Composite wrapping system based on retinol and artemisinin, preparation method and cosmetics

Abstract

The invention relates to a composite wrapping system based on retinol and artemisinin, a preparation method and cosmetics, the system comprises artemisinin through hydroxypropyl beta-cyclodextrin and sulfobutyl beta-cyclodextrin (1: 1) to form an inner core, retinol is embedded in a bacillus subtilis inner membrane vesicle-DOTAP phospholipid bilayer to form an intermediate layer, polysaccharide sandwich structures are modified on two sides, and the composite wrapping system is prepared from a composite wrapping material, and the problems of easy oxidation, poor compatibility and high irritation of the two are solved. During preparation, firstly, cyclodextrin artemisinin nanoparticles are prepared through a supercritical COtechnology, and then the cyclodextrin artemisinin nanoparticles are assembled into a composite system through vesicle extraction, retinol embedding, polysaccharide coating and membrane fusion. The system is high in stability (the activity retention rate is larger than 90% after the system is stored at 25 DEG C for 30 days), the transdermal efficiency is improved by 35 times compared with that of free components, the anti-aging and anti-inflammatory synergistic interaction is achieved, and the system can be used for preparing anti-aging and anti-inflammatory cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic active ingredient delivery, and specifically to a system, preparation method, and cosmetics for improving the stability, compatibility, and transdermal efficiency of retinol and artemisinin through composite encapsulation technology. Background Art

[0002] Retinol, as an active anti-aging ingredient, has the disadvantages of being easily oxidized and inactivated, having poorly soluble formulas, and being highly irritating. Artemisinin has anti-inflammatory effects, but has poor water solubility, insufficient photothermal stability, and low bioavailability.

[0003] When the two are mixed, the peroxide bridge structure of artemisinin will trigger the self-oxidation of retinol, causing retinol to degrade into inactive retinoic acid. At the same time, the peroxide bridge of artemisinin will be reduced and broken to form inactive derivatives, resulting in the loss of the efficacy of both.

[0004] However, traditional single encapsulation technologies (such as liposome or cyclodextrin encapsulation) are difficult to solve the compatibility and delivery efficiency problems of multiple components at the same time. A composite delivery system is urgently needed to overcome the above defects. Summary of the Invention

[0005] The purpose of the present invention is to address the problems in the prior art that single encapsulation technology cannot simultaneously solve the problems of poor stability of retinol and artemisinin, easy redox reaction failure when mixed, and low delivery efficiency. A composite encapsulation system, preparation method and cosmetics based on retinol and artemisinin are provided. Artemisinin is encapsulated in the inner cavity of cyclodextrin to form nanoparticles, and retinol is embedded in the outer bilayer of natural fermented phospholipids and modified with a double-layer polysaccharide sandwich structure to construct a double-encapsulation composite system, isolate the active ingredients from the external environment, solve their stability and compatibility problems, and synergistically improve the transdermal absorption efficiency and anti-aging and anti-inflammatory effects.

[0006] In order to achieve the above application objectives, the present invention adopts the following technical solution: a composite encapsulation system based on retinol and artemisinin includes: The core structure is a nanoparticle formed by cyclodextrin inclusion of artemisinin; The intermediate vesicle structure is composed of a natural phospholipid bilayer embedded with retinol, and the surface of the natural phospholipid bilayer is positively charged; The outer layer structure modifies the polysaccharide layer on both the inner and outer sides of the natural phospholipid bilayer through electrostatic adsorption to form a sandwich protection ring; Among them, artemisinin and retinol repel each other due to redox reaction, and the polysaccharide layer physically isolates the contact between the two.

[0007] Furthermore, the cyclodextrin is a mixture of hydroxypropyl β-cyclodextrin and sulfobutyl β-cyclodextrin in a mass ratio of 1:1.

[0008] Furthermore, the natural phospholipid bilayer of the intermediate vesicle structure is derived from the inner membrane vesicle of Bacillus subtilis, and DOTAP is added to make the surface positively charged.

[0009] Furthermore, the polysaccharide layer is selected from at least one of chitosan, hyaluronic acid, alginate or dextran.

[0010] Furthermore, the mass ratio of cyclodextrin to artemisinin is 2:1~3:1.

[0011] Furthermore, the mass ratio of Bacillus subtilis inner membrane vesicles to DOTAP is 1:1-3:1, and the mass ratio of retinol to the mixture of Bacillus subtilis inner membrane vesicles and DOTAP is 10:1-2:1.

[0012] A method for preparing the composite packaging system as described above comprises the following steps: (1) Preparation of cyclodextrin inclusion complex: Cyclodextrin and artemisinin are reacted under supercritical CO2 conditions at a reaction temperature of 35-50°C, a pressure of 10-30 MPa, and a reaction time of 2-6 hours to obtain a cyclodextrin inclusion complex; (2) Preparation of vesicle complexes: natural phospholipid vesicles were mixed with cationic lipid DOTAP, and retinol was embedded in the phospholipid bilayer; (3) Polysaccharide modification: The protonated polysaccharide solution and the vesicle complex obtained in step (2) are ultrasonically self-assembled under pH 3-6 to form a sandwich polysaccharide layer; (4) System assembly: The cyclodextrin inclusion complex and the polysaccharide-modified vesicle complex are membrane-fused in a pH 6.5-7.4 buffer solution and freeze-dried to form.

[0013] Furthermore, in step (1), the temperature of the supercritical CO2 reaction is 40°C, the pressure is 15 MPa, and the reaction time is 4 hours.

[0014] Furthermore, the number of cycles of ultrasonic self-assembly in step (3) is 30-100 times, and the mass ratio of polysaccharide, vesicle complex and cross-linking agent sodium tripolyphosphate is 1:1:0.1.

[0015] An anti-aging cosmetic comprising the above-mentioned composite encapsulation system, wherein artemisinin and retinol are sequentially released in skin cells: Retinol is released first, activating the collagen synthesis pathway of fibroblasts; Artemisinin is released later, inhibiting the inflammatory response triggered by retinol.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Stability is significantly improved, solving the problem of easy inactivation of ingredients In existing technologies, retinol and artemisinin have poor stability when present alone or mixed. However, the present invention utilizes a composite encapsulation system to form a multi-layered protective structure. The outer polysaccharide-phospholipid sandwich isolates oxygen and UV rays, the inner cyclodextrin core contains artemisinin, and the middle phospholipid bilayer embeds retinol, effectively preventing oxidative degradation of both.

[0017] Experimental data show that after storage at 25°C for 30 days, the pure retinol content is only 3.99%. However, the stability of retinol in the composite system of the present invention is 23 times higher than that of the free form, and the activity retention rate is >90%, significantly extending the shelf life of the ingredient.

[0018] 2. The transdermal absorption efficiency is greatly improved, and the bioavailability is enhanced Traditional single-encapsulation technologies have low delivery efficiency. However, the vesicle membrane of this invention is similar to the lipid structure of the skin, which can promote transmembrane penetration. Franz diffusion experiments show that the composite system has a 35-fold higher transdermal efficiency than the free components, which can more effectively deliver retinol and artemisinin to the target skin area and improve bioavailability.

[0019] 3. Synergistically enhance anti-aging and anti-inflammatory effects to achieve dual-pathway effects In the prior art, mixing the two is prone to redox reaction, resulting in loss of efficacy. The present invention avoids direct contact between the two by layered packaging, while exerting a synergistic effect: Retinol binds to the retinoic acid receptor (RAR / RXR) in the cell nucleus, upregulates the TGF-β signaling pathway, stimulates fibroblasts to secrete type I and type III collagen, and repairs the structure of the dermis.

[0020] Artemisinin inhibits matrix metalloproteinases (MMPs) to reduce collagen degradation, activates TGF-β to promote collagen and elastin synthesis, and improves skin sagging. At the same time, it inhibits the activity of nuclear transcription factor NF-κB, reduces the release of proinflammatory cytokines, and alleviates the inflammatory response caused by retinol stimulation.

[0021] In vitro experiments show that the complex system can promote fibroblast collagen synthesis by 40% compared with a single component, and the anti-wrinkle effect is significantly improved.

[0022] 4. Reduce irritation and improve safety Retinol itself is highly irritating, but the present invention reduces its direct contact with the skin through a composite packaging system. The chicken embryo chorioallantoic membrane test HETCAM score is ≤0.9, proving that the irritation of retinol to the skin can be reduced, thereby improving product safety.

[0023] 5. Solve ingredient compatibility issues and achieve efficient coexistence Traditional technologies cannot solve the redox reaction problem when retinol and artemisinin are mixed. The present invention uses a three-layer composite structure of "cyclodextrin artemisinin nanoparticles-phospholipid vesicle retinol-double-layer polysaccharide" to isolate the contact between the two, prevent the oxidation reaction caused by peroxide bridges, and achieve stable coexistence and synergistic effect of the two in the same system. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a schematic diagram of the composite architecture of the present invention; Figure 2 is a transmission electron microscope (TEM) image of the composite system morphology of the present invention; Figure 3 2 is a graph showing the content changes of various components of the embodiment of the present invention at different time periods at 25° C.; Figure 4 This is a transmittance curve diagram of retinol and retinol artemisinin complex according to an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention are within the scope of protection of the present invention.

[0026] Example 1 like Figure 1 and Figure 2 As shown, the composite encapsulation system based on retinol and artemisinin includes: The core structure is a nanoparticle formed by cyclodextrin inclusion of artemisinin; In this embodiment, the cyclodextrin is a complex of hydroxypropyl β-cyclodextrin and sulfobutyl β-cyclodextrin in a ratio of 1:1, and the mass ratio of cyclodextrin to artemisinin is 2:1 to 3:1.

[0027] The intermediate vesicle structure is composed of a natural phospholipid bilayer embedded with retinol, and the surface of the natural phospholipid bilayer is positively charged; In this example, the intermediate vesicle structure consists of a phospholipid bilayer formed by Bacillus subtilis inner membrane vesicles and 1,2-dioleoyl-3-trimethylammonium propane chloride (DOTAP). Retinol is embedded in the phospholipid bilayer, and the presence of DOTAP imparts a positive charge to the bilayer. The mass ratio of Bacillus subtilis inner membrane vesicles to DOTAP is 1:1 to 3:1, and the mass ratio of retinol to the mixture of the vesicles and DOTAP is 10:1 to 2:1.

[0028] The outer layer structure modifies the polysaccharide layer on both the inner and outer sides of the natural phospholipid bilayer through electrostatic adsorption to form a sandwich protection ring; In this embodiment, the polysaccharide is one or more of chitosan, hyaluronic acid, alginic acid, or dextran. Artemisinin and retinol repel each other due to the redox reaction, and the polysaccharide layer physically isolates the two from contact.

[0029] Example 2 Based on the same concept, this embodiment proposes a method for preparing the composite packaging system of Example 1, and the specific steps are as follows: (1) Preparation of cyclodextrin artemisinin nanoparticles: Cyclodextrin (a 1:1 mixture of hydroxypropyl β-cyclodextrin and sulfobutyl β-cyclodextrin, taking into account the molecular weight and electron-nuclear binding of artemisinin) and Artemisia annua extract (containing ≥75% artemisinin) were weighed at a mass ratio of 2:1-3:1 (excess cyclodextrin prevented artemisinin from escaping). Both were dried separately in a vacuum environment (40°C, 24 hours) to avoid moisture interference. Artemisinin and cyclodextrin are mixed in proportion and placed in a reactor. Liquid CO2 is introduced into the reactor, and the temperature is raised (temperature: 35-50°C, slightly above the critical temperature to avoid decomposition of artemisinin) and pressurized (pressure: 10-30 MPa, the higher the pressure, the greater the CO2 density and the stronger the dissolving ability) to a supercritical state; Turn on the stirring pump for 2-6 hours to allow CO2 to fully contact the mixture, promote the dissolution of artemisinin and its entry into the CD cavity (the internal cavity structure of cyclodextrin (CD)). After the reaction is completed, the pressure is slowly released to normal pressure, CO2 is gasified, and the hydrophobic cavity of cyclodextrin is used to include artemisinin, and the inclusion complex is precipitated in the form of powder.

[0030] (2) Preparation of vesicular retinol complex: Bacillus subtilis was fermented and cultured (LB medium, 37°C, 200 rpm, 24 h), and the cells were collected by centrifugation. The outer membrane was removed by lysozyme-SDS-DTT treatment, and the inner membrane vesicles were extracted by ultracentrifugation (100,000 × g, 1 h). The inner membrane vesicles and 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP) were mixed in a ratio of 1:1-3:1. Retinol was dissolved in a Tween 80-ethanol water complex solution and uniformly dispersed with the vesicles and DOTAP in a ratio of 10:1-2:1 (w / w). High-pressure microfluidization technology was used to pass the mixed system through a micron-scale interactive chamber under ultra-high pressure conditions of 120MPa. The resulting high shear force, cavitation effect and turbulence were utilized to achieve efficient embedding of retinol molecules into the vesicle bilayer. Due to the presence of DOTAP, the final vesicle-retinol complex was positively charged.

[0031] (3) Preparation of double-layer polysaccharide sandwich structure: Polysaccharides (chitosan / hyaluronic acid / alginate / dextran) are coated on both sides of the positively charged vesicle bilayer through electrostatic interaction to form a sandwich structure. The specific steps are as follows: The polysaccharide was dissolved in 0.1 M Tris-HCl (pH 3-6) to be protonated to form a negative charge, and then sonicated with the vesicle retinol complex and sodium tripolyphosphate in step (2) at a ratio of 1:1:0.1 for 5 seconds and then stopped for 10 seconds, and the mixture was terminated after 30-100 cycles.

[0032] (4) Assembly of double-wrapped composite system: The above-mentioned complex is dispersed into a thin film by removing the solvent through a rotary evaporator, and the cyclodextrin artemisinin nanoparticles and the vesicle retinol complex dispersed into a thin film are mixed in a mass ratio of 1:1-1:3. The nanoparticles are loaded into the interior of the vesicles through membrane fusion in a pH 6.5-7.4 phosphate buffer solution, and the excess water is removed by freeze-drying to obtain a composite encapsulation system of retinol and artemisinin.

[0033] In this embodiment, the raw materials involved are as follows: Cyclodextrin: hydroxypropyl β-cyclodextrin and sulfobutyl β-cyclodextrin mixed at a ratio of 1:1 (purchased from Sigma-Aldrich); Artemisia annua extract: artemisinin content ≥75% (purchased from Yunnan Botanical Pharmaceutical Co., Ltd.); Bacillus subtilis: ATCC 6633 strain (purchased from China Industrial Microorganism Culture Collection Center); 1,2-Dioleoyl-3-trimethylammonium propane chloride (DOTAP): purity ≥98 (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.); Polysaccharides: chitosan (degree of deacetylation ≥ 90%), alginate, dextran (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), hyaluronic acid (purchased from Shandong Baifu Freda Pharmaceutical Co., Ltd.); Solvents: Tween 80, anhydrous ethanol, and phosphate buffer (pH 6.5-7.4) were purchased from Sinopharm Group.

[0034] The equipment is as follows: Supercritical CO2 reactor (HA121-50-01), microfluidizer (ATS-1000), ultracentrifuge (Beckman Coulter Optima XPN), and rotary evaporator (Buchi R-210).

[0035] Specific implementation case 1 Based on the above two embodiments, this embodiment proposes a practical preparation case: Weigh 20g of the cyclodextrin mixture and 10g of Artemisia annua extract and dry them separately under vacuum (40°C for 24 hours). Place the mixture in a reactor, introduce liquid CO₂ to a pressure of 15 MPa, raise the temperature to 40°C (supercritical state), stir at 300 rpm, and react for 4 hours. After the reaction is complete, slowly release the pressure to ambient pressure (taking 1 hour) to obtain cyclodextrin artemisinin nanoparticles.

[0036] Bacillus subtilis was cultured in LB medium (37°C, 200 rpm, 24 h), and the cells were collected by centrifugation. The outer membrane was removed by lysozyme-SDS-DTT treatment, and the inner membrane vesicles were extracted by ultracentrifugation (100,000×g, 1 h). The inner membrane vesicles and 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP) were mixed in a 1:1 ratio. Retinol was dissolved in a Tween 80-ethanol-water complex solution and evenly dispersed with the vesicles and DOTAP in a 5:1 ratio. The mixture was then passed through a microfluidizer to obtain a vesicular retinol complex. Chitosan was dissolved in 0.1 M Tris-HCl and mixed with the previous vesicular retinol complex and sodium tripolyphosphate in a 1:1:0.1 ratio, with sonication for 5 s and rest for 10 s. After 60 cycles, the vesicular retinol complex was obtained.

[0037] The above complex was dispersed into a thin film by removing the solvent using a rotary evaporator. 1.00 g of cyclodextrin artemisinin nanoparticles and 2.00 g of vesicular retinol complex were weighed and dissolved in 50 ml of pH 6.5 phosphate buffer. The mixture was stirred for 15 minutes. The nanoparticles were loaded into the vesicles through membrane fusion and freeze-dried to obtain a composite encapsulation system of retinol and artemisinin.

[0038] Specific implementation case 2 Based on the same concept, this embodiment also proposes an actual preparation case, which differs from the specific implementation case 1 in that: 1. Weigh 30 g of cyclodextrin mixture and 10 g of Artemisia annua extract; 2. Introduce liquid CO2 to a pressure of 25 MPa, raise the temperature to 45°C (supercritical state), and the reaction time is 6 hours; 3. Mix the inner membrane vesicles and 1,2-dioleoyl-3-trimethylammonium propane chloride (DOTAP) in a ratio of 3:1; 4. Dissolve retinol in Tween 80-ethanol water composite solution, disperse it evenly with vesicles and DOTAP at a ratio of 8:1, and pass it through a microfluidizer; 5. Weigh 1.00 g of cyclodextrin artemisinin nanoparticles and 1.00 g of vesicular retinol complex.

[0039] The remaining steps and parameters are exactly the same.

[0040] Specific implementation case three Based on the same concept, this embodiment also proposes an actual preparation case, which differs from the specific implementation case 1 in that: 1. Weigh 25 g of cyclodextrin mixture and 10 g of Artemisia annua extract, and dry them separately in a vacuum environment (35°C, 24 hours); 2. Introduce liquid CO2 to a pressure of 10 MPa and the reaction time is 2 hours; 3. Mix the inner membrane vesicles and 1,2-dioleoyl-3-trimethylammonium propane chloride (DOTAP) in a ratio of 2:1; 4. Dissolve retinol in a Tween 80-ethanol water complex solution, disperse it evenly with vesicles and DOTAP at a ratio of 3:1, and pass it through a microfluidizer; 5. Weigh 1.00 g of cyclodextrin artemisinin nanoparticles and 3.00 g of vesicular retinol complex.

[0041] The remaining steps and parameters are exactly the same.

[0042] In summary, based on specific implementation cases 1 to 3, the stability investigation structure of the present invention is as follows: Table 1 and Figure 3 : Table 1 Content changes of each component under different conditions

[0043] From Table 1 and Figure 3 It can be seen that the contents of pure retinol, specific implementation case one, specific implementation case two, and specific implementation case three under 25°C conditions after 30 days are 3.99%, 91.86%, 80.35%, and 88.35%, respectively. It can be seen that the retinol raw material is very unstable, especially in high temperature environments, most of the retinol is lost; the retinol content after encapsulation is significantly increased, indicating that the encapsulated retinol is more stable.

[0044] In order to verify the effect of the present invention, a transdermal experiment was also conducted: The Franz diffusion cell method was used to test the cumulative permeability (%) of retinol and retinol-artemisinin complex on the back skin of rats over a period of 0-24 hours. Retinol (concentration 2 mg / mL) and retinol-artemisinin complex (effective retinol concentration 2 mg / mL) were set up separately, with three replicates per group. The specific testing process is as follows: The skin used in the experiment was the abdominal skin of 6-week-old female Wistar rats. After the rats were killed, the subcutaneous tissue layer was removed, rinsed repeatedly with clean water, cut into appropriate sizes, placed in physiological saline, and stored at 4°C for later use. The composition of the permeate used in the Franz diffusion cell is: 40% propylene glycol, 59.5% 0.01MpH=9.0PBS buffer, 0.5% tocopherol; 2mL was administered to the diffusion cell. At corresponding time intervals, 0.5mL of the receiving cell solution was aspirated, and then the corresponding volume of permeate was added; to prevent retinol degradation, the Franz diffusion cell should be protected from light, and the content test should be performed immediately after aspirating the receiving cell solution. The formula for skin permeability is as follows:

[0045] Among them, C0.5, C2, and C4 represent the concentrations of retinol in the sampling solution at the sampling points of 0.5 h, 2 h, and 4 h, respectively.

[0046] The experimental results are as follows Figure 4 As shown in the figure, compared with unencapsulated retinol, the transdermal efficiency of the retinol-artemisinin complex is 35% higher than that of free retinol. The retinol-artemisinin complex has the effect of improving the skin penetration ability of retinol.

[0047] Furthermore, in order to further verify the effect of the present invention, a type I collagen content detection experiment (anti-wrinkle efficacy evaluation) was also carried out. Fibroblasts in the skin are responsible for synthesizing the extracellular matrix, including collagen and elastin. These components are crucial for maintaining the skin's structure and stability. Type I collagen (COL I) accounts for 70%-80% of the skin's dry weight, supporting the skin's contours, enhancing its firmness, and making it appear plumper and fuller. A gradual decrease in type I collagen content can lead to the formation of wrinkles. Therefore, this test uses UVA to irradiate human skin fibroblasts and evaluate the anti-wrinkle efficacy of the sample by detecting changes in type I collagen content. The specific process is as follows: 1. Cell Culture and Model Construction Human skin fibroblasts were digested and passaged into 60 mm cell culture dishes and cultured in a cell culture incubator at 37°C and 5% CO2 until the density reached more than 70%. The negative control group, positive control group, and sample group were treated with 5J / cm 2 UVA irradiation was performed three times, with an interval of 2 hours between each exposure, to construct the cell model. Afterwards, the negative control group was replaced with blank culture medium, and the positive control group and sample group were replaced with cell culture medium containing 10 ng / mL TGF-β1 and 0.005% supramolecular retinol essence, respectively. The cells were placed in an incubator and cultured for another 48 hours. The cell culture medium of each group was centrifuged at 4000 g for 10 minutes, and the supernatant was collected for subsequent testing. Three parallel experiments were set up for each group.

[0048] 2. Testing Process The skin used in this experiment was from the abdomen of 6-week-old female Wistar rats. After the rats were sacrificed, the subcutaneous tissue layer was removed, the cells were rinsed repeatedly with clean water, and the cells were cut into appropriate pieces. The cells were placed in normal saline and stored at 4°C until further use. The permeabilization solution used in the Franz diffusion cell consisted of 40% propylene glycol, 59.5% 0.01M pH 9.0 PBS buffer, and 0.5% tocopherol. A 2 mL dose was injected into the diffusion cell. At appropriate intervals, 0.5 mL of the receiving cell solution was aspirated, and the corresponding volume of permeabilization solution was added. To prevent retinol degradation, the Franz diffusion cell was protected from light, and retinol content was measured immediately after aspiration.

[0049] 3. Detection of Type I Collagen ELISA kit was used to detect the type Ⅰ collagen content in human skin fibroblasts.

[0050] Type I collagen content was assessed: The supernatant of the centrifuged cell culture medium was diluted with PBS to an appropriate multiple for subsequent testing. The assay was performed according to the instructions for the Human COL I ELISA kit from the Nanjing Jiancheng Bioengineering Institute. The standard curve was fitted using a logistic curve, and significance was analyzed using IBM SPSS.

[0051] Calculate protein increase rate:

[0052] Wherein, B is the protein content of the negative control group; Bx is the protein content of the positive control group or sample group.

[0053] 4. The experimental results are shown in Table 2 below: Table 2 Type I collagen content in human skin fibroblasts

[0054] As shown in Table 2, compared with the blank control group, the type I collagen content in the negative control group decreased significantly, indicating that the stimulation conditions were effective; compared with the negative control group, the type I collagen content in the retinol group increased significantly, with an increase rate of 28.91%; compared with the negative control group, the increase rate in the retinol-artemisinin complex group was 40.47%; compared with the retinol group, the promotion rate of the retinol-artemisinin complex system on fibroblast collagen synthesis was 40% higher than that of the single component.

[0055] For ease of understanding, the following supplementary explanations are given for professional terms that are not explained in detail in this invention: 1. Supercritical CO2 Definition: Supercritical CO2 (sCO2) refers to carbon dioxide (CO2) at temperatures and pressures above its critical point (31.1°C, 7.38 MPa), exhibiting a unique physical state between gas and liquid. This state combines the high diffusivity and low viscosity of a gas with the high density and strong solubility of a liquid, making it valuable for applications in a wide range of fields.

[0056] The present invention is applied as follows: supercritical CO2 is used as a solvent to dissolve artemisinin at 35-50°C and 10-30MPa and promote its entry into the cyclodextrin cavity. The characteristic of CO2 leaving no residue after gasification is utilized to form pure cyclodextrin artemisinin nanoparticles, thus avoiding the destruction of the active ingredients by traditional organic solvents.

[0057] 2. High-pressure microfluidization Definition: High-pressure microfluidization is a technology that uses ultra-high pressure (100–400 MPa) to force liquid materials through micron-sized channels to achieve particle breakage, emulsification or dispersion under extreme shear force, cavitation effect and high-speed collision.

[0058] The present invention is applied by processing a mixture of retinol and vesicles-DOTAP through a microfluidizer, and using mechanical force to embed retinol into the phospholipid bilayer of the inner membrane vesicles of Bacillus subtilis to form a stable nanoscale vesicle-retinol complex, thereby improving dispersion uniformity and membrane embedding efficiency.

[0059] 3. Franz Diffusion Cell Method Definition: An in vitro transdermal test device consisting of a supply cell and a receiving cell with a skin sample sandwiched between them, used to measure the permeation rate and amount of active ingredients through the skin.

[0060] Application of the present invention: The transdermal efficiency of the composite system was tested by the Franz diffusion cell method, using rat abdominal skin as a model. The results showed that its transdermal efficiency was 35 times higher than that of free retinol, confirming the promoting effect of the similarity between the vesicle membrane and skin lipids on permeation.

[0061] 4. ELISA kit (enzyme-linked immunosorbent assay) Definition: A kit that utilizes the principle of antigen-antibody specific binding to quantitatively detect target proteins through a colorimetric reaction with an enzyme marker.

[0062] Application of the present invention: Using Human COLⅠ ELISA kit to detect the content of type I collagen secreted by human skin fibroblasts, verify the promoting effect of the composite system on collagen synthesis (increase rate 40.47%), and thus evaluate the anti-wrinkle efficacy.

[0063] 5. NLRP3 inflammasome Definition: An intracellular multiprotein complex that participates in the body's inflammatory response. Excessive activation can lead to the release of pro-inflammatory cytokines, causing skin inflammation (such as erythema and stinging).

[0064] The present invention relates to the following: when retinol is converted into excessive retinoic acid (RA), it may induce activation of the NLRP3 inflammasome, while artemisinin reduces the release of pro-inflammatory factors by inhibiting NF-κB activity, thereby alleviating the inflammatory response, reflecting the safety design of the composite system.

[0065] 6. TGF-β signaling pathway Definition: A cell signaling pathway mediated by transforming growth factor-β (TGF-β) that is involved in regulating cell proliferation, differentiation, and extracellular matrix synthesis.

[0066] Effects of the present invention: Both retinol and artemisinin can activate the TGF-β signaling pathway. The former stimulates fibroblasts to secrete type I and type III collagen, while the latter inhibits MMP (matrix metalloproteinase) to reduce collagen degradation, synergistically promoting skin repair and anti-aging.

[0067] 7. MMP (Matrix Metalloproteinase) Definition: A family of zinc-dependent proteases whose primary function is to degrade the extracellular matrix (e.g., collagen, elastin).

[0068] The present invention relates to: artemisinin reduces the degradation of collagen fibers in the skin by inhibiting the activity of MMP, forming a synergistic effect with retinol's promotion of collagen synthesis, maintaining skin elasticity and improving sagging.

[0069] 8. DOTAP (1,2-dioleoyl-3-trimethylammonium propane chloride) Definition: A cationic liposome material containing quaternary ammonium groups in its structure, which can make the liposomes positively charged.

[0070] Application of the present invention: After being mixed with the inner membrane vesicles of Bacillus subtilis in proportion, the vesicle retinol complex is made positively charged, which facilitates the formation of a double-layer sandwich structure with negatively charged polysaccharides (such as chitosan) through electrostatic interaction, thereby enhancing the stability of the composite system and the integrity of the protective layer.

[0071] 9. Lysozyme-SDS-DTT Combined Treatment Definition: A cell disruption method that uses three reagents in combination: Lysozyme: destroys the peptidoglycan structure of bacterial cell walls; SDS (sodium dodecyl sulfate): dissolves cell membrane proteins; DTT (dithiothreitol): reduces protein disulfide bonds and promotes membrane dissociation.

[0072] The invention is used for stripping the outer membrane of Bacillus subtilis so as to extract the inner membrane vesicles by subsequent ultrasonic crushing and ultracentrifugation, thereby providing raw materials for the preparation of vesicle-retinol complexes.

[0073] 10. Microfluidizer Processing Pressure Definition: The fluid pressure applied during microfluidization homogenization, measured in MPa, directly affects particle dispersion and membrane embedding efficiency.

[0074] Parameters of the present invention: In the embodiment, a pressure of 120 MPa is used to uniformly embed retinol into the vesicle membrane layer to form a stable nanoscale complex, thereby ensuring the consistency of subsequent polysaccharide coating and composite system assembly.

[0075] The parts not described in detail in the present invention are prior art, so the present invention does not describe them in detail.

[0076] It is to be understood that the term "one" should be understood as "at least one" or "one or more", that is, in one embodiment, the number of an element may be one, while in another embodiment, the number of the elements may be multiple, and the term "one" should not be understood as a limitation on the quantity.

[0077] Although this document uses a lot of professional terms, it does not exclude the possibility of using other terms. These terms are used only to more conveniently describe and explain the essence of the present invention; interpreting them as any additional limitations is contrary to the spirit of the present invention.

[0078] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that is the same or similar to the present invention falls within the scope of protection of the present invention.

Claims

1. A composite encapsulation system based on retinol and artemisinin, characterized in that: include: The core structure is a nanoparticle formed by cyclodextrin inclusion of artemisinin; The intermediate vesicle structure is composed of a natural phospholipid bilayer embedded with retinol, and the surface of the natural phospholipid bilayer is positively charged; The outer layer structure modifies the polysaccharide layer on both the inner and outer sides of the natural phospholipid bilayer through electrostatic adsorption to form a sandwich protection ring; The artemisinin and retinol repel each other due to redox reaction, and the polysaccharide layer physically isolates the contact between the two.

2. The composite wrapping system according to claim 1, characterized in that: The cyclodextrin is a mixture of hydroxypropyl β-cyclodextrin and sulfobutyl β-cyclodextrin in a mass ratio of 1:

1.

3. The composite wrapping system according to claim 1 or 2, characterized in that: The natural phospholipid bilayer of the intermediate vesicle structure is derived from the inner membrane vesicle of Bacillus subtilis, and DOTAP is added to make the surface positively charged.

4. The composite wrapping system according to claim 1, characterized in that: The polysaccharide layer is selected from at least one of chitosan, hyaluronic acid, alginate or dextran.

5. The composite wrapping system according to claim 2, characterized in that: The mass ratio of the cyclodextrin to the artemisinin is 2:1 to 3:

1.

6. The composite wrapping system according to claim 3, characterized in that: The mass ratio of the Bacillus subtilis inner membrane vesicles to DOTAP is 1:1-3:1, and the mass ratio of the retinol to the mixture of the Bacillus subtilis inner membrane vesicles and DOTAP is 10:1-2:

1.

7. A method for preparing the composite packaging system according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Preparation of cyclodextrin inclusion complex: Cyclodextrin and artemisinin are reacted under supercritical CO2 conditions at a reaction temperature of 35-50°C, a pressure of 10-30 MPa, and a reaction time of 2-6 hours to obtain a cyclodextrin inclusion complex; (2) Preparation of vesicle complexes: natural phospholipid vesicles were mixed with cationic lipid DOTAP, and retinol was embedded in the phospholipid bilayer; (3) Polysaccharide modification: The protonated polysaccharide solution and the vesicle complex obtained in step (2) are ultrasonically self-assembled under pH 3-6 to form a sandwich polysaccharide layer; (4) System assembly: The cyclodextrin inclusion complex and the polysaccharide-modified vesicle complex are membrane-fused in a pH 6.5-7.4 buffer solution and freeze-dried to form.

8. The preparation method according to claim 7, characterized in that The temperature of the supercritical CO2 reaction in step (1) is 40°C, the pressure is 15 MPa, and the reaction time is 4 hours.

9. The preparation method according to claim 7, characterized in that The number of cycles of ultrasonic self-assembly in step (3) is 30-100 times, and the mass ratio of polysaccharide, vesicle complex and cross-linking agent sodium tripolyphosphate is 1:1:0.

1.

10. An anti-aging cosmetic, characterized by: The composite encapsulation system according to any one of claims 1 to 6, wherein the artemisinin and the retinol are released sequentially in skin cells: Retinol is released first, activating the collagen synthesis pathway of fibroblasts; Artemisinin is released later, inhibiting the inflammatory response triggered by retinol.

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