An engineered extracellular vesicle and methods of making and uses thereof

Abstract

The application provides an engineered extracellular vesicle and a preparation method and use thereof, and belongs to the field of biological medicines.The engineered extracellular vesicle is an extracellular vesicle loaded with a miR-1246 mimic, which is obtained by loading the miR-1246 mimic into the extracellular vesicle by an electroporation method.The engineered extracellular vesicle has a significant protective effect on UVB-induced photoaging damage of HaCaT cells, can effectively promote the proliferation and migration of HaCaT cells, reduce the positive expression rate of aging-related beta-galactosidase, and reduce the UVB-induced aging of HaCaT cells;meanwhile, the engineered extracellular vesicle can effectively inhibit the UVB-induced inflammatory response, significantly enhance the antioxidant capacity of cells, and relieve the UVB-induced oxidative stress damage.The engineered extracellular vesicle can be widely applied to the preparation of products against skin photoaging, and provides a new strategy and industrialization technical scheme for the prevention and treatment of skin photoaging.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an engineered extracellular vesicle, its preparation method, and its uses. Background Technology

[0002] Skin photoaging, also known as extrinsic aging, refers to premature aging of the skin caused by prolonged exposure to ultraviolet (UV) radiation. It is a core skin pathological process distinct from natural physiological aging (intrinsic aging). Compared to natural aging, photoaging progresses more rapidly and manifests with more pronounced skin damage, primarily including rough skin, increased wrinkles, sagging, uneven pigmentation, telangiectasia, and impaired skin barrier function. Furthermore, photoaging not only affects appearance, but long-term cumulative UV damage also increases the risk of malignant skin tumors, posing a serious threat to skin health.

[0003] In the ultraviolet (UV) spectrum, UVA and UVB are the core factors inducing photoaging of the skin. UVB primarily acts on the epidermis, damaging keratinocyte DNA and inducing skin inflammation and pigmentation abnormalities. UVA, on the other hand, has stronger penetrating power, reaching deep into the dermis and directly damaging collagen and elastin fibers. From a molecular perspective, UV radiation can cause skin cells to produce large amounts of reactive oxygen species (ROS), triggering oxidative stress, which in turn upregulates the expression of matrix metalloproteinases (MMPs), accelerates collagen degradation and inhibits its synthesis, disrupting the metabolic balance of the skin matrix. Simultaneously, UV radiation can activate inflammatory signaling pathways and induce abnormal cell apoptosis, ultimately leading to skin structural damage and photoaging lesions. Clinically, photoaging is more common in people who are exposed to prolonged sunlight, work outdoors, or live in areas with high UV radiation, and the risk increases with age.

[0004] Currently, various anti-photoaging interventions have been developed in the clinical and skincare fields. However, all methods have significant limitations, making it difficult to achieve comprehensive protection and deep repair against photoaging. Firstly, sun protection is the primary means of blocking UV damage and can be divided into physical and chemical sunscreens. Physical sunscreens have a heavy feel, easily clog pores, and can trigger breakouts; chemical sunscreens are unstable, easily photodegraded, and some ingredients pose sensitizing and potential toxicity risks. Furthermore, sun protection only blocks UV rays and cannot repair existing photodamage or reverse aging. Secondly, retinoids (such as all-trans retinoic acid and adapalene) can promote collagen synthesis and improve photoaging, but they are highly irritating and can easily cause adverse reactions such as dryness, redness, and peeling, and increase skin photosensitivity. Their suitability is limited, and they are not suitable for long-term use. Third, antioxidants (such as vitamin C, vitamin E, coenzyme Q10, resveratrol, etc.) can reduce oxidative damage by scavenging free radicals, but they generally suffer from poor stability, easy inactivation, low transdermal absorption, and poor bioavailability. They only act on the skin surface, resulting in weak and short-lasting repair effects. Fourth, medical aesthetic procedures (such as laser treatments, chemical peels, and injectable fillers) can improve existing wrinkles and pigmentation with quick results, but they are invasive or minimally invasive procedures with risks of infection, pigmentation, and scarring. They are also expensive, have long recovery periods, and only improve surface symptoms, failing to fundamentally block the photoaging process, leading to a high recurrence rate. Fifth, natural plant extracts are highly safe, but their complex composition, low content of effective active substances, poor batch stability, and difficulty in transdermal penetration of large molecular components limit their effective repair effects and industrial application.

[0005] Recent studies have found that specific miRNAs can target and regulate core photoaging pathways such as oxidative stress, collagen metabolism, inflammation, and apoptosis, and can repair skin photodamage at the gene level, making them highly promising new anti-aging targets. However, pure miRNAs have extremely poor stability, are easily hydrolyzed by enzymes, have weak transdermal absorption, and short in vivo half-life, making them unsuitable for direct skin intervention and greatly limiting their application. Extracellular vesicles are natural nanocarriers with advantages such as good biocompatibility, no immunogenicity, strong transdermal absorption, high stability, and long-lasting sustained release. They can effectively encapsulate and protect nucleic acid-based active substances, improving their bioavailability. However, current technologies have not yet developed mature miRNA-engineered extracellular vesicle systems, lacking effective technical solutions for using miRNA-modified vesicles to target and intervene in skin photoaging.

[0006] Therefore, given the numerous bottlenecks in existing technologies, there is an urgent need to develop a new, highly efficient, low-toxicity, and reversible active intervention system for skin photoaging, providing a novel technical approach and solution for the root-cause prevention and treatment of skin photoaging. Summary of the Invention

[0007] The purpose of this invention is to provide an engineered extracellular vesicle, its preparation method, and its uses.

[0008] This invention provides an engineered extracellular vesicle, wherein the engineered extracellular vesicle is an extracellular vesicle loaded with a miR-1246 mimic, which is obtained by loading the miR-1246 mimic into the extracellular vesicle via electroporation; the electroporation voltage is 300~450V, the resistance is 100~300Ω, the capacitance is 50~250μF, and the number of electroporations is 1~2.

[0009] Furthermore, the sequence of the miR-1246 mimic is shown in SEQ ID NO.1; the extracellular vesicles are milk-derived extracellular vesicles; the electroporation voltage is 400V, the resistance is 200Ω, the capacitance is 125μF, and the number of electric shocks is 1.

[0010] The present invention also provides a method for preparing engineered extracellular vesicles, comprising the following steps: mixing a miR-1246 mimic solution with an extracellular vesicle suspension to obtain a mixture; then subjecting the mixture to electroporation to allow the miR-1246 mimic to enter the extracellular vesicle; after electroporation, performing ultrafiltration replacement and sterilization to obtain extracellular vesicles loaded with miR-1246 mimic.

[0011] Furthermore, the protein concentration in the engineered extracellular vesicles is 220-230 ng / cell.

[0012] Furthermore, the mass ratio of protein to miR-1246 mimic in the extracellular vesicle suspension is (2090~2100):1. Preferably, it is 2093.8:1.

[0013] Further, the ultrafiltration replacement includes the following steps: using an ultrafiltration tube to concentrate the electroporated mixture to half of its original volume, then adding an equal volume of physiological saline to the ultrafiltration tube for replacement, concentrating it to half volume again, then discarding the eluent in the outer sleeve, rinsing the outer sleeve with physiological saline, and discarding the residual liquid; repeating the above ultrafiltration replacement steps 3 to 5 times; the sterilization is sterilization through a 0.22 μm filter membrane.

[0014] The present invention also provides a pharmaceutical composition, which is a formulation prepared by adding engineered extracellular vesicles as active ingredients and pharmaceutically acceptable excipients.

[0015] Furthermore, the formulation is a liquid formulation, wherein the concentration of engineered extracellular vesicles is 5~40 μg / mL, preferably 20 μg / mL.

[0016] The present invention also provides a cosmetic composition which is prepared by using engineered extracellular vesicles as active ingredients and adding cosmetically acceptable excipients.

[0017] Furthermore, the cosmetic composition is a liquid composition, wherein the concentration of engineered extracellular vesicles is 5~40 μg / mL, preferably 20 μg / mL.

[0018] Further, the cosmetic composition comprises the following ingredients in weight percentages: 0.3-0.7 wt% engineered extracellular vesicles, 0.05-0.15 wt% polysaccharide moisturizers, 4-6 wt% polyol moisturizers, 2-4 wt% moisturizing penetration enhancers, 1-3 wt% skin barrier repair active ingredients, 0.3-0.7 wt% antioxidants, 0.1-0.3 wt% anti-inflammatory and soothing active ingredients, 0.3-0.7 wt% preservatives, and the balance being water.

[0019] Further, the cosmetic composition comprises the following ingredients in weight percentages: 0.5 wt% engineered extracellular vesicles, 0.1 wt% polysaccharide moisturizer, 5 wt% polyol moisturizer, 3 wt% moisturizing penetration enhancer, 2 wt% skin barrier repair active ingredient, 0.5 wt% antioxidant, 0.2 wt% anti-inflammatory and soothing active ingredient, 0.5 wt% preservative, and the balance being water.

[0020] Further, the polysaccharide moisturizer includes at least one of sodium hyaluronate, hyaluronic acid, acetylated sodium hyaluronate, β-glucan, trehalose, and chitosan; the polyol moisturizer includes at least one of glycerin, propylene glycol, 1,3-propanediol, sorbitol, xylitol, erythritol, and mannitol; the moisturizing penetration enhancer includes at least one of butylene glycol, 1,2-pentanediol, hexanediol, caprylyl glycol, and polyethylene glycol; and the skin barrier repair active ingredient includes niacinamide, panthenol, ceramide, phytosterols, cholesterol, and... The product contains at least one of palmitoyl tripeptide-5 and asiaticoside; the antioxidants include at least one of vitamin E, vitamin E acetate, vitamin C, ascorbyl glucoside, ferulic acid, glutathione, resveratrol, tea polyphenols, and astaxanthin; the anti-inflammatory and soothing active ingredients include at least one of dipotassium glycyrrhizate, ammonium glycyrrhizate, centella asiatica extract, and chamomile extract; and the preservatives include at least one of phenoxyethanol, ethylhexylglycerin, caprylyl glycol, hexanediol, 1,2-pentanediol, benzyl alcohol, potassium sorbate, and sodium dehydroacetate.

[0021] The present invention also provides the use of engineered extracellular vesicles in the preparation of pharmaceuticals and / or cosmetics for the prevention and / or treatment of photoaging of the skin.

[0022] Furthermore, in the drug or cosmetic, the concentration of engineered extracellular vesicles is 5~40 μg / mL, preferably 20 μg / mL.

[0023] Furthermore, the drug or cosmetic is a drug or cosmetic used to promote the proliferation and / or migration of HaCaT cells, reduce the positive expression rate of aging-related β-galactosidase (SA-β-Gal), inhibit ultraviolet light-induced inflammatory responses, and alleviate ultraviolet light-induced oxidative stress damage.

[0024] This invention utilizes electroporation to load miR-1246 mimics into extracellular vesicles, creating a miRNA-engineered extracellular vesicle—miR-1246-MEVs nanodelivery system. This system exhibits significant protective effects against UVB-induced photoaging damage in HaCaT cells, effectively promoting HaCaT cell proliferation and migration, reducing the positive expression rate of senescence-associated β-galactosidase (SA-β-Gal), and alleviating UVB-induced HaCaT cell senescence. Simultaneously, it effectively inhibits UVB-induced inflammatory responses, significantly enhances cellular antioxidant capacity, and alleviates UVB-induced oxidative stress damage. In particular, compared with 5 μg / mL miR-1246-MEVs, 10 μg / mL miR-1246-MEVs and 40 μg / mL miR-1246-MEVs, treatment with 20 μg / mL miR-1246-MEVs significantly promoted HaCaT cell proliferation, suggesting that a concentration of 20 μg / mL miR-1246-MEVs can enhance the proliferative activity of epidermal keratinocytes and accelerate the self-repair and renewal of the epidermis after UVB damage, thereby playing a role in resisting skin photoaging damage.

[0025] The present invention also screened the electroporation parameters and proved that electroporation loading under the conditions of 400V voltage, 200Ω resistance, 125μF capacitance and 1 electric shock can achieve the best balance between loading efficiency (72.3%) and vesicle integrity (93.2%). Increasing or decreasing the voltage, resistance, capacitance or number of electric shocks is not conducive to loading efficiency or vesicle integrity.

[0026] The extracellular vesicles loaded with miR-1246 mimics of this invention have a clear therapeutic mechanism in anti-photoaging of the skin, achieving effective prevention and treatment of skin photoaging, and can be widely used in the preparation of anti-photoaging products; at the same time, this invention also uses milk-derived extracellular vesicles, which are safe, accessible, and can be produced on a large scale; this invention provides a new strategy and industrialization technology solution for the prevention and treatment of skin photoaging.

[0027] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0028] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0029] Figure 1 Characterization of milk-derived extracellular vesicles loaded with miR-1246 mimics: A is nanoparticle tracking analysis; B is transmission electron microscopy detection; C is nanoflow cytometry detection.

[0030] Figure 2 Different UVB irradiation intensities (0, 20, 40, 60, 80, 100 mJ / cm²) 2 Statistical graph of HaCaT cell survival rate after treatment (**P<0.01, compared with 0 mJ / cm) 2 (Compared to the irradiated group).

[0031] Figure 3 This is a statistical graph showing the survival rate of HaCaT cells after treatment with milk-derived extracellular vesicles loaded with different concentrations of miR-1246 mimics (5, 10, 20, 40 μg / mL miR-1246-MEVs) for 24 h and 48 h (*P<0.05, **P<0.01, compared with the control group; #P<0.05, ##P<0.01, compared with the 20 μg / mL miR-1246-MEVs group and the 5 μg / mL, 10 μg / mL, and 40 μg / mL miR-1246-MEVs groups).

[0032] Figure 4 These are the control group, model group (UVB), and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm²) in the Transwell cell migration assay. 2 Statistical plot of cell migration number (UVB) (**P<0.01, compared with control group; ##P<0.01, compared with model group).

[0033] Figure 5 The control group, model group (UVB), and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm) were tested in the β-galactosidase staining experiment. 2 Representative images of UVB-stained cells and statistical graph of positive cell rate (**P<0.01, compared with control group; ##P<0.01, compared with model group).

[0034] Figure 6The ELISA assay was used to detect the control group, model group (UVB), and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm). 2 Statistical graph of IL-1β, IL-18 and TNF-α content in UVB cell culture supernatant (*P<0.05, **P<0.01, compared with control group; #P<0.05, compared with model group).

[0035] Figure 7 The enzyme detection methods included the control group, model group (UVB), and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm). 2 Statistical graphs of SOD activity, MDA content and GSH-PX activity in UVB cells (**P<0.01, compared with control group; #P<0.05, compared with model group). Detailed Implementation

[0036] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0037] Example 1: Preparation of milk-derived extracellular vesicles loaded with miR-1246 1.1 Extraction of milk-derived extracellular vesicles Take 1L of fresh milk and centrifuge it sequentially at 4℃: centrifuge at 2,000×g for 20min to remove fat globules and cell debris, centrifuge at 10,000×g for 30min to remove large vesicles and protein aggregates, and collect the supernatant; then filter it through a 0.22μm filter membrane, discard the precipitate, and ultracentrifuge the supernatant (100,000×g, 4℃, 70min), discard the supernatant, resuspend the precipitate in PBS and wash it once, then ultracentrifuge it again (100,000×g, 4℃, 70min), discard the supernatant, and resuspend the obtained precipitate in an appropriate amount of PBS to obtain a milk-derived extracellular vesicle (MEV) suspension; the protein concentration was determined to be 1046.90 ng / μL by the BCA method, and the suspension was aliquoted and stored at -80℃ for later use.

[0038] 1.2 Electroporation loading and ultrafiltration purification of miR-1246 (1) The dry powder of miR-1246 mimic (miR-1246 mimic, hsa-miR-1246, sequence as shown in SEQ ID NO.1) was centrifuged at 6000 rpm for 1 min to allow the powder to settle at the bottom of the tube. Then, 3 mL of enzyme-free water was added to dissolve it. After tightening the cap, the tube was shaken to mix. The mixture was then centrifuged at 2000 g for 1 min at 4 °C. Finally, a 0.5 mg / mL miR-1246 mimic solution was prepared. (2) Mix the miR-1246 mimic solution with the MEVs suspension at a volume ratio of 1:1 to obtain a mixture; (3) Add the mixture to the dried electroporation cup, place it in the electroporation tank, and use an electroporation instrument to perform electroporation. Set the parameters: voltage 400V, resistance 200Ω, capacitance 125μF, and electroporation once. After electroporation, immediately place the electroporation cup in an ice bath for 5 minutes. (4) The mixture after electroporation was subjected to ultrafiltration replacement: the extracellular vesicles and the solution medium were separated using a 100kD ultrafiltration tube, the mixture was concentrated to half of its original volume, and then an equal volume of physiological saline was added to the ultrafiltration tube for replacement. The mixture was concentrated to half its original volume again. The elution solution in the outer tube was then discarded, the outer tube was washed with 1 mL of physiological saline, and the residual liquid was aspirated. The above ultrafiltration replacement steps were repeated for a total of 3 times to completely remove the unloaded free miRNA in the solution. (5) Collect the concentrated extracellular vesicles, sterilize them by passing them through a 0.22 μm filter membrane, and obtain milk-derived extracellular vesicles MEVs loaded with miR-1246 mimic (miR-1246-MEVs); the protein concentration was determined to be 226.24 ng / μL by BCA method.

[0039] Comparative Example 1: Milk-derived extracellular vesicles loaded with non-targeted miRs and their characterization Referring to Example 1, the only difference is that miR-1246 mimic was replaced with NC mimic (negative control, sequence as shown in SEQ ID NO.2), resulting in milk-derived extracellular vesicles (NC-MEVs) loaded with NC; the BCA protein concentration was 205.15 ng / μL.

[0040] Table 1 Nucleic Acid Sequence List Example 2: Preparation of an essence containing milk-derived extracellular vesicles loaded with miR-1246 (1) Add sodium hyaluronate to deionized water and stir at room temperature until completely dissolved to form phase A; (2) Add glycerol, butanediol and nicotinamide to phase A, stir well, heat to 75~80℃, and keep warm for 15min to sterilize; (3) Cool down to below 40°C, add miR-1246-MEVs suspension, vitamin E, dipotassium glycyrrhizate and phenoxyethanol, and stir gently until well mixed; (4) Adjust the pH to 5.5-6.5 using citric acid / sodium citrate buffer; (5) After standing to defoam, and after passing the inspection, it is filled to obtain an essence containing milk-derived extracellular vesicles loaded with miR-1246. The content of each raw material is shown in Table 2.

[0041] Table 2 shows the ingredient content of the essence containing milk-derived extracellular vesicles loaded with miR-1246. The following experimental examples demonstrate the beneficial effects of the present invention.

[0042] Example 1: Characterization of milk-derived extracellular vesicles loaded with miR-1246 1.1 Experimental Methods (1) Loading efficiency experiment: Total RNA was extracted from miR-1246-MEVs, MEVs-NC and MEVs using the Trizol method. Reverse transcription and real-time quantitative PCR (qRT-PCR) were performed using miR-1246-specific primers (forward primer, sequence shown in SEQ ID NO.3) and universal reverse primers. U6 was used as an internal control. -ΔΔCt The relative expression level is calculated using this method.

[0043] (2) The particle size distribution and particle concentration of miR-1246-MEVs, MEVs-NC and MEVs were characterized by nanoparticle tracking analysis (NTA) and nanoflow cytometry, respectively. The proportion of extracellular vesicles positive for surface markers of miR-1246-MEVs, MEVs-NC and MEVs was analyzed by nanoflow cytometry combined with fluorescent labeling. The morphology of miR-1246-MEVs, MEVs-NC and MEVs was observed by transmission electron microscopy (TEM).

[0044] 1.2 Experimental Results The loading efficiency experiment results showed that the relative expression level of miR-1246 in the miR-1246-MEVs group was upregulated by 85.3 times compared with the original MEVs group and by 82.7 times compared with the MEVs-NC group, indicating that miR-1246 has been successfully and efficiently loaded into MEVs by electroporation.

[0045] The removal rate of the NC gene after ultrafiltration was 94.05% (relative to exogenous reference cel-miR-39, sequence shown in SEQ ID NO.4), indicating that unloaded free mimics were effectively removed.

[0046] Figure 1 Nanoparticle tracking analysis of A showed that the concentration of MEVs particles was 2.89 × 10⁻⁶. 11 Particles / mL, mode particle size 156.2 nm; miR-1246-MEVs particle concentration 2.38 × 10⁻⁶. 9Particles / mL, mode particle size 163.8 nm; MEVs-NC particle concentration 8.37 × 10⁻⁶ 9 The particle size distribution was 144.9 nm, with a mode particle size of 144.9 nm. The particle sizes of MEVs, miR-1246-MEVs, and MEVs-NC were all distributed in the range of 140-170 nm, which are consistent with the characteristics of extracellular vesicles. This indicates that miR-1246-MEVs were successfully prepared in this invention, and neither electroporation nor ultrafiltration replacement processes led to significant degradation or aggregation of vesicles.

[0047] Figure 1 Transmission electron microscopy (TEM) of B revealed that both MEVs and miR-1246-MEVs exhibited typical cup-shaped or disc-shaped structures, with complete morphology, spherical shape, and uniform size. The morphology of miR-1246-MEVs and MEVs-NC was not significantly different from that of the original MEVs, indicating that the electroporation loading conditions and subsequent ultrafiltration replacement process did not cause significant damage to the physical structure of the vesicles.

[0048] Figure 1 Nano-flow cytometry (NanoFCM) results for C showed that the proportion of membrane-bound particles reached 99.3%, indicating that MEVs, miR-1246-MEVs, and MEVs-NC are vesicle structures encapsulated by phospholipid membranes.

[0049] Experiment Example 2: Screening Experiment of Electroporation Loading Parameters and UVB Irradiation Intensity 2.1 Experimental Methods (1) Screening of electroporation loading parameters: miR-1246-MEVs were prepared according to Example 1, with the only difference being: different electroporation loading parameters (voltage, resistance, capacitance, number of electric shocks) were set, and the final loading efficiency (refer to Experiment 1.1 (1)) and vesicle integrity (nanoflow cytometry combined with fluorescent labeling analysis) were detected.

[0050] (2) UVB irradiation intensity screening: Human immortalized keratinocytes (HaCaT cells) in logarithmic growth phase were taken, washed with PBS, digested with 0.25% trypsin, and the cell suspension was collected. The cells were centrifuged at 250×g for 5 min, the supernatant was discarded, and the cells were resuspended in an appropriate amount of DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were counted using an automated cell counter and the cell density was adjusted to 1×10⁻⁶. 5 Cells were seeded at 100 μL / well in 96-well plates (margin wells were filled with sterile PBS) and incubated at 37°C, 5% CO2 for 24 h to allow cell adhesion. After cell adhesion, the cells were randomly divided into 7 groups, with 4 replicates per group: zeroing group (cell-free, containing only culture medium), 0 mJ / cm², and 0 mJ / cm². 2 Irradiation group (control group), 20 mJ / cm2 Irradiated group, 40 mJ / cm 2 Irradiated group, 60 mJ / cm 2 Irradiated group, 80 mJ / cm 2 Irradiated group and 100mJ / cm 2 Irradiation group: Before irradiation, the culture medium was discarded, cells were washed twice with sterile PBS, a small amount of PBS was added to cover the cells, and the cells were irradiated under a UVB light source (wavelength 311 nm). The irradiation dose was controlled by adjusting the irradiation time. After irradiation, the PBS was discarded, and fresh culture medium was added for 24 h of further culture. Cell viability was then detected using the CCK-8 assay: CCK-8 reagent was diluted 1:10 in serum-free culture medium, 110 μL of CCK-8 working solution was added to each well, and the cells were incubated at 37℃ and 5% CO2 for 2 h. The OD value of each well was measured at 450 nm using a microplate reader. One-way ANOVA was performed using SPSS 27.0 statistical software. P < 0.05 was considered statistically significant.

[0051] 2.2 Experimental Results As shown in Table 3, when the electroporation voltage is in the range of 300~400V, the loading efficiency increases with increasing voltage; above 400V, vesicle integrity decreases sharply, and loading efficiency decreases due to vesicle rupture. When the resistance is less than 200Ω, the loading efficiency is significantly reduced. When the capacitance is less than 125μF, the loading efficiency is also significantly reduced; when it is greater than 125μF, both loading efficiency and vesicle integrity are reduced simultaneously. Furthermore, a single electroporation shock can achieve 72.3% high-efficiency loading and 93.2% high vesicle integrity; increasing the number of shocks will damage the vesicles.

[0052] Experimental results show that the optimal parameters for electroporation loading of miR-1246 mimic into MEVs are: voltage 400V, resistance 200Ω, capacitance 125μF, and one electric shock. Under this parameter combination, the loading efficiency reaches 72.3% and the vesicle integrity reaches 93.2%, achieving the best balance between efficient loading and structural preservation.

[0053] Table 3 Electroporation loading parameters, loading efficiency, and vesicle integrity like Figure 2 As shown, with 0mJ / cm 2 Compared to the irradiated group (control group, survival rate 100.00%), 20 mJ / cm 2 The cell survival rate in the irradiated group was 97.97%, with no statistically significant difference; while the 40mJ / cm² group... 2 Irradiated group (93.00%), 60 mJ / cm 2 Irradiated group (92.50%), 80 mJ / cm 2Irradiated group (91.85%) and 100 mJ / cm 2 The cell viability of the irradiated group (90.63%) was significantly reduced (P<0.01), showing a clear dose-dependent effect. Considering the stability of the model and the feasibility of subsequent experiments, 40 mJ / cm² was selected. 2 The irradiation intensity was used to establish the subsequent photoaging model.

[0054] Experiment Example 3: Screening Experiment for the Concentration of Milk-Derived Extracellular Vesicles Loaded with miR-1246 3.1 Experimental Methods HaCaT cells in logarithmic growth phase were harvested, digested, counted, and their density adjusted to 1×10⁻⁶. 5 Cells were seeded at 100 μL / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. After cell adhesion, the cells were divided into 5 groups with 4 replicates per group: control group (no drug), 5 μg / mL miR-1246-MEVs group, 10 μg / mL miR-1246-MEVs group, 20 μg / mL miR-1246-MEVs group, and 40 μg / mL miR-1246-MEVs group. The corresponding concentration of miR-1246-MEVs was added to each group, and the cells were cultured for a longer period. CCK-8 assays were performed at 24 h and 48 h after drug administration. One-way ANOVA was performed using SPSS 27.0 software. P < 0.05 was considered statistically significant.

[0055] 3.2 Experimental Results like Figure 3 As shown, at 48 h, the cell viability rates of the 5 μg / mL miR-1246-MEVs group, the 10 μg / mL miR-1246-MEVs group, the 20 μg / mL miR-1246-MEVs group, and the 40 μg / mL miR-1246-MEVs group were 99.96%, 103.26%, 111.13%, and 105.05%, respectively. Compared with 24 h, the cell viability rates of each group increased, indicating that miR-1246-MEVs can promote HaCaT cell proliferation.

[0056] Specifically, at both 24h and 48h, the cell survival rate of the 20μg / mL miR-1246-MEVs group was significantly higher than that of the control group, the 5μg / mL miR-1246-MEVs group, the 10μg / mL miR-1246-MEVs group, and the 40μg / mL miR-1246-MEVs group, indicating that 20μg / mL miR-1246-MEVs is the optimal concentration for promoting HaCaT cell proliferation.

[0057] As is well known to those skilled in the art, keratinocytes are the main constituent cells of the epidermis, and their proliferative capacity directly determines the epidermal renewal rate and the integrity of the barrier function. UVB irradiation can significantly inhibit HaCaT cell proliferation, induce cell cycle arrest and cellular senescence, which is an important cellular basis for the development of skin photoaging. The above experimental results show that treatment with a concentration of 20 μg / mL miR-1246-MEVs can significantly promote HaCaT cell proliferation, suggesting that a concentration of 20 μg / mL miR-1246-MEVs can enhance the proliferative activity of epidermal keratinocytes, accelerate the self-repair and renewal of the epidermis after UVB damage, and thus exert an anti-photoaging effect on the skin.

[0058] Experiment Example 4: Transwell Cell Migration Assay 4.1 Experimental Methods HaCaT cells in logarithmic growth phase were harvested, digested, counted, and the cell density was adjusted to 4 × 10⁻⁶. 5 Cells were seeded at 2 mL / well in 6-well plates and cultured at 37°C with 5% CO2. After cell attachment, the cells were divided into 3 groups, with 3 replicates per group: control group (normal culture, no treatment), model group (40 mJ / cm²), and model group (40 mJ / cm²). 2 UVB irradiation) and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm²) 2 (UVB irradiation); the intervention group was first given 20 μg / mL miR-1246-MEVs, cultured for 48 h, and then subjected to 40 mJ / cm² irradiation. 2 UVB irradiation was performed simultaneously on the model group with the same amount of UVB irradiation. After irradiation, cells were cultured for another 24 hours, collected from each group, washed with PBS, resuspended in pre-warmed serum-free medium, counted, and the cell density was adjusted to 4 × 10⁶ cells / year. 4 Cells / mL; then, 700 μL of complete culture medium containing 10% FBS was added to the lower chamber of a 24-well plate, and the Transwell chamber (8.0 μm pore size) was placed into the well. 200 μL of cell suspension was added to the upper chamber, and the plate was cultured for another 48 h. The chamber was carefully removed, the liquid in the upper chamber was aspirated, and the cells were fixed with pre-cooled ethanol at room temperature for 30 min. Subsequently, 0.1% crystal violet staining solution (0.5% crystal violet methanol solution diluted 1:4 with ultrapure water) was prepared, and the cells were stained at room temperature for 30 min. The chamber was gently rinsed several times with PBS, the chamber was removed, the liquid in the upper chamber was aspirated, and the unmigrated cells and staining solution on the bottom membrane surface of the upper chamber were carefully wiped away with a moistened cotton swab. After appropriate air drying, the cells were photographed under an inverted microscope (200×), and the number of migrating cells was counted in 5 randomly selected fields of view. One-way ANOVA was performed using SPSS 27.0 statistical software. P < 0.05 was considered statistically significant.

[0059] 4.2 Experimental Results like Figure 4As shown, the number of migrating cells in the control group was 224.78±16.69 per field of view, while the number in the model group was 158.67±5.68 per field of view, which was significantly lower than that in the control group (P<0.01), indicating that UVB irradiation can significantly inhibit the migration ability of HaCaT cells. The number of migrating cells in the intervention group was 189.33±16.15 per field of view, which was still lower than that in the control group, but significantly higher than that in the model group (P<0.01), indicating that miR-1246-MEVs can effectively restore the migration ability of photoaged HaCaT cells and has the ability to promote skin damage repair.

[0060] Experiment Example 5: β-Galactosidase Staining Experiment 5.1 Experimental Methods HaCaT cells in logarithmic growth phase were harvested, digested, counted, and their density adjusted to 2 × 10⁻⁶. 5 Cells / mL, seeded at 1 mL / well in a 12-well plate, divided into two groups: control group (normal culture, no treatment) and model group (40 mJ / cm²). 2 UVB irradiation) and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm²) 2 UVB irradiation), with 3 replicates per group; after cell adhesion, the intervention group was added with 20 μg / mL miR-1246-MEVs, cultured for 48 h, and then subjected to UVB irradiation (40 mJ / cm²). 2 The model groups were simultaneously irradiated with the same amount of UVB and cultured for another 24 hours after irradiation. After culture, the culture medium was discarded, the cells were washed once with PBS, and 1 mL of β-galactosidase staining fixative was added to each well. The cells were fixed at room temperature for 15 min, the fixative was discarded, and the cells were washed three times with PBS for 3 min each time. The β-galactosidase staining working solution (10 μL staining solution A + 10 μL staining solution B + 930 μL staining solution C + 50 μL X-Gal solution) was prepared according to the kit instructions. 500 μL of the solution was added to each well, wrapped in aluminum foil to protect from light, and incubated overnight at 37°C (16-20 h). After incubation, the cells were observed and photographed under an inverted microscope. The blue precipitate was a positive signal of SA-β-Gal. Five fields of view were randomly selected to count the number of positive cells and the total number of cells, and the positive rate was calculated. One-way ANOVA was performed using SPSS 27.0 software. P < 0.05 was considered statistically significant.

[0061] 5.2 Experimental Results like Figure 5As shown, only a small number of cells in the control group showed blue positive staining; the number of blue positive cells in the model group increased significantly, and the positive rate of SA-β-Gal was significantly increased (P<0.01), indicating that UVB irradiation can significantly induce HaCaT cell senescence; the number of blue positive cells in the intervention group was significantly reduced compared with the model group, and the positive rate of SA-β-Gal was significantly decreased (P<0.01), indicating that miR-1246-MEVs can effectively alleviate UVB-induced HaCaT cell senescence and have the effect of delaying skin cell senescence.

[0062] Experiment Example 6: ELISA detection of inflammatory factors 6.1 Experimental Methods HaCaT cells in logarithmic growth phase were harvested, digested, counted, and their density adjusted to 2 × 10⁻⁶. 5 Cells / mL, seeded at 1 mL / well in a 12-well plate, divided into two groups: control group (normal culture, no treatment) and model group (40 mJ / cm²). 2 UVB irradiation) and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm²) 2 UVB irradiation), with 3 replicates per group; after cell adhesion, the intervention group was added with 20 μg / mL miR-1246-MEVs, cultured for 48 h, and then subjected to UVB irradiation (40 mJ / cm²). 2 The model groups were simultaneously irradiated with the same amount of UVB, and then cultured for another 24 hours. After culture, the cell culture supernatant from each well was collected for ELISA detection. The contents of IL-1β, IL-18, and TNF-α in the cell culture supernatant of each group were detected using an ELISA kit. The kit was operated strictly according to the instructions. The absorbance value at 450 nm was measured within 10 minutes using an ELISA reader. The concentration of each sample was calculated according to the standard curve (quadratic regression equation). One-way ANOVA was performed using SPSS 27.0 statistical software. P < 0.05 was considered statistically significant.

[0063] 6.2 Experimental Results like Figure 6 As shown, compared with the control group, the levels of IL-1β (P<0.01), IL-18 (P<0.01), and TNF-α in the cell culture supernatant of the model group were significantly increased (P<0.05), indicating that UVB irradiation can significantly activate the inflammatory response of HaCaT cells. Compared with the model group, the levels of IL-1β and IL-18 in the intervention group were significantly decreased (P<0.05), and the level of TNF-α was significantly reduced, indicating that miR-1246-MEVs can effectively inhibit the release of UVB-induced inflammatory factors and reduce the inflammatory response in skin photoaging damage.

[0064] Experiment 7: Enzymatic detection of cellular antioxidant markers SOD, MDA, and GSH-PX 7.1 Experimental Methods HaCaT cells in logarithmic growth phase were harvested, digested, counted, and their density adjusted to 2 × 10⁻⁶. 5 Cells / mL, seeded at 1 mL / well in a 12-well plate, divided into two groups: control group (normal culture, no treatment) and model group (40 mJ / cm²). 2 UVB irradiation) and intervention group (20 μg / mL miR-1246-MEVs + 40 mJ / cm²) 2 UVB irradiation), with 3 replicates per group; after cell adhesion, the intervention group was added with 20 μg / mL miR-1246-MEVs, cultured for 48 h, and then subjected to UVB irradiation (40 mJ / cm²). 2 The model groups were simultaneously irradiated with the same amount of UVB and cultured for another 24 hours after irradiation. After culture, the culture medium was discarded, the samples were washed twice with PBS, and RIPA lysis buffer (containing 1% PMSF) was added to each well. The samples were lysed on ice for 30 minutes, and the lysate was collected. The samples were centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was used as the total protein extract. The protein concentration was determined by the Coomassie Brilliant Blue method. The following were measured using kits from Nanjing Jiancheng Bioengineering Institute: superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione peroxidase (GSH-PX) activity. The operation of each kit was strictly followed according to the instructions. The OD values ​​at the corresponding wavelengths were measured using an ELISA reader. The results of each index were calculated according to the formula. One-way ANOVA was performed using SPSS 27.0 statistical software. P < 0.05 was considered statistically significant.

[0065] 7.2 Experimental Results like Figure 7 As shown, compared with the control group, the SOD activity and GSH-PX activity in the model group cells were significantly reduced (P<0.01), and the MDA content was significantly increased (P<0.01), indicating that UVB irradiation can significantly damage the antioxidant defense system of HaCaT cells, leading to oxidative stress damage. Compared with the model group, the SOD activity and GSH-PX activity in the intervention group were significantly restored (P<0.05), and the MDA content was lower than that in the model group, indicating that miR-1246-MEVs can effectively alleviate UVB-induced oxidative stress damage by enhancing the activity of cellular antioxidant enzymes and reducing the accumulation of lipid peroxidation products.

[0066] In summary, this invention utilizes miR-1246 mimics to load milk-derived extracellular vesicles via electroporation, thus preparing a miRNA-functionalized nanodelivery system, miR-1246-MEVs. Furthermore, it was used as an active ingredient to prepare an essence. Screening experiments showed that electroporation loading under conditions of 400V, 200Ω, 125μF, and one electroporation cycle achieved an optimal balance between loading efficiency (72.3%) and vesicle integrity (93.2%). Cellular experiments demonstrated that miR-1246-MEVs significantly protected against UVB-induced photoaging damage in HaCaT cells, effectively promoting HaCaT cell proliferation and migration, reducing the positive expression rate of senescence-associated β-galactosidase (SA-β-Gal), and alleviating UVB-induced HaCaT cell senescence. Simultaneously, it effectively inhibited UVB-induced inflammatory responses, significantly enhanced cellular antioxidant capacity, and alleviated UVB-induced oxidative stress damage. Specifically, 20 μg / mL miR-1246-MEVs was the optimal concentration for promoting HaCaT cell proliferation, with significantly higher cell survival rates than those of 5 μg / mL, 10 μg / mL, and 40 μg / mL miR-1246-MEVs. This indicates that a concentration of 20 μg / mL miR-1246-MEVs can enhance the proliferative activity of epidermal keratinocytes, accelerating the self-repair and renewal of the epidermis after UVB damage, thereby exerting a protective effect against photoaging damage. This invention provides a novel strategy for the prevention and treatment of skin photoaging.

Claims

1. An engineered extracellular vesicle, characterized in that, The engineered extracellular vesicles are extracellular vesicles loaded with miR-1246 mimics. They are obtained by loading miR-1246 mimics into the extracellular vesicles via electroporation. The electroporation voltage is 300~450V, the resistance is 100~300Ω, the capacitance is 50~250μF, and the number of electroporations is 1~2.

2. The engineered extracellular vesicles according to claim 1, characterized in that, The sequence of the miR-1246 mimic is shown in SEQ ID NO.1; the extracellular vesicles are milk-derived extracellular vesicles; the electroporation voltage is 400V, the resistance is 200Ω, the capacitance is 125μF, and the number of electric shocks is 1.

3. A method for preparing engineered extracellular vesicles according to any one of claims 1-2, characterized in that, Includes the following steps: The miR-1246 mimic solution was mixed with the extracellular vesicle suspension to obtain a mixture. Then, the mixture was subjected to electroporation to allow the miR-1246 mimic to enter the extracellular vesicles. After electroporation, ultrafiltration and sterilization were performed to obtain extracellular vesicles loaded with miR-1246 mimic.

4. The method according to claim 3, characterized in that, The mass ratio of protein to miR-1246 mimic in the extracellular vesicle suspension is (2090~2100):

1.

5. A pharmaceutical composition, characterized in that, It is a formulation made with engineered extracellular vesicles as described in any one of claims 1 to 2 as the active ingredient, plus pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The formulation is a liquid formulation, wherein the concentration of engineered extracellular vesicles is 5~40 μg / mL, preferably 20 μg / mL.

7. A cosmetic composition, characterized in that, It is made by adding cosmetically acceptable excipients to engineered extracellular vesicles as described in any one of claims 1 to 2 as active ingredients.

8. The cosmetic composition according to claim 7, characterized in that, The cosmetic composition is a liquid composition, wherein the concentration of engineered extracellular vesicles is 5~40 μg / mL, preferably 20 μg / mL.

9. Use of the engineered extracellular vesicles according to any one of claims 1 to 2 in the preparation of medicaments and / or cosmetics for the prevention and / or treatment of photoaging of the skin.

10. The use according to claim 9, characterized in that, In the drug or cosmetic, the concentration of engineered extracellular vesicles is 5-40 μg / mL, preferably 20 μg / mL.

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