Collagen composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a collagen composition as well as a preparation method and application thereof. According to the invention, PLGA (poly (lactic-co-glycolic acid)) nanoparticles are respectively or simultaneously loaded with COL3 and COL17, and are used for treating androgen-induced alopecia. When PLGA loading is not used, the treatment effect of COL17 combined with COL3 is better than that of single use of COL17 or COL3; after the PLGA is loaded, the curative effect of COL17 or COL3 is improved, the hair growth effect of COL17 and COL3 is obviously better than that of single medication, and especially, the PLGA-loaded COL17 or COL3 has a synergistic effect in the aspects of promoting the length of hair, the number of hair follicles and the thickness of skin. The medicine is carried to hair follicles through the PLGA, so that a synergistic treatment effect can be generated, the problems of high medicine resistance, multiple side reactions, easiness in relapse after medicine withdrawal and the like of a current anti-hair-loss product can be solved, and the application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a collagen composition, a preparation method and uses thereof. Background Art

[0002] Androgenetic alopecia (AGA) is the most common type of hair loss. It is a progressive hair follicle miniaturization disorder that begins in adolescence or late adolescence and affects both men and women. Although AGA does not affect physical health, it seriously affects the patient's mental health and quality of life. Early diagnosis and treatment can significantly slow the progression of hair loss and significantly improve the patient's quality of life and mental health. The pathogenesis of androgenetic alopecia is complex and currently unclear, but is mainly believed to be related to factors such as hormonal imbalance, gene mutation, aging, and autoimmunity.

[0003] An ideal treatment for androgenic alopecia remains lacking. Currently, only oral finasteride and topical minoxidil are approved for market. However, both finasteride and minoxidil have drawbacks, including long treatment cycles, numerous adverse reactions, relapses upon discontinuation, and poor patient compliance. Finasteride is commonly used to treat male androgenic alopecia by blocking the conversion of testosterone to dihydrotestosterone, thereby preventing hair follicle miniaturization. However, to maintain efficacy, it requires daily, long-term administration. Side effects include decreased libido, erectile dysfunction, mood disorders, and gynecomastia. Long-term use may even induce type 2 diabetes, non-alcoholic fatty liver disease, dry eyes, and potential renal impairment. Minoxidil primarily exerts its therapeutic effects through its metabolite, minoxidil sulfate. Therefore, its efficacy is affected by differences in sulfotransferase activity across patients. Topical minoxidil solutions may cause adverse reactions, including itchy and dry skin, contact dermatitis, and facial hirsutism. In order to solve the problems of strong drug resistance, many side effects, and easy recurrence after discontinuation of various anti-hair loss products, it is urgent to develop an anti-hair loss method with high biosafety.

[0004] Type XVII collagen (COL17) is a transmembrane, non-fibroblastic collagen that regulates skin aging and maintains the activity of hair follicle stem cells, offering promising applications in biomedicine and medical aesthetics. A 2016 study published in Science offered new insights into the treatment of androgenic alopecia, noting that maintaining COL17 expression in hair follicle stem cells can significantly prevent or alleviate hair loss by preventing follicle aging. However, COL17 is present in extremely low concentrations in the human body, making its extraction extremely difficult and prohibitive for mass production. Furthermore, animal-derived collagen products pose biosafety risks, such as immunogenicity and potential exposure to viruses and diseases.

[0005] In addition, type III collagen (COL3) is an important part of connective tissue. During wound healing and collagen development, the proportion of COL3 is usually large, reflecting its great potential in promoting wound healing and skin rejuvenation.

[0006] However, since the scalp surface is often covered with oil and hair and is exposed to the outside, it is greatly affected by the environment. How to efficiently deliver drugs and act on hair follicles is the key to the problem.

[0007] Therefore, how to combine COL17 and COL3 and effectively act on hair follicles for preventing or treating hair loss is a problem and challenge facing this field. Summary of the Invention

[0008] In response to the problems of the prior art, the present invention provides a collagen composition and a preparation method thereof, the purpose of which is to combine COL17 and COL3 and act on hair follicles to effectively prevent or treat hair loss.

[0009] The present invention provides a collagen composition, wherein the collagen composition is composed of COL17 and COL3; the COL17 and COL3 are loaded separately or simultaneously into poly(lactic acid-co-glycolic acid) copolymer microspheres;

[0010] The mass ratio of COL17 to COL3 is 1-2:1-2.

[0011] Preferably, the mass ratio of COL17 to COL3 is 1:1.

[0012] Preferably, the mass ratio of the collagen composition to the poly(lactic-co-glycolic acid) microspheres is 45-48:200.

[0013] The present invention provides the collagen composition described in any one of the above items, which is prepared by the following steps:

[0014] COL17 and / or COL3 are mixed with polylactic acid-glycolic acid copolymer microspheres to obtain the product.

[0015] Preferably, the poly(lactic-co-glycolic acid) microspheres are prepared according to a method comprising the following steps:

[0016] Step 1, mixing polylactic acid-glycolic acid with an organic reagent to prepare colostrum;

[0017] Step 2: colostrum is mixed with water and then dripped into a stabilizer aqueous solution to prepare a double emulsion;

[0018] Step 3: Mix the emulsion with water to obtain the product.

[0019] Preferably, the mixing in steps 1 and 2 is performed under oscillation conditions, with an oscillation speed of 600-1000 rpm;.

[0020] And / or, the conditions for dropping the stabilizer into the aqueous solution in step 2 include: temperature of 0-4°C, stirring speed of 6000-9000 r / min, stirring time of 10-20 s, and the stabilizer is selected from polyvinyl alcohol, gelatin, polyvinyl pyrrolidone, and polysorbate;

[0021] And / or, the mixing in step 3 is stirring at a speed of 400-600 r / min for 4-6 hours and standing for 0.5-1 hour.

[0022] Preferably, in step 1, the mass percentage of polylactic acid-glycolic acid in the colostrum is 18-25%; the organic reagent is selected from at least one of dichloromethane, chloroform, ethyl acetate, toluene, and cyclohexane;

[0023] And / or, in step 2, the mass volume ratio of polylactic acid-glycolic acid in colostrum to the water and the stabilizer aqueous solution is 50-70 mg:0.8-1 ml:40-50 ml, and the mass concentration of the stabilizer aqueous solution is 1.5-2.5%.

[0024] And / or, in step 3, the mass volume ratio of the polylactic acid-glycolic acid in the emulsion to the water is 50-70 mg: 250-300 ml.

[0025] Preferably, COL17 and / or COL3 are prepared using genetic engineering technology.

[0026] Preferably, the amino acid sequence of COL17 is shown as SEQ ID NO.1; the amino acid sequence number of COL3 is UniProtKB / Swiss-Prot: P02461.4.

[0027] The present invention provides a method for preparing any of the above-mentioned collagen compositions, comprising the following steps:

[0028] COL17 and / or COL3 are mixed with polylactic acid-glycolic acid copolymer microspheres to obtain the product.

[0029] The present invention also provides use of any of the above-mentioned collagen compositions in the preparation of a medicament for preventing and / or treating androgenic alopecia.

[0030] The present invention uses poly(lactic-co-glycolic acid) nanospheres (PLGA) loaded separately or simultaneously with COL3 and COL17 for the treatment of androgen-induced alopecia. The present invention optimizes the ratio of the raw materials and the preparation method. Experimental results demonstrate that PLGA, as a delivery system, enhances the hair growth effect of collagen drugs in animals. Without PLGA loading, COL17 and / or COL3 exhibit some efficacy in treating alopecia. However, after PLGA loading, when rhCOL17 and rhCOL3 were mixed and administered at a 1:1 concentration ratio, the hair growth effect was superior to that of either drug alone; in particular, a synergistic effect was observed in promoting hair length, increasing hair follicle number, and increasing skin thickness. By delivering drugs to hair follicles via PLGA, the present invention not only achieves a synergistic therapeutic effect but also addresses the challenges of current anti-hair loss products, such as strong drug resistance, numerous side effects, and recurrence upon discontinuation of treatment. The application prospects are promising.

[0031] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0032] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the preparation of PLGA;

[0034] Figure 2 This is a scanning electron micrograph of drug-loaded microspheres PLGA-rhCOL17, with a scale of 5 μm;

[0035] Figure 3 This is a scanning electron micrograph of drug-loaded microspheres PLGA-rhCOL17, with a scale of 2 μm;

[0036] Figure 4 The particle size distribution of drug-loaded microspheres PLGA-rhCOL17 was measured by a laser particle size analyzer;

[0037] Figure 5 This is the zeta potential result of drug-loaded microspheres PLGA-rhCOL17 measured by dynamic light scattering;

[0038] Figure 6 The back images of mice in each group on treatment day 0;

[0039] Figure 7 The back images of mice in each group on the 21st day of treatment;

[0040] Figure 8 The hair growth over time in the hair-depilating areas on the backs of mice in each group was captured by dermatoscope in Experimental Example 2;

[0041] Figure 9 The following are hematoxylin-eosin (HE) staining images of the hair loss area on the back of mice in each group in Experimental Example 2;

[0042] Figure 10 The back images of mice in each group during the treatment process in Experimental Example 3;

[0043] Figure 11 The back images of mice in each group during the treatment process in Experimental Example 4;

[0044] Figure 12 The hair growth over time in the depilated areas on the backs of mice in each group was captured by dermatoscope in Experimental Example 4;

[0045] Figure 13 Statistical graph of hair length of mice in each group in Experimental Example 4;

[0046] Figure 14 HE staining of the hair-deprived area tissues on the backs of mice in each group in Experimental Example 4;

[0047] Figure 15 This is a statistical chart of the number of hair follicles in each group of mice in Experimental Example 4;

[0048] Figure 16 Statistical graph of skin thickness of mice in each group in Experimental Example 4;

[0049] Figure 17 This is a diagram showing the expression of protein markers of the germinal pathway in each group of mice in Experimental Example 4. DETAILED DESCRIPTION

[0050] In the following examples and experimental examples, reagents and materials not otherwise specified are commercially available.

[0051] In this invention, the concentration of collagen-encapsulated PLGA microspheres is calculated based on the concentration of the encapsulated collagen. For example, 6 mg / ml of PLGA-rhCOL17 means that the concentration of rhCOL17 in the PLGA-rhCOL17 is 6 mg / ml; 10 mg / ml of COL3+COL17@PLGA means that the total concentration of COL3 and COL17 in the COL3+COL17@PLGA is 10 mg / ml.

[0052] Example 1 A collagen composition

[0053] This embodiment provides a composition of COL17 and COL3 and a preparation method thereof

[0054] 1. Preparation of recombinant collagen

[0055] (1) Preparation of rhCOL17

[0056] Recombinant human COL17 (rhCOL17) was produced using genetic engineering and proteomics techniques. The present invention prioritizes the recombinant sequence and expression system of rhCOL17. The specific preparation method follows the steps described in patent application number 2021105204999. The amino acid sequence of rhCOL17 is SEQ ID NO. 1, which is as follows:

[0057] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPPGPK GDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSGPPGPPGPQGPKGDKGDPGVPGALGIPGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ (233 amino acids in total).

[0058] (2) Preparation of rhCOL3

[0059] Type III collagen (rhCOL3) was produced using genetic engineering and proteomics techniques. The specific preparation method followed the steps described in patent application number 2021116128490. The amino acid sequence of rhCOL3 is numbered UniProtKB / Swiss-Prot: P02461.4. The amino acid sequence of rhCOL3 can be found at NCBI: https: / / www.ncbi.nlm.nih.gov / protein / P02461.4.

[0060] 2. Preparation of collagen-encapsulated PLGA microspheres PLGA-rhCOL17 and PLGA-rhCOL3

[0061] (1) Preparation of blank PLGA microspheres

[0062] The preparation method of PLGA microspheres is as follows Figure 1 Shown: 2g polyvinyl alcohol (molecular weight M w89,000-98,000 g / mol; Sigma-Aldrich, product number 341584; referred to as PVA) was added with 100 ml of deionized water, placed on a heat-collecting magnetic stirrer, slowly heated to 90°C, and stirred continuously until the PVA was completely dissolved to prepare a 2% PVA external phase water;

[0063] 60 mg of PLGA (molecular weight 30,000-60,000 g / mol; Sigma-Aldrich, product number P2191) was placed in a 15 ml centrifuge tube and transferred to a fume hood. 3 ml of dichloromethane was added and shaken at high speed until the PLGA was completely dissolved to prepare 20% colostrum.

[0064] Add 0.9 ml of deionized water to the colostrum, shake at high speed for 10 seconds, quickly transfer the resulting solution to a 5 ml syringe, quickly and evenly add it dropwise to 30 ml of 2% PVA external phase water, and stir with a magnetic stirrer at 8000 rpm for 15 seconds under ice bath to prepare a double emulsion;

[0065] Transfer the emulsion solution to a beaker containing 270 ml of deionized water and place it on a magnetic stirrer at 500 rpm for 4 hours to evaporate the dichloromethane. After the solvent evaporates, stop stirring and let the mixture stand for 0.5 hours to obtain a white precipitate, which is the blank PLGA microspheres. Collect the white precipitate in a centrifuge tube, wash it with double-distilled water, and centrifuge it at 4000 rpm for 5 minutes at room temperature. Repeat the washing process three times by pipetting. Freeze-dry the collected blank PLGA microspheres in a vacuum freeze dryer for 24 hours and store them at -20°C.

[0066] (2) Preparation of PLGA-rhCOL17 / PLGA-rhCOL3

[0067] Under sterile conditions, dilute lyophilized collagen powder (rhCOL17 or rhCOL3) with secondary water to prepare a collagen solution at a concentration of 10 mg / ml. Mix 0.2 g of blank PLGA microspheres with 5 ml of the collagen solution, shake well, and let stand for 30 minutes. Collect the microspheres to prepare PLGA-rhCOL17 or PLGA-rhCOL3. Freeze-dry and store at -20°C.

[0068] (3) Preparation of collagen composition

[0069] This embodiment provides a collagen composition, which can be used simultaneously or separately, and the amount of PLGA-rhCOL17 and PLGA-rhCOL3 used is a mass ratio of 1:1.

[0070] As a preferred solution, the collagen composition is an aqueous solution containing 3 mg / ml PLGA-rhCOL17 and 3 mg / ml PLGA-rhCOL3 used simultaneously.

[0071] Example 2 A collagen composition

[0072] Collagen-encapsulated PLGA microspheres COL3+COL17@PLGA were prepared according to the method of Example 1, except that the collagen solution was prepared by mixing 20 mg of rhCOL17 collagen lyophilized powder and 10 mg of rhCOL3 collagen lyophilized powder, and diluting with 5 ml of water, so that the total collagen concentration was 6 mg / ml.

[0073] Example 3 A collagen composition

[0074] Collagen-encapsulated PLGA microspheres COL3+COL17@PLGA were prepared according to the method of Example 1, except that the collagen solution was prepared by mixing 10 mg of rhCOL17 collagen lyophilized powder and 20 mg of rhCOL3 collagen lyophilized powder, and diluting with 5 ml of water, so that the total collagen concentration was 6 mg / ml.

[0075] Example 4 A collagen composition

[0076] Collagen-encapsulated PLGA microspheres COL3+COL17@PLGA were prepared according to the method of Example 1, except that the collagen solution was prepared by mixing 15 mg of rhCOL17 collagen lyophilized powder and 15 mg of rhCOL3 collagen lyophilized powder, and diluting with 5 ml of water, so that the total collagen concentration was 6 mg / ml.

[0077] Example 5 A collagen composition

[0078] Collagen-encapsulated PLGA microspheres COL3+COL17@PLGA were prepared according to the method of Example 1, except that the collagen solution was prepared by mixing 25 mg of rhCOL17 collagen lyophilized powder and 25 mg of rhCOL3 collagen lyophilized powder, and diluting with 5 ml of water, so that the total collagen concentration was 10 mg / ml.

[0079] The technical solution of the present invention is further illustrated by experiments below. The samples rhCOL17, rhCOL3, PLGA-rhCOL17, PLGA-rhCOL3 and COL3+COL17@PLGA in the following experimental examples were prepared by the methods of Examples 1-5, respectively.

[0080] Experimental Example 1 Characterization of drug-loaded microspheres

[0081] 1. Experimental Methods

[0082] The samples tested in this experimental example were PLGA-rhCOL17 and PLGA-rhCOL3 prepared according to the method of Example 1, and COL3+COL17@PLGA prepared according to the method of Example 5.

[0083] 1. Characterization of particle size

[0084] The drug-loaded microspheres were characterized by scanning electron microscopy and laser particle size analyzer, respectively.

[0085] 2. Determination of drug encapsulation efficiency

[0086] Encapsulation efficiency (%) = actual encapsulated drug amount / initial drug amount × 100%

[0087] 2. Experimental Results

[0088] Characterization results such as Figure 1-5 As shown, the drug-loaded microspheres have a uniform particle size of approximately 200 nm. The encapsulation efficiency of COL17 is 92.19%, the encapsulation efficiency of COL3 is 95.87%, and the encapsulation efficiency of COL17 + COL3 is 92.36%. This demonstrates that the preparation method of the present invention can produce nano-sized collagen materials with high encapsulation efficiency.

[0089] Experimental Example 2: PLGA loaded with COL17 and COL3 for the treatment of hair loss

[0090] 1. Experimental Methods

[0091] 1. Establishment of an animal model of hair loss

[0092] Male C57 mice aged 6-7 weeks were selected as experimental animals.

[0093] The experimental mice were anesthetized by intraperitoneal injection of 4% chloral hydrate solution. A patch of approximately 2х4 cm was selected at 0.5 cm from the proximal end of the tail. 2 Use a pet-grade shaver to shave the hair on the back of each mouse, with its long side parallel to the depilatory area of the spine. Shave the hair on the back of each mouse short enough to barely show the back skin. Apply depilatory cream evenly to the shaved area. Soak a cotton ball with saline and use the moistened cotton ball to remove the depilatory cream and any remaining hair.

[0094] One day after hair removal, dihydrotestosterone (DHT) suspension was uniformly injected subcutaneously at multiple sites. Using a 1ml syringe, multiple injections were made subcutaneously in the hairless area on the back of the mouse, ensuring that the needle puncture depth reached the dermis. The dose was 10 mg / kg / day for 4 consecutive weeks.

[0095] 2. Experimental Grouping

[0096] This study was divided into eight groups: untreated, blank control, rhCOL17, rhCOL3, rhCOL17+rhCOL3, PLGA-rhCOL17, PLGA-rhCOL3, and PLGA-rhCOL17+PLGA-rhCOL3. In the untreated group, mice with alopecia received no treatment and were fed normally. In the experimental groups, drugs were administered via microneedle in a 0.2 ml volume. Dosing was once daily for 21 days. The rhCOL17 and rhCOL3 groups received 6 mg / ml of rhCOL17 and rhCOL3, respectively. The rhCOL17+rhCOL3 group received a combination of 0.1 ml of 3 mg / ml rhCOL17 and 0.1 ml of 3 mg / ml rhCOL3. The PLGA-rhCOL17 group received 6 mg / ml of PLGA-rhCOL17 and the PLGA-rhCOL3 group received 6 mg / ml of PLGA-rhCOL3. The PLGA-rhCOL17+PLGA-rhCOL3 group received a combination of 0.1 ml of 3 mg / ml PLGA-rhCOL17 and 0.1 ml of 3 mg / ml PLGA-rhCOL3. A blank control group received PBS in the same manner.

[0097] 3. Determination of hair length

[0098] The hair-removed areas on the backs of each group of mice were observed using a dermatoscope. The measurement method was as follows: Hair growth, skin surface structure (such as changes in sebaceous glands, blood vessels, and the stratum corneum) on the backs of the mice were observed using a handheld dermatoscope to detect signs of inflammation, damage, or regeneration. The data were quantitatively analyzed using ImageJ to further assess hair recovery.

[0099] 4. Determination of hair follicle number, hair follicle size and skin thickness

[0100] The dorsal skin of the mouse was carefully incised using surgical scissors and forceps. The removed sample was immediately fixed in a 10% neutral formalin solution for 24 hours, then dehydrated with a gradient of alcohol and then transparentized with xylene. After transparency, the tissue was transferred to molten paraffin for embedding and then cut into 5μm thick sections using a microtome. Histological sections were stained with hematoxylin and eosin (HE), which clearly demonstrated the structure of the various layers of the skin.

[0101] ① Number of hair follicles: within a certain area (1cm 2 ) and count the number of hair follicles. Pay attention to the different developmental stages of the hair follicles (such as the resting phase, the growth phase, and the catagen phase).

[0102] ② Hair follicle size: Use microscopic image processing software for quantitative analysis, measure the diameter or area of hair follicles, and compare the changes in hair follicle size among mice in different groups.

[0103] ③ Skin thickness: Measure the thickness of the epidermis + dermis of the mouse back skin, and measure the thickness of 3 points in one field of view and take the average value.

[0104] 2. Experimental Results

[0105] 1. The effect of drugs on hair length

[0106] The pictures of mice in each group before and after treatment are as follows: Figure 6 、 7 The hair length results are shown in Figure 8 As shown in Tables 1 and 2, compared to the blank control group, both the rhCOL17 and rhCOL3 groups showed longer hair, with the rhCOL17+rhCOL3 group showing comparable growth effects to the rhCOL17 group. When rhCOL17 was loaded with PLGA, hair length was further increased, demonstrating that PLGA loading facilitates the hair growth-promoting effect of rhCOL17. When PLGA-rhCOL17 and PLGA-rhCOL3 were co-administered at a 1:1 ratio, hair length in mice was significantly increased, with a stronger growth effect than either component alone at equal concentrations, demonstrating a synergistic effect.

[0107] The above results show that when PLGA is not used for loading, the effect of rhCOL17 and rhCOL3 in promoting hair growth is weak; however, after using PLGA for loading, when the two components of PLGA-rhCOL17 and PLGA-rhCOL3 are used at equal concentrations, the hair length growth effect is significantly improved after combined use, resulting in a synergistic effect.

[0108] Table 1 Statistics of hair length

[0109]

[0110] Note: p is the result compared with the blank control group, and “-” means p>0.05.

[0111] Table 2 Dunnett t (two-sided) test results

[0112]

[0113] 2. Effects of drugs on the number of hair follicles, hair follicle size, and skin thickness

[0114] The results are as follows Figure 9As shown, the results of the number of hair follicles per unit, hair follicle size, and skin thickness can be obtained from the back tissue sections. When PLGA-rhCOL17 and PLGA-rhCOL3 are mixed and administered at a concentration ratio of 1:1, the hair growth effect is the best.

[0115] These results demonstrate that PLGA, as a delivery system, promotes the hair growth effect of collagen drugs in animals. The best hair growth effect was achieved when PLGA-rhCOL17 and PLGA-rhCOL3 were mixed and administered at a 1:1 concentration ratio.

[0116] Experimental Example 3 Screening of the ratio of PLGA loaded with COL17 and COL3

[0117] COL3+COL17@PLGA were prepared according to the methods of Examples 2-4. These materials were then subjected to the following assays to identify the optimal ratio of COL3 and COL17 for combined treatment of hair loss. Since the above experiments have demonstrated that PLGA as a delivery system can achieve better hair growth, we will no longer conduct experiments using only COL17 and / or COL3.

[0118] 1. Experimental Methods

[0119] 1. Establishment of an animal model of hair loss

[0120] A hair loss mouse model was established according to the steps described in Experimental Example 2, Experimental Method 1.

[0121] 2. Experimental Grouping

[0122] This experiment was divided into four groups: a control group, a COL3:COL17 = 2:1 group, a COL3:COL117 = 1:2 group, and a COL3:COL17 = 1:1 group. The total collagen concentration in each group was 6 mg / ml. In the COL3:COL17 = 2:1 group, the COL3 concentration was 4 mg / ml and the COL17 concentration was 2 mg / ml; in the COL3:COL117 = 1:2 group, the COL3 concentration was 2 mg / ml and the COL17 concentration was 4 mg / ml; and in the COL3:COL17 = 1:1 group, the COL3 and COL17 concentrations were both 3 mg / ml. The dosing volume for each group was 0.2 ml. Dosing was performed once daily for 21 days via microneedle delivery. The control group received a PBS injection using the same dosing method.

[0123] 3. Measurement of hair length, number of hair follicles, hair follicle size and skin thickness

[0124] The hair length was measured according to the steps described in Experimental Method 3 of Experimental Example 2; the number of hair follicles, the size of hair follicles and the skin thickness were measured according to the steps described in Experimental Method 4 of Experimental Example 2.

[0125] 2. Experimental Results

[0126] 1. The effect of drugs on hair growth

[0127] During the treatment, the hair growth of mice in each group was as follows Figure 10 As shown in Table 3, after 21 days of treatment, hair growth was observed in all groups. Compared to the control group, the COL3:COL17 = 2:1 group showed a 77.9% increase in hair growth, the COL3:COL117 = 1:2 group showed a 93.0% increase in hair growth, and the COL3:COL17 = 1:1 group showed a 123% increase in hair growth. These results indicate that a COL3:COL17 = 1:1 ratio is most effective for hair growth.

[0128] Table 3 Effects of drugs on hair growth

[0129]

[0130] 2. Effects of drugs on hair follicles and skin thickness

[0131] The effects on hair follicles during treatment are shown in Table 4. Compared to the control group, the COL3:COL17 = 2:1 group saw a 49.0% increase in hair follicles, the COL3:COL117 = 1:2 group saw a 91.9% increase in hair follicles, and the COL3:COL17 = 1:1 group saw a 149% increase in hair follicles. These results indicate that a COL3:COL17 = 1:1 ratio is most effective in promoting hair follicle growth.

[0132] Table 4 Effects of drugs on the number of hair follicles

[0133]

[0134] The results of the effects on skin thickness are shown in Table 5. Compared to the control group, the skin thickness increased by 14.3% in the COL3:COL117 = 2:1 group, 24.7% in the COL3:COL117 = 1:2 group, and 48.4% in the COL3:COL17 = 1:1 group. The results show that the COL3:COL17 = 1:1 ratio has the greatest effect on promoting skin thickness.

[0135] Table 5 Effects of drugs on skin thickness

[0136]

[0137] The above results show that when PLGA is loaded with COL17 and COL3 at the same time for the treatment of hair loss, the COL3:COL17=1:1 ratio has the best effect in promoting hair growth, hair follicles and skin thickness.

[0138] Experimental Example 4: PLGA loaded with COL17 and COL3 for the treatment of hair loss

[0139] Since the above experimental examples have demonstrated that PLGA as a delivery system can achieve better hair growth effects, we will no longer conduct experiments using only COL17 and / or COL3. We prepared COL3+COL17@PLGA according to the method of Example 4, and prepared PLGA-rhCOL17 (abbreviated as COL17@PLGA) and PLGA-rhCOL3 (abbreviated as COL3@PLGA) according to the method of Example 1. These materials were subjected to the following assays to evaluate the efficacy of the combination of COL3 and COL17 in treating hair loss.

[0140] 1. Experimental Methods

[0141] 1. Establishment of an animal model of hair loss

[0142] A hair loss mouse model was established according to the steps described in Experimental Example 2, Experimental Method 1.

[0143] 2. Experimental Grouping

[0144] This experiment was divided into four groups: a control group, a COL3@PLGA group, a COL17@PLGA group, and a COL3+COL17@PLGA group. The COL3@PLGA group received 5 mg / ml of COL3@PLGA, the COL17@PLGA group received 5 mg / ml of COL17@PLGA, and the COL3+COL17@PLGA group received 6 mg / ml of COL3+COL17@PLGA. The dosing volume was 0.2 ml. Dosing was via microneedle delivery, once daily for 21 days. The control group received PBS injections using the same dosing method.

[0145] 3. Measurement of hair length, number of hair follicles, hair follicle size and skin thickness

[0146] The hair length was measured according to the steps described in Experimental Method 3 of Experimental Example 2; the number of hair follicles, the size of hair follicles and the skin thickness were measured according to the steps described in Experimental Method 4 of Experimental Example 2.

[0147] 4. Determination of hair keratin markers, VDR pathway markers, canonical Wnt pathway markers, hair follicle stem cells and homeostasis markers

[0148] The hairless and non-hairless skin tissues from the back of the mouse were added to TRIzol reagent respectively, minced, and prepared into tissue homogenates on a homogenizer. Total RNA was extracted and purified using the RNeasy Plus Mini kit strictly according to the instructions. About 1 μg of RNA was used as a template and cDNA was synthesized using a reverse transcription kit. 1 μl of cDNA was used as a template and SYBR Green reagent was added to amplify the target gene. The primer sequences required for qRT-PCR are as follows. PCR reaction conditions: 95°C, 5 min; 95°C, 10 s, 60°C, 30 s, 40 cycles. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as the internal reference gene, and 2 -ΔΔCt The relative mRNA expression levels of hair keratin genes keratin 31 (Ha1), keratin 84 (Krt2-16), VDR / β-catenin pathway-related gene S100a3, canonical Wnt signaling pathway protein Wnt10b, and hair follicle stem cell markers Lxh2 and Sox9 were calculated by the method.

[0149] 2. Experimental Results

[0150] 1. The effect of drugs on hair growth

[0151] During the treatment, the hair growth of mice in each group was as follows Figure 11-13 As shown in Table 6, after 21 days of treatment, hair growth was observed in all groups. Compared to the control group, hair growth in the COL17@PLGA group increased by 55.9%, in the COL17@PLGA group by 36.7%, and in the COL3+COL17@PLGA group by 62.6%. These results indicate that COL3+COL17@PLGA has the greatest hair growth effect in areas of hair loss.

[0152] Table 6 Effects of drugs on hair growth

[0153]

[0154] 2. Effects of drugs on hair follicles and skin thickness

[0155] Effects on hair follicles Figure 14 、 15 As shown in Table 7, compared with the control group, the number of hair follicles increased by 3.66% in the COL3@PLGA group, 16.4% in the COL17@PLGA group, and 63.7% in the COL3+COL17@PLGA group. These results demonstrate that COL3+COL17@PLGA is most effective in increasing the number of hair follicles in areas of hair loss, significantly surpassing the combined effects of COL3@PLGA and COL17@PLGA, demonstrating a synergistic effect.

[0156] Table 6 Effects of drugs on the number of hair follicles

[0157]

[0158] Effects on skin thickness Figure 14 、 16 As shown in Table 7, compared with the control group, skin thickness increased by 39.6% in the COL3@PLGA group, 5.49% in the COL17@PLGA group, and 46.4% in the COL3+COL17@PLGA group. These results demonstrate that COL3+COL17@PLGA is most effective in promoting skin thickness growth in areas of hair loss, and this effect is superior to the combined effects of COL3@PLGA and COL17@PLGA, demonstrating a synergistic effect.

[0159] Table 7 Effects of drugs on skin thickness

[0160]

[0161] 3. Effects of drugs on protein markers of hair growth pathways

[0162] The results are as follows Figure 17 As shown in the figure: The COL3+COL17@PLGA group had the highest expression level of hair keratin, indicating the best hair growth and differentiation; the levels of VDR and Wnt pathway markers were high, and the expression of the VDR / β-catenin pathway gene S100a3 was upregulated, suggesting that the promotion of hair follicle terminal differentiation is related to the upregulation of this pathway expression; the upregulation of the canonical Wnt signaling pathway marker Wnt10b suggested that the Wnt signaling pathway may be involved in the improvement of hair follicle homeostasis and hair follicle cycle regulated by COL17; the expression of hair follicle stem cells and homeostasis markers was high, indicating that the hair follicles in the COL3+COL17@PLGA group were in a relatively stable growth cycle.

[0163] These results demonstrate that, compared with COL3@PLGA and COL17@PLGA, COL3+COL17@PLGA significantly improves hair length, follicle number, and surface thickness in areas of androgenic alopecia, as well as the expression of protein markers involved in hair growth pathways. In particular, it exhibits a synergistic effect in promoting hair follicle number and skin thickness.

[0164] The above examples and experimental examples demonstrate that the present invention utilizes PLGA loaded with COL3 and COL17, either separately or simultaneously, for the treatment of androgen-induced alopecia. Experimental results demonstrate that, without PLGA loading, rhCOL17 and / or rhCOL3 are less effective in treating alopecia. However, PLGA loading further enhances the hair growth effect of the collagen drug in animals. When rhCOL17 and rhCOL3 are coadministered at a 1:1 concentration ratio, the hair growth effect is superior to that achieved with either drug alone. In particular, significant synergistic effects are observed in promoting hair length, increasing hair follicle number, and increasing skin thickness.

Claims

1. A collagen composition, characterized in that: The collagen composition is composed of COL17 and COL3; the COL17 and COL3 are loaded separately or simultaneously into poly(lactic acid-co-glycolic acid) microspheres; The mass ratio of COL17 to COL3 is 1-2:1-2.

2. The collagen composition according to claim 1, characterized in that: The mass ratio of COL17 to COL3 is 1:

1.

3. The collagen composition according to claim 1, wherein: The mass ratio of the collagen composition to the polylactic acid-glycolic acid copolymer microspheres is 45-48:

200.

4. The collagen composition according to any one of claims 1 to 3, characterized in that Prepared according to the following steps: COL17 and / or COL3 are mixed with polylactic acid-glycolic acid copolymer microspheres to obtain the product.

5. The collagen composition according to claim 4, characterized in that The polylactic acid-co-glycolic acid microspheres are prepared according to a method comprising the following steps: Step 1, mixing polylactic acid-glycolic acid with an organic reagent to prepare colostrum; Step 2: colostrum is mixed with water and then dripped into a stabilizer aqueous solution to prepare a double emulsion; Step 3: Mix the emulsion with water to obtain the product.

6. The collagen composition according to claim 5, characterized in that: The mixing in steps 1 and 2 is performed under oscillation conditions, with an oscillation speed of 600-1000 rpm; And / or, the conditions for dropping the stabilizer into the aqueous solution in step 2 include: temperature of 0-4°C, stirring speed of 6000-9000 r / min, stirring time of 10-20 s, and the stabilizer is selected from polyvinyl alcohol, gelatin, polyvinyl pyrrolidone, and polysorbate; And / or, the mixing in step 3 is stirring at a speed of 400-600 r / min for 4-6 hours and standing for 0.5-1 hour.

7. The collagen composition according to claim 5, characterized in that: In step 1, the mass percentage of polylactic acid-glycolic acid in the colostrum is 18-25%; the organic reagent is selected from at least one of dichloromethane, chloroform, ethyl acetate, toluene, and cyclohexane; And / or, in step 2, the mass volume ratio of polylactic acid-glycolic acid in colostrum to the water and the stabilizer aqueous solution is 50-70 mg:0.8-1 ml:40-50 ml, and the mass concentration of the stabilizer aqueous solution is 1.5-2.5%. And / or, in step 3, the mass volume ratio of the polylactic acid-glycolic acid in the emulsion to the water is 50-70 mg: 250-300 ml.

8. The collagen composition according to claim 4, characterized in that: COL17 and / or COL3 are prepared by using genetic engineering technology.

9. The method for preparing the collagen composition according to any one of claims 1 to 8, characterized in that: It includes the following steps: COL17 and / or COL3 are mixed with polylactic acid-glycolic acid copolymer microspheres to obtain the product.

10. Use of the collagen composition according to any one of claims 1 to 8 in the preparation of a medicament for preventing and / or treating androgenic alopecia.

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