Adipose-derived stem cell exosome compound loaded with silver nanoparticles and application of adipose-derived stem cell exosome compound

Abstract

The invention discloses an adipose-derived stem cell exosome compound loaded with silver nanoparticles and application of the adipose-derived stem cell exosome compound loaded with the silver nanoparticles, and ADSC-Exos loaded with the silver nanoparticles is developed by integrating AgNPs into an exosome of ADSCs. In vivo, the Exo-AgNPs can accelerate healing of infected wounds by reducing bacterial colonization and promoting collagen deposition, angiogenesis and M2 type macrophage polarization. In vitro, the Exo-AgNPs activates the functions of fibroblasts and vascular endothelial cells, and shows an excellent antibacterial property. In addition, the Exo-AgNPs induces polarization of M2 type macrophages and inhibits secretion of proinflammatory cytokines by activating autophagy. The results show that Exo-AgNPs is expected to become an ideal biological material for treating infected wounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a silver nanoparticle-loaded adipose stem cell exosome complex and application thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, serving as a barrier against pathogens and physical trauma. When the skin is damaged, bacteria can easily colonize the wound site, leading to persistent inflammation and non-healing wounds. Infected wounds can cause severe pain, significant economic burden, and put patients at risk of amputation or even death. In recent years, the misuse of antibiotics and the emergence of drug-resistant bacteria have made it more difficult to treat infected wounds. Silver nanoparticles (AgNPs) are considered a highly effective broad-spectrum antibacterial agent that can kill a variety of bacteria by damaging the bacterial cell wall, reducing dehydrogenase activity, and inducing ROS production. Due to its excellent antibacterial properties, AgNPs are considered a potential nanomaterial for treating infected wounds and chronic wounds.

[0003] In addition to antibacterial properties, ideal wound treatment also needs to consider other factors that promote wound healing, such as promoting angiogenesis, improving dermal and epidermal regeneration, and modulating inflammation.

[0004] Therefore, in order to solve the above-mentioned problems in the prior art, it is necessary to develop a drug for treating infected wounds and chronic wounds. SUMMARY

[0005] The present application aims to provide a silver nanoparticle-loaded adipose stem cell exosome complex and application thereof, which has the characteristics of promoting regeneration, antibacterial and anti-inflammatory, and shows good prospects in the preparation of drugs for infected wound healing with excellent biological safety and curative effect.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect of the present application, a silver nanoparticle-loaded adipose stem cell exosome complex is provided, which is obtained by co-incubating stem cell exosomes (ADSCs) with silver nanoparticles (AgNPs), wherein the silver nanoparticles are embedded in the interior or surface of the stem cell exosomes.

[0007] Further, the particle size of the complex is 90-110 nm.

[0008] Further, the complex expresses exosome markers CD9, CD63, CD81, TSG101, and does not express Calnexin.

[0009] In a second aspect of the present application, a preparation method of the silver nanoparticle-loaded adipose stem cell exosome complex is provided, which comprises: The AgNPs suspension is obtained by chemical reduction through sodium citrate and AgNO3 solution; The stem cell exosome (ADSCs) is co-incubated with the AgNPs suspension, and then replaced with an exosome-free serum medium for continuous culture; the supernatant is collected by centrifugation, and the Exo-AgNPs complex is obtained by separation and washing of the precipitate.

[0010] Further, the concentration of AgNPs in the AgNPs suspension is 0.5-5 mg / L. If the concentration of AgNPs is less than 0.5 mg / L, it is easy to cause insufficient loading efficiency; if the concentration of AgNPs is greater than 5 mg / L, it is easy to cause significant increase in cytotoxicity. The concentration of AgNPs is preferably 2 mg / L, and the exosome yield or cell activity is optimal.

[0011] Further, the preparation method of the stem cell exosome (ADSCs) comprises: obtaining ADSCs from the subcutaneous adipose tissue of the mouse inguinal region, and culturing using a high-sugar DMEM medium containing fetal bovine serum and penicillin-streptomycin solution.

[0012] Further, the cell density in the stem cell exosome is 2×10 5 cells / mL-5×10 4 cells / mL. If the cell density is too low (<5×10 4 cells / mL), the exosome secretion is insufficient; and if the cell density is too high (>2×10 5 cells / mL), the nutrition competition is intensified.

[0013] Cell state: 3-6 generation ADSCs, fusion degree 80% (logarithmic growth phase), density reference: 1×10 5 cells / mL (conventional 6-well plate culture system).

[0014] In a third aspect of the present application, the application of the silver nanoparticle-loaded adipose stem cell exosome complex in the preparation of a drug for promoting the healing of an infected wound is provided.

[0015] Further, the complex promotes collagen deposition, angiogenesis, and M2 macrophage polarization.

[0016] In a fourth aspect of the present application, a drug for promoting the healing of an infected wound is provided, comprising the silver nanoparticle-loaded adipose stem cell exosome complex.

[0017] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: The application provides a silver nanoparticle-loaded fat stem cell exosome compound Exo-AgNPs, which breaks through the limitation of traditional single-function materials by activating a PI3K / autophagy pathway and simultaneously achieving regeneration promotion, antibiosis and immune regulation through structural innovation (AgNPs inlay loading), so that the healing efficiency is increased by 40-55%, and the healing time is shortened by 50% (compared with a single use group). The application provides an integrated treatment scheme for infected wounds, and has the following advantages: (1) Broad-spectrum antibacterial ability: the MIC of the application to Staphylococcus aureus and Escherichia coli is 0.8 μg / μL (A), which is more than 10 times higher than the antibacterial efficiency of ordinary exosomes (Exos). Figure 6

[0018] (2) Good debridement effect in vivo: in an infected wound model, the bacterial colonization of the Exo-AgNPs group is reduced by 80% (E), which is significantly better than that of the AgNPs single use group (only reduced by 50%). Figure 3

[0019] (3) Reversing AgNPs toxicity and accelerating tissue reconstruction.

[0020] (4) The application finds that Exo-AgNPs activate autophagy-mediated immune regulation through the PI3K pathway, which is a brand-new mechanism. Flow cytometry shows that the proportion of M2 type macrophages is increased by 72% (D), which is nearly 1 time higher than that of the Exos single use group (40%). The M1 type marker (CD86 fluorescence intensity, Figure 7 D-E). Figure 4 BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Characterization of ADSCs. (A) Morphology of ADSCs isolated from BALB / c mice. Scale bar = 100 µm. (B) Alizarin red staining of ADSCs after osteogenic differentiation. (C) Oil red O staining of ADSCs after adipogenic differentiation. (D) Alcian blue staining of ADSCs after chondrogenic differentiation. (E) Flow cytometry detection of markers CD44, CD90, CD105, CD29, CD31, and CD34 on ADSCs.

[0022] Figure 2 ​​​For the isolation and characterization of Exo-AgNPs. (A) Flow chart of Exo-AgNPs isolation. (B) TME images of AgNPs. Scale bar = 100 nm. (C) In vitro viability test of ADSCs after 48 h incubation with different concentrations of AgNPs. (D) TME images of Exos and Exo-AgNPs. Scale bar = 200 nm. Particle size distribution of Exos and Exo-AgNPs. (E) Western blot analysis of surface markers (CD9, CD63, CD81, TSG101 and Calnexin) of Exos and Exo-AgNPs. (F) Representative confocal images of PKH26-labeled Exo-AgNPs after 24 h co-culture with NIH-3T3 cells. Scale bar = 10 µm. *p value < 0.05, **p value < 0.01, ***p value < 0.001.

[0023] Figure 3 For Exo-AgNPs to improve wound healing and inhibit bacterial colonization in vivo. (A) Photographs of wound sites of control group, Exos group, AgNPs group and Exo-AgNPs group at 0, 3, 7 and 14 days after treatment. Scale bar = 5 mm. (B) Simulation of wound area. Scale bar = 5 mm. (C) Wound closure rates of control group, Exos group, AgNPs group and Exo-AgNPs group at 0, 3, 7 and 14 days after treatment. (D-E) Bacterial colony count detection of wound extracts of control group, Exos group, AgNPs group and Exo-AgNPs group at 14 days after treatment. (F) H&E staining of wound tissues of control group, Exos group, AgNPs group and Exo-AgNPs group at 14 days after treatment. Scale bar = 200 µm. *p value < 0.05, **p value < 0.01, ***p value < 0.001.

[0024] Figure 4To determine whether Exo-AgNPs promote collagen formation, angiogenesis, and M2 polarization of macrophages in vivo. (A) Masson's trichrome staining of wound tissues at day 14 post-treatment in control, Exos, AgNPs, and Exo-AgNPs groups. Scale bar = 200 pm. (B) IF staining of CD31 and a-SMA in wound tissues at day 14 post-treatment in control, Exos, AgNPs, and Exo-AgNPs groups. Scale bar = 200 pm. (C) Quantitative analysis of the number of blood vessels at the wound site. (D) IF staining of Ml -type macrophage marker CD86 and M2-type macrophage marker CD206 in wound tissues at day 14 post-treatment in control, Exos, AgNPs, and Exo-AgNPs groups. Scale bar = 200 pm. (E) Quantitative analysis of CD86 and CD206 cell fluorescence (DAPI fluorescence as a reference). *p value < 0.05, **p value < 0.01, ***p value < 0.001.

[0025] Figure 5 To determine whether Exo-AgNPs promote cell migration and angiogenesis in vitro. (A) Wound healing experiment of NIH-3T3 cells treated with PBS, Exos (100 pg / mL), and Exo-AgNPs (100 pg / mL). Scale bar = 200 pm. (B) Transwell experiment of NIH-3T3 cells treated with PBS, Exos, and Exo-AgNPs. Scale bar = 100 pm. (C) Quantitative analysis of the wound closure rate in the wound healing experiment and the number of migrated cells in the Transwell experiment of NIH-3T3 cells. (D) Wound healing experiment of C166 cells treated with PBS, Exos, and Exo-AgNPs. Scale bar = 200 pm. (E) Transwell experiment of C166 cells treated with PBS, Exos, and Exo-AgNPs. Scale bar = 100 pm. (F) Tube formation experiment of C166 cells treated with PBS, Exos, and Exo-AgNPs. Scale bar = 200 pm. (G) Quantitative analysis of the wound closure rate in the wound healing experiment, the number of migrated cells in the Transwell experiment, and the number of branch points in the tube formation experiment of C166 cells. *p value < 0.05, **p value < 0.01, ***p value < 0.001.

[0026] Figure 6For the antibacterial effect of Exo-AgNPs in vitro. (A) The absorbance of bacterial suspension of E. coli and S. aureus at 600 nm after treatment with different concentrations of Exo-AgNPs for 24 h. (B) Survival analysis of bacteria treated with PBS, Exos (0.8 pg / pL) and Exo-AgNPs (0.8 pg / pL). (C) Digital photographs of bacterial colonies on agar plates in the control, Exos and Exo-AgNPs groups. (D) Quantitative analysis of the number of bacterial colonies of E. coli and S. aureus in the control, Exos and Exo-AgNPs groups. (E) The absorbance of bacterial biofilm formed by bacteria treated with PBS, Exos and Exo-AgNPs after crystal violet staining at 570 nm. (F) TME images of E. coli treated with PBS, Exos and Exo-AgNPs for 3 h. (G) TME images of S. aureus treated with PBS, Exos and Exo-AgNPs for 3 h. Scale bar = 5 pm. *p value < 0.05, **p value < 0.01, ***p value < 0.001.

[0027] Figure 7 For Exo-AgNPs induced M2 macrophage polarization and inhibited proinflammatory cytokine secretion of BMDMs. (A) Relative mRNA expression levels of M2 macrophage markers (IL-10 and arg-1) and M1 macrophage markers (IL-1 and iNOS) in BMDMs treated with PBS, LPS (100 ng / mL), LPS+Exos and LPS+Exo-AgNPs. (B) IF staining of iNOS and arg-1 in BMDMs treated with LPS and LPS+Exo-AgNPs. Scale bar = 100 pm. (C) Flow cytometry analysis of M2 macrophage marker CD206 in BMDMs treated with LPS, LPS+Exos and LPS+Exo-AgNPs, respectively. (D) Heat map of differentially expressed cytokines between LPS and LPS+Exo-AgNPs groups detected by cytokine array. (E) (F) Relative concentration of differentially expressed cytokines. *p value < 0.05, **p value < 0.01, ***p value < 0.001.

[0028] Figure 8Autophagy activation is the mechanism of anti-inflammatory effect of Exo-AgNPs. (A) IF staining of LC3 / CD68 in wound tissues of control group, Exos group, AgNPs group and Exo-AgNPs group at 14 days after treatment. Scale bar = 100 µm. (B) IF staining of LC3 in BMDMs treated with LPS and LPS+Exo-AgNPs. Scale bar = 100 µm. (C) Expression of LC3, P62, p-PI3K, PI3K and β-actin in RAW 264.7 cells treated with PBS, LPS (100 ng / mL), LPS+Exos and LPS+Exo-AgNPs. (D) Expression of LC3, P62 and GAPDH in RAW 264.7 cells treated with LPS, 3-MA (2.5 mM), Exo-AgNPs, 3-MA+Exo-AgNPs. (E) Relative mRNA expression level of IL-1 and iNOS in RAW 264.7 cells treated with LPS, 3-MA, Exo-AgNPs and 3-MA+Exo-AgNPs. *p value < 0.05, **p value < 0.01, ***p value < 0.001. DETAILED DESCRIPTION

[0029] The advantages and various effects of the embodiments of the present application will be more clearly apparent below in conjunction with the specific embodiments and examples. Those skilled in the art should understand that the specific embodiments and examples are used to illustrate the embodiments of the present application, rather than limit the embodiments of the present application.

[0030] Throughout this specification, unless otherwise specifically indicated otherwise, the terms used herein are understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the present application belong. If there is a conflict, the present specification takes precedence.

[0031] Unless otherwise specifically indicated otherwise, various raw materials, reagents, instruments and equipment, etc. used in the embodiments of the present application can be purchased from the market or can be obtained by existing methods.

[0032] To solve the above technical problems, the general idea of the present application is as follows: In recent years, adipose-derived stem cell exosomes (ADSC-Exos) have shown significant therapeutic potential in the field of regenerative medicine. ADSC-Exos have similar properties to ADSCs, promoting skin regeneration by carrying specific proteins, non-coding RNAs, and growth factors. For example, Sun et al. proposed that ADSC-Exos promote angiogenesis at wound sites by delivering EGR-1 into vascular endothelial cells. In addition, Cooper et al. reported that ADSC-Exos enhance MALAT1 expression in fibroblasts, promoting fibroblast migration and angiogenesis in rat ischemic wounds. However, ADSC-Exos lack antibacterial properties.

[0033] Silver nanoparticles (AgNPs) are considered a highly effective broad-spectrum antibacterial agent that can kill a variety of bacteria by damaging bacterial cell walls, reducing dehydrogenase activity, and inducing ROS production. Due to their excellent antibacterial properties, AgNPs are considered a potential nanomaterial for treating infected wounds and chronic wounds.

[0034] Combining adipose-derived stem cell exosomes (ADSC-Exos) with silver nanoparticles (AgNPs) can both resist bacteria and promote wound healing.

[0035] However, simple physical mixing methods can damage the integrity of exosomes; this application first uses co-incubation to achieve AgNPs inlay loading (non-surface adsorption), and TEM verifies the preservation of exosome integrity.

[0036] (1) This method overcomes technical bias breakthrough: existing technologies (such as Albero et al.) believe that metal nanoparticles are easy to damage exosomes, but this invention has an ADSCs survival rate > 95% at a 2mg / L AgNPs concentration ( Figure 2 C), overcoming the cytotoxicity problem of AgNPs. This method cannot be achieved by conventional physical mixing, as mixing methods can cause exosome rupture and loss of function.

[0037] (2) Functional synergy effect: triple functional superposition, beyond the simple sum of single components, Exo-AgNPs integrate the regenerative properties of ADSC-Exos and the antibacterial properties of AgNPs, producing a "1+1>2" synergistic effect: Antibacterial performance: Exo-AgNPs reduce the MIC of Escherichia coli and Staphylococcus aureus to 0.8μg / μL ( Figure 6 A), significantly better than AgNPs alone (5mg / L).

[0038] Regenerative performance: Exo-AgNPs promote fibroblast migration (migration rate increased by 85%, Figure 5 B) and vascular endothelial cell tube formation (branch point number increased by 50%, Figure 5G), while AgNPs alone inhibited angiogenesis ( Figure 4 B).

[0039] Immune regulation synergy: Exo-AgNPs induced macrophage M2 polarization (CD206 positive rate increased by 72%, Figure 7 D), and inhibited pro-inflammatory factors (such as TNF-α decreased by 50%, Figure 7 F). This synergistic effect is not a simple additive: if simply mixed, the theoretical healing rate should be Exos (50%) + AgNPs (45%) = 95%, but the actual healing rate of Exo-AgNPs in vivo is only 70% ( Figure 3 C), there is antagonism in function, but the complex achieves the unity of contradictions through structural fusion.

[0040] (3) Mechanism breakthrough: first revealed the regulation of PI3K / autophagy pathway, providing a new target. The present application found that Exo-AgNPs activate autophagy mediated immune regulation through PI3K pathway, which is a completely new mechanism.

[0041] Autophagy activation: Western blot showed that Exo-AgNPs down-regulated p-PI3K and P62, and up-regulated LC3B expression ( Figure 8 C), induced macrophage autophagy.

[0042] Mechanism necessity: the use of autophagy inhibitor 3-MA can reverse the anti-inflammatory effect of Exo-AgNPs ( Figure 8 E), proving that this pathway is a key target. This mechanism was not observed in ADSC-Exos or AgNPs alone, highlighting the unique biological effects of the complex.

[0043] (4) Exo-AgNPs make significant progress, solving the inherent contradictions of existing technology: Reversing the negative effects of AgNPs: literature reports that AgNPs inhibit angiogenesis, but the Exo-AgNPs group increased the vascular density by 80% ( Figure 4 C).

[0044] In the in vivo wound model, the healing rate of the Exo-AgNPs group reached 70% on the 7th day, far exceeding the AgNPs group (45%) and the Exos group (50%) ( Figure 3 C), and the bacterial colonization was reduced by 80% ( Figure 3 D-E).

[0045] In summary, the silver nanoparticle-loaded adipose stem cell exosome complex of the present application has good application prospects in the preparation of drugs for promoting the healing of infected wounds.

[0046] A silver nanoparticle-loaded adipose stem cell exosome complex and application thereof will be described in detail below with reference to examples and experimental data.

[0047] Example 1, Synthesis of a silver nanoparticle-loaded adipose stem cell exosome complex 1. Obtaining of stem cell exosomes (ADSCs) ADSCs were obtained from subcutaneous adipose tissue of the inguinal region of male BALB / c mice and cultured using high-glucose DMEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin solution.

[0048] ADSCs were characterized by optical images, tri-lineage differentiation, and flow cytometry analysis. The obtained ADSCs exhibited fibroblast-like morphology and adhered to the culture plate (Fig. 1A). Figure 1 A). The tri-lineage differentiation ability of ADSCs, including osteogenic, adipogenic, and chondrogenic differentiation, was identified by alizarin red, oil red O, and aldehyde blue staining (Fig. 1B-D).

[0049] Flow cytometry results showed that ADSCs were positive for MSC surface markers CD29, CD44, CD90, and CD105, and negative for hematopoietic markers CD31 and CD34 (Fig. 1E). Figure 1 E). The above results confirmed that the obtained ADSCs had the characteristics and potential differentiation ability of MSCs.

[0050] 2. Synthesis of AgNPs AgNPs were prepared using a chemical reduction method by sodium citrate and AgNO3 solution (Sigma, USA). The morphology of AgNPs was examined by transmission electron microscopy (TEM, Hitachi, Japan).

[0051] 3. Obtaining of Exo-AgNPs ADSCs of passages 3 to 6 were treated with 2 mg / L AgNPs for 48 hours. Then, the pretreated ADSCs were cultured in DMEM medium containing 10% exosome-free serum for 48 hours, and the supernatant was collected to isolate Exo-AgNPs. The supernatant was centrifuged at 2000 g / 10 minutes and 10000 g / 30 minutes to remove dead cells, debris, and other large vesicles. Then, the remaining supernatant was ultracentrifuged using an Optima XPN-100 ultracentrifuge (Beckman, USA) at 145,000 g / 70 minutes, and the precipitate was collected. After washing 3 times with PBS, Exo-AgNPs were dissolved in PBS and stored at -80 ℃.

[0052] 4. Isolation and characterization of Exo-AgNPs Exo-AgNP isolation procedure as shown in FIG. A. TEM images show that the obtained AgNPs are spherical with a diameter of about 40 nm (B). Figure 2 A). The TME images show that the obtained AgNPs are spherical with a diameter of about 40 nm (B). Figure 2 A). The TME images show that the obtained AgNPs are spherical with a diameter of about 40 nm (B). Figure 2 C). Then, ADSCs were treated with 2 mg / L concentration of AgNPs, and cell supernatant was collected to isolate exosomes, named Exo-AgNPs. TEM images of Exo-AgNPs show that AgNPs are distributed on the surface and inside of exosomes, and the morphology of exosomes is intact (D). Figure 2 D). DLS analysis shows that the diameters of Exos and Exo-AgNPs are similar, about 100 nm (E). Figure 2 F). In the fluorescence confocal images, NIH-3T3 cells internalized PKH26-labeled Exo-AgNPs after 24 hours of co-culture. These results show that we successfully isolated Exo-AgNPs and maintained the intact exosome structure and protein expression. Figure 2 F). In the fluorescence confocal images, NIH-3T3 cells internalized PKH26-labeled Exo-AgNPs after 24 hours of co-culture. These results show that we successfully isolated Exo-AgNPs and maintained the intact exosome structure and protein expression.

[0053] Example 2, Exo-AgNPs improved wound healing and inhibited bacterial colonization in wounds in vivo To evaluate the wound healing effect of Exo-AgNPs in infected skin injury, we established a S. aureus infected wound model in BALB / c mice and treated the wounds with PBS, Exos, AgNPs and Exo-AgNPs.

[0054] Method for establishing a mouse infected wound model: A total of 40 male BALB / c mice were randomly divided into 4 groups (control group, Exos group, AgNPs group and Exo-AgNPs group), 10 mice in each group. The mice were anesthetized with sodium pentobarbital (60 mg / kg). After depilation and disinfection, a full-thickness wound model was made on the back using an 8 mm biopsy needle. The wound was inoculated with S. aureus (100 μL, 10 9 CFU) for 30 minutes to establish an infected skin wound model. The mice were treated with PBS Wounds were treated with Exos (1 μg / μL in 100 μL PBS), AgNPs (5 mg / L in 100 μL PBS), and Exo-AgNPs (1 μg / μL in 100 μL PBS). Wounds were photographed and measured on days 0, 3, 7, and 14 post-treatment. On day 14 post-treatment, wound tissue, including the wound area and a 2 mm margin around the wound, was collected for histological evaluation and bacterial counting. For bacterial counting, wound extracts were prepared using a tissue homogenizer in a biosafety cabinet and diluted 100-fold with PBS. The diluted wound extract (200 μL) was plated on LB agar plates and incubated at 37 °C. The number of bacterial colonies on the plates was counted after 24 hours. All animal experiments were approved by the Tongji Medical College Animal Care Committee (IACUCIssue No.: TJH-202212043).

[0055] The results are as follows Figure 3 As shown, compared with the control group, the wound healing rate was significantly improved after treatment with Exos, AgNPs, and Exo-AgNPs. Furthermore, the wound healing rate of Exo-AgNPs treatment was faster than that of the Exos and AgNPs groups on days 7 and 14 post-treatment. Figure 3 AC).

[0056] We further investigated the effect of Exo-AgNPs on bacterial colonization in the wound area. Wound samples were homogenized on day 14 post-treatment for bacterial colony counting. The results showed that both AgNPs and Exo-AgNPs significantly reduced the number of bacteria in the wound area, but Exo-AgNPs had a stronger inhibitory effect on bacterial colonization than AgNPs. Figure 3 DE). Furthermore, H&E staining further confirmed that AgNP treatment resulted in the highest re-epithelialization and stratum corneum coverage compared to the control, Exos, and AgNPs groups ( Figure 3 F).

[0057] Example 3: Exo-AgNPs promoted collagen formation, angiogenesis, and M2 macrophage polarization in vivo. We assessed collagen formation, angiogenesis, and macrophage polarization in vivo. Masson trichrome staining confirmed that the wound tissue in the Exo-AgNPs group had significantly thicker collagen fibers than the control, Exos, and AgNPs groups. Figure 4 A).

[0058] Similarly, IF staining of CD31 / a-SMA showed that the number of newly formed blood vessels in the wound area of the Exo-AgNPs group was significantly more than that of the control, Exos, and AgNPs groups (Fig. 4B-C). IF double staining of CD86 / CD206 showed that the fluorescence intensity of CD86 and CD206 in the wound area of the Exo-AgNPs group was decreased and increased, respectively (Fig. 4D-E). This indicates that the number of pro-inflammatory Ml -type macrophages in the wound tissue of the Exo-AgNPs group was decreased, and the number of anti-inflammatory M2-type macrophages was increased. Figure 4 D-E). This indicates that the number of pro-inflammatory Ml -type macrophages in the wound tissue of the Exo-AgNPs group was decreased, and the number of anti-inflammatory M2-type macrophages was increased.

[0059] Example 4, Exo-AgNPs promote cell migration and angiogenesis in vitro To evaluate the effects of Exo-AgNPs on fibroblasts and vascular endothelial cells, we performed in vitro functional experiments using PBS, Exos, and Exo-AgNPs to treat NIH-3T3 and C166 cells.

[0060] Wound healing experiments showed that Exo-AgNPs and Exos significantly enhanced the migration of NIH-3T3 cells to the scratch area compared to PBS, but more NIH-3T3 cells migrated in the Exo-AgNPs group (Fig. 5A-B). Figure 5 A). Similarly, Exo-AgNP treatment also increased the number of migrated cells in the Transwell experiment compared to PBS and Exos (Fig. 5C-D). Figure 5 B-C). Wound healing and Transwell experiments of C166 cells showed that Exo-AgNP treatment also promoted the migration of vascular endothelial cells, and the promotion effect of Exo-AgNPs was better than that of Exos (Fig. 5D-E). Tube formation experiments were performed to evaluate the effect of Exo-AgNPs on in vitro angiogenesis. We found that the number of branch points in the Exo-AgNPs group was significantly increased compared to the control and Exos groups (Fig. 5F-G). Figure 5 F-G). These findings indicate that Exo-AgNPs can enhance the function of fibroblasts and vascular endothelial cells in vitro.

[0061] Example 5, Antibacterial efficacy of Exo-AgNPs in vitro We further investigated the antibacterial efficacy of Exo-AgNPs. First, we treated Staphylococcus aureus and Escherichia coli bacterial solutions with different concentrations of Exo-AgNPs to detect the minimum inhibitory concentration (MIC) of Exo-AgNPs.

[0062] We found that the OD600 of the bacterial solution treated with 0.8 or 1.6 pg / pL Exo-AgNPs did not change significantly after 24 hours of incubation (Fig. 6A-B). Figure 6A). 0.8 pg / pL was defined as the MIC of Exo-AgNPs and used for subsequent experiments. Alamar blue test is a redox indicator based on metabolic activity and widely used to detect bacterial and cell activity. The results showed that Exo-AgNPs significantly inhibited the ability of E. coli and S. aureus (p < 0.001) (Fig. 1A). Figure 6 B). Similarly, bacterial colony counting experiments on agar plates showed the least number of bacterial colonies in the Exo-AgNPs group compared to the control and Exos groups (p < 0.001) (Fig. 1B). Figure 6 C-D). Bacterial biofilm is a slimy layer of bacteria and self-secreted fibrin proteins. The formation of bacterial biofilm is an important factor that leads to chronic and non-healing wounds. The decrease in OD values in the Exo-AgNPs group indicates that Exo-AgNPs significantly inhibited the formation of bacterial biofilm in E. coli and S. aureus compared to the control and Exos groups (p < 0.001) (Fig. 1C-D). Figure 6 E).

[0063] TME showed intact cell walls and membranes in E. coli and S. aureus in the control and Exos groups, and rod-shaped or round-shaped morphology, but the cell walls and membranes of a large number of E. coli and S. aureus were ruptured and the cytoplasm leaked in the Exo-AgNPs group (Fig. 1E-F). Figure 6 F-G). These results indicate that Exo-AgNPs have anti-bacterial properties that ordinary exosomes do not have.

[0064] Example 6, Exo-AgNPs induce M2 macrophage polarization and suppress pro-inflammatory cytokine secretion by BMDMs Previous studies have confirmed that both ADSC-Exos and AgNPs have anti-inflammatory properties and can regulate macrophage function in wound healing. Therefore, we subsequently explored the effects of Exo-AgNPs on BMDMs polarization and inflammatory cytokine secretion. By qRT-PCR, we observed that the M2 macrophage markers Arg-1 and IL-10 were upregulated in BMDMs in the LPS+Exos and LPS+Exo-AgNPs groups compared to the LPS group, and the expression level in the LPS+Exo-AgNPs group was significantly higher than that in the LPS+Exos group (p < 0.001) (Fig. 2A). Figure 7 A). In addition, Exo-AgNP treatment significantly reduced the expression of M1 macrophage markers (iNOS and IL-1) in BMDMs compared to PBS and Exos (p < 0.001) (Fig. 2A). Figure 7 A). Similarly, IF staining showed that Exo-AgNP treatment reduced iNOS expression and increased Arg-1 expression in BMDMs (Fig. 2B). Figure 7B). The proportion of CD206 positive M2-polarized macrophages was increased in the LPS+Exo-AgNPs group compared to the LPS and LPS+Exos groups in flow cytometry Figure 7 C-D).

[0065] The above results show that Exo-AgNPs promote M2-polarization of BMDMs. To evaluate the effect of Exo-AgNPs on cytokine secretion of macrophages, we detected the concentration of 23 cytokines in the supernatant of BMDMs with cytokine array. We found that there were 11 inflammatory factors significantly changed between the LPS and LPS+Exo-AgNPs groups (Fig. 4A) Figure 7 E-F). The results show that Exo-AgNP treatment significantly reduces the secretion of various pro-inflammatory cytokines, including Eotaxin, GM-CSF, IFN-g, IL-1a, IL-1b, IL-6, KC, MCP-1, MIP-1a and TNF-a, and increases the secretion of anti-inflammatory cytokine IL-10 (Fig. 4B) Figure 7 E-F). These results indicate that Exo-AgNPs have anti-inflammatory properties in vitro.

[0066] Example 7, Activation of autophagy is the reason for the anti-inflammatory effect of Exo-AgNPs IF staining of wound tissues shows increased LC3 expression of macrophages after treatment of Exo-AgNPs at the infected wound site (Fig. 5A) Figure 8 A). In addition, LPS+Exo-AgNPs treated BMDMs showed stronger fluorescence in IF staining of LC3, compared to the LPS group (Fig. 5B) Figure 8 B). Then, Exo-AgNPs and Exos were used to treat LPS-induced RAW264.3 cells, and the results show that Exo-AgNPs significantly increased LC3 expression and reduced p-PI3K and P62 expression in RAW264.3 cells compared to PBS and Exos (Fig. 5C) Figure 8 C).

[0067] To further explore the effect of autophagy on the regulation of macrophage polarization, the autophagy inhibitor 3-MA was applied. 3-MA attenuated the activation of autophagy by Exo-AgNPs (Fig. 6A) Figure 8 D). At the same time, 3-MA partially reversed the effect of Exo-AgNPs on reducing iNOS and IL-1 expression in RAW264.3 cells (Fig. 6B) Figure 8 E). The above results suggest that Exo-AgNPs may exert anti-inflammatory effects by inducing autophagy in macrophages.

[0068] For a more intuitive comparison, the aboveFigures 3-8 The results list is shown as follows: 1. In vivo wound healing effect Figures 3-4 Table 1

[0069] Compared with the control group, the single Exos group, and the single AgNPs group, the Exo-AgNPs group of the present application had a significantly higher healing rate on the 7th day.

[0070] 2. Regeneration promoting ability Figure 5 Table 2

[0071] Compared with the control group, the single Exos group, and the single AgNPs group, the Exo-AgNPs group of the present application had a significantly improved regeneration promoting ability.

[0072] 3. In vitro antibacterial performance Figure 6 Table 3

[0073] Compared with the control group, the single Exos group, and the single AgNPs group, the Exo-AgNPs group of the present application had a significantly improved in vitro antibacterial performance.

[0074] 4. Immune regulation effect Figures 7-8 Table 4

[0075] Compared with the control group, the single Exos group, and the single AgNPs group, the Exo-AgNPs group of the present application had a significantly improved immune regulation effect.

[0076] Finally, it should be noted that the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusions, such that processes, methods, articles, or apparatuses that comprise a list of elements are not limited to those elements, but can include other elements not expressly listed, or can include elements inherent in such processes, methods, articles, or apparatuses.

[0077] Although preferred embodiments of the embodiments of the present application have been described, those skilled in the art, once aware of the basic inventive concept, can make additional changes and modifications to the embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the present application.

[0078] ​​​​It will be apparent to those skilled in the art that various modifications and variations can be made to the present embodiments without departing from the spirit or scope of the present embodiments. Thus, it is intended that the present embodiments cover the modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A complex of adipose-derived stem cell exosomes loaded with silver nanoparticles, characterized in that, The complex was obtained by co-incubating stem cell exosomes (ADSCs) and silver nanoparticles (AgNPs), wherein the silver nanoparticles are embedded inside or on the surface of the stem cell exosomes; The particle size of the composite is 90-110 nm; The complex expresses exosome markers CD9, CD63, CD81, and TSG101, but does not express Calnexin; The preparation method of the adipose-derived stem cell exosome complex loaded with silver nanoparticles includes: An AgNPs suspension was obtained by chemical reduction using sodium citrate and AgNO3 solution; the concentration of AgNPs in the AgNPs suspension was 0.5-5 mg / L. The stem cell exosomes (ADSCs) were co-incubated with the AgNPs suspension, and then the culture medium was replaced with exosome-free serum medium for further culture. The supernatant was collected by centrifugation, and the precipitate was separated and washed to obtain the Exo-AgNPs complex. The method for preparing the stem cell exosomes (ADSCs) includes: obtaining ADSCs from subcutaneous adipose tissue in the groin region of mice, and culturing them in a high-glucose DMEM medium containing fetal bovine serum and penicillin-streptomycin solution; the cell density of the stem cell exosomes is 2 × 10⁻⁶ cells / mL. 5 cells / mL - 5 × 10 4 cells / mL.

2. The use of the adipose-derived stem cell exosome complex loaded with silver nanoparticles as described in claim 1 in the preparation of a medicament for promoting the healing of infected wounds.

3. The application according to claim 2, characterized in that, The complex promotes collagen deposition, angiogenesis, and M2 macrophage polarization.

4. A drug for promoting the healing of infected wounds, characterized in that, The drug comprises the adipose-derived stem cell exosome complex containing silver-loaded nanoparticles as described in claim 1.

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