Composition for promoting hair regeneration after chemotherapy as well as preparation method and application of composition

Abstract

The invention provides a composition for promoting hair regeneration after chemotherapy as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: modifying the surface of a gold nanocage with polydopamine, loading the modified gold nanocage on an exosome, complexing magnesium ions and zinc ions to prepare a modified exosome, preparing a nano drug-loaded liposome from curcumin, resveratrol and epigallocatechin gallate, mixing the nano drug-loaded liposome with the modified exosome to prepare a multi-layer liposome, and preparing the multi-layer liposome from the nano drug-loaded liposome. And mixing with a penetration enhancer, and adding into a temperature-sensitive hydrogel system to prepare the composition for promoting hair regeneration after chemotherapy. The composition for promoting hair regeneration after chemotherapy has a good transdermal absorption promoting effect, can slowly release drugs, promotes hair follicles to be converted from a resting stage to a growing stage under the synergistic effect of the drugs, regulates and controls multiple pathways (such as anti-apoptosis, angiogenesis promotion and anti-inflammation) at the same time, is matched with a complex pathological mechanism of hair regeneration after chemotherapy, and can promote hair regeneration after chemotherapy. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a composition for promoting hair regrowth after chemotherapy, a preparation method thereof, and an application thereof. Background Art

[0002] One of the most common side effects in tumor patients after receiving chemotherapy drugs is hair loss, which usually occurs 1-2 weeks after chemotherapy. Hair loss after chemotherapy has a psychological impact on a large number of patients after chemotherapy, and the impact is more obvious and prominent in female patients. Hair loss after chemotherapy has a different origin from androgen-induced hair loss, and there is no effective treatment drug. Currently, clinically, only a medical ice cap at -15°C can be used to cool the scalp during chemotherapy to reduce the blood flow in the scalp capillaries, thereby reducing the apoptosis of hair follicle cells. However, due to its cumbersome operation and poor experience, it is less used.

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

[0004] There is no relevant report on the use of exosome drugs for the treatment and prevention of hair loss caused by chemotherapy drugs. The exosomes in traditional technologies have been widely used in the field of hair loss (such as patent CN115227721A), mainly for androgenetic alopecia, seborrheic alopecia, and recurrent hair loss after hair transplantation, and have not been applied to the treatment and prevention of hair loss after chemotherapy. Summary of the Invention

[0005] The purpose of the present invention is to provide a composition for promoting hair regrowth after chemotherapy, a preparation method thereof, and an application thereof, which has a good effect of promoting transdermal absorption, can slowly release drugs, and under the synergistic action of drugs, can promote the transformation of hair follicles from the resting phase to the growth phase, and at the same time regulate multiple pathways (such as anti-apoptosis, promoting angiogenesis, and anti-inflammatory), matching the complex pathological mechanism of hair regrowth after chemotherapy, and has broad application prospects.

[0006] The technical solution of the present invention is realized as follows: The present invention provides a preparation method of a composition for promoting hair regeneration after chemotherapy. After the surface of gold nanocages is modified with polydopamine, they are loaded on exosomes, complexed with magnesium ions and zinc ions to obtain modified exosomes. Curcumin, resveratrol, and epigallocatechin gallate are used to prepare nano-drug-loaded liposomes, which are mixed with the modified exosomes to obtain multi-layered liposomes, and then mixed with a penetration enhancer and added to a thermosensitive hydrogel system to prepare a composition for promoting hair regeneration after chemotherapy.

[0007] As a further improvement of the present invention, it includes the following steps: S1. Preparation of modified nano-gold cages: Add gold nanocages to a Tris-HCl solution, add dopamine hydrochloride, heat and stir for reaction, centrifuge, wash, and dry to obtain modified nano-gold cages; S2. Preparation of nano-drug-loaded liposomes: Dissolve lecithin, cholesterol, curcumin, resveratrol, and epigallocatechin gallate in a dichloromethane-ethanol mixed solution, add a vitamin B5-PBS buffer solution, stir and mix, rotate and evaporate to remove dichloromethane and ethanol, perform ultrasonic treatment, and freeze-dry to obtain nano-drug-loaded liposomes; S3. Preparation of modified exosomes: Add exosomes to water, add NHS and EDC, stir for activation, add the modified nano-gold cages, stir and incubate, add magnesium salts and zinc salts, stir for complexation, dialyze, and freeze-dry the non-permeated solution to obtain modified exosomes; S4. Preparation of multi-layered liposomes: Dissolve lecithin and cholesterol in a dichloromethane-ethanol mixed solution, add a PBS buffer solution containing nano-drug-loaded liposomes and modified exosomes, stir and mix, reduce pressure and rotate to evaporate to remove dichloromethane and ethanol, perform ultrasonic treatment, and freeze-dry to obtain multi-layered liposomes; S5. Preparation of thermosensitive hydrogel: Dissolve chitosan in an acid solution, add poloxamer P407 and P188, add the multi-layered liposomes and a penetration enhancer, stir and mix evenly to obtain a composition for promoting hair regeneration after chemotherapy.

[0008] As a further improvement of the present invention, in step S1, the pH value of the Tris-HCl solution 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 h; in step S2, the mass ratio of lecithin, cholesterol, curcumin, resveratrol, epigallocatechin gallate, and vitamin B5 is 15-25:5-10:2-3:1-2:0.5-1:2-3; the volume ratio of dichloromethane to ethanol in the dichloromethane-ethanol mixed solution is 10-15:5-10.

[0009] As a further improvement of the present invention, in step S3, the exosomes are mesenchymal stem cell exosomes, and the mass ratio of the exosomes, NHS, EDC, modified nanocages, magnesium salts, and zinc salts is 10:1-2:1-2:3-5:0.8-1.2:0.5-1. The stirring activation time is 20-40 min, the temperature of the stirring incubation reaction 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; in step S4, the mass ratio of lecithin, cholesterol, nano-drug-loaded liposomes, and modified exosomes is 20-30:10-15:5-10:3-5, and the volume ratio of dichloromethane to ethanol in the dichloromethane-ethanol mixed solution is 10-15:5-10.

[0010] As a further improvement of the present invention, in step S5, the acid solution is a 1-3 wt% acetic acid or lactic acid solution, and the mass ratio of chitosan, poloxamer P407, poloxamer P188, multilamellar liposomes, and penetration enhancer is 15-25:5-8:0.5-1:7-10:1-2. The structural formula of the penetration enhancer is as shown in Formula I: Formula I.

[0011] As a further improvement of the present invention, the preparation method of the penetration enhancer is as follows: T1. React epieucalyptol with salicylic acid to obtain an intermediate, the structure of which is as follows: ; T2. React the intermediate with oleic acid to obtain a product.

[0012] As a further improvement of the present invention, in step T1, the molar ratio of epieucalyptol to salicylic acid is 1-1.1:1, and a catalyst is also added. The catalyst is concentrated sulfuric acid, and the addition amount is 2-4 wt% of salicylic acid. The solvent for the reaction is toluene, and the reaction conditions are heating under reflux with stirring for 8-10 h.

[0013] As a further improvement of the present invention, in step T2, the molar ratio of the intermediate to oleic acid is 1:1-1.1, and a catalyst is also added. The catalyst is concentrated sulfuric acid, and the addition amount is 2-4 wt% of salicylic acid. The solvent for the reaction is toluene, and the reaction conditions are heating under reflux with stirring for 10-12 h.

[0014] The present invention further protects a composition for promoting hair regrowth after chemotherapy prepared by the above preparation method.

[0015] The present invention further protects the application of the above composition for promoting hair regrowth after chemotherapy in the preparation of a drug for treating alopecia after chemotherapy.

[0016] The present invention has the following beneficial effects: The multilamellar liposomes prepared in the present invention and a penetration enhancer are mixed and added to a thermosensitive hydrogel. The thermosensitive hydrogel is in a liquid state at room temperature. After being coated on the scalp surface, it becomes a gel state under the condition of body temperature, so that the multilamellar liposomes and the penetration enhancer can be in full contact with the scalp. At the same time, it also has a sustained release effect, so that the drug can be continuously released for a long time, maintaining an effective drug concentration and reducing the frequency of drug administration. For example, by adopting a special sustained release microsphere technology, the active ingredient is encapsulated in the microspheres to slowly release the drug and extend the action time of the drug at the hair follicle site.

[0017] In addition, the penetration enhancer prepared in the present invention has epieucalyptol, oleic acid and salicylic acid as the structural main body, overcoming the problems that salicylic acid is prone to cause dermatitis and skin sensitivity problems, and that oleic acid and epieucalyptol are insoluble in water, etc. For the prepared penetration enhancer, epieucalyptol affects the arrangement of cutin lipid liquid crystals and changes its phase structure. Salicylic acid can stimulate subcutaneous blood circulation and dilate pores, thereby enhancing drug absorption. Oleic acid can enhance the disturbing effect of eucalyptol on the liquid crystal structure. The three synergistically reduce the cutin layer barrier function through different targets, thereby promoting the transdermal absorption of drugs.

[0018] The multilamellar liposomes prepared additionally in the present invention can more effectively deliver the active ingredient to the hair follicle site, improving the utilization rate and efficacy of the drug. The carrier of the liposome can protect the active ingredient from being degraded, and at the same time has better transdermal absorption performance, enabling the drug to reach the root of the hair follicle more deeply to play a role. At the same time, it also improves the water solubility of the drug, improves the biocompatibility and bioavailability of the drug, and thus greatly improves the drug efficacy.

[0019] Curcumin extracted from turmeric promotes the entry of hair follicle stem cells from the G0 phase into the proliferation cycle by activating the Wnt / β-catenin pathway. When compounded with vitamin B5, the proliferation rate of hair follicle stem cells is increased and the hair regeneration speed is accelerated. 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 factors such as IL-6 and TNF-α; Zn 2+ As a cofactor of superoxide dismutase (SOD), it enhances the ROS scavenging ability in hair follicle cells, reducing the content of malondialdehyde (MDA). Epigallocatechin gallate anti-inflammates and promotes hair follicle repair through mechanisms such as inhibiting the NF-κB pathway and activating the caspase cascade reaction. The participation of Mg 2+ can activate the skin mechanosensitive ion channel Piezo1, making the hair follicle stem cells Ca 2+The internal flow increases by 2 times, and the proliferation rate is enhanced.

[0020] As a good drug carrier, on the one hand, the CD9 / CD63 protein on the surface of exosomes specifically binds to the integrin α6β4 on the surface of hair follicle stem cells, making the drug accumulation amount at the hair follicle site 6-8 times higher than that of free drugs; on the other hand, it enters the dermal papilla cells through endocytosis, releases the loaded drugs, activates the β-catenin pathway, and promotes the conversion of hair follicles from the catagen phase to the anagen phase. At the same time, it has good biocompatibility, targeting and pleiotropy, can regulate multiple pathways simultaneously (such as anti-apoptosis, pro-angiogenesis, anti-inflammatory), and matches the complex pathological mechanism of hair regeneration after chemotherapy.

[0021] In addition, the exosomes of the present invention are also loaded with gold nanocages. Due to the good near-infrared light response of gold nanoparticles, the temperature on the gold surface will increase under the stimulation of near-infrared light, which can promote the opening of hair follicle pores, facilitate the entry of drugs into the hair follicles, play a good role in reversing the exhaustion of the growth phase and promoting hair regeneration, and can also activate heat shock proteins (HSPs) in cells to promote the transformation of hair follicles from the telogen phase to the anagen phase.

[0022] The composition for promoting hair regeneration after chemotherapy prepared by the present invention has a good effect of promoting transdermal absorption, can slowly release drugs, promotes the transformation of hair follicles from the telogen phase to the anagen phase under the synergistic action of drugs, regulates multiple pathways simultaneously (such as anti-apoptosis, pro-angiogenesis, anti-inflammatory), matches the complex pathological mechanism of hair regeneration after chemotherapy, and has broad application prospects. Detailed implementation manners

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] Gold nanocages, Xianfeng Nano, with an average particle size of 60 nm; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0025] Preparation Example 1 Preparation of Penetration Enhancer The method is as follows: T1. Add 0.1 mol of epieucalyptol and 0.1 mol of salicylic acid to 200 mL of toluene, add concentrated sulfuric acid, the addition amount of the concentrated sulfuric acid is 2 wt% of the salicylic acid, heat under reflux and stir for reaction for 8 h, remove the solvent under reduced pressure, recrystallize with ethanol, filter, wash, and dry to obtain an intermediate; ESI-MS calculated value: C 22 H 31 O3(M+H) + 343.22, measured value: 343.2, and the yield is 65%.

[0026] NMR result: 1 H NMR(300 MHz, CDCl3) δ 7.82 (d, J =6.2 Hz, 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).

[0027] The synthesis route is as follows:

[0028] T2. Add 0.1 mol of the intermediate and 0.1 mol of oleic acid to 200 mL of toluene, add concentrated sulfuric acid, the addition amount of the concentrated sulfuric acid is 2 wt% of the salicylic acid, heat under reflux and stir for reaction for 10 h, remove the solvent under reduced pressure, recrystallize with ethanol, filter, wash, and dry to obtain a product; ESI-MS calculated value: C 40 H 63 O4(M+H) + 607.46, measured value: 607.5, and the yield is 71%.

[0029] NMR result: 1 H NMR(300 MHz, CDCl3) δ 7.92 (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).

[0030] The synthesis route is as follows:

[0031] Preparation Example 2 Preparation of the penetration enhancer The method is as follows: T1. Add 0.11 mol of epieucalyptol and 0.1 mol of salicylic acid to 200 mL of toluene, add concentrated sulfuric acid, and the addition amount of the concentrated sulfuric acid is 4 wt% of the salicylic acid. Heat under reflux and stir for reaction for 10 h, remove the solvent under reduced pressure, recrystallize with ethanol, filter, wash, and dry to obtain an intermediate; the yield is 69%.

[0032] T2. Add 0.1 mol of the intermediate and 0.11 mol of oleic acid to 200 mL of toluene, add concentrated sulfuric acid, and the addition amount of the concentrated sulfuric acid is 4 wt% of the salicylic acid. Heat under reflux and stir for reaction for 12 h, remove the solvent under reduced pressure, recrystallize with ethanol, filter, wash, and dry to obtain a product; the yield is 74%.

[0033] Preparation Example 3 Preparation of Penetration Enhancer The method is as follows: T1. Add 0.105 mol of epieucalyptol and 0.1 mol of salicylic acid to 200 mL of toluene, add concentrated sulfuric acid, and the addition amount of the concentrated sulfuric acid is 3 wt% of the salicylic acid. Heat under reflux and stir for reaction for 9 h, remove the solvent under reduced pressure, recrystallize with ethanol, filter, wash, and dry to obtain an intermediate; the yield is 67%.

[0034] T2. Add 0.1 mol of the intermediate and 0.105 mol of oleic acid to 200 mL of toluene, add concentrated sulfuric acid, and the addition amount of the concentrated sulfuric acid is 3 wt% of the salicylic acid. Heat under reflux and stir for reaction for 11 h, remove the solvent under reduced pressure, recrystallize with ethanol, filter, wash, and dry to obtain a product; the yield is 72%.

[0035] Comparative Preparation Example 1 Compared with Preparation Example 3, the difference is that step T2 is not carried out.

[0036] Specifically as follows: Add 0.105 mol of epieucalyptol and 0.1 mol of salicylic acid to 200 mL of toluene, add concentrated sulfuric acid, and the addition amount of the concentrated sulfuric acid is 3 wt% of the salicylic acid. Heat under reflux and stir for reaction for 9 h, remove the solvent under reduced pressure, recrystallize with ethanol, filter, wash, and dry to obtain an intermediate, which is the penetration enhancer. Example 1

[0037] This example provides a composition for promoting hair regrowth after chemotherapy, including the following steps: S1. Preparation of modified nanogold cages: Add 7 mg of gold nanocages to 20 mL of Tris-HCl solution with a pH value of 8.5, add 2 mg of dopamine hydrochloride, heat to a temperature of 45 °C, stir for reaction for 2 h, centrifuge, wash, and dry to obtain modified nanogold cages; S2. Preparation of nano-drug-loaded liposomes: Dissolve 15 mg of lecithin, 5 mg of cholesterol, 2 mg of curcumin, 1 mg of resveratrol, and 0.5 mg of epigallocatechin gallate in 20 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 10:5). Dropwise add 10 mL of a PBS buffer solution with a pH of 7.4 containing 2 mg of vitamin B5, stir and mix for 30 min, remove dichloromethane and ethanol by rotary evaporation, perform ultrasonic treatment at 1000 W for 10 min, and then freeze-dry to obtain nano-drug-loaded liposomes; S3. Preparation of modified exosomes: Add 10 mg of exosomes to 50 mL of water, add 1 mg of NHS and 1 mg of EDC, stir and activate for 20 min, add 3 mg of modified nanocages, incubate at 36 °C and 100 r / min for 24 h, add 0.08 mg of magnesium chloride and 0.05 mg of zinc chloride, stir and complex for 1 h, dialyze with a dialysis bag with a pore size of 5000 Da for 3 d, and freeze-dry the non-permeated liquid to obtain modified exosomes; S4. Preparation of multilamellar liposomes: Dissolve 20 mg of lecithin and 10 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 10:5), add 20 mL of a PBS buffer solution with a pH of 7.4 containing 5 mg of nano-drug-loaded liposomes and 3 mg of modified exosomes, stir and mix for 30 min, remove dichloromethane and ethanol by reduced-pressure rotary evaporation, perform ultrasonic treatment at 1000 W for 15 min, and then freeze-dry to obtain multilamellar liposomes; S5. Preparation of thermosensitive hydrogel: Dissolve 15 mg of chitosan in 20 mL of 1 wt% acetic acid solution, add 5 mg of poloxamer P407 and 0.5 mg of P188, add 7 mg of multilamellar liposomes and 1 mg of the penetration enhancer prepared in Preparation Example 1, stir and mix for 30 min to obtain a composition for promoting hair regeneration after chemotherapy. Example 2

[0038] This example provides a composition for promoting hair regeneration after chemotherapy, including the following steps: S1. Preparation of modified nanocages: Add 10 mg of gold nanocages to 20 mL of a Tris-HCl solution with a pH of 9.5, add 4 mg of dopamine hydrochloride, heat to a temperature of 55 °C, stir and react for 4 h, centrifuge, wash, and dry to obtain modified nanocages; S2. Preparation of nano-drug-loaded liposomes: Dissolve 25 mg of lecithin, 10 mg of cholesterol, 3 mg of curcumin, 2 mg of resveratrol, and 1 mg of epigallocatechin gallate in 20 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 15:10). Add 10 mL of a PBS buffer solution with a pH of 7.4 containing 3 mg of vitamin B5, stir and mix for 30 min, rotary evaporate to remove dichloromethane and ethanol, perform ultrasonic treatment at 1000 W for 10 min, and freeze-dry to obtain nano-drug-loaded liposomes; S3. Preparation of modified exosomes: Add 10 mg of exosomes to 50 mL of water, add 2 mg of NHS and 2 mg of EDC, stir and activate for 40 min, add 5 mg of modified nanocages, incubate at 38 °C and 200 r / min for 36 h, add 0.12 mg of magnesium sulfate and 0.1 mg of zinc sulfate, stir and complex for 2 h, dialyze with a dialysis bag with a pore size of 8000 Da for 3 d, and freeze-dry the non-permeated solution to obtain modified exosomes; S4. Preparation of multilamellar liposomes: Dissolve 30 mg of lecithin and 15 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 15:10). Add 20 mL of a PBS buffer solution with a pH of 7.4 containing 10 mg of nano-drug-loaded liposomes and 5 mg of modified exosomes, stir and mix for 30 min, rotary evaporate under reduced pressure to remove dichloromethane and ethanol, perform ultrasonic treatment at 1000 W for 15 min, and freeze-dry to obtain multilamellar liposomes; S5. Preparation of thermosensitive hydrogel: Dissolve 25 mg of chitosan in 20 mL of 3 wt% lactic acid solution, add 8 mg of poloxamer P407 and 1 mg of P188, add 10 mg of multilamellar liposomes and 2 mg of the penetration enhancer prepared in Preparation Example 2, stir and mix for 30 min to obtain a composition for promoting hair regeneration after chemotherapy. Example 3

[0039] This example provides a composition for promoting hair regeneration after chemotherapy, including the following steps: S1. Preparation of modified nanocages: Add 8 mg of gold nanocages to 20 mL of a Tris-HCl solution with a pH of 9, add 3 mg of dopamine hydrochloride, heat to a temperature of 50 °C, stir and react for 3 h, centrifuge, wash, and dry to obtain modified nanocages; S2. Preparation of nano-drug-loaded liposomes: Dissolve 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 in 20 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 13:7). Add 10 mL of a PBS buffer solution with a pH of 7.4 containing 2.5 mg of vitamin B5, stir and mix for 30 min, remove dichloromethane and ethanol by rotary evaporation, perform ultrasonic treatment at 1000 W for 10 min, and then freeze-dry to obtain nano-drug-loaded liposomes; S3. Preparation of modified exosomes: Add 10 mg of exosomes to 50 mL of water, add 1.5 mg of NHS and 1.5 mg of EDC, stir and activate for 30 min, add 4 mg of modified nanocages, incubate at 37 °C and 150 r / min for 30 h, add 0.1 mg of magnesium nitrate and 0.07 mg of zinc nitrate, stir and complex for 1.5 h, dialyze with a dialysis bag with a pore size of 6000 Da for 3 d, and freeze-dry the non-permeated liquid to obtain modified exosomes; S4. Preparation of multilayer liposomes: Dissolve 25 mg of lecithin and 12 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 13:7). Add 20 mL of a PBS buffer solution with a pH of 7.4 containing 7 mg of nano-drug-loaded liposomes and 4 mg of modified exosomes, stir and mix for 30 min, remove dichloromethane and ethanol by reduced-pressure rotary evaporation, perform ultrasonic treatment at 1000 W for 15 min, and then freeze-dry to obtain multilayer liposomes; S5. Preparation of thermosensitive hydrogel: Dissolve 20 mg of chitosan in 20 mL of a 2 wt% lactic acid solution, add 6 mg of poloxamer P407 and 0.6 mg of P188, add 8 mg of multilayer liposomes and 1.5 mg of the penetration enhancer prepared in Preparation Example 3, stir and mix for 30 min to obtain a composition for promoting hair regeneration after chemotherapy.

[0040] Comparative Example 1 Compared with Example 3, the difference is that the penetration enhancer is prepared from Comparative Preparation Example 1.

[0041] Comparative Example 2 Compared with Example 3, the difference is that no penetration enhancer is added in step S5.

[0042] Specifically as follows: S5. Preparation of thermosensitive hydrogel: Dissolve 20 mg of chitosan in 20 mL of a 2 wt% lactic acid solution, add 6 mg of poloxamer P407 and 0.6 mg of P188, add 9.5 mg of multilayer liposomes, stir and mix for 30 min to obtain a composition for promoting hair regeneration after chemotherapy.

[0043] Comparative Example 3 Compared with Example 3, the difference lies in that vitamin B5 was not added in step S2.

[0044] Specifically as follows: S2. Preparation of nano-drug-loaded liposomes: Dissolve 20 mg of lecithin, 7 mg of cholesterol, 5 mg of curcumin, 1.5 mg of resveratrol, and 0.7 mg of epigallocatechin gallate in 20 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 13:7), add dropwise 10 mL of PBS buffer solution with pH = 7.4, stir and mix for 30 min, remove dichloromethane and ethanol by rotary evaporation, perform ultrasonic treatment at 1000 W for 10 min, and freeze-dry to obtain nano-drug-loaded liposomes.

[0045] Comparative Example 4 Compared with Example 3, the difference lies in that curcumin was not added in step S2.

[0046] Specifically as follows: S2. Preparation of nano-drug-loaded liposomes: Dissolve 20 mg of lecithin, 7 mg of cholesterol, 1.5 mg of resveratrol, and 0.7 mg of epigallocatechin gallate in 20 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 13:7), add dropwise 10 mL of PBS buffer solution with pH = 7.4 containing 5 mg of vitamin B5, stir and mix for 30 min, remove dichloromethane and ethanol by rotary evaporation, perform ultrasonic treatment at 1000 W for 10 min, and freeze-dry to obtain nano-drug-loaded liposomes.

[0047] Comparative Example 5 Compared with Example 3, the difference lies in that modified gold nanocages were not added in step S3.

[0048] Specifically as follows: S3. Preparation of modified exosomes: Add 10 mg of exosomes to 50 mL of water, add 0.1 mg of magnesium nitrate and 0.07 mg of zinc nitrate, stir and complex for 1.5 h, dialyze with a dialysis bag with a pore size of 6000 Da for 3 d, and freeze-dry the non-permeated liquid to obtain modified exosomes.

[0049] Comparative Example 6 Compared with Example 3, the difference lies in that magnesium nitrate and zinc nitrate were not added in step S3.

[0050] Specifically as follows: S3. Preparation of modified exosomes: Add 10 mg of exosomes to 50 mL of water, add 1.5 mg of NHS and 1.5 mg of EDC, stir and activate for 30 min, add 4 mg of modified nanocages, incubate at 37 °C and 150 r / min for 30 h, dialyze with a dialysis bag with a pore size of 6000 Da for 3 d, and lyophilize the non-permeated solution to obtain modified exosomes.

[0051] Comparative Example 7 Compared with Example 3, the difference is that no nano-drug-loaded liposomes were added in step S4.

[0052] Specifically as follows: S4. Preparation of multilamellar liposomes: Dissolve 25 mg of lecithin and 12 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 13:7), add 20 mL of a PBS buffer solution with a pH of 7.4 containing 11 mg of modified exosomes, stir and mix for 30 min, remove dichloromethane and ethanol by rotary evaporation under reduced pressure, perform ultrasonic treatment at 1000 W for 15 min, and lyophilize to obtain multilamellar liposomes.

[0053] Comparative Example 8 Compared with Example 3, the difference is that no modified exosomes were added in step S4.

[0054] Specifically as follows: S4. Preparation of multilamellar liposomes: Dissolve 25 mg of lecithin and 12 mg of cholesterol in 50 mL of a dichloromethane-ethanol mixed solution (the volume ratio of dichloromethane to ethanol is 13:7), add 20 mL of a PBS buffer solution with a pH of 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, perform ultrasonic treatment at 1000 W for 15 min, and lyophilize to obtain multilamellar liposomes.

[0055] Comparative Example 9 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 were added to 20 mL of water to obtain a composition for promoting hair regeneration after chemotherapy.

[0056] Test Example 1 Anti-inflammatory effect Take SPF-grade NIH male mice as experimental mice, divide them into 14 groups with 10 mice in each group, use normal saline as the blank group, use fluocinonide acetate ointment as the positive drug group, and use the compositions for promoting hair regeneration after tumor chemotherapy prepared in Examples 1-3 and Comparative Examples 1-9 as the experimental groups.

[0057] Experimental method: Apply xylene evenly to both the inner and outer surfaces of the right auricle of each mouse to induce inflammation, with a dose of 100 μL / mouse. Do not treat the left ear and use it as a blank control group. 30 minutes after xylene-induced inflammation, except for the negative control group, animals in each group were given the corresponding test substance on the right ear, with a dose of 50 μL / mouse. When administering the drug, pay attention to evenly applying it to both the inner and outer surfaces of the right auricle. Animals in the blank control group were given distilled water on the right auricle, with a dose of 0.1 mL / mouse. After administration, irradiate the administration site with near-infrared light (808 nm) for 5 - 10 minutes. 1 hour after administering the test substance, euthanize the mice by cervical dislocation, cut off both auricles, wash the test substance on the right auricle with physiological saline, and dry it. Overlap the bilateral auricles, punch out ear pieces from the left and right ears with a puncher with a diameter of 8 mm, weigh the weight difference between the two ear pieces respectively, and calculate the swelling value. The swelling value = m 右耳耳片 - m 左耳耳片。

[0058] The results are shown in Table 1.

[0059] Table 1

[0060] Note: * Compared with the blank group, P < 0.05.

[0061] 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.

[0062] Test Example 2 Select C57BL / 6 mice, female, 6 - 8 weeks old, weighing 18 - 20 g, with pink back skin, and randomly divide them into a model group, Examples 1 - 3 groups, and Comparative Examples 1 - 9 groups, with 10 mice in each group.

[0063] All mice had normal diet during the experiment.

[0064] Induction of growing hair follicles: After anesthetizing all mice with ether, depilate the resting hair on the back of the mice using the rosin / wax depilation method to induce growing hair. After depilation, observe the skin color change and hair growth situation at the depilated area on the back of the mice. From the 1st day after depilation, mice in Examples 1 - 3 and Comparative Examples 1 - 9 groups were given 2 mL of the composition for promoting hair regeneration after tumor chemotherapy per mouse every day and evenly applied it to the depilated area. After each administration, irradiate the administration site with near-infrared light (808 nm) for 5 - 10 minutes. The model group was given an equal amount of physiological saline.

[0065] Administration of chemotherapy drugs: On the 9th day after depilation, when all hair follicles in the depilated areas were in the VI growth stage, the mice were intraperitoneally injected with a single dose of cyclophosphamide at 120 mg / kg to establish an animal model of hair loss after chemotherapy. Observe the hair loss and hair regeneration situations in the depilated areas on the back of the mice after continuing to administer the drugs.

[0066] Post-experiment treatment: On the 13th day after hair plucking, 5 mice in the experimental group and the model group were sacrificed by cervical dislocation. Samples were taken from the same back area parallel to the spine, embedded in OCT, frozen in liquid nitrogen, and 8-μm frozen sections were prepared. HE staining was performed, and histological changes in hair follicles were observed under a light microscope. Hair follicles were staged according to the morphological staging criteria of Muller-Rover. For each mouse, 60 hair follicles were randomly selected to calculate the average hair cycle score (HCS: growth stage VI was 100; early regression stage was 200; mid-regression stage was 300; late regression stage was 400), and the percentages of hair follicles in growth stage VI, early regression stage, mid-regression stage, and late regression stage were calculated.

[0067] The results are shown in Table 2.

[0068] Table 2

[0069] 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.

[0070] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a composition for promoting hair regrowth after chemotherapy, characterized in that, The modified nano-gold cages were prepared by modifying the surface of gold nano-cages with polydopamine, which were loaded on exosomes, complexed with magnesium ions and zinc ions to obtain modified exosomes. Nano-drug-loaded liposomes were prepared from curcumin, resveratrol, and epigallocatechin gallate, and mixed with the modified exosomes to obtain multi-layered liposomes, which were then mixed with a penetration enhancer and added to a thermosensitive hydrogel system to obtain a composition for promoting hair regrowth after chemotherapy.

2. The preparation method according to claim 1, characterized in that, It includes the following steps: S1. Preparation of modified nano-gold cages: Gold nano-cages were added to a Tris-HCl solution, dopamine hydrochloride was added, and the mixture was heated and stirred for reaction, centrifuged, washed, and dried to obtain modified nano-gold cages; S2. Preparation of nano-drug-loaded liposomes: Lecithin, cholesterol, curcumin, resveratrol, and epigallocatechin gallate were dissolved in a dichloromethane-ethanol mixed solution, and a vitamin B5-PBS buffer solution was added dropwise, stirred and mixed, and dichloromethane and ethanol were removed by rotary evaporation, followed by ultrasonic treatment and freeze-drying to obtain nano-drug-loaded liposomes; S3. Preparation of modified exosomes: Exosomes were added to water, NHS and EDC were added, stirred and activated, modified nano-gold cages were added, stirred and incubated, magnesium salts and zinc salts were added, stirred and complexed, dialyzed, and the non-permeated solution was freeze-dried to obtain modified exosomes; S4. Preparation of multi-layered liposomes: Lecithin and cholesterol were dissolved in a dichloromethane-ethanol mixed solution, a PBS buffer solution containing nano-drug-loaded liposomes and modified exosomes was added, stirred and mixed, and dichloromethane and ethanol were removed by reduced-pressure rotary evaporation, followed by ultrasonic treatment and freeze-drying to obtain multi-layered liposomes; S5. Preparation of thermosensitive hydrogel: Chitosan was dissolved in an acid solution, poloxamer P407 and P188 were added, and multi-layered liposomes and a penetration enhancer were added, stirred and mixed evenly to obtain a composition for promoting hair regrowth after chemotherapy.

3. The preparation method according to claim 2, wherein In step S1, the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of the gold nano-cages 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 h; in step S2, the mass ratio of lecithin, cholesterol, curcumin, resveratrol, epigallocatechin gallate, and vitamin B5 is 15-25:5-10:2-3:1-2:0.5-1:2-3; 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 exosomes described in step S3 are mesenchymal stem cell exosomes. The mass ratio of the exosomes, NHS, EDC, modified nanocages, 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 reaction temperature for stirring incubation is 36-38 °C at 100-200 r / min, and 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. In step S4, the mass ratio of lecithin, cholesterol, nano-drug-loaded liposomes and modified exosomes is 20-30:10-15:5-10:3-5. The volume ratio of dichloromethane to ethanol in the dichloromethane-ethanol mixed solution is 10-15:5-10.

5. The preparation method according to claim 2, wherein The acid solution described in step S5 is a 1-3 wt% acetic acid or lactic acid solution, and the mass ratio of chitosan, poloxamer P407, poloxamer P188, multilamellar liposomes and penetration enhancer is 15-25:5-8:0.5-1:7-10:1-2. The structural formula of the penetration enhancer is shown in Formula I: Formula I.

6. The preparation method according to claim 5, characterized in that, The preparation method of the penetration enhancer is as follows: T1. React epieucalyptol with salicylic acid to obtain an intermediate with the following structure: ； T2. React the intermediate with oleic acid to obtain a product.

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

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 also added. The catalyst is concentrated sulfuric acid, and the addition amount is 2-4 wt% of salicylic acid. The reaction solvent is toluene, and the reaction conditions are heating under reflux with stirring for 10-12 h.

9. A composition for promoting hair regrowth after chemotherapy prepared by the preparation method according to any one of claims 1-8.

10. Use of the composition for promoting hair regrowth after chemotherapy according to claim 9 in the preparation of a drug for treating alopecia after chemotherapy.

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