Using Modified Cell Particles Combined with Botulinum Toxin to Improve Skin Anti-Aging

HEK293T cells (CL-0005, Procell Life Science & Technology, Wuhan, China) and human skin fibroblasts (HFF-1; BFN60808577, Shanghai Yubo Biotechnology Co., Ltd., Shanghai, China) were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; BL304A, Biosharp, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; 12664025C, Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (BL505A, Biosharp, China). Human adipose-derived mesenchymal stem cells (ADMSCs) were derived from a liposuction specimen of a 30-year-old healthy woman. After resting under sterile conditions for 30 min to allow stratification, 6 mL of middle-layer adipose tissue was mixed with 2 mL of phosphate-buffered saline (PBS) and 2.5 mg of type I collagenase (BS163, Biosharp, China). The mixture was then digested at 37 °C with shaking at 120 rpm for 60 min. The digestion was terminated by adding 2 mL low-glucose DMEM (BL305A, Biosharp, China), followed by centrifugation at 247× g for 10 min. The cell pellet was resuspended and filtered through a 70 μm strainer. The filtrate was adjusted to 4 mL and transferred into a T25 culture flask, which was then incubated at 37 °C in a 5% CO₂ atmosphere. Medium was replaced after 24 h. Cells were passaged to P1 after 48 h and expanded to P2. Flow cytometry was used to characterize P3 ADMSCs. Following trypsinization (BL512A, Biosharp, China), 5 × 10⁴ cells per tube were incubated with antibodies against CD19 (11-0199-42, Thermo Fisher Scientific, Waltham, MA, USA), CD105 (17-1057-42, Thermo Fisher Scientific), CD44 (17-0441-82, Thermo Fisher Scientific), and CD11b (11-0118-42, Thermo Fisher Scientific) at 37 °C for 1 h and analyzed using an FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

The SV2C gene was amplified by polymerase chain reaction (PCR) using specific primers: forward primer 5′-AGGTCGACTCTAGAGGATCCCGCCACCATGGAAGACTCTTACAAGGATAG-3′ and reverse primer 5′-TCCTTGTAGTCCATACCCATCAGAACCTGGGTTCGTGTGTGTC-3′. The amplicon was cloned into the GV492 vector (GeneChem, Shanghai, China) between BamHI and AgeI restriction sites to generate the GV492-SV2C expression plasmid. The final construct was confirmed by sequencing and stored at −80 °C.

Lentivirus was produced according to a previously described method [20]. First, 20 µg of GV492-SV2C, 15 µg of pHelper 1.0, 10 µg of pHelper 2.0, and 1 mL of transfection reagent were mixed and incubated for 15 min at room temperature before being added to HEK293T cells. After 6 h, the medium was replaced, and cells were cultured for an additional 72 h. The supernatant was collected by centrifugation at 4200× g for 10 min, filtered through a 0.45 µm membrane, and concentrated by ultrafiltration at 5145 g, 4 °C, for 2 h. After a second centrifugation at 2058× g for 5 min, the virus was aliquoted and stored at −80 °C. Viral titer was determined using fluorescence detection. ADMSCs were infected with the lentivirus for 72 h, then selected with 2 mL medium containing 6 µg/mL puromycin. After one day, the medium was changed, and the cells were cultured for two passages. SV2C expression was assessed by fluorescence microscopy, Western blot, and immunofluorescence. For BTX-A binding analysis, 100 µL of 3 U/mL botulinum toxin A (BTX-A; HengLi^®, Lanzhou Bioproduction Institute, Lanzhou, China) was added to the SV2C-overexpressing ADMSCs, and BTX-A binding was evaluated by immunofluorescence.

Total RNA was extracted using TRIzol reagent (15596026CN, Invitrogen, Waltham, MA, USA) and quantified with a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific). First-strand cDNA was synthesized from total RNA using the Hifair 1st Strand cDNA Synthesis SuperMix (11120ES60, Yeasen Biotech, Shanghai, China). qPCR was conducted using Hieff qPCR SYBR Green Premix (11202ES08, Yeasen Biotech, China) with the following primers: β-actin forward 5′-GGCGGCACCACCATGTACCCT-3′ and reverse 5′-AGGGGGCCGGACTCGTCATACT-3′; SV2C forward 5′-CAAGACGGGATGTCAGATTACC-3′ and reverse 5′-AAAGCACCCATAGAGCCACCTA-3′. PCR conditions were as follows: pre-denaturation at 94 °C for 3 min, followed by 22 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 5 min.

Exosomes were isolated from the culture supernatant of ADMSCs using differential ultracentrifugation as described previously [25]. ADMSCs were cultured in serum-free medium (DMEM/F12, Biosharp) for 72 h. The collected supernatant was sequentially centrifuged at 300× g, 2000× g, and 10,000× g for 10 min each at 4 °C to remove cells and debris. The resulting supernatant was filtered through a 0.22 µm microporous membrane. The filtrate was concentrated by centrifugation at 5000× g for 30 min and then subjected to ultracentrifugation at 100,000× g for 70 min. The pellet was resuspended in sterile PBS and stored at −80 °C. For morphological analysis, transmission electron microscopy (TEM) was performed. Exosomes were centrifuged at 2470× g for 10 min at 4 °C, and the supernatant was diluted at 1:10,000. A drop of diluted exosome solution was applied vertically onto a copper grid and air-dried under a heat lamp. The grid was then stained with 1% phosphotungstic acid for 70 s and dried again before observation under 60 kV using an HT-7800 electron microscope (Hitachi, Tokyo, Japan). Particle size and concentration were measured using a ZetaView^® PMX120 nanoparticle tracking analyzer (Particle Metrix, Meerbusch, Germany). To confirm the expression of exosomal surface marker CD63 and its interaction with BTX-A, flow cytometry analysis was performed. Polystyrene microspheres (Microspheres, ThermoFisher, Waltham, MA, USA) were used as capture carriers. FITC-conjugated anti-CD63 antibody (ab18235, Abcam, Cambridge, UK) was bound to biotinylated microspheres, allowing specific binding of CD63 on the exosomal surface. To detect surface-bound BTX-A, PE-conjugated anti-BTX-A antibody (ab252711, Abcam) was prepared using the Lightning-Link PE/R-Phycoerythrin Conjugation Kit (ab102918, Abcam). After staining in the dark, fluorescence signals from the FITC and PE channels were measured using a flow cytometer, allowing for the evaluation of exosome capture efficiency and verification of BTX-A binding. This method is protected by a Chinese national invention patent CN116165376A. Purified exosomes were labeled with red fluorescent dye PKH26 (MINI26-1KT, Sigma-Aldrich, St. Louis, MO, USA). The labeling reaction was terminated by adding an equal volume of exosome-free fetal bovine serum, followed by ultracentrifugation to remove excess unbound PKH26 dye and re-isolate the labeled exosome.

Human skin fibroblast cells (HFF-1) were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; BL304A, Biosharp) supplemented with 10% fetal bovine serum. When cell confluence reached approximately 80%, the cells were trypsinized and seeded into 6-well plates at a density of 3 × 10⁵ cells per well. Cellular senescence was induced using D-galactose at a final concentration of 40 mg/mL for 48 h. After induction, the medium was replaced with serum-free DMEM. Cells were treated according to experimental grouping as follows: 100 μL of 3 U/mL BTX-A (BTX-A group), 2.0 × 10⁷ particles/mL exosomes (EXO group), or an equivalent dose of the EXO^SV2C-BTX-A complex (2.0 × 10⁷ particles/mL containing 3 U/mL BTX-A). Control wells received equal volumes of PBS. Cells were cultured for an additional 24 h before analysis. To ensure consistency in exosome dosage, exosome concentrations were quantified prior to treatment using nanoparticle tracking analysis, and appropriate dilutions were made to standardize the number of exosomal particles between the EXO and EXO^SV2C-BTX-A groups.

To assess the effects of different treatments on HFF-1 cell viability, cells were seeded into 96-well plates and randomly divided into four groups: PBS, EXO, BTX-A, and EXOSV2C-BTX-A. Upon cell attachment, the culture medium was replaced with serum-free DMEM. Each group received 10 μL of 3 U/mL BTX-A solution, 2.0 × 10⁷ particles/mL exosome suspension, or an equivalent dose of the EXO-BTX-A complex; PBS was added to the control group. At 6, 12, 24, and 48 h post-treatment, 10 μL of CCK-8 reagent (C0038, Beyotime, Shanghai, China) was added to each well. Plates were incubated at 37 °C for 2 h, and absorbance was measured at 450 nm using a microplate reader (Synergy HTX, BioTek, Winooski, VT, USA) to reflect cellular metabolic activity.

Sterile 1000 μL pipette tips were used to create a vertical scratch at the center of the cell monolayer under a biosafety cabinet. Detached cells were gently washed away with PBS, and the medium was replaced with serum-free DMEM containing 100 μL of 3 U/mL BTX-A, 2.0 × 10⁷ particles/mL exosomes, or an equivalent amount of the EXO-BTX-A complex. The control group received an equal volume of PBS. Images of the scratch area were captured at 0, 12, and 24 h. ImageJ software (version 1.53; NIH, Bethesda, MD, USA) was used to calculate the wound closure rate as a measure of cell migration: Migration area (%) = (S₀ − S_n)/S₀ × 100%, where S₀ is the initial scratch area and S_n is the scratch area at time n.

An in vivo model of skin aging was established according to a previously published method [26,27]. Sixty 8-week-old male Kunming mice were housed at 25 °C with a 12-h light/dark cycle and given free access to food and water for a 2-week acclimation period. Mice were randomly divided into five groups (n = 12 per group): normal control (untreated), D-galactose only, D-galactose + EXO, D-galactose + BTX-A, and D-galactose + EXO^SV2C-BTX-A. Except for the control group, all mice received daily subcutaneous injections of D-galactose (1000 mg/kg, B21893, YuanYe Bio, Shanghai, China) into the dorsal skin for 42 consecutive days to induce aging. Starting from Day 15, mice in the treatment groups received daily subcutaneous injections of one of the following (100 μL per injection) for 28 days: exosome (2.0 × 10⁷ particles/mL), BTX-A (3 U/mL), EXO^SV2C-BTX-A complex (2.0 × 10⁷ particles/mL, 3 U/mL BTX-A). On Day 42, mice were euthanized under anesthesia, and dorsal skin tissues were collected from the injection sites for histological and molecular biological analyses. Blood samples were also collected for hematological and serum biochemical testing. To ensure consistent exosome dosing, the particle concentration of exosomes was quantified using nanoparticle tracking analysis prior to administration, and the dosage was adjusted accordingly to ensure equal exosome amounts between the EXO and EXO^SV2C-BTX-A groups. All animal procedures were approved by the Animal Care and Use Committee of Hainan Medical University (Approval No. HYLL-2023-026, Date: 24 February 2023).

To evaluate the toxicity differences between BTX-A and the EXO^SV2C-BTX-A complex, a two-stage dose-escalation experiment was performed. In the first stage, BTX-A was administered via intraperitoneal injection at doses of 1, 2, 4, 8, 16, 32, 64, and 128 U/kg. For each dose, mice were randomly divided into two groups: BTX-A and EXO-BTX-A (2.0 × 10⁷ particles/mL exosomes), with 10 mice in each group. All mice received a single intraperitoneal injection and were monitored continuously for 4 days for signs of toxicity and mortality. Mortality rates were recorded to estimate the 0% lethal dose (LD₀) and 100% lethal dose (LD₁₀₀). In the second stage, doses were refined within the LD₀–LD₁₀₀ range and set at 32, 40, 44, 48, 52, 56, 64, 68, and 72 U/kg, with 10 mice per dose group. The same treatment and observation procedure was followed. The number of deaths within 4 days was recorded, and toxicological parameters were analyzed using the BL-420 biological function experimental system (Chengdu Taimeng Technology Ltd., Chengdu, China). LD₅₀ and the 95% confidence interval (CI) were calculated to evaluate whether exosome encapsulation could mitigate BTX-A toxicity. Additionally, after 4 days of acute toxicity treatment, mice from the EXO group (2.0 × 10⁷ particles/mL), BTX-A group (at LD₅₀ dose), and EXO^SV2C-BTX-A group (at LD₅₀ dose) were sacrificed for tissue analysis. Lung, liver, and kidney tissues were collected for morphological evaluation, and blood samples were obtained for complete blood count and serum biochemical analysis to assess systemic toxicity differences among the groups further.

To evaluate oxidative stress injury in mouse dorsal skin tissue, malondialdehyde (MDA) levels were measured using a commercial assay kit (A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Fresh tissue samples were homogenized according to the kit instructions, and protein concentration was determined using the bicinchoninic acid (BCA) method. MDA content was normalized to total protein. Absorbance at 532 nm was measured using a microplate reader, and MDA concentrations were calculated based on a standard curve and expressed as nmol/mg protein.

To assess histological structure and collagen deposition in skin, tissue sections were prepared and stained as previously described [25]. Fresh dorsal skin samples were collected at the end of the experiment and fixed in 4% paraformaldehyde at 4 °C for 24 h. Tissues were processed via standard dehydration, paraffin embedding, and sectioning at a thickness of 5 μm. Hematoxylin and eosin (H&E) staining was performed according to the manufacturer's instructions (C0105S, Beyotime, China) to evaluate tissue architecture and inflammatory infiltration. Fontana-Masson Stain (ab150669, Abcam) was used to assess collagen fiber content and distribution. Images were captured using an inverted microscope (CKX53, OLYMPUS, Tokyo, Japan) for qualitative and quantitative analyses.

Western blot analysis was conducted as described previously [28]. To detect the expression levels of target proteins, cells were lysed on ice for 5 min using RIPA buffer supplemented with phenylmethylsulfonyl fluoride (PMSF). Supernatants were collected by centrifugation, and protein concentrations were measured using a BCA protein assay kit (20201ES, Yeasen, China). Equal amounts of protein were denatured at 100 °C for 8 min, separated on 5% stacking and 12% separating SDS-PAGE gels, and transferred onto nitrocellulose (NC) membranes. After blocking with 5% bovine serum albumin at room temperature for 1 h, membranes were incubated overnight with primary antibodies at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the Tannon 5200Multi chemiluminescence imaging system (Tannon, Shanghai, China). The primary antibodies used were rabbit anti-SV2C (ab33892, Abcam, 1:1000), rabbit anti-Collagen I (AF7001, Affinity Biosciences, Cincinnati, OH, USA; 1:800), and rabbit anti-β-tubulin (ab18207, Abcam, 1:800). The secondary antibody was HRP-conjugated goat anti-rabbit IgG (BL003A, Biosharp, 1:20,000). β-Tubulin served as the internal control.

Immunofluorescence staining was used to detect SV2C and Collagen I expression, following previously established protocols [29]. Once cells reached 80% confluence, the culture media were discarded, and the cells were washed three times with PBS. They were then fixed in 4% paraformaldehyde for 30 min and blocked at room temperature for 1 h. Cells were then incubated overnight at 4 °C with primary antibodies. After rewarming for 30 min the next day, cells were incubated with fluorescent secondary antibodies for 90 min at room temperature in the dark. The secondary antibodies included donkey anti-rabbit IgG (ab150076, Abcam) and goat anti-rabbit IgG (ab150073, Abcam). After three washes, nuclei were counterstained using DAPI-containing antifade mounting medium (R20889, YuanYe Bio, China). Images were captured using an inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan). To evaluate cellular senescence, senescence-associated β-galactosidase (SA-β-gal) staining was performed. Fibroblasts cultured in 6-well plates were washed twice with PBS and stained according to the manufacturer's instructions (C0602, Beyotime, China). SA-β-gal-positive cells were identified as blue-stained cells under a light microscope.

All statistical analyses were performed using GraphPad Prism version 8.0.2. Each experiment was independently repeated at least three times. Data are presented as mean ± standard error of the mean (SEM). For comparisons between two groups, statistical significance was determined using a two-tailed Student's t-test. For comparisons among more than two groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey's post hoc test. A p-value less than 0.05 (p < 0.05) was considered statistically significant.

To obtain exosomes capable of binding BTX-A via SV2C-mediated interactions, we aimed to construct exosomes with high SV2C expression on their surface (Figure 1A). We first designed and constructed the GV492-SV2C-GFP recombinant plasmid (Figure 1B). Agarose gel electrophoresis confirmed that the SV2C band was 2225 bp, consistent with the expected size, verifying successful plasmid construction (Figure 1C). Colony PCR further confirmed positive transformants (476 bp, samples 5–12) (Figure 1D). Transfection of the SV2C plasmid into HEK293T cells resulted in green fluorescence, indicating successful transfection (Figure 1E). Fluorescence-based quantification showed a maximum viral titer of 2.5 × 10⁸ TU/mL. qPCR analysis revealed significantly elevated SV2C expression in transfected HEK293T cells compared to controls (Figure 1F). These results demonstrate that the SV2C-overexpressing lentiviral vector was successfully constructed and efficiently transduced HEK293T cells.

Primary-cultured human ADMSCs exhibited a spindle-shaped morphology and orderly alignment, consistent with the characteristics of mesenchymal stem cells (Figure 2A). Flow cytometry showed that 99.51% of cells were CD105-positive and CD19-negative, while 98.59% were CD44-positive and CD11b-negative, confirming the identity of ADMSCs (Figure 2B). Following lentiviral transduction with SV2C, cells displayed green fluorescence under microscopy, whereas untransduced controls did not, indicating successful transduction (Figure 2C). After puromycin selection and two passages, purified SV2C-overexpressing ADMSCs were obtained (Figure 2D). RT-PCR and Western blotting confirmed significantly elevated SV2C mRNA and protein levels in the transduced ADMSCs (Figure 2E,F). Immunofluorescence staining further confirmed robust intracellular SV2C expression in the SV2C group compared to the empty vector and blank controls (Figure 2G). Following incubation with 3 U/mL BTX-A, immunofluorescence analysis demonstrated that SV2C-overexpressing ADMSCs bound BTX-A more effectively than low-expressing controls (Figure 2H). These findings confirm the successful generation of SV2C-overexpressing ADMSCs and their enhanced binding capability to BTX-A.

To generate an efficient exosome–toxin complex, engineered exosomes were isolated from SV2C-overexpressing ADMSCs using ultrafiltration and differential centrifugation. Transmission electron microscopy revealed cup-shaped membrane vesicles exhibiting characteristic exosomal morphology (Figure 3A). After co-incubation with 3 U/mL BTX-A for 1 h, the resulting EXO^SV2C-BTX-A complex was analyzed using bead-assisted flow cytometry. Due to the nanoscale size of exosomes, conventional flow cytometry is insufficient; therefore, fluorescent bead-based capture technology was employed to overcome this limitation. FITC-labeled antibodies were used to detect CD63, and PE-labeled antibodies were used to detect BTX-A, with FITC and PE fluorescence intensities reflecting CD63 expression and BTX-A content, respectively. EXO^SV2C-BTX-A exhibited significantly higher PE fluorescence than the EXO group, indicating enhanced BTX-A binding, while CD63 levels were comparable between the two groups, suggesting similar exosome quantities (Figure 3B). Nanoparticle tracking analysis showed mean diameters of 122 ± 2.96 nm (median: 118.6 nm) for EXO and 235.2 ± 5.08 nm (median: 235.8 nm) for EXO^SV2C-BTX-A, indicating that SV2C overexpression and BTX-A binding increased exosomal size (Figure 3C). Cellular uptake assays demonstrated that PKH26-labeled exosomes were efficiently internalized by live HFF-1 fibroblasts, as indicated by the presence of red fluorescence after 2 h of co-culture. No fluorescence was observed in fixed HFF-1 cells, suggesting active uptake by viable cells rather than passive binding (Figure 3D). CCK-8 assays revealed that EXO treatment enhanced the viability and proliferation of HFF-1 cell. Although proliferation in the EXO^SV2C-BTX-A group was slightly reduced compared to EXO alone, it remained significantly higher than the BTX-A group, indicating that exosome encapsulation alleviated BTX-A-induced cytotoxicity (Figure 3E). Similarly, scratch assays indicated that EXO^SV2C-BTX-A enhanced fibroblast migration compared to BTX-A alone (Figure 3F). Collectively, these findings demonstrate that SV2C-expressing exosomes can effectively bind BTX-A, reduce its cytotoxicity, and enhance the compatibility of fibroblasts.

D-galactose (D-gal) is a commonly used inducer of cellular aging, known to accelerate senescence through oxidative stress and the accumulation of advanced glycation end products (AGEs) [30,31]. In this study, senescence of HFF-1 cells induced by D-gal was confirmed via β-Galactosidase staining. Treatment with EXO, BTX-A, or EXO^SV2C-BTX-A significantly reduced the number of senescent cells (Figure 4A), with the EXO^SV2C-BTX-A group showing a significantly lower proportion of senescent cells compared to both the EXO and BTX-A groups (Figure 4B). Type I collagen accounts for approximately 80% of total human collagen, and its reduction is a key biomarker of skin aging [31]. Immunofluorescence analysis revealed that D-gal treatment substantially suppressed type I collagen expression in HFF-1 cells, while all three interventions—EXO, BTX-A, and EXO^SV2C-BTX-A—effectively alleviated collagen degradation and promoted collagen synthesis (Figure 4C,D). Quantitative protein analysis further confirmed that EXO^SV2C-BTX-A treatment resulted in the highest type I collagen levels, suggesting superior synergistic anti-aging efficacy (Figure 4E). Taken together, these results suggest that EXO^SV2C-BTX-A significantly suppresses D-gal-induced cellular senescence and promotes type I collagen synthesis.

To further assess the anti-aging effects of EXO^SV2C-BTX-A in vivo, we evaluated skin appearance in mice induced to age by D-gal. Normal mice exhibited smooth, taut skin and dense, glossy fur, whereas D-gal-treated mice exhibited hair thinning, reduced skin elasticity, and dermal sagging (Figure 5A). Treatment with EXO, BTX-A, or EXO^SV2C-BTX-A led to visible improvements in skin texture and fur condition. However, high-dose BTX-A and EXO^SV2C-BTX-A induced toxicity (e.g., "wasp waist"). To determine whether exosomal encapsulation alleviated BTX-A toxicity, we conducted a dose-escalation lethality assay. The calculated LD₅₀ for the EXO^SV2C-BTX-A group was 59.89 U/kg, which was higher than the BTX-A group (50.99 U/kg), and also exceeded the safety threshold of 44 U/kg for BTX-A (Figure 5B), indicating reduced acute toxicity via exosomal encapsulation. In terms of safety, no gross pathological abnormalities were observed in the liver, lung, or kidney of normal mice 4 days after treatment with EXO, BTX-A, or EXO^SV2C-BTX-A (Figure S1A). However, hematological analysis revealed that BTX-A significantly reduced white blood cells, lymphocytes, and red blood cells, as well as hemoglobin, while neutrophils were elevated, indicating systemic toxicity. In contrast, the EXO^SV2C-BTX-A group showed no such abnormalities, and values remained close to normal ranges (Figure S1B). Serum biochemical analysis further demonstrated that BTX-A elevated liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), while EXO^SV2C-BTX-A-treated mice showed better liver safety in these parameters (Figure S1C). In D-gal-induced aging mice, D-gal treatment itself did not significantly disrupt most hematological or biochemical parameters, except for a mild reduction in platelet count and an elevated AST level, indicating a subtle trend towards hepatic toxicity. Neither EXO nor BTX-A exacerbated these changes, suggesting acceptable systemic tolerance (Figure S2). MDA levels in skin tissues—a marker of oxidative stress—were significantly reduced by all treatments, with the lowest level observed in the EXO^SV2C-BTX-A group (Figure 5C). Type I collagen content was significantly higher in the EXO and EXO^SV2C-BTX-A groups compared to the D-gal+PBS group (Figure 5D). HE staining showed epidermal thickening, keratinization, and reduced dermal appendages in D-gal mice, which were attenuated by EXO and EXO^SV2C-BTX-A treatment (Figure 5E,F). Masson staining confirmed that EXO^SV2C-BTX-A prevented collagen degradation and improved fiber alignment (Figure 5G). These findings indicate that EXO^SV2C-BTX-A alleviates D-gal-induced skin aging by reducing oxidative stress, enhancing collagen synthesis, and preserving skin structure, while offering superior systemic safety and lower toxicity compared to free BTX-A.