PDRN-silk fibroin compounded plant external vesicle liposome as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetic liposomes, and relates to a PDRN-silk fibroin compounded plant external vesicle liposome as well as a preparation method and application thereof. The preparation method of the liposome comprises the following steps: preparing plant external vesicles; mixing the active component with alcohol, adding a water-soluble silk fibroin solution, and stirring and shearing to obtain an active component-silk fibroin compound; dissolving hydrogenated phospholipid and cholesterol in an organic solvent, and carrying out rotary evaporation to remove the organic solvent to obtain a lipid membrane; adding a PDRN solution and water into the lipid membrane, stirring and mixing, adding plant external vesicles and an active ingredient-silk fibroin compound, stirring, shearing and emulsifying to obtain a primary emulsion; and carrying out high-pressure homogenization treatment on the primary emulsion to obtain a liposome suspension, and filtering to obtain the liposome. The prepared liposome is small in particle size, low in PDI, high in zeta potential absolute value, excellent in stability, good in heat resistance, excellent in transdermal performance, high in active ingredient delivery efficiency and excellent in skin repairing and anti-aging effects.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic liposome technology, and relates to a PDRN-silk fibroin complex plant exovesicle liposome, its preparation method, and its application. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) possess significant cell repair, anti-inflammatory, and tissue regeneration-promoting effects; however, they are extremely unstable in vitro, easily degraded by nucleases, and due to their large molecular weight and strong hydrophilicity, they are difficult to penetrate the stratum corneum of the skin. While traditional liposomes can encapsulate PDRNs, they suffer from poor stability, easy leakage, and low transdermal efficiency.

[0003] Plant exovesicles (PEVs) are novel nanocarriers discovered in recent years, possessing natural biocompatibility and low immunogenicity. However, their loose structure results in low loading rates for hydrophobic active substances, and their protective ability against PDRN is limited when used alone. Silk fibroin (SF) exhibits good film-forming and bioadhesive properties, but direct mixing can easily lead to phase separation.

[0004] In the existing technology, there is a lack of a stable delivery system that can organically combine the high-efficiency repair ability of PDRN, the stable scaffolding effect of silk fibroin, and the natural targeting of plant exovesicles, and can simultaneously and efficiently encapsulate active ingredients. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a PDRN-silk fibroin composite plant extravesicular liposome, its preparation method, and its applications.

[0006] In one aspect, the present invention provides a method for preparing plant exovesicle liposomes composed of PDRN-silk fibroin, comprising the following steps: (1) Preparation of plant exovesicles; (2) The active ingredient is mixed with alcohol to obtain a mixture, a solution of water-soluble silk fibroin is added, and the mixture is stirred and sheared to obtain an active ingredient-silk fibroin complex; wherein, the alcohol includes at least one of ethylene glycol, propylene glycol, glycerol, and butylene glycol; (3) Dissolve hydrogenated phospholipids and cholesterol in an organic solvent, remove the organic solvent by rotary evaporation, and obtain a lipid membrane; (4) Add PDRN solution (a solution containing PDRN) and water to the lipid membrane, stir and mix, then add plant exovesicles and active ingredient-silk fibroin complex, stir, and then shear emulsify to obtain a primary emulsion; (5) The primary emulsion was subjected to high-pressure homogenization to obtain a liposome suspension, which was then filtered to obtain PDRN-silk fibroin complex plant vesicle liposomes.

[0007] Preferably, the active ingredient includes a water-soluble or hydrophobic active ingredient, and more preferably a hydrophobic active ingredient. The active ingredient includes, but is not limited to, at least one of the following: curcumin, retinol, tocopherol and its derivatives, resveratrol, ascorbate tetraisopalmitate, hesperidin, quercetin, bisabolol, lutein, medium / high molecular weight polysaccharides, polypeptides, recombinant proteins, natural plant oils, panthenol, and phenylethyl resorcinol. The active ingredient may further optionally include curcumin and retinol.

[0008] Preferably, the method for preparing plant exovesicles includes: taking plant tissue, washing and chopping it, adding buffer solution for shearing homogenization and high-pressure homogenization, then centrifuging to remove the precipitate and ultrafiltration to concentrate the extract to obtain plant exovesicles; wherein the plant includes at least one of microalgae, hydrilla, and brown algae.

[0009] Preferably, the hydrogenated phospholipids include at least one of hydrogenated lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated egg yolk phospholipid, plant-based hydrogenated lecithin, hydrogenated phosphatidylcholine, and hydrogenated phosphatidylethanolamine.

[0010] Preferably, the organic solvent includes at least one of ethanol, chloroform, and ethyl acetate.

[0011] Preferably, the active ingredients also include spirulina extract, carnosine, peptide derivatives, and Euglena macrantha extract.

[0012] Preferably, the weight ratio of Spirulina extract to Euglena extract is 1:0-1.5.

[0013] Preferably, the buffer solution pH is 6-8 during the preparation of plant exovesicles. The weight ratio of plant tissue to buffer solution is 1:5-20. Ultrafiltration concentration is a process of obtaining a retentate by ultrafiltration using an ultrafiltration membrane. This process is a concentration process, wherein the ultrafiltration membrane used has a molecular weight cutoff of 80 kDa to 350 kDa.

[0014] Preferably, in step (2), the weight ratio of the active ingredient to the alcohol is 1:10-100. The concentration of water-soluble silk fibroin is 1-3 wt%, and the weight ratio of water-soluble silk fibroin to the active ingredient is 1:0.5-2.

[0015] Preferably, in step (3), the weight ratio of hydrogenated lecithin to cholesterol is (2-6):(0.5-1.5). The weight ratio of the total mass of hydrogenated lecithin and cholesterol to the organic solvent is 1:5-15.

[0016] Preferably, in step (4), the weight ratio of lipid membrane, active ingredient-silk fibroin complex, PDRN solution, plant exovesicles and water is 1:(2-3):(2-4):(0.5-1.5):(4-7).

[0017] Preferably, the spirulina extract can be obtained by first extracting with high-concentration ethanol (70-85 wt%), filtering, distilling off the ethanol from the filtrate, adding ethyl acetate for extraction, concentrating the extract to remove the ethyl acetate, and drying.

[0018] Preferably, the Euglena extract can be extracted with low-concentration ethanol (20-35 wt%), the ethanol is removed by distillation, filtered, and the filtrate is retained by an ultrafiltration membrane with a molecular weight cutoff of not less than 2000 Da. The retained product is then dried.

[0019] On the other hand, the present invention also provides the application of the above-mentioned Spirulina extract and Euglena granulosus extract in synergistically enhancing the inhibition of tyrosinase and whitening. Further preferably, the weight ratio of Spirulina extract to Euglena granulosus extract is 1:0.8-1.5.

[0020] On the other hand, the present invention also provides a PDRN-silk fibroin complex plant extravesicular liposome prepared according to the preparation method described above.

[0021] On the other hand, the present invention also provides the application of the PDRN-silk fibroin complex plant exovesicle liposomes in the preparation of cosmetics.

[0022] On the other hand, the present invention also provides other methods for preparing PDRN-silk fibroin complex plant exovesicle liposomes, for example, the following steps: Preparation of the alcohol phase: In a reaction vessel, a polyol (such as butanediol) is used as a solvent to completely dissolve the hydrogenated phospholipids, forming a homogeneous and transparent alcohol phase; Preparation of the aqueous phase: In another reaction vessel, PDRN, silk fibroin, active ingredients, plant exovesicles and pure water are mixed to prepare the aqueous phase; Mixing and film formation: While continuously stirring (e.g., magnetic stirring) the alcohol phase, slowly add the aqueous phase to the alcohol phase using a syringe or dropper. This process will spontaneously form liposomes. Post-processing: After mixing, shear homogenization and high-pressure homogenization are performed sequentially to make the nanoparticles smaller and more uniform in size.

[0023] The advantages of this invention compared to the prior art are as follows: This invention utilizes alcohols to prepare a pre-complex system of silk fibroin and active ingredients. This allows the alcohol solvent to not only dissolve the active ingredients (e.g., hydrophobic active ingredients) but also to moderately open the secondary structure of silk fibroin, exposing more hydrophobic binding sites. Under shear stress, the hydrophobic active ingredients are anchored to the hydrophobic microregions of silk fibroin, forming a stable active ingredient-silk fibroin complex. This complex not only improves the dispersibility of the hydrophobic components but also utilizes the network structure of silk fibroin to prevent the aggregation and precipitation of the active ingredients, facilitating subsequent entry into the liposome bilayer membrane to prepare stable liposomes.

[0024] This invention introduces plant-derived extravesicles during the lipid membrane hydration stage. These vesicles are rich in plant-derived phospholipids and sterols, and their membrane structure is highly similar to that of artificial lipid membranes (hydrogenated phospholipids, cholesterol, etc.). During shear emulsification, the plant-derived extravesicles do not simply encapsulate the artificial lipid membrane but undergo partial fusion and intercalation. The natural phytosterols in the plant-derived extravesicles act as membrane fluidity regulators, compensating for the excessive rigidity of hydrogenated phospholipids. Simultaneously, a natural interfacial layer is formed on the liposome surface, significantly improving the liposomes' affinity and permeability to the skin.

[0025] This invention employs a combined use of PDRN, silk fibroin, and plant extracellular vesicles within liposomes. PDRN is primarily located within the water core of the liposome and the silk fibroin network; the hydrophobic active ingredient is situated in the lipid bilayer and the hydrophobic region of the silk fibroin; and the plant extracellular vesicles are embedded on the membrane surface and within the membrane. The gel network formed by the silk fibroin physically blocks nucleases from contacting PDRN; the antioxidant environment provided by the plant extracellular vesicles reduces oxidative damage to PDRN; and the liposome bilayer membrane provides the primary barrier. The synergistic effect of these three components extends the half-life of PDRN and enables the co-delivery of water-soluble (PDRN) and lipid-soluble (e.g., hydrophobic active ingredients) active substances, solving the problem of the unstable coexistence of water- and oil-soluble active substances in traditional liposomes.

[0026] The Spirulina extract and Euglena granulosus extract obtained by the specific process selected in this invention both have excellent tyrosinase inhibition activity and can effectively whiten the skin; the combination of Spirulina extract and Euglena granulosus extract has a synergistic effect in enhancing the inhibition of tyrosinase and the whitening effect; and the tyrosinase inhibition activity is stronger when the weight ratio of Spirulina extract and Euglena granulosus extract is in the range of 1:0.8-1.5.

[0027] The liposomes prepared by this invention have small particle size, low PDI, high absolute value of zeta potential, excellent stability, good heat resistance, excellent transdermal performance, and high efficiency in delivering active ingredients.

[0028] The liposomes prepared by this invention can effectively promote cell repair, have a highly efficient HaCaT cell barrier repair function, and have excellent skin repair effects.

[0029] The liposomes prepared by this invention can effectively promote the secretion of type I collagen by cells, have a highly effective skin-firming effect, and can effectively maintain skin elasticity and prevent skin aging. Attached Figure Description

[0030] Figure 1 : Zeta potential of liposomes.

[0031] Figure 2 The growth rate of liposome particle size after 30 days.

[0032] Figure 3 : Cumulative penetration of active ingredient (retinol) over 24 hours.

[0033] Figure 4 Tyrosinase inhibition rate. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] PDRN solution: Guaisource PDRN, Hemei Biotechnology. Microalgae: Chlorella sorokinense. Elodea: Elodea roximatelyum. Water-soluble silk fibroin: Product No. W718600 (6900-9200 Da), Maclean's.

[0036] Example 1 Liposome 1: The preparation method is as follows: Step (1) Preparation of plant exovesicles: Take 50g of microalgae, wash and chop them with water, add 7.5 times the weight of PBS buffer (pH 7.4), shear homogenize at 30℃ and 8000rpm for 10 minutes, then homogenize three times at 35℃ and 800bar (2 minutes each time), then centrifuge at 12000g for 30 minutes, remove the precipitate, and concentrate the supernatant through a 120kDa ultrafiltration membrane to obtain the retentate (solid content 21.7wt%), thus obtaining plant exovesicles; Step (2): Mix 4.5g of the active ingredient retinol with alcohol (ethylene glycol and 1,3-butanediol in a weight ratio of 3:1) at a weight ratio of 1:15 at room temperature and 150 rpm for 15 minutes to obtain a mixture. Add a 1.2wt% solution of water-soluble silk fibroin (water-soluble silk fibroin to active ingredient in a weight ratio of 1:1.2) and stir and shear at 30℃ and 1200 rpm for 30 minutes to obtain the active ingredient-silk fibroin complex. Step (3): Hydrogenated phospholipids and cholesterol (total weight of hydrogenated phospholipids and cholesterol is 12g) and ethyl acetate (84g) in a weight ratio of 3:1 are stirred and mixed at 150rpm for 10 minutes at room temperature to obtain a mixture. The organic solvent ethyl acetate is removed by rotary evaporation to obtain a lipid membrane. Step (4): Add 30g PDRN solution and 55g water to 10g lipid membrane, stir and mix at 800rpm for 20 minutes, then add 7.5g plant exovesicles and 25g active ingredient-silk fibroin complex, stir at 800rpm for 20 minutes, then shear emulsify at 10000rpm for 30 minutes to obtain primary emulsion. Step (5): The primary emulsion is subjected to high-pressure homogenization (800 bar, 5 cycles, 1.5 minutes each time) to obtain a liposome suspension. The suspension is then filtered through a 0.22-micron filter membrane to obtain PDRN-silk fibroin complex plant vesicle liposomes.

[0037] Liposome 2: The preparation method is as follows: Step (1) Preparation of plant exovesicles: Take 50g of *Hydrilla verticillata*, wash and chop it with water, add 8 times the weight of PBS buffer (pH 7.4), homogenize at 12000rpm for 15 minutes at room temperature, then homogenize three times at 800bar (2 minutes each time) at room temperature, then centrifuge at 12000g for 28 minutes, remove the precipitate, and ultrafilter the supernatant through a 120kDa ultrafiltration membrane to obtain the retentate, thus obtaining plant exovesicles; Step (2): Mix 4.5g of active ingredient (curcumin: resveratrol: hesperidin weight ratio 1:1:1) with alcohol (ethylene glycol and glycerol weight ratio 5:1) at a weight ratio of 1:17.5, at room temperature and 150rpm for 20 minutes to obtain a mixture. Add a 1.3wt% solution of water-soluble silk fibroin (water-soluble silk fibroin to active ingredient weight ratio 1:1.15), and stir and shear at 30℃ and 1100rpm for 35 minutes to obtain the active ingredient-silk fibroin complex. Step (3): Hydrogenated phospholipids and cholesterol (total weight of hydrogenated phospholipids and cholesterol is 13g) in a weight ratio of 4:1 are mixed with chloroform (95g) at room temperature and stirred at 180rpm for 12 minutes to obtain a mixture. The organic solvent chloroform is removed by rotary evaporation to obtain a lipid membrane. Step (4): Add 35g PDRN solution and 60g water to 11g lipid membrane, stir and mix at 700rpm for 30 minutes, then add 8.7g plant exovesicles and 28g active ingredient-silk fibroin complex, stir at 850rpm for 20 minutes, then shear emulsify at 12000rpm for 25 minutes to obtain primary emulsion. Step (5): The primary emulsion is subjected to high-pressure homogenization (800 bar, 4 cycles, 2 minutes each time) to obtain a liposome suspension. The suspension is then filtered through a 0.22-micron filter membrane to obtain PDRN-silk fibroin complex plant vesicle liposomes.

[0038] Liposome 3: Compared to liposome 1, in step (2), the alcohol is replaced with an equal amount of solvent PEG-400. Specifically, step (2) is as follows: 4.5g of the active ingredient retinol and polyethylene glycol-400 were mixed at a weight ratio of 1:15 at room temperature and 150rpm for 15 minutes to obtain a mixture. A solution of 1.2wt% water-soluble silk fibroin (water-soluble silk fibroin to active ingredient weight ratio of 1:1.2) was added, and the mixture was stirred and sheared at 30℃ and 1200rpm for 30 minutes to obtain the active ingredient-silk fibroin complex.

[0039] Liposome 4: Compared to liposome 1, silk fibroin is not used in step (2), and the amount is made up with water. The specific step (2) is as follows: 4.5g of the active ingredient retinol was mixed with alcohol (ethylene glycol and 1,3-butanediol in a weight ratio of 3:1) at a weight ratio of 1:15 at room temperature and 150 rpm for 15 minutes to obtain a mixture. Water was added (the amount of water was approximately 69.44 times the amount of the active ingredient), and the mixture was stirred and sheared at 30°C and 1200 rpm for 30 minutes to obtain the active ingredient complex.

[0040] Liposome 5: Compared to liposome 1, step (4) does not use plant exovesicles, and the dosage is made up with water. The specific step (4) is as follows: Add 30g of PDRN solution and 55g of water to 10g of lipid membrane, stir and mix at 800rpm for 20 minutes, then add 7.5g of water and 25g of active ingredient - silk fibroin complex, stir at 800rpm for 20 minutes, and then shear emulsify at 10000rpm for 30 minutes to obtain primary emulsion.

[0041] Liposome 6: Compared to liposome 1, the order of raw material addition in step (4) is different. Specifically, step (4) is as follows: Add 30g PDRN solution, 55g water, 7.5g plant exovesicles and 25g active ingredient-silk fibroin complex to 10g lipid membrane, stir at 800rpm for 40 minutes, and then shear emulsify at 10000rpm for 30 minutes to obtain primary emulsion.

[0042] Liposome 7: Compared to liposome 1, the amount of external vesicles used is too low, so the amount is made up with water. Step (4) is as follows: Add 30g PDRN solution and 55g water to 10g lipid membrane, stir and mix at 800rpm for 20 minutes, then add 2.5g plant exovesicles, 5g water and 25g active ingredient-silk fibroin complex, stir at 800rpm for 20 minutes, then shear emulsify at 10000rpm for 30 minutes to obtain primary emulsion.

[0043] Example 2 The liposomes obtained above were then tested.

[0044] Test 1: Particle size, zeta potential, and PDI test Instrument: Malvern ZS90 nanoparticle size and zeta potential analyzer.

[0045] Test procedure: Take an appropriate amount of the prepared liposomes and dilute them 100 times with ultrapure water. Test the diluted sample at 25℃ using an instrument to obtain the average particle size (Mean Size, nm), polydispersity index (PDI), and zeta potential (mV). Perform three parallel tests for each group and take the average value.

[0046] After storing the prepared liposomes at 45°C for 30 days, the average particle size (Mean Size, nm), polydispersity index (PDI), and zeta potential (mV) were tested according to the above method. Each group was tested in parallel for 3 days and the average value was taken.

[0047] The test results are shown in Table 1 and Figure 1-2 .

[0048] Table 1: Liposome particle size, PDI and zeta potential Group Initial particle size (nm) Initial PDI Initial Zeta (mV) Particle size (nm) after 30 days PDI after 30 days Particle size growth rate after 30 days Liposome 1 118.2 0.135 -32.4 123.5 0.158 4.48% Liposome 2 124.5 0.150 -31.1 131.4 0.176 5.54% Liposome 3 148.1 0.247 -24.5 175.8 0.310 18.70% Liposome 4 172.8 0.294 -17.8 245.3 0.452 41.96% Liposome 5 132.4 0.183 -29.3 155.9 0.233 17.75% Liposome 6 156.3 0.268 -23.2 198.7 0.348 27.13% Liposome 7 141.0 0.216 -25.9 168.2 0.281 19.29% Test 2: Transdermal Transmission Test The transdermal performance of liposomes was tested according to the Franz diffusion cell transdermal absorption test described in GB / T27818-2011 "In vitro test methods for skin absorption of chemicals". The procedure was as follows: A piece of pigskin (25cm × 20cm, approximately 0.8mm thick) was fixed between the supply and receiving chambers of the Franz diffusion cell. A magnetic stirrer at 500rpm and a constant temperature water bath at 37℃ were activated. Liposome samples were applied to the surface of the pigskin in the chamber for transdermal testing. The receiving solution was 20% PEG-400 PBS solution (pH 7.2) with 0.01mol / L PBS buffer added. The receiving solution was collected at 12 and 24 hours, filtered, and the retinol content was determined by HPLC. The transdermal absorption rate (μg / cm³) was calculated. 2 Each group was repeated three times, and the average value was taken. The results are shown in Table 2 and 3. Figure 3 .

[0049] Table 2: Transdermal absorption at different times <![CDATA[Group / (μg / cm 2 )]]> 12-hour cumulative permeation 24-hour cumulative infiltration Transdermal efficiency ratio relative to liposome 1 over 24 hours Liposome 1 26.87 48.62 / Liposome 3 17.29 31.47 64.73% Liposome 4 11.84 22.34 45.95% Liposome 5 20.12 36.71 75.50% Liposome 6 14.56 26.58 54.67% Liposome 7 18.19 33.23 68.35% According to the test results in Table 1-2 above, the present invention uses a specific alcohol to prepare the active ingredient-silk fibroin complex and adds plant vesicles. The feeding sequence is optimized by first hydrating the lipid membrane and then adding the vesicles and complex, thereby preparing liposomes with excellent performance. The resulting liposomes have small particle size, the most uniform distribution (lowest PDI), stable potential, low particle size change after high temperature storage, and high transdermal absorption of active ingredients.

[0050] Compared to liposome 1, liposome 3 does not use the ethylene glycol, propylene glycol, glycerol, and butylene glycol of the present invention as a mixed carrier for the active ingredient-silk fibroin complex. The initial particle size and PDI of liposome 3 are larger than those of liposome 1, the absolute value of the Zeta potential is reduced, the particle size growth rate after thermal storage is increased, and the transdermal permeability of the active ingredient is reduced. This is because the alcohols selected in the present invention can effectively "moderately open the secondary structure of silk fibroin", so that the active ingredient is effectively encapsulated and the stability of the liposome is increased.

[0051] In step (2), liposome 4 does not use silk fibroin. The resulting liposome 4 has an increased initial particle size and PDI, a low absolute value of Zeta potential, low thermal stability, and poor transdermal effect. This indicates that silk fibroin plays an excellent role in the system of the present invention. Liposome 4 does not have the "network structure" formed by silk fibroin to anchor and disperse hydrophobic active ingredients. These ingredients are very easy to aggregate and precipitate in liposomes, resulting in system instability, low encapsulation rate, and thus seriously affecting transdermal efficiency.

[0052] In step (4) of liposome 5, plant exovesicles are not used. Their initial particle size is large, stability is reduced, and transdermal permeability is also decreased. The main reason is the absence of plant exovesicles, which causes the liposomes to lose their role as a "natural interface layer" and "membrane fluidity regulator," resulting in an overly rigid liposome membrane structure (due to hydrogenated phospholipids), decreased affinity for the skin, and reduced transdermal efficiency. This verifies the crucial role of plant exovesicles in improving transdermal performance.

[0053] In liposome 6, all components, including PDRN, water, plant vesicles, and the complex, were added to the lipid membrane at once. The resulting liposomes had a larger initial particle size, reduced stability, and decreased transdermal permeability compared to liposome 1. The main reason for this is that liposome 1 uses a feeding sequence of "first hydration to form empty liposomes, then adding plant vesicles and the complex," which allows plant vesicles to better integrate and intercalate with the lipid membrane and enables efficient encapsulation of the active complex. However, the change in the feeding sequence results in plant vesicles being embedded inside or the active complex being unevenly distributed, thus disrupting the ordered structure of the liposomes.

[0054] Compared to liposome 1, liposome 7 reduces the amount of plant exovesicles used. Tests showed that the particle size, PDI, zeta, stability, and transdermal performance of liposome 7 were all lower than those of liposome 1. This indicates that plant exovesicles need to be selected within the scope defined in this invention to exert their effective efficacy, thereby improving the stability of liposomes and the delivery of active ingredients.

[0055] Test 3: Skin Repair Test The HaCaT cell barrier repair assay was used to verify the skin repair effect. The specific tests are as follows: Experimental principle: CCK8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt. Under the action of the carrier 1-MethoxyPMS, it can be reduced by dehydrogenases in the mitochondria of cells to a highly water-soluble yellow formazan product. The amount of formazan produced is directly proportional to the number of viable cells. Its absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) scanner can indirectly reflect the number of viable cells.

[0056] SLS Damage Model: Sodium lauryl sulfate (SLS) is a surfactant widely used in cleaning products, cosmetics, and personal care products. SLS is an anionic surfactant that can cause aberrations in cell membrane proteins. The skin damage repair efficacy of the tested cosmetic or cosmetic ingredient is evaluated by adding it to the model.

[0057] Experimental content a. Reagents Positive control group reagent: The positive control is ectoine.

[0058] Experimental test samples: liposomes 1-2.

[0059] b. Cell culture Cell thawing: Remove the cryopreservation tubes and quickly place them in a 37°C water bath, shaking until thawed. Transfer the thawed cells to centrifuge tubes (containing DMEM medium) under a clean bench. Place the centrifuge tubes in a centrifuge, balance the volume, and set the program to 800 rpm for 3 minutes. After centrifugation, discard the supernatant, add an appropriate amount of DMEM medium (containing 10% serum), gently pipette, and add to 10 mL of DMEM medium containing 10% serum in a T75 culture flask. Mix well and incubate statically in a cell culture incubator.

[0060] Cell passage: Two days after cell resuscitation, the T75 culture flask was removed and observed under a microscope. When the cell density was about 85%, the supernatant was discarded in a clean bench, the cells were washed once with PBS, and 3 mL of trypsin was added. After incubation for 5 minutes, 3 mL of LDM medium was added to stop the digestion. The cells were pipetted from the bottom of the culture dish and collected into a centrifuge tube. After centrifugation (using the same procedure as before), the supernatant was discarded, and 2 mL of complete culture medium was added to suspend the cells. After mixing with a pipette tip, 400 μL was added to a T75 culture flask containing 10 mL of LDM medium. After mixing, the flask was placed in a cell culture incubator for static culture.

[0061] Cell cryopreservation: Remove the T75 culture flask, discard the culture medium, wash once with PBS, add 3 mL of trypsin, incubate for 5 min in a static incubator, then add 3 mL of LDMMEM culture medium to stop the digestion. Pipette the cells from the bottom of the culture dish and collect them into centrifuge tubes. Centrifuge using the same procedure as before, discard the supernatant, add 5 mL of cell cryopreservation buffer (serum:DMSO:culture medium = 2:1:7) to resuspend the cells, and then add 1 mL of cell cryopreservation buffer to each cryopreservation tube. Place the cryopreservation tubes in a programmed cooling box and store at -80°C.

[0062] c. Test substance testing Cell plating: Remove the culture flask, discard the used culture medium, add 3 mL of trypsin, incubate at 37°C for 2 min, add 3 mL of culture medium to stop digestion, gently blow off the cells from the bottom of the culture dish, collect the culture medium into a centrifuge tube, place the centrifuge tube in a centrifuge and balance it, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 5 mL of LDM medium, resuspend the cells, take out 180 μL, add 20 μL of trypan blue, count the cells using a counterscan cell counter, and finally add the culture medium containing cells to a 96-well plate. The final cell density is 4.5 × 10⁶ cells / well. 4 Cells / well. After plating, incubate in a cell culture incubator for 24 hours.

[0063] Induction and sample loading: When cells reached approximately 80% confluence, the culture medium was discarded. 180 μL of DMEM high-glucose medium was added to the wells of the control group; DMEM high-glucose medium containing 100 μg / mL SLS was added to the wells of the model group; 0.25 wt% liposomes + 100 μg / mL SLS + DMEM high-glucose medium was added to the liposome group; and DMEM high-glucose medium containing 100 ng / mL ectoine + 100 μg / mL SLS was added to the positive control group. Each group was repeated in triplicate. Incubation was performed for 24 hours.

[0064] Sample collection and detection: After incubation for 24 hours, the culture medium was discarded, and 100 μL of culture medium and 10 μL of CCK8 detection solution were added to each well. The wells were then placed in an incubator and incubated for 4 hours. After incubation, cell viability was detected using a microplate reader at a wavelength of 450 nm, and the relative cell proliferation rate of each group compared to the control group was calculated. The results are shown in Table 3.

[0065] Table 3: Relative Cell Proliferation Rate

[0066] According to the test results in Table 3, the liposomes prepared by this invention can effectively promote cell repair, have a highly efficient HaCaT cell barrier repair function, and have excellent skin repair effects.

[0067] Test 4: Skin Firming Test The anti-aging and skin-firming effects were verified by testing the ability of HSF cells to secrete collagen. The specific tests are as follows: Experimental principle: Type I collagen is a major component of the extracellular matrix of dermal cells. It is synthesized intracellularly by dermal fibroblasts, secreted extracellularly, and then polymerized to form collagen fibers under the action of terminal procollagen peptidase, after the telopeptides separate. Human dermal fibroblasts can be used as a cell model to study the enhancement of type I collagen content in cosmetics. By measuring the upregulation rate of type I collagen content after administration of the test substance, compared with the blank control and the test substance, the efficacy of the test substance in promoting collagen synthesis can be evaluated. The determination of type I collagen content uses an enzyme-linked immunosorbent assay (ELISA). The principle is as follows: type I collagen specifically binds to collagen antibodies coated on an ELISA plate, which then bind to substrate-labeled anti-type I collagen antibodies. The substrate is catalyzed by the enzyme to generate a colored product. The type I collagen content is positively correlated with the intensity of the colored product. The optical density (OD value) is measured at a wavelength of 450 nm using an ELISA reader to calculate the type I collagen content.

[0068] Experimental content a. Reagents Cell culture medium: 90% DMEM (low glucose) + 10% FBS (Gibco fetal bovine serum) + 1% penicillin-streptomycin bispecific antibody.

[0069] Positive control group reagent: The positive control is TGF-β1.

[0070] Experimental test samples: liposomes 1-2.

[0071] b. Cell culture Cell thawing: Remove the cryopreservation tubes and quickly place them in a 37°C water bath, shaking until thawed. Transfer the thawed cells to centrifuge tubes (containing DMEM medium) under a clean bench. Place the centrifuge tubes in a centrifuge, balance the volume, and set the program to 800 rpm for 3 minutes. After centrifugation, discard the supernatant, add an appropriate amount of DMEM medium (containing 10% serum), gently pipette, and add to 10 mL of T75 culture flask containing 10% DMEM serum medium. Mix well and incubate statically in a cell culture incubator.

[0072] Cell passage: Two days after cell resuscitation, the T75 culture flask was removed and observed under a microscope. When the cell density was about 90%, the supernatant was discarded in a clean bench, the cells were washed once with PBS, and 3 mL of trypsin was added. After incubation for 60 seconds, 3 mL of LDM medium was added to stop the digestion. The cells were pipetted from the bottom of the culture dish and collected into a centrifuge tube. After centrifugation (using the same procedure as before), the supernatant was discarded, and 2 mL of complete culture medium was added to suspend the cells. The cells were then pipetted and 1 mL of the mixture was added to a T75 culture flask containing 10 mL of LDM medium. After mixing, the flask was placed in a cell culture incubator for static culture.

[0073] Cell cryopreservation: Remove the T75 culture flask, discard the culture medium, wash once with PBS, add 3 mL of trypsin, incubate for 60 seconds, then add 3 mL of LDMMEM culture medium to stop the digestion. Pipette the cells from the bottom of the culture dish and collect them into centrifuge tubes. Centrifuge using the same procedure as before, discard the supernatant, add 2 mL of cell cryopreservation buffer (serum:DMSO:culture medium = 2:1:7) to resuspend the cells, and then add 1 mL of cell cryopreservation buffer to each cryopreservation tube. Place the cryopreservation tubes in a programmed cooling box and store at -80°C.

[0074] c. Test substance testing Cell plating: Remove the T75 culture flask, discard the culture medium, wash once with PBS, add 3 mL of trypsin, incubate for 60 seconds, then add 3 mL of LDM medium to stop digestion. Pipette the cells from the bottom of the culture dish and collect them in centrifuge tubes. Centrifuge using the same procedure as before, discard the supernatant, add 2 mL of LDM medium, resuspend the cells, and take 180 μL. Add 20 μL of trypan blue staining solution and count the cells using a CountStar cell counter. Finally, add the culture medium containing the cells to 24-well plates. The final cell density is 3.5 × 10⁻⁶ cells / well. 4 Cells / well. After plating, incubate in a cell culture incubator for 24 hours.

[0075] d. Adding samples Normal sample loading: When cells reach approximately 70% confluence, discard the culture medium. Add 180 μL of cell culture medium to the wells of the blank control group, add cell culture medium containing 0.25 wt% liposomes to the liposome group, and add cell culture medium containing 100 ng / mL LGF-β1 to the positive control group. Each group is set up in triplicate. Incubate for 24 hours.

[0076] UV-induced sample loading: When cells reach approximately 70% confluence, discard the culture medium. Add 180 μL of cell culture medium to the wells of the blank control group, add cell culture medium containing 0.25 wt% liposomes to the liposome group, and add cell culture medium containing 100 ng / mL LGF-β1 to the positive control group. Irradiate both the liposome and positive control groups with UV light (5 J / cm², irradiation intensity should not be too high), while the blank control group is not irradiated with UV light. After irradiation, discard the culture medium. Add 180 μL of low-glucose DMEM medium to the wells of the blank control group, add low-glucose DMEM medium containing 0.25 wt% liposomes to the liposome group, and add low-glucose DMEM medium containing 100 ng / mL LGF-β1 to the positive control group.

[0077] e. Sample collection After culturing in an incubator for 24 hours, the cell culture base was collected in 1.5 mL centrifuge tubes and stored at -20°C.

[0078] f. Type I collagen content detection According to the instructions for testing type I collagen, the concentration of type I collagen was measured, the type I collagen content of the sample was calculated, and the type I collagen upregulation rate (the increase in type I collagen content compared to the blank group) was calculated. The results are shown in Table 4.

[0079] Table 4: Upregulation rate of type I collagen

[0080] According to the test results in Table 4, the liposomes prepared by this invention can effectively promote the secretion of type I collagen by cells, have a highly effective skin-tightening effect, and can effectively maintain skin elasticity and prevent skin aging.

[0081] Example 3 Spirulina extract: Take 5g of dried spirulina powder, add 75wt% ethanol solution at a material-to-liquid ratio of 1g:30g, stir and extract at 45℃ and 100rpm for 3 hours, filter with gauze, then filter with a 0.22μm pore size filter membrane, concentrate the filtrate at 40℃ and 10KPa under reduced pressure for 40 minutes to remove ethanol, and obtain the concentrate. Add 3 times the amount of ethyl acetate to the concentrate, stir and extract at 30℃ and 80rpm for 3 hours, then let stand at room temperature for 1 hour, take the separated organic phase, concentrate at 40℃ and 12KPa under reduced pressure for 1 hour to remove ethyl acetate, and then dry under vacuum at 45℃ and 0.5KPa for 2 hours to obtain spirulina extract.

[0082] Euglena gracilis extract: Take 5g of Euglena gracilis powder, add 25wt% ethanol solution at a material-to-liquid ratio of 1g:35g, stir and extract at 50℃ and 120rpm for 3.5 hours, concentrate the filtrate at 40℃ and 10KPa reduced pressure for 30 minutes to remove ethanol, filter with gauze, and then filter with a 0.22μm pore size membrane. Use an ultrafiltration membrane with a molecular weight cutoff of 3000Da to obtain the retentate. Freeze-dry the retentate to a water content of 2.37wt% to obtain Euglena gracilis extract.

[0083] The test products were obtained using the combinations shown in Table 5.

[0084] Table 5: Composition of the Test Products Groups of test products Composition (weight ratio) Test Product A Spirulina extract Test product B Euglena extract Test product C Spirulina extract and Euglena extract at a ratio of 1:0.1 Test product D Spirulina extract and Euglena extract at a ratio of 1:0.3 Test product E Spirulina extract and Euglena extract at a ratio of 1:0.8 Test product F Spirulina extract and Euglena extract in a 1:1.2 ratio Test product G Spirulina extract and Euglena extract in a 1:1.5 ratio Test product H Spirulina extract and Euglena extract in a 1:3 ratio Test Product I Spirulina extract and Euglena extract in a 1:8 ratio The performance of the above-mentioned test products was tested as follows: A 0.1% Tween-60 aqueous solution was used as the dilution solvent; the test product AI was diluted to a total weight concentration of 0.1% using the dilution solvent and mixed with an equal volume of tyrosinase solution (enzyme concentration 200 U / mL) to obtain the test sample; resveratrol was diluted to a concentration of 120 μM using the same dilution solvent and mixed with an equal volume of tyrosinase solution (enzyme concentration 200 U / mL) to obtain the positive control test sample; the test samples were incubated at 37℃ with shaking for 10 minutes, and then 8 times the volume of the test sample (20 mg / mL) of L-tyrosine solution was added. The reaction was continued at 37℃ with shaking for another 10 minutes, and the absorbance was measured at a wavelength of 475 nm to calculate the tyrosinase inhibition rate; the blank group consisted of a mixture of a 0.1% Tween-60 aqueous solution and an equal volume of tyrosinase solution (enzyme concentration 200 U / mL) as the test sample and tested according to the above method. Each group was tested in triplicate, and the average value was taken. The formula for calculating the tyrosinase inhibition rate is 100% × (A0 - A1) / A0; A0 is the absorbance obtained from the blank group test, and A1 is the absorbance of the test product and the positive control group. The results are shown in Table 6 and... Figure 4 .

[0085] Table 6: Tyrosinase Inhibition Group Tyrosinase inhibition rate Positive control 74.39% Test Product Group A 65.42% Test Product Group B 62.78% Test Product Group C 64.13% Test product group D 67.01% Test Product Group E 72.34% Test product group F 76.92% Test Product Group G 73.48% Test product group H 66.25% Test Product Group I 63.57% As shown in Table 4, the Spirulina extract and Euglena granulosus extract obtained by the corresponding processes in this invention both exhibit excellent tyrosinase inhibition activity and can effectively whiten skin. When using the same total extract concentration, the inhibitory activity efficiency of groups D to H, which combine the two extracts, is higher than that of groups A and B acting alone. This proves that the combination of the two extracts has a synergistic effect in enhancing tyrosinase inhibition and whitening effects. Furthermore, the tests show that groups E, F, and G have higher inhibition rates, meaning that within the weight ratio of Spirulina extract to Euglena granulosus extract of 1:0.8-1.5, the tyrosinase inhibition activity and whitening effect are stronger.

[0086] In addition, the above-mentioned Spirulina extract and Euglena granulosus extract can be used to prepare liposomes, and the specific process is as follows: Step (1) Preparation of plant exovesicles 1: Take 50g of microalgae, wash and chop them with water, add 7.5 times the weight of PBS buffer (pH 7.4), shear homogenize at 30℃ and 8000rpm for 10 minutes, then homogenize three times at 35℃ and 800bar (2 minutes each time), then centrifuge at 12000g for 30 minutes, remove the precipitate, and concentrate the supernatant through a 120KDa ultrafiltration membrane to obtain the retentate (solid content 21.7wt%), thus obtaining plant exovesicles; Step (2): Mix 4.5g of active ingredients (spirulina extract and Euglena extract in a weight ratio of 1:1.2) with alcohol (ethylene glycol and 1,3-butanediol in a weight ratio of 3:1) at a weight ratio of 1:15, at room temperature and 150 rpm for 15 minutes to obtain a mixture. Add a 1.2wt% solution of water-soluble silk fibroin (water-soluble silk fibroin to active ingredients in a weight ratio of 1:1.2), and stir and shear at 30℃ and 1200 rpm for 30 minutes to obtain the active ingredient-silk fibroin complex. Step (3): Hydrogenated phospholipids and cholesterol (total weight of hydrogenated phospholipids and cholesterol is 12g) and ethyl acetate (84g) in a weight ratio of 3:1 are stirred and mixed at 150rpm for 10 minutes at room temperature to obtain a mixture. The organic solvent ethyl acetate is removed by rotary evaporation to obtain a lipid membrane. Step (4): Add 30g PDRN solution and 55g water to 10g lipid membrane, stir and mix at 800rpm for 20 minutes, then add 7.5g plant exovesicles and 25g active ingredient-silk fibroin complex, stir at 800rpm for 20 minutes, then shear emulsify at 10000rpm for 30 minutes to obtain primary emulsion. Step (5): The primary emulsion is subjected to high-pressure homogenization (800 bar, 5 cycles, 1.5 minutes each time) to obtain a liposome suspension. The suspension is then filtered through a 0.22-micron filter membrane to obtain PDRN-silk fibroin complex plant vesicle liposomes.

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing plant exovesicle liposomes composed of PDRN-silk fibroin, characterized in that, Includes the following steps: (1) Preparation of plant exovesicles; (2) The active ingredient is mixed with alcohol to obtain a mixture, a solution of water-soluble silk fibroin is added, and the mixture is stirred and sheared to obtain an active ingredient-silk fibroin complex; wherein, the alcohol includes at least one of ethylene glycol, propylene glycol, glycerol, and butylene glycol; (3) Dissolve hydrogenated phospholipids and cholesterol in an organic solvent, remove the organic solvent by rotary evaporation, and obtain a lipid membrane; (4) Add PDRN solution and water to the lipid membrane, stir and mix, then add plant exovesicles and active ingredient-silk fibroin complex, stir, and then shear emulsify to obtain primary emulsion; (5) The primary emulsion was subjected to high-pressure homogenization to obtain a liposome suspension, which was then filtered to obtain PDRN-silk fibroin complex plant vesicle liposomes.

2. The preparation method according to claim 1, characterized in that, The active ingredients include at least one of curcumin, retinol, tocopherol and its derivatives, resveratrol, ascorbate tetraisopalmitate, hesperidin, quercetin, bisabolol, lutein, medium / high molecular weight polysaccharides, polypeptides, recombinant proteins, natural plant oils, panthenol, and phenylethyl resorcinol.

3. The preparation method according to claim 1, characterized in that, The method for preparing plant exovesicles includes: taking plant tissue, washing and chopping it, adding buffer solution for shearing homogenization and high-pressure homogenization, then centrifuging to remove the precipitate and ultrafiltration to concentrate the extract to obtain plant exovesicles; wherein the plant includes at least one of microalgae, hydrilla, and brown algae.

4. The preparation method according to claim 1, characterized in that, The active ingredients include curcumin and / or retinol.

5. The preparation method according to claim 1, characterized in that, Hydrogenated phospholipids include at least one of hydrogenated lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated egg yolk phospholipid, plant-based hydrogenated lecithin, hydrogenated phosphatidylcholine, and hydrogenated phosphatidylethanolamine.

6. The preparation method according to claim 1, characterized in that, Organic solvents include at least one of ethanol, chloroform, and ethyl acetate.

7. The preparation method according to claim 2, characterized in that, The active ingredients also include spirulina extract, carnosine, peptide derivatives, and Euglena buergeriana extract.

8. The preparation method according to claim 7, characterized in that, The weight ratio of Spirulina extract to Euglena extract is 1:0-1.

5.

9. A plant extravesicular liposome composed of PDRN-silk fibroin prepared by the preparation method according to any one of claims 1-8.

10. The application of a PDRN-silk fibroin complex plant exovesicle liposome according to claim 9 in the preparation of cosmetics.

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