CN113750047A

CN113750047A - Multifunctional nano liposome, preparation method and application

Info

Publication number: CN113750047A
Application number: CN202010465849.1A
Authority: CN
Inventors: 魏启春; 韩旻; 陈婕键; 铃木实; 王冬春; 耿长冉
Original assignee: China Boron Xiamen Medical Equipment Co ltd; Nanjing University of Aeronautics and Astronautics; Zhejiang University ZJU; Kyoto University NUC
Current assignee: China Boron Xiamen Medical Equipment Co ltd; Nanjing University of Aeronautics and Astronautics; Zhejiang University ZJU; Kyoto University NUC
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2021-12-07

Abstract

The invention provides a multifunctional nano liposome and a preparation method thereof, wherein the multifunctional nano liposome comprises adriamycin, carborane, cationic liposome, plasmid and polypeptide. The multifunctional liposome can realize boron neutron capture-immune-chemotherapy combined treatment, can play a role in radiotherapy sensitization of BNCT by knocking out glioma CD47, and is a combined treatment nano drug delivery system with great application prospect.

Description

Multifunctional nano liposome, preparation method and application

Technical Field

The invention belongs to the field of medicines, and particularly relates to a multifunctional nano liposome, a preparation method and application thereof in brain glioma boron neutron capture-immunization-chemotherapy combined treatment.

Background

Malignant glioma is the most common tumor of the central nervous system, and the current standard treatment is radiotherapy, synchronous temozolomide chemotherapy and auxiliary chemotherapy after tumor resection safely in the largest range. The median survival time for newly diagnosed grade III and IV gliomas is 12-24 months and 8-15 months, respectively. It has been reported that the median survival of glioblastoma is 14.6 months, and the 5-year survival rate is only 9.8%. The traditional tumor combination treatment mode mainly comprises sequential administration of a plurality of different therapeutic drugs, but the administration mode has a large burden for doctors and patients. Most of malignant gliomas recur after treatment, and the recurrent malignant gliomas further lack effective treatment means, so that a new treatment strategy needs to be explored.

Boron Neutron Capture Therapy (BNCT), currently used mainly for the treatment of brain gliomas and melanomas, has proven to be a promising approach for the treatment of gliomas. In Swedish clinical research, the median survival time of a glioblastoma operation which is treated by BNCT only is 14.2 months, the normal radiotherapy dosage of 20-30 Gy is obtained after BNCT treatment by Japanese institute of medical university, and the median survival time reaches 23.5 months, so that the result is encouraging. However, at the present stage, the single or combined BNCT cannot realize revolutionary breakthrough, and a new strategy of combined treatment needs to be explored.

Disclosure of Invention

The invention aims to provide a multifunctional nano liposome, a preparation method and application thereof in boron neutron capture-immune-chemotherapy combined treatment.

In a first aspect of the present invention, a multifunctional nanoliposome is provided, which comprises adriamycin, carborane, cationic liposome, plasmid and polypeptide.

In another preferred embodiment, the cationic liposome and plasmid have a charge ratio of greater than 2, preferably 2-50, more preferably 2-10.

In another preferred embodiment, the cationic liposome comprises (2, 3-dioleoxypropyl) trimethylammonium chloride, 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine, cholesterol, and distearoylphosphatidylacetamide-polyethylene glycol 5000.

In another preferred embodiment, the cationic liposome comprises (2, 3-dioleoxypropyl) trimethyl ammonium chloride, 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine, cholesterol and distearoyl phosphatidyl acetamide-polyethylene glycol 5000 in a molar ratio of 1: 1-3: 0.5-2: 0.02-0.05; preferably 0.8:1:0.5: 0.023.

Further, the functional nanoliposome can be used for boron neutron capture therapy, and boron in carborane is¹⁰B。

In a second aspect of the present invention, there is provided a method for preparing the multifunctional nanoliposome according to the first aspect, the method comprising the steps of:

(i) dissolving adriamycin in anhydrous dimethyl sulfoxide, adding triethylamine and 1-bromomethyl o-carborane under the protection of nitrogen, stirring at room temperature, and adding excessive diethyl ether to completely precipitate; collecting the precipitate, dialyzing with dimethyl sulfoxide and distilled water as dialysate, and lyophilizing under reduced pressure to obtain adriamycin boron cage compound;

(ii) dissolving the adriamycin boron cage compound, (2, 3-dioleoxypropyl) trimethyl ammonium chloride, 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine), cholesterol and distearoyl phosphatidyl acetamide-polyethylene glycol 5000 in chloroform, and removing the organic solvent to form a uniform film; adding a sucrose aqueous solution (containing diethyl pyrocarbonate) into the film for hydration treatment to form uniform emulsion, and filtering after ultrasonic treatment to obtain a drug-loaded cationic liposome;

(iii) mixing and incubating the drug-loaded cationic liposome solution and the plasmid to obtain a drug-loaded cationic liposome/plasmid complex;

(iv) and mixing the drug-loaded cationic liposome/plasmid compound with iRGD polypeptide to obtain the multifunctional nano-liposome.

In another preferred embodiment, the charge ratio of the drug-loaded cationic liposome and the plasmid is greater than 2, preferably 2-50, more preferably 2-10.

In another preferred embodiment, the carborane contains boron¹⁰B, preferably containing a content of 95% or more¹⁰B。

In another preferable example, the molar ratio of the adriamycin, the triethylamine and the 1-bromomethyl o-carborane is 1: 1-3.

In another preferred embodiment, the drug-loaded cationic liposome is obtained by filtration through a 0.22 μm water membrane in said step (ii).

In another preferred embodiment, the molar ratio of the iRGD polypeptide to the nanoliposome is 1-10%, preferably 5%.

In another preferred embodiment, the (2, 3-dioleyloxypropyl) trimethylammonium chloride: 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine): cholesterol: the molar ratio of distearoyl phosphatidyl acetamide-polyethylene glycol 5000 is 1: 1-3: 0.5-2: 0.02-0.05, preferably 0.8:1:0.5: 0.023.

In a third aspect of the invention, there is provided a use of the multifunctional nanoliposome according to the first aspect in the preparation of a medicament for boron neutron capture therapy, specifically a medicament for boron neutron-immune-chemotherapy combination therapy, especially a medicament for boron neutron capture-immune-chemotherapy combination therapy for glioma.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 is a schematic view of the multifunctional nanoliposome of the present invention.

FIG. 2 shows the variation of particle size and surface potential of cationic liposome/plasmid complex with different N/P ratio.

FIG. 3 is a graph showing the particle size distribution of liposomes (DOX-CB @ lipo-pDNA) prepared in example.

FIG. 4 is an electron micrograph of the particle size of liposomes (DOX-CB @ lipo-pDNA) prepared in example.

FIG. 5 is a diagram of agarose gel electrophoresis blocking experiments of cationic liposome/plasmid complexes with different N/P ratios.

FIG. 6 shows the viability of liposomes prepared in the examples with different N/P ratios after 24 hours incubation with GL261 cells.

FIG. 7 shows the cell viability of liposome lipo2000 complexes incubated with GL261 cells for 24 hours at different N/P ratios.

FIG. 8 is a fluorescence plot of GL261 cells after 48 hours of transfection with different transfection agents.

Fig. 9 is a graph comparing transfection efficiency after transfection of GL261 cells with different agents,. P < 0.001.

Fig. 10 shows CD47 expression levels of G261 cells after 72 hours of transfection with different agents, P <0.05 and P < 0.01.

FIG. 11 is a graph of confocal fluorescence of different preparations incubated with macrophages for 8 hours after transfection of GL 26172 hours.

FIG. 12 shows fluorescence profiles of ex vivo (a) and major organs (b) 24 hours after injection of plasmid/polypeptide complex DiR @ lipo-iRGD into tail vein of in situ-loaded GL261 tumor-bearing mice, and (c) after brain tissue dissection.

FIG. 13 is an ex vivo fluorescence profile of free DiR (a, c) or DiR @ lipo (b, d) injected in tail vein of GL261 tumor-bearing mice in situ for in vivo and major organs.

Figure 14 is a graph of survival curves for groups of mice after treatment.

Detailed Description

The inventor provides a multifunctional nano liposome for boron neutron capture-immune-chemotherapy combination treatment through extensive and intensive research. The invention constructs a multifunctional nano liposome drug delivery system by combining CD47 blocking immunotherapy on the basis of a self-synthesized novel boron capture agent with cell nucleus tropism, and explores the boron neutron capture-immunity-chemotherapy combined treatment effect in an in-situ glioma animal model. In the experiment, the realization of immunotherapy is mainly based on the targeted delivery capability of the iRGD mixed liposome, and the CRISPR/Cas9 gene knockout plasmid aiming at the CD47 gene is delivered to tumor tissue cells as target as possible. Because normal tissue cells lack a series of 'eat me' signals specific to tumor cells, even if the 'eat me' signals of the normal tissue cells are reduced, strong immune response is relatively difficult to initiate. The nano-liposome platform is utilized to simultaneously realize the boron neutron capture-immunization-chemotherapy combined treatment, the survival rate of tumor-bearing mice after the treatment is high, the curative effect is clear, and the nano-liposome platform is a combined treatment nano drug delivery system with great application prospect. On the basis of this, the present invention has been completed.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. All patents, patent applications, and publications cited herein are incorporated by reference in their entirety unless otherwise indicated.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter claimed. In this application, the use of the singular also includes the plural unless specifically stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the use of "or", "or" means "and/or" unless stated otherwise. Furthermore, the term "comprising" as well as other forms, such as "includes," "including," and "containing," are not limiting.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals, and treatises, are hereby incorporated by reference in their entirety.

In addition to the foregoing, the following terms, when used in the specification and claims of this application, have the meanings indicated below, unless otherwise specifically indicated.

In this application, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

In the present application, CB is 1-bromomethyl orthocarborane, lipo represents cationic liposome, DOX-CB @ lipo represents drug-loaded cationic liposome, lipo-pDNA represents cationic liposome/plasmid complex, DOX-CB @ lipo-pDNA represents drug-loaded cationic liposome/plasmid complex, DOX-CB @ lipo-iRGD represents drug-loaded cationic liposome/polypeptide complex, lipo-pDNA-iRGD represents cationic liposome/plasmid/polypeptide complex, and DOX-CB @ lipo-pDNA-iRGD represents multifunctional nanoliposome according to the first aspect of the present invention. The term BSH is mercaptoundecanediodecadiborate.

As used herein, the term "treatment" and other similar synonyms include the following meanings:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease or condition, but has not been diagnosed as having the disease or condition;

(ii) inhibiting the disease or disorder, i.e., arresting its development;

(iii) alleviating, i.e., resolving the state of, the disease or condition; or

(iv) Alleviating the symptoms caused by the disease or disorder.

The main advantages of the invention include:

the multifunctional nano-liposome has the advantages of simple preparation process, good repeatability, uniform dispersion of liposome/plasmid compound, small particle size (150 nm) and good capability of compressing nucleic acid when the N/P ratio is more than 2. The particle size of the prepared multifunctional liposome is 145.63 +/-1.42 nm, the potential is 2.32 +/-0.14 mV, and the multifunctional liposome is a spheroidal particle under a transmission electron microscope; the killing effect on GL261 cells is obviously lower than that of commercial liposome lipo2000, and the gene recombination efficiency and the transfection efficiency are both superior to that of lipo2000 and PEI. The prepared multifunctional nano liposome has cytotoxicity less than lipo2000-pD NA series compound, and embodies the advantage of relative safety of the constructed multifunctional nano liposome. GL261 cells have reduced expression of CD47 after transfection with multifunctional liposome and promote phagocytosis thereof by macrophages. The multifunctional liposome shows good brain targeting characteristics on a GL261 in-situ brain glioma mouse model, the survival rate of tumor-bearing mice after treatment is high, and the multifunctional nano liposome has a clear synergistic anti-brain glioma effect after combined treatment of a combined nucleus drug delivery driving efficient neutron capture effect and chemotherapy, so that the multifunctional nano drug delivery system has an application prospect. As a novel nano-carrier, the radiation sensitization effect of BNCT can be exerted by knocking out glioma CD 47.

The present invention is further illustrated by the following examples. It is to be understood that the following description is only of the most preferred embodiments of the present invention and should not be taken as limiting the scope of the invention. In the following examples, the experimental methods without specific conditions, usually according to the conventional conditions or according to the conditions suggested by the manufacturers, can be modified by those skilled in the art without essential changes, and such modifications should be considered as included in the protection scope of the present invention. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

EXAMPLE 1 preparation of multifunctional Nanosiliposomes

Preparation of empty cationic liposomes (lipo):

accurately weighing phospholipid material and cholesterol according to the molar ratio of DOTAP to DOPE to CHOL to DSPE-PEG5000(2, 3-dioleoxypropyl) trimethyl ammonium chloride: 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine): cholesterol: distearoyl phosphatidyl acetamide-polyethylene glycol 5000 ═ 0.8:1:0.5:0.023, total mass about 30mg, dissolved in chloroform, water bath ultrasonic assisted dissolution, rotary evaporation for 2h under 40 ℃ negative pressure condition, organic solvent is thoroughly removed, and a uniform film is formed.

Preparing 5% sucrose aqueous solution (containing DEPC diethyl pyrocarbonate), preheating to 50 ℃, and adding 5mL of the aqueous solution into the film in the previous step for hydration treatment to form uniform emulsion. Performing water bath ultrasonic treatment for 30min, performing ultrasonic treatment (250W) for 3min to obtain clear and transparent liquid, and filtering the solution with 0.22 μm water membrane for 2 times to obtain no-load cationic liposome (lipo) solution.

Preparation of drug-loaded cationic liposome (DOX-CB @ lipo):

dissolving doxorubicin DOX in anhydrous dimethyl sulfoxide, adding triethylamine under the protection of nitrogen, stirring for 2 hours, adding 1-bromomethyl o-carborane CB, stirring for 48 hours at room temperature, and adding excessive diethyl ether to completely precipitate; the molar ratio of the adriamycin to the triethylamine to the 1-bromomethyl o-carborane is 1:2: 1; collecting the precipitate, dialyzing with dimethyl sulfoxide as dialysate for 24 hr, dialyzing with distilled water as dialysate for 48 hr, and lyophilizing under reduced pressure to obtain DOX-CB.

Weighing 3mg of adriamycin boron cage compound DOX-CB; and weighing about 30mg of phospholipid and cholesterol (the molar ratio of the components is DOTAP: DOPE: CHOL: DSPE-PEG5000 is 0.8:1:0.5:0.023), completely dissolving the phospholipid and the cholesterol in trichloromethane, performing ultrasonic dissolution in a water bath, performing rotary evaporation for 2 hours at the temperature of 40 ℃ under the negative pressure condition, and completely removing the organic solvent to form a uniform film.

Preparing 5% sucrose aqueous solution (containing diethyl pyrocarbonate DEPC), preheating to 50 ℃, and adding 5mL of the aqueous solution into the film in the previous step for hydration treatment to form uniform emulsion. Performing water bath ultrasound for 30min, performing ultrasonic treatment (250W) on the probe for 4min to form red transparent liquid, and finally filtering the solution for 2 times by using a 0.22 mu m water film to obtain the drug-loaded cationic liposome (DOX-CB @ lipo).

Plasmid extraction and purification:

in the experiment, the

Plasmid DNA was extracted and purified using the hipore plasmid maxiPrep Kit. The method comprises the following specific steps: 1) preparing 200mL of liquid culture medium of the plum bacterium enrichment Broth (Luria-Bertani Broth), sterilizing, and adding ampicillin (with a final concentration of 100 μ g/mL) when the temperature of the culture medium is reduced to about 65 ℃; 2) extracting 100 μ L of glycerol bacterial liquid, adding into LB culture medium, shake culturing at 37 deg.C for 12h, centrifuging at 12000g for 3min, and removing supernatant; 3) adding 10mL of a resuspension buffer solution (Resuspensionbuffer) solution (containing RNaseA), and oscillating the suspended thallus for precipitation, wherein no visible bacterium block is ensured in the process; 4) adding 10mL of blue solution LB, turning and mixing the solution up and down for 6 times in a mild way, fully cracking the thalli, standing the solution for 5min, and changing the color of the solution from semi-transparent to transparent blue solution to prompt complete cracking; 5) adding 14mL of yellow solution NB, carefully mixing for 4-6 times until a compact yellow coagulated floc is formed, and standing for 3min at room temperature; 6) centrifuging at 12000g for 15min, carefully sucking supernatant, adding into centrifugal column, centrifuging at 8000g for 3min, and discarding eluate; 7) adding 5mL of eluent, standing at room temperature for 10min, centrifuging at 8000g for 3min, and discarding the effluent; 8) adding 5mL of Washing Buffer solution (Washing Buffer), standing at room temperature for 1min, centrifuging at 8000g for 2min, and discarding the effluent; 9) then adding 5mL of WB solution, centrifuging for 2min at the rotating speed of 8000g, and discarding the effluent liquid; 10) centrifuging at 8000g for 5min to completely remove residual washing buffer solution; 11) standing at room temperature for 10min to completely volatilize ethanol in the centrifugal column; 12) placing the centrifugal column in a clean 50mL centrifugal tube, adding 1mL elution buffer solution (preheating treatment at 70 ℃) into the central area of the centrifugal column, and standing for 10min at room temperature; 13) centrifuging at 8000g rotation speed for 3min, eluting DNA, collecting part of DNA, testing nucleic acid purity and concentration on Nanodrop machine, and storing the rest part at-20 deg.C.

Preparation of cationic liposome/plasmid Complex (DOX-CB @ lipo-pDNA):

and (3) diluting the plasmid with DEPC water, mixing with the empty-load cationic liposome solution (containing DEPC) with the same volume, continuously blowing for 1min, and standing at room temperature for 20min to obtain the cationic liposome/plasmid complex (lipo-pDNA).

Similarly, after the plasmid solution and the drug-loaded cationic liposome solution (containing DEPC) are mixed and incubated in equal volume, the corresponding drug-loaded cationic liposome/plasmid complex (DOX-CB @ lipo-pDNA) can be prepared.

Preparing a multifunctional nano liposome (DOX-CB @ lipo-pDNA-iRGD):

accurately weighing iRGD, preparing into aqueous solution, mixing with the prepared nano liposome solution uniformly, standing at room temperature for 30min to obtain corresponding multifunctional nano liposome (lipo-iRGD, lipo-pDNA-iRGD, DOX-CB @ lipo-iRGD and DOX-CB @ lipo-pDNA-iRGD), wherein the molar ratio of the iRGD polypeptide to the nano liposome is 5%.

Example 2 multifunctional Nanosomal characterization Studies

According to the above operation steps, cationic liposome/plasmid complexes (lipo-pDNA) with different ratios of cationic liposome and plasmid, namely N/P ratios (0, 1,2, 3, 4, 5, 10, 20, 30, 40 and 50) are prepared, characteristic parameters such as particle size (size), potential (zeta) and polymer dispersion coefficient (PDI) of each sample are measured on a particle size and surface potential measuring instrument, all experiments are carried out 3 times in parallel, and the influence of the N/P ratio on the main characteristic parameters of the complexes is examined.

As shown in Table 1, the N/P ratio of 0 represents a pure plasmid solution, and the plasmid is a double-stranded DNA nucleic acid structure containing a large amount of negatively charged phosphate groups and having a potential of-25.25. + -. 12.63mV, and a particle size of 562.97. + -. 40.24nm, which is probably due to the tendency of the particles in solution to aggregate to form large particles. When the N/P ratio is 1, the particle size of the composite is 225.25 +/-43.21 nm, and the surface potential is converted into positive electricity (2.41 +/-0.82 mV); when the N/P ratio is more than 2, the particle size of the composite is basically less than 150nm, and the surface potential is electropositive. FIG. 2 is a graph showing the particle size and surface potential of cationic liposome/plasmid complex (lipo-pDNA) under different N/P ratio conditions, and it can be found that the N/P ratio is 2, which is basically the inflection point of the complex, when the particle size of the complex is smaller and the potential is stable.

Preparing aqueous solution of nanoliposome (DOX-CB @ lipo, DOX-CB @ lipo-iRGD, lipo, DOX-CB @ lipo-pDNA and DOX-CB @ lipo-pDNA-iRGD, wherein the N/P of liposome compound containing plasmid is 2), using a particle size and surface potential tester to machine, respectively testing the characteristic parameters of particle size, potential, polymer dispersion coefficient and the like of each sample, and testing 3 times in parallel.

As shown in Table 2, the average particle size of several liposomes was 100-150 nm, the polydispersity was small, and the liposomes were relatively stable. When the drug-loaded cationic liposome solution is directly mixed with the iRGD polypeptide, the particle size of the prepared drug-loaded liposome/polypeptide compound DOX-CB @ lipo-iRGD is 138.13 +/-2.05 nm, the measured potential value is 0.23 +/-0.31 mV at the moment, and the particle size is almost neutral, so that the mixed interaction of the iRGD polypeptide and the liposome is proved, the abundant positive charges on the surface of the liposome can be effectively shielded, the feasibility of in vivo administration is greatly improved, and the influence on the whole particle size of the compound is small. In a liposome complex (DOX-CB @ lipo-pDNA-iRGD) carrying drugs and plasmids, the liposome complex is also found to be almost neutral in electricity (2.32 +/-0.14 mV), the particle size is only 145.63 +/-1.42 nm, the particles are uniformly dispersed (PDI is 0.176 +/-0.007), and the interaction between the iRGD polypeptide and the liposome is demonstrated again, so that abundant positive charges on the surface of the liposome can be obviously shielded, and the influence on the overall particle size of the complex is small.

FIG. 3 shows that the drug-loaded cationic liposome is further mixed with plasmid to form complex DOX-CB @ lipo-pDNA, and the particle size of the liposome is slightly larger (133.57 +/-3.11 nm) due to the plasmid wrapping, and the distribution is uniform. And (3) dropwise adding the DOX-CB @ lipo-pDNA liposome complex solution on a copper net for adsorbing for several minutes, adsorbing excessive water by using filter paper, fully drying, and observing the appearance under a transmission electron microscope. As shown in FIG. 4, a number of spheroidal particles were seen under the mirror, with a particle size of about 150 nm.

TABLE 1 cationic liposome/plasmid Complex particle size and surface potential profiles with different N/P ratios

TABLE 2 particle size and surface potential of several liposomes prepared (N/P is 2)

EXAMPLE 3 agarose gel electrophoresis retardation experiment

The binding ability of the cationic liposome vector to plasmid DNA can be judged by agarose gel electrophoresis experiments. Firstly, preparing 1% TAE agarose solution (agarose concentration is 2%), heating in a microwave oven to aid dissolution, adding a small amount of dye Gel Red (Gel Red) (the final concentration is 0.1%) after the temperature of the solution is reduced, preparing agarose Gel on a mould, carefully placing the agarose Gel on an electrophoresis tank, and adding TAE electrophoresis buffer until the agarose Gel is submerged.

According to the above operation steps, a series of cationic liposome/plasmid complexes (lipo-pDNA) with different N/P ratios (0.2, 0.4, 1, 1.6 and 2) are prepared, 4. mu.L of Loading buffer and 20. mu.L of liposome/plasmid complexes are repeatedly blown and uniformly mixed to form a solution with a total volume of 24. mu.L, and a DNAmarker group (M) and a pure plasmid group (N) are additionally set as controls. Carefully adding each sample into each sample groove of the agarose gel, replacing one sample adding head after adding one sample to prevent pollution, and simultaneously paying attention to the fact that the sample does not leak. After the sample adding is finished, electrifying for electrophoresis, setting the voltage to be 100V, slowly moving the sample from the negative electrode (black electrode end) to the positive electrode (red electrode end), stopping electrophoresis when bubbles appear in the electrophoresis buffer solution, and observing DNA under a gel imaging system when bromophenol blue moves to a position of about 1cm below the gel.

The agarose gel electrophoresis blocking experiment result is shown in figure 5, the cationic liposome/plasmid compound has DNA electrophoresis blocking phenomenon already when the N/P ratio is 1.6, and the phenomenon is more obvious when the N/P ratio is 2, which shows that under the condition that the N/P ratio is more than 1.6, the cationic liposome/plasmid compound can effectively compress and protect carried plasmid DNA, and is beneficial to in vivo and in vitro transfection.

Example 4 cytotoxicity Studies

GL261 cells in logarithmic growth phase were inoculated onto sterile 96-well cell culture plates (no cells were seeded on the outer periphery of the well plate, 200. mu.L of sterile PBS phosphate buffered saline (phosphate buffer saline) was added to each well) at a rate of 3X 103 cells per well, and the plates were placed in a cell incubator and incubated at 37 ℃ under 5% CO2 for 24 hours. The old culture was removed and 100. mu.L of fresh culture containing liposome/plasmid complexes (lipo-pDNA, lipo-pDNA-iRGD, DOX-CB @ lipo-pDNA and DOX-CB @ lipo-pDNA-iRGD) were added, respectively, and the N/P ratios of the liposome/plasmid complexes of each group were set up with a series of gradients (0.4, 1, 1.6, 2, 4, 10, 20 and 40). Liposome lipo2000(Life Technologies) was used as an experimental control, and liposomes/plasmid complex cultures were configured at different N/P ratios according to the procedures described in the product instructions, and the distribution was expressed as lipo2000-pDNA (2), lipo2000-pDNA (3), lipo2000-pDNA (4), and lipo2000-pDNA (5), and were incubated with GL261 cells for co-culture.

Placing the Cell culture plate after adding the medicine into a Cell culture box, incubating and culturing for 24h, sucking out the culture solution containing the medicine, adding the culture solution containing 10% CCK8 Cell Counting Kit-8 and the Cell proliferation and toxicity detection Kit, incubating for 2h, and shaking for 5 min. And finally, measuring the absorbance value (OD value) on an enzyme-labeling instrument, wherein the wavelength is 450nm, and each concentration of the drug is provided with 6 compound holes. The in vitro cytotoxicity of each preparation was evaluated by calculating the cell viability by the following formula:

cell survival rate ═ (a-B)/(C-B) × 100%

Wherein A represents the absorbance value of the sample group, B represents the absorbance value of the blank control culture solution, and C represents the absorbance value of the negative control group.

As shown in FIG. 6, the liposome/plasmid complexes (lipo-pDNA, lipo-pDNA-iRGD, DOX-CB @ lipo-pDNA and DOX-CB @ lipo-pDNA-iRGD) had substantially no toxic effect on GL261 cells at N/P ratios less than (or equal to) 1. The cell survival rate is higher than 50% when the N/P ratio is 2.

Using the commercial product liposome lipo2000 of Life Technologies as a control, 4 kinds of complexes of different liposome/plasmid ratios were prepared according to the product specification procedures, and lipo2000-pDNA (2), lipo2000-pDNA (3), lipo2000-pDNA (4) and lipo2000-pDNA (5) were sequentially prepared from small to large in N/P ratio, and the results of the cytotoxic effect on GL261 are shown in FIG. 7. The four liposome/plasmid complexes have greater cytotoxic effects, and the greater the N/P ratio, the greater the toxicity. Experiments show that under the condition that the N/P ratio is 2, the cytotoxicity effects of the prepared liposome/plasmid complexes (lipo-pDNA, lipo-pDNA-iRGD, DOX-CB @ lipo-pDNA and DOX-CB @ lipo-pDNA-iRGD) are smaller than that of lipo2000-pDNA series complexes, and the constructed multifunctional nano-liposome has the advantage of relative safety.

Example 5 Gene recombination experiment

A series of liposome/plasmid complexes lipo-pDNA (N/P ratios 1, 1.6, 2, 4, respectively) were prepared according to the experimental procedure described above. A complex of lipo2000 and plasmid lipo2000-pDNA (2) was prepared following the procedure in the 4.2.3 cytotoxicity study. Preparing a plasmid solution (100 mu g/mL) and a PEI Polyetherimide (polyethylenide) solution (130 mu g/mL) by using 5% sucrose water, mixing the two solutions in equal volume, continuously and uniformly blowing for 2min, and standing for 15min at room temperature to obtain the cation/plasmid composite (PEI-pDNA). GL261 cells in logarithmic growth phase were seeded at a cell density of 5X 104 cells per well in a 24-well cell culture plate, and cultured at 37 ℃ under 5% CO2 conditions for 18 h. The culture medium was discarded and rinsed 2 times with sterile PBS. Adding 500 mu L of serum-free basic culture solution into each hole, then adding 40 mu L of freshly prepared liposome/plasmid complex or cation/plasmid complex, fully shaking up, then continuing to culture for 6h, replacing with fresh serum-containing complete culture solution, continuing to culture, taking out a cell culture plate at 48h, observing the GFP expression condition of cells in each hole under an inverted fluorescence microscope, adjusting a 10-fold objective lens, and taking fluorescence pictures of a bright field and a green fluorescence channel.

As shown in fig. 8, gene recombination in each of the preparation groups was reflected by the expression level of GFP green fluorescent protein under the mirror, and in the figure, (a) lipo2000-pDNA (2), (b) PEI-pDNA, (c) lipo-pDNA (N/P ═ 1), (d) lipo-pDNA (N/P ═ 1.6), (e) lipo-pDNA (N/P ═ 2), and (f) lipo-pDNA (N/P ═ 4). As can be seen from the figure, the experiment group lipo-pDNA (N/P ═ 2) had the most and strongest green fluorescence signal under the field of fluorescence microscope, showing the best gene transfection efficiency. The fluorescence expression of lipo-pDNA (N/P-4) was the second best in the experimental group. For the lipo-pDNA experimental group with the N/P ratio less than 2, almost no green fluorescence signal exists in the visual field, which indicates that the N/P ratio is too low to be beneficial to gene transfection. In the case of two classical positive control preparations lipo2000-pDNA (2) and PEI-pDNA in cell transfection studies, the green fluorescence signal in the visual field was very weak, indicating poor transfection effect. In GL261 brain glioma cell transfection, the transfection effect of the nanoliposome prepared in the examples is superior to that of the commercial products lipo2000 and PEI.

EXAMPLE 6 Gene transfection efficiency assay

GL261 cells in logarithmic growth phase were seeded at a cell density of 5X 104 cells per well in a 24-well cell culture plate, and cultured at 37 ℃ under 5% CO2 conditions for 18 h. The medium was discarded and rinsed 2 times with sterile PBS, and 500 μ L of serum-free basal medium was added to each well. Liposome/plasmid complexes lipo2000-pDNA (2), lipo-pDNA (N/P ═ 2) and lipo-pDNA (N/P ═ 4) were freshly prepared according to the procedure of 4.2.4 gene recombination experiments, then 40. mu.L of the liposome/plasmid complexes were added to the cell culture plates, followed by culture for 6h after shaking up, replaced with fresh serum-containing complete culture medium, followed by culture, the cell culture plates were removed at 24h, and pure culture medium blank controls and plasmid incubation controls were set in the experiments. The transfected cells were discarded from the medium, rinsed 3 times with sterile PBS, and 200. mu.L of cell lysate was added to each well of the plate, and shaken for 40min at room temperature on a shaker. After repeatedly and carefully blowing the lysate, the liquid was transferred to an EP tube, centrifuged at 4 ℃ for 20min at 14000rpm, and the supernatant was aspirated into a new EP tube. Adding luciferase detection reagent substrates with equal volume amount into an EP tube respectively, mixing uniformly, immediately putting each sample into a chemiluminescence apparatus after adding the substrates for 1min, measuring relative light intensity (RLU), and keeping out of the sun during the experiment.

Meanwhile, a gradient protein concentration standard curve is established according to the operation steps of the BCA protein concentration determination kit product instruction, and 5 mu L of supernatant in each sample is respectively taken to determine the corresponding protein concentration. Finally, the transfection efficiency of each sample was evaluated in relative light intensity per mg of protein (RLU per mg of protein), and the experiments were performed in 3 replicates.

FIG. 9 shows the confocal fluorescence profiles of different formulations incubated with macrophages for 8 hours after transfection of GL 26172 hours: (A) blank group, (B) pDNA, (C) lipo2000-pDNA (2) and (D) lipo-pDNA. The gene transfection efficiency of the liposome/plasmid complex lipo-pDNA (N/P ═ 2) is the highest, and the gene transfection efficiency is remarkably different from that of a positive control preparation lipo2000-pDNA (2); lipo-pDNA (N/P ═ 4) gene transfection was the second most efficient, and plasmid DNA alone was the least efficient. This experiment again demonstrates that the nanoliposome prepared in the examples has good gene transfection ability.

Example 7 flow cytometry detection of cellular CD47 expression levels after transfection

GL261 cells in logarithmic growth phase were inoculated at a cell density of 1X 105 cells per well in a 6-well cell culture plate, and placed in a cell culture chamber at 37 ℃ in 5% CO₂Culturing under the condition for 18 h. The medium was discarded and rinsed 2 times with sterile PBS, and 2mL of serum-free basal medium was added to each well. Preparing liposome/plasmid complex lipo2000-pDNA (2), lipo-pDNA (N/P ═ 2) and lipo-pDNA (N/P ═ 4) according to the operation steps of gene recombination experiments, then adding 160 microliter of liposome/plasmid complex into a cell culture plate respectively, shaking up fully, then culturing for 6h, replacing with fresh serum-containing complete culture solution, culturing continuously, taking out the cell culture plate at 72h, setting pure culture solution blank control and plasmid incubation control group in the experiment, and determining the experiment in parallel for 3 times. And (3) discarding a culture solution of the transfected cells, rinsing the transfected cells for 3 times by using sterile PBS, digesting and collecting the cells by using pancreatin, centrifuging the cells, incubating the cells with anti-mouse CD16/32 for 10min in the absence of light on ice, washing the cells with PBS for 3 times, incubating the cells with an APC anti-mouse CD47 antibody for 30min in the absence of light on ice, washing the cells with PBS for 3 times, and placing the cells in an ice box to detect the expression quantity of the CD47 of each sample cell on a flow cytometer in time.

As shown in FIG. 10, the cellular CD47 expression level of the simple plasmid group and the blank control group (blank) without treatment were not substantially different, and the cellular CD47 expression level of the complex lipo2000-pDNA (2) was significantly reduced. The expression level of CD47 in cells of lipo-pDNA (N/P ═ 2) group was lower than that of lipo2000-pDNA (2), and the two groups had significant statistical difference. The expression level of CD47 in lipo-pDNA (N/P ═ 4) group cells is also lower than that of lipo2000-pDNA (2), and the two have statistical difference, which shows that the nano liposome prepared in this chapter has good transfection effect, and provides possibility for realizing in vivo administration immune activation function.

Example 8 cell phagocytosis assay

Extracting and culturing primary macrophages of mice:

1) SPF grade C57BL/6 mice 5 weeks old were sacrificed by cervical dislocation and soaked in 75% glacial ethanol for 5 min. Performing operation asepsis operation in a super clean bench, taking down the femur and tibia of a mouse, carefully removing muscles attached to the femur, washing with sterile PBS for several times, and placing in RPMI1640 culture solution;

2) the ophthalmic scissors cut two ends of the bone, a 5mL sterile syringe sucks fresh culture solution to wash the bone marrow until the washing solution turns from bright red to colorless, and the collected bone marrow cell suspension is centrifuged for 4min at the rotating speed of 1000 rmp. Discarding supernatant, adding erythrocyte lysate for treating for 5min, adding 3 times of fresh culture solution to stop lysis, and centrifuging at 1000rm rotation speed for 4min to obtain white cell precipitate;

3) the collected primary bone marrow cells are resuspended in a proper amount of culture solution, and M-CSF (10ng/mL) is added for co-incubation and culture, half of the culture solution is changed every 3 days, and the culture is carried out for 5-7 days to stimulate and differentiate into mouse macrophages.

Macrophage phagocytosis assay:

liposome/plasmid complexes lipo2000-pDNA (2) and lipo-pDNA (N/P ═ 2) were freshly prepared according to the protocol of the gene recombination experiments, followed by transfection of GL261-GFP, and the cells were harvested for use 72 h. Pure culture medium blank control and plasmid incubation control groups were set in the experiment.

The obtained primary mouse bone marrow cells are re-suspended by a complete culture medium, evenly inoculated in a confocal dish, and replaced by a fresh culture solution after stimulated differentiation for 5-7 days. Then adding transfected GL26-GFP cells, and placing the cells in a cell incubator for co-culture for 8 h. After removing floating cells by PBS washing, 4% paraformaldehyde was fixed at room temperature for 20min, and PBS washing was performed 3 times. Permeabilizing with 0.25% Triton X-10 cells for 20min, washing with PBS 3 times, blocking with goat serum for 25min, adding 3% BSA diluted F4/80 primary antibody diluent (dilution ratio 1: 30), incubating at room temperature for 1h, washing with PBS 3 times, incubating at room temperature for 1h with red fluorescent secondary antibody diluent (dilution ratio 1: 200), and washing with PBS 3 times. And finally, incubating the cells at room temperature for 10min by using a DAPI staining solution to mark cell nuclei, washing the cells for 3 times by using PBS, dripping an anti-quenching sealing piece, taking a cell fluorescence picture by using an inverted confocal microscope after sealing a cover glass, and observing the phagocytosis condition of macrophages in each sample to tumor cells.

FIG. 11 is a confocal fluorescence plot of 8 hours co-incubation with macrophages after 26172 hours without transfection of GL with the formulation: (A) blank, (B) pDNA, (C) lipo2000-pDNA (2), (D) lipo-pDNA; DIC (bright field light microscope), DAPI (4', 6-diamidino-2-phenylindole), GFP (green fluorescent protein), F4/80, MERGE (overlapping fields of view). GL261 tumor cells were fluorescently labeled green and macrophages were fluorescently labeled red. In the simple plasmid group, there was little finding that tumor cells were phagocytosed; only one tumor cell was also found to be phagocytosed by macrophages in the placebo group (MERGE channel is marked by white arrow in the figure). After GL261 cells transfected by liposome/plasmid complex lipo2000-pDNA (2) were incubated with macrophages, phagocytosis of macrophages was clearly observed in the under-the-lens field (indicated by white arrows on MERGE channels in the figure). The phenomenon that GL261 cells after lipoid/plasmid complex lipo-pDNA (N/P ═ 2) transfection are phagocytized by macrophages is more obvious (MERGE channel is marked by white arrow in the figure), which shows that the expression level of CD47 gene of tumor cells is reduced after lipoid transfection, and then the phagocytosis of the cells by the macrophages is increased. The nano-liposome prepared by the invention shows good application prospect in promoting the phagocytic function of cells.

Example 9 tumor-bearing mouse in vivo fluorescence imaging

And (2) preparing a cationic liposome (DiR @ lipo) physically wrapping the fluorescent dye DiR with the drug-loaded cationic liposome, and uniformly mixing part of the cationic liposome with the polypeptide iRGD solution to obtain a liposome/polypeptide compound DiR @ lipo iRGD. Establishing a GL261 in-situ tumor-bearing mouse model, dividing tumor-bearing mice into 3 groups after 2 weeks, respectively injecting 0.2mL of free DiR solution, DiR @ lipo solution and DiR @ lipo-iRGD into tail veins, and controlling the DiR injection dosage among all groups to be consistent. After 24h, the isoflurane is inhaled to anaesthetize the mouse and is placed in a living body imaging system of the small animal for observation, the exposure time among various times is kept consistent, and the operation process is protected from light. The air needle is used for killing the mouse, the main organs of the mouse, such as brain, heart, liver, spleen, lung, kidney and the like, are taken out, the surface of the mouse is washed with normal saline to remove floating blood, filter paper is used for sucking dry, and the ex vivo organs are immediately placed in a living body imaging system of the small animal for observation. Mouse ex vivo brain tissue treated with the DiR @ lipo-iRGD formulation was coronal dissected along the cell seeding hole and fluorescence pictures were taken at the same exposure time.

As shown in FIG. 12, the fluorescence distribution of the brain region of tumor-bearing mice in the DiR @ lipo-iRGD experimental group is obvious, which indicates that the preparation delivery efficiency is high. After the mouse is dissected, fluorescence imaging is carried out on an isolated organ, and the result also shows that the plastid/polypeptide compound DiR @ lipo-iRGD has good brain tumor drug delivery capacity.

As shown in FIG. 13, the DiR @ lipo group and the free DiR group tumor-bearing mouse brains had almost no fluorescence signal, because the DiR dye had no specific organ targeting, while the DiR @ lipo group could be taken up and excreted out of the body very quickly in positive polarity. From the results of tumor-bearing mouse living fluorescence imaging experiments, the liposome/polypeptide compound prepared by the invention has obvious brain targeting property, and provides a good delivery system for in vivo drug delivery of brain glioma.

Example 10 boron neutron capture-immuno-chemotherapy combination therapy in vivo anti-tumor Effect test

According to the experimental operation, the C57BL/6 mouse is injected with GL261-LUC cells intracranially to establish an in-situ tumor-bearing mouse model, the mouse is injected with D-luciferin potassium working solution in the abdominal cavity after 6 days, living body bioluminescence imaging is carried out in an animal living body imaging system, and the tumor-bearing mouse which is successfully modeled is selected. The tumor-bearing mice are randomly divided into nine groups of 7-8 mice, and the treatment process of the mice treated by each group is as follows:

(ii) DOX-CB @ lipo-iRGD-in situ-N (+): injecting 10 mu L DOX-CB @ lipo-iRGD intratumorally every day on 6 th, 7 th and 8 th days after molding, and irradiating by thermal neutrons after 24 h;

② DOX-CB @ lipo-pDNA-iRGD-in situ-N (+): injecting 10 mu L DOX-CB @ lipo-pDNA-iRGD intratumorally on 6 th, 7 th and 8 th days after molding, radiating thermal neutrons after 24h, and injecting 10 mu L lipo-pDNA-iRGD in situ on 13 th, 16 th and 19 th days;

(iii) DOX-CB @ lipo-iRGD-iv-N (+): injecting 100 mu L DOX-CB @ lipo-iRGD into tail vein every day 6, 7 and 8 days after molding, and performing thermal neutron irradiation after 24 h;

(iv) lipo-pDNA-iRGD-in situ-N (+): injecting lipo-pDNA-iRGD 10 μ L into tumor every day on 6, 7 and 8 days after molding, radiating with thermal neutron after 24h, and injecting lipo-pDNA-iRGD 10 μ L into situ every day on 13, 16 and 19 days;

DOX + CB @ lipo-iRGD-iv-N (+): injecting 100 mu L DOX + CB @ lipo-iRGD into tail vein every day 6, 7 and 8 days after molding, and performing thermal neutron irradiation after 24 h;

sixthly, BSH-N (+): injecting 100 mu L BSH solution into tail vein after 9 days of molding, and performing thermal neutron irradiation after 2 h;

n (+): performing thermal neutron irradiation on the 9 th day after the molding;

(viii) N (-): making blank control group after molding, and not irradiating;

ninthly, sham: intracranial injection of 5 μ L PBS as sham control group without irradiation.

The preparation method of the preparation DOX + CB @ lipo-iRGD is basically the same as that of DOX-CB @ lipo-iRGD except that the preparation DOX and CB are directly encapsulated with reagents. In each group, the concentration of DOX-CB is 0.3mg/mL, the concentration of DOX + CB is 0.3mg/mL, the concentration of BSH is 20mg/mL, the concentration of plasmid is 100 mu g/mL, and the ratio of liposome to plasmid N/P is 2. The experimental groups needing irradiation in the thermal neutron irradiation process are all irradiated for 2 hours. After the irradiation is finished, the mice are continuously raised, the mental states of the mice in each group are observed every day, the dead bodies of the mice are treated according to the animal center standard when the mice die, the survival time is recorded by taking the molding day as the first day, and the survival curve of each experimental group is drawn at the 66 th day.

As shown in fig. 14, the thermal neutron irradiation group (c) and the blank control group (c) mice bearing the brain tumor die within 30 days, which indicates that the GL261 in-situ brain glioma rapidly progresses without treatment, endangers life, and well simulates the dangerous biological characteristics of clinical brain glioma. The survival condition of the mice after the experimental group ninthly false operation treatment is good, the experimental operation does not cause obvious acute postoperative complications to the mice, and the interference of the operation factors to the experiment is eliminated.

The preparation used in the experimental group III is loaded with single DOX and CB reagents, the boron trapping agent is not proved to have the cell nucleus tropism, and the preparation used in the experimental group III is loaded with the cell nucleus tropism boron trapping agent DOX-CB compound. The survival rate of the experimental group ③ tumor-bearing mice is obviously higher than that of the experimental group fifthly, which is probably related to the more obvious neutron capture treatment effect of the nucleus tropism boron capture agent. The survival rates of the mice with tumors in the experimental group I and the experimental group III are not greatly different and basically equal to the survival rates of the mice with tumors in the BSH experimental group. On one hand, the bioavailability achieved by the current brain targeting drug delivery technology is lower than 10% ID/g because the intravenous injection efficiency is still not high. Even if the dose of the tail vein injection group is about 1 order of magnitude higher than that of the intratumoral injection preparation group, the concentration of the preparation reaching the target area of the brain tumor is not greatly different from that of the intratumoral injection method, so that the final curative effect is equivalent; on the other hand, BSH is one of two boron drugs clinically applied to BNCT, the administration dosage is extremely high and is 100mg/kg, the dosage of the multifunctional nanoliposome tested in the experiment is far lower than the value, and the difference of the mouse growth rate is found to be small after 66 days of observation, which shows that the multifunctional nanoliposome has a definite synergistic anti-glioma effect after the combination of the nuclear drug delivery driving high-efficiency neutron capture effect and the chemotherapy combination therapy.

The experimental group II adopts intratumoral administration to realize combined trial treatment of boron neutron capture-immunization-chemotherapy, the survival rate of tumor-bearing mice after treatment is extremely high, the curative effect is definite, only 1 mouse dies by the time point of 66 days, the mice die within 30 days after modeling, and failure caused by drug leakage in the administration process cannot be eliminated. There is a clear indication in the literature that blockade of CD47 drives macrophage-mediated immunotherapy, but it is more recommended to use it in combination with other therapies. In this experiment, the survival of animals with immunotherapy alone (experimental group iv) was less than 50%, which may be associated with the limited ability of blocking CD47 alone to activate immunity. It is worth pointing out that the constructed multifunctional nanoliposome can deliver the CRISPR/Cas9 gene knockout plasmid of CD47 gene to tumor tissue cells in a targeted manner as far as possible, and the condition that the gene is not off target is avoided. However, because normal tissue cells lack serial 'eat me' signals specific to tumor cells, even if the 'eat me' signals of the normal tissue cells are reduced due to the fact that partial multifunctional liposome is off-target, strong immune response is relatively difficult to trigger. The multifunctional nano-liposome prepared by the invention not only has good synergistic treatment effect, but also reduces the toxic and side effects of the drug as much as possible, and is a combined therapy nano-drug delivery system with application prospect.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A multifunctional nano liposome is characterized by comprising adriamycin, carborane, cationic liposome, plasmid and polypeptide.

2. The multifunctional nanoliposome of claim 1, wherein the charge ratio of the cationic liposome and the plasmid is greater than 2.

3. The multifunctional nanoliposome of claim 1, wherein the cationic liposome comprises (2, 3-dioleoxypropyl) trimethylammonium chloride, 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol 5000.

4. The multi-functional nanoliposome of claim 1, wherein the boron in the carborane comprises¹⁰B。

5. A method of preparing the multifunctional nanoliposome of claim 1, wherein the method comprises the steps of:

6. The method of claim 5, wherein the drug-loaded cationic liposome and the plasmid have a charge ratio of greater than 2.

7. The method of claim 5, wherein the molar ratio of the doxorubicin, triethylamine and 1-bromomethyl o-carborane is 1:1 to 3.

8. The method of claim 5, wherein in step (ii) the drug-loaded cationic liposomes are obtained by 0.22 μm water membrane filtration.

9. The method of claim 5, wherein the molar ratio of (2, 3-dioleyloxypropyl) trimethylammonium chloride: 1, 2-dioleoyl-sn-propanetriyl-3-phosphatidylethanolamine): cholesterol: the molar ratio of distearoyl phosphatidyl acetamide-polyethylene glycol 5000 is 1: 1-3: 0.5-2: 0.02-0.05.

10. Use of the multifunctional nanoliposome of claim 1 in the preparation of a medicament for boron neutron capture therapy.