Soluble microneedle for mediating mesoporous polydopamine nanoparticles loaded with glucocorticoid and preparation method thereof

Abstract

The invention provides a soluble microneedle for mesoporous polydopamine nanoparticles carrying glucocorticoid drugs, which is prepared by an ultrasonic vibration method, consists of a neatly arranged microneedle array, and contains mesoporous polydopamine nanoparticles carrying glucocorticoids and two soluble polymers. The invention also provides a new method for preparing mesoporous polydopamine nanoparticles carrying glucocorticoids: dopamine hydrochloride is used as a template, stirred overnight to obtain mesoporous polydopamine nanoparticles, ultrasonically dispersed in methanol to make them uniformly dispersed, glucocorticoid methanol solution is added dropwise under oil bath heating, after sufficient stirring, unbound glucocorticoids are removed by centrifugation, and dried to obtain mesoporous polydopamine nanoparticles carrying glucocorticoids. The soluble microneedle prepared by the invention has good mechanical properties and easy dissolution characteristics, and is experimentally confirmed to be able to promote rapid wound healing. The preparation method is simple, the conditions are mild, and it is conducive to large-scale industrial production.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, in particular to a soluble microneedle for mediating mesoporous polydopamine nanoparticles carrying glucocorticoid and a preparation method thereof. Background Art

[0002] Microneedle drug delivery is a minimally invasive transdermal drug delivery method that combines the advantages of subcutaneous injection and skin patch. After the microneedle pierces the stratum corneum of the skin epidermis, a tiny channel is formed. The drug enters the skin along this channel, increasing its permeability and accumulation in specific parts; at the same time, the microneedle does not touch the dermis where the pain nerves are distributed, so it will not cause pain or skin damage; therefore, microneedle drug delivery is a safe, effective, and painless transdermal drug delivery method. Microneedles have been widely used in the field of transdermal drug delivery due to their advantages such as high efficiency, safety, and painlessness.

[0003] Soluble microneedles are microneedles made of soluble and degradable polymer materials. During the drug delivery process, the drug is released after the polymer material in the soluble microneedle dissolves or degrades. Soluble microneedles have better biocompatibility and greater drug loading capacity than insoluble microneedles, and have received more and more attention and research.

[0004] Nanoparticles mainly include latex, polymer, ceramic particles, metal particles and carbon particles. Non-toxic and harmless nanoparticles with good biosafety can be used as drug carriers to improve the utilization of drugs, and have become a research hotspot in recent years. Mesoporous polydopamine nanoparticles have good biocompatibility, near-infrared absorption properties, scavenging oxidative free radicals, mesoporous structures and rich functional groups on the surface, which can be used to load glucocorticoid drugs and improve the bioavailability of glucocorticoids.

[0005] Oral ulcers are common and frequently occurring diseases on the oral mucosa. They affect a wide range of people, including men, women and people of all ages. As the etiology is unclear, the current clinical treatment is mainly local symptomatic treatment. However, due to the special oral environment, local medication will be affected by the flushing of saliva, the movement of the chewing muscles and the involuntary swallowing movement. The drug stays on the oral mucosa for a short time, the time to act on the lesion is insufficient, and the local effective drug concentration is low, so the efficacy is poor. Triamcinolone acetonide injection is used to directly inject the ulcer base locally. Ulcers are inherently painful when they occur. The pain caused by local injection leads to poor patient compliance, thereby reducing the treatment effect.

[0006] Chinese patent publication number CN115252535A discloses a soluble microneedle loaded with glucocorticoids, which is formed by a backing layer and a microneedle array, and contains glucocorticoid drugs and a soluble polymer. However, in the process of disease treatment, due to the poor water solubility and low bioavailability of glucocorticoid drugs themselves, the amount of drugs that can enter the skin or mucous membrane is small, and it is difficult to effectively exert the therapeutic effect of glucocorticoid drugs, resulting in excessive use of glucocorticoid drugs; and long-term use of glucocorticoid drugs will lead to a series of drug side effects. The above-mentioned invention only uses a soluble polymer in the soluble microneedle, which does not give full play to the combined drug function of the microneedle preparation type. Summary of the invention

[0007] The purpose of the present invention is to provide a soluble microneedle mediated by mesoporous polydopamine nanoparticles loaded with glucocorticoids and a preparation method thereof, so as to solve the problems in the prior art that the bioavailability of glucocorticoid drugs is low, resulting in a large initial dose of the drug or a long use cycle, or causing a series of side effects, and a single soluble polymer does not fully utilize the combined use of microneedle carriers.

[0008] In order to achieve the technical solution for the purpose of the present invention, the present invention provides a soluble microneedle for mesoporous polydopamine nanoparticles carrying glucocorticoid drugs, including a backing layer and a microneedle array, the backing layer and the microneedle array are integrally formed, and contain mesoporous polydopamine nanoparticles carrying glucocorticoid drugs and two soluble polymers.

[0009] Further, the glucocorticoid drugs are independently selected from triamcinolone acetonide, betamethasone 21-sodium phosphate, betamethasone 17,21-dipropionate or dexamethasone sodium phosphate, or a combination thereof. In the present invention, in a preferred embodiment, the glucocorticoid drugs are selected from triamcinolone acetonide.

[0010] In the present invention, the drug loading of the microneedle is specifically limited, and it can be a safe measurement range in a biocompatibility experiment. Preferably, the mass concentration of the mesoporous polydopamine nanoparticles carrying glucocorticoid drugs is 0.1-2 mg / mL. In a preferred embodiment of the present invention, the mass concentration of the mesoporous polydopamine nanoparticles carrying glucocorticoid drugs is 100 μg / mL.

[0011] The soluble polymer needs to have good water solubility and biocompatibility materials, and is selected from hyaluronic acid, sodium hyaluronate, bletilla striata polysaccharide, gelatin, fibroin (silk protein), dextrin, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, chondroitin, dextran, sodium alginate, pullulan, maltose, poly-γ-glutamic acid or polyvinyl pyrrolidone, or a combination thereof. Preferably, the soluble polymer is selected from sodium hyaluronate and bletilla striata polysaccharide. Generally, low molecular weight polymers can be quickly dissolved in water. Therefore, the present invention preferably uses sodium hyaluronate with a weight average molecular weight of 5 to 100 kDa, and the more preferred weight average molecular weight of sodium hyaluronate is 5 to 10 kDa.

[0012] The mass concentration of sodium hyaluronate is 300 mg / mL.

[0013] In the technical solution of the soluble microneedles of the mesoporous polydopamine nanoparticles carrying glucocorticoid drugs, the shape of the microneedles and the density of the microneedles in the microneedles can be adjusted by adjusting the size of the microneedle mold according to the actual application requirements. The microneedle body is pyramidal, conical, or quasi-conical. In order to maintain the mechanical strength of the microneedle, the microneedle length is 300-1000μm, the ratio of the bottom diameter of the microneedle to the height of the microneedle is 1:(2-3), and the distance between adjacent microneedle tips is 500-1200μm. Preferably, the microneedle length is 700μm, the bottom diameter is 300μm, and the distance between adjacent microneedle tips is 600μm.

[0014] Accordingly, the present invention provides a method for preparing soluble microneedles of mesoporous polydopamine nanoparticles carrying glucocorticoid drugs, which specifically comprises the following steps:

[0015] S1. Add 0.36 g of F-127 (F127) and 417 μL 1,3,5-Trimethylbenzene (TMB, 1,3,5-trimethylbenzene) solution. At room temperature, magnetic stirring was performed for 30-40 minutes, followed by the addition of 60 mg of dopamine hydrochloride and 90 mg of Tris (hydroxymethyl) aminomethane (TRIS, trishydroxymethylaminomethane) and overnight reaction. The crude product was collected, washed at least 3 times with an ethanol / acetone mixture (v / v = 2:1), and the template was removed by centrifugation. Finally, mesoporous polydopamine nanoparticles (MPDA) were obtained and dried in a vacuum oven.

[0016] S2. The mesoporous polydopamine nanoparticles prepared in S1 are uniformly dispersed in methanol under ultrasonic vibration; in an oil bath at 45°C and under magnetic stirring at 900 r / min, a glucocorticoid methanol solution is added dropwise, the ratio of mesoporous polydopamine nanoparticles: glucocorticoid = 1:(0.5-2), and the reaction is maintained for 3-6 hours; the crude product is then washed with methanol at least 3 times to remove unbound glucocorticoid drugs; and finally, vacuum drying is performed to obtain glucocorticoid-loaded mesoporous polydopamine nanoparticles.

[0017] S3, uniformly dispersing the glucocorticoid-loaded mesoporous polydopamine nanoparticles in a double-distilled aqueous solution under ultrasonic vibration, and then adding two soluble polymers to obtain a soluble polymer aqueous solution containing the glucocorticoid-loaded mesoporous polydopamine nanoparticles;

[0018] S4, placing the microneedle mold into an ultrasonic cleaning machine for cleaning;

[0019] S5. Fill the cleaned mold with ultrapure water, place it in a vacuum environment for 15-30 minutes, and then suck out excess water from the mold;

[0020] S6, filling the microneedle mold with the soluble polymer aqueous solution containing the mesoporous polydopamine nanoparticles loaded with glucocorticoid obtained in S3;

[0021] S7, placing the microneedle mold obtained in step S6 in an ultrasonic cleaning machine, and taking it out after ultrasonic vibration for 10-45 minutes;

[0022] S8. Dry and peel at room temperature to obtain soluble microneedles containing mesoporous polydopamine nanoparticles loaded with glucocorticoid.

[0023] In step S1, the washing times of the mesoporous polydopamine nanoparticles should be determined according to the specific centrifugation speed and time, solvent volume and other factors, at least 3 times, and finally the supernatant should be nearly colorless.

[0024] In step S2, the washing times of the glucocorticoid-loaded mesoporous polydopamine nanoparticles should be determined according to the specific centrifugation speed and time, solvent volume and other factors, at least 3 times, and finally the supernatant should be free of glucocorticoid.

[0025] When preparing mesoporous polydopamine nanoparticles loaded with glucocorticoid, the encapsulation efficiency and drug loading efficiency are calculated by measuring the remaining mass of glucocorticoid in the supernatant. Preferably, in step S2, the ratio of mesoporous polydopamine particles to glucocorticoid is 1:1.

[0026] In step S4, the mold is ultrasonically cleaned to remove contaminants in the microneedle aperture of the mold, so that the soluble aqueous solution containing the drug can be fully filled into the mold. There is no special requirement for the time of ultrasonic cleaning, as long as the cleaning purpose is met. Preferably, the ultrasonic cleaning time is 10-30 minutes, and the more preferred cleaning time is 15 minutes.

[0027] Preferably, the vacuum condition in step S5 is -0.08 MPa at room temperature. A suitable vacuum environment is very important for the soluble aqueous solution of the drug of the present invention to be fully filled into the mold, and inappropriate pretreatment conditions will make the microneedle have insufficient mechanical strength.

[0028] Ultrasonic vibration can make the soluble polymer aqueous solution containing the drug fill the entire mold, so that the microneedle obtains sufficient mechanical strength. In the actual preparation process, the appropriate ultrasonic vibration time can be selected according to the geographical environment. Preferably, in step S7, the ultrasonic vibration time is 10-45 minutes. When the indoor environment is relatively dry and the temperature is high, the ultrasonic vibration time is not less than 10 minutes. When the indoor environment is cold and humid, the ultrasonic vibration time can be appropriately extended. But the grasp of time mainly depends on the change of water temperature when the ultrasonic cleaning machine vibrates. If the time is too long, the ultrasonic machine generates heat, causing the microneedle solution in the microneedle mold to dry, which is not conducive to the microneedle solution entering the needle body part of the mold, and is even more unfavorable for demolding.

[0029] In step S6 and step S7, the microneedles and the backing layer are filled at one time. It can also be done in a step-by-step filling manner. In one embodiment, the soluble polymer aqueous solution containing the drug prepared in step S3 can be first filled into the mold, the excess solution can be scraped off, and the mold can be placed in an ultrasonic cleaning machine, and ultrasonic vibration can be performed for 30 minutes before being taken out; then the soluble polymer aqueous solution containing the drug (to make the backing layer) is filled into the mold, the excess solution can be scraped off, and the mold can be placed in an ultrasonic cleaning machine, and ultrasonic vibration can be performed for 20-30 minutes before being taken out; and then the same step S8 is performed.

[0030] In addition, in step S8, the drying and peeling time is 24-72 hours. Furthermore, the drying time is 24-48 hours.

[0031] Accordingly, the soluble microneedles of mesoporous polydopamine nanoparticles carrying glucocorticoid drugs provided by the present invention can be used to treat oral ulcers. Oral ulcers mainly include recurrent aphthous ulcers, traumatic ulcers, oral ulcers caused by radiotherapy and chemotherapy, and oral ulcers caused by Behcet's disease.

[0032] Compared with the prior art, the present invention has the following significant advantages:

[0033] 1. Compared with the existing microneedles, the soluble microneedles provided by the present invention that mediate the mesoporous polydopamine nanoparticles that carry glucocorticoid drugs increase the concentration of glucocorticoids entering the mucosal tissue and improve the bioavailability of glucocorticoids by virtue of the good drug-loading capacity of the mesoporous polydopamine nanoparticles; the mesoporous polydopamine nanoparticles can scavenge oxidative free radicals to assist in the treatment of oral ulcers. In addition, the microneedle drug delivery system contains two soluble polymers to give full play to the combined drug use effect. During the treatment process, a small amount of glucocorticoids can achieve the treatment goal, reduce the side effects of the drug, reduce costs, and increase patient compliance;

[0034] 2. The soluble microneedle of the mesoporous polydopamine nanoparticles loaded with glucocorticoid provided by the present invention has good mechanical strength and rapid dissolution characteristics, which are conducive to the microneedle piercing the oral mucosa and rapidly releasing drugs. The use of the microneedle can effectively replace local injection under the mucosa, reduce the pain of patients, and achieve rapid and painless drug delivery; at the same time, the soluble polymer used in the microneedle is a polymer material with good biocompatibility and has the potential to be a safe carrier for drug delivery. The mesoporous polydopamine nanoparticles loaded with glucocorticoid used in the microneedle have verified their safe concentration range through cell experiments, and animal experiments have confirmed that the two soluble polymers interact with the mesoporous polydopamine nanoparticles loaded with glucocorticoid in vivo to promote rapid wound healing;

[0035] 3. The method for preparing soluble microneedles of mesoporous polydopamine nanoparticles loaded with glucocorticoid provided by the present invention is simple to operate, with mild conditions, and is conducive to large-scale industrial production. At the same time, the drug loading amount can be flexibly adjusted according to actual application requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the preparation method of the present invention for preparing soluble microneedles for mesoporous polydopamine nanoparticles carrying glucocorticoid drugs;

[0037] Figure 2 The pictures of the preparation of mesoporous polydopamine nanoparticles and mesoporous polydopamine nanoparticles loaded with glucocorticoids of the present invention are as follows:

[0038] in Figure 2 A is a transmission electron microscope image and a particle size distribution diagram of the mesoporous polydopamine nanoparticles prepared in step S1 of Example 1; Figure 2 B is a transmission electron microscopy image and a particle size distribution diagram of the mesoporous polydopamine nanoparticles loaded with glucocorticoid prepared in step S2 of Example 1; Figure 2 C is the encapsulation efficiency and drug loading rate of mesoporous polydopamine nanoparticles and glucocorticoids loaded at different mass ratios; Figure 2D is a transmission electron microscopy image of the glucocorticoid-loaded mesoporous polydopamine nanoparticles prepared in Example 1;

[0039] Figure 3 This is a biosafety experiment of nanoparticles and soluble microneedles at different concentrations:

[0040] in Figure 3 A is a picture of the biosafety experiment of mesoporous polydopamine nanoparticles; Figure 3 B is a picture of the biosafety experiment of mesoporous polydopamine nanoparticles loaded with glucocorticoids; Figure 3 C is a picture of the biosafety of soluble microneedles loaded with mesoporous polydopamine nanoparticles; Figure 3 D is a picture of the biosafety experiment of soluble microneedles mediated by mesoporous polydopamine nanoparticles loaded with glucocorticoids;

[0041] Figure 4 These are pictures of microneedles prepared under different preparation conditions:

[0042] in Figure 4 A is a photo of the microneedles produced in Example 1; Figure 4 B is a photo of the microneedles produced in Example 2; Figure 4 C is a photo of the microneedles produced in Example 3; Figure 4 D is a picture of the microneedles made in Example 2; Figure 4 E is a picture of the microneedle made in Example 3;

[0043] Figure 5 This is a scanning electron microscope image of the sodium hyaluronate-bletilla striata polysaccharide soluble microneedle mediating the mesoporous polydopamine nanoparticles loaded with glucocorticoid of the present invention;

[0044] Figure 6 Schematic diagram of the application of the soluble microneedle prepared by the present invention;

[0045] Figure 7 This is a diagram showing the transdermal effect of the soluble microneedle of the present invention;

[0046] Figure 8 This is a dissolution experiment diagram of the soluble microneedle of the present invention;

[0047] FIG9 is a graph showing the therapeutic effect of the soluble microneedles of the mesoporous polydopamine nanoparticles loaded with glucocorticoid provided by the present invention applied to a rat oral ulcer model:

[0048] in Fig.9AThe present invention provides general photographs of a negative control group without treatment at the time of modeling, the first day after modeling, the third day after treatment, the fifth day after treatment, and the seventh day after treatment, a positive control group of 0.1% triamcinolone acetonide oral ointment for topical application, a sodium hyaluronate-bletilla striata polysaccharide microneedle group, a sodium hyaluronate-bletilla striata polysaccharide microneedle group loaded with mesoporous polydopamine nanoparticles, and a sodium hyaluronate-bletilla striata polysaccharide microneedle group mediated by mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide; Fig. 9B It is a schematic diagram of the therapeutic effects of the negative control group without treatment on the third day, the fifth day and the seventh day of treatment, the positive control group of topical application of 0.1% triamcinolone acetonide oral ointment, the blank sodium hyaluronate-bletilla striata polysaccharide microneedle group, the sodium hyaluronate-bletilla striata polysaccharide microneedle group loaded with mesoporous polydopamine nanoparticles, and the sodium hyaluronate-bletilla striata polysaccharide microneedle group mediated by mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide;

[0049] Fig.10 This is a H&E staining pathological picture of the present invention applied to animal experiments. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0051] The main instruments used in the embodiments of the present invention are: magnetic stirrer (Dalong Xingchuang Experimental Instrument Co., Ltd., Beijing, China); ultrasonic cleaning machine (Xinzhi Biotechnology Co., Ltd., Zhejiang, China); scanning electron microscope (VEGA3, TESCAN, Czechia); vacuum drying oven (Beijing Zhongxing Weiye Century Instrument Co., Ltd.);

[0052] The main reagents used in the examples of the present invention: 1,3,5-trimethylbenzene (TMB, Mw 120.19 g / mol, AR, 97%), 1,3,5-Trimethylbenzene (Tris, Mw 121.14 g / mol, ≥99.9%), dopamine hydrochloride (Mw 189.64 g / mol, 98%) and triamcinolone acetonide (TA) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China); Purchased from Sigma (USA); sodium hyaluronate (weight average molecular weight 5–10 kDa, Focus Freda Biotechnology Co., Ltd., Shandong, China); polysaccharide from Bletilla striata (weight average molecular weight 390–460 kDa, ≥98%, Xi'an Zelang Biotechnology Co., Ltd., Xi'an, China); double distilled water (self-made in the laboratory); polydimethylsiloxane mold (PDMS, microneedle length 700 μm, bottom diameter 300 μm, adjacent microneedle tip spacing 600 μm, Y70, Shenzhen Xunlei Cloud Information Technology Co., Ltd.). Methanol, ethanol, and acetone chemical reagents were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used directly.

[0053] The CCK-8 cell experiment used in the present invention is as follows: normal human oral epithelial keratinocytes (self-extracted in the laboratory); the complete culture medium includes Dulbecco's Modified Eagle Medium (DMEM, Biological Industries, Israel), 10% fetal bovine serum (FBS, Solarbio, Beijing, China) and 1% penicillin / streptomycin (Biological Industries, Israel). Cell counting kit-8 (CCK-8) was purchased from Absin Bioscience Inc. (Shanghai, China).

[0054] Animal model used in the examples of the present invention: Adult Sprague Dawley rats (SD, male, ~150 g) were purchased from Jinan Pengyue Experimental Animal Breeding Center (Shandong, China) and maintained in a specific pathogen-free (SPF) state at the Qingdao University Experimental Animal Center. The animal protocol was reviewed by the Ethics Committee of Qingdao Stomatological Hospital Affiliated to Qingdao University (Contract Authorization 2021KQYX031). All experiments were performed in accordance with the instructions of the Guide for the Care and Use of Laboratory Animals. Microneedle preparation:

[0055] Example 1

[0056] According to Figure 1 The steps shown are for microneedle preparation:

[0057] S1. Add 0.36 g of F-127 (F127) and 417 μL 1,3,5-Trimethylbenzene (TMB) solution. Magnetic stirring was performed at room temperature for 30-40 minutes, followed by the addition of 60 mg dopamine hydrochloride and 90 mg Tris (hydroxymethyl) aminomethane (TRIS) and overnight reaction. The crude product was collected, washed at least 3 times with an ethanol / acetone mixture (v / v = 2:1), and the template was removed by centrifugation. Finally, mesoporous polydopamine nanoparticles (MPDA) were obtained and dried in a vacuum oven.

[0058] S2. The mesoporous polydopamine nanoparticles prepared in S1 are uniformly dispersed in methanol under ultrasonic vibration; in an oil bath at 45°C and under magnetic stirring at 900 r / min, a glucocorticoid methanol solution is added dropwise, the ratio of mesoporous polydopamine nanoparticles: glucocorticoid = 1:(0.5-2), and the reaction is maintained for 3-6 hours; the crude product is then washed with methanol at least 3 times to remove unbound glucocorticoid drugs; and finally, vacuum drying is performed to obtain glucocorticoid-loaded mesoporous polydopamine nanoparticles.

[0059] S3, uniformly dispersing the glucocorticoid-loaded mesoporous polydopamine nanoparticles in a double-distilled aqueous solution under ultrasonic vibration, and then adding two soluble polymers to obtain a soluble polymer aqueous solution containing the glucocorticoid-loaded mesoporous polydopamine nanoparticles;

[0060] S4, placing the microneedle mold into an ultrasonic cleaning machine for cleaning;

[0061] S5. Fill the cleaned mold with ultrapure water, place it in a vacuum environment for 15-30 minutes, and then suck out excess water from the mold;

[0062] S6, filling the microneedle mold with the soluble polymer aqueous solution containing the mesoporous polydopamine nanoparticles loaded with glucocorticoid obtained in S3;

[0063] S7, placing the microneedle mold obtained in step S6 in an ultrasonic cleaning machine, and taking it out after ultrasonic vibration for 10-45 minutes;

[0064] S8. Dry and peel at room temperature to obtain soluble microneedles containing mesoporous polydopamine nanoparticles loaded with glucocorticoid.

[0065] like Figure 2 As shown in A, the obtained mesoporous polydopamine nanoparticles are observed by transmission electron microscopy (TEM), and the mesoporous polydopamine nanoparticles have a monodisperse spherical structure and an obvious mesoporous structure on the surface; the particle size ranges from 142 to 531 nm.

[0066] like Figure 2As shown in Figure B, the obtained glucocorticoid-loaded mesoporous polydopamine nanoparticles were observed by transmission electron microscopy (TEM), and the glucocorticoid-loaded mesoporous polydopamine nanoparticles had a monodisperse spherical structure, and the surface mesoporous structure was flatter than that of the mesoporous polydopamine nanoparticles; the particle size ranged from 142nm to 717nm. The increase in the particle size of the glucocorticoid-loaded mesoporous polydopamine nanoparticles means that some glucocorticoids are loaded on the surface of the mesoporous polydopamine nanoparticles.

[0067] like Figure 2 As shown in C, different mass ratios of mesoporous polydopamine nanoparticles and glucocorticoid were used to prepare mesoporous polydopamine nanoparticles loaded with glucocorticoid. The encapsulation efficiency and drug loading were comprehensively compared, and 1:1 was finally selected as the best.

[0068] like Figure 4 As shown in A, the obtained backing layer and microneedles contain drug ingredients, and the needle body is complete and uniform, and is brown in color. Figure 5 As shown, under a scanning electron microscope (SEM), each microneedle is seen to be upright, complete, and in the cone shape of the mold.

[0069] Example 2

[0070] The preparation operation method is basically the same as that of Example 1, except that the microneedle matrix solution in step S3 contains only two soluble polymers, sodium hyaluronate and bletilla striata polysaccharide, and does not contain mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide.

[0071] like Figure 4 As shown in B, during the production of the microneedle patch, the microneedle matrix solution only contained two soluble polymers, sodium hyaluronate and bletilla striata polysaccharide, and did not contain mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide. After the microneedles were demolded, they were intact and transparent under the microscope.

[0072] Example 3

[0073] The preparation operation method is basically the same as that of Example 1, except that the microneedle matrix solution contains two soluble polymers, sodium hyaluronate and bletilla striata polysaccharide, and 100 μg / mL mesoporous polydopamine nanoparticles.

[0074] like Figure 4 As shown in C, during the microneedle manufacturing process, the microneedle matrix solution contains two soluble polymers, sodium hyaluronate and bletilla striata polysaccharide, and blank mesoporous polydopamine nanoparticles. After demolding, the microneedle morphology is intact, the needle body is intact, and it is brown in color.

[0075] Comparative Example 1

[0076] The preparation operation method is basically the same as that of Example 1, except that the speed of the magnetic stirrer in step S2 is 400 r / min.

[0077] like Figure 2 As shown in D, the low speed of magnetic stirring makes the prepared mesoporous polydopamine nanoparticles loaded with glucocorticoids uneven in diameter and irregular in shape, and the surface mesoporous structure cannot fully load the drug.

[0078] Comparative Example 2

[0079] The preparation operation method is basically the same as that of Example 1, except that the vacuum condition in step S3 is changed to -0.07 MPa.

[0080] like Figure 4 As shown in D, an inappropriate vacuum environment causes bubbles to form in the prepared microneedles, which makes the microneedles easily break and affects their normal use.

[0081] Comparative Example 3

[0082] The preparation operation method is basically the same as that of Example 2, except that the ratio of hyaluronic acid (HA): bletilla striata polysaccharide (BSP) is changed to 1:2.

[0083] like Figure 4 As shown in E, when the microneedle is demolded, due to the low concentration of hyaluronic acid, the microneedle patch cannot be completely dried within 24-72 hours, and demolding requires a long time.

[0084] Biosafety Experiments:

[0085] The mesoporous polydopamine nanoparticles and glucocorticoid-loaded mesoporous polydopamine nanoparticles prepared in steps S1, S2 and S8 of Example 1, the soluble microneedles of mesoporous polydopamine nanoparticles loaded with glucocorticoid, and the soluble microneedles loaded with mesoporous polydopamine nanoparticles prepared in Example 3 were tested for biosafety using the standard CCK-8 test method. Human oral keratinocytes (HOK) were cultured at 3×10 3 The cells were inoculated into a 96-well plate at a density of 100 cells and cultured in complete medium at 37°C and 5% CO2 for 24 hours. The cell culture medium was replaced with the test preparation diluted with the culture medium and cultured for 24 hours. The culture medium was discarded and the cells were cultured for 1-2 hours with 100 μL of CCK-8 reagent (10%) solution per well. Subsequently, the optical density (OD) value of the well was read at a wavelength of 450 nm using a microplate reader (800TS, Bio-Tek, China). The cell viability was determined using pure culture medium as the control group.

[0086] like Figure 3As shown in A and 3B, mesoporous polydopamine nanoparticles and mesoporous polydopamine nanoparticles loaded with glucocorticoid have good biosafety within a mass concentration of 100 μg / mL; Figure 3 As shown in C and 3D, the soluble microneedles loaded with mesoporous polydopamine nanoparticles and the soluble microneedles loaded with mesoporous polydopamine nanoparticles mediated glucocorticoid have good biosafety when the mass concentration is within 2 mg / mL.

[0087] Microneedle mechanical strength test:

[0088] The soluble microneedle patch prepared in Example 1 was inserted vertically and evenly into the ventral part of the rat tongue for 3 minutes, and then gently removed. Figure 7 As shown, there are uniform pinholes on the ventral side of the rat tongue.

[0089] Microneedle solubility test:

[0090] The soluble microneedle patch prepared in Example 1 was inserted vertically and evenly into the tongue abdomen of three rats for 15 seconds, 30 seconds, 1 minute, and 3 minutes. The removed microneedle patch was placed under an ordinary optical microscope to observe the dissolution of the microneedle. Figure 8 As shown, 15 seconds after the microneedle was inserted, the needle tip part dissolved rapidly, close to 30% of the total length of the needle. After 30 seconds, the microneedle body dissolved to about 50% of the total length. After 1 minute, the microneedle body dissolved to about 70% of the total length. After 3 minutes, only the base of the microneedle remained. Therefore, the microneedle prepared by the present invention can be quickly dissolved within 3 minutes.

[0091] Efficacy evaluation:

[0092] Subjects: Male adult SD rats. The oral ulcer rat model was established by chemical cauterization with phenol solution as follows: the rats were anesthetized by injection of 1% sodium pentobarbital solution, and then the ventral tongue mucosa of the rats was cauterized with 90% phenol solution for 60 seconds. The next day, the ulcer surface formation of the rats was examined and photographed with a digital camera.

[0093] Animal grouping: Rats with established oral ulcers were divided into untreated negative control group (3 rats), 0.1% triamcinolone acetonide oral ointment topical positive control group (3 rats), sodium hyaluronate-bletilla striata polysaccharide microneedle group (3 rats), sodium hyaluronate-bletilla striata polysaccharide microneedle group loaded with mesoporous polydopamine nanoparticles (3 rats), and sodium hyaluronate-bletilla striata polysaccharide microneedle group mediated by mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide (3 rats).

[0094] Except for the negative control group (Control), the preparation methods of the other groups are as follows:

[0095]

[0096]

[0097] Treatment options:

[0098] The negative control group received no treatment;

[0099] 0.1% triamcinolone acetonide oral ointment topical positive control group (Ning Zhi ) Treat as follows: Apply an appropriate amount to the ulcer once on the first day and do not treat for the remaining 6 days;

[0100] The sodium hyaluronate-Bletilla striata polysaccharide microneedle group (HA / BSP MNs) was given microneedle pressure to press the ulcer for 3 minutes, once on the first day, and no treatment was given for the remaining 6 days;

[0101] Sodium hyaluronate-Bletilla striata polysaccharide microneedle group loaded with mesoporous polydopamine nanoparticles (MPDA-HA / BSP MNs) Figure 6 As shown, the corresponding microneedles and membranes were used to press the ulcer for 3 minutes, once on the first day, and no treatment was given for the remaining 6 days; the sodium hyaluronate-Bletilla striata polysaccharide microneedle group mediated by mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide (TA@MPDA-HA / BSP MNs) was used to press the ulcer for 3 minutes with the corresponding microneedles and membranes, once on the first day, and no treatment was given for the remaining 6 days.

[0102] Experimental results: The ulcer surface was examined with a digital camera and photographed for further analysis on days 0, 1, 3, 5, and 7. On day 7, all rats were sacrificed and the tongue tissues were taken for histological observation.

[0103] As shown in Figure 9, the treatment effect of microneedles is shown: Fig.9A As shown, the ulcers in the untreated group have not healed, the area is still large, and the epithelium is still missing; the ulcer area of ​​the positive control group of 0.1% triamcinolone acetonide oral ointment topical application and the sodium hyaluronate-bletilla polysaccharide microneedle group is slightly smaller than before, but there is still a lot of mucosal surface exudation; the sodium hyaluronate-bletilla polysaccharide microneedle group loaded with mesoporous polydopamine nanoparticles has a smaller ulcer area on the seventh day, and no obvious exudation is observed; the sodium hyaluronate-bletilla polysaccharide microneedle group mediated by mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide has basically healed the ulcer on the seventh day, the mucosal surface is intact, and there is no obvious inflammatory reaction, indicating that the sodium hyaluronate-bletilla polysaccharide microneedle mediated by mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide has a better therapeutic effect. Fig. 9B As shown, it is a schematic diagram of the effects on the third, fifth, and seventh days after microneedle treatment. The treatment effect of the sodium hyaluronate-bletilla striata polysaccharide microneedle group mediated by mesoporous polydopamine nanoparticles loaded with triamcinolone acetonide is always better than that of the other groups.

[0104] like Fig.10As shown, H&E staining of rat tongue tissue after sacrifice showed that the untreated group showed epithelial sloughing and a large number of neutrophils and plasma cells infiltrating in the submucosal layer, both of which are inflammatory cells. The fibrin layer was in direct contact with the ulcer area, resulting in a slower wound healing process and no evidence of collagen regeneration and epithelialization. In the 1% triamcinolone acetonide oral ointment group, the epithelium of the tissue lacked continuity and the stratum corneum was still missing. In the HA / BSP MNs group, the epithelial healing lacked continuity, the basement membrane was thickened, and a small amount of inflammatory cells infiltrated. In the MPDA-HA / BSP MNs group and the TA@MPDA-HA / BSPMNs group, we can clearly see that the ulcer had no significant inflammatory response, the epithelium healed, the basement membrane was intact, and the collagen regeneration was better compared with the control group. Compared with normal tissue, the basement membrane of the MPDA-HA / BSPMNs group was slightly thickened, while the TA@MPDA-HA / BSPMNs group had the best healing and tended to normal tissue histologically. This is because the microneedles are composed of mesoporous polydopamine nanoparticles loaded with glucocorticoids, sodium hyaluronate and two soluble polymers, Bletilla striata polysaccharide, both of which play a role in promoting wound healing.

[0105] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A soluble microneedle for mediating mesoporous polydopamine nanoparticles loaded with glucocorticoid, characterized in that: The invention is composed of an array of neatly arranged microneedles, containing mesoporous polydopamine nanoparticles loaded with glucocorticoids and two soluble polymers; the glucocorticoids are selected from triamcinolone acetonide, and the soluble polymers are selected from sodium hyaluronate and bletilla striata polysaccharide; the length of the microneedles on the microneedles is 300-1000 μm, the ratio of the bottom diameter of the microneedles to the height of the microneedles is 1:(2-3), and the distance between adjacent microneedle tips is 500-1200 μm.

2. The method for preparing a soluble microneedle of mesoporous polydopamine nanoparticles carrying glucocorticoid according to claim 1, characterized in that: The specific steps include: S1. Add 0.36 g Pluronic® F-127 and 417 μL 1,3,5-Trimethylbenzene solution to a mixed solution of 60 mL ethanol and 65 mL deionized water; stir magnetically for 30 minutes at room temperature, then add 60 mg dopamine hydrochloride and 90 mg Tris(hydroxymethyl)aminomethane, and keep the reaction for 24 hours; collect the crude product 1, wash it at least 3 times with an ethanol / acetone mixture, and centrifuge it; finally, obtain mesoporous polydopamine nanoparticles, and dry them in a vacuum oven, wherein the volume ratio of the ethanol / acetone mixture is: v / v = 2:1; S2, uniformly dispersing the mesoporous polydopamine nanoparticles prepared in S1 in a methanol solution under ultrasonic vibration; adding a glucocorticoid solution dissolved in methanol dropwise under magnetic stirring at 600-900 r / min in an oil bath at 45°C, and maintaining the reaction for 2-4 hours; The crude product 2 is then washed with methanol at least 3 times to remove unbound glucocorticoid; and finally vacuum dried to obtain mesoporous polydopamine nanoparticles loaded with glucocorticoid, wherein the ratio of mesoporous polydopamine nanoparticles to glucocorticoid is 2:1, or 1:1, or 1:2; S3, uniformly dispersing the glucocorticoid-loaded mesoporous polydopamine nanoparticles in a double-distilled aqueous solution under ultrasonic vibration, and then adding two soluble polymers to obtain a soluble polymer aqueous solution containing the glucocorticoid-loaded mesoporous polydopamine nanoparticles; S4, placing the microneedle mold into an ultrasonic cleaning machine for cleaning; S5. Fill the cleaned mold with ultrapure water, place it in a vacuum environment for 15-30 minutes, and then suck out excess water from the mold; S6, filling the microneedle mold with the soluble polymer aqueous solution containing the mesoporous polydopamine nanoparticles loaded with glucocorticoid obtained in S3; S7, placing the microneedle mold obtained in step S6 in an ultrasonic cleaning machine, and taking it out after ultrasonic vibration for 10-45 minutes; S8. Dry and peel at room temperature to obtain soluble microneedles containing mesoporous polydopamine nanoparticles loaded with glucocorticoid.

3. The preparation method according to claim 2, characterized in that: In the step S2, the reaction conditions are 45° C., 900 r / min, 3 hours, and the ratio of mesoporous polydopamine nanoparticles to glucocorticoid is 1:

1.

4. The preparation method according to claim 2, characterized in that: In step S3, the soluble polymer is 30% sodium hyaluronate and 15% bletilla striata polysaccharide.

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