Cross-linked cell membrane mimicking skin structure and function hydrogel, and preparation method and application thereof

Abstract

The application discloses a cross-linked cell membrane skin-imitating structure and function hydrogel, and a preparation method and application thereof. The cross-linked cell membrane skin-imitating structure and function hydrogel is prepared by using an olefin double bond modified extracellular vesicle as a cross-linking agent, using acrylamide as a monomer, and generating a cross-linked network based on a cell membrane through a free radical polymerization reaction between acrylamide and the olefin double bond on the extracellular vesicle, so as to obtain a polyacrylamide biomimetic hydrogel cross-linked by the cell membrane. The biomimetic hydrogel has antibacterial effect and shows strong ability to induce maturation and activation of dendritic cells. In the application, a copper-free catalytic click reaction between the alkyne double-end PEG and the azide modified extracellular vesicle is used to realize construction of a double cross-linked network of the biomimetic hydrogel, and the biomimetic hydrogel with adjustable structure and function is prepared. Therefore, the biomimetic hydrogel and the preparation method thereof have important value and good application prospect.

Description

Technical Field

[0001] This invention relates to a cross-linked cell membrane skin-like structure and function hydrogel, its preparation method and application, belonging to the field of biomimetic biomaterials technology. Background Technology

[0002] As the largest human organ with high mechanical strength, strong antibacterial capabilities, and immune capacity, the skin is in direct contact with the external environment and serves as a crucial physical and immune barrier protecting the body from various aggressors. There is an urgent need for methods that can mimic the structure or function of the skin to prepare promising materials applicable to tissue regeneration, wearable devices, soft robotics, health monitoring, and intelligent medical diagnostics. To mimic skin function, most research is based on hybridization, primarily by introducing multiple inorganic or conductive nanomaterials into different gel materials, such as hydrogels, organic gels, and ionic liquid gels. To date, relying on this strategy, scientists have developed biomimetic skin materials with numerous powerful functions, such as multimodal sensing capabilities, sweating ability, enhanced mechanical strength, ultrafast self-healing, controllable color change, and stable bioadhesion. To mimic skin structure, previous research has mainly integrated components or layers with different characteristics into a coherent system. For example, to achieve specific skin functions, modules such as gradient pore frameworks, micro-artificial devices, and neural-like nanonetworks have been integrated into matrix materials to mimic sweat glands and sensory receptors in the skin. Despite these remarkable achievements, methods for preparing biomaterials with skin-like functions, especially those with advanced mechanical and biological properties, that can mimic skin structure have been rarely reported.

[0003] Skin comprises a three-layered cellular structure: the epidermis, dermis, and subcutaneous layer, composed of keratinocytes, fibroblasts, and adipocytes, respectively. These cells are embedded in the extracellular matrix (ECM), which consists of multiple layers of lipids, collagen, elastin, and hyaluronic acid. Essentially, skin can be simplified as a composite hydrogel material in which numerous cells are connected via the ECM. The multiple interactions between the isolated cell membrane compartments and their surrounding ECM endow the network with good mechanical strength. Simultaneously, because living cells within the skin can release a variety of bioactive substances, this composite hydrogel material possesses unique biological properties, such as antibacterial and immunomodulatory activities, forming a protective barrier against external invasion. Therefore, given the enormous potential for preparing hydrogel materials that simultaneously mimic the structure and function of skin, synthetic strategies for constructing cell membrane-based networks are extremely attractive. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to obtain a dual biomimetic hydrogel material that can simultaneously simulate the structure and function of skin.

[0005] To address the aforementioned technical problems, this invention provides a cross-linked cell membrane biomimetic hydrogel with a skin-like structure and function. The hydrogel uses extracellular vesicles modified with olefin double bonds as a cross-linking agent and acrylamide as a monomer. A cell membrane-based cross-linking network is generated through a free radical polymerization reaction between acrylamide and the olefin double bonds on the extracellular vesicles, thereby obtaining a polyacrylamide biomimetic hydrogel SFSHs with a cell membrane cross-linking network.

[0006] Preferably, the extracellular vesicles can be of bacterial or mammalian cell origin; the extracellular vesicles can also be replaced by liposomes (artificial vesicles) with a similar structure to extracellular vesicles.

[0007] Preferably, the extracellular vesicles are bacterial extracellular vesicles; the olefin double bond modified extracellular vesicles are obtained by incubating distearate phosphatidylethanolamine-polyethylene glycol-acrylamide (DSPE-PEG-AM) with extracellular vesicles, and inserting them into the vesicle cell membrane through the hydrophilic-hydrophobic interaction of distearate phosphatidylethanolamine-polyethylene glycol-acrylamide, thereby obtaining olefin double bond modified extracellular vesicles OMV-AM.

[0008] Preferably, the hydrogel SFSHs further includes an enhanced cross-linking network, which is formed by cross-linking alkyne-terminated PEG and azide-modified extracellular vesicles through a copper-free catalytic click reaction, wherein the azide-modified extracellular vesicles are prepared by incubating extracellular vesicles with an azide reagent.

[0009] Preferably, the alkyne-terminated PEG is dibenzocyclooctyne-polyethylene glycol-dibenzocyclooctyne (DBCO-PEG-DBCO), and the azidating agent is cholesterol-polyethylene glycol-azide (Chol-PEG-N3).

[0010] Preferably, the extracellular vesicles in the enhanced cross-linking network are derived from Escherichia coli or Staphylococcus epidermidis. In this case, the extracellular vesicles derived from Staphylococcus epidermidis are cross-linked with PEG-terminated alkynes via a copper-free catalytic click reaction after being modified by azide function, forming an enhanced cross-linking network that promotes biofilm formation.

[0011] This invention also provides a method for preparing the above-mentioned cross-linked cell membrane skin-like structure and functional hydrogel, comprising the following steps:

[0012] Step 1: Extract extracellular vesicles from bacteria;

[0013] Step 2: The extracellular vesicles extracted in Step 1 were incubated with distearate-phosphatidylethanolamine-polyethylene glycol-acrylamide (DSPE-PEG-AM) to prepare olefin double bond modified extracellular vesicles OMV-AM;

[0014] Step 3: Using acrylamide as a monomer and the olefin double-bond modified extracellular vesicles obtained in Step 2 as a crosslinking agent, polyacrylamide hydrogels with a cell membrane crosslinking network (SFSHs) are prepared by initiating free radical polymerization between the double bonds on acrylamide and extracellular vesicles under the action of an initiator and a catalyst; or, extracellular vesicles are incubated with an azide reagent to obtain azide-modified extracellular vesicles OMV-N3, and then OMV-N3 is mixed with the olefin double-bond modified extracellular vesicles OMV-AM obtained in Step 2, and then acrylamide, initiator, alkyne double-terminated PEG and catalyst are added in sequence to carry out a polymerization reaction to obtain a double-network skin-like hydrogel containing an enhanced crosslinking network.

[0015] Preferably, the initiator in step 3 is ammonium persulfate (APS), and the catalyst is N,N,N',N'-tetramethylethylenediamine (TEMED).

[0016] Preferably, the extracellular vesicles in step 1 are derived from Escherichia coli.

[0017] Preferably, the extracellular vesicles modified by azidation in step 3 are derived from Escherichia coli or Staphylococcus epidermidis.

[0018] Preferably, in step 2: the incubation temperature is 25–50°C (optimal is 37°C), and the incubation time is 0.5–1 hour (optimal is <1 hour); the concentration of vesicle particles in the extracellular vesicles is 1 × 10⁻⁶. 9 ~3×10 12 The solid content of (DSPE-PEG-AM) is 0.8-8 wt% (most preferably 2 wt%).

[0019] This invention also provides the application of the above-mentioned cross-linked cell membrane skin-like structure and functional hydrogel in the preparation of smart skin biomaterials.

[0020] This invention develops skin-mimicking hydrogels (SFSHs) that are both structurally and functionally biomimetic through cell membrane crosslinking. Figure 1Distearate phosphatidylethanolamine-polyethylene glycol-acrylamide (DSPE-PEG-AM) is inserted into the vesicle cell membrane via hydrophilic-hydrophobic interactions to construct olefin double-bond modified extracellular vesicles. Using these vesicles as crosslinking agents, a cell membrane-based crosslinking network is generated through the free radical polymerization of acrylamide. Compared to typical methylene diacrylamide-crosslinked polyacrylamide hydrogels, the resulting network exhibits significantly increased mechanical strength due to the attachment of multiple polymer chains to the mechanically deformable cell membrane crosslinking agent, i.e., double-bond functionalized vesicles, as the vesicles can dissipate stretching energy through deformation. Since extracellular vesicles contain many bioactive substances inherent in their parent cells, the resulting SFSHs also exhibit specific antibacterial effects and a strong ability to stimulate dendritic cell maturation and activation. This invention further demonstrates the versatility of the method by introducing a second network between alkyne-terminated PEG and azide-modified extracellular vesicles, achieving crosslinking via a copper-free catalytic click reaction, and ultimately controlling the structure and function of the SFSHs. Given the flexibility of this method, this biomimetic strategy of the present invention can provide reference and guidance for the preparation of skin biomaterials and other biomaterials with dual structures and tunable functions.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a cell membrane crosslinking strategy for developing hydrogels with skin-like structure and function. To prepare a crosslinked cell membrane network, this invention employs free radical polymerization of acrylamide, using olefin double-bond modified extracellular vesicles as crosslinking agents. Compared to traditional methylenediacrylamide-crosslinked polyacrylamide hydrogels, SFSHs exhibit significantly enhanced mechanical strength. This is attributed to multiple polymer chains attached to a mechanically deformable cell membrane crosslinking agent, i.e., double-bond functionalized vesicles, which can dissipate energy through vesicle deformation. Due to the abundance of bioactive substances in the extracellular vesicles, the SFSHs prepared in this invention also exhibit specific antibacterial effects and a strong ability to induce dendritic cell maturation and activation. The method of this invention is versatile and tunable. This invention achieves the construction of a biomimetic hydrogel double crosslinking network through a copper-free catalytic click reaction between alkyne-terminated PEG and azide-modified extracellular vesicles, preparing multifunctional SFSHs. Therefore, this biomimetic strategy based on cell membrane crosslinking provides direction and reference for the preparation of advanced skin-like biomaterials. Attached Figure Description

[0023] Figure 1 Here is a schematic diagram of the present invention: (A) An illustration of skin, which is essentially a cell membrane-separated hydrogel connected to the cell membrane, possessing high mechanical strength, strong antibacterial ability and immune function; (B) The synthetic route of SFSHs; (C) The unique properties of SFSHs mediated by cross-linked extracellular vesicles.

[0024] Figure 2 Characterization of OMV-AM and SFSHs: (A) FCM histograms of OMVs and OMV-AM after co-incubation with SH-Cy5 at 37°C for 3 hours; (B) MFI of Cy5-labeled OMVs and OMV-AM as determined by FCM analysis; (C) Representative LSCM image of Cy5-labeled OMV-AM, scale bar: 25 μm; (D) TEM image of OMV-AM, scale bar: 50 nm; (E) Mean size and (F) zeta potential of OMVs and OMV-AM; (G) Photographs of control polyacrylamide hydrogels and SFSHs; (H) SEM image of SFSHs after freeze-drying, magnification: ×90, scale bar: 100 μm; (IK) SEM image after freeze-drying, where (I) control polyacrylamide hydrogels and SFSHs have (J) 1×10 9 and (K)1×10 12 OMV-AM of particles / mL, magnification: ×10000, scale bar: 1 μm; (L) Representative 3D LSCM plots of SFSHs prepared from Cy5 labeled OMVs, with error bars indicating standard deviation (n=3).

[0025] Figure 3 For the optimization of SFSHs: (A) Tensile stress and strain curves of SFSHs formed by OMV-AM prepared at different reaction temperatures, (B) Tensile stress, (C) Tensile modulus and (D) Tensile strain; (E) Tensile stress and strain curves of SFSHs formed by OMV-AM prepared under different reaction times and operating procedures, (F) Tensile stress, (G) Tensile modulus and (H) Tensile strain; (I) Tensile stress and strain curves of SFSHs formed by OMV-AM prepared under different particle concentrations, (J) Tensile stress, (K) Tensile modulus and (L) Tensile strain; (M) Tensile stress and strain curves of SFSHs formed under different solid contents of crosslinking agent DSPE-PEG-AM, (N) Tensile stress, (O) Tensile modulus and (P) Tensile strain; Long strip samples (25×4×2 mm) were used for testing; Error bars represent standard deviation (n=3).

[0026] Figure 4Enhanced mechanical strength of SFSHs: (A) Photographs of SFSHs under different treatment conditions, including stretching, twisting, knotting, and compression followed by release. (B) Tensile stress-strain curves of SFSHs and control polyacrylamide hydrogels, (C) tensile stress, (D) tensile modulus, and (E) tensile strain, tested using strip samples (25 × 4 × 2 mm); (F) Compressive stress-strain curves, (G) compressive stress, and (H) compressive modulus of SFSHs and control polyacrylamide hydrogels, tested using cylindrical samples (8 × 5 mm), error bars indicating standard deviation (n = 3); (I) SEM images of unstretched and (J) fully stretched and cold-dried SFSHs, scale bar: 200 nm; (K) Aspect ratio of vesicles embedded in SFSHs before and after stretching, and (L) mean aspect ratio; Data were analyzed using ImageJ software on approximately 200 samples; Statistical analysis was performed using unpaired Student's t-test between two groups, with p-values, ****p < 0.0001.

[0027] Figure 5 To demonstrate the antibacterial and immunomodulatory activity of SFSHs: (A) Survival curves of *Salmonella typhimurium* (STm) after co-incubation with free OMVs, control polyacrylamide hydrogel, and SFSHs at the same dose, measured by recording the MFI of expressed mCherry. Untreated STm served as the control group; (B) Survival curves of STm after co-incubation with different doses of SFSHs; (C) After co-incubation with free OMVs, control polyacrylamide hydrogel, and SFSHs for 3 hours and (D) 6 hours, the number of surviving STm was obtained by bacterial plate counting; (E) Photographs of culture plates containing surviving STm after co-incubation with free OMVs, control polyacrylamide hydrogel, and SFSHs, showing 10... 6 times (above) and 10 7 Results of dilution (down-diluted); expression levels of (F,I)MHC II, (G,J)CD80, and (H,K)CD86 on DC2.4 cells after co-incubation with PBS, free OMVs, control polyacrylamide hydrogels, and SFSHs for 20 and 40 hours, error bars represent standard deviation (n=4); statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test, and p-values ​​were obtained, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0028] Figure 6Schematic diagrams illustrating the expansion of the application of the present invention's method by introducing a second OMV crosslinking network to form an interpenetrating double network structure: (A) Synthesis route of the second PEG-OMV hydrogel, using OMV-N3 as a crosslinking agent and linear DBCO-PEG-DBCO as a prepolymer to prepare an OMV crosslinked PEG network via a catalyst-free azide-alkynyl click reaction; (B) Schematic diagram of expanding the versatility of SFSHs by introducing different matrices; (C) Schematic diagram of expanding the versatility of SFSHs by introducing different cell membranes.

[0029] Figure 7 For the general applicability of SFSHs: (A) FCM histograms of OMV and OMV-N3 after incubation with DBCO-FITC at 25°C for 0.5 hours; (B) MFI values ​​of FITC-labeled OMV and OMV-N3; (C) LSCM image of FITC-labeled OMV-N3, scale bar: 25 μm; (D) mean size and (E) zeta potential of OMVs and OMV-N3; (F) 3D model of PEG-OMV hydrogel formed by FITC-labeled OMVs. LSCM images; (G) Compression stress-strain curves of PEG-OMV hydrogels containing different concentrations of OMV-N3 particles, (H) Compression stress and (I) Compression modulus; (J) 3D LSCM images of DN1 prepared with Cy5-labeled OMV-AM and FITC-labeled OMV-N3; (K) Compression stress-strain curves of SN and DN1, (L) Compression stress and (M) Compression modulus, cylindrical samples (8×5 mm) were used for compression testing; (N) Sliding distance of SN and DN1 on different matrices; (O) MFI value and (P) LSCM images of SN and DN1 after co-incubation with BSA-FITC (150 μg / ml) for 10 min, scale bar: 10 μm; (Q) LSCM images of SN and DN1 after co-incubation with NIH / 3T3 cells for 24 hours, blue: cell nucleus (Hoechst 33342), scale bar: 10 μm; (R) SE with PBS and free OMV SE Biofilm formation after 24 hours of co-incubation with SN or DN2 is shown in the upper and lower photos, respectively. Error bars represent standard deviations (n=3 or 5). Statistical analysis was performed using unpaired Student's t-test between two groups or one-way ANOVA and Tukey's multiple comparison test between multiple groups. p-values ​​were obtained, *p<0.05, **p<0.01; ns, no significant difference.

[0030] Figure 8(A) Representative TEM image of OMVs, scale bar: 200 nm; (B) Size distribution of OMVs measured by DLS; (C) NTA curve of concentrated OMVs; (D) Total protein concentration of OMVs with different particle numbers as measured by BCA protein quantification kit.

[0031] Figure 9 A representative SEM image of the freeze-dried control polyacrylamide hydrogel, magnification: ×90, scale bar: 100 micrometers;

[0032] Figure 10 The growth curves of STm after co-incubation with different doses of free OMVs were obtained by recording the MFI of mCherry expressed by STm.

[0033] Figure 11 FCM histograms of DC2.4 cells after incubation with PBS, free OMVs, control polyacrylamide hydrogel, and SFSHs for 20 and 40 hours, respectively.

[0034] Figure 12 FCM scatter plot showing the expression levels of (A) MHC II, (B) CD80 and (C) CD86 in DC2.4 cells after incubation with PBS, free OMVs, control polyacrylamide hydrogel and SFSHs for 20 hours;

[0035] Figure 13 FCM scatter plot showing the expression levels of (A) MHC II, (B) CD80 and (C) CD86 in DC2.4 cells after incubation with PBS, free OMVs, control polyacrylamide hydrogel and SFSHs for 40 hours;

[0036] Figure 14 The synthetic route for DBCO-PEG-DBCO is shown. Detailed Implementation

[0037] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0039] The materials and strains involved in the following embodiments of the present invention, as well as the preparation, characterization, and performance testing of the related materials, are as follows:

[0040] 1. Materials and strains

[0041] Escherichia coli Nissle 1917 (EcN), Salmonella Typhimurium (STm), and Staphylococcus epidermidis (SE) were purchased from the China General Microbiological Culture Collection Center (Beijing, China). DC2.4 cells (mouse dendritic cell line) and NIH / 3T3 cells (mouse fibroblast cell line) were obtained from the American Type Culture Collection (ATCC). Fluorescent dyes N-hydroxysuccinimide (NHS) ester-functionalized anthocyanin 5 (Cy5) (Cy5-NHS), NHS ester-functionalized fluorescein isothiocyanate (FITC) (FITC-PEG-NHS), dibenzocyclooctene (DBCO) modified FITC (DBCO-FITC), sulfinylated Cy5 (SH-Cy5), and Hoechst 33342 were purchased from Lumiprobe Corporation. Distearate-phosphatidylethanolamine-polyethylene glycol (2kDa)-acrylamide (DSPE-PEG-AM) and cholesterol-polyethylene glycol (2kDa)-azide (Chol-PEG-N3) were supplied by Shanghai Pengshuo Biotechnology Co., Ltd. Acrylamide (99%), N,N'-methylenebisacrylamide (MBAA), ammonium persulfate (APS, 99.99%), 2,2'-azobis(2-methylpropionitrile) (AIBN, ≥95%), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 99%), and N,N-isopropylethylamine (DIEA, ≥99%) were supplied by Sigma. Dibenzocyclooctylene (DBCO acid, ≥95%) was purchased from Xi'an Ruixi Biotechnology Co., Ltd. Amino-polyethylene glycol (10kDa)-amino (NH2-PEG-NH2) was purchased from Xiamen Sinobond Co., Ltd. Plasmid pBBR1MCS2-Tac-mCherry (kanamycin resistant) and phosphate-buffered saline (PBS, 1×, pH 7.4) were provided by Sangon Biotech Co., Ltd. Anhydrous N,N-dimethylformamide (DMF, 99.8%) and diethyl ether (Et2O, ≥99.7%) were provided by Sinopharm Chemical Reagent Co., Ltd. Other reagents and solvents were used as is.

[0042] 2. Purification and characterization of OMVs (Outer membrane vesicles)

[0043] EcN was cultured in Luria Bertani (LB) medium at 37°C with a kanamycin concentration of 100 μg / mL. After overnight culture, the overnight medium was diluted 100-fold with fresh LB medium containing 100 μg / mL kanamycin and 3 μg / mL ampicillin, and cultured for another 2 days. After centrifugation at 9000 rpm for 30 min, the resulting supernatant was filtered through a 0.45 μm filter at 4°C to remove bacterial cells. The resulting particulate precipitate was then obtained by ultracentrifugation (170000 g, 4°C, 1 h). The OMVs were washed once with sterile PBS and concentrated to 1 mL by ultracentrifugation (170000 g, 4°C, 1 h). Finally, the concentrated solution was stored at -80°C for subsequent experiments. The mean size and zeta potential of the OMVs were measured by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS, UK). The number concentration of OMVs was determined by nanoparticle tracking analysis (NTA, Malvern NanoSight NS300, UK). The total protein concentration of concentrated OMVs was determined using the dicaprotonic acid (BCA) method (Beyotime, China). The morphology of OMVs was characterized by transmission electron microscopy (TEM, Hitachi, Japan). Prior to TEM imaging, OMV samples were negatively stained with 1% (w / v) phosphotungstic acid for 1 minute. For visualization of the results, OMVs were labeled with Cy5-NHS (red) or FITC-PEG-NHS (green) as needed and characterized by laser scanning confocal microscopy with a 63× oil objective (LCSM, Leica TCS SP8, Germany).

[0044] 3. Preparation and Characterization of OMV-AM

[0045] Preparation of OMVs with olefin double bond surface modification: Concentrated OMVs were incubated with 2% DSPE-PEG-AM at 37°C with shaking (600 rpm) for 0.5 h. The success of the modification was verified by flow cytometry (FCM, CytoFLEX, Beckman-Colter, US) and LSCM analysis. The obtained OMV-AM was co-incubated with AIBN and SH-Cy5 at 37°C with shaking (600 rpm) for 3 h. The size and zeta potential changes of the OMV-AM were measured by DLS. The morphology of the OMV-AM was characterized by transmission electron microscopy.

[0046] 4. Preparation and optimization of SFSHs

[0047] Using OMV-AM solution as solvent, acrylamide (284 mg, 4 mmol), initiator APS (5‰ w / w), and catalyst TEMED (5‰ w / w) were added sequentially in a nitrogen glove box to obtain a prepolymer solution at room temperature. After polymerization in a polytetrafluoroethylene (PTFE) mold for 30 minutes, SFSHs were obtained and used in subsequent experiments. Polyacrylamide hydrogels were obtained by crosslinking with the small molecule crosslinking agent MBAA without the addition of OMVs as a control. To optimize the preparation conditions, the reaction conditions for modifying OMV-AM were systematically investigated, including temperature (25℃, 37℃, or 50℃), time (0.5 h or 1 h), and operating method (with or without ultrasound). Factors affecting the crosslinking density, including the number of vesicle particles (1 × 10⁻⁶), were also studied. 9 Up to 1×10 12 The density of double bonds modified on OMVs was also measured (the solid content of DSPE-PEG-AM ranged from 0.8% to 8%). For visualization of the results, SFSHs were fabricated with OMV-AM labeled with Cy5-NHS and observed by three-dimensional (3D) LCSM imaging.

[0048] 5. SEM characterization of SFSHs

[0049] The microstructure and morphology of SFSHs after lyophilization at different OMV-AM concentrations were observed using scanning electron microscopy (SEM, Zeiss 1550VP FESEM, Germany) at an accelerating voltage of 3 kV. To verify the vesicle crosslinking points, the samples were stretched to four times their initial length and then lyophilized. All samples were sputter-coated with gold before testing.

[0050] 6. Mechanical property testing

[0051] All mechanical tests on the hydrogel were performed on a universal testing machine (Instron-3342, US). Compression tests were conducted at a strain rate of 5 mm / min using cylindrical samples measuring 8 × 5 mm, with compressive strains of 90% or 95%. Tensile tests were conducted at a strain rate of 100 mm / min using rectangular samples measuring 25 × 4 × 3 mm. Three parallel samples were used for each test.

[0052] 7. Antibacterial properties of SFSHs

[0053] STm expressing mCherry were cultured overnight at 37°C with shaking (200 rpm) in LB medium containing 100 μg / ml kanamycin. Bacteria were collected and washed with PBS, then diluted until the optical density (OD) at 600 nm was approximately 1. Subsequently, 50 μl of bacterial suspension and SFSHs were added to 96-well plates, and finally, LB medium containing 100 μg / ml kanamycin was added to a final volume of 200 μl. The mixture was incubated at 37°C with gentle shaking for 12 h. The mean fluorescence intensity (MFI) of surviving STm expressing mCherry at 590 nm was recorded every 0.5 h using a microplate reader (HIMF, BioTek, USA). Untreated STm and STm treated with the same dose of free OMVs or conventional polyacrylamide hydrogel served as controls. The effects of different doses (10%, 15%, or 20%) of free OMVs or SFSHs were also investigated. The number of surviving STm was further determined by bacterial plate counting. Add 50 μl STm(OD) 600 =1) and 300 μl of SFSHs were added to 1 ml of LB medium and incubated at 37 °C with shaking (200 rpm). At 3 h and 6 h, 50 μl of each sample was taken and counted on LB agar plates containing 100 μg / mL kanamycin. Each sample was tested in triplicate.

[0054] 8. Immunological activity of SFSHs

[0055] The effects of SFSHs on dendritic cell maturation and activation were investigated in vitro. DC2.4 cells were cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) antibiotics (penicillin-streptomycin, PS) in a cell culture incubator (37°C, 5% CO2). Cells were sputtered at 10... 5 Cells were seeded at a density of 10 cells / well in 24-well plates and treated for 20 h and 40 h with PBS (10 μl), free OMVs (10 μl), conventional polyacrylamide hydrogel (10 μl), or SFSHs (10 μl), respectively. Cells were then collected and washed with PBS. For specific labeling, anti-mouse antibodies were dissolved in PBS containing 0.5% bovine serum albumin (BSA) and co-incubated with cells on ice for 1 h. The anti-mouse antibodies used included anti-CD86-APC (24F, BioLegend), CD80-PE (16-10A1, eBioscience), and IA / IE(MHC-II)-PE / Cy7 (M5 / 114.15.2, BioLegend). After washing three times with PBS, cells were analyzed by FCM.

[0056] 9. Preparation and Characterization of OMV-N3

[0057] Preparation of azide-functionalized OMVs: Concentrated OMVs were incubated with Chol-PEG-N3 (19 μM) at 37 °C with shaking (600 rpm) for 1 h. FCM and LSCM analyses were performed to confirm successful modification. The obtained OMV-N3 was then incubated with DBCO-FITC at 25 °C with shaking (600 rpm) for 0.5 h. The size and zeta potential of the OMV-N3 were measured by DLS.

[0058] 10. Preparation and characterization of PEG-OMV hydrogels

[0059] Using OMV-N3 solution as a solvent, DBCO-PEG-DBCO was added at a stoichiometric ratio (molar ratio 1:1) according to the amount of Chol-PEG-N3 added. The PEG-OMV hydrogel was then prepared in a PTFE mold at room temperature within minutes. For visualization, PEG-OMV hydrogels were prepared using FITC-PEG-NHS-labeled OMV-N3 and observed using 3D LCSM. Compression tests were performed according to the method described above.

[0060] 11. Preparation and Characterization of DN1

[0061] Using a mixed solution of OMV-AM and OMV-N3 as a solvent, acrylamide (284 mg, 4 mmol), APS (5‰ w / w), DBCO-PEG-DBCO, and TEMED (5‰ w / w) were added sequentially in a nitrogen glove box to obtain a prepolymer solution at room temperature. After polymerization in a PTFE mold for 30 minutes, a polyacrylamide-OMV / PEG-OMV dual-network hydrogel (DN1) was obtained. DBCO-PEG-DBCO was added in a stoichiometric ratio (molar ratio 1:1) based on the amount of Chloro-PEG-N3. For visualization, OMV-AM and OMV-N3 were labeled with Cy5.5-NHS and FITC-PEG-NHS, respectively, before hydrogel preparation and observed by 3D LCSM. Compression tests were performed according to the method described above.

[0062] 12. Evaluation of antifouling performance

[0063] To evaluate the antifouling ability of hydrogels, their lubrication properties and anti-adhesion properties against proteins and cells were investigated. For lubrication properties, cylindrical samples (5×5 mm) were placed on a vertical substrate. The sliding distances of polyacrylamide-OMV single-network hydrogels (SN) and DN1 on different substrates (glass, polystyrene (PS), aluminum (Al), and PTFE) were recorded, repeated three times. To analyze anti-protein adhesion properties, SN and DN1 were incubated with BSA-FITC solution (150 μg / ml) for 10 min. After washing twice with PBS, the samples were observed using LCSM, and the microfiltration efficiency (MFI) of adhered BSA-FITC was recorded at 520 nm using a microplate reader. To analyze anti-cell adhesion properties, NIH / 3T3 fibroblasts were cultured in a cell culture incubator (37°C, 5% CO2) in Dulbecco modified Eagle medium (DMEM) containing 10% FBS and 1% PS. SN and DN1 were first spread on the bottom of a confocal culture dish, and 5×10⁻⁶ samples were then incubated. 5 NIH / 3T3 cells were seeded in confocal dishes and incubated for 24 hours, after which culture medium and unattached cells were removed. Samples were stained with 10 μg / ml Hoechst 33342 for 10 minutes, washed three times, and then imaged by LCSM.

[0064] 13. Enhance SE biofilm formation

[0065] SEs were cultured in LB medium containing 5 μg / ml erythromycin overnight at 37°C. The cultures were then diluted 200-fold with fresh LB medium for subsequent experiments. OMVs were collected from the SEs and modified with azide groups (OMVs). SE -N3). OMVs secreted by Nissle 1917 cells are represented by double bonds (OMV). EcN -AM) functionalization. Utilizing OMV. SE -N3 and OMV ECN -AM was used to prepare polyacrylamide-OMV ECN / PEG-OMV SE Dual-network hydrogel (DN2). 200 μl of SE suspension was transferred to a 96-well plate, and 10 μl of free OMV was added to each well. SE Polyacrylamide-OMV ECN Hydrogel (SN) and DN2. The mixture was incubated at 37°C for 24 hours, the supernatant was gently removed, the plate was washed twice with PBS, and dried at 37°C. 200 μl of methanol was added and incubated for 10 min, the methanol was aspirated, and the plate was dried again. 100 μl of 1% crystal violet was transferred to each well and stained for 30 min, followed by washing 10 times with PBS. Finally, 200 μl of 33% glacial acetic acid was added to each well, and the OD value of each well at 595 nm was measured using a microplate reader.

[0066] 14. Statistical Analysis

[0067] All statistical analyses were performed using GraphPad Prism 8.0. Results are expressed as mean ± standard deviation (SD). p-values ​​were given by unpaired Student's t-test between two groups or one-way ANOVA or Tukey's multiple comparison test between multiple groups. Significance was defined as p < 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001. No significance was found in ns.

[0068] Example

[0069] This embodiment provides a method for preparing and evaluating the performance of a cross-linked cell membrane-mimicking skin-like hydrogel:

[0070] 1. Preparation and design of cell membrane cross-linking networks

[0071] Given the widespread use of polyacrylamide as an ECM analog in the development of skin-inspired materials, and its advantages of good biocompatibility and ease of preparation, polyacrylamide was chosen as the matrix in this embodiment of the invention. On the other hand, extracellular vesicles were chosen as cell membrane microdomains because they possess a lipid bilayer structure and a large number of bioactive substances inherited from the maternal cell. To mimic the structure of skin, extracellular vesicles were used as multivalent cross-linking agents to cross-link polyacrylamide chains, forming a cell membrane microdomain cross-linking network. Given the readily available availability of *Escherichia coli* Nissle 1917, spherical nanoprotein liposomes, i.e., outer membrane vesicles (OMVs), generated by vesicle formation of its outer membrane were used as model extracellular vesicles to prepare SFSHs. OMVs were extracted and purified from the culture medium by ultracentrifugation. Figure 8 As shown in A and B, the obtained OMVs exhibit a spherical structure with an average size of approximately 180 nanometers. In this embodiment of the invention, OMV concentrates with different particle numbers were quantified by nanoparticle tracking analysis (NTA), and the corresponding total protein concentration was determined using a dioctanoic acid (BCA) protein quantification kit. Figure 8 (C and D). Typically, OMVs solutions contain 1.44 × 10⁻⁶. 12 Each particle per ml contains a total protein concentration of 2 mg / ml.

[0072] To act as a crosslinking agent, olefin double bonds were modified onto the vesicle surface via supramolecular hydrophilic-hydrophobic interactions between DSPE-PEG-AM and the lipid bilayer of OMVs. The acrylamide-functionalized OMV (designated OMV-AM) was validated using a thiol-ene addition reaction mediated by SH-Cy5 fluorescent dye. Flow cytometry (FCM) analysis showed that after surface functionalization with olefin double bonds, the fluorescence peak of OMV-AM shifted significantly to a higher intensity, with the mean fluorescence intensity (MFI) increasing approximately 3-fold. Figure 2 A and B). Laser scanning confocal microscopy (LSCM) showed that the OMV initially had no fluorescence, but after modification, the vesicles emitted obvious fluorescence. Figure 2 C). Both FCM and LSCM results indicate the successful preparation of olefin double-bond functionalized vesicles OMV-AM. Transmission electron microscopy (TEM) images show that the spherical morphology of OMV-AM did not change significantly. Figure 2 D). After modification, the average particle size of OMV-AM increased from 187.9 nm to 230.7 nm, and the zeta potential increased from -16.4 mV to -9.5 mV. Figure 2 E and F).

[0073] SFSHs were prepared via free radical polymerization using acrylamide, OMV-AM, ammonium persulfate (APS), and N,N,N',N'-tetramethylethylenediamine (TEMED) as monomers, crosslinking agents, initiators, and catalysts, respectively. Figure 2 G). Polyacrylamide hydrogels were prepared as a control using N,N'-methylenebisacrylamide (MBAA) as a conventional small molecule crosslinking agent without the addition of OMV. The microstructure and morphology of SFSHs were observed using scanning electron microscopy (SEM). Figure 2 H and Figure 9 As shown, both freeze-dried SFSHs and the control polyacrylamide hydrogel exhibited porous 3D structures at low magnification. With increasing magnification, significant differences in morphology became apparent between the two hydrogels, with the control hydrogel exhibiting a layered structure in the thickness direction. Figure 2 I), while SFSHs have a large number of dispersed nanospheres in their cross-section ( Figure 2 J). When the concentration of OMV-AM is from 1×10 9 Particles / ml increased to 1×10 12 At particle size / ml, SEM results showed that the hydrogel cross-section contained a large number of vesicles. Figure 2 K), highlighting the formation of the vesicle cross-linking network structure. As shown in three-dimensional (3D) LSCM imaging, OMV labeled with N-hydroxysuccinimide (NHS) ester-functionalized Cy5 fluorescent dye (Cy5-NHS) is uniformly dispersed in the hydrogel network. Figure 2 This confirms the successful preparation of SFSHs with biomimetic cell microregion structures.

[0074] 2. SFSHs-enhanced mechanical properties

[0075] In this invention, the reaction conditions for OMV modification were optimized, including temperature, time, and operating methods. Under the same experimental conditions, SFSHs prepared by incubating OMV with DSPE-PEG-AM at 37°C exhibited the best tensile properties, including the highest tensile stress, modulus, and strain, which were superior to SFSHs prepared by incubation at 25°C and 50°C. Figure 3 AD). At 37°C, as the incubation time of OMVs increased from 0.5 hours to 1 hour, these mechanical properties of SFSHs all decreased ( Figure 3 EH). The experiment also found that ultrasonic treatment during incubation reduced the tensile properties of SFSHs. That is, without ultrasonic treatment, OMV-AM obtained by incubating OMV-AM and DSPE-PEG-AM at 37°C for 0.5 hours could be used to prepare SFSHs with optimal mechanical strength. Therefore, it is speculated that these conditions provide moderate perturbation to the lipid bilayer membrane of OMVs, which can promote the insertion of DSPE-PEG-AM into the vesicle cell membrane driven by supramolecular hydrophilic-hydrophobic interactions. It is well known that the crosslinking density of the polymer network determines the mechanical properties of the hydrogel, meaning that the mechanical strength of SFSHs largely depends on the number of double bonds in the network. Therefore, in this embodiment of the invention, the effects of the number of vesicle OMV particles and the density of double bonds modified on the OMVs were investigated to optimize the mechanical properties of SFSHs. Figure 3 As shown in the IL, with other experimental conditions remaining unchanged, as the OMV-AM concentration increased from 1×10 9 Particles / ml increased to 1×10 12 Particle size / ml, tensile stress and strain were improved. Furthermore, with the same number of vesicles, the tensile modulus of SFSHs increased with increasing DSPE-PEG-AM solid content from 0.8% to 8%, while the tensile strain decreased with increasing double bond density on OMV-AM. Figure 3 MP). After screening, at an OMV-AM concentration of 1.5 × 10⁻⁶, [the desired concentration was found to be suitable]. 12With a combination of 2% DSPE-PEG-AM solid content and a specific gravity of 1 / ml, SFSHs exhibited the highest tensile strength, with a tensile stress of 192.98 ± 11.98 kPa, a tensile modulus of 18.21 ± 1.35 kPa, and a tensile strain of 1170.98 ± 68.76%. Visually, regardless of twisting or knotting, the resulting hydrogel could be stretched to 10 times its original length, and its hardness was sufficient to withstand a massive load of up to 1 kg without any damage. Figure 4 A).

[0076] Subsequently, the properties of SFSHs were compared with those of conventional polyacrylamide hydrogels to reveal the potential mechanism of increased mechanical strength. Similarly, the control polyacrylamide hydrogel was crosslinked with MBAA having an equal amount of double bonds with OMV-AM. As expected, SFSHs exhibited typical "J-shaped" tensile stress-strain curves with a non-linear relationship, with maximum tensile stress and strain of 204.48 kPa and 1268.22% ( ) respectively. Figure 4 B). Compared with the control polyacrylamide hydrogel, the tensile stress, modulus, and strain of SFSHs increased by 4.39 times, 1.08 times, and 2.38 times, respectively. Figure 4 CE). Similar enhancements were also observed in studies of the compressive properties of SFSHs, as shown by steeper compressive stress-strain curves. Figure 4 F), OMV-AM crosslinking improved the compressive properties of SFSHs. At 95% compressive strain, the compressive stress and modulus of SFSHs were approximately 3865.27±169.97 kPa and 26.16±2.48 kPa, respectively, which were 1.31 times and 1.6 times that of the control hydrogel. Figure 4 (G and H). The above results indicate that replacing small molecule crosslinking agents with OMV-AM can enhance the mechanical properties of polyacrylamide hydrogels. We hypothesize that this enhancement can be explained by deformable crosslinking points, i.e., the spherical surface of OMV is connected to numerous polymer chains. Unlike traditional crosslinking points, which are chemical bonds, vesicle crosslinking points can deform to accommodate network stretching, thereby improving the mechanical toughness of the hydrogel. This is similar to the cell-ECM network in skin, where cells can adapt to stretching by deforming due to the fluidity of the cell membrane. Our hypothesis was validated using SEM images. Figure 4 As shown in I and J, the OMVs in SFSHs deform into flattened shapes in the direction of gel stretching. Further quantitative analysis revealed that the aspect ratio of the crosslinked OMVs in the stretched lyophilized SFSHs increased from 1.41±0.36 to 1.73±0.35, and the average major axis increased from 285±69 nm to 332±86 nm. Figure 4(K and L). This finding confirms that during stretching, the polymer network obtained by OMV crosslinking can effectively dissipate stretching energy through vesicle deformation, thereby improving the mechanical properties of SFSHs.

[0077] 3. Antibacterial properties of SFSHs

[0078] OMVs carry a variety of bioactive substances from the parent cell and play a key role in resisting or inhibiting competitive bacteria. Note that Nissle 1917 cells secrete microproteins with potent antibacterial activity, inhibiting the proliferation of pathogenic Salmonella typhimurium (STm). To assess the antibacterial activity of SFSHs, STm was co-incubated with them for different time periods, and STm growth was monitored. Untreated STm and STm co-incubated with conventional polyacrylamide hydrogel served as controls. Growth curves were obtained by recording the MFI of mCherry expressed in surviving STm. As expected, we found that OMVs secreted by Nissle 1917 cells inhibited the growth of STm (…). Figure 5 A). Similarly, SFSHs exhibited significant antibacterial activity, as evidenced by the slow growth of STm over time. In contrast, the control polyacrylamide hydrogel showed almost no bactericidal effect, as co-incubated STm exhibited nearly identical growth curves to untreated STm. The ability of SFSHs to inhibit STm growth can be simply attributed to OMVs, which inherit antibacterial microproteins secreted by Nissle 1917 cells. Furthermore, unlike free OMVs, STm proliferation slowed as the volume of co-incubated SFSHs increased from 10% (volume / volume) to 20%. Figure 5 B and Figure 10 This indicates that the antibacterial effect of SFSHs is dose-dependent. The number of surviving STm cells was further quantified by bacterial plate counting. Figure 5 (CE). Clearly, co-incubation with 22% SFSH resulted in the lowest colony count, a 55.67-fold reduction compared to untreated STm. After only 3 hours of co-incubation with 22% SFSH, the number of surviving STm decreased to 1.79%, indicating a potent killing ability against pathogens. Similar to how innate skin immune cells (such as T lymphocytes) can release cytotoxins to kill pathogens, embedded OMVs carrying microproteins can confer satisfactory antibacterial activity to SFSH. Considering the versatility of DSPE-PEG-AM functionalized OMVs, by selecting different types of OMVs, SFSHs with different or even specific antibacterial activities can be introduced.

[0079] 4. Immunological activity of SFSHs

[0080] Since SFSHs contain a large number of OMVs, we next investigated their immunogenicity. Dendritic cells (DCs), as the most potent and specialized antigen-presenting cells (APCs), and the only APCs capable of activating naive T cells, play a decisive and guiding role in adaptive immune responses. DCs present specific antigens to T cells and stimulate the large-scale proliferation of antigen-specific T cells, thereby eliminating pathogens characterized by specific antigens. Given that DC maturation is crucial for the performance of its functions (such as T cell activation and polarization), we investigated the immunostimulatory capacity of SFSHs for DC maturation and activation. Notably, DC-induced T cell activation is mediated by two main pathways: the binding of the major histocompatibility complex (MHC) on DCs to T cell receptors, and the binding between co-stimulatory molecules (CD80 and CD86) on DCs and CD28 on T cells. After incubating DC2.4 cells with SFSHs for a specified time, the expression levels of MHC II, CD80, and CD86 were detected by FCM analysis. PBS, free OMVs, and conventional polyacrylamide hydrogel were used as controls. Figure 5 FK and Figure 11-13 As shown, compared with the PBS and polyacrylamide hydrogel groups, the expression levels of MHC II, CD80, and CD86 were significantly increased in the SFSHs group, while the free OMV group only showed enhancement in CD80 and CD86 after 20 hours of incubation. The SFSHs-induced expression levels of MHC II, CD80, and CD86 were 1.24, 1.11, and 1.07 times higher than those in the PBS-treated group, respectively. With the incubation time extended to 40 hours, the expression of MHC II, CD80, and CD86 in DC 2.4 cells treated with SFSHs further increased and was significantly higher than in other groups. Calculations showed that the expression of MHC II, CD80, and CD86 increased by 5.37, 2.19, and 2.20 times, respectively, compared with the PBS group. These results confirm that SFSHs can promote the expression of the major histocompatibility complex MHC II and upregulate co-stimulatory molecules on DCs, such as CD80 and CD86. SFSHs had the strongest ability to activate DCs and promote antigen processing and presentation among all groups.

[0081] 5. SFSHs introduce versatility across different matrices

[0082] After confirming the successful preparation of SFSHs via crosslinking OMVs, this invention explores the versatility of this method to adjust the structure and function of the resulting hydrogels. Considering that cells are connected by different matrices, such as the multilayered lipids, collagen, elastin, and hyaluronic acid in the skin, this invention expands the application of this method by introducing a second OMV crosslinking network to form an interpenetrating double network structure. As a proof of concept, a second PEG network was formed via a copper-free azido-yetne click reaction. Figure 6 A). Azide-modified OMV (OMV-N3) and dibenzocyclooctene (DBCO)-terminated linear PEG (DBCO-PEG-DBCO) were selected as clickable crosslinking agents and prepolymers of the second network, respectively. OMV-N3 was prepared by simple co-incubation with OMVs at 37°C for 1 hour via hydrophilic-hydrophobic self-assembly, while DBCO-PEG-DBCO was synthesized via an amide condensation reaction of DBCO-acid with amino-PEG (10kDa)-amino (NH2-PEG-NH2). Figure 14 The successful preparation of OMV-N3 was verified by FCM and LSCM with the aid of DBCO-modified fluorescein isothiocyanate (DBCO-FITC). The fluorescence peak of OMV-N3 showed a significant shift in intensity, with the MFI being 6.34 times higher than that of the original OMVs. Figure 7 A and B); LSCM images show that OMV-N3 was successfully fluorescently labeled (A and B); Figure 7 C). The average particle size of OMV-N3 decreased to 165.8 nm, and the zeta potential increased to -7.8 mV. Figure 7 (D and E). Before constructing the dual-network structure, we first investigated the feasibility of forming a PEG-OMV network via a click reaction. As expected, the solutions of DBCO-PEG-DBCO and OMV-N3 rapidly formed a hydrogel within minutes of mixing. Typical 3D LSCM images clearly show that a large number of OMVs labeled with the fluorescent dye FITC-PEG-NHS are uniformly dispersed in the hydrogel. Figure 7 F). Compressive stress increases significantly with increasing OMV-N3 particle concentration. Figure 7 GI). It is worth noting that, from ~1.5×10 12 The OMV-N3 crosslinked hydrogel with particle size / ml exhibited a maximum compressive stress of 473.11±98.78 kPa and a maximum compressive modulus of 260±89.81 Pa at 90% compressive strain. Then, based on the orthogonal characteristics between free radical polymerization and click reaction, we constructed a polyacrylamide-OMV / PEG-OMV dual-network hydrogel (DN1) via a simple one-pot method. Figure 6B). As shown in the 3D LSCM image, both Cy5-NHS-labeled OMV-AM (red) and FITC-PEG-NHS-labeled OMV-N3 (green) are present in the hydrogel. Figure 7 J) indicates the formation of OMV-AM / polyacrylamide and OMV-N3 / DBCO-PEG-DBCO networks.

[0083] Compared with OMV-AM / polyacrylamide monolayer hydrogel, the obtained DN1 exhibits significantly enhanced compressive stress-strain behavior. Figure 7 The compressive stress and modulus were 2813.72±316.69 kPa and 17.71±5.61 kPa, respectively, representing increases of 1.52 times and 6.73 times. Furthermore, compared to SN, DN1 exhibited superior lubrication properties, with longer sliding distances on various substrates such as glass, polystyrene (PS), aluminum (Al), and polytetrafluoroethylene (PTFE). Figure 7 In particular, DN1 achieved a significant sliding distance of up to 110 mm on the PTFE surface, while SN showed almost no sliding. The protein adhesion on the DN1 surface was significantly reduced, further validating this anti-adhesion property. Figure 7 O). After co-incubating DN1 with 150 μg / ml BSA-FITC protein solution for 10 minutes, the fluorescence intensity on DN1 was reduced by approximately 4.75 times compared to that on SN. Figure 7 P); similarly, after co-incubation with NIH / 3T3 cells for 24 hours, the number of cells adhering to DN1 cells was significantly reduced (P); Figure 7 Q), these anti-adhesion and antifouling properties are attributed to the hydrophilicity and low surface energy of the PEG network.

[0084] 6. The universality of SFSHs in introducing different cell membrane microregions

[0085] On the other hand, given that different types of cell membrane microdomains are embedded in the skin, this embodiment of the invention further expands the versatility of this method by developing a dual-network structure cross-linked by two different types of OMVs. Figure 6 C). Collect OMVs derived from Staphylococcus epidermidis (SE) cells and modify them with azide groups (OMVs). SE -N3), while the OMV secreted by Nissle 1917 cells is functionalized with double bonds (OMV). EcN -AM). Prepared under the same conditions as described above using polyacrylamide-OMV. EcN and PEG-OMV SEA hydrogel composed of a network (DN2). SE is a common symbiotic bacterium on human skin. Therefore, we hypothesized that a double-network hydrogel formed by SE-derived vesicles could promote the colonization of symbiotic bacteria on the surface. Given that biofilm formation is a key mechanism for bacterial colonization, the effect of DN2 on SE biofilm formation was evaluated. SE was compared with PBS and free OMV. SE Polyacrylamide-OMV ECN After incubation of the hydrogel (SN) and DN2 for 24 hours, the optical density (OD) of the formed biofilm was measured at 595 nm using a microplate reader. As expected, the OMV was significantly higher than that of the untreated SE. SE It significantly enhanced biofilm formation, increasing it by 2.07 times ( Figure 7 Interestingly, DN2 exhibits similarities to OMV. SE The biofilm-promoting capacity was comparable, 1.85 times that of the SN group. Direct observation of the culture dishes verified the above conclusions; extensive biofilm formation was observed at the bottom of the culture dishes in the DN2 group, rather than the SN group. This enhanced biofilm formation can be assessed using OMV. SE To explain the existence of OMV SE A large number of bioactive substances were inherited from the parental SE cells. These findings validate the universality of cell membrane microdomain cross-linking methods for regulating the structure and function of SFSHs.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A cross-linked hydrogel that mimics the structure and function of the cell membrane of skin, characterized in that, The hydrogel is a polyacrylamide biomimetic hydrogel SFSHs with a cell membrane crosslinking network, which is generated by free radical polymerization between acrylamide and the olefin double bonds on the extracellular vesicles, using olefin double bonds modified with olefin double bonds as crosslinking agents and olefin double bonds as monomers.

2. The crosslinked cell-membrane mimicking hydrogel of claim 1, wherein, The extracellular vesicles are bacterial extracellular vesicles; the olefin double bond modified extracellular vesicles are obtained by incubating extracellular vesicles with distearate phosphatidylethanolamine-polyethylene glycol-acrylamide (DSPE-PEG-AM), and the distearate phosphatidylethanolamine-polyethylene glycol-acrylamide inserts into the vesicle cell membrane through hydrophilic-hydrophobic interactions, thereby obtaining olefin double bond modified extracellular vesicles OMV-AM.

3. The hydrogel with cross-linked cell membrane mimicking skin structure and function as described in claim 1, characterized in that, The hydrogel SFSHs also include an enhanced cross-linking network, which is formed by cross-linking alkyne-terminated PEG and azide-modified extracellular vesicles through a copper-free catalytic click reaction. The azide-modified extracellular vesicles are formed by incubating extracellular vesicles with an azide reagent.

4. The hydrogel with cross-linked cell membrane mimicking skin structure and function as described in claim 3, characterized in that, The alkyne-terminated PEG is dibenzocyclooctyne-polyethylene glycol-dibenzocyclooctyne (DBCO-PEG-DBCO), and the azidating agent is cholesterol-polyethylene glycol-azide (Chol-PEG-N3).

5. The hydrogel with cross-linked cell membrane mimicking skin structure and function as described in claim 3 or 4, characterized in that, The extracellular vesicles in the enhanced cross-linking network are derived from Escherichia coli or Staphylococcus epidermidis. Among them, the extracellular vesicles derived from Staphylococcus epidermidis are cross-linked with PEG at both ends of alkyne through a copper-free catalytic click reaction after being modified by azide function, forming an enhanced cross-linking network with enhanced biofilm formation ability.

6. A method for preparing the cross-linked cell membrane-mimicking skin-like hydrogel according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: Extract extracellular vesicles from bacteria; Step 2: The extracellular vesicles extracted in Step 1 were incubated with distearate-phosphatidylethanolamine-polyethylene glycol-acrylamide (DSPE-PEG-AM) to prepare olefin double bond modified extracellular vesicles OMV-AM; Step 3: Using acrylamide as a monomer and the extracellular vesicles modified with olefin double bonds obtained in Step 2 as crosslinking agents, cell membrane crosslinked polyacrylamide hydrogels (SFSHs) are prepared by initiating free radical polymerization between acrylamide and the double bonds on the extracellular vesicles under the action of an initiator and a catalyst. Alternatively, extracellular vesicles can be incubated with an azide reagent to obtain azide-modified extracellular vesicles OMV-N3. OMV-N3 can then be mixed with OMV-AM prepared in step 2, and acrylamide, an initiator, alkyne-terminated PEG, and a catalyst can be added sequentially to carry out a polymerization reaction to obtain a double-network skin-like hydrogel containing an enhanced cross-linking network.

7. The method for preparing the cross-linked cell membrane-mimicking skin structure and function hydrogel as described in claim 6, characterized in that, The initiator in step 3 is ammonium persulfate (APS), and the catalyst is N,N,N',N'-tetramethylethylenediamine (TEMED).

8. The method for preparing the cross-linked cell membrane-mimicking skin structure and function hydrogel as described in claim 7, characterized in that, The extracellular vesicles in step 1 are derived from Escherichia coli.

9. The method for preparing the cross-linked cell membrane-mimicking skin structure and function hydrogel as described in claim 7, characterized in that, The extracellular vesicles modified by azidation in step 3 are derived from Escherichia coli or Staphylococcus epidermidis.

10. The application of the cross-linked cell membrane-mimicking skin structure and function hydrogel according to any one of claims 1 to 4 in the preparation of smart skin biomaterials.

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