Rhodomonas mucilaginosa-sourced extracellular vesicles as well as hydrogel composition and application thereof

Abstract

The invention discloses an extracellular vesicle derived from rhodomonas mucilaginosa and a hydrogel composition of the extracellular vesicle. The extracellular vesicle is obtained by carrying out ultrafiltration concentration and gradient ultracentrifugation on a culture bacterium solution. Compared with an original culture solution or a viable bacterium preparation thereof, the treatment efficiency and the safety of the extracellular vesicles are remarkably improved. Based on the extracellular vesicle, the invention also provides a hydrogel composition loaded with the extracellular vesicle, and the hydrogel composition is constructed from the extracellular vesicle, type I collagen, carboxymethyl chitosan and four-arm polyethylene glycol benzaldehyde in a chemical crosslinking manner, and has good adhesiveness, self-repairing ability and injectability. The extracellular vesicle, the hydrogel composition and the hydrogel preparation of the extracellular vesicle can serve as a medicine or a medicine carrier to be applied to atopic dermatitis, can specifically target 2-type inflammatory factors, and relieve pathological changes such as skin lesion phenotype, epidermis thickening, mast cell infiltration and inflammatory factor expression increase of an MC903-induced atopic dermatitis mouse model.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial extracellular vesicle application, specifically discloses extracellular vesicles of Roseomonas mucilaginosa, and further proposes a hydrogel composition loaded with extracellular vesicles of Roseomonas mucilaginosa and application thereof. Background Art

[0002] Atopic dermatitis (AD) is a recalcitrant, chronic, and relapsing inflammatory skin disease that severely impacts patients' quality of life. Its pathogenesis remains incompletely elucidated. Studies have shown that multiple factors, including genetic susceptibility, skin barrier dysfunction, immune imbalance, and abnormal skin microbiome, play a crucial role in the development and progression of AD. Among these, the type 2 inflammatory response, driven by Th2 (T helper 2) cells, is considered central to AD pathogenesis. The type 2 inflammatory cytokines interleukin-4 (IL-4) and thymic stromal lymphopoietin (TSLP) play a crucial role in the development and progression of AD. Currently, clinical treatment for AD primarily relies on topical glucocorticoids and calcineurin inhibitors as first-line medications. For moderate to severe disease, combined immunosuppressive agents or targeted therapies are often required. Although these drugs offer significant short-term benefits, their efficacy is limited, and long-term use can induce severe side effects. Therefore, there is an urgent need to develop more advanced and efficient treatment methods.

[0003] In recent years, abnormal skin microecology has been shown to play a key role in promoting the development of AD, mainly manifested by the dominant colonization of pathogenic Staphylococcus aureus, accompanied by the presence of "Rosemonas mucilaginosa ( Roseomonasmucosa, R. mucosa )” is a manifestation of the decline in the diversity and abundance of skin commensal bacteria. Some studies have shown that transplanting Roseomonas muciniphila into AD lesions can improve the skin microecological balance and thereby alleviate AD. However, clinical trials have shown that the strain's colonization efficiency on patient skin is low, seriously affecting its efficacy. Furthermore, the preparation and storage of live bacterial preparations require high technical requirements and may pose potential infection risks, further limiting their clinical application.

[0004] Extracellular vesicles (EVs) are a class of nanoscale vesicles secreted by commensal bacteria that contain a variety of bioactive substances, such as proteins, lipids, and nucleic acids. Symbiotic EVs are characterized by structural stability, rich functionality, and prolonged circulation, playing crucial roles in physiological and pathological processes such as host immune regulation, cellular metabolism, angiogenesis, and barrier repair. Therefore, further exploring the therapeutic potential of EVs derived from the skin commensal Roseomonas mucicola (RS EVs) in AD is of great scientific interest. However, direct administration of EVs often suffers from limited absorption efficiency and a short duration of action, significantly impacting their efficacy. Therefore, developing efficient EV delivery systems is crucial to improving the transdermal absorption efficiency and prolonging the duration of action of EVs. Hydrogels, similar in structure to the extracellular matrix, possess a rich network and porous structure, as well as strong water-retention and moisturizing capabilities. Some hydrogel components also possess natural antimicrobial and antioxidant properties, making them ideal topical preparations for dermatological diseases. Given the disease characteristics of AD, developing a hydrogel topical preparation that can efficiently deliver RS EVs is of great significance for breaking through the bottleneck of skin commensal bacteria in treating AD. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention provides extracellular vesicles (EVs) from Roseomonas mucilaginosa, prepared by ultrafiltration, concentration, and gradient ultracentrifugation of a culture of Roseomonas mucilaginosa, deposited with CGMCC No. 25967. The present invention further provides a hydrogel composition loaded with EVs from Roseomonas mucilaginosa, a highly adhesive, injectable, self-healing hydrogel composition that enables sustained and efficient delivery of EVs from Roseomonas mucilaginosa, thereby improving the therapeutic efficacy of AD.

[0006] To achieve the above technical objectives, the present invention provides an extracellular vesicle of Roseomonas mucilaginosa, which is prepared by ultrafiltration concentration and gradient ultracentrifugation of a culture broth of Roseomonas mucilaginosa with a deposit number of CGMCC No. 25967.

[0007] The present invention further proposes a hydrogel composition loaded with extracellular vesicles of Roseomonas mucilaginosa. The hydrogel composition comprises a hydrogel and extracellular vesicles of Roseomonas mucilaginosa loaded in the hydrogel. The hydrogel is formed by chemically cross-linking type I collagen, carboxymethyl chitosan and four-arm polyethylene glycol benzaldehyde.

[0008] The mass ratio of type I collagen, carboxymethyl chitosan and four-arm polyethylene glycol benzaldehyde is 6: (1-3): 3, preferably 2: 1: 1.

[0009] The final concentration of the Roseomonas extracellular vesicles in the hydrogel composition is 1×10 8 Number of particles / mL~1×10 10 Particle number / mL. The particle number is the number of extracellular vesicle particles.

[0010] The particle size of the extracellular vesicles of Roseomonas mucilaginosa is not greater than 500 nm, preferably 50-300 nm.

[0011] The present invention further provides a method for preparing the hydrogel composition, comprising the following steps: (1) Take the formulated amount of type I collagen, carboxymethyl chitosan, and four-arm polyethylene glycol benzaldehyde and add them to PBS buffer respectively, and mix them evenly in a water bath at 30-37°C to obtain type I collagen solution, carboxymethyl chitosan solution, and four-arm polyethylene glycol benzaldehyde solution respectively; (2) The extracellular vesicles of Roseomonas mucilaginosa are dissolved in the type I collagen solution obtained in step (1), and then uniformly mixed with the carboxymethyl chitosan solution and the four-arm polyethylene glycol benzaldehyde solution to obtain a hydrogel composition loaded with extracellular vesicles.

[0012] Specifically, the extracellular vesicles of Roseomonas mucilaginosa are prepared by the following method: S1: First, the Roseomonas mucilaginosa is inoculated into R2A medium for activation culture. Preferably, the culture conditions are constant temperature culture at 30-32°C for 20-24 hours. Then, a single Roseomonas mucilaginosa colony DL-1 is picked and inoculated into TSB liquid culture medium. The rotation speed is 180-200 rpm and cultured at 30-32°C for 12-18 hours to obtain an activated Roseomonas mucilaginosa culture liquid. S2: The activated Roseomonas muciniphila in step S1 is inoculated into TSB liquid medium for cultivation, and the supernatant is collected by centrifugation to obtain a bacterial solution containing extracellular vesicles. Preferably, the activated Roseomonas muciniphila is inoculated into TSB liquid medium and cultured on a thermostatic shaker at 30-32°C and 180-200 rpm for 12-18 hours, and then centrifuged at 2000-2500 g for 15-20 minutes, and the supernatant is collected to obtain a bacterial solution containing extracellular vesicles. S3: Centrifuge the extracellular vesicle-containing bacterial solution collected in S2 at 10,000-12,000 g and 2-4°C for 10-15 min. Collect the supernatant and filter it through a 0.22 μm filter membrane to further remove bacteria and large particles. Collect the filtrate. S4: The filtrate obtained in S3 was concentrated by ultrafiltration through a 10 kD filter membrane to obtain a high-concentration extracellular vesicle bacterial solution; S5: Centrifuge the high-concentration extracellular vesicle solution at 10,000-20,000 g at 2-4°C for 30-60 min and collect the supernatant. S6: Centrifuge the supernatant obtained in S5 at 100,000-130,000 g and 2-4°C for 60-90 min. The resulting precipitate is the extracellular vesicles of Roseomonas mucilaginosa.

[0013] Preferably, in step S1, the R2A medium components include: tryptone 0.25~0.5 g / L, acid hydrolyzed casein 0.5~0.8 g / L, yeast 0.5~1.0 g / L, soluble starch 0.5~0.8 g / L, dipotassium hydrogen phosphate 0.3~0.6 g / L, magnesium sulfate 0.1~0.15 g / L, sodium pyruvate 0.3~0.4 g / L, agar 12.0~15.0 g / L, glucose 0.5~0.8 g / L, and a pH value of 7.2±0.2; the TSB liquid medium components include: tryptone 17.0~20.0 g / L, soy peptone 3.0~5.0 g / L, sodium chloride 5.0~6.0 g / L, dipotassium hydrogen phosphate 2.5~4.5 g / L, glucose 2.5~5.0 g / L, and a pH value of 7.3± 0.2.

[0014] The present invention also discloses a hydrogel preparation comprising the hydrogel composition and a pharmaceutically acceptable carrier.

[0015] The present invention further proposes the use of the hydrogel composition or hydrogel preparation in preparing a drug carrier, a drug or a derivative preparation for treating atopic dermatitis.

[0016] The hydrogel composition or hydrogel preparation specifically targets type 2 inflammatory factors and reduces the expression level of inflammatory factor mRNA in human immortalized keratinocytes induced by tumor necrosis factor α and interferon-γ, wherein the inflammatory factors include but are not limited to any one or more combinations of tumor necrosis factor α, interleukin 6, interleukin 4 and thymic stromal lymphopoietin.

[0017] The hydrogel composition or hydrogel preparation alleviates the severity of the skin lesion phenotype, the epidermal thickness of the lesion site, and the mast cell infiltration of the lesion site in an MC903-induced atopic dermatitis mouse model, and reduces the mRNA transcription level of inflammatory factors in the lesion site.

[0018] Specifically, the extracellular vesicles can selectively inhibit the transcription of pro-inflammatory factor mRNA in human immortalized keratinocytes (HaCat) cells induced by tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ).

[0019] The extracellular vesicles exhibited superior anti-inflammatory effects compared to Roseomonas mucilaginosa culture supernatant. Compared to Roseomonas culture fluid, the extracellular vesicles were more potent in inhibiting TNF-α and IFN-γ-induced pro-inflammatory cytokine mRNA transcription in HaCat cells under the same conditions. The extracellular vesicles demonstrated superior anti-inflammatory efficacy and safety compared to a live Roseomonas mucilaginosa preparation. Compared to a live Roseomonas preparation, the extracellular vesicles were more potent in inhibiting TNF-α and IFN-γ-induced pro-inflammatory mRNA transcription in HaCat cells under the same conditions, without inducing the transcription of the corresponding pro-inflammatory mRNAs.

[0020] Preferably, the pro-inflammatory factors include any one or more combinations of TNF-α, IL-6, IL-4, and TSLP.

[0021] Specifically, the hydrogel topical preparation loaded with extracellular vesicles of Roseomonas mucilaginosa can repair the destruction of the skin barrier in the lesion area of the AD mouse model induced by calcipotriol (MC903).

[0022] Furthermore, the hydrogel topical preparation loaded with extracellular vesicles of Roseomonas mucilaginosa can alleviate the disease phenotype of the MC903-induced AD mouse model, including but not limited to reducing the severity of skin lesions, reducing the epidermal thickness of the lesion site, and inhibiting the differentiation of Th2 cells and the secretion of type 2 inflammatory cytokine IL-4.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The present invention provides an extracellular vesicle of the skin commensal bacterium Roseomonas muciniphila, which has a better inhibitory effect on type 2 inflammation than live bacterial preparations and culture supernatants of Roseomonas muciniphila, does not cause the expression of pro-inflammatory factors, and has higher safety. It is a new type of nanoscale substance with biological activity.

[0024] (2) The present invention further provides a hydrogel topical preparation loaded with extracellular vesicles of Roseomonas mucilaginosa. The hydrogel has high adhesion, injectability and self-repairing ability. When applied to AD lesion areas, it can not only effectively moisturize and promote the recovery of skin barrier function, but also achieve stable and continuous output of extracellular vesicles of Roseomonas mucilaginosa, enhance the inhibition of type 2 helper T cell (Th2) differentiation and the expression of type 2 inflammatory cytokine IL-4, and has the potential to develop new AD treatment preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Characterization results of extracellular vesicles of Roseomonas mucilaginosa (A. Transmission electron microscopy image; B. Particle size and concentration distribution); Figure 2For different concentrations (0~1×10 10 Toxic effects of extracellular vesicles of Roseomonas mucilaginosa on HaCaT cells and mouse mononuclear macrophage leukemia cells (A. HaCaT cells; B. Raw264.7 cells); Figure 3 For different concentrations (1×10 8 ~1×10 10 Effects of extracellular vesicles of Roseomonas muciniphila (RS EVs) on the mRNA transcription levels of pro-inflammatory factors in HaCaT cells induced by TNF-α and IFN-γ (A. Relative mRNA expression level of TNF-α; B. Relative mRNA expression level of IL-6; C. Relative mRNA expression level of IL-4; D. Relative mRNA expression level of TSLP); Figure 4 Effects of equal amounts of Roseomonas mucilaginosa culture supernatant (Sup.), extracellular vesicles (EVs) of Roseomonas mucilaginosa, and live cell preparation (RS) of Roseomonas mucilaginosa on the mRNA transcription levels of pro-inflammatory factors in HaCat cells induced by TNF-α and IFN-γ (A. Relative mRNA expression level of TNF-α; B. Relative mRNA expression level of IL-6; C. Relative mRNA expression level of IL-4; D. Relative mRNA expression level of TSLP); Figure 5 Schematic diagram of the cross-linking pattern of hydrogel loaded with mucilaginous rose extracellular vesicles; Figure 6 The following are scanning electron micrographs of hydrogels prepared with different mass ratios of ingredients; Figure 7 This is a scanning electron micrograph of the hydrogel loaded with extracellular vesicles of Roseomonas mucilaginosa; Figure 8 Characterization diagram of the physical properties of hydrogel loaded with extracellular vesicles of Roseomonas mucilaginosa (A. Self-healing function of hydrogel; B. Adhesion of hydrogel; C. Plasticity of hydrogel); Figure 9 To construct the MC903-induced AD model, the treatment flow chart, the clinical phenotype of skin lesions in AD mice after treatment, and the changes in ear thickness (A. Treatment flow chart; B. Changes in ear thickness before and after treatment; C. Clinical phenotype of skin lesions after treatment); Figure 10 To investigate the effects of hydrogels loaded with extracellular vesicles of Roseomonas muciniphila on the pathological changes of skin lesions in MC903-induced AD mice; Figure 11To investigate the effect of hydrogel loaded with extracellular vesicles of Roseomonas muciniphila on the mRNA expression level of the skin barrier-related protein filaggrin in the lesions of MC903-induced AD mice; Figure 12 The hydrogel loaded with extracellular vesicles of Roseomonas mucilaginosa can be used to inhibit the Th2 cell subset (IL4 + CD4 + T cells) (A. Flow cytometry gating strategy; B. CD4 + IL4 + Th2 lymphocyte subsets in CD4 + CD8 - percentage of T cells). DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0027] The Roseomonas mucilaginosa used in the following examples has a deposit number of CGMCC No. 25967, and its detailed information is disclosed in CN 116103205B.

[0028] Example 1 Extraction and characterization of extracellular vesicles of Roseomonas mucilaginosa.

[0029] 1) Activation and cultivation of Roseomonas mucilaginosa The -80°C glycerol-frozen Roseomonas mucilaginosa was inoculated into R2A medium and then activated in a 32°C constant temperature incubator for 24 h. Subsequently, a single Roseomonas mucilaginosa colony was picked and inoculated into TSB liquid medium at a speed of 200 rpm and cultured at 32°C for 16 h to obtain an activated seed suspension. The seed suspension was inoculated again into TSB liquid medium at a volume fraction of 5% and cultured at 32°C and 200 rpm for another 16 h to obtain the target bacterial suspension. The number of target bacterial suspensions can reach 5×10 9 CFU / mL or above.

[0030] The R2A culture medium components include: 0.25 g / L tryptone, 0.5 g / L acid hydrolyzed casein, 0.5 g / L yeast, 0.5 g / L soluble starch, 0.3 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 0.3 g / L sodium pyruvate, 12.0 g / L agar, 0.5 g / L glucose, and a pH value of 7.2 ± 0.2.

[0031] The TSB liquid culture medium components include: 17.0 g / L tryptone, 3.0 g / L soy peptone, 5.0 g / L sodium chloride, 2.5 g / L potassium hydrogen phosphate, and 2.5 g / L glucose, with a pH value of 7.3 ± 0.2.

[0032] 2) Extraction of extracellular vesicles from Roseomonas mucilaginosa The bacterial suspension of Roseomonas mucilaginosa obtained in step 1 was centrifuged at 2500 g and 4 °C for 20 min to remove most of the bacterial precipitate; the supernatant was collected and centrifuged at 12000 g and 4 °C for 15 min, and then the supernatant was filtered through a 0.22 μm filter membrane to further remove the bacterial components in the sample; the obtained filtrate was ultrafiltered and concentrated using a 10 kD ultrafiltration centrifuge tube, and then the obtained bacterial suspension was centrifuged in an ultracentrifuge at 20000 g and 4 °C for 60 min. The supernatant was collected and centrifuged again at 120000 g and 4 °C for 75 min to obtain the precipitate, which was the extracellular vesicles of Roseomonas mucilaginosa. After resuspending with sterile PBS, it was stored at -80 °C.

[0033] 3) Transmission electron microscopy observation of extracellular vesicles of Roseomonas mucilaginosa 15 μL of the extracellular vesicles obtained in step 2 were taken and placed on a copper mesh (ordinary carbon support membrane) for 1 min. The suspension was then blotted dry with filter paper. Then 15 μL of 2% uranyl acetate staining solution was taken and stained at room temperature for 1 min. The suspension was blotted dry with filter paper. The stained sample was placed under a lamp and baked for 10 min. It was then observed and photographed under a transmission electron microscope. The morphology was as follows: Figure 1 As shown in A, the extracellular vesicles of this mucilaginous Roseomonas are round "cup-and-disc" shaped.

[0034] 4) Analysis of extracellular vesicle nanoparticles of Roseomonas mucilaginosa The sample pool was cleaned with deionized water and the instrument (NanoSightNS300) was calibrated using polystyrene microspheres (100 nm). The extracellular vesicle sample obtained in step 2) was diluted 5000 times with PBS and then injected for detection. Each sample was tested three times in succession. The specific results are shown in the figure. Figure 1 As shown in B. The size distribution of the extracellular vesicles of Roseomonas mucilaginosa is 0~500 nm and is not zero. The original concentration is 1.14 × 10 12 Number of particles / mL.

[0035] Example 2 Evaluation of the cytotoxic effects of extracellular vesicles of Roseomonas mucilaginosa on HaCaT cells and Raw264.7 cells.

[0036] 1) HaCaT cells were cultured at a density of 1 × 10 4Each well was seeded in a 96-well plate containing 1 mL of culture medium and the cells were plated with a final concentration of 1 × 10 4 ~ 1 × 10 10 The cells were treated with extracellular vesicles with a specific number of particles / mL. Sterile PBS solution was added as the negative control group, and culture medium without cells and extracellular vesicles (DMEM medium containing 10% FBS) was used as the blank control. Six replicate wells were set for each concentration, and the culture dishes were placed in a 5% CO2, 37°C incubator for incubation for 48 h. 10 μL of CCK8 solution was added to each well, and the culture dishes were placed in a 5% CO2, 37°C incubator for further incubation for 1-4 h. The absorbance at 450 nm was measured using a microplate reader to calculate the cell viability. The results are shown in Figure 2. Figure 2 As shown in A, different concentrations of extracellular vesicles of Roseomonas mucilaginosa had no significant inhibitory effect on the viability of HaCaT cells, suggesting that the extracellular vesicles of Roseomonas mucilaginosa at 1 × 10 4 ~ 1 × 10 10 The toxicity to cells within the particle number / mL concentration range is very low or non-toxic.

[0037] 2) Raw264.7 cells were plated at 5 × 10 4 Each well was seeded in a 96-well plate containing 1 mL of culture medium and the cells were plated with a final concentration of 1 × 10 4 to 1 × 10 10 The cells were treated with extracellular vesicles with a certain number of particles / mL. Sterile PBS solution was added as the negative control group, and the culture medium without cells and extracellular vesicles (DMEM culture medium containing 10% FBS) was used as the blank control. Six replicate wells were set for each concentration, and the culture dishes were placed in a 5% CO2, 37°C incubator for incubation for 48 h. 10 μL of CCK8 solution was added to each well, and the culture dishes were placed in a 5% CO2, 37°C incubator for further incubation for 1-4 h. The absorbance at 450 nm was measured using a microplate reader to calculate the cell viability. The results are shown in Figure 2. Figure 2 As shown in B, different concentrations of extracellular vesicles of Roseomonas mucilaginosa had no significant inhibitory effect on the viability of RAW264.7 cells, indicating that the extracellular vesicles of Roseomonas mucilaginosa at 1 × 10 4 ~ 1 × 10 10 The toxicity to cells within the particle number / mL concentration range is very low or non-toxic.

[0038] Example 3 Effects of extracellular vesicles of Roseomonas mucilaginosa on the transcriptional levels of pro-inflammatory factors in HaCat cells induced by TNF-α and IFN-γ.

[0039] 1) HaCaT cells were cultured at a density of 1 × 10 5The cells were seeded into 12-well plates in DMEM medium containing 10% FBS, cultured overnight at 5% CO2 and 37°C, and then cultured with 1 × 10 8 Particle number / mL, 1 × 10 9 Particle count / mL and 1 × 10 10 The cells were pretreated with extracellular vesicles (EVs) at a final concentration of 10 ng / mL for 4 h, and then stimulated with TNF-α and IFN-γ at a final concentration of 10 ng / mL. The groups pretreated with PBS solution and stimulated with TNF-α and IFN-γ served as the experimental control group, and the groups pretreated with PBS solution and stimulated with EVs served as the blank control group. Six replicate wells were set up in each group. After continuous stimulation for 24 h, the cells were collected for total RNA extraction.

[0040] 2) Total RNA was extracted from the collected cells using the TRIZOL kit, and the RNA concentration and purity were measured using a NanoDrop spectrophotometer. The total RNA was reverse transcribed into cDNA using a reverse transcription reagent, and the mRNA expression levels of type 2 inflammation-related cytokines TNF-α, IL-6, IL-4, and TSLP were detected using the SYBR Green kit on an ABI Prism real-time quantitative PCR instrument. β-actin was used as an internal reference, and the relative expression of each cytokine mRNA was calculated based on the 2 -ΔΔCt The primer sequences for the specific cytokines and β-actin to be tested are shown in Table 1.

[0041] Table 1 Sequences of cytokines to be tested and internal reference primers

[0042] 3) Effects of extracellular vesicles of Roseomonas mucilaginosa on the transcriptional levels of pro-inflammatory factors in HaCaT cells induced by TNF-α and IFN-γ Figure 3 As shown in the results, ① compared with the blank control group, the transcriptional levels of pro-inflammatory factors such as TNF-α, IL-6, IL-4 and TSLP in HaCaT cells were increased to varying degrees by 10 ng / mL TNF-α and 10 ng / mL IFN-γ stimulation, and the differences between the two groups were statistically significant, indicating that a typical AD cell model had been successfully induced; ② compared with the experimental control group, the use of higher concentrations (1 × 10 9 Particle count / mL and 1 × 10 10 Pretreatment of HaCat cells with extracellular vesicles of Roseomonas mucilaginosa (1 × 10 particles / mL) significantly inhibited the increase in the transcription levels of pro-inflammatory factors such as TNF-α, IL-6, IL-4 and TSLP in the AD cell model. The differences between the groups were statistically significant. 9The inhibition effect of the treatment group with the concentration of 1 × 10 8 Pretreatment with extracellular vesicles (EVs) (number of particles / mL) had a significant inhibitory effect on the transcription level of TNF-α in the cell model, but had a weaker inhibitory effect on the increased transcription levels of IL-6, IL-4 and TSLP pro-inflammatory factors, and the differences between the groups were not statistically significant.

[0043] Example 4 Effects of the culture supernatant (Sup.) of Roseomonas mucilaginosa, extracellular vesicles (RSEVs) of Roseomonas mucilaginosa, and live bacterial preparation (RS) of Roseomonas mucilaginosa on the mRNA transcription levels of pro-inflammatory factors in HaCat cells induced by TNF-α and IFN-γ.

[0044] 1) HaCaT cells were cultured at a density of 1 × 10 5 Cells were seeded at 100 μg / well in a 12-well plate and cultured overnight in DMEM supplemented with 10% FBS at 5% CO₂ and 37°C. Cells were then pretreated with equal amounts of culture supernatant, extracellular vesicles of R. mucronata, and live bacterial preparations for 4 h. Cells were then stimulated with TNF-α and IFN-γ at a final concentration of 10 ng / mL. The groups pretreated with TSB medium and stimulated with TNF-α and IFN-γ served as experimental controls, while the groups pretreated with TSB medium and stimulated served as blank controls. Six replicates were set up in each group. After 24 h of stimulation, cells were harvested for total RNA extraction. For total RNA extraction, refer to Step 2 of Example 3.

[0045] 2) Effects of supernatant (Sup.), extracellular vesicles (RS EVs) and live bacterial preparation (RS) on the transcriptional levels of pro-inflammatory factors in HaCaT cells induced by TNF-α and IFN-γ Figure 4As shown in the results, ① compared with the blank control group, stimulation with 10 ng / mL TNF-α and 10 ng / mL IFN-γ increased the transcriptional levels of proinflammatory cytokines such as TNF-α, IL-6, IL-4, and TSLP in HaCaT cells to varying degrees, and the differences between the two groups were statistically significant, indicating that a typical AD cell model had been successfully induced; ② compared with the experimental control group, pretreatment with supernatant and extracellular vesicles significantly inhibited the increase in the transcriptional levels of proinflammatory cytokines TNF-α, IL-4, and TSLP in the AD cell model, and the differences between the groups were statistically significant. Among them, the extracellular vesicle-treated group also significantly inhibited the increase in IL-6 transcriptional levels, and the inhibitory effect on the transcriptional levels of proinflammatory cytokines TNF-α, IL-4, and TSLP was significantly stronger than that of the supernatant; ③ compared with the extracellular vesicle-treated group, pretreatment with live bacterial preparations significantly upregulated the expression of proinflammatory cytokines TNF-α, IL-6, IL-4, and TSLP, suggesting a potential risk of infection.

[0046] Example 5 Exploration of hydrogel preparation conditions and characterization.

[0047] 1) Hydrogel Preparation: 0.6 g, 0.3 g, and 0.3 g of type I collagen, carboxymethyl chitosan, and four-arm polyethylene glycol benzaldehyde, respectively, were added to 10 mL of PBS buffer and dissolved in a water bath at 30-37°C for 15 min to prepare collagen solution, carboxymethyl chitosan solution, and four-arm polyethylene glycol benzaldehyde solution. These solutions were then uniformly mixed in a mass ratio of 6:1:3, 6:2:3, and 6:3:3 to obtain hydrogels a, b, and c. Figure 5 Schematic diagram of the chemical cross-linking process of hydrogel loaded with mucilaginous rose extracellular vesicles.

[0048] 2) Scanning electron microscopy was used to observe the microscopic morphology. The obtained hydrogel scanning electron microscopy image is shown below: Figure 6 The results showed that hydrogels a, b, and c all had porous structures, among which hydrogel a had the largest pore size, followed by hydrogel b, and hydrogel c had the smallest pore size, suggesting that hydrogel c had a better sustained release effect, thereby achieving sustained release of extracellular vesicles.

[0049] Example 6 Preparation, characterization and physical characteristics of hydrogels loaded with extracellular vesicles of Roseomonas mucilaginosa.

[0050] 1) Preparation of hydrogel loaded with extracellular vesicles of Roseomonas mucilaginosa: The extracellular vesicles of Roseomonas mucilaginosa were dissolved in the type I collagen solution obtained in step 1) of Example 5, and then uniformly mixed with a carboxymethyl chitosan solution and a four-arm polyethylene glycol benzaldehyde solution in a mass ratio of 6:3:3 to obtain a hydrogel composition loaded with extracellular vesicles, wherein the final concentration of the extracellular vesicles of Roseomonas mucilaginosa was 1×10 9 Number of particles / ml.

[0051] 2) Scanning electron microscopy was used to observe the microscopic morphology. The obtained hydrogel scanning electron microscopy image is shown below: Figure 7 The results showed that the hydrogel loaded with extracellular vesicles of R. mucilaginosa exhibited a porous structure with a pore size of approximately 20-100 μm, which could effectively load R. mucilaginosa.

[0052] 3) The hydrogel loaded with extracellular vesicles of Rosebasidiomyces mucilaginosa has self-healing ability, good adhesion and injectability: a "maple leaf" shaped hydrogel was prepared using a rubber mold, and then the two cut hydrogels were brought close together again. Figure 8 (A) shows that as time goes by, the cut hydrogel can repair and heal itself again, demonstrating a good self-repair effect; the adhesion of the hydrogel is tested using 10ml centrifuge tubes filled with different volumes of liquid. Figure 8 (B) shows that the hydrogel of the present invention can stably adhere to 10ml centrifuge tubes filled with different volumes of liquid, showing good adhesion. A certain volume of hydrogel solution was extracted using a 1ml syringe to draw the English letter "A". Figure 8 (C) shows that the hydrogel of the present invention is injectable, suggesting that hydrogel dressings of different sizes or shapes can be designed according to the size of the skin lesions in clinical applications; Example 6 Therapeutic effect of hydrogel loaded with extracellular vesicles of Roseomonas mucicola on MC903-induced AD mouse model.

[0053] 1) Construction and intervention of animal models: Figure 9 As shown in Figure 2, 6-8 week-old C57BL / 6 or Balb / c mice with relatively uniform body weight were randomly divided into five groups, namely AD model group, positive drug treatment group, extracellular vesicle treatment group, hydrogel treatment group and Roseomonas mucilaginosa-loaded extracellular vesicle treatment group. Each group was set up with five biological replicates. The specific treatment methods are as follows (eg Figure 9 A): AD model group: Starting from the experimental day, 1 nmol of MC903 (dissolved in 20 μL of anhydrous ethanol) was evenly applied to both ears of each mouse at 9:00 am every day for 17 consecutive days; Positive drug treatment group: Starting from the experimental day, 1 nmol MC903 (dissolved in 20 μL anhydrous ethanol) was evenly applied to both ears of each mouse at 9:00 am every day, and an appropriate amount of hydrocortisone butyrate cream was evenly applied to each ear at 17:00 pm every day for 17 consecutive days; Extracellular vesicle treatment group: Starting from the experimental day, 1 nmol MC903 (dissolved in 20 μL anhydrous ethanol) was evenly applied to both ears of each mouse at 9:00 am every day, and extracellular vesicles (1 × 109 Particle number / ml, 20 μl volume / ear), and treatment for 17 consecutive days.

[0054] Hydrogel treatment group: Starting from the experimental day, 1 nmol of MC903 (dissolved in 20 μL of anhydrous ethanol) was evenly applied to both ears of each mouse at 9:00 am every day, and hydrogel (20 μL volume / ear) was evenly applied to each ear at 17:00 pm every day for 17 consecutive days.

[0055] For the hydrogel-treated group, 1 nmol of MC903 (dissolved in 20 μL of anhydrous ethanol) was evenly applied to both ears of each mouse at 9:00 a.m. every day, and 1 nmol of MC903 (dissolved in 20 μL of anhydrous ethanol) was evenly applied to each ear of each mouse at 17:00 p.m. every day. 9 Particle number / ml, 20 μl volume / ear), and treatment for 17 consecutive days.

[0056] 2) Determination of mouse ear thickness: Vernier calipers were used to precisely measure the thickness of both ears of mice at the beginning of modeling (D1) and on the 4th day (D4), 7th day (D7), 10th day (D10), 13th day (D13), 15th day (D15) and 17th day (D17) after modeling. The average value of both sides represented the degree of swelling of both ears of mice. The results are shown as follows: Figure 9 As shown in Figure B, at the observation endpoint (D17), the ears of the MC903 modeling group were significantly thickened, while the positive drug treatment group of topical hydrocortisone butyrate cream, the extracellular vesicle treatment group, the hydrogel treatment group and the hydrogel treatment group loaded with extracellular vesicles could significantly alleviate the MC903-induced thickening of the mouse ears, and all had statistical differences. Among them, the hydrogel treatment group loaded with extracellular vesicles had the best effect.

[0057] 3) Clinical phenotype analysis of mouse skin lesions: At the end of the observation period, the skin lesions of the mice were photographed. Figure 9 As shown in Figure C, the ears of mice in the AD modeling group developed erythema and swelling, and obvious scales were visible on the surface. The positive drug treatment group of topical hydrocortisone butyrate cream, the extracellular vesicle treatment group, the hydrogel treatment group, and the extracellular vesicle-loaded hydrogel treatment group could significantly improve the MC903-induced erythema, swelling, and scales in the ears of mice, among which the extracellular vesicle-loaded hydrogel treatment group had the best effect.

[0058] 4) Analysis of pathological changes in the skin lesions of mice: After euthanasia at the end of observation, the mice were euthanized and the ear specimens were obtained. They were embedded in paraffin and sliced, and then stained with hematoxylin-eosin. The staining method was as follows: ① Place the paraffin sections in an oven at 60 ℃ for 2 h; ② Dewax the paraffin sections with conventional xylene and ethanol to water; ③ Stain with hematoxylin for 10 min, then rinse with running water to remove the residual color; ④ Separate with 0.5% hydrochloric acid ethanol for 1~2 s, then rinse with running water; ⑤ Return to blue with 1% ammonia water, and examine the degree of cell nucleus separation under a fiberscope after rinsing with running water; ⑥ Stain with 1% eosin for 30 s, and quickly wash with distilled water; ⑦ Wash quickly with 80%, 90% and 95% ethanol solutions, each level for 10 s, and monitor the color contrast of the cell nucleus and cytoplasm under a microscope; ⑧ Wash with 100% ethanol twice, each time for 1~2 min; Wash with xylene twice, each time for 1~2 min; ⑨ Seal with neutral gum, and observe the staining results under a microscope. The results are as follows Figure 10 As shown in the data, the epidermis of the AD model group mice was significantly thickened, and a large number of inflammatory cells infiltrated in the dermis. The positive drug treatment group, the hydrogel treatment group, and the hydrogel treatment group loaded with extracellular vesicles could all improve the MC903-induced epidermal thickening and dermal inflammatory cell infiltration in the mice, among which the improvement effect of the hydrogel treatment group loaded with extracellular vesicles was the most significant.

[0059] 5) Analysis of gene transcription levels of skin barrier-related proteins (filaggrin) in mouse lesional areas: The fresh ear specimens obtained in step 4) were placed in a sterile, enzyme-free grinding tube and ground with grinding beads. Total RNA from the lesional tissue was then extracted using a TRIZOL kit. The total RNA was reverse transcribed into cDNA using a reverse transcription kit. The target gene mRNA transcription level was detected using an ABI 7900 real-time quantitative PCR instrument using a SYBR Green kit. β-actin was used as an internal reference. The relative expression of the target gene mRNA was calculated based on 2 -ΔΔCt The calculated results are as follows Figure 11 Compared with the filaggrin gene transcription levels in the skin lesions of mice in the AD model group, the filaggrin gene transcription levels were significantly increased in the hydrocortisone butyrate cream-positive drug treatment group, the hydrogel treatment group, and the hydrogel treatment group loaded with extracellular vesicles. Among them, the filaggrin gene transcription level in the hydrogel treatment group loaded with extracellular vesicles increased most significantly. However, there was no statistical difference between the group treated with extracellular vesicles of Roseomonas mucilaginosa alone and the AD model group.

[0060] 6) Mouse spleen CD4 + CD8 -Flow cytometric analysis of Th2 lymphocyte subsets in T cells: Euthanize the mice in step 4) to obtain fresh spleen tissue. Gently grind the tissue into a single-cell suspension and collect the cell pellet after centrifugation. Resuspend the cell pellet in red blood cell lysis buffer and allow it to lyse the red blood cells. After 3 minutes, add an appropriate amount of PBS to terminate lysis and filter through a 70 μM filter. Centrifuge and collect the cell pellet. Resuspend the cells in a volume of RPMI1640 medium containing 10% FBS and count them using a cell counter to adjust the cell concentration to 5 × 10 7 Cells / mL, 100 μL of cell suspension was transferred to a flow cytometry tube, and cell stimulation and intracellular and extracellular antigen staining were performed according to the flow cytometry steps before detection. Specific flow cytometry antibodies are shown in the figure below, and the flow cytometry gating strategy is shown in Figure 12 A, flow cytometry analysis results Figure 12 As shown in Figure B. Compared with the AD model group, the hydrocortisone butyrate cream positive drug treatment group, the extracellular vesicle treatment group, and the extracellular vesicle-loaded hydrogel group all significantly reduced the proportion of Th2 lymphocyte subsets in the spleen of the MC903-induced AD mouse model, among which the extracellular vesicle-loaded hydrogel had the most significant reduction. There was no statistically significant difference between the hydrogel treatment group and the AD model group.

[0061] In summary, the present invention provides a hydrogel external preparation loaded with extracellular vesicles of Roseomonas mucilaginosa, and its preparation method and application. Among them, the extracellular vesicles of Roseomonas mucilaginosa can inhibit the increase in the transcription levels of proinflammatory factors such as TNF-α, IL-6, IL-4 and TSLP in HaCaT cells induced by TNF-α and IFN-γ, especially can specifically reduce the expression of type 2 inflammatory cytokines TSLP and IL-4, which is more targeted and accurate for the treatment of AD; in addition, its inhibitory efficiency and safety are significantly higher than those of Roseomonas mucilaginosa DL-1 culture supernatant and live bacterial preparations. In addition, the present invention uses three organic components, type I collagen, carboxymethyl chitosan and four-arm polyethylene glycol benzaldehyde, to construct a highly adhesive, injectable and self-repairing hydrogel through chemical double cross-linking, which can achieve stable and continuous output of extracellular vesicles of Roseomonas mucilaginosa, and enhance RS EVs to inhibit the differentiation of type 2 helper T cells and the expression of type 2 inflammatory cytokine IL-4. Compared with the long-term use of glucocorticoids or live bacterial preparations, the hydrogel topical preparation loaded with extracellular vesicles of Roseomonas mucilaginosa provided by the present invention has the potential to be developed into a new AD treatment preparation.

Claims

1. An extracellular vesicle derived from Roseomonas mucilaginosa, characterized in that: The extracellular vesicles are prepared by ultrafiltration concentration and gradient ultracentrifugation of a culture solution of Roseomonas mucilaginosa with a deposit number of CGMCC No. 25967.

2. A hydrogel composition loaded with extracellular vesicles of Roseomonas mucilaginosa, characterized in that: The hydrogel composition comprises a hydrogel and the extracellular vesicles of Roseomonas mucilaginosa according to claim 1 loaded in the hydrogel. The hydrogel is formed by chemically cross-linking type I collagen, carboxymethyl chitosan and four-arm polyethylene glycol benzaldehyde.

3. The hydrogel composition according to claim 2, characterized in that The mass ratio of the type I collagen, carboxymethyl chitosan and four-arm polyethylene glycol benzaldehyde is 6: (1-3):

3.

4. The hydrogel composition according to claim 2, characterized in that The final concentration of the Roseomonas extracellular vesicles in the hydrogel composition is 1×10 8 Number of particles / mL~1×10 10 Particle number / mL; the particle size of the extracellular vesicles of Roseomonas mucilaginosa is not greater than 500 nm.

5. The method for preparing the hydrogel composition according to any one of claims 2 to 4, characterized in that: The steps include: (1) Take the formulated amount of type I collagen, carboxymethyl chitosan, and four-arm polyethylene glycol benzaldehyde and add them to PBS buffer respectively, and mix them evenly in a water bath at 30-37°C to obtain type I collagen solution, carboxymethyl chitosan solution, and four-arm polyethylene glycol benzaldehyde solution respectively; (2) The extracellular vesicles of Roseomonas mucilaginosa are dissolved in the type I collagen solution obtained in step (1), and then uniformly mixed with the carboxymethyl chitosan solution and the four-arm polyethylene glycol benzaldehyde solution to obtain a hydrogel composition loaded with extracellular vesicles.

6. The preparation method according to claim 5, characterized in that The extracellular vesicles of Roseomonas mucilaginosa are prepared by the following method: S1: first inoculating the Roseomonas mucilaginosa into R2A medium for activation culture; S2: inoculating the activated Roseomonas mucilaginosa in step S1 into TSB liquid culture medium, collecting the supernatant by centrifugation to obtain a bacterial solution containing extracellular vesicles; S3: Centrifuge the extracellular vesicle-containing bacterial solution collected in S2 at 10,000-12,000 g and 2-4°C for 10-15 min, collect the supernatant, and filter the filtrate through a 0.22 μm filter membrane. S4: The filtrate obtained in S3 was concentrated by ultrafiltration through a 10 kD filter membrane to obtain a high-concentration extracellular vesicle bacterial solution; S5: Centrifuge the high-concentration extracellular vesicle solution at 10,000-20,000 g at 2-4°C for 30-60 min and collect the supernatant. S6: Centrifuge the supernatant obtained in S5 at 100,000-130,000 g and 2-4°C for 60-90 min. The resulting precipitate is the extracellular vesicles of Roseomonas mucilaginosa.

7. The preparation method according to claim 6, characterized in that In step S1, the R2A medium components include: tryptone 0.25~0.5 g / L, acid hydrolyzed casein 0.5~0.8 g / L, yeast 0.5~1.0 g / L, soluble starch 0.5~0.8 g / L, dipotassium hydrogen phosphate 0.3~0.6 g / L, magnesium sulfate 0.1~0.15 g / L, sodium pyruvate 0.3~0.4 g / L, agar 12.0~15.0 g / L, glucose 0.5~0.8 g / L, pH value is 7.2±0.2; the TSB liquid medium components include: tryptone 17.0~20.0 g / L, soy peptone 3.0~5.0 g / L, sodium chloride 5.0~6.0 g / L, dipotassium hydrogen phosphate 2.5~4.5 g / L, glucose 2.5~5.0 g / L, pH value is 7.3± 0.

2.

8. A hydrogel preparation, characterized in that The invention comprises the hydrogel composition according to any one of claims 2 to 4 and a pharmaceutically acceptable carrier.

9. Use of the extracellular vesicles of Roseomonas mucilaginosa according to claim 1, the hydrogel composition according to any one of claims 2 to 4, or the hydrogel preparation according to claim 8 in the preparation of a drug carrier, a drug, or a derivative preparation for treating atopic dermatitis.

10. The use according to claim 9, characterized in that The extracellular vesicles, hydrogel compositions or hydrogel preparations can specifically target type 2 inflammatory factors and reduce the expression level of inflammatory factor mRNA in human immortalized keratinocytes induced by TNF-α and IFN-γ. The inflammatory factors include but are not limited to any one or more combinations of tumor necrosis factor α, interleukin 6, interleukin 4 and thymic stromal lymphopoietin; the extracellular vesicles, hydrogel compositions or hydrogel preparations can alleviate the severity of the skin lesion phenotype, the epidermal thickness of the lesion site, and the mast cell infiltration of the lesion site in the MC903-induced atopic dermatitis mouse model, and reduce the mRNA transcription level of inflammatory factors in the lesion site.

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