A hierarchical composite hair regrowth delivery system, its preparation method and application

Abstract

This invention provides a multi-layered composite hair growth delivery system, its preparation method, and its application, comprising: a biotin-bonded cyclodextrin three-dimensional network polymer backbone, Bacillus-derived natural extracellular vesicles embedded within the pores of the biotin-bonded cyclodextrin three-dimensional network polymer backbone, saw palmetto fruit extract embedded in the phospholipid bilayer of the Bacillus-derived natural extracellular vesicles, and myristoyl tetrapeptide-12 encapsulated within the Bacillus-derived natural extracellular vesicles. The biotin-bonded cyclodextrin three-dimensional network polymer backbone serves as the outer support carrier, and the Bacillus-derived natural extracellular vesicles serve as the inner active ingredient encapsulation carrier, co-loading the lipid-soluble saw palmetto fruit extract and the hydrophilic myristoyl tetrapeptide-12 to achieve time-controlled release, multi-layered transdermal delivery, and synergistic effects.

Description

Technical Field

[0001] This invention relates to the field of scalp care technology, and in particular to a multi-layered composite hair growth delivery system, its preparation method, and its application. Background Technology

[0002] Scalp care, hair follicle repair, and hair loss prevention and hair growth products are rapidly evolving towards precise delivery, time-sequential efficacy, gentleness and safety, and deep transdermal absorption. Consumer and industry demands for scalp care products have evolved from traditional basic cleansing and oil control to a comprehensive upgrade towards a full-chain, precise, and time-sequential efficacy realization encompassing "scalp microenvironment repair - targeted hair follicle activation - hair growth promotion." This places higher demands on the delivery efficiency, stability, hair follicle targeting, biocompatibility, and safety of active ingredients. However, most existing scalp care products suffer from problems such as unstable co-loading of multiple active ingredients, mismatch between release timing and scalp physiological efficacy, low transdermal absorption efficiency, easy degradation and inactivation of active ingredients, and insufficient hair follicle targeting, making it difficult to meet users' actual needs.

[0003] The scalp's physiological structure dictates its stringent requirements for active ingredient delivery systems: the scalp's stratum corneum lipid barrier is dense, the hair follicle infundibulum is elongated, and the core efficacy target, the dermal papilla layer of the hair follicle, is located deep in the dermis. Conventional active ingredients struggle to penetrate the scalp barrier to reach the target area, resulting in generally insufficient transdermal absorption. Furthermore, the core efficacy logic of hair loss prevention and hair growth promotion has a natural temporal sequence—it requires first controlling oil, reducing inflammation, and soothing the scalp surface to improve the scalp microenvironment before delivering growth-promoting ingredients deep into the hair follicles to activate them and promote hair growth. Existing technologies cannot match this physiological sequence, leading to a significant reduction in product efficacy.

[0004] Cyclodextrin, with its unique molecular structure of being hydrophilic on the outside and hydrophobic on the inside, is the most commonly used encapsulation carrier for active ingredients in scalp care products. Current technology has achieved covalent bonding between biotin and cyclodextrin to form a three-dimensional polymeric framework, which improves the structural strength and drug-carrying capacity of the carrier to some extent, making it suitable for encapsulating lipid-soluble plant extracts or peptides. However, traditional cyclodextrin and its modified framework still have core drawbacks in practical applications of scalp care products: First, it cannot achieve non-competitive co-loading of lipid-soluble oil-controlling and soothing ingredients with hydrophilic hair-growth-promoting peptides. The two are prone to competition for inclusion sites, resulting in large fluctuations in encapsulation rates. In water-based formulations such as shampoos and hair growth serums, this easily leads to ingredient leakage, aggregation, and precipitation, resulting in extremely poor shelf-life stability. Second, the release sequence is completely uncontrollable; co-loaded ingredients are mostly released simultaneously, failing to match the scalp care principle of "first improving the scalp's surface microenvironment, then deeply activating hair follicles." The physiological efficacy logic is flawed. The simultaneous release of oil-controlling and anti-inflammatory ingredients and hair growth-promoting ingredients not only fails to achieve synergistic effects, but also makes it easy for the growth-promoting ingredients to fail to function due to the unimproved microenvironment of scalp inflammation and excessive sebum secretion. Thirdly, the transdermal and targeted capabilities are insufficient. The cyclodextrin carrier alone lacks the ability to target and recognize hair follicles, making it difficult to penetrate the dense stratum corneum barrier of the scalp. It cannot accurately deliver the active ingredients to the dermal papilla layer of the hair follicle, resulting in most of the active ingredients remaining on the scalp surface and being quickly lost, with extremely low bioavailability.

[0005] In summary, existing scalp care products still face technical bottlenecks in four core dimensions: differentiated and stable co-loading of multi-nature active ingredients, precise control of release timing, deep transdermal delivery to hair follicles, and long-term stable protection of active ingredients. Single delivery carriers or conventional formula compounding systems can no longer meet the development requirements of high-end anti-hair loss and hair growth and scalp repair products. Summary of the Invention

[0006] The purpose of this invention is to provide a multi-layered composite hair growth delivery system, its preparation method, and its application. The system uses a biotin-bonded cyclodextrin three-dimensional network polymer backbone as the outer support carrier, Bacillus-derived natural extracellular vesicles as the inner active ingredient carrier, and co-loads lipid-soluble saw palmetto fruit extract and hydrophilic myristoyl tetrapeptide-12 to achieve time-controlled release, multi-layered transdermal delivery, and synergistic effects.

[0007] To achieve the above objectives, this technical solution provides a hierarchical composite hair growth delivery system, comprising: a biotin-bonded cyclodextrin three-dimensional network polymer backbone, Bacillus-derived natural extracellular vesicles embedded within the pores of the biotin-bonded cyclodextrin three-dimensional network polymer backbone, saw palmetto fruit extract embedded in the phospholipid bilayer of the Bacillus-derived natural extracellular vesicles, and myristoyl tetrapeptide-12 encapsulated within the Bacillus-derived natural extracellular vesicles.

[0008] This solution specifically selects saw palmetto fruit extract and myristoyl tetrapeptide-12 as active ingredients in the tiered delivery system. It should be noted that saw palmetto fruit extract is a core lipid-soluble active ingredient for current hair loss prevention, oil control, and scalp soothing. It can specifically inhibit 5-reductase activity, reduce excessive sebum secretion, antagonize the atrophic effect of androgens on hair follicles, and soothe scalp inflammation. It is a core active ingredient in seborrheic alopecia care and is widely used in hair loss prevention and hair growth products. However, it has extremely high lipid solubility and extremely poor water solubility, easily agglomerating and precipitating in water-based scalp care products. The active ingredients are easily oxidized and inactivated, and it cannot independently penetrate the scalp lipid barrier, resulting in extremely low scalp retention and hair follicle targeting rates. Simply relying on cyclodextrin cavity inclusion cannot fundamentally solve the core problems of its stability and deep targeted delivery.

[0009] Myristoyl Tetrapeptide-12 is a clinically proven hydrophilic small molecule peptide that promotes hair growth. It can specifically activate the vitality of hair follicle dermal papilla cells, promote keratin synthesis, prolong the hair follicle growth phase, and improve the atrophy of hair follicles. It is the core active ingredient for hair follicle repair and hair growth promotion. However, it is easily degraded by proteases on the scalp surface, is highly sensitive to the pH of the formula and storage temperature, and its strong hydrophilicity makes it difficult to penetrate the scalp lipid barrier and reach the target area of ​​the hair follicle. Existing cyclodextrin encapsulation or physical adsorption methods have problems such as rapid leakage, poor sustained release effect, and insufficient hair follicle targeting rate. The actual efficacy in scalp care products is greatly reduced, and long-lasting effects cannot be achieved.

[0010] This solution utilizes the principle of "like dissolves like" to insert the active ingredients of saw palmetto fruit extract into the phospholipid bilayer of Bacillus vesicles, forming a stable mixed membrane structure with lipid molecules. This completely avoids competition for inclusion sites with hydrophilic components, fundamentally solving the problems of poor water solubility and easy aggregation and precipitation in water-based systems. Furthermore, the hydrophilic myristoyl tetrapeptide-12 is completely encapsulated in the internal hydrophilic cavity of Bacillus vesicles, achieving complete physical spatial isolation from lipid-soluble components.

[0011] Specifically, addressing the core application pain points of saw palmetto fruit extract, such as easy oxidation and degradation, low transdermal absorption efficiency, poor hair follicle targeting, and insufficient scalp retention, this solution uses a phospholipid bilayer to form the first physical barrier for the saw palmetto fruit extract inserted within it, completely isolating it from oxygen and free radicals in the external environment and preventing the oxidative degradation of active ingredients. The outer dense biotin-bonded cyclodextrin three-dimensional network polymer backbone forms the second barrier, further isolating it from light, heat, and oxidative impurities in the formulation system. Under this dual protection, the shelf-life activity retention rate of saw palmetto fruit extract can be greatly improved compared to traditional cyclodextrin encapsulation. Furthermore, the phospholipid bilayer of Bacillus vesicles is highly homologous to the lipid barrier structure of the scalp stratum corneum. It can efficiently penetrate the dense lipid barrier of the scalp through lipid fusion and cell bypass penetration, completely solving the problem that saw palmetto fruit extract cannot penetrate the scalp barrier on its own. The scalp retention rate is greatly improved compared with traditional addition methods. The biotin-bonded cyclodextrin three-dimensional network polymer backbone itself has high affinity for hair follicles and targeted recognition ability. It can guide the entire delivery system to migrate directionally to the hair follicle infundibulum, greatly increasing the enrichment concentration of saw palmetto fruit extract around the hair follicle. It can accurately exert the effects of inhibiting 5α-reductase and antagonizing androgen-induced hair follicle atrophy, completely solving the problems of rapid loss of active ingredients on the scalp surface and extremely low hair follicle targeting rate under traditional methods.

[0012] Addressing the core pain points of myristoyl tetrapeptide-12—its susceptibility to protease degradation, high sensitivity to pH / temperature, strong hydrophilicity making it difficult to penetrate the lipid barrier, rapid leakage followed by poor slow release, and inability to achieve long-lasting effects on hair follicles—this solution completely encapsulates myristoyl tetrapeptide-12 within the closed hydrophilic cavity of Bacillus vesicles. A phospholipid bilayer forms a dense physical barrier, completely isolating it from proteases on the scalp surface and hydrogen ions in the formulation system, preventing contact between the peptides and degrading enzymes, thus fundamentally solving its problems of easy enzymatic degradation and inactivation. The outer biotin-bonded cyclodextrin three-dimensional network polymer backbone provides secondary protection, isolating the peptide activity from the effects of temperature and pH fluctuations during storage, significantly extending its activity retention period compared to traditional cyclodextrin encapsulation. Simultaneously, leveraging the lipid homology fusion capability of the Bacillus vesicle phospholipid bilayer, the hydrophilic tetrapeptide-12, which originally could not penetrate the scalp lipid barrier, is encapsulated within the vesicle for cross-keratinization delivery, completely solving the core problem of hydrophilic peptides' inability to penetrate the scalp lipid barrier.

[0013] In some embodiments, the mass ratio of myristoyl tetrapeptide-12 to saw palmetto fruit extract is (0.5~5):1.

[0014] In some embodiments, the biotin-bonded cyclodextrin three-dimensional network polymer framework is a three-dimensional network polymer framework formed by covalent bonding of cyclodextrin as structural unit and biotin as covalent linker arm. It has the characteristics of high mechanical strength, structural stability and good biocompatibility. As an outer support carrier, it can fix Bacillus vesicles, control diffusion rate, isolate the external environment and provide physical protection for inner components.

[0015] In some embodiments, the biotin in the biotin-bonded cyclodextrin three-dimensional network polymer backbone is selected as D-biotin. It should be noted that D-biotin, also known as vitamin B7 (or vitamin H), is a water-soluble B vitamin. The reason for specifically choosing D-biotin in this scheme is that: ① The D-biotin molecule contains specific carboxyl and amino functional groups, which form stable amide bonds with the hydroxyl groups of cyclodextrin. Compared to conventional biotin, the bonding stability is significantly improved, preventing the backbone from dissociating in water-based systems. ② The molecular spatial configuration of D-biotin is perfectly compatible with the glucose units of cyclodextrin, guiding the orderly cross-linking of cyclodextrin molecules to form a three-dimensional network with precisely controllable pore sizes. This perfectly matches the chimerism requirements of Bacillus-derived vesicles, achieving uniform vesicle dispersion, no aggregation, and no leakage. ③ D-biotin itself has scalp affinity, improving the overall biocompatibility of the backbone. Simultaneously, its targeting ability helps guide the vesicles to migrate directionally to the hair follicle, unlike conventional biotin in existing technologies which only serves as a connecting medium without additional functions. This framework serves as an outer support carrier, which can fix Bacillus vesicles, control the diffusion rate, isolate the external environment, and provide physical protection for the inner components.

[0016] In some embodiments, D-biotin is covalently bonded to cyclodextrin after activation by EDC·HCl / NHS.

[0017] In some embodiments, the cyclodextrin in the biotin-bonded cyclodextrin three-dimensional network polymer backbone is one or a combination of β-cyclodextrin or hydroxypropyl β-cyclodextrin. This is because the molecular cavity size of the two is matched with that of D-biotin, which can form a stable inclusion structure. Furthermore, hydroxypropyl modification can significantly improve water solubility and biocompatibility, meeting the requirements for three-dimensional backbone construction and hair follicle delivery.

[0018] In some embodiments, the purity of cyclodextrin is ≥98%, which can effectively avoid impurities occupying reaction sites and interfering with its cross-linking with D-biotin to form a network, ensuring a regular three-dimensional polymer skeleton structure and uniform pore size. At the same time, it avoids impurities inducing system aggregation and precipitation and scalp irritation, ensuring stable Bacillus vesicle loading, batch-to-batch performance and application safety.

[0019] In some embodiments, the mass ratio of cyclodextrin to biotin in the biotin-bonded cyclodextrin three-dimensional network polymer backbone needs to be controlled within the range of (3~10):1.

[0020] When β-cyclodextrin is selected, (4~5):1 is preferred because its hydroxyl groups have less steric hindrance and a lower ratio can form stable crosslinks; when hydroxypropyl-β-cyclodextrin is selected, (5~7):1 is preferred to overcome the steric hindrance caused by hydroxypropyl and ensure bonding efficiency and backbone function.

[0021] In some embodiments, the pore diameter of the biotin-bonded cyclodextrin three-dimensional network polymer backbone is (30-300 nm). At this time, the biotin-bonded cyclodextrin three-dimensional network polymer backbone has excellent hydrophilic swelling properties and carries a negative charge in the pH range of 5.0-7.0, which can further assist the uniform dispersion of vesicles through electrostatic repulsion.

[0022] In some embodiments, the Bacillus-derived natural extracellular vesicles are natural phospholipid bilayer vesicles taken from the Bacillus fermentation broth, possessing a naturally differentiated structure with an outer phospholipid bilayer and an inner hydrophilic cavity.

[0023] In some embodiments, the particle size of the natural extracellular vesicles derived from Bacillus is 80~200nm, the particle size polydispersity index is ≤0.3, the zeta potential is between -10 mV and -30 mV, and there are no deformed or broken vesicles.

[0024] It should be noted that the biotin-bonded cyclodextrin three-dimensional network polymer framework forms a three-dimensional network framework with uniform pore size and precisely controllable inner diameter. The inner diameter of its pores forms a perfect single-particle confinement space with the vesicle particle size of the natural extracellular vesicles derived from Bacillus. The inner diameter of the pore is slightly larger than the vesicle particle size, just enough to accommodate a single vesicle. This prevents vesicles from moving freely and leaking due to excessively large pores, and also prevents the membrane structure from rupturing due to excessively small pores that compress the vesicles. The uniform pores formed by ordered cross-linking enable the single-particle dispersion and intercalation of vesicles. Each pore contains only one vesicle, fundamentally avoiding contact, fusion, and aggregation between vesicles, ensuring the uniformity and long-term stability of the intercalation. This is a core advantage that ordinary irregularly cross-linked cyclodextrin frameworks cannot achieve. Therefore, when natural extracellular vesicles derived from Bacillus are mixed with the biotin-bonded cyclodextrin three-dimensional network polymer framework, the natural extracellular vesicles derived from Bacillus can be stably embedded inside the pores of the cyclodextrin polymer framework without aggregation or leakage.

[0025] Furthermore, the biotin-bonded cyclodextrin three-dimensional network polymer backbone has a large number of hydroxyl and amide polar groups on its surface, while the phospholipid bilayer of Bacillus-derived natural extracellular vesicles has phospholipid polar heads, carboxyl groups, amino groups, etc. on its outer surface. The two can form a large number of high-density intermolecular hydrogen bonds, which act like countless molecular anchors, firmly fixing the Bacillus-derived natural extracellular vesicles inside the backbone mesh, greatly improving the stability of the intercalation. At the same time, both the backbone and the vesicles are weakly negatively charged, and the electrostatic repulsion is moderate. This will not cause strong electrostatic repulsion between vesicles, which would affect the uniformity of intercalation, nor will it be enough to overcome the hydrogen bond anchoring and spatial confinement effect to push the vesicles out of the mesh. The weak negative charge can also avoid strong electrostatic aggregation between vesicles and between vesicles and the backbone, so that the vesicles maintain a stable dispersion state within the mesh, achieving a balance of "intercalation without aggregation and fixation without compression".

[0026] This formulation utilizes Bacillus-derived natural extracellular vesicles, which possess a triple function of active ingredient encapsulation, transdermal penetration enhancement, and scalp soothing and repair. Specifically, its phospholipid bilayer structure enables dual loading of saw palmetto fruit extract within the membrane and myristoyl tetrapeptide-12 within the encapsulation cavity; its negatively charged nanosurfaces achieve highly efficient transdermal penetration through hair follicle targeting, lipid fusion, and bypass pathways; and its membrane components and encapsulated active ingredients synergistically exert anti-inflammatory, barrier repair, regeneration-promoting, and anti-androgenic effects, achieving scalp soothing and repair.

[0027] In some embodiments, the mass ratio of Bacillus-derived natural extracellular vesicles to the biotin-bonded cyclodextrin three-dimensional network polymer backbone is (1~5):(5~20).

[0028] Preferably, the mass ratio of the two is 1:(3~8).

[0029] As mentioned earlier, the tiered composite hair growth delivery system provided by this solution can deliver hair follicles in stages on the scalp. The outer layer, an optimized biotin-bonded cyclodextrin three-dimensional network polymer framework, precisely controls the overall release rhythm of vesicles due to its uniform mesh and stable structure. Simultaneously, D-biotin's targeting ability assists the vesicles in their directional migration to the hair follicles. Bacillus-derived natural extracellular vesicles, acting as the core carrier and transdermal carrier of active ingredients, first release saw palmetto fruit extract from the phospholipid bilayer, achieving scalp surface soothing, oil control, and anti-inflammation within 1-2 hours. The scalp microenvironment is optimized through the rapid release of saw palmetto fruit extract on the scalp surface. Subsequently, natural extracellular vesicles derived from Bacillus subtilis penetrate the scalp barrier under the synergistic effect of D-biotin and Bacillus subtilis components. Myristoyl tetrapeptide-12 is slowly released from the hydrophilic cavities, continuously acting on the hair follicles to promote hair growth. This achieves a time-series synergistic and deep-layer effect, meaning that myristoyl tetrapeptide-12 is slowly released deep into the hair follicles. This synergistic mechanism differs from the simple efficacy superposition in existing technologies and is the result of the combined action of an optimized framework, vesicles, and active ingredients.

[0030] Secondly, this solution provides an application of a tiered, multi-functional hair regrowth delivery system, which is applied to care products, including scalp care products, hair follicle repair products, or functional skincare products. Specifically, this solution provides scalp care products, hair follicle repair products, or functional skincare products that include the aforementioned tiered, multi-functional hair regrowth delivery system. The saw palmetto fruit extract in this system acts on the scalp surface, while myristoyl tetrapeptide-12 acts deep within the hair follicles.

[0031] In some embodiments, the care product contains a layered composite hair growth delivery system comprising 1% to 10% of the total mass.

[0032] In some embodiments, scalp care products are anti-hair loss and hair growth liquid, anti-hair loss shampoo, post-operative scalp repair gel, scalp repair essence, oil-controlling and soothing spray, or scalp cleansing milk, which can achieve the effect of first controlling oil and soothing, and then promoting hair growth; hair follicle repair products are hair follicle activating essence, scalp microneedle infusion agent, or postpartum hair loss repair essence, which can improve hair follicle atrophy and enhance hair follicle vitality.

[0033] Thirdly, this solution provides a method for preparing a hierarchical composite hair growth delivery system, including the following steps: S1: Dissolve cyclodextrin to obtain a cyclodextrin solution, add D-biotin to the cyclodextrin solution and stir, then add a compound activator and adjust the pH to 5.5~6.5, place at 25~35℃ and 100~180r / min in the dark for 2~6h to obtain a biotin-cyclodextrin prepolymerized backbone solution containing a biotin-bonded cyclodextrin three-dimensional network polymer backbone; S2: Bacillus vesicle suspension extracted from Bacillus fermentation broth; S3: Take the Bacillus vesicle suspension and add PBS to adjust the pH to 7.0~7.2. Then add myristoyl tetrapeptide-12 and stir at 80~150 r / min for 0.5~1 h to obtain a single-drug-loaded Bacillus vesicle suspension. Heat the single-drug-loaded Bacillus vesicle suspension to 28~35℃ and add the dissolved saw palmetto extract solution. Stir for 1~2 h under the dark to obtain a dual-drug-loaded Bacillus vesicle suspension. S4: The dual-drug-loaded Bacillus vesicle suspension was added dropwise to the biotin-cyclodextrin prepolymerized backbone solution and homogenized under high pressure microfluidic treatment to obtain a hierarchical composite germinal delivery system.

[0034] To facilitate the preservation of the hierarchical composite hair growth delivery system, this solution further includes the following steps: S5: The hierarchical composite hair growth delivery system is dried in a freeze vacuum dryer and then pulverized through a mesh sieve to obtain a uniform powdered composite delivery system.

[0035] Regarding step S1 for preparing the biotin-cyclodextrin prepolymerized backbone solution: In some embodiments, cyclodextrin with a purity of ≥98% is selected and added to purified water or a water-alcohol mixture according to the specified ratio, with a mass concentration controlled at 10%~30%; the stirring speed is controlled at 150~250 r / min, and the mixture is continuously stirred for 30~60 min under a constant temperature water bath at 30~50℃ until the cyclodextrin is completely dissolved, resulting in a clear, precipitate-free cyclodextrin solution.

[0036] In some embodiments, the volume ratio of water to ethanol in the aqueous-ethanol mixed solvent is 7:3 to 9:1.

[0037] To ensure that D-biotin can be stably covalently bonded to cyclodextrin, this method involves adding D-biotin with a purity ≥99% to the cyclodextrin solution and stirring. Subsequently, a compound activator is added and the pH is adjusted to 5.5~6.5. The solution is then reacted at 25~35℃ and 100~180 r / min in the dark for 2~6 h to obtain a biotin-cyclodextrin prepolymerized backbone solution containing a biotin-bonded cyclodextrin three-dimensional network polymeric backbone.

[0038] In some embodiments, the compound activator is a mixture of EDC·HCl and NHS, wherein the molar ratio of EDC·HCl to NHS is 1:1 to 1.2:1, and the amount of the compound activator is 1.0 to 1.5 times the mass of D-biotin. This can efficiently activate the carboxyl group of D-biotin and stabilize the reaction intermediate, improving its amide bonding efficiency with cyclodextrin. It avoids incomplete reaction and insufficient grafting rate caused by a low activator ratio, and also prevents side reactions and impurity residues caused by excessive dosage, ensuring appropriate cross-linking of the polymerization backbone, regular structure, and excellent batch stability.

[0039] This method involves sampling and analyzing the biotin-cyclodextrin prepolymerized framework solution. The residual amount of free D-biotin in the system is determined using ultraviolet spectrophotometry or high-performance liquid chromatography (HPLC), and the covalent bond degree between D-biotin and cyclodextrin is calculated. The reaction is terminated when the bond degree between D-biotin and cyclodextrin is ≥85%, resulting in a biotin-cyclodextrin prepolymerized framework solution with uniform pore size and excellent stability.

[0040] Regarding step S2 for preparing the Bacillus vesicle suspension: In this method, the Bacillus fermentation broth is centrifuged at low temperature to remove bacterial cells and large particulate impurities, and the supernatant is collected. The supernatant is then subjected to multi-stage ultracentrifugation to obtain a vesicle suspension. After centrifugation of the vesicle suspension again, it is filtered through a 0.22μm filter membrane to obtain a clear Bacillus vesicle suspension.

[0041] In some embodiments, the conditions for low-temperature centrifugation are 4°C, 300×g, and centrifugation for 15 min.

[0042] In some embodiments, the conditions for multi-stage ultracentrifugation are: centrifugation at 4°C and 1000~3000g for 10~20 min; centrifugation at 4°C and 8000~12000g for 20~30 min; and ultracentrifugation at 4°C and 100000~150000g for 60~120 min.

[0043] In some embodiments, the conditions for re-centrifuging the vesicle suspension are 4°C, 8000~12000g for 20~30 min.

[0044] Regarding step S3 for preparing the dual-drug-loaded Bacillus vesicle suspension: This method first controls the encapsulation of myristoyl tetrapeptide-12 in natural Bacillus-derived extracellular vesicles to obtain single-drug-loaded Bacillus vesicles, and then uses the similar compatibility of single-drug-loaded Bacillus vesicles and saw palmetto fruit extract to load saw palmetto fruit extract to obtain dual-drug-loaded Bacillus vesicles.

[0045] Specifically, in some embodiments, a suspension of Bacillus vesicles with a particle size of 80-200 nm was taken, and the pH was adjusted to 7.0-7.2 with PBS. Then, myristoyl tetrapeptide-12 was added and the mixture was stirred at 80-150 r / min for 0.5-1 h. The mixture was then ultrasonically dispersed for 5 min every 20 min, with an ultrasonic power of 100-300 W and a pulse mode of 2-3 seconds of operation followed by 2-3 seconds of interval. This ensured that the hydrophilic myristoyl tetrapeptide-12 was completely encapsulated in the hydrophilic cavity inside the natural extracellular vesicles of Bacillus spp., resulting in single-drug-loaded vesicles with an encapsulation rate of ≥85%.

[0046] The purpose of using ultrasound-assisted methods in this scheme is twofold: firstly, to utilize the cavitation effect and mechanical vibration of ultrasound to improve the permeability of vesicle membranes and promote the diffusion of hydrophilic myristoyl tetrapeptide-12 into the hydrophilic cavities inside the vesicles, thereby improving the encapsulation efficiency; secondly, to use intermittent ultrasound treatment to avoid the thermal effects caused by continuous ultrasound that could damage the vesicle structure and peptide activity, while also preventing vesicle aggregation and maintaining its uniform dispersion in the system.

[0047] In some embodiments, the power of the ultrasound is 200~300W.

[0048] In some embodiments, the single-drug-loaded Bacillus vesicle suspension is heated to 28-35°C, and a dissolved saw palmetto extract solution is added while maintaining the temperature. The suspension is then stirred for 1-2 hours under dark conditions to obtain a dual-drug-loaded Bacillus vesicle suspension.

[0049] Specifically, by utilizing the principle of "like dissolves like," the fatty acid and phytosterols and other fat-soluble active ingredients in the saw palmetto fruit extract spontaneously insert into the phospholipid bilayer of the vesicles, forming a stable mixed membrane structure with the phospholipid molecules. After filtration, dual-drug-loaded vesicles are obtained, with an extract insertion rate of ≥95%. The two active ingredients are distributed in layers without interfering with each other.

[0050] Regarding step S4 in the preparation of the hierarchical composite germinal delivery system: In some embodiments, a dual-drug-loaded Bacillus vesicle suspension is added dropwise to a biotin-cyclodextrin prepolymerized backbone solution while maintaining a low stirring speed of 80-120 r / min. After the addition is complete, high-pressure microfluidic homogenization is performed. The homogenization conditions are a homogenization pressure of 10,000-30,000 psi (approximately 69-207 MPa) and a homogenization cycle of 1-5 times to obtain a hierarchical composite germinal delivery system.

[0051] Regarding step S5 of this plan: In some embodiments, the hierarchical composite hair growth delivery system is placed in a freeze vacuum dryer and pre-frozen at -40~-30℃ for 2~3 hours, then vacuum drying is started, controlling the vacuum degree to 10~30Pa and the drying temperature to -20~-10℃, and freeze vacuum drying is continued for 8~12 hours until the moisture content is ≤5%. After pulverizing, it is passed through an 80~100 mesh sieve to obtain a uniform powdered composite delivery system, which is then sealed and stored.

[0052] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects: 1. This invention creatively transforms Bacillus fermentation products into natural Bacillus extracellular vesicles and embeds them into a biotin-bonded cyclodextrin three-dimensional network polymeric framework, forming a hierarchical delivery system of vesicles (first level) and polymeric framework (second level). The Bacillus vesicles possess triple functions of encapsulation, penetration enhancement, and repair. Combined with biotin's targeted recognition, the entire complex system can be delivered more efficiently to the depths of the hair follicle. In other words, the relationship between vesicles and framework in this hierarchical delivery system is not a simple mixture, but rather an embedded, spatially stabilized relationship. This hierarchical delivery system achieves a sequential release of saw palmetto fruit extract (soothing and oil-controlling) followed by myristoyl tetrapeptide-12 (promoting hair growth) through the barrier effect of the structural characteristics of the natural Bacillus extracellular vesicles themselves.

[0053] 2. Utilizing the natural structure of the lipid bilayer and hydrophilic internal cavity of the biotin-bonded cyclodextrin three-dimensional network polymeric framework, lipid-soluble saw palmetto fruit extract and hydrophilic myristoyl tetrapeptide-12 are encapsulated, completely avoiding competitive encapsulation and achieving a stable encapsulation rate far exceeding that of traditional cyclodextrin carriers. Simultaneously, the optimized biotin-bonded cyclodextrin three-dimensional network polymeric framework has uniform pores, preventing vesicle aggregation and further enhancing encapsulation stability. The lipid bilayer of Bacillus-derived natural extracellular vesicles protects the saw palmetto fruit extract from oxidation and protects the peptides from enzymatic degradation; at the same time, the external cyclodextrin polymeric framework further enhances the physicochemical stability of the vesicles in cosmetic formulations, preventing vesicle rupture or fusion during storage. Attached Figure Description

[0054] Figure 1 This is a physical image of the powdered hierarchical composite hair growth delivery system prepared according to the present invention.

[0055] Figure 2 This is a diagram showing the results of the safety evaluation.

[0056] Figure 3 This is a graph showing the results of an ATP production test in human dermal papilla cells.

[0057] Figure 4 This is a diagram showing the results of an in vitro transdermal penetration experiment.

[0058] Figure 5 This is a graph showing the results of an in vitro sustained-release experiment. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0060] Example 1: Preparation of biotin-bonded cyclodextrin polymer backbone: Take 20g of hydroxypropyl β-cyclodextrin with a purity ≥98% and add it to a mixed solvent of 80mL purified water and 20mL ethanol (water:ethanol = 8:2). Dissolve in a 40℃ constant temperature water bath with stirring at 200r / min for 30min to obtain a clear solution. Add 3g of D-biotin with a purity ≥99%, stir and disperse, then add EDC·HCl / NHS compound activator (molar ratio 1:1.2, total amount 1.2 times the mass of D-biotin), adjust the pH to 6.0, and react at 30℃ and 150r / min in the dark for 4h. After the reaction is complete, dialyze the solution for 48h using a dialysis bag with a molecular weight cutoff of 3500Da to remove unreacted small molecules and activator, obtaining a biotin-cyclodextrin prepolymerized backbone solution.

[0061] Example 2: Extraction and purification of natural vesicles derived from Bacillus: The Bacillus subtilis fermentation broth was centrifuged at 300×g for 15 min at 4°C to remove bacterial cells. The supernatant was then centrifuged sequentially at 2000g for 15 min at 4°C, 10000g for 25 min at 4°C, and ultracentrifuged at 120000g for 90 min at 4°C. The precipitate was collected, resuspended in PBS, and centrifuged at 10000g for 25 min at 4°C. The supernatant was then filtered through a 0.22 μm filter membrane to obtain a vesicle suspension with a particle size distribution of 80–200 nm.

[0062] Example 3: Layered encapsulation of dual active ingredients in Bacillus vesicles: The vesicle suspension prepared in Example 2 was adjusted to pH 7.2 with PBS. Peptides were added at a dry weight ratio of vesicle to myristoyl tetrapeptide-12 (50:1). The mixture was stirred at 100 rpm for 40 min, with ultrasonic dispersion (250 W) for 5 min every 20 min during this period. Unencapsulated peptides were removed by ultrafiltration centrifuge (100 kDa) to obtain single-drug-loaded vesicles. The resulting peptide-encapsulated vesicles had an encapsulation efficiency of 87.6%.

[0063] The above-mentioned single-drug-loaded vesicle suspension was heated to 32°C, and stirred at 150 r / min. A saw palmetto fruit extract solution was added dropwise, maintaining a final extract-to-vesicle mass ratio of 1:50. Stirring continued for 1.5 h under dark conditions. After the reaction was complete, uninserted extract was removed by ultrafiltration to obtain dual-drug-loaded vesicles. HPLC analysis showed that the insertion rate of the saw palmetto fruit extract was 88.4%.

[0064] Example 4: Construction of a hierarchical composite hair growth delivery system and preparation of lyophilized powder: The dual-drug-loaded Bacillus vesicle suspension prepared in Example 3 was slowly added dropwise to the biotin-cyclodextrin prepolymerized backbone solution prepared in Example 1, with a vesicle to backbone solid mass ratio of 1:3. The mixture was stirred at a low speed of 100 r / min for 30 min. The mixture was then processed using a high-pressure microfluidic homogenizer (15000 psi, 3 cycles). The resulting composite system was placed in a freeze dryer and pre-frozen at -35°C for 2.5 h. Vacuum drying was then initiated, controlling the vacuum level at 20 Pa and the drying temperature at -15°C, and continued for 10 h until the moisture content was ≤5%. The mixture was then pulverized and passed through a 100-mesh sieve to obtain a powdered, hierarchical composite hair growth delivery system.

[0065] Example 5: Safety Evaluation Human immortalized dermal papilla cells were cultured in DMEM-F12 medium to a density of over 80%, passaged into 6-well plates, and cultured at 37 ℃ and 5% CO2 until the density reached 70%. The original medium was discarded, and the cells were washed three times with PBS. Different concentrations of sample solution (the tiered composite delivery sample prepared in Example 4, diluted with medium) were added to each well. The sample solution was the powdered tiered composite hair growth delivery system of Example 4, 100 μL per well. For the calibration group, 100 µL of PBS was added to cell-free wells, and the cells were incubated at 37 ℃ and 5% CO2 for 24 h. After incubation, the original medium was discarded, and each well was washed three times with PBS. 120 µL of 1 mg / mL MTT diluted in medium was added to each well, and the cells were cultured for another 6 h. Finally, the original medium in 96-well plates was discarded, and each well was washed three times with PBS. 150 µL of DMSO was added to each well to fully dissolve and crystallize the cells. The OD at 490 nm was measured using a microplate reader, and cell viability was calculated. The cell viability was obtained as shown in the figure. Figure 2 As shown, the results indicate that the hierarchical composite hair growth delivery system prepared in Example 4 of this invention has a cell survival rate of >90% in the concentration range of 0~1000 ppm, with no obvious cytotoxicity, and meets the safety requirements for cosmetic raw materials.

[0066] Example 6: Test of ATP production in human dermal papilla cells: Human immortalized dermal papilla cells were cultured in DMEM-F12 medium to a density of over 80%, passaged into 6-well plates, and cultured at 37°C with 5% CO2 until the density reached 70%. The original medium was discarded, and the cells were washed three times with PBS. 100 μL of serum-free DMEM-F12 medium was added to each well, and the cells were cultured for another 12 hours to starve them. The starved medium was then discarded, and 100 μL of the corresponding blank medium, free active ingredient, and the tiered composite delivery sample solution prepared in Example 4 were added to each well. The cells were then incubated at 37°C with 5% CO2 for 24 hours. After incubation, the medium was discarded, and the cells were washed three times with PBS. The lysis buffer from the ATP assay kit was added to each well to ensure complete cell lysis. ATP levels were measured according to the ATP assay kit instructions, and the experimental results are shown below. Figure 3 As shown in the figure. The results show that the hierarchical composite hair growth delivery system prepared in Example 4 of the present invention significantly improved the ATP production rate of human dermal papilla cells (reaching 160.2%), which is significantly better than the free active ingredient group (131.5%). This indicates that the system effectively protects the active ingredients and synergistically promotes the energy metabolism of dermal papilla cells through time-controlled release and hierarchical delivery.

[0067] Example 7: In vitro transdermal permeation experiment (Franz diffusion cell method): The skin used in the experiment was from a one-month-old Bama miniature pig. Subcutaneous tissue was removed, and the stratum corneum, epidermis, dermis, and hair follicles were retained. The skin was rinsed thoroughly with water and cut into appropriate sizes. The experimental temperature was 32±1℃, and the medium used was isotonic phosphate buffer with pH=7.4.

[0068] Pigskin was sandwiched between the supply and receiving cells of the diffusion cell with the epidermis facing upwards and the dermis facing downwards to simulate the actual penetration of the test sample into the skin from the outside. 3 mL of the test solution (containing an equal amount of active ingredient), free active ingredient, mono-loaded active ingredient vesicles (acyclic dextrin backbone), and the layered composite delivery sample prepared in Example 4 were injected into the supply cell, with three replicates for each test solution. 15 mL of PBS buffer was added to the receiving cell as the receiving solution, and air bubbles were removed. The cell was then placed in a constant temperature water bath transdermal testing instrument at 32±1℃ and a rotation speed of 350 r / min for 24 h. 1.5 mL of the receiving solution was collected at 1 h, 2 h, 4 h, 8 h, and 24 h, and the same volume of receiving solution was replenished simultaneously. The content of myristoyl tetrapeptide-12 was determined, and the results are shown below. Figure 4 As shown.

[0069] The results showed that the hierarchical composite hair growth delivery system of this invention (experimental group) significantly improved the cumulative transdermal penetration rate of active ingredients in the skin. The transdermal penetration rate of myristoyl tetrapeptide-12 increased from 11.09% in the free group to 29.32%, an increase of 2.6 times. This result indicates that the synergistic penetration-enhancing effect of the D-biotin-cyclodextrin backbone and Bacillus vesicles effectively overcomes the stratum corneum barrier, achieving deep delivery of active ingredients.

[0070] Example 8 In vitro sustained-release experiment (dialysis bag method): In vitro release experiments used free active ingredient, monopeptide vesicles (acyclic dextrin backbone), and the hierarchical composite delivery sample of this invention as test samples, and compared them under the same conditions. The samples were placed in dialysis bags and immersed in pre-prepared release medium (phosphate buffer, pH 7.4) at room temperature, while being magnetically stirred at 400 rpm / min for 24 h. At selected time points, 1 mL of the release solution was taken from the release medium and an equal volume of fresh release medium was added to maintain a constant volume. The concentration of myristoyl tetrapeptide-12 in the release medium at different time points was determined by high-performance liquid chromatography (HPLC), and the results are shown below. Figure 5 As shown.

[0071] The results showed that the release curve of myristoyl tetrapeptide-12 in the hierarchical composite germinal delivery system of this invention exhibited obvious biphasic sustained-release characteristics: no obvious burst release in the initial stage, a slow and sustained release process, and a significantly lower cumulative release rate over 24 hours compared to free myristoyl tetrapeptide-12 and the single-loaded active ingredient vesicle group. This indicates that the confinement and anchoring effect of the outer biotin-bonded cyclodextrin three-dimensional network polymer backbone on the vesicles and the membrane barrier effect of the inner Bacillus vesicles formed a synergistic controlled release, effectively delaying the release rate of the active ingredient and achieving the hierarchical delivery and time-sequential controlled release effect described in this invention.

[0072] Example 9: Final Product Stability Testing (Accelerated Testing) The hierarchical composite hair growth delivery system powder prepared in Example 4 was sealed in an aluminum foil bag. Accelerated testing was conducted at 40℃ ± 2℃ under light exposure for 3 months. Samples were taken at 0, 1, 2, and 3 months to determine the following indicators: appearance, moisture content, particle size, and active ingredient retention rate. The stability test results of the hierarchical composite hair growth delivery system powder are shown in Table 1 below: Table 1: Stability Test Results of the Powder in the First-Level Composite Hair Growth Delivery System

[0073] The results showed that after being placed at 40℃ under accelerated light conditions for 3 months, the appearance, moisture content, and particle size of the hierarchical composite hair growth delivery system of this invention did not change significantly. The retention rate of active ingredients was greater than 90%, indicating that the system has excellent resistance to high temperature and light environments, the active ingredients are effectively protected, and the system has good long-term stability.

[0074] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A hierarchical composite hair regrowth delivery system, characterized in that, include: Biotin-bonded cyclodextrin three-dimensional network polymer backbone, Bacillus-derived natural extracellular vesicles embedded in the pores of the biotin-bonded cyclodextrin three-dimensional network polymer backbone, saw palmetto fruit extract embedded in the phospholipid bilayer of Bacillus-derived natural extracellular vesicles, and myristoyl tetrapeptide-12 encapsulated in Bacillus-derived natural extracellular vesicles.

2. The hierarchical composite hair growth delivery system according to claim 1, characterized in that, The mass ratio of myristoyl tetrapeptide-12 to saw palmetto fruit extract is (0.5~5):

1.

3. The hierarchical composite hair growth delivery system according to claim 1, characterized in that, The biotin-bonded cyclodextrin three-dimensional network polymer backbone is a three-dimensional network polymer backbone formed by covalent bonding of cyclodextrin as the structural unit and biotin as the covalent linker arm, wherein the biotin is selected as D-biotin.

4. The hierarchical composite hair growth delivery system according to claim 3, characterized in that, D-Biotin is covalently bonded to cyclodextrin after activation by EDC·HCl / NHS.

5. The hierarchical composite hair growth delivery system according to claim 3, characterized in that, The cyclodextrin in the biotin-bonded cyclodextrin three-dimensional network polymer backbone is one or a combination of β-cyclodextrin or hydroxypropyl β-cyclodextrin.

6. The hierarchical composite hair regrowth delivery system according to claim 1, characterized in that, The particle size of natural extracellular vesicles derived from Bacillus is 80~200nm, and the polydispersity index is ≤0.

3.

7. A nursing product, characterized in that, The product includes a multi-layered hair growth delivery system as described in any one of claims 1 to 6, wherein the saw palmetto fruit extract in the multi-layered hair growth delivery system acts on the surface of the scalp, and myristoyl tetrapeptide-12 acts on the deep hair follicles, and the care product is a scalp care product, a hair follicle repair product, or a functional skin care product.

8. A method for preparing a hierarchical composite hair growth delivery system, characterized in that, Includes the following steps: S1: Dissolve cyclodextrin to obtain a cyclodextrin solution, add D-biotin to the cyclodextrin solution and stir, then add a compound activator and adjust the pH to 5.5~6.5, place at 25~35℃ and 100~180r / min in the dark for 2~6h to obtain a biotin-cyclodextrin prepolymerized backbone solution containing a biotin-bonded cyclodextrin three-dimensional network polymer backbone; S2: Bacillus vesicle suspension extracted from Bacillus fermentation broth; S3: Take the Bacillus vesicle suspension and add PBS to adjust the pH to 7.0~7.

2. Then add myristoyl tetrapeptide-12 and stir at 80~150 r / min for 0.5~1 h to obtain a single-drug-loaded Bacillus vesicle suspension. Heat the single-drug-loaded Bacillus vesicle suspension to 28~35℃ and add the dissolved saw palmetto extract solution. Stir for 1~2 h under the dark to obtain a dual-drug-loaded Bacillus vesicle suspension. S4: The dual-drug-loaded Bacillus vesicle suspension was added dropwise to the biotin-cyclodextrin prepolymerized backbone solution and homogenized under high pressure microfluidic treatment to obtain a hierarchical composite germinal delivery system.

9. The method for preparing the hierarchical composite hair growth delivery system according to claim 8, characterized in that, Includes the following steps: S5: The hierarchical composite hair growth delivery system is dried in a freeze vacuum dryer and then pulverized through a mesh sieve to obtain a uniform powdered composite delivery system.

10. The method for preparing the hierarchical composite hair growth delivery system according to claim 8, characterized in that, The compound activator is a mixture of EDC·HCl and NHS, wherein the molar ratio of EDC·HCl to NHS is 1:1 to 1.2:1, and the amount of compound activator is 1.0 to 1.5 times the mass of D-biotin.

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