A protein carrier-based transdermal delivery system for active ingredients to hair follicles

Abstract

This invention provides a transdermal follicular delivery system for active ingredients based on a protein carrier. The system comprises a composite structure of a small molecule active ingredient, a bioactive protein, and a polymer shell, with a size of 40-150 nm and a polymer shell thickness of 10-70 nm. The zeta potential of this protein carrier-based transdermal follicular delivery system is positively charged. The small molecule active ingredient is loaded within the bioactive protein, and the polymer shell coats the bioactive protein to form a protein nanogel. Both the small molecule active ingredient and the bioactive protein have beneficial effects on hair follicles. When this transdermal follicular delivery system is applied to the surface of a hair follicle or skin, the functional small molecules are delivered transdermally along with the nanogel to the interior of the hair follicle and surrounding tissues, where they are released in a controlled manner, thus effectively exerting their effects.

Description

Technical Field

[0001] This invention relates to the fields of biomedical technology and transdermal delivery material composite technology, specifically to a transdermal delivery system for active ingredients based on a protein carrier. Background Technology

[0002] Hair is an important part of a person's appearance. Abnormal hair loss, gray hair, and other undesirable hair growth conditions not only weaken the body's protection but also significantly affect people's mental health. Hair-related diseases are caused by changes in the hair follicles and their surrounding complex environment. Therefore, research on delivering active ingredients to hair follicles to treat related diseases is receiving increasing attention.

[0003] Hair follicles are skin appendages formed by the indentation of the epidermis into the dermis. They are relatively complete structures composed of various cell groups, including keratinocytes, melanocytes, and follicular mesenchymal stem cells. The upper part of the hair follicle is the constant part, including the infundibulum and isthmus; the lower part is the bulb, containing the dermal papilla, which is the growth point of the hair and follicle. The infundibulum at the upper end of the hair follicle is an open structure. During transdermal drug delivery, a clear hair follicle opening allows drugs to bypass the physical barrier of the epidermis and penetrate directly. However, in the scalp and facial areas, the total area of ​​hair follicle openings accounts for only 10% of the total skin area, and some hair follicles are blocked by shed keratinocytes and sebum, preventing drug penetration. The upper part of the hair follicle is surrounded by a large number of immune cells, including Langerhans cells. The immune barrier formed by these immune cells recognizes and clears drugs penetrating into the surrounding tissues. Furthermore, to ensure the delivered drug remains within or around the hair follicle, the size range of the drug is also limited. Particles that are too large (greater than 500 nm) cannot reach the deeper layers of the hair follicle, while particles that are too small (less than 10 nm) penetrate directly through the bottom of the hair follicle, resulting in a short residence time and insufficient effectiveness. Therefore, although some active drugs targeting hair follicle and hair problems are currently available, including retinoids, antioxidants, and minoxidil, their effectiveness is limited. How to efficiently deliver drugs transdermally to hair follicles and make them effective remains a research challenge and key focus.

[0004] Currently, the main clinical or commercial technologies for promoting the delivery of active ingredients to hair follicles rely on penetration enhancers. However, the use of commonly used penetration enhancers such as ethanol may affect the activity of some small molecule components, and the addition of penetration enhancers is detrimental to skin and hair follicle growth and development, posing risks of irritation and allergies. Microneedling is a relatively new treatment for hair follicle diseases. It involves creating micro-channels in the skin around the hair follicle using mechanical force, allowing medication to bypass the skin barrier and be efficiently delivered deep into the follicle. However, frequent use of microneedling can damage the local skin, causing inflammation and negatively impacting hair follicle growth and development.

[0005] The inventors' previous patents CN202311704896.7, CN202410225688.7, and CN202410286148.X respectively relate to a tyrosinase-based nanotransdermal delivery system for treating vitiligo, a blue calyx methyl ether nanotransdermal delivery system for skin anti-inflammation, and a blue calyx methyl ether nanotransdermal delivery system for skin anti-inflammation. This patent targets the hair follicle transdermal delivery environment, selecting a nanosystem size suitable for delivery within the hair follicle, optimizing the hydrophilic and hydrophobic properties of the polymer shell material, and preferentially selecting active proteins and active small molecules. Summary of the Invention

[0006] To overcome the shortcomings of existing drug delivery technologies for hair follicle diseases, such as low delivery efficiency and uncontrollable impact on hair follicle growth and development, this invention uses protein molecules as nanotemplates and prepares protein nanogels through polymer encapsulation to construct a non-invasive and highly efficient transdermal delivery system for hair follicles. Utilizing the hydrophobicity of the protein's interior, it can load active small molecule drugs with hydrophobic structures, achieving highly efficient delivery of active small molecules to the hair follicle. The protein gel used in this transdermal delivery system has a specific size; by controlling the loading amount of small molecules and the thickness of the polymer shell, the size of the nanogel carrier can be controlled, which is beneficial for efficient penetration and retention. By customizing the surface properties of the polymer shell of the protein, the delivery system can effectively penetrate the physical and immune barriers of the skin, penetrating through the surrounding skin to the depth of the hair follicle and then directly penetrating into the hair follicle to efficiently reach the deepest part of the hair follicle and exert its physiological function.

[0007] Furthermore, protein nanogels exhibit minimal irritation to the skin and hair follicles, and do not disrupt the normal physiological functions of the follicles and skin during delivery. Protein nanogels protect the stability of active ingredients and effectively prevent irritation to the skin and hair follicles caused by some active ingredients. The excellent penetration efficiency of nanogels allows active ingredients to reach deep into the hair follicle and surrounding tissues efficiently, thus achieving effective improvement of hair problems with lower concentrations of active ingredients, effectively avoiding the waste and irritation associated with high-concentration drugs. This loading method is universally applicable to active small molecules with hydrophobic structures.

[0008] This invention provides the following technical solutions:

[0009] A transdermal follicular delivery system for active ingredients based on a protein carrier is characterized by a composite structure of a small molecule active ingredient, a bioactive protein, and a polymer shell, with a size of 40-150 nm and a polymer shell thickness of 10-70 nm. The zeta potential of this protein carrier-based transdermal follicular delivery system is positively charged. The small molecule active ingredient is loaded in the bioactive protein, and the polymer shell coats the bioactive protein. The small molecule active ingredient and the bioactive protein exhibit beneficial activity on hair follicles.

[0010] The active ingredient transdermal delivery system for the protein carrier of the present invention can be simply referred to as a bioactive protein nanogel.

[0011] The protein carrier-based transdermal delivery system for active ingredients in hair follicles has a positive zeta potential; small molecule active ingredients are loaded in bioactive proteins, and a polymer shell coats the bioactive proteins. The small molecule active ingredients and bioactive nanogels have beneficial effects on hair follicles.

[0012] Furthermore, the small molecule active ingredient combines with bioactive protein molecules through adsorption, hydrophilic-hydrophobic interactions, electrostatic interactions, hydrogen bonds, etc., and its structure includes one or more of the following features: positively charged groups, hydrophobic groups, and groups that can form hydrogen bonds.

[0013] Furthermore, the small molecule active ingredients can exert physiological functions targeting hair follicle problems. Examples include, but are not limited to: for hormone-induced hair loss, the small molecule active ingredients include sulforaphane; for fine hair and poor hair follicle development, the small molecule active ingredients include vitamin A; for oxidative stress problems within hair follicles, the small molecule active ingredients include at least one of carotenoids, hesperidin, and vitamin E; and for hair follicle inflammation problems, the small molecule active ingredients include at least one of resveratrol, lycopene, and astaxanthin.

[0014] Furthermore, the bioactive protein has a size of 0.5-20 nm, preferably 3-10 nm. Bioactive protein molecules themselves have difficulty penetrating the skin barrier to enter the hair follicle. Even if a few protein molecules do enter the follicle, their size limitation restricts their transport capacity, making it difficult for them to reach the depths of the follicle and exert their effects. In addition, the microenvironment inside the hair follicle is unfavorable for the long-term effectiveness of proteins; even if delivered into the follicle, the protein will be inactivated in a short period. The poor solubility of most active small molecules already limits their individual application. Furthermore, small molecules also suffer from low delivery efficiency within the hair follicle; even if they enter, they risk inactivation or directly penetrating the follicle without retention to exert their effects. This invention increases the size of the delivery system by polymer-coating the protein, thus changing the surface potential to positive. The increased size allows the nanogel to be delivered within the hair follicle in accordance with the ratchet effect, facilitating the delivery of active ingredients to living cells deep within the follicle to exert their effects, rather than remaining on the surface of the pore or directly penetrating the follicle. Furthermore, the polymer layer protects the activity of the protein and its internal small molecules, allowing the small molecules to be slowly released from the protein, which is beneficial for the protein nanogel to exert its therapeutic effect deep within the hair follicle for a longer period.

[0015] Furthermore, examples of bioactive proteins that address hair follicle issues include, but are not limited to: for insufficient hair pigmentation, bioactive proteins include tyrosinase; for oxidative stress within hair follicles, bioactive proteins include at least one of horseradish peroxidase, peroxidase, and superoxide dismutase; for hair follicle inflammation, bioactive proteins include at least one of lysozyme, papain, and bromelain; for excessive sebum secretion, bioactive proteins include lipase; and for hair loss caused by excessive androgen secretion, bioactive proteins include 3β-hydroxysteroid dehydrogenase.

[0016] Furthermore, the zeta potential of the protein carrier-based active ingredient follicle-targeted transdermal delivery system is +3 to +7 mV. A positive surface potential facilitates the interaction between the nanogel and the negatively charged lipid components of the pores and skin barrier, weakening the barrier effect and promoting the delivery of the nanogel.

[0017] Furthermore, the polymer shell is obtained by monomer polymerization, and the coating method includes in-situ polymerization or coating the surface of bioactive protein nanogel by electrostatic attraction; or the polymer shell is a natural polymer that is coated on the surface of bioactive protein nanogel by hydrogen bonding or electrostatic interaction.

[0018] Furthermore, the polymer shell is obtained by monomer polymerization. When the polymer shell is coated on the surface of the bioactive protein nanogel by in-situ polymerization, the monomer is selected from vinylpyrrolidone, acrylamide, N-propenylsuccinimide, 2-methacryloyloxyethylphosphocholine, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide, and acrylate; the acrylate is selected from at least one of methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, propyl methacrylate, and butyl methacrylate; when the polymer shell is coated on the surface of the protein by electrostatic interaction, the polymer shell is a positively charged polymer, at least one of poly(acrylamine hydrochloride), poly(L-lysine), polyethyleneimine, poly(L-histidine), N,N-dimethylaminoethyl methacrylate, and polymethacrylamidopropyltrimethylammonium chloride; or the polymer shell is a natural polymer, including at least one of polysaccharides and chitosan.

[0019] Furthermore, the mass ratio of the small molecule active ingredient to the bioactive protein is 1:1-10, preferably 1:2-5.

[0020] A second objective of this invention is to provide a method for preparing the above-mentioned protein carrier-based transdermal delivery system for active ingredients, which is one of the following methods:

[0021] When the polymer is obtained by monomer polymerization, it is called in-situ polymerization, which includes the following steps:

[0022] (S1) Add a surface-modified monomer solution to a bioactive protein solution to obtain a surface-modified protein solution;

[0023] (S2) Add a small molecule active ingredient solution to the surface-modified protein solution obtained in step (S1), wherein the small molecule active ingredient is loaded in the bioactive protein;

[0024] (S3) Add functional monomers, positively charged monomers, crosslinking agents and initiators to initiate in-situ polymerization and obtain a transdermal delivery system for active ingredients based on protein carriers.

[0025] or,

[0026] When the shell is obtained by the self-assembly of a positively or negatively charged polymer on the protein surface, the following steps are included:

[0027] (T1) Under the conditions of water bath at 50-70℃ and stirring, add bioactive protein solution to polymer solution with positive or negative charge, stir, add small molecule active ingredient solution under ice water bath conditions, add salt solution, stir, ultrafiltration and centrifugation to obtain active ingredient hair follicle transdermal delivery system based on protein carrier.

[0028] Further, in step (S1), the concentration of the bioactive protein solution is 1-5 mg / mL, and it is a buffer solution with a pH of 7.2-7.6, such as a phosphate buffer solution; the surface-modifying monomer is selected from at least one of N-propenylsuccinimide, N-(3-aminopropyl)methacrylamide hydrochloride, and maleimide, and the role of the surface-modifying monomer is to modify the surface of the protein through interactions such as electrostatic adsorption and chemical bonding; the concentration of the surface-modifying monomer solution is 1-5 wt%, and the solvent is at least one of dimethyl sulfoxide, ethanol, propanol, and ethylene glycol; the mass ratio of bioactive protein to surface-modifying monomer is 1:0.05-0.1, for example, 1:0.075.

[0029] Further, in step (S2), the concentration of the small molecule active ingredient solution is 0.5-5 mg / mL, and the solvent is at least one of water, methanol, ethanol, and ethylene glycol; after adding the small molecule active ingredient solution, the mixture is stirred at 300-500 r / min for 10-60 min.

[0030] Further, in step (S3), the functional monomer is selected from at least one of vinylpyrrolidone, acrylamide, 2-methacryloyloxyethyl phosphocholine, and 2-hydroxyethyl acrylate; the positively charged monomer is selected from at least one of 2-(dimethylamino)methacrylate, (3-acrylamidopropyl)trimethylammonium chloride hydrochloride, and N-(3-aminopropyl)methacrylamide hydrochloride; the crosslinking agent is selected from at least one of N,N'-methylenebisacrylamide, glyceryl dimethacrylate, and glycerol 1,3-diglyceryl alcohol diacrylate; the initiator is a water-soluble initiator, specifically selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate; 2-5 mg of initiator is added per milligram of bioactive protein.

[0031] Further, in step (S3), the molar ratio of bioactive protein, functional monomer, and positively charged monomer is 1:11000-52000:1000-6000, and the amount of cross-linking agent is 5-10% of the molar amount of functional monomer.

[0032] Furthermore, in step (S3), a co-initiator, such as sodium bisulfite or tetramethylethylenediamine, is added. 5-10 mg of initiator is added per milligram of protein.

[0033] A third objective of this invention is to provide the use of the above-mentioned protein carrier-based transdermal delivery system for active ingredients in the preparation of drugs for the prevention or treatment of diseases including bacterial hair follicle infection, seborrheic folliculitis, seborrheic alopecia, and androgenetic alopecia.

[0034] Compared with the prior art, the present invention achieves the following beneficial effects:

[0035] I. This invention modifies proteins with polymerizable double-bond compounds, loads small-molecule active ingredients, and then initiates a polymerization reaction in situ on the protein surface, coating the protein surface with a polymer protective layer. This polymer protective layer is bio-friendly and has little or no skin irritation. During transdermal delivery, by controlling the surface properties and thickness of this polymer protective layer, the permeability of the protein nanogel in hair follicles and surrounding skin tissue can be effectively controlled, allowing the nanogel to effectively reach the location where the active ingredients need to exert their effects. The polymer layer of this invention not only provides effective protection for the internal bioactive protein nanogel but also does not affect the bioactive protein nanogel or the physiological activity of the bioactive small molecules loaded within it. It specifically addresses hair follicle problems and significantly improves upon the previous shortcomings of poor small-molecule delivery efficiency.

[0036] Second, for common active ingredients that are irritating to the skin and hair follicles, when loaded into protein nanogels, the nanogels not only promote their penetration depth but also reduce their irritation to the skin, making them suitable for a wider range of people.

[0037] Third, all raw materials used in this invention are bio-friendly and can be used as reagents for pharmaceuticals, thus enabling their application in transdermal delivery systems for drugs and other beneficial components. The transdermal delivery system of this invention has a simple preparation method, high yield, and can be industrially mass-produced. Attached Figure Description

[0038] Figure 1 This is a SEM image of the polyethylene glycol-3β-hydroxysteroid dehydrogenase nanogel (PEG-nHSD@Res) from Example 1.

[0039] Figure 2 This is a SEM image of the chitosan-superoxide dismutase nanogel (CS-nSOD@Ast) from Example 2.

[0040] Figure 3 This is a SEM image of the polyvinylpyrrolidone-bromelain nanogel (PVP-nBM@VA) of Example 3.

[0041] Figure 4 This describes the hair follicle growth and development of PVP-nBM@VA in Example 3 during a 20-day dosing cycle. Detailed Implementation

[0042] The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] In the embodiments of this invention, the concentration of the protein nanogel solution is expressed as protein mass.

[0045] Example 1

[0046] 0.15 mg of N-propenylsuccinimide was added to 2 mL of a 1 mg / mL solution of 3β-hydroxysteroid dehydrogenase (HSD), wherein the N-propenylsuccinimide was dissolved in dimethyl sulfoxide. The mixture was thoroughly mixed and reacted for 2 h to obtain a 3β-hydroxysteroid dehydrogenase (aHSD) with polymerizable double bonds modified on its surface.

[0047] To a 2 mL solution of 3β-hydroxysteroid dehydrogenase with a surface double bond modified by 1 mg / mL, 0.45 mL of a methanol solution of resveratrol with a concentration of 0.5 mg / mL was added dropwise while stirring at 350 r / min. After thorough mixing, resveratrol-loaded 3β-hydroxysteroid dehydrogenase (aHSD@Res) was obtained.

[0048] aHSD@Res, polyethylene glycol methyl ether acrylate, aminopropyl methacrylamide hydrochloride, and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:8000:1000:800. The protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, an initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per milligram of protein. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, polyethylene glycol-3β-hydroxysteroid dehydrogenase nanogel loaded with resveratrol (PEG-nHSD@Res) was obtained.

[0049] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to obtain a high-concentration mother liquor with a purified concentration of 5.7 mg / mL, which was then refrigerated.

[0050] Example 2 Preparation of chitosan-superoxide dismutase nanogel

[0051] Six mL of a 1 mg / mL chitosan solution was sonicated at 70 W for 10 min and allowed to stand to allow excess heat to build up. Superoxide dismutase (SOD) solution was added in a 60°C water bath, and the SOD concentration was adjusted to 1 mg / mL. The reaction was allowed to proceed for 5 min. A 0.5 mg / mL astaxanthin-acetone solution was then added dropwise to the mixture at 300 rpm in a 60°C water bath, and stirring was continued for 5 min. Finally, 1 mL of a 1 mg / mL sodium tripolyphosphate solution was added dropwise in an ice-water bath at 300 rpm. After reacting for 30 min, astaxanthin-loaded chitosan-superoxide dismutase nanogel (CS-nSOD@Ast) was obtained.

[0052] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to obtain a high-concentration mother liquor with a purified concentration of 6.1 mg / mL, which was then refrigerated.

[0053] The size of the nanogels was characterized using dynamic light scattering (DLS): a protein nanogel solution with a concentration of 1 mg / mL was added to the sample cell, and the particle size and zeta potential of the protein nanogels in the system were measured at room temperature using a Malvern Nano Zs90 nanoparticle size potentiometer. The results are summarized in Table 1.

[0054] Example 3: Preparation of polyvinylpyrrolidone-bromelain nanocapsules

[0055] Add 0.5 mg of N-propenylsuccinimide, which is dissolved in dimethyl sulfoxide, to 4 mL of bromelain (BM) solution with a concentration of 0.5 mg / mL. Mix thoroughly and react for 4 h to obtain bromelain (aBM) with polymerizable double bonds modified on the surface.

[0056] Add 0.6 mL of a 0.3 mg / mL ethanol solution of vitamin A to 4 mL of a 0.5 mg / mL solution of bromelain with surface double bonds while stirring at 300 r / min. After thorough mixing, vitamin A-loaded bromelain (aBM@VA) is obtained.

[0057] aBM@VA, N-vinyl-2-pyrrolidone, aminopropyl methacrylamide hydrochloride, and N,N'-methylenebisacrylamide were mixed in a molar ratio of 1:6000:1000:600. The protein concentration was diluted to 1 mg / mL with pH 7.4 phosphate buffer. Then, an initiator was added at a ratio of 3.8 mg of ammonium persulfate and 7.6 mg of tetramethylethylenediamine per milligram of protein. In situ free radical polymerization was initiated at room temperature. After 4 h of reaction, vitamin A-loaded polyvinylpyrrolidone-bromelain nanogel (PVP-nBM@VA) was obtained.

[0058] The obtained complex solution was ultrafiltered in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to obtain a high-concentration mother liquor with a purified concentration of 6.1 mg / mL, which was then refrigerated.

[0059] Table 1. Protein nanogel particle size and zeta potential

[0060]

[0061] The morphology of the nanogel was characterized using transmission electron microscopy (TEM): The morphology and dispersion of the polyethylene glycol-3β-hydroxysteroid dehydrogenase nanogel (PEG-nHSD@Res) from Example 1 were observed under a TEM, and the results are as follows: Figure 1 The morphology and dispersion of the chitosan-superoxide dismutase nanogel (CS-nSOD@Ast) in Example 2 were observed, and the results are as follows: Figure 2 The morphology and dispersion of the polyvinylpyrrolidone-bromelain nanogel (PVP-nBM@VA) in Example 3 were observed, and the results are as follows: Figure 3 .

[0062] Application Example 1: 3β-Hydroxysteroid Dehydrogenase Activity Assay

[0063] 3β-hydroxysteroid dehydrogenase activity test in polyethylene glycol-3β-hydroxysteroid dehydrogenase nanogel (PEG-nHSD@Res) in Example 1: The original 3β-hydroxysteroid dehydrogenase solution and the PEG-nHSD@Res solution in Example 1 were taken and the concentration was adjusted to 1 mg / mL. The activity was measured using an ELISA kit, and the results are shown in Table 2.

[0064] Table 2 Relative Activity of HSD

[0065]

[0066] The PEG-nHSD@Res prepared in Example 1 retained approximately 93.7% of the protein activity, indicating that the enzyme activity loss caused by the modification of 3β-hydroxysteroid dehydrogenase and the preparation of the polymer shell by the method of the present invention is minimal and within an acceptable range.

[0067] Application Example 2: Superoxide Dismutase Activity Assay

[0068] Superoxide dismutase (SOD) activity was determined using a UV spectrophotometer. 3 mL of 50 mmol / mL Tris-HCl buffer (pH 8, containing 1 mmol / mL EDTA) was added to the reference cell. In the sample cell, 2.4 mL of 50 mmol / mL Tris-HCl buffer (pH 8, containing 1 mmol / mL EDTA), 0.3 mL of 0.2 mmol / mL pyrogallol, and 0.3 mL of pristine SOD or enzyme nanogel solution were added. The sample cell was then placed in the UV spectrophotometer, and the change in absorbance at 420 nm was recorded for the first 10 minutes. The slope of the fitted straight line was used to represent the relative activity of SOD. The results are shown in Table 3.

[0069] Table 3 Relative activity of SOD

[0070]

[0071] This indicates that the CS-nSOD@Ast prepared in Example 2 still retains 85.7% of its activity, suggesting that the loss of enzyme activity caused by the modification of superoxide dismutase and the preparation of the polymer shell is within an acceptable range.

[0072] Application Example 3: Bromelain Activity Test

[0073] Superoxide dismutase (SOD) activity was determined using a UV spectrophotometer. 1 mL of raw bromelain solution or enzyme nanogel solution was mixed with 3 mL of 0.6% casein solution and stirred for 10 min. Then, 3 mL of trichloroacetic acid was added to precipitate the protein, and the reaction was stopped after 10 min. The mixture was centrifuged at 4000 rpm for 3 min, and the supernatant was collected. 1 mL of the supernatant was mixed with 5 mL of 0.4 mol / L sodium carbonate solution, and then 1 mL of Folin-Ciocalteu reagent was added for color development. Using a system containing 1 mL of phosphate buffer, 5 mL of 0.4 mol / L sodium carbonate solution, and 1 mL of Folin-Ciocalteu reagent as a reference, the absorbance (A) of the system containing the reaction supernatant was measured at 700 nm as the relative activity.

[0074] Table 4 Relative Activity of BM

[0075]

[0076] The results show that the PVP-nBM@VA prepared in Example 3 still retains 87.3% of its activity, indicating that the loss of enzyme activity caused by the modification of bromelain and the preparation of the polymer shell is within an acceptable range.

[0077] Application Example 4: Sustained Release of Active Ingredients

[0078] Two mL of the protein nanogel prepared in Examples 1-3 (5 mg / mL) was placed in a dialysis bag with a molecular weight cutoff of 3000. The dialysis bag was then immersed in phosphate buffer at pH 6, and the entire system was shaken at 37°C. The sustained-release time was 48 h. During this 48 h periodically, samples were taken and the concentration of the loaded small molecule drug was measured using a UV spectrophotometer to calculate the sustained-release effect of the composite system on the small molecule drug. The results are summarized in Table 5. The functional small molecule was slowly released by the protein nanogel within 24 hours, which is beneficial for the transdermal system to reach the deep hair follicle and surrounding tissues, allowing the functional small molecule to exert its effect.

[0079] Table 5. Release of small molecule drugs by nanogels

[0080]

[0081] Application Example 5: Verification of the effectiveness of small molecule active ingredients

[0082] Anti-inflammatory capacity assay of polyethylene glycol-3β-hydroxysteroid dehydrogenase nanogel (PEG-nHSD@Res): NO concentration in inflammatory cells was detected using a Griess reagent kit. HaCaT cells (1*10⁵ cells / well) were seeded in 24-well plates. After 12 h of complete cell adhesion, the original culture medium was aspirated. All groups were treated with 100 μL of serum-free medium, the negative control group with 10 μL of serum-free medium, the positive control group with 10 μL of PBS buffer, the low-concentration experimental group with 10 μL of 0.01 mg / mL PEG-nHSD@Res nanogel solution, the medium-concentration experimental group with 10 μL of 0.02 mg / mL PEG-nHSD@Res nanogel solution, and the high-concentration experimental group with 10 μL of 0.04 mg / mL PEG-nHSD@Res nanogel solution. After co-incubation for 2 h, the original culture medium was removed from all groups. The negative control group was given 110 μL of fresh culture medium, while the other groups were given 100 μL of fresh culture medium and 10 μL of interleukin-1β at a concentration of 10 ng / mL. After co-incubation for 24 h, the results were measured according to the Griess kit instructions. The results are summarized in Table 6.

[0083] Table 6. Intracellular anti-inflammatory capacity of PEG-nHSD@Res increases with increasing concentration.

[0084]

[0085] The results show that PEG-nHSD@Res nanogel has good anti-inflammatory ability, and this ability increases with increasing concentration.

[0086] Application Example 7: Animal Experiments

[0087] Example 3: Determination of the ability of polyvinylpyrrolidone-bromelain nanogel (PVP-nBM@VA) to promote hair follicle growth and development: The effect of PVP-nBM@VA on hair follicle growth and development was investigated using a Balb / c mouse model. Twelve female mice were randomly divided into two groups. Hair was removed from the back of both mice using an electric shaver, taking care not to damage the original hair follicles. Every two days, 200 μL of the nanogel solution (experimental group) or PBS buffer (control group) was applied to the shaved area. The experimental group received 300 units / mL of PVP-nBM@VA, while the control group received 20 mM pH=6 buffer solution. The solution was applied evenly to the shaved area. After 20 days of continuous administration, skin sections were taken from the back of the mice to observe hair follicle growth. The results are as follows: Figure 4 The statistical results of hair follicle growth are shown in Table 7.

[0088] Table 7. Statistics on hair follicle length and number

[0089]

[0090] The results showed that mice treated with PVP-nBM@VA had significantly better hair follicle length and average number than the control group during the 20-day treatment period, indicating that PVP-nBM@VA promoted both the increase in hair follicle number and length.

Claims

1. A method for preparing a transdermal follicular delivery system for active ingredients based on a protein carrier, comprising the following steps: (S1) Add a surface-modified monomer solution to a bioactive protein solution to obtain a surface-modified protein solution; The bioactive protein is bromelain; the surface-modifying monomer is N-propenylsuccinimide; the mass ratio of the small molecule active ingredient to the bioactive protein is 1:1-10; (S2) Add a small molecule active ingredient solution to the surface-modified protein solution obtained in step (S1). The small molecule active ingredient is loaded in the bioactive protein. The small molecule active ingredient is vitamin A. (S3) Add functional monomers, positively charged monomers, crosslinking agents, and initiators to initiate in-situ polymerization, obtaining a transdermal follicular delivery system for active ingredients based on a protein carrier; the functional monomer is vinylpyrrolidone; the positively charged monomer is N-(3-aminopropyl)methacrylamide hydrochloride; the crosslinking agent is N,N'-methylenebisacrylamide; the molar ratio of bioactive protein, functional monomer, and positively charged monomer is 1:11000-52000:1000-6000, and the amount of crosslinking agent is 5-10% of the molar amount of the functional monomer; The protein carrier-based transdermal delivery system for active ingredients in hair follicles has a composite structure of small molecule active ingredient, bioactive protein, and polymer shell, with a size of 40-150 nm and a polymer shell thickness of 10-70 nm. The zeta potential of the protein carrier-based transdermal delivery system for active ingredients in hair follicles is positively charged. The small molecule active ingredient is loaded in the bioactive protein, and the polymer shell coats the bioactive protein.

2. The production method according to claim 1, characterized by, The bioactive protein has a size of 0.5-20 nm.

3. The preparation method according to claim 2, characterized in that, The bioactive protein has a size of 3-10 nm.

4. The method of claim 1, wherein, The zeta potential of the protein carrier-based transdermal delivery system for active ingredients is +3 to +7 mV.

5. The preparation method according to claim 1, characterized in that, The mass ratio of small molecule active ingredients to bioactive proteins is 1:2-5.

6. The preparation method according to claim 1, characterized in that, In step (S1), the concentration of the bioactive protein solution is 1-5 mg / mL, and it is a buffer solution with a pH of 7.2-7.6; the concentration of the surface-modified monomer solution is 1-5 wt%, and the mass ratio of bioactive protein to surface-modified monomer is 1:0.05-0.

1.

7. The preparation method according to claim 1, characterized in that, In step (S2), the concentration of the small molecule active ingredient solution is 0.5-5 mg / mL, and the solvent is at least one of methanol and ethanol.

8. The preparation method according to claim 1, characterized in that, In step (S3), the initiator is a water-soluble initiator selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate; 2-5 mg of initiator is added per milligram of protein.

9. The use of the protein carrier-based transdermal delivery system for active ingredients prepared by the method according to any one of claims 1-8 in the preparation of drugs for the prevention or treatment of seborrheic alopecia or androgenic alopecia.

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