Nano-drug for treating glioma as well as preparation method and application of nano-drug

Abstract

The invention provides a nano-drug for treating glioma as well as a preparation method and application of the nano-drug, and particularly, the nano-drug comprises a nano-carrier and a carrier drug. The nano-drug can interact with a receptor on a brain endothelial cell, enters a brain tumor microenvironment through an endocytosis way, promotes antigen presenting cell maturation, activates T cell anti-tumor immune response, and effectively inhibits tumor growth.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a bionic nano drug composition and a preparation method thereof, and application of the bionic nano drug composition in preparing drugs for treating brain gliomas. Background Art

[0002] Glioma is a primary malignant tumor of the central nervous system, accounting for about 40% to 45% of intracranial tumors. Glioma has the characteristics of high morbidity, high mortality, high recurrence rate, and low cure rate. The treatment of glioma is mainly surgical resection, which usually needs to be combined with radiotherapy and chemotherapy. Due to the high malignancy of glioma, no clear boundaries, and very easy invasion to other parts of the brain, it is difficult to completely eradicate it by surgical resection. Temozolomide is a first-line drug for the treatment of glioma. It has the advantages of easy penetration of the blood-brain barrier, good tolerance, no superimposed toxicity with other drugs, and synergistic efficacy with radiotherapy. However, due to the complex brain structure, strong tumor heterogeneity, and high invasiveness, it is difficult for therapeutic drugs to effectively accumulate at the tumor site, and the treatment of glioma is still very difficult.

[0003] In recent years, the immunotherapy of gliomas has attracted widespread attention. Emerging immunotherapy methods such as immune checkpoint inhibitors, chimeric antigen receptor T cells, peptide vaccines and dendritic cell vaccines have been used in the treatment of brain gliomas. Although many emerging immunotherapies can inhibit tumor growth in mouse models, the results of clinical trials are not satisfactory, and the immunotherapy of gliomas is still very difficult. Compared with other tumor types, gliomas, as typical "cold tumors", have fewer tumor-infiltrating lymphocytes and other immune effector cells. Even if T cell responses are induced by vaccination and other methods, the number of antigen-specific T cells is still relatively low, and immune cells often show an exhausted phenotype. The regulation of the brain tumor microenvironment involves multiple immunosuppressive mechanisms at the molecular and cellular levels. In-depth research on the complex immunosuppressive microenvironment of gliomas can help researchers understand the pathogenesis of brain tumors and provide new ideas for the design of glioma therapeutic drugs. However, how therapeutic drugs can pass through the complex structure of the brain, penetrate the blood-brain barrier, and reverse the complex immunosuppressive microenvironment of gliomas is still very difficult. Summary of the invention

[0004] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a drug for treating glioma. To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a nano drug composition, the nano drug composition comprises a nano carrier and a carrier drug, the nano carrier comprises a first phospholipid, an apolipoprotein mimetic, and a second phospholipid modified with a functional group, the carrier drug comprises a polypeptide, the polypeptide comprises a polypeptide with blood-brain barrier targeting ability, and / or a glioma-specific antigen peptide, the polypeptide with blood-brain barrier targeting ability comprises iRGD, and / or angiopep-2, and the glioma-specific antigen peptide is EphA 2671-679 .

[0006] In the present invention, the second phospholipid modified with functional groups is a phospholipid modified with small molecule functional groups at the active site. The introduction of these functional groups allows the phospholipid to further react with oligonucleotides, small molecules, polypeptides, etc. to form a phospholipid-drug conjugate.

[0007] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0008] In one embodiment of the present invention, the EphA 2671-679 The sequence is shown in SEQ ID NO.10.

[0009] In one embodiment of the present invention, the polypeptide having blood-brain barrier targeting ability is angiopep-2. As a more preferred embodiment, the sequence of angiopep-2 is shown in SEQ ID NO.13.

[0010] In one embodiment of the present invention, the molar ratio of angiopep-2 to the second phospholipid modified with a functional group is 1:10-5:1. As a preferred embodiment, the molar ratio of angiopep-2 to the second phospholipid modified with a functional group is 0.75:1.

[0011] In one embodiment of the present invention, the EphA 2671-679 The molar ratio of the EphA to the second phospholipid modified with the functional group is 1:10-5:1. As a preferred embodiment, the EphA 2671-679 The molar ratio of the first phospholipid to the second phospholipid modified with the functional group is 0.75:1.

[0012] In one embodiment of the present invention, the drug carrier further comprises an oligonucleotide.

[0013] In the present invention, the oligonucleotides include but are not limited to antisense oligonucleotides (ASO), siRNA (small interfering RNA), microRNA and aptamer.

[0014] In one embodiment of the present invention, the oligonucleotide comprises a CpG oligonucleotide and / or a siRNA. As a preferred embodiment, the sequence of the CpG oligonucleotide is shown in SEQ ID NO. 11. As a preferred embodiment, the siRNA is a siRNA that specifically interferes with SOCS1 mRNA, and more preferably, the sequence of the siRNA that specifically interferes with SOCS1 mRNA is shown in SEQ ID NO. 12.

[0015] In one embodiment of the present invention, the ratio of the CpG oligonucleotide to the nanocarrier is 0.05-5nmol CpG oligonucleotide / mg nanocarrier. As a preferred embodiment, the ratio of the CpG oligonucleotide to the nanocarrier is 0.40nmol CpG oligonucleotide / mg nanocarrier. In a specific embodiment of the present invention, the nanocarrier is a nanocarrier (THDL) loaded with Ang.

[0016] In one embodiment of the present invention, the ratio of the siRNA to the nanocarrier is 0.05-5nmol siRNA / mg nanocarrier. As a preferred embodiment, the ratio of the siRNA to the nanocarrier is 0.15nmol siRNA / mg nanocarrier. In a specific embodiment of the present invention, the nanocarrier is a nanocarrier (THDL) loaded with Ang.

[0017] In one embodiment of the present invention, the oligonucleotide contains cholesterol modification.

[0018] In one embodiment of the present invention, the present invention performs 2′-fluoropyrimidine modification on siRNA′, that is, 2′-OH of the pyrimidine nucleotide on the siRNA is replaced with 2′-F.

[0019] In one embodiment of the present invention, the carrier drug further comprises one or more of mRNA, antibodies, and small molecule drugs.

[0020] In one embodiment of the present invention, the small molecule drugs include anti-tumor drugs, developing tracer dyes, and photoacoustic and photothermal agents.

[0021] In the present invention, the anti-tumor drugs include but are not limited to doxorubicin, temozolomide, docetaxel, paclitaxel, oxaliplatin, bortezomib, mitoxantrone, and lomustine.

[0022] In the present invention, the developing tracer dyes include but are not limited to indocyanine green, DiD, Dil, and Cy7.

[0023] In one embodiment of the present invention, the apolipoprotein mimetic is an ApoA-I mimetic peptide.

[0024] In the present invention, apolipoprotein A1 (ApoA1, ApoA-I) is the main protein component of plasma high-density lipoprotein.

[0025] In one embodiment of the present invention, the molar ratio of the first phospholipid to the second phospholipid modified with functional groups is 99:1-50:50. As a preferred embodiment, the molar ratio of the first phospholipid to the second phospholipid modified with functional groups is 9:1.

[0026] In one embodiment of the present invention, the first phospholipid is selected from hydrogenated soybean phosphatidylcholine, dierucoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, dierucoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1,2-dicinnamoyl-sn -glycerol-3-phosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylglycerol, 1,2-palmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidic acid, 1,2-dioleoylglycerol-3-phosphoethanolamine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, (2,3-dioleoyl-propyl)-trimethylamine, fat egg yolk lecithin PC-98T, egg yolk lecithin PL-100M, egg yolk phosphatidylglycerol, soybean lecithin, or one or more thereof. In a specific embodiment of the present invention, the first phospholipid is 1,2-dicinnamoyl-sn-glycerol-3-phosphatidylcholine.

[0027] As a preferred embodiment, the functionalized functional group includes but is not limited to maleimide, 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester, and DBCO. More preferably, the functionalized functional group is maleimide.

[0028] In one embodiment of the present invention, the second phospholipid is selected from hydrogenated soybean phosphatidylcholine, dierucoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, dierucoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1,2-dicinnamoyl-sn-glycero-3-phosphatidylcholine, 1-palmitoyl- One or more of 2-oleoylphosphatidylglycerol, 1,2-palmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidic acid, 1,2-dioleoylglycerol-3-phosphoethanolamine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, (2,3-dioleoyl-propyl)-trimethylamine, egg yolk lecithin PC-98T, egg yolk lecithin PL-100M, egg yolk phosphatidylglycerol, and soybean lecithin. In a specific embodiment of the present invention, the second phospholipid is 1,2-dioleoylglycerol-3-phosphoethanolamine.

[0029] In one embodiment of the present invention, the second phospholipid modified with functional groups is prepared by condensation reaction of 1,2-dioleoylglycerol-3-phosphoethanolamine and succinimidyl trans-4-(N-maleimidomethyl)cyclohexane ester.

[0030] In one embodiment of the present invention, the sequence of the ApoA-I mimetic peptide is shown as SEQ ID NO. 1 to 9. As a preferred embodiment, the sequence of the ApoA-I mimetic peptide is shown as SEQ ID NO. 1 or SEQ ID NO. 9.

[0031] In the present invention, the nanocarrier has a spherical structure and an average particle size of 8-30 nm.

[0032] In a second aspect, the present invention provides a pharmaceutical preparation, wherein the pharmaceutical preparation comprises the nanopharmaceutical composition described in the first aspect of the present invention, and a pharmaceutically acceptable excipient, carrier or diluent.

[0033] In some embodiments, the pharmaceutical formulation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal.

[0034] In a third aspect, the present invention provides a method for preparing the nano drug composition according to the first aspect of the present invention, the method comprising:

[0035] (1) preparing nanocarriers;

[0036] (2) connecting the polypeptide to a second phospholipid modified with a functional group to obtain a polypeptide-phospholipid complex;

[0037] (3) Adding the polypeptide-phospholipid complex to the nanocarrier and filtering.

[0038] In one embodiment of the present invention, step (2) is as follows:

[0039] The polypeptide and the second phospholipid modified with the functional group are weighed, completely dissolved in ultra-dry DMF, dialyzed, frozen in liquid nitrogen and then dried to obtain a polypeptide-phospholipid complex.

[0040] In one embodiment of the present invention, step (1) is as follows:

[0041] The first phospholipid and the second phospholipid modified with functional groups are dissolved in an organic solvent, suspended to dryness, and vacuumed. The obtained lipid membrane is hydrated with a buffer solution, ultrasonicated, and an apolipoprotein mimic dissolved in the buffer solution is added. The "heating-cooling-heating" operation is cycled three times, and the nanocarrier is obtained after filtering, ultrafiltration, concentration, and freeze-drying.

[0042] As a preferred embodiment, the molar ratio of the apolipoprotein mimetic to the lipid membrane is 1:2-1:20. In a specific embodiment of the present invention, the sequence of the apolipoprotein mimetic is as shown in SEQ ID NO.1, and the molar ratio of the apolipoprotein mimetic to the lipid membrane is 1:7.5.

[0043] As a preferred embodiment, the organic solvent is DCM.

[0044] As a preferred embodiment, preferably, the buffer is PBS buffer.

[0045] In one embodiment of the present invention, the method further comprises mixing a cholesterol-modified oligonucleotide with the nanocarrier.

[0046] In a fourth aspect, the present invention provides the following application:

[0047] (1) Use of the nano drug composition described in the first aspect of the present invention in the preparation of anti-tumor drugs;

[0048] (2) Use of the nanomedicine composition described in the first aspect of the present invention in the preparation of drugs for treating brain diseases;

[0049] (3) Application of SOCS1 as a target in screening and / or preparing drugs for treating glioma.

[0050] In one embodiment of the present invention, the tumor is a glioma.

[0051] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0052] The present invention combines glioma blood-brain barrier penetration, tumor microenvironment targeting and tumor microenvironment regulation, prepares a biomimetic nanocarrier with targeting ability, and proposes SOCS1 as a new target for glioma treatment through bioinformatics analysis, and loads siRNA that silences SOCS1 on the nanocarrier. Combined with the characteristics of the glioma immunosuppressive microenvironment, oligonucleotides and polypeptides with glioma microenvironment regulation capabilities are loaded on the nanocarrier to prepare a multifunctional nanomedicine. The nanomedicine can efficiently induce the maturation of bone marrow-derived dendritic cells in vitro and silence SOCS1 in dendritic cells. When the nanomedicine is injected into glioma-bearing mice through the tail vein, the nanocarrier efficiently penetrates the blood-brain barrier and delivers the drug to the brain tumor. The drug in the tumor microenvironment silences SOCS1mRNA, promotes DC maturation and activates T cell anti-tumor immune response, effectively inhibiting tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The expression of SOCS1 was analyzed by bioinformatics. Figure a is a statistical diagram of the expression level of SOCS1 in patients with gliomas of different grades, and Figure b is a statistical diagram of the survival of glioma patients with different SOCS1 expression levels.

[0054] Figure 2 The expression of SOCS1 in clinical tumor samples;

[0055] Figure 3 Figure 2 is the expression of SOCS1 in orthotopic glioma model mice, where Figure a is the SOCS1 mRNA level and Figure b is the SOCS1 protein level;

[0056] Figure 4 Ultra-high resolution transmission electron microscopy results for nanomedicine;

[0057] Figure 5 It is the result of hydrated particle size of nano drug;

[0058] Figure 6 The biodistribution of nanomedicine in mice; Figure a shows the results of biochemiluminescence and brain tissue fluorescence imaging, Figure b shows the distribution statistics of fluorescent dyes in various tissues, and Figure c shows the fluorescence imaging pictures of the main tissues of mice;

[0059] Figure 7 The results of detecting the ability of nanomedicine to target the brain are shown in Figure a, where Figure b is the result of NIR-II imaging and RT-qPCR verification of SOCS1 levels in brain tissue;

[0060] Figure 8 The results of detecting the regulation of SOCS1 expression in APCs by nanomedicine, among which, Figure a is the confocal imaging result of BMDCs, and Figure b is the statistical result of SOCS1 mRNA level in BMDCs;

[0061] Fig. 9 The results of nanomedicine promoting BMDCs are shown in Figure a, where Figure a is the flow cytometry analysis result of BMDCs 24 hours after drug treatment, and Figure b is the statistical result of mature BMDCs;

[0062] Fig.10 The nanomedicine activates the anti-tumor immune response in mice. Figure a shows the CD8 + CD3 + T cell flow cytometry analysis results, Figure b is CD8 + CD3 + T cell percentage statistical results;

[0063] Fig.11 This is a study on the mechanism of nanomedicine regulating the tumor microenvironment in mice. Figure a is the KEGG pathway enrichment analysis of the whole transcriptome sequencing results of mouse brain tissue after drug treatment, Figure b is the heat map of JAK / STAT signaling pathway clustering analysis, and Figure c is the immunoblot analysis of protein expression in mouse brain tissue;

[0064] Fig.12 The ability of nanomedicine to inhibit tumor growth in glioma model mice, where Figure a shows the establishment of the tumor model and drug treatment, Figure b shows the IVIS bioluminescence imaging image, and Figure c shows the change in bioluminescence intensity;

[0065] Fig.13 The ability of nanomedicine to inhibit tumor growth in an orthotopic glioma model, where Figure a is the Kaplan-Meier survival curve of tumor-bearing mice in different treatment groups, Figure b is the weight change curve of treated mice, and Figure c is the H&E staining picture of brain tissue sections;

[0066] Fig.14 It is a synthetic route for functionalized phospholipids;

[0067] Fig.15 It is a synthetic route for peptide-phospholipid complexes. DETAILED DESCRIPTION

[0068] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0069] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0070] Unless otherwise specified, experimental methods, detection methods, and preparation methods not described in detail in the present invention all adopt conventional techniques in the technical field.

[0071] Example 1: Analysis of SOCS1 expression levels

[0072] Given the key role of SOCS1 in anti-tumor immunity, bioinformatics was used to analyze the SOCS1 mRNA level in glioma samples. The corresponding data for GBM were downloaded from the Cancer Genome Atlas Program (TCGA) website, and the data were processed using R language to analyze the RNA sequencing results isolated from the TCGA database. Similarly, to analyze the relationship between SOCS1 expression level and the course of glioma patients, RNA sequencing data of GBM patients were downloaded from the Chinese Glioma Genome Atlas (CGGA) website, and the expression of SOCS1 in tumor samples of glioma patients of different WHO grades was analyzed, as well as the relationship between SOCS1 expression level and the average median survival of patients.

[0073] An orthotopic glioma mouse model was established. RT-qPCR was used to compare the SOCS1 mRNA levels in the brain tissues of healthy (WT) and GBM (GL261-luc) mice. Western blotting was used to compare the SOCS1 protein levels in the brain tissues of healthy and GBM mice.

[0074] result:

[0075] (1) Figure 1 , Figure 2 As shown, the results of bioinformatics analysis and patient tumor sample analysis showed that the expression level of SOCS1 in tumor tissues of WHO grade IV glioma patients was significantly higher than that of grade II and grade III patients.

[0076] (2) Figure 3 The SOCS1 mRNA and protein levels in tumor tissues of GBM mice were shown to be higher than those in WT mice.

[0077] SOCS1 may serve as a potential new target for glioma immunotherapy.

[0078] Example 2 Functionalized phospholipid synthesis

[0079] 145.6 mg DOPE and succinimidyl trans-4-(N-maleimidomethyl) cyclohexyl ester (MCC, 50.5 mg) were added to a Schlenk bottle, and the sample was dissolved in 8 mL of dichloromethane after vacuuming. Then 82.5 μL of triethylamine (Et3N) was added and the reaction was carried out in the dark for 5 h. Impurities were removed by column chromatography (DCM:CH3OH=10:1), and the light yellow solid obtained was the product PEMC. The structure is shown in Figure 2. 1 H NMR and mass spectrometry confirmed. Fig.14 shown.

[0080] result:

[0081] 1 H NMR(400MHz,DCM-d)δ6.71(s,2H),5.45-5.29(m,4H),5.23(s,1H),4.38(s,1H),4.14(s,1H ),4.04-3.61(m,4H),3.48(s,1H),3.36(d,J=6.7Hz,2H),3.12(qd,J=7.3,4.6Hz,1H),2.58 (s,1H),2.39-2.23(m,4H),2.15(d,J=13.1Hz,1H),2.03(q,J=6.3Hz,8H),1.87(s,1H),1.6 4-1.55(m,4H),1.46-1.40(m,2H),1.30(d,J=11.4Hz,40H),0.99(m,2H),0.92-0.87(m,6H).

[0082] Mass Spectrometry C 53 H 91 N2O 11 P[MH] - :calcd.:961.6288; found:961.6251.

[0083] Example 3 Synthesis of polypeptide-phospholipid complex

[0084] Weigh 30 mg of the polypeptide angiopep-2 (Ang) and 10.6 mg of PEMC, completely dissolve them in ultra-dry DMF, and stir at room temperature for 6 hours. Similarly, weigh 24 mg of the polypeptide EphA2 (Ag) and 14 mg of PEMC, completely dissolve them in ultra-dry DMF, and stir at room temperature for 6 hours. After the reaction is completed, transfer the solution to a regenerated fiber dialysis bag (1000D) and dialyze in sterile water for 24 hours, changing the water several times during the period. After the dialysis is completed, transfer the sample to a 50 mL centrifuge tube, freeze it in liquid nitrogen, and then freeze-dry it. The white powder obtained is the polypeptide phospholipid complex. The sequence of angiopep-2 is TFFYGGSRGKRNNFKTEEYC (SEQ ID NO.13). The synthetic route is as follows Fig.15 shown.

[0085] Example 4 Nano drug synthesis

[0086] Preparation of synthetic high-density lipoprotein sHDL: 27.12 mg DMPC and 4.42 mg PEMC (molar ratio = 9:1) were dissolved in 30 mL DCM. The solvent was quickly spin-dried and then vacuumed for 2 hours. The obtained lipid film was hydrated with 10 mM PBS, and the solution was gently shaken on a shaker at a speed of 150 rpm. After reacting for 1 hour, the mixture was ultrasonicated in a 40°C water bath for 10 minutes, followed by probe ultrasonication for 2 minutes (on 1s, off 2s). ApoA-I mimetic peptide 1 dissolved in PBS was added to the above mixture (ApoA-I mimetic peptide 1 / lipid membrane = 1:7.5), the solution was heated at 50°C for 10 minutes, and then cooled at 4°C for 10 minutes, and the "heating-cooling-heating" operation was cycled three times. The solution was filtered through a 0.22 μm water system membrane and then ultrafiltered through a 10KD ultrafiltration tube. The concentrated solution was frozen to obtain sHDL.

[0087] Table 1 ApoA1 mimetic peptide sequences

[0088]

[0089]

[0090] Ultra-high resolution transmission electron microscopy characterization: The morphology of sHDL was observed using an ultra-high resolution transmission electron microscope. sHDL was diluted with ultrapure water to a suitable concentration for use. The phosphotungstic acid negative staining solution was evenly dripped onto a copper mesh (ordinary microgrid membrane, 300 mesh), and the excess liquid was absorbed by filter paper. After drying, the sHDL solution was evenly spread on the copper mesh and characterized after drying. The results are shown in Figure 2. Figure 4 As shown in Figure 2, HR-TEM observed that sHDL showed a round structure. Figure 5 As shown, in aqueous solution, the hydrated particle size of sHDL was 10.21±0.48 nm and the polydispersity index (PDI) was 0.19±0.02.

[0091] Modification of sHDL by peptides: lipid-peptide complexes (peptide / PEMC = 1.5:1, molar ratio) were added to the sHDL solution and reacted on a shaker at 25°C for 2 h at a shaker speed of 150 rpm / min. After the reaction, the solution was filtered through a 0.22 μm water system membrane and ultrafiltered using a 10KD ultrafiltration centrifuge tube.

[0092] Nucleic acid modification: To load nucleic acid onto sHDL, cholesterol-modified oligonucleotides were mixed with sHDL and reacted on a shaker at 25°C for 1 h.

[0093] In the present invention, by analyzing the data from The Cancer Genome Atlas Program (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases, it was found that SOCS1 was highly expressed in glioma patients, and the expression level of SOCS1 in tumor tissues of WHO IV grade glioma patients was significantly higher than that of low-grade patients. The present invention uses SOCS1 as a new target for glioma immunotherapy. In order to relieve the inhibitory effect of SOCS1 on DC maturation and T cell presentation, RNAi technology is used to silence SOCS1. siRNA that specifically interferes with SOCS1 mRNA is selected as part of the active ingredient of the nanomedicine. The present invention selects siSOCS1 as a specific siRNA. Its sequence is: 5′-GAGAACCUGGCGCGCAUCCCUCUUA-3′ (SEQID NO.12)

[0094] In order to better regulate the immune microenvironment, glioma-specific antigen peptides and immune adjuvants were selected as components of nanomedicine. 2671-679 (EphA2, Ag) is the preferred antigen peptide and CpG oligonucleotide is the immune adjuvant. 2671-679 The sequence of CpG is CSSFSHHNIIRL (SEQ ID NO. 10). 1826 The sequence is 5′-TCCATGACGTTCCTGACGTT-3′ (SEQ ID NO. 11).

[0095] Loading these drugs into the above-mentioned nanocarriers and changing the ratio of different components provide a flexible design space for achieving personalized nanomedicine customization.

[0096] Verify the nucleic acid loading effect by agarose gel electrophoresis: weigh 0.7g agarose and dissolve it with 35mL TBE, and melt the solution in a microwave oven at high heat for 2min. When the sample cools to about 60℃, add 3.5μL YeaRed Nucleic Acid GelStain dye. After mixing, pour the gel on the gel plate, leave it at room temperature for 30min, load the sample and perform electrophoresis. Use 20bp DNALadder as the molecular weight control, and the loading volume is 9μL. Electrophoresis: 120V, image in the gel imaging system after 45min of electrophoresis.

[0097] Results: The drug loaded with Ang was named THDL, the drug loaded with Ag was named sHDL@Ag, and the drug loaded with both Ang and Ag was named THDL@Ag. The drug loaded with both peptide Ang and Ag, as well as nucleic acid CpG and siSOCS1 was named THDL@Ag@CpG-siSOCS1. Figure 5 As shown in the figure, peptide and nucleic acid drugs are modified onto the surface of sHDL by covalent linkage and cholesterol insertion. After modification of peptide and nucleic acid, the particle size of various nanoparticles is still kept at about 10nm.

[0098] Example 5 Establishment of mouse tumor model

[0099] C57BL / 6J mice (female; 4-8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Tsinghua University. All animal experiments were approved by the Animal Ethics Committee of Tsinghua University.

[0100] Tumor model establishment: C57BL / 6J mice (female, 8 weeks) were used to establish an intracranial glioma model. GL261-Luc cells in the logarithmic growth phase were washed three times with pre-cooled PBS, digested with 0.25% trypsin, and resuspended with pre-cooled PBS. After counting, the cells with adjusted density were placed in a 2mL centrifuge tube and stored on ice. Avertin (0.12mL / 10g) was injected into the peritoneal cavity of the mouse, and the hair on the head was removed after anesthesia, and it was fixed on a brain stereotaxic instrument. After the mouse head was disinfected with iodine, the skin was cut open along the midline with a scalpel, and the right striatum (1.00mm in front, 1.00mm in the right) was found as the injection site with the bregma as the zero point. Injection of cells: Use a syringe to draw 20μL of cell suspension, insert the needle 2mm, slowly inject cells into the skull, and slowly withdraw the needle after a short pause after the injection. Carefully suture the wound and place the mouse on an electric heating pad until it wakes up. Tumor growth was monitored by bioluminescence imaging.

[0101] Biochemiluminescence imaging: Biochemiluminescence imaging was performed every 6 days starting from the inoculation of tumor cells (day 0). 2+and Ca 2+ ) Prepare D-luciferin potassium salt at a concentration of 15 mg / mL. The solution was sterilized by filtration through a 0.2 μm filter membrane and stored at -80°C. Avertin (0.12 mL / 10 g) was injected intraperitoneally before imaging. After the mouse was anesthetized, 200 μL of D-luciferin potassium salt solution was injected intraperitoneally. The mouse was placed in the mask of the IVIS imager, and the gas anesthesia machine was turned on for imaging.

[0102] NIR-II imaging: Six tumor-bearing mice were randomly divided into two groups and injected with sHDL@ICG or THDL@Ag@ICG with the same ICG loading amount via the tail vein. NIR-II imaging was performed immediately after drug injection.

[0103] Results: The results of the test on the ability of nanomedicine to target the brain are as follows Figure 6 As shown, the tumor cells in the experimental mice grew well. The FAM fluorescence signal in the brain tissue of the mice in the siSOCS1 group was weak, while the fluorescence signals of the sHDL@Ag@siSOCS1 and THDL@Ag@siSOCS1 groups were significantly stronger than those of the siSOCS1 group. This indicates that the nanocarrier can deliver siSOCS1 to the brain tissue.

[0104] like Figure 7 As shown in the figure, ICG rapidly accumulated in the liver after being injected into mice. Compared with ICG, the signal of THDL@Ag@ICG in the liver was significantly weakened, while the signal in the brain was significantly enhanced.

[0105] Compared with the PBS group, the expression level of SOCS1 mRNA in the THDL@Ag@siSOCS1 group was significantly downregulated. THDL can efficiently cross the BBB and deliver drugs to brain tissue, and the siRNA drug loaded on THDL significantly downregulated the expression of the target gene.

[0106] Example 6 Extraction of BMDCs and study of drug effects

[0107] Extraction of bone marrow-derived dendritic cells (BMDCs): 4-week-old female C57BL / 6J mice were killed by cervical dislocation and soaked in 75% alcohol. The femur and tibia of the mice were removed from the clean bench. The bones were washed with PBS (containing 2% double antibody), the two ends of the femur were carefully cut off, and the bone marrow cavity was rinsed with RPMI 1640 medium. The medium was collected and filtered, centrifuged at 2000rpm / min, and the supernatant was discarded to obtain bone marrow cells. 3mL of red blood cell lysis buffer was added to the cells, and the cells were centrifuged at 1500rpm / min after standing for 1min, and the supernatant was discarded. The cells were resuspended in RPMI 1640 medium, the cell morphology was observed under a microscope, and the cells were evenly plated on a 6-well plate after counting. 20ng / mL GM-CSF and 10ng / mL IL-4 were added to the cells the next day, and half of the medium was replaced and fresh stimulation factors were added every two days. On the 6th day, non-adherent cells were collected for subsequent experiments.

[0108] Evaluation of BMDCs' ability to absorb nanomaterials: BDMCs were seeded in confocal culture dishes, and different forms of naked drugs were added to the dishes. The siRNA concentration in each sample was 200 nM. After 4 h of co-incubation, Hoechst 33342 and lysotracker were used to stain the nucleus and lysosomes, and the cells were fixed with 4% paraformaldehyde, and fluorescence images were collected by confocal microscopy.

[0109] RNAi experiment: BMDCs were seeded in 24-well plates and treated with 100 nM siRNA in different forms. After 24 hours, mRNA was isolated and purified using an RNA extraction kit. The experimental operation was performed strictly according to the instructions. Nanodrop 2000 was used to determine RNA concentration, and the PrimeScript reverse transcription (RT) kit and oligo-dT primer were used to reverse transcribe the mRNA into cDNA. Green Master Mix Instructions Add cDNA, SOCS1 or GAPDH primers, buffer and other reagents to the PCR tube, mix well and perform RT-qPCR reaction. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous housekeeping gene, and the CT was compared and the SOCS1 mRNA level of each group was calculated.

[0110] BMDCs stimulation experiment: BMDCs were seeded in 24-well plates, and then different drugs were co-incubated with cells for 24 hours. Cells and culture medium were collected for flow cytometry and ELISA assays. Cells were co-incubated with CD16 / 32 (dilution ratio of 1:100) for 10 minutes to block nonspecific binding, followed by the addition of anti-CD11c-FITC (dilution ratio of 1:200), anti-CD80-PE (dilution ratio of 1:300) and anti-CD86-APC (dilution ratio of 1:300) for 30 minutes. The test was performed on the machine, and the maturity of BMDCs was analyzed using FlowJo software. ELISA test: The cell supernatant was processed according to the instructions to detect the secretion level of IL-6.

[0111] Results: Figure 8 As shown in the figure, siSOCS1 loaded on sHDL enters cells through endocytosis under the action of sHDL. Ang modification of THDL may promote the binding of nanoparticles to LRP-1 on BMDCs, which is beneficial for the delivery of siSOCS1 to BMDCs. THDL can deliver siRNA into BMDCs, and siSOCS1 loaded on THDL can effectively play the RNA interference function.

[0112] like Fig. 9 As shown, THDL@Ag@CpG-siSOCS1 significantly promoted the maturation of DCs.

[0113] Example 7 Immune Index Detection

[0114] On the 18th day after the mice were inoculated with cells, the mice in each drug-treated group were euthanized, and blood, brain tissue, and spleen were collected. The brain tissue was washed in pre-cooled PBS and homogenized with a tissue grinder. The homogenate was filtered through a 100μm filter, centrifuged at 350g for 5 minutes, and the supernatant was discarded. The cells were resuspended in PBS and centrifuged again. Dispase neutral protease and Collagenase D were added to the HEPES solution to make a lysis solution, and the cells were lysed in the lysis solution at 37°C for 1 hour. The cells were separated with Precoll separation solution, and the cells at the bottom layer were collected. The cells were divided into two parts, one of which was used for IFN-γ + CD8 + CD3 + and GzB + CD8 + CD3 + T cell detection, another for CD8 + CD3 + T cell detection.

[0115] CD8 + CD3 +T cell flow cytometry: resuspend the cells in 100 μL PBS (containing 0.1% BSA), and block nonspecific binding with 1 μL anti-CD16 / 32. After 10 minutes, add 1 μL anti-CD3-PE, 1 μL anti-CD4-APC, and 1 μL anti-CD8-FITC to the solution, and stain for 30 minutes at room temperature in the dark. Add 100 μL 4% paraformaldehyde to the solution, vortex mix, and store at 4°C for loading.

[0116] IFN-γ + CD8 + CD3 + and GzB + CD8 + CD3 + T cell detection: After counting the cells, disperse them in RPMI 1640 medium, add 1×BFA, and collect the cells after incubation at 37℃ for 5h. 1μL anti-CD16 / 32 blocks nonspecific binding. After 10min, add 1μL anti-CD3-PE and 1μL anti-CD8-FITC to the solution, and stain at room temperature in the dark for 30min. Add 1mL PBS (containing 0.1% BSA) to the solution, vortex to mix, and centrifuge at 350g for 5min. Discard the supernatant, retain about 100μL of sample at the bottom, then add 500μL Fixation / Permeabilization working solution (fixation permeabilization solution), mix well and react in the dark. After 50min, add 1mL Permeabilization Buffer to the solution, mix well, centrifuge at 450g for 5min, discard the supernatant, and repeat the washing of cells once. Add 5μL anti-IFNγ-APC or 5μL anti-Granzyme B-PerCP / Cyanine 5.5 to the cells, mix well and react in the dark. After 50 minutes, add 1mL Permeabilization Buffer to the solution, mix well and centrifuge at 4℃ and 450g for 5 minutes. Discard the supernatant, keep about 100μL sample at the bottom, add 100μL 4% paraformaldehyde to the solution, vortex mix well and store at 4℃ for loading.

[0117] result:

[0118] like Fig.10 As shown in the figure, the percentage of CD8+ tumor-infiltrating T lymphocytes in the PBS group was 40.97%, and the percentage of CD8+ T cells increased significantly after drug treatment. In the THDL@Ag group, this value increased to 54.73%, and in the THDL@Ag@siSOCS1 group, it increased to 72.77%.

[0119] like Fig.11As shown, after THDL@Ag@CpG-siSOCS1 treatment, multiple cancer-related genes in the brain tissue of tumor-bearing mice were affected. In the JAK-STAT signaling pathway, THDL@Ag@CpG-siSOCS1 can significantly affect the expression of a series of major genes involved in tumor progression and cancer invasion.

[0120] Example 8 Nanomedicine effectively inhibits glioma growth

[0121] The mouse tumor model was established by the method described in Example 5, and tumor growth was monitored by bioluminescence imaging. After inoculation of GL261-Luc cells, the drug was administered by tail vein injection on the 8th, 12th and 16th days. The corresponding doses of the drugs were: 3.75 mg sHDL / mouse, 0.3515 mg PEMC@Ang / mouse, 7.5 μg siRNA / mouse, 10.07 μg CpG / mouse, and 150 μL PBS dissolved the drug. On the 21st day, some mice were euthanized, and the main tissues were collected for pathological section analysis. The remaining mice continued to be raised, the status of the mice was observed daily, and the survival rate of the mice was continuously monitored.

[0122] like Figure 12-13 As shown, the nanodrug inhibited tumor growth in an orthotopic glioma model. The median survival of the PBS group was 27 days, and THDL@Ag@CpG-siSOCS1 effectively extended the median survival of tumor-bearing mice to 37.5 days.

[0123] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A nano drug composition, wherein: The nano drug composition comprises a nano carrier and a drug carrier, wherein the nano carrier comprises a first phospholipid, an apolipoprotein mimetic, and a second phospholipid modified with a functional group. The drug carrier includes a polypeptide, the polypeptide includes a polypeptide with blood-brain barrier targeting ability and / or a glioma-specific antigen peptide, the polypeptide with blood-brain barrier targeting ability includes iRGD and / or angiopep-2, and the glioma-specific antigen peptide is EphA 2671-679 , Preferably, the EphA 2671-679 The sequence is shown in SEQ ID NO.10, Preferably, the polypeptide having blood-brain barrier targeting ability is angiopep-2. Preferably, the sequence of angiopep-2 is as shown in SEQ ID NO.

13. Preferably, the molar ratio of angiopep-2 to the second phospholipid modified with the functional group is 1:10-5:

1. Preferably, the molar ratio of angiopep-2 to the second phospholipid modified with the functional group is 0.75:

1. Preferably, the EphA 2671-679 The molar ratio of the EphA to the second phospholipid modified with the functional group is 1:10-5:

1. Preferably, the EphA 2671-679 The molar ratio of the first phospholipid to the second phospholipid modified with the functional group is 0.75:

1.

2. The nano drug composition according to claim 1, wherein The drug carrier also includes oligonucleotides, Preferably, the oligonucleotide comprises a CpG oligonucleotide and / or siRNA, Preferably, the sequence of the CpG oligonucleotide is as shown in SEQ ID NO.11, Preferably, the siRNA is an siRNA that specifically interferes with SOCS1 mRNA. Preferably, the sequence of the siRNA that specifically interferes with SOCS1 mRNA is as shown in SEQ ID NO.

12. Preferably, the ratio of the CpG oligonucleotide to the nanocarrier is 0.05-5 nmol CpG oligonucleotide / mg nanocarrier, preferably, the ratio of the CpG oligonucleotide to the nanocarrier is 0.40 nmol CpG oligonucleotide / mg nanocarrier, Preferably, the ratio of the siRNA to the nanocarrier is 0.05-5 nmol siRNA / mg nanocarrier, preferably, the ratio of the siRNA to the nanocarrier is 0.15 nmol siRNA / mg nanocarrier.

3. The nano drug composition according to claim 2, wherein: The oligonucleotide contains a cholesterol modification.

4. The nano drug composition according to claim 1, wherein The carrier drug also includes one or more of mRNA, antibodies, and small molecule drugs.

5. The nano drug composition according to claim 4, wherein The small molecule drugs include anti-tumor drugs, developing tracer dyes, and photoacoustic and photothermal reagents.

6. The nano drug composition according to any one of claims 1 to 5, wherein: The apolipoprotein mimetic is an ApoA-I mimetic peptide, Preferably, the molar ratio of the first phospholipid to the second phospholipid modified with the functional group is 99:1-50:

50. Preferably, the molar ratio of the first phospholipid to the second phospholipid modified with the functional group is 9:

1. Preferably, the first phospholipid is selected from hydrogenated soybean phosphatidylcholine, dierucoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, dierucoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1,2-dicinnamoyl-sn-glycerol- 3-phosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylglycerol, 1,2-palmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidic acid, 1,2-dioleoylglycerol-3-phosphoethanolamine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, (2,3-dioleoyl-propyl)-trimethylamine, egg yolk lecithin PC-98T, egg yolk lecithin PL-100M, egg yolk phosphatidylglycerol, soybean lecithin, one or more thereof, Preferably, the first phospholipid is 1,2-dicinnamoyl-sn-glycero-3-phosphatidylcholine, Preferably, the functionalized functional group includes maleimide, 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester, DBCO, preferably, the functionalized functional group is maleimide, Preferably, the second phospholipid is selected from hydrogenated soybean phosphatidylcholine, dierucoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, dierucoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1,2-dicinnamoyl-sn-glycerol- 3-phosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylglycerol, 1,2-palmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidic acid, 1,2-dioleoylglycerol-3-phosphoethanolamine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, (2,3-dioleoyl-propyl)-trimethylamine, egg yolk lecithin PC-98T, egg yolk lecithin PL-100M, egg yolk phosphatidylglycerol, soybean lecithin, one or more thereof, Preferably, the second phospholipid is 1,2-dioleoylglycerol-3-phosphoethanolamine, Preferably, the second phospholipid modified with functional groups is prepared by condensation reaction of 1,2-dioleoylglycerol-3-phosphoethanolamine and succinimidyl trans-4-(N-maleimidomethyl)cyclohexane ester.

7. The nano drug composition according to claim 6, wherein The sequence of the ApoA-I mimetic peptide is shown in SEQ ID NO.1-9, Preferably, the sequence of the ApoA-I mimetic peptide is shown as SEQ ID NO.1 or SEQ ID NO.

9.

8. A pharmaceutical preparation, wherein The pharmaceutical preparation comprises the nano drug composition according to any one of claims 1 to 7.

9. A method for preparing the nano drug composition according to any one of claims 1 to 7, wherein: The method includes: (1) preparing nanocarriers; (2) connecting the polypeptide to a second phospholipid modified with a functional group to obtain a polypeptide-phospholipid complex; (3) adding the polypeptide-phospholipid complex to the nanocarrier and filtering; Preferably, step (2) is as follows: Weigh the peptide and the second phospholipid modified with functional groups, completely dissolve them in ultra-dry DMF, dialyze, freeze with liquid nitrogen and then dry to obtain the peptide-phospholipid complex. Preferably, step (1) is as follows: The first phospholipid and the second phospholipid modified with functional groups are dissolved in an organic solvent, and then the mixture is spin-dried and vacuumed. The obtained lipid film is hydrated with a buffer solution, ultrasonicated, and an apolipoprotein mimic dissolved in the buffer solution is added. The "heating-cooling-heating" operation is repeated three times, and the nanocarrier is obtained after filtering, ultrafiltration, concentration, and freeze-drying. Preferably, the organic solvent is DCM, Preferably, the buffer is PBS buffer.

10. Applications as follows: (1) Use of the nano drug composition according to any one of claims 1 to 7 in the preparation of anti-tumor drugs; (2) Use of the nanomedicine composition according to any one of claims 1 to 7 in the preparation of drugs for treating brain diseases; (3) Application of SOCS1 as a target in screening and / or preparing drugs for treating glioma.

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