External composition for hair growth regulation and preparation method thereof

Abstract

The invention relates to an external composition for hair growth regulation and a preparation method thereof, and belongs to the technical field of cosmetics and medicines. The composition disclosed by the invention comprises nano mesoporous carbon for enhancing permeation, various surface functional modifications on the nano mesoporous carbon, minoxidil, protein fragments, polypeptides, oligopeptides, amino acids, dutasteride, bimatoprost, traditional Chinese medicine extracts, vitamins, caffeine, nucleosides, growth factors and various auxiliary functional components. Wherein the pore diameter of mesopores of the nano mesoporous carbon for enhancing permeation is 2-30 nm, the total pore volume of the mesopores is 1.5-3.5 cm < 3 > / g, and the particle size of the nano mesoporous carbon is 50-200 nm. The prepared nano mesoporous carbon is granular, controllable in size, stable in structure, free of irritation to skin, capable of keeping inert to various active ingredients in the composition, capable of enhancing absorption and permeation of other ingredients on the skin, wide in application range, capable of compounding various types of active ingredients and high in biological safety.

Description

Technical Field

[0001] The present invention relates to the fields of cosmetics and medicine, specifically a topical composition for regulating hair growth and its preparation method, particularly for the treatment of androgenic alopecia (AGA). This topical composition, through its nano-mesoporous carbon material, effectively enhances the transdermal performance of the active ingredient, strengthens scalp adsorption, and provides sustained release, significantly improving the therapeutic effect. Background Art

[0002] Androgenetic alopecia (AGA) is the most common type of hair loss. Treatment options include topical medications such as minoxidil and oral medications such as finasteride. However, existing treatments face challenges such as poor transdermal absorption, poor efficacy duration, and side effects. In recent years, nanotechnology has been increasingly applied to drug delivery systems, but most nanocarriers still suffer from low drug loading and poor stability.

[0003] Among the current mainstream medications, the topical medication minoxidil is the most common treatment option. Originally developed as a blood pressure medication, it was later found to promote hair growth. It is available over-the-counter, but requires continuous use, and efficacy varies from person to person. Poor skin penetration is a major limitation. Other topical agents, such as tretinoin, prostaglandin analogs, and corticosteroids, have varying effectiveness and may have side effects including skin irritation. Finasteride, the primary oral medication, inhibits 5α-reductase to reduce dihydrotestosterone (DHT) production, the primary cause of AGA. While effective in men, it is unsuitable for women due to its potential teratogenicity, and men may experience side effects such as decreased libido. Dutasteride is similar, but has more common side effects. Other treatments include platelet-rich plasma (PRP) therapy, which stimulates hair growth by injecting the patient's own platelet concentrate. Some studies have shown promise, but results have been inconsistent, and PRP therapy is highly invasive and costly. Low-intensity laser therapy (LLLT) can stimulate hair growth, but its mechanism is unclear and its long-term effectiveness is uncertain. Hair transplant surgeries such as follicular unit transplantation (FUT) and follicular unit extraction (FUE) are effective, but they are invasive and expensive, making them unsuitable for all patients.

[0004] AGA treatment, particularly for topical medications, faces multiple challenges in transdermal delivery: ① The skin barrier: The stratum corneum, the outermost layer of the skin, is the primary barrier, limiting drug penetration. ② Scalp specificity: The drug must be precisely delivered to the scalp to minimize systemic side effects. ③ Sustained release: The drug must be delivered continuously to reduce dosing frequency and maintain efficacy. ④ Scalp adhesion: The formulation must adhere well to the scalp, especially in areas of thinning hair, to prevent it from washing away.

[0005] In recent years, the application of nanotechnology in drug delivery systems has increased significantly, showing particular promise in transdermal delivery. By improving drug solubility, enhancing skin penetration, and controlling release rates, these technologies can effectively overcome the limitations of traditional formulations. Common nanocarriers include: ① Liposomes: Composed of phospholipids, they can encapsulate both hydrophilic and hydrophobic drugs, improving skin penetration and providing sustained release. However, they are expensive, have poor stability, and a short shelf life. ② Solid lipid nanoparticles (SLNs): Composed of solid lipids, they offer excellent stability and can enhance drug penetration, but they have low drug loading, potential cytotoxicity, and are also expensive. ③ Polymer nanoparticles: Made of biocompatible polymers, they allow for customized release profiles, but they also have low drug loading and are relatively expensive. Although these nanocarriers have found application in the treatment of AGA, they still face challenges such as high cost, low yield, low drug loading, poor stability, and insufficient biocompatibility, necessitating the exploration of novel materials.

[0006] Nano-mesoporous carbon materials, due to their unique physicochemical properties, have become a hot topic in the drug delivery field. Their key advantages include high surface area, which significantly increases drug loading capacity; adjustable pore size, which facilitates controlled drug release rate; excellent biocompatibility, low toxicity, and enhanced skin permeation, allowing active ingredients to more effectively penetrate the skin. Furthermore, the surface of nano-mesoporous carbon materials can be functionalized to enhance drug loading and release control. For example, they can penetrate the stratum corneum to deliver protein drugs, serving as transdermal delivery platforms to enhance drug penetration and provide sustained release. Given their high surface area and adjustable pore size, nano-mesoporous carbon materials can be loaded with minoxidil or other hair growth-promoting active ingredients, such as tretinoin, prostaglandin analogs, or stem cell factors, for the treatment of AGA. The high surface area increases drug loading, while the adjustable pore size enables sustained release. Furthermore, their excellent biocompatibility ensures long-term safety and reduces side effects. Their high surface energy enhances scalp adsorption, prolongs drug contact time, and improves transdermal performance, addressing the inconsistent efficacy of existing treatments.

[0007] In summary, the background technology details the current status and challenges of AGA treatment, the application potential of nanotechnology in drug delivery, and the advantages of carbon-based nanomaterials, especially nano-mesoporous carbon materials, in transdermal delivery. The present invention aims to develop a novel topical composition by utilizing the unique properties of nano-mesoporous carbon materials to significantly enhance the therapeutic effect of AGA.

[0008] The mesoporous carbon carrier-based hair growth treatment composition provided by this invention combines efficient transdermal drug delivery with sustained-release effects, filling the unmet technical gap in the market for treating androgenic alopecia. Due to its innovative application in drug delivery, this invention is expected to have broad market prospects in functional cosmetics and pharmaceutical products.

[0009] This product has significant competitive advantages over existing treatment options on the market. Through collaboration with pharmaceutical companies, this invention can be further developed into a hair growth product with even higher efficacy, advancing the treatment of androgenic alopecia. Summary of the Invention

[0010] The present invention aims to provide a novel topical composition for regulating hair growth and a method for preparing the same. First, nano-mesoporous carbon is synthesized using Chlorella as a template. Surface functional modification is then performed on the nano-mesoporous carbon. Finally, the nano-mesoporous carbon is mixed with one or more active ingredients and auxiliary functional ingredients to form a topical composition. The present invention aims to provide a novel topical composition for regulating hair growth and a method for preparing the same. First, nano-mesoporous carbon is synthesized using Chlorella as a template. ... also provides a variety of novel active ingredients.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a topical composition for regulating hair growth, characterized in that it comprises nano-mesoporous carbon for enhancing penetration, multiple surface functional modifications on the nano-mesoporous carbon, minoxidil, protein fragments, polypeptides, oligopeptides, amino acids, dutasteride, bimatoprost, traditional Chinese medicine extracts, vitamins, caffeine, nucleosides, growth factors, and multiple functional ingredients.

[0013] Furthermore, the topical composition for hair growth regulation is characterized in that the mesopore diameter of the nano-mesoporous carbon for enhancing penetration is 2-30 nm, and the total mesopore volume is 1.5-3.5 cm 3 / g, carbon particle size 50~200nm.

[0014] Furthermore, the external composition for regulating hair growth is characterized in that the nano-mesoporous carbon for enhancing penetration contains oxygen-containing functional groups, and the oxygen-containing functional groups include at least one of carbonyl, hydroxyl and epoxy groups.

[0015] Furthermore, the nano-mesoporous carbon for enhancing permeation is characterized in that the nano-mesoporous carbon has a variety of surface functional modifications, including at least one of polyethylene glycol (PEG), chitosan, polylysine, polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polydimethylaminoethyl methacrylate (PDMAEMA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethyleneimine (PEI), polyphosphate (PPE), polyethylene glycol polyacrylamide (PEG-PAA), polyethylene ethyleneamine (PEHA), polyacrylate (PAA) and its copolymers, carboxymethyl cellulose (CMC), hydroxyethyl cellulose, polyquaternary ammonium salts, sodium carboxymethyl cellulose, chitosan derivatives, sodium tetradecyl sulfate, lecithin, and cell penetrating peptides.

[0016] Furthermore, the external-use composition for regulating hair growth is used in the preparation of cosmetics, skin care products, shampoo, conditioner, and essence.

[0017] Furthermore, the method for preparing nano-mesoporous carbon for enhancing permeability is characterized by comprising the following steps:

[0018] S1: Preparation and nanostructure of biological templates:

[0019] Chlorella vulgaris is selected, as it is easy to cultivate on a large scale and is readily available. The algae is grown in a suitable culture medium and harvested by centrifugation and filtration. The algae are then washed multiple times with deionized water to remove residual culture medium. The washed Chlorella is then freeze-dried or oven-dried at low temperatures to produce dry algae powder.

[0020] S2: Nano-processing:

[0021] ① High-energy ball milling: Place the dried algae powder in a ball mill with an appropriate amount of grinding balls and mill at 300–500 rpm for 8 hours to achieve initial particle size reduction. ② Ultrasonic-assisted dispersion: Suspend the milled algae powder in an appropriate amount of ethanol and use a high-power ultrasonicator (20 kHz for 30 minutes) to further break down algae cell fragments to a size of 50–200 nm. ③ Purification: Purify the algae powder dispersion using a 200 nm pore size filter membrane to collect algae nanotemplates within the target size range (50–200 nm).

[0022] S3: Preparation of composite precursor:

[0023] Prepare a phenol-formaldehyde resin precursor solution and adjust the pH to an acidic pH of 2–4 to promote polymerization. Add an appropriate amount of metal ions (such as Zn²⁺ or Mg²⁺) as auxiliary structure modifiers.

[0024] S4: Nanoalgae template loading:

[0025] The algae nano-template obtained in step 1 is evenly dispersed in the precursor solution, and high-speed stirring and ultrasonic-assisted mixing are used to ensure that the algae template is fully and evenly mixed with the precursor to form a self-assembled mixed system.

[0026] S5: Microwave-assisted cross-linking and gelation:

[0027] The mixed system was placed in a microwave reactor, and microwave rapid heating (power 300W, time 5 minutes) was used to promote rapid cross-linking and gelation of the phenol-formaldehyde precursor, forming an ordered structure around the algae template and embedding it into the natural template.

[0028] S6: Plasma treatment:

[0029] The gelled sample was placed in a plasma (inert environment) device (argon plasma) for 20 minutes to remove organic residues on the surface and introduce micropores on the material surface, which facilitates the subsequent formation and control of the pore structure.

[0030] S7: High temperature carbonization:

[0031] The plasma-treated solid precursor is carbonized in an inert atmosphere (nitrogen or argon) at high temperature, increasing the temperature at 5°C / min to 1800°C and holding for 1 hour. At this high temperature, the organic precursor is converted into carbon material, while the algae template and part of the organic template are pyrolyzed, leaving behind the template's predetermined pore structure.

[0032] S8: Post-processing:

[0033] After carbonization, the sample is washed with an acid (dilute hydrochloric acid) of appropriate concentration to remove residual metal ions and decomposition products. The acid-washed sample is then placed in a supercritical CO2 extraction apparatus and treated at an appropriate temperature (40–50°C) and pressure for 1 hour to completely remove the template residue and further fine-tune the mesopore size (target 2–30 nm). CO2 activation treatment is then performed at a higher temperature (800–900°C) for a specified time to form more mesopores. Finally, a low-concentration oxygen plasma treatment is performed for 12 hours to remove oxygen-containing functional groups from the nano-mesoporous carbon material. The sample is then crushed in a high-energy ball mill for 30 minutes to obtain nano-mesoporous carbon material with a particle size range of 50–200 nm for enhanced permeability.

[0034] Furthermore, the external-use composition for regulating hair growth is in the form of a lotion, cream, gel, essence, spray or powder.

[0035] Furthermore, the topical composition for regulating hair growth is characterized in that the protein fragments include: osteopontin fragments, whey protein fragments, collagen fragments, keratin fragments, adiponectin fragments, and recombinant protein fragments.

[0036] Furthermore, the topical composition for regulating hair growth is characterized in that the polypeptide includes: a polypeptide containing the core active sequence SVVYGLR (S: Serine, V: Valine, Y: Tyrosine, G: Glycine, L: Leucine, R: Arginine) in osteopontin, the polypeptide can be modified by acylation (Ac-) at the N-terminus and amidation (-NH2) at the C-terminus; the polypeptide can be modified by phosphorylation (pY-) of amino acids; the polypeptide can be linked to biotin at the N-terminus or C-terminus; the polypeptide can be linked to PEG; the polypeptide can form a disulfide bond by introducing cysteine (C) at the N-terminus and C-terminus, so that the polypeptide forms a cyclic structure (such as C-SVVYGLR-C).

[0037] Furthermore, the topical composition for regulating hair growth is characterized in that the polypeptide may also include: a polypeptide containing at least one of the core sequences SIVYGLR, SVVFGLR, SVVYGLK, VVYGLR, SVVYGL, and SVVYGLRRP, wherein the polypeptide may be modified by acylation (Ac-) at the N-terminus and amidation (-NH2) at the C-terminus; the polypeptide may be modified by phosphorylation (pY-) of amino acids; the polypeptide may be linked to biotin at the N-terminus or C-terminus; the polypeptide may be linked to PEG; the polypeptide may form a disulfide bond by introducing C: cysteine (Cysteine) at the N-terminus and C-terminus, so that the polypeptide forms a cyclic structure.

[0038] Furthermore, the topical composition for regulating hair growth is characterized in that the oligopeptide includes at least one of: Copper Tripeptide-1 (GHK-Cu), Acetyl Tetrapeptide-3, Biotin Tripeptide-1, Myristoyl Pentapeptide-17, and Palmitoyl Tetrapeptide-20.

[0039] Furthermore, the topical composition for regulating hair growth is characterized in that the amino acids include: S: Serine, V: Valine, Y: Tyrosine, G: Glycine, L: Leucine, R: Arginine, D: Aspartic Acid, P: Proline, T: Threonine, I: Isoleucine, H: Hydroxyproline, and K: Lysine. One or more of the following:

[0040] Furthermore, the topical composition for regulating hair growth is characterized in that the polypeptide may also include: one or more of: collagen peptides (Collagen Peptides), keratin peptides (Keratin Peptides), BPC-157 (Body Protective Compound-157), and APN5 polypeptide.

[0041] Furthermore, the external-use composition for regulating hair growth described in 1 is characterized in that the traditional Chinese medicine includes at least one of: Platycladus orientalis leaves, Angelica sinensis, Polygonum multiflorum, Chuanxiong, Astragalus membranaceus, Salvia miltiorrhiza, ginger, Angelica dahurica, Rehmannia root, Lycium barbarum, Licorice, and ginseng.

[0042] Furthermore, the topical composition for regulating hair growth is characterized in that the vitamins include at least one of vitamin E, vitamin C, panthenol (vitamin B5), and niacinamide (vitamin B3).

[0043] Furthermore, the external composition for regulating hair growth is characterized in that the nucleoside comprises at least one of adenosine, adenosine liposomes, and 5'-methylthioadenosine.

[0044] Furthermore, the topical composition for regulating hair growth is characterized in that the growth factor includes at least one of vascular endothelial growth factor (VEGF), fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor-1, and hepatocyte growth factor.

[0045] Furthermore, the topical composition for regulating hair growth comprises multiple functional ingredients including: β-cyclodextrin, retinoic acid (retinoic acid), red clover extract, camphor, limonene, urea, salicylic acid, glycerin, transdermal peptides, hyaluronic acid, sodium hyaluronate and its cross-linked polymers, sucrose, laurocapram, alkyl betaine, lauryl betaine, isopropyl palmitate, isopropyl methyl palmitate, menthol, carbomer, xanthan gum, polyacrylamide, phenoxyethanol, parabens, polysorbate 80, ethylenediaminetetraacetic acid (EDTA), etc.

[0046] Furthermore, the topical composition for regulating hair growth, wherein the multiple functional ingredients also include ethanol, isopropyl alcohol, dimethyl sulfoxide (DMSO), decyl methyl sulfoxide, oleic acid, linoleic acid, lauryl alcohol, menthol, propylene glycol, petrolatum, silicone oil, monoglyceride or diglyceride, medium chain triglyceride, glyceryl stearate, glyceryl stearate citrate, cetearyl alcohol, ethylhexyl hydroxystearate, caprylic / capric triglyceride, allantoin (0.5-1%) or dipotassium glycyrrhizate.

[0047] Use of the external-use composition for regulating hair growth according to any one of claims 1 to 15 in a drug for treating androgenic alopecia. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 TEM image of nano-mesoporous carbon in Example 1 Figure 2 Pore diameter and total pore volume of nano-mesoporous carbon in Example 1 Figure 3 The hair growth records on the 15th day in Example 3 are from left to right: experimental group, control group 1 and control group 2 Figure 4 The hair growth records on the 30th day in Example 3 are from left to right: experimental group, control group 1 and control group 2 Figure 5 The hair growth records on the 15th day in Example 4 are from left to right: experimental group, control group 1 and control group 2 Figure 6 The hair growth records on the 30th day in Example 4 are from left to right: experimental group, control group 1 and control group 2 Figure 7 The hair growth records on the 15th day in Example 5 are from left to right: experimental group, control group 1 and control group 2 Figure 8 The hair growth records on the 30th day in Example 5 are from left to right: experimental group, control group 1 and control group 2 Figure 9The hair growth records on the 15th day in Example 6 are from left to right: experimental group, control group 1 and control group 2 Figure 10 Hair growth on day 30 in Example 6, from left to right are experimental group, control group 1 and control group 2 Figure 11 The hair growth records for 15 days in Example 7 are from left to right: experimental group, control group 1 and control group 2 Figure 12 Hair growth on day 30 in Example 7, from left to right are experimental group, control group 1 and control group 2 Figure 13 The hair growth records for 15 days in Example 8 are from left to right: experimental group, control group 1 and control group 2 Figure 14 Hair growth on the 30th day in Example 8, from left to right are the experimental group, control group 1 and control group 2 Figure 15 The hair growth records for 15 days in Example 9 are from left to right: experimental group, control group 1 and control group 2 Figure 16 Hair growth on the 30th day in Example 9, from left to right are the experimental group, control group 1 and control group 2 Figure 17 Hair growth after 15 days in Example 10, from left to right are the experimental group, control group 1 and control group 2 Figure 18 Hair growth on the 30th day in Example 10, from left to right are the experimental group, control group 1 and control group 2 DETAILED DESCRIPTION

[0049] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0050] Example 1 Preparation of 50-200 nm nano-mesoporous carbon

[0051] S1: Chlorella preparation:

[0052] To prepare Chlorella, separate the algae using centrifugation and filtration, then wash them multiple times with deionized water to remove residual culture medium. The washed Chlorella is freeze-dried at low temperatures to produce dry algal powder. The freeze-drying process is as follows: ① Freeze the Chlorella at -80°C for 24 hours to ensure complete freezing of the water and the formation of ice crystals, preparing for subsequent sublimation. ② Place the frozen sample in a freeze dryer set to -20°C and a pressure of 0.01 mbar (approximately 1 Pa) for 24 hours. During this stage, the ice transforms directly from a solid to a gaseous state through sublimation, removing most of the water. ③ A secondary drying stage is performed at 30°C for another 24 hours to remove any remaining bound water.

[0053] S2: Nano-processing:

[0054] ① High-energy ball milling: Place the dried algae powder from S1 into a ball mill, add an appropriate amount of grinding balls, and mill at 400 rpm for 8 hours to achieve initial particle size reduction. ② Ultrasonic-assisted dispersion: Suspend the milled algae powder in 500 mL of ethanol and use a high-power ultrasonicator (20 kHz, 30 minutes) to further break down algae cell fragments to a size of 50–200 nm. ③ Purification: Filter and purify the algae dispersion using a 200 nm pore size filter membrane to collect algae nanotemplates within the target size range.

[0055] S3: Preparation of composite precursor:

[0056] Prepare a phenol-formaldehyde (1:1) resin precursor solution and adjust the pH to 2–4 using dilute hydrochloric acid to promote polymerization. Add 5% zinc sulfate and 5% magnesium sulfate, with the zinc and magnesium ions acting as auxiliary structural modifiers.

[0057] S4: Nanoalgae template loading:

[0058] 10 g of the algae nanotemplate obtained in step S1 was evenly dispersed into the precursor solution prepared in step S3 (the mass ratio of algae nanotemplate to phenol-formaldehyde resin was 1:1). High-speed stirring and ultrasonic-assisted mixing were used to ensure that the algae template was fully and evenly mixed with the precursor to form a self-assembled mixed system.

[0059] S5: Microwave-assisted cross-linking and gelation:

[0060] The mixed system was placed in a microwave reactor, and microwave rapid heating (power 300W, time 5 minutes) was used to promote rapid cross-linking and gelation of the phenol-formaldehyde precursor, forming an ordered structure around the algae template and embedding it into the natural template.

[0061] S6: Plasma treatment:

[0062] The gelled sample was placed in a plasma (inert atmosphere) apparatus (argon plasma, 500W, 50sccm, 1Pa) for 20 minutes. This removed organic residues from the surface and introduced micropores into the material surface, facilitating the subsequent formation and control of the pore structure.

[0063] S7: High temperature carbonization:

[0064] The plasma-treated solid precursor is carbonized in an inert atmosphere (nitrogen) at high temperature, increasing the temperature at 5°C / min to 1800°C and holding for one hour. At this high temperature, the organic precursor is converted into carbon material, while the algae template and part of the organic template are pyrolyzed, leaving behind the template's predetermined pore structure.

[0065] S8: Post-processing:

[0066] After carbonization, the sample was washed with dilute hydrochloric acid to remove residual metal ions and decomposition products. The acid-washed sample was placed in a supercritical carbon dioxide extraction device and treated at an appropriate temperature of 50°C and 20MPa pressure for 1 hour to completely remove residual impurities and further fine-tune the mesopore size (target 2-30nm). CO2 activation treatment was performed at 800°C for 1h to form more mesopores. Finally, 10sccm oxygen plasma 100W was used for treatment for 12h to obtain oxygen-containing functional groups on the nano-mesoporous carbon material. A high-energy ball mill was used to crush it again for 30min to obtain a nano-mesoporous carbon material with a particle size in the range of 50-200nm for enhanced penetration. The nano-mesoporous carbon material obtained by this implementation has a size of 50~200nm, and its microstructure is as follows Figure 1 As shown in the TEM photo, the mesopore volume is ~3cm 3 / g, the pore size is mainly distributed in the range of 2~50nm, such as Figure 2 As shown in .

[0067] Example 2 Transdermal Experiments of Nano-mesoporous Carbon Materials and Nano-carbon Black for Enhanced Permeation

[0068] The transdermal permeation performance of the experimental group (1) using the nano-mesoporous carbon prepared in Example 1 and minoxidil, the control group (2) using 50-200 nm nanocarbon black (ECP600) and minoxidil, and the control group (3) using pure minoxidil were compared on the skin model of Bama miniature pigs to evaluate the enhanced permeation effect of nano-mesoporous carbon as a drug carrier.

[0069] Skin samples: Back skin of Bama miniature pigs (Sus scrofa domestica), 6 months old, half male and half female, approximately 1.5–2 mm thick (including epidermis and dermis), 2.5 cm diameter discs, subcutaneous fat removed, washed with saline, and stored at 4°C. Skin integrity was checked before use.

[0070] (1) Experimental equipment and reagents:

[0071] Franz diffusion cell: effective diffusion area 1.77 cm², receptor chamber volume 12 mL. Constant temperature water bath: 37°C ± 0.5°C. Magnetic stirrer: 300 rpm. Receptor solution: phosphate buffered saline (PBS, pH 7.4) containing 0.5% Tween 80 (to enhance minoxidil solubility). Analytical equipment: High-performance liquid chromatography (HPLC) with UV detection (wavelength 254 nm). Other equipment: micropipettes, disposable syringes, filter membrane (0.45 μm), scalpel, forceps, and saline. Franz diffusion cell assembly: Secure the skin sample between the donor and receptor chambers of a Franz diffusion cell, with the epidermis facing the donor chamber, ensuring there are no bubbles or wrinkles. Add 12 mL of PBS (containing 0.5% Tween 80) to the receptor chamber and stir with a magnetic stirrer at 300 rpm. Place the diffusion cell in a constant temperature water bath at 37°C and equilibrate for 30 minutes.

[0072] (2) Experimental groups and skin model allocation:

[0073] The pigskins of 24 Bama miniature pigs were randomly divided into three groups, with 12 pieces in each group. The specific groups are as follows:

[0074] ① Nano-mesoporous carbon material plus minoxidil group (experimental group): The nano-mesoporous carbon material from Example 1 was uniformly dispersed at a concentration of 1% w / v in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol. Then, 5% w / v minoxidil was added and mixed thoroughly. ② Nano-carbon black material (ECP600) plus minoxidil group (control group 1): 50-200 nm nano-carbon black (ECP600) was uniformly dispersed at a concentration of 1% w / v in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol. Then, 5% w / v minoxidil was added and mixed thoroughly. Commercial minoxidil group (control group 2): Minoxidil was dispersed at a concentration of 5% w / v in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol and mixed thoroughly. For both the experimental and control groups, 200 μL of the prepared solution was evenly applied to the skin surface of the donor. Twelve parallel samples (n=12) were set up for each group to ensure statistical reliability. The donor chamber lid was closed to prevent solvent evaporation.

[0075] (3) Sampling and experimental duration:

[0076] 1 mL samples were removed from the receptor compartment at 0.5, 1, 2, 4, 8, 12, and 24 hours, and immediately replaced with an equal volume of PBS (containing 0.5% Tween 80). The samples were filtered through a 0.45 μm filter and stored at 4°C until HPLC analysis. The experiment lasted 24 hours, maintained at 37°C and stirred at 300 rpm.

[0077] (4) Skin residue analysis:

[0078] After the experiment, the diffusion cell was disassembled, and the skin surface was wiped with a cotton swab dipped in saline to remove any unpenetrated drug. The skin sample was minced and placed in 10 mL of methanol. Ultrasonic extraction was performed for 30 minutes (40 kHz, 100 W). The extract was filtered (0.45 μm), and the residual minoxidil content in the skin was determined by HPLC.

[0079] (5) Drug concentration detection:

[0080] HPLC detection, C18 column (4.6 × 250 mm, 5 μm). Mobile phase: methanol: water (70:30, v / v), flow rate: 1 mL / min. Detection wavelength: 254 nm. Retention time: approximately 5.5 minutes. Minoxidil concentration was measured (range 1-1000 μg / mL, R² > 0.999). Skin retention: calculated based on the minoxidil concentration in the skin extract (μg / g skin).

[0081] (6) Experimental results and analysis:

[0082] 1. Cumulative permeation (Q):

[0083] Minoxidil group: The cumulative permeation amount in 24 hours was approximately 140-200 μg / cm².

[0084] Nanocarbon black / minoxidil group: The cumulative permeation amount in 24 hours was approximately 170-215 μg / cm².

[0085] Nano-mesoporous carbon / minoxidil group: The cumulative permeation amount over 24 hours was approximately 330-400 μg / cm² (approximately twice that of the control group, attributed to the permeation enhancement effect of nano-mesoporous carbon).

[0086] Trend: The nano-mesoporous carbon / minoxidil group showed a faster permeation rate in the early stage (0.5-4 hours) and entered a steady state after 6 hours.

[0087] 2. Permeation flux (J):

[0088] Minoxidil group: Steady-state flux was approximately 8-10 μg / cm² / h.

[0089] Nanocarbon black / minoxidil group: Steady-state flux was approximately 10-12 μg / cm² / h.

[0090] Nano-mesoporous carbon / minoxidil group: Steady-state flux was approximately 16-20 μg / cm² / h (approximately twice that of the control group).

[0091] Results: The high surface area and porous structure of nano-mesoporous carbon enhanced the solubility and skin diffusion of minoxidil, similar to the effects of nanocarriers reported in the literature. Nanocarbon black also had a certain enhancement effect, but the effect was less pronounced. This may be because nanocarbon black lacks mesopores and adsorbs a relatively small amount of minoxidil, leaving a large amount of minoxidil in a free state, which is not enhanced by the permeation of free minoxidil. Two comparative experiments were conducted to verify this: Nano-mesoporous carbon and minoxidil were mixed according to the aforementioned ratios, and nanocarbon black (ECP600) and minoxidil were mixed. The dispersions were then centrifuged at high speed. After removal of the precipitate, the resulting solution was evaporated to dryness. The nano-mesoporous carbon and minoxidil groups showed almost no residual solution after evaporation, while the nanocarbon black (ECP600) and minoxidil groups showed a large amount of minoxidil powder remaining in the container after evaporation, indicating that the nanocarbon black (ECP600) was unable to adequately adsorb the minoxidil molecules.

[0092] 3. Permeability coefficient (Kp):

[0093] Minoxidil group: Kp is about 1.6-2.0×10 -4 cm / h.

[0094] Nanocarbon black / minoxidil group: Kp is about 2.0-2.4×10 -4 cm / h.

[0095] Nano-mesoporous carbon / minoxidil group: Kp is about 3.2-4.0×10 -4 cm / h.

[0096] 4. Skin retention:

[0097] Minoxidil group: The average residual amount of minoxidil in the skin was 73 μg / g.

[0098] Nanocarbon black / minoxidil group: The average minoxidil residue in the skin was 93 μg / g.

[0099] Nano-mesoporous carbon / minoxidil group: The average residual minoxidil in the skin was 161 μg / g (nanomesoporous carbon can promote the retention of drugs in the skin).

[0100] Example 3 Comparative Verification of the Effects of Minoxidil-Based External Use Composition and Minoxidil on Male Bald Mice

[0101] The nano-mesoporous carbon prepared in Example 1 was used to prepare a minoxidil-based external composition. Minoxidil was selected as the active ingredient and compounded with the nano-mesoporous carbon. The hair growth effect of minoxidil was compared with that of minoxidil in male bald mice.

[0102] 1. Preparation of male bald mice:

[0103] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0104] 2. Experimental Preparation:

[0105] (1) Experimental groups and mouse allocation:

[0106] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0107] Nano-mesoporous carbon material plus minoxidil group (experimental group): The nano-mesoporous carbon in Example 1 was uniformly dispersed in 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol at a concentration of 1% w / v, and then 5% w / v minoxidil was added and mixed evenly.

[0108] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0109] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0110] The random number table method was used to group the animals and ensure that there were no significant differences in body weight and shaved areas among the groups (p>0.05, one-way analysis of variance).

[0111] (2) Preparation of experimental reagents:

[0112] Preparation steps:

[0113] 1% nano-mesoporous carbon material was added to 10 mL of ethanol and ultrasonically dispersed for 30 minutes at 200 W and 25°C in an ultrasonic cleaner (Kunshan Ultrasonic, model KQS-250, frequency 40 kHz) to obtain a uniform dispersion A. 5% minoxidil was dissolved in dispersion A and magnetically stirred (IKA, model C-MAG HS 7) at 500 rpm for 30 minutes until completely dissolved, resulting in a uniform dispersion. After mixing, propylene glycol and glycerol were added in appropriate proportions, and stirring was continued for 15 minutes. The mixture was then diluted to volume with deionized water in appropriate proportions and stirred for 10 minutes to obtain the final composition (minoxidil concentration 5% w / v, nano-mesoporous carbon material concentration 1% w / v). The composition was filtered (0.22 μm microporous membrane), placed in a brown sealed bottle, and stored at 4°C in the dark until further use. A minoxidil control was prepared similarly, except that only minoxidil was added without nano-mesoporous carbon. The matrix of the blank control group was prepared with the same solvent ratio and did not contain active ingredients.

[0114] (3) Experimental equipment and materials:

[0115] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL of fluid. Digital camera (Canon EOS 80D with 50 mm lens): Photograph hair growth. Microscope (Olympus BX53 with ImageJ software): Analyze hair density and length.

[0116] 3. Experimental steps and procedures:

[0117] (1) Dosage regimen:

[0118] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL per dose at 3:00 PM daily for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the entire shaved area is evenly covered and avoiding dripping. After dosing, the mouse was caged individually for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the back of the mouse was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0119] (2) Observation and recording:

[0120] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0121] (3) Data collection and processing:

[0122] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0123] 4. Experimental results analysis:

[0124] (1) Hair recovery rate:

[0125] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0126] Day 15 (as Figure 3 shown):

[0127] Experimental group: The recovery rate was about 30%, the hair was evenly distributed, and the coverage area was high.

[0128] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0129] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0130] Day 30 (if Figure 4 shown):

[0131] Experimental group: The recovery rate exceeded 90%, the hair grew thick and even, and basically returned to its pre-shaving state.

[0132] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0133] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0134] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was significantly higher than that of the control groups 1 and 2 (p<0.01), and that of the control group 1 was higher than that of the control group 2 (p<0.05).

[0135] (2) Skin safety:

[0136] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0137] (3) Discussion of the results:

[0138] Comparison of efficacy: The nano-mesoporous carbon material plus minoxidil group showed superior hair recovery rates at both 15 and 30 days compared to the commercial minoxidil group, indicating that the nano-mesoporous carbon material enhanced the transdermal delivery and scalp absorption of minoxidil. The nano-mesoporous carbon material may deliver minoxidil via the hair follicle pathway, prolonging its retention time on the scalp and synergizing with the permeation-enhancing effects of ethanol and propylene glycol, significantly improving efficacy. Its high specific surface area and sustained-release properties also increased drug utilization. Control performance: The commercial minoxidil group effectively promoted hair growth, but the effect was inferior to that of the experimental group. The blank group, relying solely on natural recovery, had the worst effect.

[0139] Example 4 Comparative Verification of the Effects of a Peptide-Based Topical Composition and Minoxidil on Male Bald Mice

[0140] The nano-mesoporous carbon prepared in Example 1 was used to prepare a polypeptide-based topical composition. Peptides Ac-SVVYGLR, SVVYGLR-KKK, and PEG-SVVYGLR containing the osteopontin core sequence SVVYGLR were selected as active ingredients and composited with the nano-mesoporous carbon. The hair growth effect was compared with that of minoxidil in male bald mice.

[0141] 1. Preparation of male bald mice:

[0142] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0143] 2. Experimental Preparation:

[0144] (1) Experimental groups and mouse allocation:

[0145] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0146] Nano-mesoporous carbon material plus polypeptide group (experimental group): The nano-mesoporous carbon in Example 1 was used, PEI20KD and PEG-PLA were added to modify the nano-mesoporous carbon, the active ingredients were Ac-SVVYGLR, SVVYGLR-KKK and PEG-SVVYGLR, and the solvent was 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol.

[0147] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0148] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0149] The random number table method was used to group the animals and ensure that there were no significant differences in body weight and shaved areas among the groups (p>0.05, one-way analysis of variance).

[0150] (2) Preparation of experimental reagents:

[0151] Preparation steps:

[0152] The nano-mesoporous carbon in Example 1 was first dispersed in deionized water at a concentration of 1% w / v. 1% w / v PEI20KD and 1% PEG-PLA were then added to modify the nano-mesoporous carbon. After uniform mixing, the free PEI20KD and PEG-PLA were removed by filtration and washing. The nano-mesoporous carbon was then redispersed in water at a pH of 10 at a concentration of 0.3% w / v. 0.1% w / v of Ac-SVVYGLR, SVVYGLR-KKK, and PEG-SVVYGLR were added, and the mixture was stirred thoroughly for 1 hour. After filtration, the mixture was vacuum-dried at room temperature. The resulting composite was uniformly dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 0.1% w / v of nano-mesoporous carbon to obtain a topical composition. The composition was filtered (0.22 μm microporous membrane), placed in a brown sealed bottle, and stored at 4°C in the dark for later use. The minoxidil control group and the blank control group were prepared in the same manner as in Example 2.

[0153] (3) Experimental equipment and materials:

[0154] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL. Digital camera (Canon EOS 80D with 50 mm lens): Record photos of hair growth.

[0155] 3. Experimental steps and procedures:

[0156] (1) Dosage regimen:

[0157] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL daily at 3:00 PM for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the solution covers the entire shaved area and avoiding dripping. After dosing, the mouse was individually caged for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the mouse's back was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Treatment Groups: Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0158] (2) Observation and recording:

[0159] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0160] (3) Data collection and processing:

[0161] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0162] 4. Experimental results analysis:

[0163] (1) Hair recovery rate:

[0164] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0165] Day 15 (as Figure 5 shown):

[0166] Experimental group: The recovery rate was about 35%, the hair was evenly distributed, the color was uniform, and the coverage area was high.

[0167] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0168] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0169] Day 30 (if Figure 6 shown):

[0170] Experimental group: The recovery rate exceeded 90%, the hair grew thick and even, and basically returned to its pre-shaving state.

[0171] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0172] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0173] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was significantly higher than that of the control groups 1 and 2 (p<0.01), and that of the control group 1 was higher than that of the control group 2 (p<0.05).

[0174] (2) Skin safety:

[0175] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0176] (3) Discussion of the results:

[0177] Comparison of efficacy: The nano-mesoporous carbon material plus peptide group showed superior hair recovery rates compared to the commercial minoxidil group at both 15 and 30 days, indicating that the nano-mesoporous carbon material plus peptide combination achieved excellent transdermal delivery and scalp adsorption. The nano-mesoporous carbon material may deliver peptides via the hair follicle pathway, prolonging drug retention on the scalp and synergizing with the permeation-enhancing effects of ethanol and propylene glycol, significantly improving efficacy. Furthermore, its high specific surface area and sustained-release properties also increased drug utilization. Control performance: The commercial minoxidil group effectively promoted hair growth, but the effect was inferior to that of the experimental group. The blank group, relying solely on natural recovery, had the worst effect.

[0178] Example 5 Comparative Verification of the Effects of a Topical Composition Based on Oligopeptides and Multiple Auxiliary Active Ingredients and Minoxidil on Male Bald Mice

[0179] The nano-mesoporous carbon prepared in Example 1 was used to prepare an oligopeptide-based topical composition, and the oligopeptide components were: acetyl tetrapeptide-3, biotin tripeptide-1 (Bio-GHK), tripeptide-1 copper (GHK-Cu, blue copper peptide), and auxiliary active ingredients: caffeine, nicotinamide and adenosine liposomes were used as active ingredients to be compounded with the nano-mesoporous carbon, and the hair growth effect was compared with minoxidil in male bald mice.

[0180] 1. Preparation of male bald mice:

[0181] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0182] 2. Experimental Preparation:

[0183] (1) Experimental groups and mouse allocation:

[0184] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0185] Nano-mesoporous carbon material plus polypeptide group (experimental group): The nano-mesoporous carbon in Example 1 was used, and PEI25KD, chitosan and polylactic acid-glycolic acid copolymer PLGA were added to modify the nano-mesoporous carbon. The active ingredients were acetyl tetrapeptide-3, biotin tripeptide-1 (Bio-GHK), and tripeptide-1 copper (GHK-Cu, blue copper peptide). The auxiliary ingredients were caffeine, nicotinamide, and adenosine liposomes. The solvent was 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol.

[0186] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0187] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0188] The random number table method was used to group the animals and ensure that there were no significant differences in body weight and shaved areas among the groups (p>0.05, one-way analysis of variance).

[0189] (2) Preparation of experimental reagents:

[0190] Preparation steps:

[0191] The nano-mesoporous carbon in Example 1 was first dispersed in deionized water at a concentration of 1% w / v, and then 0.5% w / v PEI25KD, 0.5% w / v chitosan and 1% PLGA were added to modify the nano-mesoporous carbon. After mixing evenly, the free PEI25KD, chitosan and PLGA were removed by filtration and washing. The nano-mesoporous carbon was redispersed in a 50% water + 50% ethanol solution at a concentration of 1% w / v, and acetyl tetrapeptide-3 (0.1%), biotin tripeptide- 1 (0.2%) and 0.2% each of tripeptide-1 copper, after thorough mixing, filtered and dried under vacuum at room temperature. The complex was re-dissolved in water at a concentration of 0.5% w / v nano-mesoporous carbon, and then 1% caffeine, 1% nicotinamide, and 1% adenosine liposomes were added. The mixture was stirred thoroughly for 1 hour, filtered and dried under vacuum at room temperature. The resulting complex was uniformly dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 0.5% w / v nano-mesoporous carbon to obtain a topical composition. The composition was filtered (0.22 μm microporous membrane), placed in a brown sealed bottle, and stored in the dark at 4°C until use. The minoxidil control group and the blank control group were prepared in the same manner as in Example 2.

[0192] (3) Experimental equipment and materials:

[0193] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL. Digital camera (Canon EOS 80D with 50 mm lens): Record photos of hair growth.

[0194] 3. Experimental steps and procedures:

[0195] (1) Dosage regimen:

[0196] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL daily at 3:00 PM for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the solution covers the entire shaved area and avoiding dripping. After dosing, the mouse was individually caged for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the mouse's back was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Treatment Groups: Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0197] (2) Observation and recording:

[0198] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0199] (3) Data collection and processing:

[0200] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0201] 4. Experimental results analysis:

[0202] (1) Hair recovery rate:

[0203] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0204] Day 15 (as Figure 7 shown):

[0205] Experimental group: The recovery rate was about 15%, with sparse hair covering only a small area near the middle.

[0206] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0207] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0208] Day 30 (if Figure 8 shown):

[0209] Experimental group: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0210] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0211] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0212] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was close to that of the control group 1 and significantly higher than that of the control group 2 (p<0.01), and the control group 1 was higher than the control group 2 (p<0.05).

[0213] (2) Skin safety:

[0214] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0215] (3) Discussion of the results:

[0216] Comparison of efficacy: The hair recovery rate of the nano-mesoporous carbon material plus peptide group was very close to that of the commercial minoxidil group at 15 and 30 days, indicating that the combination of nano-mesoporous carbon material, oligopeptide, and auxiliary active ingredients achieved good transdermal delivery and scalp adsorption. The nano-mesoporous carbon material may deliver peptides through the hair follicle pathway, prolonging the drug's residence time on the scalp, synergizing with the permeation-enhancing effects of ethanol and propylene glycol, significantly improving efficacy. Its high specific surface area and sustained-release function also improve drug utilization.

[0217] Control performance: The commercial minoxidil group effectively promoted hair growth, which was similar to the experimental group. The blank group relied solely on natural recovery and had the worst effect.

[0218] Example 6 Comparative Verification of the Effects of a Topical Composition Based on Traditional Chinese Medicine and Minoxidil on Male Bald Mice

[0219] The nano-mesoporous carbon prepared in Example 1 was used to prepare a topical composition for regulating hair growth. The following Chinese herbal extracts were selected: Platycladus orientalis leaves, Angelica sinensis, Polygonum multiflorum, Ligusticum chuanxiong, Astragalus membranaceus, Salvia miltiorrhiza, ginger, Angelica dahurica, Rehmannia root, Lycium barbarum, and Ginseng. Auxiliary active ingredients: vitamin E, vitamin C, panthenol (vitamin B5), licorice extract (glycyrrhizic acid and glycyrrhetinic acid), β-cyclodextrin, and polysorbate 80 were used as auxiliary functional components to be compounded with the nano-mesoporous carbon. The hair growth effect was compared with that of minoxidil in male bald mice.

[0220] 1. Preparation of male bald mice:

[0221] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0222] 2. Experimental Preparation:

[0223] (1) Experimental groups and mouse allocation:

[0224] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0225] Nano-mesoporous carbon material plus polypeptide group (experimental group): The nano-mesoporous carbon in Example 1 was used, and PVP, polylysine and PEG2000 were added to modify the nano-mesoporous carbon. The active ingredient was a 20% ethanol extract of traditional Chinese medicine: Platycladus orientalis leaves, Angelica sinensis, Polygonum multiflorum, Chuanxiong, Astragalus, Salvia miltiorrhiza, ginger, Angelica dahurica, Rehmannia root, Lycium barbarum, and ginseng, plus auxiliary functional ingredients of vitamin E, vitamin C, panthenol (vitamin B5), licorice extract (glycyrrhizic acid and glycyrrhetinic acid), β-cyclodextrin, and polysorbate 80. The solvent ratio was 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol.

[0226] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0227] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0228] The random number table method was used to group the animals and ensure that there were no significant differences in body weight and shaved areas among the groups (p>0.05, one-way analysis of variance).

[0229] (2) Preparation of experimental reagents:

[0230] Preparation steps:

[0231] The nano-mesoporous carbon in Example 1 was first dispersed in deionized water at a concentration of 1% w / v, and then 0.5% w / v of PVP, 0.5% w / v of polylysine and 0.5% of PEG2000 were added to modify the nano-mesoporous carbon. After uniform mixing, the free PVP, polylysine and PEG2000 were removed by filtration and washing. After drying, the nano-mesoporous carbon was redispersed in a solvent of 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol at a concentration of 1% w / v, and a 20% concentration of the Chinese medicine ethanol extract was added: the extract ratio of 40% Platycladus orientalis was 40%. , 30% Chinese angelica, 10% Polygonum multiflorum, 10% Chuanxiong, 2% Astragalus, 2% Salvia miltiorrhiza, 2% ginger, 1% Angelica dahurica, 1% Rehmannia root, 1% Lycium barbarum, and 1% ginseng. The total amount of Chinese herbal extract added was 10% w / v of the total solution. After thorough mixing, 0.5% w / v vitamin E, 0.5% w / v vitamin C, 1% w / v panthenol (vitamin B5), 2% each of glycyrrhizic acid and glycyrrhetinic acid, 0.5% w / v β-cyclodextrin, and 1% w / v polysorbate 80 were added. The mixture was stirred thoroughly for 1 hour to obtain a topical composition. The composition was filtered (0.22 μm microporous membrane), placed in a brown sealed bottle, and stored in the dark at 4°C until use. The minoxidil control group and the blank control group were prepared in the same manner as in Example 2.

[0232] (3) Experimental equipment and materials:

[0233] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL. Digital camera (Canon EOS 80D with 50 mm lens): Record photos of hair growth.

[0234] 3. Experimental steps and procedures:

[0235] (1) Dosage regimen:

[0236] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL daily at 3:00 PM for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the solution covers the entire shaved area and avoiding dripping. After dosing, the mouse was individually caged for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the mouse's back was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Treatment Groups: Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0237] (2) Observation and recording:

[0238] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0239] (3) Data collection and processing:

[0240] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0241] 4. Experimental results analysis:

[0242] (1) Hair recovery rate:

[0243] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0244] Day 15 (as Figure 9 shown):

[0245] Experimental group: The recovery rate was about 10%, and the hair was sparse and scattered.

[0246] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0247] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0248] Day 30 (if Figure 10 shown):

[0249] Experimental group: The recovery rate was about 50%, with hair covering most areas and uniform color.

[0250] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0251] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0252] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was slightly lower than that of the control group 1, and significantly higher than that of the control group 2 (p<0.01), and the control group 1 was higher than the control group 2 (p<0.05).

[0253] (2) Skin safety:

[0254] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0255] (3) Discussion of the results:

[0256] Comparison of efficacy: The hair recovery rate in the nano-mesoporous carbon material plus peptide group was slightly lower than that in the commercial minoxidil group at 15 and 30 days, but still showed significant efficacy, indicating that the combination of nano-mesoporous carbon material, traditional Chinese medicine extracts, and auxiliary active ingredients achieved excellent transdermal delivery and scalp adsorption. The nano-mesoporous carbon material may deliver peptides through the hair follicle pathway, prolonging the drug's residence time on the scalp, synergizing with the permeation-enhancing effects of ethanol and propylene glycol, significantly improving efficacy. At the same time, its high specific surface area and sustained-release function also improved drug utilization. Control performance: The commercial minoxidil group effectively promoted hair growth, slightly better than the experimental group. The blank group, relying solely on natural recovery, had the worst effect.

[0257] Example 7 Comparative Verification of the Effects of the Topical Composition Based on Protein Fragments and Minoxidil on Male Bald Mice

[0258] The nano-mesoporous carbon prepared in Example 1 was used to prepare a topical composition based on protein fragments. Protein fragments were selected as active ingredients: osteopontin fragments, whey protein fragments, collagen fragments, keratin fragments, and adiponectin fragments. Auxiliary active ingredients: Chinese herbal extracts (glycyrrhizic acid and glycyrrhetinic acid) and retinoic acid (retinoic acid) were used as active ingredients to compound with the nano-mesoporous carbon, and the hair growth effect was compared with minoxidil in male bald mice.

[0259] 1. Preparation of male bald mice:

[0260] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0261] 2. Experimental Preparation:

[0262] (1) Experimental groups and mouse allocation:

[0263] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0264] Nano-mesoporous carbon material plus polypeptide group (experimental group): The nano-mesoporous carbon in Example 1 was used, and PEI40KD, chitosan and PEG-PAA were added to modify the nano-mesoporous carbon. The active ingredients were osteopontin fragments, whey protein fragments, collagen fragments, keratin fragments, and adiponectin fragments. The auxiliary ingredients were glycyrrhizic acid and glycyrrhetinic acid, and retinoic acid (retinoic acid). The solvent was 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol.

[0265] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0266] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0267] The random number table method was used to group the animals and ensure that there were no significant differences in body weight and shaved areas among the groups (p>0.05, one-way analysis of variance).

[0268] (2) Preparation of experimental reagents:

[0269] Preparation steps:

[0270] The nano-mesoporous carbon in Example 1 was first dispersed in deionized water at a concentration of 1% w / v, and then 1% w / v CMC, 0.5% w / v chitosan and 1% PEG-PAA were added to modify the nano-mesoporous carbon. After mixing evenly, the free CMC, chitosan and PEG-PAA were removed by filtration and washing. The nano-mesoporous carbon was redispersed in a 50% water + 50% ethanol solution at a concentration of 1% w / v, and osteopontin fragments, whey protein fragments, collagen fragments, and keratin fragments were added. The mixture was thoroughly mixed, filtered, and dried under vacuum at room temperature. The complex was re-dissolved in water at a concentration of 1% w / v nano-mesoporous carbon, and then 2% glycyrrhizic acid, 2% glycyrrhetinic acid, and 0.2% retinoic acid (Tretinoin) were added. The mixture was stirred thoroughly for 1 hour, filtered, and dried under vacuum at room temperature. The resulting complex was uniformly dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 0.5% w / v nano-mesoporous carbon to obtain a composition for external use. The composition was filtered (through a 0.22 μm microporous membrane), placed in a brown sealed bottle, and stored at 4°C in the dark until use. The minoxidil control group and the blank control group were prepared in the same manner as in Example 2.

[0271] (3) Experimental equipment and materials:

[0272] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL. Digital camera (Canon EOS 80D with 50 mm lens): Record photos of hair growth.

[0273] 3. Experimental steps and procedures:

[0274] (1) Dosage regimen:

[0275] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL daily at 3:00 PM for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the solution covers the entire shaved area and avoiding dripping. After dosing, the mouse was individually caged for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the mouse's back was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Treatment Groups: Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0276] (2) Observation and recording:

[0277] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0278] (3) Data collection and processing:

[0279] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0280] 4. Experimental results analysis:

[0281] (1) Hair recovery rate:

[0282] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0283] Day 15 (as Figure 11 shown):

[0284] Experimental group: The recovery rate was about 30%, and the hair in the middle area was significantly longer.

[0285] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0286] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0287] Day 30 (if Figure 12 shown):

[0288] Experimental group: The recovery rate was about 60%. The hair had covered most areas, but was relatively sparse, especially in the middle and upper areas.

[0289] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0290] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0291] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was close to that of the control group 1 and significantly higher than that of the control group 2 (p<0.01), and the control group 1 was higher than the control group 2 (p<0.05).

[0292] (2) Skin safety:

[0293] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0294] (3) Discussion of the results:

[0295] Comparison of Effects: The hair recovery rate in the nano-mesoporous carbon material plus protein fragment group was superior to that in the commercial minoxidil group at 15 days, but hair density was not as high as that in the minoxidil group at 30 days. This indicates that the combination of nano-mesoporous carbon material, protein fragment, and auxiliary active ingredients works faster and achieves a more pronounced hair growth effect early on. However, its stimulating effect on hair growth is sparse, and later on, it is not as dense as that of minoxidil. Control Performance: Both the experimental and commercial minoxidil groups effectively promoted hair growth, with the experimental group showing greater early results and the minoxidil group showing greater long-term results. The blank group, relying solely on natural recovery, had the worst results.

[0296] Example 8 Comparative Verification of the Effects of a Peptide-Based Topical Composition and Minoxidil on Male Bald Mice

[0297] The nano-mesoporous carbon prepared in Example 1 was used to prepare a topical composition for regulating hair growth. The polypeptides SIVYGLR, SDVYGLR, and SVVYGLRRP were selected as active ingredients and composited with the nano-mesoporous carbon. Caffeine, vitamin E, vitamin C, panthenol (vitamin B5), adenosine, and EDTA were used as auxiliary ingredients to compare the hair growth effects with minoxidil on male bald mice.

[0298] 1. Preparation of male bald mice:

[0299] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0300] 2. Experimental Preparation:

[0301] (1) Experimental groups and mouse allocation:

[0302] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0303] Nano-mesoporous carbon material plus polypeptide group (experimental group): The nano-mesoporous carbon in Example 1 was used, and PEI20KD, PLGA and polylysine were added to modify the nano-mesoporous carbon. The active ingredients were SIVYGLR, SDVYGLR and SVVYGLRRP. The auxiliary active ingredients were caffeine, vitamin E, vitamin C, panthenol (vitamin B5), adenosine and EDTA. The solvent was 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol.

[0304] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0305] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0306] The random number table method was used to group the animals and ensure that there were no significant differences in body weight and shaved areas among the groups (p>0.05, one-way analysis of variance).

[0307] (2) Preparation of experimental reagents:

[0308] Preparation steps:

[0309] The nano-mesoporous carbon in Example 1 was first dispersed in deionized water at a concentration of 1% w / v, and then 1% w / v PEI20KD, 1% PLGA and 0.5% polylysine were added to modify the nano-mesoporous carbon. After mixing evenly, the free PEI20KD, PLGA and polylysine were removed by filtration and washing. The nano-mesoporous carbon was redispersed in water with a pH of 10 at a concentration of 0.3% w / v. SIVYGLR, SDVYGLR and SVVYGLRRP were added at 0.15% w / v each, and the mixture was stirred and mixed for 1 hour. After filtration, the mixture was vacuum dried at room temperature. The resulting composite was uniformly dispersed in 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol at a concentration of 0.2% w / v of nano-mesoporous carbon. Caffeine 2%, vitamin E 0.5%, vitamin C 0.5%, panthenol (vitamin B5) 1%, adenosine 1% and EDTA 1% were added and stirred to obtain a composition for external use. The composition was filtered (0.22 The minoxidil control group and the blank control group were prepared in the same manner as in Example 2.

[0310] (3) Experimental equipment and materials:

[0311] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL. Digital camera (Canon EOS 80D with 50 mm lens): Record photos of hair growth.

[0312] 3. Experimental steps and procedures:

[0313] (1) Dosage regimen:

[0314] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL daily at 3:00 PM for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the solution covers the entire shaved area and avoiding dripping. After dosing, the mouse was individually caged for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the mouse's back was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Treatment Groups: Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0315] (2) Observation and recording:

[0316] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0317] (3) Data collection and processing:

[0318] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0319] 4. Experimental results analysis:

[0320] (1) Hair recovery rate:

[0321] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0322] Day 15 (as Figure 13 shown):

[0323] Experimental group: The recovery rate was about 30%, the hair was evenly distributed, the color was uniform, and the coverage area was high.

[0324] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0325] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0326] Day 30 (if Figure 14 shown):

[0327] Experimental group: The recovery rate exceeded 75%, the hair had covered most areas, and the length was significantly longer than that of the control group, almost returning to the state before shaving.

[0328] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0329] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0330] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was significantly higher than that of the control groups 1 and 2 (p<0.01), and that of the control group 1 was higher than that of the control group 2 (p<0.05).

[0331] (2) Skin safety:

[0332] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0333] (3) Discussion of the results:

[0334] Comparison of efficacy: The hair recovery rate in the nano-mesoporous carbon material plus peptide group was superior to that in the commercial minoxidil group at both 15 and 30 days, indicating that the nano-mesoporous carbon material plus peptide combination achieved excellent transdermal delivery and scalp adsorption. The nano-mesoporous carbon material may deliver peptides through the hair follicle pathway, prolonging the drug's retention time on the scalp, synergizing with the permeation-enhancing effects of ethanol and propylene glycol, significantly improving efficacy. Furthermore, its high specific surface area and sustained-release properties also enhance drug utilization.

[0335] Control performance: The commercial minoxidil group effectively promoted hair growth, but the effect was inferior to that of the experimental group. The blank group relied solely on natural recovery and had the worst effect.

[0336] Example 9 Comparative Verification of the Effects of a Peptide-Based Topical Composition and Minoxidil on Male Bald Mice

[0337] The nano-mesoporous carbon prepared in Example 1 was used to prepare a topical composition for regulating hair growth. Collagen peptides, keratin peptides, BPC-157 (Body Protective Compound-157), and APN5 polypeptide were selected as active ingredients to be compounded with the nano-mesoporous carbon. Red clover extract and dipotassium glycyrrhizate were selected as auxiliary ingredients. The hair growth effect was compared with that of minoxidil in male bald mice.

[0338] 1. Preparation of male bald mice:

[0339] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0340] 2. Experimental Preparation:

[0341] (1) Experimental groups and mouse allocation:

[0342] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0343] Nano-mesoporous carbon material plus polypeptide group (experimental group): The nano-mesoporous carbon in Example 1 was used, and PEI25KD, PLA and polylysine were added to modify the nano-mesoporous carbon. The active ingredients were collagen peptides (Collagen Peptides), keratin peptides (Keratin Peptides), BPC-157 (Body Protective Compound-157) and APN5 polypeptide. The auxiliary ingredients were red clover extract and dipotassium glycyrrhizate. The solvent was 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol.

[0344] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0345] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0346] Grouping using random numbers:

[0347] Preparation steps:

[0348] The nano-mesoporous carbon in Example 1 was first dispersed in deionized water at a concentration of 1% w / v, and then 1% w / v PEI20KD, 1% PLA and 0.5% polylysine were added to modify the nano-mesoporous carbon. After mixing evenly, the free PEI20KD, PLA and polylysine were removed by filtration and washing. The nano-mesoporous carbon was redispersed in water with a pH of 10 at a concentration of 0.3% w / v. Collagen peptides (Collagen Peptides), keratin peptides (Keratin Peptides), BPC-157 (Body Protective Compound-157) and APN5 peptide were added at 0.3% w / v, and the mixture was stirred for 1 hour. After filtration, the mixture was vacuum dried at room temperature. The resulting composite was uniformly dispersed in 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol at a concentration of 0.2% w / v of nano-mesoporous carbon. 1% red clover extract and 1% dipotassium glycyrrhizate were added and mixed evenly to obtain a composition for external use. The composition was filtered (0.22 The minoxidil control group and the blank control group were prepared in the same manner as in Example 2.

[0349] (3) Experimental equipment and materials:

[0350] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL. Digital camera (Canon EOS 80D with 50 mm lens): Record photos of hair growth.

[0351] 3. Experimental steps and procedures:

[0352] (1) Dosage regimen:

[0353] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL daily at 3:00 PM for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the solution covers the entire shaved area and avoiding dripping. After dosing, the mouse was individually caged for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the mouse's back was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Treatment Groups: Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0354] (2) Observation and recording:

[0355] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0356] (3) Data collection and processing:

[0357] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0358] 4. Experimental results analysis:

[0359] (1) Hair recovery rate:

[0360] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0361] Day 15 (as Figure 15 shown):

[0362] Experimental group: The recovery rate was about 20%, and the hair was evenly dense, covering the central area.

[0363] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0364] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0365] Day 30 (if Figure 16 shown):

[0366] Experimental group: The recovery rate exceeded 95%, the hair was basically fully restored, and the length was significantly longer than the control group.

[0367] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0368] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0369] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was significantly higher than that of the control groups 1 and 2 (p<0.01), and that of the control group 1 was higher than that of the control group 2 (p<0.05).

[0370] (2) Skin safety:

[0371] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0372] (3) Discussion of the results:

[0373] Comparison of effects: The hair recovery rate of the nano-mesoporous carbon material plus polypeptide group was superior to that of the commercial minoxidil group at both 15 and 30 days. In particular, the hair condition was almost completely restored at 30 days, indicating that the nano-mesoporous carbon material plus polypeptide combination achieved good transdermal delivery and scalp adsorption effects. The nano-mesoporous carbon material may deliver polypeptides through the hair follicle pathway, prolonging the drug's residence time on the scalp, synergizing with the permeation-enhancing effects of ethanol and propylene glycol, and significantly improving the efficacy. At the same time, its high specific surface area and sustained-release function also improved drug utilization. Control performance: The commercial minoxidil group effectively promoted hair growth, but the effect was inferior to that of the experimental group. The blank group, which relied solely on natural recovery, had the worst effect.

[0374] Example 10 Comparative Verification of the Effect of Using Nanocarbon Black Instead of Nano-mesoporous Carbon on Male Bald Mice

[0375] Nanocarbon black (ECP600) was used instead of nano-mesoporous carbon to prepare a minoxidil-based topical composition. Minoxidil was selected as the active ingredient and compounded with nanocarbon black (ECP600). The hair growth effect of the composition was compared with that of minoxidil in male bald mice.

[0376] 1. Preparation of male bald mice:

[0377] Thirty-six male BALB / c mice, aged 6-8 weeks, were used to establish an androgenic alopecia (AGA) mouse model. Each mouse received a daily, continuous injection of testosterone propionate (2.5 mg / kg) at multiple sites in the depilatory area of the neck and back (initiated on day 1) to establish the model. Mice were acclimated to a standard rodent chow diet and drinking water ad libitum, housed one week prior to the experiment. These conditions complied with the Regulations for the Care of Laboratory Animals. Prior to the experiment, all mice were shaved to simulate alopecia. Mice were anesthetized with 2% isoflurane using a small animal anesthesia machine (RWD Life Science) and kept in a tranquil state. A 2 cm × 2 cm (approximately 4 cm²) area of the back was shaved using an electric shaver (Philips, model QP2520) located slightly to the left of the midline of the back. Clean the shaved area with warm saline (37°C) to remove any remaining hair and skin debris, then gently dry with sterile gauze. Observe for 24 hours after shaving to ensure there is no obvious skin damage or inflammation. Experiments can begin after the mice have resumed normal activity.

[0378] 2. Experimental Preparation:

[0379] (1) Experimental groups and mouse allocation:

[0380] Thirty-six mice were randomly divided into three groups, with 12 mice in each group. The specific groups were as follows:

[0381] Nanocarbon black (ECP600) material plus minoxidil group (experimental group): Nanocarbon black (ECP600) of 50-200 nm was evenly dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 1% w / v, and then 5% w / v minoxidil was added and mixed evenly.

[0382] Commercial minoxidil group (control group 1): minoxidil was dispersed in 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol at a concentration of 5% w / v and mixed evenly.

[0383] Blank control group (control group 2): a matrix solution containing no active ingredient (i.e., a mixture of 40% ethanol, 50% propylene glycol, 5% water, and 5% glycerol) was used.

[0384] The random number table method was used to group the animals and ensure that there were no significant differences in body weight and shaved areas among the groups (p>0.05, one-way analysis of variance).

[0385] (2) Preparation of experimental reagents:

[0386] Preparation steps:

[0387] 1% nanocarbon black (ECP600) was added to 10 mL of ethanol and ultrasonically dispersed for 30 minutes at 200 W and 25°C in an ultrasonic cleaner (Kunshan Ultrasonic, model KQS-250, frequency 40 kHz) to obtain a uniform dispersion A. 5% minoxidil was dissolved in dispersion A and magnetically stirred (IKA, model C-MAG HS 7) at 500 rpm for 30 minutes until completely dissolved, resulting in a uniform dispersion. After mixing thoroughly, propylene glycol and glycerol were added in appropriate proportions and stirring continued for 15 minutes. The mixture was then brought to volume with deionized water in appropriate proportions and stirred for 10 minutes to obtain the final composition (minoxidil concentration 5% w / v, nanocarbon black (ECP600) concentration 1% w / v). The composition was filtered (0.22 μm microporous membrane), placed in a brown sealed bottle, and stored at 4°C in the dark until further use. The minoxidil control group was prepared similarly, but only minoxidil was added without nanocarbon black (ECP600). The blank control group matrix was prepared with the same solvent ratio and without active ingredients.

[0388] (3) Experimental equipment and materials:

[0389] Micropipette (Eppendorf, 1-200 μL): Accurately administer 0.2 mL of fluid. Digital camera (Canon EOS 80D with 50 mm lens): Photograph hair growth. Microscope (Olympus BX53 with ImageJ software): Analyze hair density and length.

[0390] 3. Experimental steps and procedures:

[0391] (1) Dosage regimen:

[0392] Dosage and Frequency: Each mouse received a single injection of testosterone propionate at 10:00 AM daily and a single dose of 0.2 mL per dose at 3:00 PM daily for 30 consecutive days. Dosing Method: Use a micropipette to evenly apply 0.2 mL of the corresponding solution to the shaved area (4 cm²) of the mouse, covering an area of approximately 2 cm x 2 cm. Gently apply the solution using a sterile soft silicone tip, ensuring that the entire shaved area is evenly covered and avoiding dripping. After dosing, the mouse was caged individually for 5 minutes. After the solution dried naturally, it was returned to its original cage. Before each dosing, the back of the mouse was thoroughly cleaned with clean water mixed with shower gel and then deionized water to remove any residue from the previous dosing. Experimental Group: Application of nano-mesoporous carbon material plus minoxidil combination. Control Group 1: Application of commercial 5% minoxidil solution. Control Group 2: Application of blank matrix solution.

[0393] (2) Observation and recording:

[0394] Observation time points: Day 0 (before administration), Day 5, Day 10, Day 15, Day 20, Day 25, and Day 30. Recording content: Photographs of hair growth in the shaved area were taken using a digital camera, using a fixed light source (5000K natural light) and distance (20 cm). Hair color, recovery rate, and growth rate were visually observed, and any redness, swelling, or irritation of the skin was recorded.

[0395] (3) Data collection and processing:

[0396] Images were analyzed using ImageJ software to determine hair recovery rate (%), defined as the ratio of the area and amount of hair growth to the total amount and area of hair growth in the shaved area before shaving. Statistical analysis was performed using SPSS 25.0 software, using one-way analysis of variance (ANOVA) to compare differences between groups. P < 0.05 was considered significant.

[0397] 4. Experimental results analysis:

[0398] (1) Hair recovery rate:

[0399] Day 0: There was no hair in the shaved area in the three groups, and the recovery rate was 0%, which was consistent with the baseline (p>0.05).

[0400] Day 15 (as Figure 17 shown):

[0401] Experimental group: The recovery rate was about 8%, with very little hair and random coverage.

[0402] Control group 1: The recovery rate was about 15%, the hair was sparse, and the coverage area was concentrated in the middle area.

[0403] Control group 2: The recovery rate was about 5%, with only a small amount of villi visible in the middle area.

[0404] Day 30 (if Figure 18 shown):

[0405] Experimental group: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0406] Control group 1: The recovery rate was about 70%, with hair covering most areas and with uniform color.

[0407] Control group 2: The recovery rate was about 15%, the hair was shorter and grew slowly.

[0408] Statistical analysis: On the 15th and 30th days, the recovery rate of the experimental group was significantly higher than that of the control groups 1 and 2 (p<0.01), and that of the control group 1 was higher than that of the control group 2 (p<0.05).

[0409] (2) Skin safety:

[0410] During the 30-day observation period, there was no obvious redness, swelling, itching or peeling in the shaved areas of the three groups of mice, indicating that the topical composition is safe and non-irritating to the skin.

[0411] (3) Discussion of the results:

[0412] Effect comparison: The hair recovery rate of the nano-carbon black group plus minoxidil group was significantly worse at 15 days, and was similar to that of the commercial minoxidil group at 30 days, indicating that nano-carbon black could not significantly enhance the transdermal delivery and scalp absorption of minoxidil.

[0413] Example 11 Nano-mesoporous carbon skin safety test

[0414] 1. Experimental purpose:

[0415] The purpose of this experiment was to evaluate the safety of nano-mesoporous carbon materials on the skin, verify their biocompatibility on the skin, and ensure that the materials were non-toxic, non-irritating, and non-allergenic. The experimental results provide a scientific basis for the safety of nano-mesoporous carbon materials in skin-contact applications, supporting their potential application in related fields.

[0416] 2. Experimental Design

[0417] The study involved 60 BALB / c mice, half male and half female, aged 6-8 weeks, divided into six groups. The negative control (NC) group consisted of 10 mice, treated with no material; the positive control (PC) group consisted of 10 mice, treated with 0.1% SDS as a mild irritant; experimental group 1 (low dose LD) consisted of 10 mice, treated with a 0.05% mass concentration of nano-mesoporous carbon material; experimental group 2 (medium dose MD) consisted of 10 mice, treated with a 0.5% mass concentration of nano-mesoporous carbon material; experimental group 3 (high dose HD) consisted of 10 mice, treated with a 2% mass concentration of nano-mesoporous carbon material; and the solvent control (VC) group consisted of 10 mice, treated with the same solvent as in experiments 1-3. The experimental process includes the following steps: first, the nano-mesoporous carbon material is dispersed in a solvent of 40% ethanol, 50% propylene glycol, 5% water and 5% glycerol; then the hair on the back of the mouse is shaved to expose a 2 cm² skin area; then the material is applied to the skin once a day, spreading it evenly for 30 days; finally, the skin condition of the mouse is recorded every day, the body weight is measured every week, and skin tissue samples are collected on the 14th and 30th days, and blood samples are collected on the 30th day.

[0418] 3. Detection method:

[0419] In the experiment, the Draize scoring system was used to evaluate skin irritation, with a score range of 0 to 4, where 0 indicates no irritation and 4 indicates severe erythema, edema, or ulceration. Histological analysis used HE staining to observe skin tissue structure, with a score range of 0 to 3, where 0 indicates normal and 3 indicates severe inflammation or tissue damage. Biochemical analysis detected serum ALT, AST, Cr, and BUN indicators. The normal reference ranges are ALT 10-40 U / L, AST 10-40 U / L, Cr 0.5-1.2 mg / dL, and BUN 10-30 mg / dL, respectively. The mouse ear swelling test (MEST) was used to evaluate allergic reactions; an ear swelling rate of less than 20% indicates non-sensitization. These parameters are used to comprehensively evaluate the safety of nano-mesoporous carbon materials.

[0420] 4. Experimental results:

[0421] (1) In the skin irritation evaluation, the Draize score of the negative control group (NC) was 0.0 ± 0.0, the positive control group (PC) was 1.5 ± 0.5, the low dose (LD), medium dose (MD), and high dose (HD) of the experimental group were 0.1 ± 0.1, 0.2 ± 0.1, and 0.3 ± 0.2, respectively, and the solvent control group (VC) was 0.0 ± 0.0. The scores of the experimental group were not significantly different from those of the negative control group and were much lower than those of the positive control group, indicating that the nano-mesoporous carbon material had no skin irritation.

[0422] (2) In histological analysis, the histological score of the negative control group was 0.0 ± 0.0, that of the positive control group was 1.2 ± 0.4, that of the low-dose, medium-dose, and high-dose experimental groups was 0.1 ± 0.1, 0.2 ± 0.1, and 0.2 ± 0.1, respectively, and that of the solvent control group was 0.0 ± 0.0. The scores of the experimental groups were similar to those of the negative control group, with no inflammatory cell infiltration and the stratum corneum remaining intact, indicating that the material did not cause skin tissue lesions.

[0423] (3) In the biochemical analysis, the ALT of the negative control group was 24 ± 4 U / L, AST was 27 ± 5 U / L, Cr was 0.7 ± 0.1 mg / dL, and BUN was 19 ± 2 mg / dL; the ALT of the positive control group was 26 ± 5 U / L, AST was 29 ± 6 U / L, Cr was 0.8 ± 0.2 mg / dL, and BUN was 20 ± 3 mg / dL; the ALT of the low-dose, medium-dose, and high-dose experimental groups were 25 ± 5 U / L, 26 ± 4 U / L, and 27 ± 6 U / L, respectively; the AST was 28 ± 6 U / L, 29 ± 5 U / L, and 30 ± 7 U / L; the Cr was 0.8 ± 0.2 mg / dL, 0.7 ± 0.1 mg / dL, and 0.8 ± 0.2 mg / dL; the BUN was 20 ± 3 mg / dL, 21 ± 2 In the solvent control group, ALT was 24 ± 3 U / L, AST was 27 ± 4 U / L, Cr was 0.7 ± 0.1 mg / dL, and BUN was 19 ± 2 mg / dL. Biochemical parameters in the experimental group were not significantly different from those in the negative control group and were all within the normal range, indicating that the material did not affect liver and kidney function.

[0424] (4) In the allergic evaluation, the ear swelling rate was 3 ± 1% in the negative control group, 35 ± 5% in the positive control group, 5 ± 2% in the low-dose, 6 ± 2% in the medium-dose, and 7 ± 3% in the high-dose experimental groups, and 4 ± 1% in the solvent control group. The ear swelling rate in the experimental groups was less than 20%, similar to that in the negative control group, indicating that the material was non-allergenic.

[0425] (5) In terms of overall health, the weight gain of mice in all groups was normal, and there was no abnormal behavior or death. The skin of mice in the experimental group did not show abnormalities such as erythema, edema, or desquamation.

[0426] Conclusion: Experimental results demonstrate that the nano-mesoporous carbon material exhibits excellent biocompatibility and safety on the skin. Specifically, the Draize score showed no significant difference from the negative control group, indicating no skin irritation. Histological structure was normal, with no inflammation or damage. Biochemical parameters were within normal ranges, with no impact on liver and kidney function. Ear swelling was less than 20%, indicating no allergenicity. The patient was in good general health, with normal weight gain and no abnormal behavior. Therefore, this material is suitable for skin contact applications.

Claims

1. A topical composition for hair growth regulation, characterized in that It contains nano-mesoporous carbon for enhancing penetration, various surface functional modifications on nano-mesoporous carbon, as well as minoxidil, peptides, oligopeptides, amino acids, protein fragments, dutasteride, bimatoprost, Chinese medicine extracts, vitamins, caffeine, nucleosides, growth factors, and various functional ingredients.

2. The external use composition for regulating hair growth according to claim 1, characterized in that The mesopore diameter of the nano-mesoporous carbon for enhancing penetration is 2-30 nm, and the total mesopore volume is 1.5-3.5 cm 3 / g, carbon particle size 50~200 nm.

3. The external-use composition for regulating hair growth according to claim 1, characterized in that The nano-mesoporous carbon for enhancing permeation contains oxygen-containing functional groups, and the oxygen-containing functional groups include at least one of carbonyl, hydroxyl and epoxy groups.

4. The nano-mesoporous carbon for enhancing permeability according to claims 1 to 3, characterized in that: The nano-mesoporous carbon has a variety of surface functional modifications, including polyethylene glycol (PEG), chitosan, polylysine, polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polydimethylaminoethyl methacrylate (PDMAEMA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethyleneimine (PEI), polyphosphate (PPE), polyethylene glycol polyacrylamide (PEG-PAA), polyethylene ethyleneamine (PEHA), polyacrylate (PAA) and its copolymers, carboxymethyl cellulose (CMC), hydroxyethyl cellulose, polyquaternary ammonium salts, sodium carboxymethyl cellulose, chitosan derivatives, sodium tetradecyl sulfate, lecithin, and at least one of cell penetrating peptides.

5. Use of the external-use composition for regulating hair growth according to any one of claims 1 to 3 in the preparation of cosmetics, skin care products, shampoo, conditioner, and essence.

6. The method for preparing nano-mesoporous carbon for enhancing permeability according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Preparation and nanostructured biotemplates Chlorella vulgaris is selected, as it is easy to cultivate on a large scale and is readily available. The algae is grown in a suitable culture medium and harvested by centrifugation and filtration. The algae are then washed multiple times with deionized water to remove any residual culture medium. The washed algae are then freeze-dried or oven-dried at low temperatures to produce dry algae powder. S2: Nano-processing ① High-energy ball milling: Place the dried algae powder in a ball mill, add an appropriate amount of grinding balls, and ball mill at a speed of 300-500 rpm for 8 hours to initially break up the algae powder particles. ② Ultrasonic-assisted dispersion: Suspend the ball-milled algae powder in an appropriate amount of ethanol and use a high-power ultrasonic device (20 kHz, continuous ultrasound for 30 minutes) to further break up the algae cell fragments to the 50-200nm level. ③ Purification: Purify the algae powder dispersion by filtration (using a filter membrane with a pore size of 200nm) to collect algae nanotemplates within the target size range (50-200nm); S3: Preparation of composite precursors Prepare a phenol-formaldehyde resin precursor solution and adjust the pH to 2–4 in an acidic environment to promote precursor polymerization. Add appropriate amounts of metal ions (such as Zn²⁺ or Mg²⁺) as auxiliary structure regulators. S4: Nanoalgae template loading The algae nano-template obtained in step 1 is evenly dispersed in the precursor solution, and high-speed stirring and ultrasonic-assisted mixing are used to ensure that the algae template is fully and evenly mixed with the precursor to form a self-assembled mixed system; S5: Microwave-assisted cross-linking and gelation The mixed system was placed in a microwave reactor and rapidly heated by microwaves (power 300W, time 5 minutes) to promote rapid cross-linking and gelation of the phenol-formaldehyde precursor, forming an ordered structure around the algae template and embedding it into the natural template. S6: Plasma treatment The gelled sample was placed in a plasma (inert environment) device (argon plasma) for 20 minutes to remove organic residues on the surface and introduce micropores on the material surface, which facilitates the subsequent formation and regulation of the pore structure. S7: High temperature carbonization The plasma-treated solid precursor is carbonized in an inert atmosphere (nitrogen or argon) at a high temperature, increasing the temperature at 5°C / min to 1800°C and holding for 1 hour. At this high temperature, the organic precursor is converted into carbon material, while the algae template and part of the organic template are pyrolyzed, leaving behind the template's predetermined pore structure. S8: Post-processing: After carbonization, the sample is washed with an acid (dilute hydrochloric acid) of appropriate concentration to remove residual metal ions and decomposition products. The acid-washed sample is then placed in a supercritical CO2 extraction apparatus and treated at an appropriate temperature (40–50°C) and pressure for 1 hour to completely remove the template residue and further fine-tune the mesopore size (target 2–30 nm). CO2 activation treatment is then performed at a higher temperature (800–900°C) for a specified time to form more mesopores. Finally, a low-concentration oxygen plasma treatment is performed for 12 hours to remove oxygen-containing functional groups from the nano-mesoporous carbon material. The sample is then crushed in a high-energy ball mill for 30 minutes to obtain nano-mesoporous carbon material with a particle size range of 50–200 nm for enhanced permeability.

7. The external-use composition for regulating hair growth according to claims 1 to 5, in the form of an emulsion, cream, gel, essence, spray or powder.

8. The external-use composition for regulating hair growth according to claim 1, characterized in that The protein fragments include: osteopontin fragments, whey protein fragments, collagen fragments, keratin fragments, adiponectin fragments, and recombinant protein fragments.

9. The external-use composition for regulating hair growth according to claim 1, characterized in that The polypeptide includes: a polypeptide containing the core active sequence SVVYGLR (S: Serine, V: Valine, Y: Tyrosine, G: Glycine, L: Leucine, R: Arginine) in osteopontin, the polypeptide can be modified by acylation (Ac-) at the N-terminus and amidation (-NH2) at the C-terminus; the polypeptide can be modified by phosphorylation (pY-) of amino acids; the polypeptide can be linked to biotin at the N-terminus or C-terminus; the polypeptide can be linked to PEG; the polypeptide can form a cyclic structure (such as C-SVVYGLR-C) by introducing cysteine (C) at the N-terminus and C-terminus to form a disulfide bond.

10. The external-use composition for regulating hair growth according to claim 1, characterized in that The polypeptide may also include: a polypeptide containing at least one of the core sequences SIVYGLR, SVVFGLR, SVVYGLK, VVYGLR, SVVYGL, and SVVYGLRRP; the polypeptide may be modified by acylation (Ac-) at the N-terminus and amidation (-NH2) at the C-terminus; the polypeptide may be modified by phosphorylation (pY-) of amino acids; the polypeptide may be linked to biotin at the N-terminus or C-terminus; the polypeptide may be linked to PEG; the polypeptide may form a cyclic structure by introducing C: cysteine (Cysteine) at the N-terminus and C-terminus to form a disulfide bond.

11. The external-use composition for regulating hair growth according to claim 1, characterized in that The oligopeptides include at least one of: Copper Tripeptide-1 (GHK-Cu), Acetyl Tetrapeptide-3, Biotin Tripeptide-1, Myristoyl Pentapeptide-17, and Palmitoyl Tetrapeptide-20.

12. The external-use composition for regulating hair growth according to claim 1, characterized in that The amino acids include one or more of: S: Serine, V: Valine, Y: Tyrosine, G: Glycine, L: Leucine, R: Arginine, D: Aspartic Acid, P: Proline, T: Threonine, I: Isoleucine, H: Hydroxyproline, and K: Lysine.

13. The external-use composition for regulating hair growth according to claim 1, characterized in that: The polypeptides may also include: one or more of collagen polypeptides (Collagen Peptides), keratin polypeptides (Keratin Peptides), BPC-157 (Body Protective Compound-157), and APN5 polypeptides.

14. The external-use composition for regulating hair growth according to claim 1, characterized in that The traditional Chinese medicine comprises at least one of: Platycladus orientalis leaf, angelica sinensis, Polygonum multiflorum, Chuanxiong rhizome, Astragalus membranaceus, Salvia miltiorrhiza, ginger, Angelica dahurica, Rehmannia root, wolfberry fruit, liquorice and ginseng.

15. The external-use composition for regulating hair growth according to claim 1, characterized in that The vitamins include at least one of vitamin E, vitamin C, panthenol (vitamin B5), and niacinamide (vitamin B3).

16. The external-use composition for regulating hair growth according to claim 1, characterized in that: The nucleoside comprises at least one of adenosine, adenosine liposomes, and 5'-methylthioadenosine.

17. The external-use composition for regulating hair growth according to claim 1, characterized in that The growth factor comprises at least one of vascular endothelial growth factor (VEGF), fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor-1, and hepatocyte growth factor.

18. The external composition for regulating hair growth according to claim 1, wherein the plurality of functional ingredients comprises: β-cyclodextrin, tretinoin (retinoic acid), red clover extract, camphor, limonene, urea, salicylic acid, glycerin, transdermal peptide, hyaluronic acid, sodium hyaluronate and its cross-linked polymer, sucrose, lauroyl azetidine, alkyl betaine, lauryl betaine, isopropyl palmitate, isopropyl methyl palmitate, menthol, carbomer, xanthan gum, polyacrylamide, phenoxyethanol, parabens, polysorbate 80, ethylenediaminetetraacetic acid (EDTA), etc.

19. The topical composition for regulating hair growth according to claim 1, wherein the multiple functional ingredients further include ethanol, isopropyl alcohol, dimethyl sulfoxide (DMSO), decyl methyl sulfoxide, oleic acid, linoleic acid, lauryl alcohol, menthol, propylene glycol, petrolatum, silicone oil, monoglyceride or diglyceride, medium-chain triglycerides, allantoin (0.5-1%) or dipotassium glycyrrhizate, glyceryl stearate, glyceryl stearate citrate, cetearyl alcohol, ethylhexyl hydroxystearate, and caprylic / capric triglyceride.

20. Use of the external composition for regulating hair growth according to any one of claims 1 to 15 in treating androgenic alopecia.

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