Extracellular vesicles of roseburia hominis and hydrogel compositions and uses thereof

Abstract

The application discloses a kind of extracellular vesicles of Roseomonos mucosa origin and its hydrogel composition, the extracellular vesicles are obtained by ultrafiltration concentration and gradient ultracentrifugation to culture bacteria liquid.For compared with original culture liquid or its live bacteria preparation, the treatment efficiency, safety of extracellular vesicle of the application are significantly improved.Based on the vesicle, the application also provides a kind of hydrogel composition loaded with the extracellular vesicle, which is constructed by extracellular vesicle, type I collagen, carboxymethyl chitosan and four-arm polyethylene glycol benzaldehyde by chemical crosslinking method, with good adhesion, self-repairing ability and injectability.The extracellular vesicle, hydrogel composition and hydrogel preparation thereof of the application can be applied to atopic dermatitis as drug or drug carrier, can specifically target type 2 inflammatory factors, alleviate the pathological changes such as skin lesion phenotype, epidermal thickening, mast cell infiltration and inflammatory factor expression increase of MC903 induced atopic dermatitis mouse model.

Description

Technical Field

[0001] This invention relates to the field of bacterial extracellular vesicle application technology, specifically disclosing extracellular vesicles of *Rhodopseudomonas myxobolus*, and further proposing a hydrogel composition loaded with *Rhodopseudomonas myxobolus* extracellular vesicles and its application. Background Technology

[0002] Atopic dermatitis (AD) is a stubborn, chronic, relapsing inflammatory skin disease that severely impacts patients' quality of life. To date, its pathogenesis remains incompletely understood. Studies have shown that multiple factors, including genetic susceptibility, skin barrier dysfunction, immune dysregulation, and abnormal skin microecology, play crucial roles in the occurrence and development of AD. Among these, the type 2 inflammatory response, dominated by Th2 cells (T helper 2 cells), is considered the core pathogenic factor of AD. Type 2 inflammatory cytokines interleukin-4 (IL-4) and thymic stromal lymphopoietin (TSLP) play important roles in the occurrence and development of AD. Currently, clinical treatment for AD primarily uses topical corticosteroids and calcineurin inhibitors as first-line drugs. For moderate to severe cases, immunosuppressants or targeted therapy are often required in combination. Although these drugs show significant short-term effects, their efficacy is limited, and long-term use can induce serious toxic 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 Alzheimer's disease (AD), mainly manifested as the dominant colonization of pathogenic Staphylococcus aureus, accompanied by *Rosemonas myxoma* (…). Roseomonasmucosa, R.mucosa The decline in the diversity and quantity of skin symbiotic bacteria is exemplified by *Rosemonas mucosa*. Some studies have shown that transplanting *Rosemonas mucosa* to lesions in Alzheimer's disease (AD) patients can improve the skin's microecological balance, thereby alleviating AD. However, clinical trial results show that the colonization efficiency of the strains on patients' skin is low, seriously affecting its efficacy. In addition, the preparation and storage of live bacterial preparations require high technical skills and may pose potential infection risks, further limiting their clinical application.

[0004] Extracellular vesicles (EVs) are nanoscale vesicles secreted by symbiotic bacteria, containing various bioactive substances such as proteins, lipids, and nucleic acids. Symbiotic EVs are characterized by structural stability, rich functionality, and long circulation time, playing a crucial role in physiological and pathological processes such as host immune regulation, cell metabolism, angiogenesis, and barrier repair. Therefore, in-depth exploration of the therapeutic effects of EVs derived from the dermal symbiotic bacterium *Roseomonas mucinus* (RS EVs) on Alzheimer's disease (AD) has significant scientific value. However, the absorption efficiency of direct administration of extracellular vesicles is often limited, and the duration of action is short, severely affecting efficacy. Therefore, developing efficient extracellular vesicle delivery systems is crucial to improving the transdermal absorption efficiency of EVs and prolonging their duration of action. Hydrogels, with structures similar to the extracellular matrix, possess rich networks and porous structures, as well as strong water retention and moisturizing capabilities. Some hydrogel components also have natural antibacterial and antioxidant properties, making them an ideal topical preparation for dermatological diseases. Given the characteristics of Alzheimer's disease, developing a hydrogel topical formulation that can efficiently deliver RS ​​EVs is of great significance for overcoming the bottleneck in the treatment of Alzheimer's disease with dermal commensal bacteria. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide *Roseomonas myxobolus* extracellular vesicles, which are prepared by ultrafiltration concentration and gradient ultracentrifugation of a culture broth containing *Roseomonas myxobolus* (CGMCC No. 25967). The present invention further proposes a hydrogel composition loaded with *Roseomonas myxobolus* extracellular vesicles, which is a highly adhesive, injectable, self-healing hydrogel composition that enables continuous and efficient delivery of *Roseomonas myxobolus* extracellular vesicles, thereby improving the treatment efficiency of Alzheimer's disease (AD).

[0006] To achieve the above-mentioned technical objectives, the present invention provides extracellular vesicles of *Rhodopseudomonas myxobolus*, which are prepared by ultrafiltration concentration and gradient ultracentrifugation of the culture broth of *Rhodopseudomonas myxobolus* with accession number CGMCC No. 25967.

[0007] The present invention further proposes a hydrogel composition for loading extracellular vesicles of Rosomonas mucinus, the hydrogel composition comprising a hydrogel and extracellular vesicles of Rosomonas mucinus loaded in the hydrogel, the hydrogel being formed by chemical cross-linking of type I collagen, carboxymethyl chitosan and tetra-arm polyethylene glycol benzaldehyde.

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

[0009] The final concentration of the *Rosemonas* extracellular vesicles in the hydrogel composition was 1 × 10⁻⁶. 8 Particle count / mL ~1×10 10 Particle count / mL. The particle count refers to the number of extracellular vesicle particles.

[0010] The extracellular vesicles of *Rhodopseudomonas mucilaginosa* have a particle size of no more than 500 nm, preferably 50-300 nm.

[0011] The present invention further proposes a method for preparing the above-mentioned hydrogel composition, comprising the following steps:

[0012] (1) Take the prescribed amount of type I collagen, carboxymethyl chitosan and tetra-armed polyethylene glycol benzaldehyde and add them to PBS buffer. Mix them evenly in a water bath at 30~37℃ to obtain type I collagen solution, carboxymethyl chitosan solution and tetra-armed polyethylene glycol benzaldehyde solution respectively.

[0013] (2) Dissolve the extracellular vesicles of *Rhodopseudomonas mucilaginosa* in the type I collagen solution obtained in step (1), and then mix them evenly with carboxymethyl chitosan solution and tetra-arm polyethylene glycol benzaldehyde solution to obtain a hydrogel composition loaded with extracellular vesicles.

[0014] Specifically, the extracellular vesicles of *Rhodopseudomonas mucilaginosa* are prepared by the following method:

[0015] S1: First, the *Rhodopseudomonas myxobolus* is inoculated into R2A medium for activation culture. Preferably, the culture conditions are constant temperature culture at 30-32 ℃ for 20-24 h. Then, a single *Rhodopseudomonas myxobolus* colony DL-1 is picked and inoculated into TSB liquid medium at 180-200 rpm for 30-32 ℃ for 12-18 h to obtain activated *Rhodopseudomonas myxobolus* bacterial culture.

[0016] S2: The activated *Rhodopseudomonas myxobolus* from step S1 is inoculated into TSB liquid medium and cultured. The supernatant is collected by centrifugation to obtain a bacterial culture containing extracellular vesicles. Preferably, the activated *Rhodopseudomonas myxobolus* is inoculated into TSB liquid medium and cultured continuously on a constant temperature shaker at 30-32 ℃ and 180-200 rpm for 12-18 h. After centrifugation at 2000-2500 g for 15-20 min, the supernatant is collected to obtain a bacterial culture containing extracellular vesicles.

[0017] S3: Centrifuge the bacterial culture containing extracellular vesicles collected in S2 at 10000~12000 g at 2~4 ℃ for 10~15 min, collect the supernatant and filter it through a 0.22 μm filter membrane to further remove bacterial cells and large particulate impurities, and collect the filtrate;

[0018] S4: The filtrate obtained in S3 was concentrated by ultrafiltration through a 10 kD membrane to obtain a high-concentration extracellular vesicle bacterial culture;

[0019] S5: Centrifuge the high concentration of extracellular vesicle bacterial culture at 10,000-20,000 g at 2-4 ℃ for 30-60 min and collect the supernatant;

[0020] S6: Centrifuge the supernatant obtained from S5 at 100,000~130,000 g at 2~4 ℃ for 60~90 min. The resulting precipitate is the extracellular vesicle of Rosomonas myxobolus.

[0021] Preferably, in step S1, the R2A culture medium comprises: 0.25-0.5 g / L tryptone, 0.5-0.8 g / L acid-hydrolyzed casein, 0.5-1.0 g / L yeast, 0.5-0.8 g / L soluble starch, 0.3-0.6 g / L dipotassium hydrogen phosphate, 0.1-0.15 g / L magnesium sulfate, 0.3-0.4 g / L sodium pyruvate, 12.0-15.0 g / L agar, 0.5-0.8 g / L glucose, and a pH of 7.2 ± 0.2; the TSB liquid culture medium comprises: 17.0-20.0 g / L tryptone, 3.0-5.0 g / L soybean peptone, 5.0-6.0 g / L sodium chloride, 2.5-4.5 g / L dipotassium hydrogen phosphate, 2.5-5.0 g / L glucose, and a pH of 7.3 ± 0.2. 0.2.

[0022] The present invention also discloses a hydrogel formulation comprising the above-described hydrogel composition and a pharmaceutically acceptable carrier.

[0023] The present invention further proposes the use of the above-mentioned hydrogel composition or hydrogel formulation in the preparation of drug carriers, drugs or derivatives for the treatment of atopic dermatitis.

[0024] The hydrogel composition or hydrogel formulation specifically targets type 2 inflammatory factors and reduces the expression levels of tumor necrosis factor α and interferon-γ-induced human immortalized keratinocyte inflammatory factor mRNA. 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.

[0025] The hydrogel composition or hydrogel formulation alleviates the severity of skin lesion phenotype, epidermal thickness at the lesion site, and mast cell infiltration at the lesion site in a mouse model of MC903-induced atopic dermatitis, and reduces the mRNA transcription level of inflammatory factors at the lesion site.

[0026] Specifically, the extracellular vesicles can selectively inhibit the transcription of pro-inflammatory factor mRNAs in human immortalized keratinocytes (HaCat) induced by tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ).

[0027] The extracellular vesicles exhibited superior anti-inflammatory effects compared to the supernatant of *Rosemonas mucinus* culture. Under the same conditions, the extracellular vesicles showed a stronger ability to inhibit TNF-α and IFN-γ-induced transcription of pro-inflammatory factor mRNAs in HaCat cells compared to *Rosemonas mucinus* culture medium.

[0028] The extracellular vesicles exhibit superior anti-inflammatory efficacy and safety compared to the live Rosomonas myxoma preparation. Compared to the live Rosomonas myxoma preparation, the extracellular vesicles demonstrate a stronger ability to inhibit TNF-α and IFN-γ-induced transcription of pro-inflammatory cytokine mRNAs in HaCat cells under the same conditions, without inducing the transcription of the corresponding pro-inflammatory cytokine mRNAs.

[0029] Preferably, the pro-inflammatory factors include any one or more combinations of TNF-α, IL-6, IL-4, and TSLP.

[0030] Specifically, the topical hydrogel formulation loaded with *Rhodopseudomonas mucinus* extracellular vesicles can repair the skin barrier damage at the site of skin lesions in an AD mouse model induced by calcipotriol (MC903).

[0031] Furthermore, the topical hydrogel formulation loaded with *Rosamol* extracellular vesicles can alleviate the disease phenotype of MC903-induced AD mouse model, including but not limited to reducing the severity of skin lesions, reducing the thickness of the epidermis at the lesion site, and inhibiting the differentiation of Th2 cells and the secretion of type 2 inflammatory cytokine IL-4.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0033] (1) The present invention provides an extracellular vesicle of the skin symbiotic bacterium Roseola myxobolus, which has a better type 2 inflammation inhibition efficiency than the live bacterial preparation of Roseola myxobolus and the culture supernatant, and does not cause the expression of pro-inflammatory factors, and has higher safety. It is a novel nanoscale substance with biological activity.

[0034] (2) The present invention further provides a topical hydrogel formulation loaded with extracellular vesicles of Rosomonas mucinus. The hydrogel has high adhesion, injectability and self-repair ability. When applied to the AD lesion area, 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 Rosomonas mucinus, enhance the inhibition of type 2 helper T cell (Th2) differentiation and type 2 inflammatory cytokine IL-4 expression, and has the potential to develop new AD treatment agents. Attached Figure Description

[0035] Figure 1 Characterization results of extracellular vesicles of *Rosamys simplex* (A. Transmission electron microscopy image; B. Particle size and concentration distribution map);

[0036] Figure 2 For different concentrations (0~1×10) 10 The toxic effects of extracellular vesicles of *Rosamolulus mucinus* (particle count / ml) on HaCaT cells and mouse mononuclear macrophages cells (Raw264.7) (A. HaCaT cells; B. Raw264.7 cells).

[0037] Figure 3 For different concentrations (1×10) 8 ~1×10 10 Effects of extracellular vesicles (RS EVs) of *Rosamolulus mucilaginosus* on the mRNA transcription levels of pro-inflammatory factors induced by TNF-α and IFN-γ in HaCaT cells (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).

[0038] Figure 4 The effects of equal volumes of *Roseoma myxobolus* culture supernatant (Sup.), *Roseoma myxobolus* extracellular vesicles (EVs), and *Roseoma myxobolus* live bacterial preparation (RS) on the mRNA transcription levels of TNF-α and IFN-γ-induced pro-inflammatory factors in HaCat cells (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).

[0039] Figure 5 A schematic diagram of the cross-linking pattern of a hydrogel loaded with mucus rose extracellular vesicles;

[0040] Figure 6Scanning electron microscope images of hydrogels prepared with different mass ratios of components;

[0041] Figure 7 Scanning electron microscope image of a hydrogel loaded with extracellular vesicles of Rosomonas mucinus;

[0042] Figure 8 Characterization of the physical properties of hydrogels loaded with extracellular vesicles of Rosomonas mucinus (A. self-healing function of hydrogels; B. adhesiveness of hydrogels; C. plasticity of hydrogels).

[0043] Figure 9 To construct the MC903-induced AD model, the clinical phenotype of skin lesions in AD mice after treatment and changes in ear thickness were shown in the diagram (A. Processing flowchart; B. Changes in ear thickness before and after treatment; C. Clinical phenotype of skin lesions after treatment).

[0044] Figure 10 The effect of hydrogels loaded with extracellular vesicles of Rosomonas mucinus on pathological changes in skin lesions in MC903-induced AD mice;

[0045] Figure 11 The effect of hydrogels loaded with extracellular vesicles of Rosomonas mucinus on the mRNA expression level of filaggrin, a skin barrier-associated protein, in skin lesions of MC903-induced AD mice;

[0046] Figure 12 Hydrogels loaded with *Rosamolaria mucosa* extracellular vesicles were used to target Th2 cell subsets (IL4) in the spleen of MC903-induced AD mice. + CD4 + The effects of T cells (A. flow cytometry gating strategy; B. CD4) + IL4 + Th2 lymphocyte subsets in CD4 + CD8 - (Percentage of T cells). Detailed Implementation

[0047] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0048] The *Rhodopseudomonas mucilaginosa* used in the following examples has the accession number CGMCC No. 25967, and its detailed information is disclosed in CN 116103205B.

[0049] Example 1: Extraction and characterization of extracellular vesicles of *Rhodopseudomonas myxobolus*.

[0050] 1) Activation and culture of *Roseomonas myxobolus*

[0051] *Rosemonas myxomonas*, frozen at -80 ℃ with glycerol, was inoculated onto R2A medium and activated in a 32 ℃ incubator for 24 h. Then, a single *Rosemonas myxomonas* colony was picked and inoculated into TSB liquid medium at 200 rpm for 16 h to obtain an activated seed suspension. This seed suspension was then inoculated again into TSB liquid medium at a 5% (v / v) ratio and cultured at 32 ℃ for another 16 h at 200 rpm to obtain the target bacterial suspension. The target bacterial suspension concentration reached 5 × 10⁻⁶. 9 CFU / mL or higher.

[0052] 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 of 7.2 ± 0.2.

[0053] The TSB liquid culture medium consists of: 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH of 7.3 ± 0.2.

[0054] 2) Extraction of extracellular vesicles from *Rosamolulus myxobolus*

[0055] The *Rhodopseudomonas myxobolus* bacterial suspension 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. Then, the supernatant was filtered through a 0.22 μm filter membrane to further remove bacterial cell components from the sample. The resulting filtrate was concentrated by ultrafiltration using a 10 kD ultrafiltration centrifuge tube. Subsequently, the bacterial suspension was centrifuged at 20000 g and 4 °C for 60 min in an ultracentrifuge. The supernatant was collected and centrifuged again at 120000 g and 4 °C for 75 min. The precipitate obtained was the *Rhodopseudomonas myxobolus* extracellular vesicles. After resuspending in sterile PBS, it was stored at -80 °C.

[0056] 3) Transmission electron microscopy observation of extracellular vesicles of *Roseomonas myxobolus*

[0057] Take 15 μL of the extracellular vesicles obtained in step 2 and place them on a copper mesh (ordinary carbon support membrane) for 1 min. Blot dry with filter paper. Then, take 15 μL of 2% uranium acetate staining solution and stain at room temperature for 1 min. Blot dry the copper mesh with filter paper. After staining, place the sample under a lamp for 10 min and observe and photograph it under a transmission electron microscope. The morphology is as follows: Figure 1As shown in Figure A, the extracellular vesicles of this *Roseomonas mucilaginosus* are round and "cup-and-plate" shaped.

[0058] 4) Analysis of extracellular vesicle nanoparticles of *Roseomonas myxobolus*

[0059] The sample cell was cleaned with deionized water, and the instrument (NanoSight NS300) was calibrated using polystyrene microspheres (100 nm). The extracellular vesicle samples obtained in step 2) were diluted 5000-fold with PBS before injection for detection. Three consecutive test results were collected for each sample. Specific results are shown below. Figure 1 As shown in B, the extracellular vesicle size distribution of *Roseomonas myxomonas* ranges from 0 to 500 nm, and is not zero. The original concentration was measured to be 1.14 × 10⁻⁶. 12 Particle count / mL.

[0060] Example 2 Evaluation of the cytotoxic effects of extracellular vesicles of *Rhodopseudomonas myxobolus* on HaCaT and Raw264.7 cells.

[0061] 1) HaCaT cells were cultured at a rate of 1 × 10⁻⁶ 4 Inoculate one cell per well into a 96-well plate containing 1 mL of culture medium, and use a final concentration of 1 × 10⁻⁶. 4 ~ 1 × 10 10 Extracellular vesicles were treated with a concentration of vesicles per mL. A negative control group was prepared with sterile PBS solution, and a blank control group was prepared with DMEM medium (containing 10% FBS) containing no cells or extracellular vesicles. Each concentration was tested in six replicates. Culture dishes were incubated at 37°C with 5% CO2 for 48 h. 10 μL of CCK8 solution was added to each well, and the culture dishes were incubated at 37°C with 5% CO2 for another 1–4 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Results are shown below. Figure 2 As shown in Figure A, different concentrations of *Roseomonas myxomonas* extracellular vesicles had no significant inhibitory effect on HaCaT cell viability, suggesting that *Roseomonas myxomonas* extracellular vesicles at concentrations of 1 × 10⁻⁶ have a relatively high inhibitory effect on HaCaT cell viability. 4 ~ 1 × 10 10 Within the particle number / mL concentration range, it has little or no toxicity to cells.

[0062] 2) Raw264.7 cells were cultured at a rate of 5 × 10⁻⁶. 4 Inoculate one cell per well into a 96-well plate containing 1 mL of culture medium, and use a final concentration of 1 × 10⁻⁶. 4 Up to 1 × 10 10Extracellular vesicles were treated with cell counts / mL, with sterile PBS as the negative control and DMEM medium (containing 10% FBS) as the blank control. Six replicates were set up for each concentration. The culture dishes were incubated at 37°C with 5% CO2 for 48 h. 10 μL of CCK8 solution was added to each well, and the culture dishes were incubated at 37°C with 5% CO2 for another 1–4 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Results are shown below. Figure 2 As shown in Figure B, different concentrations of *Roseola myxomonas* extracellular vesicles had no significant inhibitory effect on the viability of RAW264.7 cells, indicating that the extracellular vesicles of *Roseola myxomonas* at concentrations of 1 × 10⁻⁶ cells have a relatively high inhibitory effect on the viability of RAW264.7 cells. 4 ~ 1 × 10 10 Within the particle number / mL concentration range, it has little or no toxicity to cells.

[0063] Example 3: Effects of extracellular vesicles of *Roseola myxobolus* on the transcriptional levels of pro-inflammatory factors in HaCat cells induced by TNF-α and IFN-γ.

[0064] 1) HaCaT cells were cultured at a rate of 1 × 10⁻⁶ 5 Seeds were inoculated per well in 12-well plates and cultured overnight in DMEM medium containing 10% FBS at 37 °C with 5% CO2. Subsequently, the culture was inoculated with a final concentration of 1 × 10⁻⁶ cells / well. 8 Particle count / mL, 1 × 10 9 Particle count / mL and 1 × 10 10 Extracellular vesicles were pretreated for 4 h with a particle count / mL, followed by stimulation with TNF-α and IFN-γ at a final concentration of 10 ng / mL. The experimental control group was the group pretreated with PBS solution and stimulated with TNF-α and IFN-γ solution, while the blank control group was the group pretreated with PBS solution and stimulated with TNF-α and IFN-γ solution. Each group was set up with 6 replicates. After continuous stimulation for 24 h, cells were collected for total RNA extraction.

[0065] 2) Total RNA was extracted from the collected cells using the TRIZOL kit. RNA concentration and purity were measured using a NanoDrop spectrophotometer. Total RNA was reverse transcribed into cDNA using a reverse transcription reagent. 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 control. The relative expression levels of each cytokine mRNA were determined according to 2... -ΔΔCt Calculations were performed. The primer sequences for the specific cytokines to be tested and β-actin are shown in Table 1.

[0066] Table 1 Sequences of the cytokines to be tested and the internal reference primers

[0067]

[0068] 3) The effect of extracellular vesicles of *Rosemonas myxomonas* on the transcriptional levels of pro-inflammatory factors induced by TNF-α and IFN-γ in HaCaT cells, such as... Figure 3 As shown, ① compared with the blank control group, stimulation with 10 ng / mL TNF-α and 10 ng / mL IFN-γ both increased the transcription levels of pro-inflammatory factors 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, stimulation with higher concentrations (1 × 10⁻⁶ ng / mL IFN-γ) increased the transcription levels of pro-inflammatory factors 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; 9 Particle count / mL and 1 × 10 10 Pretreatment of HaCat cells with extracellular vesicles of *Rosamolica mucilaginosa* (particle count / mL) significantly inhibited the increase in transcription levels of pro-inflammatory factors such as TNF-α, IL-6, IL-4, and TSLP in an AD cell model, with statistically significant differences between groups. Specifically, 1 × 10⁻⁶ granules / mL significantly reduced the pretreatment level. 9 The treatment group with a particle count / mL concentration showed better inhibitory effects, but a lower concentration (1 × 10⁻⁶) was used. 8 Pretreatment with extracellular vesicles (number of particles / mL) significantly inhibited TNF-α transcription levels 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, with no statistically significant differences between groups.

[0069] Example 4: Effects of culture supernatant of *Roseoma myxobolus* (Sup.), extracellular vesicles (RSEVs) of *Roseoma myxobolus*, and live bacterial preparation of *Roseoma myxobolus* (RS) on the mRNA transcription levels of pro-inflammatory factors in HaCat cells induced by TNF-α and IFN-γ.

[0070] 1) HaCaT cells were cultured at a rate of 1 × 10⁻⁶ 5Cells were seeded per well in 12-well plates and cultured overnight in DMEM medium containing 10% FBS at 37°C and 5% CO2. Cells were then pretreated for 4 h with equal volumes of *Rhodopseudomonas myxobolus* culture supernatant, *Rhodopseudomonas myxobolus* extracellular vesicles, and *Rhodopseudomonas myxobolus* live bacterial preparation, respectively. Following this, cells were stimulated with TNF-α and IFN-γ at final concentrations of 10 ng / mL and 10 ng / mL, respectively. The control group consisted of cells pretreated with TSB medium and stimulated with TNF-α and IFN-γ, while the blank control group consisted of cells pretreated with TSB medium and stimulated with TSB medium. Each group had 6 replicates. After 24 h of continuous stimulation, cells were collected for total RNA extraction. The total RNA extraction procedure is described in Example 3, step 2).

[0071] 2) Effects of supernatant (Sup.), extracellular vesicles (RS EVs), and live bacterial preparations (RS) on the transcriptional levels of pro-inflammatory factors induced by TNF-α and IFN-γ in HaCaT cells, such as... Figure 4 As shown, ① compared with the blank control group, stimulation with 10 ng / mL TNF-α and 10 ng / mL IFN-γ could increase the transcription levels of pro-inflammatory factors 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 could significantly inhibit the increase in transcription levels of pro-inflammatory factors such as 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 treatment group also significantly inhibited the increase in IL-6 transcription levels, and the inhibitory effect on the transcription levels of pro-inflammatory factors such as TNF-α, IL-4 and TSLP was significantly enhanced compared with that of the supernatant; ③ compared with the extracellular vesicle treatment group, pretreatment with live bacteria preparations significantly upregulated the expression of pro-inflammatory factors TNF-α, IL-6, IL-4 and TSLP, suggesting a potential risk of infection.

[0072] Example 5: Exploration and characterization of hydrogel preparation conditions.

[0073] 1) Hydrogel preparation: Take 0.6g, 0.3g, and 0.3g of type I collagen, carboxymethyl chitosan, and tetra-armed polyethylene glycol benzaldehyde, respectively, and add them to 10mL of PBS buffer. Dissolve them in a water bath at 30~37℃ for 15min to prepare collagen solution, carboxymethyl chitosan solution, and tetra-armed polyethylene glycol benzaldehyde solution. Mix them evenly in the following mass ratios: 6:1:3, 6:2:3, and 6:3:3 to obtain hydrogel a, hydrogel b, and hydrogel c. Figure 5 A schematic diagram illustrating the chemical cross-linking process of a hydrogel loaded with mucus rose extracellular vesicles.

[0074] 2) The microstructure was observed using scanning electron microscopy (SEM), and the resulting SEM images of the hydrogel are shown below. Figure 6 As shown in the figure. The results showed that hydrogels a, b, and c were all porous structures, with hydrogel a having the largest pore size, followed by hydrogel b, and hydrogel c having the smallest pore size, suggesting that hydrogel c has a better sustained-release effect to achieve continuous release of extracellular vesicles.

[0075] Example 6: Preparation, characterization and physical properties of hydrogels loaded with extracellular vesicles of Rosomonas mucinus.

[0076] 1) Preparation of hydrogel loaded with *Rhodotorula mucosae* extracellular vesicles: *Rhodotorula mucosae* extracellular vesicles were dissolved in the type I collagen solution obtained in step 1) of Example 5, and then uniformly mixed with carboxymethyl chitosan solution and tetra-arm polyethylene glycol benzaldehyde solution at a mass ratio of 6:3:3 to obtain the hydrogel composition loaded with extracellular vesicles. The final concentration of *Rhodotorula mucosae* extracellular vesicles was 1×10⁻⁶. 9 Particle count / ml.

[0077] 2) The microstructure was observed using scanning electron microscopy (SEM), and the resulting SEM images of the hydrogel are shown below. Figure 7 As shown in the figure. The results showed that the hydrogel loaded with extracellular vesicles of *Rhodopseudomonas myxobolus* had a porous structure with a pore size of approximately 20–100 μm, and could effectively load *Rhodopseudomonas myxobolus*.

[0078] 3) The hydrogel loaded with extracellular vesicles of *Basilosoma mucilaginosa* exhibits self-healing ability, good adhesion, and injectability: A "maple leaf" shaped hydrogel was prepared using a rubber mold, and then two cut hydrogels were brought together again. Figure 8 (A) shows that the hydrogel can self-repair and heal over time, demonstrating a good self-healing effect; the adhesiveness of the hydrogel was tested using 10ml centrifuge tubes containing different volumes of liquid. Figure 8 (B) This invention demonstrates that the hydrogel can stably adhere to 10ml centrifuge tubes containing different volumes of liquid, exhibiting good adhesion; a certain volume of hydrogel solution was drawn using a 1ml syringe, and the letter "A" was drawn. 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;

[0079] Example 6: The therapeutic effect of hydrogel loaded with *Rhodopseudomonas mucinus* extracellular vesicles on an MC903-induced AD mouse model.

[0080] 1) Construction and intervention of animal models: such as Figure 9As shown, 6-8 week old C57BL / 6 or Balb / c mice with relatively uniform body weight were randomly divided into 5 groups: AD modeling group, positive drug treatment group, extracellular vesicle treatment group, hydrogel treatment group, and *Rhodopseudomonas mucinosa*-loaded extracellular vesicle treatment group. Each group had 5 biological replicates. Specific treatment methods are as follows (e.g., ...). Figure 9 A):

[0081] AD modeling 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.

[0082] Positive drug 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 an appropriate amount of hydrocortisone butyrate cream was evenly applied to each ear at 5:00 pm every day for 17 consecutive days.

[0083] Extracellular vesicle 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 extracellular vesicles (1 × 10⁻⁶) were evenly applied to each ear at 5:00 PM every day. 9 Particle count / ml, 20μl volume / ear), treated continuously for 17 days.

[0084] 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 5:00 pm every day for 17 consecutive days.

[0085] Hydrogel treatment group loaded with *Rhodopseudomonas mucinosa* extracellular vesicles: 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 loaded with *Rhodopseudomonas mucinosa* extracellular vesicles (1 × 10⁻⁶) was evenly applied to each ear at 5:00 PM every day. 9 Particle count / ml, 20μl volume / ear), treated continuously for 17 days.

[0086] 2) Measurement of mouse ear thickness: The thickness of both mouse ears was precisely measured using calipers at the beginning of modeling (D1) and on days 4 (D4), 7 (D7), 10 (D10), 13 (D13), 15 (D15), and 17 (D17) after modeling. The average value of both ears was used to represent the degree of ear swelling. The results are as follows: Figure 9As shown in B, at the observation endpoint (D17), the ears of the MC903 model group were significantly thickened. The positive drug treatment group treated with topical hydrocortisone butyrate cream, the extracellular vesicle treatment group, the hydrogel treatment group, and the hydrogel treatment group loaded with extracellular vesicles could all significantly alleviate the thickening of the ears of mice induced by MC903, and all showed statistical differences. Among them, the hydrogel treatment group loaded with extracellular vesicles had the best effect.

[0087] 3) Clinical phenotype analysis of mouse skin lesions: At the observation endpoint, photographs were taken of the mouse skin lesion sites, and the results are as follows: Figure 9 As shown in Figure C, the AD model group mice developed erythema and swelling in their ears, with obvious scaling on the surface. The topical hydrocortisone butyrate cream positive drug treatment group, extracellular vesicle treatment group, hydrogel treatment group, and extracellular vesicle-loaded hydrogel treatment group all significantly improved MC903-induced erythema, swelling, and scaling in the mouse ears, with the extracellular vesicle-loaded hydrogel treatment group showing the best effect.

[0088] 4) Pathological changes in mouse skin lesions: Mice were euthanized at the end of the observation period, and ear specimens were obtained. These specimens were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The staining method was as follows: ① Paraffin sections were baked in an oven at 60 ℃ for 2 h; ② Paraffin sections were dewaxed to water using xylene and ethanol as usual; ③ Hematoxylin was stained for 10 min, followed by rinsing with running water to remove residual stain; ④ 0.5% hydrochloric acid and ethanol were used for color separation for 1-2 s, followed by rinsing with running water; ⑤ 1% ammonia was used for blue reversion, followed by rinsing with running water and examination of the degree of color separation of cell nuclei under a fiber microscope; ⑥ 1% eosin was stained for 30 s, followed by rapid rinsing with distilled water; ⑦ Rapid rinsing with 80%, 90%, and 95% ethanol solutions for 10 s each, with microscopic monitoring of the color contrast between cell nuclei and cytoplasm; ⑧ Washing twice with 100% ethanol for 1-2 min each time; washing twice with xylene for 1-2 min each time; ⑨ Mounting with neutral resin and observing the staining results under a microscope. Results are as follows: Figure 10 As shown, the epidermis of mice in the AD model group was significantly thickened and a large number of inflammatory cells were infiltrated in the dermis. The hydrogel treatment group and the hydrogel treatment group loaded with extracellular vesicles, both treated with topical hydrocortisone butyrate cream, could improve the epidermal thickening and dermal inflammatory cell infiltration induced by MC903 in mice. Among them, the hydrogel treatment group loaded with extracellular vesicles showed the most significant improvement.

[0089] 5) Analysis of transcriptional levels of skin barrier-related proteins (filaggrin) in mouse skin lesions: Fresh ear specimens obtained in step 4) were placed in sterile, enzyme-free grinding tubes, and grinding beads were added to grind the tissue. Total RNA was then extracted from the lesion tissue using a TRIZOL kit, and reverse transcribed into cDNA using a reverse transcription kit. The mRNA transcriptional level of the target gene was detected using a SYBR Green kit on an ABI 7900 real-time quantitative PCR instrument, with β-actin as an internal control. The relative expression level of the target gene mRNA was determined according to 2... -ΔΔCt The calculated results are as follows: Figure 11 As shown in the figure, compared with the transcription level of filaggrin gene in the skin lesions of AD model mice, the transcription levels of filaggrin gene were significantly increased in the positive drug treatment group (hydrocortisone butyrate cream), the hydrogel treatment group, and the hydrogel treatment group loaded with extracellular vesicles. Among them, the transcription level of filaggrin gene was most significantly increased in the hydrogel treatment group loaded with extracellular vesicles, while there was no statistically significant difference between the treatment group using only *Rhodopseudomonas mucinosa* extracellular vesicles and the AD model group.

[0090] 6) Mouse spleen CD4 + CD8 - Flow cytometry analysis of Th2 lymphocyte subsets in T cells: Fresh spleen tissue was obtained from mice euthanized in step 4), and gently ground into a single-cell suspension. The cell pellet was collected after centrifugation. The cell pellet was resuspended in erythrocyte lysis buffer and allowed to lyse the erythrocytes. After 3 min, an appropriate amount of PBS was added to terminate lysis, and the mixture was filtered through a 70 μM filter. The cell pellet was then collected by centrifugation. The cells were resuspended in a specific volume of RPMI 1640 medium containing 10% FBS and counted using a cell counter. The cell concentration was adjusted to 5 × 10⁻⁶ cells / year. 7 Cells / mL, 100 μL of cell suspension was transferred to a flow cytometry tube. Cell stimulation and intracellular / extracellular antigen staining were performed according to flow cytometry procedures before detection. Specific flow cytometry antibodies are shown in the figure below, and the flow cytometry gating strategy is described in [link to flow cytometry]. Figure 12 A, the flow cytometry analysis results are as follows: Figure 12 As shown in B. Compared with the AD model group, the treatment group using hydrocortisone butyrate cream, the extracellular vesicle treatment group, and the hydrogel loaded with extracellular vesicles all significantly reduced the proportion of Th2 lymphocyte subsets in the spleen of the MC903-induced AD mouse model. Among them, the reduction was most significant with the hydrogel loaded with extracellular vesicles, while there was no statistically significant difference between the hydrogel treatment group and the AD model group.

[0091] In summary, this invention provides a topical hydrogel formulation loaded with *Roseomonas myxoma* extracellular vesicles, its preparation method, and its application. Specifically, the extracellular vesicles of *Roseomonas myxoma* can inhibit the increase in transcriptional levels of pro-inflammatory factors such as TNF-α, IL-6, IL-4, and TSLP induced by TNF-α and IFN-γ in HaCaT cells, particularly specifically reducing the expression of type 2 inflammatory cytokines TSLP and IL-4, making the treatment of Alzheimer's disease (AD) more targeted and precise. Furthermore, its inhibitory efficiency and safety are significantly higher than those of *Roseomonas myxoma* DL-1 culture supernatant and live bacterial preparations. Moreover, this invention utilizes three organic components—type I collagen, carboxymethyl chitosan, and tetra-arm polyethylene glycol benzaldehyde—through a chemical double crosslinking process to construct a highly adhesive, injectable, self-healing hydrogel. This hydrogel enables stable and continuous output of *Roseomonas myxoma* extracellular vesicles, enhancing the inhibition of type 2 helper T cell differentiation and type 2 inflammatory cytokine IL-4 expression by RS EVs. Compared with long-term use of glucocorticoids or live bacterial preparations, the hydrogel topical preparation of *Rhodopseudomonas mucinosa* loaded with extracellular vesicles provided by this invention has the potential to be developed into a novel AD treatment.

Claims

1. The use of a hydrogel composition loaded with *Rhodopseudomonas mucinosa* extracellular vesicles in the preparation of a medicament for treating atopic dermatitis, characterized in that, The hydrogel composition comprises a hydrogel and *Rhodopseudomonas myxobolus* extracellular vesicles loaded in the hydrogel. The hydrogel is formed by chemical cross-linking of type I collagen, carboxymethyl chitosan, and tetra-armed polyethylene glycol benzaldehyde. The *Rhodopseudomonas myxobolus* extracellular vesicles are prepared by ultrafiltration concentration and gradient ultracentrifugation of a culture broth of *Rhodopseudomonas myxobolus* with accession number CGMCC No. 25967. The mass ratio of type I collagen, carboxymethyl chitosan, and tetra-armed polyethylene glycol benzaldehyde is 6:(1~3):

3. The particle size of the *Rhodopseudomonas myxobolus* extracellular vesicles is no greater than 500 nm. The extracellular vesicles of *Rhodopseudomonas mucilaginosa* were prepared by the following method: S1: First, inoculate the *Rhodopseudomonas mucilaginosa* in R2A medium for activation culture; S2: Inoculate the activated *Rhodopseudomonas mucilaginosus* from step S1 into TSB liquid medium and culture. Centrifuge to collect the supernatant to obtain a bacterial culture containing extracellular vesicles. S3: Centrifuge the bacterial culture containing extracellular vesicles collected in S2 at 10000~12000 g at 2~4 ℃ for 10~15 min, collect the supernatant and filter it through a 0.22 μm filter membrane to collect the filtrate; S4: The filtrate obtained in S3 is concentrated by ultrafiltration through a 10 kD filter membrane to obtain a high concentration of extracellular vesicle bacterial culture; S5: Centrifuge the high concentration of extracellular vesicle bacterial culture at 10,000-20,000 g at 2-4 ℃ for 30-60 min and collect the supernatant; S6: Centrifuge the supernatant obtained from S5 at 100,000~130,000 g and 2~4 ℃ for 60~90 min. The resulting precipitate is the extracellular vesicle of Rosomonas mucilaginosus. The R2A culture medium comprises: 0.25–0.5 g / L tryptone, 0.5–0.8 g / L acid-hydrolyzed casein, 0.5–1.0 g / L yeast, 0.5–0.8 g / L soluble starch, 0.3–0.6 g / L dipotassium hydrogen phosphate, 0.1–0.15 g / L magnesium sulfate, 0.3–0.4 g / L sodium pyruvate, 12.0–15.0 g / L agar, 0.5–0.8 g / L glucose, and a pH of 7.2 ± 0.2; the TSB liquid culture medium comprises: 17.0–20.0 g / L tryptone, 3.0–5.0 g / L soybean peptone, 5.0–6.0 g / L sodium chloride, 2.5–4.5 g / L dipotassium hydrogen phosphate, 2.5–5.0 g / L glucose, and a pH of 7.3 ± 0.

2. The hydrogel composition loaded with *Rhodopseudomonas mucinus* extracellular vesicles can alleviate the severity of skin lesions, epidermal thickness, and mast cell infiltration in a mouse model of MC903-induced atopic dermatitis, and reduce the mRNA transcription level of inflammatory factors in the skin lesions.

2. The application according to claim 1, characterized in that, The final concentration of the *Rosemonas* extracellular vesicles in the hydrogel composition was 1 × 10⁻⁶. 8 Particle count / mL ~1×10 10 Particle count / mL.

3. The application according to claim 1, characterized in that, The preparation method of the hydrogel composition includes the following steps: (1) Take the prescribed amount of type I collagen, carboxymethyl chitosan and tetra-armed polyethylene glycol benzaldehyde and add them to PBS buffer. Mix them evenly in a water bath at 30~37℃ to obtain type I collagen solution, carboxymethyl chitosan solution and tetra-armed polyethylene glycol benzaldehyde solution respectively. (2) Dissolve the extracellular vesicles of *Rhodopseudomonas mucilaginosa* in the type I collagen solution obtained in step (1), and then mix them evenly with carboxymethyl chitosan solution and tetra-arm polyethylene glycol benzaldehyde solution to obtain a hydrogel composition loaded with extracellular vesicles.

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