Chlorotoxin-melittin nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to chlorotoxin-melittin nanoparticles as well as a preparation method and application thereof. The method comprises the following steps: weighing chlorotoxin, dissolving the chlorotoxin in a PBS buffer solution, adding 2-imino sulfane hydrochloride, fully mixing, and incubating at room temperature to form a CTX-SH solution; the preparation method comprises the following steps: weighing dimyristoyl phosphatidylcholine, adding a trichloromethane solution, uniformly mixing, then adding a cholesterol oleate solution and a phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide solution, and sufficiently and uniformly mixing; drying the solution, resuspending with a PBS (Phosphate Buffer Solution), and fully dissolving by ultrasonic; adding a thiolated CTX-SH solution into the solution, incubating for 1-3 days, and carrying out ultrafiltration purification to form a CTX-NP nanoparticle solution; preparing a melittin solution, dropwise adding the melittin solution into the CTX-NP nanoparticle solution, incubating for 10-24 hours, and performing ultrafiltration purification. The invention has the advantages of targeting brain glioma, inhibiting the growth of in-situ brain glioma and improving the immunosuppressive microenvironment of brain glioma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug delivery carriers, in particular to a chlorotoxin-melittin nanoparticle and a preparation method and application thereof. BACKGROUND Glioma is one of the common primary malignant tumors in the central nervous system. The current clinical treatment method mainly relies on surgical resection, supplemented by radiotherapy and chemotherapy. Due to the strong invasiveness of glioma growth, surgical resection is often incomplete, which easily leads to recurrence. At the same time, the treatment method combined with radiotherapy and chemotherapy can produce relatively serious side effects, such as lymphocyte reduction. This traditional therapy is very limited for improving the long-term survival rate of glioma patients, and the median survival time of patients is only 15 months, and the 5-year survival rate is about 5%. The blood-brain barrier is the main obstacle for treating glioma. Only gas and small lipid-soluble small molecule substances less than 400 Da can pass through the blood-brain barrier by passive diffusion, and a small part of substances can pass through the blood-brain barrier by active transport system, while about 98% of substances cannot pass through the blood-brain barrier. Although the blood-brain barrier can maintain the stability of the central nervous system, its permeability also hinders the entry of therapeutic drugs into the central nervous system. In order to achieve the effective concentration required for treatment at the target site in the brain, it is necessary to significantly increase the drug concentration in the blood circulation or prolong the time of administration. However, this approach may increase the risk of systemic toxicity, thereby bringing potential safety hazards. Therefore, the existence of the blood-brain barrier poses a great challenge to the development of central nervous system disease therapy and drug brain delivery. Developing a drug-loaded system that helps chemotherapy drugs to cross the blood-brain barrier and deliver to the glioma area, and proposing an effective method to kill glioma cells are one of the effective ways to improve the treatment effect. Nanoparticles, as a good and effective drug delivery carrier, have a diameter in the range of 1-1000 nm, which can effectively prolong the circulation time of drugs in the body. The lipid nanoparticles approved by the US Food and Drug Administration (FDA) have good biocompatibility, relatively stable structure, low toxicity, etc. By improving the biodistribution of drugs in the whole body and prolonging the circulation half-life of drugs, the limitations of traditional chemotherapy methods are overcome. However, unmodified nanoparticles usually lack adaptability in size, surface properties, etc., which makes it difficult for them to cross the blood-brain barrier. Therefore, by modifying the surface of nanoparticles with polypeptides, the introduction of specific functional molecules can increase the affinity of nanoparticles with specific receptors or transport proteins on the blood-brain barrier, which is of great significance for promoting their crossing of the blood-brain barrier and realizing the targeted combination of glioma cells.

[0002] In recent years, focused ultrasound combined with microbubble technology has been used as a non-invasive method to temporarily and reversibly open the blood-brain barrier, but there are still some challenges, and specific targeting drugs need to be developed for tumor markers to ensure that therapeutic drugs can be specifically delivered to tumor sites without affecting healthy brain tissue to avoid potential nerve damage.

[0003] Nanoparticle surface antibody conjugation has potential benefits for blood-brain barrier drug delivery, but there are still some limitations and challenges that must be addressed. One of the main limitations is the potential immunogenicity and antibody-mediated toxicity, which can be caused by the interaction between antibody-conjugated nanoparticles and the immune system. SUMMARY The present application provides a chlorotoxin-melittin nanoparticle and a preparation method and application thereof to solve the above problems. The first object of the present application is to provide a chlorotoxin-melittin nanoparticle prepared by the following method: S1. Thiolation of chlorotoxin: add 2-iminothiolane hydrochloride to the chlorotoxin solution, mix well, incubate at room temperature, and form a CTX-SH solution; The chlorotoxin solution is obtained by dissolving chlorotoxin in a PBS buffer solution containing ethylenediaminetetraacetic acid; S2. Add dimyristoyl phosphatidylcholine to a chloroform solution, mix well, then add a cholesteryl oleate solution, a phosphatidylethanolamine-polyethylene glycol 2000-maleimide solution, and / or a DiR-BOA solution, and mix well; S3. Dry the solution obtained in step S2, resuspend it with a PBS solution, and fully dissolve it by ultrasonic vibration; The steps S1 and S2-S3 are not limited in order and can be performed simultaneously; S4. Add the CTX-SH solution obtained in S1 to the solution of step S3, incubate for 1-3 days, and purify by ultrafiltration to form a CTX-NP nanoparticle solution; S5. Add melittin solution dropwise to the CTX-NP nanoparticle solution, incubate for 10-24 h, and purify by ultrafiltration to form a chlorotoxin-melittin nanoparticle.

[0004] Further, the molar ratio of chlorotoxin solution to 2-iminothiolane hydrochloride in step S1 is (10±5):1; preferably, the molar ratio of chlorotoxin solution to 2-iminothiolane hydrochloride is 10:1.

[0005] Further, the incubation time in step S1 is ≥1 h.

[0006] Further, the PBS buffer solution in the S1 contains 5mM ethylenediamine tetraacetic acid, pH=8.0. The ethylenediamine tetraacetic acid acts as a metal chelator, which can remove the interference of metal ions in the thiolation process of CTX.

[0007] Further, the drying in the step S3 is nitrogen blowing drying or vacuum drying.

[0008] Further, the incubation time in the step S4 is 2 days.

[0009] Further, in the S5, the incubation time is 12±3 h.

[0010] Further, the amount ratio of the amount of melittin contained in the melittin solution, the amount of chlorotoxin contained in the chlorotoxin solution, the amount of the solution of dimyristoyl phosphatidylcholine and chloroform, the amount of cholesteryl oleate, the amount of phosphatidylethanolamine-polyethylene glycol 2000-maleimide, and the amount of melittin contained in the melittin solution is (3-5) mg:(0.8-1.5) mL:(0.1-0.4) mg:(0.3-0.5) mg:(1-4) mg.

[0011] Further, the chlorotoxin-melittin nanoparticle is spherical, and the average particle size is 80-100 nm.

[0012] The application also protects the use of the chlorotoxin-melittin nanoparticle in the preparation of glioma targeting drugs.

[0013] Compared with the prior art, the application can achieve the following beneficial effects: The chlorotoxin-melittin nanoparticle prepared by the application has the effects of crossing the blood-brain barrier to target brain glioma, inhibiting the growth of in situ brain glioma, and promoting the infiltration of various immune cells (CD3 + T cells, B cells, macrophages, etc.) in glioma; can realize the technical effects of crossing the blood-brain barrier, targeting killing of brain glioma, and reversing the immunosuppressive microenvironment of glioma into an immunologically active microenvironment; and the chlorotoxin-melittin nanoparticle prepared by the application has good biological safety verified by animal experiments, is conducive to the preparation of glioma targeting drugs, and provides a great prospect for glioma targeting drugs. BRIEF DESCRIPTION OF DRAWINGS Figure 1 is a nanoparticle in vitro characterization result figure provided according to an embodiment of the application; (A) a particle size diagram of CTX-NP nanoparticles; (B) a particle size diagram of chlorotoxin-melittin nanoparticles (CTX-melittin-NP); (C) a nanoparticle characterization parameter result figure; (D) a fluorescence imaging result figure of CTX-NP and CTX-melittin-NP added with different concentrations of DiR-BOA fluorescent dye; and (E) a fluorescence signal diagram of different nanoparticles after incubation with serum for 12 h and 24 h.

[0014] Figure 2 FIG. 5 is a confocal imaging result and fluorescence signal intensity comparison result diagram of different nanoparticles incubated with GL261 and CT26 cells according to an embodiment of the present application; (A) PBS buffer solution; (B) NP nanoparticles; (C) a-melittin-NP nanoparticles; (D) CTX-NP nanoparticles; (E) chlorotoxin-melittin-NP; (F) fluorescence signal intensity comparison result diagram of different nanoparticles incubated with GL261 and CT26 cells.

[0015] Figure 3 FIG. 6 is a result diagram of the killing ability of chlorotoxin-melittin-NP with different concentrations of melittin on GL261 cells according to an embodiment of the present application.

[0016] Figure 4 FIG. 7 is a result diagram of the cell survival rate of chlorotoxin-melittin-NP with different concentrations of melittin incubated with GL261 and bEnd.3 cells according to an embodiment of the present application. Figure 5 FIG. 8 is a result diagram of different nanoparticles in the body targeting glioma according to an embodiment of the present application.

[0017] Figure 6 FIG. 9 is a result diagram of the fluorescence imaging of the brain of an experimental mouse after injection of different nanoparticles (A) and the quantification result of the ratio of the tumor area to the whole brain area in different nanoparticle groups (B) according to an embodiment of the present application. Figure 7 FIG. 10 is a result diagram of the influence of chlorotoxin-melittin-NP on CD3+ T cell infiltration in the microenvironment of brain glioma according to an embodiment of the present application. Figure 8 FIG. 11 is a result diagram of the influence of chlorotoxin-melittin-NP on B cell infiltration in the microenvironment of brain glioma according to an embodiment of the present application. Figure 9 FIG. 12 is a result diagram of the influence of chlorotoxin-melittin-NP on macrophage infiltration in the microenvironment of brain glioma according to an embodiment of the present application. Figure 10 FIG. 13 is a result diagram of the microscope imaging of the degree of organ damage of chlorotoxin-melittin-NP according to an embodiment of the present application. DETAILED DESCRIPTION Hereinafter, embodiments of the present application will be described with reference to the accompanying drawings. In the following description, the same modules are denoted by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, detailed descriptions thereof will not be repeated. In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and specific examples. It should be understood that the specific examples described herein are only used to explain the present application, and do not constitute a limitation on the present application. If no specific technology or condition is specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained from the market.

[0018] Chlorotoxin, CTX (product number: DC23913; manufacturer: Shanghai Yuxi Biological Technology Co., Ltd.; polypeptide sequence: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR; NCBI sequence number: UniProtKB / Swiss-Prot: P45639.1) of the present application, 1 mL, 1 mg / mL, chlorotoxin solution dissolved in PBS (containing 5 mM ethylenediaminetetraacetic acid (EDTA), PH=8.0); 2-iminothiolane hydrochloride (Traut's Reagent, product number: 1811774; manufacturer: Shanghai McLean Biochemical Science and Technology Co., Ltd., dissolved in PBS (containing 5 mM EDTA, PH=8.0)); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, product number: 850345P, manufacturer information: Avanti Polar Lipids Company) Cholesteryl oleate (CO, product number: C9253; manufacturer: Sigma-Aldrich Company), the solution of which is dissolved in chloroform.

[0019] DSPE-PEG2000-Mal (phosphatidylethanolamine-polyethylene glycol 2000-maleimide; product number: DC80021; manufacturer: Shanghai Yuxi Biological Technology Co., Ltd.) is dissolved in a chloroform solution to obtain a DSPE-PEG2000-Mal solution.

[0020] The polypeptide sequence information of chlorotoxin is MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR, SEQ ID NO: 1; provided by Shanghai Yuxi Biological Technology Co., Ltd.

[0021] Melittin is D4F-melittin (consists of a segment of alpha-helix polypeptide D4F, linking sequence of amino acid sequence GSG and melittin).

[0022] D4F-melittin polypeptide is synthesized by Shanghai Chupe Biotechnology Co., Ltd.; the sequence is: DWFKAFYDKVAEKFKEAFGSGGIGAVLKVLTTGLPALISWIKRKRQQ-NH2; SEQ ID NO: 2.

[0023] A chlorotoxin-melittin nanoparticle is prepared by the following method: S1. Thiolation treatment of chlorotoxin: add 2-iminothiolane hydrochloride to the chlorotoxin solution, mix well, incubate at room temperature to form a CTX-SH solution; The chlorotoxin solution is obtained by dissolving chlorotoxin in a PBS buffer solution containing ethylenediaminetetraacetic acid; S2. Add dimyristoyl phosphatidylcholine to a chloroform solution, mix well, then add a cholesteryl oleate solution, phosphatidylethanolamine-polyethylene glycol 2000-maleimide and / or DiR-BOA solution, and mix well; S3. Dry the solution obtained in step S2, resuspend with a PBS solution, and fully dissolve by ultrasonic vibration; The steps S1 and S2-S3 are not limited in order and can be performed simultaneously; S4. Add the CTX-SH solution obtained in S1 to the solution of step S3, incubate for 1-3 days, not limited to 1 day, 2 days, 3 days, preferably incubate for 2 days; and purify by ultrafiltration to form a CTX-NP nanoparticle solution; S5. Add melittin solution dropwise to the CTX-NP nanoparticle solution, incubate for 10-24 h, not limited to 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, and purify by ultrafiltration to form a chlorotoxin-melittin nanoparticle. Preferably, the incubation time is 12±3 h.

[0024] Further, the molar ratio of chlorotoxin solution to 2-iminothiolane hydrochloride in step S1 is (10±5):1; not limited to 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 6:1, 5:1; preferably, the molar ratio of chlorotoxin solution to 2-iminothiolane hydrochloride is 10:1.

[0025] Further, the incubation time in step S1 is ≥1 h.

[0026] Furthermore, the PBS buffer solution in S1 contains 5 mM ethylenediaminetetraacetic acid (EDTA) with a pH of 8.0. EDTA acts as a metal chelating agent, removing interference from metal ions during the thiolation process of CTX.

[0027] Furthermore, the drying in step S3 is nitrogen blowing or vacuum drying.

[0028] Furthermore, the ratio of the amount of melittin in the dimyristoyl phosphatidylcholine, chloroform solution, cholesterol oleate, phosphatidylethanolamine-polyethylene glycol 2000-maleimide, and melittin solution is (3-5) mg: (0.8-1.5) mL: (0.1-0.4) mg: (0.3-0.5) mg: (1-4) mg.

[0029] Example 1 A method for preparing chlortoxin-bee venom peptide nanoparticles specifically includes the following steps: S1. Thiolization treatment of chloramphenicol: Weigh 1 mg of chloramphenicol (CTX) and dissolve it in 1 mL of PBS buffer solution to obtain a chloramphenicol (CTX) solution. Add 1720 μL of 0.2 mg / mL 2-iminothione hydrochloride (Traut's Reagent) solution to the chloramphenicol (CTX) solution, mix thoroughly, and incubate at room temperature for more than 1 hour to form a CTX-SH solution; The PBS buffer solution contains 5 mM ethylenediaminetetraacetic acid (EDTA); the molar ratio of chloramphenicol (CTX) solution to 2-iminothionine hydrochloride (Traut's Reagent) solution is 10:1. S2. Weigh 4 mg of myristoyl phosphatidylcholine (DMPC) into an EP tube, add 1 mL of chloroform solution, mix thoroughly, then add 26 μL of C2O solution with a concentration of 10 mg / mL dissolved in chloroform and 38.4 μL of phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-Mal) solution with a concentration of 10 mg / mL, and mix thoroughly. S3. Dry the solution obtained in step S2, resuspend it in PBS solution, and fully dissolve it by ultrasonic vibration; the drying method is to first blow it with nitrogen to form a film, and then perform vacuum drying. Before drying, it can be transferred to a round-bottom test tube; after drying, resuspend it in PBS buffer solution and fully dissolve it by ultrasonic vibration. The order of steps S1 and S2-S3 is not limited, and they can be performed simultaneously. S4. The thiolated CTX-SH solution in step S1 was added to the solution in step S3, incubated at 4°C for 2 days, and purified by ultrafiltration to form a CTX-NP nanoparticle solution; S5. A melittin solution 1 mL with a concentration of 2 mg / mL was prepared using a PBS solution, and was added dropwise to the CTX-NP nanoparticle solution, incubated for 12 h, and purified by ultrafiltration to form a chlorotoxin-melittin nanoparticle (CTX-melittin-NP). Specifically, the ultrafiltration purification was performed using a low-speed refrigerated centrifuge at a temperature of 4°C and a speed of 2600 rpm. The nanoparticle solution after incubation was filtered using a 0.22 μm filter membrane and transferred to a 30 kDa ultrafiltration tube (Millipore). The low-speed refrigerated centrifuge was pre-cooled to 4°C, and the concentrated centrifuge tube was balanced and placed in the centrifuge. The centrifuge parameters were set as follows: 2600 rpm, 4°C, 50 min. After one centrifugation, the filtrate in the lower layer was discarded, and the upper layer tube was filled with PBS and centrifuged again. The above steps were repeated twice to ensure that the unbound melittin could be removed. After centrifugation, the upper liquid in the centrifuge tube was transferred to a 1.5 mL EP tube and stored at 4°C. Example 2 A method for preparing a chlorotoxin-melittin nanoparticle (containing a fluorescent dye), specifically comprising the following steps: S1. Thiolation of chlorotoxin: 1 mg of chlorotoxin (CTX) was dissolved in 1 mL of a PBS buffer solution to obtain a chlorotoxin (CTX) solution. 1720 μL of a 2-iminothiolane hydrochloride (Traut’s Reagent) solution with a concentration of 0.2 mg / mL was added to the chlorotoxin (CTX) solution, mixed thoroughly, and incubated at room temperature for more than 1 h to form a CTX-SH solution. The PBS buffer solution contains 5 mM ethylenediaminetetraacetic acid (EDTA) and has a pH of 8.0. The molar ratio of the chlorotoxin (CTX) solution to the 2-iminothiolane hydrochloride (Traut’s Reagent) solution is 10:1. S2. 4 mg of dimyristoyl phosphatidylcholine (DMPC) was weighed into an EP tube, 1 mL of chloroform solution was added, mixed thoroughly, and then 13 μL of a CO solution with a concentration of 10 mg / mL dissolved in chloroform, 38.4 μL of a DSPE-PEG2000-Mal solution with a concentration of 10 mg / mL, and 40 μL of a DiR-BOA solution with a concentration of 10 mg / mL were added and mixed thoroughly. S3. The solution obtained in step S2 is dried, resuspended with PBS solution, and fully dissolved by ultrasonic vibration; the drying method is to first blow dry with nitrogen to form a thin film, and then vacuum drying, which can be transferred to a round-bottom test tube before drying; after drying, resuspend with PBS buffer solution, and fully dissolve by ultrasonic vibration; The steps S1 and S2-S3 are not limited in order, and can also be performed simultaneously; S4. The thiolated CTX-SH solution in step S1 is added to the solution in step S3, incubated at 4°C for 2 days, and purified by ultrafiltration to form a (DiR-BOA) CTX-NP nanoparticle solution; S5. A 2 mg / mL melittin (D4F-melittin) solution 2 mL prepared with PBS is added dropwise to the CTX-NP nanoparticle solution, incubated for 12 h, and purified by ultrafiltration to form a fluorescent dye-containing chlorotoxin-melittin nanoparticle ((DiR-BOA) CTX-melittin-NP). Example 3 According to the preparation method of Example 1, after the preparation of CTX-NP nanoparticles (i.e. steps S1-S4) and CTX-melittin-NP nanoparticles (i.e. steps S1-S5), the unbound CTX molecules in the system are removed by ultrafiltration using a low-speed refrigerated centrifuge, and the morphology, particle size, fluorescence intensity, serum stability and other properties are characterized in vitro, and the results are shown in Figure 1 The transmission electron microscopy and dynamic light scattering (DLS) test results show that the CTX-NP and CTX-melittin-NP nanoparticles are spherical, with an average particle size of 80.78 nm and 96.45 nm, respectively, and the measured zeta potential is -3.53 mV and 0.77 mV, respectively Figure 1 (Fig. 3A-C); fluorescence imaging of CTX-NP and CTX-melittin-NP with different concentrations of lipophilic near-infrared imaging dye (DiR-BOA fluorescent dye) shows that Figure 1 (Fig. 3D) shows that the fluorescence signal intensity of the nanoparticles decreases with the decrease of the concentration of DiR-BOA. In order to explore the feasibility of intravenous injection of nanoparticles, it is necessary to determine their stability in serum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) was used to detect the stability of CTX-NP and CTX-melittin-NP in serum, and the results are shown in The fluorescence signal intensity of CTX-NP, CTX-melittin-NP co-incubated with mouse serum at different time (12h, 24h) was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, namely SDS-PAGE, and whole fluorescence imaging. The results showed that the fluorescence signal intensity of the nanoparticle group co-incubated with mouse serum at different time gradients was relatively consistent. After the nanoparticle group was lysed by Tween-20, the basic structure was destroyed, the hydrophobic core wrapped DiR-BOA fluorescent dye was released, the fluorescence signal was dispersed to the whole lane, and the band was diffusely distributed, indicating that the CTX-NP nanoparticle and CTX-melittin-NP nanoparticle respectively incubated with serum still maintained good stability Figure 1 Example 4 Example 4 Different groups of nanoparticle solutions (PBS buffer solution as solvent) were set: NP nanoparticles, α-melittin-NP nanoparticles, CTX-NP nanoparticles, CTX-melittin-NP nanoparticles (all as treatment groups), and PBS buffer solution as a blank control group; 20 μM of DiR-BOA fluorescent dye was added to the nanoparticle solution for subsequent experiments. The preparation method of the NP nanoparticle is as follows: S2. 4 mg of dimyristoyl phosphatidylcholine (DMPC) was weighed in an EP tube, 1 mL of chloroform solution was added, and it was fully mixed, then 26 μL of CO solution with a concentration of 10 mg / mL and 38.4 μL of DSPE-PEG2000-Mal solution with a concentration of 10 mg / mL were added, and it was fully mixed; S3. The solution obtained in step S1 was blown dry with nitrogen, and after forming a thin film, vacuum drying was performed, resuspension was performed with PBS solution, and full dissolution was performed by ultrasonic vibration; S5. Subsequent ultrafiltration purification formed the NP nanoparticle solution. The preparation method of the α-melittin-NP nanoparticle is as follows: S2. 4 mg of dimyristoyl phosphatidylcholine (DMPC) was weighed in an EP tube, 1 mL of chloroform solution was added, and it was fully mixed, then 26 μL of CO solution with a concentration of 10 mg / mL was added, and it was fully mixed; S3. The solution obtained in step S1 was blown dry with nitrogen, and after forming a thin film, vacuum drying was performed, resuspension was performed with PBS solution, and full dissolution was performed by ultrasonic vibration; S5. Prepare a solution of melittin (D4F-melittin) at a concentration of 2 mg / mL or 2.5 mg / mL, and add it dropwise to the nanoparticle solution after ultrasonic dissolution, incubate at 4°C for 12 h, and purify by ultrafiltration to form melittin nanoparticles (a-melittin-NP). I. Confocal imaging experiment of chlorotoxin-melittin nanoparticles after incubation with GL261 and CT26 cells The nanoparticle solutions of different groups (according to the calculation, each group of nanoparticles was added to the culture medium to ensure that each group contained 20 μM DiR-BOA, such as 600 μL of culture medium containing 20 μM DiR-BOA was added to each well in a 24-well plate) were incubated with GL261 and CT26 cells for 1.5 h, and then confocal imaging was performed. The imaging results are shown in Figure 2 As shown in B and D of Figure 2 , compared with the NP group without CTX, the CTX-NP group detected stronger fluorescence signals in the cells, indicating that CTX-NP has better targeting ability for GL261 cells. In the CT26 cells with low expression of matrix metalloproteinase 2 (MMP2), the amount of CTX-NP uptake was less, and the intensity of DiR-BOA fluorescence signal in the cells was weaker, indicating that the amount of CTX nanoparticle uptake by tumor cells was determined by the difference in expression of CTX specific receptor MMP2. The a-melittin-NP and CTX-melittin-NP groups modified the melittin polypeptide on the nanoparticles, which has the effect of punching holes in the cell membrane and directly killing tumor cells. As shown in C and E of Figure 2 , although part of the CT26 cells have a certain uptake of a-melittin-NP and CTX-melittin-NP (due to the punching of melittin polypeptide on part of the cells leading to non-specific uptake), the fluorescence signal in GL261 cells is still significantly higher than that in CT26 group. As shown in F of Figure 2 , in the CTX-NP and CTX-melittin-NP groups, the fluorescence intensity of nanoparticles taken up by GL261 cells was 3.3 times and 3.4 times that of CT26 cells, respectively. II. Experiment of chlorotoxin-melittin nanoparticles on the killing ability of brain glioma cells and vascular endothelial cells Killing effect of different concentrations of melittin on cells: different concentrations of CTX-melittin-NP (melittin concentration: 2.5 μM, 5 μM, 7.5 μM, 10 μM, 25 μM, 50 μM, 100 μM) were co-incubated with GL261 cells for 3 h, and then the cells were replaced with fresh medium, and cultured for 24 h. The survival rate of GL261 cells was detected by MTS kit. The in vitro killing experiment results are shown in Figure 3As shown, when the concentration of melittin polypeptide loaded in CTX-melittin-NP was 10 μM, nearly 80% of GL261 cells could be effectively killed; when the concentration reached 25 μM, about 90% of GL261 cells died. In addition, the CTX-melittin-NP with different concentrations of melittin above were co-incubated with GL261 cells and bEnd.3 cells for 3 h respectively, and then the cell medium was changed and the cells were cultured for 24 h. The survival rate of GL261 cells and bEnd.3 cells was detected by CCK-8 kit. The in vitro killing experiment results are shown in FIG. 6. Figure 4 As shown, when the concentration of melittin polypeptide loaded in CTX-melittin-NP was 25 μM, more than 70% of GL261 cells could be effectively killed, which was similar to the result of Figure 3 The results were similar, and the mortality rate of bEnd.3 cells was less than 40%. The above results showed that CTX-melittin-NP had the ability to efficiently kill brain glioma cells, and had less killing effect on vascular endothelial cells, which laid a foundation for the subsequent in vivo treatment of brain glioma experiments.

[0030] Five groups of C57BL / 6 mice (n=5-6 per group) were selected. GL261 mouse glioma cells were intracranially inoculated into the mice. On day 10 post-inoculation, 150 μL of NP nanoparticles, α-melittin-NP nanoparticles, CTX-NP nanoparticles, and CTX-melittin-NP nanoparticle solutions were intravenously injected (each 150 μL of α-melittin-NP, CTX-NP, and CTX-melittin-NP nanoparticles contained 0.4 mg of melittin and 50 μg of CTX; NP nanoparticles, which did not contain melittin or CTX, were diluted to the same volume as α-melittin-NP nanoparticles before injection). To facilitate visualization and comparison of the signal intensity of each nanoparticle, the amount of DiR-BOA loaded on each nanocarrier was kept constant (40 μL). (nmol); 24 hours after nanoparticle injection, mice were dissected, and in vitro tissue analysis was performed using whole-body fluorescence imaging to observe the intensity and distribution of nanoparticle signals and compare the aggregation effects of different nanoparticles in the glioma region. Whole-body fluorescence imaging results showed that significant tumor signals were observed in the brain regions of all groups of mice, indicating successful establishment of the mouse glioma model. Further detection of the DiR-BOA signal of the nanoparticles revealed that no DiR-BOA signal was observed in the glioma regions of the intravenously injected NP and α-melittin-NP nanoparticle groups. However, for the CTX-modified CTX-NP and CTX-melittin-NP nanoparticle groups, DiR-BOA signals of varying intensities were observed in the glioma regions, indicating that the CTX-modified nanoparticles significantly aggregated in the glioma region. This demonstrates that modifying nanoparticles with CTX can significantly enhance their ability to cross the blood-brain barrier and target gliomas. Figure 5 ). III. Experiments on the In-situ Growth of Gliomas by Chlorine Toxin-Meliosteum Nanoparticles Using a stereotaxic instrument (Shenzhen Ruiward Life Technology Co., Ltd.), locate the anterior fontanelle point and inject 5×10⁻⁶ ions of a substance 2-3 mm to its right, 4-5 mm longitudinally backward, to a depth of 3 mm. 43 μL PBS solution of GL261-mcherry cells; mouse brain glioma orthotopic model was constructed, and 5-6 tumor-bearing mice in different groups were injected with 150 μL of PBS, NP, α-melittin-NP, CTX-NP, CTX-melittin-NP on the 10th day, 13th day, 16th day, and 19th day of glioma growth, respectively (among them, the α-melittin-NP group, the CTX-NP group, and the CTX-melittin-NP group, the melittin content was 0.4 mg and the CTX content was 50 μg in 150 μL of each injection volume. The NP group did not contain melittin or ctx, and was diluted to the same volume as the α-melittin-NP for injection); then the mouse brains were dissected on the 22nd day for whole-body fluorescence imaging, and the mCherry fluorescence protein signal obtained by imaging was used to represent the signal of the tumor (red fluorescent dye mCherry fluorescence protein: Ex: 562 / 40 nm, Em: 624 / 40 nm). The whole-body fluorescence imaging results are shown in FIG. 1A (the left column is the bright field image of the mouse brain, the middle column is the glioma GL261-mCherry fluorescence image of the mouse brain, and the right column is the bright field and fluorescence superimposed image of the mouse brain), and compared with other treatment groups, the mCherry fluorescence intensity representing the glioma signal in the CTX-melittin-NP treatment group of the mouse brain is weaker and the area is smaller, indicating that the growth of the glioma in this group is inhibited. The ratio of the glioma area to the whole brain area was calculated, and the quantitative results are shown in FIG. 1B, which shows that the glioma area ratio of the CTX-melittin-NP group is significantly lower than that of the other treatment groups, and compared with the PBS group, the inhibition rate is 94.9%. It is shown that intravenous injection of CTX-melittin-NP can effectively inhibit the growth of orthotopic brain glioma in vivo. Figure 6 Figure 6 Mouse brain glioma orthotopic model (GL261-mCherry mouse glioma cell strain): mCherry is a red fluorescent protein. The mCherry fragment (double enzyme digestion: Alf II and EcoR I) was constructed into a PiggyBac (PB) transposon vector to obtain a PB transposon vector plasmid expressing mCherry fluorescent protein.

[0031] ​​The PB transposon vector plasmid containing the mCherry sequence and the helper plasmid expressing PBase transposase are co-transfected into GL261 glioma cells; the transfected glioma cells are cultured, and single clone cell strains stably expressing mCherry are screened by site-specific digestion. The transfected GL261 glioma cells are placed in RPMI1640 culture medium containing 10% fetal bovine serum and cultured at 37°C in a 5% CO2 environment. The transfected GL261 glioma cells are digested, and the digested cells are plated in a 96-well plate. After culturing for several days, single clone cell clusters expressing mCherry fluorescent protein are screened under a fluorescence microscope, and the single clone cell clusters are aspirated into a new well plate for culture. After culturing for several days, single clone cell clusters with mCherry red fluorescence are screened under a fluorescence microscope, and it is ensured that each well has only one red fluorescent cell cluster. The culture is continued for 1-2 weeks, and finally a GL261 glioma cell strain stably expressing mCherry fluorescent protein marker is screened, with an emission rate of 95% or more. This is a mouse brain glioma in situ model (GL261-mCherry mouse glioma cell strain).

[0032] IV. Effect of tetrachlorotoxin-melittin nanoparticles on cell infiltration in the brain glioma microenvironment 1. The immune cells T cells in the brain glioma immune microenvironment were labeled using CD3 fluorescently labeled antibodies (Alexa Fluor® 647 anti-mouse CD3 antibodies) to explore the changes in CD3+ T cell infiltration in the brain glioma microenvironment of mice after CTX-melittin-NP treatment (results shown in Figure 7 Figs. 5A-E). According to the confocal imaging results, it can be observed that there are more CD3+ T cells in the core region of the brain glioma after CTX-melittin-NP treatment, with a cell density of 1160 cells / mm2, which is about 3.0 times that of the PBS group (389 cells / mm2), 1.3 times that of the NP group (867 cells / mm2), 1.3 times that of the a-melittin-NP group (927 cells / mm2), and 1.4 times that of the CTX-NP group (843 cells / mm2) (as shown in Figure 7 Fig. 5F). Different mouse samples may have significant changes in cell density due to individual differences and other reasons, but do not affect the final conclusion of the observation.

[0033] 2. The immune cells B cells in the brain glioma immune microenvironment were labeled using B220 fluorescently labeled antibodies (Alexa Fluor® 647 anti-mouse / human CD45R / B220 antibodies) to explore the changes in B220+ B cell infiltration in the brain glioma microenvironment of mice after CTX-melittin-NP treatment (results shown in Figure 8(Middle AE). Confocal imaging results showed that B-cell infiltration in the core of gliomas in mice treated with CTX-melittin-NP increased, with a cell density of 52 cells / mm², approximately 2.4 times that of the PBS group (18 cells / mm²), 1.5 times that of the NP group (29 cells / mm²), 2.7 times that of the α-melittin-NP group (19 cells / mm²), and 3.0 times that of the CTX-NP group (17 cells / mm²). Figure 8 (F). 3. Macrophages in the immune microenvironment of gliomas were labeled using an F4 / 80 fluorescently labeled antibody (Alexa Fluor® 647 anti-mouse F4 / 80 antibody) to investigate the distribution of macrophages in the glioma microenvironment. Figure 9 (Middle AE). Confocal microscopy imaging results showed that a large number of F4 / 80 labeled macrophages were distributed in the glioma microenvironment of mice treated with CTX-melittin-NP, with a cell density of 1605 cells / mm². 2 Approximately 668 cells / mm from the PBS group. 2 2.4 times that of the NP group (1074 cells / mm) 2 The concentration was 1.5 times higher in the α-melittin-NP group (1184 cells / mm²). 2 1.4 times that of the CTX-NP group (841 cells / mm) 2 1.9 times that of ) Figure 9 In the CTX-melittin-NP group, a large number of macrophages co-localized with GL261 cells in the core of gliomas after CTX-melittin-NP treatment, exhibiting phagocytosis of tumor cells. Quantitative results showed that the proportion of macrophages phagocytizing tumor cells in the CTX-melittin-NP group was 67% of the total macrophages, which was 2.3 times, 1.9 times, 1.7 times, and 2.3 times that of the PBS group (29%), NP group (36%), α-melittin-NP group (41%), and CTX-NP group (29%), respectively. Figure 9 (E). The above results indicate that a large number of macrophages were recruited and engulfed tumor cell fragments in the glioma microenvironment of mice after treatment, which has a certain promoting effect on glioma treatment. 4. Biosafety verification of chloramphenicol-bee venom peptide nanoparticles The experiment was divided into five groups, with 5-6 C57BL / 6 mice in each group.

[0034] The nanoparticle solutions of different groups were respectively administered (intravenous injection) to the experimental mice (the administration method was consistent with the previous one. On the 10th day, the 13th day, the 16th day, and the 19th day of glioma growth, 150 μL of PBS, NP, α-melittin-NP, CTX-NP, and CTX-melittin-NP were respectively injected into different groups (among them, the α-melittin-NP group, the CTX-NP group, and the CTX-melittin-NP group each contained 0.4 mg of melittin and 50 μg of CTX in 150 μL of volume. The NP group did not contain melittin or CTX, and was diluted to the same volume as the α-melittin-NP group for injection); 72 h after the last administration, the mice were anesthetized using Avertin anesthetic and dissected, and the heart, liver, spleen, lung, kidney, and other organs were collected for polyformaldehyde (PFA) fixation for hematoxylin-eosin staining (HE) sectioning, hematoxylin staining of the nucleus, and eosin staining of the cytoplasm. By comparing the tissue sections of the control group and the treatment group, it was evaluated whether the mouse main organs had obvious cell damage, necrosis, inflammatory infiltration, fibrosis, or other abnormal morphological changes. The prepared sections were imaged using an upright section scanning microscope, as shown in FIG. 8, and it was observed that the mouse organs after CTX-melittin-NP treatment had no obvious damage, indicating that CTX-melittin-NP had good biological safety. Figure 10 Principle: The specific targeting of CTX to tumors is closely related to the expression of matrix metalloproteinase 2 (MMP2) and chloride channel 3 (CLC3) on the surface of tumor cells. Among them, CLC3 is involved in cytoskeleton remodeling, thereby regulating cell morphology and movement during cell migration. MMP2 is a member of the metalloproteinase family and plays an important role in physiological processes such as angiogenesis, tumor cell invasion, and metastasis, and is highly expressed in glioma cells and lowly expressed in normal brain tissue. CTX has the ability to target glioma and cross the blood-brain barrier. The reason may be that it has a small molecular weight and a compact structure, making it easy to pass through the blood-brain barrier and narrow extracellular matrix. On the other hand, it can interact with specific receptors on certain cells that make up the blood-brain barrier. For example, CTX can interact with annexin A2 (AnxA2) on the membrane of brain microvascular endothelial cells, the main component of the blood-brain barrier, to promote receptor-mediated transcytosis of endothelial cells. Some studies have found that treating brain microvascular endothelial cells with a clathrin inhibitor can effectively inhibit the ability of CTX to cross the blood-brain barrier. ​The chlorotoxin CTX with glioma cell MMP2 receptor specificity in the application is combined with the alpha-melittin-NP nanoparticle of melittin peptide which has shown good anti-tumor and immune regulation effect in the early stage, to form the lipid nanoparticle co-modified by chlorotoxin and melittin (CTX-melittin-NP). In summary, the chlorotoxin-melittin nanoparticle CTX-melittin-NP prepared in the application can realize the strategy of 'three birds with one stone' of crossing the blood-brain barrier, targeting killing brain glioma and reversing the immunosuppressive microenvironment of glioma into the immune-activated microenvironment. It should be understood that the steps can be reordered, added, or deleted using the various forms of flow shown above. For example, the steps described in the present disclosure can be executed in parallel, in sequence, or in a different order, as long as the desired results of the technical solutions of the present disclosure can be achieved, and the present disclosure is not limited herein. The specific embodiments described above do not constitute a limitation on the protection scope of the present application. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A chlorotoxin-melittin nanoparticle, characterized in that, Prepared by the following method: S1. Thiolation of chlorotoxin: 2-iminothiolane hydrochloride was added to the chlorotoxin solution, mixed well, and incubated at room temperature to form a CTX-SH solution; The chlorotoxin solution was prepared by dissolving chlorotoxin in a PBS buffer solution containing ethylenediaminetetraacetic acid; S2. Dimyristoyl phosphatidylcholine was added to a chloroform solution, mixed well, and then cholesteryl oleate solution, phosphatidylethanolamine-polyethylene glycol 2000-maleimide, and / or DiR-BOA solution were added, mixed well; S3. The solution obtained in step S2 was dried, resuspended in a PBS solution, and dissolved by ultrasonic vibration; The steps S1 and S2-S3 are not limited in order and can be performed simultaneously; S4. The CTX-SH solution obtained in step S1 was added to the solution of step S3, incubated for 1-3 days, and purified by ultrafiltration to form a CTX-NP nanoparticle solution; S5. Melittin solution was added dropwise to the CTX-NP nanoparticle solution, incubated for 10-24 h, and purified by ultrafiltration to form a chlorotoxin-melittin nanoparticle.

2. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The molar ratio of chlorotoxin solution to 2-iminothiolane hydrochloride in step S1 is (10±5):1; preferably, the molar ratio of chlorotoxin solution to 2-iminothiolane hydrochloride is 10:

1.

3. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The incubation time in step S1 is ≥1 h.

4. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The PBS buffer solution in step S1 contains 5 mM ethylenediaminetetraacetic acid, pH=8.

0.

5. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The drying in step S3 is nitrogen blowing or vacuum drying.

6. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The incubation time in step S4 is 2 days.

7. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The incubation time in step S5 is 12±3 h.

8. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The amount of melittin contained in the dimyristoyl phosphatidylcholine, chloroform solution, cholesteryl oleate, phosphatidylethanolamine-polyethylene glycol 2000-maleimide, and melittin solution is (3-5) mg:(0.8-1.5) mL:(0.1-0.4) mg:(0.3-0.5) mg:(1-4) mg.

9. The chlorotoxin-melittin nanoparticle of claim 1, wherein, The chlorotoxin-melittin nanoparticle is spherical with an average particle size of 80-100 nm.

10. Use of the chlorotoxin-melittin nanoparticle of any one of claims 1-9 in the preparation of a glioma-targeting drug.

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