A bionic quick-dissolving microneedle patch and its preparation method and application

Abstract

The present invention relates to a bionic instant microneedle patch and a preparation method and application thereof, belonging to the field of biomedical technology; the bionic instant microneedle patch comprises a microneedle body and a backing; the microneedle body comprises an immunosuppressant, a bionic nano-melanocyte and a needle body polymer; the backing is prepared from a backing polymer solution. The instant microneedle patch of the present invention has the functions of rapid color restoration, reducing inflammation and immunoregulation. After the microneedle penetrates the skin, the needle tip dissolves rapidly, releasing immunosuppressants, ROS scavenging components and nano-melanin, achieving rapid color restoration and immunoregulation of the skin at the vitiligo site, saving medication time, and achieving rapid color restoration of the depigmented area; the instant microneedle patch of the present invention releases immunosuppressants to shape the immunosuppressive microenvironment of bionic tumors, achieving melanocyte escape from autoimmune killing, improving the efficacy of vitiligo, and in the treatment of other autoimmune diseases, has good clinical application transformation prospects.

Description

Technical Field

[0001] The present invention relates to a bionic instant micro - needle patch, a preparation method thereof and an application, belonging to the technical field of biomedicine. Background Art

[0002] Vitiligo is a common skin depigmentation disease, which is closely related to the immune - system - mediated death of melanocytes and affects about 0.1 - 2% of the global population. Autoimmune reactions are caused by factors such as immune system disorders, genetic factors or chemicals, specifically attacking melanocytes and leading to their death, thus forming vitiligo. Although vitiligo is a non - life - threatening chronic disease, overly strong autoimmune reactions usually lead to various complications. In addition, the exposed white patches can seriously affect the psychological state of patients. Currently, the clinical treatment of vitiligo mainly includes physical covering agents, immunosuppressants, hormones, transplantation and phototherapy, etc. However, these strategies are restricted by the multiple pathogenesis mechanisms of vitiligo and cannot be completely cured.

[0003] Reactive oxygen species (ROS) is an important factor inducing vitiligo, which can cause oxidative stress damage and inflammatory cascade reactions in melanocytes. Under normal circumstances, melanin is produced by melanocytes and transported to surrounding keratinocytes, and plays various biological functions including pigmentation, photoprotection and ROS scavenging. However, the lack of melanin in the vitiligo area makes keratinocytes more vulnerable to oxidative damage caused by various stimuli, exacerbating inflammation and immune responses.

[0004] Endogenous CD8+T - cell - mediated killing is the main cause of melanocyte death. Clinical statistics show that the risk of vitiligo patients developing melanoma is significantly lower than that of the general population, while the risk of melanoma patients developing vitiligo is more than 7 times higher than that of the normal population. Since melanoma cells share antigens (Melan - A, gp100 and tyrosinase) with melanocytes, 2 - 18% of melanoma patients develop vitiligo after immunotherapy, which instead verifies good immune efficacy. The tumor microenvironment can inhibit the activity of T lymphocytes due to high - level lactic acid, such as inhibiting the activation of CD8+T cells and reducing immune clearance ability, and enhancing the activity of regulatory T cells (Tregs) to exacerbate immunosuppression. In addition, the programmed death ligand 1 (PD - L1) molecule expressed on the surface of tumor cells binds to PD - 1 on the surface of T lymphocytes, reducing the activity of T cells, inhibiting the proliferation of T cells, inducing apoptosis and disintegration of T cells, and enabling cancer cells to achieve self - rescue, thus realizing immune escape. Summary of the Invention

[0005] The purpose of the present invention is to provide a bionic instant micro - needle patch, a preparation method thereof and an application to solve the technical problems existing in the above - mentioned prior art.

[0006] The technical solution provided by the present invention is as follows:

[0007] One of the objectives of the present invention is to provide a bionic instant dissolving microneedle patch, which includes microneedle bodies and a backing; the microneedle bodies include an immunosuppressant, bionic nano-melanocytes, and a needle body polymer; the backing is prepared from a backing polymer solution.

[0008] Based on the above technical solutions, the present invention can be further improved as follows:

[0009] Further, the immunosuppressant is lactic acid, sucralose, itaconic acid, anti-IL-2 receptor monoclonal antibody, FTYZO, rapamycin, mycophenolate mofetil, tacrolimus, or cyclosporine; the needle body polymer consists of a support component and a disintegrant; the concentration of the bionic nano-melanocytes is 1 - 2000 μg / mL; the mass fraction of the backing polymer solution is 3 - 60%.

[0010] Further, the support component is cyclodextrin or maltodextrin; the disintegrant is starch, carboxymethyl cellulose, fucoidan, polyvinylpyrrolidone, cellulose, or sodium carboxymethyl starch; the backing polymer is polyvinyl alcohol, chitosan, hyaluronic acid, or sodium alginate.

[0011] Further, the bionic nano-melanocytes are made by engineering cell membranes coating bionic melanin nanoparticles.

[0012] Further, the bionic melanin nanoparticles are polydopamine bionic melanin nanoparticles, polydopa bionic melanin nanoparticles, or cuttlefish ink bionic melanin nanoparticles, and the engineering cell membranes are melanoma cell B16F10 cell membranes, melanocyte membranes, or regulatory T cell membranes.

[0013] Further, the preparation method of the polydopa bionic melanin nanoparticles includes the following steps: First, dissolve polydopa in deionized water to obtain a polydopa solution, and then place it in a water bath and heat it with stirring to react to obtain polydopa bionic melanin nanoparticles.

[0014] Further, the concentration of the polydopa solution is 0.1 - 10 mg / mL.

[0015] Further, the temperature of the heating and stirring reaction is 60 - 100 °C, and the reaction time is 4 - 48 hours.

[0016] Further, the engineering cell membranes overexpress PD-L1, CD3L1, FGF21, galectin-3, LSECtin, FGL1, galectin-8, galectin-9, or CTLA4.

[0017] Further, the engineered cell membrane overexpresses PD-L1 by treating living cells with IFN-γ, Poly(I:C) or chemically modifying PD-L1 on the cell surface through EDC / NHS.

[0018] The second object of the present invention is to provide a method for preparing the bionic instant dissolving microneedle patch as described above, comprising the following steps: adding an immunosuppressant and bionic nano-melanocytes into a needle body polymer, mixing evenly to obtain a premixed solution, injecting the premixed solution into a microneedle mold, centrifuging to fill the mold cavity with the premixed solution, immediately and evenly covering a backing polymer solution after removing the excess premixed solution at the needle tip, and then drying and demolding to obtain the bionic instant dissolving microneedle patch.

[0019] Further, the drying time is 6-36 h, the temperature is 4-36 °C, and the humidity is 5-60%.

[0020] The third object of the present invention is to provide an application of the bionic instant dissolving microneedle patch as described above in the preparation of a microneedle patch for treating autoimmune diseases.

[0021] Further, the autoimmune disease includes vitiligo.

[0022] The technical solution provided by the present invention has the following beneficial effects compared with the prior art:

[0023] (1) The bionic nano-melanocytes of the present invention can simulate natural melanocytes to provide melanin, playing a role in light protection and free radical scavenging.

[0024] (2) The instant dissolving microneedle patch of the present invention has the functions of rapid repigmentation, reducing inflammation and immune regulation. The needle tip of the microneedle body is constructed by an open network structure. After the microneedle pierces the skin, the needle tip quickly dissolves, releasing the immunosuppressant, ROS scavenging component and nano-melanin, realizing rapid repigmentation and immune regulation of the skin at the vitiligo site, saving the medication time and achieving rapid repigmentation of the depigmented area.

[0025] (3) The instant dissolving microneedle patch of the present invention releases an immunosuppressant and transfers the engineered cell membrane overexpressing an immune checkpoint inhibitor to damaged melanocytes, shaping an immunosuppressive microenvironment of a bionic tumor, effectively inhibiting the activation of specific CD8+ T cells, realizing the escape of melanocytes from auto-immune killing, improving the curative effect of vitiligo, and having good clinical application and transformation prospects in the treatment of other autoimmune diseases. Description of the Drawings

[0026] Figure 1 is a transmission electron microscope image of polydopamine bionic melanin nanoparticles;

[0027] Figure 2 is a transmission electron microscope image of bionic nano-melanocytes;

[0028] Figure 3 For the particle size distribution of polydopamine biomimetic melanin nanoparticles and biomimetic nano-melanocytes;

[0029] Figure 4 For the absorption spectrum of polydopamine biomimetic melanin nanoparticles;

[0030] Figure 5 For the DPPH free radical scavenging of polydopamine biomimetic melanin nanoparticles;

[0031] Figure 6 For the ABTS free radical scavenging of polydopamine biomimetic melanin nanoparticles;

[0032] Figure 7 For the fluorescence image after co-incubation of biomimetic nano-melanocytes and Hacat cells;

[0033] Figure 8 For the transmission electron microscope image after co-incubation of biomimetic nano-melanocytes and Hacat cells;

[0034] Figure 9 Live / dead staining images of Hacat cells after treatment with biomimetic nano-melanocytes at different concentrations;

[0035] Figure 10 For the CCK-8 assay of the survival rate of Hacat cells;

[0036] Figure 11 For the schematic diagram of the preparation of the rapid-dissolving microneedle patch;

[0037] Figure 12 For the optical image of the rapid-dissolving microneedle;

[0038] Figure 13 For the scanning electron microscope image of the rapid-dissolving microneedle;

[0039] Figure 14 For the laser confocal 3D scanning image of the rapid-dissolving microneedle;

[0040] Figure 15 For the force-displacement curve of the rapid-dissolving microneedle;

[0041] Figure 16 For the trypan blue staining image of mouse skin after application of the rapid-dissolving microneedle patch;

[0042] Figure 17 For the laser confocal scanning image of mouse skin at different depths after application of the rapid-dissolving microneedle patch;

[0043] Figure 18 For the microscopic observation image after application of the rapid-dissolving microneedle patch;

[0044] Figure 19 In vitro release curves of BNM and Lac in the fast-dissolving microneedles;

[0045] Figure 20 Optical images of the skin after application of the fast-dissolving microneedle patch;

[0046] Figure 21 Continuous observation of the skin in the treatment area of vitiligo mice;

[0047] Figure 22 Melanin content in the skin of the treatment area of vitiligo mice;

[0048] Figure 23 Masson-Fontana staining of the skin on the 7th day of treatment;

[0049] Figure 24 Masson-Fontana staining of the skin on the 21st day of treatment. Detailed implementation mode

[0050] The principles and features of the present invention are described below in conjunction with examples. The examples given are only for explaining the present invention and are not used to limit the scope of the present invention.

[0051] Example 1: Preparation and performance detection of polydopamine biomimetic nano-melanocytes

[0052] 1. Preparation of polydopamine biomimetic nano-melanocytes

[0053] Dissolve 2 mg / mL L-DOPA in deionized water and stir at 80 °C for 24 hours to prepare polydopamine biomimetic melanin nanoparticles; break B16F10 cells overexpressing PD-L1 by repeated freezing and thawing, and obtain cell membranes by differential centrifugation. The cell membranes and polydopamine biomimetic melanin nanoparticles are co-extruded to prepare polydopamine biomimetic nano-melanocytes.

[0054] 2. Morphological characterization of polydopamine biomimetic nano-melanocytes

[0055] The above-mentioned polydopamine biomimetic nano-melanocytes were observed by transmission electron microscopy and the particle size distribution was analyzed using a DLS particle size analyzer. The experimental results are as Figures 1 - 3 shown.

[0056] As Figure 1 shown, the polydopamine biomimetic melanin nanoparticles are spherical and have uniform sizes; as Figure 2 shown, the polydopamine biomimetic nano-melanocytes exhibit a bilayer structure, and the polydopamine biomimetic melanin nanoparticles are wrapped by cell membranes; as Figure 3 shown, the average particle size of the polydopamine biomimetic melanin nanoparticles is 102 nm, and the average particle size of the polydopamine biomimetic nano-melanocytes increases slightly.

[0057] 3. Characterization of the properties of polydopamine biomimetic melanin nanoparticles

[0058] The absorbance value of the above-mentioned polydopamine biomimetic melanin nanoparticles was detected by ultraviolet-visible spectrophotometry. At the same time, the free radical scavenging ability of the polydopamine biomimetic melanin nanoparticles was detected using DPPH and ABTS. The experimental results are as Figures 4 - 6 shown

[0059] As Figure 4 shown, the polydopamine biomimetic melanin nanoparticles are similar to natural melanin and have the ability of broad-spectrum light absorption; as Figure 5 shown, the polydopamine biomimetic melanin nanoparticles have the ability to scavenge DPPH free radicals, and the average scavenging rate of DPPH is 82.41% at 200 μg / mL; as Figure 6 shown, the polydopamine biomimetic melanin nanoparticles have the ability to scavenge ABTS free radicals, and the average scavenging rate of ABTS is 92.61% at 200 μg / mL

[0060] In summary, the prepared polydopamine biomimetic nano-melanin cells have uniform particle size and have the abilities of broad-spectrum light absorption and free radical scavenging

[0061] Example 2: Evaluation of the efficacy of polydopamine biomimetic nano-melanin cells in transferring melanin to protect keratinocytes

[0062] 1. Ability of polydopamine biomimetic nano-melanin cells to transfer melanin

[0063] The polydopamine biomimetic nano-melanin cells prepared in Example 1 were co-incubated with human keratinocyte Hacat; the polydopamine biomimetic nano-melanin cells were labeled with Di I, and after co-incubation with Hacat cells, fluorescence microscopy imaging was performed, and the melanin in the cells was observed by transmission electron microscopy. The experimental results are as Figures 7 - 8 shown

[0064] As Figure 7 shown, the Hacat cells labeled with DAPI were co-localized with the polydopamine biomimetic nano-melanin cells labeled with Di I, proving the interaction between Hacat cells and polydopamine biomimetic nano-melanin cells; as Figure 8 shown, polydopamine biomimetic melanin nanoparticles were observed in Hacat cells, proving that polydopamine biomimetic nano-melanin cells can mimic natural melanocytes to transfer melanin into keratinocytes

[0065] 2. Evaluation of the efficacy of polydopamine biomimetic nano-melanin cells in protecting keratinocytes

[0066] An oxidative stress or ultraviolet damage model of Hacat cells was constructed using H2O2 and UV-B. After co-incubation with polydopamine biomimetic nano-melanocytes at different concentrations, Hacat cells were stained with calcein / PI, and the viability of Hacat cells was detected by CCK-8. The experimental results are as Figures 9 - 10 shown.

[0067] As Figure 9 shown, with the increase in the concentration of polydopamine biomimetic nano-melanocytes, the dead cells labeled with PI in Hacat cells after H2O2 and UV-B stimulation were significantly reduced, and the number of live cells increased; as Figure 10 shown, polydopamine biomimetic nano-melanocytes significantly increased the viability of Hacat cells after H2O2 and UV-B stimulation.

[0068] In summary, polydopamine biomimetic nano-melanocytes can mimic natural melanocytes to provide melanin for keratinocytes, thereby protecting Hacat cells from cell damage mediated by external stimuli through broad-spectrum light absorption and free radical scavenging ability. Therefore, polydopamine biomimetic nano-melanocytes can replace melanocytes for repigmentation in vitiligo areas.

[0069] Example 3: Preparation and performance detection of rapid-dissolving microneedle patches

[0070] 1. Preparation of rapid-dissolving microneedle patches

[0071] As Figure 11 shown, 30 wt% maltodextrin, 2 wt% fucoidan, 500 μg / mL of the polydopamine nano-melanocytes prepared in Example 1, and 100 mM lactic acid were mixed evenly to obtain a premixed solution. The premixed solution was added to a microneedle mold and centrifuged at 3500 rpm for 2 min to fill the needle cavity with the premixed solution; the excess premixed solution outside the needle cavity was removed, and then immediately covered with a 20 wt% PVA (polyvinyl alcohol) solution. It was dried at 50% humidity at room temperature for 24 h and separated from the mold to obtain a rapid-dissolving microneedle patch.

[0072] 2. Morphological characterization of rapid-dissolving microneedle patches

[0073] The above rapid-dissolving microneedle patches were observed by a camera or a scanning electron microscope. At the same time, the polydopamine nano-melanocytes in the premixed solution were labeled with a lipophilic dye Di I, and the prepared rapid-dissolving microneedle patches were observed by Z-axis scanning with a confocal laser microscope. The experimental results are as Figures 12 - 14 shown.

[0074] As Figure 12 shown, the body of the rapid-dissolving microneedle showed an obvious bilayer structure, the tip was black, the base was transparent, and the morphology was uniform; as Figure 13As shown, the instant micro-needle body is pyramid-shaped, with a bottom side length of 300 μm and a height of 600 μm; as Figure 14 shown, DiI-labeled polydopamine nano-melanocytes are concentrated in the tip of the instant micro-needle body.

[0075] 3. Skin-puncturing ability of the instant micro-needle body

[0076] The mechanical strength of the instant micro-needle body was detected by a compression experiment using a universal testing machine. The instant micro-needle patch was prepared into a 10*10 micro-needle array and placed on the universal testing machine to detect the curve of pressure and displacement of the instant micro-needle body after being compressed. The experimental results are as Figure 15 shown.

[0077] The skin-puncturing ability of the instant micro-needle body was studied using the back skin of C57BL / 6 mice. After anesthetizing the C57BL / 6 mice, the back hair was thoroughly cleaned with depilatory cream to keep the back skin dry. The instant micro-needle body was inserted into the back skin of the mice, pressed with a force of about 15 N for 1 min, the undissolved backing was removed, and after staining with 0.2% trypan blue for 20 min, observation was carried out. The experimental results are as Figure 16 shown.

[0078] The instant micro-needle body loaded with DiI-labeled polydopamine nano-melanocytes was inserted into the mouse skin, and 3D laser confocal scanning of the skin was performed for observation. The experimental results are as Figure 17 shown.

[0079] As Figure 15 shown, a single micro-needle tip can withstand a force of 0.6 N; as Figure 16 shown, after the micro-needles were inserted into the skin, the micro-needle tip parts were all stained with trypan blue; as Figure 17 shown, the micro-needles can penetrate the skin up to 490 μm.

[0080] 4. Dissolution and release performance of the instant micro-needles

[0081] The instant micro-needles were placed in simulated body fluid, and the nano-melanocytes and lactic acid released into the solution were detected at different times. The experimental results are as Figures 18 - 19 shown.

[0082] The skin of mice after the instant micro-needles punctured the skin. The experimental results are as Figure 20 shown.

[0083] As Figure 18 shown, the micro-needles dissolved rapidly within 1 min after being inserted into the skin; as Figure 19 shown, the nano-melanocytes and lactic acid loaded in the micro-needles were rapidly released within 100 s in vitro; as Figure 20 shown, the nano-melanocytes released after the micro-needles dissolved were observed in the skin.

[0084] In summary, the instant dissolving microneedles have sufficient mechanical strength to enable rapid dissolution after skin puncture and release of the nano-melanocytes and lactic acid loaded at the needle tips.

[0085] Example 4: Efficacy evaluation of the instant dissolving microneedle patch for the treatment of vitiligo

[0086] In this example, C57BL / 6 mice were used. In the in vivo treatment experiment, first, a vitiligo mouse model was constructed by continuously applying 40% monobenzone cream for 60 days; the successfully modeled vitiligo mice were respectively treated with tacrolimus cream, Lac / IMN, BNM / IMN, and BNM+Lac / IMN, and compared with the untreated group, with normal mice as the control, and the degree of repigmentation in the treated area was observed. The melanin content of the skin was calculated by detecting the OD450 absorbance value of the supernatant after grinding. The mouse skin in the treated area was stained with Masson-Fontana. The experimental results are as Figures 21 - 24 shown.

[0087] As Figure 21 shown, on the 7th day, only the skin in the treatment areas of the BNM / IMN group and the BNM+Lac / IMN group was black. After 28 days of treatment, the hair grown in the untreated group lacked melanin, while the hair colors of each treatment group were improved to varying degrees. The hair color of the BNM+Lac / IMN group was closest to that of the control group, and was basically completely repigmented; as Figure 22 shown, the BNM+Lac / IMN group had the highest melanin content, about 78% of that of the control group, higher than that of the tacrolimus group; as Figure 23 shown, on the 7th day, the hair follicles of all groups were in the resting phase, and no growing hair follicles were observed. Exogenous melanin in BNM was observed in the BNM / IMN and BNM+Lac / IMN groups; as Figure 24 shown, on the 21st day, growing hair follicles were observed in the hair follicles of all groups. Almost no melanin was observed in most of the hair follicles in the untreated group, and a small amount of melanin was present in some hair follicles in the tacrolimus group. In contrast, the melanin in the hair follicles of the BNM / IMN group and the BNM+Lac / IMN group was closer to that of the control group.

[0088] This shows that the instant dissolving microneedle patch prepared by the present invention has good effects in the treatment of vitiligo and can be used to prepare a microneedle patch for vitiligo repigmentation.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A bionic fast-dissolving microneedle patch, comprising a microneedle body and a backing, characterized in that: The microneedle body comprises an immunosuppressant, biomimetic nano-melanocytes and a needle body polymer; the backing is prepared from a backing polymer solution; The immunosuppressant is lactic acid; the needle body polymer is composed of a supporting component and a disintegrant; the concentration of the biomimetic nano-melanocytes is 1-2000 μg / mL; the mass fraction of the backing polymer solution is 3-60%; The supporting component is maltodextrin; the disintegrant is fucoidan; the backing polymer is gelatin, polyvinyl alcohol, chitosan, hyaluronic acid or sodium alginate; The preparation method of the bionic fast-dissolving microneedle patch comprises the following steps: adding immunosuppressants and bionic nano-melanocytes into a needle body polymer, mixing to obtain a premixed solution, injecting into a microneedle mold, centrifuging to allow the premixed solution to fill the mold cavity, removing excess needle tip premixed solution and immediately and evenly covering the needle tip with a backing polymer solution, then drying and demolding to obtain the bionic fast-dissolving microneedle patch.

2. A bionic fast-dissolving microneedle patch according to claim 1, characterized in that: The bionic nano-melanocyte is made of bionic melanin nanoparticles coated with an engineered cell membrane, and the engineered cell membrane is a melanoma cell B16F10 cell membrane or a melanocyte cell membrane.

3. A bionic fast-dissolving microneedle patch according to claim 2, characterized in that: The bionic melanin nanoparticles are polydopamine bionic melanin nanoparticles, polydopa bionic melanin nanoparticles or cuttlefish juice bionic melanin nanoparticles.

4. A method for preparing a bionic fast-dissolving microneedle patch according to any one of claims 1 to 3, characterized in that: The following steps are involved: Immunosuppressants and bionic nanomelanocytes are added to the needle body polymer, mixed to obtain a premixed solution, injected into the microneedle mold, centrifuged to allow the premixed solution to fill the mold cavity, and immediately and evenly covered with the backing polymer solution after removing excess needle tip premixed solution, followed by drying and demolding to obtain a bionic instant microneedle patch.

5. The method for preparing a bionic fast-dissolving microneedle patch according to claim 4, characterized in that: The drying time is 6-36 hours, the temperature is 4-36° C., and the humidity is 5-60%.

6. Use of a bionic fast-dissolving microneedle patch as claimed in any one of claims 1 to 3 in the preparation of a microneedle patch for treating autoimmune diseases, wherein the autoimmune disease is vitiligo.

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