A tat peptide modified phyllose liposome and its preparation method and application

Abstract

The application discloses a TAT peptide modified photoredox enzyme liposome and a preparation method and application thereof, and the method comprises the following steps: S1: mixing and dispersing phosphatidylserine, dipalmitoyl phosphatidylglycerol, phosphatidic acid and glycerol to obtain a phospholipid-glycerol mixed phase; S2: dissolving photoredox enzyme and carboxymethyl chitosan, and adding the photoredox enzyme and carboxymethyl chitosan into the phospholipid-glycerol mixed phase to obtain photoredox enzyme liposomes; and S3: mixing and dispersing TAT peptides and the photoredox enzyme liposomes to obtain the TAT peptide modified photoredox enzyme liposomes; the TAT peptide modified photoredox enzyme liposomes have the advantages of mildness, non-stimulation and low cost, are non-toxic and have biological safety, have a storage stability of more than 90 days, and can effectively promote transdermal absorption of active substances in biological drugs, and thus the application range is widened.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a TAT peptide-modified photolyase liposome, its preparation method, and its application. Background Technology

[0002] Photolysin is a DNA repair enzyme that precisely identifies damaged DNA fragments in skin cells. Using blue light and near-ultraviolet energy, it converts pyrimidine dimers formed due to photodamage into two normal thymine compounds, thus completing DNA repair. Simultaneously, it reduces reactive oxygen species (ROS) production, maintains normal cell vitality, promotes cell regeneration, and prevents UV-induced erythema and aids in sunburn repair. However, photolysin has a large molecular weight, limiting transdermal absorption and thus its activity is not fully realized, restricting its application in biopharmaceuticals.

[0003] Currently, physical methods for transdermal absorption of photolyzed enzymes mainly focus on microneedles, ultrasound electrophoresis, and iontophoresis. These methods deliver drug particles through particle accelerators or gradient field-induced convection, which increases the drug delivery rate but requires advanced technology and is often used in the medical field. Alternatively, chemical methods can be used, such as by using various chemicals and biochemical agents, to dissolve the skin barrier function and increase penetration. However, some of these agents can cause adverse skin irritation, allergies, and toxicity.

[0004] Researchers have gradually shifted their focus to milder biomass drug delivery systems, including microemulsions, infiltrates, delivery systems, liposomes, and liposomes. Among these, liposome technology is relatively mature, capable of encapsulating water-soluble drugs in the inner phase, and has been successfully applied to the delivery of various active substances. However, it still suffers from severe tissue and biomembrane barrier penetration problems, low cellular uptake, and low bioavailability. TAT peptide (Trans-Activator of Transcription Peptide) is a short-chain cationic polypeptide rich in basic amino acids, possessing a strong ability to penetrate cell membranes. Luo et al. grafted TAT peptide onto maleimide-phospholipid-polyethylene glycol, successfully preparing TAT-modified liposomes loaded with tanshinone B. Experiments showed that TAT peptide modification significantly improved the cellular uptake efficiency of liposomes (J Liposome Res, 2020, 30(1):93-106). However, this method has high costs and has not systematically evaluated its potential toxicity and biosafety, and is currently only used in research.

[0005] In conclusion, to promote the transdermal absorption of photolyzed enzymes, it is necessary to develop a cost-effective, low-skin-irritant, and highly bioavailable method for enhancing transdermal penetration. Summary of the Invention

[0006] The main objective of this invention is to provide a TAT peptide-modified photolyase liposome, its preparation method, and its application, aiming to promote the transdermal absorption of photolyase and solve the technical problem of limited transdermal absorption of photolyase.

[0007] To achieve the above objectives, the first aspect of the present invention provides a method for preparing TAT peptide-modified photolyzable liposomes, comprising the following steps:

[0008] S1: Phosphatidylserine, dipalmitoylphosphatidylglycerol, phosphatidic acid and glycerol are mixed and dispersed to obtain a phosphatidyl-glycerol mixed phase;

[0009] S2: Dissolve photolyase and carboxymethyl chitosan, and add them to the phospholipid-glycerol mixed phase to obtain photolyase liposomes;

[0010] S3: Mix and disperse the TAT peptide with the photolyase liposome to obtain the TAT peptide-modified photolyase liposome.

[0011] Anionic liposomes are a class of lipid nanoparticles carrying a negative surface charge, their charge characteristic derived from the anionic lipid components used in their formulation. These liposomes maintain structural stability through electrostatic repulsion and exhibit unique biological behaviors in drug delivery. Current anionic lipid components mainly include phosphatidylserine (PS), dipalmitoylphosphatidylglycerol (DPPG), and phosphatidic acid (PA). PS is naturally found in the inner layer of cell membranes and contains a phosphaserine group (-PO4). - DPPG is the main component of anionic liposomes and was used in the first FDA-approved nanomedicine: doxorubicin liposome (Doxil®). DPPG consists of a glycerol backbone, two saturated palmitoyl groups (C16:0), and a glycerol phosphate head group, exhibiting low immunogenicity and high biocompatibility, making it suitable for in vivo drug delivery systems. Phosphatidic acid (PA): The phosphate group is directly linked to the glycerol backbone, resulting in a high negative charge density. Because these anionic liposomes carry a negative charge and are repelled by cell membranes, they require endocytosis or targeted modification.

[0012] Carboxymethyl chitosan (CMCS) is a derivative of chitosan modified by carboxymethylation. It has excellent biocompatibility, pH sensitivity, mucosal adhesion and water solubility. It is widely used in the functional modification of liposomes to improve their stability, targeting and drug release performance. It coats the surface of liposomes through electrostatic adsorption or hydrogen bonding to form a protective layer, reducing oxidation, aggregation and drug leakage.

[0013] TAT peptide is a functional short peptide derived from the transcriptional activator protein of human immunodeficiency virus (HIV-1), known for its strong cell penetration ability. Its core functional domain is usually an 11-amino acid sequence YGRKKRRQRRR, and its high content of arginine (Arg) and lysine (Lys) endows it with a strong positive charge, with a net charge of +6 to +8 at physiological pH.

[0014] This invention prepares stable photolyase liposomes by using three anionic lipids—phosphatidylserine, dipalmitoylphosphatidylglycerol, and phosphatidic acid—in synergistic with carboxymethyl chitosan. The photolyase liposomes are then modified with TAT peptide to obtain a highly permeation-enhancing stable liposome formulation.

[0015] The inventive concept of this invention is as follows: DPPG has a high phase transition temperature T m The presence of polystyrene (PS) provides a rigid framework for liposomes, and the introduction of PS increases membrane permeability. However, the hydrophobicity difference between the saturated chains of DPPG and the unsaturated chains of PS makes them prone to lateral phase separation. Introducing PA with a high charge density can disrupt phase separation, promote uniform lipid distribution, and form a more stable anionic liposome precursor. Carboxymethyl chitosan coats the liposome surface through electrostatic adsorption or hydrogen bonding, forming a protective layer that further increases the stability of the anionic liposomes. Furthermore, the spontaneous binding of negatively charged anionic liposomes with positively charged TAT peptides avoids the cumbersome processes and high costs associated with previous TAT ​​peptide-modified liposomes.

[0016] In summary, this invention focuses on the regulation of anionic lipid ratios, with carboxymethyl chitosan and TAT peptides as auxiliary modifications. Through spontaneous cross-linking between the components, it achieves multiple objectives of "process simplification, cost optimization, and high permeability enhancement," providing a novel solution for transdermal absorption of photolyzed macromolecular proteins.

[0017] Furthermore, the mass ratio of phosphatidylserine, dipalmitoylphosphatidylglycerol, and phosphatidic acid is (4~6):(2~4):(1~3). The inventors have discovered that within this ratio range, the charge repulsion and membrane rigidity among the three components can be balanced.

[0018] Furthermore, step S1 more specifically includes:

[0019] Phosphatidylserine, dipalmitoylphosphatidylglycerol, and phosphatidic acid were premixed at a temperature of 45-55°C, a stirring speed of 200-400 rpm / min, and a stirring time of 12-15 min. The mixture was then mixed and dispersed with glycerol to obtain a phosphatidyl-glycerol mixed phase.

[0020] Furthermore, step S2 more specifically includes:

[0021] Photolysin, carboxymethyl chitosan, and water are mixed and dissolved, then added to the phospholipid-glycerol mixture, along with a preservative. The mixture is first homogenized and dispersed, then processed using a microfluidic process to obtain the photolysin liposomes. Preferably, the preservative is hexanediol and pentanediol, which primarily exert their antibacterial effect by disrupting the osmotic pressure of microbial cell membranes.

[0022] Furthermore, the parameters of the microjet process include: a pressure of 950 bar and 3 cycles.

[0023] Furthermore, the photolyzed enzyme liposomes exhibit a normal particle size distribution within the range of 30–150 nm. This particle size range demonstrates good uniformity and represents the optimal particle size for use as a nanomedicine carrier.

[0024] Furthermore, step S3 more specifically includes:

[0025] TAT peptide was dissolved in water, mixed with the photolyase liposome, and dispersed by a homogenizer to obtain the TAT peptide-modified photolyase liposome.

[0026] Furthermore, the homogenizer has a rotation speed of 8000~10000 rpm / min and a homogenization time of 1~5 min.

[0027] The second aspect of this invention provides a TAT peptide-modified photolyase liposome, obtained by any of the above preparation methods, comprising the following components: 4-6 parts of phosphatidylserine, 2-4 parts of dipalmitoylphosphatidylglycerol, 1-3 parts of phosphatidic acid, 35-60 parts of glycerol, 0.1-2 parts of photolyase, 0.5-3 parts of carboxymethyl chitosan, 0.1-2 parts of TAT peptide, 2-8 parts of hexanediol, 1-5 parts of pentanediol, and the balance being water.

[0028] The third aspect of this invention proposes the application of TAT peptide-modified photolyase liposomes in biopharmaceuticals and daily chemical products, wherein the weight percentage of the TAT peptide-modified photolyase liposomes in biopharmaceuticals and daily chemical products is 0.5% to 2%.

[0029] Beneficial effects:

[0030] The present invention aims to provide a method for preparing TAT peptide-modified photolyase liposomes. The method involves using different anionic phospholipids, namely PS, DPPG, and PA, to prepare photolyase liposomes, thereby endowing them with negative charge properties. At the same time, carboxymethyl chitosan is introduced to increase the stability of the liposomes. Subsequently, a strongly positively charged TAT peptide is added. Through the attraction between positive and negative charges, the TAT peptide is attached to the surface of the liposomes, which enhances the transmembrane penetration of the liposomes, promotes the transdermal absorption of photolyase, and fully exerts its activity.

[0031] This invention utilizes PS, DPPG, PA, and CMCS to prepare stable photolyase liposomes. By linking liposomes with TAT peptides based on their charge properties, it promotes transdermal photolyase transdermal absorption, improves the bioavailability of traditional liposomes, and expands the application range of photolyase. Evaluation shows that the TAT peptide-modified photolyase liposomes of this invention are mild and non-irritating, low-cost, non-toxic, and biosafety in nature, with a storage stability of over 90 days. Attached Figure Description

[0032] Figure 1 This is a comparison diagram of the average particle size of Example 1 and Comparative Example 1 of the present invention;

[0033] Figure 2 This is a comparison diagram of the Zeta potentials of Embodiment 1 and Comparative Example 1 of the present invention;

[0034] Figure 3 This is a comparison chart of the polydispersion coefficients of Example 1 and Comparative Example 1 of the present invention;

[0035] Figure 4 This is a comparison chart of the NIH / 3T3 cell survival rates in Example 1 and Comparative Examples 1-2 of the present invention;

[0036] Figure 5 This is a comparison chart of NIH / 3T3 cell migration rates in Example 1 and Comparative Examples 1-2 of the present invention;

[0037] Figure 6 The accumulation and permeation curves of normal mouse skin in Example 1 and Comparative Examples 1-5 of this invention are shown.

[0038] Figure 7 The retention rate of Example 1 and Comparative Examples 1-5 in normal mouse skin is shown.

[0039] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0040] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0041] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0042] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0043] Unless otherwise specified, all percentages (%) below are by weight.

[0044] The specific implementation method is as follows:

[0045] Examples 1-3 and Comparative Example 1

[0046] See Table 1 for the basic formula.

[0047] Table 1:

[0048]

[0049] (2) Preparation process:

[0050] S1: According to the above basic formula, phosphatidylserine, dipalmitoylphosphatidylglycerol, and phosphatidic acid in phase A are premixed at a temperature of 45~50℃, a stirring speed of 200~400 rpm / min, and a stirring time of 12~15 min. Then, they are mixed and dispersed with glycerol to disperse phospholipids in glycerol, thus obtaining a phospholipid-glycerol mixed phase.

[0051] S2: Mix the photolyase, carboxymethyl chitosan, and water in phase B, and after they dissolve, add them to the phospholipid-glycerol mixed phase, and add the preservatives hexanediol and pentanediol from phase C. After initial dispersion using a homogenizer (9000 rpm / min, 3 min), prepare liposomes by high-pressure microfluidic process. The microfluidic process is 950 bar, 3 cycles, to obtain photolyase liposomes.

[0052] S3: Mix the TAT peptide in phase D with water to dissolve the TAT peptide, and finally mix it with the photolyase liposome and disperse it by homogenizer (9000 rpm / min, 1 min) to obtain the TAT peptide-modified photolyase liposome.

[0053] The only difference between Examples 1-3 is the ratio of PS, DPPG, and PA in the formulation; the other conditions and preparation methods are the same.

[0054] Comparative Example 1 is similar to Example 1, except that Comparative Example 1 does not have TAT peptide modification.

[0055] Comparative Example 2

[0056] The difference between this comparative example and Example 1 is that its formulation is a 1% aqueous solution of photolyase.

[0057] Comparative Example 3

[0058] The only difference between this comparative example and Example 1 is that the mass ratio of phosphatidylserine, dipalmitoylphosphatidylglycerol and phosphatidic acid is 3:1:4.

[0059] Comparative Example 4

[0060] The only difference between this comparative example and Example 1 is that carboxymethyl chitosan is replaced with chitosan; the other components and preparation methods are the same as in Example 1.

[0061] Comparative Example 5

[0062] The only difference between this comparative example and Example 1 is that the TAT peptide is replaced with the R9 peptide; the other components and preparation methods are the same as in Example 1.

[0063] Experimental Examples 1, 2, and 3 and Control Examples 1-5

[0064] Experimental Examples 1, 2, and 3 illustrate the application of Example 1 in basic essence formulas at different proportions.

[0065] Experimental control examples 1-5 respectively illustrate the application of control examples 1-5 in basic essence formulas.

[0066] (1) Basic formula:

[0067] The basic essence formula is shown in Table 2.

[0068] Table 2:

[0069]

[0070] (2) Preparation process

[0071] First, weigh phase A material and heat it to 60°C until there is no precipitate in the mixture. Then, set it to room temperature for later use. Accurately weigh phase B and phase C materials. Then, mix phases A, B, and C and disperse them using a homogenizer. The homogenization conditions are set as follows: speed 8000 rpm / min, time 3 min. The basic essences corresponding to experimental examples 1-3 and experimental control examples 1-5 are obtained.

[0072] Experiment 1: Long-term stability test

[0073] Since liposomes are usually stored at 4°C, the present invention uses 25°C to accelerate the stability test of Examples 1, 2, and 3, and the results are shown in Table 3.

[0074] Table 3:

[0075]

[0076] The results showed that Examples 1-3 and Comparative Examples 1 and 5 could maintain 90 days of storage stability under accelerated storage conditions at room temperature, indicating that the prepared liposomes had good stability. Furthermore, replacing the TAT peptide with the equally positively charged R9 peptide did not affect its stability, meeting the requirements for routine cosmetic applications. Comparative Example 3 showed that exceeding the mass ratio of phosphatidylserine, dipalmitoylphosphatidylglycerol, and phosphatidic acid would lead to liposome demulsification and precipitation. This is because the three components need to be in a specific ratio to maintain charge balance among the liposomes and promote their stability. Comparative Example 4 showed that carboxymethyl chitosan could promote the stability of liposomes. However, under neutral conditions, the amino groups of chitosan are deprotonated, the positive charge essentially disappears, and the molecules become electrically neutral or slightly negatively charged, preventing adsorption onto the liposome surface and leading to demulsification, stratification, and other stability issues.

[0077] Experiment 2: Liposome Characterization Test

[0078] (1) The particle size of Example 1 and Comparative Example 1 was detected using a nanoparticle size analyzer. The test results are as follows: Figure 1 As shown.

[0079] (2) The potentials of Example 1 and Comparative Example 1 were analyzed using potential analysis, and the test results are as follows: Figure 2 As shown.

[0080] (3) The polydispersity index (PDI) of Example 1 and Comparative Example 1 was analyzed using a polydispersometer. The test results are as follows: Figure 3 As shown.

[0081] Depend on Figure 1 - The average particle size diagram shows that the average particle sizes of the photolyase liposomes without TAT peptide modification and those with TAT peptide modification are 84.5 nm and 89 nm, respectively, indicating that the introduction of TAT peptide does not affect the particle size of the liposomes.

[0082] Figure 2 -Zeta potential plots show that the charges in both Experimental Example 1 and Comparative Example 1 are below -20 mV, specifically -25.3 mV and -26.6 mV, respectively. The introduction of the positively charged TAT peptide into the photolyase liposome increases its charge, indicating to some extent that the two are successfully linked.

[0083] Depend on Figure 3 - The polydispersity index (PDI) comparison chart shows that the PDI of Comparative Example 1 and Example 1 are 0.276 and 0.291, respectively. This indicates that the introduction of TAT peptide slightly increases the PDI of liposomes, and their particle size uniformity is lower than that of Comparative Example 1. This result is consistent with the particle size analysis, indicating that the introduction of TAT peptide will slightly decrease the uniformity of liposomes, but will not affect their stability (in conjunction with stability observation).

[0084] Experiment 3: Liposome Activity Analysis

[0085] Toxicity and cell migration activity were tested in Example 1, Comparative Example 1, and Comparative Example 2 using NIH / 3T3 cells (mouse embryonic fibroblast cell line).

[0086] Toxicity testing: The CCK-8 assay was used to detect mitochondrial dehydrogenase activity, indirectly reflecting the number of viable cells. Figure 4 The cell survival results show that each component of the liposome of the present invention is non-toxic to NIH / 3T3 cells, and the survival rate of the sample with a content in the range of 0.156%~2.5% is >100%, indicating that the liposome of the present invention can stimulate cell metabolism and has proliferative activity.

[0087] Cell migration activity assay: A scratch assay was used, with 1 μg / ml bFGF (basic fibroblast growth factor) as the positive control. The migration rates of different sample groups were compared at 6, 24, and 40 h. The results are shown below. Figure 5 As shown, the migration rate of Example 1 is greater than or equal to that of the bFGF group and is superior to that of Comparative Examples 1 and 2. This indicates that the cell activity of Example 1 is significantly higher than that of the other groups and comparable to that of the positive control group. This proves that the TAT peptide-modified photolyase liposome of the present invention can synergistically exert the effects of each component, has strong migration-promoting activity, and can further promote cell growth.

[0088] Experiment 4: Liposome Transdermal Absorption Test

[0089] The TK-6H1 Valia-Chien dual-chamber transdermal diffusion assay system was used. Ex vivo cryopreserved skin was fixed at the junction of the diffusion chambers, with the stratum corneum facing the supply chamber. The supply chambers were filled with samples from Examples 1, 1, 2, 3, 4, and 5, respectively. The receiving chamber was filled with receiving fluid (physiological saline). The diffusion chamber was connected to a peristaltic pump and a thermostatically heated magnetic stirrer via a water bath jacket to maintain continuous stirring. The water bath temperature was set at 36.0 ± 0.5 °C, and the stirring speed was 200 r / min. Samples were taken at 0.5, 1, 2, 4, 6, 8, 10, and 24 hours. After each transfer of 1 mL of receiving medium, isothermal physiological saline was immediately added to the original volume. The total protein concentration of the samples was determined using a BCA kit, and the cumulative protein permeation Q was calculated using the following formula. n :

[0090]

[0091] In the formula Q n This indicates the cumulative transdermal absorption rate per unit area (μg / cm²). 2 ), C nC represents the protein concentration (μg / mL) at the nth sampling. i V represents the protein concentration (μg / mL) at the i-th sampling. i A represents the sampling volume (1 mL), and A represents the effective contact area (1.13 cm²). 2 ).

[0092] Subsequently, the excised skin was removed, and the skin surface was repeatedly rinsed with physiological saline until the surface proteins were clean. The skin at the treatment site was placed in a 10 mL centrifuge tube, cut into fragments, and then 4 mL of ultrapure water was added. The mixture was sonicated in a water bath for 30 min, allowed to stand for 10 min, and the supernatant was collected. The collagen concentration was determined using a BCA kit, and the skin retention rate of collagen (A) was calculated. s ):

[0093]

[0094] In the formula, V0 is the volume of ultrapure water (4.0 mL), C is the measured collagen concentration (mg / mL), and A... s Skin retention rate (%), m s The weight of mouse skin (μg).

[0095] Transdermal dose test results refer to Figure 6 As can be seen, the results of Comparative Examples 1 and 2 show that the amount of photolyase penetrating mouse skin when used alone is very small. However, after encapsulation with liposomes, the permeability of photolyase increased by 0.6 times, indicating that liposome encapsulation technology can improve the permeability of photolyase. Referring to the comparison between Example 1 and Comparative Examples 1-2, it can be seen that the permeability of photolyase liposomes modified with TAT peptide increased by 0.36 times, indicating that the introduction of TAT peptide can further promote the skin penetration of photolyase, and the permeability increased by 1.13 times compared with the use of photolyase alone. Moreover, the transdermal absorption rate of liposomes in Comparative Examples 3-5 is not as good as that in Example 1.

[0096] Reference Figure 7 In the retention experiment in mouse skin, the skin retention rates of Comparative Example 1 and Example 1 were 0.8 times and 3.3 times higher than those of Comparative Example 2, respectively, indicating that liposome encapsulation significantly increased the retention of photolyase in mouse skin, and the retention was further enhanced after modification with TAT peptide.

[0097] Experiment 5: Basic Applications of TAT Peptide-Modified Photolysin Liposomes

[0098] (1) Security testing:

[0099] Thirty volunteers were recruited, 15 men and 15 women, aged 20-50 years. A closed patch test method was used. Equal amounts (0.02g-0.025g) of the test sample (Example 3) were placed in a patch applicator, and hypoallergenic adhesive tape was applied to the volunteers' arms, gently pressing to ensure even application to the skin. The patch was left on for 24 hours. A blank control group received distilled water. After 24 hours, the patch applicator was removed, and skin reactions were observed and recorded at 0.5h, 24h, and 48h.

[0100] The severity of adverse skin reactions is shown in Table 4.

[0101] Table 4:

[0102]

[0103] The results showed that 0 out of 30 people had a positive reaction. According to the "Cosmetic Safety Technical Specifications" (2015 edition), Experiment 3 will not cause adverse skin reactions in this batch of test subjects, proving that the liposomes of the present invention are gentle and non-irritating to the skin.

[0104] (2) Soothing efficacy evaluation test:

[0105] 120 participants aged 18-60 years will be recruited. Participants should rest for 30 minutes in a constant temperature and humidity environment (50%±10%, 21℃±1℃) before the test. Test areas will be marked on the flexor surfaces of both forearms, randomly assigned to either the left or right forearm, with a minimum test area of ​​(3×3) cm². 2 The test area was randomly divided into a test area, a negative control area, and a blank control area. 0.02–0.025 ml of the inducer (3% sodium dodecyl sulfate solution) was placed on a filter paper and placed inside the patch applicator chamber. The patch applicator containing the inducer was then applied to the subject's designated test area using hypoallergenic adhesive tape. The patch was gently pressed with the palm of the hand to ensure even application to the skin, and left for 24 hours. The 6-hour period after removing the patch applicator was the skin erythema induction period, during which no treatment was performed. Based on the area settings, the tester applied appropriate amounts of sample to the designated test area and negative control area on the flexor surfaces of both forearms. The blank control area did not use the product. The sample was applied three times daily, with an interval of at least 4 hours between applications, for a total of 10 days.

[0106] After the skin erythema induction period (6 hours after the tester was removed), the subjects sat quietly in a constant temperature and humidity environment for 30 minutes. The skin erythema index a* and heme E1 values ​​in the test area were measured using Delfin SkinColcorCatch. The measured value was D0. The measurement values ​​on the 5th and 10th days of the product were recorded. The rate of change of skin heme content was calculated according to the following formula.

[0107]

[0108] The effects of subject application of experimental cases 1-3 and experimental control cases 1-5 on skin hemoglobin content are shown in Table 5.

[0109] Table 5:

[0110]

[0111] As shown in the table above, Experimental Examples 1-3 and Control Examples 2-5 can all reduce the hemoglobin content in the skin to varying degrees. Control Examples 2-4 indicate that photolyase itself has a soothing effect, but the prepared liposomes are unstable, preventing them from fully exerting their efficacy. Control Example 5 shows that replacing TAT peptide with R9 peptide results in more stable liposomes, but its redness-reducing effect is not as good as that of TAT peptide. Furthermore, the membrane-penetrating effect of R9 peptide in the system of this invention is not as good as that of TAT peptide. Experimental Examples 1-3 show better redness-reducing effects than the other groups, indicating that the essence containing TAT peptide-modified photolyase has a soothing effect, and this soothing effect increases with the increase in the amount of liposomes obtained in Example 1.

[0112] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method of preparing a TAT peptide-modified pho tosynthetic liposome, characterized by, The method comprises the following steps: S1: mixing and dispersing phosphatidylserine, dipalmitoyl phosphatidylglycerol, phosphatidic acid and glycerol to obtain a phospholipid-glycerol mixed phase; S2: dissolving photolyase and carboxymethyl chitosan, and adding them into the phospholipid-glycerol mixed phase to obtain photolyase liposomes; S3: mixing and dispersing TAT peptide and the photolyase liposomes to obtain the TAT peptide modified photolyase liposomes. The mass ratio of the phosphatidylserine, dipalmitoyl phosphatidylglycerol and phosphatidic acid is (4-6):(2-4):(1-3).

2. The method for preparing TAT peptide-modified photolyase liposomes according to claim 1, characterized in that, The step S1 more specifically comprises: The phosphatidylserine, dipalmitoyl phosphatidylglycerol and phosphatidic acid are pre-mixed at a temperature of 45-55°C and a stirring speed of 200-400 rpm / min for 12-15 min, and then mixed and dispersed with glycerol to obtain the phospholipid-glycerol mixed phase.

3. The method for preparing TAT peptide-modified photolyase liposomes according to claim 1, characterized in that, The step S2 more specifically comprises: The photolyase, carboxymethyl chitosan and water are mixed and dissolved, and then added into the phospholipid-glycerol mixed phase and a preservative, and then mixed and dispersed by a homogenizer, and then the photolyase liposomes are obtained by a micro-jet process.

4. The method for preparing TAT peptide-modified photolyase liposomes according to claim 3, characterized in that, The parameters of the micro-jet process include a pressure of 950 bar and 3 cycles.

5. The method for preparing TAT peptide-modified photolyase liposomes according to claim 1 or 3, characterized in that, The particle size of the photolyase liposomes is normally distributed in the range of 30-150 nm.

6. The method for preparing TAT peptide-modified photolyase liposomes according to claim 1, characterized in that, The step S3 more specifically comprises: The TAT peptide is dissolved in water, mixed with the photolyase liposomes and dispersed by a homogenizer to obtain the TAT peptide modified photolyase liposomes.

7. The method for preparing TAT peptide-modified photolyase liposomes according to claim 3 or 6, characterized in that, The rotating speed of the homogenizer is 8000-10000 rpm / min, and the homogenization time is 1-5 min.

8. A TAT peptide-modified phyllosemase liposome obtained by the production method according to any one of claims 1 to 7, characterized in that, The method comprises the following components: phosphatidylserine 4-6 parts, dipalmitoyl phosphatidylglycerol 2-4 parts, phosphatidic acid 1-3 parts, glycerol 35-60 parts, photolyase 0.1-2 parts, carboxymethyl chitosan 0.5-3 parts, TAT peptide 0.1-2 parts, hexanediol 2-8 parts, pentanediol 1-5 parts, and the rest is water.

9. Use of the TAT peptide-modified p hot o dyne liposome of claim 8 in a cosmetic product, characterized in that, The weight percentage of the TAT peptide modified photolyase liposomes in the daily chemical product is 0.5%-2%.

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