Bacteria-algae fusion vesicle loaded copper sulfide nanoparticles and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine and health, in particular to a nano-particle with copper sulfide loaded on bacterium-algae fused vesicles as well as a preparation method and application of the nano-particle. The invention provides nano-particles with copper sulfide loaded on bacterium-algae fusion vesicles. The nano-particles are prepared from the following components in parts by mass: 30 to 75 parts of bacterium-algae fusion vesicles, 20 to 50 parts of copper sulfide nano-particles and 5 to 20 parts of antitumor drugs. The raw materials used in the method have good biocompatibility and biodegradability and are low in cost, the preparation method is simple, and the prepared bacterium-algae fusion vesicle loaded copper sulfide nano-drug is good in stability, small in size, capable of being accumulated at a recurrent tumor position in a targeting mode and good in treatment effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and health, and particularly relates to a kind of bacteria-algae fusion vesicle loaded copper sulfide nanoparticles and its preparation method and application. BACKGROUND

[0002] For most solid tumors, surgical treatment is still the most effective treatment, however, due to the complexity of tumor characteristics, drug resistance and strong invasion, the probability of postoperative recurrence and metastasis is high. Some related clinical studies have confirmed that the immune function status of surgery is a positive factor for postoperative metastasis-free survival, the higher the body immune level, the longer the postoperative immune function failure time, and the longer the protection time of tumor recurrence, so activating the immune system and long-term tumor immune memory is the key to preventing tumor recurrence. Immune cell death (ICD) as a form of cell death that activates the innate immune system to eliminate cancer cells and maintain long-term immune vigilance, has become a particularly effective immune stimulation mechanism, which may remodel the tumor microenvironment and enhance immunogenicity, thereby initiating systemic anti-tumor immunity.

[0003] The reactive oxygen species (ROS) generated in photodynamic therapy (PDT) can trigger ICD by causing oxidative stress in tumor cells, while photothermal therapy (PTT) can effectively destroy tumor cells and promote the release of tumor-associated antigens, thereby enhancing the ICD effect. The combination of the two can produce a spatiotemporal synergistic effect, synergistically amplifying the ICD response. Since recurrent lesions are often distributed in small nodules, traditional nanocarriers are limited by abnormal vascular structures and dense matrix barriers, and have low intratumoral enrichment efficiency. Therefore, developing a delivery system that can precisely target recurrent lesions, overcome the microenvironment barrier and synergistically enhance ICD is a key path to break through the clinical treatment dilemma. SUMMARY

[0004] The purpose of the present application is mainly to solve the problem of low treatment effect of single therapy and the limitation of tumor inhibition microenvironment, and to provide a synergistic therapy system based on bacteria-algae fusion vesicles and CuS nanoparticles, which can achieve efficient inhibition of recurrent tumors.

[0005] To achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides a kind of bacteria-algae fusion vesicle loaded copper sulfide nanoparticles, including the following mass fraction components: bacteria-algae fusion vesicle: 30-75 parts, copper sulfide nanoparticles: 20-50 parts, antitumor drug: 5-20 parts.

[0007] The bacterium-alga fusion vesicle is prepared by membrane fusion method from outer membrane vesicles extracted from Escherichia coli and spirulina exosome; the copper sulfide nanoparticles are hollow mesoporous copper sulfide nanoparticles prepared by hydrothermal synthesis method; and the antitumor drug is loaded into the hollow mesoporous structure of the copper sulfide nanoparticles.

[0008] Preferably, the antitumor drug is one of a chemotherapeutic drug, a small molecule inhibitor and a STING agonist.

[0009] Preferably, the chemotherapeutic drug is doxorubicin, cisplatin or fluorouracil; the small molecule inhibitor is pyrotinib or 3-bromopyruvic acid; and the STING agonist is cyclic dinucleotide.

[0010] Preferably, the Escherichia coli is at least one of Escherichia coli BL21, Escherichia coli DH5α, Escherichia coli MG1655 and Escherichia coli Nissle1917.

[0011] The application provides a preparation method of the copper sulfide bacterium-alga fusion vesicle.

[0012] (1) centrifuging Escherichia coli liquid, collecting supernatant, discarding supernatant after centrifugation, and resuspending the precipitate;

[0013] (2) centrifuging spirulina liquid, collecting precipitate, resuspending, and then performing ultrasonic cell disruption, centrifuging to obtain supernatant, discarding supernatant after centrifugation, and resuspending the precipitate;

[0014] (3) mixing the resuspended precipitate of step (1) and the resuspended precipitate of step (2), performing ultrasonic treatment and extrusion to obtain bacterium-alga fusion vesicles;

[0015] (4) mixing, stirring, water-bathing, centrifuging, washing and vacuum drying a copper chloride dihydrate solution, polyvinylpyrrolidone, a sodium hydroxide solution, a hydrazine hydrate solution and sodium sulfide nonahydrate to obtain copper sulfide nanoparticles;

[0016] (5) mixing an antitumor drug and the copper sulfide nanoparticles obtained in step (4), performing ultrasonic treatment, stirring, centrifuging, washing and vacuum drying to obtain drug-loaded copper sulfide nanoparticles;

[0017] (6) mixing, ultrasonic treating, extruding, centrifuging and resuspending the products obtained in steps (3) and (5) to obtain copper sulfide bacterium-alga fusion vesicles.

[0018] Preferably, the mass ratio of the resuspended precipitate of step (1) to the resuspended precipitate of step (2) in step (3) is 0.8-1.2:0.8-1.2; the ultrasonic treatment is performed at a power of 65-75 W for 8-12 min; and the extrusion is performed by continuously passing through a 0.22 μm filter membrane for 8-13 times.

[0019] Preferably, the ratio of the copper chloride dihydrate solution, polyvinylpyrrolidone, sodium hydroxide solution, hydrazine hydrate solution and sodium sulfide nine hydrate in step (4) is 80-120 μL: 0.2-0.3 g: 20-30 mL: 6-7 μL: 150-250 μL.

[0020] The application provides application of the copper sulfide-loaded chlorella fusional vesicle in preparation of an antitumor drug.

[0021] The application provides an antitumor drug comprising the copper sulfide-loaded chlorella fusional vesicle.

[0022] Preferably, the copper sulfide-loaded chlorella fusional vesicle is used in combination with photodynamic therapy, photothermal therapy, chemical dynamics therapy, immunotherapy or metabolic regulation technology.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The copper sulfide-loaded chlorella fusional vesicle of the application is a nanometer drug, and is prepared by fusing an outer membrane vesicle (OMV) of escherichia coli and an exosome (SP-EVs) of spirulina through a membrane fusion technology to construct a hybrid vesicle (OMEVs). The hybrid vesicle retains lipopolysaccharide (LPS), a characteristic component of the outer membrane of gram-negative bacteria, and is specifically combined with neutrophils through a TLR4 receptor, thereby laying a foundation for "hitchhiking" delivery. Meanwhile, the hybrid vesicle inherits the photosynthetic system characteristics of spirulina, and chlorophyll derivatives produce reactive oxygen species under the excitation of a 660 nm laser, and the hybrid vesicle can also express catalase to decompose endogenous H2O2 in tumors to produce oxygen, thereby relieving the restriction of the tumor microenvironment. This design solves the problem that a single carrier cannot simultaneously meet the targeting, photosensitivity and catalytic functions.

[0025] The application realizes synergistic effect through a triple timing control strategy, generates 1O2 by using photodynamic therapy to directly kill tumor cells, synchronously catalyzes the decomposition of H2O2 in tumors to reverse PDT resistance, further induces tumor cell apoptosis by using the photothermal conversion of CuS NPs, continuously releases 3-BP to inhibit hexokinase, block the glycolysis pathway, reduce the levels of ATP and lactic acid, reduce the expression of heat shock proteins (HSPs), reverse tumor heat resistance, and significantly enhance the effects of PTT / CDT. The multi-mode synergistic therapy of PDT+PTT+CDT+immunotherapy+metabolic regulation is used to synergistically kill tumors and break through the limitations of single therapy.

[0026] The copper sulfide-loaded chlorella fusional vesicle nanometer drug has good biocompatibility, biodegradability and low cost, the preparation method is simple, the prepared copper sulfide-loaded chlorella fusional vesicle nanometer drug has good stability and small size, can be accumulated at a recurrent tumor site, and has good therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only constitute the embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative effort on the basis of the provided drawings.

[0028] Figure 1 Transmission electron microscopy (TEM) images of the products OMEVs (a), CuS (b), C-B@OMEVs (c) in Example 1;

[0029] Figure 2 Temperature changes of C-B@OMEVs with different concentrations under laser irradiation (808 nm, 1.5 W·cm -2 , 10 min) (a) and temperature changes of C-B@OMEVs (100 μg / mL) under laser irradiation with different power densities (808 nm, 10 min) (b) in Example 2;

[0030] Figure 3 Heating and cooling cycle curves of C-B@OMEVs (200 μg / mL) under on / off 808 nm laser irradiation (a) and time constant of the heating and cooling curves (b) in Example 2;

[0031] Figure 4 In vitro drug release curve of C-B@OMEVs in Example 2;

[0032] Figure 5 O2 generation of C-B@OMEVs NPs in Example 2; 1

[0033] Figure 6 Catalase activity of SP-EVs, 1917-OMVs, OMEVs and C-B@OMEVs in Example 2;

[0034] Figure 7 Cell live staining of HUVEC cells after incubation with different preparations for 24 h in Example 2 (scale: 100 μm);

[0035] Figure 8 Cell survival rate of 4T1 cells treated with C-B@OMEVs in Example 2. DETAILED DESCRIPTION

[0036] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application. ​

[0037] Experimental instrument name and model: Japan Shimadu UV-Vis2700 ultraviolet spectrophotometer; Japan Hitachi HT7700 transmission electron microscope; British MarvinNano-ZS 90 laser particle size analyzer; American BioTek SynergyH1 multifunctional enzyme label instrument; Germany Leica SP8 laser confocal microscope; Beijing layer waves flow cytometer.

[0038] Example 1

[0039] A preparation method of a bacterium-alga fusion vesicle loaded with copper sulfide nanoparticles, the steps of the preparation method described in the embodiment are:

[0040] 1. Escherichia coli Nissle1917 (purchased from Shanghai Shenguo Biological Engineering Co., Ltd.) was inoculated into LB medium at a ratio of 5%, and when the optical density value at 600 nm reached about 1.5, the bacterial solution was centrifuged at 12000xg for 10 min, the supernatant was collected, and then 0.45μm filter membrane was used for suction filtration. Then use the ultra-high-speed centrifuge at 4℃, with a speed of 200,000xg, centrifuge for 6h, pour off the supernatant, resuspend the precipitate in PBS, and obtain the outer membrane vesicle (OMV) of Escherichia coli.

[0041] 2. Spirulina (purchased from Shanghai Guangyu Biological Technology Co., Ltd.) was inoculated into the culture medium at a ratio of 1:4, and after seven days of culture, the fresh algal liquid was centrifuged at 12000rpm for 10min, the supernatant was poured off and the precipitate was taken, an appropriate amount of PBS was added for resuspension, and an ultrasonic cell crusher was used with a power of 72W, with a mode of 5s on and 6s off, and the ultrasonic was 40min. Then centrifuge at 12000rpm for 10min, then use the ultra-high-speed centrifuge at 4℃, with a speed of 140,000xg, centrifuge for 2h, repeat the above operation twice. Then pour off the supernatant, resuspend the precipitate in PBS, and obtain the spirulina exosome (SP-EVs).

[0042] 3. Mix the prepared Escherichia coli outer membrane vesicle (OMV) and spirulina exosome (SP-EVs) at a protein ratio of 1:1, then ultrasonic (ultrasonic power 500W) treatment for 10min, and pass through 0.22μm filter membrane 11 times in succession to obtain the fusion vesicle (OMEVs) of Escherichia coli outer membrane vesicle and spirulina exosome.

[0043] 4. At room temperature, with magnetic stirring, 100 μL of a 67.5 mg / mL copper chloride dihydrate solution and 0.24 g of polyvinylpyrrolidone (PVP-K40) were dispersed in 25 mL of deionized water and stirred for 5 min. Then, 25 mL of sodium hydroxide solution with pH = 9 was added and stirred for 5 min. Subsequently, 6.4 μL of 85% hydrazine hydrate solution was added and stirred for 5 min. Finally, 200 μL of a 320 mg / mL sodium sulfide nonahydrate solution was added, and the mixture was calcined at 60 °C for 2 h. After centrifugation at 12000 rpm for 10 min at room temperature, the product was washed three times with deionized water to obtain copper sulfide nanoparticles (CuS NPs).

[0044] 5. Disperse 2.0 mg of copper sulfide nanoparticles in 4 mL of 15.0 mg / mL 3-bromopyruvic acid solution and sonicate (500 W) for 30 min. Then, stir the mixture at room temperature for 24 h. Centrifuge at 10000 rpm for 10 min, and wash three times with PBS to remove free 3-bromopyruvic acid, finally obtaining copper sulfide nanoparticles loaded with 3-bromopyruvic acid.

[0045] 6. The copper sulfide nanoparticles loaded with 3-bromopyruvic acid and the fusion vesicles obtained in step 3 were mixed at a mass ratio of 1:1, then sonicated (ultrasonic power 500W) for 30 min, passed through a 0.22 μm filter membrane 11 times, and then centrifuged at 10000 rpm for 5 min. Finally, copper sulfide nanoparticles loaded with bacterial-algal fusion vesicles were obtained.

[0046] Transmission electron microscopy (TEM) images and elemental analysis diagrams of the components of the copper sulfide-loaded vesicle nanoparticles prepared in this embodiment are shown below. Figure 1 .from Figure 1 As can be observed in a, the prepared macrophyte fusion vesicles are all spherical or nearly spherical, with obvious phospholipid bilayer structure and a size of 150-200 nm. Figure 1 Figure b shows that the prepared copper sulfide nanoparticle drug carrier CuS has a hollow mesoporous structure, and the hollow structure is clear, small in size, and uniform in morphology, with a size of 150-200 nm. Figure 1 In the image, c represents the product CB@OMEVs after fusion vesicle encapsulation. A membrane layer can be seen, proving that the fusion vesicles were successfully loaded.

[0047] Example 2

[0048] This embodiment performs functional tests on the copper sulfide-loaded nanoparticles of the bacterial-algae fusion vesicles prepared in Example 1, as detailed below:

[0049] 1. In vitro photothermal performance experiment

[0050] The copper sulfide nanoparticles loaded with the fused algae-bacteria vesicles prepared in Example 1 were formulated into solutions with concentrations of 0 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. Then, an 808 nm near-infrared laser at 1.5 W / cm² was used. 2 The solution was irradiated at the specified energy density for 10 minutes, with the temperature recorded every 20 seconds. Results are shown below. Figure 2 According to a Figure 2 As shown in Figure a, when the solution concentrations were 0 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 200 μg / mL, the temperature increased by 1.6℃, 10.1℃, 12.4℃, 16.9℃, 24.3℃, and 31.8℃ respectively within 10 minutes. The results indicate that the nanoparticles possess excellent photothermal conversion properties, and this photothermal conversion effect is concentration-dependent. A solution of copper sulfide-loaded vesicles from the algal-bacterial fusion nanoparticles prepared in Example 1 was prepared with a concentration of 100 μg / mL, and then tested using an 808 nm near-infrared laser at different power densities of 0.5 W / cm². 2 1.0W / cm 2 1.5W / cm 2 Irradiation was performed for a total of 10 minutes, and the temperature of the solution was recorded every 20 seconds. Figure 2 As shown in Figure b, different power densities of 0.5 W / cm² are obtained. 2 1.0W / cm 2 1.5W / cm 2 At the specified times, the temperature increased by 14.4℃, 19.3℃, and 24.3℃, respectively. The results indicate that the photothermal conversion effect of these nanoparticles exhibits laser power dependence. These results demonstrate that CB@OMEVs NPs possess excellent photothermal properties and can be used for anti-tumor therapy via photothermal therapy.

[0051] 2. Photothermal conversion efficiency measurement experiment

[0052] The CB@OMEVs NPs prepared in Example 1 were used to prepare a solution with a concentration of 200 μg / mL, and then an 808 nm near-infrared laser at 1.5 W / cm² was used. 2 The photothermal stability of the nanomedicine was evaluated by subjecting the solution to five laser on / off cycles at a power density of [specific value]. In each cycle, the nanomedicine solution was irradiated with laser for 10 min, then the laser was turned off for 10 min, and the solution temperature was recorded every 20 s. Temperature change curves were plotted over these cycles. The results are as follows: Figure 3 As shown in figure a, the photothermal stability of CB@OMEVs NPs is demonstrated. When the temperature reaches the highest equilibrium temperature, the laser is turned off, and the solution temperature is recorded every 20 seconds. The results are shown in [Figure a]. Figure 3 b, τs The value is equal to 480.5, and the calculated photothermal conversion efficiency of the nanoparticles is 41.58%.

[0053] 3. In vitro drug release status

[0054] To investigate the in vitro drug release of copper sulfide-loaded nanoparticles from algal-bacterial fusion vesicles, 20.0 mg of the copper sulfide-loaded nanoparticles prepared in Example 1 were dispersed in 2.0 mL of PBS buffer and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The dialysis bag was immersed in 5.0 mL of pH 7.4 PBS buffer, and the entire release system was placed in a constant-temperature shaker at 37°C and 100 rpm. At corresponding time points (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 36, 38, 48, 60, 72 h), after thorough mixing, 1 mL of the release medium was drawn off and replaced with an equal volume of PBS buffer. After 36 h, the dialysis bag was removed, and the nanoparticles were released using 808 nm (1.5 W / cm²) at a concentration of 1.5 W / cm². 2 ) and 660nm (0.5W / cm 2 The laser irradiation lasted for 5 minutes. The content of 3-bromopyruvic acid was then detected using a UV spectrophotometer. The cumulative drug release was calculated using the following formula, and the release curve was plotted.

[0055]

[0056] The results are as follows Figure 4 As shown, the release kinetics of 3-bromopyruvate from CB@OMEVs NPs exhibited a slow curve, with only a modest cumulative release of 17.64% observed over 36 hours. Subsequently, CB@OMEVs NPs were subjected to 660 / 808 nm laser irradiation for 5 minutes. After a 2-hour interval, approximately 69.49% of the encapsulated 3-bromopyruvate was observed to be released, indicating that laser irradiation can trigger drug release. This release characteristic can enhance drug accumulation at the tumor site and improve the antitumor effect.

[0057] 4. Measurement 1 O2 generation capacity

[0058] In order to study CB@OMEVs NPs 1 To assess O2 generation capacity, 2 mL of CB@OMEVs NPs with a protein concentration of 200 μg / mL (SP-EVs) was added to 30 μL of 1 mg / mL ABDA solution, and the results were measured using a 660 nm microscope (0.5 W / cm²). 2 ), 808nm (1.5W / cm) 2The solution was irradiated with laser light for 0, 5, 10, 15, 20, 25, and 30 min, and the change in absorbance at 410 nm was observed. The results are as follows: Figure 5 As shown, an 808nm laser (0.5W cm⁻¹) was used. 2 ) irradiation

[0059] CB@OMEVs had no significant effect on the absorbance of ABDA at 400 nm; however, after irradiation with a 660 nm laser for 30 min, the absorbance of ABDA decreased by 35.33%. These results indicate that CB@OMEVs NPs possess excellent... 1 The ability to generate O2 can produce enough ROS to effectively kill tumors.

[0060] 5. Catalase activity and oxygen production capacity

[0061] To investigate the catalase activity of CB@OMEVs NPs, the activities of SP-EVs, 1917-OMVs (outer membrane vesicles of E. coli 1917), OMEVs, and CB@OMEVs were measured using a catalase assay kit. The results are as follows: Figure 6 As shown, with

[0062] Compared to SP-EVs, the catalase activities of OMEVs and CB@OMEVs NPs were not significantly affected by the preparation process. The results indicate that CB@OMEVs NPs possess excellent catalase activity, capable of decomposing H2O2 in the tumor microenvironment to generate O2, alleviating the hypoxic tumor microenvironment and enhancing the antitumor effect.

[0063] 6. In vitro biocompatibility

[0064] To assess the biocompatibility of CB@OMEVs NPs, the biocompatibility of CB@OMEVs NPs with human umbilical vein endothelial cells (HUVECs) was detected using a Calcein AM / PI live / dead staining assay to evaluate their in vitro cytotoxicity. HUVECs in logarithmic growth phase were cultured at 3 × 10⁻⁶ cells / year. 5 Inoculate the culture medium at a rate of / well, incubate in a six-well plate for 24 h, remove the old medium, and then add the following in sequence: OMV (200 μg / mL), SP (200 μg / mL), OMEVs (200 μg / mL), CuS (250 μg / mL), CuS-3BP (250 μg / mL), 3-BP (20 μg / mL), and...

[0065] Cells were inoculated with CB@OMEVS (250 μg / mL) for 24 hours, after which the drug-containing medium was discarded. After washing three times with PBS, 1 mL of pre-prepared Calcein AM / PI staining working solution was added, and the cells were incubated at room temperature in the dark for 30 minutes. Cell labeling was observed and photographed under a fluorescence microscope. Calcein AM (Ex / Em: 495 nm / 520 nm) showed green fluorescence, and PI (Ex / Em: 530 nm / 620 nm) showed red fluorescence.

[0066] The results are as follows Figure 7 As shown, CB@OMEVs NPs did not exhibit any significant cytotoxicity to HUVEC cells even at a high concentration of 500 μg / mL, demonstrating that CB@OMEVs NPs have good biocompatibility.

[0067] 7. In vitro cell inhibition effect

[0068] To evaluate the therapeutic effects of PDT and PTT on the viability of tumor cells in vitro, the following methods were used:

[0069] The CCK-8 assay was used to investigate the cytotoxic effect on 4T1 cells. 4T1 cells were cultured at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 μg / mL in 96-well plates and cultured for 24 hours. Then, they were cultured for another 6 hours in an incubator with medium containing different concentrations of CB@OMEVs NPs (40, 80, 100, 250, 500 μg / mL). Each group was divided into 3 replicates. The cells were then irradiated with different lasers, 660 (0.5W, 5 min) and 808 (1.5W, 5 min), and incubated for another 24 hours. Subsequently, 10 μL of CCK-8 solution was added to each well under light-protected conditions, and the cells were returned to the incubator for 1 hour. The OD value of each well was then measured at 450 nm using a microplate reader, and the cell viability was calculated.

[0070]

[0071] The results are as follows Figure 8As shown, CB@OMEVs NPs (250 μg / mL) induced 29.99% and 39.09% cell death under single laser irradiation at 660 and 808 nm, respectively, compared to the control group. However, dual laser irradiation at 660 / 808 nm induced 76.64% cell death, while irradiation at 808 / 660 nm induced 58.21% cell death, indicating that changing the laser irradiation sequence can improve cell death rate. This is because reactive oxygen species (ROS) generated in PDT can trigger ICD by inducing oxidative stress in tumor cells, while PDT can effectively destroy tumor cells and promote the release of tumor-associated antigens, thereby enhancing the ICD effect. In conclusion, the CB@OMEVs NPs + 660 / 808 nm group has a stronger inhibitory effect on 4T1 tumor cells, demonstrating stronger in vitro antitumor activity.

[0072] In summary, the algal-bacterial fusion vesicles loaded with copper sulfide nanomedicine prepared in this invention are small in size, highly safe, and exhibit targeted efficacy against recurrent tumors. This invention first synthesizes a near-infrared responsive hollow mesoporous copper sulfide nanoparticle, utilizing its mesoporous structure to encapsulate the small molecule inhibitor 3-bromopyruvic acid. Subsequently, algal-bacterial fusion vesicles targeting neutrophils and ROS-producing cells are prepared, and these vesicles are used to encapsulate the drug-loaded copper sulfide nanoparticles, resulting in nanomedicines (CB@OMEVs) with recurrent tumor-targeting capabilities. Ultimately, this achieves a combined therapeutic effect of photothermal therapy, photodynamic therapy, immunotherapy, and metabolic regulation. Existing data indicate that the CB@OMEVs developed in this invention have great potential as a highly efficient and accurate nanotherapeutic agent for inducing photothermal therapy, photodynamic therapy, and immunotherapy, exhibiting excellent anti-tumor effects.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanoparticle containing copper sulfide loaded in a fusion of bacteria and algae vesicles, characterized in that, The composition includes the following components in parts by weight: 30-75 parts of fused algae and bacteria vesicles, 20-50 parts of copper sulfide nanoparticles, and 5-20 parts of antitumor drugs; The bacterial-algal fusion vesicles are prepared by membrane fusion of outer membrane vesicles extracted from Escherichia coli and Spirulina exosomes; the copper sulfide nanoparticles are hollow mesoporous copper sulfide nanoparticles prepared by hydrothermal synthesis; the antitumor drug is loaded into the hollow mesoporous structure of the copper sulfide nanoparticles.

2. The copper sulfide-loaded bacterial-algal fusion vesicle according to claim 1, characterized in that, The antitumor drug is one of the following: chemotherapy drugs, small molecule inhibitors, and STING agonists.

3. The copper sulfide-loaded bacterial-algal fusion vesicle according to claim 2, characterized in that, The chemotherapy drug is doxorubicin, cisplatin, or fluorouracil; the small molecule inhibitor is pyrotinib or 3-bromopyruvic acid; and the STING agonist is a cyclic dinucleotide.

4. The copper sulfide-loaded bacterial-algal fusion vesicle according to claim 1, characterized in that, The *Escherichia coli* is at least one of *Escherichia coli* BL21, *Escherichia coli* DH5α, *Escherichia coli* MG1655, and *Escherichia coli* Nissle1917.

5. The method for preparing copper sulfide-loaded bacterial-algal fusion vesicles according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Centrifuge the Escherichia coli culture, collect the supernatant, discard the supernatant after centrifugation, and resuspend the precipitate. (2) Centrifuge the Spirulina liquid, collect the precipitate, resuspend it, use an ultrasonic cell disruptor to probe the supernatant, centrifuge to collect the supernatant, discard the supernatant after centrifugation, and resuspend the precipitate. (3) Mix the precipitate after resuspension in step (1) and the precipitate after resuspension in step (2), sonicate and squeeze to obtain bacterial-algal fusion vesicles; (4) Copper chloride dihydrate solution, polyvinylpyrrolidone solution, sodium hydroxide solution, hydrazine hydrate solution and sodium sulfide nonahydrate were mixed, stirred, subjected to water bath, centrifuged, washed and vacuum dried to obtain copper sulfide nanoparticles; (5) Mix the antitumor drug with the copper sulfide nanoparticles obtained in step (4), sonicate, stir, centrifuge, wash, and vacuum dry to obtain drug-loaded copper sulfide nanoparticles. (6) The products obtained in steps (3) and (5) are mixed, sonicated, squeezed, centrifuged and resuspended to obtain copper sulfide-loaded bacterial-algal fusion vesicles.

6. The method for preparing copper sulfide-loaded bacterial-algal fusion vesicles according to claim 5, characterized in that, The mass ratio of the precipitate after resuspension in step (1) to the precipitate after resuspension in step (2) in step (3) is 0.8-1.2:0.8-1.2; the ultrasonic power is 65-75W and the time is 8-12min; the extrusion is to continuously pass through the 0.22μm filter membrane 8-13 times.

7. The method for preparing copper sulfide-loaded bacterial-algal fusion vesicles according to claim 5, characterized in that, The ratio of copper chloride dihydrate solution, polyvinylpyrrolidone, sodium hydroxide solution, hydrazine hydrate solution and sodium sulfide nonahydrate in step (4) is 80-120 μL: 0.2-0.3 g: 20-30 mL: 6-7 μL: 150-250 μL.

8. The use of the copper sulfide-loaded bacterial-algal fusion vesicles according to any one of claims 1 to 4 or the copper sulfide-loaded bacterial-algal fusion vesicles prepared by the method according to any one of claims 5 to 7 in the preparation of antitumor drugs.

9. An antitumor drug, characterized in that, Includes copper-sulfide-loaded bacterial-algal fusion vesicles as described in any one of claims 1 to 4, or copper-sulfide-loaded bacterial-algal fusion vesicles prepared by the method described in any one of claims 5 to 7.

10. The antitumor drug according to claim 9, characterized in that, The copper sulfide-loaded bacterial-algal fusion vesicles are used in conjunction with photodynamic therapy, photothermal therapy, chemokinetic therapy, immunotherapy, or metabolic regulation techniques.

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