Organic ionic liquid formed by mixing double medicines and application of organic ionic liquid

Abstract

The invention discloses an organic ionic liquid formed by mixing double drugs and application thereof.The organic ionic liquid with different molar ratios is prepared from imatinib (IM) and 3-bromopyruvic acid (BP), and the optimal molar ratio is obtained according to in-vitro toxicity experiments and theoretical calculation on tumor cells; the organic ionic liquid with the proportion is encapsulated into engineered bacterium exosome-liposome hybrid vesicles (IM-BP-coated HV-Pep-PEG), and a double-drug co-delivery system is constructed. And a better synergistic treatment effect is achieved while the dosage is reduced. Meanwhile, the outer membrane vesicle part of the carrier can reverse a tumor immune microenvironment, promote tumor cell immunogenic death, and realize an anti-tumor effect of immunotherapy-assisted double-drug synergistic treatment.

Description

Technical Field

[0001] This invention relates to the design of organic ionic liquids, the preparation of bio-based vesicles, the synthesis of multifunctional PEG polymers, and click chemistry, specifically to an organic ionic liquid formed by a mixture of two drugs and its applications. Background Technology

[0002] Tyrosine kinases (TKs) are important targets for tumor signaling regulation. Based on the presence or absence of cell membrane receptors, they can be divided into non-receptor tyrosine kinases and receptor tyrosine kinases. Tyrosine kinase inhibitors (TKIs) selectively block the abnormal activation of tyrosine kinases, inhibiting multiple signaling pathways related to tumor cell growth, proliferation, differentiation, and apoptosis, and have played a significant role in cancer treatment in recent years. The first-generation TKI, imatinib (IM), was approved by the U.S. Food and Drug Administration in 2001 for the treatment of chronic myeloid leukemia. Since then, the development of TKI-based targeted drugs has flourished, with drugs such as gefitinib, erlotinib, afatinib, and osimertinib gradually being added to clinical use, the vast majority of which are used for cancer treatment. Although TKIs have achieved good therapeutic effects, drug resistance remains a challenging issue in clinical treatment. 10-40% of patients cannot use these drugs due to primary resistance, and almost all patients develop secondary resistance after 1-2 years of long-term use. Imatinib's main targets are the BCR-ABL fusion protein, the stem cell factor receptor c-KIT, and the platelet-derived growth factor receptor (PDGFR). It specifically recognizes the binding site of BCR-ABL protein to ATP, competitively inhibiting BCR-ABL protein phosphorylation, thereby inhibiting its own and downstream pathways such as Ras / Erk, PI3K / Akt, Stat5, CrkL, and c-Myc. While it has achieved good clinical results, statistics show that approximately 25% of patients develop resistance after long-term IM treatment. Discontinuation due to resistance affects the effectiveness of subsequent treatments and can lead to withdrawal syndrome.

[0003] ATP-binding cassette proteins (such as ABCB1, ABCC1, and ABCG2) are transport proteins located on the cell membrane surface that mediate increased efflux of various chemotherapeutic drugs. ABCB1 protein itself has a high ATP hydrolysis rate, which continues to increase in the presence of substrates and most inhibitors and modulators, reaching levels up to 1 μmol ATP / min / mg protein. In clinical settings, ABCB1 inhibitors suffer from limited efficacy and significant adverse reactions, possibly because the drug efflux function of ABCB1 is replaced by other transport proteins.

[0004] Due to the abnormal growth, proliferation, and invasion behaviors of tumor cells, the tumor microenvironment (TME) exhibits significant differences from normal cells. This includes differences in cell types such as endothelial cells, fibroblasts, and immune cells, as well as extracellular components such as cytokines, growth factors, hormones, and extracellular matrix. The most prominent features include dynamic hypoxia, high concentrations of redox components, elevated intracellular adenosine triphosphate (ATP) and lactate levels, and extracellular acidosis. Transferrin receptor (TfR) transports iron into cells by binding to the plasma glycoprotein transferrin, playing a crucial role in mediating cellular iron uptake. Transferrin receptor 1 (TFR1) has been shown to be highly expressed on the surface of various cells, including those in breast cancer, lung cancer, and colon cancer. Clinically, TfR1 is used as a tumor marker for the early diagnosis and treatment of breast cancer. Matrix metalloproteinases (MMPs) are key regulators of cell-cell and cell-extracellular matrix communication. Their role is to release bioactive peptides and growth factors, and to degrade proteins on the cell surface and outside the cell, thus having a decisive influence on cell growth and proliferation. In malignant tumors, the expression and activity of MMPs are significantly altered. Summary of the Invention

[0005] Purpose of the Invention: Dual-drug synergistic therapy for tumors presents key technical challenges. While there exists an optimal dosage range between drugs, precisely controlling the molar ratio between them during actual treatment is difficult. In nanodelivery systems, physical encapsulation methods struggle to control the loading of different drugs, and methods such as polymer prodrugs and host-guest interactions are complex and difficult to generalize. This invention targets the broad category of drug combinations consisting of hydrophobic drugs (organic bases) and hydrophilic drugs (organic acids), developing an organic ionic liquid capable of precisely adjusting the molar ratio between the two drugs. This liquid is then encapsulated in multifunctional hybrid vesicles, thereby achieving precise synergistic therapy for multidrug-resistant tumor models through optimal co-delivery dosage.

[0006] Technical Solution: The organic ionic liquid formed by the mixture of two drugs of the present invention comprises the organic base imatinib and the organic acid 3-bromopyruvic acid. The hydrophobic organic base has four nitrogen-containing cationization sites, which can ionize with hydrophilic organic acids of different equivalent ratios of 1:1 to 4 in aqueous solution. Preferably, the equivalent ratio of 1:1 to 4 is a molar ratio.

[0007] The method for preparing the organic ionic liquid includes the following steps: dispersing imatinib in an organic solvent, dissolving 1-4 times the molar mass of 3-bromopyruvic acid in an equal volume of organic solvent, mixing the two thoroughly, adding ethyl acetate dropwise, and stirring to obtain a clear and transparent solution; rotary evaporating the organic solvent to obtain a yellow solid 3-bromopyruvic acid-imatinib (IM-BP), which is the organic ionic liquid formed by the mixture of the two drugs.

[0008] Hybrid nanovesicle materials for targeting drug-resistant tumor cell lines, serving as efficient carriers of the organic ionic liquid, are composed of engineered bacterial outer membrane vesicles and liposomes. Organic ionic liquids can be incorporated into the materials during preparation for encapsulation.

[0009] The nanovesicle material is prepared by cloning the gene encoding transferrin T12 into a plasmid vector and then introducing it into engineered bacteria. The engineered bacteria are fed 6-azidogalactose and inducing T12 expression with isopropyl β-D-1-thiogalactosidase. The bacterial culture is collected, centrifuged to remove bacterial cells, concentrated using an ultrafiltration tube, and then ultracentrifuged again to obtain bacterial outer membrane vesicle precipitates. 3-bromopyruvate-imatinib is encapsulated in liposomes. The liposomes loaded with 3-bromopyruvate-imatinib are mixed with the outer membrane vesicles, and repeatedly extruded using a liposome extruder with a polycarbonate porous membrane to collect the resulting hybrid membrane vesicles. Pep-PEG dry powder is mixed with the obtained vesicles, shaken, and modified onto the vesicle surface through a click reaction to obtain the hybrid nanovesicles. Preferably, the liposomes are repeatedly extruded 18-30 times using a liposome extruder with a polycarbonate porous membrane. Preferably, the structure of Pep-PEG is DBCO-cr9GALGLPXe8-PEG, where each letter in GALGLP corresponds to a common amino acid abbreviation, X represents 6-Aminocaproic acid, e8 represents 8 D-type amino acids E, r9 represents 9 D-type amino acids R, and c represents D-type amino acid C. Since the vast majority of naturally occurring amino acids are L-type amino acids, D-type amino acids are represented by lowercase letters.

[0010] The hybrid nanovesicle material is wherein the bacteria is Escherichia coli.

[0011] The preparation method described above, specifically the preparation process of the outer membrane vesicles, is as follows: ClyA-Flag-T12 BL21 Escherichia coli is inoculated into LB broth medium, and the bacterial culture OD... 600 When the pH value was 0.6-0.8, isopropyl β-D-1-thiogalactosidase (IPTG) and 6-azidogalactose were added, respectively. After overnight incubation, bacterial outer membrane vesicles were obtained by centrifugation and ultracentrifugation. The role of adding isopropyl β-D-1-thiogalactosidase (IPTG) was to induce T12 expression, and the role of adding 6-azidogalactose was to modify the polysaccharide structure of the membrane surface.

[0012] The preparation method described above, wherein the synthesis steps of Pep-PEG are as follows: mPEG-COOH, N-hydroxysuccinimide (NHS), and N,N'-dicyclohexylcarbodiimide (DCC) are dissolved in anhydrous N,N-dimethylformamide (DMF) and reacted for 20-30 h; after the reaction is completed, the mixture is filtered through a 0.1-0.3 μm filter, and anhydrous diethyl ether is allowed to settle, yielding a white solid, which is then vacuum dried to obtain mPEG-NHS; preferably, mPEG-COOH, N-hydroxysuccinimide (NHS), and N,N'-dicyclohexylcarbodiimide (DCC) are dissolved in anhydrous N,N-dimethylformamide (DMF) in a molar ratio of 1:1.2:1.2.

[0013] Fmoc-e8XPLGLAGr9c, dibenzocyclooctylene DBCO-maleimide, and methylmorpholine were dissolved in N,N-dimethylformamide and reacted. mPEG-NHS was dissolved in an organic solvent and then added to the above reaction system. The reaction was allowed to proceed overnight. After the reaction was completed, anhydrous diethyl ether precipitated to obtain a white solid, which was then dried under vacuum to obtain the final product.

[0014] The application of imatinib and 3-bromopyruvic acid in the preparation of drugs for treating malignant tumors, including breast cancer and glioma. Preferably, the molar ratio of imatinib to 3-bromopyruvic acid is 1:1 to 4. More preferably, the molar ratio of imatinib to 3-bromopyruvic acid is 1:1, 1:2, 1:3, or 1:4.

[0015] The application of the aforementioned nanovesicle material in the preparation of drugs for bioorthogonal catalytic therapy.

[0016] The application of the aforementioned nanovesicle material in the preparation of drugs for treating malignant tumors.

[0017] Further preferably, the organic ionic liquid formed by the mixture of two drugs is used for tumor treatment, wherein the two drugs are the organic base imatinib and the organic acid 3-bromopyruvic acid; the hydrophobic organic base has four nitrogen-containing cationization sites, which can be ionized with hydrophilic organic acids of different equivalents of 1:1~4 in aqueous solution, thereby improving the water solubility and efficacy of the organic base; the biological effects of this drug combination have a direct upstream and downstream relationship, 3-bromopyruvic acid can reverse the multidrug resistance of tumors, enhance the sensitivity of tumor drug-resistant cell lines to imatinib, thereby inducing tumor cell apoptosis and immunogenic death.

[0018] This invention designs a hybrid nanovesicle material for targeting drug-resistant tumor cell lines. The nanovesicle material is composed of engineered bacterial outer membrane vesicles and liposomes. Organic ionic liquids can be incorporated during the preparation process to achieve effective encapsulation of the drug solution.

[0019] The aforementioned nanovesicle material possesses tumor-targeting, tumor microenvironment-responsive, and macrophage repolarization-stimulating functions. E. coli, through gene editing, can express the transferrin T12 fragment on outer membrane vesicles, actively targeting the highly expressed T12 receptor in 4T1 drug-resistant cell lines. Engineered bacteria, fed with 6-azidogalactose, imbue the sugar molecules on the outer membrane vesicles with azid sites, which can be modified with DBCO-coated multifunctional small peptide-polyethylene glycol (Pep-PEG). The structure of Pep-PEG is DBCO-cr9GALGLPXe8-PEG, which can reduce the endotoxicity of lipopolysaccharide (LPS) on outer membrane vesicles during in vivo circulation. The matrix metalloproteinase (MMP9), highly expressed in the tumor microenvironment, can cleave the GL site in the small peptide, removing PEG from the vesicle surface while retaining the polycationic r9 fragment linked to DBCO-c, thereby enhancing cellular uptake. The LPS component on the vesicles is immunogenic, capable of stimulating M2 phenotype macrophages to repolarize to the M1 phenotype, assisting organic ionic liquids in tumor immunotherapy.

[0020] The method for preparing the hybrid nanovesicles includes the following steps: separating azide-containing bacterial outer membrane vesicles secreted by *E. coli* ClyA-Flag-T12 BL21 using an ultracentrifuge; mixing liposomes loaded with IM-BP with the outer membrane vesicles, repeatedly extruding the mixture 21 times using a liposome extruder with a polycarbonate porous membrane, and collecting the resulting hybrid membrane vesicles; mixing Pep-PEG dry powder with the resulting vesicles, shaking at 200 rpm for 1 h, and modifying the vesicle surface via a click reaction. The system is then placed in a dialysis bag (Mw=8000) and dialyzed in 1×PBS to remove any drug not loaded into the vesicles.

[0021] The preparation method described above, specifically the preparation process of the outer membrane vesicles, is as follows: ClyA-Flag-T12 BL21 Escherichia coli is inoculated into 500 mL of LB broth (30 μg / mL Kana), and the bacterial culture OD... 600 When the value was 0.6-0.8, 1 M isopropyl β-D-1-thiogalactosidase (IPTG) was added to induce T12 expression and 6-azido-galactose to modify the polysaccharide structure of the membrane surface. After overnight culture, bacterial outer membrane vesicles were obtained by centrifugation and ultracentrifugation.

[0022] The preparation method described above, specifically the preparation process of the hybrid vesicles, is as follows: Liposome dry powder is dissolved in chloroform / methanol (v:v=1:2), and the organic solvent is removed by rotary evaporation to obtain a dry lipid membrane. A pre-prepared IM-BP solution is added to obtain drug-loaded liposomes. Subsequently, the liposomes and bacterial membrane are hybridized, and the mixture is repeatedly extruded 21 times using a liposome extruder with a 100 nm pore size polycarbonate porous membrane. The resulting hybrid vesicles are then collected.

[0023] The synthesis steps of Pep-PEG are as follows: mPEG-COOH, N-hydroxysuccinimide (NHS), and N,N'-dicyclohexylcarbodiimide (DCC) are dissolved in anhydrous N,N-dimethylformamide (DMF) at a molar ratio of 1:1.2:1.2, and the reaction is carried out for 24 h. After the reaction is completed, the mixture is filtered through a 0.2 μm nylon filter, and anhydrous diethyl ether is allowed to settle, yielding a white solid, which is then dried under vacuum to obtain mPEG-NHS.

[0024] Fmoc-e8XPLGLAGr9c, dibenzocyclooctylene (DBCO)-maleimide, and methylmorpholine were dissolved in DMF and reacted for 4 h. mPEG-NHS was dissolved in DMF (40% piperidine) and then added to the above reaction system, and the reaction was allowed to proceed overnight. After the reaction was complete, anhydrous diethyl ether precipitated, yielding a white solid, which was then dried under vacuum, thus completing the DBCO conversion of PEG.

[0025] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: Organic ionic liquids with different molar ratios of imatinib (IM) and 3-bromopyruvic acid (BP) were prepared, and the optimal molar ratio was determined based on in vitro toxicity experiments on tumor cells and theoretical calculations. This ratio of organic ionic liquid was encapsulated into engineered bacterial exosome-liposome hybrid vesicles (IM-BP@HV-Pep-PEG) to construct a dual-drug co-delivery system. This achieves better synergistic therapeutic effects while reducing drug dosage. Simultaneously, the outer membrane vesicle portion of this carrier can reverse the tumor immune microenvironment, promote immunogenic tumor cell death, and achieve the anti-tumor effect of synergistic dual-drug therapy in conjunction with immunotherapy. The principle of MMP-9-responsive small peptide-PEG-modified engineered bacterial hybrid vesicles co-delivering imatinib and 3-bromopyruvic acid for synergistic treatment of multidrug-resistant tumors is as follows... Figure 3 As shown. Attached Figure Description

[0026] Figure 1 The table shows the cytotoxicity of (A) IM and (B) BP on 4T1 cells; and the synergistic index of IM and BP on 4T1 cells calculated by the Chou-Talalay method.

[0027] Figure 2 Table showing the cytotoxicity of (A) IM and (B) BP on U87 MG cells; and the synergistic index of IM and BP on U87 MG cells calculated by the Chou-Talalay method.

[0028] Figure 3 A schematic diagram of engineered bacterial hybrid vesicles modified with MMP-9 small peptide-PEG to co-deliver imatinib and 3-bromopyruvate for the synergistic treatment of multidrug-resistant tumors;

[0029] Figure 4 Structural characterization of the organic ionic liquid IM-BP; (A is a schematic diagram of the reaction; B is the Fourier transform infrared (FTIR) spectrum of IM, BP, and IM-BP; C is the proton nuclear magnetic resonance spectrum of IM, BP, and IM-BP, with DMSO-d6 as the solvent;).

[0030] Figure 5 Thermogravimetric analysis plots of IM, BP, and IM-BP;

[0031] Figure 6 Transmission electron microscope (TEM) images; (A) TEM image of OMVs, scale bar: 200 nm; (B) TEM image of HV-Pep-PEG, scale bar: 200 nm; (C) Characterization results of average hydrated particle size of HV, HV-Pep-PEG, IM-BP@HV, and IM-BP@HV-Pep-PEG after 16 h of dialysis; (D) Trend of average hydrated particle size of HV and HV-Pep-PEG over 96 h.

[0032] Figure 7 The zeta potentials of HV, IM-BP@HV, IM-BP@HV-Pep-PEG, and IM-BP@HV-Pep-PEG+MMP-9;

[0033] Figure 8 To evaluate the cytotoxicity of IM-BP@HV-Pep-PEG against 4T1 / MDR;

[0034] Figure 9 For IM-BP, IM-BP@HV, IM-BP@HV-Pep-PEG, and tamoxifen, the effects on (A) 4T1 cells and (B) 4T1 / MDR cells;

[0035] Figure 10 To evaluate the therapeutic effect of IM-BP@HV-Pep-PEG on 4T1 / MDR tumor-bearing mice in vivo;

[0036] Figure 11 The cytotoxicity of HV-Pep-PEG on bEnd.3 cells;

[0037] Figure 12 The relative contents of IM, IM-BP, IM-BP@HV, and IM-BP@HV-Pep-PEG in the upper chamber (A) and lower chamber (B) of Transwell;

[0038] Figure 13Fluorescence imaging of HV-Pep-PEG uptake in U87 MG cells. DAPI channel (blue) excitation wavelength: 358 nm, emission wavelength: 461 nm; DiI channel (orange) excitation wavelength: 480 nm, emission wavelength: 570 nm; scale bar: 50 μm;

[0039] Figure 14 Cytotoxicity assessment of IM-BP@HV-Pep-PEG against U87 MG. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] All reagents and materials described in the examples are commercially available. 3-Bromopyruvic acid was purchased from Leyen, imatinib from Sigma-Aldrich, and MMP-9 from Prospec.

[0042] Example 1

[0043] (1) Synergistic effect of imatinib and 3-bromopyruvic acid on 4T1 and 4T1 / MDR To evaluate whether there is a synergistic effect between imatinib (IM) and 3-bromopyruvate (BP), the cytotoxicity of each drug was first assessed using the CTG (CellTiter-Glo) assay. The results are as follows: Figure 1 As shown in Figures AB, IM and BP were combined in different ratios and co-incubated with 4T1 cells. The Combination Index (CI) was calculated using the Chou-Talalay method to determine the synergistic effect of different drug ratios and cell numbers. Figure 1 As shown in C, calculations revealed that when the ratio of IM to BP is 1:4 and 4:1, the two exhibit strong or moderate synergy; when the ratio is 3:1, the main effects are synergy and cumulative; and when the ratio is 1:3 and 1:1, the main effects are antagonistic.

[0044] By increasing the expression of ABCB1 protein in 4T1 cells through transfection, 4T1 / MDR cells with multidrug resistance were obtained, and resistance to IM was verified in cell experiments. Figure 1 As shown in AB, the half-maximal inhibitory concentration (IC50) of 4T1 for IM is... 50 The IC of 4T1 / MDR for IM is 53.63 μM. 50 The concentration was 96.91 μM, calculated according to the Resistance Index (RI) formula.

[0045] The RI was calculated to be 1.81. After evaluating the cytotoxicity of single drugs against 4T1 / MDR cells, IM and BP were combined in different ratios and co-incubated with 4T1 / MDR cells, and the CI was calculated using the same method. Figure 1 As shown in Figure C, IM and BP showed good synergy in 4T1 / MDR cells at all ratios, with the best synergistic effect observed when the IM:BP ratio was 1:4.

[0046] (2) Synergistic effect of 3-bromopyruvic acid and imatinib on U87 MG To evaluate whether imatinib (IM) and 3-bromopyruvate (BP) have a synergistic effect on U87 MG cells, the cytotoxicity of each drug was first assessed using the CTG (CellTiter-Glo) assay. The results are as follows: Figure 2 As shown in AB, its IC 50 The concentrations were 40.16 μM and 71.41 μM, respectively. IM and BP were combined in different ratios and co-incubated with U87 MG cells. The Combination Index (CI) was calculated using the Chou-Talalay method to determine the synergistic effect of different drug ratios on different numbers of cells. Figure 2 As shown in Figure C, calculations revealed that when the ratio of IM to BP was 1:4, 1:3, and 4:1, it exhibited excellent synergistic effects in inhibiting the proliferation of U87 MG cells.

[0047] Example 2

[0048] (1) Synthesis of bromopyruvate-imatinib Imatinib was dispersed in isopropanol, and 4 molar amounts of 3-bromopyruvic acid were dissolved in an equal volume of methanol. The two solutions were mixed thoroughly. A small amount of ethyl acetate was added dropwise, and the mixture was stirred for 5 hours to obtain a clear and transparent solution. The organic solvent was then rotary evaporated, and the resulting yellow solid was 3-bromopyruvic acid-imatinib (IM-BP).

[0049] Characterization: 1 mg each of IM, BP, and IM-BP were co-ground with 200 mg of potassium bromide (KBr) to form a fine powder. The powder was then thoroughly mixed and dried under a heat lamp to remove moisture. The dried powder mixture was placed in a tableting mold and subjected to appropriate pressure for several minutes to obtain a translucent tablet. Fourier transform infrared spectroscopy (FTIR) was used for detection. Figure 4 As shown, compared with the raw materials, the synthesized product IM-BP shows a new peak υ. N-H + (1492 cm) -1 This indicates that the interaction between the two is the protonation of the N atom in IM; furthermore, BP's υC=O (1728 cm) -1 The change occurs, with a redshift of υ. C=O (1638 cm) -1 This phenomenon indicates that due to the protonation of N in IM, the lone electron of O in BP forms a conjugation effect with the adjacent carbonyl group. These results demonstrate that IM and BP successfully form a copolymer.

[0050] Take 50 mg each of IM, BP, and IM-BP, dissolve them in 600 μL of deuterated dimethyl sulfoxide (DMSO-d6), and characterize their structures using a 400 MHz proton nuclear magnetic resonance spectrometer. 1 ¹H-NMR (400 MHz, DMSO-d6): δ 9.28 (s, ¹H), 7.54 (s, 2H), 7.41 (s, ¹H), 7.21 (s, 2H). The presence of characteristic signals identical to those in IM indicates successful IM-BP synthesis. ¹H-NMR (400 MHz, DMSO-d6): δ 10.25 (s, ¹H), 8.71 (s, ¹H), 7.47 (s, 2H). The appearance of new characteristic signals not present in IM and BP suggests the presence of NH4+ in the product. + The integral area ratio is 1:1.02:1.97, indicating that the binding ratio of IM to BP in the product is 1:4, and the structure is as follows. Figure 4 As shown.

[0051] Take 10 mg each of IM, BP, and IM-BP, and perform thermogravimetric analysis (TGA) to record the relationship between sample mass change and temperature. Figure 5 As shown, the first weight loss of IM-BP was at 30-107℃, which was due to the evaporation of adsorbed water in the sample; the second weight loss accounted for 27.2%, which was due to the decomposition of BP components; and the third weight loss accounted for 42.8%, which was due to the joint decomposition of IM and BP components. Since the BP components decomposed first and absorbed heat, the decomposition rate of IM components in the third weight loss was reduced, which proves that IM-BP has higher thermal stability compared with the original drug.

[0052] (2) Extraction of outer membrane vesicles of ClyA-Flag-T12 BL21 Escherichia coli Escherichia coli BL21 was purchased from the American Collection of Standard Microbial Cultures (Baltimore, USA). To construct a T12-expressing strain, the gene encoding ClyA-Flag-T12 was cloned into the pET-30a plasmid vector. Western blotting was used to verify the expression of T12 in the bacteria and outer membrane vesicles, confirming successful construction.

[0053] ClyA-Flag-T12 BL21 *E. coli* were fed 50 μM of 6-azidogalactose to introduce azide sites into the sugar molecules on the outer membrane vesicles. These sites are suitable for DBCO-mediated modification with multifunctional small peptide-polyethylene glycol (Pep-PEG). After culturing *E. coli* to the logarithmic growth phase, they were inoculated into 500 mL LB broth (30 μg / mL Kana) and incubated at 37°C. o Shake at 200 rpm (C). Take a small amount of bacterial culture for testing; OD... 600 When the value was 0.6-0.8, the addition of 1M isopropyl β-D-1-thiogalactosidase (IPTG) induced T12 expression. At 30... o Incubate overnight at 180 rpm with shaking at C.

[0054] The extraction method for outer membrane vesicles is referenced in Feng Q et al. Engineered Bacterial OuterMembrane Vesicles as Controllable Two-Way Adaptors to Activate Macrophage Phagocytosis for Improved Tumor Immunotherapy [J]. Adv. Mater., 2022, 34:e2206200. The bacterial culture was centrifuged at 1800 ×g for 10 min to remove bacterial cells, then concentrated using a 100 kDa ultrafiltration tube. o Bacterial outer membrane vesicles were obtained by ultracentrifugation at 150,000 ×g for 2 h at C. The precipitate was resuspended in sterile 1×PBS, aliquoted, and incubated at -80°C. o Store in a refrigerator (C).

[0055] Characterization: A suitable amount of freshly extracted outer membrane vesicles (OMVs) were used to prepare IM-BP@lipo via lipid thin-film hydration. OMVs with an equal protein content were added, and IM-BP@HV was obtained through co-extrusion. After surface modification with PEG, IM-BP@HV-Pep-PEG was obtained. A suitable amount of sample was dropped onto a copper grid, allowed to dry naturally in a cool place, and then observed using a transmission electron microscope (TEM). Figure 6 As shown in Figure A, the OMVs have a particle size of approximately 170 nm.

[0056] (3) Synthesis of PEG-Pep-DBCO mPEG-COOH, N-hydroxysuccinimide (NHS), and N,N'-dicyclohexylcarbodiimide (DCC) were dissolved in anhydrous N,N-dimethylformamide (DMF) at a molar ratio of 1:1.2:1.2, and the reaction was carried out for 24 h. After the reaction was completed, the solution was filtered through a 0.2 μm nylon filter, and anhydrous diethyl ether was allowed to settle, yielding a white solid, which was then vacuum dried to obtain mPEG-NHS. PEG2000 was used as the mPEG.

[0057] Fmoc-e8XPLGLAGr9c, dibenzocyclooctylene (DBCO)-maleimide, and methylmorpholine were dissolved in DMF and reacted for 4 h. mPEG-NHS was dissolved in DMF (40% piperidine) and then added to the above reaction system, and the reaction was allowed to proceed overnight. After the reaction was complete, anhydrous diethyl ether precipitated, yielding a white solid, which was then dried under vacuum, thus completing the DBCO conversion of Pep-PEG.

[0058] (4) Preparation and drug loading of hybrid membrane vesicles The liposome powder (commercial phospholipid) was dissolved in chloroform / methanol (v:v=1:2) at 40 °C. o Rotary evaporation was performed under C20 water bath heating until the organic solvent was completely removed to obtain a dry lipid film. Pre-prepared IM-BP solution was added to obtain drug-loaded liposomes, which were then sonicated to ensure uniform dispersion. The liposomes were then frozen in liquid nitrogen for 5 min and then incubated at 37°C. o Heating in a C-water bath until completely dissolved, repeating the above steps 6 times to obtain liposomes with higher drug loading capacity. The liposomes and bacterial membranes were hybridized, and the mixture was repeatedly extruded 21 times using a liposome extruder with a 200 nm pore size polycarbonate porous membrane. The resulting hybrid membrane vesicles were collected, and the extruder was cleaned. The polycarbonate porous membrane was replaced with one containing 100 nm pores, and the extrusion was repeated 21 times to obtain drug-loaded hybrid membrane vesicles of the ideal particle size. PEG powder was mixed with the obtained vesicles, and the mixture was shaken at 200 rpm for 1 h to modify the vesicle surface with PEG via a click reaction. The system was placed in a dialysis bag (Mw=8000) and dialyzed in 1×PBS to remove unloaded drug from the vesicles.

[0059] The preparation method for empty liposomes is the same as above, except that the IM-BP solution is replaced with 1×PBS.

[0060] Characterization: OMVs themselves suffer from low yield and insufficient drug loading. To overcome these shortcomings, we introduced liposomes into this system to form HV carriers. For example... Figure 6As shown in Figure B, the HV particle size is approximately 200 nm. The particle sizes of HV, HV-Pep-PEG, IM-BP@HV, and IM-BP@HV-Pep-PEG were determined using dynamic light scattering (DLS) technology. The system was dialyzed for 16 h prior to measurement to remove excess IM-BP. Results are shown below. Figure 6 As shown in Figure C, dialysis induces the aggregation of lipid carriers, resulting in an undesirable increase in particle size and posing a risk of damaging the lipid membrane and altering surface properties. This phenomenon was confirmed in the HV and IM-BP@HV groups, while no significant increase in particle size was observed in the HV-Pep-PEG and IM-BP@HV-Pep-PEG groups. The HV and HV-Pep-PEG groups were incubated at room temperature for 96 hours for continuous particle size monitoring. Figure 6 As shown in Figure D, there is a significant difference between the two. The particle size of HV remained below 200 nm for the first 24 hours, then continued to increase, reaching approximately 450 nm by 72 hours, and remained at this level thereafter. The particle size of HV-Pep-PEG continued to increase, but the rate of increase was extremely slow, never exceeding 200 nm within 96 hours. This result indicates that PEG can prevent the aggregation of nanocarriers and maintain particle size stability over a long period.

[0061] To characterize the surface potential of the carrier, IM-BP@HV-Pep-PEG was co-incubated with MMP-9 for 24 h. MMP-9 was then removed from the system by dialysis, and the surface potential of each group was detected by DLS. Figure 7 As shown, the surface potential of HV increased from 2.4 mV to 10.0 mV after loading with IM-BP, and decreased to 0.6 mV after PEG modification, indicating that PEG has the ability to reduce the surface charge of the carrier and make it electropositive. Electropositive ionic nanoparticles have a certain adsorption effect on tumor cells, while the surface of OMVs contains a large amount of electronegative LPS. PEG modification can effectively shield LPS, making HV generally electropositive, thereby enhancing the targeting ability of HV to tumor cells. After co-incubation with MMP-9, the surface potential increased to 14.7 mV, because the PEG layer was cleaved to expose cationic groups, further enhancing the targeting ability.

[0062] (5) Cell culture This phase of the experiment used mouse breast cancer cells (4T1) and human kidney epithelial cells (293T). The culture media were DMEM high-glucose medium and 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin, respectively. The culture conditions were 37°C. oC, CO2 concentration 5%. Observe cell status every 24 h, and passage at a ratio of 1:3 when cell density is approximately 90%. Digest and resuspend cells in the appropriate culture medium, count, and then dilute to the appropriate concentration for use.

[0063] (6) Construction and culture of 4T1 / MDR cells The pECMV-Abcb1b-m-FLAG-EGFP recombinant plasmid was transformed into [a specific plasmid] using the heat shock method. E.coil In DH5α competent cells, the liquid plasmid was first centrifuged at 5000 rpm for 1 min, while the frozen competent cells were thawed on ice to an ice-water mixture. 1 μL of plasmid was added to 50 μL of competent cells, and the mixture was gently tapped at the bottom of the EP tube to mix. The tube was then incubated on ice for 25 min before being placed at 42°C. o Heat the mixture in a water bath at 37°C for 45 seconds, then immediately return it to ice and let it stand for 2 minutes. Add 700 μL of fresh LB broth (antibiotic-free) and heat at 37°C. o Resuscitate the cells on a shaker (200 rpm) for 60 min. Finally, centrifuge the culture medium (5000 rpm, 1 min), resuspend approximately 100 μL of the supernatant by gentle pipetting, and spread it onto an LB agar plate containing 30 μg / mL Kana. Incubate at 37°C. o Incubate overnight in a C incubator.

[0064] When extracting recombinant plasmids, it is necessary to amplify a large number of bacterial cells containing the recombinant plasmids. First, the frozen bacterial cells containing pECMV-Abcb1b-m-FLAG-EGFP... E. coli DH5α bacterial culture was added to 200 mL LB liquid medium (30 μg / mL Kana) and placed on a shaker (37°C). o Incubate overnight at 200 rpm (C). Refer to the endotoxin-free plasmid large-scale extraction kit (TIANGEN) for specific plasmid extraction procedures. After extraction, measure the absorbance at 260 / 280 nm and the plasmid concentration using a UV spectrophotometer. Store the recombinant plasmid at -20°C. o C is for use in subsequent experiments.

[0065] 4T1 cells were seeded in cell culture dishes and transfected when the cell density reached approximately 40%, following the LipoTRF reagent instructions. 4T1 / MDR cells were obtained, and subsequent cell culture was performed using 1640 medium containing 1 μg / mL imatinib to maintain drug resistance.

[0066] (7) Cytotoxicity evaluation 4T1 and 4T1 / MDR cells were cultured for in vitro tumor cell killing efficacy evaluation. When the cell density was approximately 90%, the culture medium was removed, and the cells were washed with a small amount of sterile 1×PBS to remove dead cells. EDTA-trypsin solution was then added, and the cells were incubated for 3 min to digest. Digestion was terminated by adding an equal volume of 1640 culture medium, and the cells were collected after gentle pipetting. The cells were centrifuged at 500 × g for 3 min, resuspended in a small amount of fresh 1640 culture medium, counted using a cell counting chamber, and then analyzed at 1×10⁻⁶. 3 The cells were seeded at a density of 1:1 in each well of a 96-well plate and cultured for 24 h. After the cells adhered and grew, the culture medium was replaced with a medium containing different concentrations of the drug, and the cells were incubated for another 48 h. The culture medium in each well was then replaced with fresh medium, 20 μL of CTG reagent was added, and the plates were shaken in the dark for 10 min, then allowed to stand for 5 min. The autoluminescence was then measured using a microplate reader.

[0067] Cell viability was evaluated using the CTG method, such as Figure 8 As shown, the calculated IC50 of IM-BP@HV-Pep-PEG on 4T1 / MDR cells was... 50 It was 86.71 μg / mL.

[0068] In the experiment, the IC50 of IM, IM-BP, and IM-BP@HV-Pep-PEG on 4T1 / MDR cells was obtained through cytotoxicity assessment. 50 The content of IM was calculated. As shown in Table 1, it can be found that BP salting can effectively reduce the dosage of IM. The use of HV-Pep-PEG carrier can further reduce the dosage of IM through targeting, revealing its advantages in reducing drug dosage and reducing drug toxicity and side effects. It is expected to reduce the difficulty of subsequent tumor treatment by slowing down the progression of drug resistance.

[0069] Table 1. IM content at half-maximal inhibitory concentration (IC50) of IM, IM-BP, and IM-BP@HV-Pep-PEG in 4T1 / MDR cells.

[0070]

[0071] (8) Determination of ABCB1 content 4T1 and 4T1 / MDR cells were collected, counted using a cell counting chamber, and then analyzed at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 12-well plates and incubated overnight. The next day, when good cell adhesion and appropriate cell density were observed, PBS, IM-BP, IM-BP@HV, IM-BP@HV-Pep-PEG, and tamoxifen were added, and the cells were incubated for 24 hours. Total protein content was determined using the BCA method, and ABCB1 content was detected using an ABCB1 protein assay kit.

[0072] The experiment used the BCA method to determine the total amount of protein in cells and the ABCB1 protein content in cells to determine the content of ABCB1 protein in cells using an ABCB1 protein kit. The relative content of ABCB1 protein in cells was then calculated. The results are as follows: Figure 9 As shown, both the experimental groups and the clinical ABCB1 protein inhibitor tamoxifen inhibited ABCB1 protein expression. In 4T1 cells, tamoxifen exhibited a stronger inhibitory effect on ABCB1 protein expression; in 4T1 / MDR cells, IM-BP@HV-Pep-PEG showed significantly better inhibitory efficacy than tamoxifen. After co-incubation with IM-BP@HV-Pep-PEG, the proportion of ABCB1 protein in the total protein in 4T1 / MDR cells was 0.045‰, lower than the level in the untreated 4T1 cells. These results indicate that IM-BP@HV-Pep-PEG has a superior inhibitory effect on ABCB1 protein expression and more effectively reverses the drug-resistant phenotype. This finding provides important experimental evidence for targeted therapy of drug-resistant tumors.

[0073] (9) In vivo anti-tumor experiment Balb / c mice were purchased from Qizhen (Suzhou) Model Animal Research Co., Ltd. All animal experiments were conducted in accordance with the "Safety Work Procedures" approved by the Ethics Committee on Human Specimens and Animal Experiments of Soochow University.

[0074] A subcutaneous tumor model was established by injecting 100 μL of 4T1 / MDR cell suspension into the axilla of each mouse. The tumor volume was calculated using the following formula:

[0075] When the tumor volume reaches 80 mm 2 Mice were randomly divided into groups (n=5) and injected intravenously with 100 μL of PBS, IM-BP, IM-BP@HV, and IM-BP@HV-Pep-PEG (BP content in samples was uniformly 10 mg / kg) on ​​days 4, 7, 10, and 13, respectively. Tumor volume and mouse weight changes were monitored every other day using calipers and an electronic balance. To further investigate the in vivo antitumor effect of the material, tumor tissue sections were obtained after mouse sacrifice and analyzed by terminal deoxynucleotidyl transferase-mediated dUTP end-labeling (TUNEL) staining. Simultaneously, hematoxylin and eosin (H&E) staining was used to analyze sections of the mouse heart, liver, spleen, lung, kidney, and tumors, and imaging was performed under a microscope.

[0076] like Figure 10As shown in Figure A, the first administration was designated as day 0. A total of five administrations were administered on days 0, 3, 6, 9, and 12. Mouse body weight and tumor volume were recorded a total of seven times on days 0, 2, 4, 6, 8, 10, 12, and 14. Changes in mouse body weight are shown below. Figure 10 As shown in Figure B, the overall trend in each group was upward, indicating that none of the treatment groups had significant toxicity to mice. Changes in mouse tumor volume are shown in Figure B. Figure 10 As shown in Figure C, the tumor volume in the PBS-injected group increased to 912 mm within 14 days. 3 The IM-BP and IM-BP@HV injection groups showed limited inhibitory effects on mouse tumors, with significant and continuous growth in tumor volume. The IM-BP@HV-Pep-PEG treatment group showed no significant change in tumor volume in the first 12 days, but a slight increase in volume on day 14, demonstrating a good overall inhibitory effect. Compared with the control group, the tumor growth inhibition rate (TGI) was 83.2%. Figure 10 D represents the comparison of tumor volumes removed from each group on day 14. Figure 10 E represents the comparison of tumor weight, showing that IM-BP@HV-Pep-PEG has a significantly better tumor-inhibiting effect than IM-BP and IM-BP@HV. This experiment verifies the effectiveness of IM-BP@HV-Pep-PEG in inhibiting tumor growth in vivo.

[0077] Example 3 Antitumor experiment on glioblastoma (1) Cell culture Human glioblastoma cells (U87 MG) and mouse brain microvascular endothelial cells (bEnd.3) were used in this stage of the experiment. The culture medium was MEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. The culture conditions were 37°C and 5% CO2. Cell status was observed every 24 hours, and when the cell density reached approximately 90%, the cells were passaged at a 1:2 ratio. The cells were digested and resuspended in MEM medium, counted, and diluted to an appropriate concentration before use.

[0078] (2) Cytotoxicity evaluation When the cell density was approximately 90%, the culture medium was removed, and the cells were washed with a small amount of sterile 1×PBS to remove dead cells. Then, EDTA-trypsin solution was added, and the cells were incubated for 2 minutes for digestion. Digestion was terminated by adding an equal volume of MEM culture medium, and the cells were collected after gentle pipetting. The cells were centrifuged at 500 × g for 3 minutes, resuspended in a small amount of fresh MEM culture medium, counted using a cell counting chamber, and then analyzed at 1×10⁻⁶ cells / mL. 3The cells were seeded at a density of 1:1 in each well of a 96-well plate and cultured for 24 h. After cell adhesion and growth, the culture medium was replaced with medium containing different concentrations of the drug, and incubation continued for 48 h. The culture medium in each well was then replaced with fresh medium, 20 μL CTG reagent was added, and the plates were shaken in the dark for 10 min, then incubated for 5 min. The autoluminescence was measured using a microplate reader.

[0079] To verify the biosafety of HV-Pep-PEG across the blood-brain barrier, it was co-incubated with bEnd.3 cells, and cell viability was assessed using the CTG assay. Results are as follows: Figure 11 As shown, at an HV-Pep-PEG concentration of 1000 μg / mL, the viability of bEnd.3 cells was approximately 74%. Cell viability increased with decreasing concentration, reaching approximately 99% at concentrations below 31.25 μg / mL, indicating that it had virtually no effect on normal cellular physiological activities. These experimental results demonstrate that HV-Pep-PEG does not damage the blood-brain barrier, confirming its biosafety.

[0080] (3) Verification of the ability of hybrid membrane vesicles to penetrate the blood-brain barrier With 1×10 4 bEnd.3 cells were seeded in the upper chamber of a Transwell (0.4 μm pore size) and U87MG cells were seeded in the lower chamber. After 5 days of culture, a TEER ≥ 150 Ω / cm was measured. 2 This indicates that a dense layer has formed, allowing for subsequent experiments. IM, IM-BP, IM-BP@HV, and IM-BP@HV-Pep-PEG were added to the upper chamber, and after incubation for 24 h, the lower layer of culture medium and cells were collected. The IM content was detected by high-performance liquid chromatography (HPLC). The mobile phase used was 30% acetonitrile and 70% 0.02 M potassium dihydrogen phosphate aqueous solution, the flow rate was 0.8 mL / min, and the column temperature and injection temperature were 30 °C. o C.

[0081] To investigate the blood-brain barrier penetration ability of HV-Pep-PEG, we used Transwell chambers. bEnd.3 cells were seeded in the upper chamber to simulate the blood-brain barrier, and U87 MG cells were seeded in the lower chamber to simulate gliomas. IM, IM-BP, IM-BP@HV, and IM-BP@HV-Pep-PEG were added to the upper chamber, respectively, and incubated for 24 h. The blood-brain barrier penetration ability of each drug group was measured by detecting the drug concentration in the lower chamber. The results are as follows: Figure 12As shown in Figures AB, IM and IM-BP were almost unable to enter the lower chamber of the Transwell through bEnd.3 cells; IM-BP@HV and IM-BP@HV-Pep-PEG could enter the lower chamber of the Transwell via the carrier transport portion, at 57.7% and 58.5%, respectively. These results demonstrate that HV-Pep-PEG possesses the ability to deliver drugs across the blood-brain barrier.

[0082] (4) Validation of the targeting ability of hybrid membrane vesicles to U87 MG cells The uptake of HV and HV-Pep-PEG by U87 MG cells was verified using confocal microscopy. Cells were loaded at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density in laser confocal microscopy dishes and cultured for 24 hours to allow cell adhesion. HV and HV-Pep-PEG were co-incubated with DiI dye for 1 hour, followed by dialysis to remove excess dye. HV and HV-Pep-PEG were then co-incubated with cells for different times (0.5, 1, 2, and 4 hours), followed by washing three times with pre-chilled PBS, fixation with 4% paraformaldehyde for 10 minutes, washing three times with pre-chilled PBS, and finally, staining of the nuclei with DAPI, followed by washing three times with pre-chilled PBS. Images were obtained using a Zeiss confocal microscope. All procedures were performed in the dark.

[0083] To investigate the uptake behavior of HV-Pep-PEG by U87 MG cells, we used confocal laser scanning microscopy to photograph endocytosis after co-incubation for 0.5, 1, 2, and 4 h. The results are as follows: Figure 13 As shown, no uptake was observed after 0.5 hours of co-incubation. After 1 hour, a small amount of the carrier was observed to adhere to the cell surface, which gradually increased thereafter. After 4 hours, a large amount of the carrier was observed to be taken up by the cells, and some of it had entered the cell interior, indicating that HV-Pep-PEG can be effectively taken up by U87 MG cells.

[0084] (5) Killing effect of IM-BP@HV-Pep-PEG on U87 MG cells To evaluate the cytotoxic effect of IM-BP@HV-Pep-PEG on U87 MG cells, cell viability was assessed using the CTG assay after 48 h of co-incubation. Figure 14 As shown, IM-BP@HV-Pep-PEG has an effect on the IC50 expression of U87 MG cells. 50 The concentration was 27.31 μg / mL, lower than the IC50 of 4T1 / MDR cells. 50 The value indicates that U87 MG exhibits high sensitivity to IM-BP@HV-Pep-PEG.

[0085] The effects of IM, IM-BP, and IM-BP@HV-Pep-PEG treatments on the IC50 of U87 MG cells were obtained through experiments. 50 The content of IM was calculated, and the results are shown in Table 2. The use of BP synergy and HV-Pep-PEG carrier can effectively reduce the dosage of IM, which may reduce the effect on the central nervous system.

[0086] Table 2. IM content at half-maximal inhibitory concentration (IC50) of IM, IM-BP, and IM-BP@HV-Pep-PEG in U87 MG cells.

[0087] .

Claims

1. An organic ionic liquid formed by mixing two drugs, characterized in that, The two drugs are the organic base imatinib and the organic acid 3-bromopyruvic acid; the hydrophobic organic base has four nitrogen-containing cationization sites and can be ionized with hydrophilic organic acids of different equivalent ratios of 1:1~4 in aqueous solution.

2. A method for preparing the organic ionic liquid according to claim 1, characterized in that, The process includes the following steps: dispersing imatinib in an organic solvent, dissolving 1-4 molar amounts of 3-bromopyruvic acid in an equal volume of organic solvent, mixing the two thoroughly, adding ethyl acetate dropwise, and stirring to obtain a clear and transparent solution; rotary evaporating the organic solvent to obtain a yellow solid 3-bromopyruvic acid-imatinib, which is the organic ionic liquid formed by the mixture of the two drugs.

3. A hybrid nanovesicle material for targeting drug-resistant tumor cell lines as a highly efficient carrier of the organic ionic liquid of claim 2, characterized in that, The nanovesicle material is composed of engineered bacterial outer membrane vesicles and liposomes, and organic ionic liquids can be incorporated during the preparation process for encapsulation.

4. The nanovesicle material according to claim 2, characterized in that, The nanovesicle material was prepared by cloning the gene encoding transferrin T12 into a plasmid vector and then introducing it into engineered bacteria. The engineered bacteria were fed 6-azidogalactose and T12 expression was induced by the addition of isopropyl β-D-1-thiogalactosidase. The bacterial culture was collected, centrifuged to remove bacterial cells, concentrated using an ultrafiltration tube, and then ultracentrifuged to obtain bacterial outer membrane vesicle precipitate. 3-bromopyruvate-imatinib was encapsulated in liposomes. The liposomes loaded with 3-bromopyruvate-imatinib were mixed with the outer membrane vesicles, and the mixture was repeatedly extruded using a liposome extruder with a polycarbonate porous membrane to collect the resulting hybrid membrane vesicles. Pep-PEG dry powder was mixed with the obtained vesicles, shaken, and modified onto the vesicle surface through a click reaction to obtain the hybrid nanovesicles.

5. The hybrid nanovesicle material according to claim 4, characterized in that, The bacteria in question is Escherichia coli.

6. The hybrid nanovesicle material according to claim 5, characterized in that, The specific preparation process of the outer membrane vesicles is as follows: ClyA-Flag-T12 BL21 Escherichia coli culture is inoculated into LB broth medium, and the bacterial solution OD... 600 When the value is 0.6-0.8, isopropyl β-D-1-thiogalactosidase and 6-azido-galactose are added respectively. After overnight incubation, bacterial outer membrane vesicles are obtained by centrifugation and ultracentrifugation.

7. The hybrid nanovesicle material according to claim 5, characterized in that, The synthesis steps of Pep-PEG are as follows: mPEG-COOH, N-hydroxysuccinimide, and N,N'-dicyclohexylcarbodiimide are dissolved in anhydrous N,N-dimethylformamide and reacted for 20-30 h; after the reaction is completed, the mixture is filtered through a 0.1-0.3 μm filter, precipitated with anhydrous diethyl ether, and a white solid is obtained. The solid is then dried under vacuum to obtain mPEG-NHS. Fmoc-e8XPLGLAGr9c, dibenzocyclooctylene DBCO-maleimide, and methylmorpholine were dissolved in N,N-dimethylformamide and reacted. mPEG-NHS was dissolved in an organic solvent and then added to the above reaction system. The reaction was allowed to proceed overnight. After the reaction was completed, anhydrous diethyl ether precipitated to obtain a white solid, which was then dried under vacuum to obtain the final product.

8. The application of imatinib and 3-bromopyruvic acid in the preparation of drugs for treating malignant tumors; wherein the malignant tumors include breast cancer and glioma.

9. Use of the nanovesicle material of claim 3 in the preparation of drugs in the field of bioorthogonal catalytic therapy.

10. Use of the nanovesicle material according to claim 3 in the preparation of drugs for treating malignant tumors.

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