Active oxygen responsive microneedle with separable base and preparation method thereof

Abstract

The invention relates to a preparation method of an active oxygen responsive microneedle with a separable base, which comprises the following steps: mixing 3-(acrylamide) phenylboronic acid, epigallocatechin gallate and N-vinyl pyrrolidone, and standing to obtain an E-A compound solution; dissolving cyclosporine and an initiator in the E-A compound solution to obtain a microneedle precursor solution; placing the microneedle precursor solution on the surface of a microneedle mold, and performing in-situ polymerization to form a microneedle body; paving a mixed aqueous solution of poly (N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol on the microneedle mold substrate forming the microneedle body, and drying to form a microneedle substrate; and stripping the microneedle body with the microneedle substrate from the microneedle mold to obtain the active oxygen responsive microneedle with the separable base. According to the product prepared by the preparation method, drug release can be adjusted by sensing ROS of an inflammation part, deep penetration of the drug is realized, controllable separation between a backing layer and a needle head can be realized, and discomfort of a patient is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of ophthalmic preparations, and more specifically, to an active oxygen responsive microneedle with a detachable base and a preparation method thereof. Background Art

[0002] Microneedle technology is an emerging minimally invasive strategy that can achieve effective treatment, convenient drug delivery, and controlled drug release. According to the mode of action, the current microneedles are divided into solid microneedles, coated microneedles, hollow microneedles, soluble microneedles, hydrogel microneedles, and frozen microneedles. Among the various types of microneedles, hydrogel microneedles are composed of stimuli-responsive materials and can intelligently adjust the drug release rate according to the severity of the disease. However, most reported hydrogel microneedles use water-soluble polymers as the main materials, such as hyaluronic acid, gelatin, and polyvinyl alcohol, which cannot load hydrophobic drugs. However, about 40% of approved drugs and about 90% of drugs under development are hydrophobic drugs. In addition, these polymers generally have high viscosity, which is not conducive to needle filling. Therefore, it is imperative to develop new microneedles that can deliver both hydrophilic and hydrophobic drugs and are easy to fill. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a base-detachable active oxygen responsive microneedle and a preparation method thereof. The base-detachable active oxygen responsive microneedle prepared by the preparation method provided by the present invention can regulate drug release by sensing ROS at the inflammatory site to achieve deep drug penetration, and can achieve controllable separation between the backing layer and the needle tip, thereby reducing the patient's discomfort.

[0004] The present invention provides a method for preparing an active oxygen responsive microneedle with a detachable base, comprising the following steps:

[0005] a) mixing 3-(acrylamido)phenylboronic acid, epigallocatechin gallate and N-vinylpyrrolidone, and allowing to stand to obtain an EA complex solution;

[0006] b) dissolving cyclosporine and an initiator in the EA complex solution obtained in step a) to obtain a microneedle precursor solution; placing the microneedle precursor solution on the surface of a microneedle mold and performing in-situ polymerization to form a microneedle body;

[0007] c) spreading a mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol on a microneedle mold substrate forming a microneedle body, and drying to form a microneedle substrate; finally, peeling off the microneedle body with the microneedle substrate from the microneedle mold to obtain an active oxygen responsive microneedle with a detachable base.

[0008] Preferably, the amount of 3-(acrylamide)phenylboronic acid used in step a) is 1 mol% to 4 mol% of N-vinyl pyrrolidone; the amount of epigallocatechin gallate used is 0.5 mol% to 2 mol% of N-vinyl pyrrolidone; and the molar ratio of 3-(acrylamide)phenylboronic acid to epigallocatechin gallate is 2:1.

[0009] Preferably, the mixing process in step a) is specifically as follows:

[0010] dissolving 3-(acrylamide)phenylboronic acid in N-vinylpyrrolidone to obtain a first solution;

[0011] dissolving epigallocatechin gallate in N-vinylpyrrolidone to obtain a second solution;

[0012] Mixing the first solution and the second solution for 10s to 60s to obtain a mixed solution;

[0013] The above dissolving and mixing processes are carried out under ultrasonic conditions of 10kHz to 20kHz.

[0014] Preferably, the standing temperature in step a) is 20° C. to 30° C., and the standing time is 1 h to 3 h.

[0015] Preferably, in step b), the amount of cyclosporine used is 0.5 mol% to 1.5 mol% of N-vinyl pyrrolidone; and the initiator is photoinitiator 2959, and the amount used is 0.5 mol% to 1.5 mol% of N-vinyl pyrrolidone.

[0016] Preferably, the microneedle mold in step b) is a PDMS mold having a 5×8 array;

[0017] The process of placing on the surface of the microneedle mold is specifically as follows:

[0018] The microneedle precursor solution is placed on the surface of the microneedle mold, and the bubbles in the liquid are removed by vacuuming for 1 to 10 minutes, so that the solution fills the needle of the microneedle mold, and then the excess solution on the surface of the microneedle mold is removed.

[0019] Preferably, the in-situ polymerization in step b) is performed by irradiating the microneedle mold under a 360nm to 370nm ultraviolet lamp for 20min to 40min to polymerize, crosslink and solidify the small molecules in the microneedle precursor solution to form a microneedle body.

[0020] Preferably, the concentration of the mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol in step c) is 10wt% to 20wt%, wherein the mass ratio of poly(N-isopropylacrylamide-n-butyl acrylate) to polyvinyl alcohol is 1:1.

[0021] Preferably, the drying temperature in step c) is 30° C. to 40° C., and the drying time is 6 h to 18 h.

[0022] The present invention also provides an active oxygen responsive microneedle with a detachable base, which is prepared by the preparation method described in the above technical solution.

[0023] The invention provides an active oxygen responsive microneedle with a detachable base and a preparation method thereof. The preparation method comprises the following steps: a) mixing 3-(acrylamide)phenylboronic acid, epigallocatechin gallate and N-vinyl pyrrolidone, and allowing to stand to obtain an EA complex solution; b) dissolving cyclosporine and an initiator in the EA complex solution obtained in step a) to obtain a microneedle precursor solution; placing the microneedle precursor solution on the surface of a microneedle mold and performing in-situ polymerization to form a microneedle body; c) spreading a mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol on a microneedle mold substrate forming a microneedle body, and drying to form a microneedle substrate; and finally peeling the microneedle body with the microneedle substrate from the microneedle mold to obtain an active oxygen responsive microneedle with a detachable base. Compared with the prior art, the preparation method provided by the present invention selects specific types of raw materials and cooperates with specific process steps to achieve better overall interaction. The prepared active oxygen responsive microneedles with detachable bases can regulate drug release by sensing ROS in the inflammatory site, achieve deep drug penetration, and can achieve controllable separation between the backing layer and the needle, reducing the patient's discomfort. At the same time, the preparation method provided by the present invention has a simple process, low cost, and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a typical digital photo of a microneedle patch;

[0025] Figure 2 Typical images of three types of microneedles under a stereo microscope, with a scale bar of 400 μm;

[0026] Figure 3 is the SEM image of the microneedle;

[0027] Figure 4 It is the top view fluorescence image of the microneedles loaded with rhodamine-B and FITC;

[0028] Figure 5 Front view (left) and side view fluorescence images (right) of microneedles loaded with rhodamine-B and FITC;

[0029] Figure 6 The images of mouse skin after microneedle puncture with three ratios;

[0030] Figure 7A typical image of H&E staining after pressing microneedles into the skin;

[0031] Figure 8 This is a photo of the microneedle patch being separated from the base after being pressed on the back skin of a BALB / c mouse;

[0032] Fig. 9 A schematic diagram of the working process of the active oxygen responsive microneedle with a detachable base provided by the present invention;

[0033] Fig.10 The in vitro cumulative release of CsA from microneedles with three different concentration ratios at pH 7.4 and the typical HPLC chromatograms (n=3, mean±SD). The retention time of CsA was 11.2min.

[0034] Fig.11 CE-MN under different H2O2 concentrations 0.01 In vitro cumulative release of CsA and typical HPLC chromatogram (n=3, mean±SD), the retention time of CsA was 11.2min;

[0035] Fig.12 CE-MN under different H2O2 concentrations 0.01 In vitro cumulative release of EGCG (n=3, mean±SD). DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The present invention provides a method for preparing an active oxygen responsive microneedle with a detachable base, comprising the following steps:

[0038] a) mixing 3-(acrylamido)phenylboronic acid, epigallocatechin gallate and N-vinylpyrrolidone, and allowing to stand to obtain an EA complex solution;

[0039] b) dissolving cyclosporine and an initiator in the EA complex solution obtained in step a) to obtain a microneedle precursor solution; placing the microneedle precursor solution on the surface of a microneedle mold and performing in-situ polymerization to form a microneedle body;

[0040] c) spreading a mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol on a microneedle mold substrate forming a microneedle body, and drying to form a microneedle substrate; finally, peeling off the microneedle body with the microneedle substrate from the microneedle mold to obtain an active oxygen responsive microneedle with a detachable base.

[0041] The invention firstly mixes 3-(acrylamide)phenylboronic acid, epigallocatechin gallate and N-vinylpyrrolidone (NVP), and then allows the mixture to stand to obtain an EA complex solution.

[0042] The present invention has no particular limitation on the sources of the 3-(acrylamido)phenylboronic acid, epigallocatechin gallate and N-vinylpyrrolidone (NVP), and commercially available products known to those skilled in the art may be used.

[0043] In the present invention, the amount of 3-(acrylamide)phenylboronic acid is preferably 1 mol% to 4 mol% of N-vinyl pyrrolidone, specifically 1 mol%, 2 mol%, and 4 mol%; the amount of epigallocatechin gallate is preferably 0.5 mol% to 2 mol% of N-vinyl pyrrolidone, specifically 0.5 mol%, 1 mol%, and 2 mol%; the molar ratio of 3-(acrylamide)phenylboronic acid to epigallocatechin gallate is preferably 2:1.

[0044] In the present invention, the mixing process is preferably specifically as follows:

[0045] dissolving 3-(acrylamide)phenylboronic acid (powder) in N-vinylpyrrolidone to obtain a first solution;

[0046] dissolving epigallocatechin gallate in N-vinylpyrrolidone to obtain a second solution;

[0047] Mixing the first solution and the second solution for 10s to 60s to obtain a mixed solution;

[0048] The above dissolution and mixing process is carried out under ultrasonic conditions of 10kHz to 20kHz; in addition, in order to ensure sufficient dissolution in the above dissolution process, the ultrasonic time is preferably 10min to 30min.

[0049] In the present invention, the standing temperature is preferably 20° C. to 30° C., the time is preferably 1 h to 3 h; and it is preferably carried out in a dark place.

[0050] In a preferred embodiment of the present invention, 3 concentrations of EA complex solutions (0.25 mol%, 0.5 mol%, 1 mol%, relative to the molar percentage of NVP) were obtained.

[0051] After obtaining the EA complex solution, the present invention dissolves cyclosporine and an initiator in the obtained EA complex solution to obtain a microneedle precursor solution; and the microneedle precursor solution is placed on the surface of a microneedle mold for in-situ polymerization to form a microneedle body.

[0052] The present invention has no particular limitation on the sources of the cyclosporine and the initiator, and commercially available products known to those skilled in the art may be used.

[0053] In the present invention, the amount of cyclosporine is preferably 0.5 mol% to 1.5 mol% of N-vinyl pyrrolidone; the initiator is preferably photoinitiator 2959, and the amount is preferably 0.5 mol% to 1.5 mol% of N-vinyl pyrrolidone.

[0054] In the present invention, the microneedle mold is preferably a PDMS mold with a 5×8 array.

[0055] In the present invention, the process of placing on the surface of the microneedle mold is preferably specifically as follows:

[0056] The microneedle precursor solution is placed on the surface of the microneedle mold, and the bubbles in the liquid are removed by vacuuming for 1 to 10 minutes, so that the solution fills the needle of the microneedle mold, and then the excess solution on the surface of the microneedle mold is removed.

[0057] In the present invention, the in-situ polymerization method is preferably to place the microneedle mold under a 360nm-370nm ultraviolet lamp for 20min-40min to polymerize, crosslink and solidify the small molecules in the microneedle precursor solution to form a microneedle body.

[0058] After the microneedle body is formed, the present invention spreads a mixed aqueous solution of poly (N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol on a microneedle mold substrate that forms the microneedle body, and forms a microneedle substrate after drying; finally, the microneedle body with the microneedle substrate is peeled off from the microneedle mold to obtain an active oxygen responsive microneedle with a detachable base.

[0059] In the present invention, the concentration of the mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol is preferably 10wt% to 20wt%, wherein the mass ratio of poly(N-isopropylacrylamide-n-butyl acrylate) to polyvinyl alcohol is preferably 1:1.

[0060] In the present invention, the drying temperature is preferably 30° C. to 40° C., and the drying time is preferably 6 h to 18 h; and the drying is preferably carried out in an oven.

[0061] The present invention develops a base-detachable hydrogel microneedle with reactive oxygen species (ROS) response, selects low-viscosity N-vinyl-2-pyrrolidone (NVP) as the main monomer (also serving as a solvent), selects a borate bond complex formed by epigallocatechin gallate (EGCG) and 3-(acrylamide)phenylboronic acid as a cross-linking agent, and obtains a microneedle body with ROS sensitivity by ultraviolet polymerization; at the same time, the present invention also uses a mixture of poly(N-isopropylacrylamide-co-butyl acrylate) (PNIPAM-B) and polyvinyl alcohol (PVA) to form a temperature-sensitive detachable base. At present, in the prior art, the realization of the base detachable microneedle is by first pouring a layer of 15wt% PNIPAM-B aqueous solution, and then pouring a layer of 20wt% polyvinyl alcohol aqueous solution after drying; while the present invention mixes the two first and then pours them, reducing the steps and thus simplifying the process. In addition, the present invention selects low-viscosity, low-molecular-weight monomers as basic materials and performs in-situ polymerization in the microneedle mold, which is more conducive to industrial production, so the cost is low and has broad application prospects.

[0062] The present invention also provides an active oxygen responsive microneedle with a detachable base, which is prepared by the preparation method described in the above technical solution.

[0063] The key points of the present invention are: the construction of active oxygen responsive microneedles based on NVP materials and their application in drug delivery, the selection of four specific compounds, NVP, 3APBA, EGCG and photoinitiator 2959, and the preparation of microneedles by ultraviolet polymerization under specific ratios; the microneedle products obtained by the preparation method provided by the present invention can be used to encapsulate and deliver a variety of ophthalmic drugs including cyclosporine, tanamycin, amphotericin B, dexamethasone, etc., and have broad application prospects. At the same time, the present invention selects temperature-sensitive poly (N-isopropylacrylamide-butyl acrylate) as a detachable layer, realizes the controllable separation between the backing layer and the needle, and reduces the patient's discomfort. Therefore, the present invention provides a cost-effective home solution for the treatment of dry eye.

[0064] The invention provides an active oxygen responsive microneedle with a detachable base and a preparation method thereof. The preparation method comprises the following steps: a) mixing 3-(acrylamide)phenylboronic acid, epigallocatechin gallate and N-vinyl pyrrolidone, and allowing to stand to obtain an EA complex solution; b) dissolving cyclosporine and an initiator in the EA complex solution obtained in step a) to obtain a microneedle precursor solution; placing the microneedle precursor solution on the surface of a microneedle mold and performing in-situ polymerization to form a microneedle body; c) spreading a mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol on a microneedle mold substrate forming a microneedle body, and drying to form a microneedle substrate; and finally peeling the microneedle body with the microneedle substrate from the microneedle mold to obtain an active oxygen responsive microneedle with a detachable base. Compared with the prior art, the preparation method provided by the present invention selects specific types of raw materials and cooperates with specific process steps to achieve better overall interaction. The prepared active oxygen responsive microneedles with detachable bases can regulate drug release by sensing ROS in the inflammatory site, achieve deep drug penetration, and can achieve controllable separation between the backing layer and the needle, reducing the patient's discomfort. At the same time, the preparation method provided by the present invention has a simple process, low cost, and broad application prospects.

[0065] In order to further illustrate the present invention, the following examples are provided for detailed description. The raw materials used in the following examples of the present invention are all commercially available.

[0066] Example 1

[0067] CE-MN 0.0025 Preparation of microneedles:

[0068] (1) Take 1.72 mg of 3-(acrylamido)phenylboronic acid (3APBA) powder and dissolve it in 100 μL N-vinylpyrrolidone (NVP) solution at a molar ratio of 1 mol%. Take 2.06 mg of epigallocatechin gallate (EGCG) and dissolve it in 100 μL NVP solution at a molar ratio of 0.5 mol%. Then, after the two solutions are mixed in equal volumes for 30 seconds, they are allowed to stand in the dark at room temperature for 2 hours to obtain an EA complex solution with a concentration of 0.25 mol%. The above dissolution and mixing process is carried out under ultrasonic conditions (16 kHz, 20 min) to promote dissolution.

[0069] (2) Prepare the microneedle body by in-situ polymerization process: 10.6 mg of cyclosporine (CsA) and 4 mg of photoinitiator 2959 (1 mol% each) were dissolved in 200 μL of EA mixture solution to obtain a microneedle precursor solution. The solution was placed on the surface of a PDMS mold with a 5×8 array, and the bubbles in the liquid were removed by vacuum for 5 minutes, so that the solution filled the needle head of the microneedle mold. After removing the excess solution on the mold surface, the mold was placed under a 365nm ultraviolet lamp for 30 minutes (60W) to polymerize the small molecules in the solution, cross-link and solidify to form a microneedle body.

[0070] (3) Preparation of substrate solution: 150 mg of poly (N-isopropylacrylamide-butyl acrylate) powder (PNIPAM-B, Mw: 30000 Da, butyl acrylate: 12 mol%) was dissolved in 1 mL of deionized water and placed at 4°C for 48 h until the polymer was completely dissolved in water; 150 mg of polyvinyl alcohol powder (PVA) was dissolved in 1 mL of deionized water and stirred at 60°C overnight to completely dissolve the PVA. The two solutions were precooled in a cold room and mixed in equal volumes in a cold room to obtain a PNIPAM-B / PVA solution.

[0071] (4) In a cold room, spread the PNIPAM-B / PVA solution on the mold base layer, and place the mold in an oven at 35°C to dry overnight to obtain a thermosensitive microneedle base. Finally, gently peel the microneedle patch from the PDMS mold to obtain the CE-MN 0.0025 Microneedling.

[0072] Example 2

[0073] CE-MN 0.005 Preparation of microneedles:

[0074] (1) Take 3.44 mg of 3-(acrylamido)phenylboronic acid (3APBA) powder and dissolve it in 100 μL N-vinylpyrrolidone (NVP) solution at a molar ratio of 1 mol%. Take 4.12 mg of epigallocatechin gallate (EGCG) and dissolve it in 100 μL NVP solution at a molar ratio of 0.5 mol%. Then, after the two solutions are mixed in equal volumes for 30 seconds, they are allowed to stand in the dark at room temperature for 2 hours to obtain an EA complex solution with a concentration of 0.25 mol%. The above dissolution and mixing process is carried out under ultrasonic conditions (16 kHz, 20 min) to promote dissolution.

[0075] (2) Prepare the microneedle body by in-situ polymerization process: 10.6 mg of cyclosporine (CsA) and 4 mg of photoinitiator 2959 (1 mol% each) were dissolved in 200 μL of EA mixture solution to obtain a microneedle precursor solution. The solution was placed on the surface of a PDMS mold with a 5×8 array, and the bubbles in the liquid were removed by vacuum for 5 minutes, so that the solution filled the needle head of the microneedle mold. After removing the excess solution on the mold surface, the mold was placed under a 365nm ultraviolet lamp for 30 minutes (60W) to polymerize the small molecules in the solution, cross-link and solidify to form a microneedle body.

[0076] (3) Preparation of substrate solution: 150 mg of poly (N-isopropylacrylamide-butyl acrylate) powder (PNIPAM-B, Mw: 30000 Da, butyl acrylate: 12 mol%) was dissolved in 1 mL of deionized water and placed at 4°C for 48 h until the polymer was completely dissolved in water; 150 mg of polyvinyl alcohol powder (PVA) was dissolved in 1 mL of deionized water and stirred at 60°C overnight to completely dissolve the PVA. The two solutions were precooled in a cold room and mixed in equal volumes in a cold room to obtain a PNIPAM-B / PVA solution.

[0077] (4) In a cold room, spread the PNIPAM-B / PVA solution on the mold base layer, and place the mold in an oven at 35°C to dry overnight to obtain a thermosensitive microneedle base. Finally, gently peel the microneedle patch from the PDMS mold to obtain the CE-MN 0.005 Microneedling.

[0078] Example 3

[0079] CE-MN 0.01 Preparation of microneedles:

[0080] (1) Take 6.88 mg of 3-(acrylamido)phenylboronic acid (3APBA) powder and dissolve it in 100 μL N-vinylpyrrolidone (NVP) solution at a molar ratio of 1 mol%. Take 8.24 mg of epigallocatechin gallate (EGCG) and dissolve it in 100 μL NVP solution at a molar ratio of 0.5 mol%. Then, after the two solutions are mixed in equal volumes for 30 seconds, they are allowed to stand in the dark at room temperature for 2 hours to obtain an EA complex solution with a concentration of 0.25 mol%. The above dissolution and mixing process is carried out under ultrasonic conditions (16 kHz, 20 min) to promote dissolution.

[0081] (2) Prepare the microneedle body by in-situ polymerization process: 10.6 mg of cyclosporine (CsA) and 4 mg of photoinitiator 2959 (1 mol% each) were dissolved in 200 μL of EA mixture solution to obtain a microneedle precursor solution. The solution was placed on the surface of a PDMS mold with a 5×8 array, and the bubbles in the liquid were removed by vacuum for 5 minutes, so that the solution filled the needle head of the microneedle mold. After removing the excess solution on the mold surface, the mold was placed under a 365nm ultraviolet lamp for 30 minutes (60W) to polymerize the small molecules in the solution, cross-link and solidify to form a microneedle body.

[0082] (3) Preparation of substrate solution: 150 mg of poly (N-isopropylacrylamide-butyl acrylate) powder (PNIPAM-B, Mw: 30000 Da, butyl acrylate: 12 mol%) was dissolved in 1 mL of deionized water and placed at 4°C for 48 h until the polymer was completely dissolved in water; 150 mg of polyvinyl alcohol powder (PVA) was dissolved in 1 mL of deionized water and stirred at 60°C overnight to completely dissolve the PVA. The two solutions were precooled in a cold room and mixed in equal volumes in a cold room to obtain a PNIPAM-B / PVA solution.

[0083] (4) In a cold room, spread the PNIPAM-B / PVA solution on the mold base layer, and place the mold in an oven at 35°C to dry overnight to obtain a thermosensitive microneedle base. Finally, gently peel the microneedle patch from the PDMS mold to obtain the CE-MN 0.01 Microneedling.

[0084] According to the experimental needs, the present invention selected a 5×8 array of PDMS molds to investigate the drug-loaded microneedles (CE-MN) with three different EA concentrations. 0.0025 Microneedle, CE-MN 0.005 Microneedle, CE-MN 0.01 Microneedle), for the target polymerization system, the monomer is NVP, EA complex, the initiator is photoinitiator 2959, CsA is doped into it, and the photoinitiated polymerization is carried out under ultraviolet light, and the cross-linking and curing form a needle body. The microneedle morphology is photographed with a digital camera, and the following is obtained: Figure 1 As shown in the picture, the microneedles form a regular 5×8 array. The microneedles were observed using a stereo microscope. The optical images of the microneedles with three different concentration ratios are shown in Figure 2 As shown in FIG. 1 , the needle body is a regular and uniform cone with a diameter of about 300 μm, a height of about 600 μm, and a spacing of about 500 μm between the needle bodies. The microneedle is scanned using a field emission scanning electron microscope to obtain an image of the microneedle as shown in FIG. Figure 3 As shown, the needle morphology is consistent with the optical image.

[0085] To demonstrate the independence between the needle and the detachable base, rhodamine-B and FITC were added to the precursor solutions of the microneedle and base, respectively, and observed by stereo fluorescence microscopy. Figures 4-5 As shown, Rhodamine-B can only be observed in the microneedle body, while FITC is evenly distributed throughout the PNIPAM-B / PVA basal layer.

[0086] The penetration of microneedles is crucial to the efficiency of drug delivery. The present invention evaluates the ability of microneedle patches to penetrate mouse skin. Figure 6 As shown in the figure, trypan blue aggregates appeared in the needle holes of the skin punctured by the three types of microneedles, indicating that the three types of microneedles can effectively destroy the stratum corneum of the skin. The present invention further confirmed this through H&E staining, and obvious gaps appeared on the skin surface after the microneedles penetrated ( Figure 7 ).

[0087] In order to investigate the detachable effect of the microneedle patch, after pressing the patch on the skin, an ice pack filled with cold water (~10°C) was placed on the microneedles. The microneedle base melted at low temperature and separated from the needle body ( Figure 8-Figure 9 );in, Fig. 9 The above process is demonstrated, the microneedle can penetrate the epidermis and separate from the base under low temperature conditions, and the ROS in the inflammatory microenvironment triggers the breaking of the connecting bond in the needle body to achieve the intelligent release of the loaded drug. Based on the above results, the present invention successfully prepares a microneedle patch with a detachable function and capable of piercing the skin.

[0088] Given that borate bonds can be continuously formed and broken under mild conditions, the present invention illustrates whether CE-MN can respond to ROS to achieve controlled release. Theoretically, under ROS conditions, the phenyl borate bonds in the EA complex will break rapidly, thereby releasing the loaded drug. The present invention first determined the release rate of three different concentration ratios of microneedles under pH = 7.4 (normal physiological conditions) by high performance liquid chromatography. 2 mL of buffer was added to a centrifuge tube, and the microneedles were placed therein. The drug release rate was measured by taking the supernatant at regular time points, and the results were as follows: Fig.10 Results of CE-MN 0.0025 The release of CsA was relatively fast, and the release was basically completed within 8 hours. As the proportion of cross-linking agent increased, the release rate of the microneedle gradually decreased. CE-MN 0.005 The CsA released within 48 h reached 81.9%. 0.01 Only 62.6% was released within 48 h, which was slower than other CE-MNs and consistent with the expected sustained release effect. 0.01 The microneedles were used in subsequent experiments and were named CE-MN in the following.

[0089] H2O2 was added to the PBS buffer to simulate the high ROS environment under inflammatory conditions. The rate of CsA release from CE-MN in PBS containing 0.2 mM H2O2 increased significantly, and 91.1% of CsA was released within 48 h; with the increase of H2O2 concentration, the release rate of CsA was further accelerated ( Fig.11 In addition, the present invention also uses the Folin phenol method to detect the release kinetics of EGCG in CE-MN. Consistent with the above results, EGCG also exhibits a faster release rate in PBS buffer containing H2O2 ( Fig.12 ). These results indicate that CE-MNs are highly sensitive to the ROS microenvironment.

[0090] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an active oxygen responsive microneedle with a detachable base, characterized in that: The following steps are involved: a) mixing 3-(acrylamido)phenylboronic acid, epigallocatechin gallate and N-vinylpyrrolidone, and allowing to stand to obtain an EA complex solution; b) dissolving cyclosporine and an initiator in the EA complex solution obtained in step a) to obtain a microneedle precursor solution; placing the microneedle precursor solution on the surface of a microneedle mold and performing in-situ polymerization to form a microneedle body; c) spreading a mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol on a microneedle mold substrate forming a microneedle body, and drying to form a microneedle substrate; finally, peeling off the microneedle body with the microneedle substrate from the microneedle mold to obtain an active oxygen responsive microneedle with a detachable base.

2. The preparation method according to claim 1, characterized in that: In step a), the amount of 3-(acrylamide)phenylboronic acid used is 1 mol% to 4 mol% of N-vinyl pyrrolidone; the amount of epigallocatechin gallate used is 0.5 mol% to 2 mol% of N-vinyl pyrrolidone; and the molar ratio of 3-(acrylamide)phenylboronic acid to epigallocatechin gallate is 2:

1.

3. The preparation method according to claim 1, characterized in that: The mixing process in step a) is specifically as follows: dissolving 3-(acrylamide)phenylboronic acid in N-vinylpyrrolidone to obtain a first solution; dissolving epigallocatechin gallate in N-vinylpyrrolidone to obtain a second solution; Mixing the first solution and the second solution for 10s to 60s to obtain a mixed solution; The above dissolving and mixing processes are carried out under ultrasonic conditions of 10kHz to 20kHz.

4. The preparation method according to claim 1, characterized in that: The standing temperature in step a) is 20° C. to 30° C., and the standing time is 1 h to 3 h.

5. The preparation method according to claim 1, characterized in that: In step b), the amount of cyclosporine used is 0.5 mol% to 1.5 mol% of N-vinyl pyrrolidone; the initiator is photoinitiator 2959, and the amount used is 0.5 mol% to 1.5 mol% of N-vinyl pyrrolidone.

6. The preparation method according to claim 1, characterized in that: The microneedle mold in step b) is a PDMS mold with a 5×8 array; The process of placing on the surface of the microneedle mold is specifically as follows: The microneedle precursor solution is placed on the surface of the microneedle mold, and the bubbles in the liquid are removed by vacuuming for 1 to 10 minutes, so that the solution fills the needle of the microneedle mold, and then the excess solution on the surface of the microneedle mold is removed.

7. The preparation method according to claim 1, characterized in that: The in-situ polymerization in step b) is carried out by placing the microneedle mold under a 360nm-370nm ultraviolet lamp for 20min-40min to polymerize, crosslink and solidify the small molecules in the microneedle precursor solution to form a microneedle body.

8. The preparation method according to claim 1, characterized in that: The concentration of the mixed aqueous solution of poly(N-isopropylacrylamide-n-butyl acrylate) and polyvinyl alcohol in step c) is 10wt% to 20wt%, wherein the mass ratio of poly(N-isopropylacrylamide-n-butyl acrylate) to polyvinyl alcohol is 1:

1.

9. The preparation method according to claim 1, characterized in that: The drying temperature in step c) is 30° C. to 40° C., and the drying time is 6 h to 18 h.

10. An active oxygen responsive microneedle with a detachable base, characterized in that: The preparation method is described in any one of claims 1 to 9.

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