A nano-drug-loaded hydrogel catheter coating and a preparation method and application thereof

Abstract

The present application belongs to the field of medical material surface functionalization, and particularly relates to a kind of nano drug-loaded hydrogel catheter coating and its preparation method and application.The present application utilizes PLGA to load antibiotic amikacin sulfate AMK or antibacterial peptide FK13-a1, synthesizes nanoparticle NP-AM and nanoparticle NP-FK, prepares NP-AM / FK@OMV nanoparticle complex with EcN OMV as carrier, then adds poloxamer-hyaluronic acid composite hydrogel to obtain NP-AM / FK@OMV-P / H nano drug-loaded hydrogel, and forms coating after coating and drying.The nano drug-loaded hydrogel coating provided by the present application has the characteristics of enzyme response, antibacterial, prevention of biofilm formation and low toxicity, has great application potential in inhibiting medical catheter surface biofilm, and has important significance for preventing and treating CAUTI, prolonging the use time of indwelling catheter, and reducing the discomfort of patients caused by frequent replacement of catheter.

Description

Technical Field

[0001] This invention belongs to the field of surface functionalization of medical materials, specifically relating to a nano-drug-loaded hydrogel urinary catheter coating, its preparation method, and its application. Background Technology

[0002] Nearly 150 million cases of urinary tract infection (UTI) occur globally each year, with bacterial infection being one of the main causes. Approximately 70-80% of complicated UTIs are related to indwelling catheters.

[0003] Catheter-associated urinary tract infection (CAUTI) is a highly prevalent hospital-acquired infection. The most common pathogen is urinary tract pathogenic Escherichia coli (UPEC), which adheres to the catheter surface via adhesins secreted by type I fimbriae (FimH) to form a biofilm, which is the main cause of CAUTI.

[0004] When CAUTI occurs, it can cause local or systemic symptoms such as pyelonephritis, local and systemic bacteremia, urinary tract stones, bladder cancer, and even death. Preventing CAUTI solely through nursing interventions, such as shortening catheter usage time, using sterile equipment and techniques for catheter insertion, and other alternative methods, is not significantly effective in reducing the incidence of CAUTI. Currently, the main methods for CAUTI prevention are still the use of antibiotics and / or functional catheters, which involve impregnating the catheter with an antibacterial coating. This coating, which may contain preservatives, antibacterial agents, and metal ions, modifies the surface to reduce infection and prolong catheter life. Developing novel nanoparticle-loaded hydrogel catheter coatings that resist bacterial colonization or biofilm formation shows great promise.

[0005] Bacterial outer membrane vesicles (OMVs) are spherical, bilayered lipid nanostructures with a diameter of 20–300 nm, naturally secreted by all Gram-negative bacteria and some Gram-positive bacteria during normal growth. Numerous studies have explored the application of OMVs in various biomedical fields, such as vaccine adjuvants, tumor immunotherapy, drug carriers, and antibacterial active substances. Utilizing the highly active EcN OMVs of the bacterium UPEC, which has high similarity to its parent bacterium EcN, OMVs can be used as nanocarrier materials to deliver antibiotics. This not only improves UPEC's drug uptake and sensitivity but also prevents the delivered drug from being intercepted by the bacterial outer membrane, significantly enhancing the efficacy of antibiotic treatment and reducing drug resistance, demonstrating enormous application potential in the antibacterial field.

[0006] Amikacin sulfate, an aminoglycoside antibiotic clinically used to treat complicated urinary tract infections, was selected. Its antibacterial mechanism targets the 30S subunit of the ribosome, inhibiting bacterial protein synthesis and disrupting bacterial cell wall integrity, leading to bacterial death. However, due to its toxic side effects, strict dosage control is required in clinical application. Through preliminary screening, the research group discovered that combining amikacin with the short α-helical antimicrobial peptide FK13-a1 derived from LL-37 can enhance its antibacterial effect, reduce antibiotic dosage, and decrease toxic side effects.

[0007] To address diverse scenarios and meet usage and infection prevention needs, the requirements for the functionality of implant materials are becoming increasingly stringent, demanding properties such as adjustable release, self-adaptation, and intelligent response. This is crucial for developing a diverse range of antimicrobial coating systems. Therefore, leveraging the advantages of bacterial outer membrane vesicles (OMVs) and the combined use of antibiotics and antimicrobial peptides in the antimicrobial field, developing a coating capable of antimicrobial and antibiofilm formation to resist the adhesion of multidrug-resistant E. coli and inhibit the formation of bacterial biofilms on the catheter surface is of great significance for preventing CAUTI, prolonging the use time of indwelling catheters, reducing medical costs, alleviating the economic burden on patients, and reducing the discomfort caused by frequent catheter changes. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention aims to provide a nano-drug-loaded hydrogel catheter coating, its preparation method, and its applications. This invention utilizes a nano-drug-loaded system of antimicrobial peptide FK13-a1 synergistic with antibiotic AMK, employing a poloxamer (PF127)-hyaluronic acid (HA) hydrogel as a HAase-responsive intelligent antimicrobial coating physically adsorbed onto the surface of Foley catheter materials. This achieves more precise and efficient bactericidal effects while simultaneously providing antifouling properties. It effectively inhibits the adhesion of multidrug-resistant E. coli and suppresses the formation of bacterial biofilms on the catheter surface, preventing CAUTI, extending the lifespan of indwelling catheters, reducing medical costs, alleviating the economic burden on patients, and reducing the discomfort caused by frequent catheter changes.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first objective of this invention is to provide a method for preparing a nanoparticle drug-loaded hydrogel urinary catheter coating. This method utilizes PLGA to encapsulate the antibiotic amikacin sulfate (AMK) or the antimicrobial peptide FK13-a1 to synthesize NP-AM and NP-FK nanoparticles. Using EcN OMV as a carrier, an NP-AM / FK@OMV nanoparticle composite is prepared. This composite is then co-mixed with a poloxamer-hyaluronic acid composite hydrogel to obtain an NP-AM / FK@OMV-P / H nanoparticle drug-loaded hydrogel. After coating, the coating is dried to form a coating layer. The specific preparation method includes the following steps:

[0011] Separation and purification of S1, EcN OMV;

[0012] S2. Using the W / O / W emulsification-solvent evaporation method, polylactic acid-glycolic acid copolymer (PLGA) was used to encapsulate the antibiotic amikacin sulfate AMK or the antimicrobial peptide FK13-a1 to synthesize nanoparticles NP-AM and NP-FK.

[0013] S3. Encapsulate the NP-AM and NP-FK nanoparticles synthesized in step S2 with the EcN OMV extracted in step S1 to obtain the NP-AM / FK@OMV nanoparticle composite.

[0014] S4. Preparation of sterile poloxamer-hyaluronic acid composite hydrogel;

[0015] S5. The NP-AM / FK@OMV nanoparticle composite obtained in step S3 is mixed into the poloxamer-hyaluronic acid composite hydrogel to form NP-AM / FK@OMV-P / H nanoparticle drug-loaded hydrogel.

[0016] S6. Using an impregnation coating process, uniformly coat the NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel from step S5 onto the clean inner wall of the catheter, and then vacuum dry to form a coating.

[0017] Preferably, the amino acid sequence of the antimicrobial peptide FK13-a1 is shown in SEQ ID NO.1.

[0018] WKRIVRRIKRWLR-NH2 (SEQ ID NO. 1).

[0019] Preferably, the bacterial outer membrane vesicles EcN OMV described in step S1 are derived from... E. coli Nissle 1917, strain number: DSM 6601; the EcN OMV separation and purification includes ultrafiltration concentration-ultracentrifugation, taking samples from the logarithmic growth phase. E. coli Nissle The culture was expanded to 500 mL in 1917, and then filtered and concentrated to 1 / 5 to 1 / 7 of the original volume. EcN OMV was purified by low-temperature ultracentrifugation, and the precipitate was then resuspended in 1 mL of sterile PBS to obtain EcN OMV resuspension.

[0020] Preferably, the polylactic acid-glycolic acid copolymer PLGA in step S2 is composed of lactic acid LA and glycolic acid GA in a ratio of 50:50;

[0021] The W / O / W emulsification-solvent evaporation method for synthesizing nanoparticles NP-AM and NP-FK includes the following steps: Under ice bath conditions, polymer PLGA is dissolved in dichloromethane (DCM), and FK13-a1 or AMK is dissolved in 0.5% (w / v) polyvinyl alcohol (PVA) to prepare an inner aqueous phase solution. The inner aqueous phase is added dropwise to the dichloromethane solution containing PLGA to form a W / O emulsion. Subsequently, it is added to a 1% (w / v) sterile aqueous solution of PVA for further emulsification to form a W / O / W emulsion. The dichloromethane is evaporated, centrifuged, and the precipitate is washed with sterile water. The nanoparticle precipitate obtained by centrifugation is resuspended in an appropriate amount of sterile water, transferred to a centrifuge tube, pre-frozen, and freeze-dried to obtain lyophilized powders of nanoparticles NP-AM and NP-FK.

[0022] Preferably, step S3 includes preparing the EcN OMV extracted in step S1 into a resuspension, taking a certain proportion of the lyophilized nanoparticles NP-AM and NP-FK synthesized in step S2, resuspending them in OMV resuspension instead of sterile water, stirring evenly, and sonicating in a water bath to form a nanoparticle dispersion, thereby obtaining a suspension of the OMV-coated nanoparticle composite NP-AM / FK@OMV.

[0023] Preferably, the ratio of the NP-AM nanoparticles to the NP-FK lyophilized powder is NP-AM:NP-FK = 1:1~4 (w / w).

[0024] Preferably, the poloxamer used in step S4 is PF127 (Pluronic ® F-127), with a mass fraction of 20% (w / v); the hyaluronic acid has a molecular weight of 40-100 kDa and a mass fraction of 1% (w / v);

[0025] The preparation of sterile poloxamer-hyaluronic acid composite hydrogel includes the following steps: 20% (w / v) poloxamer is added to water and dissolved in an ice bath. After complete dissolution, 1% (w / v) hyaluronic acid powder is added and stirred evenly to disperse the solution. The solution is allowed to stand overnight at 0-4℃ to defoam, sterilized, and stored at 4℃ for later use.

[0026] Preferably, the preparation of the NP-AM / FK@OMV-P / H nanoparticle drug-loaded hydrogel in step S5 includes: adding the NP-AM / FK@OMV nanoparticle composite suspension obtained in step S3 dropwise to the poloxamer-hyaluronic acid hydrogel while stirring, to form a uniformly colored and particle-dispersed NP-AM / FK@OMV-P / H nanoparticle drug-loaded composite hydrogel.

[0027] A second objective of this invention is to provide a nano-drug-loaded hydrogel catheter coating NP-AM / FK@OMV-P / H prepared by the method described above.

[0028] Another object of the present invention is to provide the application of the above-described nano-drug-loaded hydrogel catheter coating NP-AM / FK@OMV-P / H in inhibiting biofilms on the surface of medical catheters.

[0029] A schematic diagram of the synthesis of the NP-AM / FK@OM-P / H nano-drug-loaded hydrogel coating of this invention is shown below. Figure 1 As shown. This invention utilizes PLGA to encapsulate the antibiotic amikacin sulfate (AMK) or the antimicrobial peptide FK13-a1 to synthesize NP-AM and NP-FK nanoparticles. Using EcN OMV as a carrier, an NP-AM / FK@OMV nanoparticle composite is prepared, which is then co-mixed with poloxamer-hyaluronic acid composite hydrogel to obtain NP-AM / FK@OMV-P / H drug-loaded nanogel. After coating and drying, a coating layer is formed. This invention prepares a medical coating of drug-loaded nanogel. By adding OMV as the "coating" of the nanoparticles, it achieves a "targeted" bactericidal effect, compared with the control strain. E. coli ATCC 25922, this coating has a more significant effect on multidrug-resistant Escherichia coli strains. Therefore, the OMV nano-drug delivery system provided by this invention is applied to inhibit biofilm on the surface of medical catheters, exhibiting enzymatic responsiveness, antibacterial properties, prevention of biofilm formation, and low toxicity.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) This invention developed a nano-drug delivery system of antimicrobial peptide FK13-a1 synergistic with antibiotic AMK and used poloxamer (PF127)-hyaluronic acid (HA) hydrogel as a HAase-responsive smart antimicrobial coating to physically adsorb onto the surface of Foley catheter material. This can achieve more precise and efficient bactericidal effect, and at the same time has antifouling properties. It realizes the adhesion of multidrug resistant Escherichia coli and inhibits the formation of bacterial biofilm on the surface of the catheter, thus achieving the effect of preventing and treating CAUTI.

[0032] (2) The antibacterial and anti-biofilm studies of the NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating prepared in this invention were conducted. It was found that the NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating showed a significant effect in inhibiting the formation of biofilm on the surface of medical catheters. At the same time, it also reduced the toxicity of the antibiotic AMK and improved its narrow therapeutic window.

[0033] (3) The NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating prepared by the method of the present invention is applied to the surface of catheter materials such as urinary catheters. This can prolong the service time of indwelling urinary catheters, reduce medical costs, alleviate the economic burden on patients and the discomfort caused by frequent replacement of urinary catheters. Attached Figure Description

[0034] Figure 1 A schematic diagram of the synthesis of NP-AM / FK@OM-P / H nanoparticle drug-loaded hydrogel coating;

[0035] Figure 2 NTA plot of EcN OMV;

[0036] Figure 3 SDS-PAGE electrophoresis image of EcN OMV;

[0037] Figure 4 MIC, MBC, and FIC of FK13-a1, AMK-treated strains GIM1.457, and ATCC 25922;

[0038] Figure 5 Standard curves for the determination of NP-FK and NP-AM content, encapsulation efficiency, and drug loading rate;

[0039] Figure 6 DLS and Zeta potential results for NP, NP-AM, NP-FK, NP-AM / FK@OMV, and OMV;

[0040] Figure 7 TEM images for EcN OMV, NP-AM, NP-FK, and NP-AM / FK@OMV;

[0041] Figure 8 SEM image of the NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating;

[0042] Figure 9 The contact angle of the NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating;

[0043] Figure 10 The cumulative in vitro release rate of the NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating in different media;

[0044] Figure 11 Evaluation of the hemolytic properties of the nano-drug-loaded hydrogel coating on mouse blood, rat blood, and human blood;

[0045] Figure 12 Evaluation of the cytotoxicity of the nano-drug-loaded hydrogel coating against NIH3T3, L02, and HK-2;

[0046] Figure 13 Antibacterial effect diagram of NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating;

[0047] Figure 14 Bactericidal effect diagram of NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating under fluorescence microscope;

[0048] Figure 15 Anti-biofilm effect diagram of NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating;

[0049] Figure 16 Result diagram of the in vivo preventive effect of NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating on biofilm formation. Detailed implementation manners

[0050] The above content of the present invention will be further described in detail through the following specific implementation manners in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples.

[0051] Experimental animals and materials

[0052] 1. Mice: SPF-grade C57BL / 6 female wild-type mice were all purchased from the Guangdong Provincial Center for Medical Laboratory Animals. The production license number of experimental animals: SCXK (Guangdong) 2022-0002, and the animal use license code of the experimental unit: SYXK (Guangdong) 2017-0125.

[0053] 2. Rats: SPF-grade SD rats were purchased from Guangzhou Jinwei Biotechnology Co., Ltd. The production license number of experimental animals: SYXK (Beijing) 2019-0010.

[0054] 3. Mouse embryonic fibroblasts NIH3T3, human renal cortical proximal tubular epithelial cells HK-2, and human normal hepatocytes L02 were all purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0055] 4. BCA protein concentration assay kit was purchased from Beyotime Biotechnology Co., Ltd. in Shanghai, model P0010.

[0056] Example 1 Isolation and purification of EcN OMV

[0057] Ultrafiltration concentration-ultracentrifugation method: Bacterial culture cultured to the late logarithmic growth phase was placed in a centrifuge tube and centrifuged at 4°C and 4000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter to remove residual bacteria and cell debris. The filtrate was placed in a Millipore 15 mL ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and concentrated by centrifugation at 4°C and 4000 rpm for 5 min, approximately concentrating to 1 / 5 to 1 / 7 of the original volume. The precipitate was then ultracentrifuged at 4°C and 100,000 × g for 3 h using a BECKMAN COULTER ultracentrifuge (rotor: SW 32 Ti). The precipitate was then resuspended in 1 mL of sterile PBS. The resulting EcN OMV can be stored for short-term at -20°C and long-term at -80°C.

[0058] Figure 2 The NTA analysis results indicate that the average particle size of OMV is mainly concentrated between 100 and 200 nm.

[0059] Example 2: EcN OMV SDS-PAGE electrophoresis

[0060] The protein concentration of the OMV extract prepared in Example 1 was determined according to the instructions of the BCA protein concentration assay kit (Beyotime, Shanghai Beyotime Biotechnology Co., Ltd., model P0010). An appropriate amount of sample was taken and mixed at a ratio of 1 (10×Loading Buffer): 9 (protein sample). The mixture was then heated in a metal bath at 95°C for 10 min to denature the sample before it could be used for loading.

[0061] Prepare a 12% separating gel and inject it into the gel holder. After solidification, add a 5% stacking gel and insert a comb. Pour electrophoresis buffer into the electrophoresis tank, install the formed gel onto the electrophoresis apparatus, and remove the comb to load the samples. The sample loading volume for each group is approximately 30 μg. Maintain a constant voltage of 80V for 20 min, then adjust the voltage to 120V and electrophoresis for 1 h until the bands reach the bottom of the gel.

[0062] The gel was stained with Coomassie Brilliant Blue R250 staining solution for 1 hour, then the destaining solution was changed to remove the gel background until it was transparent and the bands were clear. Finally, the gel bands were photographed using a gel imaging system.

[0063] Figure 3 The SDS-PAGE electrophoresis results showed the presence of OMV marker protein bands OmpA (35kDa) and OmpC and OmpF (38kDa), further indicating that EcN-OMV was successfully extracted.

[0064] Example 3 Synthesis of NP-loaded drug nanoparticles (NP-AM, NP-FK)

[0065] Under ice bath conditions, 100 mg of polymer PLGA (50:50) was completely dissolved in 4 mL of dichloromethane. An internal aqueous phase solution was prepared by dissolving 10 mg of FK13-a1 or 10 mg of AMK in 200 μL of 0.5% (w / v) PVA. This internal aqueous phase was added dropwise to the PLGA-dichloromethane solution, and the mixture was sonicated for 2 min, 3 s intervals with a 2 s interval, at an amplitude of 30%, to form a primary W / O milky white emulsion. The primary W / O emulsion was then added dropwise to 15 mL of 1% (w / v) polyvinyl alcohol (PVA) solution while stirring. After homogenization, further emulsification was performed to form a W / O / W emulsion. The mixture was magnetically stirred at 37°C and 600 rpm for 3 h to allow complete evaporation of the dichloromethane. The mixture was then centrifuged at 4°C and 8000 rpm for 30 min. The precipitate was washed twice with sterile water, repeating the above steps. The precipitate obtained by centrifugation was resuspended in an appropriate amount of sterile water, ultrasonically dispersed in a water bath for 5 min, transferred to a centrifuge tube, pre-frozen at -80℃ overnight, and freeze-dried for 24 h to obtain lyophilized powders of nanoparticles NP-AM (AMK) and NP-FK (FK13-a1). Empty nanoparticles (NP) can be prepared using the same process.

[0066] Figure 4 The MIC, MBC, and FIC results for FK13-a1, AMK-treated strains GIM1.457, and ATCC 25922 indicate that the antimicrobial peptide FK13-a1 has a synergistic antimicrobial effect against the antibiotic AMK.

[0067] The FK13-a1 antimicrobial peptide, whose amino acid sequence is shown in SEQ ID NO.1, can be synthesized by commercial companies. In this invention, the synthesis and purification were entrusted to Jier Biochemical (Shanghai) Co., Ltd.

[0068] WKRIVRRIKRWLR-NH2 (SEQ ID NO. 1).

[0069] Example 4: Standard curve for the determination of FK13-a1 and AMK content

[0070] Following the instructions of the BCA kit, standard curves were prepared for FK13-a1 concentration gradients of 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.025, and 0 mg / mL. Absorbance was measured at 570 nm using a microplate reader. OD values ​​were then used as the analytical parameters. 570 nm Using the vertical axis as the ordinate and the standard concentration gradient as the horizontal axis, a linear regression equation is fitted to plot the protein content standard curve.

[0071] Take an appropriate amount of 1 mg / mL AMK solution into a 10 mL EP tube, add 2 mL of 10 g / L ninhydrin solution and 1.5 mL of 0.1 mol / L pH 6.5 acetate-sodium acetate solution, mix well, and heat in a boiling water bath (99.9℃) for 75 min. Remove and cool to room temperature, then dilute to the 10 mL mark with ddH2O. Using an AMK-free blank as a reference, measure the absorbance at a wavelength of 402 nm. Prepare AMK concentration gradients of 0.4, 0.3, 0.2, 0.1, 0.05, and 0 mg / mL, and measure the absorbance at 402 nm using UV spectrophotometry. (Using ABS...) 402 nm Using the vertical axis as the ordinate and the gradient concentration of the standard as the horizontal axis, a linear regression equation is fitted to plot the standard curve of AMK content.

[0072] Calculate the encapsulation efficiency and drug loading rate using the following formulas:

[0073]

[0074]

[0075] Figure 5 The standard curves for FK13-a1 and AMK showed good linearity. The encapsulation efficiency of synthesized PLGA loaded with 10 mg AMK (NP-AM) nanoparticles was 82.94±0.44%, and the drug loading rate was 9.20±0.05%. The encapsulation efficiency of synthesized PLGA loaded with 10 mg FK13-a1 (NP-FK) nanoparticles was 75.91±3.21%, and the drug loading rate was 8.11±0.34%.

[0076] Example 5: OMV encapsulation of NP-AM and NP-FK nanoparticles yields NP-AM / FK@OMV composites.

[0077] Weigh out a certain proportion (≈1:1~4) of lyophilized NP-AMK and NP-FK13-a1 nanoparticle powders, and resuspend them in OMV resuspension instead of sterile water (nanoparticle:OMV mass ratio ≈10:1). Repeatedly pipetting, stirring, and sonicating in a water bath for 10 min to form a nanoparticle dispersion. A dispersion of OMV-coated nanoparticle composites NP-AM@OMV and NP-FK@OMV is obtained.

[0078] Figure 6 The DLS results indicate that OMV was successfully encapsulated on the surface of NP-AM and NP-FK nanoparticles, resulting in an NP-AM / FK@OMV nanodrug delivery system with good stability and dispersibility.

[0079] Figure 7The TEM images show the overall structure of OMV(a) encapsulating nanoparticles NP-AM(b) and NP-FK(c), further proving that OMV was successfully coated on the surface of nanoparticles NP-AM and NP-FK, forming NP-AM / FK@OMV nanoparticle composites (d).

[0080] Example 6 Preparation of sterile poloxamer-hyaluronic acid hydrogel

[0081] Weigh 10 g of poloxamer PF127, add 45 mL of ddH2O, stir and dissolve completely under ice bath conditions, then add 0.5 g of hyaluronic acid powder, mix thoroughly and stir until a uniform clear solution is obtained, place at 4°C overnight to allow it to fully swell and eliminate air bubbles, then sterilize for later use.

[0082] Example 7 Synthesis of Nanoparticle Drug-Loaded Hydrogels

[0083] The nanoparticle composites NP-AM@OMV and NP-FK@OMV encapsulated in OMV were prepared into a 5 mL NP-AM / FK@OMV suspension and added to the above PF127-HA hydrogel. The suspension was stirred in an ice bath to form a uniformly dispersed hydrogel solution.

[0084] Example 8: Nanoparticle-loaded hydrogel coating

[0085] The silicone catheter was ultrasonically cleaned sequentially with anhydrous ethanol and double-distilled water for 15 minutes each. After removal, it was soaked in 75% alcohol overnight, disinfected by UV irradiation for 30 minutes, and then allowed to dry in a clean bench for later use. Subsequently, it was immersed in NP-AM / FK@OMV-P / H hydrogel and slowly extracted to coat the surface with a uniform layer of drug-loaded hydrogel. After slightly drying at room temperature, it was placed in a 50℃ oven for 1 hour and then dried at room temperature for 24 hours to form a coating. Thus, the NP-AM / FK@OMV-P / H hydrophilic nano-drug-loaded hydrogel coating was obtained on the silicone surface.

[0086] After impregnation and coating, a dense hydrogel structure is formed, which can obtain a uniform nano-drug-loaded hydrogel coating with certain hydrophilicity. Figure 8 (a) shows the morphology of the surface of the NP-AM / FK@OMV-P / H hydrogel; Figure 8 (b) shows the internal hierarchical structure of the NP-AM / FK@OMV-P / H hydrogel; Figure 8 (c) shows the cross-section of the NP-AM / FK@OMV-P / H hydrogel coating and its internal structure. Figure 9 This shows the water contact angle of the conduit surface with and without coating (left) versus with coating (right).

[0087] Example 9: Cumulative in vitro release rate of NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating in different media

[0088] Use a punch to punch holes in the silicone tubes to create appropriately sized silicone sheets (approximately 6 mm in diameter). Then, take a 24-well plate and immerse the silicone sheets coated with PF127 / HA, NP-FK@OMV-P / H, NP-AM@OMV-P / H, and NP-AM / FK@OMV-P / H, respectively, in pH 6.0 PBS (control group), PBS containing HAase (88 U / mL or 175 U / mL), or PBS containing E. coli GIM1.457 (concentration of 10). 3 Or 10 5 The sample was incubated in 2 mL of 0.01 mol / L, pH 6.0 PBS at 37°C for 48 h. At regular time points (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h), and 300 μL of the corresponding fresh release medium solution was added. The concentrations of FK13-a1 and AMK, and the cumulative release rates of FK13-a1 and AMK were determined using the following method.

[0089] FK13-a1 content determination method: A standard curve was prepared. Samples collected at different time points (1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 mg / mL) were added to 96-well plates. The absorbance (OD) value at 280 nm was measured using a full-wavelength microplate reader (Bio Tek Elx808). Each group contained 100 μL, with 3 replicates. The concentration was plotted on the x-axis, and the OD value was plotted on the y-axis. 280 nm Plot a standard curve with the values ​​on the ordinate, calculate the concentration of FK13-a1, and plot the cumulative release curve of FK13-a1.

[0090] AMK content determination method: The complex formed by AMK and alkaline copper tartrate reagent has maximum absorption at 570 nm. A standard curve was prepared. 100 μL of samples collected at concentrations of 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, and 0.05 mg / mL, and at different time points, were added to each well of a 96-well plate, followed by 200 μL of alkaline copper tartrate solution. The plate was incubated at 37℃ for 10 min, and the OD value at 570 nm was measured using a microplate reader. Each group had three replicates. The values ​​were plotted with concentration on the x-axis and OD value on the y-axis. 570 nm Plot a standard curve with the values ​​on the ordinate, calculate the concentration of AMK, and depict the cumulative release curve of AMK.

[0091] Formula for calculating cumulative release rate:

[0092]

[0093]

[0094] Q n C: Cumulative drug release at the nth sampling point; n : Drug concentration at the nth sampling point; V0: Total volume of the release medium; V: Volume of each sample taken.

[0095] Figure 10 (A) shows the cumulative release curves of antimicrobial peptide FK13-a1 in different release media in the PF127 / HA, NP-FK@OMV-P / H and NP-AM@OMV-P / H coating groups over 48 h. Figure 10 (B) shows the cumulative release curves of antibiotic AMK in the PF127 / HA, NP-AM@OMV-P / H, and NP-AM@OMV-P / H coated groups over 48 h in different release media. Compared with the PBS group, the cumulative release rates of antimicrobial peptide FK13-a1 and antibiotic AMK were higher in the presence of HAase and E. coli GIM1.457 than in the PBS group.

[0096] Example 10 Evaluation of the hemolytic properties of the nano-drug-loaded hydrogel coating

[0097] After anticoagulating mouse, rat, or human blood, centrifuge at 1000 g, 4°C for 15 min, and collect the bottom layer of red blood cells. Repeat the above steps three times with 200 μL PBS. Mix 150 μL of drug suspension from each material group (OMV, 1 mg / mL NP, 1 mg / mL NP-FK, 1 mg / mL NP-AM, PF127 / HA, NP-AM@OMV-P / H, NP-FK@OMV-P / H, NP-AM / FK@OMV-P / H) with 150 μL of 2% (red blood cell to PBS volume ratio) red blood cell PBS suspension to form the experimental group. The positive control group was treated with 1% Triton X-100, and the blank control group was treated with PBS. Incubate at 37°C for 1 h, centrifuge at 1200 g, 4°C for 15 min, and observe by photography. Alternatively, the supernatant can be aspirated and analyzed by OD. 570 nm Measure the amount of hemoglobin released and calculate the hemolysis rate (%).

[0098]

[0099] Figure 11 The results showed that, compared with the positive control group (1% Triton X-100), none of the material groups showed significant hemolysis of red blood cells from the three sources, and none produced a hemolytic effect compared with PBS.

[0100] Example 11 Evaluation of the cytotoxicity of the nano-drug-loaded hydrogel coating against NIH3T3, L02, and HK-2.

[0101] The cytotoxicity of different coating groups was tested using the CCK-8 assay. NIH3T3, L02, and HK-2 cells in good growth condition were washed with PBS, digested with trypsin, and prepared into single-cell suspensions according to standard cell culture methods, with accurate cell counting. The cell concentration was adjusted to 1×10⁶ cells using culture medium. 5 Cells were seeded at 100 μL per well in 96-well plates and cultured at 37°C for 24 h in 5% CO2 to allow cell adhesion. The culture medium was discarded, and drug-containing culture media (50 μL hydrogel stock solution + 50 μL culture medium) for PF127 / HA, NP-AM@OMV-P / H, NP-FK@OMV-P / H, and NP-AM / FK@OMV-P / H were added to each well, with four replicates per group, a blank group (100 μL culture medium only), and a negative control group (100 μL culture medium containing only cells). Cells were incubated at 37°C for 48 h in 5% CO2. The solution in the wells was discarded, and the cells were gently washed twice with sterile PBS. 90 μL of culture medium and 10 μL of CCK-8 solution were added to each well, and the cells were incubated at 37°C for 2 h in 5% CO2. The absorbance (OD) of each well was measured at 450 nm using a microplate reader, and the results were recorded and the cell viability (%) was calculated.

[0102]

[0103] Figure 12 The results showed that NP-AM@OMV-P / H exhibited mild cytotoxicity to normal human renal cortical proximal tubular epithelial cells HK-2 and normal hepatocytes L02. However, when used in combination with the antimicrobial peptide FK13-a1, the cell survival rate was relatively improved and the cytotoxicity was reduced. None of the coating groups showed significant cytotoxicity to NIH3T3 cells.

[0104] Example 12 Antibacterial effect of NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating

[0105] CON and silicone sheets coated with PF127 / HA, NP-FK@OMV-P / H, NP-AM@OMV-P / H, and NP-AM / FK@OMV-P / H were respectively mixed with 1 mL of E. coli culture (1×10⁻⁶). 6 The bacterial culture (CFU / mL) was added to a 12-well plate and incubated at 37°C. At the corresponding time points (9 h, 12 h, 24 h), the plate was removed, and the liquid in the corresponding well was collected into an EP tube. The bacterial culture was diluted with sterile PBS at a ratio of 1000×(1 μL + 999 μL) and spread onto LB agar plates. The plates were incubated overnight at 37°C. The growth of bacterial colonies on the surface of different coating groups was observed over time, and the bacterial colonies formed on the surface of the agar plates were counted.

[0106] Figure 13No bacteria grew in the NP-AM / FK@OMV-P / H coating group during the co-incubation experiment, proving that it has a sustained antibacterial effect.

[0107] Set up group CON and PF127 / HA, NP-FK@OMV-P / H, NP-AM@OMV-P / H, and NP-AM / FK@OMV-P / H coating groups. Dilute the bacterial stock solution to OD. 600 =0.05(1×10 6 For each well, a sterile 12-well plate was incubated at 37°C for 24 h. Approximately 0.5 mL of the pre-coated layer for each material and 1 mL of bacterial suspension were added to each well. The plate was then incubated. 100 μL of the liquid from each well was transferred to a 1.5 mL centrifuge tube and centrifuged at 3000 g for 10 min at 4°C. The supernatant was removed, and the bacterial spores were resuspended in PBS and washed three times. The washed bacterial spores were resuspended in 100 μL of physiological saline, followed by the addition of 1 μL of 100× staining solution (1 μL DAMO + 2 μL EthD-Ⅲ + 8 μL 0.9% NaCl). The plate was stained at room temperature in the dark for 20 min, then centrifuged at 3000 g for 10 min at 4°C to remove excess staining solution from the supernatant. 5 μL of the bacterial spores and 1 drop of anti-fluorescence quencher were placed on a sterile glass slide and covered with a coverslip. The fluorescence staining of each group of bacteria was observed using a fluorescence microscope. Dead bacteria stained with EthD-III appear red in the field of view, while live bacteria stained with DAMO appear green. ImageJ software was used to analyze the fluorescence intensity. The experiment must be conducted in the dark throughout the fluorescence staining process.

[0108] Figure 14 In the fluorescence microscope field of view, both the NP-AM@OMV-P / H and NP-AM / FK@OMV-P / H coating groups were able to kill most of the Escherichia coli.

[0109] Example 13: Anti-biofilm effect of NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating

[0110] The 16 Fr Foley catheters were cut into 1 cm segments, thoroughly washed with sterile autoclaved water and 75% ethanol, air-dried, and then immersed in gels PF127 / HA, NP-AM@OMV-P / H, NP-FK@OMV-P / H, and NP-AM / FK@OMV-P / H to form a coating. The coated tubes were then placed in 48-well plates and 1 mL of 1×10⁻⁶ gel was added. 6 CFU / mL E. coliThe bacterial culture (LB broth) completely submerged the tubes, and the incubation was carried out at 37°C for 24 h. The tube samples were then removed and gently rinsed twice with PBS buffer to remove any unattached free bacteria. Subsequently, the tube samples from each group were placed in EP tubes containing 1 mL of PBS solution and sonicated in a water bath for 10 min to detach the bacterial biofilm adhering to the tube walls. Serially diluted PBS solutions containing bacteria were spread onto LB agar plates and incubated overnight at 37°C. Colony counts were then performed on the surface of the agar plates.

[0111] Figure 15 The results showed that in the in vitro anti-biofilm efficacy verification, the number of bacteria adhering to the catheter in the NP-AM / FK@OMV-P / H coating group was relatively less than that in other groups (a. Control; b. PF127 / HA; c. NP-FK@OMV-P / H; d. NP-AM@OMV-P / H; e. NP-AM / FK@OMV-P / H).

[0112] Example 14: In vivo prevention of biofilm formation by NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel coating

[0113] Catheter biofilm colony counting: Three urinary catheters from each group were randomly selected and transferred to 1.5 mL sterile EP tubes. The catheters were gently rinsed once with 500 μL sterile PBS, the PBS was discarded, and the catheters were transferred to new 1.5 mL EP tubes. 500 μL of fresh sterile PBS was added for soaking, and the tubes were sonicated in a water bath for 10 min to disperse the biofilm. After 10-fold serial dilution, 100 μL of each concentration was taken and spread on LB agar plates. The plates were incubated overnight at 37°C, and the number of colonies was counted. The number of bacteria in the original solution was calculated.

[0114] Scanning electron microscopy (SEM): Urinary catheters from three mice in each group were randomly selected. Sterile PBS was added to disposable bacterial culture dishes, and the collected catheters were gently rinsed and air-dried. They were then transferred to 1.5 mL sterile EP tubes, and 2.5% glutaraldehyde solution was added to cover the catheters. Fixation was performed for 2 h, the 2.5% glutaraldehyde solution was removed, and the catheters were rinsed three times with sterile PBS, which was then discarded. The catheters were dehydrated, dried, cut, and sputter-coated with gold. The morphology of the bacterial biofilm on the inner wall of the catheters was observed under an SEM microscope, with several fields of view randomly selected for imaging.

[0115] Figure 16 The NP-AM / FK@OMV-P / H coating group showed significant antibacterial and anti-biofilm effects. Compared with the Model group, there was almost no bacterial adhesion and colonization in the catheter (a. Control; b. Model; c. PF127 / HA; d. NP-AM@OMV-P / H; e. NP-FK@OMV-P / H; f. NP-AM / FK@OMV-P / H).

[0116] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a nano-drug-loaded hydrogel urinary catheter coating, characterized in that, NP-AM and NP-FK nanoparticles were synthesized by encapsulating the antibiotic amikacin sulfate (AMK) or the antimicrobial peptide FK13-a1 using PLGA. Using EcN OMV as a carrier, an NP-AM / FK@OMV nanoparticle composite was prepared. This composite was then co-mixed with a poloxamer-hyaluronic acid composite hydrogel to obtain an NP-AM / FK@OMV-P / H drug-loaded nanogel. After coating and drying, a coating layer was formed. The specific preparation method includes the following steps: Separation and purification of S1, EcN OMV; S2. Using the W / O / W emulsification-solvent evaporation method, polylactic acid-glycolic acid copolymer (PLGA) was used to encapsulate the antibiotic amikacin sulfate AMK or the antimicrobial peptide FK13-a1 to synthesize nanoparticles NP-AM and NP-FK. S3. Encapsulate the NP-AM and NP-FK nanoparticles synthesized in step S2 with the EcN OMV extracted in step S1 to obtain the NP-AM / FK@OMV nanoparticle composite. S4. Preparation of sterile poloxamer-hyaluronic acid composite hydrogel; S5. The NP-AM / FK@OMV nanoparticle composite obtained in step S3 is mixed into the poloxamer-hyaluronic acid composite hydrogel to form NP-AM / FK@OMV-P / H nanoparticle drug-loaded hydrogel. S6. The NP-AM / FK@OMV-P / H nano-drug-loaded hydrogel from step S5 is uniformly coated onto the clean inner wall of the catheter using an impregnation coating process, and then vacuum dried to form a coating. The bacterial outer membrane vesicles EcN OMV mentioned in step S1 are derived from E. coli Nissle 1917, strain number: DSM6601; the isolation and purification of EcN OMV includes ultrafiltration concentration-ultracentrifugation method, taking E. coli Nissle 1917 in the logarithmic growth phase and expanding the culture to 500 mL, then filtering and concentrating to 1 / 5 to 1 / 7 of the original volume, purifying EcN OMV by low-temperature ultracentrifugation, and then resuspending the obtained precipitate in 1 mL of sterile PBS to obtain EcN OMV resuspension.

2. The method for preparing the nano-drug-loaded hydrogel catheter coating according to claim 1, characterized in that, The amino acid sequence of the antimicrobial peptide FK13-a1 is shown in SEQ ID NO.

1.

3. The method for preparing the nano-drug-loaded hydrogel catheter coating according to claim 1, characterized in that, The polylactic acid-glycolic acid copolymer PLGA described in step S2 is composed of lactic acid LA and glycolic acid GA in a ratio of 50:50; The W / O / W emulsification-solvent evaporation method for synthesizing nanoparticles NP-AM and NP-FK includes the following steps: Under ice bath conditions, polymer PLGA is dissolved in dichloromethane (DCM), and FK13-a1 or AMK is dissolved in 0.5% (w / v) polyvinyl alcohol (PVA) to prepare an inner aqueous phase solution. The inner aqueous phase is added dropwise to the dichloromethane solution containing PLGA to form a W / O emulsion. Subsequently, it is added to a 1% (w / v) sterile aqueous solution of PVA for further emulsification to form a W / O / W emulsion. The dichloromethane is evaporated, centrifuged, and the precipitate is washed with sterile water. The nanoparticle precipitate obtained by centrifugation is resuspended in an appropriate amount of sterile water, transferred to a centrifuge tube, pre-frozen, and freeze-dried to obtain lyophilized powders of nanoparticles NP-AM and NP-FK.

4. The method for preparing the nano-drug-loaded hydrogel catheter coating according to claim 1, characterized in that, Step S3 includes preparing the EcN OMV extracted in step S1 into a resuspension, taking a certain proportion of the lyophilized nanoparticles NP-AM and NP-FK synthesized in step S2, resuspending them in OMV resuspension instead of sterile water, stirring evenly, and sonicating in a water bath to form a nanoparticle dispersion, thus obtaining a suspension of OMV-coated nanoparticle composite NP-AM / FK@OMV.

5. The method for preparing the nano-drug-loaded hydrogel catheter coating according to claim 4, characterized in that, The ratio of the NP-AM nanoparticles to the NP-FK lyophilized powder is NP-AM:NP-FK = 1:1~4 (w / w).

6. The method for preparing the nano-drug-loaded hydrogel catheter coating according to claim 1, characterized in that, The poloxamer mentioned in step S4 is of type PF127; the molecular weight of the hyaluronic acid is 40-100 kDa; The preparation of sterile poloxamer-hyaluronic acid composite hydrogel includes the following steps: 20% (w / v) poloxamer is added to water and dissolved in an ice bath. After complete dissolution, 1% (w / v) hyaluronic acid powder is added and stirred evenly to disperse the solution. The solution is allowed to stand overnight at 0-4℃ to defoam, sterilized, and stored at 4℃ for later use.

7. The method for preparing the nano-drug-loaded hydrogel catheter coating according to claim 1, characterized in that, The preparation of the NP-AM / FK@OMV-P / H nanoparticle drug-loaded hydrogel in step S5 includes: adding the NP-AM / FK@OMV nanoparticle composite suspension obtained in step S3 dropwise to the poloxamer-hyaluronic acid hydrogel while stirring, to form a uniformly colored and particle-dispersed NP-AM / FK@OMV-P / H nanoparticle drug-loaded composite hydrogel.

8. A nano-drug-loaded hydrogel catheter coating NP-AM / FK@OMV-P / H prepared by the preparation method according to any one of claims 1-7.

9. The application of the nano-drug-loaded hydrogel catheter coating NP-AM / FK@OMV-P / H as described in claim 8 in the preparation of biofilm materials that inhibit the surface of medical catheters.

Images