Fusion membrane of staphylococcus epidermidis vesicles and curcumin-derived exosome-like vesicles and preparation method and application thereof

Abstract

The application discloses a fusion membrane of Staphylococcus epidermidis vesicles and curcumin-derived exosome-like vesicles and a preparation method and application thereof. The application fuses Staphylococcus epidermidis vesicles and curcumin-derived exosome-like vesicles to obtain HMVs, and verifies the antibacterial effect of the HMVs through in-vitro experiments, and then the HMVs are loaded in hyaluronic acid microneedles. The HMVs MNs nano system can be locally administered by pressing on acne lesions, and the in-vivo experiments on Balb / c mice prove that the system has relatively remarkable antibacterial and anti-inflammatory effects. Therefore, the HMVs MNs have relatively good application potential in treating acne.

Description

Technical Field

[0001] This invention relates to the field of vesicle fusion technology, specifically to a fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles, its preparation method, and its application. Background Technology

[0002] Currently, drug treatment for acne has certain limitations. Oral antibiotics are a commonly used systemic treatment, but due to the increasing resistance rate of pathogens, both domestic and international acne guidelines have issued warnings about antibiotic resistance. Oral retinoids are currently the only oral medications targeting the four key pathophysiological processes of acne pathogenesis, but isotretinoin has common adverse reactions, such as dry skin and mucous membranes, musculoskeletal pain, elevated blood lipids, abnormal liver enzymes, and dry eyes; long-term use before puberty may cause premature epiphyseal closure, bone hyperplasia, and osteoporosis; it has a clear teratogenic effect; and long-term use may be associated with depression or suicidal tendencies. Therefore, systemic drug treatment is unlikely to achieve satisfactory clinical results and has serious side effects. Topical medications, due to the skin barrier function, cannot be delivered through the skin to achieve therapeutic effects in moderate to severe acne. Furthermore, *Propionibacterium acnes* can secrete extracellular polysaccharides to form biofilms, leading to bacterial resistance to common antibiotics and host inflammatory cells. Therefore, in order to avoid adverse reactions caused by systemic medication, local treatment methods targeting the affected area are safer, and there is an urgent need to explore more effective treatment methods that reach the therapeutic level in order to improve the effectiveness of local treatment for acne. Summary of the Invention

[0003] The technical problem to be solved by this invention is to propose a fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles, which can be loaded into microneedles to treat acne, thereby improving the treatment effect and reducing the occurrence of adverse reactions.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0005] A fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles is obtained by fusing Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles; wherein...

[0006] The Staphylococcus epidermidis vesicles are the precipitate obtained by further centrifuging the supernatant (to remove bacterial cells) after Staphylococcus epidermidis culture;

[0007] The turmeric-derived exosome-like vesicles were obtained by centrifuging the homogenate obtained from grinding turmeric (removing impurities) to obtain the supernatant (removing turmeric cells), centrifuging the supernatant again to obtain the precipitate, and then purifying it by sucrose density gradient centrifugation.

[0008] In this invention, Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles are fused by ultrasonic treatment.

[0009] The method for preparing the fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles of the present invention includes the following steps:

[0010] S1. Preparation of Staphylococcus epidermidis Extracellular vesicles (SE-EVs)

[0011] Single colonies of Staphylococcus epidermidis were inoculated into tryptic soy broth (TSB) and cultured overnight at 37°C and 220 rpm. The culture medium was centrifuged at 6000×g to remove bacterial cells, and the supernatant was further centrifuged at 20000×g to collect the precipitate, which was then resuspended in 1×PBS solution for later use.

[0012] S2. Preparation of turmeric-derived vesicles (TNVs)

[0013] Fresh turmeric was washed with clean water, sterilized under ultraviolet light, and homogenized using a grinder. The homogenate was first filtered to remove fibers and impurities, and then the filtrate was centrifuged at 3000×g and then at 10000×g. The supernatant was further centrifuged at 100000×g, and the resulting precipitate was resuspended in 1×PBS solution to obtain crude turmeric-derived exosome-like vesicles. Subsequently, turmeric-derived exosome-like vesicles were purified by sucrose density gradient centrifugation.

[0014] The sucrose density gradient centrifugation was performed at 8%, 30%, and 45% sucrose density gradients to purify the product. Then, the bands between the 30% and 45% sucrose density layers were collected, and finally, sucrose was removed by ultrafiltration to obtain turmeric-derived exosome-like vesicles.

[0015] In this invention, Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles are mixed in a volume ratio of 1:1 and subjected to ultrasonic treatment to obtain Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membranes (HMVs).

[0016] This invention also proposes a microneedle (MNs) for treating acne, the preparation method of which includes the following steps:

[0017] Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane and sodium hyaluronate were dissolved in 1×PBS solution and stirred until completely dissolved to obtain a mixed solution. The mixed solution was then dropped onto a PDMS microneedle mold.

[0018] The PDMS microneedle mold was centrifuged at 3000×g, and after complete filling, it was dried in a desiccator to obtain the final product.

[0019] In recent years, a series of non-pharmacological treatments have been developed and applied to the treatment of infections, including photodynamic therapy (PDT) and photothermal therapy (PTT), as well as ultrasound-triggered sonodynamic therapy (SDT). Among these methods, sonodynamic therapy is considered one of the most promising approaches to treating bacterial infections due to its excellent tissue penetration and significant efficacy against drug-resistant bacteria and biofilms. Ultrasound, as a safe imaging technology widely used in the biomedical field, can also excite specific ultrasound-responsive materials to generate reactive oxygen species (ROS). Under ultrasound irradiation, sonosensitive agents generate various ROS through cavitation and sonoluminescence effects, including hydroxyl radicals (•OH), singlet oxygen (¹O2), and superoxide anions (O2•⁻). These ROS can react with important biomolecules (proteins, lipids, DNA) in bacterial cells, leading to protein denaturation, lipid peroxidation, and damage to genetic material, thereby effectively killing bacteria. Ultrasonic dynamic therapy also generates mechanical effects such as liquid microjets, microflow shock waves, and shear forces, which can form pores in bacterial cell membranes, disrupt bacterial cell structure, increase drug permeability, and interfere with biofilm formation and homeostasis. Simultaneously, the localized temperature increase (thermal effect) caused by ultrasonic cavitation disrupts the cytoskeleton and bacterial biomolecular structure, further enhancing the antibacterial effect of ultrasonic dynamic therapy.

[0020] This invention is the first in China and abroad to explore the sonic-dynamic antibacterial efficacy of turmeric exosome-like vesicles against *C. acnes*. As a natural sonic sensitizer, curcumin has significant anti-inflammatory and antibacterial effects, capable of generating ROS under ultrasound to kill pathogens. However, its low solubility (<1 μg / mL) and poor targeting ability limit its application. Exosome-like vesicles extracted from turmeric rhizomes are rich in curcumin, which retains the anti-inflammatory effects of turmeric while improving its solubility. Furthermore, turmeric exosome-like vesicles are more readily available.

[0021] To enhance the targeting effect of turmeric exosome-like vesicles on *Propionibacterium acnes*, this invention uses *Staphylococcus epidermidis* vesicles as the delivery system. *Staphylococcus epidermidis*, as a dermal commensal bacterium, has been shown to inhibit the pathogenicity of *Propionibacterium acnes* through multiple mechanisms: (1) competitively consuming carbon sources such as glycerol and metabolizing short-chain fatty acids (SCFAs) to directly inhibit *Propionibacterium acnes* proliferation; (2) secreting succinic acid to inhibit TLR and TNF-α, alleviating the inflammatory cascade; and (3) inducing miR-143 expression through LTA, targeting and inhibiting TLR2 protein synthesis, thus blocking *Propionibacterium acnes*-mediated inflammatory signals. Furthermore, *Staphylococcus epidermidis* vesicles improve disease progression by inhibiting inflammatory signaling pathways. Based on the fact that *Staphylococcus epidermidis* and *Propionibacterium acnes* are both Gram-positive bacteria with similar cell membrane components, *Staphylococcus epidermidis* vesicles may have a homologous targeting effect on *Propionibacterium acnes*. Therefore, *Staphylococcus epidermidis* vesicles are selected as the delivery system to enhance the targeting effect of turmeric-derived exosome-like vesicles on *Propionibacterium acnes*.

[0022] The role of microneedles (MNs): Due to their non-invasive, low-pain, and highly efficient transdermal delivery characteristics, they have become an innovative strategy for overcoming the skin barrier. Their mechanism of action relies on physically penetrating the stratum corneum to form transient microporous channels, which are then quickly closed by the skin's elastic recoil, avoiding the wound risks associated with traditional injections. Soluble MNs, in particular (such as the hyaluronic acid microneedle patch used in this invention), achieve precise drug delivery through biodegradability, while avoiding the risk of metal needle residue.

[0023] This invention prepared microneedles containing Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles (HMVs), a nanomedicine delivery system. First, Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles were fused to obtain HMVs. Their antibacterial effects were verified through in vitro experiments. Then, they were loaded into hyaluronic acid microneedles. This HMVs MNs nanosystem can be used for local drug delivery by pressing on acne lesions. In vivo experiments in Balb / c mice demonstrated significant antibacterial and anti-inflammatory effects. Therefore, HMVs MNs have good potential for treating acne. Attached Figure Description

[0024] Figure 1Preparation and characterization of HMVs. a) Schematic diagram of HMV preparation process. b) Particle size distribution of SE-EVs, TNVs, and HMVs. c) Zeta potential of SE-EVs, TNVs, and HMVs. d) Fluorescence spectra of SE-EVs and TNVs labeled with FRET dye pairs (DIO and DiI) after sonication with different protein mass ratios. e) Transmission electron microscopy images of SE-EVs, TNVs, and HMVs. Scale bar = 100 nm. f) Fluorescence images of HMVs prepared by physical mixing or sonication, where TNVs are labeled with DiI (red) and SE-EVs are labeled with DiO (green). Scale bar = 100 μm. Particle size stability assessment after 7 days of storage at 4℃ (n=3).

[0025] Figure 2 Characterization of antibacterial activity. (a) Singlet oxygen (1O2) generation capacity of TNVs and HMVs was detected by SOSG method. (b) Singlet oxygen (1O2) generation capacity of TNVs and HMVs was detected by DPBF method. (c) Flow cytometry analysis and (d) representative images showing the uptake of Propionibacterium acnes in different samples after incubation for 2.5 hours (n=3). (e) Colony count of Propionibacterium acnes after treatment of different samples. (fg) The clearance effect of PBS dilution (1×PBS), TNVs and HMVs on Propionibacterium acnes biofilm under ultrasonic or non-ultrasonic conditions was evaluated by crystal violet staining (n=3). (h) Three-dimensional reconstruction images of Propionibacterium acnes biofilm of different samples treated under ultrasonic or non-ultrasonic conditions after staining with live / dead staining kit.

[0026] Figure 3 Preparation and characterization of microneedles. (a) Schematic diagram of HMVs microneedle preparation process. (b) Microscopic images of blank microneedles and TNVs microneedles, scale bar = 200 μm. (c) Antibacterial efficacy of TNVs-MNs and HMVs-MNs after storage at 4℃ for 1, 3, and 7 days (n = 3). (d) Histological observation of porcine skin tissue after microneedle puncture with crystal violet staining, showing cross-sectional images of microneedle puncture, scale bar = 100 μm.

[0027] Figure 4 To evaluate the in vivo anti-acne effect of microneedles. (a) Schematic diagram of the experimental procedure. (b) Quantitative analysis of skin thickness and (c) Representative images of the wound areas in each group (n=3). (d) Quantitative colony counts of Propionibacterium acnes infected mouse skin tissue in different treatment groups and (e) Representative plate images (n=3). (f) Immunofluorescence staining images of Propionibacterium acnes in acne lesions, scale bar = 100 μm.

[0028] Figure 5The in vivo anti-inflammatory effects of HMVs and MNs. (a) Hematoxylin-eosin (H&E) staining images of mouse skin tissue sections after different treatments. Representative images of the expression of TNF-α (bc), IL-1β (de), and TGF-β (fg) in mouse acne lesions, and quantitative results obtained by ImageJ analysis (n = 3). Scale bar = 100 μm.

[0029] Figure 6 In vitro characterization of healing-promoting and anti-inflammatory properties. (a) HaCaT cell migration ability was detected by scratch assay. (b) Quantitative analysis of HaCaT cell migration rate within 48 hours (n=3). (c) Schematic diagram of Transwell migration assay. (d) Representative images of migrating cells and (g) quantitative analysis results (n=3). (e) Representative confocal fluorescence images of DCFH-DA staining and (f) quantitative analysis by flow cytometry (n=3). (h) qPCR detection of iNOS gene expression level of macrophages 12 hours after treatment with SE-EVs, TNVs, and HMVs (n=3). (i) NO secretion of macrophages 12 hours after treatment with Griess assay (n=3). Detailed Implementation

[0030] To enable those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below with reference to the accompanying drawings.

[0031] Example 1

[0032] The preparation process of the Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane of the present invention is as follows:

[0033] S1. For turmeric-derived vesicles (TNVs), fresh turmeric (purchased online, originating from Guangxi, China) was rinsed with water, exposed to ultraviolet light for 30 minutes, and homogenized using a grinder. The homogenate was first filtered through medical gauze to remove fibers and impurities, and then centrifuged sequentially at 3000×g for 30 minutes and 10000×g for 60 minutes. The supernatant was further centrifuged at 100000×g for 60 minutes, and the resulting precipitate was resuspended in 1×PBS solution. The crude turmeric-derived vesicles were then purified by sucrose density gradient centrifugation (8%, 30%, 45%). The bands between the 30% and 45% sucrose layers were collected, and the sucrose was removed by ultrafiltration.

[0034] S2. For *S. epidermidis* extracellular vesicles (SE-EVs), a single *S. epidermidis* (ATCC 12228) colony was inoculated into TSB medium and cultured overnight at 37°C and 220 rpm. Bacterial cells were removed by centrifugation at 6000×g for 5 minutes, and the supernatant was further centrifuged at 20000×g for 2 hours to collect the precipitate, which was then resuspended in 1×PBS solution.

[0035] S3. Mix Staphylococcus epidermidis vesicles and curcuma-derived exosome-like vesicles at a volume ratio of 1:1 and sonicate for 10 minutes to obtain Staphylococcus epidermidis vesicle-curcuma-derived exosome-like vesicle fusion membranes (HMVs). The preparation process is as follows: Figure 1 a.

[0036] Preparation of microneedles (HMVs MNs) loaded with Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membranes.

[0037] First, 400 μg of Staphylococcus epidermidis vesicle-curcumin-derived exosome-like vesicle fusion membrane and 400 mg of sodium hyaluronate (NaHA) were dissolved in 800 μL of 1×PBS solution and stirred until completely dissolved. Then, 200 μL of the solution was dropped onto a PDMS microneedle mold. The mold was centrifuged at 3000×g for 1 hour to ensure complete filling, and then dried at room temperature for 12 hours using a desiccant. In addition, different microneedles (TNVs MNs, SE-EVs MNs) were prepared by replacing 400 μg of Staphylococcus epidermidis vesicle-curcumin-derived exosome-like vesicle fusion membrane with 400 μg of curcumin-derived exosome-like vesicles and Staphylococcus epidermidis vesicles, respectively.

[0038] Characterization of SE-EVs, TNVs, and HMVs

[0039] Particle size and zeta potential were measured using a Zeta potential and particle size analyzer (Malvern Zetasizer Pro, UK). To observe the morphology of the samples, they were first negatively stained with 1% (mass / volume) uranium acetate and then imaged under a transmission electron microscope (Hitachi HT7800).

[0040] SE-EVs labeled with DiO (cell membrane green fluorescent probe) and TNVs labeled with DiI (cell membrane orange fluorescent probe) were physically mixed or sonicated, and then their fluorescence colocalization was examined using a confocal microscope (Olympus FV3000, Japan).

[0041] In the Foster resonance energy transfer (FRET) study, the masses of DiO (excitation / emission = 484 / 501 nm) and DiI (excitation / emission = 550 / 567 nm) labeled TNVs were kept constant, while the masses of SE-EVs were varied. After sonication, the fluorescence spectra of the samples were observed and recorded using a fluorescence spectrophotometer (China Taikepu FL-970), with the excitation wavelength set to 488 nm.

[0042] The generation of reactive oxygen species (ROS) was detected using 1,3-diphenylisobenzofuran (DPBF). 1 mg / mL of TNVs or HMVs were added to DPBF to achieve a final concentration of 40 μl / mL. The sonication conditions were as follows: 2 minutes per cycle, frequency 1.0 MHz, duty cycle 50%, and intensity 1.5 w / cm². Absorbance spectra were recorded every 2 minutes using a UV-Vis spectrophotometer, and absorbance values ​​at 410 nm were used for quantitative analysis.

[0043] 2',7'-Dichlorodihydrofluorescein (SOSG) was also used to detect the generation of reactive oxygen species (ROS). 1 mg / mL of TNVs or HMVs was added to 10 μM SOSG. Sonication was performed under the following conditions: 2 minutes per cycle, frequency 1.0 MHz, duty cycle 50%, and intensity 1.5 w / cm². The excitation wavelength was set to 488 nm, and the fluorescence spectrum was scanned every 2 minutes using a fluorescence spectrophotometer. Quantitative analysis was performed by measuring the fluorescence intensity at 525 nm.

[0044] Stability assay of SE-EVs, TNVs and HMVs in 1×PBS solution

[0045] SE-EVs, TNVs, and HMVs at a concentration of 100 μg / ml were dispersed in 1×PBS solution. The stability of these nanoparticles was observed. The particle size of the nanoparticles was measured every other day for 7 consecutive days.

[0046] The results show:

[0047] The hydrodynamic diameter of the HMVs obtained after fusion is approximately 200 nm. Figure 1 b), consistent with the results of transmission electron microscopy observations ( Figure 1 e). After TNVs and SE-EVs are fused, the Zeta potential of HMVs falls between the two ( Figure 1 c). Confocal imaging showed no obvious co-localization in the physically mixed group, while the HMVs group prepared by ultrasound showed orange fluorescence (indicating successful membrane fusion). To verify the fusion process of TNVs and SE-EVs, fluorescence resonance energy transfer (FRET) experiments were performed. Figure 1d). DiO-labeled SE-EVs and DiI-labeled TNVs were mixed at different mass ratios and then sonicated. As the proportion of TNVs increased, DiO fluorescence emission increased while DiI fluorescence emission decreased, indicating an increase in the distance between the two dyes. This confirmed the successful fusion of TNVs and SE-EVs. Figure 1 f). The hydrodynamic diameter of the sample dispersed in 1×PBS solution remained relatively stable within one week of storage at 4°C. Figure 1 g).

[0048] Example 2

[0049] In vitro antibacterial activity of Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane

[0050] As a sonosensitive agent, curcumin's antibacterial efficacy relies on its ability to generate reactive oxygen species (ROS) under ultrasonic irradiation. To ensure effective eradication of overproliferated Propionibacterium acnes at acne lesions, we systematically evaluated the sonodynamic activities of TNVs and HMVs. These were determined using SOSG and DPBF assays. Figure 2 ab) Determination of singlet oxygen ( 1 The generation of O2 was found to increase with prolonged ultrasound time. 1 O2 production gradually increased. Although the sonodynamic activity of HMVs was slightly lower than that of TNVs, it was still significantly higher than that of the control group. We ultimately selected 50% duty cycle, 4 minutes of irradiation time, and 1 w / cm² ultrasound conditions as the parameters for the in vitro antibacterial experiment.

[0051] To investigate the mechanism of action of HMVs in bactericidal activity, we co-incubated DiI-labeled TNVs and HMVs with bacteria for 2.5 hours and then performed flow cytometry analysis. The results showed that, based on the fact that both *Staphylococcus epidermidis* and *Propionibacterium acnes* are Gram-positive bacteria, HMVs can enter bacterial cells more rapidly. Figure 2 c). Confocal observation showed that DiI-labeled HMVs and Hoechst-stained bacteria exhibited high colocalization, while no obvious colocalization was observed in the TNVs group. Figure 2 d), confirming that the SE-EVs component in HMVs can promote their internalization by Propionibacterium acnes.

[0052] Under ultrasonic conditions (50% duty cycle, 4 minutes, 1 w / cm²), 100 μg / mL TNVs reduced bacterial count by three orders of magnitude. Notably, the same concentration of HMVs exhibited even stronger bactericidal activity, reducing Propionibacterium acnes count from 1 × 10⁻⁶ to 10⁻⁶. 8 CFU / mL decreased to 1×10 4 CFU / mL Figure 2 e). Given that the pathogenicity of Propionibacterium acnes is mainly related to its biofilm formation. We further examined the samples' ability to remove biofilm. In the control group without samples, sonication alone was sufficient to remove some of the biofilm. Figure 2 (fg) indicates that ultrasound can disrupt the dense structure of biomembranes. With the aid of ultrasound, TNVs can remove 50% of the biomembrane, while HMVs increase the removal rate to 80%. The weakening of green fluorescence in the three-dimensional reconstruction images of live and dead cell staining directly reflects the disruption of the biomembrane structure. Figure 2 h).

[0053] Studies have shown that TNVs possess excellent sonodynamic activity, and the ROS they generate can effectively kill planktonic bacteria and biofilms. When hybridized with SE-EVs to form HMVs, the enhanced targeting ability further improves their efficacy against Propionibacterium acnes.

[0054] Example 3

[0055] Preparation and characterization of microneedles

[0056] NaHA, a naturally derived polysaccharide, has been widely used in wound repair due to its excellent biocompatibility. This invention selects NaHA as a microneedle matrix material to achieve transdermal drug delivery. Figure 3 The microneedle fabrication flowchart shown in figure a illustrates the process of mixing SE-EVs, TNVs, or HMVs with NaHA, injecting the mixture into a PDMS mold, and then drying to obtain a microneedle array. The fabricated microneedles exhibit a regular pyramidal tip structure. Figure 3 (b) This design effectively enhances its penetration ability into the stratum corneum of the skin. Experiments have confirmed that drug-loaded TNVs and HMVs retain significant antibacterial activity in microneedles, and this activity can be maintained for up to 7 days. Figure 3 c).

[0057] For skin puncture performance, microneedles were applied to pigskin and firmly pressed with thumb pressure for 2 minutes. After removing the microneedles, 0.05% (w / v) crystal violet solution was applied for 3 minutes, and then excess solution was removed with a cotton swab. The puncture area was then photographed using a digital camera. The treated pigskin was embedded in OCT embedding medium, sectioned using a cryostat (Leica CM1950, Germany), and examined under a fluorescence microscope. The punctured pigskin was fixed, prepared into cryosections, and observed under a microscope (Nikon, Japan). Evaluation through ex vivo pig skin puncture experiments showed that the prepared microneedles could achieve a puncture depth of 200 μm. Figure 3 d), fully meeting the needs of transdermal drug delivery.

[0058] Example 4

[0059] In vivo antibacterial activity of Staphylococcus epidermidis vesicles-turmeric-derived exosome-like vesicle fusion membrane microneedles (HMVs MNs)

[0060] This invention establishes a mouse acne model to evaluate the efficacy of HMVs and MNs. A Propionibacterium acnes infection model was established using male BALB / c mice (8-10 weeks old, weighing 25-30g). All experimental procedures were approved by the Laboratory Animal Management and Use Committee of Sun Yat-sen University (SYSU-IACUC-2024-002662). Propionibacterium acnes (ATCC6919) was transferred from the bacterial strain bank to MHA medium (Mueller-Hinton Agar, MHA), placed in an anaerobic bag, and incubated at 37℃ for 72 hours to activate the strain. The bacterial colony surface was rinsed with 1 mL of sterile saline, and the bacterial suspension was transferred to a 1.5 mL sterile Eppendorf tube, centrifuged for 3 minutes, the supernatant was discarded, and 1 mL of sterile saline was added again. After thorough mixing with a vortex mixer, the bacterial concentration was adjusted to 1×10⁻⁶. 7 CFU / mL. One day prior to inoculation, a 2×3 cm section was cut from the back of male Balb / c mice. 2 For long hair covering an area, apply depilatory cream and wipe it off with a cotton swab dipped in water after 5 minutes. Repeat the depilation process if the hair is not completely removed. After drying, return the mice to their cages. D-2 mice will be inoculated after the depilated area on their backs is checked for inflammation. Before inoculation, mice will be anesthetized by intraperitoneal injection of 3% sodium pentobarbital. Inoculate the bacterial suspension intradermally at a dose of 100 μL into the center of the depilated area on the back. Mix the bacterial suspension thoroughly before each inoculation, and immediately after injection, press a cotton swab against the injection site to prevent leakage.

[0061] Intradermal injection of Propionibacterium acnes (1×10) on day D-2 7 CFU / site), typical acne lesions (0.4-0.6 cm in diameter) formed after 48 hours. Balb / c mice were randomly divided into 6 groups (n=3): Blank (1×PBS solution), Control (untreated), MNs (blank microneedles), TNVs MNs, SE-EV MNs, and HMVs MNs groups. Treatment involved microneedle patches (3 minutes) combined with ultrasound (1 MHz, 50%, 1.5 w / cm², 3 minutes). Observation results showed that the skin condition in the Blank group was normal, with no obvious lesions; the lesions in the Control group continued to enlarge as the disease progressed; although the physical effect of microneedle puncture was observed in the MNs group, it did not show a significant therapeutic effect due to the lack of active ingredients. In contrast, the drug-loaded microneedle groups (TNVs MNs, SE-EV MNs, and HMVs MNs) all showed varying degrees of therapeutic effect, with the HMVs MNs group showing the most significant efficacy. Figure 4c). Statistical analysis showed that from day 3 of treatment, there was a significant difference in the diameter of skin lesions between the HMVs MNs group and the Control group (D3: P < 0.01), and the difference became more pronounced over time (D5: P < 0.001; D7: P < 0.0001). Figure 4 (b) This remarkable therapeutic effect stems from the dual mechanism of action of HMVs-MNs: on the one hand, the microneedle array effectively penetrates the stratum corneum barrier, precisely delivering drug-loaded nanoparticles to the dermis; on the other hand, ultrasound-activated nanoparticles release a large amount of reactive oxygen species (ROS), achieving highly efficient killing of Propionibacterium acnes. In particular, the fusion membrane of the two vesicles (HMVs) combines the acoustic properties of curcumin with the targeting ability of Staphylococcus epidermidis exosomes, exhibiting a synergistically enhanced therapeutic effect.

[0062] The diffusion plate method was used to perform quantitative bacterial analysis on the treated skin tissue. Figure 4 e). Experimental results showed no significant difference in bacterial load between the MNs group and the Control group (P>0.05), indicating that simple microneedle physical treatment does not have antibacterial effect. In contrast, both the TNVs MNs group and the SE-EV MNs group showed significant antibacterial effects, with bacterial counts significantly lower than the Control group (P<0.01), confirming that single-drug treatment has certain antibacterial activity. Notably, the HMVs MNs group exhibited the strongest antibacterial effect (P<0.001). This result is related to changes in lesion diameter ( Figure 4 c) They showed a good correlation, jointly confirming the superiority of the HMV synergistic treatment regimen.

[0063] In immunofluorescence staining, Propionibacterium acnes expression is presented as a green fluorescent signal. Figure 4 f). In the Blank group, only a small amount of dark green fluorescence was observed in the hair follicles, while nonspecific green fluorescence signals appeared in the muscle layer, indicating that the expression level of Propionibacterium acnes was low in the untreated state. In the Control group, green fluorescence was significantly enhanced in the hair follicles and dermal fat layer, indicating that Propionibacterium acnes was actively expressed in these areas. In the MNs, TNVs, MNs, and SE-EV MNs groups, green fluorescence in the hair follicle areas was reduced, but a large amount of green fluorescence was still present in the dermal fat layer, suggesting that microneedling treatment reduced the expression of Propionibacterium acnes in the hair follicles to some extent, but had limited inhibitory effect on Propionibacterium acnes in the deep dermis. In the HMVs MNs group, green fluorescence in the hair follicles was significantly weakened, and almost no green fluorescence signal was observed in the dermal fat layer, indicating that the expression of Propionibacterium acnes was significantly inhibited in this group.

[0064] Example 5

[0065] In vivo anti-inflammatory and repair-promoting effects of Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane

[0066] Histopathological H&E staining ( Figure 5 a) This further revealed the ameliorative effect of HMVs-MNs on the pathological changes of acne. The Blank group showed normal skin structure, normal epidermal thickness, intact sebaceous glands and hair follicles, and no obvious inflammatory cell infiltration. The Control group, however, exhibited typical acne pathological features: significantly thickened epidermis, accompanied by obvious hyperkeratosis and follicular opening dilation; sebaceous gland hyperplasia and hypertrophy; and abundant inflammatory cell infiltration in the dermis. The MNs group showed similar pathological manifestations to the Control group, confirming that simple physical puncture could not improve the inflammatory state. Although the TNVs-MNs and SE-EV-MNs groups improved epidermal thickness to some extent, significant sebaceous gland hyperplasia and inflammatory cell infiltration remained. The HMVs-MNs group showed the best pathological improvement: epidermal thickness returned to near normal levels, the degree of sebaceous gland and hair follicle dilation was significantly reduced, and the number of inflammatory cell infiltrations was significantly decreased. This histological evidence corroborates the aforementioned antibacterial experimental results, confirming that HMVs-MNs effectively improved the pathological process of acne by inhibiting Propionibacterium acnes infection and reducing the inflammatory response.

[0067] The inflammatory response induced by Propionibacterium acnes infection mainly activates the TLR-2 / TLR-4 pathway of keratinocytes through its bacterial components peptidoglycan and lipoteichoic acid, thereby promoting the release of pro-inflammatory factors such as TNF-α and IL-1β, leading to the spread of inflammation around the pilosebaceous unit. Experimental data showed that compared with the Control group, the SE-EV MNs group exhibited a certain degree of downregulation of TNF-α expression (P<0.01), which may be related to the inhibitory effect of Staphylococcus epidermidis-derived exosomes on the inflammatory signaling pathway induced by Propionibacterium acnes. Notably, the anti-inflammatory effects of the MNs group and the TNVs MNs group were comparable (P<0.001), indicating that the anti-inflammatory effect of curcumin alone is limited (Figure 5c). The HMVs MNs group showed the strongest anti-inflammatory effect, with TNF-α and IL-1β expression levels close to those of the Blank group (Figure 5b-e). This result is highly consistent with the inflammation reduction observed in histopathology, confirming the significant advantage of the hybrid membrane vesicle microneedle system in regulating the inflammatory response.

[0068] In terms of tissue repair, the TGF-β signaling pathway plays a core regulatory role in skin wound repair by coordinating processes such as inflammatory cell recruitment, fibroblast proliferation, and collagen synthesis. Microneedling therapy creates controllable micro-damage channels in the dermis through physical puncture, which not only promotes drug delivery but also stimulates the production of collagen and elastin, accelerating skin remodeling. Experimental results showed that all microneedling treatment groups significantly increased TGF-β expression levels (P<0.05), with single-drug-loaded microneedles (TNVs MNs and SE-EV MNs) showing stronger TGF-β induction capacity (P<0.01). Notably, the TGF-β expression level was highest in the HMVs MNs group (P<0.001), further confirming the unique advantage of this combined treatment strategy in simultaneously achieving repair-promoting efficacy (Figure 5g).

[0069] Example 6

[0070] In vitro characterization of healing-promoting and anti-inflammatory properties

[0071] Wound healing assay: Human keratinocytes (HaCaT) were cultured at 5.0 × 10⁶ cells per well. 5 Cells were cultured overnight in 6-well plates at a density of [insert density here], and then cultured further in a 5% CO2, 37°C incubator. Cells were scratched with a pipette tip, washed with PBS, and then cultured in serum-free DMEM medium for 48 hours. Cell migration was quantified using ImageJ (National Institutes of Health) software and microscopic images taken every 24 hours using a microscope (Nikon, Japan).

[0072] Cell migration assay: Corning Transwell chambers were used. HaCaT cells were seeded in the upper chamber of a 24-well plate, with TNVs, SE-EVs, and HMVs (40 μg / mL) added, respectively. DMEM medium containing 10% fetal bovine serum was added to the lower chamber. After 24 hours of culture, non-migrated cells were gently removed with a cotton swab, fixed with 4% paraformaldehyde, and stained with crystal violet. Migrating cells were observed under a microscope (Nikon, Japan), and the number of migrating cells in each image was quantitatively analyzed using ImageJ software.

[0073] Given the favorable therapeutic effect of HMVs-MNs in a mouse acne model, we further evaluated their regulatory effects on HaCaT cells and RAW264.7 cells. HaCaT cell migration ability is an important indicator for assessing wound repair. The results showed that at a concentration of 40 μg / mL, both SE-EVs and TNVs promoted the lateral migration of HaCaT cells, while HMVs exhibited a stronger pro-migration effect. Figure 6ab). Transwell assays further confirmed that HMVs significantly enhanced the migration ability of HaCaT cells (ab). Figure 6 cg).

[0074] Intracellular reactive oxygen species (ROS) detection: HaCaT cells were subjected to a 5×10⁻⁶ ppm irradiation. 5 Cellular ROS were seeded at a density in confocal culture dishes and co-cultured overnight with TNVs, SE-EVs, and HMVs (40 μg / mL). Subsequently, each culture dish was incubated with 100 mM H2O2 for 30 minutes, and ROS generation was detected using the DCFH-DA probe. Fluorescence images were observed using a confocal microscope (OLYMPUS FV3000, Japan), and intracellular ROS levels were quantitatively analyzed using flow cytometry (Beckman CytoFLEX, Germany).

[0075] During acne development, ROS-induced oxidative stress damages skin tissue and leads to cell death. After constructing a HaCaT cell oxidative stress model through H2O2 treatment, DCFH-DA fluorescent labeling showed that intracellular ROS (green fluorescence) was reduced in the SE-EVs and TNVs groups, while almost no fluorescence signal was detected in the HMVs group. Figure 6 e). Flow cytometry results are consistent with this. Figure 6 f).

[0076] RT-qPCR analysis: Human keratinocytes (HaCaT) cells were cultured in 6-well plates at 5.0 × 10⁻⁶ cells / well. 5 Cells were cultured overnight at high density, and then further cultured in a 5% CO2, 37°C incubator. Cells were treated with SE-EVs, TNVs, and HMVs (40 μg / mL), and cultured for 24 hours with LPS (Thermo Fisher Scientific) at a final concentration of 1 μg / mL. Total RNA was extracted using an RNA extraction kit (Novozymes, China), and cDNA was synthesized using reverse transcription reagent (Novozymes, China). Quantitative real-time PCR was performed using the Roche Light Cycle 480 SYBR Green Promix Pro Taq HS qPCR kit (Accurate Biology, China). Amplification conditions were set according to the manufacturer's instructions. All experiments were repeated three times, and changes in expression levels were analyzed using the 2−ΔΔCT method. The iNOS forward and reverse primer pairs are shown in Table 1.

[0077] Table 1. RT-qPCR primers encoding iNOS and GAPDH genes

[0078]

[0079] Immune cells such as macrophages play a crucial role in the progression of acne. Stimulation with LPS and other substances can induce macrophages to highly express inducible nitric oxide synthase (iNOS), thereby releasing the pro-inflammatory mediator nitric oxide (NO). Studies have found that HMVs can significantly inhibit iNOS gene transcription and reduce NO release levels, confirming their anti-inflammatory properties. Figure 6 hi).

[0080] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. Application of fusion membranes of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles in the preparation of topical acne treatment drugs; In application, the drug is combined with ultrasonic sterilization, and the ultrasonic conditions are: 50% duty cycle, 4 minutes of irradiation time and 1 w / cm². The fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles was obtained by mixing Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles in a volume ratio of 1:1 and then fusing them by ultrasonic treatment. The method for preparing the fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles includes the following steps: S1. Preparation of Staphylococcus epidermidis vesicles The cultured Staphylococcus epidermidis was centrifuged at 6000×g to remove bacterial cells. The supernatant was then further centrifuged at 20000×g to collect the precipitate, which was then resuspended in 1×PBS solution for later use. S2. Preparation of turmeric-derived exosome-like vesicles After washing and sterilizing the fresh turmeric, it was homogenized using a grinder. The homogenate was first filtered to remove fibers and impurities, and then the filtrate was centrifuged at 3000×g and 10000×g respectively. The obtained supernatant was further centrifuged at 100,000×g, and the resulting precipitate was resuspended in 1×PBS solution to obtain crude curcumin-derived exosome-like vesicles. Subsequently, sucrose density gradient centrifugation was performed to purify the curcumin-derived exosome-like vesicles. S3. Mix Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles at a volume ratio of 1:1 and sonicate to obtain a Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane. The sucrose density gradient centrifugation was performed at 8%, 30%, and 45% sucrose density gradients to purify the product. Then, the bands between the 30% and 45% sucrose density layers were collected. Finally, the sucrose was removed by ultrafiltration to obtain turmeric-derived exosome-like vesicles.

2. The application as described in claim 1, characterized in that, The drug is prepared into a microneedle for acne treatment, which kills Propionibacterium acnes in the hair follicle area and the dermal fat layer.

3. The application as described in claim 2, characterized in that, The preparation method of microneedles for acne treatment includes the following steps: Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane and sodium hyaluronate were dissolved in 1×PBS solution and stirred until completely dissolved to obtain a mixed solution. The mixed solution was then dropped onto a PDMS microneedle mold. The PDMS microneedle mold was centrifuged at 3000×g, and after complete filling, it was dried in a desiccator to obtain the final product.

4. The application as described in claim 3, characterized in that, 400 μg of Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane and 400 mg of sodium hyaluronate were dissolved in 800 μL of 1×PBS solution and stirred until completely dissolved to obtain a mixed solution. Then, 200 μL of the mixed solution was dropped onto a PDMS microneedle mold.

5. Application of the fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles in the preparation of topical Propionibacterium acnes bactericide; Propionibacterium acnes bactericide combined with ultrasonic sterilization, the ultrasonic conditions are: 50% duty cycle, 4 minutes irradiation time and 1 w / cm²; The fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles was obtained by mixing Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles in a volume ratio of 1:1 and then fusing them by ultrasonic treatment. The method for preparing the fusion membrane of Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles includes the following steps: S1. Preparation of Staphylococcus epidermidis vesicles The cultured Staphylococcus epidermidis was centrifuged at 6000×g to remove bacterial cells. The supernatant was then further centrifuged at 20000×g to collect the precipitate, which was then resuspended in 1×PBS solution for later use. S2. Preparation of turmeric-derived exosome-like vesicles After washing and sterilizing the fresh turmeric, it was homogenized using a grinder. The homogenate was first filtered to remove fibers and impurities, and then the filtrate was centrifuged at 3000×g and 10000×g respectively. The obtained supernatant was further centrifuged at 100,000×g, and the resulting precipitate was resuspended in 1×PBS solution to obtain crude curcumin-derived exosome-like vesicles. Subsequently, sucrose density gradient centrifugation was performed to purify the curcumin-derived exosome-like vesicles. S3. Mix Staphylococcus epidermidis vesicles and turmeric-derived exosome-like vesicles at a volume ratio of 1:1 and sonicate to obtain a Staphylococcus epidermidis vesicle-turmeric-derived exosome-like vesicle fusion membrane. The sucrose density gradient centrifugation was performed at 8%, 30%, and 45% sucrose density gradients to purify the product. Then, the bands between the 30% and 45% sucrose density layers were collected. Finally, the sucrose was removed by ultrafiltration to obtain turmeric-derived exosome-like vesicles.

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