An anti-aging and repairing nanoliposome based on microfluidic technology and its preparation method

Abstract

This invention relates to an anti-aging and repairing nanoliposome based on microfluidic technology and its preparation method, belonging to the field of cosmetic technology. The nanoliposomes comprise the following components: hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, S-type serotonin, acetyl hexapeptide-8, hydroxypinazone retinate (HPR), tetrahydrocurcumin, sodium DNA, sea fennel callus culture filtrate, and water. The preparation method includes: S1 oil phase preparation, S2 aqueous phase preparation, S3 colostrum preparation, S4 dynamic high-pressure microfluidic homogenization, and S5 post-treatment and stabilization. The liposomes have a uniform particle size (approximately 100 nm), high encapsulation efficiency, good stability and transdermal permeability, and can achieve anti-aging effects through synergistic effects of anti-wrinkle, antioxidant, and repair pathways, making them suitable for anti-aging and repairing cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to an anti-aging and repairing nanoliposome based on microfluidic technology and its preparation method. Background Technology

[0002] With the deepening research into the mechanisms of skin aging and the ever-increasing demands of consumers for the efficacy of anti-aging skincare products, the development of efficient, stable, and transdermal delivery systems for anti-aging active ingredients has become a research hotspot in the cosmetics field. Skin aging is a complex biological process involving multiple mechanisms such as collagen loss in the dermis, degradation of elastic fibers, damage to the extracellular matrix, accumulation of oxidative stress, and release of inflammatory factors. Targeting these mechanisms, a variety of anti-aging active ingredients have emerged on the market, including but not limited to Pro-Xylane, peptides, retinol derivatives, antioxidants, and plant stem cell extracts.

[0003] However, existing anti-aging ingredients face numerous technical bottlenecks in application. First, most active ingredients have low transdermal absorption efficiency. For example, S-type Pro-Xylane, a patented ingredient of L'Oréal Group, can promote the synthesis of glycosaminoglycans and improve skin firmness, but its large molecular weight makes it difficult to penetrate the skin's stratum corneum barrier; Acetyl hexapeptide-8, a neurotransmitter inhibitory peptide, can reduce expression lines by inhibiting catecholamine release, but peptides are highly hydrophilic, have poor skin permeability, and low bioavailability; HPR hydroxypinazone retinyl ester, a stable derivative of retinol, although less irritating than traditional retinol, still suffers from photosensitivity and unsatisfactory transdermal absorption.

[0004] Secondly, many anti-aging ingredients are unstable and easily degraded and inactivated. Tetrahydrocurcumin, as a potent antioxidant, has a phenolic hydroxyl structure that is easily oxidized; sodium DNA, as a cell repair component, is easily destroyed by enzymatic hydrolysis; and the active substances in the filtrate of sea fennel callus culture are also easily affected by environmental factors, resulting in reduced activity. These instabilities cause the product's efficacy to gradually weaken during storage, making it difficult to guarantee stable effects throughout the shelf life.

[0005] Third, multiple active ingredients may interact when combined, affecting their individual efficacy. For example, retinol derivatives and peptides may react under certain conditions, reducing their respective activities; differences in solubility (hydrophilicity and hydrophobicity) of different ingredients also make it difficult to achieve ideal compatibility and stability in the same formulation.

[0006] To address the aforementioned issues, various carrier systems, such as liposomes, nanoemulsions, and polymer nanoparticles, have been employed in existing technologies. Among these, liposomes have attracted widespread attention due to their excellent biocompatibility and transdermal penetration enhancement. However, traditional liposome preparation methods, such as thin-film methods, reverse-phase evaporation methods, and ethanol injection methods, have significant drawbacks: the prepared liposomes exhibit wide particle size distributions, low encapsulation efficiency, poor stability, and are difficult to mass-produce industrially. More importantly, liposomes prepared by traditional methods typically have particle sizes larger than 200 nm, limiting their transdermal efficiency, especially their ability to reach deep target sites within the skin.

[0007] Dynamic high-pressure microfluidics, as an emerging nanodispersion technology, has been applied in the pharmaceutical and food industries. However, in the field of anti-aging skincare products, especially in the preparation of nanoliposomes co-encapsulating multiple complex ingredients, there is still room for technological optimization. Currently, there are no reports on nanoliposomes using hydrogenated lecithin, polyglycerol-10 laurate, and α-tocopherol as composite wall materials to simultaneously encapsulate multiple anti-aging active ingredients such as S-type boswellic acid, acetyl hexapeptide-8, HPR hydroxypinazone retinate, tetrahydrocurcumin, sodium DNA, and sea fennel callus culture filtrate.

[0008] Therefore, developing a nanoliposome system that can efficiently encapsulate multiple anti-aging active ingredients, with small particle size, narrow distribution, high stability, and excellent transdermal properties, has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0009] In order to overcome the shortcomings of the existing technology, the present invention aims to provide an anti-aging and repairing nanoliposome based on microfluidic technology and its preparation method. The liposome has the technical effects of strong transdermal permeability, good stability, and multi-component synergistic anti-aging and skin repair.

[0010] This invention is achieved through the following technical solution: an anti-aging and repairing nanoliposome based on microfluidic technology, comprising the following components by mass percentage: hydrogenated lecithin 2.0-5.0%, polyglycerol-10 laurate 2.0-3.5%, α-tocopherol 0.2-0.6%, S-type boswellicin 1.5-5%, acetyl hexapeptide-8 0.05-0.2%, hydroxypinazone retinate HPR 0.1-0.3%, tetrahydrocurcumin 0.1-0.3%, sodium DNA 0.05-0.3%, sea fennel callus culture filtrate 0.05-0.4%, with the balance being water.

[0011] Preferably, the anti-aging and repairing nanoliposomes based on microfluidic technology comprise the following components by weight percentage: hydrogenated lecithin 3.0-4.5%, polyglycerol-10 laurate 2.5-3.0%, α-tocopherol 0.4-0.5%, S-type boswellicin 2-4%, acetyl hexapeptide-8 0.1-0.15%, hydroxypinazone retinate HPR 0.15-0.25%, tetrahydrocurcumin 0.2-0.3%, sodium DNA 0.1-0.3%, sea fennel callus culture filtrate 0.2-0.4%, and the balance being water.

[0012] On the other hand, the present invention provides a method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology, which includes the following steps:

[0013] S1: Oil phase preparation

[0014] Weigh out hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR, and tetrahydrocurcumin according to the specified amounts, and add them sequentially to anhydrous ethanol. Heat at 45-55℃ and stir at 150-200 rpm to dissolve. The ratio of the amount of anhydrous ethanol to the total mass of hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR, and tetrahydrocurcumin is (1.5-2.5):1mL / g.

[0015] S2: Aqueous phase preparation

[0016] Weigh out the S-type bosine, acetyl hexapeptide-8, sodium DNA, and sea fennel callus culture filtrate according to the specified amounts, and add them to water in sequence. Heat at 45-55℃ and stir at 150-200 rpm to dissolve. The volume ratio of water to anhydrous ethanol in step S1 is (1-2):1.

[0017] S3: Colostrum Preparation

[0018] Keep both the oil phase and the aqueous phase at 45-55℃. Slowly add the oil phase to the aqueous phase at a rate of 5-10 mL / min through a constant pressure dropping funnel. During the addition process, simultaneously turn on the high shear disperser and set the speed to 7000-9000 rpm for shear emulsification. After the oil phase is added, continue high shear dispersion for 5-30 min to form a milky white primary emulsion.

[0019] S4: Dynamic High-Pressure Microfluidic Homogenization

[0020] Turn on the jacket cooling system of the dynamic high-pressure micro-jet homogenizer and set the cooling water temperature to 25-30℃ (to avoid high temperature damage to active ingredients, such as acetyl hexapeptide-8, caused by homogenization).

[0021] Transfer the colostrum to the homogenizer feed tank and set the homogenization pressure to 120-130 MPa (too low a pressure will not achieve nanoscale particle size, and too high a pressure will easily cause liposome rupture). After homogenizing twice, add the same volume of water at 25-30℃, mix well, and then homogenize twice more.

[0022] S5: Post-processing and stabilization

[0023] The homogenized nanoliposomes were transferred to a vacuum degasser and degassed and had ethanol removed for 0.5-2 hours at 30-40℃ and a vacuum of <0.1MPa. The remaining water was then added and the mixture was homogenized. The nanoliposomes were then filtered through a 0.22μm microporous membrane and filled into containers.

[0024] The microporous filter membrane is a polyethersulfone microporous filter membrane.

[0025] Preferably, the heating temperatures in steps S1 and S2 are the same, preferably 50-55°C.

[0026] Beneficial effects

[0027] This invention provides an anti-aging and repairing nanoliposome based on microfluidic technology and its preparation method. The liposome has the technical effects of strong transdermal permeability, good stability, and synergistic anti-aging and skin repair through multiple components.

[0028] Synergistic enhancement of liposome film-forming properties and particle size uniformity. Hydrogenated lecithin, as the core raw material of the lipid bilayer and the "basic framework" of liposomes, is prone to particle size inhomogeneity when used alone due to its limited emulsifying ability. Polyglycerol-10-laurate, a nonionic emulsifier, assists in the orderly arrangement of lecithin and reduces oil-water interfacial tension; α-tocopherol not only scavenge free radicals in the system (preventing HPR oxidative degradation) but also inserts into the hydrogenated lecithin bilayer to regulate membrane fluidity (avoiding excessive rigidity of low-temperature membranes and looseness of high-temperature membranes). The synergistic effect of these three agents simultaneously enhances both chemical and physical stability. The three agents synergistically improve encapsulation efficiency, reduce particle size, and enhance transdermal absorption capacity.

[0029] Anti-wrinkle, antioxidant, and repair pathways work synergistically to combat aging. 1) Anti-wrinkle pathway: Dual-target improvement of "dynamic wrinkles + static wrinkles". S-type Pro-Xylane can activate fibroblasts and promote the synthesis of type I / III collagen and glycosaminoglycans (GAGs), specifically improving static wrinkles. Acetyl hexapeptide-8: Reduces muscle contraction by inhibiting the release of neurotransmitters (such as acetylcholine), specifically improving dynamic wrinkles. 2) Anti-photoaging pathway: Dual protection of "keratinocyte regulation + anti-inflammatory and antioxidant". HPR (hydroxypinazone retinate): Can act directly on the skin, promote normal differentiation of keratinocytes, and reduce keratin buildup caused by photoaging; Tetrahydrocurcumin: Can inhibit the release of inflammatory factors (IL-6, TNF-α); at the same time, its antioxidant capacity can neutralize UV-induced free radicals. 3) Barrier repair pathway: Synergistic enhancement of "immediate moisturizing + long-term regeneration". Sodium DNA (deoxyribonucleic acid): Can immediately replenish the moisture of the stratum corneum, repair the damaged stratum corneum barrier structure, and reduce moisture loss. Sea fennel callus culture filtrate: can promote epidermal renewal and barrier regeneration, and long-term use can enhance the skin's own barrier function. In summary, the effective components in this invention can achieve significant anti-aging effects through multi-pathway synergy of anti-wrinkle, antioxidant, and repair mechanisms. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some preferred embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the embodiments are conventional or common methods in the art, and the materials and reagents used are commercially available unless otherwise specified.

[0032] The raw materials used in the examples and comparative examples are described below: Hydrogenated lecithin: purchased from Lipoid Kosmetik AG; Sodium DNA: Purchased from Baihong Synthetic Biotechnology (Yantai) Co., Ltd.; S-type Bosein: purchased from Shanghai Yunluo Biotechnology Co., Ltd.; Sea fennel callus culture filtrate: purchased from SEPPIC SA.

[0033] Unless otherwise specified, all components and raw materials used in the embodiments and comparative examples of this invention are commercially available, and the same type of components and raw materials are used in each parallel experiment.

[0034] Example 1 A method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology includes the following steps:

[0035] S1: Oil phase preparation Weigh out hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR, and tetrahydrocurcumin according to the specified amounts, and add them sequentially to anhydrous ethanol. Heat at 50°C and shear and stir at 180 rpm to dissolve (forming a semi-transparent homogeneous mixture). The ratio of the amount of anhydrous ethanol to the total mass of hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR, and tetrahydrocurcumin is 1.5:1 mL / g.

[0036] S2: Aqueous phase preparation Weigh out the S-type bosine, acetyl hexapeptide-8, sodium DNA, and sea fennel callus culture filtrate, and add them sequentially to water (deionized water). Heat at 50°C and magnetically stir at 180 rpm for 5 minutes to dissolve. The volume ratio of water to anhydrous ethanol in step S1 is 1.4:1.

[0037] S3: Colostrum Preparation Keep both the oil and aqueous phases at 50°C. Slowly add the oil phase to the aqueous phase at a rate of 8 mL / min using a constant pressure dropping funnel. During the addition, simultaneously turn on the high-shear disperser and set the speed to 8000 rpm for shear emulsification. After the oil phase is added, continue high-shear dispersion for 10 min to form a milky white primary emulsion. (The particle size of the primary emulsion is measured using a dynamic light scattering instrument. The particle size should be 1.5-3.0 μm, and the PDI (polydispersity index) should be <0.4 (too large a particle size or too high a PDI will increase the difficulty of subsequent homogenization)).

[0038] S4: Dynamic High-Pressure Microfluidic Homogenization Turn on the jacket cooling system of the dynamic high-pressure micro-jet homogenizer and set the cooling water temperature to 25℃. Transfer the colostrum to the homogenizer feed tank, set the homogenization pressure to 125MPa, homogenize twice, add the same volume of water at 30℃, mix well, and then homogenize twice more.

[0039] S5: Post-processing and stabilization The homogenized nanoliposomes were transferred to a vacuum degasser and degassed and had ethanol removed for 1 hour at 40°C and a vacuum of <0.1 MPa. The remaining water was then added to bring the solution to 100% and mixed thoroughly. The mixture was then filtered through a 0.22 μm polyethersulfone microporous membrane and filled into vials.

[0040] Examples 2-7 Examples 2-7 were prepared using the same method as Example 1, except that the amounts of each component were adjusted (see Table 1).

[0041] Example 8 The difference between Example 8 and Example 1 lies in the amount of anhydrous ethanol used in step S1 and the amount of water used in step S2. The adjustments are as follows: the mass ratio of anhydrous ethanol to the total mass of hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR, and tetrahydrocurcumin is 2.5:1 mL / g; the volume ratio of water to anhydrous ethanol used in step S1 is 1:1.

[0042] Example 9 The difference between Example 9 and Example 1 lies in the amount of anhydrous ethanol used in step S1 and the amount of water used in step S2. The adjustments are as follows: the mass ratio of anhydrous ethanol to the total mass of hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR, and tetrahydrocurcumin is 1.5:1 mL / g; the volume ratio of water to anhydrous ethanol used in step S1 is 2:1.

[0043] Comparative Example 1 Comparative Example 1 was prepared in the same way as Example 1, except that it lacked the component polyglycerol-10 laurate.

[0044] Comparative Example 2 Comparative Example 2 was prepared using the same method as Example 1, except that it lacked the component α-tocopherol.

[0045] Comparative Example 3 Comparative Example 3 was prepared using the same method as Example 1, except that it lacked the component S-type Bosein.

[0046] Comparative Example 4 Comparative Example 4 was prepared using the same method as Example 1, except that it lacked the component acetyl hexapeptide-8.

[0047] Comparative Example 5 Comparative Example 5 was prepared using the same method as Example 1, except that it lacked the component HPR.

[0048] Comparative Example 6 Comparative Example 6 was prepared using the same method as Example 1, except that it lacked the component tetrahydrocurcumin.

[0049] Comparative Example 7 Comparative Example 7 was prepared using the same method as Example 1, except that it lacked the component sodium DNA.

[0050] Comparative Example 8 Comparative Example 8 was prepared in the same way as Example 1, except that it lacked the sea fennel callus culture filtrate.

[0051] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is that the amount of anhydrous ethanol used in step S1 is different. It is adjusted to be: the ratio of the amount of anhydrous ethanol to the total mass of hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR and tetrahydrocurcumin is 1:1 mL / g.

[0052] Comparative Example 10 The difference between Comparative Example 10 and Example 1 is that the amount of anhydrous ethanol used in step S1 is different. It is adjusted to: the ratio of the amount of anhydrous ethanol to the total mass of hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR and tetrahydrocurcumin is 3:1 mL / g.

[0053] Comparative Example 11 The difference between Comparative Example 11 and Example 1 is that the amount of water used in step S2 is different. It is adjusted so that the volume ratio of water to anhydrous ethanol in step S1 is 0.7:1.

[0054] Comparative Example 12 The difference between Comparative Example 12 and Example 1 is that the amount of water used in step S2 is different. It is adjusted to: the volume ratio of water to anhydrous ethanol in step S1 is 2.5:1.

[0055] Comparative Example 13 The only difference between Comparative Example 13 and Example 1 is step S4, which is adjusted as follows: S4: Dynamic High-Pressure Microfluidic Homogenization Turn on the jacket cooling system of the dynamic high-pressure micro-jet homogenizer and set the cooling water temperature to 25℃. Transfer the colostrum to the homogenizer feed tank, set the homogenization pressure to 125MPa, homogenize and circulate twice (without adding the same volume of 30℃ water), and then homogenize and circulate twice more.

[0056] Table 1. Distribution ratio of Examples 1-7 and Comparative Examples 1-8 (mass percentage)

[0057] Note: " / " indicates that the component was not added (missing); Examples 8-9 and Comparative Examples 9-13 have the same group ratio as Example 1.

[0058] Table 2 Comparison of preparation methods (process parameters) between Examples 1, 8, and 9 and Comparative Examples 9-13

[0059] Performance verification test

[0060] 1. Establishment of a method for detecting encapsulation efficiency

[0061] HPR was selected as the detection index for the encapsulation efficiency (denoted as encapsulation efficiency 1) of the effective components (tetrahydrocurcumin and HPR) in the oil phase, and a standard curve was established: y = 75.31x + 34.27, r = 0.9997, with a linear range of 0.03 mg / L to 200 mg / L. Chromatographic conditions: Wondasil C18 Superb (4.6 mm × 250 mm, 5 μm); column temperature: 30℃; injection volume: 10 μL; detection wavelength: 358 nm. Mobile phase: acetonitrile (A) to mobile phase B in a 3:7 ratio, flow rate 1 mL / min. Sample preparation: The sample was centrifuged at 5000 g using a high-speed centrifuge. 100 μL of the supernatant was collected, 400 μL of methanol was added, and the sample was centrifuged again. 50 μL of the supernatant was collected, diluted with 450 μL of methanol, and then injected.

[0062] S-type Bosein was selected as the detection index for the encapsulation efficiency (denoted as encapsulation efficiency 2) of the effective components in the aqueous phase (S-type Bosein, acetyl hexapeptide-8, sodium DNA, and filtrate from sea fennel callus culture). High-performance liquid chromatography with electro-atomization detector (HPLC-CAD) was used, and a standard curve was established: y = 508.31x - 427.22, r = 0.9995, with a linear range of 0.05 mg / L to 150 mg / L. An amide column (4.6 mm × 250 mm, 5 μm) was selected, with an injection volume of 10 μL; column temperature of 30℃, flow rate of 1.0 mL / min; and CAD detector parameters: nebulization temperature of 50℃, filtration constant of 3.6 s, and sampling frequency of 5 Hz. Mobile phase A was water (containing 0.1% acetic acid by volume), and mobile phase B was acetonitrile (containing 0.1% acetic acid by volume), with a volume ratio of mobile phase A to mobile phase B of 2:8. Sample preparation: Centrifuge the sample at 5000g using high speed, collect 200μL of the supernatant, add 300μL of ethanol, centrifuge again, collect 50μL of the supernatant, dilute with 450μL of 50% ethanol solution, and then inject the sample (the injection result is the determined content). Encapsulation efficiency = (1 - determined content / theoretical value) × 100%.

[0063] 2. Results of encapsulation efficiency and particle size determination

[0064] Table 3 Encapsulation efficiency and particle size of Examples 1-9 and Comparative Examples 1-13

[0065] Note: " / " indicates that the data is missing because it could not be measured.

[0066] According to the data in Table 3, 1) the anti-aging and repairing nanoliposomes prepared in Examples 1-9 all have good encapsulation efficiency and particle size. Among them, the encapsulation efficiency 1 is between 90.2% and 97.6%, the encapsulation efficiency 2 is between 83.3% and 89.3%, the particle size is between 94.1 and 105.5 nm, and the PDI is less than 0.18, indicating that the particle size is relatively uniform. 2) Polyglycerol-10-laurate is a nonionic emulsifier. Its hydrophilic groups (polyglycerol chains) can form hydrogen bonds with the aqueous phase, while its lipophilic groups (laurate chains) embed into the hydrophobic regions of lecithin, assisting in the orderly arrangement of hydrogenated lecithin and improving membrane fluidity, reducing oil-water interfacial tension, thereby improving the encapsulation efficiency and reducing particle size of liposomes. Compared to Example 1, Comparative Example 1, due to the lack of polyglycerol-10-laurate in the liposome membrane material, resulted in a significant decrease in encapsulation efficiency (72.3%, 65.8%) and an increase in particle size (156.4 nm, PDI: 0.32). 3) α-Tocopherol is a viscous liquid at room temperature. It can insert into the hydrogenated lecithin bilayer to regulate membrane fluidity (avoiding excessive rigidity of the membrane at low temperatures and looseness of the membrane at high temperatures). Compared to Example 1, Comparative Example 2, due to the lack of α-tocopherol in the membrane material, resulted in changes in encapsulation efficiency and particle size. 4) Comparative Examples 3-8 have the same membrane material composition as Example 1 (same preparation method), the only difference being the reduction of a certain effective component, so the effect on encapsulation efficiency and particle size is not significant. 5) Compared to Example 1, the amount of anhydrous ethanol used in step S1 of the preparation method in Comparative Example 9 is reduced, resulting in a relatively viscous oil phase, leading to poor membrane fluidity and difficulty in forming liposomes, thus causing a significant decrease in encapsulation efficiency and an increase in particle size. 6) The amount of anhydrous ethanol used in step S1 of the preparation method in Comparative Example 10 is increased, leading to a relative increase in the amount of aqueous phase used in step S2. Consequently, during the liposome preparation process, the oil phase has difficulty effectively encapsulating the aqueous phase, resulting in a significant decrease in encapsulation efficiency, but no significant effect on particle size. 7) Due to the significant reduction in water usage in step S2 of Comparative Example 11, the initial emulsion viscosity and particle size are too large, causing blockage of the homogenizer and making homogenization difficult (therefore, encapsulation efficiency and particle size data are lacking). 8) In Example 12, the significantly increased water usage in step S2 made it difficult for the oil phase to effectively encapsulate the aqueous phase, resulting in a decrease in encapsulation efficiency (mainly affecting encapsulation efficiency 2). 9) In Comparative Example 13, in step S4: after two homogenization cycles, no additional volume of 30°C water was added, and the homogenization cycle was directly repeated twice. Since the liposomes formed in the first two homogenization cycles had already effectively encapsulated the aqueous phase, the lack of aqueous phase outside the liposomes led to liposome rupture during the subsequent two homogenization cycles, significantly affecting encapsulation efficiency and particle size.

[0067] In summary, the amount of anhydrous ethanol used in step S1, the amount of water used in step S2, and whether water is added during the homogenization process in step S4 can all have a significant impact on particle size and encapsulation efficiency.

[0068] 3. Long-term stability test Long-term stability: Temperature: 25±2℃, RH 50%±5%, record encapsulation efficiency 1, encapsulation efficiency 2, particle size, and appearance at 0, 3, 6, 12, and 18 months.

[0069] Table 4 Long-term stability data

[0070] Continued table

[0071] According to Table 4, 1) the encapsulation efficiency and particle size of Examples 1-9 did not show significant changes within 18 months. 2) Comparative Example 1 showed turbidity and precipitation after 3 months, indicating that the lack of polyglycerol-10 laurate in the liposome membrane material of Comparative Example 1 could affect the stability of the liposomes. 3) The encapsulation efficiencies of Comparative Example 2 decreased by 6% and 7% respectively, and the particle size changed, indicating that α-tocopherol in the membrane material can regulate the fluidity of the liposome membrane and thus improve the stability of the liposomes. 4) Comparative Example 11 lacked relevant data because the liposomes were not successfully prepared; the encapsulation efficiencies of Comparative Examples 3-10 and 12-13 did not show significant changes, while the particle size of Comparative Example 13 showed a significant change. 4) In summary, based on the stability data of Examples 1 and Comparative Examples 1 and 2, it can be seen that hydrogenated lecithin, polyglycerol-10 laurate, and α-tocopherol, as membrane materials for liposomes, can effectively synergistically improve the encapsulation efficiency and stability.

[0072] 4. Cell anti-aging test Experimental Methods: DMEM culture medium containing 1% of the samples (Examples 1-9, Comparative Examples 1-10, 12, 13) was prepared. A positive control group was set up, with DMEM culture medium containing 1% of the positive control substance. The positive control substance was an aqueous solution (suspension) of S-type serotonin, acetyl hexapeptide-8, HPR, tetrahydrocurcumin, sodium DNA, and filtrate from sea fennel callus culture. The amounts of each effective component were the same as in Example 1. The model group directly used DMEM culture medium containing an equal volume of sterile deionized water. HaCaT cells in the logarithmic growth phase were harvested and cultured at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 cell / mL in 24-well plates, and 16 wells were selected for subsequent experiments. 2 mL of cell suspension was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. Both the model and experimental groups were exposed to ultraviolet light: UVA at an intensity of 2000 μW / cm² for 1 hour, and UVB at an intensity of 700 μW / cm² for 3 minutes.

[0073] After irradiation, discard all old culture medium from all wells: add 2 mL of sample solution to each well in the experimental group, and add 2 mL of serum-free DMEM culture medium to each well in the model group and normal control group, and continue culturing in the incubator for 16 h. After culturing, discard all culture medium, wash cells twice with PBS; add 1.5 mL of LDFH-DA solution to each well, and return to the cell culture incubator at 37°C for 30 min, gently mixing every 5 min to ensure sufficient binding of the probe to the cells.

[0074] After incubation, the DCFH-DA probe solution in the wells was discarded. Cells were washed twice with preheated serum-free medium, and 1 mL of serum-free medium was added to each well. Incubation was carried out at 37°C for 10 min. Cells were then washed once with PBS, digested with trypsin, washed twice with PBS, and finally resuspended in 300 μL PBS. Before flow cytometry analysis, the cell suspension was filtered through a 100-mesh filter. Dual-channel detection was performed using the FL1-H channel of the flow cytometer. 10,000 cells were collected for each sample for data analysis. The ROS clearance rate was calculated as follows: ROS clearance rate = (Model group detection value - Experimental group detection value) / Model group detection value × 100%.

[0075] Table 5. Cellular ROS clearance rate (%)

[0076] According to the data in Table 5: 1) Examples 1-9 all showed good anti-aging effects, with ROS scavenging rates ranging from 81.9% to 87.4%. 2) Compared with Example 1, the positive control group showed that liposomes could effectively encapsulate the active components (S-type Boswellia, acetyl hexapeptide-8, HPR, tetrahydrocurcumin, sodium DNA, and sea fennel callus culture filtrate) and transport them into cells, achieving effective antioxidant and anti-aging effects. 3) In Comparative Example 1, the lack of polyglycerol-10 laurate in the membrane material resulted in poor liposome membrane fluidity and low encapsulation rate, leading to a reduction in the effective components transported into cells and a significant decrease in ROS scavenging rate. 4) Compared with Example 1, Comparative Example 2 lacked α-tocopherol, which has antioxidant properties; Comparative Example 5 lacked HPR; and Comparative Example 6 lacked tetrahydrocurcumin, all showing a significant decrease in antioxidant performance. This indicates that tetrahydrocurcumin, HPR, and α-tocopherol can play important antioxidant and anti-aging roles in the composition. However, the antioxidant properties of Examples 3, 4, and 7 also decreased to varying degrees compared to Example 1. This indicates that the effective components in the examples can achieve synergistic antioxidant and anti-aging effects through combination, rather than relying solely on the antioxidant activity of a single effective component. 5) The preparation processes of Comparative Examples 9, 10, 12, and 13 differed from those of Example 1, resulting in differences in the encapsulation efficiency and particle size of the prepared liposomes, which in turn led to differences in the intracellular transport capacity of the liposomes. This may be the reason for the decrease in antioxidant and anti-aging efficacy.

[0077] 4. Human patch test Human patch tests were conducted in accordance with the "Cosmetic Safety Technical Specifications" (2015 edition) to verify the irritation of sensitive skin to individuals using Examples 1-9, Comparative Examples 1-10, and 12-13. The grading criteria for adverse skin reactions are shown in Table 6.

[0078] 210 volunteers were recruited (each participating in the testing of three samples), aged 20-53 years, with an average age of 29 years. Using a suitable patch tester, a closed patch test method was employed. An equal volume of 25 μL of the test substance was placed in the patch tester, which was then applied to the volunteer's arm with hypoallergenic adhesive tape. Gently pressing the tape ensured even application to the skin. The test substance was removed after 24 hours. Skin reactions were observed for 48 hours after patch removal (time points were set at 0.5, 24, and 48 hours). Adverse skin reactions were recorded and are shown in Table 7.

[0079] Table 6. Grading Standards for Adverse Skin Reactions

[0080] Table 7. Results of adverse skin reactions (number of people)

[0081] According to the test results in Table 7, Examples 1-9, Comparative Examples 1-10, and 12-13 all showed negative reactions, with no adverse skin reactions. This indicates that the anti-aging and repairing nanoliposomes prepared by the microfluidic technology in Examples 1-9 of this invention have the characteristics of being non-irritating and non-sensitizing.

[0082] 5. Skin elasticity test and wrinkle change test

[0083] 210 women aged 30-45 years, with an average age of 36.4 years, were recruited and randomly divided into 21 groups. Each morning and before bed, 2 mL of the sample was applied evenly to the face, and the skin between the right eye and cheekbone was gently massaged for 1 minute. This area, which is covered with fine wrinkles, was designated as the test area.

[0084] The skin elasticity of volunteers was measured using a Corneometer MPA580 skin elasticity tester before and after 28 days of continuous product use. The skin elasticity change rate (%) was calculated and recorded using the following formula: Skin elasticity change rate (%) = [(28-day skin elasticity value - 0-day skin elasticity value) / 0-day skin elasticity value] × 100%.

[0085] The PRIMOS rapid 3D skin imaging system was used to detect skin wrinkles in volunteers before and after 28 days of continuous product use. After the detection, the skin wrinkle change rate (%) was calculated and recorded using the following formula: Skin wrinkle change rate (%) = [(0-day skin wrinkle value - 28-day skin wrinkle value) / 0-day skin wrinkle value] × 100%.

[0086] Table 8. Record of Skin Elasticity Change Rate (%) and Wrinkle Change Rate (%)

[0087] According to the data in Table 8: 1) In Examples 1-9, the skin elasticity change rate and skin wrinkle change rate were between 38.5%-43.4% and 18.3%-19.8%, respectively, showing excellent effects in improving skin elasticity, anti-wrinkle, and anti-aging compared to the comparative examples. 2) In Comparative Example 1, the lack of polyglycerol-10 laurate in the membrane material resulted in poor liposome membrane fluidity and low encapsulation rate, which in turn led to a reduction in the effective components transported into the cells by the liposomes, resulting in a significant decrease in the skin elasticity change rate and skin wrinkle change rate. 3) Compared with Example 1, Comparative Example 3 lacked S-type BOXER, Comparative Example 4 lacked acetyl hexapeptide-8, Comparative Example 5 lacked HPR, Comparative Example 7 lacked sodium DNA, and Comparative Example 8 lacked sea fennel callus culture filtrate. All of these examples showed a significant decrease in the rate of change in skin elasticity and the rate of change in skin wrinkles. This indicates that S-type BOXER, acetyl hexapeptide-8, HPR, sodium DNA, and sea fennel callus culture filtrate play the main anti-wrinkle and anti-aging effects in this invention, and that the components can mutually promote and enhance these effects. 4) The preparation processes of Comparative Examples 9, 10, 12, and 13 differed from those of Example 1, resulting in differences in the encapsulation efficiency and particle size of the prepared liposomes. This, in turn, led to differences in the intracellular transport capacity of the liposomes, which may be the reason for the decreased anti-wrinkle and anti-aging effects.

[0088] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An anti-aging and repairing nanoliposome based on microfluidic technology, characterized in that, It contains the following components by weight percentage: hydrogenated lecithin 2.0-5.0%, polyglycerol-10 laurate 2.0-3.5%, α-tocopherol 0.2-0.6%, S-type boswellicin 1.5-5%, acetyl hexapeptide-8 0.05-0.2%, hydroxypinazone retinate HPR 0.1-0.3%, tetrahydrocurcumin 0.1-0.3%, sodium DNA 0.05-0.3%, sea fennel callus culture filtrate 0.05-0.4%, and the balance being water.

2. The anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 1, characterized in that, It contains the following components by weight percentage: hydrogenated lecithin 3.0-4.5%, polyglycerol-10 laurate 2.5-3.0%, α-tocopherol 0.4-0.5%, S-type boswellicin 2-4%, acetyl hexapeptide-8 0.1-0.15%, hydroxypinazone retinate HPR 0.15-0.25%, tetrahydrocurcumin 0.2-0.3%, sodium DNA 0.1-0.3%, sea fennel callus culture filtrate 0.2-0.4%, and the balance being water.

3. The anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 1, characterized in that, The water is deionized water.

4. The method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 1, characterized in that, It includes the following steps: S1: oil phase preparation; S2: aqueous phase preparation; S3: primary emulsion preparation; S4: dynamic high-pressure microfluidic homogenization; S5: post-treatment and stabilization.

5. The method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 4, characterized in that, The specific steps of step S1 are as follows: Weigh hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, hydroxypinazone retinate (HPR), and tetrahydrocurcumin according to the specified amounts, and add them sequentially to anhydrous ethanol. Heat at 45-55°C and stir at 150-200 rpm to dissolve. The ratio of the volume of anhydrous ethanol to the total mass of hydrogenated lecithin, polyglycerol-10 laurate, α-tocopherol, HPR, and tetrahydrocurcumin is (1.5-2.5):1 mL / g.

6. The method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 4, characterized in that, The specific steps of step S2 are as follows: Weigh out the S-type bosine, acetyl hexapeptide-8, sodium DNA, and sea fennel callus culture filtrate according to the specified amounts, and add them to water in sequence. Heat at 45-55℃ and stir at 150-200 rpm to dissolve. The volume ratio of water to anhydrous ethanol used in step S1 is (1-2):

1.

7. The method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 4, characterized in that, The specific steps of step S3 are as follows: Keep the temperature of both the oil phase and the aqueous phase at 45-55℃, and slowly drip the oil phase into the aqueous phase at a rate of 5-10 mL / min through a constant pressure dropping funnel; during the dripping process, simultaneously turn on the high shear disperser and set the rotation speed to 7000-9000 rpm for shear emulsification; after the oil phase is completely dripped, continue high shear dispersion for 5-30 min to form a milky white primary emulsion.

8. The method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 4, characterized in that, The specific steps of step S4 are as follows: turn on the jacket cooling system of the dynamic high-pressure micro-jet homogenizer and set the cooling water temperature to 25-30℃; transfer the initial emulsion to the homogenizer feed tank, set the homogenization pressure to 120-130MPa, homogenize and circulate twice, add the same volume of water at 25-30℃, mix well, and then homogenize and circulate twice more.

9. The method for preparing anti-aging and repairing nanoliposomes based on microfluidic technology according to claim 4, characterized in that, The specific steps of step S5 are as follows: the homogenized nanoliposomes are transferred to a vacuum degasser and degassed and ethanol is removed at 30-40℃ and vacuum degree <0.1MPa for 0.5-2h. The remaining water is added and mixed well. The mixture is then filtered through a 0.22μm microporous membrane and filled into containers.

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