Glutathione-depleted engineered red blood cell membrane hybrid vesicles, methods of making and uses thereof

Abstract

The application discloses a glutathione consumption type engineered red blood cell membrane hybrid vesicle as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The preparation method comprises the following steps: mixing cinnamyl aldehyde dimers, platinum nanoparticles, lactic acid oxidase and red blood cell membranes, performing ultrasonic crushing, and then adding distearoyl phosphatidyl ethanolamine-polyethylene glycol-mannose for mixing reaction to obtain the glutathione consumption type engineered red blood cell membrane hybrid vesicle. The glutathione consumption type engineered red blood cell membrane hybrid vesicle provided by the application has excellent structural stability and targeted delivery capacity, has integrated synergistic effects of anti-tumor and anti-infection, has good biocompatibility, and provides a safe and efficient new strategy for postoperative treatment of bladder cancer and infection prevention and control.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a glutathione-consuming engineered erythrocyte membrane hybrid vesicle, its preparation method, and its application. Background Technology

[0002] Currently, common treatments for bladder carcinoma in situ (CIS) include surgical resection, radiotherapy, and chemotherapy. However, these methods have limitations, such as poor targeting, significant side effects, high recurrence rates, and postoperative infection problems. Most existing drug delivery systems utilize carriers such as liposomes and polymer nanoparticles. While these carriers can improve drug delivery efficiency, they still suffer from insufficient targeting, difficulty in penetrating biomembranes, and failure to improve the immune microenvironment. Especially in postoperative infection prevention and control, existing anti-infective drug delivery systems face challenges such as short drug retention time and poor immune response. Therefore, there is an urgent need for a multifunctional drug delivery system that can simultaneously address both tumor treatment and postoperative infection. Summary of the Invention

[0003] The purpose of this invention is to provide a glutathione-consuming engineered erythrocyte membrane hybrid vesicle, its preparation method, and its application, to solve the problems existing in the prior art. The glutathione-consuming engineered erythrocyte membrane hybrid vesicle provided by this invention has a synergistic effect of integrated anti-tumor and anti-infection, good biocompatibility, and provides a safe and efficient new strategy for postoperative treatment and infection control of bladder cancer.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing glutathione-consuming engineered erythrocyte membrane hybrid vesicles, comprising the following steps: Cinnamaldehyde dimer, platinum nanoparticles, lactate oxidase and erythrocyte membrane were mixed and ultrasonically broken down. Then, distearate phosphatidylethanolamine-polyethylene glycol-mannose was added and mixed to obtain the glutathione-consuming engineered erythrocyte membrane hybrid vesicle.

[0005] Furthermore, the cinnamaldehyde dimer is obtained by condensing cinnamaldehyde and diethyltriamine.

[0006] Furthermore, the molar ratio of cinnamaldehyde to diethyltriamine is 2:1.

[0007] Furthermore, the condensation reaction is carried out at a temperature of 60°C for 12 hours.

[0008] Furthermore, the platinum nanoparticles are prepared by a reduction reaction using hexachloroplatinic acid hexahydrate as the platinum source.

[0009] Furthermore, the reduction reaction uses polyvinylpyrrolidone as a stabilizer and sodium borohydride as a reducing agent.

[0010] Furthermore, the mixed reaction is carried out under ice-water bath conditions for 6 hours.

[0011] The present invention also provides a glutathione-consuming engineered erythrocyte membrane hybrid vesicle prepared according to the above preparation method.

[0012] The present invention also provides the application of the above-mentioned glutathione-consuming engineered erythrocyte membrane hybrid vesicles in any one of (1)-(3): (1) Preparation of drugs for treating bladder cancer; (2) Preparation of antibacterial drugs; (3) Prepare drugs for the prevention and / or treatment of urinary tract infections after bladder cancer surgery.

[0013] The present invention also provides a drug having at least one of the functions described in (1)-(3), wherein the active ingredient of the drug includes the above-described glutathione-consuming engineered erythrocyte membrane hybrid vesicles; (1) Treatment of bladder cancer; (2) Antibacterial; (3) Prevention and / or treatment of urinary tract infections after bladder cancer surgery.

[0014] The present invention discloses the following technical effects: This invention modularly integrates cinnamaldehyde dimer (CAD), platinum nanozyme (Pt), lactate oxidase (LOx), and mannose-modified erythrocyte membranes to prepare glutathione-consuming engineered erythrocyte membrane hybrid vesicles, exhibiting excellent structural stability, targeted delivery capability, and biosafety. The material possesses a uniform core-membrane structure with controllable particle size and potential, and a well-defined elemental composition. It can responsively disintegrate in a high-glutathione environment, achieving lesion-specific drug release. In vitro experiments show that it combines highly efficient peroxidase-like activity with lactate oxidase cascade catalytic ability, significantly consuming glutathione and continuously generating reactive oxygen species. It exhibits significant inhibitory effects on bladder cancer cell proliferation and apoptosis induction, and can regulate the expression of apoptosis-related proteins. Simultaneously, the vesicles possess broad-spectrum antibacterial activity against common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, as well as drug-resistant bacteria, and can effectively inhibit biofilm formation. In vivo experiments showed that it could significantly inhibit bladder cancer tumor growth and cause significant tumor tissue damage; in a urinary tract infection model, it could effectively reduce bacterial load, alleviate bladder tissue inflammation and pathological damage, and reduce the levels of pro-inflammatory factors such as IL-6. In summary, the vesicle material provided by this invention has a synergistic effect of integrated anti-tumor and anti-infection, good biocompatibility, and provides a safe and efficient new strategy for postoperative treatment and infection control of bladder cancer. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 A schematic diagram of the preparation process of M-RBCM / CA@Pt-LOx; Figure 2 Transmission electron microscopy (TEM) images of CAD, CA@Pt, CA@Pt-LOx and M-RBCM / CA@Pt-LOx nanoparticles; Figure 3 Element mapping analysis diagram for M-RBCM / CA@Pt-LOx; Figure 4 Particle size (B) and zeta potential (A) of CAD, CA@Pt, CA@Pt-LOx and M-RBCM / CA@Pt-LOx nanoparticles are shown in the figure. Figure 5 SDS-PAGE protein electrophoresis images of different samples; Figure 6 A statistical graph showing the relative consumption rate of glutathione in different samples; Figure 7 TEM images of M-RBCM / CA@Pt-LOx before and after GSH processing; Figure 8 The graph shows the MTT assay results of different samples inhibiting the proliferation of NIH-3T3 cells. Figure 9 Fluorescence microscopy images and quantitative analysis diagrams of 5637 bladder cancer cells after different sample treatments, showing their viability and death. Among them, A is a Calcein-AM / PI double-staining fluorescence image, with green fluorescent markers for live cells and red fluorescent markers for dead cells, and Merge is the image overlay result; B is a quantitative statistical diagram of cell apoptosis rate. Figure 10 Western blotting data showing the expression of apoptosis-related proteins in 5637 bladder cancer cells after different samples were treated with these samples. Figure 11 The graphs show the quantitative analysis of the in vitro bactericidal detection and inhibition rate of different samples against Escherichia coli and Staphylococcus aureus; where A is the result of the plate inhibition experiment; B is a statistical graph of the inhibition rate of different samples against Escherichia coli; and C is a statistical graph of the inhibition rate of different samples against Staphylococcus aureus. Figure 12 In vivo bioluminescence imaging of different samples used to treat mouse bladder carcinoma in situ; Figure 13 HE staining images of tumor tissues from different groups; Figure 14 HE staining images of bladder tissue sections from different groups; Figure 15 Immunofluorescence analysis of IL-6 in bladder tissues from different groups of urinary tract infections. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] This invention modularly integrates cinnamaldehyde dimer (CAD), platinum nanozyme (Pt), lactate oxidase (LOx), and mannose-modified erythrocyte membranes to prepare glutathione-consuming engineered erythrocyte membrane hybrid vesicles M-RBCM / CA@Pt-LOx (see schematic diagram of the preparation process). Figure 1It achieves four-dimensional synergy of metabolic intervention, cascade catalysis, immune targeting and physical membrane permeation, and has shown excellent effects in the treatment of bladder carcinoma in situ and the prevention and treatment of postoperative urinary tract infections, with good biosafety.

[0023] Example 1 This embodiment provides a glutathione-consuming cascade nanozyme-integrated engineered erythrocyte membrane hybrid vesicle, which is obtained through the following preparation steps: (1) CAD synthesis Cinnamaldehyde and diethyltriamine were dissolved in 50 mL of anhydrous ethanol at a molar ratio of 2:1, and the condensation reaction was carried out at 60 °C for 12 h. The product was purified by washing twice with n-hexane to obtain a yellow viscous oil. Finally, it was freeze-dried to obtain CAD.

[0024] (2) Synthesis of platinum nanozymes Platinum nanoparticles (Pt NPs) were prepared using polyvinylpyrrolidone (PVP) as a stabilizer and sodium borohydride (NaBH4) as a reducing agent. 600 μL of a 20 mM solution of hexachloroplatinic acid hexahydrate (H2PtCl6·6H2O) was slowly added to 50 μL of freshly prepared 0.1 M NaBH4 solution with continuous stirring. The mixture was then stirred at a constant temperature for 4 h to ensure complete reaction. After the reaction was complete, the reaction mixture was transferred to a 100 kDa ultrafiltration tube and centrifuged at 1000 rpm for 5 min. Unreacted impurities and small molecules were removed by ultrafiltration, and the precipitate in the ultrafiltration tube was collected to obtain platinum nanoparticles.

[0025] (3) Preparation of red blood cell membranes Whole blood from Kunming mice was collected in anticoagulant tubes and centrifuged at 3000 rpm for 5 min. After centrifugation, the supernatant plasma, leukocytes, and platelet components were carefully removed, retaining the bottom erythrocyte precipitate. The erythrocyte precipitate was then washed three times with PBS, centrifuged after each wash, and the supernatant was discarded to thoroughly remove impurities. The washed erythrocytes were directly dissolved in pre-chilled lysis buffer (0.01 M, pH 8.0 Tris-HCl buffer containing 0.02 mM PMSF), with a lysis buffer volume 40 times that of the erythrocyte precipitate. The mixture was incubated at 4°C for 2 h for lysis. After lysis, the mixture was centrifuged at 4°C and 1000 g for 30 min, the supernatant was discarded, and the bottom precipitate was collected. The precipitate was washed three more times with PBS, and the erythrocyte concentration was determined to be 20 mg / mL using a BCA protein quantification curve. The erythrocyte membrane was aliquoted into 100 μL tubes and stored at -80°C for later use.

[0026] (4) Preparation of M-RBCM / CA@Pt-LOx Prepare stock solutions of CAD (50 mg / mL), Pt NPs (10 mg / mL), and LOx (100 U / mL) under suitable conditions. Pipette 100 μL of each of the following stock solutions: CAD stock solution, Pt NPs stock solution, LOx stock solution, and erythrocyte suspension into a centrifuge tube containing 5 mL of ultrapure water. Gently invert the centrifuge tube to mix thoroughly. A mixed reaction system was obtained. This system was placed in an ultrasonic homogenizer at 100W for 3 minutes. Then, distearate-phosphatidylethanolamine-polyethylene glycol-mannose (DSPE-PEG-Mannose) was added and placed in an ice-water bath with continuous stirring for 6 hours. After stirring, the reaction system was extruded through a liposome extruder and filtered through a 200nm filter to remove large particles and unreacted aggregates. The extruded solution was transferred to a refrigerated centrifuge at 4℃ and 5000rpm for 5 minutes. After centrifugation, the supernatant was carefully discarded, and the precipitate at the bottom was the target product. The precipitate was collected to obtain M-RBCM / CA@Pt-LOx.

[0027] Characterization of M-RBCM / CA@Pt-LOx: Transmission electron microscope image of the product prepared in Example 1 is shown below. Figure 2 .from Figure 2 As can be seen, the CAD nanoparticles exhibit a uniform spherical or near-spherical structure with a narrow particle size distribution and clear interparticle boundaries, demonstrating good monodispersity. After loading Pt nanoparticles, clear dot-like or sheet-like high electron density regions appeared on the surface of the spherical support of CA@Pt. After LOx enzyme modification, the overall particle size of CA@Pt-LOx increased slightly, and the surface loading was more uniformly distributed. Some nanoparticles exhibited a chain-like or beaded arrangement, and the final constructed M-RBCM / CA@Pt-LOx nanohybrid exhibited a clear spherical core-membrane structure.

[0028] from Figure 3 As can be seen from the data, the glutathione-consuming engineered hybrid vesicle M-RBCM / CA@Pt-LOx prepared in this embodiment contains six elements: C, N, O, S, P, and Pt. This further proves that the synthesis of M-RBCM / CA@Pt-LOx was successful.

[0029] The particle size and zeta potential analysis diagrams for CAD, CA@Pt, CA@Pt-Lox, and M-RBCM / CA@Pt-LOx are shown below. Figure 4 .from Figure 4As can be seen, in terms of particle size, the initial hydrated particle size of CAD, CA@Pt, CA@Pt-Lox, and M-RBCM / CA@Pt-LOx increased from 130 nm to approximately 180 nm. After modification with LOx enzyme, the particle size further increased to approximately 230 nm, and the final constructed M-RBCM / CA@Pt-LOx nanohybrid reached a particle size of approximately 240 nm. This is closely related to the coating of the M-RBCM cell membrane layer. The phospholipid bilayer structure of the cell membrane significantly increased the hydrated diameter of the particles, and also confirmed the successful construction of the biomimetic cell membrane coating layer. Regarding zeta potential, the zeta potential of CAD, CA@Pt, CA@Pt-Lox, and M-RBCM / CA@Pt-LOx decreased from +25 mV to approximately -10 mV, and further decreased to approximately -25 mV. The zeta potential of the final product M-RBCM / CA@Pt-LOx stabilized at approximately -20 mV. This change is mainly due to the effective neutralization of some surface negative charges by the cell membrane coating.

[0030] SDS-PAGE protein electrophoresis confirmed successful LOX encapsulation and successful erythrocyte membrane coating: Four types of samples—CAD, CA@Pt, CA@Pt-LOx, and RBCM / CA@Pt-LOx—were taken and adjusted to the same protein concentration using PBS buffer. An appropriate amount of LOX standard and red blood cell membrane were also taken as positive controls. 5×SDS loading buffer was added to each sample and control, and after thorough mixing, the mixture was boiled in a 95℃ water bath for 5 minutes to fully denature the proteins. The mixture was then cooled to room temperature before use.

[0031] The gels were prepared using the one-step PAGE color gel ultra-rapid preparation kit from Wuhan Saiwei Biotechnology Co., Ltd. A 12% separating gel and a 5% stacking gel were prepared and solidified sequentially. The separating gel was then prepared and the stacking gel was prepared. The sample comb was inserted, and after the stacking gel had completely solidified, the sample comb was removed. The gel was then placed in the electrophoresis tank, and Tris-glycine electrophoresis buffer was added, ensuring that the buffer covered the upper and lower surfaces of the gel.

[0032] Sample loading: Using a micropipette, aspirate the prepared samples and controls separately and slowly add them into the sample wells of the gel, with a loading volume of 10 μL per well.

[0033] Electrophoresis procedure: Set the electrophoresis parameters. The voltage for the stacking gel stage is 80V. After the bromophenol blue indicator enters the separating gel, adjust the voltage to 120V and continue electrophoresis until the bromophenol blue indicator reaches the bottom of the gel (about 90 minutes). Turn off the power to terminate the electrophoresis.

[0034] Staining and destaining: Remove the polyacrylamide gel after electrophoresis and place it in a clean dish. Wash three times with pure water for 20 seconds each time. After pouring out the pure water, add 20 mL of Coomassie Brilliant Blue Ultrafast Staining Solution (enough to submerge the gel) and vortex for staining for 30 minutes. Pour out the staining solution and wash with pure water for 1 hour.

[0035] Results Observation and Analysis: The destained gel was placed on a white plate, and gel images were taken. The protein bands of each sample were observed and analyzed. Using the LOX standard band as a reference, it was determined whether specific bands corresponding to LOX appeared in the CA@Pt-LOx and RBCM / CA@Pt-LOx samples.

[0036] like Figure 5 As shown, specific bands corresponding to LOX and erythrocyte membranes appeared in the M-RBCM / CA@Pt-LOx sample, proving that LOX was successfully loaded and erythrocyte membranes were successfully coated.

[0037] Example 1 of effect verification Detection principle: Glutathione (GSH) can react with dithionitrobenzoic acid (DTNB) to produce 2-nitro-5-mercaptobenzoic acid and glutathione disulfide (GSSG). 2-nitro-5-mercaptobenzoic acid is a yellow product with maximum light absorption at 412 nm.

[0038] Verify the ability of each experimental sample to consume glutathione: 1) Preparation of 200μM reduced glutathione standard solution: Weigh 3.1mg of reduced glutathione, dissolve it in a small amount of anhydrous ethanol, and then add pure water to make up to 50mL. 2) Preparation of 4mM DTNB reagent: Weigh 15.8mg of dithionitrobenzoic acid (DTNB), and dilute to 10mL with 0.01M PBS (pH: 7.2-7.4) solution. Store at 4℃ and use immediately after preparation.

[0039] 3) Preparation of GSH standard curve: Add the corresponding reagents according to Table 1, mix well, react at 25℃ for 30 min, and measure the absorbance of the colorimetric solution at 405 nm. Plot the standard curve with GSH concentration as the x-axis and absorbance as the y-axis.

[0040]

[0041] 4) Sample Detection: 100 μL of different sample solutions were mixed with 100 μL of GSH solution (200 μM) and reacted at room temperature for 30 min. Then, 50 μL of DTNB solution (4 mM) was added and mixed, and reacted at 25℃ for 30 min. The absorbance of each solution at 405 nm was measured and substituted into the standard curve to calculate the GSH consumption. PBS was used as a negative control, and 0.1 M hydrogen peroxide (H2O2) solution was used as a positive control. The experimental samples were CAD, CA@Pt, CA@Pt-LOx, and RBCM / CA@Pt-LOx. The formula for calculating GSH consumption is as follows: .

[0042] from Figure 6 It can be seen that the relative GSH consumption rate of M-RBCM / CA@Pt-LOx remains at around 70%, which is comparable to that of CA@Pt-LOx, indicating that the responsiveness of nanoparticles to GSH is well preserved after coating with RBCM film.

[0043] TEM images of M-RBCM / CA@Pt-LOx before and after GSH treatment are shown below. Figure 7 ,from Figure 7 As can be seen, before GSH treatment, M-RBCM / CA@Pt-LOx nanoparticles exhibit clear and complete spherical or near-spherical structures with uniform particle size, clear boundaries, and no obvious aggregation or structural damage. After GSH treatment, significant aggregation, fusion, and disintegration occur, forming numerous irregular fragmented or flocculent structures with blurred particle boundaries and a loose and disordered overall morphology. These morphological changes indicate that M-RBCM / CA@Pt-LOx nanoparticles have a significant responsiveness to high concentrations of GSH. This responsive characteristic is of great significance for achieving specific drug release in the tumor microenvironment: in normal tissues, the GSH concentration is low, and the nanoparticles can maintain structural stability; while in tumor cells and bacteria, high concentrations of GSH can induce rapid disintegration, releasing the loaded LOx, thereby improving therapeutic efficiency and reducing systemic toxicity.

[0044] Example 2 of effect verification The biosafety performance of the M-RBCM / CA@Pt-LOxt nanoparticles prepared in Example 1 was evaluated: The MTT assay was used to assess the cell viability of NIH3T3 cells at different concentrations (0, 50, 100, 200, 400, 800 μg / mL) of CAD, CA@Pt, CA@Pt-LOx, and M-RBCM / CA@Pt-LOx. Cells were seeded at a density of 8000 cells per well in 96-well plates and incubated at 37°C for 24 h in 5% CO2. Cells were then treated with the appropriate sample and concentration, and incubated for another 12 h at 37°C. After incubation, the cell suspension was discarded, and adherent cells were stained with 20 μL of MTT. Cells were then incubated at 37°C in a 5% CO2 incubator for 4 h. At the end of the incubation period, 150 μL of DMSO was added to each well, and the 96-well plates were shaken for 20 min. Subsequently, the absorbance (OD) of the cells at 492 nm was measured using enzyme-labeled markers. 492 ), thereby calculating cell viability.

[0045] .

[0046] The effect of M-RBCM / CA@Pt-LOx nanoparticles on cell viability of NIH-3T3 cells is shown in [reference needed]. Figure 8 .from Figure 8 It can be seen that the cell survival rate of the M-RBCM / CA@Pt-LOx group at each concentration is close to or exceeds 90%, which is significantly higher than that of the CA@Pt-LOx group and comparable to that of the CAD and CA@Pt groups, showing good in vitro biocompatibility.

[0047] Example 3 of effect verification The experiment used 5637 bladder cancer cells in the logarithmic growth phase, and distributed them at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μg / mL in 24-well plates and cultured for 24 h. After cell attachment, the old culture medium was removed, and each well was washed twice with 1 mL PBS. The 24-well plates were divided into five groups, and fresh culture medium containing CAD, CA@Pt, CA@Pt-LOx, and M-RBCM / CA@Pt-LOx (all at 10 μg / mL) was added to each well, and the cells were cultured for another 12 h. After the drug reached the specified action time, 10×Assay Buffer was diluted with deionized water to prepare 1×Assay Buffer, and the cells were washed twice. 1 mL of 1×Assay Buffer and 1 μL of Calcein-AM (stock solution) were added to each well, mixed well, and incubated at 37°C in the dark for 20 min. Subsequently, 5 μL of PI stock solution was added to the stained cells, and staining was performed at room temperature in the dark for 5 min. After staining, the staining solution was aspirated, and the cells were washed twice with PBS. Finally, the yellow-green fluorescence of live cells and the red fluorescence of dead cells were observed and detected using a fluorescence inverted microscope.

[0048] The effect of M-RBCM / CA@Pt-LOx on the death of 5637 bladder cancer cells is shown in […]. Figure 9 .from Figure 9 The results show that the apoptosis rate in the control group was 2%; the apoptosis rate in the CAD-treated group increased to 15%; the apoptosis rate in the CA@Pt group further increased to 48%; the apoptosis rate in the CA@Pt-LOx group increased to 58%; and the apoptosis rate in the M-RBCM / CA@Pt-LOx group was the highest, reaching 75%. These results indicate that the apoptosis-inducing effect of M-RBCM / CA@Pt-LOx on 5637 bladder cancer cells increased progressively. The M-RBCM / CA@Pt-LOx nanodelivery system most significantly induced apoptosis in bladder cancer cells.

[0049] The effect of M-RBCM / CA@Pt-LOx on the expression of apoptosis-related proteins in 5637 bladder cancer cells is shown in the figure. Figure 10 .from Figure 10 It can be seen that M-RBCM / CA@Pt-LOx can synergistically induce apoptosis in 5637 bladder cancer cells by inhibiting the PI3K / AKT signaling pathway, downregulating the expression of the anti-apoptotic protein Bcl-2, activating the Caspase-3 apoptosis pathway and upregulating P53 protein.

[0050] Example 4 of effect verification Prepare 1 mL sample solutions with a nanoparticle concentration of 500 μg / mL, including CAD, CA@Pt, CA@Pt-LOx, and M-RBCM / CA@Pt-LOx. Then, add 10 μL of bacterial suspension (10 μg / mL) to each sample group. 8 After thorough mixing (CFU / mL), incubate in a constant temperature shaker at 37°C and 180 rpm for 3 hours. After the reaction, dilute each reaction solution according to an appropriate ratio, spread evenly on LB broth agar plates, and incubate in an inverted position overnight at 37°C. Finally, count the single colonies on the plates and calculate the bacterial survival rate using the following formula: Bacterial survival rate = CFU (sample group) / CFU (blank control group) × 100%.

[0051] The results of the antibacterial efficacy verification of glutathione-consuming engineered erythrocyte membrane hybrid vesicles are shown in [the table below]. Figure 11 .from Figure 11 It can be seen that in the M-RBCM / CA@Pt-LOx group: there was no colony growth at all. The erythrocyte membrane coating and mannose improved the biocompatibility and targeting of the material, and further enhanced the broad-spectrum bactericidal effect against Gram-negative and Gram-positive bacteria.

[0052] Tumor burden in mice under the CAD, CA@Pt, CA@Pt-Lox, and M-RBCM / CA@Pt-LOx groups as observed by a small animal imaging system. Figure 12 .from Figure 12 It can be seen that the signal of M-RBCM / CA@Pt-LOx was comparable to that of other groups on Day 0; the signal was significantly weakened after Day 4, almost approached the background level on Day 8, and remained at a very low signal on Day 14, indicating that tumor growth was strongly inhibited or even nearly eliminated.

[0053] H&E staining images of the antitumor effects of CAD, CA@Pt, CA@Pt-Lox, and M-RBCM / CA@Pt-LOx on bladder cancer mice are shown below. Figure 13 .from Figure 13 It can be seen that, compared with the other four groups, the M-RBCM / CA@Pt-LOx treatment group had the best anti-tumor effect, and the tumor cells almost completely disappeared.

[0054] HE staining images of bladder tissue in CAD, CA@Pt, CA@Pt-Lox, and M-RBCM / CA@Pt-LOx groups during urinary tract infections are shown below. Figure 14 .from Figure 14 It can be seen that, compared with the other four groups, the bladder tissue morphology of the M-RBCM / CA@Pt-LOx group is basically the same as that of the normal group, with intact mucosal structure and no obvious inflammatory infiltration or tissue edema, showing the best tissue protection and inflammation relief effect.

[0055] The results of IL-6 immunofluorescence analysis in bladder tissue from urinary tract infections in the CAD, CA@Pt, CA@Pt-Lox, and M-RBCM / CA@Pt-LOx groups are shown below. Figure 15 .from Figure 15 It can be seen that the M-RBCM / CA@Pt-LOx hybrid vesicles can significantly inhibit the expression of pro-inflammatory factors and effectively alleviate the inflammatory response of bladder tissue.

[0056] The above results indicate that the glutathione-consuming engineered erythrocyte membrane vesicles prepared in this invention exhibit excellent antitumor and antibacterial activities both in vivo and in vitro, and have good biosafety, showing potential for application in postoperative synergistic treatment of bladder cancer.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a glutathione-consuming engineered erythrocyte membrane hybrid vesicle, characterized in that, Includes the following steps: Cinnamaldehyde dimer, platinum nanoparticles, lactate oxidase and erythrocyte membrane were mixed and ultrasonically disrupted. Then, distearate phosphatidylethanolamine-polyethylene glycol-mannose was added and mixed to obtain the glutathione-consuming engineered erythrocyte membrane hybrid vesicle.

2. The preparation method according to claim 1, characterized in that, The cinnamaldehyde dimer is obtained by condensing cinnamaldehyde and diethyltriamine.

3. The preparation method according to claim 2, characterized in that, The molar ratio of cinnamaldehyde to diethyltriamine is 2:

1.

4. The preparation method according to claim 2, characterized in that, The condensation reaction was carried out at a temperature of 60°C for 12 hours.

5. The preparation method according to claim 1, characterized in that, The platinum nanoparticles were prepared by a reduction reaction using hexachloroplatinic acid hexahydrate as the platinum source.

6. The preparation method according to claim 1, characterized in that, The reduction reaction uses polyvinylpyrrolidone as a stabilizer and sodium borohydride as a reducing agent.

7. The preparation method according to claim 1, characterized in that, The mixed reaction was carried out under ice-water bath conditions for 6 hours.

8. A glutathione-consuming engineered erythrocyte membrane hybrid vesicle prepared by the preparation method according to any one of claims 1-7.

9. The use of a glutathione-consuming engineered erythrocyte membrane hybrid vesicle as described in claim 8 in any one of (1)-(3): (1) Preparation of drugs for treating bladder cancer; (2) Preparation of antibacterial drugs; (3) Prepare drugs for the prevention and / or treatment of urinary tract infections after bladder cancer surgery.

10. A medicament having at least one of the functions described in (1)-(3), characterized in that, The active ingredient of the drug includes the glutathione-consuming engineered erythrocyte membrane hybrid vesicles as described in claim 8. (1) Treatment of bladder cancer; (2) Antibacterial; (3) Prevention and / or treatment of urinary tract infections after bladder cancer surgery.

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