Composition for promoting hair regeneration after chemotherapy, preparation method and application thereof

Abstract

The present invention provides a composition for promoting hair regeneration after chemotherapy, as well as its preparation method and application, belonging to the field of medical technology. Gold nanocages are surface-modified with polydopamine and loaded onto exosomes to complex magnesium and zinc ions to produce modified exosomes. Curcumin, resveratrol, and epigallocatechin gallate are then mixed with the modified exosomes to produce multilamellar liposomes. The liposomes are then mixed with a permeation enhancer and added to a thermosensitive hydrogel system to produce a composition for promoting hair regeneration after chemotherapy. The composition exhibits excellent transdermal absorption and slow drug release. The synergistic effects of the drugs promote the transition of hair follicles from the resting phase to the anagen phase, while simultaneously modulating multiple pathways (such as anti-apoptosis, angiogenesis, and anti-inflammation) to address the complex pathological mechanisms of hair regeneration after chemotherapy, offering broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition for promoting hair regeneration after chemotherapy, and a preparation method and application thereof. Background Art

[0002] One of the most common side effects of chemotherapy treatment for cancer patients is hair loss within one to two weeks. This psychological impact is particularly pronounced in women, and it is a distinct problem. The cause of post-chemotherapy hair loss differs from that caused by androgens, and there is no effective treatment. Currently, the only clinical option during chemotherapy is to apply a -15°C medical ice cap to the scalp to reduce capillary blood flow and thus reduce hair follicle cell apoptosis. However, this procedure is cumbersome and lacks user experience, making it less commonly used.

[0003] Common chemotherapy drugs mainly block the cell cycle, and a large proportion of them will cause hair loss. Hair growth mainly goes through the growth phase, the resting phase and the regression phase, of which about 90% of hair is in the growth phase. Chemotherapy-induced hair loss mainly affects the hair follicles in the growth phase. The main feature of the hair follicles in the growth phase is the proliferation of the epithelial chamber, and the hair matrix cells show the greatest proliferation activity in order to form the hair shaft. Chemotherapy drugs can cause the mitotic activity of the hair matrix cells to suddenly stop, resulting in the reduction and narrowing of the proximal end of the keratinized hair shaft, causing the hair tube to rupture, causing hair loss. The speed of hair loss is fast and the amount is large (80%-90%), which often occurs within a few days to a few weeks after chemotherapy.

[0004] There are no reports on the use of exosomes as a drug for the treatment and prevention of chemotherapy-induced hair loss. While exosomes have been widely used in traditional hair loss treatments (e.g., patent CN115227721A), they are primarily used for androgenic alopecia, seborrheic alopecia, and post-transplant hair loss, but not for the treatment and prevention of chemotherapy-induced hair loss. Summary of the Invention

[0005] The purpose of the present invention is to propose a composition for promoting hair regeneration after chemotherapy, as well as its preparation method and application. The composition has good transdermal absorption promotion effect, can slowly release drugs, and promotes the transformation of hair follicles from the resting phase to the growth phase under the synergistic effect of drugs. It also regulates multiple pathways (such as anti-apoptosis, angiogenesis promotion, and anti-inflammation), matching the complex pathological mechanism of hair regeneration after chemotherapy, and has broad application prospects.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present invention provides a method for preparing a composition for promoting hair regeneration after chemotherapy. The gold nanocages are surface-modified with polydopamine and loaded onto exosomes. Magnesium ions and zinc ions are complexed to prepare modified exosomes. Curcumin, resveratrol, and epigallocatechin gallate are used to prepare nano-drug-loaded liposomes, which are mixed with the modified exosomes to prepare multilamellar liposomes. The liposomes are then mixed with a permeation enhancer and added to a thermosensitive hydrogel system to prepare the composition for promoting hair regeneration after chemotherapy.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Preparation of modified gold nanocages: Gold nanocages were added to a Tris-HCl solution, dopamine hydrochloride was added, the reaction was heated with stirring, centrifuged, washed, and dried to obtain modified gold nanocages;

[0010] S2. Preparation of drug-loaded nanoliposomes: Phosphatidylcholine, cholesterol, curcumin, resveratrol, and epigallocatechin gallate were dissolved in a dichloromethane-ethanol mixture. Vitamin B5-PBS buffer solution was added dropwise, stirred, and the dichloromethane and ethanol were removed by rotary evaporation. The mixture was sonicated and freeze-dried to prepare drug-loaded nanoliposomes.

[0011] S3. Preparation of modified exosomes: Exosomes were added to water, followed by NHS and EDC, and activated with stirring. Modified gold nanocages were added, and the mixture was incubated with stirring. Magnesium and zinc salts were added, and the mixture was complexed with stirring. The mixture was dialyzed, and the non-permeated solution was freeze-dried to obtain modified exosomes.

[0012] S4. Preparation of multilamellar liposomes: Phosphatidylcholine and cholesterol were dissolved in a dichloromethane-ethanol mixture, and PBS buffer containing drug-loaded nanoliposomes and modified exosomes was added. The mixture was stirred, and the dichloromethane and ethanol were removed by rotary evaporation under reduced pressure. The mixture was sonicated and freeze-dried to prepare multilamellar liposomes.

[0013] S5. Preparation of thermosensitive hydrogel: Chitosan was dissolved in acid solution, and poloxamer P407 and P188 were added, along with multilamellar liposomes and a permeation enhancer. The mixture was stirred and mixed to obtain a composition for promoting hair regeneration after chemotherapy.

[0014] As a further improvement of the present invention, the pH value of the Tris-HCl solution in step S1 is 8.5-9.5, the mass ratio of the gold nanocages and dopamine hydrochloride is 7-10:2-4, the temperature of the heating and stirring reaction is 45-55°C, and the time is 2-4 hours; the mass ratio of lecithin, cholesterol, curcumin, resveratrol, epigallocatechin gallate, and vitamin B5 in step S2 is 15-25:5-10:2-3:1-2:0.5-1:2-3; and the volume ratio of dichloromethane and ethanol in the dichloromethane-ethanol mixed solution is 10-15:5-10.

[0015] As a further improvement of the present invention, the exosomes in step S3 are mesenchymal stem cell exosomes, the mass ratio of the exosomes, NHS, EDC, modified nanogold cage, magnesium salt and zinc salt is 10:1-2:1-2:3-5:0.8-1.2:0.5-1, the stirring activation time is 20-40 min, the stirring incubation reaction temperature is 36-38 ° C, 100-200 r / min, the incubation reaction time is 24-36 h, the stirring complexation time is 1-2 h, the magnesium salt is magnesium chloride, magnesium sulfate or magnesium nitrate, and the zinc salt is zinc chloride, zinc sulfate or zinc nitrate; the mass ratio of lecithin, cholesterol, nano-drug-loaded liposomes and modified exosomes in step S4 is 20-30:10-15:5-10:3-5, and the volume ratio of dichloromethane and ethanol in the dichloromethane-ethanol mixed solution is 10-15:5-10.

[0016] As a further improvement of the present invention, the acid solution in step S5 is a 1-3 wt% acetic acid or lactic acid solution, and the mass ratio of the chitosan, poloxamer P407, poloxamer P188, multilamellar liposomes and permeation enhancer is 15-25:5-8:0.5-1:7-10:1-2. The structural formula of the permeation enhancer is shown in Formula I: Formula I.

[0017] As a further improvement of the present invention, the preparation method of the penetration enhancer is as follows:

[0018] T1. Epieucalyptol and salicylic acid are reacted to produce an intermediate having the following structure:

[0019] ;

[0020] T2. React the intermediate with oleic acid to obtain the product.

[0021] As a further improvement of the present invention, the molar ratio of epieucalyptol to salicylic acid in step T1 is 1-1.1:1, and a catalyst is added, wherein the catalyst is concentrated sulfuric acid, and the addition amount is 2-4wt% of salicylic acid. The solvent of the reaction is toluene, and the reaction conditions are heating under reflux and stirring for 8-10h.

[0022] As a further improvement of the present invention, the molar ratio of the intermediate to oleic acid in step T2 is 1:1-1.1, and a catalyst is added, wherein the catalyst is concentrated sulfuric acid, and the addition amount is 2-4wt% of salicylic acid. The solvent of the reaction is toluene, and the reaction conditions are heating under reflux and stirring for 10-12 hours.

[0023] The present invention further protects a composition for promoting hair regeneration after chemotherapy, which is prepared by the above preparation method.

[0024] The present invention further protects the use of the above-mentioned composition for promoting hair regeneration after chemotherapy in the preparation of a drug for treating hair loss after chemotherapy.

[0025] The present invention has the following beneficial effects:

[0026] The present invention combines the prepared multilamellar liposomes and a penetration enhancer into a thermosensitive hydrogel. This thermosensitive hydrogel is liquid at room temperature and, after being applied to the scalp, gels at body temperature, allowing the multilamellar liposomes and penetration enhancer to fully contact the scalp. Simultaneously, the hydrogel exhibits a sustained-release effect, allowing the drug to be released over a prolonged period, maintaining an effective drug concentration and reducing the frequency of medication use. For example, a special sustained-release microsphere technology is employed to encapsulate the active ingredient within the microspheres, slowly releasing the drug and prolonging its duration of action within the hair follicles.

[0027] In addition, the permeation enhancer prepared by the present invention uses epieudecanol, oleic acid and salicylic acid as main structural components, thereby overcoming the problems that salicylic acid easily causes dermatitis and skin sensitivity, and the problems that oleic acid and epieudecanol are insoluble in water. In the prepared permeation enhancer, epieudecanol affects the arrangement of lipid liquid crystals in the stratum corneum to change its phase structure, salicylic acid can stimulate subcutaneous blood circulation and expand pores, thereby enhancing drug absorption, and oleic acid can enhance the disturbing effect of eudecanol on the liquid crystal structure. The three ingredients synergistically reduce the barrier function of the stratum corneum through different targets, thereby promoting transdermal absorption of drugs.

[0028] The multilamellar liposomes prepared in the present invention can more effectively deliver active ingredients to the hair follicles, thereby improving the utilization and efficacy of the drug. The liposome carrier can protect the active ingredients from degradation and has better transdermal absorption performance, allowing the drug to reach the roots of the hair follicles more deeply to exert its effect. At the same time, the water solubility of the drug is improved, and the biocompatibility and bioavailability of the drug are improved, thereby greatly improving the drug efficacy.

[0029] Curcumin, extracted from turmeric, activates the Wnt / β-catenin pathway, promoting hair follicle stem cells to enter the proliferation cycle from the G0 phase. When combined with vitamin B5, it increases the proliferation rate of hair follicle stem cells and accelerates hair regeneration. Curcumin can also inhibit GSK-3β phosphorylation, stabilize β-catenin protein and promote its nuclear translocation, activating downstream proliferation-related genes such as CyclinD1. Resveratrol blocks NF-κB nuclear translocation by inhibiting IκB kinase (IKK), reducing the secretion of pro-inflammatory cytokines such as IL-6 and TNF-α. 2+ As a cofactor of superoxide dismutase (SOD), it enhances the ROS scavenging capacity in hair follicle cells and reduces the content of malondialdehyde (MDA). Epigallocatechin gallate inhibits inflammation and promotes hair follicle repair by inhibiting the NF-κB pathway and activating the caspase cascade. 2+ The participation of can activate the skin mechanosensitive ion channel Piezo1, so that the hair follicle stem cells Ca 2+ The inflow increased by 2 times and the proliferation rate increased.

[0030] Exosomes serve as excellent drug carriers. Firstly, the CD9 / CD63 proteins on their surface specifically bind to the integrin α6β4 on the surface of hair follicle stem cells, resulting in drug accumulation in the hair follicles 6-8 times higher than free drug. Secondly, they enter the dermal papilla cells via endocytosis, release the loaded drug, and activate the β-catenin pathway, promoting the transition of hair follicles from the catagen phase to the anagen phase. Furthermore, they possess excellent biocompatibility, targeted properties, and pleiotropic effects, simultaneously modulating multiple pathways (such as anti-apoptosis, angiogenesis, and anti-inflammation), matching the complex pathological mechanisms of hair regeneration after chemotherapy.

[0031] In addition, the exosomes of the present invention are also loaded with gold nanocages. Since gold nanocages have good near-infrared light response, the temperature of the gold surface will increase under the stimulation of near-infrared light, thereby promoting the opening of hair follicle pores and facilitating the entry of drugs into the hair follicles, playing a good role in reversing growth phase failure and promoting hair regeneration. It can also activate intracellular heat shock proteins (HSPs) and promote the transformation of hair follicles from the resting phase (Telogen) to the growth phase (Anagen).

[0032] The composition for promoting hair regeneration after chemotherapy prepared by the present invention has a good transdermal absorption-promoting effect, can slowly release drugs, and promotes the transformation of hair follicles from the resting phase to the growth phase under the synergistic effect of drugs. It also regulates multiple pathways (such as anti-apoptosis, angiogenesis promotion, and anti-inflammation), matching the complex pathological mechanism of hair regeneration after chemotherapy, and has broad application prospects. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0034] Gold nanocages, Xianfeng Nano, average particle size 60 nm; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0035] Preparation Example 1 Preparation of permeation enhancer

[0036] Here’s how:

[0037] T1. 0.1 mol of epieucalyptol and 0.1 mol of salicylic acid were added to 200 mL of toluene, and concentrated sulfuric acid was added in an amount of 2 wt % of salicylic acid. The mixture was stirred under reflux for 8 h, and the solvent was removed under reduced pressure. The mixture was recrystallized from ethanol, filtered, washed, and dried to obtain an intermediate; ESI-MS calculated value: C 22 H 31 O3(M+H) + 343.22, found: 343.2, yield: 65%.

[0038] NMR results: 1 H NMR (300MHz, CDCl3) δ7.82 (d, J =6.2Hz, 1H), 7.32 (m, 1H), 6.89-6.94 (m, 2H), 5.05 (br, 1H), 1.92-2.21 (m, 5H) , 1.72 (s, 3H), 1.45-1.52 (m, 4H), 1.42 (s, 6H), 1.32-1.40 (m, 4H), 1.22 (s, 3H).

[0039] The synthetic route is as follows:

[0040]

[0041] T2. 0.1 mol of the intermediate and 0.1 mol of oleic acid were added to 200 mL of toluene, and concentrated sulfuric acid was added at a concentration of 2 wt % based on the amount of salicylic acid. The reaction was stirred and refluxed for 10 h. The solvent was removed under reduced pressure, and the product was recrystallized from ethanol, filtered, washed, and dried to obtain the product. ESI-MS calculated value: C 40 H 63 O4(M+H) + 607.46, found: 607.5, yield: 71%.

[0042] NMR results: 1 H NMR (300MHz, CDCl3) δ7.92 (m, 1H), 7.45 (m, 1H), 7.22-7.3 (m, 2H), 5.42 (m, 2H), 2.23 (t, 2H), 1.94-2.12 ( m, 9H), 1.7-1.82 (m, 7H), 1.56 (m, 2H), 1.48 (s, 6H), 1.33-1.38 (m, 6H), 1.29-1.30 (m, 21H), 0.92 (m, 3H).

[0043] The synthetic route is as follows:

[0044]

[0045] Preparation Example 2 Preparation of penetration enhancer

[0046] Here’s how:

[0047] T1. 0.11 mol of epieucalyptol and 0.1 mol of salicylic acid were added to 200 mL of toluene, followed by concentrated sulfuric acid (4 wt % relative to the salicylic acid). The reaction was heated under reflux with stirring for 10 hours. The solvent was removed under reduced pressure, and the mixture was recrystallized from ethanol, filtered, washed, and dried to obtain an intermediate in a yield of 69%.

[0048] T2. 0.1 mol of the intermediate and 0.11 mol of oleic acid were added to 200 mL of toluene. Concentrated sulfuric acid was added at a rate of 4 wt % relative to the salicylic acid. The reaction was heated under reflux with stirring for 12 h. The solvent was removed under reduced pressure, and the mixture was recrystallized from ethanol, filtered, washed, and dried to obtain the product in a yield of 74%.

[0049] Preparation Example 3 Preparation of permeation enhancer

[0050] Here’s how:

[0051] T1. 0.105 mol of epieucalyptol and 0.1 mol of salicylic acid were added to 200 mL of toluene. Concentrated sulfuric acid was added at a concentration of 3 wt % based on the salicylic acid. The reaction was heated under reflux with stirring for 9 hours. The solvent was removed under reduced pressure, and the mixture was recrystallized from ethanol, filtered, washed, and dried to obtain an intermediate; the yield was 67%.

[0052] T2. 0.1 mol of the intermediate and 0.105 mol of oleic acid were added to 200 mL of toluene. Concentrated sulfuric acid was added at a concentration of 3 wt % based on the salicylic acid. The reaction was heated under reflux with stirring for 11 hours. The solvent was removed under reduced pressure, and the mixture was recrystallized from ethanol, filtered, washed, and dried to obtain the product in a yield of 72%.

[0053] Comparative Preparation Example 1

[0054] Compared with Preparation Example 3, the difference is that step T2 is not performed.

[0055] The details are as follows:

[0056] 0.105 mol of epieucalyptol and 0.1 mol of salicylic acid were added to 200 mL of toluene, and concentrated sulfuric acid was added in an amount of 3 wt % of the salicylic acid. The mixture was heated under reflux with stirring for 9 h, and the solvent was removed under reduced pressure. The mixture was recrystallized with ethanol, filtered, washed, and dried to obtain an intermediate, which was a penetration enhancer. Example 1

[0057] This embodiment provides a composition for promoting hair regeneration after chemotherapy, comprising the following steps:

[0058] S1. Preparation of modified gold nanocages: 7 mg of gold nanocages were added to 20 mL of Tris-HCl solution (pH 8.5), followed by 2 mg of dopamine hydrochloride. The mixture was heated to 45°C and stirred for 2 h. The reaction was centrifuged, washed, and dried to obtain modified gold nanocages.

[0059] S2. Preparation of drug-loaded nanoliposomes: 15 mg of lecithin, 5 mg of cholesterol, 2 mg of curcumin, 1 mg of resveratrol, and 0.5 mg of epigallocatechin gallate were dissolved in 20 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio of 10:5). 10 mL of PBS buffer (pH 7.4) containing 2 mg of vitamin B5 was added dropwise. The mixture was stirred for 30 minutes. The dichloromethane and ethanol were removed by rotary evaporation. The mixture was sonicated at 1000 W for 10 minutes and freeze-dried to prepare drug-loaded nanoliposomes.

[0060] S3. Preparation of modified exosomes: 10 mg of exosomes were added to 50 mL of water, followed by 1 mg of NHS and 1 mg of EDC. The mixture was stirred for activation for 20 min. 3 mg of modified gold nanocages were added and the reaction was incubated at 36°C, 100 rpm, for 24 h. 0.08 mg of magnesium chloride and 0.05 mg of zinc chloride were added and the mixture was stirred for 1 h. The mixture was dialyzed using a 5000 Da dialysis bag for 3 days, and the retentate was freeze-dried to obtain the modified exosomes.

[0061] S4. Preparation of multilamellar liposomes: Dissolve 20 mg of lecithin and 10 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio, 10:5). Add 20 mL of PBS buffer (pH 7.4) containing 5 mg of drug-loaded nanoliposomes and 3 mg of modified exosomes. Stir and mix for 30 minutes. Remove the dichloromethane and ethanol by rotary evaporation under reduced pressure. Ultrasonicate at 1000 W for 15 minutes and freeze-dry to prepare multilamellar liposomes.

[0062] S5. Preparation of thermosensitive hydrogel: 15 mg of chitosan was dissolved in 20 mL of 1 wt% acetic acid solution, 5 mg of poloxamer P407 and 0.5 mg of P188 were added, 7 mg of multilamellar liposomes and 1 mg of the permeation enhancer prepared in Preparation Example 1 were added, and the mixture was stirred for 30 minutes to prepare a composition for promoting hair regeneration after chemotherapy. Example 2

[0063] This embodiment provides a composition for promoting hair regeneration after chemotherapy, comprising the following steps:

[0064] S1. Preparation of modified gold nanocages: 10 mg of gold nanocages were added to 20 mL of Tris-HCl solution (pH 9.5), followed by 4 mg of dopamine hydrochloride. The mixture was heated to 55°C and stirred for 4 h. The reaction was centrifuged, washed, and dried to obtain modified gold nanocages.

[0065] S2. Preparation of drug-loaded nanoliposomes: 25 mg of lecithin, 10 mg of cholesterol, 3 mg of curcumin, 2 mg of resveratrol, and 1 mg of epigallocatechin gallate were dissolved in 20 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio, 15:10). 10 mL of PBS buffer (pH 7.4) containing 3 mg of vitamin B5 was added dropwise. The mixture was stirred for 30 minutes. The dichloromethane and ethanol were removed by rotary evaporation. The mixture was sonicated at 1000 W for 10 minutes and freeze-dried to prepare drug-loaded nanoliposomes.

[0066] S3. Preparation of modified exosomes: 10 mg of exosomes were added to 50 mL of water, followed by 2 mg of NHS and 2 mg of EDC. The mixture was stirred for activation for 40 min. 5 mg of modified gold nanocages were added and the reaction was incubated at 38°C, 200 rpm, for 36 h. 0.12 mg of magnesium sulfate and 0.1 mg of zinc sulfate were added and the mixture was stirred for 2 h. The mixture was dialyzed using an 8000 Da dialysis bag for 3 days, and the retentate was freeze-dried to obtain the modified exosomes.

[0067] S4. Preparation of multilamellar liposomes: Dissolve 30 mg of lecithin and 15 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio, 15:10). Add 20 mL of PBS buffer (pH 7.4) containing 10 mg of drug-loaded nanoliposomes and 5 mg of modified exosomes. Stir and mix for 30 minutes. Remove the dichloromethane and ethanol by rotary evaporation under reduced pressure. Ultrasonicate at 1000 W for 15 minutes and freeze-dry to prepare multilamellar liposomes.

[0068] S5. Preparation of thermosensitive hydrogel: 25 mg of chitosan was dissolved in 20 mL of 3 wt % lactic acid solution, 8 mg of poloxamer P407 and 1 mg of P188 were added, 10 mg of multilamellar liposomes and 2 mg of the permeation enhancer prepared in Preparation Example 2 were added, and the mixture was stirred for 30 minutes to prepare a composition for promoting hair regeneration after chemotherapy. Example 3

[0069] This embodiment provides a composition for promoting hair regeneration after chemotherapy, comprising the following steps:

[0070] S1. Preparation of modified gold nanocages: 8 mg of gold nanocages were added to 20 mL of Tris-HCl solution (pH 9), followed by 3 mg of dopamine hydrochloride. The mixture was heated to 50°C and stirred for 3 h. The reaction was centrifuged, washed, and dried to obtain modified gold nanocages.

[0071] S2. Preparation of drug-loaded nanoliposomes: 20 mg of lecithin, 7 mg of cholesterol, 2.5 mg of curcumin, 1.5 mg of resveratrol, and 0.7 mg of epigallocatechin gallate were dissolved in 20 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio of 13:7). 10 mL of PBS buffer (pH 7.4) containing 2.5 mg of vitamin B5 was added dropwise. The mixture was stirred for 30 minutes. The dichloromethane and ethanol were removed by rotary evaporation. The mixture was sonicated at 1000 W for 10 minutes and freeze-dried to prepare drug-loaded nanoliposomes.

[0072] S3. Preparation of modified exosomes: 10 mg of exosomes were added to 50 mL of water, followed by 1.5 mg of NHS and 1.5 mg of EDC. The mixture was stirred for activation for 30 min. 4 mg of modified gold nanocages were added and the reaction was incubated at 37°C, 150 rpm, for 30 h. 0.1 mg of magnesium nitrate and 0.07 mg of zinc nitrate were added and the mixture was stirred for 1.5 h. The mixture was dialyzed using a 6000 Da dialysis bag for 3 days. The filtrate was freeze-dried to obtain the modified exosomes.

[0073] S4. Preparation of multilamellar liposomes: Dissolve 25 mg of lecithin and 12 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio, 13:7). Add 20 mL of PBS buffer (pH 7.4) containing 7 mg of drug-loaded nanoliposomes and 4 mg of modified exosomes. Stir and mix for 30 minutes. Remove the dichloromethane and ethanol by rotary evaporation under reduced pressure. Ultrasonicate at 1000 W for 15 minutes and freeze-dry to prepare multilamellar liposomes.

[0074] S5. Preparation of thermosensitive hydrogel: 20 mg of chitosan was dissolved in 20 mL of 2 wt % lactic acid solution, 6 mg of poloxamer P407 and 0.6 mg of P188 were added, 8 mg of multilamellar liposomes, and 1.5 mg of the permeation enhancer prepared in Preparation Example 3 were added, and the mixture was stirred for 30 minutes to prepare a composition for promoting hair regeneration after chemotherapy.

[0075] Comparative Example 1

[0076] Compared with Example 3, the difference is that the penetration enhancer is prepared by Comparative Preparation Example 1.

[0077] Comparative Example 2

[0078] Compared with Example 3, the difference is that no penetration enhancer is added in step S5.

[0079] The details are as follows:

[0080] S5. Preparation of thermosensitive hydrogel: 20 mg of chitosan was dissolved in 20 mL of 2 wt% lactic acid solution, 6 mg of poloxamer P407 and 0.6 mg of P188 were added, and 9.5 mg of multilamellar liposomes were added. The mixture was stirred for 30 minutes to prepare a composition for promoting hair regeneration after chemotherapy.

[0081] Comparative Example 3

[0082] Compared with Example 3, the difference is that vitamin B5 is not added in step S2.

[0083] The details are as follows:

[0084] S2. Preparation of drug-loaded nanoliposomes: 20 mg of lecithin, 7 mg of cholesterol, 5 mg of curcumin, 1.5 mg of resveratrol, and 0.7 mg of epigallocatechin gallate were dissolved in 20 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol was 13:7). 10 mL of a pH 7.4 PBS buffer solution was added dropwise, and the mixture was stirred for 30 min. The dichloromethane and ethanol were removed by rotary evaporation, and the mixture was sonicated at 1000 W for 10 min. The mixture was freeze-dried to prepare drug-loaded nanoliposomes.

[0085] Comparative Example 4

[0086] Compared with Example 3, the difference is that curcumin is not added in step S2.

[0087] The details are as follows:

[0088] S2. Preparation of drug-loaded nanoliposomes: 20 mg of lecithin, 7 mg of cholesterol, 1.5 mg of resveratrol, and 0.7 mg of epigallocatechin gallate were dissolved in 20 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol was 13:7). 10 mL of a PBS buffer solution (pH = 7.4) containing 5 mg of vitamin B5 was added dropwise. The mixture was stirred for 30 min, and the dichloromethane and ethanol were removed by rotary evaporation. The mixture was sonicated at 1000 W for 10 min and freeze-dried to prepare drug-loaded nanoliposomes.

[0089] Comparative Example 5

[0090] Compared with Example 3, the difference is that no modified gold nanocage is added in step S3.

[0091] The details are as follows:

[0092] S3. Preparation of modified exosomes: 10 mg of exosomes were added to 50 mL of water, along with 0.1 mg of magnesium nitrate and 0.07 mg of zinc nitrate. The mixture was stirred for 1.5 h and dialyzed using a 6000 Da dialysis bag for 3 days. The filtrate was freeze-dried to obtain modified exosomes.

[0093] Comparative Example 6

[0094] Compared with Example 3, the difference is that magnesium nitrate and zinc nitrate are not added in step S3.

[0095] The details are as follows:

[0096] S3. Preparation of modified exosomes: 10 mg of exosomes were added to 50 mL of water, along with 1.5 mg of NHS and 1.5 mg of EDC. The mixture was stirred for activation for 30 min. 4 mg of modified gold nanocages were added and the reaction was incubated at 37°C, 150 rpm, for 30 h. The mixture was dialyzed using a 6000 Da dialysis bag for 3 days, and the filtrate was freeze-dried to obtain the modified exosomes.

[0097] Comparative Example 7

[0098] Compared with Example 3, the difference is that no drug-loaded nano liposomes are added in step S4.

[0099] The details are as follows:

[0100] S4. Preparation of multilamellar liposomes: Dissolve 25 mg of phosphatidylcholine and 12 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio, 13:7). Add 20 mL of PBS buffer (pH 7.4) containing 11 mg of modified exosomes and stir for 30 min. Remove the dichloromethane and ethanol by rotary evaporation under reduced pressure. Ultrasonicate at 1000 W for 15 min and freeze-dry to prepare multilamellar liposomes.

[0101] Comparative Example 8

[0102] Compared with Example 3, the difference is that no modified exosomes are added in step S4.

[0103] The details are as follows:

[0104] S4. Preparation of multilamellar liposomes: Dissolve 25 mg of phosphatidylcholine and 12 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixture (dichloromethane:ethanol volume ratio of 13:7). Add 20 mL of PBS buffer (pH = 7.4) containing 11 mg of nano-drug-loaded liposomes, stir and mix for 30 min, remove dichloromethane and ethanol by rotary evaporation under reduced pressure, sonicate at 1000 W for 15 min, and freeze-dry to prepare multilamellar liposomes.

[0105] Comparative Example 9

[0106] Compared with Example 3, the difference is that in step S5, 8 mg of multilamellar liposomes and 1.5 mg of the penetration enhancer prepared in Preparation Example 3 are added to 20 mL of water to prepare a composition for promoting hair regeneration after chemotherapy.

[0107] Test Example 1 Anti-inflammatory effect

[0108] SPF-grade NIH male mice were used as experimental mice and divided into 14 groups, with 10 mice in each group. Normal saline was used as a blank group, fluocinonide ointment was used as a positive drug group, and the compositions for promoting hair regeneration after tumor chemotherapy prepared in Examples 1-3 and Comparative Examples 1-9 were used as experimental groups.

[0109] Experimental methods:

[0110] The inside and outside surfaces of the right auricle of each mouse were evenly smeared with xylene to induce inflammation, with a dose of 100 μL / mouse. The left ear was not treated as a blank control group. 30 minutes after xylene induced inflammation, the corresponding test substance was given to the right ear of each group of animals, except for the negative control group, at a dose of 50 μL / mouse. When administering, pay attention to evenly apply it to the inside and outside surfaces of the right auricle. Distilled water was given to the right auricle of the blank control group animals at a dose of 0.1 mL / mouse. After administration, the administration site was irradiated with near-infrared light (808 nm) for 5-10 minutes. One hour after administration of the test substance, the mice were killed by cervical dislocation, the auricles on both sides were cut off, the test substance on the right auricle was washed with saline, and then dried. The bilateral auricles were overlapped, and the left and right ear pieces were punched out with a puncher with a diameter of 8 mm. The weight difference of the two ear pieces was weighed respectively, and the swelling value was calculated. The swelling value = m 右耳耳片 -m 左耳耳片。

[0111] The results are shown in Table 1.

[0112] Table 1

[0113]

[0114] Note: * compared with the blank group, P < 0.05.

[0115] As can be seen from the above table, the compositions for promoting hair regeneration after tumor chemotherapy prepared in Examples 1-3 of the present invention have good anti-inflammatory effects.

[0116] Test Example 2

[0117] Female C57BL / 6 mice, 6-8 weeks old, weighing 18-20 g, with pink back skin, were selected and randomly divided into a model group, Example 1-3 groups, and Comparative Example 1-9 groups, with 10 mice in each group.

[0118] All mice were fed a normal diet during the experiment.

[0119] Induction of anagen hair follicles:

[0120] All mice were anesthetized with ether and then plucked using the rosin / wax method to remove telogen hair from their backs. Anagen hair was then induced. Skin color changes and hair growth at the plucked areas were observed after pluck. Starting from the first day after pluck, mice in Example 1-3 and Comparative Examples 1-9 groups were given a daily dose of 2 mL of a composition for promoting hair regrowth after tumor chemotherapy, evenly applied to the plucked area. Following each dose, the treated area was irradiated with near-infrared light (808 nm) for 5-10 minutes. The model group received an equal volume of normal saline.

[0121] Chemotherapy drugs are given:

[0122] Nine days after hair plucking, when all hair follicles in the plucking area were in stage VI growth, mice were intraperitoneally injected with a single dose of cyclophosphamide (120 mg / kg) to establish a mouse model of post-chemotherapy hair loss. Hair loss and hair regrowth in the plucking area on the back of the mice were observed with continued dosing.

[0123] Post-experimental processing:

[0124] On the 13th day after hair plucking, five mice in each of the experimental and model groups were killed by cervical dislocation. Hair samples were collected from the same site of the back parallel to the spine, embedded in OCT, frozen in liquid nitrogen, and 8 μm frozen sections were prepared. Hair follicle histology was observed under a light microscope and stained with hematoxylin and eosin. Hair follicles were staged according to the Muller-Rover hair follicle morphological staging system. Sixty follicles were randomly selected from each mouse to calculate the average hair cycle score (HCS: 100 for growth stage VI, 200 for early catagen, 300 for mid-catagen, and 400 for late catagen) and the percentages of follicles in growth stage VI, early catagen, mid-catagen, and late catagen.

[0125] The results are shown in Table 2.

[0126] Table 2

[0127]

[0128] As can be seen from the above table, the compositions for promoting hair regeneration after tumor chemotherapy prepared in Examples 1-3 of the present invention can significantly promote hair regeneration after chemotherapy.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a composition for promoting hair regeneration after chemotherapy, characterized in that: The surface of a gold nanocage is modified with polydopamine to produce a modified nanogold cage, which is then loaded onto exosomes and complexed with magnesium and zinc ions to produce modified exosomes. Curcumin, resveratrol, and epigallocatechin gallate are then added to prepare drug-loaded nanoliposomes, which are then mixed with the modified exosomes to produce multilamellar liposomes. The liposomes are then mixed with a permeation enhancer and added to a thermosensitive hydrogel system to produce a composition for promoting hair regeneration after chemotherapy; the exosomes are mesenchymal stem cell exosomes. The structural formula of the penetration enhancer is shown in Formula I:

2. The preparation method according to claim 1, characterized in that The following steps are involved: S1. Preparation of modified gold nanocages: Adding gold nanocages to a Tris-HCl solution, adding dopamine hydrochloride, heating and stirring to react, centrifuging, washing, and drying to obtain modified gold nanocages; S2. Preparation of drug-loaded nanoliposomes: Phosphatidylcholine, cholesterol, curcumin, resveratrol, and epigallocatechin gallate were dissolved in a dichloromethane-ethanol mixture, vitamin B5-PBS buffer solution was added dropwise, the mixture was stirred, the dichloromethane and ethanol were removed by rotary evaporation, the mixture was sonicated, and the mixture was freeze-dried to prepare drug-loaded nanoliposomes. S3. Preparation of modified exosomes: exosomes were added to water, NHS and EDC were added, and the mixture was stirred for activation. Modified gold nanocages were added and incubated with stirring. Magnesium salt and zinc salt were added and the mixture was stirred for complexation. The mixture was dialyzed, and the non-permeated solution was freeze-dried to obtain modified exosomes. S4. Preparation of multilamellar liposomes: Phosphatidylcholine and cholesterol were dissolved in a dichloromethane-ethanol mixture, and PBS buffer containing drug-loaded nanoliposomes and modified exosomes was added. The mixture was stirred and mixed. The dichloromethane and ethanol were removed by rotary evaporation under reduced pressure. The mixture was sonicated and freeze-dried to prepare multilamellar liposomes. S5. Preparation of thermosensitive hydrogel: dissolve chitosan in acid solution, add poloxamer P407 and P188, add multilamellar liposomes and a permeation enhancer, stir and mix evenly to prepare a composition for promoting hair regeneration after chemotherapy.

3. The preparation method according to claim 2, characterized in that The pH value of the Tris-HCl solution in step S1 is 8.5-9.5, the mass ratio of the gold nanocages to dopamine hydrochloride is 7-10:2-4, the temperature of the heating and stirring reaction is 45-55°C, and the time is 2-4 hours; the mass ratio of lecithin, cholesterol, curcumin, resveratrol, epigallocatechin gallate, and vitamin B5 in step S2 is 15-25:5-10:2-3:1-2:0.5-1:2-3; and the volume ratio of dichloromethane to ethanol in the dichloromethane-ethanol mixed solution is 10-15:5-10.

4. The preparation method according to claim 2, characterized in that The mass ratio of the exosomes, NHS, EDC, modified nanogold cages, magnesium salts and zinc salts in step S3 is 10:1-2:1-2:3-5:0.8-1.2:0.5-1, the stirring activation time is 20-40 min, the stirring incubation reaction temperature is 36-38 ° C, 100-200 r / min, the incubation reaction time is 24-36 h, the stirring complexation time is 1-2 h, the magnesium salt is magnesium chloride, magnesium sulfate or magnesium nitrate, and the zinc salt is zinc chloride, zinc sulfate or zinc nitrate; the mass ratio of lecithin, cholesterol, nano-drug-loaded liposomes and modified exosomes in step S4 is 20-30:10-15:5-10:3-5, and the volume ratio of dichloromethane and ethanol in the dichloromethane-ethanol mixed solution is 10-15:5-10.

5. The preparation method according to claim 2, characterized in that The acid solution in step S5 is a 1-3 wt% acetic acid or lactic acid solution, and the mass ratio of the chitosan, poloxamer P407, poloxamer P188, multilamellar liposome and permeation enhancer is 15-25:5-8:0.5-1:7-10:1-2.

6. The preparation method according to claim 5, characterized in that The preparation method of the penetration enhancer is as follows: T1. Epieucalyptol and salicylic acid react to obtain an intermediate having the following structure: T2. The intermediate is reacted with oleic acid to obtain the product.

7. The preparation method according to claim 6, characterized in that In step T1, the molar ratio of epieucalyptol to salicylic acid is 1-1.1:1, and a catalyst is added, wherein the catalyst is concentrated sulfuric acid, and the addition amount is 2-4wt% of salicylic acid. The solvent of the reaction is toluene, and the reaction conditions are heating under reflux and stirring for 8-10h.

8. The preparation method according to claim 6, characterized in that In step T2, the molar ratio of the intermediate to oleic acid is 1:1-1.1, and a catalyst is added. The catalyst is concentrated sulfuric acid, and the addition amount is 2-4wt% of salicylic acid. The solvent of the reaction is toluene, and the reaction conditions are heating under reflux and stirring for 10-12 hours.

9. A composition for promoting hair regeneration after chemotherapy, obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the composition for promoting hair regeneration after chemotherapy according to claim 9 in the preparation of a medicament for treating hair loss after chemotherapy.

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