Self-assembled collagen composite microneedle patch and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a self-assembled collagen composite microneedle patch, a preparation method and application. The microneedle patch comprises a microneedle body matrix of array structure and nanoparticles embedded on the tip of the microneedle body matrix, the microneedle body matrix is a soluble array structure microneedle body constructed by recombinant type III collagen, and the nanoparticles are formed by self-assembly of baicalin and arginine in an aqueous phase through intermolecular forces. The application uses baicalin and arginine as raw materials, combines the soluble microneedle containing recombinant type III collagen after self-assembly of molecules, and finally forms a composite microneedle patch with hair follicle targeting function, solves the problem of uneven dispersion caused by poor solubility in the aqueous phase matrix of the microneedle, and discards the synthetic adjuvant in the traditional nanocarrier which may affect the skin safety and the hair follicle microenvironment, thereby guaranteeing the biocompatibility of the preparation from the source.

Description

Technical Field

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

[0002] Androgenetic alopecia (AGA) is a common hair loss disorder caused by a combination of genetic factors and abnormal androgen metabolism. Clinically, it manifests as progressive miniaturization of hair follicles, leading to thinning, thinning, and even hair loss, severely impacting patients' appearance, mental health, and quality of life. It has become a significant public health concern in dermatology and regenerative medicine. However, current first-line clinical drugs such as finasteride and minoxidil generally have limitations, including significant side effects, high relapse rates, and large individual variability in efficacy, falling far short of meeting patients' clinical needs. Recent studies have shown that AGA is not merely a localized skin disease; it also has potential links to systemic diseases such as metabolic syndrome and cardiovascular disease. Therefore, in-depth research into the pathogenesis of AGA and the development of novel treatment strategies have significant scientific and clinical value.

[0003] Existing drug-loaded soluble microneedle systems for treating androgenetic alopecia include: Xu S.; Zhou L.; Zhao H.; Li S. Advances in transdermal delivery systems for treating androgenetic alopecia. Pharmaceutics. 2025, 17(8), 984-1007; Xiong S.; Li Z.; Jiao S.; Xiao T.; Wu Y.; Chen C.; Guo S.; Li X.; Pan Z.; Li J.; Xu Y. Black phosphorus nanosheets encapsulated microneedle for multifunctional therapy for androgenic alopecia. J. Nanobiotechnology. 2025, 23(1), 147-167; Xiong S.; Ying W.; Xiao T.; Liu Z.; Fu H.; Chai G.; Xu Y. Hair follicle-targeted baicalin nanocrystal delivery using microneedles for long-acting treatment of androgenetic alopecia. alopecia. Int. J. Pharm. 2026, 693, 126675-126692. Typically, drugs, small molecules, or biological agents are first prepared into nanoformulations, and then loaded into a microneedle matrix through physical mixing or simple encapsulation. Ultimately, the physical puncture action of the microneedles promotes transdermal drug absorption, aiming to improve local delivery efficiency and reduce systemic side effects. However, traditional nanocarriers pose safety risks; their excipients may affect skin barrier function or disrupt the homeostasis of the hair follicle microenvironment. Furthermore, due to the lack of an efficient carrier system for solubilizing and protecting baicalin, the drug is prone to degradation during preparation, storage, and release, resulting in large batch-to-batch variations in actual delivered doses, low bioavailability, and difficulty in achieving effective drug release. To improve the drug loading and release behavior of baicalin, existing technologies often rely on synthetic polymers or organic solvents as carriers or excipients. However, these exogenous synthetic components may induce local immune responses or inflammation, and their metabolites or residual solvents also pose potential safety risks, thereby interfering with the physiological homeostasis of the hair follicle microenvironment.More importantly, current research mainly focuses on the single aspect of drug delivery, while the pathological process of androgenetic alopecia involves multiple dimensions: on the one hand, it manifests as structural degradation of the hair follicle microenvironment, such as an imbalance in extracellular matrix remodeling; on the other hand, it is accompanied by cellular functional decline, such as impaired mitochondrial autophagy. Delivering drugs with only a single mechanism of action is insufficient to simultaneously reverse microenvironmental aging and cellular dysfunction, thus limiting efficacy and failing to achieve the original regeneration of hair follicle structure.

[0004] In summary, although existing technologies have improved transdermal efficiency through microneedles, they still have significant shortcomings in terms of efficient and stable drug loading, formulation biosafety, and synergistic regulation of multidimensional pathological processes in the hair follicle microenvironment. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a self-assembled collagen composite microneedle patch, its preparation method, and its application. The microneedle patch uses baicalin and arginine as raw materials to construct BA / Arg nanomedicine through molecular self-assembly; it is further combined with soluble microneedles containing recombinant type III collagen to ultimately form a composite delivery platform with hair follicle targeting function. This solves the problem of uneven dispersion caused by poor solubility in the aqueous microneedle matrix, eliminates synthetic excipients in traditional nanocarriers that may affect skin safety and the hair follicle microenvironment, and ensures the biocompatibility of the formulation from the source. This green process significantly improves the safety of topical medication for treating androgenetic alopecia while ensuring therapeutic efficacy, achieving efficient delivery.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] The first objective of this invention is to provide a self-assembled collagen composite microneedle patch, comprising a base, an array of microneedles, and nanoparticles embedded in the tips of the microneedles. The base is attached to the tail of the microneedles. The microneedle matrix is ​​a soluble array of microneedles constructed from recombinant type III collagen. The nanoparticles are formed by self-assembly of baicalin and arginine in an aqueous phase through intermolecular forces. The molar ratio of baicalin to arginine is 1:1 to 4.

[0008] This invention aims to provide a composite microneedle patch capable of stably loading and efficiently delivering hydrophobic drugs in an aqueous phase. Its core structure comprises a composite structure with a dual relationship of "self-assembled nanoparticles" and "matrix support." Using baicalin and arginine as raw materials, stable nanoparticles spontaneously assemble in the aqueous phase through intermolecular forces such as hydrogen bonds, π-π stacking, and electrostatic interactions. These self-assembled particles constitute the drug-active core of the system, responsible for encapsulating and protecting the hydrophobic active ingredient baicalin, and imparting good water dispersibility and cell penetration. Then, soluble microneedles constructed from recombinant type III collagen are arranged in an array on a microneedle mold backing layer, forming a patch-like drug delivery method. The self-assembled nanoparticles are dispersed and loaded within the microneedle tip matrix, forming the composite microneedle patch. This invention utilizes a self-assembled nanoscale "drug carrier" as its core, uniformly encapsulated within a "soluble microneedle framework" formed by rhCol III, serving as its outer shell. The microneedles, acting as macroscopic drug delivery devices, penetrate the stratum corneum of the skin. The nano-self-assembled system, as a submicron-scale drug-carrying unit, is responsible for further delivering and releasing the drug deep into the hair follicle after the microneedles dissolve, achieving efficient loading and stable delivery of the hydrophobic drug baicalin. This self-assembled nanosystem encapsulates the drug within a nanoscale hydrophilic interface, solving the problem of uneven dispersion caused by poor solubility in the aqueous matrix of microneedles.

[0009] In some embodiments, the particle size of the nanoparticles is 100 nm to 200 nm.

[0010] In some embodiments, the method for preparing nanoparticles includes the following steps: Baicalin was added to an aqueous solution of arginine and then subjected to ultrasound to drive self-assembly in the aqueous phase through intermolecular forces. After drying to remove the solvent, nanoparticles were obtained.

[0011] In some embodiments, the concentration of the arginine aqueous solution is 0.1 mg / mL to 0.4 mg / mL, the ultrasonic power is 80 W to 100 W, and the time is 30 min to 60 min.

[0012] In some embodiments, after ultrasonication, most of the solvent is removed by rotary evaporation at 55°C for 30 min, and then vacuum drying at 40°C for 24 h is performed to obtain nanoparticles.

[0013] A second objective of this invention is to provide a method for preparing the aforementioned self-assembled collagen composite microneedle patch, such as... Figure 1 As shown, it includes the following steps: S1. Disperse the nanoparticles in an aqueous solution containing recombinant type III collagen, add hyaluronic acid, and stir to form a semi-solid gel solution.

[0014] S2. Inject the semi-solid gel solution into a polydimethylsiloxane mold with a microneedle array structure, centrifuge to fill the cavity of the microneedle tip with the semi-solid gel solution, repeat until the cavity of the microneedle tip is completely filled with the semi-solid gel solution, use centrifugation to precipitate the nanoparticles to the tip, then inject the base matrix solution, centrifuge again and solidify, peel off to obtain a self-assembled collagen composite microneedle patch.

[0015] In some embodiments, the mass-to-volume ratio of nanoparticles to recombinant type III collagen aqueous solution is 1 mg to 5 mg: 1 mL, and the mass concentration of recombinant type III collagen solution is 0.5 wt.% to 20 wt.%.

[0016] Understandably, the amount of nanoparticles used can be selected according to the amount of baicalin used for hair regeneration in androgenetic alopecia. The amount of baicalin used is a commonly used amount in the art that has a good effect on hair regeneration, but the drug loading capacity is still limited. This invention disperses and loads self-assembled nanoparticles into the microneedle tip matrix. The microneedle tip matrix can be adjusted by the corresponding preparation mold to achieve carrier-free loading of baicalin. It can control the amount of baicalin used and load it as needed, thereby achieving efficient loading.

[0017] In some embodiments, the mass ratio of nanoparticles to hyaluronic acid is 1 to 5:250, and the molecular weight of hyaluronic acid is 52 kDa.

[0018] In some embodiments, the substrate matrix solution is formed by adding hyaluronic acid with a molecular weight of 52 kDa, hyaluronic acid with a molecular weight of 200 kDa to 400 kDa, and dextran 40 to water in a mass ratio of 1:1:1 and allowing them to swell overnight. The concentrations of the hyaluronic acid with a molecular weight of 52 kDa, the hyaluronic acid with a molecular weight of 200 kDa to 400 kDa, and dextran 40 in the substrate matrix solution are all 10 wt.

[0019] In some embodiments, the centrifugation speed is 3000 rpm to 4000 rpm, the time is 3 min to 5 min, the curing temperature is 4°C, and the time is 24 h to 48 h.

[0020] This invention utilizes an all-aqueous, green preparation process to ultimately form a composite delivery system with hair follicle-targeting function. The all-aqueous process avoids the risk of skin irritation caused by residual organic solvents. Recombinant type III collagen, as a component of the human extracellular matrix, is not only non-immunogenic but also actively participates in and guides normal tissue repair. It eliminates the need for synthetic excipients in traditional nanocarriers that may affect skin safety and the hair follicle microenvironment, ensuring the biocompatibility of the formulation from the source. This green process significantly improves the safety of topical medication for treating androgenetic alopecia while ensuring therapeutic efficacy, achieving efficient delivery. The process route is simple, the conditions are mild, and it has good reproducibility, making it easy to scale up production.

[0021] A third objective of this invention is to provide the application of the above-mentioned self-assembled collagen composite microneedle patch in the preparation of a drug for androgenetic alopecia.

[0022] Compared with the prior art, the present invention has the following advantages: The self-assembled collagen composite microneedle patch provided by this invention is based on a composite structure with a dual relationship between "self-assembled nanoparticles" and "matrix support." Using baicalin and arginine as raw materials, stable nanoparticles are spontaneously assembled in an aqueous phase through intermolecular forces such as hydrogen bonds, π-π stacking, and electrostatic interactions. This self-assembled nanosystem encapsulates the drug within a nanoscale hydrophilic interface, loading baicalin and endowing it with good water dispersibility and cell permeability. The self-assembled nanoparticles are then dispersed and loaded onto a soluble array structure constructed from recombinant type III collagen. The needle tip is attached to the needle tail of the microneedle body through the base to form a composite microneedle patch. The microneedle, as a macroscopic drug delivery device, is responsible for penetrating the stratum corneum of the skin. The nano-self-assembled body, as a submicron-level drug-carrying unit, is responsible for further delivering and releasing the drug into the depths of the hair follicle after the microneedle dissolves. This achieves efficient loading and stable delivery of the hydrophobic drug baicalin, solving the problem of uneven dispersion caused by its poor solubility in the aqueous matrix of microneedles. It also eliminates the synthetic excipients in traditional nanocarriers that may affect skin safety and the microenvironment of the hair follicle, ensuring the biocompatibility of the formulation from the source.

[0023] This invention utilizes an all-aqueous, green preparation process to ultimately form a composite delivery system with hair follicle-targeting function. The all-aqueous process avoids the risk of skin irritation caused by residual organic solvents. Recombinant type III collagen, as a component of the human extracellular matrix, is not only non-immunogenic but also actively participates in and guides normal tissue repair. It eliminates the need for synthetic excipients in traditional nanocarriers that may affect skin safety and the hair follicle microenvironment, ensuring the biocompatibility of the formulation from the source. This green process significantly improves the safety of topical medication for treating androgenetic alopecia while ensuring therapeutic efficacy, achieving efficient delivery. The process route is simple, the conditions are mild, and it has good reproducibility, making it easy to scale up production. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the preparation of BA / Arg self-assembled nanoparticles according to the present invention.

[0025] Figure 2 The images show the physical specimens and properties of the BA / Arg self-assembled nanoparticles of this invention. Figure 2 In the diagram, Figure A shows the appearance, Figure B shows the Tyndall effect, and Figure C shows the particle size.

[0026] Figure 3 This is a microscopic morphology diagram of the BA / Arg self-assembled nanoparticles of the present invention.

[0027] Figure 4 This is a differential scanning calorimeter of the BA / Arg self-assembled nanoparticles of the present invention.

[0028] Figure 5 This is the 1H NMR spectrum of the BA / Arg self-assembled nanoparticles of this invention.

[0029] Figure 6 This is a structural model diagram of the two small molecules BA and Arg in molecular dynamics simulations of this invention. Figure 6 Figure A shows the structural models of BA and Arg, both displayed in VDW mode, while Figure B is a representative snapshot of the self-assembly process.

[0030] Figure 7 This is a diagram illustrating the self-assembly mechanism analysis of BA and Arg molecules using molecular dynamics simulations in this invention. Figure 7 In the diagram, Figures A through B show the detailed process of intermolecular interactions during the self-assembly of BA and Arg nanoparticles.

[0031] Figure 8 This is an in vitro release curve of the BA / Arg self-assembled nanoparticles of the present invention.

[0032] Figure 9 This is a graph showing the fluorescence colocalization of C6-labeled mitochondria in different groups and the quantitative analysis of the fluorescence intensity of internalized C6. Figure 9 In the diagram, Figure A shows the fluorescence colocalization of mitochondria labeled with C6 in different groups, and Figure B shows the quantitative analysis of the fluorescence intensity of internalized C6.

[0033] Figure 10 The figure shows the anti-aging results of BA, Arg, and BA / Arg self-assembled nanoparticles in a dihydrotestosterone-induced AGA cell model. Figure 10 In the image, Figure A is a visual representation of aging-related β-galactosidase staining, and Figure B is a semi-quantitative representation of the area of ​​aging-related β-galactosidase-positive staining regions.

[0034] Figure 11The image shows the ultrastructure of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, which is the blank control of this invention.

[0035] Figure 12 This is an ultrastructure diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, representing the DHT control of this invention.

[0036] Figure 13 This is an ultrastructural diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, representing the DHT+BA control of this invention.

[0037] Figure 14 This is an ultrastructural diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, representing the DHT+Arg control of this invention.

[0038] Figure 15 This is an ultrastructural diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model of the present invention, using DHT+BA / Arg.

[0039] Figure 16 The figure shows the ATP content detection results of BA, Arg, and BA / Arg self-assembled nanoparticles in the dihydrotestosterone-induced AGA cell model of the present invention.

[0040] Figure 17 The image shows the mitochondrial membrane potential staining results of BA, Arg, and BA / Arg self-assembled nanoparticles in a dihydrotestosterone-induced AGA cell model.

[0041] Figure 18 This is a schematic diagram of the construction process of BA / Arg@rhCol MNs of the present invention.

[0042] Figure 19 The image shows the cell viability results of microneedles prepared from recombinant type III collagen in different proportions of the present invention after co-incubation with human dermal papilla cells for 24 hours.

[0043] Figure 20 This document presents a digital camera morphology visualization, microneedle parameter diagram, mechanical strength and insertion depth diagram of BA / Arg@rhCol MNs according to Embodiment 1 of the present invention. Figure 20 In the diagram, Figure A is a visual representation of the digital camera's shape, Figure B is a schematic diagram of the microneedle parameters, Figure C is a displacement-fracture force diagram, and Figure D is a diagram of the insertion depth.

[0044] Figure 21 This is a graph showing the in vitro release behavior of BA / Arg@rhCol MNs in Example 1 of the present invention.

[0045] Figure 22This is a visual representation of the hair regeneration effect of BA / Arg@rhCol MNs in a testosterone-induced AGA mouse model according to Example 1 of the present invention. Figure 22 In the table, A represents the normal control group, B represents the model group, C represents the BA / Arg nanoparticle group, D represents the blank microneedle group (Blank MNs), E represents the microneedle group (BA / Arg@rhCol MNs), and F represents the positive control minoxidil group (MXD). Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] It should be noted that the technical terms used in this invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0048] This invention provides a self-assembled collagen composite microneedle patch. Through structural design and process innovation, it integrates nano-self-assembly technology with a soluble microneedle platform to achieve efficient and stable drug loading, green and safe preparation, and multi-dimensional synergistic repair of the hair follicle microenvironment. The composite microneedle patch includes an array-structured microneedle matrix and nanoparticles embedded at the tips of the microneedle matrix. The microneedle matrix is ​​a soluble array-structured microneedle body constructed from recombinant type III collagen. The nanoparticles are formed by self-assembly of baicalin and arginine in an aqueous phase through intermolecular forces.

[0049] This invention enables the efficient loading and stable delivery of the hydrophobic drug baicalin. A self-assembled nanosystem encapsulates the drug within a nanoscale hydrophilic interface. Based on a microneedle array design, it achieves a synergistic effect of "physical penetration-drug release-multidimensional regulation." 1. Physical penetration and barrier overcoming: When the microneedle array is applied to the skin surface, its needle tip can penetrate the dense stratum corneum painlessly and minimally invasively, forming temporary microchannels in the skin and physically bypassing the main transdermal barrier.

[0050] 2. Matrix Dissolution and Targeted Release: The microneedles rapidly dissolve in the skin's tissue fluid, releasing the embedded baicalin / arginine self-assembled nanomedicine. This process delivers the drug directly to the superficial dermis, specifically the hair follicle-rich area.

[0051] 3. Nanocarrier-mediated hair follicle targeting and cell delivery: The released baicalin / arginine nanoparticles, due to their suitable nanoscale size, promote the efficient uptake of baicalin by hair follicle cells such as dermal papilla cells.

[0052] 4. Multidimensional Synergistic Therapeutic Effect: The delivered active ingredients exert a dual function at the site of action: Recombinant Type III Collagen: As a key component of ECM, it directly replenishes and reshapes the dermal microenvironment surrounding the hair follicle, improving structural support. Baicalin / Arginine Nanoparticles: Baicalin exerts pharmacological activity within cells, improving cellular energy metabolism and antioxidant capacity by activating pathways such as mitochondrial autophagy, reversing hair follicle cell aging. Together, they synergistically intervene in the pathological process of androgenetic alopecia from two levels: "tissue microenvironment repair" and "cellular function recovery."

[0053] The following specific examples provide further details. Baicalin is abbreviated as BA, arginine as Arg, and recombinant type III collagen was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and its abbreviation is rhCol III.

[0054] Example 1 A method for preparing a self-assembled collagen composite microneedle patch, such as... Figure 18 As shown, it includes the following steps: Preparation of S1, BA / Arg self-assembled nanoparticles: 4 mg of Arg was accurately weighed and added to 10 mL of deionized water. After mixing thoroughly, an arginine aqueous solution with a pH of 9.45 was obtained. Then, 10 mg of BA was added, dissolved, and mixed thoroughly. At this point, the molar ratio of BA to Arg was approximately 1:1. The acidic phenolic hydroxyl groups of BA and the basic groups of Arg underwent a neutralization reaction, causing a change in the pH of the system, which decreased from 9.45 to 6.39. Subsequently, the mixture was sonicated at 100 W for 30 min, driving the spontaneous assembly of BA and Arg molecules into nanoparticles through non-covalent interactions, resulting in a nanoparticle suspension. The obtained nanoparticle suspension was then rotary evaporated at 55 °C for 30 min to remove most of the solvent, and then dried in a vacuum drying oven at 40 °C for 24 h to obtain the assembled nanoparticle solid powder, named BA / Arg self-assembled nanoparticles.

[0055] S2. Weigh 2 mg of BA / Arg self-assembled nanoparticles and disperse them in 1 mL of an aqueous solution containing 1 wt.% rhCol Ⅲ. Mix well, then add 0.25 g of hyaluronic acid with a molecular weight of 52 kDa. Mix thoroughly and allow to swell overnight to obtain a homogeneous and fully swollen semi-solid colloidal solution.

[0056] S3. Take a semi-solid gel-like solution and inject it into a polydimethylsiloxane mold with a microneedle array structure. Then, perform a centrifugal filling operation: centrifuge at 4000 rpm for 3 min, repeat 6 times, until the needle tip matrix evenly covers the entire microneedle mold, ensuring that the solution fully fills the microneedle tip cavity. After that, add 0.12 g of base matrix solution to the mold. The base matrix solution is prepared by adding HA with a molecular weight of 52 kDa, HA with a molecular weight of 200 kDa to 400 kDa, and dextran 40 to water in a mass ratio of 1:1:1 and swelling overnight. The concentrations of HA with a molecular weight of 52 kDa, HA with a molecular weight of 300 kDa, and dextran 40 are all 10 wt.%. Then, centrifuge again at 4000 rpm for 3 min, and centrifuge 40 4 times to ensure uniform distribution of the base matrix. Finally, place the centrifuged mold in a desiccator and dry at 4℃ for 48 h to obtain a self-assembled collagen composite microneedle patch, named BA / Arg@rhCol MNs composite microneedle patch.

[0057] Example 2 A method for preparing a self-assembled collagen composite microneedle patch differs from Example 1 in that the molar ratio of BA to Arg in S1 is 1:3.9.

[0058] Example 3 A method for preparing a self-assembled collagen composite microneedle patch differs from Example 1 in that the molar ratio of BA to Arg in S1 is 1:1.96.

[0059] Example 4 A method for preparing a self-assembled collagen composite microneedle patch differs from Example 1 in that the molar ratio of BA to Arg in S1 is 1:1.3.

[0060] Example 5 A method for preparing a self-assembled collagen composite microneedle patch differs from Example 1 in that the concentration of the aqueous solution of rhCol Ⅲ in S2 is 0.5 wt.%.

[0061] Example 6 A method for preparing a self-assembled collagen composite microneedle patch differs from Example 1 in that the concentration of the aqueous solution of rhCol Ⅲ in S2 is 5 wt.%.

[0062] Example 7 A method for preparing a self-assembled collagen composite microneedle patch differs from Example 1 in that the concentration of the aqueous solution of rhCol Ⅲ in S2 is 10 wt.%.

[0063] The self-assembled collagen composite microneedle patches prepared in Examples 1 to 7 were tested, and the results are shown below.

[0064] First, this invention investigated the solubility of BA and Arg at different molar ratios, such as 1:1 to 1:4. The results showed that BA dissolved completely when the molar ratio was approximately 1:1. Further investigation was conducted without the addition of arginine; in this case, baicalin exhibited extremely low water solubility, with a concentration of 13.17 μg / mL ± 1.06 μg / mL as detected by high-performance liquid chromatography, making comparison impossible. Therefore, this invention further illustrates the invention using the BA / Arg self-assembled nanoparticles prepared in Example 1 and self-assembled collagen composite microneedle patches formed with different rhCol III concentrations.

[0065] Figure 1 This is a schematic diagram illustrating the preparation of the BA / Arg self-assembled nanoparticles of this invention. Figure 1 As shown, the preparation of BA / Arg self-assembled nanoparticles was as described in Example 1 above: 4 mg of Arg was accurately weighed, then 10 mL of deionized water was added, and the mixture was stirred evenly to obtain an arginine aqueous solution with a pH of 9.45. Then, 10 mg of BA was added, dissolved, and stirred evenly. At this point, the molar ratio of BA to Arg was approximately 1:1. Subsequently, the mixture was ultrasonically treated at 100 W for 30 min to drive the spontaneous assembly of BA and Arg molecules into nanoparticles through non-covalent interactions, resulting in a nanoparticle suspension. The obtained nanoparticle suspension was then rotary evaporated at 55 °C for 30 min to remove most of the solvent, and then dried in a vacuum drying oven at 40 °C for 24 h to obtain the assembled nanoparticle solid powder, which was named BA / Arg self-assembled nanoparticles.

[0066] Figure 2 The images show the physical specimens and properties of the BA / Arg self-assembled nanoparticles of this invention. Figure 2 In the diagram, A is the appearance, B is the Tyndall effect diagram, and C is the particle size distribution diagram. Figure 2 As shown in Figure A, the prepared BA / Arg is light yellow and clear; as Figure 2 As shown in Figure B, the Tyndall effect can be observed clearly under laser pointer illumination; as... Figure 2 As shown in Figure C, its particle size is 170.43 nm ± 6.44 nm, PDI is 0.36 ± 0.06, and Zeta potential is -29.67 mV ± 0.31 mV.

[0067] Figure 3 This is a microstructure diagram of the BA / Arg self-assembled nanoparticles of this invention. (Example:) Figure 3 As shown in the figure, transmission electron microscopy reveals that the BA / Arg self-assembled nanoparticles are spherical with a particle size distribution between 150 nm and 200 nm.

[0068] Figure 4 This is a differential scanning calorimetry (DSC) spectrum of the BA / Arg self-assembled nanoparticles of this invention. Figure 4As shown, BA has an endothermic peak at 213℃; Arg has two endothermic peaks at 224℃ and 243℃, respectively, with 224℃ being an endothermic melting peak and 243℃ being a decomposition peak; the physical mixture of the two exhibits the superposition characteristics of the DSC curves of BA and Arg; while the endothermic peak of BA / Arg nanoparticles decreases to 197℃, indicating that there is a significant interaction between Arg and BA.

[0069] Figure 5 This is the 1H NMR spectrum of the BA / Arg self-assembled nanoparticles of this invention. Figure 5 As shown, for BA, its benzene ring hydrogens are as follows: H a H b H c H d The chemical shifts of the protons were 7.84 ppm, 7.46 ppm, 6.86 ppm, and 6.65 ppm, respectively. After forming BA / Arg, the proton signals shifted to higher fields, reaching 7.32 ppm, 7.17 ppm, 6.34 ppm, and 6.15 ppm, respectively. This phenomenon indicates that the guanidinium group of Arg approaches the carboxyl group of BA through electrostatic interactions, allowing guanidinium protons such as NH to enter the shielding region of the aromatic ring of the flavonoid nucleus of BA. This weakens the magnetic field actually experienced by the protons, resulting in a shift of the resonance signal to higher fields. For Arg, the methylene proton signal directly connected to the guanidinium group on its side chain was located at 3.19 ppm, and similarly shifted to higher fields to 3.04 ppm in BA / Arg. This change is attributed to the strong intermolecular interactions between the guanidinium group of Arg and BA, mainly including electrostatic attraction and π-cation interactions. These interactions effectively dispersed and neutralized part of the positive charge density on the guanidinium group, weakening its induced deshielding effect on neighboring methylene protons, ultimately leading to a shift of the proton resonance signal to higher fields. In summary, BA / Arg's 1 The characteristic absorption peaks of HNMR indicate that the two are mainly bound together through electrostatic interactions, hydrogen bonding, and π-cation interactions.

[0070] Figure 6 This is a structural model diagram of the two small molecules BA and Arg in molecular dynamics simulations of this invention. Figure 6 In the diagram, Figure A shows schematic diagrams of the structural models of BA and Arg, both displayed in VDW mode, while Figure B is a representative snapshot of the self-assembly process. Figure 6 As shown, as the simulation time increases, BA and Arg gradually self-assemble and aggregate together to form nanoparticles. At 100 ns, all BA and Arg in the system have self-assembled into a stable nanoparticle.

[0071] Figure 7 This is a diagram illustrating the self-assembly mechanism analysis of BA and Arg molecules using molecular dynamics simulations in this invention. Figure 7In the diagram, Figures A through B illustrate the detailed process of intermolecular interactions during the self-assembly of BA and Arg nanoparticles. Figure 7 As shown, BA and Arg molecules mainly interact through electrostatic interactions, hydrogen bonds, van der Waals, π-Alkyl, and π-πstacking.

[0072] Figure 8 This is an in vitro release curve of the BA / Arg self-assembled nanoparticles of this invention. Figure 8 As shown, the cumulative release rate of BA / Arg self-assembled nanoparticles reached 94.6% within 8 hours, while that of BA raw material was only 22.29%. This indicates that BA / Arg nanoparticles significantly improved the solubility of BA, thereby greatly promoting its dissolution behavior.

[0073] C6-labeled BA / Arg nanoparticles were co-incubated with human dermal papilla (hDPC) cells and their co-localization with different mitochondria was used to quantify the amount entering the cells. The experimental method is as follows: Human dermal papilla (hDPC) cells were incubated at 1×10⁻⁶ cells / cells. 5 Seed at a density of 10 cells / mL on 10 mm 2 Cells were cultured overnight in confocal microscopy dishes until fully adhered. The culture medium was then removed, and the cells were co-incubated for 4 hours with fresh medium containing either 0.1 μg / mL free C6 or 0.1 μg / mL C6-BA / Arg, designated as the free C6 group and the C6-nanoparticle group, respectively. The culture medium was then removed, and the cells were washed with ice-cold PBS, followed by MitoTracker™ Red staining for 1 hour. After washing, the cells were washed three more times with PBS, and then fixed with paraformaldehyde for 15 minutes. The nuclei were stained with DAPI after washing with PBS. After 5 minutes of incubation, the cells were washed three more times with PBS. Finally, 200 μL of PBS solution was added to each culture dish, and all samples were imaged using a laser confocal scanning microscope.

[0074] Figure 9 This is a graph showing the fluorescence colocalization of C6-labeled mitochondria in different groups and the quantitative analysis of the fluorescence intensity of internalized C6. Figure 9 In the diagram, Figure A shows the fluorescence co-localization of C6-labeled mitochondria from different groups, and Figure B shows the quantitative analysis of the fluorescence intensity of internalized C6. Figure 9 As shown in Figure A, cells treated with free C6 exhibited only weak green fluorescence, while the BA / Arg self-assembled nanoparticle group showed significant green fluorescence in the cytoplasm and cell membrane of hDPC cells, indicating that the BA / Arg self-assembled nanoparticles can significantly enhance the cellular uptake of C6. Figure 9 As shown in Figure B, quantitative analysis results showed that the intracellular fluorescence intensity of the BA / Arg group was increased by 6.02 times compared with the free C6 group.

[0075] BA, Arg, and BA / Arg self-assembled nanoparticles were used in dihydrotestosterone-induced AGA cells. The experimental method was as follows: hDPCs were stimulated with 1 μM DHT to establish an in vitro AGA cell model. hDPC cells were grown at a concentration of 2 × 10⁶ cells / year. 5 Cells were seeded at a density of cells / dish in 12-well plates and cultured for 24 h before the culture medium was removed. Then, BA (20 μM), Arg, and BA / Arg groups (containing equal concentrations of 20 μM BA, with Arg diluted proportionally) were added to each well. Incubation continued for 24 h, with DHT-stimulated and unstimulated cells serving as blank controls. The cells were then labeled as Control, DHT-stimulated, DHT+BA, DHT+Arg, and DHT+BA / Arg groups. The culture medium was then removed, and the cells were washed twice with PBS. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Next, the cells were incubated with senescence-associated β-galactosidase (SA-β-gal) staining solution at 37°C in CO2-free conditions for 12 h. After incubation, the cells were washed, and blue SA-β-gal positive cells were imaged using a cell imaging system. The average percentage of SA-β-gal positive area was calculated using ImageJ.

[0076] Figure 10 The figure shows the anti-aging results of BA, Arg, and BA / Arg self-assembled nanoparticles in a dihydrotestosterone-induced AGA cell model. Figure 10 In the image, Figure A is a visual representation of aging-related β-galactosidase staining, and Figure B is a semi-quantitative map of the area of ​​aging-related β-galactosidase-positive staining regions. Figure 10 As shown in Figure A, after DHT treatment, the number of SA-β-gal positive cells (as indicated by the blue color) increased significantly, indicating that DHT successfully induced hDPC cell senescence. In contrast, the BA, Arg, and BA / Arg groups all significantly reduced the proportion of SA-β-gal positive cells, with the BA / Arg group showing the most significant reduction. Figure 10 As shown in Figure B, quantitative analysis results showed that the BA group, Arg group, and BA / Arg group reduced the proportion of SA-β-gal positive cells by 10.39%, 13.93%, and 24.32%, respectively.

[0077] Transmission electron microscopy was used to examine the ultrastructure of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model using different formulations such as BA, Arg, and BA / Arg. The experimental method was as follows: hDPC cells were stimulated with 1 μM DHT to establish an in vitro AGA cell model. hDPC cells were seeded in 6-well plates at a density of cells / plate and cultured for 24 h before the culture medium was removed. Then, BA (20 μM), Arg, and BA / Arg groups (containing an equal concentration of 20 μM BA, with Arg diluted proportionally) were added to each well. Incubation continued for 24 h, with DHT-stimulated and unstimulated cells serving as blank controls. The cells were named as Control, DHT, DHT+BA, DHT+Arg, and DHT+BA / Arg, respectively. Subsequently, the culture medium was removed, cells were collected, washed, and centrifuged to obtain a cell pellet. Then, 2.5% glutaraldehyde solution was added to fix the cell pellet for 24 h. Then, glutaraldehyde was removed, and the sections were fixed with a 1% osmium tetroxide solution for 2 hours. Finally, the sections were stained with uranium acetate and lead citrate, and then detected by TEM.

[0078] Figure 11 The image shows the ultrastructure of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, which is the blank control of this invention. Figure 12 This is an ultrastructure diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, representing the DHT control of this invention. Figure 13 This is an ultrastructural diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, representing the DHT+BA control of this invention. Figure 14 This is an ultrastructural diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model, representing the DHT+Arg control of this invention. Figure 15 This is an ultrastructural diagram of mitochondria and autophagosomes in a dihydrotestosterone-induced AGA cell model of the present invention, using DHT+BA / Arg. Figures 11 to 15 In the diagram of mitochondrial structure, arrows indicate mitochondria; in the diagram of autophagosome structure, arrows indicate autophagosomes. For example... Figures 11 to 15 As shown, DHT treatment significantly inhibited the formation of intracellular autophagosomes and induced signs of senescence in hDPC cells, specifically mitochondrial swelling, cristae disappearance, and vacuolization. BA, Arg, and BA / Arg treatments all reversed this inhibition, promoted autophagosome formation, increased the number of intracellular autophagosomes, and improved mitochondrial morphology to round, oval, and dumbbell shapes. BA / Arg treatment showed the most significant improvement, suggesting that the effects of BA / Arg may be related to the regulation of autophagy and mitochondrial function.

[0079] The ATP content of different formulations, such as BA, Arg, and BA / Arg, in a dihydrotestosterone-induced AGA cell model was detected. The experimental method is as follows: hDPC cells were stimulated with 1 μM DHT to establish an in vitro AGA cell model. hDPC cells were then cultured at a concentration of 1 × 10⁶ cells / cells. 6 Cells were seeded at a density of 1 cell / dish in 6 cm diameter culture dishes and incubated for 24 h, after which the culture medium was discarded. Subsequently, BA (20 μM), Arg, and BA / Arg groups (containing equal concentrations of 20 μM BA, with Arg diluted proportionally) were added to each well. Incubation continued for another 24 h, with DHT-stimulated and unstimulated cells serving as blank controls. The cells were named as follows: Control (normal group), DHT-stimulated group, DHT+BA group, DHT+Arg group, and DHT+BA / Arg group. After incubation, the culture medium was removed, cells were collected, washed, and centrifuged to obtain a cell pellet. 300–500 μL of cold double-distilled water was added to the cell pellet, and the cells were homogenized or sonicated in an ice-water bath. A small amount of homogenate was used to determine the protein concentration of each group using a BCA kit. The cell homogenate was then heated in a boiling water bath for 10 min, removed, and vortexed for 1 min. The resulting samples were analyzed using a kit.

[0080] Figure 16 This image shows the ATP content detection results of BA, Arg, and BA / Arg self-assembled nanoparticles in a dihydrotestosterone-induced AGA cell model. Figure 16 As shown, DHT treatment significantly reduced intracellular ATP content by 23.72%; while BA, Arg, and BA / Arg treatments all reversed this inhibitory effect, increasing ATP content by 34.91%, 11.55%, and 49.54%, respectively.

[0081] Mitochondrial membrane potential staining in a dihydrotestosterone-induced AGA cell model was performed using different formulations such as BA, Arg, and BA / Arg. The experimental method was as follows: hDPC cells were stimulated with 1 μM DHT to establish an in vitro AGA cell model. In the experiment, hDPC cells were sputtered at a rate of 3 × 10⁶ cells / year. 5 Samples were seeded at a density of 1 sample per well in 6-well plates and incubated for 24 hours, after which the culture medium was discarded. Subsequently, BA (20 μM), Arg, and BA / Arg groups (containing an equal concentration of 20 μM BA, with Arg diluted proportionally) were added. Treatment was continued for another 24 hours, with DHT-stimulated and unstimulated samples serving as blank controls. The samples were named as follows: Normal Group (Control), Stimulated Group (DHT), DHT+BA, DHT+Arg, and DHT+BA / Arg. After incubation, the culture medium was discarded, and the samples were processed according to the Beyotime Mitochondrial Membrane Potential Detection Kit-C2006 (i.e., according to the JC-1 instruction manual). Finally, the samples were observed and detected under a confocal microscope.

[0082] Figure 17This image shows the mitochondrial membrane potential staining results of BA, Arg, and BA / Arg self-assembled nanoparticles in a dihydrotestosterone-induced AGA cell model. Figure 17 As shown, after DHT treatment, the intensity of red fluorescence in the mitochondria of hDPC cells decreased significantly, while the green fluorescence in the cytoplasm increased significantly, indicating that JC-1 exists in monomeric form. After treatment with BA, Arg, and BA / Arg, JC-1 mainly exists in the mitochondria in polymeric form. Among them, the BA / Arg group showed bright red fluorescence, while the green fluorescence in the cytoplasm was extremely weak, indicating that BA / Arg had the most significant effect on improving mitochondrial function.

[0083] The viability of hDPCs cells was tested using microbeads containing different concentrations of rhCol III from Examples 1 to 7. The experimental method is as follows: hDPC cells were cultured at a density of 8 × 10⁸ cells per well. 3 Cells were seeded at a density of 100 μL in 96-well plates and cultured for 24 h until fully adherent. Then, the culture medium was discarded, and microneedles containing different concentrations of rhCol III (as in Examples 1 and 5-7) were added and incubated for 24 h. The microneedles of different concentrations of rhCol III needed to be pre-dissolved in the culture medium. Next, 100 μL of 0.1% w / v MTT solution was added to each well, and incubation continued for 4 h. After incubation, the supernatant was discarded, and 150 μL of DMSO was added to each well, gently shaking for 15 min to completely dissolve the formazan. Finally, the absorbance (OD) value of each well was measured at 570 nm using a microplate reader, and cell viability was calculated.

[0084] Figure 19 The image shows the cell viability results of microneedles prepared with different proportions of recombinant type III collagen according to this invention, co-incubated with human dermal papilla cells for 24 hours. Figure 19 As shown, this invention investigated the effects of microneedles prepared with different concentrations of rhCol III aqueous solutions on human dermal papilla cells after dissolving in culture medium. At the same dilution ratios (0–16000), the 1 wt.% rhCol III group exhibited the strongest cell proliferation-promoting activity. Therefore, 1 wt% rhCol III is preferred for subsequent studies.

[0085] The mechanical strength and insertion depth of the BA / Arg@rhCol MNs microneedles prepared in Example 1 were tested. The experimental method is as follows: (A) The prepared BA / Arg@rhCol MNs needles were placed on a texture analyzer with the tip facing upwards, and the relevant instrument parameters were set: the descent speed of the texture analyzer probe was set to 1 mm / s, and the maximum force was limited to 100 N. Then the instrument switch was turned on, and the probe began to move downwards and apply pressure to the tip of the microneedle until it broke. During this process, the force-displacement correlation curve was recorded, and the maximum load force measured was the mechanical strength of the microneedle. (B) 10 layers of Parafilm MNs were then inserted into the microneedle.® Layers of sealing film were stacked to simulate skin, with each layer approximately 100 μm thick. During the experiment, a force of 30 N was applied to insert microneedles into the sealing film for 5 minutes to simulate the process of microneedles penetrating the skin. After the experiment, the number of pinholes formed on each layer of sealing film was recorded to calculate the insertion depth of the microneedles.

[0086] Figure 20 This document presents a digital camera morphology visualization, microneedle parameter diagram, mechanical strength and insertion depth diagram of BA / Arg@rhCol MNs according to Embodiment 1 of the present invention. Figure 20 In the diagram, A is a visual representation of the digital camera's shape, B is a schematic diagram of the microneedle parameters, C is a displacement-fracture force diagram, and D is a diagram of the insertion depth. Figure 20 As shown, BA / Arg@rhCol MNs are 10×10 arrays of conical microneedles with good and uniform morphology, and no tip breakage or missing parts were observed. Its parameters are: needle length of 800 μm, bottom side length of 450 μm, needle spacing of 500 μm, and needle tip width of 10 μm. Its mechanical strength is 95.03 N ± 1.12 N and it can effectively penetrate to the fourth membrane, indicating that its penetration depth can reach 400 μm.

[0087] The in vitro release behavior of BA / Arg@rhCol MNs prepared in Example 1 was investigated using the dialysis bag method. The experimental method is as follows: Based on the molecular weight of BA (446.37), a dialysis bag with a molecular weight cutoff of 2000 Da was selected for the experiment. Before the experiment, the dialysis bag was activated, and then the microneedles were placed inside the dialysis bag, and both ends were sealed with nylon cable ties. The sealed dialysis bag was placed in a 50 mL centrifuge tube containing 10 mL of release medium (30%, v / v PEG-400 saline solution), and a magnetic stir bar was added. The release conditions were set as follows: stirring speed 250 rpm, temperature 32℃ ± 0.5℃. 1 mL of receiving medium was sampled at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and an equal volume of blank receiving solution was immediately added. The samples were filtered through a 0.22 μm filter membrane, and the BA content was determined by high-performance liquid chromatography (HPLC).

[0088] Figure 21 This is a graph showing the in vitro release behavior of BA / Arg@rhCol MNs in Example 1 of the present invention. Figure 21 As shown, the cumulative 24-hour release of BA in the BA / Arg@rhCol MNs group was 84.12% ± 0.52%.

[0089] Using BA / Arg@rhCol MNs from Example 1, the hair regeneration effect in an AGA mouse model was tested. The experimental method is as follows: To establish the AGA mouse model, the backs of experimental mice were shaved in an area of ​​approximately 2cm × 3cm and randomly divided into 6 groups: a normal control group (Control), a model group (Model), a BA / Arg nanoparticle group, a blank microneedle group (Blank MNs), a microneedle group (BA / Arg@rhColMNs), and a positive control group (minoxidil group, MXD). Before the formal experiment, hair removal cream was applied to the modeling area on the backs of all mice, and the area was allowed to recover for 24 hours. Except for the Control group, 0.1mL of a 0.2% w / v testosterone solution in 50% ethanol was applied topically to the shaved skin on the backs of the mice in the other groups daily for 28 consecutive days to induce the AGA model. The administration regimens were as follows: the Control group received 0.1 mL of physiological saline solution topically daily; the Model group received 0.1 mL of testosterone solution topically daily; the BA / Arg group received 0.1 mL of BA / Arg solution containing 100 μM BA topically daily; the Blank MNs group and the BA / Arg@rhCol MNs group received microneedle administration, with one microneedle inserted into the skin each time, applying 30 N of pressure and maintaining it for 5 min, once every 3 days; the MXD group received 0.1 mL of 5% minoxidil solution topically daily, Zhejiang Wansheng Pharmaceutical Co., Ltd., as a positive control.

[0090] Figure 22 This is a visual representation of the hair regeneration effect of BA / Arg@rhCol MNs in a testosterone-induced AGA mouse model according to Example 1 of the present invention. Figure 22 In the diagram, A represents the normal control group, B represents the model group, C represents the BA / Arg nanoparticle group, D represents the blank microneedle group (Blank MNs), E represents the microneedle group (BA / Arg@rhCol MNs), and F represents the positive control minoxidil group (MXD). Figure 22 The results show the hair regeneration process on the backs of mice from day 0 to day 15 post-treatment. The results indicate that no hair growth was observed in the model group throughout the entire treatment period, demonstrating the successful establishment of the AGA mouse model. Looking at hair growth in each group on day 15 post-treatment, the final trend in hair regeneration effect was: Normal group > Positive control (MXD) group ≈ BA / Arg@rhCol microneedle group > BA / Arg nanoparticle group > Blank microneedle group > Model group. Notably, although the BA / Arg@rhCol microneedle group received only one-third the medication frequency of the MXD group, it achieved a comparable therapeutic effect, indicating that the microneedle combined with nanoparticle delivery system has significant advantages in promoting hair growth in the AGA mouse model and demonstrates good application potential.

[0091] In summary, this invention achieves efficient loading and stable delivery of the hydrophobic drug BA. The BA / Arg self-assembled nanosystem encapsulates the drug within a nanoscale hydrophilic interface, solving the problem of uneven dispersion caused by poor solubility in a microneedle aqueous matrix. This significantly improves drug delivery efficiency and therapeutic reliability, while the all-aqueous process avoids the risk of skin irritation caused by residual organic solvents. rhCol III, as an inherent extracellular matrix component of the human body, is not only non-immunogenic but also actively participates in and guides normal tissue repair. This enables multidimensional synergistic treatment of AGA pathological processes, breaking through the bottleneck of therapeutic efficacy.

[0092] This invention pioneers a new integrated treatment model of "delivery-repair-regulation". Microenvironment structural repair: Loaded rhCol III directly replenishes lost type III collagen in the dermis, reversing pathological ECM remodeling. Cellular functional restoration: Efficiently delivered BA significantly enhances mitochondrial autophagy activity in hair follicle cells, clearing dysfunctional mitochondria. Synergistic effect: The above two factors form a positive cycle: the improved ECM provides a better microenvironment for cell survival, promoting functional recovery; the functionally improved hair follicle cells can then secrete healthier ECM. The preparation process is simple, mild, and reproducible, facilitating large-scale production. The self-assembly process is non-chemically driven, requiring no expensive equipment, greatly reducing production barriers and quality control costs; microneedle preparation uses one-time casting and centrifugation molding, resulting in high production efficiency. The raw material cost of a single microneedle patch is controllable, possessing the potential for large-scale market promotion.

[0093] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0094] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A self-assembled collagen composite microneedle patch, characterized in that, The device includes a base, an array-structured microneedle body, and nanoparticles embedded in the tip of the microneedle body. The base is attached to the tail of the microneedle body. The microneedle body matrix is ​​a soluble array-structured microneedle body constructed from recombinant type III collagen. The nanoparticles are formed by self-assembly of baicalin and arginine in an aqueous phase through intermolecular forces. The molar ratio of baicalin to arginine is 1:1 to 4.

2. The self-assembled collagen composite microneedle patch according to claim 1, characterized in that, The particle size of the nanoparticles is 100nm to 200nm.

3. The self-assembled collagen composite microneedle patch according to claim 1, characterized in that, The preparation method of nanoparticles includes the following steps: Baicalin was added to an aqueous solution of arginine and subjected to ultrasonic treatment to drive the self-assembly of molecules in the aqueous phase through intermolecular forces. The solvent was then removed by drying to obtain nanoparticles.

4. The self-assembled collagen composite microneedle patch according to claim 3, characterized in that, The concentration of the arginine aqueous solution was 0.1 mg / mL to 0.4 mg / mL, the ultrasonic power was 80 W to 100 W, and the time was 30 min to 60 min.

5. A method for preparing a self-assembled collagen composite microneedle patch according to any one of claims 1 to 4, characterized in that, Includes the following steps: The nanoparticles were dispersed in water and mixed with an aqueous solution of recombinant type III collagen. Hyaluronic acid was added and stirred to form a semi-solid gel solution. A semi-solid gel solution is injected into a mold with a microneedle array structure. The mold is then centrifuged to allow the semi-solid gel solution to fill the cavity of the microneedle tip. A base matrix solution is then injected, and the mold is centrifuged again and then cured. The resulting self-assembled collagen composite microneedle patch is then peeled off.

6. The method for preparing the self-assembled collagen composite microneedle patch according to claim 5, characterized in that, The mass-to-volume ratio of nanoparticles to recombinant type III collagen aqueous solution is 1 mg to 5 mg: 1 mL, and the mass concentration of recombinant type III collagen solution is 0.5 wt.% to 20 wt.%.

7. The method for preparing the self-assembled collagen composite microneedle patch according to claim 5, characterized in that, The mass ratio of nanoparticles to hyaluronic acid is 1–5:250, and the molecular weight of hyaluronic acid is 52 kDa.

8. The method for preparing the self-assembled collagen composite microneedle patch according to claim 5, characterized in that, The substrate matrix solution is formed by adding hyaluronic acid with a molecular weight of 52kDa, hyaluronic acid with a molecular weight of 200kDa to 400kDa, and dextran 40 to water in a mass ratio of 1:1:1 and allowing them to swell overnight. The concentration of each of the hyaluronic acid with a molecular weight of 52kDa, hyaluronic acid with a molecular weight of 200kDa to 400kDa, and dextran 40 in the substrate matrix solution is 10wt.

9. The method for preparing the self-assembled collagen composite microneedle patch according to claim 5, characterized in that, The centrifugation speed is 3000 rpm to 4000 rpm, the time is 3 min to 5 min, the curing temperature is 4℃, and the time is 24 h to 48 h.

10. The use of the self-assembled collagen composite microneedle patch according to any one of claims 1 to 4 in the preparation of a drug for androgenetic alopecia.

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