Probiotic-nano-drug composite microneedle patch as well as preparation method and application thereof

Abstract

The invention discloses a probiotic-nano-drug composite microneedle patch as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The probiotic-nano-drug composite microneedle patch is used as a delivery system, so that the probiotics and the drug can be efficiently delivered, the whole body is prevented from being exposed, and the safety is improved; in addition, the PLGA nanoparticles can control slow release of the medicine, so that the utilization rate and the treatment effect of the medicine are remarkably improved; in addition, after the microneedle substrate layer is encapsulated, the activity of probiotics can be maintained, and the local long-acting effect is achieved; when the probiotic-nano-drug composite microneedle patch is used for treating androgenetic alopecia, the androgenetic alopecia is efficiently treated by promoting hair growth, increasing the density and diameter of hair follicles, improving microcirculation and cell proliferation and reducing the expression level of inflammatory factors; therefore, the method has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a probiotic-nanomedicine composite microneedle patch, its preparation method, and its application. Background Technology

[0002] Androgenetic alopecia (AGA) is a common chronic hair loss condition characterized by progressive shrinkage of hair follicles, leading to thinning of hair and decreased hair density on the scalp. Its main pathological mechanisms involve abnormal metabolism of androgens within the hair follicles, particularly the conversion of testosterone to the more potent dihydrotestosterone (DHT) via the action of 5α-reductase. DHT binds to androgen receptors (AR) in dermal papillary cells, disrupting the normal hair growth cycle by shortening the hair growth phase and inhibiting pro-regeneration signaling pathways (such as Wnt / β-catenin), ultimately leading to follicle atrophy and hair loss. Increasing evidence suggests that chronic perifollicular inflammation and microcirculatory disturbances significantly influence the progression of AGA. Therefore, effective treatment of androgenetic alopecia remains a challenge in the field.

[0003] Currently, commonly used clinical drugs, such as finasteride and minoxidil, have problems such as limited efficacy, significant side effects, and poor patient compliance.

[0004] Therefore, there is an urgent need for a probiotic-nanomedicine composite microneedle patch that can simultaneously regulate androgen activity, inflammation, and microvascular dysfunction to address the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic-nanomedicine composite microneedle patch, its preparation method, and its application. This addresses the problems of limited efficacy, significant side effects, and poor patient compliance associated with existing drugs for treating androgenetic alopecia.

[0006] In a first aspect, the present invention provides a probiotic-nanomedicine composite microneedle patch, comprising a backing layer and microneedles, wherein the microneedles comprise a microneedle matrix layer and a probiotic-nanomedicine composite encapsulated in the microneedle matrix layer; wherein the probiotic-nanomedicine composite is formed by electrostatic self-assembly of modified positively charged probiotics that produce nitric oxide and drug-loaded PLGA nanoparticles.

[0007] In this invention, the inventors discovered that by using a probiotic-nanomedicine composite microneedle patch as a delivery system, probiotics and drugs can be delivered efficiently, avoiding systemic exposure and improving safety. Furthermore, PLGA nanoparticles can control the slow release of drugs, thereby significantly improving drug utilization and therapeutic efficacy. Additionally, the microneedle matrix layer encapsulation maintains the activity of probiotics, achieving a localized, long-lasting effect. When this probiotic-nanomedicine composite microneedle patch is used to treat androgenetic alopecia, it effectively treats androgenetic alopecia by promoting hair growth, increasing hair follicle density and diameter, improving microcirculation and cell proliferation, and reducing the expression level of inflammatory factors.

[0008] In some embodiments, the preparation of modified, positively charged, nitric oxide-producing probiotics includes: mixing a polyallylamine hydrochloride solution with a probiotic solution, centrifuging the mixture to obtain modified, positively charged, nitric oxide-producing probiotics; the probiotics include *Westernella esculenta* (…). Weissella cibaria ).

[0009] In this invention, probiotics are modified with polyallylamine hydrochloride (PAH) to give them a positive charge, which facilitates electrostatic self-assembly with negatively charged drug-loaded PLGA nanoparticles, thereby obtaining a probiotic-nanomedicine complex with better performance.

[0010] It is understandable that the types of probiotics can be conventionally selected based on actual usage needs, as long as they can efficiently produce nitric oxide (NO). For example, in this invention, the probiotics preferably include *Westernella esculenta* (…). Weissella cibaria ).

[0011] In some embodiments, the concentration of the polyallylamine hydrochloride solution is 15-25 mg / mL, preferably 20 mg / mL; the content of the probiotic solution is 0.5 × 10⁻⁶. 8 -1.5×10 8 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL; the volume ratio of polyallylamine hydrochloride solution to probiotic solution is (0.5-1.5):(0.5-1.5), preferably 1:1.

[0012] In this invention, by controlling the reaction concentration and volume of polyallylamine hydrochloride solution and probiotic solution within a specific range, the reaction can be completed, resulting in probiotics with uniform charge distribution, which facilitates better assembly with drug-loaded PLGA nanoparticles.

[0013] In some embodiments, the mass ratio of drug to PLGA in the drug-loaded PLGA nanoparticles is 1:(1-3), preferably 1:2; the drug includes clascoterone.

[0014] In this invention, by controlling the mass ratio of drug to PLGA within a specific range, the drug can be completely loaded into PLGA nanoparticles, facilitating the slow release of the drug.

[0015] It is understandable that the type of medication can be conventionally selected based on actual needs, as long as it can effectively treat androgenetic alopecia. For example, in this invention, the medication preferably includes clascoterone.

[0016] In some embodiments, the number of modified, positively charged, nitric oxide-producing probiotics to the mass ratio of drug-loaded PLGA nanoparticles is (0.5 × 10⁻⁶). 8 -1.5×10 8 CFU: (1-3 mg), preferably 1×10⁻⁶ mg. 8 CFU: 2mg.

[0017] In this invention, by controlling the ratio of the number of probiotics to the mass of drug-loaded PLGA nanoparticles within a specific range, the drug-loaded PLGA nanoparticles can be completely adsorbed onto the surface of the probiotics, resulting in a probiotic-nanomedicine complex with better performance.

[0018] In some embodiments, the microneedle matrix layer comprises polyvinyl alcohol and sucrose, and the mass ratio of polyvinyl alcohol to sucrose is (0.5-1.5):(0.5-1.5), preferably 1:1.

[0019] In this invention, by controlling the mass ratio of polyvinyl alcohol and sucrose within a specific range, the probiotic-nanomedicine complex can be better encapsulated, further maintaining the activity of the probiotics and achieving a localized long-lasting effect.

[0020] In a second aspect, the present invention provides a method for preparing a probiotic-nanomedicine composite microneedle patch as described above, comprising the following steps: mixing a drug organic solution with a PLGA organic solution, adding the mixture dropwise to a first polyvinyl alcohol aqueous solution, emulsifying the mixture ultrasonically, sonicating the resulting emulsion under ice bath conditions, adding a second polyvinyl alcohol aqueous solution, removing the organic solvent under reduced pressure, washing, and drying to obtain drug-loaded PLGA nanoparticles; providing a modified probiotic suspension with a positive charge and producing nitric oxide and a drug-loaded PLGA nanoparticle solution, mixing the two and performing electrostatic self-assembly, centrifuging, and washing to obtain a probiotic-nanomedicine composite; providing a probiotic-nanomedicine composite solution, mixing the probiotic-nanomedicine composite solution with polyvinyl alcohol and sucrose in MRS medium to obtain a microneedle casting solution; adding the microneedle casting solution dropwise to a microneedle mold, centrifuging, and drying to obtain a probiotic-nanomedicine composite microneedle patch.

[0021] The preparation method provided by this invention is simple, and the raw materials used are cheap and readily available. The prepared probiotic-nanomedicine composite microneedle patch can efficiently deliver probiotics and drugs, thereby effectively treating androgenetic alopecia.

[0022] In some embodiments, in the step of obtaining drug-loaded PLGA nanoparticles, the concentration of the drug organic solution is 40-45 mg / mL, preferably 41.67 mg / mL; the concentration of the PLGA organic solution is 1-3 mg / mL, preferably 2 mg / mL; the mass concentration of the first polyvinyl alcohol aqueous solution is 2-4%, preferably 3%; the mass concentration of the second polyvinyl alcohol aqueous solution is 0.2-0.4%, preferably 0.3%; and the volume ratio of the drug organic solution, PLGA organic solution, first polyvinyl alcohol aqueous solution, and second polyvinyl alcohol aqueous solution is (0.1-0.2):(8-12):(2-4):(8-12); preferably 0.12:10:3:10.

[0023] In this invention, by controlling the amount of each raw material within a specific range, PLGA nanoparticles with good performance loaded with drugs can be prepared.

[0024] It is understood that the drug organic solution and PLGA organic solution are obtained by dissolving the drug and PLGA in organic solvents respectively. The type of organic solvent can be conventionally selected according to actual use needs. For example, in this invention, the organic solvent preferably includes dichloromethane.

[0025] In some embodiments, in the step of obtaining the probiotic-nanomedicine complex, the probiotic suspension content is 0.5 × 10⁻⁶. 8 -1.5×10 8 CFU / mL, preferably 1×10⁻⁶ 8 The concentration of the drug-loaded PLGA nanoparticle solution is 1-3 mg / mL, preferably 2 mg / mL; the volume ratio of the probiotic suspension to the drug-loaded PLGA nanoparticle solution is (0.5-1.5):(0.5-1.5), preferably 1:1; in the step of obtaining the probiotic-nanomedicine composite microneedle patch, the content of the probiotic-nanomedicine composite solution is 0.5 × 10⁻⁶ CFU / mL; 8 -1.5×10 8 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL; polyvinyl alcohol concentration of 15-20%, preferably 18%; sucrose concentration of 15-20%, preferably 18%.

[0026] In a third aspect, the present invention provides the application of the probiotic-nanomedicine composite microneedle patch prepared by any of the above-described probiotic-nanomedicine composite microneedle patches or the probiotic-nanomedicine composite microneedle patches prepared by any of the above-described preparation methods in any of the following: 1) preparing a drug for the prevention and / or treatment of androgenetic alopecia; 2) preparing a drug for promoting hair follicle regeneration; 3) preparing a drug for improving scalp microcirculation.

[0027] In this invention, the probiotic-nanomedicine composite microneedle patch treats androgenetic alopecia through multiple mechanisms. Specifically, the drug in the microneedle patch blocks the androgen signaling pathway, prevents hair follicle miniaturization, and promotes hair follicle regeneration; the probiotics promote angiogenesis and the formation of an anti-inflammatory microenvironment by producing NO, thereby improving scalp microcirculation; through the above synergistic effects, the treatment effect of androgenetic alopecia is significantly improved.

[0028] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention uses a probiotic-nanomedicine composite microneedle patch as a delivery system, which can efficiently deliver probiotics and drugs, avoid systemic exposure, and improve safety; furthermore, PLGA nanoparticles can control the slow release of drugs, thereby significantly improving drug utilization and therapeutic effect; in addition, the microneedle matrix layer encapsulation can maintain the activity of probiotics and achieve local long-term effects; when this probiotic-nanomedicine composite microneedle patch is used to treat androgenetic alopecia, it can effectively treat androgenetic alopecia by promoting hair growth, increasing hair follicle density and diameter, improving microcirculation and cell proliferation, and reducing the expression level of inflammatory factors; therefore, it has good application prospects. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the preparation process of the probiotic-nanomedicine composite microneedle patch and its principle for treating androgenetic alopecia in this invention. Figure 2The following are the characterization and performance test results of the WC@Cla MN microneedle patch prepared in Example 1 of this invention: (A) is a schematic diagram of the preparation process of WC@Cla MN; (B) is a SEM image of Cla@PLGA; (C) is a hydration diameter distribution map of Cla@PLGA; (D) is a TEM image of WC@Cla biohybrid; (E) is a graph showing the zeta potential change during the stepwise formation of WC@Cla; (F) is a bright-field microscopy image of the prepared WC@Cla MN; (G) is a cross-sectional SEM image of blank MN and WC@Cla MN; (H) is the mechanical strength test result of WC@Cla MN; (I) is the skin penetration efficiency result of WC@Cla MN after application to mouse skin; (J) is the growth activity test result of WC in WC@Cla biohybrid; (K) is the quantitative analysis result of the survival rate of WC in WC@Cla biohybrid; (L) is the WC@Cla biohybrid at different time points. The colony count results of WC@Cla retained in MN; (M) is the quantitative analysis result of WC@Cla survival rate in MN; data are expressed as mean ± SD (n=6) P <0.05, P <0.001); Figure 3 Immunofluorescence images of macrophage polarization mediated by different treatment groups in Example 2 of this invention; Figure 4 This is a diagram illustrating the angiogenesis of human umbilical vein endothelial cells mediated by different treatment groups in Example 2 of the present invention; Figure 5 This is a graph showing the results of different treatment groups promoting hair regeneration in mice with androgenetic alopecia in Example 3 of the present invention; Figure 6 SEM images of regenerated hair in different treatment groups after 21 days of treatment in Example 3 of the present invention; Figure 7 This is an image showing the immunofluorescence detection results of CD31 and Ki67 protein expression in different treatment groups in Example 3 of the present invention; Figure 8 This is an image showing the immunofluorescence detection results of IL-6 and TNF-α expression in different treatment groups in Example 3 of the present invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0032] In this invention, *Westernella esculenta* (… Weissella cibaria See the report in the inventors' team's published literature (Huan-Huan Chen, Fang-Sheng Fu, Qi-Wen Chen, et al. "Two-Pronged MicrobeDelivery of Nitric Oxide and Oxygen for Diabetic Wound Healing." Nano Letters23.12(2023):5595-5602.).

[0033] Please see Figure 1 This is a schematic diagram illustrating the preparation process of the probiotic-nanomedicine composite microneedle patch (WC@Cla MN) and its principle for treating androgenetic alopecia in this invention. Specifically, polyallylamine hydrochloride (PAH) is used to target *Wesella esculenta* (…). Weissella cibaria The probiotics were modified to produce positively charged, nitric oxide-producing probiotics. These probiotics were then electrostatically self-assembled with drug-loaded PLGA nanoparticles (Cla@PLGA) prepared using a single emulsion solvent evaporation method to form WC@Cla biohybrids. The WC@Cla biohybrids were then mixed with polyvinyl alcohol and sucrose and dropped onto a microneedle mold to obtain a probiotic-nanomedicine composite microneedle patch. When this probiotic-nanomedicine composite microneedle patch was applied to mice with androgenetic alopecia, the probiotic-nanomedicine complex was released from the microneedles. The drug blocked the androgen signaling pathway, preventing hair follicle miniaturization, while the NO produced by the probiotics promoted angiogenesis and the formation of an anti-inflammatory microenvironment. Through a multi-mechanism synergy, this resulted in highly effective treatment of androgenetic alopecia.

[0034] Example 1: Preparation of Probiotic-Nanomedicine Composite Microneedle Patch (WC@Cla MN) This embodiment provides a method for preparing WC@Cla MN (see details in [link]). Figure 2 A), including the following steps: 1) Preparation of PLGA nanoparticles loaded with clascoterone (Cla): 5 mg clascoterone was dissolved in 0.12 mL of dichloromethane, and 10 mg PLGA was dissolved separately in 5 mL of the same solvent. The two organic phases were mixed and added dropwise to 3 mL of polyvinyl alcohol (PVA, 3 wt%) aqueous solution, followed by emulsification by sonication at 180 W for 1 minute. The resulting emulsion was sonicated in an ice bath for 10 minutes (pulse mode, 30 W, 5 seconds on / 5 seconds off). Then, 10 mL of 0.3 wt% PVA solution was added, and the mixture was stirred under reduced pressure for 3 hours using a rotary evaporator to completely remove the organic solvent. The nanoparticles were collected by centrifugation at 15000 rpm for 20 minutes, washed twice with deionized water, and finally freeze-dried to obtain Cla@PLGA nanoparticles (SEM image shown). Figure 2 As shown in Figure B, the Cla@PLGA nanoparticles have a smooth surface and a uniform spherical morphology; the average diameter is approximately 173 nm. Figure 2 C).

[0035] 2) Preparation of WC@Cla biohybrids: First, positively charged polyallylamine hydrochloride (PAH) was used to target *Westernella esculenta* (…). Weissella cibaria WC (WC) culture was modified to introduce a positive surface charge. The WC culture was diluted with ultrapure water to a concentration of 10. 8 Add an equal volume of PAH solution (20 mg / mL) to CFU / mL. Gently vortex the mixture for 60 seconds, then centrifuge at 6000g for 3 minutes to collect PAH-modified WC cells, and wash three times with ultrapure water. Then, add 1 mL of PAH-modified WC suspension (10... 8 WC@Cla biohybrids (CFU / mL) were mixed with 2 mg of Cla@PLGA nanoparticles (dissolved in ultrapure water, concentration 2 mg / mL) and gently shaken for 60 seconds to promote electrostatic self-assembly. The resulting WC@Cla biohybrids were collected by centrifugation (6000 × g, 3 min) (TEM image shown). Figure 2 As shown in Figure D, it can be seen that Cla@PLGA nanoparticles were successfully adsorbed onto the WC surface, and then thoroughly washed with ultrapure water.

[0036] 3) Preparation of WC@Cla MN microneedle patches: WC@Cla bio-hybrid suspension (10 8 To prepare the microneedle (MN) casting solution, CFU / mL was mixed with PVA (18% w / v) and sucrose (18% w / v) in MRS medium. Approximately 100 μL of the mixture was added dropwise to the surface of the microneedle mold and centrifuged at 3500 rpm for 8 minutes to ensure complete filling of the mold cavity. The mold was then dried in a 37°C oven for 48 hours. After curing, the microneedle patch (which looks like a...) was carefully applied... Figure 2 As shown in Figure F, scanning electron microscopy of the cross-section revealed that WC cells were completely encapsulated within the microneedle matrix layer, and the pore size of the matrix was smaller than the diameter of the WC cells, which effectively prevented premature leakage of probiotics. Figure 2 G)) Remove from the mold and store in a desiccator for later use.

[0037] The zeta potentials of WC and PAH-modified WC, Cla@PLGA nanoparticles and WC@Cla biohybrids were tested, and the results are as follows: Figure 2 As shown in Figure E, the WC@Cla biohybrid was successfully synthesized.

[0038] Furthermore, the mechanical strength of the WC@Cla MN microneedle patch was tested, and the results showed that the microneedles had sufficient strength to penetrate the stratum corneum without deformation. Figure 2 H).

[0039] Furthermore, to verify the permeability of the WC@Cla MN microneedle patch, rhodamine B was incorporated into WC@Cla MN as a fluorescent tracer and applied to the skin on the back of mice. The results showed that a clear fluorescent signal of rhodamine B was detected at the insertion site and within the dermis. Figure 2 I) confirmed effective transdermal penetration and delivery.

[0040] Furthermore, the growth viability of WC in the WC@Cla biohybrid was tested. Specifically, WC bacterial suspensions and WC@Cla biohybrid suspensions of the same concentration were inoculated onto MRS agar plates and incubated at 37°C for 24 hours. The colony-forming units (CFU) on the plates were counted to assess the survival of WC in the microneedles. Figure 2 The results showed no significant difference in survival counts between WC and WC@Cla MN microneedles, indicating that PAH modification and nanoparticle assembly did not impair probiotic survival. Figure 2 K).

[0041] Furthermore, the stability of WC in the WC@Cla MN microneedle patch was tested. Specifically, WC@Cla MN was stored at 4°C, and samples were taken on days 0, 10, 20, and 30. The patch was dissolved in 2 mL of sterile physiological saline, and 50 μL was spread onto an MRS agar plate. After incubation, CFU counts were recorded to determine the stability of the probiotics during storage. The results showed that WC encapsulated in the microneedles remained stable after 20 days of storage at 4°C, and only a very small decrease in colony count was observed after the microneedles were dissolved in PBS. Figure 2 L, 2M).

[0042] The above results demonstrate that the probiotic-nanomedicine composite microneedle patch prepared in this invention possesses good structural stability, reliable mechanical integrity, and sustained probiotic activity. These properties make it promising for achieving controlled dual release of NO and Cla generated by WC, and provide a solid foundation for subsequent biological evaluation and therapeutic applications in AGA.

[0043] Example 2: Study on the immunomodulatory activity of NO released from WC in WC@ClaMN microneedle patches In this embodiment, to investigate the immunomodulatory activity of NO released from WC in the WC@Cla MN microneedle patch, WC@Cla MN was placed in the upper chamber of a Transwell system, while RAW264.7 macrophages were cultured in the lower chamber. The experiment was divided into five groups: Control group, LPS group, LPS+Cla MN treatment group, LPS+WC MN treatment group, and LPS+WC@Cla MN treatment group.

[0044] Immunofluorescence staining results showed significant differences among the treatment groups. LPS stimulation induced strong CD86 expression and reduced CD206 expression, confirming the typical M1 phenotype. In contrast, RAW264.7 cells exposed to WC MN or WC@Cla MN exhibited weakened CD86 expression and enhanced CD206 expression, with the WC@Cla MN group showing the most significant M2 polarization. These results indicate that WC-derived NO can effectively shift macrophages from a pro-inflammatory M1 state to an anti-inflammatory M2 phenotype, contributing to the establishment of a microenvironment conducive to hair follicle repair. Figure 3 ).

[0045] In addition, observations showed that human umbilical vein endothelial cells (HUVECs) treated with WC@Cla MN formed dense and well-branched capillary-like structures, indicating that WC@Cla MN has strong pro-angiogenic activity. Figure 4 ).

[0046] Example 3: Study on the promoting effect of WC@Cla MN microneedle patches on in vivo hair follicle regeneration In this embodiment, mice were used as experimental animals, and an AGA mouse model was established to evaluate the therapeutic effect of WC@Cla MN.

[0047] Specifically, an AGA model was first established using male C57BL / 6 mice: a 2×2cm mouse was selected. 2The back area of ​​the mice was shaved, and then treated daily with a 0.2% (w / v) testosterone solution (dissolved in 50% ethanol) for a total of 28 days. Mice were randomly divided into five groups: Control group, AGA group, AGA+Cla MN treatment group, AGA+WC MN treatment group, and AGA+WC@Cla MN treatment group. Pigmentation and hair regrowth were observed on days 1, 10, 16, 19, and 21 in each treatment group. The results showed that mice treated with WC MN, Cla MN, or WC@Cla MN began to show gradual skin pigmentation from around day 10, indicating that the hair follicles re-entered the anagen phase. Among all treatment groups, WC@Cla MN caused the fastest and most significant pigmentation, accompanied by a significant improvement in hair regrowth. Figure 5 ).

[0048] In addition, SEM analysis was performed on the regenerated hair of mice in each treatment group 21 days later. The results showed that the hair coverage and hair shaft diameter of the WC@Cla MN group were significantly higher than those of the groups treated with WC MN or Cla MN alone. Figure 6 ).

[0049] The above results indicate that NO produced by probiotics and the drug Cla in WC@Cla MN microneedle patches have a significant synergistic effect on hair follicle recovery.

[0050] Furthermore, on day 21, mice in each treatment group were euthanized, and skin tissue from the back of the mice was collected, fixed with 4% paraformaldehyde, and subjected to immunofluorescence staining.

[0051] To evaluate the angiogenesis and proliferative activity of mouse skin after treatment, immunofluorescence was used to detect the expression of CD31 and Ki67 proteins in each treatment group. The results showed that the WC@Cla MN group exhibited the most extensive and continuous vascular network, indicating that the blood supply around the hair follicle was effectively restored. Simultaneously, WC@Cla MN treatment restored Ki67 expression, reflecting enhanced cell division and tissue renewal within the hair follicle region. Figure 7 ).

[0052] To evaluate the regulation of the skin microenvironment in mice under each of the above treatment groups, the expression of IL-6 and TNF-α in each treatment group was detected using immunofluorescence. The results showed that WC@ClaMN significantly reduced the expression of IL-6 and TNF-α, indicating that WC@ClaMN can effectively rebalance the inflammatory environment around hair follicles. Figure 8 ).

[0053] In summary, this invention utilizes a probiotic-nanomedicine composite microneedle patch as a delivery system, enabling efficient delivery of probiotics and drugs while avoiding systemic exposure and improving safety. Furthermore, PLGA nanoparticles control the slow release of drugs, significantly improving drug utilization and therapeutic efficacy. Additionally, the microneedle matrix layer encapsulation maintains the activity of probiotics, achieving a localized, long-lasting effect. When this probiotic-nanomedicine composite microneedle patch is used to treat androgenetic alopecia, it effectively treats the condition by promoting hair growth, increasing hair follicle density and diameter, improving microcirculation and cell proliferation, and reducing the expression level of inflammatory factors.

[0054] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0055] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A probiotic-nanomedicine composite microneedle patch, characterized in that, It includes a backing layer and microneedles, wherein the microneedles include a microneedle matrix layer and a probiotic-nanomedicine complex encapsulated in the microneedle matrix layer; The probiotic-nanomedicine complex is formed by electrostatic self-assembly of modified, positively charged probiotics that produce nitric oxide and drug-loaded PLGA nanoparticles.

2. The probiotic-nanomedicine composite microneedle patch according to claim 1, characterized in that, The preparation of the modified, positively charged probiotics that produce nitric oxide includes: The polyallylamine hydrochloride solution was mixed with the probiotic solution, and after centrifugation, the modified probiotics with positive charge and nitric oxide production were obtained. The probiotics include Weissella esculenta.

3. The probiotic-nanomedicine composite microneedle patch according to claim 2, characterized in that, The concentration of the polyallylamine hydrochloride solution is 15-25 mg / mL, the content of the probiotic liquid is 0.5 x 10 8 -1.5 x 10 8 CFU / mL; and the volume ratio of the polyallylamine hydrochloride solution to the probiotic liquid is (0.5-1.5):(0.5-1.5).

4. The probiotic-nanomedicine composite microneedle patch according to claim 1, characterized in that, In the drug-loaded PLGA nanoparticles, the mass ratio of the drug to the PLGA is 1:(1-3), and the drug includes claspone.

5. The probiotic-nanomedicine composite microneedle patch according to claim 1, characterized in that, The ratio of the number of modified, positively charged probiotics that produce nitric oxide to the mass ratio of the drug-loaded PLGA nanoparticles is (0.5 × 10⁻⁶). 8 -1.5×10 8 CFU (1-3 mg).

6. The probiotic-nanomedicine composite microneedle patch according to claim 1, characterized in that, The microneedle matrix layer comprises polyvinyl alcohol and sucrose, and the mass ratio of polyvinyl alcohol to sucrose is (0.5-1.5):(0.5-1.5).

7. A method for preparing a probiotic-nanomedicine composite microneedle patch as described in any one of claims 1-6, characterized in that, Includes the following steps: After mixing the drug organic solution with the PLGA organic solution, the mixture was added dropwise to a first polyvinyl alcohol aqueous solution. After ultrasonic emulsification, the resulting emulsion was ultrasonicated under ice bath conditions. Then, a second polyvinyl alcohol aqueous solution was added. After removing the organic solvent under reduced pressure, washing, and drying, drug-loaded PLGA nanoparticles were obtained. A probiotic suspension modified to carry a positive charge and produce nitric oxide and a PLGA nanoparticle solution loaded with drugs were provided respectively. The two were mixed and electrostatically self-assembled. After centrifugation and washing, a probiotic-nanomedicine complex was obtained. A probiotic-nanomedicine complex solution is provided, and the probiotic-nanomedicine complex solution is mixed with polyvinyl alcohol and sucrose in MRS medium to obtain a microneedle casting solution; The microneedle casting solution was dropped into a microneedle mold, and after centrifugation and drying, a probiotic-nanomedicine composite microneedle patch was obtained.

8. The preparation method according to claim 7, characterized in that, In the step of obtaining drug-loaded PLGA nanoparticles, the concentration of the drug organic solution is 40-45 mg / mL, the concentration of the PLGA organic solution is 1-3 mg / mL, the mass concentration of the first polyvinyl alcohol aqueous solution is 2-4%, and the mass concentration of the second polyvinyl alcohol aqueous solution is 0.2-0.4%. The volume ratio of the drug organic solution, the PLGA organic solution, the first polyvinyl alcohol aqueous solution, and the second polyvinyl alcohol aqueous solution is (0.1-0.2):(8-12):(2-4):(8-12).

9. The preparation method according to claim 7, characterized in that, In the step of obtaining the probiotic-nanomedicine complex, the content of the probiotic suspension is 0.5 × 10⁻⁶. 8 -1.5×10 8 The concentration of the drug-loaded PLGA nanoparticle solution is 1-3 mg / mL, and the volume ratio of the probiotic suspension to the drug-loaded PLGA nanoparticle solution is (0.5-1.5):(0.5-1.5). In the step of obtaining the probiotic-nanomedicine composite microneedle patch, the content of the probiotic-nanomedicine composite solution is 0.5 × 10⁻⁶. 8 -1.5×10 8 The concentration of polyvinyl alcohol is 15-20% and the concentration of sucrose is 15-20% (CFU / mL).

10. The use of the probiotic-nanomedicine composite microneedle patch according to any one of claims 1-6 or the probiotic-nanomedicine composite microneedle patch prepared by the preparation method according to any one of claims 7-9 in any of the following: 1) To prepare drugs for the prevention and / or treatment of androgenetic alopecia; 2) Prepare drugs to promote hair follicle regeneration; 3) Prepare drugs for improving scalp microcirculation.

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