Application of miR-396-5p from cistanche deserticola extracellular vesicles in preparation of products for improving skin photoaging

Abstract

This invention relates to the application of miR-396-5p derived from Cistanche deserticola extracellular vesicles in the preparation of products for improving skin photoaging, belonging to the fields of biomedicine and skin anti-aging technology. This invention experimentally demonstrates that miR-396-5p derived from Cistanche deserticola extracellular vesicles can alleviate UV-induced damage to human dermal fibroblasts, improve cell viability, promote the expression of collagen-related factor COL1A1, inhibit the expression of matrix metalloproteinase MMP3, reduce collagen degradation, and maintain dermal extracellular matrix homeostasis, thereby improving UV-induced skin photoaging damage. This invention utilizes Cistanche deserticola extracellular vesicles to construct a natural miR-396-5p delivery system. Cistanche deserticola extracellular vesicles can effectively load, protect, and deliver miR-396-5p, improving its stability, bioavailability, cellular uptake efficiency, and transdermal delivery capability.

Description

Technical Field

[0001] This invention relates to the application of miR-396-5p derived from extracellular vesicles of Cistanche deserticola in the preparation of products that improve skin photoaging, and belongs to the fields of biomedicine and skin anti-aging technology. Background Technology

[0002] Photoaging of the skin is a degeneration of skin structure and function caused by long-term exposure to ultraviolet radiation, with UVB-induced skin damage being the most common. The main manifestations of photoaging include skin laxity, increased wrinkles, decreased elasticity, pigmentation, and impaired barrier function. Studies have shown that ultraviolet radiation can induce skin cells to produce large amounts of reactive oxygen species (ROS), activating various signaling pathways such as MAPK, thereby triggering oxidative stress and inflammatory responses. This, in turn, promotes the expression of matrix metalloproteinases (MMPs), leading to collagen degradation and accelerated cellular aging damage, thus accelerating the skin aging process.

[0003] Currently, existing technologies for improving skin aging and alleviating photoaging mainly involve topical application of antioxidants, retinoids, and growth factors. While these active ingredients have certain anti-photoaging effects, their targets are broad and can easily affect normal cellular physiological processes. They generally suffer from problems such as high irritation, insufficient stability, low bioavailability, poor targeting, and adverse reactions with long-term use, thus limiting their clinical application and promotion.

[0004] In recent years, miRNAs, as a class of endogenous non-coding small RNAs, have participated in cellular biological processes by specifically regulating the expression of target genes. Compared with traditional active ingredients, miRNAs have advantages such as well-defined targets, high regulatory efficiency, good targeting, and lower side effects, showing promising application prospects in skin tissue repair and photoaging intervention. However, free miRNAs are easily degraded by nucleases, have poor stability, and suffer from low transdermal absorption efficiency and insufficient delivery efficiency, which seriously limits their practical application.

[0005] Extracellular vesicles (exosomes) are natural nanoscale membrane vesicles that can carry various bioactive substances such as proteins, lipids, and nucleic acids, playing an important role in intercellular communication. In recent years, plant-derived extracellular vesicles have been considered natural carriers for functional nucleic acid delivery due to their natural origin, good biocompatibility, low immunogenicity, and ability to load nucleic acid molecules. Utilizing plant-derived extracellular vesicles to deliver miRNAs holds promise for improving miRNA stability and transdermal penetration, thereby enhancing their therapeutic effects on photoaging of the skin.

[0006] Therefore, the discovery of a miRNA with clear anti-photoaging activity and the construction of a safe and efficient delivery system by combining it with natural plant-derived extracellular vesicles is of great research significance for improving the stability and delivery efficiency of miRNA and enhancing its effect on improving skin photoaging. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides the application of miR-396-5p derived from extracellular vesicles of Cistanche deserticola in the preparation of products that improve skin photoaging.

[0008] The technical solution of the present invention is as follows: The use of any one or more of miR-396-5p, miR-396-5p mimics, and extracellular vesicles of Cistanche deserticola loaded with miR-396-5p in the preparation of products for improving skin photoaging, wherein the nucleotide sequence of miR-396-5p is shown in SEQ ID NO.1.

[0009] According to a preferred embodiment of the present invention, the improvement of skin photoaging includes one or more of the following: increasing skin cell vitality, reducing collagen degradation, promoting collagen synthesis, promoting COL1A1 expression, inhibiting MMP3 expression, inhibiting aging-related β-galactosidase activity, reducing inflammatory response, or delaying skin aging.

[0010] According to a preferred embodiment of the present invention, the miR-396-5p is derived from extracellular vesicles of Cistanche deserticola.

[0011] According to a preferred embodiment of the present invention, the skin photoaging is ultraviolet-induced skin photoaging.

[0012] More preferably, the ultraviolet light is UVA and / or UVB.

[0013] According to a preferred embodiment of the present invention, the method for preparing the extracellular vesicles of Cistanche deserticola loaded with miR-396-5p includes the following steps: washing Cistanche deserticola, adding PBS buffer and grinding to break it up, and separating and purifying the extracellular vesicles of Cistanche deserticola by differential centrifugation combined with sucrose density gradient centrifugation to obtain a supernatant solution containing extracellular vesicles of Cistanche deserticola loaded with miR-396-5p.

[0014] The extracellular vesicles of Cistanche deserticola loaded with miR-396-5p prepared in this invention have a particle size of 50–300 nm, an average particle size of 197.2 ± 0.5508 nm, a PDI index of 0.196 ± 0.02, and a Zeta potential of -23.3 ± 1.37 mV. These extracellular vesicles of Cistanche deserticola can protect miR-396-5p from nuclease degradation and improve its stability, biological activity, and delivery efficiency.

[0015] A composition for improving photoaging of skin, comprising one or more of the above-mentioned miR-396-5p, miR-396-5p mimics, or extracellular vesicles of Cistanche deserticola loaded with miR-396-5p.

[0016] According to a preferred embodiment of the present invention, the composition is a pharmaceutical composition, a cosmetic composition, a medical aesthetic preparation, or a skin repair preparation.

[0017] According to a preferred embodiment of the present invention, the dosage form of the composition is a gel, emulsion, serum, facial mask liquid, hydrogel, spray, or lyophilized powder.

[0018] According to a preferred embodiment of the present invention, the composition is used to prevent, delay, or improve ultraviolet-induced photoaging of the skin.

[0019] Beneficial effects: (1) This invention experimentally demonstrates that miR-396-5p derived from extracellular vesicles of Cistanche deserticola can alleviate UV-induced damage to human dermal fibroblasts, improve cell viability, and expand the application of miRNA in the prevention and treatment of skin photoaging. Compared with traditional small molecule active ingredients, miR-396-5p can exert biological functions by regulating specific target genes, and has the characteristics of clear target and precise regulation, providing a new technical solution for the precise intervention of skin photoaging.

[0020] (2) The results showed that miR-396-5p could promote the expression of collagen-related factor COL1A1, inhibit the expression of matrix metalloproteinase MMP3, reduce collagen degradation, and maintain the homeostasis of the extracellular matrix in dermal cells, thereby improving UV-induced skin photoaging damage and providing experimental evidence for miRNA to regulate collagen metabolism and improve skin photoaging.

[0021] (3) The present invention utilizes extracellular vesicles of Cistanche deserticola to construct a natural delivery system for miR-396-5p. Extracellular vesicles of Cistanche deserticola can effectively load, protect and deliver miR-396-5p, improving its stability, bioavailability, cellular uptake efficiency and transdermal delivery ability. It overcomes the problems of free miRNA being easily degraded by nucleases, poor stability and low delivery efficiency, and enhances the effect of miR-396-5p in improving skin photoaging.

[0022] (4) The extracellular vesicles of Cistanche deserticola form a natural delivery system with miR-396-5p, realizing the synergistic effect of miRNA and natural nanocarrier, providing a new idea for the delivery of functional miRNA from plant-derived extracellular vesicles for skin photoaging intervention.

[0023] (5) Through functional verification experiments, it was found that miR-396-5p is the key active ingredient that improves skin photoaging. The delivery system can effectively maintain its biological function, providing a new technical solution for skin photoaging intervention strategy based on functional miRNA.

[0024] (6) The miR-396-5p and its Cistanche deserticola extracellular vesicle delivery system provided by the present invention have good biocompatibility and application potential. They can be prepared into a variety of products such as pharmaceutical compositions, cosmetic compositions, medical aesthetic preparations and skin repair preparations for the prevention, delay and improvement of skin photoaging, and have good prospects for industrial application. Attached Figure Description

[0025] Figure 1 The results of the isolation and characterization of extracellular vesicles from Cistanche deserticola are shown in the following diagrams: a) Morphological diagram of extracellular vesicles from Cistanche deserticola; b) Particle size distribution diagram of extracellular vesicles from Cistanche deserticola; c) Zeta potential detection diagram of extracellular vesicles from Cistanche deserticola. Figure 2 A bar chart showing the relative expression levels of miR-396-5p in extracellular vesicles of Cistanche deserticola; Figure 3 A bar chart showing the experimental results of the effect of miR-396-5p on cell viability; compared with the model group. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001; Figure 4 Figure 1 shows the experimental results of the effect of miR-396-5p on the expression of MMP3 and COL1A1 proteins in cells. Figure 2 shows a Western blotting image; Figure 3 shows a bar chart of the relative expression of MMP3 protein; Figure 4 shows a bar chart of the relative expression of COL1A1 protein. Compared with the model group, ns indicates P > 0.05. This indicates that P < 0.05. This indicates that P < 0.01; Figure 5 The figure shows the experimental results of the effect of miR-396-5p on the activity of SA-β-gal in cells. Figure a shows the staining results of senescence-related SA-β-gal activity in dermal fibroblasts detected using an inverted microscope; figure b is a bar chart showing the percentage of SA-β-gal-positive cells. Compared with the model group... This indicates that P < 0.001; Figure 6 A bar graph showing the validation results of key active components in Cistanche deserticola extracellular vesicles loaded with miR-396-5p; compared with the model group, ns indicates P > 0.05. This indicates that P < 0.01. This indicates that P < 0.0001; Figure 7 A bar graph showing the stability of extracellular vesicles derived from Cistanche deserticola against miR-396-5p; compared with the model group, ns indicates P > 0.05. This indicates that P < 0.01. This indicates that P < 0.0001; Figure 8 Line graphs showing the Wrinkle Percentile test results for the test group and control group at weeks 0, 2, and 4. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0028] Unless otherwise specified, the culture media used in the following examples are at their natural pH. Quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0029] Example 1 Extraction and characterization of extracellular vesicles from Cistanche deserticola Extraction of extracellular vesicles from Cistanche deserticola: Cistanche deserticola was washed and placed in a juicer with PBS buffer (pH 7.4) for grinding and disruption. Extracellular vesicles were isolated and purified using differential centrifugation combined with sucrose density gradient centrifugation. The specific steps are as follows: Centrifuge at 300×g for 10 min at 4℃, transfer the supernatant to a new centrifuge tube, and centrifuge at 3,000×g for 20 min at 4℃ to remove large cell debris. Next, transfer the supernatant to a new centrifuge tube and centrifuge at 10,000×g for 30 min at 4℃. After discarding the precipitate, the supernatant was filtered through a 0.45 μm microporous membrane and ultrabrominated at 100,000×g for 1 h at 4 °C. The supernatant was discarded, and the obtained precipitate was resuspended in PBS buffer (pH 7.4). The precipitate was centrifuged at 10,000×g for 30 min at 4 °C. The obtained supernatant was placed above a gradient of sucrose solutions (8%, 30%, 45%, 60%) and centrifuged at 150,000×g for 2 h at 4 °C. The bands between 30% and 45% were collected, resuspended in PBS buffer (pH 7.4), and centrifuged at 150,000×g for 1 h at 4 °C. The supernatant was discarded, and the obtained precipitate was resuspended in PBS buffer. The resulting suspension was centrifuged at 10,000×g for 10 min, and the supernatant was collected to obtain the Cistanche deserticola extracellular vesicle (ChEVLP) solution. The protein concentration was determined using a BCA kit, and particle concentration was counted to determine the dosage for subsequent experiments.

[0030] Quality characterization of extracellular vesicles in Cistanche deserticola: The morphological characteristics of extracellular vesicles were observed using transmission electron microscopy. Their particle size distribution, polydispersity index (PDI), and zeta potential were detected using a nanoparticle size and zeta potential analyzer. The results are as follows: Figure 1 As shown.

[0031] from Figure 1 As can be seen from a, the extracellular vesicles of Cistanche deserticola prepared by the method of this embodiment are round or elliptical, with clear edges and regular shape, and have obvious vesicle-like lipid bilayer membrane structure characteristics.

[0032] Figure 1 In b, the horizontal axis represents particle size, and the vertical axis represents percentage. Figure 1 As can be seen from b, the average particle size of the extracellular vesicles of Cistanche deserticola prepared in this embodiment is 197.2±0.5508 nm, the PDI index is 0.196±0.02, and the dominant peak accounts for 100% of the distribution.

[0033] Figure 1 In graph c, the horizontal axis represents the magnitude of the Zeta potential, and the vertical axis represents the total count of extracellular vesicles at the corresponding potential in *Cistanche deserticola* cells. Figure 1As shown in Figure c, the surface charge of the extracellular vesicles of Cistanche deserticola is -23.3 ± 1.37 mV, indicating that the sample surface carries a strong negative charge, which can effectively prevent particle aggregation and thus ensure the long-term stability of the dispersion system. The above-mentioned spherical vesicle structure and negative Zeta potential are typical characteristics of extracellular vesicles, which is sufficient to prove that the method used in this embodiment can successfully prepare extracellular vesicles of Cistanche deserticola.

[0034] Example 2 extracellular vesicle miRNA sequencing of Cistanche deserticola Total RNA was extracted from extracellular vesicles of Cistanche deserticola using the TRIzol kit from USA Invitrogen, following the manufacturer's instructions.

[0035] miRNA sequencing: Libraries were constructed according to the manufacturer's instructions using the VAHTS Universal V10 RNA-seq Library Preparation Kit. Differential expression analysis was performed using DESeq2. Radar plots of the top 30 genes were generated using the R package gradar. GO and KEGG enrichment analyses were performed on the DEGs using R (v3.2.0) to screen for significantly enriched terms.

[0036] Table 1. Mature miR-396-5p sequence in extracellular vesicles of Cistanche deserticola

[0037] Sequencing results as follows Figure 2 As shown in Table 1, miRNA sequencing analysis of extracellular vesicles of Cistanche deserticola revealed that miR-396-5p had a high expression level of 80744.15036.

[0038] Based on the mature miR-396-5p sequence obtained from sequencing, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize miR-396-5p mimic, inhibitor miR-396-5p, and corresponding negative controls (NC mimic and inhibitor NC mimic) for subsequent cell function verification experiments.

[0039] Example 3 miR-396-5p's efficacy in improving photoaging was verified. 1. Establish a photoaging model of human dermal fibroblasts Human dermal fibroblasts (HSF, purchased from Shandong Jinyuan Biotechnology Co., Ltd.) were cultured in 6-well plates at 37℃ and 5% CO2 until they reached a density of 80% (approximately 4 × 10⁻⁶). 3 (cells / well), irradiated with UVB lamp (UV dose 320 mJ / cm²). 2A photoaging model was established by irradiating the area for 20 seconds, then irradiating it for another 20 seconds.

[0040] 2. CCK-8 cell viability assay The experiment was divided into Control, NC, Model, and miR-396-5p groups (10 / 20 / 30 / 50 / 100). The Control and NC groups used normal cells without a photoaging model, while the other groups used cells with a photoaging model. Transfection was performed using Lipofectamine™ 3000; the Control group received no treatment; the NC and Model groups were transfected with NCmimic (final concentration 50 nM); the miR-396-5p groups were transfected with different concentrations of miR-396-5p mimic (final concentrations 10 nM, 20 nM, 30 nM, 50 nM, and 100 nM). Six hours after transfection, the culture medium was replaced with basal MEM medium containing 1% FBS to reduce toxicity. Twenty-four hours after transfection, cells were collected, and cell viability was assessed according to the CCK-8 cell viability assay kit instructions.

[0041] The results are as follows Figure 3 As shown in the CCK-8 results, the cell survival rate in the Model group was significantly lower than that in the NC group, indicating that UVB irradiation can successfully induce dermal fibroblast damage and reduce the survival rate. Furthermore, miR-396-5p at concentrations below 100 nM can significantly restore the cell viability of dermal fibroblasts.

[0042] 3. Western Blot protein expression detection The experiment was divided into four groups: NC group, inhibitor NC group, model group, inhibitor miR-396-5p group, and miR-396-5p group. The NC group and inhibitor NC group used normal cells without a photoaging model, while the other groups used cells with a photoaging model. Transfection was performed using Lipofectamine™ 3000. The NC group and model group were transfected with NCmimic (final concentration 50 nM); the inhibitor NC group was transfected with inhibitor NC mimic (final concentration 50 nM); the inhibitor miR-396-5p group was transfected with miR-396-5p mimic (final concentration 50 nM) and inhibitor miR-396-5p (final concentration 50 nM); and the miR-396-5p group was transfected with miR-396-5p mimic (final concentration 50 nM). After 6 hours, the culture medium was replaced with basal MEM medium containing 1% FBS to reduce toxicity. Cells were collected 24 hours after transfection and subjected to Western blot electrophoresis to detect the expression levels of matrix metalloproteinase MMP3 and collagen COL1A1 in each group of cells and to perform quantitative analysis. ACTIN was used as an internal reference protein.

[0043] The results are as follows Figure 4 As shown in a, b, and c, the Western blot results indicated that compared with the NC group and the inhibitor NC group, the MMP3 protein level in the Model group was significantly increased, while the COL1A1 protein level was significantly decreased. This suggests that UVB damage can promote the expression of matrix metalloproteinase MMP3 and inhibit collagen COL1A1 synthesis, successfully constructing a cell photoaging damage model. After transfection with miR-396-5p, both MMP3 and COL1A1 were significantly restored, while the protein expression levels of the inhibitor miR-396-5p transfected were not significantly different from those in the model group, indicating that miR-396-5p can treat UVB-induced collagen degradation in dermal cells and alleviate skin photoaging damage.

[0044] 4. Detection of SA-β-gal cell senescence level Using the β-galactosidase staining kit, SA-β-gal cell senescence staining was performed on the Control group, NC group, Model group, and miR-396-5p groups with concentrations of 30 nM, 50 nM, and 100 nM constructed in the CCK-8 cell viability assay of this example, in accordance with the instructions.

[0045] The results are as follows Figure 5As shown in results a and b, compared with the Control and NC groups, the model group exhibited a significantly increased level of SA-β-gal positive cells, indicating that UVB can successfully induce significant senescent damage in dermal fibroblasts. Treatment with miR-396-5p at concentrations below 100 nM significantly reduced the UVB-induced SA-β-gal positive cell level, alleviating UVB-induced skin cell senescence damage. Therefore, miR-396-5p can effectively improve UVB-induced skin photoaging.

[0046] Example 4 miR-396-5p is a key active ingredient in the extracellular vesicles of Cistanche deserticola. A UVB-induced photoaging model of human dermal fibroblasts was established according to the method in Example 3. The experiment was divided into four groups: inhibitor NC group, Model group, ChEVLPs group, ChEVLPs+inhibitor miR-396-5p group, and miR-396-5p group.

[0047] The Inhibitor NC group used normal cells without a photoaging model, while all other groups used cells with a photoaging model. Transfection was performed using Lipofectamine™ 3000. The Inhibitor NC group was treated with Inhibitor NCmimic (final concentration 50 nM); the ChEVLPs group was treated with ChEVLPs at a final concentration of 40 μg / mL (prepared according to Example 1, the concentration being the protein concentration determined by the BCA kit, the same below); the ChEVLPs+Inhibitor miR-396-5p group was treated with ChEVLPs at a final concentration of 40 μg / mL and then transfected with Inhibitor miR-396-5p (final concentration 50 nM); the miR-396-5p group was transfected with miR-396-5p mimic (final concentration 50 nM). After 6 hours of incubation, the culture medium was replaced with basal MEM medium containing 1% FBS to reduce toxicity. Cells were collected 24 hours after transfection and processed according to the CCK8 kit instructions to analyze the therapeutic effects of miR-396-5p and ChEVLPs on UVB-induced dermal fibroblasts.

[0048] Table 2. Effects of different treatment groups on cell viability

[0049] The results are shown in Table 2. Figure 6As shown, the results indicate that compared with the model group, the cell viability of the ChEVLPs group was significantly improved, while there was no significant difference in the ChEVLPs+inhibitor 396 group. This suggests that the effect of Cistanche deserticola extracellular vesicles in improving photoaging damage was significantly weakened after miR-396-5p inhibition. Furthermore, there was no significant difference in cell survival rate between the ChEVLPs and miR-396-5p groups, indicating that miR-396-5p is an important active ingredient in Cistanche deserticola extracellular vesicles that exert anti-skin photoaging effects.

[0050] Example 5 Verification of the stability and delivery effect of extracellular vesicles of Cistanche deserticola on miR-396-5p A human dermal fibroblast photoaging model was established according to the method in Example 3. The experiment was divided into NC group, Model group, miR-396-5p group, miR-396-5p+RNase group, ChEVLPs group, ChEVLPs+RNase group and ChEVLPs+TritonX-100+RNase group.

[0051] In the NC group, normal cells without a photoaging model were transfected with Lipofectamine™ 3000 (final concentration 50 nM). In all other groups, a photoaging model was established, followed by transfection with Lipofectamine™ 3000. The Model group was transfected with NC mimic (final concentration 50 nM); the miR-396-5p group was transfected with miR-396-5p mimic (final concentration 50 nM); the miR-396-5p+RNase group was treated with RNase A at a final concentration of 10 μg / mL at 37°C for 30 min before transfection into cells (final concentration 50 nM); the ChEVLPs group was treated with ChEVLPs at a final concentration of 40 μg / mL; and the ChEVLPs+RNase group was treated with ChEVLPs at a final concentration of 40 μg / mL. ChEVLPs pretreated with RNase (RNase treatment conditions were the same as the miR-396-5p+RNase group) were treated with μg / mL of RNase. The ChEVLPs+Triton X-100+RNase group was first treated with 0.1% Triton X-100 (v / v) to disrupt the vesicle membrane structure, exposing miRNAs within the outer vesicles. Then, RNase A was added (RNase treatment conditions were the same as the miR-396-5p+RNase group), and cells were transfected at a final concentration of 40 μg / mL. After 6 h of treatment in each group, the culture medium was replaced with basal MEM medium containing 1% FBS to reduce toxicity. Cells were collected 24 h after transfection, and extraction was performed according to the CCK8 kit instructions to analyze the therapeutic effects of miR-396-5p and ChEVLPs on UVB-induced dermal fibroblasts.

[0052] Table 3. Effects of RNase treatment on cell viability improvement by different delivery systems.

[0053] As shown in Table 3, Figure 7As shown, compared with the model group, the cell survival rate of the miR-396-5p group was significantly improved, while there was no significant difference in miR-396-5p+RNase, indicating that the biological activity of miR-396-5p decreased significantly after RNase treatment, suggesting that free miR-396-5p is easily degraded and inactivated. At the same time, both ChEVLPs and ChEVLPs+RNase showed significant improvements compared with the model group, indicating that intact Cistanche deserticola extracellular vesicles can significantly improve cell viability. However, the cell survival rate was significantly reduced after Triton X-100 disrupted the vesicle membrane structure and treated with RNase (ChEVLPs+Triton X-100+RNase group), indicating that the vesicle membrane structure has a protective effect on the internal active RNA.

[0054] Based on the miRNA sequencing results in Example 2 and the functional verification results in Examples 3 and 4, it can be seen that miR-396-5p is an important active substance in the extracellular vesicles of Cistanche deserticola, which plays an anti-skin photoaging role. The extracellular vesicles of Cistanche deserticola can protect and deliver the miR-396-5p they are loaded with, improve their stability and biological activity, and enhance their anti-skin photoaging effect.

[0055] Example 6 Comparative validation of the transdermal delivery performance of Cistanche deserticola extracellular vesicles as a natural miR-396-5p delivery system. To evaluate the transdermal delivery capability of *Cistanche deserticola* extracellular vesicles as a natural delivery system for miR-396-5p, the final concentration of miR-396-5p in the system was set at 100 nM. Free miR-396-5p (Free-miR) and ordinary liposomes (Lipo-miR, prepared by membrane hydration and composed of soybean lecithin and cholesterol (6:1, m / m); after preparation by conventional membrane hydration, the liposomes were incubated with miR-396-5p to form a Lipo-miR complex) served as controls. The experimental group used *Cistanche deserticola* extracellular vesicles (ChEVLPs, prepared according to Example 1) loaded with miR-396-5p, corresponding to a particle concentration of 2 × 10⁻⁶. 11 The study compared the effects of different delivery systems on transdermal transport, skin tissue enrichment, and stability of miR-396-5p, using particles / mL.

[0056] Fresh pig ear skin was harvested, and after removing subcutaneous fat, it was fixed in a Franz diffusion cell with the cuticle facing the donor chamber. Equal volumes of Free-miR, Lipo-miR, and ChEVLPs-miR formulations (particle concentration 2 × 10⁻⁶) were added to the donor chamber. 11The receptor chamber was filled with PBS buffer (pH 7.4) and continuously stirred magnetically at 37°C. Receptor fluid was collected at 2, 4, 8, 12, and 24 h, and total RNA was extracted. The miR-396-5p content was detected by RT-qPCR, and the cumulative permeation was calculated. After 24 h, pig ear skin was removed, and residual samples were thoroughly washed away. Dermal tissue was separated, and total RNA was extracted. The enrichment of miR-396-5p in the dermal tissue was detected by RT-qPCR.

[0057] Table 4. Cumulative transmittance of miR-396-5p in each group within 24 hours

[0058] The results showed that the cumulative permeation of miR-396-5p in each group gradually increased over time. Compared with free miR-396-5p, ordinary liposomes could improve the transdermal efficiency of miRNA; while the extracellular vesicle group of Cistanche deserticola showed a higher cumulative permeation at all time points, with the cumulative permeation at 24h being approximately 3.2 times that of the free miRNA group and 1.7 times that of the ordinary liposome group.

[0059] Table 5. 24h skin tissue miR-396-5p enrichment

[0060] The content of miR-396-5p in skin tissue was detected by qPCR. Compared with the free miRNA group, both delivery systems could increase the enrichment level of miR-396-5p in the skin. The extracellular vesicle group of Cistanche deserticola had the highest enrichment level, which was about 3.2 times that of the free miRNA group, and significantly higher than that of the ordinary liposome group.

[0061] Example 7 A gel formulation containing Cistanche deserticola extracellular vesicles loaded with miR-396-5p to improve skin photoaging By mass percentage, it comprises the following components: extracellular vesicles of Cistanche deserticola loaded with miR-396-5p (prepared according to the method of Example 1, with a particle concentration of 2). 10 11 0.10% (particles / mL); 0.50% carbomer; 5.00% glycerol; 0.15% sodium hyaluronate; appropriate amount of triethanolamine; purified water to 100%. Add carbomer to purified water and allow it to swell completely for 4–8 h; add glycerol and sodium hyaluronate, and stir well; add extracellular vesicles of Cistanche deserticola loaded with miR-396-5p, and stir slowly at low temperature; adjust the pH to 6.0–7.0 using triethanolamine; stir well and defoam to obtain the final product.

[0062] Example 8 A Cistanche deserticola extracellular vesicle formulation for improving skin photoaging. By mass percentage, it includes: extracellular vesicles of Cistanche deserticola loaded with miR-396-5p (prepared according to the method of Example 1, with a particle concentration of 2). 10 11 The emulsion consisted of: 0.1% (particles / mL); 5.00% glycerol; 4.00% squalane; 5.00% caprylic / capric triglyceride; 2.00% cetearyl alcohol; 1.00% polyglycerol fatty acid ester; and purified water to 100%. Squalane, caprylic / capric triglyceride, and cetearyl alcohol were mixed as the oil phase and heated to 70°C. Glycerol was added to purified water as the aqueous phase and heated to 70°C. The oil phase was slowly added to the aqueous phase, and the mixture was emulsified at high speed for 10 min. After cooling to below 40°C, extracellular vesicles of *Cistanche deserticola* loaded with miR-396-5p were added. The mixture was stirred until homogeneous to obtain the emulsion preparation. The prepared emulsion was uniform and fine in appearance, milky white, without layering or precipitation, with a pH of 5.5–6.5. It exhibited good spreadability and skin compatibility, making it suitable for improving photoaging of the skin.

[0063] Example 9 A serum formulation containing Cistanche deserticola extracellular vesicles loaded with miR-396-5p to improve skin photoaging By mass percentage, it includes: extracellular vesicles of Cistanche deserticola loaded with miR-396-5p (prepared according to the method of Example 1, with a particle concentration of 2). 10 11 The following ingredients were added to the solution: 0.10% (particles / mL); 3.00% glycerol; 5.00% butylene glycol; 0.10% sodium hyaluronate; 1.00% panthenol; purified water to 100%. Glycerol and butylene glycol were added to purified water; sodium hyaluronate was added and dissolved completely; panthenol was added and stirred until homogeneous; extracellular vesicles of Cistanche deserticola loaded with miR-396-5p were added at room temperature; the solution was filtered, sterilized, and then filled into vials to obtain the essence preparation.

[0064] Example 10 A Cistanche deserticola extracellular vesicle formulation for improving skin photoaging. By mass percentage, it includes: extracellular vesicles of Cistanche deserticola loaded with miR-396-5p (prepared according to the method of Example 1, with a particle concentration of 2). 10 11The mixture consisted of: 0.10% (particles / mL); 5.00% glycerol; 5.00% butylene glycol; 0.10% sodium hyaluronate; 0.20% β-glucan; and purified water to 100%. Glycerol and butylene glycol were added to purified water and mixed thoroughly. Sodium hyaluronate and β-glucan were added and dissolved completely. Extracellular vesicles of Cistanche deserticola loaded with miR-396-5p were added and mixed thoroughly. The mixture was filtered for sterilization. The solution was then impregnated with a non-woven fabric mask substrate, sealed, and packaged to obtain the mask liquid preparation.

[0065] Example 11 A spray formulation for improving skin photoaging containing extracellular vesicles of Cistanche deserticola loaded with miR-396-5p. By mass percentage, it includes: extracellular vesicles of Cistanche deserticola loaded with miR-396-5p (prepared according to the method of Example 1, with a particle concentration of 2). 10 11 0.10% (particles / mL); 2.00% glycerol; 0.05% sodium hyaluronate; 0.50% panthenol; purified water to 100%. Add glycerol, sodium hyaluronate and panthenol to purified water; stir until completely dissolved; add extracellular vesicles of Cistanche deserticola loaded with miR-396-5p; filter to sterilize; fill into spray containers to obtain the spray formulation.

[0066] Example 12 A freeze-dried powder formulation of Cistanche deserticola containing miR-396-5p-loaded extracellular vesicles for improving skin photoaging. By mass percentage, it includes: extracellular vesicles of Cistanche deserticola loaded with miR-396-5p (prepared according to the method of Example 1, with a particle concentration of 2). 10 11 0.10% (particles / mL); 5.00% trehalose; 2.00% mannitol; phosphate buffer to 100%. Dissolve trehalose and mannitol in phosphate buffer; add extracellular vesicles of Cistanche deserticola loaded with miR-396-5p and mix gently; aliquot into sterile lyophilization bottles; pre-freeze at -80℃ for 8 h; freeze-dry under vacuum for 24–48 h; seal and store to obtain the lyophilized powder preparation. For use, reconstitute with sterile water for injection, physiological saline, or buffer.

[0067] Example 13 Human efficacy evaluation trial Test substance: The essence prepared in Example 9.

[0068] Participants: A total of 60 women, aged 35-60 years. All participants exhibited varying degrees of photoaging on their faces, including increased fine lines, skin laxity, and dullness, meeting the inclusion criteria.

[0069] Instructions for use: Following the consumer use testing methods stipulated in the "Cosmetic Efficacy Claim Evaluation Standards," 60 subjects were selected and divided into a test group and a control group. The test group applied the serum prepared in Example 9 to both sides of the face after cleansing in the morning and evening, while the control group applied deionized water. Approximately 0.5 mL was used each time, for a continuous period of 4 weeks. Subjects did not use other skincare products claiming anti-wrinkle or firming effects during the test period. Wrinkle Percentile (WPS) was obtained using the VISIA® skin image analysis system and its accompanying analysis software before use (week 0), at 2 weeks, and at 4 weeks as an indicator of skin wrinkle evaluation. A higher Wrinkle Percentile value indicates a higher proportion of subjects with better skin wrinkle condition than the same-age reference population, suggesting a more significant improvement in skin wrinkles.

[0070] The results of the Wrinkle Percentile test in the subjects are shown in Table 6. Figure 8 .

[0071] Table 6. Subject Wrinkle Percentile

[0072] The results showed that, compared with before use, after continuous use of Cistanche deserticola-derived extracellular vesicle extract loaded with miR-396-5p, the skin wrinkle score rate of the subjects in the experimental group was significantly increased, skin wrinkles were improved, and skin elasticity was significantly improved. Among them, at 4 weeks, the subjects' wrinkle percentage increased to 71%, indicating that the subjects' facial wrinkles were significantly improved compared with the same age group, and the degree of photoaging of the skin was alleviated.

[0073] The results show that the Cistanche deserticola extracellular vesicle extract containing miR-396-5p provided by the present invention can effectively improve the skin photoaging state, enhance skin elasticity, and promote the improvement of the dermal layer structure, and has good safety and application value.

[0074] Example 14 Product anti-wrinkle effect satisfaction Test substance: Facial mask prepared in Example 10 Participants: A total of 30 participants, including 5 men and 25 women, aged 35-50 years. The participants exhibited varying degrees of photoaging on their faces, including increased fine lines, skin laxity, decreased elasticity, rough skin texture, and dull skin tone, meeting the inclusion criteria.

[0075] Instructions for use: Subjects should use the mask product continuously, 3 times a week, for 15-20 minutes each time, for 4 consecutive weeks. During the testing period, other skincare products claiming anti-wrinkle or firming effects should not be used.

[0076] After the experiment, the anti-wrinkle effect of the product was evaluated through a questionnaire survey. The results are shown in Table 7.

[0077] Table 7. Satisfaction with the anti-wrinkle effect of the product

[0078] The results showed that after using the mask of the present invention for 4 weeks, more than 80% of the subjects believed that their fine lines and crow's feet were improved, and they had a high degree of recognition of the overall anti-wrinkle effect of the product. This indicates that the Cistanche deserticola extracellular vesicle mask containing miR-396-5p provided by the present invention has a good effect on improving skin photoaging and anti-wrinkle.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of any one or more of miR-396-5p, miR-396-5p mimics, and extracellular vesicles of Cistanche deserticola loaded with miR-396-5p in the preparation of products for improving skin photoaging, characterized in that, The nucleotide sequence of miR-396-5p is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The improvement of skin photoaging includes one or more of the following: increasing skin cell vitality, reducing collagen degradation, promoting collagen synthesis, promoting COL1A1 expression, inhibiting MMP3 expression, inhibiting aging-related β-galactosidase activity, reducing inflammatory response, or delaying skin aging.

3. The application as described in claim 1, characterized in that, The miR-396-5p is derived from extracellular vesicles of Cistanche deserticola.

4. The application as described in claim 1, characterized in that, The skin photoaging mentioned refers to ultraviolet-induced skin photoaging.

5. The application as described in claim 4, characterized in that, The ultraviolet light is UVA and / or UVB.

6. The application as described in claim 1, characterized in that, The method for preparing extracellular vesicles of Cistanche deserticola loaded with miR-396-5p includes the following steps: washing Cistanche deserticola, adding PBS buffer and grinding to break it up, and separating and purifying the extracellular vesicles of Cistanche deserticola by differential centrifugation combined with sucrose density gradient centrifugation to obtain a supernatant solution containing extracellular vesicles of Cistanche deserticola loaded with miR-396-5p.

7. A composition for improving photoaging of the skin, characterized in that, It includes one or more of the following: miR-396-5p as described in claim 1, miR-396-5p mimics, or extracellular vesicles of Cistanche deserticola loaded with miR-396-5p.

8. The composition according to claim 7, characterized in that, The composition is a pharmaceutical composition, a cosmetic composition, a medical aesthetic preparation, or a skin repair preparation.

9. The composition according to claim 7, characterized in that, The dosage form of the composition is a gel, emulsion, serum, facial mask liquid, hydrogel, spray, or lyophilized powder.

10. The composition according to any one of claims 7 to 9, characterized in that, The composition is used to prevent, delay, or improve ultraviolet-induced photoaging of the skin.

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