CuS-gsno@rbcms multifunctional nanoparticles and preparation and application thereof

Abstract

The application relates to a CuS-GSNO@RBCM multifunctional nanoparticle and a preparation and application thereof, and belongs to the technical field of medicine preparation. The CuS-GSNO nanoparticle is prepared by taking CuS as a carrier to load GSNO, and the CuS-GSNO@RBCM nanoparticle is prepared by coating the CuS-GSNO nanoparticle with mouse red blood cell membranes RBCM. The application effectively solves the problems of CuS-GSNO agglomeration and the poor consistency between the ROS generated by CuS through PDT and the NO released by GSNO by adopting CuS to load GSNO and then coating the CuS-GSNO with RBCM. Through the multiple synergistic effects of ROS generated by PDT, the reaction of ROS and NO to generate RNS, and the thermoelectric effect to promote the generation of RNS, the killing effect of the nanoparticle on MRSA is improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, specifically to a CuS-GSNO@RBCM multifunctional nanoparticle and its preparation and application. Background Technology

[0002] Methicillin-resistant Staphylococcus aureus (MRSA), a typical "superbug," possesses inherent resistance to β-lactam antibiotics and often forms biofilms that enhance resistance, leading to high treatment failure rates and mortality rates of 20-40%. The core of MRSA's strong resistance and pathogenicity lies in its biofilm-forming ability. Biofilms, formed by the extracellular matrix (EPS) composed of polysaccharides, proteins, and DNA, encapsulate bacteria, hindering antibiotic penetration and providing a hypoxic, highly reducing protective microenvironment. Furthermore, the bacteria within the biofilm exist as persistent organisms, insensitive to traditional antibiotics. Current research focuses on multi-mechanism synergistic strategies to eliminate MRSA through multiple pathways, including disrupting bacterial cell walls / membranes, interfering with metabolism, generating reactive oxygen species (ROS), and inhibiting biofilm formation, while simultaneously reducing the risk of resistance.

[0003] s-nitrosoglutathione (GSNO), as a natural donor of nitric oxide (NO), can decompose to produce NO in the slightly acidic environment inside bacterial biofilms. NO, as a gaseous signaling molecule, specifically downregulates the core genes involved in MRSA biofilm formation, thus preventing biofilm formation and maturation at its source. It can also inhibit biofilm formation and maturation through its own processes and reactive nitrogen derivatives (RNS, such as ONOO). - GSH (Glycosyl Oxide) targets MRSA at multiple sites and oxidizes and consumes GSH, causing bacteria to lose their main antioxidants, thereby achieving the goal of clearing MRSA. Therefore, a certain concentration of GSNO can inhibit the growth of bacterial biofilms and even kill bacteria, which is of great significance for treating infections caused by bacterial biofilms. However, GSNO is very unstable in aqueous solution, so other methods must be used to improve its stability.

[0004] Copper sulfide nanoparticles possess unique physical properties. With a particle size of only about 140 nm, this small size allows them to directly interact with bacterial cell membranes, disrupting their integrity and altering their permeability, ultimately leading to bacterial death. Furthermore, copper sulfide nanoparticles can generate reactive oxygen species (ROS) under near-infrared light excitation, including hydroxyl radicals (·OH) and superoxide anion radicals (O2). -These substances, such as proteins and nucleic acids, can oxidize intracellular biomolecules, affecting normal bacterial metabolism and physiological functions, thus exerting an antibacterial effect. However, CuS is easily "captured" by the anionic groups in EPS and cannot diffuse into the deep biofilm's resident bacterial region, only clearing surface MRSA, leading to biofilm recurrence. Summary of the Invention

[0005] The purpose of this invention is to provide a CuS-GSNO@RBCM multifunctional nanoparticle.

[0006] Another objective of this invention is to provide a method for preparing the aforementioned CuS-GSNO@RBCM multifunctional nanoparticles. By loading GSNO onto copper sulfide as a carrier and then modifying it with erythrocyte membrane RBCM, the consistency of drug efficacy between GSNO and CuS is effectively improved, and the circulation time of the drug in the blood is prolonged, thereby enhancing the clearance effect against MRSA.

[0007] The third objective of this invention is the application of the aforementioned CuS-GSNO@RBCM multifunctional nanoparticles.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A CuS-GSNO@RBCM multifunctional nanoparticle is characterized by: CuS-GSNO nanoparticles being obtained by loading GSNO onto CuS as a carrier, and then being coated with mouse erythrocyte membrane RBCM to obtain CuS-GSNO@RBCM nanoparticles.

[0010] The average particle size of the CuS-GSNO@RBCM nanoparticles is 200 nm.

[0011] Furthermore, the CuS nanoparticles are prepared by dissolving CuCl2·2H2O and polyvinylpyrrolidone (PVPK30) in deionized water, then adding NaOH solution, L-ascorbic acid solution and sodium sulfide aqueous solution in sequence, reacting at 55~65℃ for 2~3h, and then centrifuging and washing.

[0012] Furthermore, the ratio of CuCl2·2H2O, PVP, and deionized water is 34~35mg:190~210mg:100mL.

[0013] Furthermore, the volume ratio of the deionized water, NaOH solution, L-AA solution, and sodium sulfide aqueous solution is 100:4~5:4~5:4~5, the concentration of NaOH solution is 0.15~0.25 M, the concentration of L-AA solution is 0.08~0.12 M, and the concentration of sodium sulfide aqueous solution is 0.8~1.2 M.

[0014] Furthermore, the CuS-GSNO nanoparticles are obtained by mixing CuS and s-nitrosoglutathione (GSNO) at a mass ratio of 1.8~2.2:1, mixing them on a drug delivery machine for 10~12 h, and then centrifuging and washing them.

[0015] Furthermore, the RBCM coating involves mixing CuS-GSNO nanoparticles with mouse erythrocyte membranes (RBCM) at a mass ratio of 4.5~5.5:1, sonicating for 25~35 min, and then extruding the RBCM-encapsulated nanoparticles using a micro extruder. Specifically, the nanoparticles are first repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 400 nm, and then repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

[0016] A method for preparing CuS-GSNO@RBCM multifunctional nanoparticles, characterized by: preparing CuS nanoparticles, loading GSNO onto CuS nanoparticles as a carrier to prepare CuS-GSNO nanoparticles, and then coating them with RBCM to form CuS-GSNO@RBCM nanoparticles.

[0017] Furthermore, the preparation of Cu nanoparticles involves dissolving CuCl2·2H2O and polyvinylpyrrolidone (PVPK30) in deionized water, then sequentially adding NaOH solution, L-ascorbic acid solution, and sodium sulfide aqueous solution, reacting at 55~65℃ for 2~3 h, and then centrifuging and washing to obtain CuS nanoparticles.

[0018] Furthermore, the ratio of CuCl2·2H2O, PVP, and deionized water is 34~35mg:190~210mg:100mL.

[0019] Furthermore, the volume ratio of the deionized water, NaOH solution, L-AA solution, and sodium sulfide aqueous solution is 100:4~5:4~5:4~5, the concentration of NaOH solution is 0.15~0.25 M, the concentration of L-AA solution is 0.08~0.12 M, and the concentration of sodium sulfide aqueous solution is 0.8~1.2 M.

[0020] Furthermore, the preparation of CuS-GSNO nanoparticles involves mixing CuS with s-nitrosoglutathione (GSNO) at a mass ratio of 1.8~2.2:1, then mixing the mixture on a drug delivery machine for 10~12 h, followed by centrifugation and washing to obtain CuS-GSNO nanoparticles.

[0021] Furthermore, the RBCM coating involves mixing CuS-GSNO nanoparticles with mouse erythrocyte membranes (RBCM) at a mass ratio of 4.5~5.5:1, sonicating for 25~35 min, and then extruding the RBCM-encapsulated nanoparticles using a micro extruder. Specifically, the nanoparticles are first repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 400 nm, and then repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

[0022] In this invention, based on hydrophilic and electrostatic interactions, GSNO is encapsulated in hollow CuS nanoparticles to obtain CuS-GSNO nanoparticles. Under near-infrared light irradiation, the ROS generated by the CuS nanoparticles can interact with the NO produced by the decomposition of GSNO to generate a stronger and more potent peroxynitrate anion (ONOO). - This RNS can effectively disrupt bacterial biofilm structures and has a more significant bactericidal effect against drug-resistant strains of MRSA. However, after CuS is loaded with GSNO, the nanoparticles exhibit significant aggregation, leading to a decrease in photothermal conversion efficiency, and the Cu produced by CuS... 2+ This catalyzes and promotes the decomposition of GSNO to release NO, causing GSNO to be catalytically decomposed before reaching the target site, resulting in premature NO release. However, under laser irradiation, CuS reacts with Cu through the PTT effect. 2+ The dual catalytic effect causes GSNO to rapidly decompose and release a large amount of free NO. This release is out of control, and the free NO cannot match and synergize with the ROS generated by CuS via PDT, thus triggering non-targeted quenching and reducing the antibacterial effect.

[0023] In this invention, RBCM is coated onto the surface of CuS-GSNO to achieve physical isolation and Cu 2+ Capture and inhibit the aggregation of CuS-GSNO nanoparticles while regulating Cu 2+ The nanoparticles pre-decompose GSNO and, through the phase transition characteristics of RBCM, induce a reversible phase transition in RBCM under laser irradiation due to the PTT effect of CuS. This leads to the simultaneous release of ROS and NO at the target site, generating more RNS that directly acts on the bacteria, resulting in effective synergistic killing. Furthermore, the nanoparticles generate a thermal difference within the particles through thermoelectric effects, promoting charge transfer and generating current, which further promotes the production of RNS and enhances the killing effect on MRSA.

[0024] A method for preparing CuS-GSNO@RBCM multifunctional nanoparticles, characterized by comprising the following steps: Preparation of S1.CuS nanoparticles Dissolve CuCl2·2H2O and polyvinylpyrrolidone (PVPK30) in deionized water. Stir the solution for 4–6 min, then add 0.15–0.25 M NaOH solution dropwise. After adding NaOH for 5 min, add 0.08–0.12 M L-ascorbic acid (L-AA) dropwise to the solution. Stir the mixture continuously at room temperature for 4–6 min, then add 0.8–1.2 M sodium sulfide aqueous solution dropwise to the solution. React at 55–65 °C for 2–3 h with a stirring rate of 400–500 rpm. After the reaction is complete, centrifuge at 8000–10000 rpm for 10–15 seconds. The precipitate was collected and washed alternately with water and ethanol. The resulting black precipitate was then resuspended in deionized water to obtain a CuS solution for later use. The ratio of CuCl2·2H2O, PVP and deionized water was 34~35mg:190~210mg:100mL, and the volume ratio of deionized water, NaOH solution, L-AA solution and sodium sulfide aqueous solution was 100:4~5:4~5:4~5. Synthesis of S2.CuS-GSNO nanoparticles According to the mass ratio of CuS to s-nitrosoglutathione (GSNO) of 1.8~2.2:1, GSNO was added to CuS solution, and the mixture was stirred on a drug delivery machine for 10~12 h. After centrifugation, the mixture was washed 2~3 times with RO water. The precipitate was resuspended in deionized water to obtain CuS-GSNO nanoparticle solution for later use. Synthesis of S3.CuS-GSNO@RBCM Nanoparticles The CuS-GSNO nanoparticle solution was mixed with mouse erythrocyte membrane (RBCM) at a mass ratio of 4.5~5.5:1. The mixture was then sonicated at 80~100 W for 25~35 min under ice bath conditions. Subsequently, the nanoparticles were extruded using a micro extruder to obtain RBCM-encapsulated nanoparticles. Specifically, the nanoparticles were first repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 400 nm, and then repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

[0025] The application of CuS-GSNO@RBCM nanoparticles prepared by the above method in the preparation of drugs for MRSA removal.

[0026] Therefore, combining GSNO and CuS with erythrocyte membranes shows significant advantages in biomimetic drug delivery systems. By modifying the surface of nanomedicines with purified erythrocyte membranes, the circulation time of drugs in the blood can be significantly prolonged, the consumption of endogenous ROS by NO can be reduced, and the release consistency of ROS generated by NO and CuS via PDT can be improved, thereby enhancing the synergistic bactericidal effect.

[0027] The present invention has the following technical effects: This invention effectively solves the problems of CuS-GSNO agglomeration and poor consistency between ROS generated by CuS via PDT and NO released by GSNO by using CuS loaded with GSNO and then coated with RBCM. Through multiple synergistic effects such as ROS generation by PDT, ROS reaction with NO to generate RNS, and thermoelectric effect promoting RNS generation, the killing effect of nanoparticles on MRSA is improved. Attached Figure Description

[0028] Figure 1 X-ray diffraction pattern of CuS nanoparticles prepared in this invention.

[0029] Figure 2 TEM and SEM images of the CuS nanoparticles prepared in this invention.

[0030] Figure 3 TEM images of CuS-GSNO and CuS-GSNO@RBCM nanoparticles prepared in this invention.

[0031] Figure 4 Particle size distribution diagrams of different formulation groups.

[0032] Figure 5 Zeta potential diagrams for different formulation groups.

[0033] Figure 6 Qualitative and quantitative targeting binding results of RHB-labeled CuS@RBCM after co-incubation with MRSA USA300 for different times.

[0034] Figure 7 Live / dead staining images of MRSA USA300 after treatment with different formulations.

[0035] Figure 8 FM images generated by ROS after MRSA USA300 was treated with different formulation groups.

[0036] Figure 9 FM images generated by RNS after MRSA USA300 was treated with different formulation groups.

[0037] Figure 10 Changes in bacterial membrane potential after treatment with different formulations of MRSA USA300.

[0038] Figure 11 Safety testing of CuS-GSNO@RBCM nanoparticles: A. In vitro cytotoxicity test; B. Hemolytic reaction test.

[0039] Figure 12 Thermoelectric properties of CuS particles: A. Surface impedance of CuS nanoparticles under light and darkness; B. Surface current of CuS nanoparticles in laser switching cycle; C. AFM surface potential diagram of CuS nanoparticles under no light (25℃); E. AFM surface potential diagram of CuS nanoparticles under laser irradiation (55℃). Detailed Implementation

[0040] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0041] Example 1 A method for preparing NO / PDT multifunctional copper sulfide nanoparticles includes the following steps: Preparation of S1.CuS nanoparticles Dissolve 35 mg of CuCl2·2H2O and 210 mg of polyvinylpyrrolidone (PVPK30) in 100 mL of deionized water. After stirring the solution for 4 min, add 5 mL of 0.15 M NaOH solution dropwise. After adding NaOH for 4 min, add 5 mL of 0.08 M L-ascorbic acid (L-AA) dropwise to the solution. Stir the mixture continuously at room temperature for 5 min, then add 5 mL of 0.8 M sodium sulfide aqueous solution dropwise to the solution. The solution immediately turns black. React at 55 °C for 3 h with stirring at 400 rpm. After the reaction is complete, centrifuge at 8000 rpm for 15 min, collect the precipitate, wash with water and ethanol alternately, and then resuspend the obtained black precipitate in deionized water to obtain CuS solution for later use. Synthesis of S2.CuS-GSNO nanoparticles According to the mass ratio of CuS to s-nitrosoglutathione (GSNO) of 1.8:1, GSNO was added to CuS solution, and the mixture was stirred on a drug delivery machine for 10 h. After washing with RO water three times, the precipitate was resuspended in deionized water to obtain CuS-GSNO nanoparticle solution for later use. Synthesis of S3.CuS-GSNO@RBCM Nanoparticles The CuS-GSNO nanoparticle solution was mixed with mouse erythrocyte membrane (RBCM) at a mass ratio of 4.5:1 and sonicated for 25 min. Then, the nanoparticles were extruded using a micro extruder to obtain RBCM-encapsulated nanoparticles. Specifically, the nanoparticles were first extruded 11 times with a polycarbonate membrane with a pore size of 400 nm, and then extruded 11 times with a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

[0042] Example 2 A method for preparing NO / PDT multifunctional copper sulfide nanoparticles includes the following steps: Preparation of S1.CuS nanoparticles Dissolve 34 mg of CuCl2·2H2O and 200.0 mg of polyvinylpyrrolidone (PVPK30) in 100 mL of deionized water. After stirring the solution for 6 min, add 4 mL of 0.25 M NaOH solution dropwise. After adding NaOH for 6 min, add 4 mL of 0.12 M L-ascorbic acid (L-AA) dropwise to the solution. Stir the mixture continuously at room temperature for 5 min, then add 4 mL of 1.2 M sodium sulfide aqueous solution dropwise to the solution. The solution immediately turns black. React at 60 °C for 3 h with stirring. After the reaction is complete, centrifuge at 9000 rpm for 12 min, collect the precipitate, wash with water and ethanol alternately, and then resuspend the resulting black precipitate in deionized water to obtain CuS solution for later use. Synthesis of S2.CuS-GSNO nanoparticles At a mass ratio of CuS to s-nitrosoglutathione (GSNO) of 2.2:1, GSNO was added to the CuS solution and mixed on a drug delivery machine for 12 h. The mixture was then washed twice with RO water, and the resulting precipitate was resuspended in deionized water to obtain a CuS-GSNO nanoparticle solution for later use. Synthesis of S3.CuS-GSNO@RBCM Nanoparticles The CuS-GSNO nanoparticle solution was mixed with mouse erythrocyte membrane (RBCM) at a mass ratio of 5.5:1. The mixture was then sonicated at 80 W for 35 min under ice bath conditions. Subsequently, the nanoparticles were extruded using a micro extruder to encapsulate the RBCM. Specifically, the nanoparticles were first extruded 13 times with a polycarbonate membrane with a pore size of 400 nm, and then extruded 13 times with a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

[0043] Example 3 A method for preparing NO / PDT multifunctional copper sulfide nanoparticles includes the following steps: Preparation of S1.CuS nanoparticles Dissolve 34.5 mg of CuCl2·2H2O and 190 mg of polyvinylpyrrolidone (PVPK30) in 100 mL of deionized water. After stirring the solution for 5 min, add 4.5 mL of 0.2 M NaOH solution dropwise. After adding NaOH for 5 min, add 4.5 mL of 0.1 M L-ascorbic acid (L-AA) dropwise to the solution. Stir the mixture continuously at room temperature for 5 min, then add 4.5 mL of 1.0 M sodium sulfide aqueous solution dropwise to the solution. The solution immediately turns black. React at 60 °C for 3 h with stirring. After the reaction is complete, centrifuge at 10000 rpm for 10 min, collect the precipitate, wash with water and ethanol alternately, and then resuspend the resulting black precipitate in deionized water to obtain CuS solution for later use. Synthesis of S2.CuS-GSNO nanoparticles At a mass ratio of CuS to s-nitrosoglutathione (GSNO) of 2:1, GSNO was added to the CuS solution and mixed on a drug delivery machine for 12 h. The mixture was then washed twice with RO water, and the resulting precipitate was resuspended in deionized water to obtain a CuS-GSNO nanoparticle solution for later use. Synthesis of S3.CuS-GSNO@RBCM Nanoparticles The CuS-GSNO nanoparticle solution was mixed with mouse erythrocyte membrane (RBCM) at a mass ratio of 5:1 and sonicated for 30 min. Then, the nanoparticles were extruded using a micro extruder to obtain RBCM-encapsulated nanoparticles. Specifically, the nanoparticles were first extruded 11 times with a polycarbonate membrane with a pore size of 400 nm, and then extruded 11 times with a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

[0044] The CuS nanoparticles prepared in Example 3 were freeze-dried to obtain powder, and after thorough grinding, their X-ray diffraction spectra were measured using an X-ray diffractometer. Figure 1 As shown, SEM and TEM images of CuS nanoparticles are as follows: Figure 2 As shown in the image (scale bar = 200 nm), CuS exhibits good dispersion and uniform particle size. TEM images of the prepared CuS-GSNO nanoparticles and CuS-GSNO@RBCM nanoparticles are shown below. Figure 3 As shown (scale bar = 200nm).

[0045] The particle size distributions of CuS-GSNO nanoparticles and CuS-GSNO@RBCM nanoparticles are as follows: Figure 4 As shown, the particle size of CuS-GSNO nanoparticles is concentrated at around 150 nm, while the particle size of CuS-GSNO@RBCM nanoparticles increases to around 200 nm.

[0046] The zeta potentials of CuS-GSNO nanoparticles and CuS-GSNO@RBCM nanoparticles are as follows: Figure 5 As shown, the negative potential of CuS-GSNO nanoparticles increases after further coating with RBCM.

[0047] RHB-labeled CuS@RBCM was co-incubated with MRSA USA300 for different times. The affinity between the nanoparticles and bacteria was qualitatively and quantitatively analyzed using fluorescence microscopy (FM) and flow cytometry (FCM). The results are as follows: Figure 6 As shown (scale bar = 10 μm). Blank represents bright-field imaging, Rhodamine (RHB) represents the red fluorescence group, and Merged represents the merged image. It can be seen that the fluorescence of the RHB group gradually increases and brightens at 0.5 h and 1 h, visually demonstrating the enhanced binding of nanoparticles to bacteria over time, qualitatively indicating that the affinity of nanoparticles for MRSA increases over time. In the quantitative analysis, the peak fluorescence intensity of the RHB group shifts to the right over time, indicating a significant increase in the number of nanoparticles bound to bacteria.

[0048] The groups included CuS, CuS+Laser, CuS-GSNO@RBCM, and CuS-GSNO@RBCM+Laser, with 1064nm laser irradiation: The bacterial viability of CuS and CuS-GSNO@RBCM nanoparticle solutions after co-incubation with bacteria for 12 h was evaluated using a live / dead bacteria staining kit. SYTO9 was the green fluorescence channel, PI was the red fluorescence channel, and Merged was the overlay channel. The results are shown below. Figure 7 As shown (scale bar = 20 μm, G1 to G5 represent the control group, CuS group, CuS+Laser group, CuS-GSNO@RBCM group, and CuS-GSNO@RBCM+Laser group respectively), only a small number of bacteria in the CuS, CuS+Laser, and CuS-GSNO@RBCM groups were stained red, indicating that their antibacterial activity was poor. However, a large number of bacteria in the CuS-GSNO@RBCM+Laser group were stained red, indicating that the bactericidal ability of CuS-GSNO@RBCM was significantly enhanced after 1064 nm laser irradiation. That is, under the action of light, CuS promotes GSNO to produce more NO, and works synergistically with the ROS reaction produced by PDT to improve the overall bactericidal ability.

[0049] The generation of ROS in bacteria was detected using the DCFH-DA probe. The fluorescence intensity of DCF (green fluorescence) was observed using FM to assess the level of ROS generation in bacteria. Hoechst was used for blue fluorescence, and Merged was used for merged images. Results are shown below. Figure 8 As shown (scale bar = 20 μm, G1 to G5 represent the control group, CuS group, CuS+Laser group, CuS-GSNO@RBCM group, and CuS-GSNO@RBCM+Laser group, respectively), virtually no green fluorescence was observed in the CuS, CuS+Laser, and CuS-GSNO@RBCM groups, indicating very low ROS generation. However, the CuS-GSNO@RBCM+Laser group showed significant green fluorescence, indicating substantial ROS generation under these conditions. This is likely due to the PDT effect of the nanoparticles, which promotes ROS generation under 1064 nm illumination. Theoretically, the CuS+Laser group should generate more ROS, resulting in stronger fluorescence than the CuS-GSNO@RBCM group, which should be weaker due to NO consuming ROS. The experimental results are the opposite, suggesting that RBCM coating enhances the binding of nanoparticles to bacteria and the probe uptake efficiency, thereby strengthening the fluorescence signal.

[0050] The distribution of intrabacterial RNS was detected using a fluorescent O52 probe under FM conditions, with Hoechst as the blue fluorescent dye and ONOO. - It is a reactive oxygen species (RNS), which is detected by a specific fluorescent probe and reacts with ONOO. - The reaction produces green fluorescence, the intensity of which is similar to that of intracellular ONOO. - The content is directly positively correlated. Merged images are merged; experimental results are as follows: Figure 9 As shown (scale bar = 20 μm, G1 to G5 represent the control group, CuS group, CuS+Laser group, CuS-GSNO@RBCM group, and CuS-GSNO@RBCM+Laser group, respectively), almost no green fluorescence signal was observed in the CuS group alone, while a small amount of green fluorescence was observed in the CuS+Laser group. This may be attributed to the local thermal effect of CuS under illumination or the low level of ROS inducing RNS generation. In contrast, a small amount of green fluorescence was observed in the CuS-GSNO@RBCM group, while a large amount of green fluorescence was observed in the CuS-GSNO@RBCM+Laser group. These results indicate that the slow-release behavior of GSNO in CuS-GSNO@RBCM nanoparticles can promote RNS generation, and 1064 nm light stimulation significantly promotes RNS generation. This suggests that under illumination, the PTT effect of CuS and the CuS-GSNO@RBCM-regulated CuS... 2+The catalytic effect promotes the decomposition of GSNO to release NO, which matches the ROS generated by CuS via PDT, thereby generating more RNS.

[0051] A bacterial membrane potential detection kit was used to assess changes in bacterial membrane potential after nanoparticle interaction. Figure 10 (Scale bar = 20 μm) It can be seen that the green fluorescence of the CuS-GSNO@RBCM+Laser group is significantly enhanced, indicating that the bacterial inner membrane has undergone depolarization. This increases membrane permeability, allowing nanoparticles to more easily enter the bacterial interior and thus exert a better antibacterial effect. In contrast, the green fluorescence of the CuS+Laser group is relatively weak, followed by the CuS-GSNO@RBCM group. Green fluorescence is barely observed when CuS is used alone. These results indicate that CuS nanoparticles alone have little effect on bacterial membrane permeability, and the effects of CuS and the natural slow release of GSNO on membrane permeability are also relatively small. However, 1064 nm laser irradiation of CuS-GSNO@RBCM nanoparticles can enhance the depolarization ability of the bacterial inner membrane, thereby increasing membrane permeability and allowing nanoparticles to more easily enter the bacterial interior and thus exert a better antibacterial effect. This demonstrates that CuS generates ROS through PDT under light irradiation, and that CuS exhibits a PTT effect after light irradiation, while RBCM-regulated CuS... 2+ The catalytic effect promotes the decomposition of GSNO to release NO, which then reacts with ROS to generate ONOO, which has higher oxidation activity. - It plays a role in further enhancing membrane permeability.

[0052] The MTT assay was used to detect the toxicity of CuS-GSNO@RBCM nanoparticles in RAW264.7 cells, and the results are as follows: Figure 11 As shown in Figure A, after 24 h of interaction between different concentrations of CuS-GSNO@RBCM nanoparticles and cells, the cell survival rate remained above 85%, even at the maximum tested concentration of 150 μg / mL (higher than the in vivo experimental concentration), the cell survival rate remained at a normal level. These results indicate that CuS-GSNO@RBCM nanoparticles possess very low cytotoxicity.

[0053] The blood compatibility of CuS-GSNO@RBCM nanoparticles was investigated using mouse erythrocytes, and the results are as follows: Figure 11 As shown in Figure B, the hemolysis rate of CuS-GSNO@RBCM nanoparticles mixed with red blood cell suspension at different concentrations was less than 5%, and the maximum test concentration of 200 μg / mL selected here is also much higher than the in vivo administration concentration. These results indicate that CuS-GSNO@RBCM nanoparticles have good blood compatibility.

[0054] The changes in resistance and current of Cus nanoparticles before and after 1064 nm laser irradiation were studied using an electrochemical workstation. The results are as follows: Figure 12 As shown, the surface resistance of CuS nanoparticles decreased significantly after irradiation with a 1064 nm laser. The current initially increased and then decreased with the switching of the laser. This indicates that CuS nanoparticles possess excellent thermoelectric properties. Surface potential measurements of CuS nanoparticles using atomic force microscopy (AFM) compared the potentials before and after near-infrared irradiation (25℃) and after irradiation (55℃), revealing a more significant potential change at 55℃. These results collectively demonstrate the superior thermoelectric properties of CuS nanoparticles. Their unique thermoelectric conversion capability provides a powerful catalytic platform for the efficient generation of reactive oxygen species (RNS), opening a new pathway for researching novel and highly effective antibacterial treatment strategies. Through the thermoelectric effect, CuS-GSNO@RBCM nanoparticles generate a thermal difference within the particles, promoting charge transfer and thus generating current, further promoting RNS generation and enhancing the killing effect on MRSA.

Claims

1. A CuS-GSNO@RBCM multifunctional nanoparticle, characterized in that: CuS-GSNO nanoparticles were obtained by loading GSNO onto CuS as a carrier, and then CuS-GSNO@RBCM nanoparticles were obtained by coating them with mouse erythrocyte membranes.

2. The CuS-GSNO@RBCM multifunctional nanoparticle as described in claim 1, characterized in that: The CuS nanoparticles are prepared by dissolving CuCl2·2H2O and polyvinylpyrrolidone (PVPK30) in deionized water, then adding NaOH solution, L-ascorbic acid solution and sodium sulfide aqueous solution in sequence, reacting at 55-65℃ for 2-3 h, and then centrifuging and washing.

3. The CuS-GSNO@RBCM multifunctional nanoparticle as described in claim 2, characterized in that: The CuS-GSNO nanoparticles are obtained by mixing CuS and s-nitrosoglutathione at a mass ratio of 1.8~2.2:1, mixing them on a drug delivery machine for 10~12 h, and then centrifuging and washing them.

4. The CuS-GSNO@RBCM multifunctional nanoparticle as described in claim 3, characterized in that: The RBCM coating process involves mixing CuS-GSNO nanoparticles with mouse erythrocyte membranes at a mass ratio of 4.5–5.5:1, sonicating for 25–35 minutes, and then extruding the mixture using a micro extruder to obtain erythrocyte membranes that encapsulate the nanoparticles. Specifically, the nanoparticles are first repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 400 nm, and then repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

5. A method for preparing CuS-GSNO@RBCM multifunctional nanoparticles, characterized in that: First, CuS nanoparticles were prepared, then CuS-GSNO nanoparticles were prepared by loading GSNO onto CuS nanoparticles as a carrier, and finally CuS-GSNO@RBCM nanoparticles were formed by coating with RBCM.

6. The method for preparing CuS-GSNO@RBCM multifunctional nanoparticles as described in claim 5, characterized in that: The preparation of CuS nanoparticles involves dissolving CuCl2·2H2O and polyvinylpyrrolidone (PVPK30) in deionized water, then sequentially adding NaOH solution, L-ascorbic acid solution, and sodium sulfide aqueous solution. The mixture is reacted at 55-65°C for 2-3 hours, followed by centrifugation and washing to obtain CuS nanoparticles.

7. The method for preparing CuS-GSNO@RBCM multifunctional nanoparticles as described in claim 6, characterized in that: The ratio of CuCl2·2H2O, PVP, and deionized water is 34~35mg:190~210mg:100mL. The volume ratio of deionized water, NaOH solution, L-AA solution, and sodium sulfide aqueous solution is 100:4~5:4~5:4~5. The concentration of NaOH solution is 0.15~0.25 M, the concentration of L-AA solution is 0.08~0.12M, and the concentration of sodium sulfide aqueous solution is 0.8~1.2M.

8. The method for preparing CuS-GSNO@RBCM multifunctional nanoparticles as described in claim 7, characterized in that: The preparation of CuS-GSNO nanoparticles involves mixing CuS and s-nitrosoglutathione at a mass ratio of 1.8~2.2:1, then mixing them on a drug delivery machine for 10~12 h, followed by centrifugation and washing to obtain CuS-GSNO nanoparticles.

9. The method for preparing CuS-GSNO@RBCM multifunctional nanoparticles as described in claim 8, characterized in that: The RBCM coating process involves mixing CuS-GSNO nanoparticles with mouse erythrocyte membranes at a mass ratio of 4.5–5.5:1, sonicating for 25–35 minutes, and then extruding the mixture using a micro extruder to obtain erythrocyte membranes that encapsulate the nanoparticles. Specifically, the nanoparticles are first repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 400 nm, and then repeatedly extruded 11 or 13 times using a polycarbonate membrane with a pore size of 200 nm to obtain CuS-GSNO@RBCM nanoparticles.

10. The application of CuS-GSNO@RBCM multifunctional nanoparticles prepared by the method of claim 9 in the preparation of drugs for scavenging MRSA.

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