in therapeutic efficacy. Therefore, a delivery system that can simultaneously reduce oxidative stress and improve gene transfection efficiency is urgently needed. Summary of the Invention

[0003] To address the shortcomings of the prior art, the present invention provides a cryogel microneedle, its preparation method, and its application. The cryogel microneedles prepared in this invention enable transdermal delivery of antioxidant nanoparticles (CeNPs) and an anti-inflammatory gene encoded by a recombinant adeno-associated virus (rAAV2), addressing the prior art issue of low gene transfection efficiency due to elevated oxidative stress levels in the treatment of chronic inflammatory diseases.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: A method for preparing cryogel microneedles comprises the following steps: Step S1, adding 10 mL of PBS solution to 0.025 g of initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, heating and stirring in a water bath at 40-50 ° C for 15 minutes to obtain a standard solution of initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate with a mass fraction of 0.25%; Step S2: 0.1 g of methacryloylated hyaluronic acid (HAMA) was placed in a brown glass bottle, and 2 mL of the initiator phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt standard solution obtained in step S1 was added to the brown glass bottle. The mixture was stirred in the dark at room temperature for 1 h to obtain a photocrosslinker methacryloylated hyaluronic acid (HAMA) hydrogel solution with a mass fraction of 1%; Step S3, mixing the photocrosslinker methacryloylation hyaluronic acid HAMA hydrogel solution obtained in step S2 with the antioxidant nanozyme CeNPs and the recombinant adeno-associated virus rAAV2, respectively, to obtain 2 μg / mL CeNPs@HAMA hydrogel solution and rAAV2@HAMA hydrogel solution; Step S4: Inject the CeNPs@HAMA hydrogel solution obtained in step S3 into the tip of the microneedle mold, centrifuge to remove bubbles, and cross-link under blue light irradiation for 60 seconds to form a rigid needle tip matrix of the microneedle mold; inject the rAAV2@HAMA hydrogel solution obtained in step S3 into the rear end of the microneedle mold, repeat the above centrifugation and cross-linking steps to form a complete porous gel microneedle mold tip loaded with gene therapy vector; Step S5, injecting pure methacrylated hyaluronic acid (HAMA) solution into the microneedle base area, and repeating the centrifugation and cross-linking steps in step S to form a microneedle mold base; Step S6, freezing the microneedle mold containing the needle tip and the base loaded with the gene therapy vector to obtain a cryogel microneedle array; Step S7: gently demolding the cryogel microneedle array obtained in step S6 to obtain cryogel microneedles.

[0005] Furthermore, the antioxidant nanozyme CeNPs in step S3 is purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0006] Furthermore, in step S3, the mass ratio of the photocrosslinking agent methacryloylation hyaluronic acid HAMA hydrogel solution to the antioxidant nanozyme CeNPs and the recombinant adeno-associated virus rAAV2 is 500:1:1.

[0007] Furthermore, the nucleotide sequence of the anti-inflammatory gene encoded by the recombinant adeno-associated virus rAAV2 in step S3 is shown in SEQ ID NO: 1.

[0008] Furthermore, in step S4, the height of the microneedle body of the microneedle mold is 600 μm, and the distance between the needle tips of the microneedle body is 600 μm.

[0009] Furthermore, in step S4, the tip area of ​​the microneedle mold occupies 2 / 3 of the height of the microneedle body; the centrifugal force of the centrifugation is 1500xg; the intensity of the blue light is 50mW / cm², and the wavelength of the blue light is 405nm.

[0010] Furthermore, the specific process of the freezing treatment in step S6 is: first cooling to -20°C at a rate of 1°C / min and holding for 2 hours, then transferring to -80°C deep freezing for 24 hours, and finally quick freezing with liquid nitrogen.

[0011] Furthermore, the cryogel microneedle obtained in step S7 has a compression modulus of ≥1.5 MPa at room temperature and can withstand the puncture stress of skin tissue with a thickness of 300 μm.

[0012] Furthermore, a cryogel microneedle is prepared based on the above method.

[0013] Furthermore, a cryogel microneedle prepared by the above method or the use of the above cryogel microneedle in preparing a product for treating chronic inflammatory diseases.

[0014] Compared with the prior art, the present invention has the following positive and beneficial effects: The present invention uses photo-cross-linked methacryloyl hyaluronic acid (HAMA) hydrogel as the matrix material, and prepares cryogel microneedles through a regional loading process and programmed freezing technology. The microneedles can achieve spatially separated co-delivery of antioxidant nanoenzymes (CeNPs) and recombinant adeno-associated virus (rAAV2) therapeutic vectors. CeNPs are first delivered to eliminate oxidative stress, and then rAAV2 is delivered to improve gene transfection efficiency, which solves the problem of low gene transfection efficiency caused by increased oxidative stress levels in the treatment of chronic inflammatory diseases. Moreover, the cryogel microneedles prepared by the present invention have good mechanical properties (compression modulus ≥1.5MPa at room temperature) and skin penetration ability (can withstand the puncture stress of skin tissue with a thickness of 300μm). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 1 is a schematic flow chart of the method for preparing cryogel microneedles of the present invention; Figure 2 Figures b and c show the morphology of the cryogel microneedles of the present invention and the mechanical strength test results (Figure a). Figure 3 This is a graph showing the results of a test on the penetration ability of the cryogel microneedles of the present invention on the skin surface; Figure 4 This is a diagram showing the therapeutic effect of the cryogel microneedle of the present invention in treating psoriasis in mice. DETAILED DESCRIPTION

[0016] The technical solutions of the present invention are further described in detail below through examples and drawings. These examples and drawings are provided for the purpose of illustrating the present invention and are not intended to limit the present invention. All other examples obtained by persons of ordinary skill in the art based on the examples in this application without creative effort are intended to fall within the scope of protection of this application.

[0017] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0018] Example Figure 1 The figure is a flow chart of the method for preparing cryogel microneedles of the present invention, which specifically includes the following steps: Step S1, adding 10 mL of PBS solution to 0.025 g of initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, heating and stirring in a water bath at 40-50 ° C for 15 minutes to obtain a standard solution of initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate with a mass fraction of 0.25%; Step S2: 0.1 g of methacryloylated hyaluronic acid (HAMA) was placed in a brown glass bottle, and 2 mL of the initiator phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt standard solution obtained in step S1 was added to the brown glass bottle. The mixture was stirred in the dark at room temperature for 1 h to obtain a photocrosslinker methacryloylated hyaluronic acid (HAMA) hydrogel solution with a mass fraction of 1%; Step S3, mixing the photocrosslinker methacryloylation hyaluronic acid HAMA hydrogel solution obtained in step S2 with antioxidant nanozymes CeNPs at a concentration of 20 μg / mL and recombinant adeno-associated virus rAAV2 at a titer of 1×10¹² vg / mL, respectively, to obtain CeNPs@HAMA hydrogel solution and rAAV2@HAMA hydrogel solution; wherein the nucleotide sequence of the anti-inflammatory gene encoded by the recombinant adeno-associated virus rAAV2 is shown in SEQ ID NO: 1; Step S4: Inject the CeNPs@HAMA hydrogel solution obtained in step S3 into the tip of the microneedle mold. After centrifugation at 1500×g to concentrate the solution at the tip of the microneedle mold and remove bubbles, perform photocrosslinking for 60 seconds under blue light irradiation with a wavelength of 405 nm and an intensity of 50 mW / cm² to form a rigid tip matrix of the microneedle mold; wherein the tip area of ​​the microneedle mold occupies 2 / 3 of the height of the microneedle body; inject the rAAV2@HAMA hydrogel solution obtained in step S3 into the rear end of the microneedle mold (the remaining 1 / 3 of the microneedle body area), repeat the above centrifugal enrichment and bubble removal, and then perform secondary photocrosslinking (under the same crosslinking conditions as above) to form a complete porous gel microneedle tip loaded with the gene therapy vector; Step S5: injecting pure methacryloylated hyaluronic acid (HAMA) solution into the microneedle base area, centrifuging to enrich and remove bubbles, and then performing secondary photocrosslinking (under the same centrifugation and crosslinking conditions as in step S4) to form a microneedle mold base; Step S6: The microneedle mold with the formed needle tip and base is subjected to a freezing treatment. The specific process of the freezing treatment is: first, cooling to -20°C at a rate of 1°C / min and holding for 2 hours, then transferring to a deep freeze at -80°C for 24 hours, and finally quick freezing with liquid nitrogen to obtain a cryogel microneedle array with a hierarchical pore structure; Step S7: gently demould the cryogel microneedle array obtained in step S6 to obtain cryogel microneedles, the specific form of which is as follows: Figure 2 b and Figure 2 As shown in c, Figure 2 b Confocal microscopy three-dimensional reconstruction shows that the segmented microneedles have been successfully prepared. The tip segment is labeled with green fluorescent CeNPs-FITC, with an average height of approximately 400 μm, and the rear segment is labeled with red fluorescent rAAV2-A20, with an average height of approximately 200 μm. Figure 2 c Cryogenic scanning electron microscopy (Cryo-SEM) images show that the microneedle tips are well-shaped and the entire array is uniform, with no visible bubbles or morphological defects.

[0019] SEQ ID NO: 1: The cryogel microneedles prepared by the present invention have good mechanical properties (compression modulus ≥ 1.5 MPa at room temperature), specifically Figure 2 As shown in a, in order to determine the mechanical strength of different photocrosslinked hydrogel materials in pre-crosslinked cryomicroneedles, four commonly used photocrosslinked hydrogels (CMCSMA hydrogel, SilMA hydrogel, AlgMA hydrogel, GelMA hydrogel) were selected for comparison with HAMA hydrogel). Figure 2 aThe results show that HAMA hydrogel exhibits the best low-temperature mechanical strength at a concentration of 1%. The microneedles in the present invention also have good skin penetration ability (can withstand the stress of puncturing skin tissue with a thickness of 300μm). Figure 3 As shown, Figure 3 a The skin surface pressed by Cryogel MN (cryomicroneedle) shows neatly arranged micropores without obvious bleeding; Figure 3 b Histological hematoxylin and eosin (H&E) staining of skin sections showed that the microneedle tip penetrated the mouse epidermis to a depth of approximately 300 µm; Figure 3 c By comparing Cryogel MN ( Figure 3 c left panel) and 48 h after rAAV2-GFP multi-point injection ( Figure 3 c (right panel) In vivo fluorescence microscopy revealed differences in local fluorescence intensity.

[0020] Application Examples (1) C57BL / 6 mice were used to establish a psoriatic dermatitis model induced by imiquimod (IMQ). 5% IMQ cream (10 mg / mouse) was applied to the back skin of mice daily for 7 consecutive days to induce lesion formation. (2) On the third day after the establishment of the mouse psoriatic dermatitis model, the mice were treated and randomly divided into IMQ group (negative control group), microneedle treatment group (empty vector microneedle group, antioxidant nanozyme CeNPs microneedle group, recombinant adeno-associated virus rAAV2 microneedle group, and antioxidant nanozyme CeNPs combined with recombinant adeno-associated virus rAAV2 microneedle group) and tacrolimus treatment group (positive control group) (n=3); (3) The mice in the IMQ group were not treated. For the mice in the microneedle treatment group, the cryogel microneedle array was vertically pressed on the skin lesion area of ​​the mice for 60 seconds to ensure penetration of the stratum corneum. The microneedle array was replaced every 24 hours for 4 consecutive days. For the tacrolimus treatment group, tacrolimus ointment was evenly applied to the skin lesion area of ​​the mice once a day for 4 consecutive days. (4) Record the psoriasis area severity index (PASI) of mice daily, including the degree of erythema, scaling, and infiltration; (5) On the 7th day of treatment, skin lesions of mice were collected for H&E and immunofluorescence staining to measure epidermal thickness and inflammatory cell infiltration. The specific effects were as follows: Figure 4 As shown, the CeNPs@Cryogel MN group exhibited significantly improved psoriatic phenotypes compared to the Cryogel MN group. Specifically, epidermal thickness was significantly reduced, and K14 and CD31 expression was significantly decreased, indicating reduced inflammation and vascular infiltration. Similarly, the CeNPs@A20@Cryogel MN group exhibited significantly reduced psoriasis severity compared to the A20@Cryogel MN group. Based on these results, CeNPs@Cryogel MN, with or without the combined use of A20-AAV, effectively alleviated IMQ-induced psoriatic dermatitis. Furthermore, both CeNPs@Cryogel MN and CeNPs@A20@Cryogel MN demonstrated superior efficacy compared to the positive control drug, tacrolimus. Clearly, CeNPs@Cryogel MN has the potential to become a transdermal treatment for psoriatic dermatitis.

[0021] In summary, the cryogel microneedles prepared by the present invention can achieve spatially separated co-delivery of antioxidant nanoenzymes (CeNPs) and recombinant adeno-associated virus (rAAV2) therapeutic vectors, first delivering CeNPs to eliminate oxidative stress, and then delivering rAAV2 to improve gene transfection efficiency, which solves the problem of low gene transfection efficiency caused by increased oxidative stress levels in the treatment of chronic inflammatory diseases, and can be used for long-term synergistic treatment of chronic inflammatory diseases; and the cryogel microneedles prepared by the present invention have good mechanical properties (compression modulus ≥1.5MPa at room temperature), can maintain structural integrity on the skin surface, and also have good skin penetration ability (can withstand puncture stress of skin tissue with a thickness of 300μm).

[0022] Finally, it should be noted that although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that all equivalent changes and improvements made within the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing cryogel microneedles, characterized in that: The steps include: Step S1, adding 10 mL of PBS phosphate buffer to 0.025 g of initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, heating and stirring in a 40-50 ° C water bath for 15 minutes to obtain a standard solution of initiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate with a mass fraction of 0.25%; Step S2: 0.1 g of methacryloylated hyaluronic acid (HAMA) was placed in a brown glass bottle, and 2 mL of the initiator phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt standard solution obtained in step S1 was added to the brown glass bottle. The mixture was stirred in the dark at room temperature for 1 h to obtain a photocrosslinker methacryloylated hyaluronic acid (HAMA) hydrogel solution. Step S3, mixing the photocrosslinker methacryloylation hyaluronic acid HAMA hydrogel solution obtained in step S2 with the antioxidant nanozyme CeNPs and the recombinant adeno-associated virus rAAV2, respectively, to obtain a CeNPs@HAMA hydrogel solution and a rAAV2@HAMA hydrogel solution; Step S4: Inject the CeNPs@HAMA hydrogel solution obtained in step S3 into the tip of the microneedle mold, centrifuge to remove bubbles, and cross-link under blue light irradiation for 60 seconds to form a rigid needle tip matrix of the microneedle mold; inject the rAAV2@HAMA hydrogel solution obtained in step S3 into the rear end of the microneedle mold, repeat the above centrifugation and cross-linking steps to form a complete porous gel microneedle mold tip loaded with gene therapy vector; Step S5, injecting pure methacrylated hyaluronic acid (HAMA) solution into the microneedle base area, and repeating the centrifugation and cross-linking steps in step S4 to form a microneedle mold base; Step S6, freezing the microneedle mold containing the needle tip and the base loaded with the gene therapy vector to obtain a cryogel microneedle array; Step S7: gently demolding the cryogel microneedle array obtained in step S6 to obtain cryogel microneedles.

2. The method for preparing a cryogel microneedle according to claim 1, wherein: In step S3, the mass ratio of the photocrosslinker methacryloylation hyaluronic acid HAMA hydrogel solution to the antioxidant nanozyme CeNPs and the recombinant adeno-associated virus rAAV2 is 500:1:

1.

3. The method for preparing a cryogel microneedle according to claim 1, wherein: The nucleotide sequence of the anti-inflammatory gene encoded by the recombinant adeno-associated virus rAAV2 in step S3 is shown in SEQ ID NO:

1.

4. The method for preparing a cryogel microneedle according to claim 1, wherein: In the step S4, the height of the microneedle body of the microneedle mold is 600 μm, and the distance between the needle tips of the microneedle body is 600 μm.

5. The method for preparing a cryogel microneedle according to claim 4, wherein: In step S4, the tip area of ​​the microneedle mold occupies 2 / 3 of the height of the microneedle body; the centrifugal force of the centrifugation is 1500xg; the intensity of the blue light is 50mW / cm², and the wavelength of the blue light is 405nm.

6. The method for preparing a cryogel microneedle according to claim 1, wherein: The specific process of the freezing treatment in step S6 is: first cooling to -20°C at a rate of 1°C / min and holding for 2 hours, then transferring to -80°C deep freezing for 24 hours, and finally quick freezing with liquid nitrogen.

7. The method for preparing a cryogel microneedle according to claim 1, wherein: The cryogel microneedles obtained in step S7 have a compression modulus of ≥1.5 MPa at room temperature and can withstand the puncture stress of skin tissue with a thickness of 300 μm.

8. A cryogel microneedle prepared by the method according to any one of claims 1 to 8.

9. Use of the cryogel microneedle prepared by the method according to any one of claims 1 to 7 or the cryogel microneedle according to claim 8 in preparing a product for treating chronic inflammatory diseases.

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