Analgesic microneedle patch based on 3D printing as well as preparation method and application of analgesic microneedle patch

Abstract

The invention provides an analgesic microneedle patch based on 3D printing and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The preparation method of the hydrogel microneedle patch based on 3D printing comprises the following steps that 1, cold water fish skin gelatin is dissolved in DPBS, and a gelatin solution is obtained; (2) mixing methacrylic anhydride and gelatin liquid to generate polymerization reaction, so as to obtain a hydrogel material; and 3) loading an anesthetic and analgesic drug on the hydrogel material, and printing by a photocuring 3D printing method to obtain the hydrogel microneedle patch. The 3D printing technology is combined with a microneedle which is a dysmenorrhea-free skin drug delivery system, local anesthetics are loaded into the microneedle patch, on the basis of painless drug delivery, the drug delivery efficiency is improved, the drug onset time is shortened, and then the effect of effectively, rapidly and safely playing a local anesthesia role is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a 3D-printed analgesic microneedle patch and a preparation method and application thereof. Background Art

[0002] The process of radial artery puncture and catheterization is often accompanied by pain and discomfort. First, from the perspective of patient experience, traditional methods, such as direct intradermal and subcutaneous injection of lidocaine for local infiltration anesthesia, may cause additional pain and psychological pressure to patients. Topical application of lidocaine cream or transdermal patch does achieve painless drug administration, but due to the natural barrier effect of the stratum corneum of the skin, exogenous drugs are difficult to penetrate and the penetration time is long, resulting in delayed drug efficacy. Microneedles, due to their tiny needle size, can directly penetrate the stratum corneum and deliver drugs to the dermis without causing pain or tissue damage, which can significantly reduce the patient's pain, improve the patient's comfort and satisfaction, and achieve the exact treatment amount. Secondly, from the perspective of medical safety, the analgesic method of transdermal introduction of lidocaine by microneedles is safer. Since the microneedle patch only pierces the stratum corneum and epidermis of the skin and does not touch the blood vessels in the dermis, it avoids the risks of infection, bleeding, etc. that may be caused by traditional injections. In summary, the research significance of 3D printing to prepare microneedle patches lies in innovating drug delivery methods, personalized customized treatment, expanding application areas, promoting the development of medical technology, and reducing medical costs. As a new transdermal drug delivery technology, 3D printed microneedle patches have the advantages of high efficiency, safety, and convenience. By applying it to analgesia before radial artery puncture and catheterization, it can not only improve the patient's medical experience, but also provide new drug delivery ideas and solutions for other medical fields.

[0003] There are currently five types of microneedles, including solid microneedles, hollow microneedles, coated microneedles, dissolving microneedles, and hydrogel microneedles. Traditional microneedles have been widely studied and reported, while hydrogel microneedles, as a new type of microneedle, have gradually become a hot topic of research. Hydrogel-forming microneedles (HFM) are composed of swellable polymers. Their mechanism of action is different from that of the above-mentioned microneedles. They are characterized by water absorption and swelling, that is, after being inserted into the skin, the microneedle will quickly absorb tissue fluid, the hydrogel will swell and produce continuous, unblocked hydrogel channels inside, and the drug can enter the body through these channels. Drugs can be loaded into microneedles in two ways, one is to incorporate the drug into the microneedle matrix during the preparation process, and the other is to load the drug into a separate reservoir and then attach it to the hydrogel microneedle as a substrate. Both preparation methods can deliver drugs continuously over a period of time. The advantage of HFMs is that they can resist the closure of skin tissue pores after puncturing the skin. Since the material used to make the hydrogel microneedles has good biocompatibility, the micropores formed on the skin will remain open after the hydrogel microneedles are inserted into the skin until the hydrogel microneedles are removed. And due to the inherent swelling and viscoelasticity of the matrix material, no matrix material remains when the HFM patch is removed.

[0004] The materials used to prepare HFMs are non-toxic, degradable and biocompatible. Commonly used materials include natural compounds (such as gelatin) and polymer copolymers (such as poly (methyl vinyl ether-co-maleic acid) cross-linked with polyethylene glycol (PMVE / MAPEG) (ZHANG YS, KHADEMHOSSEINI A. Advances in engineering hydrogels [J / OL]. Science (New York, NY), 2017, 356 (6337): eaaf3627.). Among them, PMVE / MAPEG has excellent water absorption capacity and can be used to prepare super-swelling HFMs. These HFMs can absorb liquid and swell to 20 times their original size (RAJOLI RKR, FLEXNER C, CHIONG J, et al. Modeling the intradermal delivery of microneedle array patches for long-acting antiretrovirals using PBPK [J / OL]. European Journal of Pharmaceutics and Biopharmaceutics: Official Journal of Arbeitsgemeinschaft Fur Pharmazeutische Verfahrenstechnik eV, 2019, 144: 101-109.). HFMs pierce the skin and quickly absorb interstitial fluid, causing the hydrogel to swell, forming a continuous and unobstructed hydrogel channel for drug penetration into the skin (DONNELLY RF, SINGH TRR, ALKILANI AZ, et al. Hydrogel-forming microneedle arrays exhibit antimicrobial properties: potential for enhanced patient safety [J / OL]. International Journal of Pharmaceutics, 2013, 451 (1-2): 76-91.).

[0005] Combining 3D printing technology with microneedles, a painless transdermal drug delivery system, raises the following scientific questions: Is it possible to load local anesthetics into microneedle patches to improve drug delivery efficiency and shorten drug onset time on the basis of painless drug delivery, thereby achieving the effect of local anesthesia that is effective, rapid and safe? Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide an analgesic microneedle patch based on 3D printing, and its preparation method and application. The present invention combines 3D printing technology with microneedles, a painless transdermal drug delivery system, loads local anesthetics into the microneedle patch, improves drug delivery efficiency on the basis of painless drug delivery, shortens drug onset time, and thus achieves effective, rapid and safe local anesthesia.

[0007] The present invention provides a method for preparing a hydrogel microneedle patch based on 3D printing, comprising the following steps:

[0008] 1) Dissolve gelatin in DPBS to obtain gelatin solution;

[0009] 2) mixing methacrylic anhydride and gelatin solution, reacting to obtain a hydrogel material;

[0010] 3) Anesthetic and analgesic drugs are loaded onto the hydrogel material and printed by photocuring 3D printing to obtain a hydrogel microneedle patch.

[0011] Preferably, the gelatin in step 1) is cold water fish skin gelatin, the dissolution temperature is 55-65° C., the dissolution time is 40-80 min; and the ratio of the gelatin to DPBS is 5 g:45-55 mL.

[0012] Preferably, the reaction temperature in step 2) is 45-55° C., the reaction time is 2.5-3.5 h; and the volume ratio of methacrylic anhydride to gelatin solution is 1:(50-60).

[0013] Preferably, methacrylic anhydride is added to the gelatin solution at a rate of 0.8 to 1.2 mL / min.

[0014] Preferably, after the reaction in step 2) is completed, DPBS is added to terminate the reaction, and dialysis is performed after the reaction is terminated. The molecular weight cutoff of the dialysis bag used for dialysis is 12 to 14 kDa, and the dialysis time is 6 to 8 days.

[0015] Preferably, the anesthetic analgesic drug in step 3) is lidocaine hydrochloride, and the mass ratio of lidocaine hydrochloride to hydrogel material is 1:(1.8-2.2).

[0016] The invention provides a hydrogel microneedle patch prepared by the preparation method.

[0017] The invention provides application of the hydrogel microneedle patch in preparing an anesthesia drug delivery system.

[0018] The present invention provides application of the hydrogel microneedle patch in preparing an anesthetic for radial artery puncture and catheterization.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The 3D printing-based hydrogel microneedle patch and preparation method thereof provided by the present invention combine microneedle technology with light-curing 3D printing technology to develop a customizable hydrogel microneedle patch, which achieves minimally invasive and precise transdermal delivery of local anesthetics, and provides a new technical path for surface anesthesia. The present invention achieves full integration with the local anesthetic lidocaine through the design of cross-linked methacryloyl gelatin hydrogel microneedles. After piercing the skin, it absorbs tissue fluid and swells without dissolving, allowing the local anesthetic to be continuously released, thereby achieving an ideal surface anesthesia effect. The present invention applies light-curing 3D printed analgesic hydrogel microneedle patches to clinical scenarios for the first time, improving patients' medical experience and providing new drug delivery ideas for other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Roadmap for the preparation of hydrogel microneedle patches based on 3D printing. DETAILED DESCRIPTION

[0021] The present invention provides a preparation method of a hydrogel microneedle patch based on 3D printing, comprising the following steps: 1) dissolving gelatin in DPBS to obtain a gelatin solution; 2) mixing methacrylic anhydride with the gelatin solution to undergo a polymerization reaction to obtain a hydrogel material; 3) loading a local anesthetic on the hydrogel material, and printing the hydrogel microneedle patch by a photocuring 3D printing method.

[0022] In the present invention, gelatin is dissolved in DPBS to obtain a gelatin solution; the gelatin is preferably cold water fish skin gelatin, and the present invention has no special restrictions on the source and preparation method of the DPBS, and the Dulbecco's phosphate buffered saline solution known in the art can be used; in the present invention, the dissolution temperature is preferably 55-65°C, more preferably 58-62°C, and more preferably 60°C, and the dissolution time is preferably 40-80min, more preferably 50-70min, and more preferably 60min; the dissolution process is preferably accompanied by stirring, and the present invention has no special restrictions on the stirring speed, as long as dissolution can be achieved; in the present invention, the ratio of gelatin to DPBS is preferably 5g:45-55mL, more preferably 5g:47-53mL, and more preferably 5g:50mL.

[0023] After obtaining gelatin solution, the present invention mixes methacrylic anhydride with the gelatin solution, reacts, and obtains a hydrogel material. In the present invention, the methacrylic anhydride is preferably added to the gelatin solution at a rate of 0.8 to 1.2 mL / min, more preferably 0.9 to 1.1 mL / min, and more preferably 1.0 mL / min; the volume ratio of the methacrylic anhydride to the gelatin solution is preferably 1: (50 to 60), and more preferably 1: (52 to 58); in the present invention, the reaction temperature is preferably 45 to 55°C, more preferably 47 to 53°C, and more preferably 50°C; the reaction time is preferably 2.5 to 3.5 hours, more preferably 2.8 to 3.2 hours, and more preferably 3 hours.

[0024] After the reaction is completed, the present invention preferably adds DPBS to terminate the reaction; the temperature of adding DPBS is preferably 35-45°C, and the volume of DPBS is preferably the same as the volume of DPBS used to dissolve gelatin. The present invention preferably performs dialysis after terminating the reaction, and the molecular weight cutoff of the dialysis bag used for dialysis is preferably 12-14kDa, the dialysis time is preferably 6-8d, more preferably 7d, and the dialysis temperature is preferably 36-38°C, more preferably 37°C; during the dialysis process, ultrapure water is preferably exchanged twice a day, and the role of the dialysis is to remove residual methacrylic anhydride and other impurities. After the dialysis is completed, the dialysis product is preferably passed through a 0.44μm filter membrane to remove bacteria and other impurities, and then freeze-dried for one week to obtain a white porous foam hydrogel material GelMA.

[0025] In the present invention, the anesthetic analgesic drug is loaded on the hydrogel material, and the hydrogel microneedle patch is obtained by printing by photocuring 3D printing. In the present invention, the anesthetic analgesic drug is preferably lidocaine hydrochloride, and the mass ratio of lidocaine hydrochloride to the hydrogel material is preferably 1: (1.8-2.2), more preferably 1: (1.9-2.1), and more preferably 1: 2. In the present invention, the hydrogel material and lidocaine hydrochloride are preferably dissolved in water to prepare a LiH / GelMA solution for photocuring 3D printing.

[0026] In the present invention, the photocuring 3D printing method uses a digital light processing (DLP) printer to project blue light (405nm) with an optical pattern into the hydrogel material GelMA. First, the bottom surface pattern of the microneedle is drawn in the computer software, and then the picture is imported into the printer projection device to project the patterned digital light into GelMA. Utilizing the specific light intensity distribution of digital light (the light intensity gradually decreases from the center of the focal plane to the outside), the hydrogel material GelMA can undergo precise "static" photopolymerization under specific printing parameter conditions, thereby forming a high-quality microneedle array in one piece. Subsequently, the uncross-linked residual hydrogel material GelMA is washed with anhydrous ethanol, and the microneedle array is photocured again to ensure that the material has been fully cured, and the obtained microneedle array is dried and stored for standby use.

[0027] The present invention also provides a hydrogel microneedle patch prepared by the preparation method. The hydrogel microneedle patch provided by the present invention has good physical properties, has a very significant analgesic effect after loading lidocaine, and can effectively, quickly and safely exert the effect of local anesthesia.

[0028] The present invention also provides application of the hydrogel microneedle patch in preparing an anesthetic drug delivery system.

[0029] The present invention also provides the use of the hydrogel microneedle patch in preparing an anesthetic for radial artery puncture and catheterization.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] Hydrogel material synthesis

[0033] Preparation of GelMA: Dissolve 5g of cold-water fish skin gelatin in 50mL DPBS at 60°C and stir for 1h. Subsequently, add 1mL of methacrylic anhydride at a rate of 1mL / min and react at 50°C for 3h. After sufficient reaction, add 50mL of preheated DPBS (40°C) to terminate the reaction. Use a dialysis bag with a molecular weight cutoff of 12-14kDa to dialyze the reactants at 37°C for one week (exchange ultrapure water twice a day) to remove residual methacrylic anhydride and other impurities. After the dialyzed product is filtered through a 0.44μm membrane to remove bacteria and other impurities, it is freeze-dried for one week to obtain a white porous foam GelMA.

[0034] Preparation of lidocaine hydrochloride nano-microneedles (LiH / MNs):

[0035] Photocuring 3D printing method: lidocaine hydrochloride was dissolved in 5 mg / mL GelMA solution (LiH: GelMA = 1: 2) to prepare LiH / GelMA solution; a digital light processing (DLP) printer was used to project blue light (405 nm) with an optical pattern into the previously prepared LiH / GelMA solution. First, the bottom surface of the microneedle was drawn in the computer software, and then the picture was imported into the printer projection device to project the patterned digital light into the LiH / GelMA solution. Using the specific light intensity distribution of digital light (the light intensity gradually weakened from the center of the focal plane to the outside), GelMA can undergo precise "static" photopolymerization under specific printing parameters, thereby forming a high-quality microneedle array in one piece. Subsequently, the uncross-linked residual LiH / GelMA solution was washed with anhydrous ethanol, and the microneedle array was photocured again to ensure that the material was fully cured. The obtained microneedle array was dried and stored for later use.

[0036] Example 2

[0037] Animal experiments

[0038] In vitro transdermal drug release: Rhodamine B was used as a simulated drug and added to GelMA solution (5 mg / mL) to prepare MNs. MNs were punctured into porcine gelatin gel (10% w / v), and the real-time diffusion of Rhodamine B in bright field and fluorescent field was observed by microscopy at 0 min, 5 min, 30 min, 60 min, 120 min, 240 min, and 360 min.

[0039] In vivo transdermal drug release: In this study, the in vivo transdermal drug release of LiH / MNs and LiH patches (commercially available products) was studied by the opposite method. LiH / MNs were inserted into the dorsal skin of rats, taken out at 0h, 0.5h, 1h, 2h, 4h, 8h, 16h and 24h, and soaked in 5mL PBS at 37°C for 24h. The soaking fluid was collected to determine the residual content in the backing layer to infer the in vivo transdermal drug release. In order to determine the skin deposition of LiH, the rats were sacrificed and the dorsal skin of the rats was excised at 0h, 0.5h, 1h, 2h, 4h, 8h, 16h and 24h. The excised skin (1cm 2 ) were cut into small pieces and sonicated in 5 mL PBS at 37°C for 24 hours. The in vivo release samples of LiH patches containing the same amount (3.00 mg) of lidocaine were collected using the same method. All samples were analyzed by HPLC.

[0040] Biosafety: The cytotoxicity of microneedles to L-929 fibroblast cell line (purchased from Wuhan Pronocell Life Science Co., Ltd.) was analyzed by CCK-8 method. Specifically, L-929 fibroblast cell line was cultured in 96-well plates at 1×10 4 The cells were cultured in 100 μL cell culture medium (1% penicillin-streptomycin solution, 10% fetal bovine serum and 89% Dulbecco's modified Eagle medium) at a density of 100 μL per well. The 96-well plate was placed in a constant temperature and humidity chamber (37°C, 5% CO2) for 24 hours, and then 10 μL of GelMA and LiH / MNs extract were added to each well. After 24 hours of cell culture, 10 μL of CCK-8 solution was added to each well of the 96-well plate, placed in an incubator and continued to incubate for 3 hours, and finally, the absorbance of each well was measured by an ELISA reader (ReadMax1200, Shanghai Flash Spectrum Biotechnology Co., Ltd., Shanghai, China) at a wavelength of 450 nm.

[0041] Skin penetration performance: Take the ex vivo skin of healthy and uninjured rats, remove the subcutaneous fat, and lay it flat with the stratum corneum facing outwards. Puncture the skin vertically with LiH / MNs, and remove it after the microneedle stays on the skin for 1 minute. The skin is immediately stained with methylene blue solution, and the excess methylene blue dye is removed with isopropyl alcohol. The stained skin is photographed to detect the puncture depth.

[0042] Analgesic effect test:

[0043] The anesthetic ability of LiH / MNs prepared by the present invention was evaluated by rat behavior. The pain threshold of the rat soles was tested by the von Frey method at 0, 5, 10, 30, 60, and 120°C.

[0044] Treatment Group:

[0045] LiH / MNs group: The LiH / MNs prepared in Example 1 was applied to the skin of the soles of rats, and the timing began after the microneedles were removed.

[0046] Injection group: The same dose was injected.

[0047] Cream group: Apply 0.5 g of 5% compound lidocaine cream.

[0048] Control group: PBS buffer was injected with the same dose.

[0049] Experimental results:

[0050] In vitro transdermal drug release: Rhodamine B was used as a simulated drug. Microscopic observation showed that Rhodamine B began to diffuse gradually after 5 minutes, confirming that MNs could effectively deliver drugs.

[0051] In vivo transdermal drug release: The drug release rates at different times are shown in Table 1.

[0052] Table 1 Drug release rate at different times

[0053]

[0054]

[0055] Biosafety: The results showed that the treatment with GelMA and LiH / MNs extracts did not cause significant death of L929 fibroblasts. After 24 hours, the results showed that the survival rate of L929 cells was higher than 86%, indicating that the LiH / MNs of the present invention has good biosafety.

[0056] Skin penetration performance: From the staining results, it can be seen that LiH / MNs can achieve puncture and has no obvious toxic effect on skin tissue.

[0057] Analgesic effect: The mechanical pain threshold of the plantar surface of mice is shown in Table 2.

[0058] Table 2 Mechanical pain threshold of the plantar surface of mice in different treatment groups

[0059] Time (min) LiH / MNs group Injection group Cream group Control group 0 8.34 8.21 7.95 8.12 5 134.25 156.32 25.34 8.74 10 283.12 288.76 80.25 9.84 30 295.61 297.35 96.21 7.23 60 286.44 172.46 130.48 8.42 120 284.67 20.13 18.37 7.22

[0060] It can be seen from the above embodiments that the present invention combines 3D printing technology with the microneedle painless transdermal drug delivery system, loads local anesthetics into the microneedle patch, improves the drug delivery efficiency on the basis of painless drug delivery, shortens the drug onset time, and thus achieves the effect of local anesthesia effectively, quickly and safely.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel microneedle patch based on 3D printing, characterized in that: The following steps are involved: 1) Dissolve gelatin in DPBS to obtain gelatin solution; 2) Mixing methacrylic anhydride and gelatin solution to undergo polymerization reaction to obtain a hydrogel material GelMA; 3) The local anesthetic is loaded onto the hydrogel material and printed by photocuring 3D printing to obtain a hydrogel microneedle patch.

2. The preparation method according to claim 1, characterized in that: Step 1) the gelatin is cold water fish skin gelatin, the dissolving temperature is 55-65° C., the dissolving time is 40-80 min; the ratio of the gelatin to DPBS is 5 g:45-55 mL.

3. The preparation method according to claim 1, characterized in that: Step 2) The reaction temperature is 45-55° C., and the reaction time is 2.5-3.5 hours; the volume ratio of the methacrylic anhydride to the gelatin solution is 1:(50-60).

4. The preparation method according to claim 3, characterized in that: Methacrylic anhydride was added into the gelatin solution at a rate of 0.8-1.2 mL / min.

5. The preparation method according to claim 4, characterized in that: Step 2) After the reaction is completed, DPBS is added to terminate the reaction, and dialysis is performed after the reaction is terminated. The molecular weight cutoff of the dialysis bag used for dialysis is 12 to 14 kDa, and the dialysis time is 6 to 8 days.

6. The preparation method according to claim 5, characterized in that: Step 3) The anesthetic analgesic drug is lidocaine hydrochloride, and the mass ratio of lidocaine hydrochloride to the hydrogel material is 1:(1.8-2.2).

7. The hydrogel microneedle patch prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the hydrogel microneedle patch according to claim 7 in preparing an anesthetic drug delivery system.

9. Use of the hydrogel microneedle patch according to claim 7 in preparing an anesthetic for radial artery puncture and catheterization.

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