Retinol retinoate-ceramide lipidosome modified by hyaluronic acid and salts thereof and preparation method of retinol retinoate-ceramide lipidosome

Abstract

The invention discloses retinol retinoate-ceramide lipidosome modified by hyaluronic acid and salts thereof and a preparation method of the retinol retinoate-ceramide lipidosome, and belongs to the technical field of cosmetic nano-carriers. The preparation method comprises the steps that retinol retinoate-ceramide lipidosome is prepared, hyaluronic acid is pretreated, and the hyaluronic acid and the salts thereof are combined to prepare the retinol retinoate-ceramide lipidosome. The liposome is modified by hyaluronic acid and salts thereof in a dual-mechanism manner, and purification and stabilization are realized; the preparation method of the retinol retinoate-ceramide liposome comprises the following steps: dissolving phospholipid, a functionalized lipid component with positive ions and amino functional groups, cholesterol, retinol retinoate and ceramide in absolute ethyl alcohol to serve as an organic phase, taking a phosphate buffer solution as a water phase, dropwise adding the organic phase into the water phase under a stirring condition, stirring, performing ultrasonic treatment, washing, and drying to obtain the retinol retinoate-ceramide liposome. Performing membrane extrusion to obtain a liposome suspension; the retinol retinoate-ceramide liposome modified by hyaluronic acid and salts thereof prepared by the invention has excellent stability, skin transmittance and anti-aging and moisturizing effects, also has low irritation, and is suitable for anti-aging cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetic nanocarrier technology, specifically to a hyaluronic acid and its salt-modified retinyl retinoic acid ester-ceramide liposome and its preparation method. Background Technology

[0002] As the largest organ in the human body, the skin is the first line of defense against external environmental stimuli. With consumers' increasing demands for skincare efficacy, active ingredients with outstanding skincare benefits are receiving growing attention. Among them, ceramides and retinol and its derivatives (such as retinyl retinoic acid esters) are widely recognized as two benchmark functional ingredients.

[0003] Retinyl retinoate (RR) is a highly effective retinol derivative that is more stable than retinol and can be converted into bioactive retinoic acid in the skin through an enzymatic reaction. Retinic acid can accelerate keratinocyte renewal and promote collagen and elastin production, thus effectively reducing wrinkles and improving photoaging and pigmentation. Compared to traditional retinol and retinoic acid, retinyl retinoate has potential advantages in efficacy and gentleness, but its prominent drawbacks in practical applications remain skin irritation and chemical instability. High concentrations can easily cause skin redness, peeling, and dryness; furthermore, they are extremely sensitive to light and oxygen and are easily degraded and inactivated in conventional formulations.

[0004] Ceramides are a major component of the intercellular lipids in the stratum corneum of the skin, accounting for approximately 50%. They play a crucial role in maintaining skin barrier function, locking in moisture, and resisting external stimuli. Supplementing with ceramides can effectively repair a damaged skin barrier and improve dry, rough, and sensitive skin. However, as a lipid-soluble, high-molecular-weight substance, ceramides have low transdermal penetration efficiency, making it difficult to effectively penetrate the stratum corneum to reach their target sites, thus limiting their bioavailability. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a hyaluronic acid and its salt-modified retinol retinoic acid ester-ceramide liposome and its preparation method. The prepared hyaluronic acid and its salt-modified retinol retinoic acid ester-ceramide liposome has excellent stability, skin permeability, and anti-aging and moisturizing effects, and also has low irritation, making it suitable for anti-aging cosmetics.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing retinol retinoic acid-ceramide liposomes modified with hyaluronic acid and its salts, comprising: preparing retinol retinoic acid-ceramide liposomes, pretreating hyaluronic acid, modifying liposomes with a dual mechanism of hyaluronic acid and its salts, and purifying and stabilizing; The preparation of retinyl retinoate-ceramide liposomes involves dissolving phospholipids, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinoate, and ceramide in anhydrous ethanol as the organic phase, and using a phosphate buffer solution as the aqueous phase. Under stirring conditions of 600-1200 rpm, the organic phase is added dropwise to the aqueous phase, stirred for 30-40 min, ultrasonicated, and extruded through a membrane to obtain a liposome suspension. In the preparation of retinol retinoic acid ester-ceramide liposomes, the phospholipid is at least one of soybean phospholipid, soybean lecithin, egg yolk lecithin, and hydrogenated lecithin; The functionalized lipid component with cationic and amino functional groups is a mixture of DOTAP and DSPE-PEG-NH2, wherein the molar ratio of DOTAP to DSPE-PEG-NH2 is 1:0.9-1.1; The functionalized lipid components with cationic and amino functional groups constitute 5-20% of the total lipids by mass. The mass ratio of phospholipids, cholesterol, retinyl retinate, and ceramide is 200:20-50:10-30:20-35. The ratio of the organic phase to the aqueous phase is 1:4-10; The phosphate buffer solution has a concentration of 10 mM and a pH value of 6.8-7.4; When adding the organic phase to the aqueous phase, the adding rate is 0.3-2.0 mL / min; During ultrasonic treatment, the power is 180-220W, the parameters are pulse 2-4s / interval 6-8s, and the time is 10-20min; During membrane extrusion, it is extruded 5-6 times through an 80nm polycarbonate membrane; The pretreatment of hyaluronic acid and its salts involves adding hyaluronic acid and its salts to ultrapure water, stirring and dissolving at room temperature, and adjusting the pH to 4.5-6.0 to obtain an aqueous solution of hyaluronic acid and its salts. In the pretreated hyaluronic acid and its salts, the hyaluronic acid and its salts are at least one selected from hyaluronic acid, sodium hyaluronate, zinc hyaluronate, copper hyaluronate, strontium hyaluronate, magnesium hyaluronate, and potassium hyaluronate; The molecular weight of the hyaluronic acid and its salts is 10-200 kDa; The mass concentration of the aqueous solution of hyaluronic acid and its salts is 0.1-1%; When adjusting the pH to 4.5-6.0, use a citric acid solution with a molar concentration of 0.02-0.1M. The hyaluronic acid and its salts are modified liposomes through a dual mechanism, including electrostatic adsorption and activated covalent coupling. The electrostatic adsorption involves dropping an aqueous solution of hyaluronic acid and its salts into a liposome suspension and stirring at 150-500 rpm for 0.5-2.0 h at room temperature to form an electrostatic complex of hyaluronic acid and its salts-liposomes. In the electrostatic adsorption process, the dripping rate of the aqueous solution of hyaluronic acid and its salts is 0.5-5 mL / min; The mass ratio of the aqueous solution of hyaluronic acid and its salts to the liposome suspension is 1:2-8; The activation covalent coupling involves adding EDC·HCl to hyaluronic acid and its salt-liposome electrostatic complex, stirring at 200-600 rpm for 0.25-1 h, then adding NHS, maintaining the pH at 4.5-6.0, and continuing stirring for 1-3 h to obtain the reaction solution. In the activated covalent coupling, the molar ratio of EDC・HCl to the total carboxyl groups in hyaluronic acid and its salts in the preparation of hyaluronic acid and its salts-liposome electrostatic complexes is 0.8-2.5:1; The molar ratio of NHS to EDC·HCl is 0.8-2:1; To maintain the pH at 4.5-6.0, adjust using a citric acid solution with a molar concentration of 0.02-0.1M; The purification and stabilization process involves purifying the reaction solution using an ultrafiltration centrifuge tube and centrifuge, taking the retentate, and washing the retentate with ultrapure water. After each washing, the solution is purified using an ultrafiltration centrifuge tube and centrifuge, and the retentate is taken to obtain hyaluronic acid-modified retinyl retinoate-ceramide liposomes. The ultrafiltration centrifuge tubes used in the purification and stabilization process are 5-15 kDa ultrafiltration centrifuge tubes; When washing the retentate with ultrapure water, the washing frequency is 3-5 times; Furthermore, the obtained retinyl retinoate-ceramide liposomes are preserved after adding a preservative, wherein the preservative is at least one selected from phenoxyethanol, chlorphenesin, benzyl alcohol, parabens, sorbic acid and its salts, benzoic acid and its salts, 1,2-hexanediol, ethylhexylglycerol and octyl glycol.

[0007] A hyaluronic acid and its salt-modified retinyl ester-ceramide liposome prepared by the aforementioned preparation method.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The preparation method of hyaluronic acid and its salt-modified retinol retinoate-ceramide liposomes of the present invention, compared with traditional retinol and retinoic acid, retinol retinoate has better chemical stability and potential skin mildness while retaining high anti-aging activity. It can slowly release retinoic acid in the skin through enzymatic conversion, which avoids the strong irritation caused by high concentration of retinoic acid and achieves continuous anti-wrinkle and collagen production effects, making it more suitable for use in daily skin care product systems.

[0009] (2) The preparation method of hyaluronic acid and its salt-modified retinol retinoic acid-ceramide liposomes of the present invention uses ethanol injection method to prepare liposomes that can simultaneously encapsulate lipophilic retinol retinoic acid and ceramide, significantly improving the solubility and formulation stability of the two. The liposome structure can isolate retinol retinoic acid from the external light and oxygen environment, slowing down its degradation. At the same time, through the high compatibility between the phospholipid bilayer and the stratum corneum of the skin, it promotes transdermal delivery of active ingredients, improves bioavailability, and reduces the risk of irritation from direct contact with the skin.

[0010] (3) The preparation method of hyaluronic acid and its salts-modified retinyl retinoate-ceramide liposomes of the present invention introduces cationic lipids into the lipid membrane material, making the liposomes cationic in whole. Then, utilizing the negatively charged properties of hyaluronic acid and its salt molecules, electrostatic adsorption and binding with the liposomes are first achieved. Subsequently, the carboxyl groups of hyaluronic acid are activated using the EDC / NHS system to form stable amide bonds, thereby constructing a dual-mechanism surface modification method of "HA negative charge - liposome positive charge electrostatic adsorption + HA activation covalent coupling". Compared with traditional modification methods that rely solely on physical adsorption or single chemical coupling, the present invention can achieve efficient and robust hyaluronic acid modification in a shorter time, significantly improving the stability of the carrier structure and active ingredients, making the obtained liposomes more uniform and stable under different storage conditions, and less prone to precipitation.

[0011] (4) The preparation method of hyaluronic acid and its salts modified retinol retinoate-ceramide liposomes of the present invention, by modifying the surface of liposomes with hyaluronic acid and its salts, not only endows the formulation with good moisturizing properties, but also enhances the affinity and adhesion of liposomes to the stratum corneum, thereby improving the skin permeability of retinol retinoate and ceramide, and solving the problems of poor transdermal permeability and low utilization of traditional liposomes.

[0012] (5) Liposomes are nano- or micron-sized carriers with closed vesicle structures formed by the self-assembly of phospholipid bilayers. In the cosmetics field, liposomes can significantly improve the transdermal performance, stability, and bioavailability of active ingredients. Their phospholipid bilayer structure has high biocompatibility with the intercellular lipids of the stratum corneum. Through mechanisms such as fusion, adsorption, and intercellular transport, they can promote the penetration of encapsulated hydrophilic or hydrophobic active ingredients through the skin barrier and target delivery to the deep epidermis and even the dermis. Liposomes not only solve the technical bottleneck of low transdermal permeability of active ingredients in traditional formulations, but also prolong the duration of action through sustained-release effects. Simultaneously, by isolating irritating ingredients from the skin, they effectively reduce the potential sensitization of the formulation. Therefore, applying liposome technology is an important strategy to overcome the barriers to the delivery of cosmetic active ingredients and achieve product efficiency and functionality. To overcome the problem of poor transdermal absorption of the active ingredients (retinyl retinoate and ceramide) and to improve their stability and reduce their irritation, this invention provides a method for preparing hyaluronic acid and its salts-modified retinyl retinoate-ceramide liposomes. First, liposomes are encapsulated using liposome technology. Then, hyaluronic acid (HA) and its salts are used to modify the surface of the liposomes. On one hand, hyaluronic acid and its salts possess excellent moisturizing properties and biocompatibility; when modified on the liposome surface, they can synergistically enhance skincare effects with retinyl retinoate and ceramide, and can also improve targeting by specifically binding to CD44 receptors on skin cell surfaces. On the other hand, thanks to the excellent bioadhesion and film-forming properties of hyaluronic acid and its salts, the modified liposomes can prolong the residence time of the active ingredients on the skin surface, preventing loss. Furthermore, the hydrophilic protective layer formed on the liposome surface by hyaluronic acid and its salts effectively prevents aggregation, fusion, and leakage of contents.

[0013] (6) The hyaluronic acid and its salt-modified retinyl retinoic acid-ceramide liposomes prepared in this invention have excellent stability, skin permeability, and anti-aging and moisturizing effects, and are also low in irritation, making them suitable for anti-aging cosmetics. The particle size of the prepared liposomes is 97.34-100.82 nm, and the particle size distribution index (PDI) is 0.101-0.115. The liposomes prepared in this invention were placed at 4°C for 90 days, and remained homogeneous without precipitation. They were also placed at 25°C for 90 days, and at 40°C for 30 days, and remained homogeneous without precipitation, indicating good storage stability. Using the Franz diffusion cell method, the cumulative skin permeability of the liposomes prepared in this invention over 12 hours was 45.6%-49.8%. The moisturizing performance was evaluated using an in vitro weighing method; the moisturizing rate of the liposome samples was 83.9%-88.5% after 4 hours and 70.8%-75.2% after 8 hours. Using human dermal fibroblasts (HDF) as a model, the cell scratch assay was used to evaluate the promoting effect of the liposomes prepared in this invention on cell migration / wound repair, with a cell migration rate of 28.9%-30.1%. Furthermore, using HDF as a model, the ability of the liposomes prepared in this invention to stimulate collagen secretion was tested. The relative Col-I generation rate was calculated using a blank control group as a baseline, and the relative Col-I generation rate of the liposomes prepared in this invention was 181.3%-182.5%. Attached Figure Description

[0014] Figure 1 The cell viability results are for the culture media treated in Examples 1-4 and Comparative Examples 1-2 in Performance Test 5. Detailed Implementation

[0015] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0016] Example 1 A method for preparing hyaluronic acid-modified retinyl retinoate-ceramide liposomes, specifically comprising: (1) Preparation of retinyl retinoate-ceramide liposomes: Soybean lecithin, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinoate and ceramide were dissolved in anhydrous ethanol as the organic phase, and 10mM pH7.4 phosphate buffer solution was used as the aqueous phase. Under stirring at 900rpm, the organic phase was added dropwise to the aqueous phase at a rate of 0.5mL / min and stirred continuously for 30min. The mixture was then sonicated at 200W (pulse 3s / interval 7s) for 10min. The mixture was then extruded through an 80nm polycarbonate membrane 5 times to obtain a liposome suspension. The mass ratio of soybean lecithin, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinate, and ceramide is 180:20:40:25:20. The functionalized lipid component with cationic and amino functional groups is a mixture of DOTAP and DSPE-PEG-NH2 (molar ratio 1:1). The ratio of the organic phase to the aqueous phase is 1:5; (2) Pretreatment of hyaluronic acid: Take hyaluronic acid with a molecular weight of 80kDa and add it to ultrapure water. Stir and dissolve at room temperature. Ensure that the concentration of hyaluronic acid is 0.25% during dissolution. Adjust the pH to 5.2 with 0.05M citric acid solution to obtain an aqueous solution of hyaluronic acid. (3) Hyaluronic acid dual-mechanism modification of liposomes: ① Electrostatic adsorption: The hyaluronic acid aqueous solution from step (2) is added dropwise to the liposome suspension from step (1) at a rate of 0.5 mL / min, and stirred at 300 rpm for 1 h at room temperature to form a hyaluronic acid-liposome electrostatic complex. The mass ratio of the hyaluronic acid aqueous solution to the liposome suspension is 1:4; ② Activation of covalent coupling: EDC・HCl was added to the above hyaluronic acid-liposome electrostatic complex, and the mixture was stirred at 400 rpm for 0.5 h to activate it. Then NHS was added to maintain the pH at 5.2 (adjusted with 0.05 M citric acid solution), and stirring was continued for 2 h to achieve HA covalent modification and obtain the reaction solution. The molar ratio of EDC・HCl to the total carboxyl groups in hyaluronic acid used in the preparation of the hyaluronic acid-liposome electrostatic complex is 2:1; The molar ratio of NHS to EDC·HCl is 1.5:1; (4) Purification and stabilization: The reaction solution in step (3) was purified by a 10kDa ultrafiltration centrifuge tube and a centrifuge. The retentate was taken and washed three times with ultrapure water. After each washing, the solution was purified by a 10kDa ultrafiltration centrifuge tube and a centrifuge and the retentate was taken to obtain hyaluronic acid modified retinyl retinoate-ceramide liposomes. 0.8% phenoxyethanol was added to the obtained retinyl retinoate-ceramide liposomes and then stored.

[0017] This embodiment also provides a hyaluronic acid-modified retinyl retinoic acid ester-ceramide liposome prepared by the aforementioned preparation method.

[0018] Example 2 A method for preparing retinol retinoic acid and its salt-modified retinyl ester-ceramide liposomes, specifically comprising: (1) Preparation of retinyl retinoate-ceramide liposomes: Soybean phospholipids, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinoate and ceramide were dissolved in anhydrous ethanol as the organic phase, and 10 mM pH 7.4 phosphate buffer solution was used as the aqueous phase. Under stirring conditions of 1000 rpm, the organic phase was added dropwise to the aqueous phase at a rate of 1 mL / min and stirred continuously for 30 min. The mixture was then sonicated at 200 W (pulse 3 s / interval 7 s) for 10 min and extruded 5 times through an 80 nm polycarbonate membrane to obtain a liposome suspension. The mass ratio of soybean phospholipids, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinoic acid esters, and ceramides is 170:30:30:30:35. The functionalized lipid component with cationic and amino functional groups is a mixture of DOTAP and DSPE-PEG-NH2 (molar ratio 1:1). The ratio of the organic phase to the aqueous phase is 1:8; (2) Pretreatment of sodium hyaluronate: Sodium hyaluronate with a molecular weight of 50 kDa was added to ultrapure water and stirred at room temperature to dissolve it. The concentration of the solution was 0.2%. The pH was adjusted to 5.5 with 0.05 M citric acid solution to obtain an aqueous solution of sodium hyaluronate. (3) Sodium hyaluronate dual-mechanism modification of liposomes: ① Electrostatic adsorption: The sodium hyaluronate aqueous solution from step (2) is added dropwise to the liposome suspension from step (1) at a rate of 1 mL / min, and stirred at 300 rpm for 1 h at room temperature to form a sodium hyaluronate-liposome electrostatic complex. The mass ratio of sodium hyaluronate to liposome suspension is 1:5; ② Activation of covalent coupling: EDC・HCl was added to the above sodium hyaluronate-liposome electrostatic complex, and the mixture was stirred at 400 rpm for 0.5 h to activate it. Then NHS was added to maintain the pH at 5.5 (adjusted with 0.05 M citric acid solution), and stirring was continued for 2 h to achieve HA covalent modification and obtain the reaction solution. The molar ratio of EDC・HCl to the total carboxyl groups in sodium hyaluronate used in the preparation of the sodium hyaluronate-liposome electrostatic complex is 1.5:1; The molar ratio of NHS to EDC·HCl is 1.2:1; (4) Purification and stabilization: The reaction solution in step (3) was purified by a 10kDa ultrafiltration centrifuge tube and a centrifuge. The retentate was taken and washed 4 times with ultrapure water. After each washing, the solution was purified by a 10kDa ultrafiltration centrifuge tube and a centrifuge and the retentate was taken to obtain sodium hyaluronate retinyl retinoate-ceramide liposomes. The obtained retinyl retinoate-ceramide liposomes were added to 0.8% phenoxyethanol and stored.

[0019] This embodiment also provides a sodium hyaluronate-modified retinyl retinoate-ceramide liposome prepared by the aforementioned preparation method.

[0020] Example 3 A method for preparing retinol retinoic acid and its salt-modified retinyl ester-ceramide liposomes, specifically comprising: (1) Preparation of retinyl retinoate-ceramide liposomes: Hydrogenated lecithin, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinoate and ceramide were dissolved in anhydrous ethanol as the organic phase, and 10 mM pH 6.8 phosphate buffer solution was used as the aqueous phase. Under stirring at 800 rpm, the organic phase was added dropwise to the aqueous phase at a rate of 0.3 mL / min. The mixture was stirred for 35 min, sonicated at 180 W (pulse 2 s / interval 6 s) for 20 min, and extruded 6 times through an 80 nm polycarbonate membrane to obtain a liposome suspension. The mass ratio of hydrogenated lecithin, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinate, and ceramide is 190:10:20:10:20. The functionalized lipid component with cationic and amino functional groups is a mixture of DOTAP and DSPE-PEG-NH2 (molar ratio 1:0.9); the ratio of the organic phase to the aqueous phase is 1:6. (2) Pretreatment of hyaluronic acid and its salts: Sodium hyaluronate with a molecular weight of 200 kDa was added to ultrapure water and stirred at room temperature to dissolve. The concentration of the solution was 0.5%. The pH was adjusted to 5.0 with 0.05 M citric acid solution to obtain an aqueous solution of sodium hyaluronate. (3) Sodium hyaluronate dual-mechanism modification of liposomes: ① Electrostatic adsorption: The sodium hyaluronate aqueous solution from step (2) was added dropwise to the liposome suspension from step (1) at a rate of 0.5 mL / min, and stirred at 150 rpm for 2.0 h at room temperature to form a sodium hyaluronate-liposome electrostatic complex. The mass ratio of the sodium hyaluronate aqueous solution to the liposome suspension is 1:2; ② Activation of covalent coupling: Add EDC・HCl to the above sodium hyaluronate-liposome electrostatic complex, stir at 200 rpm for 1 h to activate, then add NHS, maintain pH at 5.0 (adjusted with 0.05M citric acid solution), and continue stirring for 3 h to achieve covalent modification and obtain the reaction solution; The molar ratio of EDC・HCl to the total carboxyl groups in sodium hyaluronate used in the preparation of the sodium hyaluronate-liposome electrostatic complex is 1.2:1; The molar ratio of NHS to EDC·HCl is 1:1; (4) Purification and stabilization: The reaction solution in step (3) was purified by a 5kDa ultrafiltration centrifuge tube and a centrifuge. The retentate was taken and washed 5 times with ultrapure water. After each washing, the solution was purified by a 5kDa ultrafiltration centrifuge tube and a centrifuge and the retentate was taken to obtain sodium hyaluronate modified retinyl retinoate-ceramide liposomes. The obtained liposomes were added to 0.8% phenoxyethanol and stored.

[0021] This embodiment also provides a sodium hyaluronate-modified retinyl retinoate-ceramide liposome prepared by the aforementioned preparation method.

[0022] Example 4 A method for preparing retinol retinoic acid and its salt-modified retinyl ester-ceramide liposomes, specifically comprising: (1) Preparation of retinyl retinoate-ceramide liposomes: Egg yolk lecithin, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinoate and ceramide were dissolved in anhydrous ethanol as the organic phase, and 10 mM pH7.4 phosphate buffer solution was used as the aqueous phase. Under stirring conditions of 1200 rpm, the organic phase was added dropwise to the aqueous phase at a rate of 2.0 mL / min and stirred continuously for 30 min. The mixture was then sonicated at 220 W (pulse 4 s / interval 8 s) for 10 min and extruded 5 times through an 80 nm polycarbonate membrane to obtain a liposome suspension. The mass ratio of egg yolk lecithin, functionalized lipid components with cationic and amino functional groups, cholesterol, retinyl retinoic acid ester, and ceramide is 160:40:50:30:35. The functionalized lipid component with cationic and amino functional groups is a mixture of DOTAP and DSPE-PEG-NH2 (molar ratio 1:1.1); the ratio of the organic phase to the aqueous phase is 1:10. (2) Pretreatment of hyaluronic acid and its salts: Sodium hyaluronate with a molecular weight of 150 kDa was added to ultrapure water and stirred at room temperature to dissolve. The concentration of the solution was 1%. The pH was adjusted to 6.0 with 0.1 M citric acid solution to obtain an aqueous solution of sodium hyaluronate. (3) Sodium hyaluronate dual-mechanism modification of liposomes: ① Electrostatic adsorption: The sodium hyaluronate aqueous solution from step (2) was added dropwise to the liposome suspension from step (1) at a rate of 5 mL / min, and stirred at 500 rpm for 1.5 h at room temperature to form a sodium hyaluronate-liposome electrostatic complex. The mass ratio of the sodium hyaluronate aqueous solution to the liposome suspension is 1:8; ② Activation of covalent coupling: EDC・HCl was added to the above sodium hyaluronate-liposome electrostatic complex, and the mixture was stirred at 600 rpm for 0.5 h to activate it. Then NHS was added to maintain the pH at 6.0 (adjusted with 0.1 M citric acid solution), and stirring was continued for 1 h to achieve covalent modification and obtain the reaction solution. The molar ratio of EDC・HCl to the total carboxyl groups in sodium hyaluronate during the preparation of the sodium hyaluronate-liposome electrostatic complex is 2.5:1; The molar ratio of NHS to EDC·HCl is 2:1; (4) Purification and stabilization: The reaction solution in step (3) was purified by a 15kDa ultrafiltration centrifuge tube and a centrifuge. The retentate was taken and washed 4 times with ultrapure water. After each washing, the solution was purified by a 15kDa ultrafiltration centrifuge tube and a centrifuge and the retentate was taken to obtain sodium hyaluronate modified retinyl retinoate-ceramide liposomes. The obtained liposomes were added to 0.8% phenoxyethanol and stored.

[0023] This embodiment also provides a sodium hyaluronate-modified retinyl retinoate-ceramide liposome prepared by the aforementioned preparation method.

[0024] Comparative Example 1 In the preparation method of sodium hyaluronate modified retinol retinoate-ceramide liposomes in Example 2, no functionalized lipid components with cationic and amino functional groups are added in step (1), and steps (2) and (3) are omitted. That is, the retinol retinoate-ceramide liposome suspension prepared in step (1) is not modified with hyaluronic acid, and the remaining operations are the same as in Example 2.

[0025] Comparative Example 2 In the preparation method of sodium hyaluronate modified retinol retinoate-ceramide liposomes in Example 2, the functionalized lipid component with cationic and amino functional groups in step (1) was replaced with an equal mass of DOTAP, and the activation covalent coupling in step (3) was omitted. That is, the retinol retinoate-ceramide liposome suspension prepared in step (1) was only modified by electrostatic adsorption of sodium hyaluronate, without covalent coupling modification. The remaining operations were the same as in Example 2.

[0026] Performance Test 1 The particle size and particle size distribution index of the retinol retinate-ceramide liposomes prepared in Examples 1-4 and Comparative Examples 1-2 were determined using a laser particle size analyzer, and the results are shown in Table 1. Table 1. Test results of particle size and particle size distribution index

[0027] Performance Test 2 The liposome samples prepared in Examples 1-4 and Comparative Examples 1-2 were sealed in brown spiral glass bottles and placed in environments of 4°C (refrigeration), 25°C (room temperature), and 40°C (accelerated), respectively. The appearance (precipitation, sedimentation, and stratification) was observed periodically, as shown in Table 2. Table 2 Appearance Changes

[0028] Performance Test 3 Using isolated porcine skin as a transdermal barrier, the skin permeability of liposome samples prepared in Examples 1-4 and Comparative Examples 1-2 was tested using the Franz diffusion cell method. The receiving solution was PBS buffer with a pH of 7.4. The samples were maintained at a constant temperature of 32°C with magnetic stirring and were sampled at 2h, 4h, 8h, and 12h. The retinol retinate content in the receiving solution was determined by high-performance liquid chromatography (HPLC), and the cumulative skin permeability over 12h was calculated. The results are shown in Table 3. Table 3. Skin permeability of retinyl retinyl esters

[0029] Performance Test 4 The moisturizing properties of liposome samples were evaluated using an in vitro weighing method. 1g of liposome samples prepared in Examples 1-4 and Comparative Examples 1-2 were uniformly spread in a 3.5cm diameter weighing dish and placed in a desiccator containing color-changing silica gel. Weighing was performed every hour to record the amount of water evaporation, and the moisturizing rate was calculated. Moisturizing rate (%) = (weight at time / initial weight) × 100%. A blank buffer solution was used as a control. Table 4 shows the results. Table 4 Moisturizing Rate

[0030] Performance Test 5 Cytotoxicity was assessed using a cell counting kit (CCK-8). HDFa was digested, counted, and seeded into 96-well cell culture plates (approximately 8 × 10⁶ cells per well). 3 Cells were cultured at 100 μL / well at 37°C and 5% CO2 for 24 h to adhere. The culture medium was then discarded, and culture medium (100 μL / well, 3 replicates per group) of samples from Examples 1-4 and Comparative Examples 1-2 diluted according to the set concentration gradients was added. Culture was continued for 48 h. After culture, CCK-8 reagent (10% of the culture medium volume) was added, and the cells were incubated in the dark for 1 h. The absorbance was measured at 450 nm using a microplate reader to calculate cell viability. Figure 1 .

[0031] Performance Test 6 Using human dermal fibroblasts (HDF) as a model, the cell scratch assay was used to evaluate the promoting effect of retinol retinoic acid ester-ceramide liposomes prepared in Examples 1-4 and Comparative Examples 1-2 on cell migration / wound repair. HDF cells were seeded in 6-well cell culture plates and cultured until cell confluence reached 90-100%. A sterile 200 μL pipette tip was used to create a uniform, straight scratch on the surface of the cell monolayer to form a "wound." The cells were gently washed with PBS to remove detached cells. A sample solution (retinol retinoic acid ester concentration of 10 μg / mL) prepared with serum-containing medium was then added, and the cells were cultured at 37°C and 5% CO2. The scratch area was photographed at 0 h and 24 h under the same field of view, and the scratch area was measured using image analysis software. Cell migration rate was calculated with 0 h as the baseline: Cell migration rate (%) = (A0 − A t ) / A0×100% (where A0 is the scratch area at 0h, A t (where t is the area of ​​the scratch), resulting in Table 5.

[0032] Table 5 Cell migration rate

[0033] Performance Test 7 Using human dermal fibroblasts (HDF) as a model, the ability of retinol retinoic acid ester-ceramide liposomes prepared in Examples 1-4 and Comparative Examples 1-2 to stimulate collagen secretion was tested. During the test, a blank control group and a sample group (the sample group consisted of retinol retinoic acid ester-ceramide liposomes prepared in Examples 1-4 and Comparative Examples 1-2) were set up. In 12-well cell culture plates, human dermal fibroblasts (HDF) were mixed with the liposomes prepared in Examples 1-4 and Comparative Examples 1-2 (final retinol retinoic acid concentration was 10 μg / mL). After culturing at 37℃ and 5% CO2 for 48 h, the production of type I collagen (Col-I) was detected using an ELISA kit. The relative production rate of Col-I was calculated based on the blank control group, and the results are shown in Table 6. Table 6. Relative Generation Rate of Col-I

[0034] The results of performance test 1 show that, compared with Comparative Examples 1 and 2, the retinol retinoic acid ester-ceramide liposomes prepared in Examples 1-4 have the advantage of uniform particle size. The results of performance test 2 show that, compared with Comparative Examples 1 and 2, the retinol retinoic acid ester-ceramide liposomes prepared in Examples 1-4 are more uniform and stable under different storage conditions and less prone to precipitation. The results of performance tests 3-7 show that, compared with Comparative Examples 1 and 2, the retinol retinoic acid ester-ceramide liposomes prepared in Examples 1-4 are more easily able to penetrate the skin barrier, have lower cytotoxicity, and exhibit good moisturizing and anti-aging effects.

[0035] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a hyaluronic acid and its salts modified retinol retinoate-ceramide liposome, characterized by, The application relates to a preparation method of retinol retinoate-ceramide liposomes, pretreatment of hyaluronic acid, a double-mechanism modified liposome of hyaluronic acid and its salts, purification and stabilization. The preparation method of the retinol retinoate-ceramide liposomes comprises the following steps: dissolving phospholipid, a functionalized lipid component with cationic and amino functional groups, cholesterol, retinol retinoate and ceramide in anhydrous ethanol as an organic phase, dissolving a phosphate buffer solution as an aqueous phase, dropping the organic phase into the aqueous phase under stirring, stirring, ultrasonic treatment and membrane extrusion to obtain a liposome suspension. The pretreatment of the hyaluronic acid and its salts comprises the following steps: adding the hyaluronic acid and its salts into ultrapure water, stirring and dissolving at room temperature, adjusting the pH to 4.5-6.0 to obtain a hyaluronic acid and its salts aqueous solution. The double-mechanism modified liposome of the hyaluronic acid and its salts comprises the following steps: electrostatic adsorption and activated covalent coupling. The electrostatic adsorption comprises the following steps: dropping the hyaluronic acid and its salts aqueous solution into the liposome suspension, stirring at room temperature to form a hyaluronic acid and its salts-liposome electrostatic complex. The activated covalent coupling comprises the following steps: adding EDC.HCl into the hyaluronic acid and its salts-liposome electrostatic complex, stirring for 0.25-1 h, then adding NHS, maintaining the pH at 4.5-6.0 and continuing to stir for 1-3 h to obtain a reaction solution. In the preparation method of the retinol retinoate-ceramide liposomes, the phospholipid is at least one of soybean phospholipid, soybean lecithin, egg yolk lecithin and hydrogenated lecithin.

2. The method for preparing hyaluronic acid and salt-modified retinyl retinoate-ceramide liposomes according to claim 1, characterized by, The functionalized lipid component with cationic and amino functional groups is a mixture of DOTAP and DSPE-PEG-NH2, wherein the molar ratio of DOTAP to DSPE-PEG-NH2 is 1:0.9-1.

1. The mass fraction of the functionalized lipid component with cationic and amino functional groups is 5-20% of the total lipid. In the preparation method of the retinol retinoate-ceramide liposomes, the mass ratio of the phospholipid, cholesterol, retinol retinoate and ceramide is 200:20-50:10-30:20-35.

3. The method for preparing hyaluronic acid and salt-modified retinyl retinoate-ceramide liposomes according to claim 1, characterized by, The ratio of the organic phase to the aqueous phase is 1:4-10. The concentration of the phosphate buffer solution is 10 mM, and the pH value is 6.8-7.

4. When the organic phase is dropped into the aqueous phase, the dropping speed is 0.3-2.0 mL / min. When the ultrasonic treatment is performed, the power is 180-220 W, the parameters are pulse 2-4 s / interval 6-8 s, and the time is 10-20 min. When the membrane extrusion is performed, the membrane extrusion is performed 5-6 times through a 80 nm polycarbonate membrane. In the pretreatment of the hyaluronic acid and its salts, the hyaluronic acid and its salts are at least one of hyaluronic acid, sodium hyaluronate, zinc hyaluronate, copper hyaluronate, strontium hyaluronate, magnesium hyaluronate and potassium hyaluronate.

4. The method for preparing hyaluronic acid and salt-modified retinyl retinoate-ceramide liposomes according to claim 1, characterized by, The molecular weight of the hyaluronic acid and its salts is 10-200 kDa. The mass concentration of the hyaluronic acid and its salts aqueous solution is 0.1-1%. When the pH is adjusted to 4.5-6.0, a citric acid solution with a molar concentration of 0.02-0.1 M is used for adjustment. ​ 5. The method for preparing hyaluronic acid and salt-modified retinyl retinoate-ceramide liposomes according to claim 1, characterized by, The speed of dropping the hyaluronic acid and its salts aqueous solution is 0.5-5 mL / min. The mass ratio of the hyaluronic acid and its salts aqueous solution to the liposome suspension is 1:2-8.

6. The method for preparing hyaluronic acid and salt-modified retinyl retinoate-ceramide liposomes according to claim 1, characterized by, In the activation covalent coupling, the molar ratio of EDC·HCl to the total amount of carboxyl groups in the hyaluronic acid and its salts in the preparation of the hyaluronic acid and its salts-liposome electrostatic complex is 0.8-2.5:

1. The molar ratio of NHS to EDC·HCl is 0.8-2:

1. The pH is maintained at 4.5-6.0 by using a 0.02-0.1 M citric acid solution.

7. The method for preparing hyaluronic acid and salt-modified retinyl retinoate-ceramide liposomes according to claim 1, characterized by, In the purification and stabilization, the reaction solution is purified by an ultrafiltration centrifuge tube and a centrifuge, the cut-off liquid is taken, the cut-off liquid is washed with ultrapure water, and after each washing, the cut-off liquid is purified by an ultrafiltration centrifuge tube and a centrifuge, and the cut-off liquid is taken, thereby obtaining the hyaluronic acid modified retinol retinoate-ceramide liposome.

8. The method for preparing hyaluronic acid and salt-modified retinyl retinoate-ceramide liposomes according to claim 7, characterized by, In the purification and stabilization, the ultrafiltration centrifuge tube used is a 5-15 kDa ultrafiltration centrifuge tube. When the cut-off liquid is washed with ultrapure water, the washing is performed 3-5 times. The obtained retinol retinoate-ceramide liposome is stored after adding a preservative, and the preservative is at least one of phenoxyethanol, chlorphenesin, benzyl alcohol, parabens, sorbic acid and its salts, benzoic acid and its salts, 1,2-hexanediol, ethylhexylglycerin, and caprylyl glycol.

9. A hyaluronic acid and its salts modified retinol retinoate-ceramide liposome prepared by the preparation method of any one of claims 1-8.

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