A nanogene composition for treating brain glioma and a preparation method thereof

Abstract

The application belongs to the technical field of biomedical engineering, and specifically discloses a nano gene composition for treating brain glioma and a preparation method thereof, which is synthesized from bovine serum albumin loaded manganese dioxide particles, PLGA, DSPE-PEG3000 and DSPE-PEG-Angiopep2 as raw materials; the average particle size of the nano gene composition is 122.18±31.3 nm. The application also discloses a preparation method of the nano gene composition. The nano gene composition provided by the application encapsulates miRNA-138 in PLGA to avoid the degradation of miRNA-138 by RNA enzymes in the body, and simultaneously utilizes the targeting property of Angiopep2 to target the delivery of the nano gene composition to tumor tissues, to deliver microRNA into the brain while reducing tumor hypoxia and increasing the sensitivity of cells to X-ray irradiation, so as to realize multidirectional comprehensive treatment of glioblastoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering, and particularly relates to a nano-gene composition for treating brain glioma and a preparation method thereof. BACKGROUND

[0002] Glioblastoma (GB) is the most common primary malignant brain tumor, accounting for 57% of glioma and 48% of primary malignant central nervous system tumors, and is also the most malignant brain glioma among astrocytic tumors.

[0003] The purpose of radiotherapy for glioblastoma is to give the maximum radiation dose to the tumor while preserving the surrounding normal brain tissue, but due to the presence of the blood-brain barrier, most drugs currently used to treat brain glioma cannot reach the tumor tissue to play a role. In addition, the current drugs for treating brain glioma cannot protect the normal brain tissue around the tumor. Therefore, developing a drug that can not only pass through the blood-brain barrier but also target glioblastoma is currently the top priority for improving radiotherapy for glioblastoma. SUMMARY

[0004] The present application aims to provide a nano-gene composition for treating brain glioma and a preparation method thereof to solve one of the above technical problems.

[0005] One of the objects of the present application is to provide a nano-gene composition for treating brain glioma, which is synthesized from bovine serum albumin loaded manganese dioxide particles, PLGA, DSPE-PEG3000 and DSPE-PEG-Angiopep2; the average particle size of the nano-gene composition is 122.18±31.3nm.

[0006] Preferably, the bovine serum albumin loaded manganese dioxide particles are synthesized from bovine serum albumin and potassium permanganate.

[0007] The second object of the present application is to provide a preparation method of the above nano-gene composition, comprising the following steps:

[0008] S1, constructing bovine serum albumin loaded manganese dioxide particles:

[0009] (1) preparing a bovine serum albumin solution;

[0010] (2) adding 0.5-1ml of a standard solution of potassium permanganate (20mmol / L) to the bovine serum albumin solution and reacting for 3-8min under continuous stirring at 200-300rpm;

[0011] (3) the test solution obtained above is placed in a centrifugal filter, centrifuged at a speed of 2000-3000 rpm for 10-20 min, 3-7 ml of ultrapure water is further added, and centrifuged at a speed of 2000-3000 rpm for 10-20 min, so as to completely remove unbound free bovine serum albumin, and bovine serum albumin-loaded manganese dioxide particles are obtained;

[0012] S2, constructing a nanogene composition:

[0013] (1) N,N-dimethylformamide (DMF) is used to dissolve PLGA, DSPE-PEG3000 and DSPE-PEG-Angiopep2, and PLGA solution, DSPE-PEG3000 solution and DSPE-PEG-Angiopep2 solution are respectively prepared, and the concentration is 15-25 mg / ml; DMF is used to dissolve cation G0-C14, and GOC14 solution is obtained, and the concentration is 3-8 mg / ml;

[0014] (2) the PLGA solution, the DSPE-PEG3000 solution, the DSPE-PEG-Angiopep2 solution and the GOC14 solution are mixed according to the volume ratio = 8: (2-4) : (2-4) : (1-2);

[0015] (3) the mixed solution is mixed with 0.5-1.4 nmol Cy5-miRNA-138mimics (0.1 nmol / μl), 10-25 μl of bovine serum albumin-loaded manganese dioxide particles prepared in S1 to form a working solution;

[0016] (4) under vigorous stirring, the working solution is added dropwise into 3-10 ml of ultrapure water to form a nanogene composition;

[0017] S1 uses bovine serum albumin (BSA) as a biological template, and uses the reduced group on the surface of the protein to reduce potassium permanganate in situ (i.e. BSA and potassium permanganate are reacted), and bovine serum albumin-loaded manganese dioxide particles are prepared. S2 is self-assembled in an aqueous phase to form a nanogene composition.

[0018] The principle and beneficial effects of the present application are:

[0019] 1. The nanogene composition provided by the present application has a significant radiotherapy sensitization effect on glioblastoma, can inhibit the proliferation of tumors, and can enhance the apoptosis of tumor cells, thereby achieving multidirectional treatment of glioblastoma, and providing a new drug and a new method with potential application prospects for the treatment of clinical glioblastoma / cerebral tumor.

[0020] The nanogene composition provided by the application comprises: miRNA-138 for inhibiting tumor growth, drug bovine serum albumin loaded manganese dioxide (MnO2) particles for treating glioblastoma / cerebroma, gene delivery carrier PLGA for loading and delivering drugs, hydrophilic and non-ionic polymer polyethylene glycol (PEG), and targeting ligand Angiopep2.

[0021] The nanogene composition provided by the application encapsulates miRNA-138 in PLGA to avoid the degradation of miRNA-138 by RNA enzymes in vivo, and simultaneously targets the nanogene composition to tumor tissues of glioblastoma / cerebroma by using the targeting property of Angiopep2.

[0022] The nanogene composition provided by the application can deliver miRNA-138 into the brain while reducing tumor hypoxia and increasing the sensitivity of cells to X-ray irradiation, so that multidirectional comprehensive treatment of glioblastoma is achieved.

[0023] The qPCR result proves that the nanogene composition can well deliver miRNA-138 into cells and successfully express, so that the expression of miRNA in U87 cells is significantly high.

[0024] The reason for synthesizing the nanogene composition by selecting miRNA-138, drug bovine serum albumin loaded manganese dioxide for treating brain glioma, PLGA, PEG, and targeting ligand Angiopep2 is that:

[0025] miRNA-138 is obviously down-regulated in clinical specimens and cell lines of glioblastoma, and overexpression of miRNA-138 can effectively inhibit the proliferation of glioblastoma cells in vitro and the tumorigenicity in vivo. Meanwhile, it has been found that miRNA-138 has a strong effect in the radiosensitization process of lung cancer cells. However, the effective delivery of genes in vivo still greatly limits the application of gene therapy in the clinic. PLGA is considered to be a good gene delivery carrier due to its high safety and sustained release, and can also improve the uptake of cells. The nanogene composition constructed in the application encapsulates miRNA-138 in the inside by the electrostatic interaction between the yin and yang particles, so that miRNA-138 is protected from the degradation of RNA enzymes.

[0026] Poly(lactide-co-glycolide), PLGA is one of the most widely used biodegradable polymers, which can form two endogenous monomers, lactic acid and glycolic acid, after hydrolysis, and can be easily metabolized by the body through the Krebs cycle, so PLGA has the advantage of minimal systemic toxicity when used as a drug delivery or biomaterial. PLGA is composed of different molecular weights and copolymers, which has been approved by the FDA and the European Medicines Agency (EMA) for use in humans. Due to the different molecular weights and copolymer ratios, the degradation time can vary from a few months to several years.

[0027] PLGA nanoparticles are endocytosed by cells through liquid endocytosis and clathrin-mediated endocytosis, and can quickly escape from lysosomes into the cytoplasm within a few minutes after incubation. The reason is that nanoparticles can interact with vesicle membranes, causing transient local instability of the vesicle membrane, causing nanoparticles to escape from lysosomes and escape into the cytoplasm. The body recognizes hydrophobic particles as foreign substances. The reticulo-endothelial system (RES) can remove these substances in the blood stream and digest and absorb them in the liver or spleen. This process is the most important biological barrier in the process of nanoparticle drug delivery. The opsonin protein in the serum binds to the nanoparticles to form opsonized particles, which adhere to macrophages, and then internalize them through phagocytosis. In order to solve these limitations, the hydrophilic and non-ionic polymer polyethylene glycol is used to modify the surface of PLGA, so that the RES cannot recognize the bovine serum albumin-loaded manganese dioxide particles. In addition, "polyethyleneglycolization" increases the blood circulation half-life of the nanogene composition by several orders of magnitude, and PEG also has good biocompatibility.

[0028] Angiopep-2: Low-density lipoprotein receptor-related protein 1 (LRP1) is not only highly expressed on the blood-brain barrier, but more importantly, it is also highly expressed on glioma cells. Angiopep2 is a 19-amino-acid-containing targeting peptide that has a high binding affinity to LRP1, so it has dual targeting properties for the blood-brain barrier and glioma cells. Therefore, Angiopep-2 is used as a targeting ligand in the nanogene composition constructed in the present application.

[0029] Due to the presence of the blood-brain barrier, most radiotherapy sensitizers cannot penetrate the blood-brain barrier to reach the inside of the tumor tissue to exert their effects. Synthesizing nanoparticles that can effectively cross the blood-brain barrier for high-precision brain tumor treatment undoubtedly has great clinical significance and is expected to revolutionize traditional brain tumor treatment and improve the clinical development of glioma precision therapy.

[0030] Among the many means of treating glioma, the position of radiotherapy is very important, radiotherapy uses X-ray irradiation to damage tumor cell DNA. Although high-energy rays can penetrate the deep brain to kill infiltrating cancer cells, due to the hypoxic microenvironment of the tumor and the inherent radioresistance of glioblastoma, the efficacy of radiotherapy is usually weakened. Tumor tissue is usually characterized by hypoxia. Therefore, hypoxia is one of the main obstacles faced by radiotherapy. Improving the local oxygen content of tumor tissue is one of the important means to improve the effect of radiotherapy. MnO2 can rapidly improve the hypoxic microenvironment by decomposing H2O2. Therefore, manganese dioxide is a very promising radiosensitizer that can further improve the efficiency of radiotherapy. However, due to the existence of the blood-brain barrier, MnO2 is seriously hindered from reaching the central nervous system, affecting its radiosensitizing effect. Therefore, the nanometer gene composition provided by the application can effectively cross the blood-brain barrier, effectively solving this clinical problem.

[0031] 2、The nanometer gene composition provided by the application is mainly targeted to tumor tissue through the (the enhanced permeability and retention, EPR) effect. The parameters in the construction process: the nanometer gene composition has a hydrophilic surface and a particle size of less than 200 nm, which is very necessary for improving the EPR effect, which may be due to the increased residence time of the nanometer gene composition in the blood.

[0032] The PEG modification on the surface of PLGA reduces the interaction of the nanometer gene composition with serum proteins in the blood circulation, avoids the phagocytosis of the nanometer gene composition by RES, and ensures a longer residence time in the body.

[0033] In addition, the kidney can filter particles smaller than 20 nm, and the liver can capture particles larger than 200 nm. Therefore, the appropriate particle size of the nanometer gene composition should be between 20-200 nm, and the average particle size of the nanometer gene composition provided by the application is 122.18±31.3 nm, which can ensure its stability in the body.

[0034] 3、The nanometer gene composition penetrates the cell membrane and enters the lysosome, and the pH value in the lysosome is very low and contains a large amount of hydrolytic enzymes. Generally, once the nanometer gene composition is internalized, it will be in the endoplasmic reticulum or lysosome. Therefore, in order to avoid the digestion of the nanometer gene composition by lysosomal enzymes or acid, the lysosome escape function of the nanometer gene composition is very important. Experiments have proved that the nanometer gene composition provided by the application can be well separated from the lysosome and has good lysosome escape ability.

[0035] 4、In view of the rationality of the inventive concept and design, the nanometer gene composition provided by the application significantly expands the bioavailability of each component, achieving an unexpected technical effect. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Particle size distribution of the nanogene composition

[0037] Figure 2 Zeta potential of the nanogene composition

[0038] Figure 3 Transmission electron microscopy of the nanogene composition

[0039] Figure 4 Cytotoxicity of the nanogene composition

[0040] Figure 5 Uptake and lysosomal escape of the nanogene composition

[0041] Figure 6 Uptake of the nanogene composition by U87 cells (***P≤0.05)

[0042] Figure 7 Expression of miRNA-138 in U87 cells (***P≤0.05)

[0043] Figure 8 Effect of the nanogene composition on HIF-1a in U87 cells (***P≤0.05)

[0044] Figure 9 Radiosensitization effect of the nanogene composition on U87 cells by CCK8 detection (***P≤0.05)

[0045] Figure 10 Radiosensitization effect of the nanogene composition on U87 cells by apoptosis detection (***P≤0.05)

[0046] Figure 11 Expression of γH2AX in U87 cells by immunofluorescence detection (***P≤0.05)

[0047] Figure 12 Expression of γH2AX in U87 cells by Western blot detection (***P≤0.05)

[0048] Figure 13 Effect of the nanogene composition on ROS in U87 cells (**P≤0.05)

[0049] Figure 14 Purity identification of brain microvascular endothelial cells

[0050] Figure 15 Blood-brain barrier transport ability of two nanogene compositions at different time points (**P≤0.05)

[0051] Figure 16 Evaluation of the targeting ability of the nanogene composition for small animal live imaging system (**P≤0.05); A. Bioluminescence of U87 cells expressing luciferase after injection of luciferin substrate solution for 10 min; B. Cy5 fluorescence image of the mouse brain in vitro;

[0052] Figure 17 Evaluation of the targeting ability of the nanogene composition for laser confocal microscope;

[0053] Figure 18 Pharmacokinetic study of the nanogene composition;

[0054] Figure 19 Radiosensitization of the nanogene composition to tumor-bearing mice (***P≤0.05);

[0055] Figure 20 Survival analysis of tumor-bearing nude mice in different treatment groups;

[0056] Figure 21 HE detection of the toxicity of the nanogene composition to tumor-bearing nude mice;

[0057] Figure 22 Effect of the nanogene composition on HIF-1a;

[0058] Figure 23 Effect of the nanogene composition on Ki67, γH2AX and Tunnel. DETAILED DESCRIPTION

[0059] The following is further described in detail through specific embodiments:

[0060] Example 1 - A nanogene composition for treating brain glioma

[0061] 1. Preparation method

[0062] S1, Construction of bovine serum albumin-loaded manganese dioxide particles

[0063] (1) Weigh 100 mg of bovine serum albumin powder (purchased from Sigma Company, USA) into a sample bottle and dissolve it in 18 ml of ultrapure water;

[0064] (2) Add 1 ml of potassium permanganate standard solution (20 mmol / L) to the bovine serum albumin solution and react for 5 min under continuous stirring at 250 rpm;

[0065] (3) The test solution obtained above was placed in a 100KD centrifugal filter (purchased from Millipore Corporation, USA) and centrifuged at 2500 rpm for 15 min, and then 5 ml of ultrapure water was added and centrifuged at 2500 rpm for 15 min to completely remove unbound free bovine serum albumin;

[0066] (4) The test solution obtained above was diluted to 1 ml with ultrapure water to obtain bovine serum albumin-loaded manganese dioxide particles.

[0067] In this step, bovine serum albumin (BSA) was used as a biological template, and the reduced groups on the surface of the protein were used to reduce potassium permanganate in situ (i.e., BSA and potassium permanganate were reacted) to prepare bovine serum albumin-loaded manganese dioxide particles.

[0068] S2, Constructing a nanogene composition:

[0069] (1) N,N-dimethylformamide (DMF) was used to dissolve PLGA, DSPE-PEG3000 and DSPE-PEG-Angiopep2 (all purchased from Xi'an Ruishi Biological Technology Co., Ltd.), and PLGA solution, DSPE-PEG3000 solution and DSPE-PEG-Angiopep2 solution were prepared, with a concentration of 20 mg / ml; DMF was used to dissolve X cation G0-C14 (for specific preparation steps, see CN112940077A), to obtain GOC14 solution, with a concentration of 5 mg / ml;

[0070] (2) The PLGA solution, DSPE-PEG3000 solution, DSPE-PEG-Angiopep2 solution and GOC14 solution were mixed according to the volume ratio = 40:14:16:9, and in this embodiment, the PLGA solution was 400 μl, the DSPE-PEG3000 was 140 μl, the DSPE-PEG-Angiopep2 solution was 160 μl, and the GOC14 solution was 90 μl; in this step, an amphiphilic liposome was formed, with the hydrophilic Angiopep-2 facing outward.

[0071] (3) The above mixture was mixed with 1 nmol Cy5-miRNA-138mimics (0.1 nmol / μl), 20 μl of bovine serum albumin-loaded manganese dioxide particles prepared in S1 to form a working solution; in this step, the amphiphilic liposome encapsulated miRNA-138 and manganese dioxide particles. Cy5-miRNA-138mimics is a miRNA-138-loaded composition prepared, which contains a tracer carrying miRNA-138 MicroRNA, designed and constructed by Guangzhou Ribo Biological Technology Co., Ltd.

[0072] (4) Under vigorous stirring (1000 rpm / min), the working solution was added dropwise into 5 ml ultrapure water to form the nanogene composition;

[0073] The nanogene composition was transferred into an ultrafiltration tube and concentrated by centrifugation at 2500 rpm, followed by centrifugal washing with 5 ml of ultrapure water twice to remove free compounds and excess organic solvents.

[0074] This step is to form a nanogene composition by self-assembly in an aqueous phase.

[0075] The following experiments were statistically analyzed using SPSS statistical software. The measurement data were subjected to normality test and homogeneity of variance test. The data conforming to normal distribution and homogeneity of variance were described as mean ± standard deviation. The difference between two groups was compared by two independent sample T test, and the data among three groups were compared by One-Sample ANOVA analysis. P≤0.05 was statistically different.

[0076] Experiment 1: Characterization of nanogene composition

[0077] (1) Measurement of particle size distribution and zeta potential of nanogene composition: The synthesized nanogene composition was measured by Malvern nanoparticle size potential instrument, the detection temperature was 25℃, the scattering angle was 90°, the nanogene composition sample was diluted to the appropriate concentration with ultrapure water, and then added into the sample cell, and each sample was repeated for 3 times.

[0078] The particle size distribution data and zeta potential results of the nanogene composition are shown in Figure 1 、 Figure 2 According to the measurement results, the average particle size of the nanogene composition is 122.18±31.3 nm, the average zeta potential is -2.78±2.85 mv, and the average PDI is 0.246±0.19.

[0079] (2) Transmission electron microscopy (TEM) was used to observe the morphology of the nanogene composition: 10 μl of nanogene composition dispersion was slowly dropped on a copper mesh, and the floating liquid was absorbed from the edge using filter paper after 5 min of precipitation; dry for 5 min at room temperature, and then observe the morphology, size and distribution of the nanogene composition (operating voltage 100 kv).

[0080] The experimental results are shown in Figure 3 The nanogene composition is elliptical or circular, no aggregation is observed, the distribution is uniform, and the particle size is basically consistent with the result measured by Dynamic Light Scattering (DLS) method.

[0081] Experiment two: detection of the Cy5-miRNA-138 mimics loading rate in the nanogene composition

[0082] (1) The nanogene composition dispersion was diluted to 1 ml with ultrapure water, and 10 μl of the diluted nanogene composition dispersion was mixed with 200 μl of DMSO; (2) 10 μl of a Cy5-miRNA-138 mimics solution (0.1 nmol / μl) was mixed with 200 μl of DMSO as a standard; (3) the fluorescence intensity (FI) of the Cy5-miRNA-138 mimics was measured using a multifunctional enzyme marker, and the EE% of the Cy5-miRNA-138 mimics was calculated as EE% = (FI nanogene composition / FI standard) x 100%.

[0083] The experimental results showed that the Cy5-miRNA-138 mimic in the nanogene composition was 26 ± 0.1%.

[0084] Experiment three: radiosensitization effect of the nanogene composition on glioma cell lines

[0085] 1. Cell culture: U87 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) were cultured in DMEM high glucose medium containing 10% fetal bovine serum in a 5% CO2, 37°C cell incubator. The cells were in good condition and were used for experiments.

[0086] 2. CCK8 assay of the cytotoxicity of the nanogene composition: (1) logarithmic growth phase U87 cells were taken, and after counting was completed, a cell suspension was prepared with DMEM complete culture medium, and the cells were seeded into a 96-well plate at a density of 5 x 10 3 cells / well; (2) the seeded 96-well plate was placed in a cell incubator for culture, and the culture conditions were 5% CO2, 37°C, and the time was 24 h; (3) after 24 h, the original culture medium was discarded and the dead cells were washed with sterile PBS, and DMEM complete culture medium (containing 10% FBS) with different volumes of nanogene composition dispersion was added to the 96-well plate in turn, the volume of the nanogene composition was 0, 0.5, 1.5, 3, 5, 7, and 10 μl, and three replicate wells were set. The same volume of DMEM complete culture medium was added to the blank control wells, and then the 96-well plate was placed in a cell incubator, and the culture conditions were 5% CO2, 37°C, and the time was 12 h; (4) after 12 h, 10 μl of CCK-8 solution (purchased from Shanghai Yisen Biotechnology Co., Ltd.) was added to each well, and the 96-well plate was placed in a cell incubator for further culture, and the culture conditions were 5% CO2, 37°C, and the time was 4 h; (5) the OD value at 450 nm was measured using a multifunctional enzyme marker.

[0087] Cell toxicity activity (%) = [A(NPs) - A(blank)] / [A(control) - A(blank)] x 100

[0088] The experimental results are shown in Figure 4 It can be seen from Figure 4 that the survival rate of U87 cells after incubation with different volumes of nanogene composition dispersion solution is high, and no obvious cell death phenomenon occurs. When the volume ratio of the solution in which the nanogene composition is fully mixed to the cell culture medium reaches 1:10, the survival of U87 cells is still higher than 90%. Therefore, the nanogene composition prepared has no obvious cytotoxicity.

[0089] 3. Analysis of the uptake ability of cells to nanogene composition and lysosome escape experiment by laser confocal microscope: (1) U87 cells in logarithmic growth phase were inoculated into 3.5 cm confocal special dishes at a density of 1 x 10 4 cells / well, and placed in a 5% CO2, 37°C cell incubator overnight; (2) Discard the original culture medium, mix 75 μl of nanogene composition dispersion with 1 ml of complete culture medium, and then add the mixture to the culture dish and place it in the cell culture incubator for 12 h; (3) Wash the U87 cells with PBS for 3 times and then fix them with 4% paraformaldehyde for 20 min; (4) After discarding the fixing solution, incubate the lysosome red fluorescent probe at room temperature for 30 min; (5) Wash with PBS for 3 times and stain with DAPI at room temperature for 5 min; (6) Wash with PBS for 2 times, and then observe and take pictures by laser confocal microscope.

[0090] The experimental results are shown in Figure 5 Under the laser confocal microscope, it can be seen that after incubation of the nanogene composition with U87 cells for 6 h, the cy5 blue fluorescence and the red fluorescence of the lysosome probe co-localize; this co-localization reaches a maximum at 12 h, indicating that the nanogene composition enters the lysosome after being taken up by the cells. With the extension of incubation time, it can be seen that the Cy5-labeled nanogene composition gradually separates from the red lysosome, indicating that the nanogene composition can be well taken up by U87 cells and escape from the lysosome, and will not be digested or degraded by the lysosome.

[0091] Experiment four: analysis of the uptake of nanogene composition by cells by flow cytometry

[0092] Take U87 cells in logarithmic growth phase, count, and inoculate 1 x 10 5 ​(1) U87 cells were seeded at a density of 1 x 105cells / well into 6-well plates and incubated overnight in a cell incubator at 5% CO2, 37°C; (2) the original culture medium was discarded, 150 μl of the nano-gene composition dispersion was mixed with 2 ml of DMEM complete culture medium, and the mixed solution was added to the culture dish and incubated in the cell incubator for 12 h; the control wells were only added with 2 ml of DMEM complete culture medium; (3) the culture medium in the 6-well plate was aspirated with a pipette, the cells were washed with PBS, digested with 0.25% trypsin, collected and resuspended with 500 μl of PBS; (4) detected by flow cytometry.

[0093] The experimental results are shown in Table 1. Figure 6 As shown in Table 1, U87 cells can well uptake the nano-gene composition.

[0094] Experiment Five: RNA extraction and qRT-PCR detection of the influence of the nano-gene composition on the expression of miRNA-138 in U87 cells

[0095] Logarithmic growth phase U87 cells were taken, counted, and seeded at a density of 1 x 105cells / well into 6-well plates and incubated overnight in a cell incubator at 5% CO2, 37°C; (2) the original culture medium was discarded, 150 μl of the nano-gene composition dispersion was mixed with 2 ml of DMEM complete culture medium, and the mixed solution was added to the culture dish and incubated in the cell incubator for 12 h; the control wells were only added with 2 ml of DMEM complete culture medium; (3) the culture medium in the 6-well plate was aspirated with a pipette, the cells were washed with PBS, digested with 0.25% trypsin, collected and resuspended with 500 μl of PBS; (4) detected by flow cytometry. 5(1) Seed cells / wells at a density of 150 μl into 6-well plates and cultured overnight in a cell culture incubator at 5% CO2 and 37°C. (2) Discard the original culture medium, mix 150 μl of the nano-gene composition dispersion with 2 ml of DMEM complete culture medium, add the mixture to a culture dish, and culture in a cell culture incubator for 12 h. Add only 2 ml of DMEM complete culture medium to the control wells. (3) Extract total RNA from cells: Add Trizol reagent to the cells in the 6-well plate, gently shake the 6-well plate to distribute Trizol evenly on the cell surface, and allow the cells to fully lyse. Then, use a pipette to transfer the lysate to an RNase-free EP tube, and let it stand at room temperature for 5 min. Add 200 μl of chloroform (1 / 5 of the volume of Trizol), shake vigorously for 15 s, avoiding vortexing, and emulsify the liquid to a pinkish-white color. Let it stand at room temperature for 5 min, and then centrifuge in a low-temperature high-speed centrifuge (12000 rpm, 4°C, 20 min). At this time, the solution in the EP tube appears in three layers: the upper layer is a colorless supernatant RNA layer, the middle layer is a white protein layer, and the bottom layer is a pink organic layer. Carefully collect the colorless supernatant with a pipette and place it in a new EP tube. Add an equal volume of isopropanol, gently invert several times to mix the solution, and let it stand at room temperature for 10 min. After centrifugation in a low-temperature high-speed centrifuge (12000 rpm, 4℃, 10 min), RNA precipitate can be seen at the bottom of the EP tube. Discard the supernatant, slowly add 1 ml of 75% alcohol, gently invert, centrifuge in a low-temperature high-speed centrifuge (12000 rpm, 4℃, 10 min), and discard the supernatant. Repeat this step once. Dry at room temperature for 10 min, add DEPC water to dissolve the RNA, and detect the RNA purity and concentration with a NanoDrop micro UV-Vis spectrophotometer. (4) miRNA reverse transcription: Reverse transcription reaction was performed using specific stem-loop primers according to the instructions of the Bulge-Loop™ miRNA qRT-PCR Primer kit from Ribo Biotech. The miRNA primers were designed and provided by Ribo Biotech. The reaction system is shown in Table 1 below:

[0096] Table 1 Reaction System

[0097] Reagent 10 μl system Total RNA Template (1 μg) X μl Bulge-Loop™ miRNA RT Primer 1 μl 5X Reverse Transcription Buffer 2 μl RTase Mix 2 μl RNase-free H2O Up to 10 μl

[0098] After mixing the above reactants, centrifuge briefly and then place in a PCR instrument. The reaction conditions are: 40℃, 60 min, 70℃, 10 min. Immediately after the reaction, quickly remove the cDNA product and store it on ice or at -20℃. miRNA quantitative PCR reaction: Using cDNA as a template, examine the expression of miRNA-138 in U87 cells from different treatment groups. Primers were designed and provided by Ribo Biotech. The reaction system is shown in Table 2 below.

[0099] Table 2 Reaction System

[0100] Reagent 20 μl system 2X SYBR green Mix 10 μl cDNA 2 μl Bulge-Loop TM miRNA Forward Primer 2 μl Bulge-Loop TM Reverse Primer 2 μl ddH2O Up to 20 μl

[0101] Mix the above reaction system gently (avoid vigorous vortexing), and detect in three steps. The reaction procedure is shown in Table 3:

[0102] Table 3 Reaction system

[0103]

[0104] The fluorescent quantitative PCR reaction is performed using a quantitative PCR instrument QuantStudio5 to detect the Ct value of each sample. All samples are detected repeatedly for three times, and the relative expression amount of miRNA is calculated according to the following formula, and is expressed as 2-ΔΔCt value. NPS is a nanoparticle.

[0105] ΔΔCt = [Ct(miRNA-138)-Ct(U6)] NPs -[Ct(miRNA-138)-Ct(U6)] 对照

[0106] The experimental results are shown in Table 4: Figure 7 It can be seen from the results that after the nanogene composition is added to U87 cells, the expression amount of miRNA-138 is obviously increased, which indicates that the exogenous miRNA-138 has been loaded into the cells by the nanogene composition, and is successfully expressed.

[0107] Experiment six: cell immunofluorescence detection of the influence of the nanogene composition on HIF-1a in U87 cells

[0108] 1. Experimental design: (1) Place sterile coverslips in 6-well plates, inoculate U87 cell suspension on the coverslips, and place them in a cell culture incubator overnight for culture, with culture conditions of 5% CO2, 37°C; (2) Discard the original culture medium, and add the mixed solution of the nanogene composition and DMEM complete culture medium to the well plate, and add only DMEM complete culture medium to the control well, and place them in a cell culture incubator for culture, with culture conditions of 5% CO2, 37°C, for 12 h; (3) Discard the liquid in the 6-well plate, wash with PBS for 3 times, and fix with 4% paraformaldehyde at room temperature for 30 min; (4) Wash with PBS for 3 times, and permeabilize with 0.1% Triton X-100 at room temperature for 15 min; (5) Add goat serum for blocking at room temperature for 30 min; (6) Discard the goat serum, add HIF-1a primary antibody (1:200), and incubate at 4°C overnight; (7) Discard the HIF-1a primary antibody, and wash with PBS for 3 times; (8) Add Cy3-labeled goat anti-rabbit IgG secondary antibody (1:500), and incubate at room temperature for 1 h; (9) Discard the secondary antibody, and wash with PBS for 3 times under light-protected conditions; (10) DAPI nuclear staining for 5 min at room temperature under light protection; (11) Wash with PBS for 3 times. Seal with mounting medium; (12) Observe and take pictures under a BX63 fully automatic intelligent fluorescence microscope.

[0109] The experimental results are shown in Table 1. Figure 8 The incubation of the nanogene composition with U87 cells significantly reduces the expression of HIF-1a, which demonstrates that the prepared nanogene composition can improve the hypoxic condition of tumor cells.

[0110] Experiment Seven: CCK8 detection of the radiotherapy sensitization effect of the nanogene composition on U87 cells

[0111] 1. Experimental design: (1) Take U87 cells in the logarithmic growth phase, count the cells, and prepare a cell suspension with DMEM complete culture medium, and inoculate the cell suspension into 96-well plates at a density of 5×10 3 cells / well, and place the inoculated 96-well plates in a cell culture incubator overnight for culture, with culture conditions of 5% CO2, 37°C; (2) Discard the original culture medium, and add the nanogene composition dispersion and DMEM complete culture medium mixture to the well plate, and add only DMEM complete culture medium to the control well, and place them in a 5% CO2, 37°C cell culture incubator for culture for 12 h; (3) After giving the cells 0 Gy, 4 Gy, and 8 Gy of irradiation, continue to culture for 24 h; (4) Add 10 μl of CCK-8 solution to each well, and continue to culture for 4 h; (5) Measure the OD value at 450 nm with a multifunctional enzyme label instrument.

[0112] Cell viability (%) = [A(NPs)-A(blank)] / [A(control)-A(blank)]x100

[0113] The experimental results are shown in Table 1. Figure 9The U87 cell activity of the simple nano-gene composition group was 90.70±1.68% in the non-irradiation group, and the difference was statistically significant. In the irradiation group, the U87 cell activity of the nano-gene composition group and the control group was compared at the doses of 4 Gy and 8 Gy: when the irradiation dose was 4 Gy, the U87 cell activity of the simple irradiation group was 93.49±4.88%, and the U87 cell activity of the nano-gene composition group was 61.73±11.26%. When the irradiation dose was 8 Gy, the U87 cell activity of the simple irradiation group was 95.4±2.93%, and the U87 cell activity of the nano-gene composition group was 45.9±0.84%, and the difference was statistically significant.

[0114] Experiment Eight: Detection of the Radiotherapy Sensitization Effect of the Nano-gene Composition on U87 Cells by Apoptosis Kit

[0115] The apoptosis kit was purchased from Jiangsu Kaiqi Biotechnology Co., Ltd.

[0116] 1. Experimental design: (1) Take the U87 cells in the logarithmic growth phase, count the cells, and prepare a cell suspension with DMEM complete culture medium, and inoculate 5x10 5 cells / well into a 6-well plate, and place the inoculated 6-well plate in a cell culture incubator overnight at 5% CO2 and 37°C; (2) discard the original culture medium, and add the nano-gene composition dispersion liquid and the DMEM complete culture medium mixture to the well plate, and add only the DMEM complete culture medium to the control well, and place it in a 5% CO2, 37°C cell culture incubator for 12 h; (3) give the cells 0 Gy and 4 Gy of irradiation, and continue to culture for 24 h; (4) trypsinize and centrifuge (2000 rmp, 5 min) to collect the cells, wash the cells with PBS twice, and collect 1-5x10 5 cells; (5) add 500 μl of Bingding Buffer to resuspend the cells; (6) add 5 μl of Annexin V-EGFP and mix; (7) add 5 μl of Propidium Iodide and mix; (8) react the above solutions at room temperature and in the dark for 15 min; (9) detect the fluorescent positive cells by flow cytometry (Ex=488 nm; Em=530 nm).

[0117] The experimental results are as follows Figure 10As shown: in the non-irradiation group, the apoptosis rate of the nanogene combination group was 10.6±1.19%, while the apoptosis rate of the control group was 6.4±0.78%. In the irradiation group, the activity of U87 cells in the nanogene combination group and the control group was compared at a dose of 4Gy: the apoptosis rate of U87 cells in the simple irradiation group was 7.4±2.00%; while the apoptosis rate of U87 cells in the nanogene combination group was 19.6±5.56%, P<0.05, which was statistically significant.

[0118] Experiment Nine: Immunofluorescence detection of the expression of U87 cell γH2AX to evaluate the radiotherapy sensitization effect of the nanogene combination on U87 cells

[0119] 1. Experimental design: (1) Sterile coverslips were placed in a 6-well plate, and U87 cell suspension was inoculated on the coverslips and cultured in a 5% CO2, 37°C cell incubator overnight; (2) The original culture medium was discarded, and the nanogene combination dispersion liquid was mixed with DMEM complete culture medium and added to the well plate, and the control well was only added with DMEM complete culture medium, and was placed in a 5% CO2, 37°C cell incubator for culture for 12h; (3) The cells were given 0Gy and 4Gy irradiation, and then the well plate was placed in the cell incubator for continuous culture for 24h; (4) The liquid in the 6-well plate was aspirated with a pipette, and washed with PBS for 3 times, and then fixed with 4% paraformaldehyde at room temperature for 30min; (5) The 4% paraformaldehyde was discarded, and washed with PBS for 3 times, and then incubated with 0.1% Triton X-100 at room temperature for 15min; (6) After the 0.1% Triton X-100 was discarded, ready-to-use goat serum was added for blocking at room temperature for 30min; (7) The goat serum was discarded, and γH2AX primary antibody (1:200) was added and placed in a 4°C refrigerator overnight, and γH2AX is a marker protein of double-stranded DNA break; (8) The γH2AX primary antibody was discarded, and washed with PBS for 3 times; (9) Alexa Fluor 488 labeled goat anti-rabbit IgG secondary antibody (1:500) was added, and incubated at room temperature in the dark for 1h; (10) The secondary antibody was discarded, and washed with PBS for 3 times in the dark; (11) DAPI was used to stain the nucleus at room temperature in the dark for 5min; (12) The DAPI was discarded, and washed with PBS for 3 times, and then mounted with mounting medium; (13) BX63 full-automatic intelligent fluorescence microscope was used for observation and photography.

[0120] The experimental results are shown in Table 9 as follows: Figure 11 It can be seen that the expression of γH2AX in the nanogene combination group was significantly higher than that in the control group, which can further confirm that the nanogene combination can increase the sensitivity of U87 cells to radiotherapy.

[0121] Experiment 10: Western blot analysis of γH2AX protein expression in U87 cells under different treatment groups to evaluate the radiosensitizing effect of the nanogene composition on U87 cells.

[0122] 1. Extraction of total cell protein: (1) Take U87 cells in the logarithmic growth phase, count the cells, and prepare a cell suspension with DMEM complete medium. 5 (1) Seed cells / well density into 6-well plates; place the seeded 6-well plates in a cell culture incubator for overnight culture under the following conditions: 5% CO2, 37℃; (2) Discard the original culture medium and add the nanogene composition dispersion and DMEM complete culture medium mixture into the well plates. Add only DMEM complete culture medium to the control wells. Place the plates in a cell culture incubator for culture under the following conditions: 5% CO2, 37℃, for 12 hours; (3) Irradiate the cells with 0 Gy and 4 Gy and continue to culture for 24 hours; (4) Take an appropriate amount of RIPA and add protease inhibitor and phosphatase inhibitor a few minutes before use to make the final concentration of both 1 nm; (5) After collecting the cells, wash the cells three times with ice-cold PBS, centrifuge and aspirate the supernatant as much as possible before adding the cells. Mix the cells by pipetting and shake on ice for 30 minutes; (6) Measure the protein concentration by BCA method.

[0123] 2. Western blot: (1) Protein denaturation: After mixing the protein sample with the loading buffer at a certain volume ratio, boil it in boiling water for 5 minutes and store it at -80℃; (2) Gel preparation: separating gel: 15%; stacking gel: 5%; after the gel solidifies, transfer it to the electrophoresis tank and add electrophoresis buffer; (3) Sample loading: calculate the volume of 50 μg protein solution according to the measured protein concentration, add it to the sample well, and add markers on both sides. 3 μl and 1.5 μl; (4) Electrophoresis: First, use 80V constant voltage electrophoresis. After the markers separate, increase the voltage to 120V for electrophoresis. Stop electrophoresis when bromophenol blue appears on the liquid surface; (5) Hydrophilic treatment of PVDF membrane: Immerse the membrane in methanol for 2 min; then immerse it in deionized water for 2 min; (6) Transfer: This experiment uses wet transfer method. Cut the gel containing the target band according to the marker. Clamp the sponge, filter paper, gel, membrane, filter paper, and sponge in the transfer clamp. Transfer the membrane at a constant current of 250mA for 30 min; (7) Immunohistochemistry: At room temperature, place the transferred PVDF membrane in 5% BSA solution for 4 h to block. Then place the cut PVDF band in the diluted primary antibody solution and shake overnight at 4℃. On the second day, the PVDF band was washed three times with TBST solution for 10 min each time, and then diluted secondary antibody (1:10000) was added and incubated at room temperature for 1 h. The band was washed three times with TBST solution for 10 min each time, and the band was washed once with TBS for 10 min. (8) ECL luminescence detection.

[0124] Experimental results are as follows Figure 12 As shown, U87 cells were incubated with the nanogene composition for 12 hours and then irradiated. The results were consistent with those obtained from immunofluorescence assays. The nanogene composition combined with 4Gy radiotherapy group showed significantly high expression of γH2AX protein, indicating that the DNA double-strand breaks in this group were significantly higher than in the other three groups (blank control group, the 4Gy irradiated control group, and the nano-anticancer gene complex combined with 0Gy irradiation group), further confirming the radiosensitizing effect of the nanogene composition.

[0125] Experiment 11: Detection of the effect of nanogene composition on ROS exacerbation in U87 cells using a reactive oxygen species (ROS) detection kit.

[0126] 1. Experimental design: (1) Take U87 cells in logarithmic growth phase and administer at a dose of 1×10 4 (1) Seed cells / wells at a density of 3.5 cm in a confocal microplate and cultured overnight in a cell culture incubator under the following conditions: 5% CO2, 37°C. (2) Discard the original culture medium, mix 75 μl of nanogene composition dispersion with 1 ml of complete culture medium, add the mixture to the culture dish, and culture in a cell culture incubator for 12 h. (3) Irradiate the cells with 4 Gy and continue culturing for 24 h. (4) For adherent cells, use in situ loading probes: dilute DCFH-DA to a final concentration of 10 μM with serum-free culture medium. Then discard the original cell culture medium, add 1 ml of DCFH-DA to each dish to a final concentration of 10 μM, and incubate in a cell culture incubator at 37°C in the dark for 20 min. Then wash the cells three times with serum-free cell culture medium to remove DCFH-DA that has not entered the cells. (5) Detection: Directly examine and photograph using laser confocal microscopy (Ex = 488 nm, Em = 525 nm).

[0127] Experimental results are as follows Figure 13 As shown, the cells of the nanogene composition group showed a significant increase in ROS production after irradiation, indicating that the nanogene composition may indirectly increase DNA double-strand breaks in tumor cells by generating more ROS.

[0128] Experiment 12: Radiosensitizing effect of synthetic compounds in tumor-bearing mice

[0129] The following experiments used SPSS statistical software for statistical analysis. Quantitative data were first tested for normality and homogeneity of variance. Data conforming to a normal distribution and with homogeneous variance were described as mean ± standard deviation. Differences between two groups were compared using the independent samples t-test, and comparisons among three groups were performed using one-sample ANOVA. A p-value < 0.05 was considered statistically significant. Survival analysis was performed using the Kaplan-Meier method.

[0130] 1. Isolation of rat brain microvessel fragments and culture of brain microvascular endothelial cells: (1) 10 Wistar rats, 12 days old, were sacrificed by cervical dislocation in a clean bench and sterilized with 75% ethanol; (2) the brain was removed by decapitation on ice and the removed brain tissue was placed in sterile ice PBS; (3) the pia mater, large blood vessels and white matter on the surface of the brain tissue were carefully removed, leaving the cerebral cortex; (4) the cerebral cortex was washed with ice PBS for 3 times, placed in DMEM complete culture medium, and cut into small pieces; (5) the cut tissue was placed in a 50 ml centrifuge tube, 10 ml of 0.1% collagenase type II containing 30 U / ml DNase I was added, mixed and incubated at 37°C for 1.5 h; (6) after digestion, the mixture was centrifuged at room temperature at 1000 r / min for 10 min; (7) the supernatant was discarded and placed in 20% BSA at 4°C, centrifuged at 1000 g for 20 min; (8) the supernatant was discarded and 0.1% collagenase / dispase containing 20 U / mL DNase I was added, mixed and incubated at 37°C for 1 h, then centrifuged at room temperature at 1000 r / min for 10 min; (9) the supernatant was discarded and the precipitate was resuspended with 2 ml of DMEM complete culture medium, then carefully placed on a 33% continuous gradient of Percoll formed by centrifugation at 3000 g for 1 h, centrifuged at 4°C at 1000 g for 10 min, then the mixture appeared to be layered, the lower layer was red blood cell layer, and the upper layer was cloud-like microvessel fragment enriched layer, which was carefully sucked with a pipette; (10) washed twice with DEME complete culture medium at room temperature at 1000 r / min for 5 min; (11) after discarding the supernatant, resuspended with ECM culture medium, then added to mouse tail glue coated culture dishes, and cultured in a 5% CO2, 37°C incubator, and the medium was changed after 24 h; (12) when the cells grow to the fusion state, they are routinely digested and passaged.

[0131] 2. Purity identification of brain microvascular endothelial cells: (1) Put sterile coverslips into 6-well plates, inoculate brain microvascular cell suspension on the coverslips, and place in a 5% CO2, 37°C cell culture box; (2) Use a pipette gun to suck the liquid in the 6-well plate, wash 3 times with PBS, and then fix with 4% paraformaldehyde at room temperature for 30 min; (3) Discard the 4% paraformaldehyde, wash 3 times with PBS, and incubate with 0.1% Triton X-100 at room temperature for 15 min; (4) After discarding the 0.1% Triton X-100, add ready-to-use goat serum and block at room temperature for 30 min; (5) Discard the goat serum, add vWF primary antibody (1:200), and place in a 4°C refrigerator overnight; (6) Discard the vWF primary antibody, wash 3 times with PBS; (7) Add Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (1:500), and incubate at room temperature in the dark for 1 h; (8) Discard the secondary antibody, wash 3 times with PBS in the dark; (9) Dye the nucleus with DAPI at room temperature in the dark for 5 min; (10) Discard the DAPI, wash 3 times with PBS, and mount with mounting medium; (11) Observe and take pictures under a BX63 full-automatic intelligent fluorescence microscope.

[0132] In this experiment, brain microvessel segments were successfully isolated and extracted from the rat cerebral cortex, and were cultured in an ECM endothelial cell-specific medium in a cell culture box at 37°C and 5% CO2. The endothelial cell marker vWF was detected by cell immunofluorescence to verify the brain microvascular endothelial cells required for establishing an in vitro blood-brain barrier model. The experimental results, as shown in Figure 14 , prove that the cell culture was successful.

[0133] 3. Establishment of an in vitro blood-brain barrier: (1) Coat the Transwell cell culture chamber (polyester transparent membrane, pore size 0.4 μm) with 2% rat tail glue; (2) Inoculate brain microvascular endothelial cells into the 12-well plate Transwell chamber at a density of 7.5 x 10 3 cells per chamber; (3) On the 15th day of inoculation, add the culture medium to the upper and lower chambers to form a liquid level difference of about 0.5 cm, and observe the liquid level change after 4 h; the control group is a blank chamber; (4) Detect the transendothelial electrical resistance (TEER) value by an electrical resistance meter; (5) The 4 h leakage test is negative, and the TEER value is greater than 250 Ω·cm 2 , indicating that the blood-brain barrier is successfully established.

[0134] In the in vitro blood-brain barrier model, the transport efficiency of the Angiopep2-modified nanogene combination was higher than that of the nanogene combination without Angiopep2 modification at each detected time point, and the difference was statistically significant, as shown in Figure 15 .

[0135] 4. Evaluation of the transport of the nanogene composition across the in vitro blood brain barrier: (1) The transport ability of the liposome composition across the blood brain barrier was evaluated using a blood brain barrier model simulated by a Transwell chamber; (2) the Angiopep2-modified nanogene composition and the nanogene composition without Ang modification were added to the corresponding chamber (Cy5-miRNA-1 nmol / ml), and 4 replicates were set for each group; (3) 200 μl of medium samples were taken from the lower chamber at 0.5 h, 1 h, 1.5 h and 2 h, and fresh medium was immediately added; (4) the cy5 fluorescence intensity of 400 μl of the sample was detected by a multifunctional enzyme label meter, and the transport efficiency of the different compositions across the blood brain barrier at each time point was calculated; (5) the integrity of the blood brain barrier was detected by a TEER resistance meter during the whole experiment.

[0136] 5. Establishment of a nude mouse intracranial glioma model: (1) 0.1% pentobarbital was injected intraperitoneally for anesthesia; (2) the head of the nude mouse was fixed on a stereotaxic instrument; (3) the skin of the nude mouse was incised along the midline after routine disinfection and draping, and the bregma was fully exposed; (4) positioning: 1.2 mm forward and 2.0 mm right to the bregma; (5) the skull was drilled without damaging the meninges; (6) 5 μl of the prepared Luc-GFP-U87 cell suspension (containing 1*10 6 6. Small animal live imaging: 10 days after the intracranial glioma model was inoculated, 6 nude mice were randomly divided into 2 groups, and the nanogene composition and the nanogene composition without Ang (the dose of Cy5-miRNA-138 was 1 nmol per mouse) were injected through the tail vein, and 24 h after the injection, the nude mice were anesthetized by intraperitoneal injection of 0.1% pentobarbital and then placed in a small animal live imaging system for observation and photography.

[0137] 6. Small animal live imaging: 10 days after the intracranial glioma model was inoculated, 6 nude mice were randomly divided into 2 groups, and the nanogene composition and the nanogene composition without Ang (the dose of Cy5-miRNA-138 was 1 nmol per mouse) were injected through the tail vein, and 24 h after the injection, the nude mice were anesthetized by intraperitoneal injection of 0.1% pentobarbital and then placed in a small animal live imaging system for observation and photography.

[0138] In order to evaluate the blood brain barrier crossing and tumor targeting ability of the nanogene composition in tumor-bearing nude mice, the nanogene composition and the nanogene composition without Ang were injected into the tail vein of the mice, respectively, and 24 h later, the nude mice were sacrificed, the brain was perfused and taken out, and the brain tissue was placed in a small animal live imaging system to detect the fluorescence intensity of Cy5 in the mouse brain. It can be seen from Figure 16 It can be seen that the fluorescence intensity in the brain of the nanogene composition group was significantly higher than that of the group without Angiopep2 modification. This result proves that the prepared nanometer can successfully cross the blood brain barrier and enrich in the brain.

[0139] The frozen brain tissue section was stained with DAPI staining solution, and after sealing, it was observed under a laser confocal microscope and photographed. Figure 17 As can be seen, the Cy5 blue fluorescence of the Angiopep2 modified nanogene composition group appeared to be enriched near and inside the green fluorescence U87 tumor, while the non-Angiopep2 modified composition group showed almost no blue nanogene composition near the green fluorescence tumor. Thus, it can be proved that the nanogene composition utilizes the affinity of Angiopep2 to the LDLR1 receptor to realize the targeting of the nanogene composition to the tumor cells after crossing the blood-brain barrier.

[0140] The experimental results are shown in Figure 18 The pharmacokinetics of the nanogene composition was studied by intravenous injection of the nanogene composition (the dose of Cy5-miRNA-mimics was 1 nmol / mouse, n = 3) into healthy mice. The results showed that the circulating half-life (t 1 / 2 ) of the nanogene composition in the blood was about 15 min, while the t 1 / 2 of Cy5-miRNA-138 mimics was less than 5 min. This longer circulation characteristic is related to the PEG modification of the nanogene composition.

[0141] 7. In vivo radiosensitization and anti-tumor effect of the nanogene composition: After the intracranial glioma model was inoculated in nude mice, they were randomly divided into 4 groups, namely the simple PBS group, the simple nanogene composition group, the PBS combined with radiotherapy group, and the nanogene composition combined with radiotherapy group, each group of 10. On the 10th day after the operation, the tumor size was recorded by live imaging, and on the 12th, 14th, and 16th day after the operation, PBS and nanogene composition were injected into the tail vein, and 12 h after the injection, 10 Gy of radiotherapy was given. Four days after the last treatment, 3 nude mice from each group were sacrificed, and their hearts, brains, spleens, lungs, kidneys, and livers were taken for HE staining. The remaining nude mice were used to observe the general condition and survival time.

[0142] The experimental results are shown in Figure 19 As can be seen, the tumor in the nanogene composition combined with radiotherapy group showed a significant reduction.

[0143] To further evaluate the therapeutic effect of the nano-gene composition combined with radiotherapy, the survival time of the tumor-bearing mice was monitored. The tumor-bearing mice in the PBS group were fed normally 24 h after the orthotopic tumor transplantation. The tumor-bearing mice in the PBS group began to show reduced activity on day 13, and 2 died on day 17, 3 died on day 22, and all died on day 29. The tumor-bearing mice in the nano-gene composition group and the nano-gene composition combined with radiotherapy group showed reduced activity and restlessness after administration, and recovered after a few seconds. The tumor-bearing mice in the nano-gene composition group died on day 25, 1 died on day 28, 2 died on day 30, and all died on day 42. The tumor-bearing mice in the PBS combined with radiotherapy group died on day 25, 3 died on day 28, and all died on day 33. The tumor-bearing mice in the nano-gene composition combined with radiotherapy group died on day 38, and the general state of the tumor-bearing mice in this group was better than that in the other three groups, with the longest survival time of 70 days. Figure 20 It can be seen that the median survival time (95% confidence interval) of the tumor-bearing mice in the PBS group, the nano-gene composition group, the PBS combined with radiotherapy group, and the nano-gene composition combined with radiotherapy group was 22 (18.096, 25.904), 30 (27.657, 32.343), 28 (26.185, 29.815), and 45 (27.543, 31.457), respectively. Compared with the other three groups, the survival time of the tumor-bearing mice in the nano-gene composition combined with radiotherapy group was significantly prolonged (P<0.05).

[0144] 8. Brain perfusion of the nude mice: (1) 0.1% pentobarbital was injected intraperitoneally for anesthesia; (2) the skin of the nude mice was cut from the sternum to expose the heart, a syringe was inserted into the left ventricle, and hemostatic forceps were used for fixation; (3) the right auricle was cut with tissue scissors; (4) then, 20 ml of ice-cold physiological saline was perfused with a syringe until the liver turned white, and clear liquid flowed out of the right auricle; (5) then, 20 ml of 4% paraformaldehyde was perfused with a syringe; (6) after decapitation, the heart, brain, spleen, lung, and kidney were taken out and stored in 4% paraformaldehyde at 4°C.

[0145] Experimental results Figure 21 As shown in FIG. 6, no obvious abnormalities were found in the important organs of the tumor-bearing mice in the PBS group and the nano-gene composition group, which confirmed that the nano-gene composition had no cytotoxicity to the nude mice, which was consistent with the results of the cell experiment.

[0146] 9. Preparation of frozen sections: (1) Dehydration of brain tissue: after the brain is taken out as described above, the brain tissue is placed in 15% sucrose 4% paraformaldehyde solution, and is kept at 4°C overnight. After the brain tissue sinks to the bottom, it is transferred to 30% sucrose 4% paraformaldehyde solution, and is kept at 4°C overnight. After the brain tissue sinks to the bottom, OCT embedding is performed; (2) OCT embedding: an OCT embedding box is used, and the brain tissue is infiltrated with OCT, and then is placed in a -80°C freezer for storage; (3) Sectioning: a freezing microtome is used to perform continuous sectioning, and the section thickness is 10 μm.

[0147] 10. Histological examination: (1) The fixed nude mouse tissue is embedded in a wax block; (2) The tissue is sectioned continuously, and the section thickness is 10 μm; (3) Section dehydration: xylene I 10 min; xylene II 10 min; anhydrous ethanol 5 min; 95% ethanol 2 min; 75% ethanol 2 min; PBS 2 min, twice; (4) HE staining: hematoxylin stock solution is used to stain the nucleus for 1 min, and tap water is used to wash and stop the reaction for about 5 min, and then the tissue is blued in PBS solution for a few seconds; eosin staining solution is used for 2 min; (5) Dehydration, transparency, and mounting: the above-described stained section is placed in 95% ethanol for 2 min, twice; anhydrous ethanol for 2 min, twice. Then the section is placed in xylene for transparency for 5 min, and is mounted with neutral balsam after the reaction is stopped; (6) Observation under a microscope, and photographing. HE detection of the toxicity of the nanocomposite to the tumor-bearing nude mouse.

[0148] Figure 22 Effect of the nanogene composition on HIF-1a, Figure 23 Effect of the nanogene composition on Ki67, γH2AX, and Tunnel. The experimental results are shown in Figure 22 and Figure 23 Immunohistochemical analysis of the brain of the tumor-bearing mouse is performed. The nanocomposition provided in the present experiment can significantly reduce the expression of HIF-1a in the brain tumor cells ( Figure 22 ), which indicates that the nanogene composition can effectively improve tumor hypoxia, and from Figure 23 it can be seen that the angiopep-2 modified pegylated manganese dioxide PLGA-MiRNA-138 nanogene composition combined with RT can significantly increase the expression of γH2AX in the tumor cells, which indicates that the nanogene composition has a significant radiotherapy sensitization effect on the glioblastoma, can inhibit the proliferation of the tumor, and can enhance the apoptosis of the tumor cells.

[0149] The preferred embodiments of the present application have been described above in detail. It should be understood that modifications and variations to the present application can be affected by those skilled in the art without departing from the scope of the application. Accordingly, it is intended that all possible modifications and alterations be included within the scope of the present application as defined by the following claims.

Claims

1. A nanogene composition for treating glioma, characterized in that, The nanogene composition was synthesized from bovine serum albumin-loaded manganese dioxide particles, PLGA, DSPE-PEG3000, DSPE-PEG-Angiopep2, and Cy5-miRNA-138 mimics; the average particle size of the nanogene composition was 122.18 nm; the nanogene composition was prepared through the following steps: S1. Manganese dioxide particles loaded with bovine serum albumin were constructed using bovine serum albumin and potassium permanganate. S2. Constructing nanogene compositions: (1) PLGA, DSPE-PEG3000 and DSPE-PEG-Angiopep2 were dissolved in N,N-dimethylformamide to prepare PLGA solution, DSPE-PEG3000 solution and DSPE-PEG-Angiopep2 solution respectively, with a concentration of 15-25 mg / ml; cationic G0-C14 was dissolved in N,N-dimethylformamide to obtain GOC14 solution with a concentration of 3-8 mg / ml; (2) Mix PLGA solution, DSPE-PEG3000 solution, DSPE-PEG-Angiopep2 solution and GOC14 solution in a volume ratio of 8:(2-4):(2-4):(1-2); (3) Mix the above mixture with 10-25 μl of manganese dioxide particles loaded with bovine serum albumin prepared by S1 with a concentration of 0.1 nmol / μl and a substance amount of 0.5-1.4 nmol Cy5-miRNA-138mimics and a substance amount of 0.5-1.4 nmol to form a working solution; (4) Add the working solution dropwise to 3-10 ml of ultrapure water to form a nanogene composition.

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