Application of combination of nano-liposome and low-temperature plasma in preparation of anti-hair loss and / or hair growth product

Abstract

The invention discloses application of combination of nano-liposome and low-temperature plasma in preparation of hair loss prevention and / or hair growth products, and belongs to the technical field of hair loss prevention and hair growth. The nano-liposome disclosed by the invention is a nano-liposome containing anti-hair-loss hair-growing components including pyrrolidinyl diaminopyrimidine oxide (kopryl), biotin tripeptide-1 and myristoyl pentapeptide-4, and the nano-liposome is combined with low-temperature plasma for use, so that the anti-hair-loss hair-growing composition is prepared. The composition can further break through a skin cuticle barrier, promote active components to penetrate through the skin cuticle and quickly enter active epidermis, and can enhance the anti-hair loss and hair growth effects of kopyltrol, biotin tripeptide-1 and myristoyl pentapeptide-4. The anti-alopecia and / or hair-growing product developed by combining the nano-liposome and the low-temperature plasma has a wide application prospect in the aspects of treating androgenetic alopecia and promoting the growth of newborn hair.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti - hair loss and hair growth promotion, and particularly relates to the application of the combination of nano - liposomes and low - temperature plasma in the preparation of anti - hair loss and / or hair growth promotion products. Background Art

[0002] Androgenetic alopecia (AGA) is the most common type of hair loss globally, which seriously affects people's quality of life and mental health. The pathogenesis of AGA is mainly mediated by androgens. Testosterone, an androgen in the body, is converted into dihydrotestosterone (DHT) by type II 5α - reductase. Under the action of DHT, hair follicles gradually atrophy and finally necrosis, resulting in hair loss. Currently, minoxidil is the main drug approved by the US Food and Drug Administration (FDA) for the treatment of AGA. Minoxidil can shorten the telogen phase of the hair growth cycle and prolong the anagen phase. However, the obvious side effects of minoxidil limit its use, mainly including contact dermatitis, headache, and hirsutism, and it is not suitable for pregnant women and patients with cardiovascular diseases. In addition, it requires long - term and frequent use to take effect, otherwise hair loss will start again.

[0003] Pyrrolidinediaminopyrimidine oxide (Kopyrrol) has a similar structure to minoxidil. This active ingredient can not only promote the proliferation of dermal papilla cells and accelerate the hair regeneration process, but also has lower toxicity compared with minoxidil, but its mechanism of action still needs further study. In addition, one of the other reasons for the poor treatment effect of AGA is that it is difficult for traditional single - drug treatment to achieve multi - target synergistic effects. The classic signal pathway regulating hair growth is the Wnt / β - catenin pathway. The β - catenin protein is mainly regulated by Wnt signals and is a positive regulator for hair follicle regeneration and maintenance of the anagen phase. In addition, human dermal papilla cells can change androgen drive by autocrine or paracrine activation or inhibition factors, affecting the growth of hair follicles or epithelial follicular cells. Currently, short - chain peptides composed of amino acids, especially biotin tripeptide - 1 and myristoyl pentapeptide - 4, have been introduced into the market as cosmetic active ingredients for promoting hair health and skin regeneration. Biotinyl tripeptide - 1 strengthens the hair structure by promoting the proliferation of follicular keratinocytes and enhancing the synthesis of adhesion molecules laminin 5 and collagen IV. Myristoyl pentapeptide - 4, as a signal peptide, promotes the growth of new hair by up - regulating the expression of keratin genes and increasing the synthesis of keratin. Therefore, combining kopyrrol with the two active peptides can achieve multi - target synergistic effects and is expected to improve the treatment effect.

[0004] In addition, the active ingredient also needs to enter the deep hair follicles of the skin to take effect. However, due to the barrier effect of the stratum corneum, it is difficult for the active ingredient to enter the action site and reach the deep hair follicles of the skin. Now, researchers have developed many effective strategies and technologies to overcome the stratum corneum barrier, including chemical enhancers, iontophoresis, electroporation, sonophoresis, nanocarriers, and microneedles. Cold atmospheric plasma (CAP) is the fourth state of matter, and the unique properties of plasma and its interactions with other states of matter provide broad prospects for research and application. CAP operating at atmospheric pressure and room temperature can avoid causing electrical or thermal damage to cells, which has attracted widespread attention in the fields of dermatological treatment, antimicrobial applications, and transdermal drug delivery systems. CAP can be used as a skin pretreatment method. The active ingredients it generates promote the formation of oxidized fatty acid derivatives in the skin, increase the hydrophilicity of fatty acids in the lipid matrix, thereby increasing skin permeability. There are also reports suggesting that the high electric field effect generated by CAP can disrupt the tight junctions between keratinocytes temporarily, change the skin barrier properties, and thus enhance the transdermal permeability and local delivery efficiency of drugs.

[0005] In addition, after the active ingredient penetrates into the hair follicles, it also needs to break through the cell membrane barrier and maintain an effective dose intracellularly for a long time to fully exert its effect. To further improve the transdermal delivery efficiency of the active ingredient and accumulate it in the hair follicle area of the dermis to reach an effective therapeutic dose, nanoliposomes are an ideal means. Nanoliposomes have a phospholipid bilayer membrane structure and can encapsulate hydrophilic, lipophilic, or amphiphilic active substances. They can bind to the skin cell membrane, thereby enhancing the transdermal absorption and endocytosis efficiency of the active substances. Nanoliposomes can promote the uptake and intracellular accumulation of the loaded active substances by target cells, achieve the slow release and long-term effect of drugs, and help improve the stability and bioavailability of drugs. Summary of the Invention

[0006] The present invention discovers that the combination of nanoliposomes containing kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4 with cold atmospheric plasma (CAP) has a good therapeutic effect on androgenetic alopecia. Based on this, the purpose of the present invention is to provide the application of the combination of anti-hair loss and hair growth nanoliposomes and cold atmospheric plasma in the preparation of anti-hair loss and / or hair growth products.

[0007] On the one hand, the present invention provides the application of the combination of anti-hair loss and hair growth nanoliposomes and cold atmospheric plasma in the preparation of anti-hair loss and / or hair growth products. The anti-hair loss and hair growth nanoliposomes described are nanoliposomes containing anti-hair loss and hair growth ingredients.

[0008] In a preferred embodiment, the anti-hair loss and hair growth nanoliposomes are nanoliposomes containing kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4.

[0009] In a preferred embodiment, the raw materials of the anti - hair - loss and hair - growth nanoliposomes include: kopyrrol, biotin tripeptide - 1, myristoyl pentapeptide - 4, lecithin, emulsifier, polyol, stabilizer, absolute ethanol, and water.

[0010] The lecithin preferably includes any one or a combination of more than one of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, etc.

[0011] The emulsifier preferably includes any one or a combination of more than one of polyoxyethylene castor oil - type emulsifiers, polyoxyethylene hydrogenated castor oil - type emulsifiers, polyglycerol - type emulsifiers, poloxamer, cocoyl glucoside, triglycerides, pyrrolidones, polyglycerate, polysorbates, etc. The polyoxyethylene hydrogenated castor oil - type emulsifiers include PEG - 20 hydrogenated castor oil, PEG - 40 hydrogenated castor oil, PEG - 60 hydrogenated castor oil, etc.; the polysorbates include polysorbate - 20, polysorbate - 60, polysorbate - 80, etc.

[0012] The polyol preferably includes any one or a combination of more than one of glycerol, propylene glycol, butylene glycol, 1,3 - propanediol, 1,2 - pentanediol, ethoxydiglycol, 1,2 - hexanediol, dipropylene glycol, isopropanol, polyethylene glycol - 200, PPG - 10 sorbitol, octyldodecanol, etc.

[0013] The stabilizer preferably includes any one or a combination of more than one of sodium cholate, Tween 80, Tween 20, Tween 60, cholesterol.

[0014] In a preferred embodiment, the anti - hair - loss and hair - growth nanoliposomes contain the following raw materials by mass percentage: 0.5% - 5% of kopyrrol, 0.05 - 0.5% of biotin tripeptide - 1, 0.05 - 0.5% of myristoyl pentapeptide - 4, 3% - 9% of lecithin, 3% - 9% of emulsifier, 50% - 70% of polyol, 0.1% - 0.4% of stabilizer, 1% - 10% of absolute ethanol, and the balance of water.

[0015] In a preferred embodiment, the particle size of the anti - hair - loss and hair - growth nanoliposomes is 40 - 500 nm, more preferably 40 - 300 nm, and even more preferably 100 - 200 nm.

[0016] In a preferred embodiment, the anti - hair - loss and hair - growth nanoliposomes are prepared by a method including the following steps:

[0017] (1) Dissolve kopyrrol in polyol to obtain a first mixed solution. The polyol is preferably propylene glycol.

[0018] (2) Dissolve lecithin and stabilizer in absolute ethanol to obtain a second mixed solution. The lecithin is preferably soy lecithin, and the stabilizer is preferably cholesterol.

[0019] (2) Dissolve biotin tripeptide-1, myristoyl pentapeptide-4, emulsifier and polyol in water to obtain a third mixed solution. The emulsifier is preferably PEG-40 hydrogenated castor oil, and the polyol is preferably glycerol.

[0020] (3) Mix the first, second and third mixed solutions and stir to obtain a liposome emulsion.

[0021] (4) Subject the liposome emulsion to nanosizing treatment to obtain the anti-hair loss and hair growth-promoting nano-liposomes.

[0022] Preferably, in step (3), the stirring conditions are stirring at 45-70 °C for 30-60 minutes.

[0023] Preferably, in step (4), the nanosizing treatment methods include high-pressure homogenization, high-speed microfluidization or high-speed shearing, etc. The high-pressure homogenization method is preferably adopted, and the conditions are preferably: pressure 600-1200 MPa, number of cycles 3-4 times.

[0024] The sources of the low-temperature plasma include dielectric barrier discharge (DBD), dielectric-barrier-discharge-like, non-dielectric-barrier discharge, etc. Further preferably, the low-temperature plasma is a DBD plasma jet, and the conditions are preferably: high-frequency pulse signal voltage 3-6 kV, frequency 15-25 kHz.

[0025] On the other hand, the present invention provides an anti-hair loss and / or hair growth-promoting product, which contains the above anti-hair loss and hair growth-promoting nano-liposomes and a low-temperature plasma source.

[0026] The present invention has the following beneficial effects: The anti-hair loss and hair growth-promoting nano-liposomes of the present invention have a uniform particle size distribution, no cytotoxicity, and good stability, and enhance the skin penetration effect of kopyrrol, biotin tripeptide-1 and myristoyl pentapeptide-4. The combination of low-temperature plasma and anti-hair loss and hair growth-promoting nano-liposomes can further break through the skin stratum corneum barrier, promote the penetration of active ingredients through the skin stratum corneum, quickly enter the viable epidermis, and enhance the anti-hair loss and hair growth-promoting effects of kopyrrol, biotin tripeptide-1 and myristoyl pentapeptide-4. The anti-hair loss and / or hair growth-promoting product developed by combining the anti-hair loss and hair growth-promoting nano-liposomes with low-temperature plasma of the present invention has broad application prospects in the treatment of androgenetic alopecia and promoting the growth of new hair. Description of the Drawings

[0027] Figure 1It is the particle size distribution diagram of KBM-NLPs.

[0028] Figure 2 It is the transmission electron microscopy image of KBM-NLPs.

[0029] Figure 3 It is the in vitro release curve of kopyrrol in KBM-NLPs.

[0030] Figure 4 It is the in vitro release curve of biotin tripeptide-1 in KBM-NLPs.

[0031] Figure 5 It is the in vitro release curve of myristoyl pentapeptide-4 in KBM-NLPs.

[0032] Figure 6 It is the result of the percutaneous penetration experiment of Rho-NLPs.

[0033] Figure 7 It is the skin state of mice after CAP treatment. Detailed implementation mode

[0034] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. In the following examples, quantitative tests are all set with at least three repeated experiments, and the results are averaged.

[0035] Example 1: Preparation and formulation study of co-delivery nanoliposomes (KBM-NLPs) containing kopyrrol, biotin tripeptide-1 and myristoyl pentapeptide-4

[0036] Investigate the effects of the content of soybean lecithin, PEG-40 hydrogenated castor oil (CO40), and glycerol on the particle size and PDI of nanoliposomes. Use Box-Behnken design-response surface to optimize the formulation of KBM-NLPs. The response surface method (RSM) is to conduct experiments, model building, and data analysis, investigate various factors interfering with the entire system, and use a mathematical model to fit and predict the optimal solution according to the investigation results.

[0037] The factors and levels established by the response surface design are as follows: A: Lecithin (3% - 9%), B: CO40 (3% - 9%), and C: Glycerol (5% - 15%). See Table 1. Using the particle size and PDI of KBM-NLPs as response values, based on the Box-Benhnken design with Design-Expert.V8.0.6.1.

[0038] Table 1 Three-factor and three-level table of response surface design

[0039]

[0040]

[0041] The prescriptions of KBM-NLPs with different ratios were designed by Box-Benhnken, and KBM-NLPs were prepared according to the following method. The measured particle size and PDI are shown in Table 2. 1% kopyrrol was dissolved in 50% propylene glycol to obtain phase A; lecithin and 0.2% cholesterol were dissolved in 5% absolute ethanol to obtain phase B; CO40, glycerol, 0.1% biotin tripeptide-1, and 0.1% myristoyl pentapeptide-4 were dissolved in water to obtain phase C; phases A, B, and C were heated and stirred at 65 °C respectively. After dissolution, the three phases were mixed and stirring was continued to obtain the primary emulsion, which was subjected to high-pressure homogenization at a homogenization pressure of 800 Mpa and 3 homogenization times to obtain KBM-NLPs. The sum of the mass percentage contents of each material in this preparation method is 100%.

[0042] Table 2 Particle size and PDI of different prescriptions in Box-Benhnken response surface experimental design

[0043]

[0044]

[0045] The optimized result of the prescription of KBM-NLPs obtained by response prediction using RSM is: A (phospholipid) is 3.89%, B (CO40) is 5.37%, and C (glycerol) is 15%.

[0046] Example 2:

[0047] Liposomes were prepared by the high-pressure homogenization method according to the method in Example 1: 1% kopyrrol was dissolved in 50% propylene glycol to obtain phase A; 3.89% soybean lecithin and 0.2% cholesterol were dissolved in 5% absolute ethanol to obtain phase B; 5.37% CO40, 15% glycerol, 0.1% biotin tripeptide-1, and 0.1% myristoyl pentapeptide-4 were dissolved in water to obtain phase C; phases A, B, and C were heated and stirred at 65 °C respectively. After dissolution, the three phases were mixed and stirring was continued to obtain the primary emulsion, which was subjected to high-pressure homogenization at a homogenization pressure of 800 Mpa and 3 homogenization times to obtain KBM-NLPs. The average particle size and PDI of the KBM-NLPs prepared according to this prescription were 45.4 nm and 0.209 respectively.

[0048] Example 3:

[0049] Prepare liposomes by high-pressure homogenization according to the method in Example 1: Dissolve 1% kopyrrol in 50% propylene glycol to obtain Phase A; dissolve 6% soybean lecithin and 0.2% cholesterol in 5% absolute ethanol to obtain Phase B; dissolve 9% CO40, 15% glycerol, 0.1% biotin tripeptide-1 and 0.1% myristoyl pentapeptide-4 in water to obtain Phase C; Heat and stir Phases A, B, and C at 65 °C respectively. After dissolution, mix the three phases and continue stirring to obtain the primary emulsion. Perform high-pressure homogenization at a homogenization pressure of 800 Mpa and 3 homogenization times to obtain KBM-NLPs. The average particle size and PDI of the KBM-NLPs prepared according to this formulation are 154.6 nm and 0.104 respectively (Group 3 in Table 2 above).

[0050] Example 4:

[0051] Prepare liposomes by high-pressure homogenization according to the method in Example 1: Dissolve 1% kopyrrol in 50% propylene glycol to obtain Phase A; dissolve 9% soybean lecithin and 0.2% cholesterol in 5% absolute ethanol to obtain Phase B; dissolve 3% CO40, 10% glycerol, 0.1% biotin tripeptide-1 and 0.1% myristoyl pentapeptide-4 in water to obtain Phase C; Heat and stir Phases A, B, and C at 65 °C respectively. After dissolution, mix the three phases and continue stirring to obtain the primary emulsion. Perform high-pressure homogenization at a homogenization pressure of 800 Mpa and 3 homogenization times to obtain KBM-NLPs. The average particle size and PDI of the KBM-NLPs prepared according to this formulation are 237.9 nm and 0.464 respectively (Group 8 in Table 2 above).

[0052] Example 5: Physicochemical property characterization of KBM-NLPs

[0053] (1) Microscopic morphology observation

[0054] Dilute the newly prepared KBM-NLPs by several times, drop them on the surface of the copper mesh, adsorb for 10 min and then air dry. Stain with 1% phosphotungstic acid for 4 min, suck off the surface staining solution, and air dry naturally. Observe the sample with a transmission electron microscope (TEM), and select a suitable area to observe the morphology of KBM-NLPs. The particle size distribution diagram of the KBM-NLPs prepared in Example 2 is as Figure 1 shown. The TEM observation is as Figure 2 shown: The appearance of KBM-NLPs is spherical or ellipsoidal, and the particle size of KBM-NLPs shown by TEM is basically consistent with the particle size result of KBM-NLPs measured by a particle size analyzer (DLS).

[0055] (2) Encapsulation efficiency and drug loading

[0056] The encapsulation efficiencies of kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4 in the KBM-NLPs prepared in Example 2 were measured by ultrafiltration centrifugation and were 94.3 ± 1.31%, 91.9 ± 0.56%, and 74.9 ± 5.27%, respectively. The drug loadings of kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4 in the KBM-NLPs were 5.09 ± 0.15%, 1.96 ± 0.01%, and 1.02 ± 0.53%, respectively. This demonstrated that the active ingredients were successfully encapsulated in the nanoliposomes.

[0057] (3) Stability evaluation of KBM-NLPs

[0058] The newly prepared KBM-NLPs were stored at 4°C. At regular intervals within 3 months, samples of KBM-NLPs were taken, and their particle size, PDI, and Zeta potential were measured and recorded. The recorded particle size, PDI, and Zeta potential of the KBM-NLPs prepared in Example 2 after storage at 4°C for 90 days are shown in Table 3: At 30 days, there were almost no changes in the average particle size, PDI, and Zeta potential of the KBM-NLPs. At 90 days, there were slight changes in the particle size, PDI, and Zeta potential of the KBM-NLPs, and their physicochemical properties were relatively stable, indicating good storage stability of the KBM-NLPs under low-temperature conditions.

[0059] Table 3 Changes in particle size, PDI, and Zeta potential of KBM-NLPs within 90 days

[0060]

[0061] Example 6: Investigation of in vitro release and transdermal performance of KBM-NLPs

[0062] (1) In vitro release

[0063] After boiling the dialysis bag with ultrapure water, it was treated with 50% ethanol solution for 24 h for standby. 1 mL of KBM-NLPs prepared in Example 2 was loaded into the dialysis bag, and both ends were sealed with a sealing clip. Then it was placed in a reagent bottle containing 80 mL of PBS solution (pH = 7.4) and oscillated in a constant temperature shaker at 37 °C and 80 rpm for 24 h. 1 mL of the sample was taken at different time points, and an equal volume of PBS solution was supplemented. At the same time, a free KBM solution (Free KBM) group was set up. First, kopyrrol was dissolved in propylene glycol, biotin tripeptide-1 and myristoyl pentapeptide-4 were dissolved in PBS, and then the kopyrrol solution and the active peptide solution were diluted with PBS to prepare a free KBM solution with the same concentrations of kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4 as in KBM-NLPs. 1 mL of the Free KBM solution was placed in the dialysis bag. After the release was completed, the concentrations of kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4 in all samples were detected by HPLC. The in vitro release curves of kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4 in the Free KBM group and the KBM-NLPs group are as Figure 3 and 4 shown in Figure 5. The cumulative release percentage of kopyrrol in the Free KBM group within the first 4 hours was 90.1 ± 1.15%. After 12 hours of release, the cumulative release rate of kopyrrol in the Free KBM group reached 89.9 ± 2.96%, and that in the KBM-NLPs group was 74.4 ± 4.13%. After 9 hours of release, the cumulative release rates of biotin tripeptide-1 and myristoyl pentapeptide-4 in the Free KBM group reached 73.06 ± 3.91% and 81.8 ± 5.4% respectively, and those in the KBM-NLPs group reached 59.38 ± 14.59% and 54.3 ± 1.3% respectively. These results indicate that nano-liposome encapsulation can slow down the release of kopyrrol, biotin tripeptide-1, and myristoyl pentapeptide-4.

[0064] (2) Ex vivo skin permeation performance

[0065] The Franz diffusion cell method was used to study the ex vivo skin permeation performance of KBM-NLPs. RhoB-NLPs used in this experiment: 5% RhoB was encapsulated in blank liposomes, and the blank liposome formulation was the same as that of KBM-NLPs in Example 2. The low-temperature plasma (CAP) used was a DBD plasma jet, with a high-frequency pulsed signal voltage of 3 - 6 kV and a frequency of 15 - 25 kHz.

[0066] The pigskin was sandwiched between the supply chamber and the receiving chamber, and 0.01g of Rhodamine B standard was dissolved in 10mL of deionized water and diluted 100 times for use. 0.5mL of free RhoB solution (Free RhoB) and RhoB-NLPs containing equal concentrations of RhoB were evenly applied to the skin surface as a control. After the pigskin skin was treated with low-temperature plasma (CAP) for 1min and 3min, RhoB-NLPs were applied to the skin surface. At a specific time point of 6h, the corresponding pigskin was removed, the treated skin was embedded, and cut into 10μm thick slices. CLSM was used to observe the penetration of RhoB in ex vivo pigskin, and the ability of CAP to promote the percutaneous penetration of liposomes was studied.

[0067] Depend on Figure 6 As shown in Table 4, at 6 hours, a small amount of RhoB can be seen in the Free RhoB group through the skin barrier into the skin tissue. In general, most of the RhoB in the Free RhoB group failed to pass through the keratin barrier, and the distribution in the deep skin tissue was small and the red fluorescence was weak, indicating that only a small amount of RhoB was transdermally absorbed into the skin tissue. Compared with the Rho-NLPs group, the transdermal penetration ability of RhoB in the Rho-NLPs+CAP group irradiated with CAP was significantly enhanced, which was much higher than that of the nanoliposomes alone. At 6 hours, the distribution area of ​​RhoB in the deep skin tissue was wider, the red fluorescence was stronger, and it was enriched in the hair follicles. The fluorescence intensity of the RhoB-NLPs+CAP 3min group was 3.06 times that of the RhoB-NLPs group, and the fluorescence intensity of the RhoB-NLPs+CAP 1min group was 1.57 times that of the RhoB-NLPs group. At the same time, the penetration depth of RhoB-NLPs+CAP 3min group into the skin was 2.0 times that of RhoB-NLPs group, and the penetration depth of RhoB-NLPs+CAP1min group into the skin was 1.2 times that of RhoB-NLPs group. Compared with the use of nanoliposomes alone, the penetration-enhancing effect of CAP combined with nanoliposomes was significantly enhanced.

[0068] Table 4 RhoB fluorescence intensity and fluorescence penetration depth

[0069]

[0070] Example 7: Construction of androgenic alopecia model and treatment with different administration methods

[0071] C57BL / 6 male mice were selected to establish a testosterone-induced androgenetic alopecia model. After one week of adaptive cultivation, one day before the experiment began, the back hair was shaved off with an electric shaver to avoid skin damage, and depilatory cream was used to further remove the hair and confirm that the back hair of C57BL / 6 male mice was in the telogen phase (the skin in the depilated area was pink). The C57BL / 6 male mice were randomly divided into 10 groups (12 mice in each group): (1) control group (untreated normal mice, Control); (2) model group (AGA model mice established by testosterone induction, Model); (3) minoxidil group (AGA mice treated with 3% minoxidil); (4) Free KBM group (AGA mice treated with Free KBM containing 1 mg / mL kopyrrol, 0.1 mg / mL biotin tripeptide-1 and 0.1 mg / mL myristoyl pentapeptide-4); (5) Blank-NLPs group (AGA mice treated with Blank-NLPs); (6) KBM-NLPs group (AGA mice treated with KBM-NLPs containing 1 mg / mL kopyrrol, 0.1 mg / mL biotin tripeptide-1 and 0.1 mg / mL myristoyl pentapeptide-4); (7) CAP 1min group (AGA mice treated with CAP on the back skin for 1 min); (8) CAP 3min group (AGA mice treated with CAP on the back skin for 3 min); (9) KBM-NLPs+CAP 1min group (AGA mice first treated with CAP on the back skin for 1 min and then treated with KBM-NLPs containing 1 mg / mL kopyrrol, 0.1 mg / mL biotin tripeptide-1 and 0.1 mg / mL myristoyl pentapeptide-4); (10) KBM-NLPs+CAP 3min group (AGA mice first treated with CAP on the back skin for 3 min and then treated with KBM-NLPs containing 1 mg / mL kopyrrol, 0.1 mg / mL biotin tripeptide-1 and 0.1 mg / mL myristoyl pentapeptide-4). In the above treatments, the cold atmospheric plasma (CAP) used was a DBD plasma jet with a high-frequency pulsed signal voltage of 3-6 kV and a frequency of 15-25 kHz; the KBM-NLPs used was a reagent containing 1 mg / mL kopyrrol, 0.1 mg / mL biotin tripeptide-1 and 0.1 mg / mL myristoyl pentapeptide-4 prepared by formulating the KBM-NLPs prepared in Example 2 with the compound solution shown in Table 5; the Free KBM and Blank-NLPs used were obtained by diluting the compound solution by the same multiple.

[0072] Apply 250 μL of 0.05% testosterone (dissolved in 75% ethanol) to the depilated area on the back of each C57BL / 6 male mouse every day. The normal group is applied with an equal volume of normal saline. After 30 min, apply 500 μL of the prepared Blank-NLPs, Free KBM, and KBM-NLPs evenly to the skin of the depilated area on the back of C57BL / 6 male mice. The Model group and the Control group are applied with an equal volume of the compound solution. In the CAP group, after treatment with CAP, then apply 500 μL of the prepared Blank-NLPs, Free KBM, and KBM-NLPs evenly to the skin of the depilated area on the back of C57BL / 6 male mice.

[0073] Table 5 Prescription Table of Compound Solution

[0074]

[0075] Observe and record the number of days when the skin of C57BL / 6 male mice in different treatment groups turns black and grows hair every day. When the experiment reaches the 14th day, the mice in the Control group have grown hair, and the back skin of the mice in the Model group is still pink, indicating that the hair of the mice is still in the resting phase, suggesting that the androgenetic alopecia model mice are successfully modeled. The skin of the mice in the Free KBM and KBM-NLPs groups has turned black or there is already some new hair, while the back skin of the mice in the Blank-NLPs group has not changed significantly, indicating that after the action of Free KBM and KBM-NLPs on the hair follicles in the depilated area, the hair follicles have begun to be induced into the growth phase. Compared with the Free KBM and KBM-NLPs groups, the skin of C57BL / 6 male mice in the group of combined use of KBM-NLPs and CAP turns black in a shorter time and has a better effect.

[0076] On the 14th day after hair removal, 6 mice in each group were sacrificed, and dorsal skin sections were taken for observation. The dermal thickness and the proportion of hair follicles in the growth phase were analyzed. The relevant results are shown in Table 6. Compared with the Model group, the hair follicle structures in the Free KBM, KBM-NLPs, and CAP groups were more complete. Compared with the groups treated with CAP and KBM-NLPs alone, the combined treatment of KBM-NLPs and CAP on C57BL / 6 male mice had a more complete hair follicle structure and a larger hair follicle diameter. At the same time, compared with the Model group, the dermal thickness increased by 69.8% in the KBM-NLPs group, by 42.1% in the CAP 3min group, and by 138.1% in the KBM+CAP 3min group, and the increase was greater than the sum of KBM-NLPs and CAP 3min. Compared with the Model group, the proportion of hair follicles increased by 428.7% in the KBM-NLPs group, by 296.5% in the CAP 3min group, and by 800.0% in the KBM+CAP 3min group, and the increase was greater than the sum of KBM-NLPs and CAP 3min. The above results indicate that the combined use of CAP and KBM-NLPs has a synergistic effect, can significantly increase the number of hair follicles and dermal thickness in C57BL / 6 male mice, and has a positive effect on maintaining the hair follicle morphology and increasing the hair follicle diameter.

[0077] After 21 days of drug administration, the remaining mice were sacrificed, and the newly grown hair in the dorsal hair removal area of each group was collected and weighed with an analytical balance to obtain the average value. As shown in Table 6, compared with the Model group, there was a highly significant difference (p<0.01) in the number of days required for the skin of C57BL / 6 mice in the combined KBM-NLPs and CAP group to grow hair, and under the action of CAP, the resting period of hair follicles could be shortened more rapidly than in the KBM-NLPs group, inducing the hair growth cycle to turn from the resting phase to the growth phase. As shown in Table 6, after the experiment, the weight of the newly grown hair in the KBM-NLPs+CAP 3min group was 2.2 times and 1.6 times that of the CAP 3min group and the KBM-NLPs group, respectively, showing a significant difference. Compared with the Model group, the hair weight increased by 625.9% in the KBM-NLPs group, by 448.1% in the CAP 3min group, and by 1085.2% in the KBM+CAP 3min group, and the increase was greater than the sum of KBM-NLPs and CAP 3min. The above results indicate that the combined use of CAP and KBM-NLPs has a synergistic effect, can significantly shorten the number of days required for the growth of newly grown hair, and increase the weight of newly grown hair in C57BL / 6 male mice.

[0078] The particle sizes of KBM-NLPs in the above experimental groups were all between 40-100 nm. At the same time, two groups of experiments were set up, namely: (11) KBM-NLPs+CAP 3 min group (the KBM-NLPs used were prepared in Example 3, and the particle size was between 100-200 nm); (12) KBM-NLPs+CAP 3 min group (the KBM-NLPs used were prepared in Example 4, and its particle size was above 200 nm). The therapeutic effects of nanoliposomes with different particle sizes combined with CAP are shown in Table 6. The results showed that the number of days of skin darkening in Group 11 was shorter than that in Group 10 and Group 12, the weight of newly generated hair in Group 11 was heavier than that in Group 10 and Group 12, the thickness of the dermis in Group 11 was thicker than that in Group 10 and Group 12, the proportion of hair follicles in the growth phase in Group 11 was higher than that in Group 10 and Group 12, and the weight of newly generated hair, the thickness of the dermis, and the proportion of hair follicles in the growth phase in Group 11 almost reached the level of normal mice in the control group. The above results indicate that nanoliposomes with a particle size below 200 nm, especially in the range of 100-200 nm, combined with low-temperature plasma have the best effect on hair follicles and achieve better therapeutic effects.

[0079] Table 6 Growth of newly generated hair in mice

[0080]

[0081]

[0082] Example 8: Skin injury and histopathological examination

[0083] Depilate the back skin of C57BL / 6 male mice. Use untreated normal mice as the control group. Treat the mice with CAP for 1 min and 3 min respectively, and observe the changes in the skin of the CAP-treated area. The changes in the back skin of the mice after CAP treatment are as Figure 7 , after CAP irradiation for 1 min and 3 min, no obvious injury occurred in the back skin within 90 min.

[0084] The above examples are only used to help illustrate the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of anti-hair loss and hair growth nanoliposomes and low-temperature plasma in the preparation of anti-hair loss and / or hair growth products, characterized in that: The anti-hair loss and hair growth nanoliposome is a nanoliposome containing anti-hair loss and hair growth ingredients.

2. The use according to claim 1, characterized in that: The anti-hair loss and hair growth nanoliposome is a nanoliposome containing pyrrolidino diaminopyrimidine oxide, biotin tripeptide-1 and myristoyl pentapeptide-4.

3. The use according to claim 1, characterized in that: The raw materials of the anti-hair loss and hair growth nanoliposomes include: pyrrolidino diaminopyrimidine oxide, biotin tripeptide-1, myristoyl pentapeptide-4, lecithin, emulsifier, polyol, stabilizer, anhydrous ethanol and water.

4. The use according to claim 3, characterized in that: The lecithin includes any one or more combinations of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, and hydrogenated egg yolk lecithin; and / or the emulsifier comprises any one or more combinations of polyoxyethylene castor oil emulsifiers, polyoxyethylene hydrogenated castor oil emulsifiers, polyglycerol emulsifiers, poloxamer, coconut glucoside, triglycerides, pyrrolidones, polyglyceryl esters, and polysorbates; And / or the polyol includes any one or more combinations of glycerol, propylene glycol, butylene glycol, 1,3-propanediol, 1,2-pentanediol, ethoxydiglycol, 1,2-hexanediol, dipropylene glycol, isopropanol, polyethylene glycol-200, PPG-10 sorbitol, and octyldodecanol; And / or the stabilizer includes any one or more combinations of sodium cholate, Tween 80, Tween 20, Tween 60, and cholesterol.

5. The use according to claim 3, characterized in that: The anti-hair loss and hair growth nanoliposome comprises the following raw materials in percentage by mass: 0.5%-5% of pyrrolidino diaminopyrimidine oxide, 0.05-0.5% of biotin tripeptide-1, 0.05-0.5% of myristoyl pentapeptide-4, 3%-9% of lecithin, 3%-9% of emulsifier, 50%-70% of polyol, 0.1%-0.4% of stabilizer, 1%-10% of anhydrous ethanol, and the balance of water.

6. The use according to claim 1, characterized in that: The particle size of the anti-hair loss and hair growth nanoliposome is 40-500nm.

7. The use according to claim 1, characterized in that: The anti-hair loss and hair growth nanoliposomes are prepared by a method comprising the following steps: (1) dissolving pyrrolidine diamino pyrimidine oxide in a polyol to obtain a first mixed solution; (2) dissolving lecithin and a stabilizer in anhydrous ethanol to obtain a second mixed solution; (2) dissolving biotin tripeptide-1, myristoyl pentapeptide-4, an emulsifier and a polyol in water to obtain a third mixed solution; (3) mixing the first, second and third mixed solutions, and stirring to obtain a liposome emulsion; (4) The liposome emulsion is subjected to nano-processing to obtain the anti-hair loss and hair growth nano-liposome.

8. The use according to claim 1, characterized in that: The sources of the low-temperature plasma include dielectric barrier discharge, quasi-dielectric barrier discharge, and non-dielectric barrier discharge.

9. A hair loss prevention and / or hair growth product, characterized in that: Contains anti-hair loss and hair growth nanoliposomes and low-temperature plasma source.

10. The hair loss prevention and / or hair growth product according to claim 9, characterized in that: The anti-hair loss and hair growth nanoliposome is the anti-hair loss and hair growth nanoliposome in the application according to any one of claims 1 to 7.

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