Method for preparing folic acid modified plant exosome anti-photoaging gel through cross-linking method

Abstract

The invention discloses a method for preparing folic acid modified plant exosome light-aging-resistant gel through a cross-linking method, and relates to the technical field of cosmetic biological materials.The method comprises the specific steps that firstly, plant exosomes are subjected to extraction purification and low-temperature plasma pretreatment; respectively activating folic acid and TAT skin penetrating peptide, coupling the folic acid and the TAT skin penetrating peptide to the surface of the exosome step by step, and purifying to obtain a co-modified exosome; the preparation method comprises the following steps: preparing a co-modified exosome, then synthesizing a difunctional cross-linking agent by using genipin and EGCG, finally modifying hyaluronic acid, adding the co-modified exosome and the cross-linking agent for composite cross-linking, and performing post-treatment to obtain the target gel. Through low-temperature plasma pretreatment and folic acid-TAT step-by-step coupling, the exosome modification efficiency and the targeted transdermal ability are improved; by combining an EGCG-genipin difunctional cross-linking agent with chemical-optical composite cross-linking, gel stability and efficacy synergy are optimized, accurate release of active ingredients is realized, and efficacy waste is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetic biomaterials, in particular to a method for preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking. BACKGROUND

[0002] Skin photoaging is a phenomenon of skin aging caused by long-term ultraviolet radiation. The core mechanism is that ultraviolet radiation induces skin cells to produce a large number of active oxygen free radicals, triggering oxidative stress, damaging DNA, proteins, and lipids, and activating matrix metalloproteinases to degrade collagen and elastic fibers, ultimately leading to increased wrinkles, decreased elasticity, and pigmentation. With the upgrading of consumer anti-aging needs, plant exosomes are gradually becoming the core raw material of anti-photoaging skincare products due to their rich natural active ingredients such as polyphenols and flavonoids, excellent antioxidant, anti-inflammatory, and skin repair functions. However, plant exosomes need to solve key technical problems such as targeted delivery, transdermal absorption, and stability in practical application, and the innovation and optimization of related preparation processes have become a research hotspot in the field of cosmetic biomaterials.

[0003] The preparation of traditional anti-photoaging plant exosome products has a fixed mode: the modification link mostly uses direct chemical activation to modify plant exosomes with single targeting or penetration, the cross-linking link mostly uses a single functional natural cross-linking agent or chemical cross-linking agent to construct a gel matrix, and the carrier design mostly uses a non-responsive gel to load exosomes. However, these traditional methods have obvious shortcomings: direct chemical activation can easily lead to inactivation of exosome active ingredients, single modification cannot balance precise targeting and transdermal absorption; natural cross-linking agents can only maintain structural stability, chemical cross-linking agents have safety hazards, and additional antioxidants can easily cause compatibility problems; non-responsive gels lead to early loss or non-targeted release of active ingredients, resulting in low efficacy utilization. These problems greatly reduce the anti-photoaging effect of existing products and make it difficult to meet consumer demand for high-efficiency and safe skincare products. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provides a method for preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking. This method activates the surface of plant exosomes through low-temperature plasma pretreatment technology, and combines step-by-step coupling of folic acid and TAT skin penetration peptides to significantly improve the targeted transdermal ability of exosomes. EGCG-geni-pen bifunctional cross-linking agent is used to achieve chemical-photocrosslinking, enhancing the stability and efficacy of the gel. This process ensures the precise release of active ingredients, solving the problem of non-targeted release of traditional gels.

[0005] To solve the above technical problems, the present application provides the following technical solution: a method for preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking, the specific steps of which are as follows: S1, Plant exosome extraction, purification and pretreatment: Fresh plant tissues were selected, and after being crushed, homogenized and purified by centrifugation, the plant exosomes were pretreated by low-temperature plasma. S2, Preparation of plant exosomes co-modified with folic acid-penetrating peptide: Folic acid and TAT skin-penetrating peptide were activated separately and coupled to the surface of pretreated plant exosomes in steps, and the folic acid-penetrating peptide co-modified plant exosomes were obtained by purification. S3, Synthesis of bifunctional crosslinking agent: Using genipin and epigallocatechin gallate as raw materials, EGCG-genipin grafted bifunctional crosslinking agent was obtained by transesterification reaction, purification and drying. S4, UV-responsive gel preparation: Hyaluronic acid was modified with o-nitrobenzyl, and folic acid-penetrating peptide was added to co-modify plant exosomes and a bifunctional cross-linking agent for composite cross-linking. The target gel was obtained after post-treatment.

[0006] Further, in step S1, the plant tissue is selected from one or more of green tea, aloe vera, ginkgo leaves, and grape seeds; centrifugal purification includes low-speed centrifugation to remove impurities, high-speed centrifugation for enrichment, and sucrose density gradient centrifugation purification; the sucrose density gradient is 10%-40%, and the density gradient centrifugation conditions are 100,000-120,000g for 60-90 minutes; the low-temperature plasma pretreatment conditions are argon atmosphere and vacuum degree 10. -3 -10 -2 Pa, processing power 50-80W, processing time 15-25s.

[0007] Furthermore, in step S1, the homogenate is prepared using PBS buffer with a pH of 7.2-7.4, and the ratio of plant tissue to PBS buffer is 1:10-1:20; the low-speed centrifugation conditions are 3000-5000 r / min for 15-20 min; the high-speed centrifugation conditions are 10000-12000 r / min for 30-40 min; and the resuspended concentration of plant exosomes before pretreatment is 1-2 mg / mL.

[0008] Furthermore, in step S2, the sequence of the TAT skin-penetrating peptide is YGRKKRRQRRR; the folic acid activation conditions are: folic acid concentration 10-20 mg / mL, molar ratio of folic acid to EDC and NHS 1:1.2-1.5:1.2-1.5, activation at 25-30℃ for 30-40 min; the TAT skin-penetrating peptide activation conditions are: concentration 5-10 mg / mL, molar ratio of TAT to EDC and NHS 1:1.2-1.5:1.2-1.5, activation at 25-30℃ for 20-30 min.

[0009] Furthermore, in step S2, the specific steps of the stepwise coupling are as follows: first, the activated folic acid is coupled with the pretreated plant exosomes, and then the activated TAT skin-penetrating peptide is coupled with the folic acid-modified plant exosomes. The stepwise coupling conditions are: the mass ratio of folic acid to plant exosomes is 1:5-1:10, and the reaction is carried out at 37°C in the dark for 1-1.5 hours. Then, the TAT skin-penetrating peptide activation solution is added at a molar ratio of folic acid to TAT of 2:1-3:1, and the reaction is continued at 37°C in the dark for 1-1.5 hours. The purification is carried out by centrifugation using a 100kDa ultrafiltration membrane at a speed of 5000-6000 r / min for 15-20 minutes, and repeated 2-3 times.

[0010] Furthermore, in step S3, the transesterification reaction conditions are as follows: the molar ratio of genipin to EGCG is 1:1 to 1:1.2; N,N-dimethylformamide is used as the solvent; 4-dimethylaminopyridine is used as the catalyst; the catalyst addition amount is 5%-8% of the mass of genipin; the reaction is carried out under reflux at 60-70℃ for 4-6 hours; purification is performed by dialyzing with a 3000Da dialysis bag for 48 hours, with water changed every 6 hours; and drying is performed by freeze drying.

[0011] Furthermore, in step S4, the o-nitrobenzyl modification conditions are as follows: the mass ratio of hyaluronic acid to 2-bromoethyl o-nitrobenzyl ether is 1:0.3-1:0.5, the pH value is 8.5-9.0, the reaction temperature is 40-50℃ for 8-10 h, and the purification is performed by dialyzing with a 5000Da dialysis bag for 24 h; the mass concentration of the UV-responsive hyaluronic acid solution is 1%-3%; and the mass fraction of folic acid-penetrating peptide co-modified plant exosomes in the gel system is 0.5%-2%.

[0012] Furthermore, in step S4, the composite crosslinking is a combination of chemical crosslinking and photocrosslinking. Specifically, the bifunctional crosslinking agent is added at 5%-10% of the mass of the UV-responsive hyaluronic acid. The first step is chemical crosslinking initiated by genipin, with a pH of 6.5-7.5, a temperature of 30-40℃, and a time of 1-2 hours. The second step is photocrosslinking initiated by UV irradiation, with irradiation conditions of a 365nm wavelength and a power of 10-20mW / cm². 2 The process involves 5-10 min for initial homogenization, followed by degassing and sterilization. The homogenization speed is 10000-15000 r / min, and the time is 5-10 min. The vacuum degassing is performed at a vacuum degree of -0.08 to 0.1 MPa for 10-15 min. The sterilization method is irradiation sterilization, with an irradiation dose of 25-30 kGy.

[0013] Compared with existing technologies, this cross-linking method for preparing folic acid-modified plant exosome anti-photoaging gel has the following advantages: I. This invention improves the modification efficiency and functional integrity of plant exosomes through a synergistic design of low-temperature plasma pretreatment and stepwise coupling of folic acid-TAT skin-penetrating peptide. Low-temperature plasma activates the surface of plant exosomes under an argon atmosphere, enriching the types and quantities of surface-active groups and providing sufficient reaction sites for subsequent dual modification. This effectively avoids the destruction of natural active ingredients within exosomes by traditional chemical activation methods. The co-modification design of folic acid and TAT skin-penetrating peptide breaks free from the functional limitations of single modification. It achieves precise targeted recognition by leveraging the specific binding of folic acid to receptors on the surface of aging skin cells, and also utilizes the membrane-penetrating properties of TAT peptide to help exosomes overcome the skin's stratum corneum barrier, allowing active ingredients to reach the dermis more efficiently and exert their effects, thus enhancing the targeting and transdermal delivery efficiency of anti-photoaging efficacy.

[0014] II. This invention innovatively integrates an EGCG-genipin grafted bifunctional crosslinking agent with a chemical-photocomposite crosslinking system, simultaneously enhancing the stability and synergistic efficacy of the gel. The bifunctional crosslinking agent covalently binds EGCG and genipin through an ester exchange reaction, retaining genipin's ability to construct a stable three-dimensional gel network while leveraging EGCG's natural antioxidant activity to synergize with plant exosomes. This avoids the problem of poor component compatibility without the need for additional antioxidants. The combined chemical and photocrosslinking approach balances the gel's storage stability and application responsiveness. During daily storage, the structure remains stable; during use, UV irradiation triggers responsive reconstruction only at photo-aged damaged areas, enabling precise on-demand release of plant exosomes. This solves the problems of non-targeted release and wasted efficacy associated with traditional gels.

[0015] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0017] Figure 1 This is a process flow diagram of the preparation method of the present invention; Figure 2 Flowchart for the preparation of plant exosomes co-modified with folic acid-penetrating peptides; Figure 3 This is a flowchart of the composite crosslinking process of UV-responsive gel. Detailed Implementation

[0018] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0019] Example 1 Raw material and equipment preparation: Ingredients: 50g fresh green tea, PBS buffer pH 7.3, folic acid, TAT skin-penetrating peptide sequence YGRKKRRQRRR, EDC, NHS, dimethyl sulfoxide, genipin, EGCG, N,N-dimethylformamide, 4-dimethylaminopyridine, hyaluronic acid, 2-bromoethyl o-nitrobenzyl ether, sterile deionized water; Equipment: High-speed refrigerated centrifuge, plasma treatment instrument, constant temperature magnetic stirrer, reflux reaction device, dialysis bags with molecular weight cutoff of 3000Da and 5000Da, freeze dryer, 365nm UV irradiator, high-speed homogenizer, vacuum degasser, and irradiation sterilization equipment.

[0020] The specific preparation steps are as follows: S1, Extraction, purification and pretreatment of plant exosomes Fresh green tea leaves were rinsed three times with sterile deionized water, drained, and then ground in a high-speed grinder to a particle size of 1-2 mm. Subsequently, 750 mL of PBS buffer was added at a material-to-liquid ratio of 1:15. The mixture was placed in an ice bath and homogenized continuously for 25 min using a high-speed homogenizer to obtain a fine green tea homogenate. The homogenate was transferred to a centrifuge tube and centrifuged at 3800 r / min for 18 min. The tissue fragments at the bottom were discarded, and the clear supernatant was collected.

[0021] Carefully transfer the collected supernatant to an ultracentrifuge tube and centrifuge at 11,000 rpm for 35 minutes. After centrifugation, collect the precipitate at the bottom of the tube and resuspend it three times with 50 mL of PBS buffer to obtain a homogeneous resuspension. Prepare a 10%-40% sucrose density gradient centrifugation buffer in advance. Slowly spread the resuspension on top of the density gradient buffer and centrifuge at 110,000 g for 75 minutes. After centrifugation, pipette the white turbid fraction with a density between 1.10 and 1.18 g / mL, which is the purified plant exosome.

[0022] The purified plant exosomes were diluted to a concentration of 1.5 mg / mL with PBS buffer and transferred to a plasma processing instrument. The processing parameters were set to argon atmosphere and a vacuum degree of 5 × 10⁻⁶. -3The equipment was started with a pressure of Pa, a processing power of 65W, and a processing time of 20s to complete the surface activation treatment. The treated plant exosome suspension was then refrigerated at 4°C for later use. Figure 1 As shown.

[0023] S2, preparation of plant exosomes co-modified with folic acid-penetrating peptide Weigh 3.75 mg of folic acid and add 0.2 mL of dimethyl sulfoxide. Stir with a magnetic stirrer until completely dissolved. Then add 4.65 mg of EDC and 3.28 mg of NHS. Incubate the reaction system at 28°C and continuously stir for 35 min to obtain the folic acid activated solution. In another beaker, weigh 1.5 mg of TAT skin-penetrating peptide and add 0.3 mL of PBS buffer. Stir to dissolve, then add 1.88 mg of EDC and 1.31 mg of NHS. Similarly, activate the solution at 28°C for 25 min to obtain the TAT activated solution.

[0024] Folic acid activation solution was slowly added dropwise to 20 mL of surface-activated plant exosome suspension, with continuous stirring during the addition. After the addition was complete, the mixture was placed in a dark environment at 37°C and stirred for 1.2 h. Then, TAT activation solution was slowly added dropwise to the reaction system, maintaining the same temperature and dark conditions, and the reaction was continued with stirring for another 1.2 h. After the reaction was complete, the reaction solution was transferred to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 5500 rpm for 18 min, discarding the supernatant. The retentate was resuspended in PBS buffer, and the ultrafiltration centrifugation was repeated three times. The final collected retentate is the folic acid-TAT co-modified plant exosome suspension. Figure 2 As shown.

[0025] S3, Synthesis of Bifunctional Crosslinking Agent Weigh 50 mg of genipin and place it in a reaction flask containing 1 mL of N,N-dimethylformamide. Stir until completely dissolved. Then add 45.8 mg of EGCG and 3 mg of 4-dimethylaminopyridine, and continue stirring until well mixed. Place the reaction flask in a 65°C oil bath, turn on the reflux device, and stir continuously for 5 hours. Observe the solution state every 30 minutes during the reaction until the solution becomes light yellow and transparent.

[0026] After the reaction was completed, the reaction solution was transferred to a 3000 Da dialysis bag and dialyzed using sterile deionized water as the dialysis solution for 48 hours, with the dialysis solution being changed every 6 hours. After dialysis, the solution in the bag was transferred to a freeze dryer and freeze-dried for 24 hours to obtain a white powdery EGCG-genipin grafted bifunctional crosslinking agent, which was then sealed and stored at 4°C for later use.

[0027] S4, UV-responsive gel preparation Weigh 2g of hyaluronic acid and add it to 100mL of sterile deionized water. Stir at 40℃ until completely dissolved. Then add 0.6g of 2-bromoethyl o-nitrobenzyl ether, adjust the pH of the system to 8.8 with 0.1mol / L NaOH solution, and maintain the reaction at 45℃ with stirring for 9h. After the reaction is complete, transfer the reaction solution to a 5000Da dialysis bag and dialyze with sterile deionized water for 24h. After dialysis, freeze-dry to obtain UV-responsive hyaluronic acid.

[0028] Weigh 2g of UV-responsive hyaluronic acid and add it to 100mL of sterile deionized water. Stir at 35℃ for 30min until completely dissolved to obtain a 2% (w / w) UV-responsive hyaluronic acid solution. Add an appropriate amount of folic acid-TAT co-modified plant exosome suspension to this solution and continue stirring for 30min to achieve a plant exosome mass fraction of 0.5% in the gel system.

[0029] Weigh 0.1 g of EGCG-genipin grafted bifunctional crosslinking agent and add it to the above solution. Adjust the pH of the system to 7.0 with 0.1 mol / L HCl solution. Stir the reaction mixture at 35℃ for 1.5 h to complete the first step of chemical crosslinking. Then, place the reaction solution in a 365 nm UV irradiator and set the irradiation power to 15 mW / cm². 2 Irradiation time was 8 minutes, followed by the second step of photocrosslinking to obtain the initial gel product.

[0030] The initial gel sample was placed in a high-speed homogenizer and homogenized at 12000 rpm for 8 minutes to achieve a uniform and fine gel texture. It was then transferred to a vacuum degasser and degassed at -0.09 MPa for 12 minutes to remove air bubbles. Finally, it was sterilized using an irradiation dose of 28 kGy to obtain a folic acid-penetrating peptide co-modified plant exosome UV-responsive anti-photoaging gel. Figure 3 As shown.

[0031] Targeted testing showed that the amount of exosomes binding to aging cells in the dermis was 1.8 times that of unmodified exosomes; the DPPH free radical scavenging rate reached 75%, demonstrating good antioxidant capacity; and the exosome release rate after UV irradiation was 55%, showing stable release performance. This gel is gentle and low-irritant, with low raw material costs, making it suitable as a raw material for skincare products targeting sensitive skin or those with basic anti-aging needs.

[0032] Example 2 Raw material and equipment preparation: Ingredients: 50g fresh green tea, PBS buffer pH 7.3, folic acid, TAT skin-penetrating peptide sequence YGRKKRRQRRR, EDC, NHS, dimethyl sulfoxide, genipin, EGCG, N,N-dimethylformamide, 4-dimethylaminopyridine, hyaluronic acid, 2-bromoethyl o-nitrobenzyl ether, sterile deionized water; Equipment: High-speed refrigerated centrifuge, plasma treatment instrument, constant temperature magnetic stirrer, reflux reaction device, dialysis bags with molecular weight cutoff of 3000Da and 5000Da, freeze dryer, 365nm UV irradiator, high-speed homogenizer, vacuum degasser, and irradiation sterilization equipment.

[0033] The specific preparation steps are as follows: S1, Extraction, purification and pretreatment of plant exosomes Fresh green tea leaves were rinsed three times with sterile deionized water, drained, and then ground in a high-speed grinder to a particle size of 1-2 mm. 750 mL of PBS buffer was then added at a material-to-liquid ratio of 1:15. The mixture was placed in an ice bath and homogenized continuously for 25 minutes using a high-speed homogenizer to obtain a fine green tea homogenate. The homogenate was transferred to centrifuge tubes and centrifuged at 3800 rpm for 18 minutes. The tissue debris precipitate at the bottom was discarded, and the clear supernatant was collected.

[0034] Carefully transfer the collected supernatant to an ultracentrifuge tube and centrifuge at 11,000 rpm for 35 minutes. After centrifugation, collect the precipitate at the bottom of the tube and resuspend it three times with 50 mL of PBS buffer to obtain a homogeneous resuspension. Prepare a 10%-40% sucrose density gradient centrifugation buffer in advance. Slowly spread the resuspension on top of the density gradient buffer and centrifuge at 110,000 g for 75 minutes. After centrifugation, pipette the white turbid fraction with a density between 1.10 and 1.18 g / mL, which is the purified plant exosome.

[0035] The purified plant exosomes were diluted to a concentration of 1.5 mg / mL with PBS buffer and transferred to a plasma processing instrument. The processing parameters were set to argon atmosphere and a vacuum degree of 5 × 10⁻⁶. -3 The surface activation treatment was completed by setting the Pa value, processing power of 65W, and processing time of 20s. The treated plant exosome suspension was then refrigerated at 4°C for later use.

[0036] S2, preparation of plant exosomes co-modified with folic acid-penetrating peptide Weigh 3.75 mg of folic acid and add 0.2 mL of dimethyl sulfoxide. Stir with a magnetic stirrer until completely dissolved. Then add 4.65 mg of EDC and 3.28 mg of NHS. Incubate the reaction system at 28°C and continuously stir for 35 min to obtain the folic acid activated solution. In another beaker, weigh 1.5 mg of TAT skin-penetrating peptide and add 0.3 mL of PBS buffer. Stir to dissolve, then add 1.88 mg of EDC and 1.31 mg of NHS. Similarly, activate the solution at 28°C for 25 min to obtain the TAT activated solution.

[0037] Folic acid activation solution was slowly added dropwise to 20 mL of surface-activated plant exosome suspension with continuous stirring. After the addition was complete, the mixture was placed in a dark environment at 37°C and stirred for 1.2 h. Then, TAT activation solution was slowly added dropwise to the reaction system, maintaining the same temperature and dark conditions, and the reaction was continued with stirring for another 1.2 h. After the reaction was completed, the reaction solution was transferred to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 5500 rpm for 18 min, discarding the supernatant. The retentate was resuspended in PBS buffer, and the ultrafiltration centrifugation was repeated 3 times. The final collected retentate was the folic acid-TAT co-modified plant exosome suspension.

[0038] S3, Synthesis of Bifunctional Crosslinking Agent Weigh 50 mg of genipin and place it in a reaction flask containing 1 mL of N,N-dimethylformamide. Stir until completely dissolved. Then add 45.8 mg of EGCG and 3 mg of 4-dimethylaminopyridine, and continue stirring until well mixed. Place the reaction flask in a 65°C oil bath, turn on the reflux device, and stir continuously for 5 hours. Observe the solution state every 30 minutes during the reaction until the solution becomes light yellow and transparent.

[0039] After the reaction was completed, the reaction solution was transferred to a 3000 Da dialysis bag and dialyzed using sterile deionized water as the dialysis solution for 48 hours, with the dialysis solution being changed every 6 hours. After dialysis, the solution in the bag was transferred to a freeze dryer and freeze-dried for 24 hours to obtain a white powdery EGCG-genipin grafted bifunctional crosslinking agent, which was then sealed and stored at 4°C for later use.

[0040] S4, UV-responsive gel preparation Weigh 2g of hyaluronic acid and add it to 100mL of sterile deionized water. Stir at 40℃ until completely dissolved. Then add 0.6g of 2-bromoethyl o-nitrobenzyl ether, adjust the pH of the system to 8.8 with 0.1mol / L NaOH solution, and maintain the reaction at 45℃ with stirring for 9h. After the reaction is complete, transfer the reaction solution to a 5000Da dialysis bag and dialyze with sterile deionized water for 24h. After dialysis, freeze-dry to obtain UV-responsive hyaluronic acid.

[0041] Weigh 2g of UV-responsive hyaluronic acid and add it to 100mL of sterile deionized water. Stir at 35℃ for 30min until completely dissolved to obtain a 2% (w / w) UV-responsive hyaluronic acid solution. Add an appropriate amount of folic acid-TAT co-modified plant exosome suspension to this solution and continue stirring for 30min to achieve a plant exosome mass fraction of 1.2% in the gel system.

[0042] Weigh 0.16g of EGCG-genipin grafted bifunctional crosslinking agent and add it to the above solution. Adjust the pH of the system to 7.0 with 0.1mol / L HCl solution, and stir the reaction mixture at a constant temperature of 35℃ for 1.5h to complete the first step of chemical crosslinking. Then, place the reaction solution in a 365nm UV irradiator and set the irradiation power to 15mW / cm². 2 Irradiation time was 8 minutes, followed by the second step of photocrosslinking to obtain the initial gel product.

[0043] The initial gel sample was placed in a high-speed homogenizer and homogenized at 12000 rpm for 8 minutes to achieve a uniform and fine gel texture. It was then transferred to a vacuum degasser and degassed at -0.09 MPa for 12 minutes to remove air bubbles. Finally, it was sterilized using an irradiation dose of 28 kGy to obtain a folic acid-penetrating peptide co-modified plant exosome UV-responsive anti-photoaging gel.

[0044] Targeted testing showed that the amount of exosomes binding to aging cells in the dermis was 3.5 times that of unmodified exosomes; the DPPH free radical scavenging rate was as high as 94%, demonstrating excellent antioxidant properties; and the exosome release rate after UV irradiation was 82%, showing outstanding responsive release efficiency. This gel achieves an optimal balance between efficacy and gentleness, is suitable for a wide range of people, and can be used as a core ingredient in mainstream anti-photoaging skincare products.

[0045] Example 3 Raw material and equipment preparation: Ingredients: 50g fresh green tea, PBS buffer pH 7.3, folic acid, TAT skin-penetrating peptide sequence YGRKKRRQRRR, EDC, NHS, dimethyl sulfoxide, genipin, EGCG, N,N-dimethylformamide, 4-dimethylaminopyridine, hyaluronic acid, 2-bromoethyl o-nitrobenzyl ether, sterile deionized water; Equipment: High-speed refrigerated centrifuge, plasma treatment instrument, constant temperature magnetic stirrer, reflux reaction device, dialysis bags with molecular weight cutoff of 3000Da and 5000Da, freeze dryer, 365nm UV irradiator, high-speed homogenizer, vacuum degasser, and irradiation sterilization equipment.

[0046] The specific preparation steps are as follows: S1, Extraction, purification and pretreatment of plant exosomes Fresh green tea leaves were rinsed three times with sterile deionized water, drained, and then ground in a high-speed grinder to a particle size of 1-2 mm. 750 mL of PBS buffer was then added at a material-to-liquid ratio of 1:15. The mixture was placed in an ice bath and homogenized continuously for 25 minutes using a high-speed homogenizer to obtain a fine green tea homogenate. The homogenate was transferred to centrifuge tubes and centrifuged at 3800 rpm for 18 minutes. The tissue debris precipitate at the bottom was discarded, and the clear supernatant was collected.

[0047] Carefully transfer the collected supernatant to an ultracentrifuge tube and centrifuge at 11,000 rpm for 35 minutes. After centrifugation, collect the precipitate at the bottom of the tube and resuspend it three times with 50 mL of PBS buffer to obtain a homogeneous resuspension. Prepare a 10%-40% sucrose density gradient centrifugation buffer in advance. Slowly spread the resuspension on top of the density gradient buffer and centrifuge at 110,000 g for 75 minutes. After centrifugation, pipette the white turbid fraction with a density between 1.10 and 1.18 g / mL, which is the purified plant exosome.

[0048] The purified plant exosomes were diluted to a concentration of 1.5 mg / mL with PBS buffer and transferred to a plasma processing instrument. The processing parameters were set to argon atmosphere and a vacuum degree of 5 × 10⁻⁶. -3 The surface activation treatment was completed by setting the Pa value, processing power of 65W, and processing time of 20s. The treated plant exosome suspension was then refrigerated at 4°C for later use.

[0049] S2, preparation of plant exosomes co-modified with folic acid-penetrating peptide Weigh 3.75 mg of folic acid and add 0.2 mL of dimethyl sulfoxide. Stir with a magnetic stirrer until completely dissolved. Then add 4.65 mg of EDC and 3.28 mg of NHS. Incubate the reaction system at 28°C and continuously stir for 35 min to obtain the folic acid activated solution. In another beaker, weigh 1.5 mg of TAT skin-penetrating peptide and add 0.3 mL of PBS buffer. Stir to dissolve, then add 1.88 mg of EDC and 1.31 mg of NHS. Similarly, activate the solution at 28°C for 25 min to obtain the TAT activated solution.

[0050] Folic acid activation solution was slowly added dropwise to 20 mL of surface-activated plant exosome suspension with continuous stirring. After the addition was complete, the mixture was placed in a dark environment at 37°C and stirred for 1.2 h. Then, TAT activation solution was slowly added dropwise to the reaction system, maintaining the same temperature and dark conditions, and the reaction was continued with stirring for another 1.2 h. After the reaction was completed, the reaction solution was transferred to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 5500 rpm for 18 min, discarding the supernatant. The retentate was resuspended in PBS buffer, and the ultrafiltration centrifugation was repeated 3 times. The final collected retentate was the folic acid-TAT co-modified plant exosome suspension.

[0051] S3, Synthesis of Bifunctional Crosslinking Agent Weigh 50 mg of genipin and place it in a reaction flask containing 1 mL of N,N-dimethylformamide. Stir until completely dissolved. Then add 45.8 mg of EGCG and 3 mg of 4-dimethylaminopyridine, and continue stirring until well mixed. Place the reaction flask in a 65°C oil bath, turn on the reflux device, and stir continuously for 5 hours. Observe the solution state every 30 minutes during the reaction until the solution becomes light yellow and transparent.

[0052] After the reaction was completed, the reaction solution was transferred to a 3000 Da dialysis bag and dialyzed using sterile deionized water as the dialysis solution for 48 hours, with the dialysis solution being changed every 6 hours. After dialysis, the solution in the bag was transferred to a freeze dryer and freeze-dried for 24 hours to obtain a white powdery EGCG-genipin grafted bifunctional crosslinking agent, which was then sealed and stored at 4°C for later use.

[0053] S4, UV-responsive gel preparation Weigh 2g of hyaluronic acid and add it to 100mL of sterile deionized water. Stir at 40℃ until completely dissolved. Then add 0.6g of 2-bromoethyl o-nitrobenzyl ether, adjust the pH of the system to 8.8 with 0.1mol / L NaOH solution, and maintain the reaction at 45℃ with stirring for 9h. After the reaction is complete, transfer the reaction solution to a 5000Da dialysis bag and dialyze with sterile deionized water for 24h. After dialysis, freeze-dry to obtain UV-responsive hyaluronic acid.

[0054] Weigh 2g of UV-responsive hyaluronic acid and add it to 100mL of sterile deionized water. Stir at 35℃ for 30min until completely dissolved to obtain a 2% (w / w) UV-responsive hyaluronic acid solution. Add an appropriate amount of folic acid-TAT co-modified plant exosome suspension to this solution and continue stirring for 30min to achieve a 2% (w / w) mass fraction of plant exosomes in the gel system.

[0055] Weigh 0.2 g of EGCG-genipin grafted bifunctional crosslinking agent and add it to the above solution. Adjust the pH of the system to 7.0 with 0.1 mol / L HCl solution, and stir the reaction mixture at a constant temperature of 35℃ for 1.5 h to complete the first step of chemical crosslinking. Then, place the reaction solution in a 365 nm UV irradiator and set the irradiation power to 15 mW / cm². 2 Irradiation time was 8 minutes, followed by the second step of photocrosslinking to obtain the initial gel product.

[0056] The initial gel sample was placed in a high-speed homogenizer and homogenized at 12000 rpm for 8 minutes to achieve a uniform and fine gel texture. It was then transferred to a vacuum degasser and degassed at -0.09 MPa for 12 minutes to remove air bubbles. Finally, it was sterilized using an irradiation dose of 28 kGy to obtain a folic acid-penetrating peptide co-modified plant exosome UV-responsive anti-photoaging gel.

[0057] Targeted testing showed that the amount of exosomes binding to aging cells in the dermis was 3.8 times that of unmodified exosomes; the DPPH free radical scavenging rate reached 96%, and the antioxidant capacity was close to the limit; after UV irradiation, the exosome release was 85%, and the response release performance was close to saturation. This gel has strong efficacy and outstanding anti-photoaging ability, making it suitable as a core ingredient for high-end functional anti-aging products.

[0058]

[0059] The three embodiments primarily adjusted the dosage of folic acid-TAT co-modified plant exosomes and EGCG-genipin grafted bifunctional cross-linking agent, while keeping other process parameters completely consistent, ultimately exhibiting significant differences in efficacy. The low-dosage gel is gentle and non-irritating, and cost-effective, making it suitable for sensitive skin or those who only require basic anti-aging; the medium-dosage version strikes a good balance between efficacy and gentleness, excelling in cell binding capacity, free radical scavenging effect, and responsive release efficiency, perfectly meeting the needs of most people for anti-photoaging skincare products; the high-dosage version maximizes efficacy, offering the highest antioxidant and targeted delivery capabilities, making it more suitable for high-end functional products.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a folic acid-modified plant exosome anti-photoaging gel by cross-linking method, characterized in that, The specific steps of the method are: S1, plant exosome extraction, purification and pretreatment: select fresh plant tissue, crush, homogenate, centrifuge and purify, and then pretreat the plant exosome with low-temperature plasma; S2, preparation of folic acid-penetrating peptide co-modified plant exosome: activate folic acid and TAT skin-penetrating peptide respectively, and couple them to the surface of the pretreated plant exosome in steps, and then purify to obtain the folic acid-penetrating peptide co-modified plant exosome; S3, synthesis of bifunctional crosslinking agent: using genipin and epigallocatechin gallate as raw materials, ester exchange reaction, purification and drying are carried out to obtain the EGCG-genipin grafted bifunctional crosslinking agent; S4, preparation of UV-responsive gel: modifying hyaluronic acid with o-nitrobenzyl, adding folic acid-penetrating peptide co-modified plant exosome and bifunctional crosslinking agent for composite crosslinking, and then post-treating to obtain the target gel.

2. The method of claim 1, wherein the method of preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking is characterized by, The plant tissue in the S1 step is selected from one or more of green tea, aloe, ginkgo leaves, and grape seeds; the centrifugal purification includes low-speed centrifugal removal of impurities, high-speed centrifugal enrichment, and sucrose density gradient centrifugal purification; the sucrose density gradient is 10%-40%, and the density gradient centrifugal conditions are 100000-120000g centrifugation for 60-90min; the low-temperature plasma pretreatment conditions are an argon atmosphere, a vacuum degree of 10 -3 -10 -2 Pa, a processing power of 50-80W, and a processing time of 15-25s.

3. The method of claim 1, wherein the method of preparing a folic acid-modified plant exosome anti-photoaging gel by cross-linking is characterized by, In the S1 step, the homogenate adopts PBS buffer with a pH value of 7.2-7.4, and the solid-liquid ratio of the plant tissue to the PBS buffer is 1:10-1:20; the low-speed centrifugation condition is 3000-5000 r / min for 15-20 min; the high-speed centrifugation condition is 10000-12000 r / min for 30-40 min; and the resuspension concentration of the plant exosome before pretreatment is 1-2 mg / mL.

4. The method of claim 1, wherein the method of preparing a folic acid-modified plant exosome anti-photoaging gel by cross-linking is characterized by, In the S2 step, the sequence of the TAT skin-penetrating peptide is YGRKKRRQRRR; the activation condition of folic acid is that the concentration of folic acid is 10-20 mg / mL, the molar ratio of folic acid to EDC and NHS is 1:1.2-1.5:1.2-1.5, and the activation is carried out at 25-30°C for 30-40 min; and the activation condition of the TAT skin-penetrating peptide is that the concentration is 5-10 mg / mL, the molar ratio of TAT to EDC and NHS is 1:1.2-1.5:1.2-1.5, and the activation is carried out at 25-30°C for 20-30 min.

5. The method of claim 1, wherein the method of preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking is characterized by, In the S2 step, the specific steps of the step-by-step coupling are as follows: first, coupling the activated folic acid with the pretreated plant exosome, and then coupling the activated TAT skin-penetrating peptide with the folic acid-modified plant exosome; the step-by-step coupling condition is that the mass ratio of folic acid to plant exosome is 1:5-1:10, the reaction is carried out at 37°C in the dark for 1-1.5 h, then TAT skin-penetrating peptide activation solution is added according to the molar ratio of folic acid to TAT of 2:1-3:1, and the reaction is continued at 37°C in the dark for 1-1.5 h; and the purification adopts 100 kDa ultrafiltration membrane centrifugation at a speed of 5000-6000 r / min for 15-20 min, and the operation is repeated for 2-3 times.

6. The method of claim 1, wherein the method of preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking is characterized by, In the S3 step, the ester exchange reaction condition is that the molar ratio of genipin to EGCG is 1:1-1:1.2, N,N-dimethylformamide is used as the solvent, 4-dimethylaminopyridine is used as the catalyst, the catalyst is added in an amount of 5%-8% of the mass of genipin, and the reaction is carried out at 60-70°C for 4-6 h; and the purification adopts 3000 Da dialysis bag dialysis for 48 h, and the water is changed every 6 h, and the drying method is freeze-drying.

7. The method of claim 1, wherein the method of preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking is characterized by, The modification condition of the o-nitrobenzyl in the S4 step is that the mass ratio of hyaluronic acid to 2-bromoethyl o-nitrobenzyl ether is 1:0.3-1:0.5, the pH value is 8.5-9.0, the reaction is carried out at 40-50 DEG C for 8-10 h, the purification is carried out by using a 5000 Da dialysis bag for 24 h, the mass concentration of the UV responsive hyaluronic acid solution is 1%-3%, and the mass fraction of the folic acid-penetratin co-modified plant exosome in the gel system is 0.5%-2%.

8. The method of claim 1, wherein the method of preparing a folic acid modified plant exosome anti-photoaging gel by cross-linking is characterized by, In the S4, the composite crosslinking is a combination of chemical crosslinking and photo-crosslinking, and the specific conditions are as follows: the amount of the bifunctional crosslinking agent is 5%-10% of the mass of the UV-responsive hyaluronic acid; the first step is chemical crosslinking initiated by genipin, the pH value is 6.5-7.5, the temperature is 30-40℃, and the time is 1-2h; the second step is photo-crosslinking initiated by UV irradiation, and the irradiation conditions are as follows: the wavelength is 365nm, the power is 10-20mW / cm 2 , and the time is 5-10min; the post-treatment includes homogenization, degassing and sterilization, the homogenization speed is 10000-15000r / min, the time is 5-10min; the vacuum degassing degree is-0.08--0.1MPa, the time is 10-15min; and the sterilization method is irradiation sterilization, and the irradiation dose is 25-30kGy.

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