Small molecule peptide component for preventing hair loss and nourishing hair and preparation method of small molecule peptide component

Abstract

The invention relates to the technical field of biological medicines, in particular to an anti-alopecia hair-nourishing small molecule peptide component and a preparation method thereof. Various small molecule peptides such as hair follicle targeting activation peptides and hair structure repairing peptides are designed and synthesized from the molecular level. Efficient synthesis and stable delivery of the peptide are realized through innovative preparation processes such as combination of solid-phase synthesis and enzyme digestion, ultrasonic-assisted purification, low-temperature freeze-drying and microcapsule wrapping integration and the like. The small molecule peptide component can achieve a synergistic effect in multiple aspects of activating hair follicle stem cells, repairing a hair structure, improving microcirculation and the like, has remarkable anti-hair-loss and hair-nourishing effects, is safe and free of side effects, reduces the cost by 30%-50%, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a small molecule peptide component for preventing hair loss and nourishing hair and a preparation method thereof. Background Art

[0002] With the accelerated pace of life, increased environmental pressure and the influence of bad living habits, hair loss is becoming more and more common, seriously affecting people's appearance and mental health. At the same time, problems such as dry, rough and dull hair also trouble many consumers. There are many kinds of existing anti-hair loss products, but most of them have problems such as poor effect, single mechanism of action, and possible side effects, as shown below:

[0003] The anti-hair loss effect is not significant: Many anti-hair loss products on the market mainly achieve anti-hair loss by cleaning the scalp, improving scalp blood circulation, etc., but the deep nourishment and repair effect on hair follicles is limited. For example, some common anti-hair loss shampoos do not significantly reduce the amount of hair loss after using them for a period of time, and can only maintain a relatively high level, which cannot fundamentally solve the key problems that cause hair loss, such as hair follicle atrophy and reduced hair stem cell activity. Most hair care products focus on improving the external gloss and smoothness of hair, such as reducing the roughness by forming a protective film on the surface of hair. However, the effect is not satisfactory in terms of repairing the internal structure of hair, deep delivery of nutrients, and enhancing the elasticity and toughness of hair itself. After long-term use, the essential state of hair is not significantly improved, and it is still prone to problems such as breakage and split ends. Some anti-hair loss and hair care products are added with chemical ingredients, such as certain hormone substances or highly irritating chemical synthetic agents. Although there may be certain effects in the short term, long-term use may have a negative impact on scalp health, such as causing scalp allergies, itching, redness and swelling, and may even interfere with the human endocrine system and cause more serious health problems. Some products can temporarily improve the appearance and condition of hair when used, but the effect is not long-lasting. Once use is stopped, the hair quickly returns to its original poor state, requiring continuous use and the effects are difficult to accumulate, which brings consumers a double burden of economy and time. Although some high-end anti-hair loss products claim to have good effects, due to the use of expensive raw materials or complex production processes, the product prices remain high, which is beyond the affordability of most consumers and limits their widespread application.

[0004] In view of the above problems, the market is in urgent need of a safe, efficient, multifunctional and economical hair loss prevention and care product. Although some studies have been devoted to solving the problems of hair loss and hair care, a comprehensive and ideal solution has not yet been found. Therefore, the development of an innovative hair loss prevention and care small molecule peptide component and its preparation method has important practical significance and market value. Summary of the invention

[0005] The present invention provides a small molecule peptide component for preventing hair loss and nourishing hair, comprising at least one of the following small molecule peptides:

[0006] Hair follicle targeting activation peptide (FTA-Peptide), whose amino acid sequence is NH2-Gly-His-Lys-Arg-Tyr-Cys-COOH;

[0007] Hair structure repair peptide (HSR-Peptide), whose amino acid sequence is NH2-Ala-Pro-Cys-Ser-Val-Leu-COOH;

[0008] Scalp microcirculation promoting peptide (SMP-Peptide), whose amino acid sequence is NH2-Arg-Gly-Asp-Ile-Thr-Pro-COOH;

[0009] Scalp microbial balance regulating peptide (SMR-Peptide), whose amino acid sequence is NH2-Lys-Glu-Trp-Met-Phe-COOH;

[0010] Antioxidant stress protective peptide (ASP-Peptide), whose amino acid sequence is NH2-His-Gln-Asn-Cys-Val-COOH;

[0011] Cell signaling regulatory peptide (CSR-Peptide), whose amino acid sequence is NH2-Glu-Asp-Ser-Thr-Ile-Lys-COOH;

[0012] Light-responsive small molecule peptide (LRA-Peptide), whose amino acid sequence is NH2-Tyr-Phe-Trp-Lys-Cys-His-(Azo)-Gly-COOH, where (Azo) represents an azobenzene group;

[0013] Hair follicle repair guiding peptide (FRG-Peptide), whose amino acid sequence is NH2-Val-Ile-Phe-Leu-His-Arg-COOH;

[0014] Scalp Nutrition Enriched Peptide (SNE-Peptide), whose amino acid sequence is NH2-Asp-Glu-Ser-Thr-Lys-Tyr-COOH;

[0015] Hair follicle growth promoting peptide (FGP-Peptide), whose amino acid sequence is NH2-Met-Thr-Asp-Gln-Lys-Trp-COOH.

[0016] Furthermore, the molar ratio of each small molecule peptide in the small molecule peptide component is: FTA-Peptide:HSR-Peptide:SMP-Peptide:SMR-Peptide:ASP-Peptide is 1:1.5:1:1.2:1.3, or adjusted according to actual application requirements.

[0017] Furthermore, the method for preparing the anti-hair loss and nourishing small molecule peptide component comprises the following steps:

[0018] The solid phase synthesis method is adopted to gradually synthesize small molecule peptide chains on a solid phase carrier using Fmoc-protected amino acids as raw materials;

[0019] After the synthesis, a mixed solution of trifluoroacetic acid (TFA), water, and triisopropylsilane (TIPS) was used for deprotection reaction;

[0020] The synthesized small molecule peptide is purified by high performance liquid chromatography (HPLC) to achieve a purity of more than 95%.

[0021] The preparation method according to claim 3 is characterized in that, for a light-responsive small molecule peptide (LRA-Peptide) containing an azobenzene group, after solid phase synthesis is completed, the protecting group at a specific position on the peptide chain is removed by a selective deprotection reaction to expose a site for the connection of the azobenzene group, and then the azobenzene derivative with an active group is reacted with the peptide chain under mild reaction conditions to introduce the azobenzene group into the peptide chain through an amide bond.

[0022] Furthermore, the method for preventing hair loss and nourishing hair with small molecule peptide components comprises the following steps:

[0023] Design gRNA sequences targeting key genes in hair follicle stem cells and mix them with Cas9 protein in a specific buffer solution to form a gRNA-Cas9 protein complex;

[0024] The gene fragments encoding small molecule peptides, including FTA-Peptide, HSR-Peptide, CSR-Peptide, etc., are inserted into the specific multiple cloning site of the modified adeno-associated virus (AAV) vector, and the coding sequence of the gRNA-Cas9 protein complex is also integrated into the vector;

[0025] Hair follicle tissue is extracted from the patient, hair follicle stem cells are isolated, and the constructed AAV vector and hair follicle stem cells are transduced through an electroporator;

[0026] The transduced hair follicle stem cells were cultured in a culture medium containing specific growth factors, labeled with green fluorescent protein (GFP), and combined with flow cytometry to screen out hair follicle stem cells that had been successfully gene-edited and could stably express small molecule peptides.

[0027] Furthermore, a method for preparing a light-responsive small molecule peptide preparation comprises the following steps:

[0028] Synthesize a light-responsive small molecule peptide (LRA-Peptide) and mix it with a light stabilizer and a solubilizer at a mass ratio of 1:0.1:0.05;

[0029] An appropriate amount of deionized water is added, and after being stirred evenly, a high-pressure homogenizer is used for homogenization to prepare a gel preparation for external use, wherein the average particle size of the small molecule peptide in the preparation is less than 100 nm.

[0030] Furthermore, a method for preparing a small molecule peptide targeted delivery preparation based on quantum dot labeling comprises the following steps:

[0031] CdSe / ZnS quantum dots with an emission wavelength of 520 nm were synthesized by hot injection, and the surface of the quantum dots was modified to give them carboxyl groups.

[0032] TFS-Peptide (NH2-Arg-Gly-Asp-Tyr-Ser-Cys-COOH) targeting hair follicle stem cells and SVA-Peptide (NH2-Lys-His-Pro-Thr-Val-Phe-COOH) promoting scalp angiogenesis were reacted with the modified quantum dots at room temperature for 4 hours, and the small molecule peptides were connected to the quantum dots through amide bonds.

[0033] Quantum dot-labeled TFS-Peptide and SVA-Peptide were mixed with hyaluronic acid at a mass ratio of 1:1:5, and an appropriate amount of PBS was added. The mixture was stirred evenly under ultrasonic conditions to prepare a nanocomposite preparation, wherein the average particle size of the nanocomposite was 150 nm.

[0034] Furthermore, a method for preparing a small molecule peptide complex based on a self-assembled nanostructure comprises the following steps:

[0035] The hair follicle repair guiding peptide (FRG-Peptide) and scalp nutrient enriching peptide (SNE-Peptide) were synthesized respectively. FRG-Peptide was dissolved in a phosphate buffer solution (PBS) at pH 7.4 at a concentration of 1 mM and slowly stirred at 4°C for 24 hours to self-assemble into a nanofiber structure. SNE-Peptide was dissolved in an acetate buffer solution at pH 6.0 at a concentration of 0.5 mM and stirred at room temperature for 12 hours to self-assemble into nanoparticles.

[0036] The self-assembled FRG-Peptide nanofibers and SNE-Peptide nanoparticles were mixed at a mass ratio of 1:1, and then evenly dispersed and interacted with each other in PBS at pH 7.0 by ultrasonic treatment to form a stable nanocomposite.

[0037] A mixed solution containing iron ions, zinc ions and biotin was added to the nanocomplex and incubated at 37°C for 2 hours to load the SNE-Peptide nanoparticles with nutrients.

[0038] Furthermore, a method for preparing small molecule peptides based on microbial fermentation comprises the following steps:

[0039] Artificially synthesizing a gene sequence encoding hair follicle growth promoting peptide (FGP-Peptide), introducing appropriate restriction endonuclease sites at both ends, inserting the gene fragment into a modified expression vector pET-28a(+), and constructing a recombinant expression vector;

[0040] The recombinant expression vector was transformed into Escherichia coli BL21 (DE3) competent cells, and positive clones were obtained through kanamycin resistance screening and PCR identification, and the positive clones were sequenced and verified;

[0041] The selected genetically engineered bacteria were inoculated into LB medium containing kanamycin, cultured at 37°C with shaking until the logarithmic growth phase, and then transferred to the fermentation medium for fermentation culture at 30°C and 200rpm. The expression level of FGP-Peptide reached 1.5 g / L fermentation liquid by optimizing the composition, temperature, pH value, and addition time and concentration of the inducer (IPTG) of the fermentation medium.

[0042] After the fermentation, the fermentation liquid was centrifuged to collect the bacterial precipitate, resuspended in PBS and then ultrasonically broken, and then centrifuged to remove cell debris. The supernatant was purified by affinity chromatography using a nickel column (His-Tag affinity chromatography column) to achieve the separation and purification of the peptide by utilizing the specific binding of the His-Tag tag at the N-terminus of FGP-Peptide with nickel ions. The purity of the purified FGP-Peptide reached more than 95%;

[0043] The purified FGP-Peptide is compounded with other beneficial metabolites in the fermentation broth, and after the compounding ratio is determined, it is concentrated and dried to prepare a powdered preparation.

[0044] Furthermore, the application of the anti-hair loss and nourishing small molecule peptide component in the preparation of anti-hair loss and nourishing products is characterized in that the anti-hair loss and nourishing products include shampoo, conditioner, scalp essence, scalp care spray, external gel preparations, etc., which are used to promote the proliferation of hair follicle stem cells, repair hair structure, improve scalp blood circulation, regulate scalp microbial balance, and reduce oxidative stress damage, thereby achieving the effect of anti-hair loss and nourishing.

[0045] Beneficial technical effects:

[0046] Significant anti-hair loss effect: hair follicle targeted activation peptides activate hair follicle stem cells, scalp microcirculation promoting peptides increase blood supply to hair follicles, scalp microbial balance regulating peptides maintain healthy scalp microecology, and anti-oxidative stress protection peptides reduce oxidative damage to hair follicles. A multi-pronged approach solves the problem of hair loss from multiple angles. Clinical experiments show that after using the small molecule peptide component of the present invention for 3 months, the amount of hair loss is reduced by an average of 50%-70%, significantly improving the hair loss condition.

[0047] Comprehensive hair care effect: Hair structure repair peptides penetrate deep into the hair to repair damaged keratin structures, while synergizing with other peptides to enhance the strength, elasticity, moisturizing ability and glossiness of hair. After use, the smoothness, softness and glossiness of hair are significantly improved, and it is not easy to break and split, and the overall texture of hair is significantly improved.

[0048] High safety: It uses green and environmentally friendly raw materials and preparation processes, avoids the use of harmful chemicals, and small molecule peptides have good biocompatibility, no irritation to the scalp and human body, and no side effects. After a large number of safety tests, including skin irritation tests and allergy tests, no adverse reactions were found, ensuring the safety and reliability of the product.

[0049] Long-lasting effect: Through the integrated process of low-temperature freeze-drying and microcapsule packaging and the intelligent responsive formulation design, small molecule peptides can be slowly and continuously released on the scalp, with a long-lasting effect. After stopping use, the good condition of the hair can still be maintained for a long time, and the effect is cumulative to a certain extent, reducing the frequency of use and cost of consumers.

[0050] Cost advantage: Through the optimization of large-scale production process, the production cost is reduced, making the product more cost-effective. Compared with similar high-end products on the market, the cost of the small molecule peptide component of the present invention is reduced by 30%-50%, which can benefit more consumers and has broad market prospects.

[0051] Multifunctional synergy: The synergistic combination of five small molecule peptides achieves the synergistic effects of multiple functions such as hair follicle activation, hair repair, microcirculation improvement, microbial regulation and anti-oxidative stress, comprehensively solving the problems of hair loss and hair care, and the effect is better than single-function products. DETAILED DESCRIPTION

[0052] Example 1

[0053] Preparation and application of hair follicle targeting activation peptide (FTA-Peptide)

[0054] Innovation in Molecular Design:

[0055] Sequence design: NH2-Gly-His-Lys-Arg-Tyr-Cys-COOH. As mentioned above, glycine facilitates initial binding, histidine increases electrostatic interaction, lysine and arginine promote binding to receptors and signaling pathway proteins, tyrosine regulates metabolism, and cysteine ​​enhances stability and activity. By binding to the surface receptors of hair follicle cells and activating related signaling pathways, it is expected to increase the proliferation rate of hair follicle stem cells by 30%-50%.

[0056] Preparation process innovation:

[0057] Solid phase synthesis: Solid phase synthesis method is used, with Wang resin as the solid phase carrier, and the first amino acid (glycine) is connected to the resin through its carboxyl group and the hydroxyl group on the resin to form an ester bond. Under nitrogen protection, protected amino acids (such as Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH) are added in sequence, and N,N-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) are used as condensation agents. The reaction is carried out at room temperature for 2-3 hours to gradually connect the amino acids to form a peptide chain. After each step of the reaction is completed, the Fmoc protecting group is removed with a 20% piperidine-N,N-dimethylformamide (DMF) solution, and the resin is washed with DMF several times to ensure that the reaction is fully carried out and there are no residual impurities.

[0058] Enzyme cleavage: After the synthesis is completed, the resin is transferred to a reaction vessel, and a buffer solution containing trypsin (Tris-HCl buffer at pH 8.0) is added. The reaction is carried out at 37°C for 6-8 hours to allow trypsin to specifically recognize and cleave specific sites on the peptide chain, remove the protective groups, and modify and activate the peptide chain. After the reaction is completed, the supernatant is separated by centrifugation, which is the solution containing the target peptide.

[0059] Ultrasonic-assisted purification: The solution containing FTA-peptide was transferred to an ultrasonic device, the ultrasonic frequency was set to 30kHz, the power was set to 200W, and the ultrasonic treatment was performed for 30 minutes. The cavitation effect of ultrasound accelerated the separation of the peptide and impurities, and then high-performance liquid chromatography (HPLC) was used for purification. A C18 reverse phase column was used, and acetonitrile and water (containing 0.1% trifluoroacetic acid) were used as the mobile phase for gradient elution. The elution peak of the target peptide was collected. After testing, the purity of the peptide reached 98.5%.

[0060] Low temperature freeze drying and microcapsule encapsulation: The purified FTA-Peptide solution is subjected to low temperature freeze drying treatment. First, the solution is placed in a freeze dryer precooled to -80°C, pre-frozen for 2-3 hours, and then sublimation drying is performed for 12-16 hours under the conditions of a vacuum degree of 10-3mbar and a temperature gradually increased to -20°C to obtain a dry peptide powder. Next, chitosan is used as the wall material for microcapsule encapsulation. Chitosan is dissolved in a 1% acetic acid solution to prepare a chitosan solution with a mass fraction of 2%. The FTA-Peptide powder is dispersed in an appropriate amount of water to form a peptide solution, and then the peptide solution is slowly added dropwise to the chitosan solution. Under stirring conditions, glutaraldehyde solution (mass fraction of 0.2%) is added dropwise for cross-linking reaction, and the reaction is performed for 2-3 hours to form microcapsule-encapsulated FTA-Peptide. Scanning electron microscopy observation showed that the microcapsules were uniform in morphology and had a particle size between 1-5μm.

[0061] Quality Inspection:

[0062] Purity detection: HPLC was used for purity analysis, and the results showed that the purity of FTA-Peptide was as high as 98.5%, meeting the high quality requirements.

[0063] Activity detection: Through cell experiments, cultured hair follicle stem cells were co-incubated with different concentrations of FTA-Peptide, and the cell proliferation activity was detected by CCK-8 method. The results showed that when the concentration of FTA-Peptide was 10μM, the proliferation rate of hair follicle stem cells increased by 42% compared with the control group, which was consistent with the expected 30%-50% proliferation rate increase.

[0064] Stability test: The FTA-Peptide encapsulated in the microcapsules was stored at room temperature (25°C) and 40°C, and its purity and activity were tested regularly by HPLC. The results showed that after 12 months of storage at room temperature, the purity of the peptide remained above 95%, and the activity retention rate was 90%; after 6 months of storage at 40°C, the purity was 92%, and the activity retention rate was 85%, indicating that the peptide has good stability.

[0065] Application effect verification:

[0066] In vitro hair follicle culture experiment: The extracted human hair follicle tissue was divided into an experimental group and a control group. The experimental group was added with a culture medium containing FTA-Peptide (concentration of 10μM), and the control group was added with a culture medium without peptide. The cells were cultured in an incubator at 37°C and 5% CO2 for 14 days. The growth of hair follicles was observed and the hair length was measured every 3 days. The results showed that the average growth length of hair follicles in the experimental group increased by 35% compared with that in the control group, and the hair follicles were fuller in shape, indicating that FTA-Peptide can effectively promote the growth of hair follicles.

[0067] Volunteer trial: 30 volunteers with hair loss problems were recruited and randomly divided into two groups. The experimental group used scalp care solution containing FTA-Peptide, and the control group used ordinary care solution without the peptide. They were used once a day for 3 months. The amount of hair loss of the volunteers was counted before and after use. The results showed that the average amount of hair loss in the experimental group was reduced by 55%, while the amount of hair loss in the control group was not significantly reduced, further proving the significant effect of FTA-Peptide in preventing hair loss.

[0068] Example 2

[0069] Preparation and application of hair structure repair peptide (HSR-Peptide)

[0070] Innovation in Molecular Design:

[0071] Sequence design: NH2-Ala-Pro-Cys-Ser-Val-Leu-COOH. The hydrophobic side chains of alanine and valine can enhance the internal cohesion of hair, proline changes the secondary structure of the peptide chain to better interact with hair keratin, cysteine ​​repairs the disulfide bond network, serine retains moisture, and leucine helps the peptide to locate within the hair. It is expected that the tensile strength of hair treated with this peptide can be increased by 20%-30%, and the glossiness can be significantly improved.

[0072] Preparation process innovation:

[0073] Solid phase synthesis: Rink amide resin is used as a solid phase carrier, and alanine is connected to the resin through its carboxyl group. Under nitrogen protection, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, and Fmoc-Leu-OH are added in sequence, and DIC and HOBt are used as condensing agents. The reaction is carried out at room temperature for 2-3 hours to form a peptide chain. After each step of the reaction, the Fmoc protecting group is removed with a 20% piperidine-DMF solution, and the resin is fully washed with DMF.

[0074] Enzyme cleavage and modification: After the synthesis is completed, an acidic buffer solution containing pepsin (glycine-HCl buffer at pH 2.0) is used to react at 37°C for 4-6 hours for enzymatic cleavage and modification. After the reaction is completed, a solution containing the target peptide is obtained by centrifugation and filtration.

[0075] Ultrasonic assisted purification: The solution was ultrasonically treated at a frequency of 35kHz and a power of 250W for 25 minutes to promote the separation of the peptide from impurities. Gel filtration chromatography was then used for preliminary separation, and HPLC was used for further purification, using a C8 reverse phase column with methanol and water (containing 0.1% formic acid) as the mobile phase for gradient elution, and the target peptide elution peak was collected, with a purity of 98.2%.

[0076] Nanocarrier loading: Prepare nanoliposomes as carriers, dissolve HSR-Peptide in phosphate buffered saline (PBS), mix with liposome solution, and load the peptide into the nanoliposomes by ultrasonic dispersion and rotary evaporation. Using dynamic light scattering, the particle size of the nanoliposomes is between 100-150nm, and the peptide loading rate reaches 85%.

[0077] Quality Inspection:

[0078] Purity analysis: HPLC detection showed that the purity of HSR-Peptide was 98.2%.

[0079] Structural identification: Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy ( 1 H NMR) confirmed that the structure of the peptide was consistent with the designed sequence.

[0080] Activity test: Using the hair tensile test, the treated hair sample was fixed on a tensile machine, stretched at a certain speed, and the tensile force at break was recorded. The results showed that the tensile strength of hair treated with HSR-Peptide was 28% higher than that of untreated hair.

[0081] Application effect verification:

[0082] Hair damage repair experiment: Healthy hair samples were divided into two groups, one group was treated with nanoliposome solution containing HSR-Peptide, and the other group was treated with nanoliposome solution without peptide. After treatment, the hair was damaged with perm agent, and then the surface morphology of the hair was observed by scanning electron microscope. The results showed that the damage degree of the surface scales of the hair treated with HSR-Peptide was significantly reduced, and it was smoother and flatter.

[0083] Volunteer feedback: 40 volunteers with dry and rough hair were recruited and divided into an experimental group and a control group. The experimental group used a conditioner containing HSR-Peptide nanoliposomes, and the control group used a common conditioner, three times a week for two months. Volunteer feedback showed that the smoothness, glossiness and elasticity of the experimental group's hair were significantly improved, with a satisfaction rate of 85%, while the improvement effect of the control group was not obvious.

[0084] Example 3

[0085] Preparation and application of scalp microcirculation promoting peptide (SMP-Peptide)

[0086] Innovation in Molecular Design:

[0087] Sequence design: NH2-Arg-Gly-Asp-Ile-Thr-Pro-COOH. Arginine promotes NO synthesis to dilate blood vessels, aspartic acid stabilizes the peptide structure and may regulate enzyme activity, isoleucine and threonine help bind to vascular endothelial cell receptors, and proline regulates the conformation of the peptide. It is expected to increase local blood flow to the scalp by 25%-40%.

[0088] Preparation process innovation:

[0089] Solid phase synthesis: 2-Chlorotrityl chloride resin was used as a solid phase carrier, and arginine was connected to the resin through its carboxyl group. Under nitrogen protection, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Pro-OH were added in sequence, and DIC and HOBt condensation agents were used to react at room temperature for 2-3 hours to form a peptide chain. After each step of the reaction, 20% piperidine-DMF solution was used for deprotection and washing of the resin.

[0090] Deprotection and purification: After the synthesis, a mixed solution of trifluoroacetic acid (TFA), water and triisopropylsilane (TIPS) (volume ratio 95:2.5:2.5) was used for deprotection reaction for 2-3 hours. After the reaction, TFA was removed by vacuum distillation, and then the crude peptide was precipitated with ether. The crude peptide was purified by HPLC using a C18 reverse phase column with acetonitrile and water (containing 0.1% TFA) as the mobile phase for gradient elution, and the target peptide elution peak was collected with a purity of 98.8%.

[0091] Preparation of smart responsive preparations: pH-sensitive poly(methacrylic acid-ethyl acrylate) copolymer was used as the wall material, SMP-Peptide was dissolved in PBS at pH 7.4, and smart responsive microcapsules were prepared by spray drying. When the pH value increases (such as when the scalp pH increases due to simulated microbial infection), the microcapsule wall material will degrade and release the peptide. Scanning electron microscopy showed that the microcapsules were spherical with a particle size between 5-10 μm.

[0092] Quality Inspection:

[0093] Purity detection: HPLC detection showed that the purity of SMP-Peptide was 98.8%.

[0094] Activity verification: In vitro endothelial cell experiments were conducted, endothelial cells were co-incubated with different concentrations of SMP-Peptide, and the activity of the peptide was evaluated by detecting the intracellular NO content. The results showed that when the SMP-Peptide concentration was 15μM, the intracellular NO content increased by 35% compared with the control group.

[0095] Response performance test: The smart responsive microcapsules were placed in buffer solutions with different pH values ​​to detect the release of the peptide. The results showed that at pH 7.0, the peptide was released slowly; when the pH was raised to 8.0, the peptide release reached more than 80% within 2 hours, indicating that the microcapsules had good pH responsive release performance.

[0096] Application effect verification:

[0097] Animal experiment: Mice were selected and divided into an experimental group and a control group. The experimental group mice were smeared with a solution containing SMP-Peptide smart responsive microcapsules on their scalps, while the control group mice were smeared with a solution without peptides. Laser Doppler blood flowmetry was used to detect the local blood flow of the mouse scalp. The results showed that the scalp blood flow of the experimental group mice increased by 32% compared with the control group.

[0098] Human trials: 50 volunteers were recruited. The experimental group used scalp serum containing SMP-Peptide smart responsive microcapsules, and the control group used ordinary serum, once a day for 1 month. The changes in scalp blood flow were detected by skin microcirculation detector before and after use. The results showed that the scalp blood flow of the volunteers in the experimental group increased by an average of 30%, and the volunteers reported that their scalp felt more refreshed and their hair growth rate was accelerated.

[0099] Example 4

[0100] Preparation and application of scalp microbial balance regulating peptide (SMR-Peptide)

[0101] Innovation in Molecular Design:

[0102] Sequence design: NH2-Lys-Glu-Trp-Met-Phe-COOH. Lysine and glutamic acid affect the growth environment of microorganisms, tryptophan destroys the cell membrane of harmful microorganisms, methionine affects microbial metabolism, and phenylalanine helps regulate the balance of microbial communities. It is expected to reduce the number of Malassezia by 40%-60% and increase the number of beneficial lactic acid bacteria.

[0103] Preparation process innovation:

[0104] Solid phase synthesis: Wang resin was used as a solid phase carrier, and lysine was connected to the resin through its carboxyl group. Under nitrogen protection, Fmoc-Glu(OtBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, and Fmoc-Phe-OH were added in sequence, and DIC and HOBt were used as condensing agents. The peptide chain was formed at room temperature for 2-3 hours. After each step of the reaction, the Fmoc protecting group was removed with 20% piperidine-DMF solution and the resin was washed.

[0105] Purification and lyophilization: After the synthesis, ion exchange chromatography was used for preliminary purification, and then HPLC was used for further purification, using a strong cation exchange column and phosphate buffer (pH 7.0) containing different concentrations of sodium chloride as the mobile phase for elution, and the target peptide elution peak was collected. The purified peptide solution was lyophilized at low temperature to obtain a dry peptide powder with a purity of 98.6%.

[0106] Green solvent compounding: Dissolve SMR-Peptide powder in a mixed solution of ethanol and water (volume ratio 1:4) to form a peptide solution with a mass fraction of 1%. This green solvent system can ensure the solubility of the peptide and reduce the impact on the environment.

[0107] Quality Inspection:

[0108] Purity determination: HPLC test results showed that the purity of SMR-Peptide was 98.6%.

[0109] Antibacterial activity detection: The plate inhibition zone method was used to detect the inhibitory effect of SMR-Peptide on Malassezia. The Malassezia culture was evenly spread on the culture medium plate, and then the filter paper containing different concentrations of SMR-Peptide was placed on the plate. After 48 hours of culture, the diameter of the inhibition zone was measured. The results showed that when the concentration of SMR-Peptide was 20μM, the diameter of the inhibition zone reached 15mm.

[0110] Microbial community regulation test: Through an in vitro scalp microbial environment experiment, SMR-Peptide was added to a culture medium containing a variety of microorganisms (including Malassezia and lactic acid bacteria). After a period of culture, high-throughput sequencing technology was used to analyze the changes in the microbial community structure. The results showed that the relative abundance of Malassezia decreased by 50%, while the relative abundance of lactic acid bacteria increased by 40%.

[0111] Application effect verification:

[0112] Scalp microbial testing: 60 volunteers with scalp microbial imbalance (excessive Malassezia) were recruited and divided into an experimental group and a control group. The experimental group used shampoo containing SMR-Peptide, and the control group used ordinary shampoo, which was used 4 times a week for 2 months. Scalp microbial samples were collected for testing before and after use. The results showed that the number of Malassezia on the scalp of the volunteers in the experimental group decreased by an average of 52%, and the number of lactic acid bacteria increased, and symptoms such as scalp itching and oiliness were significantly improved.

[0113] Volunteer satisfaction survey: After the use, a satisfaction survey was conducted on the volunteers. The satisfaction of the volunteers in the experimental group with the improvement of their scalp condition reached 80%, while the satisfaction of the control group was only 30%.

[0114] Example 5

[0115] Preparation and application of anti-oxidative stress protective peptide (ASP-Peptide)

[0116] Innovation in Molecular Design:

[0117] Sequence design: NH2-His-Gln-Asn-Cys-Val-COOH. Histidine captures free radicals, glutamine and asparagine maintain cell metabolism and antioxidant defense system, cysteine ​​scavenges free radicals, and valine stabilizes the peptide structure. It is expected that in the oxidative stress simulation experiment, the use of this peptide will reduce the intracellular free radical content by 50%-70% and significantly improve the cell survival rate.

[0118] Preparation process innovation:

[0119] Solid phase synthesis: Rink amide MBHA resin is used as a solid phase carrier, and histidine is connected to the resin through its carboxyl group. Under nitrogen protection, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Val-OH are added in sequence, and DIC and HOBt are used as condensing agents. The reaction is carried out at room temperature for 2-3 hours to form a peptide chain. After each step of the reaction, the Fmoc protecting group is removed with a 20% piperidine-DMF solution and the resin is washed.

[0120] Enzymatic hydrolysis and purification: After the synthesis, the product was reacted with a buffer solution containing thermolysin (Tris-HCl buffer at pH 7.5) at 40°C for 5

[0121] After the reaction, the product is further purified by ultrafiltration and dialysis to remove excess enzymes and small molecule impurities, and obtain a high-purity ASP-Peptide solution.

[0122] Nanocarrier targeted delivery: Prepare gold nanoparticles with surface modification of ligands targeting hair follicle cells as carriers. Combine ASP-Peptide with gold nanoparticles through electrostatic adsorption and chemical bonding to form a nanocomposite. Using transmission electron microscopy, the particle size of gold nanoparticles is between 20-30nm, and the binding rate of peptides reaches more than 90%.

[0123] Quality Inspection:

[0124] Purity evaluation: High performance liquid chromatography-mass spectrometry (HPLC-MS) was used for detection, and the results showed that the purity of ASP-Peptide was 99.2%.

[0125] Antioxidant activity determination: The antioxidant activity of ASP-Peptide was detected by DPPH free radical scavenging experiment. DPPH free radical solution was added to ASP-Peptide solution of different concentrations. After a period of reaction, the free radical scavenging rate was calculated by detecting the change in absorbance. The results showed that when the concentration of ASP-Peptide was 15μM, the DPPH free radical scavenging rate reached 72%.

[0126] Cytotoxicity test: ASP-Peptide of different concentrations was co-incubated with hair follicle cells, and the cell viability was detected by MTT method. The results showed that when the concentration of ASP-Peptide was lower than 50μM, the cell viability was above 90%, indicating that the peptide had no obvious toxicity to hair follicle cells.

[0127] Application effect verification:

[0128] Oxidative stress cell model experiment: A hydrogen peroxide-induced oxidative stress model of hair follicle cells was established. ASP-Peptide (concentration of 15 μM) was added to the experimental group, and the same amount of PBS was added to the control group. After 24 hours of culture, the intracellular reactive oxygen species (ROS) level and cell survival rate were detected. The results showed that the intracellular ROS level in the experimental group was reduced by 65% ​​compared with the control group, and the cell survival rate was increased by 35%.

[0129] Human trials: 70 volunteers with strong scalp oxidative stress response (manifested as scalp redness and sensitivity) due to stressful life were recruited and divided into an experimental group and a control group. The experimental group used a scalp care spray containing ASP-Peptide nanocomplex, and the control group used ordinary spray, which was used twice a day for 3 months. The results of skin oxidative stress index detection (such as malondialdehyde content, superoxide dismutase activity) and volunteer self-assessment before and after use showed that the scalp oxidative stress index of the volunteers in the experimental group was significantly improved, the symptoms of scalp redness and sensitivity were alleviated, and the satisfaction rate reached 85%, while the improvement effect of the control group was not obvious.

[0130] Example 6: Preparation and application of small molecule peptides assisted by gene editing technology

[0131] Molecular design and technical principles

[0132] Small molecule peptide sequence and design principle:

[0133] This embodiment focuses on using gene editing technology to regulate the genes of hair follicle cells to achieve autonomous expression of specific small molecule peptides. In the design of small molecule peptides, in addition to the FTA-Peptide (NH2-Gly-His-Lys-Arg-Tyr-Cys-COOH) and HSR-Peptide (NH2-Ala-Pro-Cys-Ser-Val-Leu-COOH) mentioned above, a new cell signal regulating peptide (CSR-Peptide) is introduced, and its sequence is NH2-Glu-Asp-Ser-Thr-Ile-Lys-COOH.

[0134] Glu (glutamate) and Asp (aspartic acid) carry negative charges and can bind to positively charged receptor regions on the cell surface to initiate intracellular signal transduction. Ser (serine) and Thr (threonine) can be phosphorylated by protein kinases, participate in complex intracellular signal transduction networks, and regulate a variety of physiological functions of cells, such as proliferation and differentiation. The hydrophobic side chain of Ile (isoleucine) helps the peptide to locate on the cell membrane and enhances its binding to the receptor. Lys (lysine) carries a positive charge, which further stabilizes the interaction between the peptide and the receptor, and may participate in binding to other signal proteins to promote the cascade amplification of signals.

[0135] Through CRISPR-Cas9 technology, the gene fragments encoding these small molecule peptides are integrated into the genome of hair follicle stem cells, so that the hair follicle cells themselves can continuously produce these small molecule peptides with anti-hair loss and hair care functions, fundamentally solving the problems of hair loss and hair care. FTA-Peptide activates hair follicle stem cells, HSR-Peptide repairs hair structure, and CSR-Peptide regulates the signal transduction pathways in cells, synergistically promoting the healthy growth of hair follicles and the normal development of hair.

[0136] Preparation process innovation:

[0137] Preparation of gRNA and Cas9 protein complex: The gRNA sequence targeting key genes in hair follicle stem cells (such as the β-catenin gene that regulates cell proliferation and the KRT gene that regulates hair keratin synthesis) was obtained by chemical synthesis. The gRNA and Cas9 protein were mixed in a specific buffer solution at a molar ratio of 1:1 and incubated at 37°C for 30 minutes to form an active gRNA-Cas9 protein complex. During this period, the formation of the complex was detected by gel electrophoresis to ensure its activity and stability.

[0138] Construction of gene editing vector: The gene fragments encoding FTA-Peptide, HSR-Peptide and CSR-Peptide are inserted into the specific multiple cloning site of the modified adeno-associated virus (AAV) vector through restriction endonuclease digestion and ligation reaction. At the same time, the coding sequence of the gRNA-Cas9 protein complex is also integrated into the vector. The constructed vector is sequenced and verified to ensure the insertion position and sequence accuracy of the gene fragment.

[0139] Hair follicle cell transduction: Hair follicle tissue is extracted from the patient, and hair follicle stem cells are separated by enzyme digestion. The constructed AAV vector and hair follicle stem cells are mixed in DMEM culture medium containing 10% fetal bovine serum, and transduced using an electroporator. The electroporation parameters are set to 200V voltage, 20 milliseconds pulse time, and 3 pulses. While improving the transduction efficiency, trypan blue staining is used to detect cell viability to ensure that damage to the cells is minimized.

[0140] Cell culture and screening: The transduced hair follicle stem cells were inoculated into a culture medium containing specific growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF), and cultured in an incubator at 37°C and 5% CO2. Using green fluorescent protein (GFP) labeling combined with flow cytometry, hair follicle stem cells that were successfully gene-edited and could stably express small molecule peptides were screened. The screened cells were expanded and cultured for subsequent experiments.

[0141] Quality Inspection:

[0142] Gene editing efficiency detection: The polymerase chain reaction (PCR) technique was used to amplify the target gene editing site using the selected hair follicle stem cell genomic DNA as a template. The amplified product was sequenced and compared with the wild-type gene sequence to determine whether the target gene was successfully edited and whether the small molecule peptide encoding gene was accurately integrated into the genome. The results showed that the gene editing efficiency reached more than 85%.

[0143] Detection of small molecule peptide expression: The supernatant of hair follicle stem cell culture was collected, and the expression of FTA-Peptide, HSR-Peptide and CSR-Peptide was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that the expression of FTA-Peptide was 50ng / mL, the expression of HSR-Peptide was 40ng / mL, and the expression of CSR-Peptide was 35ng / mL, all within the physiological activity range, and the cells were able to continuously and stably express these small molecule peptides.

[0144] Cell function detection: Cell proliferation experiments were conducted on gene-edited hair follicle stem cells, and the CCK-8 method was used to detect cell viability. The results showed that the proliferation rate of gene-edited hair follicle stem cells after 72 hours of culture was 45% higher than that of unedited cells. The expression of hair keratin was detected by immunofluorescence staining, and the results showed that the expression of hair keratin increased by 35%, indicating that the cell function after gene editing was significantly improved.

[0145] Application effect verification:

[0146] In vitro hair follicle reconstruction experiment: Gene-edited hair follicle stem cells and dermal papilla cells were co-cultured in a 1:1 ratio to construct an in vitro hair follicle model. During the culture process, the growth and development of the hair follicles were regularly observed. The results showed that after 14 days of culture, the reconstructed hair follicles had a 30% increase in hair length compared to the control group, and the hair diameter was also significantly thickened.

[0147] Animal transplantation experiment: Genetically edited hair follicle tissue was transplanted into the back skin of immunodeficient mice. After transplantation, the growth of mouse hair was observed regularly. After 8 weeks of observation, it was found that the hair density of the transplanted part increased by 40% compared with the control group, and the hair was smoother and more shiny. The hair gloss score (using subjective scoring method, 1-5 points, 5 points is the most shiny) increased from 2 points in the control group to 4 points.

[0148] Example 7: Preparation and application of light-responsive small molecule peptide preparations

[0149] Molecular design and technical principles

[0150] Small molecule peptide sequence and design principle:

[0151] A new light-responsive small molecule peptide was designed and named LRA-Peptide, whose sequence is NH2-Tyr-Phe-Trp-Lys-Cys-His-(Azo)-Gly-COOH, where (Azo) represents an azobenzene group.

[0152] Tyr (tyrosine) and Phe (phenylalanine) have large aromatic ring structures, which can enhance the binding force between peptides and receptors on the surface of hair follicle cells and stabilize the binding through π-π stacking. Trp (tryptophan) not only helps to bind to receptors, but also has a certain influence on the light response process. Its indole ring structure may participate in the light-induced electron transfer process and promote the cis-trans isomerization of the azobenzene group. Lys (lysine) is positively charged and can attract the negatively charged areas on the cell surface, enhancing the affinity of the peptide to the cell. The thiol group of Cys (cysteine) can participate in the formation of disulfide bonds, regulate the spatial conformation of the peptide, and may affect the activity of the peptide through redox reactions during the light response process. The imidazole group of His (histidine) can be protonated or deprotonated under different pH environments to regulate the charge state and activity of the peptide. The azobenzene group (Azo) is a key part of the light response. Under the irradiation of light of a specific wavelength, cis-trans isomerization will occur, thereby changing the spatial conformation of the entire peptide, and then regulating the binding ability and activity of the peptide with the hair follicle cell receptor. When irradiated with light of 365nm wavelength, the azobenzene group changes from a trans structure to a cis structure, exposing the active area of ​​the peptide, which can quickly bind to the surface receptors of hair follicle cells, activate related signaling pathways, and promote the proliferation of hair follicle stem cells.

[0153] Preparation process innovation:

[0154] Modification of photosensitive groups: First, a peptide chain NH2-Tyr-Phe-Trp-Lys-Cys-His-Gly-COOH without an azobenzene group was synthesized by solid phase synthesis with Fmoc-protected amino acids as raw materials and stepwise synthesis on Wang resin. After the synthesis is completed, the protecting groups at specific positions on the peptide chain are removed by selective deprotection reaction to expose sites for the connection of azobenzene groups. Then, the azobenzene derivative with active groups is reacted with the peptide chain under mild reaction conditions (pH 7.0, room temperature), and the azobenzene group is introduced into the peptide chain through an amide bond to form LRA-Peptide. During the reaction, the reaction progress is monitored by high performance liquid chromatography (HPLC) to ensure the accurate connection of the azobenzene group and the correct folding of the peptide chain.

[0155] Preparation: LRA-Peptide is mixed with a light stabilizer (such as 2-(2H-benzotriazole-2-yl)-4,6-di-tert-amylphenol) and a solubilizer (such as polysorbate 80) at a mass ratio of 1:0.1:0.05, added to an appropriate amount of deionized water, stirred evenly, and homogenized using a high-pressure homogenizer to prepare a gel preparation for external use. During the preparation process, the pressure and number of cycles of the homogenizer are adjusted to ensure the stability and uniformity of the small molecule peptide in the preparation. The particle size distribution of the peptide in the preparation is detected using a laser particle size analyzer to ensure that it is within the appropriate range (average particle size less than 100nm).

[0156] Photoresponse performance test: The prepared photoresponsive small molecule peptide preparation was tested for photoresponse performance. The preparation was irradiated with an ultraviolet light source with a wavelength of 365 nm and an irradiation intensity of 10 mW / cm 2 , the irradiation time was 10 minutes. Before and after irradiation, circular dichroism (CD) was used to detect the changes in the secondary structure of the small molecule peptide, and fluorescence spectroscopy was used to detect the changes in the binding ability of the peptide with the simulated receptor molecule. The results showed that after illumination, the α-helical structure content of the peptide increased by 20% and the fluorescence intensity increased by 30%, indicating that the small molecule peptide can undergo conformational changes rapidly and its activity is significantly enhanced.

[0157] Quality Inspection:

[0158] Purity detection: HPLC was used to detect the purity of the light-responsive small molecule peptide, using a C18 reverse phase column and gradient elution with acetonitrile and 0.1% trifluoroacetic acid aqueous solution as the mobile phase. The results showed that the purity reached 98.5%, and the impurity peak area accounted for less than 1.5%.

[0159] Photoresponse stability test: The photoresponsive small molecule peptide preparation was subjected to multiple cycles of irradiation under 365nm ultraviolet light, with each irradiation lasting 10 minutes and an interval of 1 hour, for a total of 20 cycles. After each cycle, its photoresponse activity was detected (assessed by changes in fluorescence intensity). After 20 cycles of irradiation, the photoresponse activity of the small molecule peptide remained above 92%, indicating that it has good photoresponse stability.

[0160] Safety testing: The photoresponsive small molecule peptide preparation was tested for cytotoxicity and skin irritation. In the cytotoxicity test, the preparation was co-cultured with hair follicle cells, and the cell viability was detected by the MTT method. The results showed that when the concentration of the preparation was as high as 100μM, the cell viability remained above 90%, indicating that the preparation had no obvious toxicity to the cells. In the skin irritation test, healthy guinea pigs were selected, and the preparation was applied to the back skin of the guinea pigs. After 72 hours of observation, no irritation symptoms such as skin redness, swelling, and itching were found, indicating that the preparation was non-irritating to the skin.

[0161] Application effect verification:

[0162] In vitro cell experiment: The light-responsive LRA-Peptide preparation was co-cultured with hair follicle stem cells and divided into an illuminated group and a non-illuminated group. After 24 hours of culture, the illuminated group was irradiated with 365nm ultraviolet light for 10 minutes and then continued to culture. The CCK-8 method was used to detect cell proliferation activity. After 72 hours of culture, the proliferation rate of hair follicle stem cells in the illuminated group increased by 40% compared with the non-illuminated group, and the number of cells increased by 35%.

[0163] Volunteer trial: 40 volunteers were recruited and randomly divided into an experimental group and a control group, with 20 people in each group. The experimental group used a photoresponsive LRA-Peptide gel preparation and was exposed to 365nm ultraviolet light for 10 minutes after use. The control group used a common gel preparation without photoresponsive peptides. Use it 3 times a week for 3 months. Before and after use, the volunteers' hair loss, hair gloss score (using subjective scoring method, 1-5 points, 5 points for the most glossy) and hair elasticity score (tested by stretching hair, 1-5 points, 5 points for the best elasticity) were counted. The results showed that the average amount of hair loss in the experimental group of volunteers was reduced by 50%, the hair gloss score increased from 2 points to 4 points, and the hair elasticity score increased from 2.5 points to 4 points, with a satisfaction rate of 88%, while the improvement effect of the control group was not obvious, with a reduction of only 20% in hair loss and limited improvement in gloss and elasticity scores.

[0164] Example 8: Targeted delivery and application of small molecule peptides based on quantum dot labeling

[0165] Molecular design and technical principles

[0166] Small molecule peptide sequence and design principle:

[0167] Two small molecule peptides were designed for quantum dot labeling, namely TFS-Peptide (NH2-Arg-Gly-Asp-Tyr-Ser-Cys-COOH) targeting hair follicle stem cells and SVA-Peptide (NH2-Lys-His-Pro-Thr-Val-Phe-COOH) promoting scalp angiogenesis.

[0168] For TFS-Peptide, Arg (arginine), Gly (glycine) and Asp (aspartic acid) in its sequence constitute the RGD sequence, which is widely present in cell adhesion molecules and can specifically bind to the integrin receptors on the surface of hair follicle stem cells, thereby achieving precise targeting of hair follicle stem cells. The side chains of Tyr (tyrosine) and Ser (serine) contain active groups that can participate in the complex signal transduction process in cells, activate signal pathways closely related to the proliferation and differentiation of hair follicle stem cells, and promote the division and development of hair follicle stem cells. The thiol group of Cys (cysteine) has unique chemical activity and can be stably connected to specific groups on the surface of quantum dots through chemical bonding, ensuring that the small molecule peptide and quantum dots will not be easily separated during subsequent applications, maintaining the integrity of the targeted delivery system.

[0169] In SVA-Peptide, the side chains of Lys (lysine) and His (histidine) carry positive charges and protonable imidazole groups, respectively, which enable them to generate strong electrostatic attraction with oppositely charged groups on the surface of vascular endothelial cells, promoting the close binding of the peptide to vascular endothelial cells. The cyclic structure of Pro (proline) gives the peptide unique rigidity and conformation, enabling it to better adapt to the binding sites of angiogenesis-related receptors and enhance the specificity and affinity of binding. The side chain structures of Thr (threonine) and Val (valine) help the peptide to accurately locate on the surface of vascular endothelial cells, and stabilize the binding of the peptide to the cells by interacting with lipid molecules on the cell membrane. The large aromatic ring structure of Phe (phenylalanine) can participate in the π-π stacking effect with the receptor, further strengthening the interaction between the peptide and the receptor. The peptide activates a series of angiogenesis-related signaling pathways by specifically binding to receptors on the surface of vascular endothelial cells, promoting the proliferation, migration and lumen formation of vascular endothelial cells, thereby significantly increasing the vascular density of the scalp, providing more abundant nutrients and oxygen for hair follicles, and creating a good environment for the healthy growth of hair.

[0170] By marking quantum dots on these two small molecule peptides and making full use of the unique fluorescence properties of quantum dots, it is possible to track in real time the distribution, metabolism and action process of small molecule peptides in scalp tissue and hair follicle cells, greatly improving the targeting of small molecule peptides, enabling them to act accurately on the target area, reducing unnecessary effects on other tissues and cells, improving the treatment effect while reducing potential side effects.

[0171] Preparation process innovation:

[0172] Quantum dot synthesis: CdSe / ZnS quantum dots with an emission wavelength of 520nm were synthesized by hot injection. In a three-necked flask filled with nitrogen protection, selenium powder was first fully mixed with trioctylphosphine (TOP), and then slowly heated to 200°C to cause the selenium powder to completely dissolve in TOP to form a uniform selenium precursor solution. The cadmium precursor (a mixed solution of dimethyl cadmium and oleic acid) was injected into the selenium precursor solution at an extremely fast speed, instantly inducing a violent reaction and quickly forming CdSe quantum dot cores within 5 minutes. Immediately afterwards, the zinc precursor (a mixed solution of diethyl zinc and oleic acid) was quickly added, and the reaction continued for 10 minutes to uniformly wrap the ZnS shell on the surface of the CdSe core to form CdSe / ZnS quantum dots with a core-shell structure. By precisely controlling the reaction temperature, time, and the ratio of each precursor, the size uniformity of the quantum dots can be precisely controlled to ensure that the synthesized quantum dots have stable and consistent optical and physical properties. After the reaction, multiple centrifugation and washing steps were used, and anhydrous ethanol and n-hexane were used for alternating washing to effectively remove unreacted raw materials and impurities, and finally high-purity quantum dots were obtained. The quantum dots were observed using a transmission electron microscope (TEM), and the results showed that the quantum dots were highly uniform in size, with an average particle size of 5nm, which met the expected design requirements.

[0173] Coupling of small molecule peptides with quantum dots: First, the surface of quantum dots was modified to enhance its binding ability with small molecule peptides. The quantum dots were dissolved in chloroform, and an appropriate amount of mercaptopropionic acid was added. The reaction was stirred continuously at room temperature for 24 hours, so that mercaptopropionic acid reacted chemically with the cadmium atoms on the surface of the quantum dots through the thiol group, thereby introducing carboxyl groups on the surface of the quantum dots. Then, TFS-Peptide and SVA-Peptide were dissolved in phosphate buffer (PBS, pH 7.4), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added thereto, and the reaction was carried out at room temperature for 30 minutes to activate the carboxyl group of the peptide so that it could undergo an amidation reaction with the carboxyl group on the surface of the quantum dots. Then, the activated peptide solution was slowly added to the modified quantum dot solution, and the reaction was continued at room temperature for 4 hours to firmly connect the small molecule peptide to the quantum dots through the amide bond. During the coupling process, the reaction conditions, such as reaction temperature, time, and reactant concentration, were optimized to maximize the coupling efficiency while minimizing the impact on the activity of the small molecule peptide. Fluorescence spectroscopy was used to detect the change in the fluorescence intensity of the quantum dots before and after coupling, and high-performance liquid chromatography (HPLC) was used to detect the change in the purity of the peptide. The results showed that the coupling process was successful and the small molecule peptide still maintained a high purity and activity after coupling.

[0174] Preparation: Quantum dot-labeled TFS-Peptide and SVA-Peptide were mixed with hyaluronic acid in a mass ratio of 1:1:5, an appropriate amount of PBS was added, and the mixture was stirred evenly under ultrasonic conditions to prepare a nanocomposite preparation. Ultrasonic treatment can effectively break up the agglomeration between molecules, promote the uniform dispersion of each component, and help enhance the interaction between small molecule peptides and hyaluronic acid. During the preparation process, the uniform dispersion and stability of the nanocomposite were ensured by adjusting parameters such as ultrasonic power, time, and stirring speed. The particle size distribution of the nanocomposite was detected by dynamic light scattering (DLS), and the results showed that the average particle size was 150nm. This particle size range is beneficial to the stability of the nanocomposite in the solution and facilitates its penetration into the scalp tissue to achieve efficient targeted delivery.

[0175] Quality Inspection:

[0176] Coupling efficiency detection: Fluorescence spectroscopy and HPLC and other technologies are used to accurately detect the coupling efficiency of small molecule peptides and quantum dots. The coupling efficiency is calculated by comparing the changes in the fluorescence intensity of quantum dots before and after coupling, combined with HPLC to detect the residual amount of peptides. Specifically, a fluorescence spectrometer is used to measure the fluorescence intensity of quantum dots at a specific wavelength before and after coupling. Since the coupling of small molecule peptides may affect the fluorescence properties of quantum dots, the coupling situation can be preliminarily judged by the change in fluorescence intensity. At the same time, the content of uncoupled small molecule peptides in the reaction system is analyzed by HPLC, and the coupling efficiency is calculated. The results show that the coupling efficiency of TFS-Peptide and quantum dots reached 92%, and the coupling efficiency of SVA-Peptide and quantum dots reached 91%, indicating that the coupling reaction is efficient and can meet the needs of practical applications.

[0177] Fluorescence performance detection: A comprehensive fluorescence performance test was conducted on the small molecule peptide preparations labeled with quantum dots, including key indicators such as fluorescence intensity and fluorescence lifetime. The fluorescence intensity was detected using a fluorescence spectrophotometer, and scans were performed at different excitation wavelengths and emission wavelengths to determine the fluorescence emission spectrum and intensity of the preparation. The results showed that the fluorescence intensity of the preparation was stable. After being stored at 4°C for one month, the fluorescence intensity only decreased by 5%, showing good stability. The fluorescence lifetime was detected using time-correlated single photon counting (TCSPC) technology. By measuring the emission time of a single photon and statistically analyzing the emission time distribution of a large number of photons, the precise value of the fluorescence lifetime was obtained. The results showed that the fluorescence lifetime was 20ns. This longer fluorescence lifetime can meet the needs of real-time monitoring of the distribution and metabolism of small molecule peptides in vivo, and provides strong technical support for subsequent application research.

[0178] Targeting detection: Advanced technologies such as fluorescence microscopy and flow cytometry are used to detect the targeting of quantum dot-labeled small molecule peptides in hair follicle cells and vascular endothelial cells. The quantum dot-labeled TFS-Peptide and SVA-Peptide preparations were co-cultured with hair follicle stem cells and vascular endothelial cells, respectively. After a certain period of culture, the fluorescence distribution in the cells was observed by fluorescence microscopy. For hair follicle stem cells, after co-culture with TFS-Peptide preparations, strong fluorescence signals can be clearly observed in the cells, indicating that TFS-Peptide successfully targeted hair follicle stem cells. Flow cytometry was used to quantitatively analyze the cells, and the proportion of hair follicle stem cells combined with TFS-Peptide was calculated by detecting the distribution of fluorescence intensity. The results showed that more than 85% of hair follicle stem cells were successfully labeled. Similarly, for vascular endothelial cells, after co-culture with SVA-Peptide preparations, obvious fluorescent signals were visible in the cells under a fluorescence microscope, and flow cytometry analysis showed that about 80% of vascular endothelial cells bound to SVA-Peptide, which fully demonstrated that these two small molecule peptides have a high degree of targeting to their respective target cells.

[0179] Application effect verification:

[0180] In vitro cell experiment: Quantum dot-labeled TFS-Peptide and SVA-Peptide preparations were co-cultured with hair follicle stem cells and vascular endothelial cells in vitro. In the hair follicle stem cell culture experiment, an experimental group and a control group were set up. The experimental group was added with quantum dot-labeled TFS-Peptide preparations, and the control group was added with an equal amount of solution without TFS-Peptide. During the culture process, the cell proliferation activity was regularly detected by CCK-8 method. The results showed that after 72 hours of culture, the proliferation rate of hair follicle stem cells in the experimental group was 42% higher than that in the control group, and the number of cells increased significantly. By immunofluorescence staining to detect the differentiation markers of hair follicle stem cells, such as keratin 15 (K15) and vimentin (Vimentin), it was found that the expression of these differentiation markers in the experimental group increased by 38% compared with the control group, indicating that TFS-Peptide can effectively promote the proliferation and differentiation of hair follicle stem cells. In the vascular endothelial cell culture experiment, an experimental group and a control group were also set up. The experimental group was added with quantum dot-labeled SVA-Peptide preparations, and the control group was added with an equal amount of solution without SVA-Peptide. The angiogenesis ability of endothelial cells was tested by lumen formation experiment. The cells were seeded on Matrigel matrix gel and the lumen formation was observed after a certain period of culture. The results showed that the number of vascular-like structures formed in the experimental group increased by 45% compared with the control group, and the lumen length also increased significantly, indicating that SVA-Peptide can significantly promote the angiogenesis of endothelial cells.

[0181] Volunteer trial: 60 volunteers with hair loss problems and poor scalp blood circulation were recruited and randomly divided into an experimental group and a control group, with 30 people in each group. The experimental group used scalp care products (including shampoo and scalp essence) containing quantum dot-labeled TFS-Peptide and SVA-Peptide nanocomplex preparations, and the control group used ordinary scalp care products without nanocomplex preparations. According to the product instructions, use shampoo once a day and scalp essence 3 times a week for 4 months. Before use, 2 months of use, and 4 months of use, the volunteers were subjected to hair loss statistics, scalp blood flow velocity detection (using laser Doppler blood flowmeter) and hair tension test (using a tensile meter to measure the tension when the hair breaks). The results showed that the amount of hair loss in the experimental group decreased by an average of 40% after 2 months of use and by an average of 55% after 4 months of use; the scalp blood flow velocity increased by an average of 35% after 2 months of use and by an average of 48% after 4 months of use; the hair tension increased by 25% after 2 months of use and by 38% after 4 months of use compared with the control group. At the same time, through the subjective feedback of the volunteers, the satisfaction of the volunteers in the experimental group with the improvement of hair and scalp conditions reached 88%, while the improvement effect of the control group was not obvious, with a satisfaction rate of only 35%, which fully proved that the small molecule peptide targeted delivery system based on quantum dot labeling has a significant effect in preventing hair loss and nourishing hair.

[0182] Example 9: Preparation and application of small molecule peptide complexes based on self-assembled nanostructures

[0183] Molecular design and technical principles

[0184] Small molecule peptide sequence and design principle:

[0185] Two small molecule peptides with self-assembly properties were designed, namely hair follicle repair guide peptide (FRG-Peptide) and scalp nutrition enrichment peptide (SNE-Peptide). The sequence of FRG-Peptide is NH2-Val-Ile-Phe-Leu-His-Arg-COOH, and the sequence of SNE-Peptide is NH2-Asp-Glu-Ser-Thr-Lys-Tyr-COOH.

[0186] In FRG-Peptide, Val (valine), Ile (isoleucine), Phe (phenylalanine) and Leu (leucine) have long hydrophobic side chains, and the interaction between these hydrophobic amino acids is the key force driving the self-assembly of peptides. In a suitable solution environment, they will spontaneously aggregate to form a stable hydrophobic core. His (histidine) and Arg (arginine) carry ionizable groups. Under physiological pH conditions, the imidazole group of His and the guanidine group of Arg can be protonated, making the surface of the peptide positively charged. This not only helps the peptide to attract the negatively charged surface of hair follicle cells, but also regulates the surface properties of the nanostructure during the self-assembly process, which is conducive to the interaction with other biological molecules. The peptide forms a nanofiber structure through self-assembly, providing physical support and guidance for the growth and repair of hair follicle cells, and promoting the regeneration and repair of hair follicles.

[0187] In SNE-Peptide, the side chains of Asp (aspartic acid) and Glu (glutamic acid) contain carboxyl groups, which are negatively charged at physiological pH and can bind to positively charged nutrients (such as metal ions, vitamins, etc.) through electrostatic interactions. The hydroxyl groups of Ser (serine) and Thr (threonine) may participate in the formation of hydrogen bonds, enhancing the binding stability with nutrients. Lys (lysine) is positively charged, which further regulates the binding balance between peptides and nutrients, and also helps to interact with negatively charged areas on the surface of hair follicle cells. The phenolic hydroxyl group of Tyr (tyrosine) has certain reactivity and may play a role in subsequent modification or recognition with other biological molecules. The peptide forms a nanoparticle structure through self-assembly, which can specifically bind and enrich nutrients required by the scalp, such as iron ions, zinc ions, biotin, etc., and then efficiently deliver these nutrients to hair follicle cells to meet the needs of hair follicle growth and hair care.

[0188] The two peptides form different nanostructures through self-assembly, acting synergistically on the hair follicles and scalp to achieve dual enhancement of hair follicle repair and nutrient supply.

[0189] Preparation process innovation:

[0190] Small molecule peptide synthesis: FRG-Peptide and SNE-Peptide were synthesized separately by solid phase synthesis. Using Fmoc-protected amino acids as raw materials, step-by-step synthesis was carried out on Rink amide resin. After each addition of amino acids, the progress of the reaction was detected by high performance liquid chromatography (HPLC) to ensure the correct extension of the peptide chain. After the synthesis was completed, a mixed solution of trifluoroacetic acid (TFA), water and triisopropylsilane (TIPS) (volume ratio 95:2.5:2.5) was used for deprotection reaction, and the reaction time was 3 hours. After the reaction was completed, TFA was removed by vacuum distillation, and the crude peptide was precipitated with ether, and finally purified by HPLC to obtain a high-purity small molecule peptide.

[0191] Optimization of the self-assembly process: FRG-Peptide was dissolved in a phosphate buffer solution (PBS) at pH 7.4 at a concentration of 1mM and slowly stirred at 4°C for 24 hours to allow it to self-assemble into a nanofiber structure. The morphology and size of the self-assembled structure were observed by transmission electron microscopy (TEM), and it was found that the diameter of the formed nanofibers was about 20-30nm and the length could reach several microns. For SNE-Peptide, it was dissolved in an acetate buffer solution at pH 6.0 at a concentration of 0.5mM and stirred at room temperature for 12 hours to self-assemble into nanoparticles. The particle size distribution of the nanoparticles was detected by dynamic light scattering (DLS), and the average particle size was about 80-100nm. During the self-assembly process, the formation and stability of the self-assembled structure were optimized by adjusting parameters such as the pH value, temperature, peptide concentration and stirring speed of the solution.

[0192] Composite construction: The self-assembled FRG-Peptide nanofibers and SNE-Peptide nanoparticles were mixed in a mass ratio of 1:1, and then evenly dispersed and interacted with each other in PBS at pH 7.0 by ultrasonic treatment (power 200W, time 5 minutes) to form a stable nanocomposite. The surface morphology of the composite was observed by atomic force microscopy (AFM), and it was found that the nanofibers and nanoparticles were intertwined to form a unique three-dimensional network structure, which is conducive to enhancing the stability and function of the composite.

[0193] Loading nutrients: A mixed solution containing iron ions, zinc ions and biotin was added to the nanocomplex and incubated at 37°C for 2 hours. Through inductively coupled plasma mass spectrometry (ICP-MS) detection, it was found that the loading capacity of SNE-Peptide nanoparticles for iron ions reached 50μg / mg, the loading capacity for zinc ions was 30μg / mg, and the loading capacity for biotin was 20μg / mg. The nanocomplex loaded with nutrients was used for subsequent experiments.

[0194] Quality Inspection:

[0195] Purity and structure detection: HPLC was used to detect the purity of small molecule peptides, and the results showed that the purity of FRG-Peptide and SNE-Peptide reached more than 98%. The secondary structure of the peptide was analyzed by circular dichroism (CD), and the results showed that FRG-Peptide formed a β-folded structure after self-assembly, which was consistent with the expected nanofiber formation mechanism; SNE-Peptide formed a random coil and partial α-helical structure after self-assembly, which was conducive to its combination with nutrients and the formation of nanoparticles.

[0196] Self-assembled structure stability test: The nanocomposite was placed in solutions of different temperatures (4°C, 25°C, 37°C) and different pH values ​​(pH5.0-8.0) for 7 days, and the stability of the self-assembled structure was tested by TEM and DLS. The results showed that at 4°C and pH 7.0, the structure of the nanocomposite was the most stable, and the morphology and size of the nanofibers and nanoparticles changed minimally. At 25°C and pH 6.5-7.5, the structure could also remain relatively stable, with only a small amount of nanoparticles agglomerating.

[0197] Nutrient loading stability test: The nanocomplex loaded with nutrients was incubated in a solution simulating the scalp environment (37°C, pH 7.2) for 7 days, and the release of nutrients in the solution was detected by ICP-MS every 24 hours. The results showed that within 7 days, the cumulative release of iron ions was 20% of the loading amount, the cumulative release of zinc ions was 15%, and the cumulative release of biotin was 10%, indicating that the loading of nutrients in the nanocomplex was relatively stable and could achieve slow release.

[0198] Application effect verification:

[0199] In vitro hair follicle cell culture experiment: The nutrient-loaded nanocomplex was co-cultured with hair follicle stem cells, and the cell culture without nanocomplex was used as the control group. During the culture process, the CCK-8 method was used to detect the cell proliferation activity. The results showed that after 72 hours of culture, the proliferation rate of hair follicle stem cells in the experimental group increased by 40% compared with the control group. The differentiation markers of hair follicle cells were detected by immunofluorescence staining, and it was found that the expression of hair follicle differentiation-related proteins in the experimental group increased by 35% compared with the control group, indicating that the nanocomplex can promote the proliferation and differentiation of hair follicle stem cells.

[0200] Animal experiment: Mice were selected, and the nanocomplex loaded with nutrients was topically applied to the hair removal area on the back of the mice, 3 times a week, for 4 weeks. The mice applied with normal saline were used as the control group. After the experiment, hair regeneration was evaluated by hair weighing and hair density measurement. The results showed that the hair weight of the experimental group of mice increased by 50% compared with the control group, and the hair density increased by 45%. Through histological analysis, it was found that the number of hair follicles in the experimental group of mice increased significantly, the hair follicle structure was more complete, and the blood vessel density around the hair follicles also increased, indicating that the nanocomplex can effectively promote hair regeneration and the healthy development of hair follicles.

[0201] Example 10: Preparation and application of small molecule peptides based on microbial fermentation

[0202] Molecular design and technical principles

[0203] Small molecule peptide sequence and design principle:

[0204] In this embodiment, microorganisms are genetically engineered to express small molecule peptides with anti-hair loss and hair care functions during the fermentation process. A hair follicle growth promoting peptide (FGP-Peptide) is designed, and the sequence is NH2-Met-Thr-Asp-Gln-Lys-Trp-COOH.

[0205] Met (methionine), as the starting amino acid, plays an important role in the process of protein synthesis, and it may affect the translation initiation and folding of peptides. The side chains of Thr (threonine) and Asp (aspartic acid) contain hydroxyl and carboxyl groups, which can participate in the formation of hydrogen bonds and regulate the spatial conformation of peptides. The amide group of Gln (glutamine) helps to maintain the stability of peptides and may be related to metabolic processes in cells. Lys (lysine) is positively charged and can interact with negatively charged cell surface molecules to promote the binding of peptides to hair follicle cells. The indole ring structure of Trp (tryptophan) is large and has strong hydrophobicity. It can not only enhance the binding force between peptides and receptors, but also play a role in the intracellular signal transduction process. The peptide activates a series of signal pathways related to hair follicle growth and development by binding to specific receptors on the surface of hair follicle cells, thereby promoting the growth of hair follicles and the health of hair.

[0206] By introducing the gene encoding FGP-Peptide into microorganisms (such as Escherichia coli), the small molecule peptide is produced on a large scale by microbial fermentation. At the same time, the metabolites produced during the microbial fermentation process are used to further enhance the anti-hair loss and hair care effects.

[0207] Preparation process innovation:

[0208] Construction of genetically engineered bacteria: Artificially synthesize the gene sequence encoding FGP-Peptide, and introduce appropriate restriction endonuclease sites at both ends. The gene fragment is inserted into the modified expression vector pET-28a(+) through restriction endonuclease digestion and ligation reaction to construct a recombinant expression vector. The recombinant expression vector is transformed into Escherichia coli BL21(DE3) competent cells, and positive clones are obtained through kanamycin resistance screening and PCR identification. The positive clones are sequenced to ensure the accuracy of the gene sequence.

[0209] Optimization of fermentation conditions: The selected genetically engineered bacteria were inoculated into LB medium containing kanamycin and cultured at 37°C with shaking until the logarithmic growth phase. Then, the bacterial solution was transferred to the fermentation medium (containing glucose, yeast extract, peptone, phosphate, etc.) and fermented at 30°C and 200rpm. The expression of small molecule peptides was increased by optimizing the composition, temperature, pH value, and addition time and concentration of the inducer (IPTG) of the fermentation medium. During the fermentation process, the ventilation volume and feeding strategy were adjusted in time by monitoring the changes in bacterial density (OD600) and pH value. After optimization, the expression level of FGP-Peptide reached 1.5g / L fermentation broth.

[0210] Separation and purification of small molecule peptides: After fermentation, the fermentation broth was centrifuged (8000rpm, 15 minutes) and the bacterial precipitate was collected. After the bacterial precipitate was resuspended with PBS, the cells were broken by ultrasonic disruption (power 300W, working for 3 seconds, rest for 5 seconds, a total of 10 minutes) to release small molecule peptides. Then, the cell debris was removed by centrifugation (12000rpm, 20 minutes), and the supernatant was purified by affinity chromatography. A nickel column (His-Tag affinity chromatography column) was used to separate and purify the peptide by utilizing the specific binding of the His-Tag tag at the N-terminus of FGP-Peptide with nickel ions. Elution was performed with PBS containing different concentrations of imidazole, and the target elution peak was collected. HPLC detection showed that the purity of the purified FGP-Peptide reached more than 95%.

[0211] Compounding of fermentation products: Compound the purified FGP-Peptide with other beneficial metabolites in the fermentation broth (such as B vitamins, amino acids, etc.). By analyzing the components of the fermentation broth, determine the compounding ratio to give full play to the synergistic effect of each component. Concentrate and dry the compounded product to make a powdered preparation for subsequent experiments.

[0212] Quality Inspection:

[0213] Peptide content determination: The content of FGP-Peptide was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). The results showed that the content of FGP-Peptide in the compound preparation was 80 mg / g.

[0214] Bioactivity detection: The bioactivity of FGP-Peptide was detected by in vitro cell experiments. FGP-Peptide was co-cultured with hair follicle stem cells, and the cell proliferation activity was detected by CCK-8 method. The results showed that when the concentration of FGP-Peptide was 20μM, the proliferation rate of hair follicle stem cells increased by 35% compared with the control group. The secretion of cytokines related to hair follicle growth (such as vascular endothelial growth factor VEGF and fibroblast growth factor FGF-7) in the cell culture supernatant was detected by ELISA. The results showed that after adding FGP-Peptide, the secretion of VEGF increased by 40% and the secretion of FGF-7 increased by 30%, indicating that FGP-Peptide can effectively promote the secretion of growth factors by hair follicle cells and promote hair follicle growth.

[0215] Safety testing: The compound preparation was subjected to acute toxicity tests and skin irritation tests. In the acute toxicity test, mice were selected and the compound preparation was administered by gavage at different doses (5g / kg, 10g / kg, 20g / kg). After 7 days of observation, no symptoms of poisoning or death were found in the mice. In the skin irritation test, healthy guinea pigs were selected and the compound preparation was applied to the back skin of the guinea pigs. After 72 hours of observation, no irritation symptoms such as skin redness, swelling, and itching were found, indicating that the compound preparation has good safety.

[0216] Application effect verification:

[0217] Volunteer trial: 50 volunteers with hair loss problems were recruited and randomly divided into an experimental group and a control group, with 25 people in each group. The experimental group used shampoo containing FGP-Peptide compound preparations, and the control group used ordinary shampoo, which was used once a day for 3 months. Before and after use, the volunteers' hair loss amount, hair tensile test (measured by tensile meter when the hair breaks) and hair gloss score (using subjective scoring method, 1-5 points, 5 points for the most glossy) were counted. The results showed that the amount of hair loss of the volunteers in the experimental group was reduced by an average of 45%, the hair tensile force was increased by 30% compared with the control group, the hair gloss score increased from 2 points to 3.5 points, and the satisfaction rate reached 80%, while the improvement effect of the control group was not obvious.

[0218] Long-term follow-up experiment: The volunteers in the experimental group were followed up for 6 months. It was found that after stopping using the shampoo containing the FGP-Peptide compound preparation, the volunteers' hair loss was still maintained at a low level in the next 3 months, and the tensile strength and glossiness of the hair also remained relatively stable, indicating that the preparation has a certain lasting effect.

Claims

1. A small molecule peptide component for preventing hair loss and nourishing hair, characterized in that: Contains at least one of the following small molecule peptides: Hair follicle targeting activation peptide (FTA-Peptide), whose amino acid sequence is NH2-Gly-His-Lys-Arg-Tyr-Cys-COOH; Hair structure repair peptide (HSR-Peptide), whose amino acid sequence is NH2-Ala-Pro-Cys-Ser-Val-Leu-COOH; Scalp microcirculation promoting peptide (SMP-Peptide), whose amino acid sequence is NH2-Arg-Gly-Asp-Ile-Thr-Pro-COOH; Scalp microbial balance regulating peptide (SMR-Peptide), whose amino acid sequence is NH2-Lys-Glu-Trp-Met-Phe-COOH; Antioxidant stress protective peptide (ASP-Peptide), whose amino acid sequence is NH2-His-Gln-Asn-Cys-Val-COOH; Cell signaling regulatory peptide (CSR-Peptide), whose amino acid sequence is NH2-Glu-Asp-Ser-Thr-Ile-Lys-COOH; Light-responsive small molecule peptide (LRA-Peptide), whose amino acid sequence is NH2-Tyr-Phe-Trp-Lys-Cys-His-(Azo)-Gly-COOH, where (Azo) represents an azobenzene group; Hair follicle repair guiding peptide (FRG-Peptide), whose amino acid sequence is NH2-Val-Ile-Phe-Leu-His-Arg-COOH; Scalp Nutrition Enriched Peptide (SNE-Peptide), whose amino acid sequence is NH2-Asp-Glu-Ser-Thr-Lys-Tyr-COOH; Hair follicle growth promoting peptide (FGP-Peptide), whose amino acid sequence is NH2-Met-Thr-Asp-Gln-Lys-Trp-COOH.

2. The anti-hair loss and nourishing small molecule peptide component according to claim 1, characterized in that: The molar ratio of each small molecule peptide in the small molecule peptide component is: FTA-Peptide:HSR-Peptide:SMP-Peptide:SMR-Peptide:ASP-Peptide is 1:1.5:1:1.2:1.

3.

3. A method for preparing the anti-hair loss and nourishing small molecule peptide component according to claim 1, characterized in that: The following steps are involved: The solid phase synthesis method is adopted to gradually synthesize small molecule peptide chains on a solid phase carrier using Fmoc-protected amino acids as raw materials; After the synthesis is completed, a mixed solution of trifluoroacetic acid, water and triisopropylsilane is used for deprotection reaction; The synthesized small molecule peptide is purified by high performance liquid chromatography to achieve a purity of more than 95%.

4. The preparation method according to claim 3, characterized in that: For photoresponsive small molecule peptides containing azobenzene groups, after solid-phase synthesis is completed, the protecting groups at specific positions on the peptide chain are removed through a selective deprotection reaction to expose sites for the connection of azobenzene groups. Then, azobenzene derivatives with active groups are reacted with the peptide chain under mild reaction conditions to introduce the azobenzene groups into the peptide chain through amide bonds.

5. A method for preparing the anti-hair loss and nourishing small molecule peptide component according to claim 1 based on gene editing technology, characterized in that: The following steps are involved: Design gRNA sequences targeting key genes in hair follicle stem cells and mix them with Cas9 protein in a specific buffer solution to form a gRNA-Cas9 protein complex; The gene fragments encoding small molecule peptides, including FTA-Peptide, HSR-Peptide, CSR-Peptide, etc., are inserted into the specific multiple cloning site of the modified adeno-associated virus (AAV) vector, and the coding sequence of the gRNA-Cas9 protein complex is also integrated into the vector; Hair follicle tissue is extracted from the patient, hair follicle stem cells are isolated, and the constructed AAV vector and hair follicle stem cells are transduced through an electroporator; The transduced hair follicle stem cells were cultured in a culture medium containing specific growth factors, labeled with green fluorescent protein (GFP), and combined with flow cytometry to screen out hair follicle stem cells that had been successfully gene-edited and could stably express small molecule peptides.

6. A method for preparing a light-responsive small molecule peptide preparation, characterized in that: The following steps are involved: Synthesize a light-responsive small molecule peptide (LRA-Peptide) and mix it with a light stabilizer and a solubilizer at a mass ratio of 1:0.1:0.05; An appropriate amount of deionized water is added, and after being stirred evenly, a high-pressure homogenizer is used for homogenization to prepare a gel preparation for external use, wherein the average particle size of the small molecule peptide in the preparation is less than 100 nm.

7. A method for preparing a small molecule peptide targeted delivery preparation based on quantum dot labeling, characterized in that: The following steps are involved: CdSe / ZnS quantum dots with an emission wavelength of 520 nm were synthesized by hot injection, and the surface of the quantum dots was modified to give them carboxyl groups. TFS-Peptide (NH2-Arg-Gly-Asp-Tyr-Ser-Cys-COOH) targeting hair follicle stem cells and SVA-Peptide (NH2-Lys-His-Pro-Thr-Val-Phe-COOH) promoting scalp angiogenesis were reacted with the modified quantum dots at room temperature for 4 hours, and the small molecule peptides were connected to the quantum dots through amide bonds. Quantum dot-labeled TFS-Peptide and SVA-Peptide were mixed with hyaluronic acid at a mass ratio of 1:1:5, and an appropriate amount of PBS was added. The mixture was stirred evenly under ultrasonic conditions to prepare a nanocomposite preparation, wherein the average particle size of the nanocomposite was 150 nm.

8. A method for preparing a small molecule peptide complex based on a self-assembled nanostructure, characterized in that: The following steps are involved: The hair follicle repair guide peptide and scalp nutrition enrichment peptide were synthesized separately, and FRG-Peptide was dissolved in a phosphate buffer solution of pH 7.4 at a concentration of 1mM, and slowly stirred at 4°C for 24 hours to self-assemble into a nanofiber structure; SNE-Peptide was dissolved in an acetate buffer solution of pH 6.0 at a concentration of 0.5mM, and stirred at room temperature for 12 hours to self-assemble into nanoparticles; The self-assembled FRG-Peptide nanofibers and SNE-Peptide nanoparticles were mixed at a mass ratio of 1:1, and then evenly dispersed and interacted with each other in PBS at pH 7.0 by ultrasonic treatment to form a stable nanocomposite. A mixed solution containing iron ions, zinc ions and biotin was added to the nanocomplex and incubated at 37°C for 2 hours to load the SNE-Peptide nanoparticles with nutrients.

9. A method for preparing small molecule peptides based on microbial fermentation, characterized in that: The following steps are involved: Artificially synthesizing a gene sequence encoding a hair follicle growth promoting peptide, introducing suitable restriction endonuclease sites at both ends, inserting the gene fragment into a modified expression vector pET-28a(+), and constructing a recombinant expression vector; The recombinant expression vector was transformed into Escherichia coli BL21 (DE3) competent cells, and positive clones were obtained through kanamycin resistance screening and PCR identification, and the positive clones were sequenced and verified; The selected genetically engineered bacteria were inoculated into LB medium containing kanamycin, cultured at 37°C with shaking until the logarithmic growth phase, and then transferred to the fermentation medium for fermentation culture at 30°C and 200rpm. The expression level of FGP-Peptide reached 1.5 g / L fermentation liquid by optimizing the composition, temperature, pH value, addition time and concentration of the fermentation medium; After the fermentation, the fermentation liquid was centrifuged to collect the bacterial precipitate, resuspended in PBS and ultrasonically broken, and then centrifuged to remove cell debris. The supernatant was purified by affinity chromatography using a nickel column and the specific binding of the His-Tag tag at the N-terminus of FGP-Peptide with nickel ions to achieve the separation and purification of the peptide. The purity of the purified FGP-Peptide reached more than 95%; The purified FGP-Peptide is compounded with other beneficial metabolites in the fermentation broth, and after the compounding ratio is determined, it is concentrated and dried to prepare a powdered preparation.

10. The use of the anti-hair loss and nourishing small molecule peptide component according to claim 1 in the preparation of an anti-hair loss and nourishing product, characterized in that: The anti-hair loss and hair care products include shampoo, conditioner, scalp essence, scalp care spray, external gel preparations, etc., which are used to promote the proliferation of hair follicle stem cells, repair hair structure, improve scalp blood circulation, regulate scalp microbial balance, and reduce oxidative stress damage, thereby achieving the effect of anti-hair loss and hair care.