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

Abstract

The invention relates to the technical field of drug delivery carriers, in particular to chlorotoxin-melittin nanoparticles as well as a preparation method and application thereof. The preparation method comprises the following steps: weighing chlorotoxin, dissolving 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 C.O solution and a phospholipid-polyethylene glycol-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 CTX-NP, 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 invention relates to the technical field of drug delivery carriers, and in particular to chlorotoxin-melittin nanoparticles and a preparation method and application thereof. Background Art

[0002] Gliomas are one of the common primary malignant tumors of the central nervous system. Currently, the commonly used treatment methods in clinical practice are mainly surgical resection, supplemented by radiotherapy and chemotherapy. Due to the highly invasive growth of gliomas, surgical resection is often incomplete in clinical practice and is very likely to cause recurrence. At the same time, the combination of radiotherapy and chemotherapy will produce relatively serious side effects, such as symptoms such as lymphocytopenia. This traditional therapy has very limited effect on improving the long-term survival rate of patients with gliomas. The median survival time of patients is only 15 months, and the 5-year survival rate is about 5%.

[0003] The blood-brain barrier is the main obstacle to the treatment of gliomas. Only gases and lipid-soluble small molecules less than 400Da can pass through the blood-brain barrier by passive diffusion. A small number of substances can pass through the blood-brain barrier through active transport systems, 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 limitations also hinder the entry of therapeutic drugs into the central nervous system. In order to achieve the effective concentration required for treatment at the brain target, it is necessary to significantly increase the drug concentration in the blood circulation or extend the time of administration. However, this approach may increase the risk of systemic toxicity, thereby posing potential safety hazards. Therefore, the existence of the blood-brain barrier poses a huge challenge to the development of therapies for central nervous system diseases and the delivery of drugs into the brain. Developing a drug delivery system that assists chemotherapeutic drugs to cross the blood-brain barrier and deliver them to the glioma area, as well as proposing a method to effectively kill glioma cells, is one of the effective ways to improve the therapeutic effect.

[0004] Nanoparticles are a good and effective drug delivery carrier with a diameter in the range of 1-1000nm, which can effectively prolong the circulation time of drugs in the body. Lipid nanoparticles approved by the U.S. Food and Drug Administration (FDA) have the characteristics of good biocompatibility, relatively stable structure, and low toxicity. They overcome the limitations of traditional chemotherapy methods by improving the biodistribution of drugs throughout the body and prolonging the circulation half-life of drugs. However, unmodified nanoparticles usually lack adaptability in terms of size, surface properties, etc., which makes it difficult for them to pass through the blood-brain barrier. Therefore, by modifying the surface of nanoparticles with peptides and introducing specific functional molecules, the affinity of nanoparticles to specific receptors or transporters on the blood-brain barrier can be increased, which is of great significance for promoting their crossing of the blood-brain barrier and achieving targeted binding to glioma cells.

[0005] 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. It is necessary to develop specific targeted drugs for tumor markers to ensure that therapeutic drugs can be specifically delivered to the tumor site without affecting healthy brain tissue to avoid potential neurological damage.

[0006] 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 may be caused by the interaction between antibody-conjugated nanoparticles and the immune system. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a chlorotoxin-melittin nanoparticle and a preparation method and application thereof.

[0008] The first object of the present invention is to provide a chlorotoxin-melittin nanoparticle, which is prepared by the following method:

[0009] S1. Thiolation treatment of chlorotoxin: weigh chlorotoxin and dissolve it in PBS buffer solution to obtain a chlorotoxin solution; add 2-iminothiolane hydrochloride to the chlorotoxin solution, mix well, and incubate at room temperature to form a CTX-SH solution;

[0010] S2. Weigh dimyristoylphosphatidylcholine, add chloroform solution, mix thoroughly, then add cholesterol ester solution, phospholipid-polyethylene glycol-maleimide solution, mix thoroughly;

[0011] S3. The solution obtained in step S2 is dried, resuspended with PBS solution, and fully dissolved by ultrasonic vibration;

[0012] S4. adding the thiolated CTX-SH solution to the solution of step S3, incubating for 1 to 3 days, and purifying by ultrafiltration to form a CTX-NP nanoparticle solution;

[0013] S5. Prepare a melittin solution, add the CTX-NP nanoparticle solution dropwise, incubate for 10 to 24 hours, and purify by ultrafiltration to form chlorotoxin-melittin nanoparticles.

[0014] Preferably, in step S1, the molar ratio of the chlorotoxin solution to the 2-iminothiolane hydrochloride solution is 10:1.

[0015] Preferably, the incubation time in step S1 is ≥ 1 h.

[0016] Preferably, in step S2, the amount of dimyristoylphosphatidylcholine added is 3-5 mg, the amount of chloroform solution added is 0.8-1.5 mL, the concentration of cholesterol ester solution is 8-15 mg / mL, and the concentration of phospholipid-polyethylene glycol-maleimide solution is 8-15 mg / mL.

[0017] Preferably, the drying in step S3 is nitrogen drying or vacuum drying.

[0018] Preferably, the incubation time in step S4 is 2 days.

[0019] Preferably, the concentration of the melittin solution in step S5 is 1.5-2.5 mg / mL, and the incubation time is 12 h.

[0020] Preferably, the PBS buffer solution contains 5 mM ethylenediaminetetraacetic acid.

[0021] Preferably, the chlorotoxin-melittin nanoparticles are spherical, and the average particle size is 80-100 nm.

[0022] The second purpose of the present invention is to provide an application of chlorotoxin-melittin nanoparticles in the preparation of glioma targeted drugs.

[0023] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0024] The chlorotoxin-melitin nanoparticles prepared by the present invention have the ability to cross the blood-brain barrier to target gliomas, inhibit the growth of in situ gliomas, and promote the expression of multiple immune cells (CD3 + The invention can achieve the technical effects of crossing the blood-brain barrier, targeting and killing brain gliomas, and reversing the immunosuppressive microenvironment of gliomas to an immune-activating one; and the chlorotoxin-melittin nanoparticles prepared by the invention have been verified by animal experiments to have good biosafety, which is conducive to the preparation of glioma-targeted drugs and provides great prospects for glioma-targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figures are the results of in vitro characterization of nanoparticles provided according to an embodiment of the present invention; (A) zate potential of CTX-NP nanoparticles; (B) zate potential of chlorotoxin-melitin nanoparticles (CTX-melittin-NP); (C) nanoparticle characterization parameters; (D) fluorescence imaging results of CTX-NP and CTX-melittin-NP with different concentrations of DiR-BOA fluorescent dye added; (E) fluorescence signal diagram of nanoparticles after incubation with serum for 12h and 24h.

[0026] Figure 2The confocal imaging results and fluorescence signal intensity comparison results of different nanoparticles provided in an embodiment of the present invention incubated with GL261 and CT26 cells; (A) PBS buffer solution; (B) NP nanoparticles; (C) α-melittin-NP nanoparticles; (D) CTX-NP nanoparticles; (E) chlorotoxin-melitin nanoparticles (CTX-melittin-NP); (F) fluorescence signal intensity comparison results of different nanoparticles incubated with GL261 and CT26 cells.

[0027] Figure 3 These are the results of the killing ability of chlorotoxin-melittin nanoparticles containing different melittin concentrations provided in the examples of the present invention on GL261 cells.

[0028] Figure 4 This is the result of in vivo targeting of brain glioma by different nanoparticles provided by the embodiments of the present invention.

[0029] Figure 5 The figures are the fluorescence imaging results of the brain of experimental mice after injection of different nanoparticles (A) and the quantitative results of the ratio of the tumor area to the whole brain area in different nanoparticle groups (B) according to the embodiments of the present invention.

[0030] Figure 6 The chlorotoxin-melittin nanoparticles provided by the embodiment of the present invention are used to treat CD3 in the microenvironment of brain glioma + The impact of T cell infiltration on outcomes.

[0031] Figure 7 This is the result of the effect of chlorotoxin-melittin nanoparticles provided in an embodiment of the present invention on B cell infiltration in the microenvironment of brain glioma.

[0032] Figure 8 This is the result of the effect of chlorotoxin-melittin nanoparticles provided in an embodiment of the present invention on the infiltration of macrophages in the microenvironment of brain glioma.

[0033] Fig. 9 This is the microscope imaging result of the damage degree of organs by chlorotoxin-melittin nanoparticles provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0034] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are represented by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, the detailed description thereof will not be repeated.

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0036] Example 1

[0037] A method for preparing chlorotoxin-melittin nanoparticles comprises the following steps:

[0038] S1. Thiolation treatment of chlorotoxin: Weigh 1 mg of chlorotoxin (CTX) and dissolve it in 1 mL of PBS buffer solution to obtain a chlorotoxin (CTX) solution; add 1720 μL of 0.2 mg / mL 2-iminothiolane hydrochloride (Traut's Reagent) solution to the chlorotoxin (CTX) solution, mix well, and incubate at room temperature for more than 1 hour to form a CTX-SH solution;

[0039] The PBS buffer solution contains 5 mM ethylenediaminetetraacetic acid (EDTA); the molar ratio of the chlorotoxin (CTX) solution to the 2-iminothiolane hydrochloride (Traut's Reagent) solution is 10:1;

[0040] S2. Weigh 4 mg of dimyristoylphosphatidylcholine (DMPC) into an EP tube, add 1 mL of chloroform solution, mix thoroughly, then add 26 μL of 10 mg / mL CO solution and 38.4 μL of 10 mg / mL phospholipid-polyethylene glycol-maleimide (DSPE-PEG2000-Mal) solution, mix thoroughly;

[0041] 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 dry, and the solution can be transferred to a round-bottom test tube before drying; after drying, the solution is resuspended with PBS buffer solution and fully dissolved by ultrasonic vibration;

[0042] S4. adding the thiolated CTX-SH solution to the solution of step S3, incubating at 4°C for 2 days, and purifying by ultrafiltration to form a CTX-NP nanoparticle solution;

[0043] S5. Prepare a 2 mg / mL D4F-melittin solution, add CTX-NP nanoparticle solution dropwise, incubate for 12 h, and purify by ultrafiltration to form chlorotoxin-melittin nanoparticles (CTX-melittin-NP).

[0044] Specifically, the melittin is D4F-melittin (composed of a segment of α-helical polypeptide D4F, an amino acid sequence GSG of a connecting sequence and melittin).

[0045] Specifically, ultrafiltration purification was performed using a low-speed refrigerated centrifuge at 4°C and 2600 rpm; the incubated nanoparticle solution was filtered using a 0.22 μm filter membrane and transferred to a 30Kd concentrated centrifuge tube; the low-speed refrigerated centrifuge was precooled to 4°C in advance, and the concentrated centrifuge tube was placed in the centrifuge after being leveled; the centrifuge parameters were set to: 2600 rpm, 4°C, 50 min; after one centrifugation, the filtrate in the lower layer was poured out, and the upper layer tube was filled with PBS and continued to centrifuge, which was repeated twice to ensure that the unbound bee venom peptide could be removed; after the centrifugation was completed, the upper layer of the centrifuge tube was transferred to a 1.5 mL EP tube and stored at 4°C.

[0046] Example 2

[0047] A method for preparing chlorotoxin-melittin nanoparticles comprises the following steps:

[0048] S1. Thiolation treatment of chlorotoxin: Weigh 0.5 mg of chlorotoxin (CTX) and dissolve it in 0.5 mL of PBS buffer solution to obtain a chlorotoxin (CTX) solution; add 860 μL of 0.2 mg / mL 2-iminothiolane hydrochloride (Traut's Reagent) solution to the chlorotoxin (CTX) solution, mix well, and incubate at room temperature for more than 1 hour to form a CTX-SH solution;

[0049] The PBS buffer solution contains 5 mM ethylenediaminetetraacetic acid (EDTA); the molar ratio of the chlorotoxin (CTX) solution to the 2-iminothiolane hydrochloride (Traut's Reagent) solution is 10:1;

[0050] S2. Weigh 2 mg of dimyristoylphosphatidylcholine (DMPC) into an EP tube, add 0.5 mL of chloroform solution, mix thoroughly, then add 13 μL of 10 mg / mL cholesterol ester (CO) solution and 19.2 μL of 10 mg / mL phospholipid-polyethylene glycol-maleimide (DSPE-PEG2000-Mal) solution, and mix thoroughly;

[0051] S3. The solution obtained in step S2 is dried with nitrogen gas, and then vacuum dried after forming a thin film, resuspended with PBS solution, and fully dissolved by ultrasonic vibration;

[0052] S4. adding the thiolated CTX-SH solution to the solution of step S3, incubating at room temperature for 1 day, and purifying by ultrafiltration to form a CTX-NP nanoparticle solution;

[0053] S5. Prepare a 2.5 mg / mL D4F-melittin solution, add CTX-NP nanoparticle solution dropwise, incubate for 12 h, and purify by ultrafiltration to form chlorotoxin-melittin nanoparticles (CTX-melittin-NP).

[0054] Specifically, the melittin is D4F-melittin, which is composed of an alpha helical polypeptide (D4F), a connecting sequence GSG and melittin.

[0055] Specifically, ultrafiltration purification was performed using a low-speed refrigerated centrifuge at a temperature of 4° C. and a rotation speed of 2600 rpm.

[0056] Example 3

[0057] According to the preparation method of Example 1, after the CTX-NP nanoparticles and CTX-melittin-NP nanoparticles were prepared, the unbound CTX molecules in the system were removed by ultrafiltration using a low-speed refrigerated centrifuge, and their morphological characteristics, particle size, fluorescence intensity, serum stability and other properties were characterized in vitro. The results are as follows Figure 1 The results of transmission electron microscopy and dynamic light scattering (DLS) showed that the CTX-NP and CTX-melittin-NP nanoparticles were spherical, with average particle sizes of 80.78nm and 96.45nm, respectively. The measured zate potentials were -3.53mV and 0.77mV, respectively. Figure 1 AC); Fluorescence imaging was performed on CTX-NP and CTX-melittin-NP with different concentrations of lipophilic near-infrared imaging dye (DiR-BOA fluorescent dye). The results were ( Figure 1 D) shows that the fluorescence signal intensity of the nanoparticles decreases as the concentration of DiR-BOA decreases, indicating that the fluorescence signal intensity of the nanoparticles is highly correlated with the concentration of the nanoparticles.

[0058] In order to explore the feasibility of intravenous injection of nanoparticles, it is necessary to determine their stability in serum. The fluorescence signal intensity of CTX-NP and CTX-melittin-NP co-incubated with mouse serum at different times (12h, 24h) was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and overall fluorescence imaging. The results showed that at different time gradients, the nanoparticle groups co-incubated with mouse serum showed relatively consistent fluorescence signal intensity; while for the nanoparticle group after cleavage by Tween-20, the basic structure of the nanoparticle group was destroyed, and the DiR-BOA fluorescent dye wrapped in the hydrophobic core was released, causing the fluorescence signal to be dispersed throughout the lane, and the bands were diffusely distributed, indicating that the structures of CTX-NP nanoparticles and CTX-melittin-NP nanoparticles were still intact after incubation with serum, and they still maintained relatively good stability ( Figure 1 Middle E).

[0059] Example 4

[0060] Different groups of nanoparticle solutions (PBS buffer solution as solvent) were set up: NP nanoparticles, α-melittin-NP nanoparticles, CTX-NP nanoparticles, CTX-melittin-NP nanoparticles (all as treatment groups), and PBS buffer solution was used as the blank control group; 20 μM DiR-BOA fluorescent dye was added to the nanoparticle solution for subsequent experiments.

[0061] The preparation method of NP nanoparticles is:

[0062] S1. Weigh 4 mg of dimyristoylphosphatidylcholine (DMPC) into an EP tube, add 1 mL of chloroform solution, mix thoroughly, then add 26 μL of 10 mg / mL CO solution and 38.4 μL of 10 mg / mL phospholipid polyethylene glycol maleimide (DSPE-PEG2000-Mal) solution, mix thoroughly;

[0063] S2. The solution obtained in step S1 is dried with nitrogen gas, vacuum dried after forming a thin film, resuspended with PBS solution, and fully dissolved by ultrasonic vibration;

[0064] S3. Subsequently, it is ultrafiltered and purified to form NP nanoparticle solution.

[0065] The preparation method of α-melittin-NP nanoparticles is as follows:

[0066] S1. Weigh 4 mg of dimyristoylphosphatidylcholine (DMPC) into an EP tube, add 1 mL of chloroform solution, mix thoroughly, and then add 26 μL of 10 mg / mL CO solution. Mix thoroughly;

[0067] S2. The solution obtained in step S1 is dried with nitrogen gas, vacuum dried after forming a thin film, resuspended with PBS solution, and fully dissolved by ultrasonic vibration;

[0068] S3. Prepare a 2 mg / mL or 2.5 mg / mL D4F-melittin solution, add it dropwise to the nanoparticle solution dissolved by ultrasound, incubate at 4°C for 12 h, and purify by ultrafiltration to form α-melittin nanoparticles (α-melittin-NP).

[0069] 1. Confocal imaging experiment after incubation of chlorotoxin-melitin nanoparticles with GL261 and CT26 cells

[0070] The nanoparticle solutions of different groups were incubated with GL261 and CT26 cells for 1.5 hours and then confocal imaging was performed. Figure 2 as shown; Figure 2 As shown in B and D, compared with the NP group without melittin killing drug, the CTX-NP group detected a stronger fluorescence signal in the cells, indicating that CTX-NP has a better ability to target GL261 cells; in CT26 cells with low expression of matrix metalloproteinase 2 (MMP2), the amount of CTX-NP taken up was less, and the intensity of the DiR-BOA fluorescence signal in the cells was weaker, indicating that the amount of CTX nanoparticles taken up by tumor cells is determined by the difference in the expression of CTX-specific receptor MMP2. The nanoparticles in the α-melittin-NP and CTX-melittin-NP groups are modified with melittin polypeptide, which has the function of punching holes in the cell membrane and directly killing tumor cells. Figure 2 As shown in Figures C and E, although some CT26 cells have a certain uptake of α-melittin-NP and CTX-melittin-NP (due to the non-specific uptake caused by the melittin peptide puncturing some cells), the fluorescence signal in GL261 cells is still significantly higher than that in the CT26 group. Figure 2 Middle (F) shows that 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.

[0071] 2. Experiment on the killing ability of chlorotoxin-melitin nanoparticles on brain glioma cells

[0072] Different concentrations of CTX-melittin-NP (melittin concentration: 2.5μM, 5μM, 7.5μM, 10μM, 25μM, 50μM, 100μM) were co-incubated with GL261 cells for 3 hours, and then the cell medium was replaced. After culturing for 24 hours, the survival rate of GL261 cells was detected by MTS kit. Figure 3 As shown in the figure, when the concentration of melittin peptide loaded in CTX-melitn-NP is 10μM, it can effectively kill nearly 80% of GL261 cells; when the concentration reaches 25μM, about 90% of GL261 cells die. The above results show that CTX-melittin-NP has the ability to effectively kill brain glioma cells, laying the foundation for the subsequent in vivo treatment of brain glioma experiments.

[0073] C57BL / 6 mice were selected and glioma cells were inoculated intracranially. On the 10th day after intracranial inoculation of glioma cells, 150 μL of NP, α-melittin-NP, CTX-NP and CTX-melittin-NP nanoparticle solutions were injected intravenously. To visualize the subsequent signals of each nanoparticle and compare the signal intensity of each nanoparticle, the amount of DiR-BOA loaded on each nanocarrier was kept constant (40 nmol). The mice were dissected 24 hours after the nanoparticle injection, and the in vitro tissue detection was performed by whole-body fluorescence imaging to observe the intensity and distribution of the nanoparticle signal and compare the aggregation effects of different nanoparticles in the brain glioma area. The whole-body fluorescence imaging results showed that obvious tumor signals could be observed in the brain area of ​​each group of mice, indicating that the mouse brain glioma model was successfully established. Further detection of the DiR-BOA signal of the nanoparticles showed that the signal was not observed in the brain glioma area of ​​the intravenously injected NP and α-melittin-NP nanoparticle groups, while for the CTX-NP and CTX-melittin-NP nanoparticle groups modified with CTX, DiR-BOA signals of different intensities were observed in the brain glioma area, that is, the nanoparticles modified with CTX were obviously gathered in the brain glioma area. This shows that modifying CTX on the nanoparticles can significantly enhance the ability of the nanoparticles to pass through the blood-brain barrier and target brain gliomas ( Figure 4 ).

[0074] 3. Experiment on the effect of chlorotoxin-melitin nanoparticles on the in situ growth of brain glioma

[0075] A brain stereotaxic instrument was used to construct an in situ model of mouse brain glioma. On the 10th, 13th, 16th, and 19th days of glioma growth, 150 μL of PBS, NP, α-melittin-NP, CTX-NP, and CTX-melittin-NP were injected into different groups, respectively (melittin: 0.4 mg / time; CTX: 50 μg / time; reaching this dose can achieve a killing effect); then on the 22nd day, the mouse brain was dissected and taken for whole-body fluorescence imaging, and the signal of mCherry fluorescent protein obtained by imaging was used to characterize the tumor signal (red fluorescent dye mCherry fluorescent protein: Ex: 562 / 40nm, Em: 624 / 40nm). The imaging results are shown in Figure 2. Figure 5 Figure A shows that compared with the other treatment groups, the mCherry fluorescence intensity representing the tumor signal in the brain of mice in the CTX-melittin-NP treatment group was weaker and the area was smaller, indicating that the growth of tumors in this group was inhibited. The ratio of the tumor area to the overall brain area was calculated, and the quantitative results were shown in Figure 2. Figure 5 As shown in B, the tumor area ratio of the CTX-melittin-NP group was significantly lower than that of the other treatment groups, and the inhibition rate was 94.9% compared with the PBS group, indicating that intravenously injected CTX-melittin-NP can effectively inhibit the growth of orthotopic brain gliomas in vivo.

[0076] Effects of chlorotoxin-melittin nanoparticles on cell infiltration in the brain glioma microenvironment

[0077] 1. Labeling CD3 by immunofluorescence technique + T cells, to investigate the CD3 + Changes in T cell infiltration (results such as Figure 6 According to the confocal imaging results, more CD3 + T cells, with a cell density of 1160 cells / mm 2 , about PBS group (389 cells / mm 2 ) was 3.0 times that of the NP group (867 cells / mm 2 ) was 1.3 times that of the α-melittin-NP group (927 cells / mm 2 ) was 1.3 times that of the CTX-NP group (843 cells / mm 2 ) is 1.4 times (e.g. Figure 6 (middle D).

[0078] 2.By using B220 antibody to label B cells in the immune microenvironment of brain glioma ( Figure 7 Confocal imaging results showed that B cell infiltration in the core of mouse glioma increased after CTX-melittin-NP treatment, with a cell density of 52 cells / mm 2 , about PBS group (18 cells / mm 2 ) was 2.4 times that of the NP group (29 cells / mm 2 ) was 1.5 times that of the α-melittin-NP group (19 cells / mm 2 ) was 2.7 times that of the CTX-NP group (17 cells / mm 2 ) is 3.0 times ( Figure 7 (middle D).

[0079] 3. Antibody labeling of macrophages carrying F4 / 80 antigen on the surface of mouse brain slices was performed by immunofluorescence to explore the distribution of macrophages in the microenvironment of brain glioma ( Figure 8 Confocal microscopy results showed that a large number of F4 / 80-labeled macrophages were distributed in the microenvironment of the mouse glioma after treatment with CTX-melittin-NP, with a cell density of 1605 cells / mm 2 , about PBS group (668 cells / mm 2 ) was 2.4 times that of the NP group (1074 cells / mm 2 ) was 1.5 times that of the α-melittin-NP group (1184 cells / mm 2 ) was 1.4 times that of the CTX-NP group (841 cells / mm 2 ) is 1.9 times ( Figure 8 In the CTX-melittin-NP group, the percentage of macrophages engulfing tumor cells was 67%, which was 2.3 times, 1.9 times, 1.7 times, and 2.3 times that of the PBS group (29%), the NP group (36%), the α-melittin-NP group (41%), and the CTX-NP group (29%). Figure 8 The above results indicate that a large number of macrophages were recruited into the microenvironment of the mouse glioma after treatment and phagocytosed tumor cell fragments, which has a certain promoting effect on the treatment of glioma.

[0080] 4. Biosafety Verification of Chlorotoxin-Melittin Nanoparticles

[0081] Different groups of nanoparticle solutions were administered to experimental mice (intravenous injection); 72 hours after the last administration, the mice were anesthetized with Avertin anesthetic and then dissected, and the heart, liver, spleen, lung, kidney and other organs were collected for polyformaldehyde (PFA) fixation and hematoxylin-eosin (HE) staining sections. Hematoxylin stained the cell nucleus and eosin stained the cytoplasm. By comparing the tissue sections of the control group and the treatment group, it was evaluated whether there was obvious cell damage, necrosis, inflammatory infiltration, fibrosis or other abnormal morphological changes. The prepared sections were imaged using an upright section scanning microscope, such as Fig. 9 As shown, it can be observed that there is no obvious damage to the organs of mice after CTX-melittin-NP treatment, indicating that CTX-melittin-NP has good biosafety.

[0082] Principle: The specific targeting of CTX to tumors is closely related to matrix metalloproteinase 2 (MMP2) and chloride channel 3 (CLC3) expressed on the surface of tumor cells. Among them, CLC3 participates in cytoskeleton reorganization, thereby regulating cell morphology and movement during cell migration. MMP2 is a member of the metalloproteinase family, which 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. On the one hand, CTX has the ability to target gliomas, and on the other hand, it has the ability to cross the blood-brain barrier. The reason may be that it has a small molecular weight and a compact structure, which makes it easy to pass through the blood-brain barrier and the narrow extracellular matrix. On the other hand, it may be because 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 brain microvascular endothelial cells, which are the main components of the blood-brain barrier, thereby promoting receptor-mediated transcytosis of endothelial cells. There are also studies that have found that the ability of CTX to cross the blood-brain barrier can be effectively inhibited by treating brain microvascular endothelial cells with clathrin inhibitors.

[0083] In the present invention, chlorotoxin CTX with glioma cell MMP2 receptor specificity is combined with bee venom peptide nanoparticles α-melittin-NP that have previously shown good anti-tumor and immunomodulatory effects to form lipid nanoparticles (CTX-melittin-NP) co-modified with chlorotoxin and bee venom peptide. In summary, the chlorotoxin-melittin peptide nanoparticles CTX-melittin-NP prepared by the present invention can achieve a "one stone, three birds" strategy of crossing the blood-brain barrier, targeted killing of brain gliomas, and reversing the immunosuppressive microenvironment of gliomas to an immune-activating type.

[0084] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0085] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Chlorotoxin-melittin nanoparticles, characterized in that: Prepared by the following method: S1. Thiolation treatment of chlorotoxin: weigh chlorotoxin and dissolve it in PBS buffer solution to obtain a chlorotoxin solution; add 2-iminothiolane hydrochloride to the chlorotoxin solution, mix well, and incubate at room temperature to form a CTX-SH solution; S2. Weigh dimyristoylphosphatidylcholine, add chloroform solution, mix thoroughly, then add cholesterol ester solution, phospholipid-polyethylene glycol-maleimide solution, mix thoroughly; S3. The solution obtained in step S2 is dried, resuspended with PBS solution, and fully dissolved by ultrasonic vibration; S4. adding the thiolated CTX-SH solution to the solution of step S3, incubating for 1 to 3 days, and purifying by ultrafiltration to form a CTX-NP nanoparticle solution; S5. Prepare a melittin solution, add the CTX-NP nanoparticle solution dropwise, incubate for 10 to 24 hours, and purify by ultrafiltration to form chlorotoxin-melittin nanoparticles.

2. The chlorotoxin-melittin nanoparticles according to claim 1, characterized in that: In the step S1, the molar ratio of the chlorotoxin solution to the 2-iminothiolane hydrochloride solution is 10:

1.

3. The chlorotoxin-melittin nanoparticles according to claim 2, characterized in that: The incubation time in step S1 is ≥ 1 h.

4. The chlorotoxin-melittin nanoparticles according to claim 3, characterized in that: In step S2, the amount of dimyristoylphosphatidylcholine added is 3-5 mg, the amount of chloroform solution added is 0.8-1.5 mL, the concentration of cholesterol ester solution is 8-15 mg / mL, and the concentration of phospholipid-polyethylene glycol-maleimide solution is 8-15 mg / mL.

5. The chlorotoxin-melittin nanoparticles according to claim 4, characterized in that: The drying in step S3 is nitrogen drying or vacuum drying.

6. The chlorotoxin-melittin nanoparticles according to claim 5, characterized in that: The incubation time in step S4 is 2 days.

7. The chlorotoxin-melittin nanoparticles according to claim 6, characterized in that: The concentration of the melittin solution in step S5 is 1.5-2.5 mg / mL, and the incubation time is 12 h.

8. The chlorotoxin-melittin nanoparticles according to claim 7, characterized in that: The PBS buffer solution contains 5 mM ethylenediaminetetraacetic acid.

9. The chlorotoxin-melittin nanoparticles according to claim 8, characterized in that: The chlorotoxin-melitin nanoparticles are spherical, and the average particle size is 80-100nm.

10. Use of the chlorotoxin-melittin nanoparticles according to any one of claims 1 to 9 in the preparation of glioma targeted drugs.

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