Freeze-dried powder entrapped with minoxidil hydrochloride mixed vesicles as well as preparation method and application of freeze-dried powder

Abstract

The invention relates to the technical field of biological medicine, in particular to minoxidil hydrochloride entrapped mixed vesicle freeze-dried powder as well as a preparation method and application thereof.The minoxidil hydrochloride entrapped mixed vesicle freeze-dried powder comprises minoxidil hydrochloride entrapped mixed membrane vesicles, a freeze-drying protective agent, a vesicle stabilizer, a plasticizer, a pH buffer system and the balance of water, and the mixed membrane vesicles are composed of stem cell membrane vesicles and phospholipid vesicles, through an optimized preparation process including high-speed shearing, high-pressure homogenization and double-layer polycarbonate filter membrane circulating extrusion, uniform dispersion and stability of the vesicles are ensured. The freeze-drying protective agent prevents the vesicles from being broken, keeps the structural integrity, and is convenient for long-term storage and transportation. The pH buffer system adjusts the pH of the preparation to 5.5-6.5, irritation to skin is reduced, patient compliance is improved, the freeze-dried powder can act on a scalp alopecia area in a coating, non-woven fabric water infiltration and coverage, microneedle introduction or subcutaneous injection mode after being dissolved, and experimental results show that the preparation remarkably improves the hair papilla cell proliferation activity to 186%-425%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a mixed vesicle lyophilized powder containing minoxidil hydrochloride, a preparation method and application thereof. BACKGROUND

[0002] At present, the treatment methods of alopecia mainly include drug therapy (oral and external use), hair transplantation, local injection (such as platelet-rich plasma), auxiliary physical therapy (such as microneedle, low-energy fractional laser therapy) and TCM syndrome differentiation and treatment. Commonly used drugs include finasteride, spironolactone, minoxidil and progestational drugs. However, these drug treatments have obvious adverse reactions, causing patients to have doubts. Although hair transplantation has become a mature treatment method, it still faces challenges due to the lack of autologous hair and the fact that alopecia is usually progressive. Other methods such as local injection, auxiliary physical therapy and TCM syndrome differentiation and treatment have not achieved ideal effects.

[0003] Minoxidil is a first-line external drug for androgenetic alopecia and alopecia areata, especially suitable for patients with mild to moderate alopecia. It promotes hair growth through multiple mechanisms: opening and expanding potassium channels, increasing blood supply to hair follicles, improving the microenvironment, prolonging the hair growth phase, shortening the resting phase, activating hair follicle stem cells (through SCF, VEGF, Wnt, etc.) and promoting hair follicle regeneration. However, minoxidil hydrochloride has poor water solubility, and in order to achieve an effective therapeutic concentration, it is usually prepared into tinctures and liniments with ethanol and propylene glycol as solvents. With the evaporation of organic solvents, a large amount of minoxidil crystals will precipitate on the skin surface, causing itching, skin rash, dandruff and allergic contact dermatitis and other drug irritant adverse reactions, and the patient's compliance is poor.

[0004] In recent years, the combined application of exosomes and other hair growth components has shown significant therapeutic effects. For example, patent CN115252647A discloses a composite external preparation for promoting hair growth, which uses exosomes to load minoxidil, which can not only exert the natural drug delivery and barrier penetration functions of exosomes, but also reduce the amount of organic solvents in the prescription, effectively improving the safety of treatment and patient compliance. In addition, patents CN112153957A and CN111617237A respectively demonstrate the application of mesenchymal stem cell exosomes and PLGA nanoparticles loaded with a new type of small molecule hair growth agent and collagen loaded in hair papilla cell exosomes, which produce better hair regeneration effects than ordinary minoxidil hydrochloride preparations in alopecia mouse models. However, exosomes have high production costs, poor stability and need to be stored at low temperatures, which has certain limitations in commercialization. SUMMARY

[0005] (I) Technical problems solved

[0006] In view of the deficiencies of the prior art, the application provides a mixed vesicle lyophilized powder for loading minoxidil hydrochloride, a preparation method and application thereof.

[0007] (II) Technical solutions

[0008] To achieve the above object, the application provides the following technical solutions: the mixed vesicle lyophilized powder for loading minoxidil hydrochloride comprises the following components: mixed membrane vesicles for loading minoxidil hydrochloride, 2-100 trillion particles per bottle, a lyophilization protective agent, 0.5-10 wt%, a vesicle stabilizer, 0.1-1.0 wt%, a plasticizer, 1.0-3.0 wt%, a pH buffer system, 0.02-2.7 wt%, and the balance is water;

[0009] The mixed membrane vesicles are mixed by stem cell membrane vesicles and phospholipid vesicles, and the stem cell membrane vesicles are derived from one of hair follicle stem cells, umbilical cord-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells;

[0010] The phospholipid of the phospholipid vesicles is soybean phospholipid or lecithin, the lyophilization protective agent is one of mannitol, trehalose and glycine, the vesicle stabilizer is vitamin E polyethylene glycol succinate or poloxamer 188, the plasticizer is 1,2-propanediol, the pH buffer system is a sodium phosphate dibasic / sodium phosphate monobasic buffer system or a citric acid / citric acid sodium buffer system, and the pH buffer system adjusts the pH of the preparation to 5.5-6.5.

[0011] Preferably, the particle size of the mixed membrane vesicles is 100-200 nm, the Zeta potential is-20 to-40 mV, the content of minoxidil hydrochloride in the lyophilized powder preparation is 0.1-1.0 wt%, and the total content of phospholipids in the suspension before lyophilization of the lyophilized powder preparation is 0.5-5.0 wt%.

[0012] Further preferably, the stem cell membrane vesicles are derived from hair follicle stem cells, the lyophilization protective agent is trehalose, the vesicle stabilizer is vitamin E polyethylene glycol succinate, and the pH buffer system is a citric acid / citric acid sodium buffer system.

[0013] Still more preferably, the preparation method of the mixed vesicle lyophilized powder for loading minoxidil hydrochloride comprises the following steps:

[0014] Step 1: weigh the raw material components according to the mass percentage: minoxidil hydrochloride, 0.1-1.0 wt%, a lyophilization protective agent, 0.5-10 wt%, phospholipids, 0.5-5.0 wt%, a vesicle stabilizer, 0.1-1.0 wt%, 1,2-propanediol, 1.0-3.0 wt%, a pH buffer system, 0.02-2.7 wt%, and the balance is batch production water;

[0015] Step 2: Add minoxidil hydrochloride and vesicle stabilizer into a suitable container, add 1,2-propanediol, heat to 50-65℃, stir to dissolve until clear, to obtain material a;

[0016] Step 3: Add phospholipid to material a, mix in a closed container at 50-65℃, to obtain material b;

[0017] Step 4: Add components of pH buffer system into a suitable container, add an appropriate amount of water, stir to dissolve until clear, check that the pH is in the range of 5.5-6.5, to obtain material c;

[0018] Step 5: Add material b to material c, high-speed shear, high-pressure homogenization, to obtain material d;

[0019] Step 6: Circulatory extrude material d through 10μm-2μm double-layer polycarbonate filter membranes 3-10 times, to remove insoluble particles and impurities, to obtain material e;

[0020] Step 7: Circulatory extrude P4-P8 stem cells through 10μm-2μm double-layer polycarbonate filter membranes 3-10 times, to obtain material f;

[0021] Step 8: Combine and mix material e and material f, to obtain material g;

[0022] Step 9: Circulatory extrude material g through 0.8μm-0.2μm double-layer polycarbonate filter membranes 3-10 times, to obtain material h;

[0023] Step 10: Add the prescription amount of lyophilization protectant into a suitable container, add the remaining amount of water to dissolve until clear, to obtain material i;

[0024] Step 11: Combine material h and material i, mix well, filter through a 0.2μm PES filter membrane, to obtain material j;

[0025] Step 12: Measure the nanoparticle size of material j, confirm that the number of dispensable particles is 2-100 trillion / bottle, the particle size is 100-200nm, and the Zeta potential is -20 to -40mV, then dispense material j into containers, semi-tighten the stopper, and freeze dry;

[0026] Step 13: After freeze drying, flush with nitrogen, tighten the stopper, cap, and subsequent packaging.

[0027] Preferably, the freeze-drying procedure in step 12 comprises:

[0028] (a) Material temperature equilibration: equilibrate at 0-10℃ for 30-60 minutes;

[0029] ​(b) pre-freezing: pre-freezing at -40 to -50℃ for 120-360 minutes;

[0030] (c) sublimation drying: drying at -20-0℃ and a vacuum degree of 10-50 Pa for 600-2000 minutes;

[0031] (d) desorption drying: drying at 0-30℃ and a vacuum degree of 5-30 Pa for 120-660 minutes.

[0032] Further preferably, the rotation speed of the high-speed shearing in step 5 is 8000-12000 r / min, and the shearing time is 5-10 minutes; the pressure of the high-pressure homogenization is 30-50 MPa, and the homogenization times is 3-5 times.

[0033] Again preferably, the stem cells in step 7 are hair follicle stem cells, the filter membrane circulation extrusion times in steps 6 and 9 are both 5-8 times, and the container in step 12 is a stoppered erlenmeyer flask.

[0034] Preferably, the freeze-drying procedure in step 12 is specifically as follows:

[0035] (a) material temperature balancing: balancing at 2-6℃ for 30-60 minutes;

[0036] (b) pre-freezing: pre-freezing at -42 to -45℃ for 240-300 minutes;

[0037] (c) sublimation drying: first drying at -10 to -15℃ and a vacuum degree of 20-30 Pa for 300-720 minutes, and then drying at 0℃ and a vacuum degree of 20-30 Pa for 300-600 minutes;

[0038] (d) desorption drying: first drying at 5-10℃ and a vacuum degree of 10-15 Pa for 120 minutes, and then drying at 30℃ and a vacuum degree of 10-15 Pa for 540 minutes.

[0039] Further preferably, the application of the freeze-dried powder of the mixed vesicles loaded with minoxidil hydrochloride is that, when used, 1-2 times the weight of the freeze-dried powder before freeze-drying of physiological saline or sterile water for injection is added to the freeze-dried powder preparation to dissolve, and the freeze-dried powder preparation is applied to the hair loss area of the scalp by coating, non-woven fabric water immersion covering, micro-needle / micro-crystal pen introduction or subcutaneous injection.

[0040] Again preferably, after the freeze-dried powder preparation is dissolved, the freeze-dried powder preparation is administered once a day, the administration amount is 1 mL each time, the continuous administration period is ≥21 days, and after the freeze-dried powder preparation is dissolved, the proliferation activity of the hair follicle cells is increased to 186%-425%.

[0041] (Three) beneficial effects

[0042] Compared with the prior art, the application provides a minoxidil hydrochloride-loaded mixed vesicle freeze-dried powder and a preparation method and application thereof, and has the following beneficial effects:

[0043] The technical solution has remarkable drug delivery efficiency, biocompatibility, stability and therapeutic effect, is suitable for hair loss treatment, and mainly comprises minoxidil hydrochloride-loaded mixed membrane vesicles, a freeze-drying protective agent, a vesicle stabilizer, a plasticizer and a pH buffer system, and the balance is water.

[0044] The stem cell membrane vesicle and phospholipid vesicle mixing technology is adopted to effectively improve the drug targeting and cell uptake efficiency. The hair follicle stem cell membrane vesicle can specifically recognize and combine with the hair papilla cells, enhance the local drug concentration and improve the curative effect.

[0045] The vesicle particle size is controlled to be 100-200 nm, and the Zeta potential is-20 to-40 mV, so that good dispersibility and stability are ensured, the agglomeration phenomenon is avoided, the blood circulation time is prolonged, and the side effects are reduced.

[0046] Mannitol, trehalose or glycine are used as the freeze-drying protective agent, can form a glassy matrix in the freeze-drying process, prevent the vesicle from being broken, maintain the structural integrity and facilitate long-term storage.

[0047] Vitamin E polyethylene glycol succinate or poloxamer 188 significantly enhances the vesicle stability, prevents drug leakage and ensures the drug activity.

[0048] Through high-speed shearing and high-pressure homogenization treatment, the uniform distribution of the vesicles is ensured, the particle size is consistent, and the preparation quality is improved.

[0049] Double-layer polycarbonate filter membranes are used for circulation extrusion to remove insoluble particles and impurities, so that the purity and safety of the preparation are ensured.

[0050] The sodium phosphate dibasic / sodium phosphate monobasic or citric acid / sodium citrate buffer system adjusts the pH of the preparation to 5.5-6.5, which is close to the physiological pH value of the skin, reduces the irritation and improves the patient compliance.

[0051] After the freeze-dried powder is dissolved, the proliferation activity of the hair papilla cells is increased to 186%-425%, the hair growth is significantly promoted, and the hair loss condition is improved.

[0052] The freeze-dried powder can be used on the scalp hair loss area through various ways such as coating, non-woven fabric water immersion covering, microneedle / microcrystal pen introduction or subcutaneous injection, and can flexibly adapt to different clinical needs. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 It is a schematic diagram of the core component system of the application;

[0054] Figure 2 It is a schematic diagram of the raw material pretreatment and initial material body of the application;

[0055] Figure 3 Preparation diagram of phospholipid vesicle and stem cell membrane vesicle of the application;

[0056] Figure 4 Preparation diagram of mixed vesicle fine-tuning and pre-treatment before freeze-drying of the application;

[0057] Figure 5 Application scheme system diagram of the application. DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the application.

[0059] Please refer to Figures 1-5 The freeze-dried powder of the mixed vesicle loaded with minoxidil hydrochloride of the application comprises the following components: mixed membrane vesicle loaded with minoxidil hydrochloride, 2-100 trillion particles per bottle, freeze-drying protective agent, 0.5-10 wt%, vesicle stabilizer, 0.1-1.0 wt%, plasticizer, 1.0-3.0 wt%, pH buffer system, 0.02-2.7 wt%, and the balance is water.

[0060] The mixed membrane vesicle is mixed by stem cell membrane vesicle and phospholipid vesicle, and the stem cell membrane vesicle is derived from one of hair follicle stem cells, umbilical cord-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells.

[0061] The phospholipid of the phospholipid vesicle is soybean phospholipid or lecithin, the freeze-drying protective agent is one of mannitol, trehalose and glycine, the vesicle stabilizer is vitamin E polyethylene glycol succinate or poloxamer 188, the plasticizer is 1,2-propanediol, the pH buffer system is a sodium phosphate dibasic / sodium phosphate monobasic buffer system or a citric acid / sodium citrate buffer system, and the pH buffer system adjusts the pH of the preparation to 5.5-6.5.

[0062] The technical solution solves the problems of poor water solubility of minoxidil hydrochloride, strong skin irritation and safety risk of stem cell treatment through the synergistic effect of stem cell membrane vesicle and phospholipid vesicle, and realizes stable storage of the preparation at room temperature by means of freeze-drying process. The specific working principle is as follows:

[0063] The mixed membrane vesicle serves as a delivery carrier of minoxidil hydrochloride, has the triple effects of targeting hair follicles, drug sustained release and microenvironment repair, and improves the bioavailability of the drug and the hair growth promotion effect.

[0064] Drug encapsulation of phospholipid vesicles: soybean phospholipid / lecithin self-assembles into bilayer vesicles in an aqueous environment, minoxidil hydrochloride, a fat-soluble drug, is encapsulated in the bilayer interlayer or the internal water phase. With the help of 1,2-propanediol, the solubility is increased from 0.2 g / 100 mL to 1.5 g / 100 mL, and the encapsulation efficiency can reach more than 85%. The vesicle particle size is 100-200 nm, which can penetrate the hair follicle sebaceous gland duct, avoiding the problem of drug crystal retention on the skin surface in traditional tincture;

[0065] Synergistic effect of stem cell membrane vesicles: membrane vesicles derived from hair follicle stem cells retain stem cell surface adhesion molecules such as CD44 and Integrin β1, which can specifically bind to hair follicle epithelial cells for targeted delivery. At the same time, they carry endogenous growth factors such as VEGF and FGF, which can improve the hair follicle microenvironment through paracrine effect and activate hair follicle stem cell proliferation. After mixing in a ratio of 1:1-3:1, mixed vesicles are formed through membrane fusion, extending the drug release period from 4 hours in traditional preparations to 12 hours, and continuously acting on the hair follicle.

[0066] Zeta potential regulation: Zeta potential of-20 to-40 mV avoids vesicle aggregation through electrostatic repulsion, ensuring that the preparation remains well dispersed after reconstitution, improving skin penetration, and Franz diffusion cell experiments show that the drug penetration amount is 3.2 times higher than that of the commercially available liniment.

[0067] Functional additives

[0068] Freeze-drying protectants such as trehalose / mannitol / glycine:

[0069] Through the water replacement mechanism, trehalose forms hydrogen bonds with membrane proteins and a glass formation mechanism, forming an amorphous glass state after freeze-drying, inhibiting the damage of ice crystals to the vesicle membrane during freeze-drying, and ensuring that the vesicle integrity retention rate is ≥90%. At the same time, it reduces the hygroscopicity of the preparation after freeze-drying, ensuring that the vesicle particle size changes by ≤10% after 12 months of storage at room temperature;

[0070] Vesicle stabilizers such as TPGS / poloxamer 188:

[0071] Embedded in the phospholipid bilayer, reducing the membrane surface tension from 35 mN / m to 28 mN / m, inhibiting vesicle aggregation and fusion; a dosage of 0.1-1.0 wt% can make the vesicles change in particle size by ≤5% at 4°C for 30 days, avoiding drug leakage;

[0072] pH buffer system, such as citric acid / sodium citrate, etc.:

[0073] The formulation pH is adjusted to 5.5-6.5, matching the physiological environment of the scalp, such as scalp pH 5.4-5.9, reducing the irritation to the skin barrier; while maintaining the stability of the vesicle membrane, the vesicle rupture rate increases by 15% and the drug leakage increases by 20% when the pH deviates from 6.0;

[0074] Plasticizers such as 1,2-propanediol:

[0075] Both solubilization and plasticization have dual effects, which not only improve the solubility of minoxidil hydrochloride in water, but also enhance the flexibility of the vesicle membrane, avoiding vesicle rupture during filter membrane extrusion.

[0076] The freeze-drying process removes free water and bound water in the formulation by low-temperature freeze-drying, converting the formulation from a liquid state to a solid freeze-dried powder, achieving room temperature storage, such as 25℃ shelf life ≥12 months, solving the storage problem of traditional liquid dosage forms which need 4℃ cold storage with a shelf life of only 3 months.

[0077] The pre-freezing stage of -40 to -50℃ makes the material completely frozen, avoiding mechanical damage to the vesicle membrane caused by ice crystal growth; the sublimation drying stage, -20-0℃, 10-50Pa, slowly removes free water to prevent vesicle collapse due to rapid water loss; the desorption drying stage, 0-30℃, 5-30Pa, removes bound water to improve the stability of the formulation; nitrogen flushing after freeze-drying further inhibits the oxidative degradation of the vesicle.

[0078] Application mechanism

[0079] After reconstitution of the freeze-dried powder, add 1-2 times the weight of normal saline / sterile water for injection, and apply it to the scalp through coating, microneedle introduction, etc.

[0080] When coating, mixed vesicles penetrate into the hair follicle root through the hair follicle duct;

[0081] Microneedle introduction can open small channels in the skin, further increasing the permeability of the vesicle by 50%;

[0082] After drug release, minoxidil hydrochloride expands the blood vessels of the hair follicle through the open potassium channel, increasing blood supply; the growth factors carried by the stem cell membrane vesicle activate the Wnt / β-catenin signaling pathway, prolonging the hair growth period and shortening the resting period; the synergistic effect of the two makes the proliferation activity of the hair papilla cells reach 186%-425%, and the hair coverage area of the alopecia mice reaches 0-1 level after 21 days, completely covering or slightly sparse.

[0083] Component optimization: hair follicle stem cell membrane vesicles, trehalose, TPGS, citric acid / sodium citrate system;

[0084] Compared with umbilical cord / fat mesenchymal stem cell membrane vesicles, the hair follicle targeting of hair follicle stem cell membrane vesicles is increased by 40%, the growth factor content is increased by 20%, and the proliferation activity of hair papilla cells is increased by 30%.

[0085] Compared with mannitol / glycine, the recovery rate of vesicles after freeze-drying is increased by 15% to 95%, and the reconstitution time is short and the operation is convenient.

[0086] Compared with poloxamer 188, TPGS has better stability effect on vesicle membrane, and the vesicle rupture rate is reduced by 10% after being placed at 4°C for 30 days, and has a certain penetration effect, and the drug skin penetration rate is increased by 15%.

[0087] Compared with disodium hydrogen phosphate / sodium dihydrogen phosphate, the pH buffering capacity of the citric acid / sodium citrate buffer system is stronger, and the skin irritation is lower.

[0088] This combination makes the preparation achieve the optimal balance in efficacy, stability and safety, and is especially suitable for sensitive skin and children.

[0089] The preferred preparation process is: high-speed shearing, such as 10000r / min, high-pressure homogenization, such as 40MPa multi-stage membrane filtration extrusion 5-8 times

[0090] Shearing at 10000r / min for 8 minutes can preliminarily disperse phospholipids and drugs, avoiding local concentration too high leading to vesicle aggregation;

[0091] High-pressure homogenization at 40MPa for 4 times can refine the particle size, improve the uniformity of phospholipid vesicles, and increase the drug encapsulation rate by 10%;

[0092] 10μm-2μm filter membrane extrusion 5 times to remove insoluble impurities such as undissolved phospholipid particles, 0.8μm-0.2μm filter membrane extrusion 5 times to accurately control the particle size to 100-200nm, the particle size distribution coefficient of variation is ≤15%, avoiding large particle size vesicles cannot penetrate the hair follicle duct;

[0093] The stem cell membrane vesicles are prepared by membrane filtration extrusion instead of traditional ultracentrifugation, the preparation time is shortened from 8 hours to 1 hour, the membrane protein retention rate is increased from 60% to 80%, the production cost is reduced by 60%, and it is suitable for large-scale production, such as single batch yield can reach 1000 bottles.

[0094] The preferred freeze-drying process is: staged temperature control freeze-drying, -42°C pre-freezing, gradient sublimation, 30°C resolution

[0095] -42°C pre-freezing for 240 minutes, compared with -40°C / 360 minutes, not only ensures that the material is completely frozen, but also shortens the pre-freezing time and improves the production efficiency;

[0096] Sublimation drying is divided into stages, -15°C→0°C, to avoid temperature rising too fast and causing the collapse of the vesicles, and to improve the integrity of the vesicles by 10%; 20-30 Pa vacuum degree can slowly remove free water to prevent ice crystals from recrystallizing;

[0097] Analytical drying at 30°C combined with 10-15 Pa high vacuum can improve the removal rate of combined water by 20% compared to 0°C analysis, and the stability of the preparation in the 40°C / 75%RH accelerated test is improved by 15% within 6 months.

[0098] Application mode is preferred: micro-needle introduction, 1 mL per day, 21-day cycle

[0099] 0.2-0.5mm micro-needle introduction can open the channel of the stratum corneum, increase the permeability of the vesicles by 50% compared to the coating method, and increase the amount of drugs reaching the root of the hair follicle by 40%, shortening the onset time from 7 days to 5 days;

[0100] 1 mL per day, 1 mL per dose, can maintain the local drug concentration in the hair follicle within the effective treatment window, avoiding skin irritation caused by excessive dose or insufficient efficacy caused by low dose;

[0101] 21-day continuous administration cycle, one small cycle of hair growth, can ensure that the hair follicle is completely transferred from the resting phase to the growth phase, and the hair coverage area is increased by 30% compared to the 14-day cycle, and the recurrence rate is reduced by 25% after drug withdrawal.

[0102] Detailed process summary

[0103] Raw material preparation and pretreatment stage

[0104] Step 1: Raw material weighing and inspection

[0105] Weigh each raw material accurately according to the formula ratio. For example, 1000 bottles / 5mL specifications: minoxidil hydrochloride 1.0kg, purity ≥99.5%, meeting the pharmaceutical standards of Chinese Pharmacopoeia, trehalose 10.0kg, food and drug grade, purity ≥99%, soybean phospholipid 5.0kg, phosphatidylcholine content ≥70%, acid value ≤30mg KOH / g, vitamin E polyethylene glycol succinate (TPGS) 1.0kg, pharmaceutical grade, PEG content ≥90%, 1,2-propanediol 3.0kg, pharmaceutical grade, purity ≥99.5%, citric acid 0.62kg, sodium citrate 2.07kg, purified water 87.31kg, meeting the standard of water for injection, electrical conductivity ≤2.1μS / cm@25℃;

[0106] Raw material inspection: high performance liquid chromatography is used to detect the purity of minoxidil hydrochloride to ensure that there is no impurity peak; thin layer chromatography is used to verify the phosphatidylcholine content in soybean phospholipid; trypan blue staining method is used to detect the activity of P4-P8 generation of hair follicle stem cells, ensuring that it is ≥90%, and unqualified raw materials need to be replaced immediately to avoid affecting the quality of the preparation.

[0107] Step 2: Stem cell pretreatment

[0108] Stem cell collection and washing: the hair follicle stem cells cultured to P4-P8 generation were digested with 0.25% trypsin-EDTA for 3-5 minutes, and the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum, and the cells were collected by centrifugation at 1000 r / min for 5 minutes; repeatedly washed with normal saline, and the supernatant was discarded after each centrifugation to remove residual culture medium and digestion solution;

[0109] Cell concentration adjustment: resuspend the cells with normal saline, count them using a hemocytometer, and adjust the cell concentration to 1 x 10<6> cells / mL; take a sample for review of viability by staining with trypan blue, and store it in a 4°C refrigerator to avoid a decrease in cell viability caused by room temperature storage for more than 30 minutes;

[0110] Filter inspection: take out 10 μm-2 μm and 0.8 μm-0.2 μm double-layer polycarbonate filters, rinse them twice with sterile normal saline under a clean bench, confirm that the filters have no damage or pinholes by light transmission inspection, and check the sealing of the extruder pipeline to avoid liquid leakage or impurity contamination during preparation.

[0111] Mixed vesicle preparation stage

[0112] Step 1: Preparation of phospholipid vesicles

[0113] Preparation of material body a: add 1.0 kg of minoxidil hydrochloride and 1.0 kg of TPGS to a 100 L stainless steel stirring tank, add 3.0 kg of 1,2-propanediol, start the stirring paddle at a speed of 500 r / min, heat to 50-65°C through the jacket, preferably 55°C, monitor the temperature in the tank in real time with a thermometer, continue stirring for 20 minutes until the solution is completely clear, check by visual inspection that there are no visible particles, and ensure that the minoxidil hydrochloride is completely dissolved to avoid the impact of undissolved drugs on the subsequent vesicle encapsulation rate;

[0114] Preparation of material body b: slowly add 5.0 kg of soybean phospholipid to material body a, close the stirring tank to prevent water evaporation from causing changes in concentration, maintain the temperature in the tank at 55°C, and increase the stirring speed to 1000 r / min, continue stirring for 30 minutes to uniformly disperse the phospholipid and form a preliminary emulsion, and take a sample to observe that there is no obvious phospholipid aggregation;

[0115] ​Material body c preparation: in another 50L stirring tank, add 20kg purified water, put in citric acid 0.62kg, sodium citrate 2.07kg, start stirring, 800r / min, until completely dissolved, use precision pH meter to detect solution pH, need to control in 5.5-6.5, preferably 6.0;If pH<5.5, supplement sodium citrate to adjust;If pH>6.5, supplement citric acid to adjust, ensure that the buffer system is stable, avoid subsequent vesicle rupture due to pH fluctuation;

[0116] Material body d preparation: slowly inject material body b into material body c through peristaltic pump, flow rate 500mL / min, open high-speed shearing machine at the same time, speed 8000-12000r / min, preferably 10000r / min, shear for 5-10 minutes, preferably 8 minutes, form coarse vesicle suspension;Then introduce the suspension into a high-pressure homogenizer, pressure 30-50MPa, preferably 40MPa, homogenize 3-5 times, preferably 4 times, take samples after each homogenization and observe until the suspension is uniform and white, without stratification, at this time the phospholipid vesicle is preliminarily formed;

[0117] Material body e preparation: transfer material body d to the extruder, first circulate extrusion 3 times through a 10μm filter membrane, then replace the 2μm filter membrane and circulate extrusion 2 times, a total of 5 times, to remove undispersed phospholipid particles and impurities;Control the flow rate to 200mL / min during extrusion to avoid pressure surge causing filter membrane damage;After extrusion, measure the particle size with a nanoparticle size analyzer, at this time the particle size should be controlled in 200-300nm, PDI≤0.3, unqualified needs to be re-extruded.

[0118] Step 2: preparation of stem cell membrane vesicles

[0119] Material body f preparation: transfer the pretreated hair follicle stem cell suspension to the extruder, install a 10μm-2μm double-layer polycarbonate filter membrane, start the extruder, pressure 0.3-0.5MPa, circulate extrusion 3-10 times, preferably 5 times;Mechanical shearing force of the filter membrane destroys the cell membrane to form stem cell membrane vesicles;After extrusion, detect the membrane protein content with BCA protein quantitative kit, and detect the specific marker on the surface of the vesicles such as CD44 and Integrinβ1 with flow cytometry, confirm that the membrane protein retention rate is≥80%;If the membrane protein content is insufficient, supplement stem cells and re-extrude.

[0120] Step 3: preparation of mixed vesicles

[0121] Material body g preparation: material body e and material body f are added into a 50L mixing tank at a volume ratio of 1:1-3:1, preferably 2:1, stirring is started at a speed of 800r / min, and mixing is carried out at room temperature for 30 minutes; during the mixing process, samples are taken every 10 minutes, and the membrane fusion is observed by using a fluorescence probe labeling method, such as DiI labeling phospholipid vesicles and DiO labeling stem cell membrane vesicles, to ensure that the two kinds of vesicles are fully fused and there is no obvious stratification;

[0122] Material body h preparation: the material body g is transferred to an extruder, 0.8μm filter membrane is installed first, and extrusion is carried out 3 times, then 0.2μm filter membrane is replaced, and extrusion is carried out 2 times, for a total of 5 times, to accurately control the particle size of the mixed vesicles to 100-200nm; after extrusion, the particle size and Zeta potential are detected by a nanoparticle size analyzer, the Zeta potential needs to be in the range of-20 to-0mV, to ensure the dispersion of the vesicles, and the PDI≤0.2; if the particle size is too large or the Zeta potential deviates from the range, the number of extrusions needs to be increased or the mixing ratio of the two kinds of vesicles needs to be adjusted until the parameters are qualified.

[0123] Freeze-drying pretreatment and sub-packaging stage

[0124] Step 1: freeze-drying protectant dissolution and mixing

[0125] Material body i preparation: 10.0kg of trehalose is added into a 100L stirring tank, the remaining purified water, about 67.31kg, is added, stirring is started at a speed of 800r / min, and stirring is carried out at room temperature for 20 minutes until the trehalose is completely dissolved; the sample is detected by a refractometer to ensure that the trehalose content meets the formula requirement of 0.5-10wt%, to avoid the concentration deviation affecting the freeze-drying effect;

[0126] Material body j preparation: the material body h is slowly injected into the material body i through a peristaltic pump, stirring is carried out at a speed of 800r / min while injecting, and mixing is carried out for 30 minutes; after the mixing is completed, the suspension is filtered through a 0.2μm PES filter membrane to remove microorganisms and small impurities; during the filtering process, the pressure is controlled to be≤0.2MPa to avoid excessive pressure causing the vesicles to be broken, and the filtered freeze-drying liquid is collected and placed in a sterile storage tank for standby.

[0127] Step 2: intermediate detection and sub-packaging

[0128] Intermediate detection: 10mL of the freeze-drying liquid is taken, the particle number is measured by a nanoparticle size analyzer, which needs to be in the range of 2-100 million per bottle, preferably 10 million per bottle, the particle size is 100-200nm, and the Zeta potential is-20 to-40mV; at the same time, the content of minoxidil hydrochloride is detected by HPLC to be 0.1-1.0wt%, to ensure that it meets the formula requirement; if any parameter is unqualified, it needs to be returned to the corresponding step for adjustment, such as supplementing mixed vesicles if the particle number is insufficient, or supplementing minoxidil hydrochloride if the content deviates;

[0129] Sub-packing operation: In the ten-thousand-level clean area, the qualified material j is sub-packed into 5 mL stopper cefalin bottles by automatic sub-packing machine, 2 mL per bottle; after sub-packing, the butyl rubber plug is pressed into the bottle opening by half with a half-pressure plug machine, leaving a ventilation channel to facilitate water vapor discharge during freeze drying; the cefalin bottles are arranged neatly on the freeze dryer tray, which needs to be sterilized in advance to avoid contamination.

[0130] Freeze-drying and packaging stage

[0131] Step 1: Freeze-drying program execution

[0132] Material temperature balance: Push the tray containing the cefalin bottles into the freeze dryer cold trap, close the freeze dryer door, turn on the refrigeration system, reduce the cold trap temperature to 0-10°C, preferably 4°C, and maintain for 30-60 minutes, preferably 45 minutes, to make the material temperature in the bottle consistent with the cold trap temperature, avoiding ice crystal breakage due to excessive temperature difference during subsequent pre-freezing;

[0133] Pre-freezing: Continue to reduce the cold trap temperature to -40 to -50°C, preferably -42°C, and maintain for 120-360 minutes, preferably 240 minutes; insert the temperature probe into the control bottle material to confirm that the material center temperature is reduced to below -40°C and completely frozen, avoiding material collapse after freeze-drying due to incomplete freezing;

[0134] Sublimation drying: Turn on the vacuum system to reduce the vacuum degree in the freeze dryer to 10-50 Pa, preferably 20 Pa, and slowly heat to -10 to -15°C, preferably -12°C, and maintain for 300-720 minutes, preferably 600 minutes, at which point the free water in the bottle begins to sublimate; then continue to heat to 0°C and maintain for 300-600 minutes, preferably 450 minutes, to further remove residual free water; monitor the vacuum degree in real time during sublimation by using a vacuum gauge; if the vacuum degree suddenly rises, check the system sealing to avoid incomplete sublimation;

[0135] Desorption drying: Maintain the vacuum degree at 10-15 Pa, first raise the cold trap temperature to 5-10°C, preferably 8°C, and maintain for 120 minutes to remove part of the bound water in the material; then heat to 30°C and maintain for 540 minutes to completely remove the remaining bound water; after drying, take a sample and test the water content with a Karl Fischer moisture meter, which should be ≤3%; if the water content exceeds the standard, extend the desorption drying time.

[0136] Step 2: Nitrogen protection and packaging

[0137] Nitrogen filling: After freeze-drying, turn off the vacuum system and fill sterile nitrogen into the freeze dryer until the pressure in the machine returns to normal pressure to avoid air entering and causing material oxidation;

[0138] Plug and capping: open the freeze-drying machine door, immediately use the full plug machine to press the rubber plug of the test tube into the bottle opening; then transfer the test tube to the capping machine, and use the aluminum-plastic combined cover to cap;

[0139] Lamp inspection and packaging: place the capped test tube under the lamp inspection machine for inspection, and the qualified standard is: the freeze-dried block is uniform and loose white block, without collapse, discoloration, foreign matter; unqualified products, such as freeze-dried block cracking, black spots need to be removed; qualified products are labeled with product name, specification, batch number, expiration date, and packed into paper box, and desiccant such as silica gel is placed in the paper box, the content is 1g / box, finally packed into aluminum-plastic bag and sealed, stored in warehouse, storage condition: below 25℃, relative humidity ≤60%, away from strong light and heat source.

[0140] Application and efficacy monitoring stage

[0141] Step 1: preparation reconstitution

[0142] Reconstitution operation: before use, in a clean environment such as a household clean table top or a medical treatment room, open the aluminum-plastic cover of the test tube, use a sterile syringe to extract 2mL of normal saline or sterilized water for injection, which meets the pharmaceutical standard, slowly inject into the freeze-dried powder bottle; gently shake the test tube to avoid violent shaking causing vesicle rupture, about 30 seconds after the freeze-dried powder is completely dissolved, forming a uniform white suspension, no visible particles or precipitates; if there are undissolved substances, check whether the reconstitution water is qualified or the freeze-dried powder is damp, unqualified preparation is prohibited.

[0143] Step 2: administration method and cycle

[0144] Administration method selection:

[0145] Mild alopecia: mild hair sparseness, visible scalp under strong light: use the coating method, use a sterile cotton swab to dip the reconstituted solution, evenly apply it to the alopecia area, gently massage for 3-5 minutes, promote vesicle penetration, 1mL per time, 1 time per day;

[0146] Moderate alopecia: visible scalp under natural light, hair is obviously sparse: use non-woven fabric water immersion covering method, dip the sterile non-woven fabric with reconstituted solution, apply it to the alopecia area, keep for 30 minutes, then remove it, the remaining liquid is absorbed by massage, 1 time per day, 1mL per time;

[0147] Severe alopecia: large area of scalp exposure, severe hair sparseness: use microneedle introduction method, use 0.2mm sterile microneedle to roll in the alopecia area 2 times a week, the force is appropriate to cause slight redness, avoid bleeding, then immediately apply the reconstituted solution and massage for 5 minutes; for the remaining 5 days, use the coating method, 1 time per day, 1mL per time;

[0148] Dosing cycle: continuous administration for 21 days, small cycle of hair growth, ensure that the hair follicles from the resting phase into the growth phase, daily fixed time administration, such as before sleep, avoid missing; if missing, the next day according to the normal dose of compensation, no need to double dose.

[0149] Step 3: efficacy monitoring

[0150] On the 7th day, initial efficacy observation:

[0151] Visual assessment: the degree of hair thinning should be reduced from level 3, obvious thinning, to level 2, moderate thinning, epidermis visible under natural light, from level 4, severe hair loss, to level 3, severe hair loss, with reduced redness of the scalp, no obvious irritation reactions such as itching and rash;

[0152] Safety monitoring: observe whether there are adverse reactions such as redness, itching, and desquamation on the scalp, if there is mild irritation, redness, and mild itching, reduce the massage time to 1 minute, if the symptoms worsen, suspend the drug and consult a professional;

[0153] On the 14th day:

[0154] Cell activity detection: take samples through skin biopsy in medical institutions, such as using CCK-8 method to detect hair papilla cell proliferation activity, which should be ≥200%, taking the pre-administration as 100% benchmark, and the cell morphology is normal without apoptosis signs;

[0155] Hair density monitoring: use a hair density instrument to randomly select 3 detection points in the hair loss area, calculate the average hair density, which should be increased by 10%-15% compared with the pre-administration;

[0156] On the 21st day:

[0157] Hair coverage evaluation: use the hair loss grading standard, level 0: completely covered, no visible epidermis; level 1: mild thinning, epidermis visible only under strong light; level 2: moderate thinning, epidermis visible under natural light; level 3: obvious thinning, epidermis exposure >50%; level 4 and above: severe hair loss, mild hair loss should reach level 1, moderate hair loss should reach level 2, and severe hair loss should reach level 3 or below;

[0158] Long-term effect suggestion: after 21 days of administration, it can be changed to administration once every 2 days, maintain for 1 month, and consolidate the effect; at the same time, record the hair growth conditions such as hair shaft diameter and growth speed, and regularly review to ensure stable efficacy;

[0159] Adverse reaction summary: statistics the incidence of adverse reactions during the 21-day administration, no serious allergic or irritation events.

[0160] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and adaptions can be effected without departing from the spirit and scope of the present application, which is not limited to the exact construction and arrangement described. It is intended, therefore, to cover all modifications and adaptions that fall within the scope of the claims and their equivalents.

Claims

1. A lyophilized powder containing minoxidil hydrochloride mixed vesicles, characterized in that, Includes the following components: The mixture of membrane vesicles containing minoxidil hydrochloride contains 2-100 trillion particles per bottle, 0.5-10 wt% lyophilization protectant, 0.1-1.0 wt% vesicle stabilizer, 1.0-3.0 wt% plasticizer, 0.02-2.7 wt% pH buffer system, and the balance is water. The mixed membrane vesicles are a mixture of stem cell membrane vesicles and phospholipid vesicles, and the stem cell membrane vesicles are derived from one of hair follicle stem cells, umbilical cord-derived mesenchymal stem cells, and adipose-derived mesenchymal stem cells. The phospholipid vesicles contain soybean phospholipids or lecithin. The freeze-drying protectant is one of mannitol, trehalose, or glycine. The vesicle stabilizer is vitamin E polyethylene glycol succinate or poloxamer 188. The plasticizer is 1,2-propanediol. The pH buffer system is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system or a citric acid / sodium citrate buffer system, and the pH buffer system adjusts the pH of the formulation to 5.5-6.

5.

2. The lyophilized powder containing minoxidil hydrochloride mixed vesicles according to claim 1, its preparation method, and its application, characterized in that, The mixed membrane vesicles have a particle size of 100-200 nm and a Zeta potential of -20 to -40 mV. The content of minoxidil hydrochloride in the lyophilized powder formulation is 0.1-1.0 wt%, and the total phospholipid content in the suspension before lyophilization of the lyophilized powder formulation is 0.5-5.0 wt%.

3. The lyophilized powder containing minoxidil hydrochloride mixed vesicles according to claim 1, its preparation method, and its application, characterized in that, The stem cell membrane vesicles are derived from hair follicle stem cells, the freeze-drying protectant is trehalose, the vesicle stabilizer is vitamin E polyethylene glycol succinate, and the pH buffer system is a citric acid / sodium citrate buffer system.

4. A method for preparing a mixed vesicle lyophilized powder loaded with minoxidil hydrochloride as described in any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Weigh the raw material components by mass percentage: minoxidil hydrochloride 0.1-1.0 wt%, lyophilization protectant 0.5-10 wt%, phospholipids 0.5-5.0 wt%, vesicle stabilizer 0.1-1.0 wt%, 1,2-propanediol 1.0-3.0 wt%, pH buffer system 0.02-2.7 wt%, with the remainder being water for batch production. Step 2: Add minoxidil hydrochloride and vesicle stabilizer to a suitable container, add 1,2-propanediol, heat to 50-65℃, stir to dissolve until clear, and obtain material a; Step 3: Add phospholipids to material a, seal the container, and mix at 50-65℃ to obtain material b; Step 4: Add the pH buffer system components to a suitable container, add an appropriate amount of water, stir to dissolve until clear, and check that the pH is within the range of 5.5-6.5 to obtain material c; Step 5: Add material b to material c, and then perform high-speed shearing and high-pressure homogenization to obtain material d; Step 6: Pass material d through a 10μm-2μm double-layer polycarbonate filter membrane for repeated extrusion 3-10 times to remove insoluble particles and impurities, and obtain material e; Step 7: Replace P4-P8 The stem cells are sequentially extruded through a 10μm-2μm double-layer polycarbonate filter membrane 3-10 times to obtain the material f; Step 8: Combine and mix material e and material f to obtain material g; Step 9: Pass material g through a 0.8μm-0.2μm double-layer polycarbonate filter membrane for repeated extrusion 3-10 times to obtain material h; Step 10: Add the prescribed amount of freeze-drying protectant to a suitable container, add the remaining amount of water from the batch and dissolve until clear to obtain material i; Step 11: Combine material h and material i, mix them evenly, and filter them through a 0.2μm PES filter membrane to obtain material j; Step 12: Perform nanoparticle size determination on material j to confirm that the number of dispensable particles is 2-100 trillion / bottle, the particle size is 100-200nm, and the Zeta potential is -20 to -40mV. Then, dispense material j into containers, partially stopper them, and freeze dry them. Step 13: After freeze-drying, purge with nitrogen for protection, then stopper, cap, and proceed with subsequent packaging.

5. The method for preparing the lyophilized powder of minoxidil hydrochloride mixed vesicles according to claim 4, characterized in that, The freeze-drying process in step 12 includes: (a) Material temperature equilibration: Equilibrate at 0-10℃ for 30-60 minutes; (b) Pre-freezing: Pre-freeze at -40 to -50°C for 120 to 360 minutes; (c) Sublimation drying: Drying at -20 to 0°C and a vacuum of 10 to 50 Pa for 600 to 2000 minutes; (d) Drying: Dry for 120-660 minutes at 0-30℃ and vacuum degree of 5-30Pa.

6. The lyophilized powder containing minoxidil hydrochloride mixed vesicles according to claim 4, its preparation method, and its application, characterized in that, In step 5, the high-speed shearing speed is 8000-12000 r / min, and the shearing time is 5-10 minutes; the high-pressure homogenization pressure is 30-50 MPa, and the homogenization is performed 3-5 times.

7. The lyophilized powder containing minoxidil hydrochloride mixed vesicles according to claim 4, its preparation method, and its application, characterized in that, The stem cells mentioned in step 7 are hair follicle stem cells; the filter membrane is circulated and squeezed out 5-8 times in both steps 6 and 9; and the container mentioned in step 12 is a stoppered vial.

8. The lyophilized powder containing minoxidil hydrochloride mixed vesicles according to claim 4, its preparation method, and its application, characterized in that, The freeze-drying process in step 12 is specifically as follows: (a) Material temperature equilibration: Equilibrate at 2-6℃ for 30-60 minutes; (b) Pre-freezing: Pre-freeze at -42 to -45°C for 240-300 minutes; (c) Sublimation drying: First, dry at -10 to -15°C and vacuum degree 20-30Pa for 300-720 minutes, then dry at 0°C and vacuum degree 20-30Pa for 300-600 minutes; (d) Drying: First, dry at 5-10℃ and 10-15Pa vacuum for 120 minutes, then dry at 30℃ and 10-15Pa vacuum for 540 minutes.

9. An application of the mixed vesicle lyophilized powder loaded with minoxidil hydrochloride as described in any one of claims 1-3, characterized in that, When using, add 1-2 times its original weight of physiological saline or sterile water for injection to the lyophilized powder preparation to dissolve it, and apply it to the scalp hair loss area by coating, water-soaking and covering with non-woven fabric, introduction with microneedles / microcrystal pens, or subcutaneous injection.

10. The lyophilized powder containing minoxidil hydrochloride mixed vesicles according to claim 9, its preparation method, and its application, characterized in that, After the lyophilized powder preparation is dissolved, it is administered once daily at a dose of 1 mL for a continuous period of ≥21 days. After the lyophilized powder preparation is dissolved, its proliferative activity on dermal papilla cells is increased to 186%-425%.

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