Liposome drug delivery system for targeted therapy of brain glioma and application thereof

Abstract

The invention discloses a lipidosome drug delivery system for targeted therapy of brain glioma and application thereof. The lipidosome drug delivery system is a lipidosome mixture prepared from a basic component, a functional component and an external water-phase buffer solution. After intravenous injection, the liposome is efficiently combined with complement protein C3 and immune globulin M in plasma to synergistically activate a complement system, actively attracts and activates neutrophils to uptake, and crosses a blood brain barrier in a mode of'building a defecation vehicle 'by utilizing the inflammatory taxis of the neutrophils. Neutrophile granulocytes are stimulated by inflammatory factors in a microenvironment after reaching a tumor brain area to form extracellular trap release lipidosome. The lipidosome realizes specific targeting through folate receptors over-expressed on the surfaces of glioma cells. The liposome shows excellent cross-blood-brain-barrier drug delivery efficiency and brain glioma curative effect in vivo, can be effectively combined with surgery, radiotherapy and the like, and provides a solution for the main problems of radiotherapy resistance, chemotherapy drug resistance and the like in clinic.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a liposomal drug delivery system for targeted treatment of glioblastoma and its application. Background Art

[0002] Glioblastoma (GBM) is the most common primary intracranial malignant tumor, characterized by a fast growth rate and a strong ability of infiltrative growth. Currently, the most commonly used treatment methods are surgery, radiotherapy and chemotherapy. Due to the often invasive and infiltrative growth of glioblastoma, the surgical difficulty is high, and the remaining tiny lesions are extremely likely to cause recurrence again, resulting in a low survival rate of patients.

[0003] In addition, almost all macromolecular drugs and more than 98% of small molecule drugs cannot cross the blood-brain barrier (BBB) and the blood-brain tumor barrier (BBTB) to enter the brain, resulting in little effect of many drugs that have good anti-cancer effects in other organs in brain tumors. At present, people use various receptor-targeted endothelial cells to achieve drug delivery to the brain tissue across the BBB, but cannot target the brain tumor area. Even if some responsive controlled release mechanisms are added, it can only reach the microenvironment near the brain tumor, but cannot target brain tumor cells, still resulting in damage to normal tissues and reduced efficacy. Therefore, it is crucial to invent a one-stop targeted delivery vector that can cross the BBB, BBTB and further target glioblastoma cells for the treatment of glioblastoma.

[0004] Neutrophils are the most abundant white blood cells in the body's blood circulation and can accurately migrate to the sites of inflammatory reactions. Therefore, they will infiltrate a large amount in brain tumor tissues. As the first line of defense in the body's immune response, neutrophils have a powerful phagocytic function, a natural and efficient ability to cross the blood-brain barrier (BBB), and clear pro-inflammatory characteristics. Therefore, neutrophils are an ideal carrier for efficiently crossing the BBB and targeting the inflammatory microenvironment of gliomas. Currently, when delivering drugs using the surface of neutrophils, it is often necessary to extract a large number of neutrophils, break and extract the cell membrane through various physical and chemical methods, and then coat it on the surface of the drug-loaded delivery system. This process is complex and costly, and various physical and chemical operations will cause losses in the membrane protein activity on the surface of neutrophils, resulting in a significant reduction or even loss of the ability to cross the BBB. To solve this problem, it is expected to target neutrophils in situ in the body. In order to endow the drug delivery system with the targeting ability of neutrophils, most studies will modify the targeting polypeptides or antibodies on the surface of neutrophils. However, after intravenous injection into the body, due to electrostatic adsorption, a layer of non-specific protein corona will be enriched on the surface of the drug delivery system, shielding the targeting ability of the polypeptides or antibodies. To solve these problems, molecules with anti-electrostatic adsorption such as polyethylene glycol (PEG) have been developed. However, while anti-protein corona, it also reduces the targeting ability to cells to a certain extent. Therefore, there is an urgent need for an innovative targeting strategy to efficiently target neutrophils and use neutrophils to deliver drugs to the brain tumor microenvironment.

[0005] As a drug delivery system that is currently the most mature in research, liposomes not only have the advantages of convenient preparation, low price, good stability, and good biodegradability, but also have the characteristics of a long action time, the ability to extend the drug action time, reduce drug toxicity, and improve drug stability. They are an ideal nano-drug delivery system and have been widely used clinically. They are a reliable choice for studying carriers targeting neutrophils. Using neutrophils, the liposome drug delivery system reaches the glioma microenvironment and is released through the process of neutrophil extracellular trap (NETs) formation. By further modifying the components targeting glioma cells on the surface of liposomes, one-stop treatment of delivering drugs into gliomas can be achieved. Summary of the Invention

[0006] The object of the present invention is to address the current difficulties and progress in cross-blood-brain barrier drug delivery. To solve the problem of low targeting efficiency of traditional drug delivery systems with target modification towards neutrophils, a one-stop liposome drug delivery system is provided that can efficiently hitchhike on neutrophils to cross the BBB and BBTB and further target glioma cells. After entering the body through the vein, a specific protein corona forms on the surface of the liposome, attracting activated neutrophils to actively uptake it. After being uptaken, it utilizes its pro-inflammatory property to cross the BBB and reach the inflammatory region of glioma. Stimulated by inflammatory factors in the microenvironment, neutrophils form NETs and release the liposome in the form of exocytosis. The FA structure on the surface of the liposome promotes the specific uptake of the liposome by glioma cells. This liposome drug delivery system has precise targeting, clear principles, excellent therapeutic effects, and potential clinical application prospects.

[0007] The technical solution of the present invention

[0008] A liposome drug delivery system for targeted treatment of glioma is prepared from a liposome mixture composed of the following components: a basic component, a functional component, and an external aqueous buffer solution.

[0009] The basic component and the functional component are collectively referred to as lipid molecules, and the molar ratio of the basic component to the functional component is 3:7; the drug content is changed according to the therapeutic concentration required for different drugs in the body, ranging from 1 to 100 mg / mL. In some embodiments, the molar ratio of the drug to the lipid molecules is 1:3 - 1:20, preferably 1:5; the volume of the external aqueous buffer solution is changed according to needs. In some embodiments, the final liposome concentration is 0.1 - 100 mg / mL, preferably 10 mg / mL, by adding the external aqueous buffer solution.

[0010] The liposome drug delivery system for targeted treatment of glioma is prepared by the conventional thin film hydration method. The solution of lipid molecules and the drug is mixed evenly in a flask, the solvent is evaporated to form a lipid film by reduced pressure distillation, vacuum dried overnight, the external aqueous buffer solution is added, ultrasonically dispersed, extruded through a polycarbonate membrane, and dialyzed and purified in the external aqueous buffer solution at 4 degrees through a dialysis bag with a molecular weight of 8k - 14 kDa to prepare the liposome drug delivery system.

[0011] In some embodiments, the functional component comprises one or both of the following two lipid molecules:

[0012] (1) An amphiphilic lipid molecule with a hydrophilic trans-phosphatidylcholine (CP) structure at one end and a lipophilic end at the other end, and the structure is shown as the general formula (2-1).

[0013]

[0014] (2) An amphiphilic lipid molecule with a hydrophilic folic acid (FA) structure at one end and a lipophilic end at the other end, and the structure is shown in the general formula (2-2).

[0015]

[0016] The term "amphiphilic lipid molecule" refers to a molecule with a hydrophilic end at one end, connected by an arbitrary linker chain in the middle, and a hydrophobic (lipophilic) end that is a saturated or unsaturated alkyl chain represented by R; wherein, the definition of the saturated or unsaturated alkyl chain is a cyclic or acyclic, straight-chain or branched-chain tail-containing, two or more tail-containing, saturated or unsaturated bond-containing, aryl, heteroaryl, aryl or heteroaryl-substituted alkyl chain with 3 to 50 carbon atoms. In the disclosed examples applicable to the present invention, the R is a saturated or unsaturated alkyl chain, and the saturated alkyl chains include: cinnamoyl, myristoyl, palmitoyl, stearoyl and erucoyl; the unsaturated alkyl chains include: oleoyl, linoleoyl, dilinoleoyl and linolenoyl; the hydrophobic alkyl chain is coupled to CP or FA via an arbitrary linker chain; wherein, the molar ratio of the two lipid molecules, the CP lipid molecule and the FA lipid molecule, contained in the above functional components is 100:0 to 0:100, preferably one of 100:0, 75:25, 50:50, 25:75, 0:100.

[0017] Mechanism of action supplement: The activation of the complement system is the main mechanism of the body's anti-infection defense, including the alternative pathway, the classical pathway and the lectin pathway. IgG and IgM that bind to pathogens activate the classical pathway to trigger C3 activation, which acts synergistically with the C3 positive feedback loop in the alternative pathway to amplify and enrich C3b protein in a cascade manner. C3b is a protein specifically recognized by the neutrophil CR3 receptor, and through this synergistic effect, neutrophils can be rapidly recruited to phagocytose pathogens with C3b enriched on their surface. Selective opsonization liposome (So-lipo) is prepared by the thin-film hydration method with the basic component cholesterol plus two functional components. Among the functional components, the CP lipid molecule can enrich the complement protein C3 and activate the alternative pathway, and the FA lipid molecule can enrich the immunoglobulins IgG and IgM and activate the classical pathway. The two form a synergistic effect, which can enrich C3b protein on the surface of the liposome and attract the phagocytosis of activated neutrophils. The results of proteomics show that at a specific ratio, the two achieve the best synergistic effect and the amount of enriched C3b reaches the peak. Through the uptake on the CP surface, the FA surface and the synergistic surface of the two, it is demonstrated at the cellular and in vivo levels that the ratio of CP lipid molecule:FA lipid molecule = 25:75 can trigger the highest level of synergistic effect of the alternative pathway and the classical pathway of the complement system and achieve the best neutrophil uptake effect. In some embodiments, the basic components include neutral lipids, cationic lipids and / or anionic lipids.

[0018] (1) Neutral lipids include cholesterol and its derivatives in steroids; phospholipids include one or more of lecithin, soy lecithin, cephalin, sphingomyelin, and hydrogenated soy lecithin; the molar percentage of the steroid in the total moles of the mixture of the base component and the functional component is 10%-90%, preferably 30%; the lipid molecule contains at least one phospholipid and cholesterol; the molar percentage of the cholesterol in the total moles of the base component can be 0.1%-90%, preferably 20%-60%; the molar percentage of the lipid molecule in the total moles of all components of the liposome mixture can be 10%-99%, preferably 20%-70%.

[0019] Among them, the phospholipids include one or more of dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), dioleoyl phosphatidylcholine (DOPC), egg yolk phosphatidylcholine (EPC), dierucoyl phosphatidylcholine (DEPC), dilauroyl phosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), palmitoyl oleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dipalmitoyl glycerophosphoglycerol (DPPG), dipalmitoyl phosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidylserine (DSPS), dipalmitoyl phosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoyl phosphatidylinositol (DMPI), or distearoyl phosphatidylinositol (DSPI);

[0020] (2) A cationic lipid or an anionic lipid selected from N,N-dioleoyl-N,N-dimethylammonium chloride, N,N-distearoyl-N,N-dimethylammonium bromide, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, N,N-dimethyl-2,3-dioleoyloxypropylamine, 3-(dilauryldimethylammonio)-N1,N1,4-tridodecyl-1-piperazineethanamine, N1-[2-(dilauryldimethylammonio)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine, 14,25-bis(tridecyl)-15,18,21,24-tetraaza-tritriacontane, 1,2-dilinoleyloxy-N,N-dimethylaminopropane, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane, 4-(dimethylamino)butyric acid heptatriaconta-6,9,28,31-tetraen-19-yl ester, and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium (DOSPA), 4-(N,N-dimethylamino)butyric acid (dilinoleoyl)methyl ester DLin-MC3-DMA, SM-102, any one of them and mixtures thereof.

[0021] In some embodiments, the external aqueous phase buffer includes physiological saline, acetate, succinate, gluconate, histidine, citrate, carbonate, PBS, HEPES or other organic acid buffers, preferably HEPES buffer; the concentration of the buffer can be a conventional concentration, preferably an isotonic concentration.

[0022] In some embodiments, the drug refers to a drug that can be encapsulated by the liposome drug delivery system, and the drug is a nucleic acid drug or an anti-tumor agent; the nucleic acid drug is selected from any one of DNA, antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme and siRNA; the anti-tumor agent includes drugs for treating tumors and infectious diseases, specifically including but not limited to drugs for treating one or more of the following diseases: breast cancer, lung cancer, melanoma, liver cancer, kidney cancer, brain tumor, myocardial infarction, stroke, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, pneumonia, nephritis and various immunomodulatory drugs.

[0023] In some embodiments, the encapsulated anti-tumor drug is selected from small molecule, nucleic acid or protein drugs that have the ability to kill glioblastoma, inhibit the growth of glioblastoma, or synergistically enhance the sensitivity of common clinical treatment strategies such as surgery, radiotherapy, chemotherapy, and immunotherapy. The small molecule drugs include, but are not limited to: paclitaxel, doxorubicin, adriamycin, lomustine, carmustine, nimustine, tirapazamine, nimorazole, temozolomide, platinum-based drugs, stiripentol, imatinib, dasatinib, ponatinib, gefitinib, icotinib, erlotinib, osimertinib, apaciclib, apatinib, fruquintinib, toceranib, cediranib, palbociclib, ribociclib, abemaciclib, talazoparib, olaparib, iniparib, niraparib, vismodegib, sonidegib, daurismo, everolimus, azd8055, azd2014, temsirolimus, al3810, scc-31, sunitinib, regorafenib, sorafenib, vandetanib, cabozantinib, lenvatinib, pazopanib, vemurafenib, axitinib, dabrafenib, linifanib, nintedanib, or any combination thereof.

[0024] Anti-tumor protein drugs include, but are not limited to: Bevacizumab, Atezolizumab, Sirolimus (albumin-bound), Interferon; Nucleic acid drugs include, but are not limited to: siRNA, miRNA, ASO targeting MGMT, MET / HGF, p38MAPK resistance pathways, oncogene inhibitors, epidermal growth factor receptor (EGFR) inhibitors, and transforming growth factor-β (TGF-β) inhibitors.

[0025] The present invention also provides the use of the liposomal drug delivery system for targeted treatment of glioblastoma in the preparation of tumor cell preparations with high folate receptor expression.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. The liposomal mixture of the present invention forms a liposomal drug delivery system by combining neutral lipids, CP lipid molecules, and FA lipid molecules, which can be used to encapsulate various types of drug molecules and maintain the performance of the active ingredients.

[0028] 2. The particle size of the liposomal drug delivery system of the present invention is 150-200 nanometers, with good stability.

[0029] 3. The liposomes prepared by the present invention solve the problem of low efficiency of traditional drug delivery systems in targeting neutrophils. On the basis of a large number of experiments, it is proved that the doping of CP lipid molecules or FA lipid molecules can attract the uptake of neutrophils. Among them, when liposomes are composed of CP lipid molecules and FA lipid molecules in a molar ratio of 25:75, they can most efficiently target neutrophils in situ in plasma and be taken up by neutrophils at the highest level, achieving the most efficient effect of crossing the blood-brain barrier.

[0030] By simulating the BBB in vitro through Transwell, it is proved that neutrophils phagocytosing liposomes still have the ability to normally cross the BBB. Through two-photon imaging, it is observed that neutrophils carrying liposomes cross the tight structure composed of vascular endothelium, basement membrane, and astrocytes, and reach the tumor microenvironment according to chemotaxis. It is proved that liposomes can hitchhike on neutrophils to efficiently reach the tumor microenvironment.

[0031] 4. After neutrophils carrying liposomes reach the inflammatory microenvironment of brain tumors, the liposomes are efficiently released through the formation of neutrophil extracellular traps, and highly express folate receptors on the surface of brain tumor cells through the surface of FA lipid molecules, so that the drugs encapsulated in the liposomes are specifically taken up by brain tumor cells. By adding FA to block folate receptors, it is proved that the targeting principle of tumor cells is that folate modified on the surface of liposomes targets folate receptors highly expressed on the surface of gliomas.

[0032] 5. Through the liposome drug delivery system provided by the present invention, it realizes efficient cross-blood-brain barrier and precise drug delivery and treatment of gliomas in one stop, avoiding problems such as the toxic and side effects of drugs on normal brain tissue cells. When loaded with paclitaxel (PTX) for chemotherapy, compared with the clinically commercialized liposome paclitaxel injection (Taxol), So-lipo-PTX can extend the survival time of mice with highly invasive CT2A gliomas; loading the radiotherapy sensitizing drug lomustine (CCNU) combined with radiotherapy can more significantly inhibit the growth of gliomas. These results indicate that So-lipo has the ability to deliver drugs and RNAs to the brain and various inflammatory disease regions, providing a potential platform for the efficient drug delivery of gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the principle of liposome targeting neutrophils to deliver drugs to gliomas.

[0034] Figure 2 It is the characterization of DOCP.

[0035] Figure 3 It is the characterization of DOAP-FA.

[0036] Figure 4For DSPE-FA characterization.

[0037] Figure 5 Show the standard curves of PTX and CCNU determined by HPLC for the determination of the encapsulation efficiency.

[0038] Figure 6 In it, the top row of figure captions indicates that the liposomes prepared when the ratio of CP lipid molecules to FA lipid molecules is 100:0 are group A, 75:25 are group B, 50:50 are group C, 25:75 are group D, and 0:100 are group E. The A, B, C, D, and E written in the small figures a-c are the corresponding grouping names. a shows the zeta potential of the prepared liposomes, b shows the particle size of the prepared liposomes, and c shows the transmission electron micrograph of the prepared liposomes.

[0039] Figure 7 Show the protein corona components enriched on the surface of liposomes with different ratios of functional components, where the small figures a, b, c, d, and e correspond to the protein corona components of groups A, B, C, D, and E in sequence. f statistically analyzes the content of C3 protein in the whole protein corona components.

[0040] Figure 8 Show the high purity of neutrophils extracted from bone marrow.

[0041] Figure 9 Show the uptake of liposomes with different ratios of functional components by activated and non-activated neutrophils.

[0042] Figure 10 In it, a shows that among the liposomes with different ratios of functional components, group D has the most uptake and release of liposomes, and b shows that there is good co-localization between the released NETs and liposomes in the liposomes with different ratios of functional components.

[0043] Figure 11 In it, a shows that in vivo, the uptake of liposomes by blood cells is mostly completed by neutrophils, b shows the statistics of group D in a, and 72.04% of the liposomes are taken up by neutrophils, and c shows that for mice induced by LPS inflammation, the uptake specificity of group D liposomes is the best.

[0044] Figure 12 Show good co-localization of neutrophils and liposomes in retinal blood vessels.

[0045] Figure 13 Show that in the case of simulating the BBB by the in vitro Transwell model, neutrophils carrying liposomes can normally cross the BBB and release liposomes, and the liposomes are further taken up by tumor cells. Figure 13In it, a shows the confocal images of uptake, and b shows the results of flow cytometry, demonstrating that group D has no effect on the migration ability of neutrophils and can efficiently deliver the liposomes of group D into tumor cells.

[0046] Figure 14 In it, a shows the results of two-photon in vivo imaging, in which the liposomes are continuously enriched in the glioma region, and b shows the statistical results of the liposome signals. The signals in the normal brain tissue and the brain tumor region are statistically analyzed. It can be seen that the signals of the liposomes gradually increase in the tumor region, while the signals in the normal brain tissue remain almost unchanged.

[0047] Figure 15 It shows that neutrophils carrying liposomes cross the blood-brain barrier and tend to the tumor site in two-photon in vivo imaging.

[0048] Figure 16 It shows the enrichment of liposomes in the brains of encephalitis model mice. The materials injected intravenously are as follows: group A, CP lipid molecule:FA lipid molecule = 100:0; group B, 75:25; group C, 50:50; group D, 25:75; group E, 0:100. The results show that the material enrichment in group D is the most in the encephalitis site.

[0049] Figure 17 It shows the enrichment of liposomes in the brains of brain tumor model mice and the liposome distribution in ex vivo organs. The materials injected intravenously are as follows: group A, CP lipid molecule:FA lipid molecule = 100:0; group D, 25:75; group E, 0:100. The results show that the material enrichment in group D is the most in the brain tumor region, and the enrichment signal is significant, approaching the liver signal.

[0050] Figure 18 It shows the enrichment of liposomes in the brain tumor region of brain slices. a shows the liposomes of group A, b shows the liposomes of group D, and c shows the liposomes of group E. It can be seen that the liposomes of group D are most significantly enriched in the brain tumor region.

[0051] Figure 19 It shows the separation of liposomes and NETs released by neutrophils in brain slices. a shows the multi-channel combined image, b shows DAPI, c shows the specific staining of NETs, and d shows the channel of liposomes labeled with DiD fluorescence.

[0052] Figure 20It shows that neutrophils carrying liposomes release liposomes by forming NETs. The time marked above the figure is the duration of LPS-induced NETs formation. Cells were fixed at 0 hour, 2 hours, 4 hours, and 6 hours respectively for NETs staining and observation. The upper row is the image obtained by confocal microscopy, and the lower row is the line co-localization analysis at the corresponding time points. The Manders co-localization coefficient is marked in the upper right corner, with a value ranging from 0 to 1. The closer the value is to 1, the higher the co-localization degree. The results show that NETs have not been fully formed at 0 hour, are fully formed at 2 hours and have a strong co-localization with liposomes (Manders co-localization coefficient 0.941), liposomes gradually release from NETs at 4 hours and the co-localization decreases, and a large amount of liposomes are released at 6 hours and the co-localization further decreases.

[0053] Figure 21 It shows the specific uptake of liposomes by various cells. a shows the experimental operation flow chart, b shows the uptake levels of liposomes by seven kinds of cells at 4 hours and 8 hours, and c shows that the uptake of liposomes by brain tumor cells can be blocked after pre-incubation with folic acid for 30 minutes, proving that this uptake is related to the expression of folic acid receptor.

[0054] Figure 22 It shows flow cytometry analysis of the uptake of liposomes by whole brain cells. a shows the experimental operation flow chart, b shows the bar chart of the uptake of liposomes by whole brain cells, and c shows the pie chart statistics of b.

[0055] Figure 23 It shows that the toxicity of So-lipo encapsulating PTX to neutrophils is much lower than that of the clinical liposomal paclitaxel injection Taxol.

[0056] Figure 24 It shows that So-lipo encapsulating PTX has selectivity for glioma cells and very low toxicity to normal cells.

[0057] Figure 25 Among them, a shows the treatment plan of So-lipo encapsulating PTX for CT2A glioma-bearing mice, and b shows the in vivo imaging map during the treatment process.

[0058] Figure 26 It shows the (a) body weight change graph, (b) survival curve, and (c) H&E, TUNEL, Ki67 staining of So-lipo encapsulating PTX for the treatment of CT2A glioma-bearing mice.

[0059] Figure 27 Among them, a shows the treatment plan of So-lipo encapsulating CCNU combined with radiotherapy (RT) for CT2A glioma-bearing mice, b shows the in vivo imaging map during the treatment process, and c shows the growth curve of brain tumors of each mouse in each group.

[0060] Figure 28 Show the (a) body weight change graph, (b) survival curve, and (c) H&E, TUNEL, and Ki67 staining of CT2A glioma mice treated with So-lipo combined RT encapsulating CCNU. Detailed implementation manners

[0061] A liposome for targeted treatment of glioma, which can deliver various small molecule drugs and siRNA, etc. At the same time, combined radiotherapy can further improve the inhibition of glioma.

[0062] 1. Experimental drugs: Paclitaxel (PTX), Lomustine (CCNU), Doxorubicin, Temozolomide, etc. are commercially available as conventional in this field, such as Shanghai Aladdin Biochemical Technology Co., Ltd.

[0063] 2. Test instruments: The instruments used in the following examples are the self-owned instrument equipment of Tianjin Medical University, and their equipment models and source information are as follows:

[0064] Ultraviolet-visible spectrum on an ultraviolet-visible spectrophotometer: Shimadzu, Japan;

[0065] Fluorescence emission spectrum on a traditional: F7000, Hitachi, Japan;

[0066] Measurement of mass spectrometry: Waters Xevo G2-XS Q-Tof mass spectrometer;

[0067] Measurement of the particle size and zeta potential of liposomes: Malvern Zetasizer, Nano series ZS, UK;

[0068] Transmission electron microscope: Talos F200C electron microscope, with an accelerating voltage of 120 kV;

[0069] Microplate reader: BioTEK, USA;

[0070] Confocal: FluoView confocal laser scanning microscope - FV1000;

[0071] IVIS Lumina imaging system: Caliper Life Sciences, USA;

[0072] High performance liquid chromatography (HPLC) analysis is carried out using a Waters Alliance e2695 chromatograph and a Symmetry C18 column (5 μm, 4.6 m × 150 mm) at 25 °C.

[0073] 3. The present invention will be further illustrated by way of examples below, but the present invention is not limited to the scope of the examples described herein. For the experimental methods without specific conditions indicated in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications. For the operating temperature and pressure, unless otherwise specified, they generally refer to room temperature and normal pressure. Among them, room temperature refers to 10 - 30 °C, and normal pressure refers to one standard atmosphere.

[0074] Example 1: Preparation of Liposome Drug Delivery System

[0075] (1) Synthesis of CP Lipid Molecules

[0076] The structure of a CP lipid is shown in the general formula (2 - 1):

[0077]

[0078] This example provides a preparation method applicable to the general formula of the CP lipid molecular structure:

[0079]

[0080] Structures applicable to the general formula (2 - 1) can all be used for liposome synthesis and targeting of glioma cells. In some specific embodiments of the present invention, CP lipid molecules with the structures shown in Table 1 below are finally obtained, including but not limited to any one of the following structures:

[0081] Table 1

[0082]

[0083] Among them, CP lipid molecule 1 is preferably named DOCP, and the mass spectrometry characterization is as Figure 2 shown, HRMS(ESI, m / z): calculated for C 43 H 81 NO8P - ([m + 2H] 2+ ) 772.5856, found: 772.5869.

[0084] (2) Synthesis of Folic Acid Lipid Molecules

[0085] The structure of an FA lipid is shown in the general formula (2 - 2):

[0086]

[0087] This example provides a preparation method applicable to the general formula of the FA lipid molecular structure:

[0088]

[0089] Structures applicable to the general formula (2-2) can all be used for the synthesis of liposomes and targeting glioma cells. In some specific embodiments of the present invention, FA lipid molecules with the structures shown in Table 2 below were finally obtained, including but not limited to any one of the following structures:

[0090] Table 2

[0091]

[0092]

[0093] Among them, FA lipid molecules 1 and 2 are preferred. FA lipid molecule 1 is named DOAP-FA, and its mass spectrometry characterization is as Figure 3 shown. HRMS(ESI,m / z): calculated for C 58 H 90 N8O9([m+H] + ) 1043.6909, found: 1043.6882. FA lipid molecule 2 is named DSPE-FA, and its mass spectrometry characterization is as Figure 4 shown. MS(ESI,m / z): calculated for C 60 H 99 N8O 13 P([m+H] + ) 1171.7, found: 1171.4.

[0094] (3) Preparation of drug-free liposome drug delivery systems

[0095] Cholesterol, CP lipid molecules, and FA lipid molecules are used. Cholesterol accounts for 30% of the total molar percentage of all components of the liposome, and the sum of the molar numbers of CP lipid molecules and FA lipid molecules accounts for 70% of the total molar percentage of all components. Among them, the ratio of CP lipid molecules and FA lipid molecules is prepared according to a gradient, and the ratios and corresponding names are: Group A: CP lipid molecules: FA lipid molecules = 100:0, Group B: 75:25, Group C: 50:50, Group D: 25:75, Group E: 0:100. The CP lipid is selected from any one of CP lipid molecule 1, CP lipid molecule 2, CP lipid molecule 3, CP lipid molecule 4, and CP lipid molecule 5 in this example (1), and among them, CP lipid 1 is preferred; the FA lipid is selected from any one of FA lipid molecule 1, FA lipid molecule 2, FA lipid molecule 3, FA lipid molecule 4, and FA lipid molecule 5 in this example (2), and among them, FA lipid 1 or 2 is preferred.

[0096] Add the solution of the above components to a 10 mL round-bottom flask. After ultrasonic mixing, perform vacuum distillation to completely evaporate the organic solvent and form a uniform lipid film. Pump with a vacuum pump for 12 h, add HEPES (10 mM, pH = 7.4) buffer and ultrasonically hydrate for 30 minutes, then extrude through a polycarbonate membrane with a pore size of 0.22 μm three times, dialyze overnight at 4 °C in a 10 kDa molecular weight dialysis bag in HEPES (10 mM, pH = 7.4) buffer, and store at 4 °C. The partial synthesis results are shown in Table 3 below, indicating that liposomes can be successfully synthesized with a small dispersion coefficient and uniform particle size of the surface liposomes.

[0097] Table 3

[0098]

[0099] (4) Preparation of Liposomal Drug Delivery System Encapsulating Lipophilic Drugs

[0100] Mix cholesterol, CP lipid molecules, and FA lipid molecules, where cholesterol accounts for 30% of the total molar percentage of all components of the liposome, and the sum of the molar numbers of CP lipid molecules and FA lipid molecules accounts for 70% of the total molar percentage of all components. Among them, the ratio of CP lipid molecules to FA lipid molecules is grouped in gradients as Group A 100:0, Group B 75:25, Group C 50:50, Group D 25:75, and Group E 0:100. The Group A, Group B, Group C, Group D, and Group E mentioned in the subsequent examples all conform to this ratio and naming method.

[0101] Mix the above components in a 10 mL round-bottom flask, add a methanol stock solution of a lipophilic drug at 20% of the molar ratio of the above lipid molecules, mix well, and perform vacuum distillation at 37 °C to form a uniform lipid film. Pump with a vacuum pump for 12 h, add HEPES (10 mM, pH = 7.4) buffer and ultrasonically hydrate for 30 minutes, then extrude through a polycarbonate membrane with a pore size of 0.22 μm three times, dialyze overnight at 4 °C in a 10 kDa molecular weight dialysis bag in HEPES (10 mM, pH = 7.4) buffer to remove the unencapsulated drug. Store at 4 °C. Determine the drug content in the liposomes by HPLC and calculate the drug encapsulation efficiency. The formula for calculating the encapsulation efficiency is: Encapsulation efficiency = Encapsulated amount / Total drug amount × 100%.

[0102] The encapsulated drugs and encapsulation efficiencies are shown in Table 4 below.

[0103] (5) Preparation of Liposomal Drug Delivery System Encapsulating Water-Soluble Drugs

[0104] Cholesterol, CP lipid molecules, and FA lipid molecules are used, where cholesterol accounts for 30% of the total molar percentage of all components of the liposome, and the sum of the molar numbers of CP lipid molecules and FA lipid molecules accounts for 70% of the total molar percentage of all components. Among them, the ratio of CP lipid molecules to FA lipid molecules is grouped in gradients as follows: Group A 100:0, Group B 75:25, Group C 50:50, Group D 25:75, and Group E 0:100.

[0105] Mix the above components in a 10 mL round-bottom flask. After mixing, perform vacuum distillation at 37 °C to form a uniform lipid film. After pumping with a vacuum pump for 12 h, dissolve the water-soluble drug (or siRNA) in HEPES buffer (or enzyme-free water), add it to the flask containing the lipid film, and ultrasonically hydrate for 30 minutes. Subsequently, extrude it 3 times through a polycarbonate membrane with a pore size of 0.22 μm, and dialyze overnight at 4 °C in a 10 kDa molecular weight dialysis bag in HEPES (10 mM, pH = 7.4) buffer to remove the unencapsulated drug. Store at 4 °C. Determine the drug content in the liposome by HPLC, and calculate the drug encapsulation efficiency. The formula for calculating the encapsulation efficiency is: Encapsulation efficiency = Encapsulated amount / Total drug amount × 100%.

[0106] The encapsulated drugs and encapsulation efficiencies are shown in Table 4 below.

[0107] (6) Determination of encapsulation efficiency

[0108] Prepare a stock solution of the drug with a known concentration, dilute it to different multiples with methanol to obtain drug solutions with different known concentrations, measure the ultraviolet absorbance of the characteristic absorption peak by HPLC, and construct a standard curve for each drug (as Figure 5 shown, taking paclitaxel PTX and carmustine CCNU as examples). Take the dialyzed liposome, add an equal volume of ethanol to break the emulsion, remove water and ethanol by rotary evaporation at 40 °C, add a certain amount of methanol and ultrasonically dissolve the drug, measure the ultraviolet absorbance of the characteristic absorption peak by HPLC, and substitute it into the standard curve to calculate the amount of drug encapsulated in the liposome. Take an equal amount of the sample before dialysis and perform the same emulsion-breaking operation. The total drug amount is calculated by HPLC. The formula for calculating the encapsulation efficiency is: Encapsulation efficiency = Encapsulated amount / Total drug amount × 100%.

[0109] Table 4

[0110] Drug Solubility Encapsulation efficiency (%) averaged over three syntheses Paclitaxel (PTX) Liposolubility 83％ Carmustine (CCNU) Liposolubility 89％ Doxorubicin Liposolubility 95％ Temozolomide Water solubility 72％ siRNA Water solubility 63％

[0111] Example 2: Performance characterization of the liposome drug delivery system

[0112] 1) Particle size and Zeta potential

[0113] Take 10 μL of the test sample prepared in Example 1 and disperse it in 1 mL of HEPES buffer in turn to detect the particle size and potential. Among them, the ratio of CP lipid molecules to FA lipid molecules in Group A is 100:0, in Group B is 75:25, in Group C is 50:50, in Group D is 25:75, and in Group E is 0:100. Each sample is placed in the potential detection cell in turn, and the Zeta detection mode is selected to test the potential (as Figure 6 a), and the results show that as the incorporation of FA molecules increases (increasing in turn from A to D), the potential gradually becomes negative; transfer the sample into the sample cell of the nanoparticle size analyzer, and select the DLS mode to test the particle size (as Figure 6 b), and the results show that all five groups of samples exhibit a relatively small hydrodynamic diameter, all around 200 nanometers.

[0114] 2) Transmission electron microscopy

[0115] Use a transmission electron microscope to observe the morphology of the samples: Take 20 μL of the test sample prepared in Example 1 and disperse it in 1 mL of deionized water in turn. Among them, the ratio of CP lipid molecules to FA lipid molecules in Group A is 100:0, in Group B is 75:25, in Group C is 50:50, in Group D is 25:75, and in Group E is 0:100. Each sample is dropped on the copper grid in turn and allowed to settle for 10 min, then use filter paper to absorb the excess liquid. After the copper grid is dried, drop 10 μL of sodium phosphotungstate solution for negative staining for 1 min, and then use filter paper to absorb the excess liquid. After the copper grid is dried, place it in the transmission electron microscope instrument to take electron micrographs (as Figure 6 c), and the results show that the liposome drug delivery system has been successfully prepared. The negatively stained liposome sphere structure can be directly seen under the transmission electron microscope, and the actual particle size is less than 200 nanometers.

[0116] Example 3: Fluorescent labeling of the liposome surface with DiO, DiD or DiR probes

[0117] Cholesterol, CP lipid molecules, and FA lipid molecules, where cholesterol accounts for 30% of the total molar percentage of all components of the liposome, and the sum of the molar numbers of CP lipid molecules and FA lipid molecules accounts for 70% of the total molar percentage of all components. Among them, the ratio of CP lipid molecules to FA lipid molecules is grouped in gradients as 100:0 in Group A, 75:25 in Group B, 50:50 in Group C, 25:75 in Group D, and 0:100 in Group E.

[0118] Mix the above components in a 10 mL round-bottom flask, add the ethanol stock solution of DiO, DiD or DiR probe at the concentration recommended in the instruction manual attached to the purchased fluorescent probe. After mixing, perform reduced-pressure distillation at 37 °C in the dark to form a uniform lipid film. Pump with a vacuum pump for 12 h, add HEPES (10 mM, pH = 7.4) buffer solution and hydrate by ultrasonic treatment for 30 minutes. Subsequently, extrude through a polycarbonate membrane with a pore size of 0.22 μm three times, and dialyze overnight at 4 °C in a 10 kDa molecular weight dialysis bag in HEPES (10 mM, pH = 7.4) buffer solution to remove unencapsulated molecules. Store in the dark at 4 °C.

[0119] Measure liposomes with different fluorescent labels using a fluorescence spectrophotometer, and determine the working concentration according to the fluorescence intensity.

[0120] Example 4: Analysis of protein corona components

[0121] Use inflammatory mouse serum to simulate the protein corona environment in plasma in vitro, and detect the adsorption of proteins in the serum by liposomes. The operation steps are as follows:

[0122] 1) Synthesize liposome drug delivery systems with different ratios according to the synthesis method in Example 1: Group A, CP lipid molecules: FA lipid molecules = 100:0; Group B, 75:25; Group C, 50:50; Group D, 25:75; Group E, 0:100;

[0123] 2) Select female C57b mice aged 6 - 8 weeks (purchased from Beijing Speyford Co., Ltd.), intraperitoneally inject LPS, collect blood from the eye socket 1 hour later, let the serum precipitate at room temperature for 2 hours, and centrifuge to obtain inflammatory serum;

[0124] 3) Mix each group of liposomes with serum respectively, vortex for 5 seconds and incubate at 37 °C for 2 hours;

[0125] 4) Centrifuge at 12000 rpm for 15 minutes at 4 °C, and the liposomes combined with the protein corona will be centrifuged down, discard the supernatant;

[0126] 5) Resuspend the precipitate with PBS buffer, transfer it to a 1.5 mL ultrafiltration tube with a molecular weight cut-off of 300 Da, and ultrafilter and wash with PBS three times to remove liposome components;

[0127] 6) Send the sample for proteomic analysis (as Figure 7 shown). The protein corona components of liposomes A - E with different ratios correspond to Figure 7 a - e respectively, and Figure 7 f summarizes the C3 protein content in the protein corona of each group. There is adsorption of C3 protein in each group of liposomes. Among them, Group D shows the best synergistic effect, that is, the highest enrichment of C3 protein content.

[0128] Example 5: Extraction experiment of mouse neutrophils

[0129] Female 6 - 8 - week - old C57b mice (purchased from Beijing Speywood Co., Ltd.) were selected to extract mouse bone marrow cells. The specific operation steps are as follows:

[0130] 1) After anesthesia, the C57b mice were euthanized, and the mice were soaked in 75% ethanol for 5 minutes for disinfection;

[0131] 2) The skin and muscle were cut open to obtain the complete femurs and tibias of the mice, which were soaked in pre - cooled 1×HBSS buffer;

[0132] 3) 20 mM EDTA and RPMI - 1640 complete medium were mixed at a ratio of 1:9 and aspirated into a syringe;

[0133] 4) The two ends of the femur or tibia were cut open, and the bone marrow components were blown out into a cell culture dish with a syringe;

[0134] 5) The bone marrow was gently dispersed and filtered through a 70 - nm cell sieve;

[0135] 6) Centrifuged at 500 g for 5 min at 4°C, the supernatant was discarded, and the cells were resuspended in 0.5 mL of 1×HBSS buffer;

[0136] 7) Isotonic solution was prepared with Percoll, and then the isotonic Percoll was prepared into three concentration gradients of 78%, 65%, and 55% with 1×HBSS. A 15 - mL centrifuge tube was rinsed with serum, and 2 mL of 78% Percoll, 2 mL of 65% Percoll, 2 mL of 55% Percoll, and 0.5 mL of cell suspension were slowly added to the 15 - mL centrifuge tube along the wall in sequence;

[0137] 8) Density gradient centrifugation was carried out at 500 g for 30 min at 25°C, and the acceleration and deceleration rate was 2;

[0138] 9) The liquid was aspirated and discarded layer by layer, and the cells between the 78% and 65% layers were collected, and washed 3 times with 1×HBSS buffer to obtain neutrophils;

[0139] 10) The neutrophils were purified by CD11b antibody and Ly6G antibody.

[0140] Figure 8 Show the purity of neutrophils (CD11b + Ly6G + ) extracted by the above - mentioned method is 95.8%.

[0141] Example 6: Experiment on the uptake of liposome drug delivery system by neutrophils

[0142] Activated neutrophils were extracted from the bone marrow of inflammatory mice, and resting neutrophils were extracted from the bone marrow of normal mice. Neutrophils were co-incubated with liposomes in vitro, and the uptake of liposomes by neutrophils was detected. The operation steps are as follows:

[0143] 1) Synthesize liposomes labeled with DiO at different ratios according to the method of Example 3;

[0144] 2) Select female C57b mice aged 6 - 8 weeks (purchased from Beijing Speyford Co., Ltd.). Inject LPS intraperitoneally as the activated neutrophil group, and inject normal saline as the normal control group. Extract mouse neutrophils according to the method of Example 5;

[0145] 3) Incubate liposomes at different ratios: Group A, CP lipid molecule: FA lipid molecule = 100:0; Group B, 75:25; Group C, 50:50; Group D, 25:75; Group E, 0:100, respectively, with activated or normal neutrophils. After 4 hours, centrifuge and wash the neutrophils, disperse them in HBSS buffer without calcium and magnesium, and analyze by flow cytometry (as Figure 9 shown).

[0146] The results showed that high - level uptake of liposomes by neutrophils was achieved in each group. To achieve specific uptake of activated neutrophils in vivo, when the uptake difference between activated and normal neutrophils is large, it has better selectivity. According to the experimental results, Group D has the best selectivity for liposome uptake among all groups.

[0147] Example 7: Experiment on the release of liposomes by neutrophils

[0148] Induce the formation of NETs in neutrophils by simulating an inflammatory environment in vitro, and observe whether liposomes can be released from NETs. The specific steps are as follows:

[0149] 1) Synthesize liposomes labeled with DiD at different ratios according to the method of Example 3;

[0150] 2) Select female C57b mice aged 6 - 8 weeks (purchased from Beijing Speyford Co., Ltd.). Inject LPS intraperitoneally as the activated neutrophil group, and extract mouse neutrophils according to the method of Example 5;

[0151] 3) Incubate liposomes at different ratios with neutrophils in 1640 complete medium containing LPS. After 4 hours, centrifuge and wash the neutrophils;

[0152] 4) Add antibodies against NETs for staining, and centrifuge and wash;

[0153] 5) Drop the cells on a coverslip and let them settle for 15 minutes. Stain with DAPI and mount the coverslip, and observe the uptake by confocal microscopy (as Figure 10As shown). The results showed that high levels of uptake of liposomes were obtained in each group of liposomes (Group A 100:0, Group B 75:25, Group C 50:50, Group D 25:75, Group E 0:100), and neutrophils were able to release liposomes through the formation of NETs. Among them, Group D had better selectivity for liposome uptake ( Figure 10 a), and there was very good co-localization between the NETs and liposomes released by neutrophils in each group ( Figure 10 b), indicating that neutrophils released liposomes through the formation of NETs after reaching the inflammatory region.

[0154] Example 8: In vivo blood cell uptake experiment

[0155] 1) Female 6-8-week-old C57b mice (purchased from Beijing Speyford Co., Ltd.) were selected. LPS was intraperitoneally injected as the neutrophil activation group, and normal saline was intraperitoneally injected as the normal control group. After 12 hours, DiO-labeled liposomes were injected into the tail vein. After 1 hour, blood was collected by eye after anticoagulation;

[0156] 2) The blood was lysed, centrifuged and washed. Specific antibodies against neutrophils (Ly6G), monocytes (Ly6C), macrophages (F4 / 80) and CD11b antibody were mixed and added to different groups of cells for staining at 37 °C for 15 minutes;

[0157] 3) Wash with PBS, and then perform flow cytometry analysis (as Figure 11 shown).

[0158] Analysis was performed on phagocytic cells in the blood. High levels of uptake of liposomes were obtained in each group of liposomes (Group A 100:0, Group B 75:25, Group C 50:50, Group D 25:75, Group E 0:100). Among them, the amount of liposomes taken up by Group D was the highest ( Figure 11 a), and 72.04% of the liposomes were taken up by neutrophils ( Figure 11 b). For better selectivity, comparing inflamed mice and non-inflammatory mice, it can be seen that the liposomes in Group D had the highest specificity and uptake amount ( Figure 11 c).

[0159] Example 9: Ex vivo imaging of neutrophil uptake of liposomes

[0160] In order to visually observe the process of neutrophil uptake of liposomes under physiological conditions in vivo, the inventor performed ex vivo imaging of in vivo retinal inflammation. The specific method is as follows:

[0161] Isoflurane-anesthetized female C57b mice aged 6 - 8 weeks (purchased from Beijing Speywood Co., Ltd.) were instilled with topical anesthetic eye drops on the eye surface, followed by mydriatic eye drops. A microinjector was used to inject a physiological saline solution containing 0.1 μg LPS into the retinal cavity of the mice, and erythromycin eye ointment was applied topically.

[0162] After 9 hours, a mixture of 200 μL of DiD-labeled liposomes prepared by the method of Example 3 and 10 μL of PE-Ly6G neutrophil antibody was injected via the tail vein.

[0163] After 30 minutes, the mice were anesthetized and perfused successively with 15 mL of PBS - 15 mL of 4% paraformaldehyde - 15 mL of PBS - 15 mL of 5 mg / mL FITC-concanavalin - 15 mL of PBS. The eyeballs were removed and fixed in 4% paraformaldehyde at room temperature for 30 minutes.

[0164] The retina was removed and spread on a slide, and confocal imaging was taken (as Figure 12 shown).

[0165] The results showed good co-localization of neutrophils and liposomes in the inflammatory area, demonstrating that neutrophils phagocytosed liposomes in the blood vessels.

[0166] Example 10: Transwell simulation of blood-brain barrier BBB

[0167] To verify that the uptake of liposomes by neutrophils does not affect their natural function of crossing the BBB, the inventors constructed a Transwell model with endothelial cells in vitro and verified that neutrophils have the ability to efficiently cross the BBB after taking up liposomes. The specific method is as follows:

[0168] The Transwell insert of a Corning 24-well Transwell plate (3 μm pore size, 6.5 mm diameter and 0.33 cm 2 polyester membrane) was pre-coated with Matrigel and seeded with 2 × 10 5 bEnd.3 cells. The integrity of the barrier was evaluated by measuring the transendothelial electrical resistance (TEER). After incubation for two weeks, the TEER value of bEnd.3 Transwell reached > 150 Ω·cm 2 , indicating the formation of tight junctions. LN229R cells were collected and 1 × 10 5 cells were seeded in the lower chamber overnight.

[0169] Mouse neutrophils were extracted according to the method of Example 5 and then co-incubated with liposomes labeled with DiO by the method in Example 3. After culturing at 37 °C for 1 hour, the neutrophils were washed three times with 1 × HBSS to remove free liposomes. Then, 2 × 10 5Neutrophils were added to the upper chamber. Serum-free medium with or without 0.5 μg / mL LPS was added to the lower chamber of the Transwell. After incubation at 37 °C for 10 minutes, the upper chamber was discarded, the contents of the lower chamber were collected and centrifuged to remove LPS. The pellet was resuspended with complete medium and further cultured with LN229R cells for 9 hours. Then, the LN229R cells were collected and analyzed using flow cytometry or confocal microscopy. In Figure 13 In Figure 13 a, it shows that the tumor cells took up the liposomes. In Figure 13 b, it can be more objectively seen by statistical methods that neutrophils can carry the liposomes to the lower layer, and the tumor cells can further take up the liposomes.

[0170] Example 11: In vivo imaging of neutrophil uptake of liposomes

[0171] To visually observe the process of neutrophil uptake of liposomes and the process of neutrophils carrying liposomes to tumor cells under physiological conditions in vivo, the inventors respectively performed ex vivo imaging of in vivo retinal inflammation and two-photon imaging inside the body. The specific methods are as follows:

[0172] 1) Two-photon imaging of in vivo brain tumor liposome enrichment model establishment: Female C57b mice aged 6 - 8 weeks (purchased from Beijing Speywood Co., Ltd.) were selected. The mice were anesthetized with isoflurane. A small hole was drilled with a dental drill at a position 2 mm to the right of the midpoint of the midline of the mouse brain. 1×10 4 GFP-CT2A cells were injected to a depth of 2 mm through a microinjector. The injection time was 3 minutes, the injection volume was 1.5 μL. After injection, the mouse was left to stand for 3 minutes, and then the injection needle was slowly withdrawn and the epidermis was sutured and disinfected. Three days later, tumors formed on the surface of the mouse brain, which were used for two-photon imaging.

[0173] Window making: A piece of skull was shaved off flat with a blade at the tumor-forming position to expose the tumor and nearby small blood vessels without damaging the dura mater. After complete hemostasis, cortex buffer was dropped, and a coverslip with a diameter of 3 mm was covered. The skull around the coverslip was dried, 1% low melting point agarose gel was dropped to preliminarily fix the coverslip. After the gel solidified, the gel outside 0.5 mm around the coverslip was removed, the skull was dried, and n-butyl cyanoacrylate bioadhesive was dropped to completely cover the gel and the edge of the coverslip.

[0174] Imaging: The mice were maintained under anesthesia with isoflurane. Saline was dropped on the window-making coverslip for imaging. The focal plane was at the tumor. One slice was scanned every 11 seconds, and while continuously imaging, the DiD-labeled liposomes prepared by the method of Example 3 were injected into the tail vein.

[0175] As Figure 14As shown, over time, the liposome signal in the tumor region gradually increases, while there is no signal enhancement in the normal brain tissue region.

[0176] 2) Two-photon imaging of neutrophils carrying liposomes across the BBB

[0177] Model establishment: Female C57b mice aged 6 - 8 weeks (purchased from Beijing Speyford Co., Ltd.) were selected. The mice were anesthetized with isoflurane. A small hole was drilled at a position 2 mm to the right of the midpoint of the midline of the mouse brain using a dental drill. 1×10 4 GFP-CT2A cells were injected to a depth of 2 mm through a microinjector over 3 minutes with an injection volume of 1.5 μL. After injection, the mice were left to stand for 3 minutes, and then the injection needle was slowly withdrawn and the epidermis was sutured and disinfected. Three days later, tumors formed on the brain surface of the mice, which were used for two-photon imaging.

[0178] Window making: A piece of skull was shaved off flat with a blade at the tumor position to expose the tumor and nearby small blood vessels without damaging the dura mater. After complete hemostasis, cortexbuffer was dropped, and a coverslip with a diameter of 3 mm was covered. The skull around the coverslip was dried, 1% low melting point agarose gel was dropped to initially fix the coverslip. After the gel solidified, the gel outside 0.5 mm around the coverslip was removed, the skull was dried, and n-butyl cyanoacrylate bioadhesive was dropped to completely cover the gel and the edge of the coverslip.

[0179] Imaging: The mice were maintained under isoflurane anesthesia. Saline was dropped on the window-making coverslip for imaging. The focusing plane was at the small blood vessels near the tumor. Scanning was performed every 11 seconds through time series, and a mixed solution of DiD-labeled liposomes and Ly6G neutrophil antibody prepared by the method of Example 3 was injected via the tail vein while continuously imaging.

[0180] As Figure 15 indicated by the arrow, a neutrophil carrying liposomes can be seen rolling from the inner wall of the blood vessel and passing through the vascular endothelium to reach the brain tissue, that is, crossing the blood-brain barrier. Over time, neutrophils carrying liposomes gradually migrate towards the inflammation and reach the tumor region.

[0181] Example 12: Experiment on the distribution of liposomes in the brain of LPS encephalitis model

[0182] 1) Female C57b mice aged 6 - 8 weeks (purchased from Beijing Speyford Co., Ltd.) were selected. The mice were anesthetized with isoflurane. A small hole was drilled at a position 2 mm to the right of the midpoint of the midline of the mouse brain using a dental drill. 0.5 μL of LPS saline solution was injected to a depth of 3 mm through a microinjector over 1 minute with an injection volume of 1 μL. Then the injection needle was slowly withdrawn and the epidermis was sutured and disinfected.

[0183] 2) After 12 h, the encephalitis model mice were randomly grouped and 200 μL of the liposomes with different ratios of functional components labeled with DiD prepared by the method of Example 3 was injected into the tail vein. The mice were anesthetized and sacrificed for perfusion 2 h after injection, and the brain tissues were collected for ex vivo imaging through the IVIS Lumina system.

[0184] As Figure 16 shown, obvious DiD fluorescence signals of liposomes in each group (Group A 100:0, Group B 75:25, Group C 50:50, Group D 25:75, Group E 0:100) were observed at the injection site. Among them, the signal of Group D was the strongest, indicating that the synergistic effect of Group D was the best at the in vivo level and the liposomes were most effectively delivered to the brain inflammation area.

[0185] Example 13: Experiment on the distribution of liposomes in the brain tumor model

[0186] 1) Establishment of the brain tumor model and ex vivo imaging

[0187] Female C57b mice aged 6 - 8 weeks (purchased from Beijing Speywood Co., Ltd.) were selected. The mice were anesthetized with isoflurane, and a small hole was drilled with a dental drill at a position 2 mm to the right of the midpoint of the brain midline of the mice. 1×10 4 CT2A cells were injected to a depth of 3 mm through a micro syringe. The injection time was 3 min, the injection volume was 1.5 μL. After injection, the mice were allowed to stand for 3 min, and then the injection needle was slowly withdrawn and the epidermis was sutured and disinfected.

[0188] After 5 days, the mice developed tumors. The brain tumor model mice were randomly grouped and 200 μL of the liposomes with different ratios of functional components labeled with DiD was injected into the tail vein. The mice were anesthetized and sacrificed for perfusion 2 h after injection, and the main organs (heart, liver, spleen, lung, kidney) and brain tissues were collected for ex vivo imaging through the IVIS Lumina system.

[0189] As Figure 17 shown, obvious DiD fluorescence signals of liposomes in each group (Group A 100:0, Group B 75:25, Group C 50:50, Group D 25:75, Group E 0:100) were observed at the tumor site of the brain tissues. Among them, the signal of Group D was the strongest, indicating that the synergistic effect of Group D was the best at the in vivo level and the liposomes were most effectively delivered to the brain inflammation area, and only a small part of the liposomes was metabolized in the liver, and the targeting effect was very good.

[0190] 2) Brain tissue sectioning and fluorescence staining

[0191] The brains of the mice in 1) above were frozen sectioned, and the sections were stained with DAPI and NETs.

[0192] Immunofluorescence staining: Take the frozen sections out from -80°C and let them return to room temperature. Moisten the sections with PBS for 15 minutes, block them with 5% BSA (Sigma) for 1 hour, then add the primary antibody against NETs (the two species are different), and incubate overnight at 4°C in the dark. After 12 hours, wash off the primary antibody with PBS, and add the corresponding fluorescent secondary antibody. After 1 hour, mount the slides and take pictures for confocal microscopy analysis.

[0193] The results are as Figure 18 shown. The signal intensity of liposomes in the brain tumor region of each group is much higher than that in normal brain tissue, and the signal of liposome D in the brain tumor region is stronger. As Figure 19 shown, taking the liposome D group as an example, the signals of liposomes and the signals of neutrophils releasing NETs are well co-localized, proving that after reaching the brain tumor region, neutrophils extrude their contents through NETs, including liposome components.

[0194] Example 14: Confocal microscopy of the formation and release of liposomes by neutrophil NETs

[0195] Induce the formation of NETs by in vitro inflammation, and use confocal microscopy to observe whether liposomes can be separated from NETs. The experimental steps are as follows: 1) Incubate the DiD-labeled liposomes synthesized by the method of Example 3 with inflammatory mouse serum for 30 min to form a protein corona, and then add the neutrophils purified by the method of Example 5 and incubate for 1 h;

[0196] 2) Fix the neutrophils with 4% paraformaldehyde at different time points, block the protein, specifically stain NETs with the CitH3 antibody, wash, stain with the secondary antibody, and finally stain with DAPI and mount the slides;

[0197] 3) Take confocal pictures of the cells. The results are as Figure 20 shown. The time marked above the figure is the duration of adding LPS to induce the formation of NETs. Fix the cells at 0 h, 2 h, 4 h, and 6 h respectively for NETs staining and observation. The upper row is the image obtained by confocal microscopy, and the lower row is the line-scan co-localization analysis at the corresponding time points. The Manders co-localization coefficient is marked in the upper right corner, with a value ranging from 0 to 1. The closer the value is to 1, the higher the co-localization degree. At the cellular level, neutrophils release liposomes by forming NETs, and liposomes can be successfully separated from NETs, providing a basis for further uptake of liposomes by tumor cells. The results show that NETs are not fully formed at 0 h, are fully formed at 2 h and have a strong co-localization with liposomes (Manders co-localization coefficient 0.941), liposomes gradually release from NETs at 4 h and the co-localization decreases, and a large amount of liposomes are released at 6 h and the co-localization further decreases.

[0198] Example 15: Specific uptake experiment of liposomes by various cells

[0199] Using normal mouse brain cells C8D1A and HT-22, mouse-derived glioma cells CT2A and GL261, and human-derived glioma cells A172, U87, and LN229R as models, the uptake levels of liposomes by these cells were examined by flow cytometry for differences. The experimental steps are as follows:

[0200] 1) Incubate liposomes with inflammatory mouse serum for 30 min to form a protein corona, then add purified neutrophils and incubate for 1 h, and centrifuge and wash the neutrophils;

[0201] 2) Add neutrophils to the well plates of various cells, add LPS to induce the formation of NETs, and incubate for 9 h;

[0202] 3) Detach the cells, fix and wash them, and perform flow analysis.

[0203] The process is as Figure 21 shown in Figure 21 a, and the results are as Figure 21 shown in b. The uptake levels of normal mouse brain cells HT-22 and C8D1A are extremely low, the uptake of mouse-derived glioma cell CT2A is relatively high, and the uptake levels of human-derived glioma cells U87, A172, and LN229R are extremely high. It is proved that there is selectivity in the uptake of liposomes by cells. Before adding various cells for uptake, adding 1 mM FA to block folate receptors can significantly reduce the uptake level of liposomes (

[0204] Example 16: Flow cytometry analysis of whole brain cells

[0205] By the method for constructing a brain tumor model in Example 13, a CT2A brain tumor model was constructed by stereotactic injection of CT2A-GFP cells into C57b mice, and a U87 brain tumor model was constructed by stereotactic injection of U87-GFP cells into nude mice. After tail vein injection of liposomes labeled with a fluorescent probe, the brain was taken to specifically stain all cells, and the uptake levels of different types of brain cells for the material were analyzed by flow cytometry. The experimental steps are as follows:

[0206] 1) Inject DiD-labeled liposomes synthesized by the method in Example 3 into the tail vein of C57BL / 6 mice with CT2A brain tumors or nude mice with U87 brain tumors. After 9 h, anesthetize the mice, perfuse with warm PBS at a flow rate of 3 mL / min, dissect to obtain the whole brain, and immerse it in ice-precooled 1×HBSS;

[0207] 2) Homogenize the brain using a homogenizer for ice sitting, sieve through a 70 nm cell sieve, perform isopycnic centrifugation using 30% Percoll, discard the uppermost lecithin disc, wash with 1×HBSS and collect the cells in the lower layer;

[0208] 3) Add various antibodies to specifically stain the cells: including CD45, CD11b, ACSA2, CC1, Thy1, CD31. The cells that can be distinguished by gating include: glioma cells (GFP), microglia, macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons, endothelial cells.

[0209] 4) Analyze the uptake of liposomes by various cells through flow cytometry.

[0210] The process is as Figure 22 shown in Figure 22 a, and the results are as Figure 22 shown in b and

[0211] Example 17: Neutrophil toxicity experiment of PTX-encapsulated liposomes

[0212] Test method: Prepare a liposomal drug delivery system encapsulating PTX by the method in Example 1, and perform a cytotoxicity experiment by the CCK-8 method. Experimental steps:

[0213] 1. Use 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Extract neutrophils from bone marrow, obtain them by density gradient centrifugation, and seed 1×10 5 cells per well after counting.

[0214] 2. Add liposomes or Taxol with a concentration gradient of PTX (100, 50, 25, 12.5, 6.125, 0 μg / mL), with a final volume of 100 μL per well.

[0215] 3. Place the 96-well plate in a 5% CO2, 37 °C incubator and culture for 4 hours.

[0216] 4. Add 12 μL of CCK-8 working solution to each well and continue to culture for 4 hours.

[0217] 5. Terminate the culture. Measure the absorbance of each well at OD490 nm using an enzyme-linked immunosorbent assay reader. The cell survival rate can be calculated by the following formula: Cell survival rate = (OD 450 (samples) / (OD 450 (control) × 100%.

[0218] From Figure 23From the cytotoxicity results of the CCK-8 method, it can be seen that under the same PTX content, liposome-encapsulated PTX has lower toxicity to neutrophils than Taxol used clinically, proving its safety for targeting neutrophils.

[0219] Example 18: Cell specificity experiment of liposomes encapsulating PTX

[0220] Prepare a liposomal drug delivery system encapsulating PTX by the method in Example 1

[0221] 1. Use DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. Incubate C8D1A or HT-22 or CT2A cells in a 37°C, 5% CO2 cell culture incubator, and pre-culture the cells until confluence before the experiment. Collect cells in the logarithmic phase, adjust the cell suspension concentration, add 100 μL to each well, and plate to adjust the density of the cells to be tested to 10,000 per well. Fill the edge wells with sterile PBS.

[0222] 2. Incubate at 5% CO2, 37°C until the cell monolayer covers the bottom of the well (96-well flat bottom plate), and add liposomes of Group D with a concentration gradient of PTX (100, 50, 25, 12.5, 6.125, 0 μg / mL).

[0223] 3. Place the 96-well plate in a 5% CO2, 37°C incubator and culture for 5 hours.

[0224] 4. Add 12 μL of CCK-8 working solution to each well and continue to culture for 0.5 hours.

[0225] 5. Terminate the culture. Measure the absorbance of each well at OD450nm using an enzyme-linked immunosorbent detector. The cell survival rate can be calculated by the following formula: Cell survival rate = (OD 450 (samples) / (OD 450 (control) × 100%.

[0226] From Figure 24 the cytotoxicity results of the CCK-8 method, it can be seen that glioma cells CT2A have a higher uptake of PTX liposomes than normal cells (C8D1A and HT-22), proving the specificity of glioma cells for this liposome.

[0227] Example 19: CT2A glioma chemotherapy experiment with liposomes encapsulating PTX

[0228] Prepare a liposomal drug delivery system encapsulating PTX by the method in Example 1

[0229] 1) Establish a brain tumor model

[0230] The brain tumor model was constructed by the method of Example 13. Female C57b mice aged 6 - 8 weeks (purchased from Beijing Speywood Co., Ltd.) were selected. The mice were anesthetized with isoflurane. A small hole was drilled at a position 2 mm to the right of the midpoint of the brain midline of the mice using a dental drill. 1×10 4 Luc-CT2A cells were injected to a depth of 3 mm through a micro syringe. The injection time was 3 min, the injection volume was 1.5 μL. After injection, the cells were allowed to stand for 3 min, and then the injection needle was slowly withdrawn and the epidermis was sutured and disinfected. The day of tumor implantation was defined as day 0.

[0231] Three days later, tumors formed in the mice. The mice with brain tumor models were randomly grouped and injected with PBS, Taxol or So-lipo-PTX via the tail vein. The PTX content in the latter two groups was quantified by HPLC to be the same. The drugs were administered once every 1 day for a total of 6 times. The dosage of PTX was 3 mg / kg body weight. As Figure 25 shown in a, it was the time axis of treatment. Every 2 days, ex vivo imaging was performed using the IVIS Lumina system. The results were as Figure 25 shown in b. Compared with the blank control group PBS and the clinical paclitaxel injection Taxol, the liposome group showed the best therapeutic effect. The body weight was monitored as Figure 26 shown in a. The body weight of the mice in the liposome group did not decrease significantly. The survival curve was as Figure 26 shown in b. The mice in the liposome group had the longest median survival time. The mice without survival curve monitoring had their brains removed after treatment for H&E, TUNEL, and Ki67 staining. Among them, the liposome group and the group with similar imaging effects had the smallest volume of tumors in the H&E sections, and showed the effect of inhibiting tumor proliferation through fluorescence staining (as Figure 26 shown in c).

[0232] Example 20: CT2A Glioblastoma Chemotherapy Experiment of CCNU-Encapsulated Liposomes

[0233] The liposome drug delivery system encapsulating CCNU was prepared by the method in Example 1

[0234] 1) Establishment of brain tumor model

[0235] The brain tumor model was constructed by the method of Example 13. Female C57b mice aged 6 - 8 weeks (purchased from Beijing Speywood Co., Ltd.) were selected. The mice were anesthetized with isoflurane. A small hole was drilled at a position 2 mm to the right of the midpoint of the brain midline of the mice using a dental drill. 1×10 4Luc-CT2A cells were injected to a depth of 3 mm for 3 minutes with an injection volume of 1.5 μL. After injection, the cells were allowed to stand for 3 minutes, and then the injection needle was slowly withdrawn. The epidermis was sutured and disinfected. The day of tumor implantation was defined as day 0. Three days later, when tumors developed in the mice, the mice with brain tumor models were randomly grouped and injected with PBS, CCNU, or So-lipo-CCNU via the tail vein. Drugs were administered once every other day, and the radiotherapy group was irradiated. A total of three doses were administered (the treatment timeline is as shown in Figure 27 a). Every two days, ex vivo imaging was performed using the IVIS Lumina system. The results are shown in Figure 27 b. The tumor growth curves of each individual were statistically analyzed, as shown in Figure 27 c. The liposome combined with radiotherapy group, So-lipo-CCNU + RT, showed the best therapeutic effect. Body weight monitoring is shown in Figure 28 a. The body weight of the mice in the So-lipo-CCNU + RT group of the liposome combined with radiotherapy group did not significantly decrease. The survival curve is shown in Figure 28 b. The mice in the So-lipo-CCNU + RT group of the liposome combined with radiotherapy had the longest median survival time. Mice without survival curve monitoring had their brains removed after treatment for H&E, TUNEL, and Ki67 staining. Among them, the liposome group and the imaging group had tumors with the smallest volume in the H&E sections and showed an effect of inhibiting tumor proliferation through fluorescence staining (as shown in Figure 28 c).

[0236] H&E and TUNEL staining

[0237] Steps for H&E staining: The mouse tumor cells were fixed in 4% paraformaldehyde (sigma) for 24 hours and then sent to Tianjin Yishengyuan Biotechnology Co., Ltd. for paraffin sectioning and H&E staining experiments. Subsequently, microscopic analysis was performed.

[0238] Steps for TUNEL staining: The mouse tumors were processed for cryosectioning. After being moistened with PBS for 15 minutes, staining was performed following the experimental manual provided by Roach Company.

[0239] Flow cytometry: The freshly harvested tumor tissues were cut into small pieces and homogenized using a GentleMACs Dissociator. Then, the tumor homogenate was filtered through a 70×10 -6 μm cell strainer to prepare a single-cell suspension. The cells were collected and diluted to 1×10 7 cells / mL. Fluorescent antibody-conjugated primary antibody was added to the cell suspension and incubated at room temperature for 30 minutes. After staining, the cells were fixed with 4% paraformaldehyde and analyzed using a flow cytometer.

[0240] Industrial applicability

[0241] The present invention provides a liposomal drug delivery system for specifically delivering small molecule drugs and siRNA into gliomas by targeting neutrophils in vivo and crossing the BBB, and its application. The liposome of the present invention can be made into corresponding pharmaceutical compositions together with appropriate active drug molecules, etc., and is suitable for industrial application.

Claims

1. A liposomal drug delivery system for targeted treatment of glioma, characterized in that, A liposome mixture prepared from the following components: a basic component, a functional component, and an external aqueous phase buffer; The basic component and the functional component are collectively referred to as lipid molecules, and the molar ratio of the basic component to the functional component is 3:7; the drug content is changed according to the therapeutic concentration required for different drugs in the body, ranging from 1 to 100 mg / mL, and the molar ratio of the drug to the lipid molecules is 1:3 - 1:20; the volume of the external aqueous phase buffer is changed as needed, and the final liposome concentration is made 0.1 - 100 mg / mL, preferably 10 mg / mL, by adding the external aqueous phase buffer; The liposome drug delivery system is prepared by the thin film hydration method, including: (1) Mix the solutions of lipid molecules and drugs evenly; (2) Form a lipid film by the reduced pressure distillation method and vacuum dry overnight; (3) Add the external aqueous phase buffer and perform ultrasonic dispersion; (4) Extrude through a polycarbonate membrane; (5) Purify by dialysis in an external aqueous phase buffer at 4°C through a dialysis bag with a molecular weight of 8k - 14kDa to prepare the liposome drug delivery system.

2. The liposomal drug delivery system for targeted treatment of glioma according to claim 1, characterized in that, The functional component contains one or both of the following two lipid molecules: (1) An amphiphilic lipid molecule with a hydrophilic trans-phosphatidylcholine (CP) structure at one end and a lipophilic end at the other end, and the structure is shown in the general formula (2-1); (2) An amphiphilic lipid molecule with a hydrophilic folic acid (FA) structure at one end and a lipophilic end at the other end, and the structure is shown in the general formula (2-2); The term "amphiphilic lipid molecule" refers to a molecule with a hydrophilic end, an arbitrary linking chain in the middle, and a hydrophobic end which is a saturated or unsaturated alkyl chain represented by R at the other end; among them, the definition of the saturated or unsaturated alkyl chain is a cyclic or acyclic, straight-chain or branched-chain-tailed, multi-tailed, saturated or unsaturated-bond-containing, aryl, heteroaryl, aryl- or heteroaryl-substituted alkyl chain with 3 to 50 carbon atoms, and the R is a saturated or unsaturated alkyl chain. The saturated alkyl chains include: cinnamoyl, myristoyl, palmitoyl, stearoyl, and erucoyl; the unsaturated alkyl chains include: oleoyl, linoleoyl, dilinoleoyl, and linolenoyl; the hydrophobic alkyl chain is coupled to CP or FA via an arbitrary linking chain; among them, the molar ratio of the two lipid molecules CP lipid molecule and FA lipid molecule contained in the above functional component is 100:0 to 0:

100.

3. The liposomal drug delivery system for targeted treatment of glioma according to claim 2, characterized in that, The molar ratio of the two lipid molecules CP lipid molecule and FA lipid molecule contained in the functional component is preferably one of 100:0, 75:25, 50:50, 25:75, 0:

100.

4. The liposomal drug delivery system for targeted treatment of glioma according to claim 1, characterized in that, The basic component includes neutral lipids, cationic lipids, and / or anionic lipids; (1) The neutral lipids include: cholesterol and its derivatives in steroids; phospholipids include one or more of lecithin, soybean phospholipid, cephalin, sphingomyelin, and hydrogenated soybean phospholipid; The molar percentage of the steroid in the total moles of the mixture of the basic component and the functional component is 10%-90%, preferably 30%; the neutral lipid contains at least one phospholipid and cholesterol; the molar percentage of the cholesterol in the total moles of the basic component is 0.1%-90%, preferably 20%-60%; the molar percentage of the lipid molecules in the total moles of all components of the liposome mixture is 10%-99%, preferably 20%-70%; (2) The cationic lipid or anionic lipid is selected from any one of N,N-dioleyl-N,N-dimethylammonium chloride, N,N-distearyl-N,N-dimethylammonium bromide, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, N,N-dimethyl-2,3-dioleyloxypropylamine, 3-(dilauryldiamino)-N1,N1,4-tridodecyl-1-piperazineethanamine, N1-[2-(dilauryldiamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine, 14,25-bis(tridecyl)-15,18,21,24-tetraaza-38-ane, 1,2-dilinoleyloxy-N,N-dimethylaminopropane, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane, 4-(dimethylamino)butyric acid heptatriaconta-6,9,28,31-tetraen-19-yl ester, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium (DOSPA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester DLin-MC3-DMA, SM-102 and mixtures thereof.

5. The liposomal drug delivery system for targeted treatment of glioma according to claim 4, wherein The phospholipids include one or more of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), dioleoylphosphatidylcholine (DOPC), egg yolk phosphatidylcholine (EPC), dierucoylphosphatidylcholine (DEPC), dilauroylphosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dipalmitoyl glycerophosphoglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoyl phosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoylphosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoylphosphatidylinositol (DMPI) or distearoylphosphatidylinositol (DSPI).

6. The liposomal drug delivery system for targeted treatment of glioma according to claim 1, characterized in that, The outer aqueous phase buffer is physiological saline, acetate, succinate, gluconate, histidine, citrate, carbonate, PBS, HEPES or other organic acid buffers, preferably HEPES buffer; the concentration of the buffer is a conventional concentration, preferably an isotonic concentration.

7. The liposomal drug delivery system for targeted treatment of glioma according to claim 1, wherein, The liposome drug delivery system can encapsulate nucleic acid drugs or anti-tumor agents.

8. The liposomal drug delivery system for targeted treatment of glioma according to claim 7, wherein The nucleic acid drugs are selected from any one of DNA, antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme and siRNA; the anti-tumor agents include drugs for treating tumors and infectious diseases, specifically including but not limited to drugs for treating one or more of the following diseases: breast cancer, lung cancer, melanoma, liver cancer, kidney cancer, brain tumor, myocardial infarction, stroke, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, pneumonia, nephritis and various immunomodulatory drugs.

9. The liposomal drug delivery system for targeted treatment of glioma according to claim 8, wherein The anti-tumor agent drugs described above are selected from small molecule, nucleic acid or protein drugs with the ability to kill glioblastoma, inhibit the growth of glioblastoma, or synergistically enhance the sensitivity in combination with commonly used clinical treatment strategies such as surgery, radiotherapy, chemotherapy, and immunotherapy; among them, the small molecule drugs include but are not limited to: paclitaxel, doxorubicin, adriamycin, lomustine, carmustine, nimustine, tirapazamine, nimorazole, temozolomide, platinum-based drugs, stiripentol, imatinib, dasatinib, ponatinib, gefitinib, icotinib, erlotinib, osimertinib, apaciclib, apatinib, fruquintinib, toceranib, cediranib, palbociclib, ribociclib, abemaciclib, talazoparib, olaparib, iniparib, niraparib, vismodegib, sonidegib, daurismo, everolimus, azd8055, azd2014, temsirolimus, al3810, scc-31, sunitinib, regorafenib, sorafenib, vandetanib, cabozantinib, lenvatinib, pazopanib, vemurafenib, axitinib, dabrafenib, linifanib, nintedanib, or any combination thereof; The anti-tumor protein drugs described above include but are not limited to: bevacizumab, atezolizumab, sirolimus, interferon; the nucleic acid drugs include but are not limited to: siRNA, miRNA, ASO targeting the MGMT, MET / HGF, p38MAPK drug resistance pathway, oncogene inhibitors, epidermal growth factor receptor inhibitors, and transforming growth factor-β inhibitors.

10. Use of the liposomal drug delivery system for targeted treatment of glioblastoma according to any one of claims 1 to 9 in the preparation of a tumor cell preparation with high folate receptor expression.

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