C57BL6 males were purchased from the model animal Institute of Nanjing Medical University (Nanjing, China). Adipose mesenchymal stem cells (ADMSCs), HEK 293T and RAW 264.7 cell were purchased from ATCC cell bank (Shanghai, China). The synthetic siRNA was purchased from GenScript (Nanjing, China), and serum, DMEM medium was purchased from Thermo Fisher (USA). CD63, CD9, Calnexin and ALIX antibodies were purchased from Abcam (USA).

Cells were cultured using DMEM medium containing 10% FBS. Lentiviral packaging plasmids containing siRNA and GFP fluorescent sequences, as well as lentiviral vectors containing puromycin, were transfected into logarithmically growing HEK 293T cells, changed 6 h later, and lentiviral supernatant 48 h after transfection was collected. Cells and cell debris were removed, and 50000 g was ultracentrifuged for 2 h to concentrate viral particles, which were resuspended using sterile PBS. At ADMSC cell density of 80%, approximately 10⁷ viral particles were added to 10⁶ cells for transfection. After 3 days, 10^-3 of puromycin was added and siRNA containing stable ADMSC cells (si-ADMSCs) were selected.

CCK-8 assay was performed to analyze cell proliferation and viability. A density of 2×10³ cells/well was seeded in 96 well plates. 10 μL of CCK-8 solution was added per well at 12, 24, 36, 48 and 60 (h), respectively, and then cell proliferation was determined at a wavelength of 450 nm using a microplate reader.

When cells reached 80% confluence, 10% exosome free FBS DMEM was used for rehydration, and cell supernatants were collected after 48 h of culture. Next, exosomes were obtained by centrifugation at 300 g for 10 min, followed by centrifugation at 10000 g for 1 h and ultracentrifugation at 110000 g for 70 min and resuspension with appropriate amounts of PBS. To know the number of exosomes, their particle size distribution and concentration were detected using a nanoparticle size tracking analyzer (NTA). The morphology of exosomes was observed using transmission electron microscopy (TEM). 30 μg of exosomal protein was used to perform Western blot (WB) analysis.

Samples of freshly ultradissociated exosomes were diluted to 1 mL with diluent C and loaded with 6 μL PKH26 to the tube, mix gently for 30 s, let stand at room temperature for 5min. The reaction was quenched by adding 10% BSA in 2 mL PBS configuration and brought to 8.5 mL with serum-free medium. 1.5 mL of sucrose solution (0.971 M) was slowly added dropwise with a gun to an ultracentrifuge tube, and the exosome-PKH26 labeling solution was added above the sucrose buffer layer. 110000 g for over dissociation 2 h at 4 °C. The medium and middle layer were carefully aspirated and exosomes were resuspended with 1x PBS and transferred inside 10 kDa MWCO ultrafiltration tubes. Add 9 mL PBS, 0.75 mL culture medium, and centrifuge at 3000 g for 40 min to a final volume of 0.5-1 mL. The stained exosomes were added dropwise to epithelial cells cultured in serum-free exosomes, and the cells were collected after 4 h of culture for observation under a fluorescence microscope. Meanwhile, the stained exosomes were applied on the surface of mouse skin, and the absorption of exosomes was photographed under a fluorescence imaging system after 4 h.

Total RNA from cells and tissues was extracted using Trizol as previously described (19). Reverse transcription and quantitative PCR primers for siRNAs were designed using miRNA design v1.01, and all primers were synthesized at GenScript Biotech (Nanjing, China). Briefly, 2 μL of total RNA was first mixed with 5 × gDNA wiper mix and incubated at 42°C for 2 min to remove contamination of genomic DNA. Incubate the mixture at 25°C for 5 min, 50°C for 15 min, and 85°C for 5 min. Finally, SYBR dye qPCR amplification was carried out on LightCyler 96 instrument. PCR program for tsRNAs included the following steps: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The threshold cycle (CT) values were determined using a fixed threshold setting.

Exosomes or tissues were homogenized by protease free Congo beads, protein lysates were prepared using an ice bath of Pierce RIPA buffer containing protease and phosphatase inhibitors for 30min, and samples were quantified using a BCA protein assay kit. Protein samples were denatured by addition of SDS at 99° C for 5 min and run at 80 V for 30 min on 12% bis-tris stacking gel and 120 V for 1 h on separating gel according to the manufacturer’s recommended protocol. Proteins were transferred to 0.2 µm PVDF membranes. Membranes were blocked with 5% nonfat milk for 1 h at room temperature, incubated with antibodies against CD63, anti-CD9 as well as anti-AlIX in 1 x TBST overnight at 4°C, washed, and incubated with the corresponding secondary antibodies for 1 h at room temperature to detect target proteins. Finally, ECL luminescent liquid was added dropwise to expose in an exposure instrument.

All animal experiments were approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University. 8 weeks old male C57BL/6 mice were randomly divided into four groups (n = 5), and an area of 3 × 3 cm² just above the back of the mice was applied with hair removal cream, scraped off after 10 min, and 0.1 mg carprofen was administered to each mouse via drinking water. After 48 h, the UVB lamp height was set to 1 m (W = 10⁷ μJ/cm²), the mice in the 4 groups were irradiated for 0, 16, 32, and 64 (min) times per week until the mice developed macroscopic skin lesions in the skin. Twenty 8-week-old C57BL/6 males were UV induced for 32 min as described above until the appearance of skin lesions and randomly assigned to three groups: (1) Skin smeared with 200 μL PBS, (2) Skin smeared with 200 μL ADMSC-EXOs (1 ug/μL), (3) Skin ameared with 200 μL si-ADMSC-EXOs. Treatments were given 3 times per week for 2 weeks.

Mice were euthanized and UV irradiated and control skin were surgically removed, fixed using 4% paraformaldehyde. Skin tissues were embedded in paraffin, sectioned (8 μM thick), UV induced inflammation, cellular infiltration and skin damage were assessed with H & E staining.

The cell supernatant and mouse skin tissues were collected and centrifuged at 1000 g for 10 min, followed by 10000 g for 30 min, and the supernatant was taken to a clean EP tube. The levels of cytokines in the samples were then analyzed using an inflammatory cytokine multi analyte ELISArray Kit (Qiagen, Germany) according to the manufacturer’s instructions. Absorbance (450 nm) was measured using a multifunctional microplate reader.

All values are expressed as mean ± SEM or mean ± SD. Statistically significant differences between the groups were analyzed using one-way or two-way analysis of variance (ANOVA). Differences with P < 0.05 were considered statistically significant. (Statistical significance was designated as ^* P < 0.05, ^** P < 0.01, ^*** P < 0.005, and ^**** P < 0.001).

Exosomal membrane is a kind of cell membrane like structure, and exosomes can be taken up by cells or tissues in the way of membrane fusion. To explore the contribution of skin uptake of ADMSC derived exosomes, we characterized ADMSC exosomes obtained by ultracentrifugation and applied the stained exosomes with epithelial cells and mice skin cells for fluorescence tracing. Transmission electron microscopy (TEM) observed that exosomes extracted from ADMSC cell culture fluid were vesicle like structures with diameters ranging between 100-200 nm ( Figure 1A ); Further analysis using nanoparticle size tracking (NTA) confirmed that the size distribution of ADMSC-EXOs was between 30-200 nm, with a peak size at 130 nm and a concentration of approximately 9×10¹¹ particles/ml ( Figure 1B ). We next performed western blot (WB) analysis of membrane proteins from ADMSC-EXOs, which express CD9, CD63, and Alix characteristic of exosomes, but not calnexin, which is part of the cell membrane skeleton ( Figure 1C ). Next, we added the PKH26 stained exosomes into culture medium of epithelial cells, and exosomes could be absorbed by epithelial cells after 4 h incubation ( Figure 1D ). Besides, the stained exosomes were applied on the surface of mouse skin tissue, and the fluorescence could be detected in cuticle and dermal tissues of skin after 4 h ( Figure 1E ). These results suggested that ADMSC derived exosomes can be taken up by epithelial cells and mouse skin, and may function as carrier for drug delivery.

The therapeutic use of siRNA is limited by the inability of RNA molecules to reach their target cells. Recently, nature’s own carriers of RNA, exosomes, are increasingly being considered as siRNA delivery vehicles due to their properties. NF-κB (nuclear factor kappa light chain enhancer of activated B cells), which is one of the key regulators of inflammatory immune responses and regulated cytokine production (e.g., interleukin-6 (IL-6) and TNF-α), plays an integral role in the pathogenesis of skin lesions. We therefore evaluated the therapeutic effects of the siRNA designed to specifically target NF-κB for the treatment of skin lesions. We constructed a genetic circuit consisting of two functional modules: the promoter module drives the transcription of siRNA, which leads to the package of saturated cytoplasmic siRNA into exosomes, while the siRNA expression cassette module maximises the expression of the siRNA guide strand and minimises the expression of undesired passenger strand. Besides, the cytomegalovirus (CMV) promoter was selected as the promoter module, and the pre-miR-155 backbone was selected as the optimal siRNA expression cassette to produce siRNA. To make the siRNA of target genes highly expressed in ADMSC exosomes, we constructed lentivirus containing these siRNA-expressed genetic circuit. After the lentivirus infected the cell, we could select the siRNA stablely expressed ADMSC cell line (si-ADMSCs). The si-ADMSC exosomes were then obtained by ultracentrifugation ( Figure 2A ). Quantitative PCR revealed that the siRNA expression of NF-κB was significantly higher in si-ADMSC exosomes (si-ADMSC-EXOs) than in normal ADMSC-EXOs ( Figure 2B ). TEM results confirmed that there was no obvious difference in the morphology between si-ADMSC-EXOs and ADMSC-EXOs, and NTA analysis also indicated that there were no significant differences in particle number and size distribution between the two types of exosomes ( Figures 2C, D ). Proteins of exosomes were also detected by WB, and the amount of protein expression of si-ADMSC-EXOs was similar with that of ADMSC-EXOs ( Figure 2E ). These results suggested the successful synthesis of siRNA overexpressed si-ADMSC-EXOs, and the characterization of si-ADMSC-EXOs and ADMSC-EXOs was not significantly different, providing the potential for gene therapy in skin lesions.

We evaluated whether the formation of NF-κB siRNA-encapsulating exosomes could reduce NF-κB expression in vitro. Macrophages play a central role in all stages of wound healing and orchestrate the wound healing process. Their functional phenotype is dependent on the wound microenvironment, which changes during healing, hereby altering macrophage phenotype. Pathological functioning of macrophages in the wound healing process can result in derailed wound healing, like the formation of ulcers, chronic wounds, hypertrophic scars and keloids. We first incubated si-ADMSC-EXOs with macrophage cells (RAW 264.7 cell lines) and epithelial cells, respectively, and detected the expression levels of the NF-κB. Significant decrease in NF-κB protein and mRNA expression was observed in both primary macrophages and epithelial cells ( Figures 3A, B ). We then explored the function of NF-κB siRNA-encapsulating exosomes in macrophage proliferation by CCK-8 assay. Compared with the mock or control group, the engineered si-ADMSC-EXOs significantly inhibited the proliferation of RAW 264.7 cells ( Figure 3C ). Next, we measured the levels of cytokines in RAW 264.7 cells through ELISA, and we found that the expression levels of IL-6 and TNF-α, but not IL-10, were decreased in RAW 264.7 cell relative to controls ( Figure 3D ). These results indicated that the biosynthesized engineered si-ADMSC-EXOs can effectively inhibit the expression of the target genes and inhibit the macrophage activation.

Above, we confirmed that si-ADMSC-EXOs could be taken up by epithelial cells and skin, while suppressing the expression of target genes and inhibiting the proliferation and cytokines of macrophages in vitro. Here, we constructed skin injury model in C57BL/6 mice by two weeks of UV irradiation and then treated by smearing ADMSC-EXOs or si-ADMSC-EXOs, respectively ( Figure 2A ), while negative control mice were treated without UV irradiation (vehicle). Comparing with vehicle group mice, two weeks of UV irradiation caused the mice acute skin redness and ulceration in both ADMSC-EXOs and si-ADMSC-EXOs group ( Figure 4A ). As expected, treating with si-ADMSC-EXOs could faster recovery the injury in skin comparing with ADMSC-EXOs group ( Figure 4A ). H&E staining was further employed to observe the skin pathological changes, and UV irradiation would change the nuclei of skin tissue into irregular morphology, and the epidermal layer became thicker, while the si-ADMSC-EXOs treatment group showed a higher rate of skin recovery than ADMSC-EXOs group ( Figure 4B ). WB analysis of the treated skin revealed that the expression of NF-κB was no significantly different between the ADMSC-EXOs and vehicle groups, but remarkably reduced in the si-ADMSC-EXOs group ( Figure 4C ). Consistently, the expression levels of IL-6 and TNF-α, but not IL-10 were decreased in si-ADMSC-EXOs group comparing to ADMSC-EXOs group ( Figure 4D ). Besides, the level of phosphorylated ERK protein (p-ERK) and c-Jun protein (p-c-Jun) were lower in si-ADMSC-EXOs group, indicating the skin inflammation was reduced ( Figure 4E ). Finally, by immunohistochemical analysis, the level of the macrophage marker F480 in the skin tissue was higher in the UV irradiated group than in the control group, whereas the level of the M2 type macrophage marker CD206 was higher after treatment with si-ADMSC-EXOs than in the other two groups ( Figures 4F, G ). These results suggested that UV irradiation promoted an increase in M1 macrophages in the skin, which secrete inflammatory cytokines; while si-ADMSC-EXOs treatment could ultimately induce M2 type macrophage activation and inhibit inflammatory responses, thereby repairing and remodeling damaged skin cells.