Biomimetic nano-delivery system based on mrsa outer vesicle coating and applications thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a biomimetic nano delivery system based on MRSA outer vesicle coating and application thereof. The application first couples chitosan oligosaccharide and monocarboxylated curcumin through an amide reaction to synthesize an amphiphilic polymer carrier, then loads vancomycin, daptomycin and other drugs through self-assembly to obtain a drug micelle, and finally coats the drug micelle with MRSA outer vesicle to successfully prepare a targeted biomimetic nano delivery system. The prepared biomimetic nano delivery system has excellent antibacterial effect, can realize targeted delivery and long-acting release of antibacterial drugs at an infection site, and is expected to overcome the bottleneck of drug resistance and insufficient drug penetration in traditional treatment, and provides a potential solution for the treatment of diseases such as periprosthetic infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomimetic nanodelivery system based on MRSA exovesicle coating and its applications. Background Technology

[0002] With the increasing aging of the global population, the number of patients with osteoarthritis is constantly rising, and prosthesis replacement surgery has become an important means of relieving pain associated with osteoarthritis. However, periprosthetic infection (PJI) is a serious complication of prosthesis replacement surgery, and its incidence increases with the number of surgeries performed.

[0003] Treatment of percutaneous joint injury (PJI) faces high recurrence and mortality rates, as well as a significant medical burden. Current treatment strategies primarily include conservative drug therapy, thorough debridement with prosthesis preservation, one- or two-stage revision surgery, joint fusion, and invasive methods such as amputation. However, the emergence of drug-resistant strains, insufficient drug penetration at the infection site, and systemic side effects have increased the difficulty of PJI treatment, urgently requiring innovative treatment strategies.

[0004] Nanoparticle delivery systems, as a novel type of microbial therapeutic system, can not only improve the targeting of drugs to tumor cells or infected tissue cells, but also enhance the sustained release of encapsulated drugs through the characteristics of the carrier, thereby improving the bioavailability and efficacy of drugs, while reducing the side effects of drugs on normal tissues of the body.

[0005] However, the emergence of drug-resistant strains, insufficient drug penetration at the infection site, and systemic side effects have increased the difficulty of treating PJI, making innovative treatment strategies urgently needed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a biomimetic nanodelivery system based on MRSA exovesicle coating and its applications.

[0007] The biomimetic nanodelivery system based on MRSA exovesicles provided by this invention includes: MRSA exovesicles and drug micelles coated thereon, wherein the drug micelles are an amphiphilic polymer composed of chitosan oligosaccharide and monocarboxylated curcumin as a carrier, and the drug is loaded onto the amphiphilic polymer through self-assembly; the drug includes, but is not limited to, any one of vancomycin, daptomycin, and teicoplanin.

[0008] Preferably, the drug is selected from either vancomycin or daptomycin.

[0009] Preferably, the particle size of the nanodelivery system is 130-160 nm.

[0010] As a further preferred embodiment, the particle size of the nanodelivery system is 150-160 nm.

[0011] The present invention also provides a method for preparing the above-mentioned biomimetic nanodelivery system based on MRSA exovesicle coating, comprising the following steps:

[0012] S1 Preparation of amphiphilic polymer: Under the catalysis of 4-dimethylaminopyridine, curcumin and glutaric anhydride undergo esterification to generate an intermediate product, single-terminal carboxylated curcumin. Then, under the catalysis of EDC / NHS, the single-terminal carboxylated curcumin is linked to the amino group of chitosan oligosaccharide via an amide bond to synthesize the amphiphilic polymer.

[0013] S2 Preparation of MRSA Exovesicles: MRSA strains were activated and cultured to OD. 600 When the bacterial culture is 1.0, the culture is collected and centrifuged at a centrifugal force of (3000~8000)×g for 10~20 min. The supernatant is filtered through a 0.45 μm sterile filter membrane, the filtrate is collected, and the MRSA exovesicle suspension is obtained by ultrafiltration and concentration for later use.

[0014] S3 Preparation of Nanodelivery System: The amphiphilic polymer obtained in S1 is used as a carrier to self-assemble and encapsulate the drug to obtain drug micelles. Then, the drug micelles are encapsulated with the MRSA exovesicle suspension prepared in S2 to obtain the biomimetic nanodelivery system based on MRSA exovesicle encapsulation.

[0015] In the above preparation method, preferably, in S1, the molar ratio of chitosan oligosaccharide to curcumin is 1:0.1~1, and the molar ratio of monocarboxylated curcumin:EDC:NHS is 1:3~8:3~6.

[0016] As a further preferred embodiment, in S1, the molar ratio of chitosan oligosaccharide to curcumin is 1:0.1~0.5, and the molar ratio of monocarboxylated curcumin:EDC:NHS is 1:4~7:4~6.

[0017] Preferably, in S2, the ultrafiltration concentration is specifically operated as follows: the filtrate is centrifuged at low speed for 3-10 min with a centrifugal force of (3000-4000)×g using an ultrafiltration tube with a relative molecular mass cutoff of 80-200 kDa, and concentrated to 1 / 8 of the original volume. Then, it is ultracentrifuged at a centrifugal force of (100000-200000)×g for 2-5 h. The supernatant is discarded, and the precipitate is resuspended with PBS buffer to obtain MRSA exovesicle suspension.

[0018] Preferably, the weight ratio of the drug to the amphiphilic polymer in S3 is 0.1~0.5:1. Furthermore, the application of a biomimetic nanodelivery system based on MRSA exovesicle coating in periprosthetic infections is also a key technical aspect protected by this invention.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention uses an amphiphilic polymer composed of chitosan oligosaccharide and monocarboxylated curcumin as a carrier to load drugs, forming drug micelles, and then uses MRSA exovesicles to encapsulate the drugs, ultimately obtaining a biomimetic nanodelivery system based on MRSA exovesicle encapsulation.

[0021] In vivo and in vitro experiments have demonstrated that the biomimetic nanodelivery system provided by this invention not only has good safety, stability, and targeting, but also excellent antibacterial effects. It can achieve targeted delivery and long-term release of antibacterial drugs at the site of infection, and is expected to overcome the bottlenecks of drug resistance and insufficient drug penetration in traditional treatments, providing a potential solution for the treatment of diseases such as periprosthetic infections. Attached Figure Description

[0022] Figure 1 This is a diagram illustrating the preparation mechanism of the amphiphilic polymer carrier in Example 1 of the present invention;

[0023] Figure 2 The pyrene fluorescence emission spectrum I in Example 1 of this invention 373 / I 384 Relationship with the logarithm of amphiphilic polymer concentration;

[0024] Figure 3 This is a scanning electron microscope image of the MRSA external vesicles in Embodiment 2 of the present invention;

[0025] Figure 4 This is an in vitro release curve of VAN in different carrier systems in Example 4 of the present invention;

[0026] Figure 5 This is a graph showing the results of the in vitro antibacterial activity evaluation experiment in Example 5 of the present invention;

[0027] Figure 6 This is a graph showing the plate count results of the in vitro antibacterial activity in Example 5 of the present invention;

[0028] Figure 7 This is a diagram showing the results of the co-focusing observation of bacterial live / dead staining experiment in Example 5 of the present invention;

[0029] Figure 8 The figure shows the experimental results of the crystal violet experiment in Example 5 of this invention to evaluate the destructive effect on the mature biofilm of MRSA;

[0030] Figure 9 This is a diagram showing the bacterial life and morphology within the biofilm in Example 5 of the present invention;

[0031] Figure 10 This is a graph showing the results of cell viability detection using the CCK-8 assay in Example 5 of this invention.

[0032] Figure 11 This is a graph showing the results of rat weight and leg circumference measurements during treatment in Example 6 of the present invention;

[0033] Figure 12 This is a graph showing the results of the plate count method for determining the bacterial load in synovial tissue in Example 6 of the present invention;

[0034] Figure 13 OD in Embodiment 6 of the present invention 600 The results of the biofilm assay on the prosthesis are shown in the figure. Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0036] The code names and names used in the following embodiments are as follows:

[0037] CUR: Curcumin, COS: Chitosan oligosaccharide, DMAP: 4-Dimethylaminopyridine, THF: Anhydrous tetrahydrofuran, TEA: Triethylamine, GA: Glutaric anhydride, CUR-COOH: Monocarboxylated curcumin, DMSO: Anhydrous dimethyl sulfoxide, EDC: 1-Ethyl-3-[3-dimethylamino]carbodiimide, NHS: N-hydroxysuccinimide, COS-CUR: Amphiphilic polymer carrier, PBS buffer: Phosphate buffer solution, MRSA: Methicillin-resistant Staphylococcus aureus, EVs: Exovesicles.

[0038] Example 1

[0039] According to the attached Figure 1 The method shown is used to prepare COS-CUR, and the specific steps are as follows:

[0040] Take 0.40 g of CUR and 0.03 g of DMAP in a reaction vessel, add 10 mL of THF to dissolve them, then add 300 μL of LTEA, stir in the dark for 15 min, take 0.14 g of GA, dissolve it in 3 mL of THF and slowly add it dropwise to the above solution, react at 70 °C in the dark for 24 h under nitrogen protection, after the reaction is completed, and obtain CUR-COOH by purification;

[0041] Weigh 50 mg of CUR-COOH into a reaction vessel, add 5 mL of DMSO to dissolve it, then add a certain amount of EDC, stir at room temperature for 1 h, add NHS, and continue stirring to activate for 4 h to obtain an active ester solution, wherein the molar ratio of CUR-COOH:EDC:NHS is 1:5:5;

[0042] A certain amount of COS was weighed into a reaction vessel and dissolved in 10 mL of solvent. The activated active ester solution was slowly added dropwise to the chitosan oligosaccharide solution under rapid stirring. The reaction was carried out at room temperature for 24 h. The reactant solution was placed in a 35 kDa dialysis bag and dialyzed against DMSO for 24 h, then against distilled water for 72 h. After freeze drying, COS-CUR was obtained. The molar ratio of COS to CUR was set to 1:0.1, 1:0.2, 1:0.5, and 1:1, respectively. The solvent was a mixture of DMSO and H2O with a volume ratio of 1:1.

[0043] The performance of the COS-CUR obtained above was verified.

[0044] (1) Calculation of CUR degree of substitution

[0045] The degree of substitution of CUR (DS, mol%) represents the proportion of CUR grafted onto the COS sugar ring, i.e., the number of CUR molecules per 100 COS sugar residues, and is determined using the following method:

[0046] Accurately weigh 1 mg of COS-CUR and dissolve it in DMSO. Using DMSO as a blank control, measure the absorbance of the solution at the maximum absorption wavelength. Substitute the absorbance into the pre-plotted CUR standard curve to obtain the concentration of CUR, and calculate the degree of substitution (DS, mol%) of CUR using the following formula:

[0047] ;

[0048] Note: m CUR This indicates the CUR content determined by the standard curve equation; m polymer Indicates the mass of the added COS-CUR polymer; M COS M represents the relative molecular weight of the sugar units in COS; CUR This indicates the relative molecular weight of CUR.

[0049] The calculated degree of substitution of CUR under different feed ratios is shown in Table 1 below.

[0050] Table 1. Degree of substitution of CUR under different feed ratios

[0051] CUR:COS molar ratio Reaction liquid state DS (%) 0.1：1 Brownish-yellow clear 3.11±0.10 0.2：1 Brownish-yellow clear 8.01±0.13 0.5：1 Very little sediment 8.42±0.08 1：1 Obvious sedimentation 10.90±0.50

[0052] As shown in the table above, the optimal molar ratio of CUR:COS is 0.2:1. This is because the synthesis process of the polymer COS-CUR involves the coupling of the amino group of COS with the single-sided carboxylated curcumin. The number of amino groups on COS affects the curcumin content coupled to the COS backbone. In addition, steric hindrance also restricts the grafting of CUR and COS.

[0053] (2) Determination of critical aggregation concentration

[0054] The critical aggregation concentration (CAC) of the synthesized polymer COS-CUR was determined using the pyrene fluorescent probe method. The results are shown in the attached figure. Figure 2 As shown.

[0055] Appendix Figure 2 As can be seen, the concentration corresponding to the intersection of the two fitted curves is 0.0166 mg / mL, which is the CAC value of COS-CUR, less than 0.050 mg / mL. This indicates that the synthesized amphiphilic polymer can self-assemble into nanoparticles at a low concentration and has a certain degree of stability.

[0056] Example 2

[0057] MRSA exovesicles were prepared using a combination of ultrafiltration concentration and ultracentrifugation. The specific method is as follows:

[0058] (1) Activation and culture of MRSA strain: A single MRSA colony was picked up using a sterile inoculation loop and inoculated into LB liquid medium. The medium was placed in a constant temperature shaking incubator and cultured for 12 h. Then, it was transferred to 400 mL of LB liquid medium to expand the culture to OD. 600 It is 1.0;

[0059] (2) Extraction of EVs by ultrafiltration concentration combined with ultracentrifugation: EVs cultured to OD 600 The bacterial culture with a concentration of 1.0 was centrifuged at 6000g and 4℃ for 15 min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane to remove residual bacteria. The filtered supernatant was then centrifuged at 3500g and 4℃ for 5 min using a Millipore ultrafiltration tube with a cutoff molecular weight of 100 kDa. The supernatant was continuously concentrated until the final liquid volume was 1 / 8 of the original volume. The concentrated supernatant was transferred to a sterile ultracentrifuge tube and ultracentrifuged at 150,000xg and 4℃ for 3 h. The supernatant was discarded, and the precipitate was resuspended in 500 μL of PBS buffer to obtain an EV suspension. The suspension was filtered through a 0.22 μm filter to remove large particulate impurities and stored at -80℃ for later use.

[0060] The extracted EVs were observed under a transmission electron microscope (TEM), and the results are shown in the attached figure. Figure 3 As shown, attached Figure 3 Image A is the unstained image, and image B is the transmission electron microscope image of EVs after negative staining with phosphotungstic acid.

[0061] Appendix Figure 3 As can be seen, the particle size range of the prepared MRSA exovesicles is about 200 nm, with a complete membrane structure, and most of them are round or elliptical.

[0062] Example 3

[0063] 3.1 Preparation of blank micelles

[0064] Take an appropriate amount of the COS-CUR lyophilized powder prepared in Example 1, dissolve it in PBS buffer (pH=7.4), stir at room temperature to fully dissolve and disperse it evenly, and use a probe to sonicate at a frequency of 150 W under ice bath conditions, sonicating for 4 min in a mode of 2 s on and 4 s off. Repeat this process three times. After sonication, the resulting micelle solution is filtered through a 0.8 μm filter membrane to obtain COS-CUR nano micelle solution.

[0065] 3.2 Preparation of COS-CUR / VAN drug-loaded nanomicelles

[0066] 10 mg of COS-CUR prepared in Example 1 was weighed and dispersed in 4 mL of DMF. The mixture was stirred for 1 h to achieve uniform dispersion. Then, 1 mL of DMF solution of VAN of different concentrations was added dropwise, and stirring was continued for more than 4 h. The DMF was evaporated at 80 °C and 100 rpm, and the polymer formed a uniform coating on the wall of the flask. The flask was placed in a vacuum drying oven at room temperature overnight. After overnight drying, 10 mL of deionized water was added, and the mixture was ultrasonically hydrated at 37 °C for 40 min. The ultrasonically hydrated sample was centrifuged at 5000 rpm for 10 min to remove unloaded solid drug. The sample was filtered through a 0.8 μm aqueous microporous membrane to obtain a COS-CUR / VAN drug-loaded nanomicelle solution. The solution was pre-frozen at -80 °C for 12 h and then lyophilized for 36 h to obtain COS-CUR / VAN solid powder.

[0067] 3.3 Fabrication of a nanodelivery system based on MRSA exovesicle coating

[0068] Weigh 10 mg of the COS-CUR / VAN drug-loaded micelle lyophilized powder prepared in 3.2, dissolve it in 5 mL of deionized water, stir for 1 h to disperse it evenly, add the EVs prepared in Example 2 and mix well. Place the solution under the probe for sonication in an ice bath, sonicate for 3 min at a frequency of 50 W with a 2s on and 4s off mode. After sonication, centrifuge the sample at 10000 rpm for 10 min to remove residual EVs. Resuspend the lower precipitate in PBS buffer to obtain vesicle-coated drug-loaded nanomicelles COS-CUR / VAN@EVs.

[0069] To obtain drug-loaded nanoparticles with high drug loading capacity, the effects of different VAN / carrier feed ratios on the drug loading and encapsulation efficiency of the nanoparticles were investigated. The effects of different VAN feed ratios on the drug loading and encapsulation efficiency of the drug-loaded nanomicelles are shown in Table 2 below, where the optimal feed ratio was 2.5:10.

[0070] The particle sizes of the different nanoparticles obtained under the optimal feed ratio are shown in Table 3 below.

[0071] Table 2. Effect of different VAN feed amounts on drug loading and encapsulation efficiency of drug-loaded nanomicelles (n=3)

[0072] VAN: Vector (w / w) DL (%) EE (%) 1.0：10 0.52±0.03 4.38±0.28 2.0：10 3.14±0.35 14.11±1.59 2.5：10 10.05±0.07 36.98±0.26 3.0：10 11.44±0.21 31.65±0.57

[0073] Table 3. Particle size, PDI, and Zeta potential of different nanoparticles (n=3)

[0074] Sample Name Particle size (nm) Dispersion Index (PDI) zata potential (mV) COS-CUR 127.1±0.60 0.217±0.017 8.51±0.16 COS-CUR / VAN 132.4±1.31 0.230±0.018 2.17±0.15 COS-CUR / VAN@EVs 153.3±2.05 0.187±0.035 -4.07±0.59

[0075] The results show that the average particle size of the blank micelles was 127.1 nm, while the average particle size of the drug-loaded micelles increased slightly to 132.4 nm. The hydrophobic portion of vancomycin entered the micelle core, causing slight swelling of the micelles. The increase was small, indicating that the micelle structure was relatively stable and that drug loading did not significantly disrupt the self-assembly of the micelles. The potential decreased slightly after drug loading but remained positive. This may be because, due to space or drug loading capacity limitations, some drug remained adsorbed on the micelle surface or existed through weak interactions, and its charge partially neutralized the original positive charge of the micelles. In addition, vancomycin molecules may have formed a "shielding layer" through physical adsorption or chemical interactions, reducing the exposure of the positive charge on the micelle surface. Drug loading may have introduced buffers or other ions, compressing the electric double layer, which may also have led to a decrease in potential. After drug-loaded micelles were coated with vesicles, the particle size increased by about 20 nm, the PDI decreased relatively, and the potential reversed, indicating successful vesicle coating with a uniform particle size distribution and increased stability.

[0076] Example 4 In vitro sustained-release experiment

[0077] The release behavior of the MRSA-coated nanodelivery system was evaluated using dynamic membrane dialysis. The release medium was PBS buffer (pH 7.4) containing 0.5% Tween 80. The results are shown in the attached figure. Figure 4 As shown.

[0078] The results showed that free vancomycin, being directly exposed to the release medium, was primarily driven by diffusion, resulting in rapid and near-complete release in the initial stage. In contrast, vancomycin release from drug micelles CUR-COS / VAN could be divided into two stages. In the early stage, due to the high drug concentration gradient between the release medium and the hydrophobic core of the nanoparticles, drug diffusion was rapid. In the later stage, due to the decrease in the drug concentration gradient between the release medium and the hydrophobic core of the nanoparticles, carrier dissolution slowed down, and release became slower. Furthermore, the encapsulation of bacterial exovesicles created an additional barrier on the outer layer of the drug-loaded micelles, further slowing down vancomycin release and effectively prolonging the drug release time, making it more suitable for the long-term treatment of periprosthetic infections.

[0079] Example 5: In vitro biological function evaluation

[0080] 5.1 Evaluation of in vitro antibacterial effect

[0081] The antibacterial properties of the components prepared in Examples 1-3 were determined using the micro-broth serial dilution method. The minimum inhibitory concentrations (MICs) of VAN, COS-CUR, COS-CUR / VAN, and COS-CUR / VAN@EVs against MRSA were also determined, as shown in the attached figure. Figure 5 As shown, Figure 5 Figure A shows that the minimum inhibitory concentration (MIC) of VAN against the airborne bacterium MRSA is 0.5 μg / mL, and the MIC of COS-CUR against MRSA is 32 μg / mL. At a drug loading of 10.05 ± 0.07%, the MICs of COS-CUR / VAN and COS-CUR / VAN@EVs are 2 μg / mL (containing approximately 0.2 ug / mL of VAN). This indicates that the blank vector COS-CUR has certain antibacterial activity, and the drug-loaded micelles, after being loaded with vancomycin, exert a synergistic antibacterial effect, with a decrease in MIC compared to VAN alone and the blank vector. Compared to COS-CUR / VAN, the encapsulation of bacterial outer membrane vesicles may have delayed drug release to some extent. Although COS-CUR / VAN@EVs did not significantly improve the MIC against planar MRSA, COS-CUR / VAN@EVs showed stronger antibacterial activity at 1 ug / mL, suggesting that the presence of outer vesicles may improve drug stability, enhance the binding of the formulation to bacteria, and have potential targeting effects. This indicates that drug-loaded micelles encapsulated in vesicles may have better antibacterial potential at low concentrations and are more advantageous in scenarios with low antibiotic sensitivity or requiring higher targeting.

[0082] In addition, the inhibition zone test ( Figure 5 B / 5C) and plate counting experiment ( Figure 6 The results showed that the blank carrier COS-CUR itself had some antibacterial activity, but its effect was weak and could only assist in enhancing the performance of antibacterial drugs. Drug-loaded micelles COS-CUR / VAN significantly improved antibacterial activity by increasing drug solubility and local concentration, and by exerting a synergistic antibacterial effect with curcumin and chitosan oligosaccharides. Encapsulation by outer membrane vesicles further enhanced the antibacterial effect of COS-CUR / VAN@EVs, indicating that its delivery and targeting capabilities were significantly superior to those of single micelles, and its antibacterial activity was enhanced. This suggests that vesicle-loaded drug micelles may provide a more effective treatment method for drug-resistant bacterial infections.

[0083] 5.2 Bacterial live / dead staining experiment

[0084] SYTO9 is a small-molecule green fluorescent dye capable of penetrating intact cell membranes. It binds to bacterial nucleic acids and emits green fluorescence, making it suitable for labeling all bacteria. PI is a basic dye with a larger structure, unable to penetrate intact cell membranes. It can only penetrate damaged or ruptured cell membranes, binding to nucleic acids and emitting red fluorescence. The SYTO9 / PI staining kit can be used to detect the integrity of bacterial cell membranes. The staining results of MRSA after treatment with different nano-formulations are shown in the attached image. Figure 7 As shown in the figure, Figure A shows MRSA live-dead chromatograms (CLSM) after treatment with different formulations, and Figure B shows the quantitative results of fluorescence intensity. The control group exhibits strong green fluorescence, while the bacteria treated with VAN, COS-CUR / VAN, and COS-CUR / VAN@EVs show significantly enhanced red fluorescence, especially the COS-CUR / VAN@EVs group. The quantitative results of fluorescence intensity are shown in the right figure, suggesting that the blank carrier COS-CUR may have caused some degree of membrane damage to bacteria, but its bactericidal effect is limited. Both free VAN and the micelle-loaded groups showed significant red fluorescence, with intensities of 69% and 74%, respectively, indicating that both had a strong bactericidal effect on bacteria. Because micelles improve the solubility and cell membrane penetration of vancomycin, the COS-CUR / VAN group had a higher bactericidal efficiency. The vesicle encapsulation improved the targeting and stability of the micelles and enhanced the interaction between the drug and bacteria. The COS-CUR / VAN@EVs group showed the strongest red fluorescence, with a red fluorescence intensity of 85%, indicating that the group had the best bactericidal effect and caused significant damage to the cell membrane of MRSA strains.

[0085] 5.3 Determination of biofilm clearance rate by crystal violet method

[0086] Crystal violet assay was used to evaluate its disruptive effect on mature MRSA biofilms; the results are attached. Figure 8 As shown in Figure A, the well plates exhibited different shades of purple after dye dissolution, and the calculated biofilm clearance rate is shown in Figure B. Low concentrations of VAN showed weak biofilm-destructive ability, and no significant difference was observed between different formulations. However, the biofilm-destructive ability of nanoparticles increased with increasing concentration. Specifically, VAN alone had limited biofilm-destructive ability, limited by its permeability and significantly affected by concentration. COS-CUR / VAN, due to its improved drug stability and synergistic antibacterial effect, outperformed free VAN at high concentrations. At a VAN concentration of 10 μg / mL, COS-CUR / VAN@EVs achieved the highest biofilm clearance rate (69.3%), significantly better than free VAN (51.2%). This indicates that the EVs coating effectively enhanced VAN permeation and antibacterial activity within the biofilm, demonstrating that EVs possess good membrane fusion and targeting capabilities. COS-CUR / VAN@EVs shows potential in anti-biofilm activity.

[0087] CLSM was used to observe the bacterial viability and morphology within the biofilm. The results are shown in the attached figure. Figure 9 As shown in Figure A, the calculated biofilm clearance rate is shown in the appendix. Figure 9 B, consistent with the results of the crystal violet staining method, further demonstrates that the COS-CUR / VAN@EVsMRS biofilm has a better clearance effect compared to free VAN.

[0088] 5.4 CCK-8 assay for cell viability and cell live / dead staining

[0089] While ensuring antibacterial properties, the biocompatibility of the material is also an important indicator for evaluating its potential clinical applications. The results of cell viability determination using the CCK-8 assay are attached. Figure 10 As shown in Figure A, within the concentration range of 0.5-200 μg / mL, the VAN group, COS-CUR group, VAN / COS-CUR group, and VAN / COS-CUR@EVs group did not significantly affect the viability of L929 cells. Bacterial live / dead staining results are attached. Figure 10 As shown in Figures B / C, cell viability was observed under an inverted fluorescence microscope using Calcein-AM (live cells, green) and PI (dead cells, red) double staining. Almost all cells showed green fluorescence, consistent with the results verified by the CCK-8 assay. This indicates that the nano-formulation has no significant cytotoxicity to host cells and exhibits good cell compatibility, supporting the possibility of further in vivo experiments to evaluate its efficacy and long-term safety in the PJI model.

[0090] Example 6 In vivo biocompatibility assessment

[0091] 6.1 PJI Model Evaluation

[0092] A rat model of PJI bacterial infection was established by implanting a titanium rod into the knee joint and inoculating it with methicillin-resistant Staphylococcus aureus (MRSA). One week post-surgery, significant redness and swelling were observed at the knee joint in the MRSA-infected group, indicating postoperative infection and inflammatory response. CT scans of the rats showed that the titanium rod was stably implanted in the femoral medullary cavity without significant displacement or fracture, and that surrounding bone was destroyed, ensuring the successful anatomical establishment of the model. Hematological tests showed significantly elevated systemic inflammatory markers. Compared to the Sham group, other groups inoculated with MRSA and implanted with titanium rods had significantly higher neutrophil percentages and white blood cell counts, consistent with the acute infection state of PJI.

[0093] The above indicators further confirm the successful establishment of the model and are consistent with the clinical characteristics associated with PJI.

[0094] The results of rat weight and leg circumference measurements during treatment are attached. Figure 11As shown in the figure, during the treatment period, the body weight of rats in all groups showed an increasing trend, indicating that the animals were generally in good health and no significant consumption due to infection or treatment occurred. The PBS group showed a slower increase in body weight, suggesting that persistent infection may cause increased metabolic burden or decreased appetite, consistent with the characteristics of chronic inflammation leading to growth restriction in the PJI animal model. After the administration was completed, there was no significant difference in body weight among the groups, indicating that the treatment groups could effectively control the infection and did not affect the growth and development of the animals, and also indicating that the treatment had no obvious toxic effects on the body. The leg circumference of all groups showed a decreasing trend, indicating that the inflammation gradually subsided, but the rate of decrease varied among the groups, reflecting to some extent the differences in the severity of infection and the treatment effect. After the treatment, obvious redness and swelling were still visible in the PBS group, indicating that the infection persisted. The leg circumference of the Sham group was smaller and the decreasing trend was obvious, indicating that the implantation of sterile titanium rods did not cause a persistent inflammatory response, further proving that infection was the key factor leading to abnormally large leg circumference. The leg circumference of other treatment groups gradually decreased, indicating that antibacterial treatment effectively relieved the inflammation caused by infection.

[0095] 6.2 Determination of bacterial load in synovium and soft tissue

[0096] Synovial tissue infection is a significant source of infection spread and persistent inflammation; synovial hyperplasia and inflammatory infiltration are frequently observed during surgical debridement. To directly assess the effectiveness of the preparation in controlling intratissue infection, the plate count method was used to determine the bacterial load in synovial tissue. The experimental results are attached. Figure 12 As shown in the figures, Figure A is a photograph of the synovial tissue soaking solution plate, and Figure B shows the statistical values ​​of the bacterial colony count on the plate. The figures show that no significant bacterial growth was observed in the Sham group plate culture, but a large number of golden-yellow bacteria were observed, indicating that MRSA proliferated vigorously in the synovial tissue in the untreated state, consistent with the pathological characteristics of infection. The bacterial counts in the VAN and COS-CUR groups decreased, but a large number of colonies remained, indicating that while free VAN can reduce the bacterial load in tissues, its effectiveness is limited due to its susceptibility to biofilms and tissue barriers, resulting in a low concentration at the target site. The COS-CUR / VAN group showed a further decrease in colony count; the COS-CUR / VAN@EVs group showed only scattered colonies, demonstrating the best antibacterial effect, which may be related to the nanocarrier enhancing the local retention, permeability, and controlled release capacity of vancomycin. Simultaneously, the chitosan oligosaccharide-curcumin copolymer (COS-CUR) itself has certain antibacterial and immunomodulatory effects, further enhancing infection control after combination with antibiotics.

[0097] 6.3 Determination of bacterial load in prosthetic biofilm

[0098] Biofilm formation on the prosthesis surface is a core issue hindering the treatment of polymyxin B (PJI). Bacteria within the biofilm are highly resistant to antibiotics and difficult to eradicate, significantly contributing to PJI treatment failure and infection recurrence. To further evaluate the inhibitory and clearance capabilities of the formulation against biofilm bacteria, the prosthesis was removed, cultured in LB medium, and OD (octane rating) was measured. 600 The biofilm bacterial load on the prosthesis was assessed, and the experimental results are attached. Figure 13 As shown in Figure A, the turbidity of the culture medium was observed; the OD of the bacterial culture was... 600 The quantitative values ​​are shown in Figure B. As can be seen from the figure, the PBS group showed the most vigorous bacterial growth and turbidity. Both the VAN and COS-CUR groups partially inhibited biofilm bacteria, but still maintained a high survival rate. The COS-CUR / VAN group significantly reduced the bacterial load within the biofilm, with lower turbidity, indicating that COS-CUR can enhance the antibacterial effect of vancomycin. The COS-CUR / VAN@EVs group, further encapsulated with EVs, exhibited the strongest antibacterial effect, with the clearest bacterial solution and OD... 600 The lowest value indicates that it can more effectively penetrate biofilms and clear bacteria from the implant surface. Combined with the aforementioned synovial tissue bacterial load measurement results, the PBS group had high levels of inflammatory factors and a heavy bacterial load, while the COS-CUR / VAN@EVs group achieved the best antibacterial effect on both synovial tissue and prosthesis surface. This shows that the nano-formulation can not only inhibit the inflammatory response but also effectively reduce the survival of bacteria in biofilms, thus providing a more advantageous strategy for the treatment of PJI.

Claims

1. The application of a biomimetic nanodelivery system based on MRSA exovesicle coating in the preparation of drugs for treating periprosthetic infections, characterized in that, The biomimetic nanodelivery system includes MRSA exovesicles and drug micelles encapsulated thereon. The drug micelles are carried by an amphiphilic polymer composed of chitosan oligosaccharide and monocarboxylated curcumin. The drug is loaded onto the amphiphilic polymer through self-assembly. The drug is vancomycin. The amphiphilic polymer was prepared by the following method: under the catalysis of 4-dimethylaminopyridine, curcumin and glutaric anhydride underwent an esterification reaction to generate the intermediate product monocarboxylated curcumin. Then, under the catalysis of EDC / NHS, the monocarboxylated curcumin was linked to the amino group of chitosan oligosaccharide via an amide bond to synthesize the amphiphilic polymer. The molar ratio of chitosan oligosaccharide to curcumin is 1:0.2; The weight ratio of vancomycin to the amphiphilic polymer is 1:

4.

2. The application as described in claim 1, characterized in that, The particle size of the biomimetic nanodelivery system is 130-160 nm.

3. The application as described in claim 1, characterized in that, The preparation method of the biomimetic nanodelivery system based on MRSA exovesicle coating includes the following steps: S1 Preparation of amphiphilic polymer: Under the catalysis of 4-dimethylaminopyridine, curcumin and glutaric anhydride undergo esterification to generate the intermediate product monocarboxylated curcumin. Then, under the catalysis of EDC and NHS, the monocarboxylated curcumin is linked to the amino group of chitosan oligosaccharide via an amide bond to synthesize the amphiphilic polymer. S2 Preparation of MRSA Exovesicles: MRSA strains were activated and cultured to OD. 600 When the bacterial culture is 1.0, the culture is collected and centrifuged at a centrifugal force of (3000~8000)×g for 10~20 min. The supernatant is filtered through a 0.45 μm sterile filter membrane, the filtrate is collected, and the MRSA exovesicle suspension is obtained by ultrafiltration and concentration for later use. S3 Preparation of a biomimetic nanodelivery system: The amphiphilic polymer obtained in S1 is used as a carrier to self-assemble and encapsulate the drug to obtain drug micelles. Then, the MRSA exovesicle suspension prepared in S2 is used to encapsulate the drug micelles to obtain the biomimetic nanodelivery system based on MRSA exovesicle encapsulation.

4. The application as described in claim 3, characterized in that, The molar ratio of monocarboxylated curcumin:EDC:NHS in S1 is 1:3~8:3~6.

5. The application as described in claim 3, characterized in that, In S2, the specific operation of ultrafiltration concentration is as follows: the filtrate is centrifuged at low speed for 3-10 min with a centrifugal force of (3000-4000)×g using an ultrafiltration tube with a relative molecular mass cutoff of 80-200 kDa, and concentrated to 1 / 8 of the original volume. Then, it is ultracentrifuged at a centrifugal force of (100000-200000)×g for 2-5 h. The supernatant is discarded, and the precipitate is resuspended with PBS buffer to obtain MRSA exovesicle suspension.

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