Preparation method and application of phenethyl caffeate photoresponse liposome

Abstract

The invention provides a preparation method and application of a phenethyl caffeate photoresponse liposome. According to the preparation method disclosed by the invention, the CAPE or a grafted compound of the CAPE and mercaptocholesterol is respectively embedded into a phospholipid bilayer to obtain the novel photoresponse liposome, and the novel photoresponse liposome can be used for wrapping an active matter, so that photoresponsiveness and efficacy are combined and have a synergistic effect. The photoresponse liposome has high light-controlled release efficiency, high thermal stability and high oxidation resistance, and has good innovation and application prospects in the field of cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics; more specifically, the present invention relates to a preparation method of caffeic acid phenethyl ester photoresponsive liposome and application thereof. Background Art

[0002] The concept of liposomes was first proposed in 1965. Researchers found that phospholipids can spontaneously form bilayer vesicles in water and named them liposomes. This discovery is considered a major breakthrough in the pharmaceutical field in the 20th century. Liposomes have attracted widespread attention as drug carriers because their main raw material phospholipids are inherent components of human cells and have good biocompatibility and immunogenicity.

[0003] The application of liposomes also plays an important role in drug delivery systems. Because their structure is similar to biological membranes, liposomes are also called artificial biological membranes, which makes it easier for them to penetrate biological barriers such as blood vessel walls and cell membranes, thereby improving drug delivery efficiency and bioavailability.

[0004] Liposomes are liposomes with a bilayer structure made of lecithin and ceramide. Their structure is the same as the skin cell membrane, so they are more friendly to the skin. In the art, they are used to encapsulate moisturizing substances such as hyaluronic acid, polyglucosidase, etc. as moisturizing substances. ‌‌

[0005] Currently in the field of cosmetics / skin care products, photoresponsive materials are mainly used in photosensitive color cosmetics, and some of them are irritating.

[0006] Therefore, it is of great significance to develop mild photoresponsive carriers that can be stimulated by ultraviolet or natural light for use in cosmetics. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation method of caffeic acid phenethyl ester light-responsive liposome and application thereof.

[0008] In a first aspect of the present invention, a method for preparing photoresponsive liposomes is provided, the method comprising: using caffeic acid phenethyl ester or a grafted complex thereof with thiol cholesterol as a membrane material, interacting with lecithin, to prepare photoresponsive liposomes; the light comprises ultraviolet light or natural light.

[0009] In one or more embodiments, the photoresponsive liposomes are obtained by a thin film dispersion method.

[0010] In one or more embodiments, in the method, caffeic acid phenethyl ester or a grafted complex thereof with thiol cholesterol is embedded in a phospholipid bilayer to obtain the liposome.

[0011] In one or more embodiments, the mass ratio of caffeic acid phenethyl ester or its grafted complex with thiol cholesterol to lecithin is 1: (8-60); preferably 1: (10-50); more preferably 1: (12-40); for example 1: 15, 1: 16, 1: 18, 1: 20, 1: 25, 1: 30, 1: 35.

[0012] In one or more embodiments, the method comprises: (1) dissolving caffeic acid phenethyl ester or a grafted complex thereof with thiol cholesterol and lecithin in an organic solvent to obtain a lipid solution; (2) removing the organic solvent in the lipid solution of (1) to leave a lipid film; (3) The lipid film of (2) is hydrated and dispersed in a dispersion to obtain a photoresponsive liposome dispersion.

[0013] In one or more embodiments, in (1), the mass ratio of caffeic acid phenethyl ester or its grafted complex with thiol cholesterol to lecithin is 1:(5-80); or (2), removing the organic solvent by evaporation; or (3), hydrated and dispersed in a dispersion liquid having a pH of 7.4±0.4 (preferably pH 7.4±0.3, pH 7.4±0.2 or pH 7.4±0.1); the dispersion liquid comprises: phosphate buffer, physiological saline or water.

[0014] In one or more embodiments, the organic solvent is a mixed solvent.

[0015] In one or more embodiments, the organic solvent includes a mixed solvent of alcohol and chloroform.

[0016] In one or more embodiments, the organic solvent is an ethanol-chloroform or methanol-chloroform mixed solvent, preferably a methanol-chloroform mixed solvent.

[0017] In one or more embodiments, in (2), the lipid solution is rotary evaporated at 40±5°C (preferably, 40±3°C, 40±2°C or 40±1°C), 17±5 rpm (preferably, 17±3 rpm, 17±2 rpm or 17±1 rpm), 0.05±0.03MPa (preferably, 0.05±0.02 MPa or 0.05±0.01 MPa) to remove the organic solvent and form the lipid film at the bottom of the container.

[0018] In one or more embodiments, in (3), a phosphate buffer solution having a pH of 7.4±0.4 is added to the flask and hydrated for 1-2 h to disperse the film.

[0019] In one or more embodiments, after step (3), the method further includes: (4) removing the precipitate in the dispersion and harvesting the supernatant, which is a purified photoresponsive liposome dispersion.

[0020] In one or more embodiments, in (4), ultrasonic treatment is performed, and the precipitate is removed by centrifugation to obtain a supernatant.

[0021] In one or more embodiments, the method for preparing the grafted complex of caffeic acid phenethyl ester and thiol cholesterol comprises: (a) dissolving caffeic acid phenethyl ester in an alcohol medium, using copper ions as a catalyst and hydrogen peroxide as an oxidant, mixing and oxidizing (at room temperature, such as 10 to 30° C.) to obtain an oxidizing solution; adjusting the oxidizing solution to neutrality, and drying to obtain an oxidized solid product; (b) dissolving the oxidized solid product and thiol cholesterol (SH-CHOL) in an organic solvent and reacting them under an alkaline environment to obtain the grafted composite.

[0022] In one or more embodiments, in (a), the copper ions include (but are not limited to) from copper chloride, for example, copper chloride is added to form copper ions in the solution.

[0023] In one or more embodiments, in (a), the amount of cupric chloride, caffeic acid phenethyl ester and hydrogen peroxide is 1: (100-900): (10-90) in mass ratio; preferably 1: (150-750): (15-75); more preferably 1: (200-500): (20-50); more preferably 1: (250-350): (25-35); for example 1: 280: 30.

[0024] In one or more embodiments, in (a), the neutrality is pH 7.0±0.3, pH 7.0±0.2 or pH 7.0±0.1.

[0025] In one or more embodiments, in (a), the mixing and oxidation are performed for 1±0.5 h, 1±0.3 h or 1±0.1 h.

[0026] In one or more embodiments, in (a), the drying is performed in an oven at 37±2°C.

[0027] In one or more embodiments, in (b), the organic solvent includes chloroform.

[0028] In one or more embodiments, in (b), the alkaline environment is pH 9±0.5, pH 9±0.3 or pH 9±0.1.

[0029] In one or more embodiments, in (b), the reaction in an alkaline environment is a static reaction at room temperature, preferably for 6±3 h, 6±2 h or 6±1 h.

[0030] In one or more embodiments, the lecithin includes (but is not limited to): soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, or a combination thereof.

[0031] In another aspect of the present invention, a photoresponsive liposome is provided, which is prepared by any of the methods described above; the light includes ultraviolet light or natural light.

[0032] In one or more embodiments, the particle size of the photoresponsive liposome decreases after irradiation (preferably irradiation at different irradiation times) by more than 5%, preferably more than 10%, more preferably more than 15%, more preferably more than 20% or more than 25%.

[0033] In one or more embodiments, the photoresponsive liposomes have high light-controlled release efficiency.

[0034] In one or more embodiments, the photoresponsive liposomes have high stability (including thermal stability).

[0035] In one or more embodiments, the photoresponsive liposomes have high antioxidant capacity.

[0036] In another aspect of the present invention, there is provided the use of the photoresponsive liposome in preparing cosmetics (including skin care products) having an encapsulation layer.

[0037] In one or more embodiments, in the application, the photoresponsive liposome is used as an encapsulation layer to encapsulate the active substance.

[0038] In another aspect of the present invention, a method for preparing cosmetics (including skin care products) having an encapsulation layer is provided, comprising encapsulating active substances (active ingredients having skin-improving or makeup-improving effects) using the photoresponsive liposomes.

[0039] In one or more embodiments, the active substance includes: a fat-soluble or water-soluble active substance, such as an active substance commonly used in cosmetics or skin care products.

[0040] In one or more embodiments, the active substance includes: an unstable active substance.

[0041] In one or more embodiments, the active substance includes (but is not limited to): small molecule peptides, blue copper peptides, palmitoyl tripeptide, vitamin B, vitamin C or niacinamide, or a combination thereof.

[0042] In another aspect of the present invention, a cosmetic (including skin care product) having an encapsulation layer is provided, which comprises: the photoresponsive liposome as the encapsulation layer, and an active substance encapsulated in the encapsulation layer.

[0043] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 , HPLC peak diagram of CAPE under different illumination conditions, characterizing the light responsiveness of CAPE and the cis-trans structural changes of CAPE standard under different illumination conditions.

[0045] Figure 2 , HPLC results of CAPE after oxidation and grafting.

[0046] Figure 3 , CAPE and SH-CHOL grafted composites 1 H NMR results.

[0047] Figure 4 , Changes in the particle size (nm) of CAPE and CAPE-SH-CHOL liposomes under different lighting conditions (ultraviolet (UV) and natural light irradiation).

[0048] Figure 5 , Changes in PDI of CAPE and CAPE-SH-CHOL liposomes under different lighting conditions (ultraviolet (UV) and natural light irradiation).

[0049] Figure 6 , Zeta potential of CAPE anionic / cationic liposomes.

[0050] Figure 7 , the release amount of GHK-Cu by liposomes under UV-340 irradiation for 0-6 h.

[0051] Figure 8 , morphology of liposomes under scanning electron microscope; (a) CAPE liposomes encapsulating GHK-Cu (b) CAPE-SH-CHOL liposomes encapsulating GHK-Cu.

[0052] Fig. 9 、In vitro antioxidant activity of liposomes; (a) ABTS total antioxidant capacity (b) Fe 2+ Reducing ability (c) DPPH free radical scavenging activity. DETAILED DESCRIPTION

[0053] After in-depth research, caffeic acid phenethyl ester (CAPE) or its grafted complex with thiol cholesterol was embedded in the phospholipid bilayer to obtain a new type of light-responsive liposome, which can be used to encapsulate active substances, thereby combining light responsiveness and efficacy and synergistically enhancing synergy. The light-responsive liposome has high light-controlled release efficiency, high thermal stability and high antioxidant capacity, and has good innovation and application prospects in the field of cosmetics.

[0054] As used in the present invention, the "light" mentioned includes ultraviolet light or natural light.

[0055] As used in the present invention, the "photoresponsive liposome" is a photoresponsive material embedded in a nanocarrier system, which induces chemical structure conversion through stimulation of an external light source, leading to the breakage, partial isomerization and conformational transition of the photoresponsive functional groups, thereby changing the nanocarrier structure to achieve targeted release of its contents.

[0056] In the present invention, the photoisomerism of CAPE is utilized to prepare light-responsive liposomes by a thin film dispersion method, which has excellent light-controlled release effect and significant antioxidant activity, and can improve the instability of active substances in cosmetic systems.

[0057] The CAPE has unique photoisomerism, and its molecular structure contains conjugated carbon-carbon double bonds. CAPE can undergo conversion between cis and trans structures under ultraviolet irradiation of a specific wavelength. The CAPE is one of the main biologically active ingredients of propolis extracts and is a cinnamic acid derivative. Although some naturally occurring cinnamic acid derivatives have been considered beneficial to hair since ancient times and have been used in plant extracts to achieve the purpose of hair conditioning, the art has not yet applied them to embedding phospholipid bilayers to prepare light-responsive liposomes. The present invention uses CAPE or its complex with cholesterol as a membrane material, and embeds them in phospholipid bilayers to prepare liposomes.

[0058] CAPE undergoes a structural change from trans to cis under natural light or ultraviolet irradiation. Its cis-trans isomerization under light is shown below: .

[0059] In the art, the aqueous phase and phospholipid bilayer of liposomes are used to load suitable drugs, which have biocompatibility and tissue targeting. Most photoresponsive groups require a certain amount of photon energy to activate photochemical reactions. Studies have found that near-infrared light has strong tissue penetration ability, but the photon energy is too low to activate photochemical reactions, which limits the application of such liposomes in cosmetics. In view of this, the present invention is optimized. In the technical solution of the present invention, a photoresponsive carrier excited by ultraviolet or natural light is provided. Ultraviolet light and low-wavelength visible light have greater energy and are more suitable for participating in the design of photoresponsive skin care products.

[0060] The present invention provides a composite system of CAPE and mercapto-cholesterol (SH-CHOL), which effectively improves the thermal stability of CAPE liposomes and the fluidity of the membrane. The other photoresponsive molecule is a grafted product of CAPE and SH-CHOL, which uses free radical catalytic oxidation to form an orthophenol structure, and reacts with SH-CHOL in an organic solvent under alkaline conditions to form a grafted complex CAPE-SH-CHOL, the structure of which is as follows: .

[0061] The study found that the photoresponsive liposomes prepared using the grafted product (CAPE-SH-CHOL) have stronger stability than CAPE.

[0062] In a preferred embodiment of the present invention, the preparation method of the photoresponsive molecule CAPE-SH-CHOL is as follows: (1) Weigh 0.28 g of caffeic acid phenethyl ester and dissolve it in 10 mL of tert-butyl alcohol. Add 0.001 g of copper chloride and 1 mL of hydrogen peroxide solution. Stir and react at room temperature for 1 h to obtain oxidation solution I.

[0063] (2) adjusting the pH of solution I to 7.0 and drying overnight to obtain a solid product of caffeic acid phenethyl ester oxidation.

[0064] (3) Weigh 0.07 g of thiocholesterol and 0.1 g of caffeic acid phenethyl ester oxide and dissolve them in 10 mL of chloroform. Add 12 μL of organic base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Seal the mixture with aluminum foil and let it react at room temperature for 6 h to obtain caffeic acid phenethyl ester-thiocholesterol graft copolymer.

[0065] In the preferred embodiment of the present invention, soybean lecithin from natural sources is used as a lipid molecule for the first time, which has high biocompatibility and a wider range of applications. Caffeic acid phenethyl ester (CAPE) or its grafted complex with thiol cholesterol is respectively embedded in the phospholipid bilayer to obtain a new type of photoresponsive liposome, which can be used to encapsulate active substances, thereby combining photoresponsiveness and efficacy and synergistically enhancing the effect. The photoresponsive liposome has high light-controlled release efficiency, high stability and high antioxidant capacity, and has good innovation and application prospects in the field of cosmetics.

[0066] In a preferred embodiment of the present invention, caffeic acid phenethyl ester and soybean lecithin are dissolved in a methanol-chloroform mixed solvent, and a liposome solution is prepared by a thin film dispersion method. More specifically, the photoresponsive liposome is prepared by the following method: (1) 0.005 g of caffeic acid phenethyl ester or caffeic acid phenethyl ester-thiocholesterol and 0.09 g of soybean lecithin were completely dissolved in a mixed solvent of 4 mL of methanol and 6 mL of chloroform to obtain a lipid solution; (2) The dissolved solution was evaporated at 40°C, 17 rpm, and 0.05 MPa to remove the organic solvent, and a lipid film was formed at the bottom of the flask; (3) Add 15 mL of pH 7.4 phosphate buffer to the flask and hydrate for 1-2 h to disperse the film; (4) After ultrasonic treatment for 10 min, centrifugation at 3,000 g for 15 min was performed to obtain the supernatant, which was the liposome dispersion.

[0067] In the present invention, the raw materials for preparing the liposome solution may include phospholipids, CAPE, CAPE-SH-CHOL and liposome dispersion, wherein the liposome dispersion may be selected from one of physiological saline, water and buffer (eg, pH=7.4 phosphate buffer).

[0068] In the present invention, the phospholipids include soybean lecithin, egg yolk lecithin or hydrogenated soybean lecithin, or a combination thereof. Although the phospholipids mentioned in the present invention are preferably used, the phospholipids may not be limited to the above-mentioned ones.

[0069] In the present invention, the types of the unstable active substances can be various, for example, they may include (but are not limited to): small molecule peptides, blue copper peptides, palmitoyl tripeptide, vitamin B, vitamin C, niacinamide, etc.

[0070] The photoresponsive liposomes provided by the present invention have the following advantages: (1) Currently, photoresponsive liposomes are used in the fields of cosmetics, optoelectronics and medicine, most of which are irritating and toxic. In the field of medicine, the near-infrared excitation mode is not suitable for skin care products to be applied on the skin. The photoresponsive liposomes prepared by the present invention are simple to prepare, with safe raw materials, and can achieve rapid release of active substances by absorbing ultraviolet light in nature.

[0071] (2) Liposomes can load a variety of active substances, have high biocompatibility and tissue targeting, and protect the activity and stability of the active substances. The present invention modifies and functionalizes liposomes to give them new functions, thereby enhancing the stability and bioavailability of active substances at lower concentrations.

[0072] (3) The liposome membrane material of the present invention is caffeic acid phenethyl ester, which is one of the key components of propolis extract and has antioxidant and anti-inflammatory activities. These activities may be retained and exerted in the lipid membrane, thereby giving the liposome itself additional efficacy.

[0073] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0074] Example 1. Preparation of CAPE and SH-CHOL grafted composite 1. Preparation of CAPE and SH-CHOL grafted composites The specific process is as follows: (1) Weigh 0.28 g of CAPE and dissolve it in 10 mL of tert-butyl alcohol, add 0.001 g of copper chloride solid (as a catalyst) and 1 mL of hydrogen peroxide solution (as an oxidant, the mass of hydrogen peroxide contained in 1 mL of the solution is 0.03 g), stir and react at room temperature for 1 h to obtain oxidizing solution I; (2) adjusting the pH of solution I to 7.0 and drying in an oven at 37°C overnight for 14 h to obtain a CAPE oxidation solid product; (3) Weigh 0.07 g SH-CHOL and 0.1 g CAPE oxidation product and dissolve them in 10 mL chloroform. Add 12 μL DBU to provide an alkaline reaction environment (about pH 9). Seal with aluminum foil and let react at room temperature for 6 h to obtain a grafted composite, called CAPE-SH-CHOL.

[0075] 2. Analysis of CAPE-SH-CHOL grafting results A tert-butanol solution of CAPE standard, CAPE oxide and CAPE-SH-CHOL with a concentration of 0.05 g / mL was prepared, and the residual amount of CAPE in each sample was detected according to the above HPLC conditions. The peak areas of the three were compared to determine the oxidation and grafting conditions of CAPE.

[0076] like Figure 2 As shown in the figure, compared with the peak area of ​​CAPE standard, the peak area of ​​CAPE after oxidation decreased by 56%, indicating that CAPE underwent chemical transformation during the oxidation process, and some CAPE molecules were oxidized to other chemical forms, resulting in a weakened response signal at the detection wavelength and a decrease in peak area. The peak area of ​​CAPE grafted decreased by 64% compared with the CAPE standard, and compared with CAPE oxidized, its peak area decreased by 19%. This result shows that CAPE and SH-CHOL undergo a grafting reaction to form a new complex, and there may be chemical transformation or conformational changes in the complex, which changes the response characteristics of CAPE in HPLC detection.

[0077] The results were analyzed using a Bruker AscendTM 600MHz Nuclear Magnetic Resonance Hydrogen Spectrum. 1 H NMR) analysis of CAPE-SH-CHOL. By comparing the 1 H NMR spectrum, identify the characteristic peaks of different hydrogen atoms, analyze the chemical shift of the characteristic peaks, and further judge the grafting results of CAPE and SH-CHOL.

[0078] The grafting analysis results are as follows Figure 3 As shown, CAPE-SH-CHOL has a new characteristic peak at 0.6-1.2 ppm, which is the signal of long-chain alkyl or methyl protons, indicating the successful introduction of the cholesterol part. At the same time, multiple absorption peaks within 2.7-2.9 ppm, corresponding to the hydrogen atoms in the CAPE molecule, are significantly weakened or even disappear in the spectrum of the grafted product, indicating that the hydrogen atoms adjacent to the benzene ring in the CAPE molecule are replaced by thiol groups, changing the chemical environment of the original hydrogen atoms. This result shows that CAPE and SH-CHOL have undergone a grafting reaction to obtain a grafted product.

[0079] Example 2. Preparation of photoresponsive liposomes 1. Preparation of photoresponsive liposomes The specific process of preparing photoresponsive liposomes using CAPE or CAPE-SH-CHOL is as follows: (1) 0.005 g CAPE or 0.017 g CAPE-SH-CHOL and 0.09 g soybean lecithin were dissolved in 10 mL of a methanol-chloroform (volume ratio 1:2) mixed solution. The parameters of the rotary evaporator were set to a water bath temperature of 40 °C, a rotation speed of 17 rpm, and a vacuum degree of 0.05 MPa. The organic solvent was removed by rotary evaporation, and a transparent and uniform film was formed at the bottom of the flask. (2) Add 15 mL of pH 7.4 phosphate buffer and hydrate for 1-2 h to disperse the film; (3) Set the ultrasonic power to 200 W for 10 min. Centrifuge at 3,000 g for 15 min and collect the supernatant, which is the liposome dispersion.

[0080] 2. Analysis of the photoresponsivity of CAPE Photoresponsiveness is caused by the photoisomerization of CAPE and is embedded in the phospholipid bilayer. Here, we first tested whether CAPE undergoes isomerization in the dark, UV and natural light.

[0081] Here are the steps: (1) Prepare a 0.5 mM CAPE alcohol solution and take three equal samples and place them in different lighting environments (darkness, natural light, and UV-340) for 6 h.

[0082] (2) CAPE isomers were separated using a Waters 1525 binary HPLC pump equipped with a Waters 2998 photodiode array detector. A Waters C18 column (4.6 × 250 mm, 5 μm) was used for reversed phase separation. The mobile phase was methanol and water in a ratio of 60:40, the flow rate was 1 mL / min, and the injection volume was 10 μL.

[0083] Figure 1 The HPLC peak diagram of CAPE under different lighting conditions. Under dark conditions, CAPE maintains its trans structure and shows a characteristic peak at 12.7 min. When CAPE is exposed to natural light or UV-340, new retention times and peak areas appear in the HPLC spectrum, indicating that the CAPE molecule undergoes cis-trans isomerization, and part of the trans structure is converted into the cis structure. The elution time of the cis structure is 11.5 min. The HPLC results show that light is the key factor in triggering the cis-trans isomerization process of CAPE, and it is excited at an ultraviolet wavelength of 340 nm. Natural light also has this characteristic.

[0084] 3. Encapsulate active ingredients with CAPE liposomes The unstable active substance blue copper peptide was selected for encapsulation. The specific process of encapsulating the active substance with CAPE liposomes is as follows: (1) CAPE and CAPE-SH-CHOL liposomes were prepared according to the method described above in this example, and 15 mL of PBS buffer containing 0.3% of Glycyl-L-Histidyl-L-Lysine-Cu (GHK-Cu) was added during the hydration step; (2) Take 5 mL of liposome sample, place it in a dialysis bag with a molecular cutoff of 10,000 Da, place it in 200 mL of deionized water, and dialyze it at 4°C for 2 h to remove free GHK-Cu and obtain liposomes encapsulating GHK-Cu.

[0085] The morphology of CAPE liposomes encapsulating GHK-Cu and CAPE-SH-CHOL liposomes encapsulating GHK-Cu under scanning electron microscopy is shown in Figure 2. Figure 8 .

[0086] Comparative Example, Preparation of Conventional Liposomes (encapsulating GHK-Cu) 0.01 g of cholesterol and 0.09 g of soybean lecithin were completely dissolved in 10 mL of methanol-chloroform (volume ratio 1:2) mixed solution, and conventional liposomes were prepared by thin film dispersion method, and 15 mL of PBS buffer containing 0.3% GHK-Cu was added in the hydration step to obtain a conventional liposome dispersion containing GHK-Cu as a control. The preparation method of conventional liposomes, the set instrument parameters, and the determination methods of encapsulation and light-controlled release properties were the same as those in Example 2.

[0087] Example 3: Photoresponsiveness, encapsulation and light-controlled release of CAPE liposomes 1. Photoresponsiveness of CAPE liposomes The liposomes prepared in Example 2 and the comparative example were irradiated with natural light and UV-340 for 0, 2, 4 and 6 h, respectively. The particle size and dispersibility of each liposome were analyzed using a Malvern Zetasizer Nano ZS90 nanoparticle size and Zeta potential analyzer.

[0088] like Figure 4The particle size detection results shown in the figure show that under two lighting conditions, the particle sizes of CAPE and CAPE-SH-CHOL liposomes prepared in Example 2 are reduced, and the most significant result is obtained after 4 h of irradiation. After 4 h of UV-340 irradiation, the particle sizes of CAPE and CAPE-SH-CHOL liposomes are reduced by 25% and 16%, respectively, showing strong photoresponsiveness; after 4 h of irradiation under natural light, the particle sizes of the two CAPE liposomes are reduced by 12% and 27%, respectively, indicating that they are also photoresponsive under natural light, and the particle size change of CAPE-SH-CHOL liposomes under natural light is particularly significant. The reduction in particle size is conducive to better absorption. In contrast, the particle size of the conventional liposomes in the comparative example did not change significantly under the two lighting conditions.

[0089] like Figure 5 As shown in the PDI results, after 4 h of irradiation, the PDI values ​​of the CAPE and CAPE-SH-CHOL liposomes prepared in Example 2 increased significantly, indicating that the particle size distribution of the liposomes changed.

[0090] 0.06% sodium dodecyl sulfate (SDS) as an anionic additive and 0.1% cetyltrimethylammonium bromide (CTAB) as a cationic additive were added to the CAPE liposomes, and the Zeta potential was measured. Figure 6 As shown, at this addition amount, the absolute values ​​of the Zeta potential of the two are stable at about 20. This result shows that the added SDS and CTAB give the liposomes corresponding charges with moderate intensity, indicating that the liposomes exist stably and play a specific function.

[0091] 2. Encapsulation of CAPE liposomes The encapsulation of CAPE photoresponsive liposomes was analyzed as follows: (1) CAPE and CAPE-SH-CHOL liposomes were prepared according to the method of "3" in Example 2, and 0.3% of blue copper peptide (Glycyl-L-Histidyl-L-Lysine-Cu, GHK-Cu) was added in the hydration step; conventional liposomes (encapsulating GHK-Cu) prepared according to the "Comparative Example" in Example 2 were used as a control; (2) Take 5 mL of liposome sample, place it in a dialysis bag with a molecular cutoff of 10,000 Da, place it in 200 mL of deionized water, and dialyze it at 4°C for 2 h to remove free GHK-Cu; (3) Prepare GHK-Cu solutions of different concentrations, measure the absorbance at a wavelength of 215 nm, and draw a linear curve between absorbance and concentration. Measure the absorbance of the liposome samples at 215 nm before and after dialysis, and calculate the encapsulation efficiency of GHK-Cu by liposomes based on the standard curve.

[0092] The encapsulation efficiency of GHK-Cu was calculated according to the following formula: Encapsulation efficiency = (W1-W2) / W1*100%; Where: W1 is the total mass concentration of active substances added into liposomes; W2 is the mass concentration of free active substances.

[0093] The results of the encapsulation efficiency of GHK-Cu by different types of liposomes are shown in Table 1.

[0094] Table 1. Encapsulation efficiency of GHK-Cu by different types of liposomes Components Encapsulation rate (%) Comparative Example (Cholesterol, Phospholipids) 79 CAPE liposomes (phospholipids) 80 CAPE-SH-CHOL liposomes (phospholipids) 80

[0095] According to Table 1, the encapsulation efficiency of GHK-Cu by the two CAPE liposomes was 80%, which was comparable to the encapsulation efficiency of conventional liposomes prepared with cholesterol.

[0096] 3. Light-controlled release of CAPE liposomes The light-controlled release of CAPE photoresponsive liposomes was analyzed as follows: Two kinds of CAPE photoresponsive liposomes encapsulating GHK-Cu were prepared according to the above method. Each liposome sample was divided into four groups. Each group of samples was exposed to UV-340 for 0, 2, 4 and 6 h, respectively. After irradiation, the samples were dialyzed for 2 h to remove free GHK-Cu. A SHIMADZU high performance liquid chromatograph was used, equipped with a SHIMADZU-LC20A-C18 column (250 x 4.6 mm, particle size 5 μm), column temperature 35 ° C, mobile phases of pure water and acetonitrile, gradient elution, injection volume 10 μL, UV detection wavelength set to 215 nm, GHK-Cu retention time of 2.99 min. By comparing the peak area of ​​GHK-Cu under different irradiation times, the release amount of GHK-Cu was calculated.

[0097] The release amount of GHK-Cu was calculated according to the following formula: Release amount = AU0-AU i / AU0*100%; Of which: AU i is the HPLC peak area of ​​GHK-Cu contained in the liposomes after dialyzation under UV irradiation for i h, where i is 2, 4 and 6; AU0 is the HPLC peak area of ​​GHK-Cu contained in the liposomes after dialyzation without UV irradiation (i.e., irradiation for 0 h).

[0098] Figure 7The results show that the photo-controlled release performance of GHK-Cu by the two CAPE liposomes is significantly better than that of the control group. After 2 h of irradiation, the two CAPE liposomes showed a high release efficiency, and their GHK-Cu release was 10% to 15% higher than that of the control group. As the irradiation time was extended to 4 h, the GHK-Cu release of the two CAPE liposomes was 20% higher than that of the control group. After continuous irradiation for 6 h, the release of GHK-Cu by the two CAPE liposomes was stable at 63%, showing good photo-controlled release, while the release of the control group was 48%.

[0099] The results showed that photoresponsive liposomes prepared with CAPE and its complexes as membrane materials significantly promoted the release of active substances under UV-340 irradiation.

[0100] Example 4. Thermal stability of CAPE photoresponsive liposomes The two CAPE anionic liposomes containing GHK-Cu were placed under three different temperature conditions, namely 0°C, 25°C, and 40°C, and observed for two weeks to observe the changes in the color and morphology of the liposomes (Table 2).

[0101] When stored at 0°C and 25°C, both remained in a translucent blue suspension state with no significant color change, indicating that they have good stability at low and room temperature.

[0102] At 40°C, CAPE liposomes showed instability, which was manifested by slight precipitation of phospholipid components and color shift to yellow. CAPE-SH-CHOL liposomes showed almost no change at the same temperature, and the color and morphology remained stable, indicating that the introduction of thiol cholesterol may enhance the structural stability of liposomes and resist the influence of high temperature on GHK-Cu.

[0103] Table 2, Comparison of properties of examples and comparative examples

[0104] Example 5: Antioxidant activity of CAPE liposomes in vitro 1. DPPH free radical scavenging ability The antioxidant activity of the two CAPE liposomes was evaluated using a DPPH free radical scavenging ability kit. For specific steps, please refer to the kit instructions.

[0105] The assay tube A1 (containing the CAPE liposome sample to be tested and the DPPH solution), the control tube A2 (containing only the DPPH solution without the sample) and the blank tube A3 (containing the liposome sample to be tested and deionized water) were set up respectively. All the test tubes were kept at room temperature in the dark for 30 minutes to ensure that the reaction was fully carried out.

[0106] After the reaction is completed, the precipitate is removed by centrifugation, and the supernatant is collected. The absorbance value A of the solution in each tube at a wavelength of 517 nm is measured using a spectrophotometer, corresponding to the measurement tube A1, the control tube A2 and the blank tube A3 respectively.

[0107] Refer to the following formula to calculate the scavenging rate of DPPH free radicals: DPPH clearance rate (%) = [(A2 + A3) - A1] / A2 × 100%.

[0108] like Fig. 9 As shown in Figure c, both CAPE liposomes exhibited significant DPPH free radical scavenging ability. When the sample was diluted 10 times, the scavenging rates of DPPH free radicals reached 22.75% and 20.76%, respectively, indicating that both CAPE liposomes have antioxidant properties.

[0109] 2. Total antioxidant capacity of ABTS and FRAP The total antioxidant capacity of the two CAPE liposomes was evaluated using a total antioxidant capacity detection kit (ABTS method and FRAP method). For specific steps, please refer to the kit instructions.

[0110] Prepare Trolox solution and solution as a positive control, and deionized water as a negative control.

[0111] like Fig. 9 As shown in (a), the two CAPE liposomes exhibited different ABTS⁺ free radical scavenging abilities, and their antioxidant effects were equivalent to 0.31 mM and 0.46 mM Trolox equivalents, respectively, showing strong antioxidant properties.

[0112] like Fig. 9 As shown in b, two CAPE liposomes The reducing ability of liposomes was quite significant, reaching 0.33 mM and 0.46 mM Trolox equivalents respectively, which also proved that the introduction of CAPE gave the liposomes themselves significantly enhanced antioxidant capacity.

[0113] Therefore, the CAPE liposomes of the present invention have both photoresponsiveness and antioxidant efficacy, and can be better applied in the field of cosmetics.

[0114] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing caffeic acid phenethyl ester light-responsive liposomes, characterized in that: The method comprises: using caffeic acid phenethyl ester or a grafted complex of caffeic acid phenethyl ester and thiol cholesterol as a membrane material, interacting with lecithin, and preparing the caffeic acid phenethyl ester light-responsive liposome; the light comprises ultraviolet light or natural light.

2. The method for preparing the caffeic acid phenethyl ester light-responsive liposome according to claim 1, wherein: The caffeic acid phenethyl ester light-responsive liposome is obtained by a thin film dispersion method.

3. The method for preparing the caffeic acid phenethyl ester light-responsive liposome according to claim 1, wherein: The method comprises: (1) dissolving caffeic acid phenethyl ester or a grafted complex thereof with thiol cholesterol and lecithin in an organic solvent to obtain a lipid solution; (2) removing the organic solvent in the lipid solution of (1) to leave a lipid film; (3) Hydrate the lipid film of (2) and disperse it in a dispersion to obtain the caffeic acid phenethyl ester photoresponsive liposome dispersion.

4. The method for preparing caffeic acid phenethyl ester light-responsive liposomes according to claim 3, wherein: (1), the mass ratio of caffeic acid phenethyl ester or its grafted complex with thiol cholesterol to lecithin is 1:(5-80); or (2), removing the organic solvent by evaporation; or (3), hydrating and dispersing in a dispersion liquid with a pH of 7.4±0.4; the dispersion liquid comprises: phosphate buffer, physiological saline or water.

5. The method for preparing the caffeic acid phenethyl ester light-responsive liposome according to claim 1, wherein: The method for preparing the grafted complex of caffeic acid phenethyl ester and mercapto cholesterol comprises: (a) dissolving caffeic acid phenethyl ester in an alcohol medium, using copper ions as a catalyst and hydrogen peroxide as an oxidant, mixing and oxidizing to obtain an oxidation solution; adjusting the oxidation solution to neutrality, and drying to obtain an oxidation solid product; (b) dissolving the oxidized solid product and mercaptocholesterol in an organic solvent and reacting them under an alkaline environment to obtain the grafted composite.

6. The method for preparing the caffeic acid phenethyl ester photoresponsive liposome according to any one of claims 1 to 5, characterized in that: The lecithin includes: soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, or a combination thereof.

7. A caffeic acid phenethyl ester photoresponsive liposome prepared by the method according to any one of claims 1 to 6.

8. Use of the caffeic acid phenethyl ester photoresponsive liposome according to claim 7 in preparing cosmetics with an encapsulation layer.

9. A method for preparing a cosmetic having an encapsulation layer, comprising encapsulating an active substance using the caffeic acid phenethyl ester photoresponsive liposomes of claim 7.

10. The method for preparing a cosmetic having a coating layer according to claim 9, characterized in that: The active ingredients include: small molecule peptides, blue copper peptides, palmitoyl tripeptide, vitamin B, vitamin C or niacinamide, or a combination thereof.

11. A cosmetic having a wrapping layer, comprising: The caffeic acid phenethyl ester photoresponsive liposome according to claim 7 is used as an encapsulating layer, and the active substance encapsulated in the encapsulating layer.

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