Conjugated linoleic acid vesicle for preventing and treating hair loss and application thereof

Abstract

The present invention discloses a conjugated linoleic acid vesicle for preventing and treating hair loss and its application; specifically, conjugated linoleic acid is used as the main vesicle material, and conjugated linoleic acid vesicles are prepared together with phospholipids and vitamin E polyethylene glycol succinate, and arginine is modified on the surface to obtain conjugated linoleic acid nanovesicles; the conjugated linoleic acid nanovesicles of the present invention have excellent transdermal penetration and retention behavior, and can accumulate in the hair follicle area, with a hair follicle targeting effect. After the conjugated linoleic acid nanovesicles are loaded with drugs, the skin retention and hair follicle targeting of the loaded drugs can be enhanced; the preparation method of the conjugated linoleic acid nanovesicles and the drug-loaded nanovesicles is simple, and can treat androgenic alopecia through multiple mechanisms, and can also alleviate the aging problem in androgenic alopecia. The drug-loaded nanovesicles obtained by using conjugated linoleic acid vesicles as drug carriers and encapsulating hair loss treatment drugs have good therapeutic effects in both male and female androgenic alopecia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a conjugated linoleic acid vesicle for preventing and treating hair loss and an application thereof. Background Art

[0002] Hair loss is a global disease that has seriously affected the mental health and quality of life of more than 50% of the population, affecting both men and women. In recent years, with the intensification of mental stress and environmental pollution, the prevalence of hair loss has increased year by year.

[0003] At present, the main drugs for treating hair loss are oral finasteride and topical minoxidil. Among them, finasteride is an oral preparation and is generally not used to treat female hair loss. Long-term use can cause side effects such as sexual dysfunction and breast hyperplasia. Minoxidil topical preparations usually contain a large amount of organic solvents such as propylene glycol, which makes some patients unable to use it due to allergies. At the same time, there are problems such as increased skin adverse reactions with prolonged use, which greatly reduces patient compliance. In addition, hair loss is also affected by many factors, and the mechanism of action of existing drugs is relatively simple, so it is difficult to achieve good therapeutic effects. For example, cell senescence plays an important role in the occurrence and development of hair loss. However, to date, there is a lack of effective hair loss treatment strategies targeting hair follicle cell senescence.

[0004] Conjugated linoleic acid is a polyunsaturated fatty acid with multiple pharmacological activities, including anti-adipogenesis, anti-atherosclerosis, restoration of vascular function, anti-inflammatory and antioxidant effects. CN 108967665A discloses a hair-enhancing agent containing conjugated linoleic acid, which can increase the density of hair follicles by promoting cell proliferation and activating hair follicles. However, free conjugated linoleic acid cannot effectively accumulate in the hair follicle area and cannot form a drug reservoir, resulting in limited effectiveness in treating hair loss. In addition, conjugated linoleic acid can usually only self-assemble into nanovesicles in an alkaline environment. However, the normal pH value of the skin is weakly acidic. Alkaline systems are not only not conducive to the absorption of active ingredients, but long-term use can also disrupt the skin's barrier function and bacterial balance, leading to skin damage and the occurrence of diseases. Summary of the Invention

[0005] The present invention proposes to construct nanovesicles using conjugated linoleic acid as the primary vesicle material. In hair loss treatment, conjugated linoleic acid can serve as both a vesicle material and an active ingredient. Conjugated linoleic acid not only exhibits pharmacological activity, such as promoting hair follicle cell proliferation and migration and alleviating the onset of hair follicle cell aging, but the constructed conjugated linoleic acid nanovesicles can also serve as nanocarriers for treating hair loss, loading various hair loss therapeutic drugs. Conjugated linoleic acid vesicles can improve the transdermal penetration and retention of conjugated linoleic acid and the drugs they carry, enhancing their accumulation and targeting within hair follicles, thereby enhancing the drug's effectiveness in treating hair loss.

[0006] The first aspect of the present invention aims to provide a nanovesicle.

[0007] The second aspect of the present invention aims to provide a method for preparing the above-mentioned nanovesicles.

[0008] The third aspect of the present invention aims to provide applications of the above-mentioned nanovesicles.

[0009] The fourth aspect of the present invention aims to provide a product comprising the above-mentioned nanovesicles.

[0010] The fifth aspect of the present invention aims to provide a method for preparing the above-mentioned product.

[0011] The technical solution adopted by the present invention is:

[0012] A first aspect of the present invention provides a nanovesicle, wherein raw materials for preparing the nanovesicle include conjugated linoleic acid, phospholipids, and vitamin E polyethylene glycol succinate.

[0013] Preferably, the phospholipid is one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, distearoylphosphatidylcholine, dioleoyl lecithin, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoyl lecithin, dipalmitoylphosphatidylcholine, and dioleoylphosphatidylcholine.

[0014] Preferably, the mass ratio of the conjugated linoleic acid, phospholipid, vitamin E polyethylene glycol succinate and arginine is 2:0.1-0.9:0.1-0.9.

[0015] Preferably, the raw materials for preparing the nanovesicles further include arginine.

[0016] Preferably, the mass ratio of the conjugated linoleic acid, phospholipid, vitamin E polyethylene glycol succinate and arginine is 2:0.1-0.9:0.1-0.9:0.1-5.

[0017] The second aspect of the present invention provides a method for preparing the above-mentioned nanovesicles, comprising the following steps:

[0018] S1: Conjugated linoleic acid, phospholipids and vitamin E polyethylene glycol succinate are weighed separately, mixed and dissolved in an organic solvent to obtain a nanovesicle solution A.

[0019] Preferably, the method further comprises:

[0020] S2: adding solution A to the arginine solution and mixing them to obtain a nanovesicle solution containing arginine.

[0021] Preferably, the mass volume ratio of the mixture to the organic solvent is 60 to 120 mg / mL.

[0022] Preferably, the organic solvent includes at least one of anhydrous ethanol, butylene glycol, and propylene glycol.

[0023] Preferably, the arginine solution is prepared by adding arginine into a solvent to obtain the arginine solution.

[0024] Preferably, the mass volume ratio of arginine to solvent is 0.1 to 12.5 mg / mL.

[0025] Preferably, the solvent for the arginine includes but is not limited to water and buffer.

[0026] Preferably, the buffer comprises PBS buffer or HEPES buffer.

[0027] Preferably, the pH of the buffer solution is 6-8.

[0028] Preferably, the mixed solution in step S2 is added dropwise to the arginine solution.

[0029] Preferably, the mixing time in step S2 is 30 min to 60 min, the rotation speed is 500 rpm to 1000 rpm, and the temperature is 10° C. to 30° C.

[0030] The third aspect of the present invention provides the use of the nanovesicles described in the second aspect of the present invention in the preparation of products for hair growth and / or prevention and treatment of hair loss.

[0031] Preferably, the hair loss includes partial hair loss, total hair loss or generalized hair loss caused by androgenic alopecia, seborrheic alopecia, anagen effluvium, telogen effluvium, drug-induced alopecia, alopecia areata, tinea capitis or hypotrichosis.

[0032] Preferably, the products include medicines and daily chemicals.

[0033] Preferably, the daily chemical products include but are not limited to hair growth liquid, hair tonic, hair conditioner, shampoo head, hair cleaning foam, hair oil, hair dryer, hair dye, hair gel, hair glaze, hair moisturizer, hair mask, hair treatment cream, eyebrow growth agent, eyelash growth agent, eyelash nutrition agent.

[0034] Preferably, the product further comprises auxiliary materials.

[0035] Preferably, the excipients include at least one of surfactants, thickeners, solubilizers, pearlescent agents, conditioners, preservatives, chelating agents, buffers, flavors, pigments, diluents, wetting agents, adhesives, disintegrants, lubricants, color or odor regulators, solvents, solubilizers, emulsifiers, antioxidants, metal complexing agents, inert gases, local analgesics, pH regulators, and isotonic or isotonic regulators.

[0036] Preferably, the dosage form of the product includes but is not limited to granules, granules, capsules, tablets, sprays, lotions, ointments, patches, injections, emulsions, aerosols, drops, gels, microneedles, controlled-release agents, and sustained-release agents.

[0037] Preferably, the administration of the product includes subcutaneous administration, intramuscular administration, intraperitoneal administration, intravenous injection, infusion, oral administration, nasal administration or other local administration, or a combination of any of the above administration methods.

[0038] The fourth aspect of the present invention provides a product, which comprises the nanovesicles described in the first aspect of the present invention.

[0039] Preferably, the product further comprises one or more other drugs for treating hair loss.

[0040] Preferably, the other drugs include minoxidil, finasteride, dutasteride, spironolactone tablets, cyproterone acetate, and / or clobetasol propionate or pharmaceutically acceptable salts thereof.

[0041] Preferably, the mass ratio of conjugated linoleic acid to other drugs in the nanovesicles is 1:0.01-1.5.

[0042] Preferably, the product further comprises auxiliary materials.

[0043] Preferably, the excipients include at least one of surfactants, thickeners, solubilizers, pearlescent agents, conditioners, preservatives, chelating agents, buffers, flavors, pigments, diluents, wetting agents, adhesives, disintegrants, lubricants, color or odor regulators, solvents, solubilizers, emulsifiers, antioxidants, metal complexing agents, inert gases, local analgesics, pH regulators, and isotonic or isotonic regulators.

[0044] Preferably, the dosage form of the product includes but is not limited to granules, granules, capsules, tablets, sprays, lotions, ointments, patches, injections, emulsions, aerosols, drops, gels, microneedles, controlled-release agents, and sustained-release agents.

[0045] Preferably, the administration of the product includes subcutaneous administration, intramuscular administration, intraperitoneal administration, intravenous injection, infusion, oral administration, nasal administration or other local administration, or a combination of any of the above administration methods.

[0046] The fifth aspect of the present invention provides a method for preparing the product according to the fourth aspect of the present invention, comprising the step of mixing the nanovesicles with other drugs.

[0047] Preferably, the method specifically comprises the following steps:

[0048] S1: Conjugated linoleic acid, phospholipids, vitamin E polyethylene glycol succinate, and other drugs are weighed separately to obtain a mixture, and the mixture is dissolved in an organic solvent to obtain a mixed solution.

[0049] Preferably, the method further comprises:

[0050] S2: Add the mixed solution to the arginine solution and mix.

[0051] Preferably, the mass volume ratio of the mixture to the organic solvent is 60 to 120 mg / mL.

[0052] Preferably, the organic solvent includes at least one of anhydrous ethanol, butylene glycol, and propylene glycol.

[0053] Preferably, the arginine solution is prepared by adding arginine into a solvent to obtain the arginine solution.

[0054] Preferably, the mass volume ratio of arginine to solvent is 0.1 to 12.5 mg / mL.

[0055] Preferably, the solvent for the arginine includes but is not limited to water and buffer.

[0056] Preferably, the buffer comprises PBS buffer or HEPES buffer.

[0057] Preferably, the pH of the buffer solution is 6-8.

[0058] Preferably, the mixed solution in step S2 is added dropwise to the arginine solution.

[0059] Preferably, the mixing time in step S2 is 30 min to 60 min, the rotation speed is 500 rpm to 1000 rpm, and the temperature is 10° C. to 30° C.

[0060] The beneficial effects of the present invention are:

[0061] In order to improve the conditions for the self-assembly of conjugated linoleic acid, the present invention provides a nanovesicle constructed based on conjugated linoleic acid. Specifically, conjugated linoleic acid vesicles are prepared using conjugated linoleic acid, phospholipids and vitamin E polyethylene glycol succinate, and arginine is modified on the surface to obtain conjugated linoleic acid nanovesicles Arg-CLAVs; the conjugated linoleic acid nanovesicles of the present invention have good transdermal penetration and retention behavior, achieving the accumulation of conjugated linoleic acid nanovesicles in the hair follicle area and targeting the hair follicles; the preparation method of conjugated linoleic acid nanovesicles is simple, and can treat androgenic alopecia through multiple mechanisms, and can alleviate the aging problem in androgenic alopecia. After using conjugated linoleic acid vesicles as carriers to encapsulate hair loss treatment drugs, the resulting preparation has good therapeutic effects in both male and female androgenic alopecia. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The particle size of nanovesicles prepared by combining different membrane materials with conjugated linoleic acid under different pH conditions.

[0063] Figure 2 Skin penetration effect of conjugated linoleic acid vesicles loaded with minoxidil.

[0064] Figure 3 The effects of conjugated linoleic acid vesicles on the proliferation (a) and migration (b, c) of human dermal papilla cells, where (c) is the image of cell migration at different time points, and (b) is the quantitative result of (c).

[0065] Figure 4 The effects of conjugated linoleic acid vesicles on the proliferation (a) and migration (b, c) of venous endothelial cells, where (c) is the image of cell migration at different time points, and (b) is the quantitative result of (c).

[0066] Figure 5 The figure shows the inhibitory effect of conjugated linoleic acid vesicles on androgen-induced hair follicle cell senescence, where A is the β-galactosidase staining result and B is the quantitative result of A.

[0067] Figure 6 Uptake of different nanovesicles loaded with coumarin 6 by human dermal papilla cells.

[0068] Figure 7 The figure shows the hair regeneration effect of minoxidil-encapsulated conjugated linoleic acid vesicles on the male androgenic alopecia model, where (a) is a photograph of the back of a male androgenic alopecia model mouse after administration of minoxidil-encapsulated conjugated linoleic acid vesicles, (b) is the statistical score of the skin color on the back of the mouse in Figure (a), and (c) is the statistical weight of the newly grown hair on the back of the mouse in Figure (a).

[0069] Figure 8The figure shows the hair regeneration effect of minoxidil-encapsulated conjugated linoleic acid vesicles on the female androgenic alopecia model, where (a) is a photograph of the back of a female androgenic alopecia model mouse after administration of minoxidil-encapsulated conjugated linoleic acid vesicles, (b) is the statistical score of the skin color on the back of the mouse in Figure (a), and (c) is the statistical weight of the newly grown hair on the back of the mouse in Figure (a).

[0070] Figure 9 H&E staining photos of the new hair of mice with alopecia model after being treated with minoxidil-conjugated linoleic acid vesicles (a) and the detection results of the follicle colonization depth of the new hair (b), skin thickness (c) and the number of hair follicles (d).

[0071] Figure 10 The inhibitory effect of conjugated linoleic acid vesicles loaded with finasteride on 5α-reductase.

[0072] Figure 11 The figure shows the hair regeneration effect of finasteride-encapsulated conjugated linoleic acid vesicles on androgenic alopecia model mice, where (a) is a photograph of the back of androgenic alopecia model mice after administration of finasteride-encapsulated conjugated linoleic acid vesicles, and (b) is the quantitative result of hair coverage.

[0073] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are all within the scope of protection of the present invention.

[0074] CLA: Conjugated Linoleic Acid.

[0075] PC: Phosphatidylcholine.

[0076] TPGS: Vitamin E polyethylene glycol succinate.

[0077] MNX: Minoxidil.

[0078] CLAVs: conjugated linoleic acid vesicles without arginine surface modification.

[0079] Arg-CLAVs: conjugated linoleic acid vesicles modified with arginine surface.

[0080] MNX@Arg-CLAVs: Arginine-modified conjugated linoleic acid vesicles encapsulating the hair loss treatment drug minoxidil.

[0081] Polydispersity index (PDI): An important concept in polymer science that reflects the dispersion and uniformity of nanoparticles. The smaller the value, the more uniform the particles.

[0082] β-galactosidase: A marker of cell senescence. The higher its expression level, the higher the degree of cell senescence.

[0083] The present invention provides conjugated linoleic acid-based nanovesicles that can encapsulate hair loss treatment drugs for the treatment of androgenic alopecia. The nanovesicles contain conjugated linoleic acid, phospholipids, vitamin E polyethylene glycol succinate, and arginine. The mass ratio of conjugated linoleic acid to phospholipids to vitamin E polyethylene glycol succinate is 2:0.1-0.9:0.1-0.9:0.1-5. The phospholipids are one or more of soy lecithin, egg yolk lecithin, hydrogenated lecithin, distearoylphosphatidylcholine, dioleoyl lecithin, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoyl lecithin, dipalmitoylphosphatidylcholine, and dioleoylphosphatidylcholine. The encapsulated hair loss treatment drugs include minoxidil, finasteride, dutasteride, and the like.

[0084] Example 1: Preparation of arginine-free conjugated linoleic acid vesicles

[0085] Conjugated linoleic acid, egg yolk lecithin, and vitamin E polyethylene glycol succinate were mixed in a mass ratio of 2:0.5:0.5 and dissolved in anhydrous ethanol at a mass-to-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother liquor. This anhydrous ethanol mother liquor was then added dropwise to a 9:1 volume ratio of PBS buffer (pH 6.5) at a stirring speed of 1000 rpm and a temperature of 30°C. The mixture was stirred for 30 minutes to obtain an arginine-free conjugated linoleic acid vesicle solution (CLAVs).

[0086] Example 2: Preparation of Arginine-Conjugated Linoleic Acid Vesicles

[0087] Conjugated linoleic acid, egg yolk lecithin, and vitamin E polyethylene glycol succinate were mixed in a mass ratio of 2:0.5:0.5 and dissolved in anhydrous ethanol at a mass-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother solution. Arginine was dissolved in a mass-volume ratio of 5.56 mg / mL in PBS buffer (pH 6.5). The anhydrous ethanol mother solution was added dropwise to the arginine-containing PBS solution at a stirring speed of 1000 rpm and a stirring temperature of 30°C. The volume ratio of PBS buffer to anhydrous ethanol was 9:1, and the mass ratio of conjugated linoleic acid to arginine was 1:1. The mixture was stirred for 30 minutes to obtain a conjugated linoleic acid vesicle solution (Arg-CLAVs).

[0088] Example 3: Preparation of arginine-conjugated linoleic acid vesicles

[0089] Conjugated linoleic acid, soy lecithin, and vitamin E polyethylene glycol succinate were mixed in a mass ratio of 2:0.5:0.5 and dissolved in anhydrous ethanol at a mass-to-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother liquor. Arginine was dissolved in a mass-to-volume ratio of 5.56 mg / mL in PBS buffer (pH 6.5). The anhydrous ethanol mother liquor was added dropwise to the arginine-containing PBS solution at a stirring speed of 1000 rpm and a stirring temperature of 30°C. The volume ratio of PBS buffer to anhydrous ethanol was 9:1, and the mass ratio of conjugated linoleic acid to arginine was 1:1. The mixture was stirred for 30 minutes to obtain a conjugated linoleic acid vesicle solution.

[0090] Example 4: Preparation of Arginine-Conjugated Linoleic Acid Vesicles

[0091] Conjugated linoleic acid, egg yolk lecithin, and vitamin E polyethylene glycol succinate were mixed in a mass ratio of 2:0.25:0.75 and dissolved in anhydrous ethanol at a mass-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother solution. Arginine was dissolved in a pH 6.5 PBS buffer at a mass-volume ratio of 5.56 mg / mL. The anhydrous ethanol mother solution was added dropwise to the arginine-containing PBS solution at a stirring speed of 1000 rpm and a stirring temperature of 30°C, with a mass ratio of conjugated linoleic acid to arginine of 1:1. The mixture was stirred for 30 minutes to obtain a conjugated linoleic acid vesicle solution.

[0092] Example 5: Preparation of Arginine-Conjugated Linoleic Acid Vesicles

[0093] Conjugated linoleic acid, egg yolk lecithin, and vitamin E polyethylene glycol succinate were mixed in a mass ratio of 2:0.75:0.25 and dissolved in anhydrous ethanol at a mass-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother solution. Arginine was dissolved in a pH 6.5 PBS buffer at a mass-volume ratio of 5.56 mg / mL. The anhydrous ethanol mother solution was added dropwise to the arginine-containing PBS solution at a stirring speed of 1000 rpm and a stirring temperature of 30°C, with a mass ratio of conjugated linoleic acid to arginine of 1:1. The mixture was stirred for 30 minutes to obtain a conjugated linoleic acid vesicle solution.

[0094] Example 6: Preparation of conjugated linoleic acid vesicles loaded with minoxidil

[0095] Conjugated linoleic acid, egg yolk lecithin, vitamin E polyethylene glycol succinate, and minoxidil were mixed in a mass ratio of 2:0.5:0.5:1.8 and dissolved in anhydrous ethanol at a mass-volume ratio of 120 mg / mL to obtain an anhydrous ethanol mother solution. Arginine was dissolved in PBS buffer at a mass-volume ratio of 5.56 mg / mL at a pH of 6.5. The anhydrous ethanol mother solution was added dropwise to the arginine-containing PBS solution at a stirring speed of 1000 rpm and a temperature of 30°C. The volume ratio of PBS buffer to anhydrous ethanol was 9:1, and the mass ratio of conjugated linoleic acid to arginine was 1:1. The mixture was stirred for 30 minutes to obtain minoxidil-encapsulated conjugated linoleic acid vesicles (MNX@Arg-CLAVs).

[0096] Example 7: Preparation of conjugated linoleic acid vesicles loaded with finasteride

[0097] Conjugated linoleic acid, egg yolk lecithin, vitamin E polyethylene glycol succinate, and finasteride were mixed in a mass ratio of 2:0.5:0.5:0.1 and dissolved in anhydrous ethanol at a mass-to-volume ratio of 77.5 mg / mL to obtain an anhydrous ethanol mother solution. Arginine was dissolved in PBS buffer at a mass-to-volume ratio of 5.56 mg / mL. The anhydrous ethanol mother solution was added dropwise to the arginine-containing PBS solution at a stirring speed of 1000 rpm and a stirring temperature of 30°C. The volume ratio of PBS buffer to anhydrous ethanol was 9:1, and the mass ratio of conjugated linoleic acid to arginine was 1:1. The mixture was stirred for 30 minutes to obtain conjugated linoleic acid vesicles (FIN@Arg-CLAVs) loaded with finasteride.

[0098] Comparative Example 1

[0099] Conjugated linoleic acid and egg yolk lecithin were mixed in a 2:1 mass ratio and then dissolved in anhydrous ethanol at a mass-to-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother liquor. The entire anhydrous ethanol mother liquor was then added dropwise to a 9:1 volume ratio of PBS buffer (pH 6.5) at a stirring speed of 1000 rpm and a temperature of 30°C. The mixture was stirred for 30 minutes to obtain an arginine-free conjugated linoleic acid vesicle solution.

[0100] Comparative Example 2

[0101] Conjugated linoleic acid and vitamin E polyethylene glycol succinate were mixed in a 2:1 mass ratio and dissolved in anhydrous ethanol at a mass-to-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother liquor. The entire anhydrous ethanol mother liquor was then added dropwise to a 9:1 volume ratio of PBS buffer (pH 6.5) at a stirring speed of 1000 rpm and a temperature of 30°C. The mixture was stirred for 30 minutes to obtain an arginine-free conjugated linoleic acid vesicle solution.

[0102] Comparative Example 3

[0103] Conjugated linoleic acid, lecithin, and vitamin E polyethylene glycol succinate were mixed in a mass ratio of 2:0.5:0.5 and dissolved in anhydrous ethanol at a mass-to-volume ratio of 75 mg / mL to obtain an anhydrous ethanol mother liquor. The entire anhydrous ethanol mother liquor was then added dropwise to a 9:1 volume ratio of PBS buffer (pH 6.5) at a stirring speed of 1000 rpm and a temperature of 30°C. The mixture was stirred for 30 minutes to obtain an arginine-free conjugated linoleic acid vesicle solution.

[0104] Effect Example 1

[0105] Conjugated linoleic acid vesicle solutions (CLA:PC:TPGS) were prepared according to the method in Example 1, wherein the pH of the PBS buffer used was 6.0, 6.5, 7.0, 7.5, and 8.0, respectively.

[0106] Conjugated linoleic acid vesicle solutions (CLA:TPGS) were prepared according to the method in Comparative Example 2, wherein the pH values ​​of the PBS buffer used were 6.0, 6.5, 7.0, 7.5, and 8.0, respectively.

[0107] Conjugated linoleic acid vesicle solutions (CLA:PC) were prepared according to the method in Comparative Example 1, wherein the pH values ​​of the PBS buffer used were 6.0, 6.5, 7.0, 7.5, and 8.0, respectively.

[0108] The particle size and polydispersity index of the three vesicle solutions were measured. The results are shown in Figure 1 The results show that the particle size and polydispersity index of the conjugated linoleic acid vesicles prepared according to the method in Example 1 are smaller than those of the vesicles prepared in Comparative Example 1 and Comparative Example 2, indicating that the quality of the conjugated linoleic acid vesicles prepared in Example 1 is better.

[0109] Effect Example 2

[0110] According to the method in Example 1, a PBS buffer solution with a pH value of 6.5 was used to prepare a conjugated linoleic acid vesicle solution (CLAVs) without arginine modification.

[0111] According to the method in Example 2, arginine-modified conjugated linoleic acid vesicle solution (Arg-CLAVs) was prepared using PBS buffer with a pH value of 6.5.

[0112] The particle size and polydispersity index of two different vesicle solutions were measured, and the results are shown in Table 1 ;

[0113] Table 1

[0114] prescription Particle size (nm) Polydispersity Index CLAVs 201.2±1.1 0.136±0.028 Arg-CLAVs 206.3±3.1 0.113±0.012

[0115] The results showed that the conjugated linoleic acid vesicles Arg-CLAVs with arginine surface modification prepared in Example 2 had a smaller polydispersity index than the vesicles CLAVs in Example 1 without arginine surface modification, indicating that the Arg-CLAVs were more uniform and the vesicle quality was better.

[0116] Effect Example 3

[0117] The treated pigskin was washed and wiped dry, cut to the required size, and fixed in a transdermal diffusion cell. Physiological saline containing 40% PEG-400 was added to the receiving cell and balanced at 32°C and 250rpm for 30 minutes. MNX@Arg-CLAVs was prepared according to Example 6. At the same time, minoxidil was weighed and dissolved in ethanol-propylene glycol-PBS buffer (3:5:2, v / v) to obtain MNX tincture. 500 μL of MNX@Arg-CLAVs or MNX tincture was added to the supply cell, and each group was repeated 5 times in parallel. At a specific time point, 1 mL of receiving solution was taken out from the receiving cell, filtered, and then the cumulative permeation of minoxidil was determined by HPLC.

[0118] At specific time points, the transdermal diffusion cell was removed, excess liquid was discarded, and the pigskin was removed. The pigskin was washed with saline, dried, and the stratum corneum was removed by tearing it 15 times with transparent tape. An appropriate amount of cyanoacrylate glue was applied to the pigskin epidermis, covered with transparent tape, and pressed to allow the glue to penetrate into the hair follicles. After the glue dried, the transparent tape was removed and soaked in methanol. Ultrasonic extraction was performed, and the minoxidil content in the hair follicles was determined by HPLC after filtration. The pigskin was chopped and soaked in methanol. Ultrasonic extraction was performed, and the minoxidil content in the hair follicles was determined by HPLC after filtration. The skin retention of minoxidil was determined by HPLC after shredding.

[0119] The results are as follows Figure 2 As shown, the results show that the conjugated linoleic acid vesicles MNX@Arg-CLAVs loaded with minoxidil prepared in Example 6 have a faster skin penetration rate, a higher retention rate in the skin, and a higher degree of accumulation in the hair follicles than the commonly used minoxidil tincture Mandi, and have hair follicle targeting, indicating that MNX@Arg-CLAVs can enter the skin faster and form a drug reservoir in the skin, with sustained effect, and can also act on the hair follicles at the lesion site.

[0120] Effect Example 4

[0121] Human dermal papilla cells were seeded in 96-well plates and allowed to adhere overnight. Conjugated linoleic acid (CLA), Arg-CLAVs, and minoxidil were diluted to the desired concentrations in culture medium, and 100 μL of drug-containing culture medium was added to each well. Drug-free culture medium served as the negative control, and minoxidil at a final concentration of 20 μg / mL served as the positive control. The final concentration of CLA and Arg-CLAVs was 200 μg / mL. Six replicates were performed in each group. After 24 hours of incubation at 37°C in the dark, the culture medium was discarded and 1% CCK-8 (v / v) was added. After incubation for 2 hours, absorbance at 450 nm was measured using a microplate reader, and cell proliferation rate was calculated.

[0122] Human dermal papilla cells were seeded in a 6-well plate. After reaching 80% to 90% confluence, three vertical lines were scratched with a pipette tip and the cells were washed with PBS. The drug was diluted to the desired concentration in culture medium. Drug-free culture medium served as the negative control, and minoxidil served as the positive control. Each group was replicated three times. The final concentration of CLA, CLAVs, and Arg-CLAVs was 200 μg / mL, and the final concentration of minoxidil was 20 μg / mL. The cells were cultured in an incubator. Scratches at fixed locations were photographed at 0, 24, and 48 hours, and cell migration rates were calculated.

[0123] The results showed that the conjugated linoleic acid vesicles Arg-CLAVs prepared in Example 2 can promote the proliferation and migration of human dermal papilla cells and contribute to the regeneration of hair follicles; compared with CLAVs without arginine surface modification, Arg-CLAVs have a better effect in promoting cell migration ( Figure 3 ).

[0124] Effect Example 5

[0125] Human umbilical vein endothelial cells were seeded in 96-well plates and allowed to adhere overnight. Conjugated linoleic acid (CLA), Arg-CLAVs, and minoxidil were diluted to the desired concentrations in culture medium, and 100 μL of drug-containing culture medium was added to each well. Drug-free culture medium served as the negative control, and minoxidil at a final concentration of 20 μg / mL served as the positive control. The final concentration of CLA and Arg-CLAVs was 50 μg / mL. Six replicates were performed in each group. After 24 hours of incubation at 37°C in the dark, the culture medium was discarded, and 1% CCK-8 (v / v) was added. After incubation for 2 hours, absorbance at 450 nm was measured using a microplate reader, and cell proliferation rate was calculated.

[0126] Human umbilical vein endothelial cells were seeded in 6-well plates. After reaching 80% to 90% confluence, three vertical lines were scratched with a pipette tip and the cells were washed with PBS. The drug was diluted to the desired concentration in culture medium. Drug-free culture medium served as the negative control, and minoxidil served as the positive control. Each group was repeated three times. The final concentration of CLA, CLAVs, and Arg-CLAVs was 50 μg / mL, and the final concentration of minoxidil was 20 μg / mL. The cells were cultured in an incubator. Scratches at fixed locations were photographed at 0 and 24 hours, and cell migration rates were calculated.

[0127] The results showed that the conjugated linoleic acid vesicles Arg-CLAVs prepared in Example 2 can promote the proliferation and migration of venous endothelial cells, help the formation of blood vessels around hair follicles, restore the nutritional supply of hair follicles, and promote hair regeneration. Compared with CLAVs without arginine surface modification, Arg-CLAVs have a better effect in promoting cell migration ( Figure 4 )

[0128] Effect Example 6

[0129] Human dermal papilla cells were seeded in 12-well plates and cultured overnight. CLA, CLAVs, and Arg-CLAVs were diluted with culture medium, and the original culture medium was replaced for 24 hours. The next day, a dihydrotestosterone stock solution was diluted with culture medium and mixed with culture medium containing CLA, CLAVs, and Arg-CLAVs, respectively, and the culture medium was replaced again. The final concentration of dihydrotestosterone was 10 μM, and the final concentrations of CLA, CLAVs, and Arg-CLAVs were all 200 μg / mL. A negative control group was cultured with culture medium alone, and a positive control group was cultured with culture medium containing dihydrotestosterone alone. Each group was replicated three times. After 48 hours, cells were stained with a β-galactosidase staining kit for 24 hours. Cells were observed and images were captured using a cell imaging system, and the β-galactosidase positivity rate was calculated.

[0130] The results showed that the conjugated linoleic acid vesicles Arg-CLAVs prepared in Example 2 can effectively reduce the expression of β-galactosidase, a cell senescence marker caused by androgens, which means that it can alleviate the senescence of hair follicle cells caused by androgens, restore the health of hair follicles, and better treat androgenic alopecia ( Figure 5 ).

[0131] Effect Example 7

[0132] Human dermal papilla cells were seeded in a laser confocal microscopy dish and cultured overnight. Coumarin 6-encapsulated CLAVs and Arg-CLAVs were prepared and diluted in culture medium. Free coumarin 6, coumarin 6-encapsulated CLAVs, or coumarin 6-encapsulated Arg-CLAVs were added to the cells and incubated at 37°C for 4 hours in the dark. The culture medium was then discarded and the cells were washed with PBS. The cell nuclei were then stained with DAPI and incubated at 37°C for 10 minutes. After staining, the cells were washed with PBS and observed under a laser confocal microscope to calculate the cellular uptake of coumarin 6.

[0133] The results showed that the conjugated linoleic acid vesicles Arg-CLAVs prepared in Example 2 can significantly promote their uptake by human dermal papilla cells; compared with CLAVs without arginine surface modification, Arg-CLAVs have a better effect in promoting cell uptake, indicating that Arg-CLAVs can enter cells faster and exert their effects ( Figure 6 ).

[0134] Effect Example 8

[0135] Male C57BL / 6J mice (6-8 weeks old) were randomly divided into five groups, with five mice in each group. After anesthesia, the mice were shaved and depilatory cream was applied to their backs. After one day of recovery, the mice were subjected to the experiment. The control group was treated with saline only. The model group was first treated with a testosterone solution, followed by a 30-minute wait before saline application. Each experimental group was first treated with a testosterone solution, followed by a 30-minute wait before applying the drug solution. The drug solution consisted of 5% minoxidil tincture (Mandi, containing 50 mg / mL minoxidil), Arg-CLAVs (5 mg / mL), or MNX@Arg-CLAVs (5 mg / mL, containing 4.5 mg / mL minoxidil). Testosterone was dissolved in an ethanol-saline solution (1:1, v / v) at a concentration of 5 mg / mL. After the start of drug administration, photographs of the depilatory areas on the backs of the mice were taken on days 1, 13, 17, 21, and 27. Skin color in the depilatory areas of the mice was scored on days 13 and 17. On the 27th day of the experiment, the mice were anesthetized, and the newly grown hair in the hair loss area was shaved off to obtain the weight of the newly grown hair.

[0136] The compositions of the commercially available 5% minoxidil tincture and MNX@Arg-CLAVs are shown in Table 2. It can be seen that the minoxidil content in the minoxidil-loaded conjugated linoleic acid vesicles MNX@Arg-CLAVs prepared in Example 6 is approximately one-tenth of that in the commercially available minoxidil tincture Mandi, and the organic solvent content is reduced by about 7 times compared to the minoxidil tincture, and does not contain the irritating raw material propylene glycol.

[0137] Table 2

[0138]

[0139] The results of animal experiments further showed that the minoxidil-loaded conjugated linoleic acid vesicles MNX@Arg-CLAVs prepared in Example 6 can significantly promote hair regeneration in the male androgenic alopecia model. The skin on the back of the mice darkens faster and the weight of the new hair is heavier. The effect is better than the common 5% minoxidil tincture Mandi ( Figure 7 ).

[0140] Effect Example 9

[0141] Female C57BL / 6J mice (6-8 weeks old) were randomly divided into four groups, each containing four mice. After anesthesia, the mice were shaved and depilatory cream was applied to their backs. After one day of recovery, the mice were subjected to the experiment. The control group was treated with saline only. The model group was first treated with a testosterone solution, followed by a 30-minute wait before applying saline. Each experimental group was first treated with a testosterone solution, followed by a 30-minute wait before applying the drug solution. The drug solution was either 5% minoxidil tincture (Mandi, containing 50 mg / mL minoxidil) or MNX@Arg-CLAVs (5 mg / mL, containing 4.5 mg / mL minoxidil). Testosterone was dissolved in an ethanol-saline solution (1:1, v / v) at a concentration of 5 mg / mL. After the start of drug administration, photographs of the depilated areas on the backs of the mice were taken on days 1, 13, 17, 26, and 34. Skin color in the depilated areas of the mice was scored on days 13 and 17. On the 34th day of the experiment, the mice were anesthetized, the newly grown hair in the hair loss area was shaved off, and the weight of the newly grown hair was measured.

[0142] The experimental results show that the minoxidil-loaded conjugated linoleic acid vesicles MNX@Arg-CLAVs prepared in Example 6 also have a good therapeutic effect in the female androgenic alopecia model, and can significantly promote hair regeneration and the darkening speed of the mouse back skin. The weight of the newly grown hair is higher, and the therapeutic effect is still better than the common 5% minoxidil tincture Mandi ( Figure 8 ).

[0143] Effect Example 10

[0144] On day 27 of the experiment, male mice were anesthetized and sacrificed. Skin from the depilated dorsal area was obtained, wrapped in foil, and fixed in 4% paraformaldehyde. The tissue was then embedded in paraffin, sectioned, and stained with H&E. The sections were observed and imaged using a cell imaging system to assess hair follicle colonization depth, skin thickness, and hair follicle number.

[0145] The results showed that the minoxidil-loaded conjugated linoleic acid vesicles MNX@Arg-CLAVs prepared in Example 6 can effectively increase the depth of hair follicle colonization, skin thickness and the number of hair follicles of new hair, so that the hair can grow healthily and vigorously ( Figure 9 ).

[0146] Effect Example 11

[0147] 5α-reductase was extracted from rat prostate. A centrifuge tube was filled with 0.25 mL of phosphate buffer, 0.70 mL of enzyme extract, 0.1 mL of testosterone solution (0.5 mg / mL), 0.35 mL of NADPH solution (1 mg / mL), and 0.6 mL of sample solution (0.25 mg / mL finasteride solution or FIN@Arg-CLAVs in Example 7). In the blank control group, the enzyme extract was replaced with phosphate buffer, while in the negative control group, the sample solution was sterile saline. The sample tube was reacted at 37°C for 30 minutes, and then dichloromethane was added to terminate the reaction. The sample tube was centrifuged at 5000 rpm for 10 minutes. The organic phase was evaporated to dryness, and the residue was dissolved in methanol. The residual testosterone content was determined by HPLC, and the inhibition rate of 5α-reductase was calculated (n = 6).

[0148] The results showed that the finasteride-loaded conjugated linoleic acid vesicles FIN@Arg-CLAVs prepared in Example 7 could effectively inhibit the activity of 5α-reductase, and the inhibition rate was significantly higher than that of the finasteride solution with the same concentration ( Figure 10 ).

[0149] Effect Example 12

[0150] Male C57BL / 6J mice (6-8 weeks) were randomly divided into 4 groups, with 4 mice in each group. After the mice were anesthetized, the hair on their backs was shaved and depilatory cream was used to remove the hair. After the mice recovered for one day, the experiments were performed on the mice. The control group was treated with normal saline only; the model group was first applied with testosterone solution, and then normal saline was applied after waiting for 30 minutes; each experimental group was first applied with testosterone solution, and then the drug solution was applied after waiting for 30 minutes. The drug solution was 0.25 mg / mL finasteride or FIN@Arg-CLAVs in Example 7. Testosterone was dissolved in ethanol-normal saline solution (1:1, V / V) at a concentration of 5 mg / mL. After the start of administration, photos of the depilatory area on the back of the mice were taken on days 1, 6, 13, 17, 22, 26, and 31. On day 31, the hair coverage of the depilatory area of ​​the mice was calculated.

[0151] The results showed that the finasteride-loaded conjugated linoleic acid vesicles FIN@Arg-CLAVs prepared in Example 7 had a good therapeutic effect in the male androgenic alopecia model, significantly promoting hair regeneration and the darkening speed of the mouse back skin. The hair coverage rate on the 31st day was significantly better than that of the finasteride solution with the same concentration ( Figure 11 ).

[0152] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A nanovesicle, wherein the raw materials for preparing the nanovesicle include conjugated linoleic acid, phospholipid, vitamin E polyethylene glycol succinate, and arginine, wherein the mass ratio of the conjugated linoleic acid, phospholipid, vitamin E polyethylene glycol succinate, and arginine is 2: 0.1-0.9: 0.1-0.9: 0.1-5; Conjugated linoleic acid vesicles were prepared using conjugated linoleic acid, phospholipids and vitamin E polyethylene glycol succinate, and arginine was modified on their surface to obtain nanovesicles.

2. The nanovesicle according to claim 1, characterized in that The phospholipid is one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, distearoylphosphatidylcholine, dioleoyl lecithin, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoyl lecithin, dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine.

3. The method for preparing the nanovesicles according to claim 1 or 2, comprising mixing conjugated linoleic acid, lecithin and vitamin E polyethylene glycol succinate and dissolving the mixture in an organic solvent to obtain a solution A; Add solution A to the arginine solution and mix.

4. Use of the nanovesicles according to claim 1 or 2 in preparing products for hair growth and / or preventing and treating hair loss.

5. The use according to claim 4, characterized in that The hair loss includes partial hair loss, total hair loss or systemic hair loss caused by androgenic alopecia, seborrheic alopecia, anagen effluvium, telogen effluvium, drug-induced alopecia, alopecia areata, tinea capitis or hypotrichosis. A product comprising the nanovesicles according to claim 1 or 2.

7. The product according to claim 6, characterized in that The product may also include one or more other drugs for treating hair loss.

8. The product according to claim 7, characterized in that The other drugs include minoxidil, finasteride, dutasteride, spironolactone tablets, cyproterone acetate and / or clobetasol propionate or pharmaceutically acceptable salts thereof.

9. The product according to claim 8, characterized in that The mass ratio of conjugated linoleic acid to other drugs in the nanovesicles is 1: 0.01-1.

5.

10. The product according to any one of claims 6 to 9, characterized in that: The products include medicines and daily chemical products.

11. The product according to claim 10, characterized in that The daily chemical products include hair growth liquid, hair tonic, hair conditioner, shampoo, hair cleansing foam, hair oil, hair dryer, hair dye, hair gel, hair moisturizer, hair mask, hair treatment cream, eyebrow growth agent, eyelash growth agent, and eyelash nutrition agent.

12. The product according to claim 11, characterized in that The product also includes auxiliary materials.

13. The product according to claim 12, characterized in that The dosage forms of the product include sprays, lotions, ointments, emulsions and gels.

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