Hybrid vesicle composition containing finasteride and nanogold as well as preparation and application of hybrid vesicle composition

Abstract

The invention discloses a hybrid vesicle composition containing finasteride and nanogold. The composition is prepared from hair follicle stem cell outer vesicles, lipidosome, finasteride and nanogold, according to the method, the treatment efficiency of finasteride on androgenetic alopecia is enhanced by utilizing the delivery property of a mediated hair recovery mechanism pathway and a homing effect of heterozygous hair follicle stem cell outer vesicles to a hair follicle microenvironment and assisting with a photothermal effect provided by nanogold light conversion, and the method can be widely applied to the fields of medical cosmetology and pharmacy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a composition and preparation method of finasteride, nanogold, hair follicle stem cell extracellular vesicles, and liposomes, and in particular to a hybrid vesicle composition containing finasteride and nanogold, and its preparation and application. Background Art

[0002] Androgenic alopecia is a non-fatal disease, but it can cause great psychological and mental stress to patients, affecting about 80% of men and 50% of women worldwide. Currently, common drugs used in clinical practice to treat androgenic alopecia, such as oral 1 mg of finasteride (approved by the U.S. Food and Drug Administration for androgenic alopecia in 1997 and launched in China in 2004), inhibit the activity of type II 5α reductase that catalyzes the conversion of testosterone to dihydrotestosterone to inhibit the shrinkage of scalp hair follicles, thereby reversing androgenic alopecia. However, this inhibition of peripheral hormone reductase is likely to be accompanied by some sexual side effects, including but not limited to decreased libido, erectile dysfunction, and decreased ejaculation volume.

[0003] Photobiomodulation therapy was first demonstrated to promote hair regeneration in the late 1960s. Current studies have shown that it can promote the regulation of adenosine triphosphate (ATP), vascular endothelial growth factor (VEGF), reactive oxygen species (ROS), and nitric oxide synthase (NOS), as well as increase the induction of intracellular transcription factors, which are reflected in mitochondrial metabolism, protein synthesis, cell migration, and proliferation. In 2007, the U.S. Food and Drug Administration approved it as an expensive commercial medical device. Most studies on the effects of photobiomodulation therapy on hair growth provide a consensus irradiation wavelength ranging from 630 nanometers to near-infrared wavelengths (about 810 nanometers). Similar treatment wavelengths are similar to the emission wavelengths converted by synthetic nanogold through visible light excitation. In addition, the thermal effect after excitation can also promote vascular dilation and enhance the efficacy of photobiomodulation therapy.

[0004] The unique structural morphology of hair follicles is necessary to maintain the hair follicle niche throughout the hair cycle. This delicately coordinated cycle requires the participation of comprehensive biological pathways and hair follicle stem and progenitor cell populations. Cell signals carried by hair follicle stem cell extracellular vesicles promote communication between hair follicle cells. Hair follicle stem cell extracellular vesicles are reported to play a role in hair regeneration, and the proteins or miRNAs in them comprehensively mediate the restoration-related mechanism pathways, such as Wnt / β-catenin, Shh and BMP pathways. However, studies have shown that direct topical application of extracellular vesicles through solution formulations will encounter obstacles from the stratum corneum, affecting their accumulation and penetration in the skin. On the one hand, by artificially inserting drug molecules into extracellular vesicles with homing effects, their highly biocompatible delivery function combined with the therapeutic effects of signal molecules in the vesicles for combined therapy has been confirmed. On the other hand, the biodegradability and biocompatibility of extracellular vesicles hinder their stability in local application in the environment.

[0005] The present invention will develop a hybrid hair follicle stem cell extracellular vesicle constructed by fusion liposomes, and use finasteride and nano-gold together to improve the loading efficiency of hair follicle stem cell extracellular vesicles for finasteride and nano-gold, the stability of refrigerated storage, and the accumulation of subcutaneous hair follicles. The purpose is to use the hair follicle delivery of finasteride, the photothermal effect of nano-gold, and the signal molecules of hair follicle stem cell extracellular vesicles to improve the efficiency of hair regeneration. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a hybrid vesicle composition containing finasteride and nano-gold as well as its preparation and application.

[0007] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0008] A hybrid vesicle composition containing finasteride and nano-gold, which consists of hair follicle stem cell extracellular vesicles, liposomes, finasteride and nano-gold.

[0009] Preferably, the mass percentages of the components are: 5-40% (calculated based on membrane protein content) of the hair follicle stem cell extracellular vesicles; 20%-80% of the liposomes; 0.5%-4% of the finasteride; and 8%-40% of the nanogold.

[0010] Preferably, the mass percentages of the components are: 6.93%-34.63% (calculated based on membrane protein content) of the hair follicle stem cell extracellular vesicles; 34.63%-74.57% of the liposomes; 0.69%-3.45% of the finasteride; and 9.73%-38.23% of the nanogold.

[0011] The present invention also provides a method for preparing the above-mentioned hybrid vesicle composition containing finasteride and nano-gold, which is characterized by being achieved specifically by the following route:

[0012] (1) Extracellular vesicles enrichment of hair follicle stem cells

[0013] Hair follicle stem cells were cultured in DMEM high-glucose medium containing 10% extracellular vesicle-free fetal bovine serum until about 90% was achieved. The culture fluid was harvested and resuspended in 200 μL PBS after gradient centrifugation and membrane filtration. The surface protein concentration was detected by the BCA method as a quantitative reference.

[0014] (2) Preparation of gold nanoparticles

[0015] Dissolve perchloric acid in 10 mL of water (Au 3+ Concentration: 0.197-0.394 mg mL -1 ), heat to boiling and dissolve 10-20 mg mL -1 Anhydrous trisodium citrate was added and the mixture was refluxed and boiled for more than 20 minutes to obtain reduced nano-gold.

[0016] (3) Preparation of hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and gold nanoparticles

[0017] 67.5 mg of lecithin, 22.5 mg of cholesterol and 0.5-5 mg of finasteride were dissolved in about 20 mL of chloroform and dried for about 30 minutes by thin film drying. After the dried lipid film was hydrated with 10 mL of distilled water, the liposomes containing finasteride were peeled and dispersed by ultrasound for about 40 minutes. 1 mL was added to about 10 mL of dispersion solution containing 1-10 mg of hair follicle stem cell extracellular vesicles, and then the nano-gold dispersion system with the sum of the volumes of the two solutions (about 20 mL) was added. After about 10 minutes of ultrasonic treatment with a probe, a hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and nano-gold was obtained.

[0018] The use of a hybrid vesicle composition containing finasteride and nanogold as described in any one of claims 1-3 is characterized in that the hair regeneration effect on mice with induced androgenic alopecia is better than that of single finasteride, nanogold and hybrid hair follicle stem cell extracellular vesicles.

[0019] The present invention has the following advantages:

[0020] By fusion liposomes to construct hybrid hair follicle stem cell extracellular vesicles, and combining finasteride and gold nanoparticles for application, the loading efficiency of hair follicle stem cell extracellular vesicles for finasteride and gold nanoparticles, the stability during refrigerated storage, and the accumulation of subcutaneous hair follicles are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1It is a representative picture of the continuous treatment of mice with androgenic alopecia by the hybrid hair follicle stem cell extracellular vesicle gel containing finasteride, the single gel control group and the positive control group in Example 1;

[0022] Figure 2 The hair regeneration area ratio of the androgenic alopecia mice treated continuously with the hybrid hair follicle stem cell extracellular vesicle gel containing finasteride in Example 1, the single gel control group and the positive control group. DETAILED DESCRIPTION

[0023] In order to better understand the purpose and function of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the accompanying drawings.

[0024] Example 1 Preparation of hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and nano-gold

[0025] 1. Extracellular vesicle enrichment of hair follicle stem cells

[0026] Hair follicle stem cells were cultured in DMEM high-glucose medium containing 10% extracellular vesicle-free fetal bovine serum until about 90% was achieved. The culture fluid was harvested and resuspended in 200 μL PBS after gradient centrifugation and membrane filtration. The surface protein concentration was detected by the BCA method as a quantitative reference.

[0027] 2. Preparation of Gold Nanoparticles

[0028] Dissolve perchloric acid in 10 mL of water (Au 3+ Concentration: 0.197-0.394 mg mL -1 ), heat to boiling and dissolve 10-20 mg mL -1 Anhydrous trisodium citrate was added and the mixture was refluxed and boiled for more than 20 minutes to obtain reduced nano-gold.

[0029] 3. Preparation of hybrid hair follicle stem cell extracellular vesicles containing finasteride and gold nanoparticles

[0030] 67.5 mg of lecithin, 22.5 mg of cholesterol and 0.5-5 mg of finasteride were dissolved in about 20 mL of chloroform and dried for about 30 minutes by thin film drying. After the dried lipid film was hydrated with 10 mL of distilled water, the liposomes containing finasteride were peeled and dispersed by ultrasound for about 40 minutes. 1 mL was added to about 10 mL of dispersion solution containing 1-10 mg of hair follicle stem cell extracellular vesicles, and then the nano-gold dispersion system with the sum of the volumes of the two solutions (about 20 mL) was added. After about 10 minutes of ultrasonic treatment with a probe, a hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and nano-gold was obtained.

[0031] Example 2 Application of hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and nano-gold

[0032] In vivo study of the anti-androgenic alopecia effect of a hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and nanogold. Using a single blank hybrid hair follicle stem cell extracellular vesicle and finasteride as the control group, and using male C57BL / 6 mice raised to 7 weeks of age as an in vivo model, the composition was semi-quantitatively measured to evaluate the hair regeneration area of ​​mice with androgenic alopecia, and its ability to treat androgenic alopecia. Under comprehensive consideration, the corresponding prescription composition was prepared according to Example 1, and it was mixed with commercial carbomer gel at a volume ratio of 1:1. Male C57BL / 6 mice raised to 7 weeks of age were shaved on the designated back area with an animal razor, and then the designated area was further depilated with an animal depilatory cream until the pink back skin was exposed. 0.5g of testosterone powder was mixed with 100mL of 50% ethanol aqueous solution, and stirred at 37°C in the dark until completely dissolved to obtain a 0.5% testosterone / ethanol solution. 0.1mL·cm was applied to the pink back area of ​​the mouse every day. -2 A 0.5% testosterone / ethanol solution was applied topically to induce androgenic alopecia. After it was completely absorbed, 0.1 mL cm was applied to the pink back area of ​​the mice. -2 The blank gel, the combination gel, the control gel or 5% minoxidil (positive control) were topically applied daily at a dose of 100 mg / kg, and the number of mice in each group was 6. The mice were housed in a specific pathogen-free environment and maintained with a regular light / dark cycle and a constant temperature (25±1°C). The hair regeneration on the back of the mice was observed daily and recorded every few days, and the area covered by hair regeneration was semi-quantitatively evaluated using ImageJ software.

[0033] The results are as follows Figure 1 Figure 2 As shown, the hair regeneration area ratio of the composition gel in Example 1 on mice with androgenic alopecia was significantly increased compared with the control group, that is, it showed better ability to promote the recovery of androgenic alopecia. Considering factors such as cost, combination ratio, drug delivery amount, etc., Example 1 is a better prescription for the composition.

[0034] The beneficial effects of the present invention are:

[0035] The efficiency of hair regeneration is improved by combining the hair follicle delivery of finasteride, the photothermal effect of gold nanoparticles, and the extracellular vesicle signaling molecules of hair follicle stem cells.

[0036] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A hybrid vesicle composition containing finasteride and nano-gold, characterized in that: The composition consists of hair follicle stem cell extracellular vesicles, liposomes, finasteride and nanogold.

2. A hybrid vesicle composition containing finasteride and nano-gold according to claim 1, characterized in that: The mass percentages of the components are: 5-40% (calculated by membrane protein content) of the hair follicle stem cell extracellular vesicles; 20%-80% of the liposomes; 0.5%-4% of the finasteride; and 8%-40% of the nano-gold.

3. A hybrid vesicle composition containing finasteride and nano-gold according to claim 2, characterized in that: The mass percentages of the components are: 6.93%-34.63% (calculated by membrane protein content) of the hair follicle stem cell extracellular vesicles; 34.63%-74.57% of the liposomes; 0.69%-3.45% of the finasteride; and 9.73%-38.23% of the nano-gold.

4. A method for preparing a hybrid vesicle composition containing finasteride and nano-gold according to any one of claims 1 to 3, characterized in that: This is achieved through the following means: (1) Extracellular vesicles enrichment of hair follicle stem cells Hair follicle stem cells were cultured in DMEM high-glucose medium containing 10% extracellular vesicle-free fetal bovine serum until about 90% was achieved. The culture fluid was harvested and resuspended in 200 μL PBS after gradient centrifugation and membrane filtration. The surface protein concentration was detected by the BCA method as a quantitative reference. (2) Preparation of gold nanoparticles Dissolve perchloric acid in 10 mL of water (Au 3+ Concentration: 0.197-0.394 mg mL -1 ), heat to boiling and dissolve 10-20 mg mL -1 Anhydrous trisodium citrate was added and the mixture was refluxed and boiled for more than 20 minutes to obtain reduced nano-gold. (3) Preparation of hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and gold nanoparticles 67.5 mg of lecithin, 22.5 mg of cholesterol and 0.5-5 mg of finasteride were dissolved in about 20 mL of chloroform and dried for about 30 minutes by thin film drying. After the dried lipid film was hydrated with 10 mL of distilled water, the liposomes containing finasteride were peeled and dispersed by ultrasound for about 40 minutes. 1 mL was added to about 10 mL of dispersion solution containing 1-10 mg of hair follicle stem cell extracellular vesicles, and then the nano-gold dispersion system with the sum of the volumes of the two solutions (about 20 mL) was added. After about 10 minutes of ultrasonic treatment with a probe, a hybrid hair follicle stem cell extracellular vesicle composition co-loaded with finasteride and nano-gold was obtained.

5. The use of a hybrid vesicle composition containing finasteride and nano-gold according to any one of claims 1 to 3, characterized in that: The hair regeneration effect on mice with androgenic alopecia induced by finasteride is better than that of single finasteride, nanogold and hybrid hair follicle stem cell extracellular vesicles.

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