A synergistic hair growth microneedle based on reactive oxygen scavenging and nitric oxide delivery, and a preparation method and application thereof

Abstract

The application provides a synergistic hair growth microneedle based on active oxygen scavenging and nitric oxide delivery and a preparation method thereof. The hair growth microneedle comprises a substrate layer and a plurality of microneedles arranged on the substrate layer, and at least comprises: an active oxygen scavenging component arranged on a needle root part of the microneedle, which is used for scavenging excessive active oxygen under a pathological oxidative environment around a hair follicle; a nitric oxide delivery component arranged on a needle tip part of the microneedle, which is used for continuously releasing nitric oxide after the microneedle is inserted into the skin; a mass ratio of the active oxygen scavenging component to the nitric oxide delivery component is (0.01-10):1; and the active oxygen scavenging component and the nitric oxide delivery component in the microneedle have a synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and transdermal drug delivery technology, specifically relating to a synergistic hair growth microneedle based on reactive oxygen species scavenging and nitric oxide delivery, its preparation method and application. Background Technology

[0002] Hair loss has become a common skin and cosmetic problem worldwide, especially androgenetic alopecia (AGA), which has a high incidence rate and is showing a trend of affecting younger people year by year. Patients not only suffer from damage to their appearance, but also experience serious psychological burdens such as anxiety, depression, and decreased self-confidence, which significantly impacts their quality of life.

[0003] Traditional views hold that AGA (Advanced Growth Anemia) is primarily caused by increased sensitivity of hair follicles to androgens, particularly dihydrotestosterone (DHT). Recent studies have further indicated that factors such as oxidative stress, immune inflammation, vascular sparseness, and even abnormal neural regulation within the hair follicle microenvironment play crucial roles in the abnormal transition of hair follicles from the anagen (growth) phase to the catagen (transitional) and telogen (resting) phases.

[0004] Under the influence of androgens, the mitochondrial function of dermal papilla cells and hair follicle stem cells is impaired, leading to the production of large amounts of reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide, and hydroxyl radicals. When ROS levels remain high, it can cause cellular DNA damage, excessive protein and lipid oxidation, triggering cellular senescence and apoptosis, thereby impairing the regenerative capacity of hair follicles and causing them to gradually miniaturize. Simultaneously, due to the reduced number of capillaries around the hair follicles, insufficient blood supply, and the presence of mild local chronic inflammation, an unfavorable microenvironment of "high ROS, low blood flow, and mild inflammation" forms around the hair follicles. This microenvironment inhibits the activation of hair follicle stem cells and the proliferation of keratinocytes, making it difficult for hair follicles to re-enter the anagen phase from the resting phase.

[0005] Currently, commonly used clinical drug treatments include topical minoxidil and oral 5α-reductase inhibitors (such as finasteride). The former mainly works by dilating blood vessels and prolonging the hair follicle growth phase, while the latter inhibits hair follicle miniaturization by reducing DHT production. However, both classes of drugs have significant limitations:

[0006] Minoxidil is a lipophilic small molecule, and its percutaneous penetration rate is low in ordinary topical formulations. It needs to be used continuously for a long time, once or twice a day, which can easily cause scalp irritation, itching, or even rebound hair loss. 5α-reductase inhibitors are systemic drugs, and long-term use may cause systemic adverse reactions such as decreased libido and erectile dysfunction. Most patients have compliance problems.

[0007] Microneedle transdermal drug delivery technology creates numerous micron-sized pores on the skin surface, allowing for painless penetration of the stratum corneum to deliver drugs directly to the subepidermal and dermal layers, increasing local drug concentration and reducing systemic exposure. Simultaneously, the slight mechanical damage caused by microneedles can activate regenerative signaling pathways such as Wnt / β-catenin, which has been shown to promote hair follicle regeneration. Studies have shown that loading minoxidil, VEGF, JAK inhibitors, and nanomedicines onto dissolving or hydrogel microneedles has yielded superior efficacy compared to traditional topical medications in animal models of hair loss.

[0008] On the other hand, nanozymes with enzyme-like activity have been widely used in recent years for reactive oxygen species (ROS) scavenging and antioxidant therapy. By mimicking the catalytic functions of superoxide dismutase (SOD) and catalase (CAT), they efficiently decompose superoxide anions and hydrogen peroxide, reducing oxidative stress. Platinum nanozymes have been shown to significantly reduce ROS levels around hair follicles in animal models of hair loss, promoting hair follicle progression and thus enabling hair regrowth from the scalp.

[0009] Besides "clearing damaging factors," utilizing "positive signaling molecules" to promote hair follicle regeneration has also attracted much attention. Nitric oxide (NO) is an important endogenous gaseous signaling molecule that can mediate vasodilation and promote angiogenesis through the cyclic guanosine monophosphate (cGMP) pathway, and participate in neural and immune regulation. Appropriate amounts of NO can increase blood flow around hair follicles, improve oxygen and nutrient supply, and promote tissue repair. However, if NO is released in the presence of high levels of reactive oxygen species (ROS), it may generate strong oxidants such as peroxynitrite, which could exacerbate cell damage. Therefore, NO delivery should be carried out within an appropriate time window and concentration range only after "ROS is effectively controlled."

[0010] Existing NO delivery systems are mostly used for repairing infected wounds and treating chronic wounds. Their design principle is to release higher levels of NO in the early stages for sterilization and inflammation reduction, and then release lower levels of NO in the later stages to promote angiogenesis and tissue healing. This approach has not yet been systematically introduced for the hair follicle microenvironment, especially in the field of hair loss treatment. Currently, there is a lack of percutaneous microneedle products that integrate ROS removal and NO delivery in a synergistic design. Summary of the Invention

[0011] The purpose of this invention is to overcome the defects of the prior art and provide a synergistic hair growth microneedle based on reactive oxygen species scavenging and nitric oxide delivery, as well as its preparation method and application. By using microneedle transdermal drug delivery technology, the pathological microenvironment around the hair follicle can be precisely regulated, thereby significantly promoting the transformation of the hair follicle from the resting phase to the growth phase, and achieving efficient and physiologically friendly hair regeneration.

[0012] This invention provides the following technical solution:

[0013] This invention provides a synergistic hair growth microneedle based on reactive oxygen species (ROS) scavenging and nitric oxide (NO) delivery. The microneedle comprises a basal layer and a plurality of microneedles disposed on the basal layer. Each microneedle includes at least: an ROS scavenging component disposed at the root of the microneedle to scavenge excess ROS in the pathological oxidative environment around the hair follicle; and a NO delivery component disposed at the tip of the microneedle to continuously release NO after the microneedle is inserted into the skin. The mass ratio of the ROS scavenging component to the NO delivery component is (0.01–10):1.

[0014] The base layer comprises one or more of the following: hyaluronic acid or its derivatives, alginate or its thiolized derivatives, gelatin, chitosan, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, polylactic acid-glycolic acid copolymer microparticles and their derivatives or combinations; the reactive oxygen species scavenging component comprises nanoenzymes with superoxide dismutase-like and / or catalase-like activities; the nitric oxide delivery component comprises at least one NO donor, which is immobilized or partially immobilized by covalent bonds or electrostatic interactions with the polymer matrix through thiol, carboxyl, or amino groups to form a NO donor-polymer network;

[0015] The polymer matrix is ​​selected from at least one of the following: thiolated alginate, hyaluronic acid, polyvinyl alcohol, gelatin, and chitosan; wherein, the thiolated alginate is obtained by grafting cysteine ​​or glutathione onto the alginate backbone through EDC / NHS coupling, and undergoes a nitrosation reaction with S-nitroso compounds such as GSNO through thiol groups to form a stable S-nitroso network, thereby achieving the slow and controlled release of NO.

[0016] Specifically, the thiol content of the thiolized alginate (SA-SH) is preferably 100–300 μmol / g, and the degree of alcoholysis of polyvinyl alcohol is 80–99%.

[0017] The existing technology still has the following problems that urgently need to be solved:

[0018] 1. Insufficient removal of reactive oxygen species targeting hair follicles: Existing oral or topical antioxidant preparations are difficult to achieve sufficient concentrations around hair follicles;

[0019] 2. Lack of precise control over NO duration and concentration: Simple topical application of NO donors or NO-releasing patches cannot balance efficacy and safety;

[0020] 3. The inability to simultaneously and synergistically regulate the "ROS-NO-vascular / immune" axis in both space and time results in the hair follicle microenvironment failing to reach the optimal overall state for hair growth;

[0021] 4. Poor medication adherence: The medication needs to be applied or taken orally repeatedly every day, making it difficult for patients to adhere to the treatment in the long term.

[0022] To address the aforementioned shortcomings, this invention constructs a soluble or hydrogel-shaped microneedle array made of biocompatible polymer materials. The needle length, spacing, and strength are adapted to the thickness of the human scalp and the distribution of hair follicles. Simultaneously, the microneedles are loaded with: reactive oxygen species scavenging components, mainly platinum nanozymes, possessing SOD / CAT-like activity, for efficiently decomposing ROS and generating a suitable amount of oxygen; and NO donors, which are immobilized or partially immobilized by binding to functional groups such as thiol and carboxyl groups, for the continuous and controllable release of NO over a certain period of time.

[0023] Simultaneously, double-layer or multi-layer microneedle structures can be employed to enrich NO donors in the needle tip region and nanozymes in the needle root or middle region, thereby forming a "spatial stratification and temporal staggered" ROS and NO regulation strategy:

[0024] Initial insertion: Microneedles mechanically stimulate the scalp and rapidly release some NO, while nanoenzymes immediately participate in ROS clearance;

[0025] Mid-to-late stage: As nanozymes continue to clear ROS and generate O2, NO exists in a low-dose sustained release, promoting angiogenesis and maintaining hair follicle regeneration signals;

[0026] By adjusting the ratio of platinum nanozyme to NO donor, the cross-linking density of polymer network, the thickness of microneedles, and the dissolution properties, an integrated synergistic hair growth system that balances efficacy and safety is obtained.

[0027] This invention utilizes transdermal drug delivery technology, allowing the microneedle material to partially or completely dissolve within 5–60 minutes under physiological conditions at 37°C, releasing the loaded active ingredients without leaving any sharp solid structures under the skin. The reactive oxygen species scavenging component and the nitric oxide delivery component in the microneedles work synergistically. A NO donor-polymer network is formed within the microneedles, enabling sustained release and time-controlled nitric oxide delivery. By controlling the mass ratio of the reactive oxygen species scavenging component to the nitric oxide delivery component to be (0.01–10):1, a safe nitric oxide concentration that promotes angiogenesis and hair follicle growth can be obtained while effectively inhibiting oxidative stress.

[0028] Specifically, hyaluronic acid has a molecular weight of 10–1500 kDa, and alginate can be obtained through Ca... 2+ Crosslinking enables reversible gelation, which can be used to regulate the NO release rate.

[0029] Furthermore, the nanozyme is selected from at least one of the following: platinum nanozyme, gold nanozyme, manganese dioxide nanoparticles or nanosheets, iron-based nanozyme, cobalt-based nanozyme, copper-based nanozyme and its derivatives or combinations thereof;

[0030] Furthermore, the particle size of the platinum nanozyme is 1–10 nm, preferably 1–5 nm.

[0031] Furthermore, the reactive oxygen species scavenging component also includes at least one small molecule or macromolecule antioxidant, wherein the antioxidant is selected from: glutathione, N-acetylcysteine, superoxide dismutase, catalase, vitamin C, vitamin E, tea polyphenols, resveratrol, and combinations thereof.

[0032] Furthermore, the NO donor is selected from: S-nitrosoglutathione, S-nitrosoN-acetylpenicillamine, diazoxide diols, metal nitroso complexes, N-nitrosylated polypeptides, N-nitrosylated polysaccharides and their derivatives or combinations thereof.

[0033] Furthermore, the microneedle has a double-layer or multi-layer structure, including at least: a first functional layer near the needle tip, enriched with the nitric oxide delivery component; a second functional layer near the needle root or base layer, enriched with the reactive oxygen species scavenging component; the mass ratio of the reactive oxygen species scavenging component to the nitric oxide delivery component is preferably (0.1-2):1.

[0034] Furthermore, the thickness of the first functional layer accounts for 5-35% of the needle length, the thickness of the second functional layer accounts for 65-95% of the needle length, the mass of the NO donor in the first functional layer accounts for 1-30% of the dry weight of the microneedle, and the mass of the nanozyme in the second functional layer accounts for 0.01-10% of the dry weight of the microneedle.

[0035] Furthermore, the geometric dimensions of the microneedles satisfy the following: needle length is 300–900 μm, preferably 400–700 μm; needle root diameter is 150–350 μm; needle tip radius is less than 15 μm; microneedle spacing is 400–800 μm; and the number of microneedles in the microneedle array on the substrate layer is 100–900.

[0036] The thickness and component content of each functional layer were designed to meet the following time-release curves: in the initial stage (0-24h), excess ROS was rapidly removed and a high concentration of NO (approximately 50-100 μM) was released; in the middle stage (24-72h), a moderate concentration of NO was maintained for sustained release (approximately 20-50 μM); and in the later stage (72h-7d), a low level of NO sustained release (< 20 μM) and a continuous antioxidant environment were maintained.

[0037] Specifically, the time-release curve is achieved through the following structural parameters: (i) a two-layer spatial stratification structure—the needle tip (first functional layer, accounting for 5–35% of the needle length) is enriched with NO donors, and the needle root (second functional layer, accounting for 65–95% of the needle length) is enriched with platinum nanozymes; (ii) regulation of thiolated alginate crosslinking density—via Ca 2+ The cross-linked network density determines the NO diffusion rate. A high cross-linking density can extend the release cycle from 24 h to 3 to 7 days. (iii) NO donor loading control - the mass of NO donor in the first functional layer accounts for 1 to 30% of the dry weight of the microneedles. The initial release peak concentration is controlled by adjusting this ratio.

[0038] Furthermore, an adhesion ring or support layer is provided around the base layer. The adhesion ring includes acrylic medical pressure-sensitive adhesive, temperature-sensitive polymer or hydrogel adhesive, which can achieve adaptive adhesion under the action of scalp temperature and sweat humidity, and is easy to peel off after treatment, reducing mechanical traction on newly grown hair.

[0039] This invention also provides a method for preparing the above-mentioned hair growth microneedles, comprising the following steps:

[0040] (1) Prepare reactive oxygen species scavenging nanoenzymes or antioxidant precursors and disperse them in a first polymer solution to obtain a reactive oxygen species scavenging functional solution;

[0041] (2) Prepare NO donor and react it with a second polymer solution containing thiol, carboxyl or amino groups to obtain NO donor-polymer precursor solution;

[0042] (3) Fill the tip area of ​​the microneedle mold with the NO donor-polymer precursor solution and pre-cur it; fill the remaining part of the microneedle mold with the reactive oxygen scavenging solution and dry or cross-link it;

[0043] (4) A base layer solution is coated on the back of the needle body, and after molding, the hair growth microneedles are demolded to obtain the hair growth microneedles.

[0044] The first polymer solution contains one or more of the following: hyaluronic acid, gelatin, polyvinyl alcohol and its derivatives, and the total polymer mass concentration of the solution is 10-30 wt%; wherein the mass ratio of hyaluronic acid to polyvinyl alcohol or gelatin is (1-3):1.

[0045] The second polymer solution uses sodium thiosulfate alginate (SA-SH) as the core component (1-3 wt%, dissolved in pH 7.4 PBS). SA-SH provides thiol groups (-SH). Under light-protected ice bath conditions, it is mixed with sodium nitrite and hydrochloric acid in an equimolar ratio (pH 2-3). After a nitrosation reaction for 40 min, the pH is adjusted back to neutral to form S-nitrosolated alginate (SA-SNO), i.e., NO donor-polymer precursor solution.

[0046] Furthermore, 1-5 wt% glycerol can be added to the first polymer solution as a plasticizer to improve the flexibility of the microneedles, and hyaluronic acid (1-5 wt%) or polyvinyl alcohol (5-10 wt%) can be added to the second polymer solution to improve film-forming properties and mechanical strength.

[0047] Further, in step (1), the reactive oxygen species scavenging nanoenzyme is prepared by aqueous phase reduction or coprecipitation. After the reaction, small molecule ions and unreacted precursors are removed by dialysis, ultrafiltration or centrifugation. In step (3), the solution is fully filled into the microneedle mold by vacuum filtration or centrifugation. The pre-curing includes cooling, photocrosslinking, ionic crosslinking or a combination thereof.

[0048] The present invention also provides the use of the above-mentioned hair growth microneedles in the preparation of drugs for treating or preventing hair loss, wherein the hair loss includes at least one of androgenetic alopecia, telogen effluvium, alopecia areata, chemotherapy-related alopecia, and non-scarring alopecia caused by other reasons.

[0049] The present invention has the following beneficial effects:

[0050] 1. Precisely improve the pathological microenvironment of hair follicles: Nanozymes efficiently decompose excess ROS around hair follicles, reduce oxidative stress, and counteract androgen-induced premature aging and apoptosis; restore local oxygen levels to a certain extent and alleviate hair follicle hypoxia; synergistically release NO, improve microcirculation, promote angiogenesis, and establish a more favorable comprehensive microenvironment for hair follicle regeneration.

[0051] 2. Innovative application of gas molecule synergistic therapy: Integrating "reactive oxygen species scavenging (anti-damage)" and "NO delivery (promoting repair and angiogenesis)" into the same microneedle system, and synergistically designing it in time and space; avoiding the adverse reactions that may be caused by simply superimposing ROS scavengers and NO donors, ensuring that NO plays a positive role in a low ROS background; and enhancing hair follicle regeneration dynamics and vascular support capacity through the coupling regulation of O2 and NO.

[0052] 3. Improved drug compliance and safety: Microneedle patches can be used once every few days with short application time each time; no hard needles remain after the microneedles are completely or substantially dissolved, resulting in high patient comfort; local drug delivery significantly reduces the risk of systemic exposure and systemic side effects, and can be used in combination with existing drugs. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 This is a schematic diagram of the overall structure of the synergistic hair growth microneedles of the present invention;

[0055] Figure 2 This is a schematic diagram illustrating the application of the microneedles of the present invention on the scalp;

[0056] Figure 3 This is a schematic diagram of the preparation process of the synergistic hair growth microneedles of the present invention;

[0057] Figure 4 This is a schematic diagram illustrating the synergistic mechanism of the microneedles of the present invention in achieving ROS removal and NO delivery around the hair follicle;

[0058] Figure 5 This is a schematic diagram of the results of in vitro cell experiments;

[0059] Figure 6 This is a schematic diagram comparing hair growth in different treatment groups in an animal model of hair loss.

[0060] Figure 7 This is a schematic diagram of the NO release curve;

[0061] Figure 8 ROS removal curve / ROS relative level change over time;

[0062] Figure 2 In the diagram, 1—basal layer / patch base; 2a—single microneedle; 4—hair follicle as a whole; 5—perifollicular blood vessels; 6—perifollicular nerve fibers; 7—stratum corneum / epidermis; 8—dermis; 9—subcutaneous tissue / subcutaneous fat layer; 10—sebaceous gland; 11—hair shaft; 12—dermal papilla; 31—nanozyme particle; 32—NO donor complex; 35—ROS; 37—O2 molecule. Detailed Implementation

[0063] 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.

[0064] The structure, preparation, application, and mechanism of action of the microneedles in this invention are described in the following embodiments. Figures 1-4 ;in Figure 1 The overall structure of the microneedle array and the double-layer cross-section design of a single microneedle are shown. The left side is a top view of the microneedle patch, showing the base layer (1), the overall microneedle array (2) and the adhesion layer (3), with the patch size (L, W) and microneedle spacing (s) marked. The right side is an enlarged cross-section view of a single microneedle, showing the tip (23), the first functional layer / NO enrichment layer (21), the second functional layer / ROS scavenging layer (22) and the root (24), as well as the NO donor complex (32) enriched in the tip region and the nanoenzyme particles (31) in the root region.

[0065] Figure 2The microneedles are shown to penetrate the scalp and release drugs through various layers of the scalp: stratum corneum / epidermis (7), dermis (8), and subcutaneous tissue / subcutaneous fat layer (9). After the microneedles are inserted, the first functional layer of the needle tip releases the NO donor complex (32), and NO diffuses around the hair follicle (4). At the same time, the needle root area containing nanoenzyme particles (31) clears excess reactive oxygen species (35) in the dermis and generates O2 molecules (37), which improves the microenvironment of blood vessels (5) and nerve fibers (6) around the hair follicle.

[0066] Figure 3 The fabrication process of microneedles is demonstrated, with the main steps including: Step 41 - Nanozyme preparation (synthesis of platinum nanozymes via aqueous phase reduction method); Step 42 - NO donor-polymer precursor preparation (sodium thiolate is nitrosated to form SA-SNO); Step 43a - NO functional layer filling (SA-SNO solution is filled into the needle tip area of ​​the PDMS mold and pre-cured); Step 43b - ROS removal layer filling (polymer solution containing platinum nanozymes is used to cover the remaining mold space); Steps 44-45 - Crosslinking / drying and molding, followed by demolding, to obtain the finished microneedle patch;

[0067] Figure 4 The mechanism of synergistic action between ROS clearance and NO delivery is demonstrated. The left side shows the state before treatment: hair follicles (4) atrophy, surrounding blood vessels (5) reduced or sparse, the level of reactive oxygen species in the hair follicle microenvironment increased (35), and the activity of hair follicle stem cells was inhibited. The right side shows the state after using the microneedles of the present invention: after the microneedles are inserted, the nanoenzyme (31) catalyzes the decomposition of reactive oxygen species (35) into O2 (37), and the NO donor complex (32) releases NO molecules (36), which synergistically promote angiogenesis with O2 (37), improve microcirculation and oxygen supply, and activate hair follicle regeneration.

[0068] Example 1: Typical preparation method of platinum nanozyme / GSNO synergistic hair growth microneedles

[0069] 1. Dissolve chloroplatinic acid hexahydrate in ultrapure water to prepare a 1 mM chloroplatinic acid solution, and stir magnetically under ice bath conditions. Slowly add freshly prepared NaBH4 solution dropwise at a NaBH4:Pt molar ratio of 5:1, controlling the addition time to 5–20 min. During the reaction, the solution color gradually changes from light yellow to dark brown, indicating the formation of platinum nanoparticles. Continue the reaction for 30 min, then stop stirring. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyze it in deionized water for 24 h, changing the dialysate every 4 h to remove unreacted precursors and small molecule byproducts.

[0070] The dialyzed solution was observed by transmission electron microscopy (TEM), which showed that the particle size was concentrated in the range of 1–5 nm and the particles were uniformly dispersed. The hydrated particle size was measured to be approximately 2–8 nm using dynamic light scattering. By measuring the decomposition rate in a system containing H₂O₂ and the changes in the fluorescent probe signal in a superoxide anion generation system, it was demonstrated that the nanoparticles have significant CAT-like and SOD-like activities.

[0071] 2. Preparation of NO donor-polymer precursor solution: Sodium alginate was dissolved in MES buffer at pH 5.0 to prepare a 1-2 wt% solution. EDC and NHS were added to activate the carboxyl groups on the sodium alginate backbone. After stirring for a certain period of time, cysteine ​​or glutathione containing thiol groups were added to introduce thiol groups into the side chain of sodium alginate. After the reaction was completed, unreacted small molecules were removed by dialyzing to obtain thiolized sodium alginate (SA-SH).

[0072] SA-SH was dissolved in ice-cold PBS solution. Nitrite and hydrochloric acid were added under light-protected conditions to adjust the pH to 2–3. An S-nitroso structure was formed at the thiol site via nitrosation. After a certain reaction time, the pH was quickly adjusted back to neutral and the solution was placed in a light-protected environment to obtain an SA-SNO solution containing a stable NO precursor. To improve film-forming properties and mechanical strength, it can be blended with a certain proportion of hyaluronic acid or polyvinyl alcohol.

[0073] 3. Microneedle mold preparation

[0074] Molds with conical cavities are fabricated using PDMS or other elastic silicone rubber materials via micro-nano fabrication technology. The microneedles are designed with a height of 600 μm, a base diameter of 250 μm, a tip angle controlled between 20 and 30°, and a spacing of 600 μm. Each mold contains 20 × 20 microcavities. The mold surface is treated with plasma or coated with materials such as polyethylene glycol to improve solution wettability and release properties.

[0075] 4. Microneedle layered casting

[0076] NO enrichment layer casting:

[0077] SA-SNO solution (which may contain a certain proportion of hyaluronic acid and glycerin) is dropped onto the surface of the PDMS mold and placed in a vacuum drying oven for 5–15 minutes to allow the solution to fully enter the tip region of the microcavity; centrifugation can also be used to accelerate the filling process. The mold is then pre-cured at 4–8°C, or ion-crosslinked by spraying with CaCl2 solution to form a preliminary gel structure of the first functional layer and fix it at the needle tip.

[0078] ROS removal layer casting:

[0079] The platinum nanozyme solution is mixed with hyaluronic acid, gelatin, PVA, etc., to prepare a solution with a polymer concentration of 10-30 wt%. Appropriate amounts of glycerol and sorbitol can be added to improve flexibility. The solution is then added to a mold, filling the remaining volume of the microcavity and the back substrate. The mold is then dried at room temperature or slightly above 12-24 hours to allow the needle and substrate to form a unified structure.

[0080] 5. Forming of the substrate and adhesion ring

[0081] An additional layer of solution or film based on PVA / PVP or medical pressure-sensitive adhesive is cast onto the back of the microneedles to form a soft substrate and annular adhesion zone. After complete drying, the microneedle array is gently peeled off from the PDMS mold to obtain the platinum nanozyme / SA-SNO synergistic hair growth microneedle patch.

[0082] 6. Evaluation of physical and release performance

[0083] The morphology of the microneedles was observed using a stereomicroscope to confirm that all microneedles were intact and had sharp tips. Compression tests were performed using a texture analyzer or a universal testing machine to determine that a single needle did not undergo plastic deformation under a load of 0.2 N. Using pigskin or ex vivo human skin as models, the dye labeling method was used to verify that the microneedles could successfully penetrate approximately 300–500 μm below the stratum corneum.

[0084] Microneedles were inserted into simulated body fluids or PBS solution, and their dissolution behavior was observed. Results showed that most microneedles dissolved within 5–30 min, with the needle body disappearing and only the back substrate remaining. NO release from the supernatant was measured using a Griess kit, and release curves were plotted. A relatively high release rate was observed in the first 24–72 h, followed by a slower tail-release phase, with the total release time lasting 3–7 days. ROS probe experiments demonstrated that the platinum nanozyme remained active during this time, continuously scavenging ROS such as H2O2.

[0085] Example 2: In vitro cell experiments to verify the protective and hair growth-promoting effects of hair follicle-associated cells.

[0086] 1. Dermal papillary cell model

[0087] Human or mouse dermal papillary cells (DPCs) were routinely cultured in DMEM medium containing 10% fetal bovine serum. An in vitro model simulating androgenetic alopecia and oxidative stress was established by adding dihydrotestosterone (DHT) and H2O2.

[0088] The following processing groups will be established:

[0089] Normal control group: No DHT / H2O2 added; Model control group: DHT and H2O2 added, no treatment given;

[0090] Nanoenzyme microneedle dissolution group: microneedle dissolution containing only platinum nanoenzymes was administered; NO microneedle dissolution group: microneedle dissolution containing only SA-SNO was administered; Synergistic hair growth microneedle dissolution group: synergistic microneedle dissolution prepared in Example 1 was administered.

[0091] The effectiveness will be evaluated through the following tests:

[0092] CCK-8 assay was used to detect cell viability; DCFH-DA fluorescent probe was used to detect intracellular ROS levels; SA-β-gal staining was used to assess the proportion of senescent cells; EdU or BrdU incorporation assay was used to evaluate cell proliferation capacity; and Western blot was used to detect the expression of β-catenin, Cyclin D1, Lef-1, and the anti-apoptotic protein Bcl-2, which are related to hair follicle growth.

[0093] Expected results: The synergistic microneedle dissolving solution group can significantly reduce ROS levels in DPCs, decrease the proportion of SA-β-gal positive cells, and increase the expression of proliferation markers and β-catenin pathway proteins, showing a more significant advantage compared to the nanozyme group or the NO group alone.

[0094] 2. Co-culture model of hair follicle stem cells and keratinocytes

[0095] A co-culture system was established using stem cells from the hair follicle protuberance area and epidermal keratinocytes, and a hair loss environment was simulated by DHT / H2O2 treatment. The following changes were observed after intervention with the lysing solutions of each treatment group: changes in the expression of stem cell surface markers (such as K15 and Sox9); changes in keratinocyte proliferation and differentiation markers (such as K10 and K14); and the expression of key proteins in the Wnt / β-catenin and Shh pathways in the co-culture system.

[0096] The synergistic microneedle group is expected to upregulate the proliferation and differentiation capacity of hair follicle stem cells and keratinocytes on the basis of inhibiting ROS, suggesting a positive regulatory effect on the hair follicle regeneration lineage.

[0097] The results of the above in vitro cell experiments are summarized in Figure 5 ,in Figure 5 (a) The results of dermal papillary cell viability in Example 2 are presented. The protective effect of microneedle lysis solution against DHT / H2O2-induced damage is evaluated by the dermal papillary cell (DPC) viability assay (CCK-8 assay). Figure 5 (b) Present the results of intracellular ROS level detection. The ROS scavenging capacity of each group was compared by using the results of intracellular reactive oxygen species level detection in DPCs (DCFH-DA fluorescent probe).

[0098] Example 3: Angiogenesis-Promoting Experiment with Vascular Endothelial Cells

[0099] HUVECs or other microvascular endothelial cells were collected and treated in groups similar to those in Example 2, with a focus on using microneedle dissolution and release solutions that matched the NO release curve.

[0100] The following experiment was conducted:

[0101] Scratch assay: to monitor the degree of cell migration promotion by different treatment groups; Matrigel lumen formation assay: to count the number of tubular structures, the number of branch points and the total tube length; qPCR or Western blot: to detect the expression of angiogenesis-related factors such as VEGF, eNOS, and Ang-1.

[0102] Experimental results are expected to show that the synergistic microneedle dissolving solution of this invention will not cause endothelial cell toxicity within the effective concentration range, but will instead significantly promote its migration and lumen formation, thereby explaining its mechanism of promoting angiogenesis and improving hair follicle blood supply in vivo.

[0103] The above experimental results on vascular endothelial cells are summarized in Figure 5 (c) The results of the Matrigel lumen formation assay (HUVECs tubular structure formation assay) demonstrate the promoting effect of each group of microneedle dissolving solutions on the tubular structure formation ability.

[0104] Example 4: Application in a mouse model of androgenetic alopecia

[0105] 1. Model building and grouping

[0106] Male C57BL / 6 mice aged 6–8 weeks were selected. The backs of the mice were shaved, and residual hair was removed with depilatory cream to induce the hair follicles on the back to enter the resting phase. Androgenetic alopecia model was established by periodically applying testosterone propionate to the back skin or by subcutaneous injection of androgens for 4 weeks.

[0107] Mice were randomly divided into the following groups, with no fewer than 8 mice in each group: Normal control group: no modeling or treatment; Model control group: no treatment after modeling; Topical minoxidil group: 2% or 5% minoxidil solution applied daily; Nanoenzyme microneedle group: microneedles containing only platinum nanoenzyme applied every 3 days; NO microneedle group: microneedles containing only SA-SNO applied every 3 days; Synergistic hair growth microneedle group: synergistic microneedles of the present invention applied every 3 days.

[0108] 2. Microneedle patch application method

[0109] After stabilizing the mice, the synergistic hair growth microneedle patch was applied to the hair removal area on the back, and a pressure of approximately 0.2–0.5 N / needle was applied and maintained for 30 seconds to ensure complete microneedle penetration. The patch was then fixed for 30 minutes to allow the microneedles to dissolve completely or partially. The patch was then removed, and the area was visually observed and photographed.

[0110] 3. Observation indicators

[0111] During the treatment, photos were taken every 3 to 7 days to record the hair regeneration on the back. Image analysis software was used to calculate indicators such as hair coverage and average grayscale change, and the hair growth rate and density of each group were compared intuitively.

[0112] The animal was euthanized after treatment, and skin tissue from its back was collected for further analysis.

[0113] HE staining: to observe changes in the number, depth, cross-sectional area, and skin thickness of hair follicles; Masson staining or immunohistochemistry: to observe the distribution of collagen fibers and blood vessels; CD31 immunolabeling: to count the number of microvessels around hair follicles; Ki67 immunofluorescence: to evaluate the degree of hair follicle cell proliferation; DHE fluorescent probe staining: to observe the ROS level around hair follicles; ELISA or qPCR: to detect the expression of inflammatory factors such as TNF-α and IL-6.

[0114] Expected results: The synergistic microneedling group showed faster hair regeneration, thicker hair shafts, deeper and more numerous hair follicles, and significantly higher CD31-positive blood vessel density and Ki67-positive cell ratio around the hair follicles compared to other treatment groups. At the same time, the DHE fluorescence intensity and inflammatory factor levels decreased significantly, indicating that the present invention reconstructs a comprehensive microenvironment conducive to hair follicle regeneration by simultaneously regulating ROS and NO.

[0115] The above in vivo experimental results are summarized in Figures 6-8 ,in Figure 6 The diagram illustrates the hair growth cycle and pathological changes in hair follicles. The left side shows the stage changes of the hair growth cycle on the back of C57BL / 6 mice (sparse → gradually increasing → peak → dense → falling out), and the right side shows the pathological process of hair follicle atrophy and reduced peripheral blood vessels in a hair loss model, as well as the promoting effect of the treatment of this invention on hair follicle regeneration. Figure 7 The NO release kinetic curves of each group are shown, and the relative NO release values ​​of the synergistic hair growth microneedle group (71), the NO donor-only microneedle group (72), and the blank microneedle group (73) are shown to change over 144 hours. The synergistic microneedle group shows a continuous upward trend. Figure 8 The ROS scavenging effects of each group are shown, displaying the changing trends of relative ROS levels over 96 hours in the model control group (81, no scavenging effect), nanozyme microneedle group (82, moderate scavenging), NO microneedle group (83, slight scavenging), and synergistic hair growth microneedle group (84, significant scavenging).

[0116] Example 5: Recommendations for Human Treatment Plans

[0117] For male patients with mild to moderate androgenetic alopecia, the following treatment plan can be designed:

[0118] 1. At the initial consultation, assess the hair loss grade, scalp condition and previous treatment history to rule out severe scarring alopecia or skin lesions.

[0119] 2. After cleaning and drying the scalp, apply the hair growth microneedle patch of the present invention to the main hair loss area and press lightly for 10-30 seconds to ensure that the microneedles are fully inserted.

[0120] 3. Keep the patch attached for 30–45 minutes to allow the microneedles to dissolve; then remove and discard the patch without additional rinsing.

[0121] 4. During the initial treatment phase, it is recommended to use it once every 3 to 4 days for 8 to 12 weeks. Depending on the hair regrowth, the maintenance phase can be extended to once every 1 to 2 weeks.

[0122] 5. It can be used in combination with topical minoxidil, oral finasteride or low-energy laser cap as needed, but the frequency should be adjusted under the guidance of a doctor to avoid superimposed skin irritation.

[0123] By regularly following up and recording patients' hair density, scalp itching, or erythema, it is expected that a reduction in hair loss and an increase in new short hair growth can be observed within 3 months, and a significant improvement in overall density and coverage can be observed within 6 to 12 months.

[0124] Example 6: Other variations

[0125] Without departing from the core idea of ​​this invention—namely, "synergistic hair growth microneedles based on reactive oxygen species scavenging and nitric oxide delivery"—those skilled in the art can make various modifications and improvements, such as:

[0126] 1. Replace some or all of the platinum nanozymes with other nanozymes with ROS scavenging capabilities, such as gold nanozymes, MnO2 nanosheets, and iron oxide nanoparticles;

[0127] 2. Replace SA-SNO with NO donors with different decomposition kinetics, and finely control the NO release curve by adjusting its chemical structure and support properties;

[0128] 3. Optimize the mechanical strength and dissolution rate of microneedles by using materials such as hyaluronic acid, alginate, chitosan, and PVA with different molecular weights and degrees of substitution;

[0129] 4. Design the microneedles as hollow or encapsulated structures, and further load small molecule drugs (minoxidil, JAK inhibitors) or protein factors (VEGF, bFGF, etc.) into them.

[0130] 5. The microneedle array can be designed in strip, fan, or irregular shapes according to the scalp area and the shape of the hair loss area to improve the fit;

[0131] 6. For women or people with mild hair loss, the dosage of nanozyme and NO donor can be reduced, or the microneedle spacing can be increased to avoid overtreatment.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microneedle for hair growth based on synergistic scavenging of reactive oxygen species and delivery of nitric oxide, characterized in that, The hair growth microneedle includes a basal layer and a plurality of microneedles disposed on the basal layer. The microneedle includes at least: a reactive oxygen species scavenging component disposed at the root portion of the microneedle to remove excess reactive oxygen species in the pathological oxidative environment around the hair follicle; and a nitric oxide delivery component disposed at the tip portion of the microneedle to continuously release nitric oxide after the microneedle is inserted into the skin. The mass ratio of the reactive oxygen species scavenging component to the nitric oxide delivery component is (0.01-10):

1. The base layer comprises one or more of the following: hyaluronic acid or its derivatives, alginate or its thiolized derivatives, gelatin, chitosan, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, polylactic acid-glycolic acid copolymer microparticles and their derivatives or combinations; the reactive oxygen species scavenging component comprises nanoenzymes with superoxide dismutase-like and / or catalase-like activities; the nitric oxide delivery component comprises at least one NO donor, which is immobilized or partially immobilized by covalent bonds or electrostatic interactions with the polymer matrix through thiol, carboxyl, or amino groups to form a NO donor-polymer network; The polymer matrix is ​​selected from at least one of the following: thiolated alginate, hyaluronic acid, polyvinyl alcohol, gelatin, and chitosan; wherein, the thiolated alginate is obtained by grafting cysteine ​​or glutathione onto the alginate backbone through EDC / NHS coupling, and undergoes a nitrosation reaction with S-nitroso compounds through thiol groups to form a stable S-nitroso network, thereby achieving the slow and controlled release of NO.

2. The hair growth microneedle as described in claim 1, characterized in that: The nanozyme is selected from at least one of the following: platinum nanozyme, gold nanozyme, manganese dioxide nanoparticles or nanosheets, iron-based nanozyme, cobalt-based nanozyme, copper-based nanozyme and its derivatives or combinations thereof; the particle size of the platinum nanozyme is 1 to 10 nm, preferably 1 to 5 nm.

3. The hair growth microneedle as described in claim 2, characterized in that: The reactive oxygen species scavenging component further includes at least one small or large molecule antioxidant, which is selected from: glutathione, N-acetylcysteine, superoxide dismutase, catalase, vitamin C, vitamin E, tea polyphenols, resveratrol, and combinations thereof.

4. The hair growth microneedle as described in claim 1, characterized in that: The NO donor is selected from: S-nitrosoglutathione, S-nitrosoN-acetylpenicillamine, diazoxide diols, metal nitroso complexes, N-nitrosylated polypeptides, N-nitrosylated polysaccharides and their derivatives or combinations.

5. The hair growth microneedle as described in claim 1, characterized in that: The microneedle has a double-layer or multi-layer structure, including at least: a first functional layer near the needle tip, which is enriched with the nitric oxide delivery component; and a second functional layer near the needle root or base layer, which is enriched with the reactive oxygen species scavenging component; the mass ratio of the reactive oxygen species scavenging component to the nitric oxide delivery component is preferably (0.1-2):

1.

6. The hair growth microneedle as described in claim 1, characterized in that: The thickness of the first functional layer accounts for 5-35% of the needle length, the thickness of the second functional layer accounts for 65-95% of the needle length, the mass of the NO donor in the first functional layer accounts for 1-30% of the dry weight of the microneedle, and the mass of the nanozyme in the second functional layer accounts for 0.01-10% of the dry weight of the microneedle.

7. The hair growth microneedle as described in claim 1, characterized in that: The geometric dimensions of the microneedles meet the following requirements: needle length is 300–900 μm, preferably 400–700 μm; needle root diameter is 150–350 μm; needle tip radius is less than 15 μm; microneedle spacing is 400–800 μm; and the number of microneedles in the microneedle array on the substrate layer is 100–900.

8. The method for preparing hair growth microneedles according to any one of claims 1 to 7, characterized in that: Includes the following steps: (1) Prepare reactive oxygen species scavenging nanoenzymes or antioxidant precursors and disperse them in a first polymer solution to obtain a reactive oxygen species scavenging functional solution; (2) Prepare NO donor and react it with a second polymer solution containing thiol, carboxyl or amino groups to obtain NO donor-polymer precursor solution; (3) Fill the tip area of ​​the microneedle mold with the NO donor-polymer precursor solution and pre-cur it; fill the remaining part of the microneedle mold with the reactive oxygen scavenging solution and dry or cross-link it; (4) Coat the back of the needle body with a base layer solution, and demold after molding to obtain the hair growth microneedles; The first polymer solution contains one or more of the following: hyaluronic acid, gelatin, polyvinyl alcohol and its derivatives, and the total polymer mass concentration of the solution is 10-30 wt%; wherein the mass ratio of hyaluronic acid to polyvinyl alcohol or gelatin is (1-3):

1. The second polymer solution uses sodium thiosulfate as the core component, which provides thiol groups. Under light-protected ice bath conditions, it is mixed with sodium nitrite and hydrochloric acid in an equimolar ratio. After a nitrosation reaction for 40 minutes, the pH is adjusted back to neutral to form S-nitrosolated alginate, i.e., NO donor-polymer precursor solution.

9. The preparation method according to claim 8, characterized in that: In step (1), the reactive oxygen species scavenging nanoenzyme is prepared by aqueous phase reduction or coprecipitation. After the reaction, small molecule ions and unreacted precursors are removed by dialysis, ultrafiltration or centrifugation. In step (3), the solution is fully filled into the microneedle mold by vacuum filtration or centrifugation. The pre-curing includes cooling, photocrosslinking, ionic crosslinking or a combination thereof.

10. The use of the hair growth microneedles according to any one of claims 1 to 7 in the preparation of a medicament for treating or preventing hair loss, characterized in that, The hair loss includes at least one of androgenetic alopecia, telogen effluvium, alopecia areata, chemotherapy-related alopecia, and non-scarring alopecia caused by other reasons.

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