Glioma targeting nanocapsule containing Cas9 / sgGDF15 compound as well as preparation method and application of glioma targeting nanocapsule

Abstract

The invention discloses a nanocapsule for targeting glioma containing a Cas9 / sgGDF15 compound as well as a preparation method and application of the nanocapsule, and belongs to the technical field of biological medicines. According to the nanocapsule disclosed by the invention, the glioma targeting peptide Ang with strong brain penetrating power is cross-linked on the surface, so that the glioma targeting property of the nanocapsule is improved; a disulfide bond is added on the surface of the nanocapsule, so that the nanocapsule can be prevented from being degraded by enzyme action in blood, and a Cas9 / sgGDF15 compound entrapped in the nanocapsule can be ruptured and accurately released in a high glutathione microenvironment at a tumor part of the nanocapsule, so that the GDF15 gene of glioma cells is efficiently edited, and the GDF15 gene in the glioma cells is specifically knocked out; the immunosuppressive microenvironment of the glioma is reversed, the anti-tumor effect of immune cells is recovered, and the aim of treating the glioma is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing a glioma-targeting nanocapsule containing a Cas9 / sgGDF15 complex and an application thereof in the treatment of glioma. Background Art

[0002] Gliomas are the most common primary central nervous system tumors. Glioblastoma Multiforme (GBM) is the most aggressive subtype, accounting for about half of glioma cases. GBM is the most malignant glioma among astrocytic tumors and belongs to WHO grade IV. The current standard treatments for tumors, such as surgical intervention, radiotherapy, and drug therapy (such as the chemotherapy drug temozolomide), have made only limited progress in improving the survival rate of GBM patients. The lack of progress in GBM treatment can be attributed to a variety of factors, such as the blood-brain barrier (BBB) ​​hindering drug delivery, cancer cells' resistance to cell death induction, and the lack of targeted therapy for specific oncogenic pathways. In addition, when exploring immune-based GBM treatments, the unique immune microenvironment of the central nervous system must also be considered.

[0003] GBM patients participating in clinical trials currently benefit limitedly from immunotherapy approaches, which is mainly attributed to the "cold" immune microenvironment of GBM tumors, which refers to the microenvironmental state in which the number of immune cells surrounding the tumor is small and the activity of these cells is low, which cannot effectively attack tumor cells. The dilemma of immunotherapy in the field of anti-cancer stems from the lack of universally applicable tumor-specific antigen targets and the existence of an immunosuppressive microenvironment that hinders the infiltration and activation of T cells in tumors. Therefore, there is an urgent need to develop innovative treatments to counteract precancerous immune responses and address tumor resistance to immunotherapy in the microenvironment, especially for the treatment of GBM.

[0004] The applicant team previously found that growth differentiation factor-15 (GDF15) plays a vital role in immune escape through in vivo screening of CRISPR libraries combined with transcriptomic analysis, and can become a new immunotherapy target for GBM. Experiments have confirmed that knocking out GDF15 in tumor cells in vivo can reshape the glioma TME (tumor microenvironment) and reverse the immunosuppressive microenvironment. As a gene editing tool, CRISPR / Cas9 can permanently destroy tumor genes, circumvent the repeated dose limitations of traditional therapies, reduce treatment costs, and improve treatment effects. Therefore, delivering the Cas9 / sgRNA complex to the glioma area, knocking out the GDF15 gene, and improving the inhibitory immune state of the glioma TME to achieve tumor suppression has become a potential new treatment method. However, due to the existence of the blood-brain barrier and the limitations of the Cas9 protein properties, the traditional CRISPR / Cas9 system delivery vector has a low targeting efficiency and cannot effectively knock out the GDF15 gene. Therefore, there is an urgent need to develop a new Cas9 / sgGDF15 complex delivery vector to effectively improve the blood-brain barrier penetration and glioma targeting capabilities. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a glioma-targeting nanocapsule containing a Cas9 / sgGDF15 complex and a preparation method and application thereof, so as to solve the technical problem that the existing CRISPR / Cas9 system delivery vector has low targeting efficiency and cannot effectively knock out the GDF15 gene.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses a glioma-targeting nanocapsule containing a Cas9 / sgGDF15 complex, which is composed of a nanocapsule outer wall encapsulating a nanocapsule inner core; wherein: The nanocapsule core contains a negatively charged Cas9 / sgGDF15 complex; The outer wall of the nanocapsule contains glioma targeting peptide Ang.

[0007] Preferably, the Cas9 / sgGDF15 complex is obtained by dissolving sgGDF15 and Cas 9 proteins in a molar ratio of 1:1 in HEPES buffer.

[0008] Further preferably, the gene sequence of sgGDF15 is as shown in SEQ ID NO: 1, specifically: CGTCAGGCGCAGGCGTAATG.

[0009] Preferably, the nanocapsule wall is polymerized by a disulfide bond crosslinker and polyethylene glycol, and the glioma targeting peptide Ang is connected to the disulfide bond group through an amidation reaction.

[0010] Preferably, the nanocapsule is spherical in shape and has a size of 100-160 nm.

[0011] More preferably, the size of the nanocapsule is 120-130 nm.

[0012] The present invention also discloses a method for preparing the above-mentioned glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex, comprising: preparing a nanocapsule inner core containing a negatively charged Cas9 / sgGDF15 complex; preparing a nanocapsule outer wall containing a glioma-targeting peptide Ang; and wrapping the nanocapsule inner core with the nanocapsule outer wall to prepare a glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex.

[0013] Preferably, preparing the nanocapsule core comprises: dissolving the positively charged Cas9 protein and the negatively charged sgGDF15 to obtain a negatively charged Cas9 / sgGDF15 complex, and then adding the negatively charged Cas9 / sgGDF15 complex to a positively charged citric acid buffer to obtain the nanocapsule core.

[0014] Preferably, the outer wall of the nanocapsule is prepared, comprising: polymerizing a disulfide bond-containing crosslinking agent with a neutral molecule polyethylene glycol having an amide group and a succinic acid functional group, and connecting the glioma targeting peptide Ang to the disulfide bond group through an amidation reaction.

[0015] The present invention provides a method for preparing the above-mentioned glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex, comprising the following steps: (1) Dissolve DLin-MC3-DMA (MC3), DSPC, cholesterol, DMG-PEG, DSPE-SS-PEG, DSPE-PEG-peptide ANG, and DSPE-PEG-Cy5.5 in 2 mL of anhydrous ethanol at a ratio of 10.5 / 60 / 13 / 1.5 / 5 / 8 / 2 and transfer to a 25 mL eggplant-shaped flask. This reaction system is the lipid component. (2) Dissolve the positively charged Cas9 protein and the negatively charged sgGDF15 to obtain the negatively charged Cas9 / sgGDF15 complex, and add it to 3 mL of citric acid buffer containing 25% ethanol and mix well; (3) Slowly adding the Cas9 / sgGDF15 complex mixture obtained in step (2) to the lipid component in step (1), mixing well and incubating for 20 minutes to form positively charged nanocapsules; (4) After equilibration at room temperature for 2 min, the hydrated lipids were extruded by sonication and extrusion using a liposome extruder through two stacked 100 nm pore size filters until the vesicle diameter determined by a nanoparticle size analyzer was about 100 nm; (5) Dialysis using a nanodialysis device to remove unloaded RNA and protein and various residual reaction components; (6) Adding trehalose lyophilization protective agent to the liposome dispersion, freeze-drying is performed to finally obtain glioma-targeting nanocapsules containing Cas9 / sgGDF15 complexes; Further preferably, in the step (1), D-Lin-MC3-DMA (dilinoleyl methyl dimethylaminobutyrate); DSPC (distearoylphosphatidylcholine); DMG-PEG (dimyristoyl-sn-glycerol-polyethylene glycol); DSPE-SS-PEG (distearoylphosphatidylethanolamine-disulfide bond-polyethylene glycol); DSPE-PEG-polypeptide Ang (distearoylphosphatidylethanolamine-polyethylene glycol-polypeptide Angiopep); DSPE-PEG-CY5.5 (distearoylphosphatidylethanolamine-polyethylene glycol-CY5.5).

[0016] Among them, D-Lin-MC3-DMA and DMG-PEG provide positive charge to the outer wall of the nanocapsule, DSPC (distearoylphosphatidylcholine) is a commonly used PC-type phospholipid, DSPE-SS-PEG plays a role in reduction response, and DSPE-PEG-CY5.5 is used to label fluorescence to facilitate subsequent in vivo tracing experiments.

[0017] Further preferably, in step (2), citric acid buffer (50 mM citrate, pH=4) is used.

[0018] Further preferably, in step (5), the dialysis device uses a polycarbonate membrane with a pore size of 30 nm.

[0019] The present invention also discloses the use of the above-mentioned glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex in the preparation of a glioma-targeting drug.

[0020] The present invention also discloses the use of the above-mentioned glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex in the preparation of a PD1 antibody enhancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a nanocapsule targeting glioma containing a Cas9 / sgGDF15 complex, which is composed of a nanocapsule outer wall encapsulating a nanocapsule inner core, wherein the inner core contains a Cas9 / sgGDF15 complex, and the glioma targeting of the nanocapsule is improved by cross-linking a target peptide Angiopep-2 (Ang) with strong brain penetration ability in the nanocapsule outer wall, and adding a disulfide bond on the surface of the nanocapsule, which not only protects the nanocapsule from degradation by enzymes in the blood, but also allows it to rupture in the high glutathione microenvironment of the tumor site, accurately releasing the encapsulated Cas9 / sgGDF15 complex therein, and then efficiently editing the GDF15 gene of glioma cells, and specifically knocking out the GDF15 gene in glioma cells. Therefore, after the nanocapsule is transferred into cells, the purpose of knocking out the GDF15 gene of glioma cells can be achieved.

[0022] The preparation of the targeted glioma nanocapsule containing the Cas9 / sgGDF15 complex provided by the present invention improves the glioma targeting of the nanocapsule by loading the glioma targeting peptide Ang on the surface of the nanocapsule; adding a disulfide bond on the surface of the nanocapsule not only protects the nanocapsule from degradation by the enzymes in the blood, but also allows the high glutathione microenvironment of the tumor site to rupture, accurately releases the packaged Cas9 / sgGDF15 complex therein, and then efficiently edits the gene of GDF15 of glioma cells, specifically knocks out the GDF15 gene in glioma cells, reverses the immunosuppressive microenvironment of glioma, restores the anti-tumor effect of immune cells, and achieves the purpose of treating glioma. The present invention avoids the defects of low loading efficiency, non-specific targeting, the risk of causing neuroinflammation, and the lack of reactive drug release mechanism existing in traditional drug delivery carriers, thereby greatly improving the therapeutic effect. Therefore, delivering the Cas9 / sgGDF15 complex to the glioma area, knocking out the GDF15 gene, and reversing the inhibitory immune microenvironment of glioma becomes a potential clinical treatment scheme.

[0023] The present invention has been verified through experiments that in the mouse glioma models of GL261 orthotopic glioma and virus-induced primary glioma, the volume of mouse glioma was significantly reduced after the nanocapsule was injected through the tail vein for 4 times; and the survival period of mice in the treatment group was significantly prolonged; T7E1 enzyme digestion method and NGS sequencing method determined that the tumor growth inhibition was caused by GDF15 gene knockout; immunohistochemical staining further confirmed that the expression level of GDF15 in tumor tissue in the treatment group was significantly reduced, and the expression level of Ki67 was also significantly reduced; the nanocapsule of the present invention is combined with PD1 antibody, and the two have a synergistic effect of inhibiting glioma. The above data show that the glioma-targeting nanocapsule containing Cas9 / sgGDF15 complex of the present invention can effectively inhibit the growth of mouse glioma and significantly prolong the survival period of mice, providing support for its application in the preparation of targeted glioma drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A is a schematic diagram of the construction process of nanocapsules loaded with Cas9 / sgRNA and the addition of Ang glioma targeting peptides on their surface; Figure 1B This is a scanning electron micrograph of a glioma-targeting nanocapsule containing a Cas9 / sgGDF15 complex; Figure 1C This is the Zeta potential detection diagram of the nanocapsule; Figure 1D This is the transmission electron microscopy image of the nanocapsules; Figure 1E Schematic diagram of glioma-targeted nanocapsule genome editing containing Cas9 / sgGDF15 complex; Figure 1F This is a graph showing the in vitro gene editing efficiency of nanocapsules for GDF15; Figure 1G Determination of the GDF15 gene editing site in GL261 cells treated with glioma-targeted nanocapsules containing Cas9 / sgGDF15 complexes; Figure 1H To establish an in vitro blood-brain barrier model to verify the targeting ability of nanocapsules to glioma cells; Fig. 1I The distribution diagram of nanocapsules with and without Ang targeting peptides in the brain and internal organs of mice with orthotopic glioma model injected into the tail vein; Figure 1J Immunofluorescence staining was used to detect the distribution of nanocapsules with and without targeting peptide Ang in tumor tissues; Figure 1K This is a scanning electron microscope image of the nanocapsule prepared by the present invention; Figure 1L The fluorescence spectra of the nanocapsule experimental group and the control group prepared by the present invention are shown in FIG.

[0025] Figure 2A Schematic diagram of the construction of GL261 orthotopic glioma model and the time point of drug administration; Figure 2 B is a quantitative analysis of the tumor volume of mice in different treatment groups after the designated treatment; Figure 2 C shows the tumor fluorescence results of C57 mice in different treatment groups after orthotopic tumor bearing of GL261-Luc cells and the tumor distribution after HE staining of whole brain sections; Figure 2 D is a quantitative analysis of tumor fluorescence levels in mice of different treatment groups during the indicated treatment periods; Figure 2 E is the statistical results of the survival of mice in different treatment groups; Figure 2 F shows the editing efficiency of the GDF15 gene in tumor tissues of mice in different treatment groups; Figure 2G To detect the editing of GDF15 gene in mouse tumors after treatment with glioma-targeted nanocapsules containing Cas9 / sgGDF15 complexes at DNA sequencing level; Figure 2H Immunohistochemical analysis of GDF15, Caspase-3, and Ki67 expression in tumor tissues resected from mice after receiving the indicated treatments; Figure 2I-2K Flow cytometry was used to detect CD206 in tumors of mice in different treatment groups after treatment. + TAM, CD86 + TAM and CD3 + CD8 + GZM + T cell content; Figure 2L Representative multiplex immunofluorescence staining of tumor tissues of mice treated with the indicated drugs in different treatment groups; Figure 3A Schematic diagram of the construction of virus-induced spontaneous glioma model and the time point of drug administration; Figure 3B The tumor distribution of spontaneous glioma mice in different treatment groups after treatment was shown in HE staining of whole brain sections; Figure 3C It is a quantitative analysis of the tumor volume of mice in different treatment groups after the specified treatment; Figure 3D The growth curves are the relative tumor volumes of mice in different treatment groups during the specified treatment period; Figure 3E It is the statistical result of the survival period of mice in different treatment groups; Figure 3F is the editing efficiency of the GDF15 gene in the tumor tissues of mice in different treatment groups; Figure 3G To detect the editing of GDF15 gene in mouse tumors after treatment with glioma-targeted nanocapsules containing Cas9 / sgGDF15 complexes at DNA sequencing level; Figure 3H Immunohistochemical analysis of GDF15, Caspase-3, and Ki67 expression in tumor tissues resected from mice after receiving the indicated treatments; Figure 3I-3K Flow cytometry was used to detect CD206 in tumors of mice in different treatment groups after treatment. + TAM, CD86 + TAM and CD3 + CD8+ GZM + T cell content; Figure 3L Representative multiplex immunofluorescence staining of tumor tissues of mice treated with the indicated drugs in different treatment groups; Figure 4A The tumor fluorescence results after designated treatment in C57 mice of different treatment groups bearing GL261-Luc cells orthotopically and the tumor distribution after HE staining of whole brain sections; Figure 4B is a quantitative analysis of tumor fluorescence levels in four groups of differently treated mice during the indicated treatment periods; Figure 4C This is the statistical result of the survival period of mice in four groups with different treatments; Figure 4D To detect the editing of GDF15 gene in tumor of mice treated with Cas9 / sgGDF15 nanocapsules combined with PD-1 monoclonal antibody at DNA sequencing level; Figure 4E Immunohistochemical analysis of GDF15, Caspase-3, and Ki67 expression in tumor tissues resected from mice after receiving the indicated treatments; Figure 4F-4H Flow cytometry was used to detect CD206 in tumors of mice in different treatment groups after treatment. + TAM, CD86 + TAM and CD3 + CD8 + GZM + T cell content; Fig. 4I Representative multiplex immunofluorescence staining of tumor tissues from mice treated with the indicated drugs in different treatment groups. DETAILED DESCRIPTION

[0026] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention provides a nanocapsule targeting glioma containing a Cas9 / sgGDF15 complex, the nanocapsule comprising a nanocapsule core containing a negatively charged Cas9 / sgGDF15 complex and a nanocapsule wall containing a glioma targeting peptide Ang; the nanocapsule wall containing the glioma targeting peptide Ang is polymerized by a disulfide bond cross-linking agent and a neutral molecule having an amide group and a succinic acid functional group, and the surface of the nanocapsule wall contains a disulfide bond functional group, and the disulfide bond functional group is connected to the glioma targeting peptide Ang through an amidation reaction. The nanocapsule targeting glioma containing a Cas9 / sgGDF15 complex of the present invention has a spherical shape and a size distribution of 100 to 160 nm.

[0028] The present invention provides a method for preparing the above-mentioned glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex, comprising the following steps: (1) Dissolve DLin-MC3-DMA (MC3), DSPC, cholesterol, DMG-PEG, DSPE-SS-PEG, DSPE-PEG-peptide ANG, and DSPE-PEG-Cy5.5 in 2 mL of anhydrous ethanol at a ratio of 10.5 / 60 / 13 / 1.5 / 5 / 8 / 2 and transfer to a 25 mL eggplant-shaped flask. This reaction system is the outer wall of the nanocapsule. (2) Dissolve the positively charged Cas9 protein and the negatively charged sgGDF15 to obtain the negatively charged Cas9 / sgGDF15 complex, which is added to 3 mL of citric acid buffer containing 25% ethanol and mixed; (3) Slowly add the Cas9 / sgGDF15 complex (the inner core of the nanocapsule) mixture obtained in step (2) to the outer wall component of the nanocapsule in step (1), mix well, and incubate for 20 minutes to form positively charged nanocapsules; (4) After equilibration at room temperature for 2 min, sonication and extrusion were performed using a liposome extruder, through two stacked 100 nm pore size filters until the vesicle diameter determined by a nanoparticle size analyzer was about 100 nm; (5) Dialysis using a nanodialysis device to remove unloaded RNA and protein and various residual reaction components; (6) adding trehalose lyophilization protective agent to the dispersion obtained in step (5), and freeze-drying to finally obtain nanocapsules targeting glioma containing Cas9 / sgGDF15 complex; In the step (1), D-Lin-MC3-DMA (dilinoleyl methyl dimethylaminobutyrate); DSPC (distearoylphosphatidylcholine); DMG-PEG (dimyristoyl-sn-glycerol-polyethylene glycol); DSPE-SS-PEG (distearoylphosphatidylethanolamine-disulfide bond-polyethylene glycol); DSPE-PEG-polypeptide Ang (distearoylphosphatidylethanolamine-polyethylene glycol-polypeptide Angiopep); DSPE-PEG-CY5.5 (distearoylphosphatidylethanolamine-polyethylene glycol-CY5.5).

[0029] The gene sequence of sgGDF15 is shown in SEQ ID NO: 1, specifically: CGTCAGGCGCAGGCGTAATG.

[0030] In the step (2), the citric acid buffer (50 mM citrate, pH=4) is used.

[0031] In the step (5), the dialysis device uses a polycarbonate membrane with a pore size of 30 nm.

[0032] At the same time, the present invention also prepared a group of control group nanocapsules, specifically comprising: (1) Dissolve DLin-MC3-DMA (MC3), DSPC, cholesterol, DMG-PEG, DSPE-SS-PEG, and DSPE-PEG-Cy5.5 in 2 mL of anhydrous ethanol at a ratio of 10.5 / 60 / 13 / 1.5 / 5 / 8 and transfer to an eggplant-shaped flask. This reaction system is the outer wall of the nanocapsule in the control group. Slowly add citric acid buffer (50 mM citrate, pH 4) containing 25% ethanol to the lipids and mix well, then incubate for 20 minutes. Treat with ultrasound and an extruder (100 nm filter membrane).

[0033] (2) Dialysis using a nanodialysis device (polycarbonate membrane, pore size 30 nm).

[0034] (3) Add a certain amount of freeze-drying protective agent and perform freeze-drying.

[0035] The difference between the control group and the above experimental group is that no DSPE-PEG-peptide ANG was added to the outer wall of the nanocapsule.

[0036] The experimental group was tested for particle size and potential, and the results are shown in Table 1 below: Table 1 Hydrodynamic size (nm) PDI Zeta potential (mV) 1st time 128.45 0.215 17.46 2nd 121.17 0.187 14.52 3rd 133.05 0.196 16.04 average value 127.557±5.99 0.199±0.01 16.007±1.47 In addition, the SEM images of the experimental group are shown in Figure 1K Further, the fluorescence spectra of the experimental group and the control group were obtained by fluorescence spectrum test as shown in Figure 1L shown.

[0037] The present invention provides an application of the above-mentioned glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex in the preparation of a glioma-targeting drug.

[0038] Example 1: Preparation of glioma-targeting nanocapsules containing Cas9 / sgGDF15 complexes and verification of in vitro gene editing efficiency 1. Construction of glioma-targeting nanocapsules containing Cas9 / sgGDF15 complex (1) Dissolve 2.4 mg DLin-MC3-DMA (MC3), 24 mg DSPC, 1.2 mg cholesterol, 1.2 mg DMG-PEG2000, 1.5 mg DSPE-SS-PEG2000, 1.5 mg DSPE-PEG-peptide Ang, and 1.2 mg DSPE-PEG-Cy5.5 in 2 mL of anhydrous ethanol and transfer to a 25 mL eggplant-shaped flask. This reaction system is the liposome component. (2) Positively charged Cas9 (20 μg) and negatively charged sgGDF15 (4.0 μg) were mixed in a 1:1 molar ratio in 500 μL HEPES buffer (10 mM, pH 7.4) by electrostatic adsorption, incubated at room temperature for 5 min to form a negatively charged Cas9 / sgGDF15 complex, and 3 mL of citric acid buffer (50 mM citrate, pH 4) containing 25% ethanol was added; (3) Slowly add the mixture containing the Cas9 / sgGDF15 complex to the lipid component, mix well, and incubate for 20 minutes to form positively charged nanocapsules; (4) After equilibration at room temperature for 2 minutes, the mixture was sonicated and extruded, and the hydrated lipids were extruded through two stacked 100 nm pore size filters using a liposome extruder until the vesicle diameter determined by a nanoparticle size analyzer was about 100 nm; (5) Dialysis using a nanodialysis device (polycarbonate membrane, pore size 30 nm) to remove unloaded RNA and protein and various residual reaction components; (6) Add trehalose lyophilization protective agent and perform freeze-drying to obtain glioma-targeting nanocapsules containing Cas9 / sgGDF15 complex.

[0039] 2. Transmission electron microscopy observation of nanocapsule GSH response 0.15 mg of glioma-targeting nanocapsules containing Cas9 / sgGDF15 complex were weighed and dissolved in normal saline and normal saline containing 10 mM GSH, respectively, and incubated at 37°C for 4 h; transmission electron microscopy was performed by Science Compass Biotech.

[0040] 3. T7 restriction enzyme digestion experiment (1) DNA extraction a. Add the nanocapsules or untreated Cas9 and sgGDF15 obtained above to the GL261 cell culture medium, shake well, and collect the cells to be tested after 48 hours; b. Add 180 μL Buffer GL, 20 μL Proteinase K, and 10 μL RNase A, digest at 56°C for 3 h, centrifuge at 12,000 rpm for 2 min, and collect the supernatant; c. Add 200 μL Buffer GB and 200 μL anhydrous ethanol, and mix by inversion; d. Add to the Spin Column, centrifuge at 12000 rpm for 2 min, and discard the liquid; e. Add 500 μL Buffer WA to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; f. Add 700 μL Buffer WB to the Spin Column, centrifuge at 12,000 rpm at room temperature for 1 min, discard the filtrate, and centrifuge the empty column for 2 min; g. Place the Spin Column in a new centrifuge tube, add 50 μL of sterile water, and let stand for 5 min; h. Centrifuge at 12000 rpm for 2 min to obtain genomic DNA, and then perform quantitative analysis using an enzyme marker; (2) T7E1 digestion detection a. PCR amplification of target fragment; b. Purification of PCR products: Purify the target fragment using ethanol; c. DNA fragment annealing, annealing system is as follows: 2 μL 10×T7E1 Buffer, 200 ng DNA fragment, 14 μL DEPC water, annealing procedure is as follows:

[0041] d. Add 0.5 μL T7E1 Enzyme and incubate at 37°C for 30 min. e. Agarose gel electrophoresis detection; (3) Agarose gel electrophoresis a. Weigh 1 g agarose powder, add it to 100 mL TAE buffer, and heat to dissolve; b. Add 10 μL of GelRed dye to the gel, mix well and pour the gel into the mold; c. Place the gel in the electrophoresis tank, pour in TAE buffer, and load the sample; d. Electrophoresis at 100 V for 40 min; e. Take out the gel and put it into the imager, take a picture and save it.

[0042] The above results are shown in Figure 1 A- Figure 1G , which shows the construction diagram of the nanocapsule targeting glioma containing the Cas9 / sgGDF15 complex of the present invention and the in vivo evaluation diagram of the glioma targeting of the nanocapsule, Figure 1A It can be seen that the present invention successfully prepared glioma-targeting nanocapsules containing Cas9 / sgGDF15 complexes; Figure 1B The average particle size of the glioma-targeting nanocapsules containing the Cas9 / sgGDF15 complex prepared in this example was measured by dynamic light scattering technology to be about 124 nm; Figure 1C The Zeta potential detection graph shows that the Zeta potential of the glioma-targeting nanocapsules containing the Cas9 / sgGDF15 complex prepared in this example is about +24.65 ± 3.79 Mv; Figure 1D The transmission electron microscopy image of the nanocapsule shows that the nanocapsule of the present invention will undergo structural disintegration in a physiological saline solution with a GSH concentration of 10 mM; Figure 1E It can be seen that the nanocapsule structure of the present invention is stable and enhances the efficiency of GDF15 gene editing; Figure 1F and Figure 1G The sequencing results showed that after the cells were treated with the nanocapsules of the present invention, the GDF15 gene of the cells was significantly edited.

[0043] Example 2: Evaluation of the targeting ability of glioma-targeting nanocapsules containing Cas9 / sgGDF15 complexes to gliomas in vitro and in vivo 1. Construction of in vitro blood-brain barrier model a. bEnd.3 cells were seeded into the upper chamber of a Tranwell chamber coated with 2% gelatin solution and cultured in DMEM containing 1% double antibody and 10% FBS for 7 days; b. GL261 cells were seeded in the lower chamber of the Tranwell chamber, and the transendothelial electrical resistance (TEER) value of the bEnd.3 monolayer exceeded 200 Ω cm 2 This indicates that the blood-brain barrier was successfully established; c. The stained nanocapsules (FITC-labeled Cas9 protein, Cy5.5-labeled sgRNA) were added to the upper chamber, and the fluorescence intensity of the liposomes in the glioma cells in the lower chamber was observed after 24 h to evaluate the penetration efficiency of the liposomes through the blood-brain barrier. 2. Construction of mouse orthotopic glioma model a. Digest and count GL261 cells expressing luciferase at 2×10^6 / cell, and resuspend in 15 μL of saline to prepare a single cell suspension; b. Weigh the C57BL / 6 mice and calculate the dose required for anesthetizing nude mice according to 0.07 mL / g. The anesthetic used in this experiment was 5% chloral hydrate. After intraperitoneal injection, the mice were completely anesthetized in about 5 minutes. The mice were fixed on a stereotaxic apparatus to prepare for surgery. c. Shave the hair of the mouse head, disinfect it, cut the midline skin of the head to expose the skull, use a stereotaxic instrument to locate the needle 2.5 mm to the right and 0.5 mm from the front edge of the sagittal suture of the mouse, and drill a hole to a depth of 2.5 mm; d. Single cell suspension was injected into the mouse brain at a rate of 2 μL / min.

[0044] e. After the injection stops, withdraw the needle by 0.5 mm every 2 minutes. f. After the needle is completely removed, use 75% alcohol cotton balls to disinfect the burr hole, suture the incision on the mouse's head with sterile sutures, and place the mouse in an incubator. 3. Small Animal In Vivo Imaging D-Luciferin working solution (15 mg / mL) was prepared with DPBS and sterile filtered with a 0.2 μm filter membrane. The mice were weighed and each group of mice was intraperitoneally injected with D-Luciferin working solution at a dose of 10 μL / g. Subsequently, each group of mice was placed in an anesthesia induction box containing isoflurane. After 7 minutes, the animals were taken out and their heads / nose were fixed in an anesthesia mask, and the animals were checked to see if they were in a state of complete anesthesia. The exposure time of the small animal imager was set to 1 s, and the animals were observed and photographed for preservation. The Living Images software was used to determine the integrated flux of photons to confirm that the in situ glioma model in mice was successfully constructed.

[0045] 4. Tail vein injection of the nanocapsules of the present invention The mice with successfully constructed glioma models were placed in the tail vein injection auxiliary instrument, and each mouse was injected with approximately 200 μL of nanocapsule solution at a dose of 2 mg / kg of Cas9 protein. The fluorescence distribution of the nanocapsules was observed with a small animal imager at 0, 1, 2, 4, 6, 8, 12, and 24 hours after injection. After the 24th hour of observation, the mice were euthanized, and the brain and major organs (heart, liver, spleen, lung, and kidney) of the mice were dissected and removed. Photos were taken to observe the distribution of nanocapsules in the brain and major organs of the mice. Finally, frozen sections were made of the brain tumor tissues of the mice, and after DAPI staining of the cell nuclei, laser confocal microscopy was used to observe the distribution of nanocapsules in tumors and normal tissues in the liver.

[0046] The above results are shown in the attached Figure 1H-1JAs shown, Figure 1H In order to establish an in vitro blood-brain barrier model to verify the targeting ability of nanocapsules to glioma cells, confocal microscopy imaging results showed that nanocapsules with targeting peptide Ang had the highest targeting efficiency; Fig. 1I To investigate the enrichment of nanocapsules in the brain tumor of mice with or without targeting peptide Ang after tail vein injection of mice with orthotopic glioma model within 24 hours, and the enrichment of nanocapsules in important organs and brain glioma 24 hours after injection. Nanocapsules with glioma targeting peptide Ang will be more enriched in brain glioma 24 hours after tail vein injection. Figure 1J This is the color image of nanocapsules with and without targeting peptide Ang in tumor tissues. The laser confocal results show that nanocapsules with glioma targeting peptide Ang can be more enriched in brain tumor sites, while nanocapsules without targeting peptide are more enriched in normal brain tissues.

[0047] Example 3: Verification of the therapeutic effect of glioma-targeting nanocapsules containing Cas9 / sgGDF15 complexes in the GL261 orthotopic glioma mouse model 1. Construction of mouse orthotopic glioma model The same method for constructing the mouse orthotopic glioma model as in Example 2 2. Treatment of orthotopic glioma model mice On the 7th day after the glioma model was established, the three groups of mice were injected with targeted peptide nanocapsules, control nanocapsules and saline through the tail vein, and the tail vein was administered 4 times, namely on the 7th day, 14th day, 21st day and 28th day. Before each administration, the mice were imaged and monitored using a small animal imager, and the tumor fluorescence value of each mouse was recorded. Two days after the last administration, the last mouse tumor imaging was performed on the 30th day after modeling, and the mice were subsequently euthanized.

[0048] 3. Tumor tissue detection Mice were dissected to obtain glioma tissues, and the tumor tissue of each mouse was divided into 4 parts. One part was used to extract DNA for PCR amplification and T7 enzyme digestion experiments; one part was used for immunohistochemistry staining to detect the expression of Ki67, Caspase-3 and GDF15 in the tissue; one part was used for flow cytometry to detect the changes in immune cells in mouse glioma; and the last part was used for immunofluorescence staining to detect the expression of major immune cell markers in the tissue.

[0049] 4. Flow cytometry Dissociate the collected tumor tissue into a single cell suspension and select appropriate fluorescent-labeled antibodies (CD3, CD8, GZMB, CD86, CD206, F4 / 80, CD11b). Mix the cells with the fluorescent antibodies and incubate them at room temperature in the dark for about 30 minutes. After incubation, wash the cells 2-3 times with PBS or other buffer to remove unbound dyes or antibodies. Then resuspend the washed cells in an appropriate buffer to keep the cells dispersed and detect them on a flow cytometer.

[0050] In this example and subsequent examples, in addition to the conventional control group injected with normal saline, an additional negative control group injected with an equal amount of glioma-targeted nanocapsules containing Cas9 / sgNC complexes was set up to illustrate the importance of precise knockout of GDF15 in tumor cells.

[0051] The results of the above experiments are shown in the attached Figure 2A-2L As shown, it shows the tumor treatment effect of the nanocapsule of the present invention in the GL261 orthotopic glioma mouse model, and FIG2A is a schematic diagram of the construction of the GL261 orthotopic glioma model and the administration time point; Figure 2B The quantitative analysis of the tumor volume of mice in different treatment groups after specified treatment shows that the tumor volume of mice treated with the Cas9 / sgGDF15 nanocapsules of the present invention is smaller than that of the other two groups. Figure 2C The tumor fluorescence results after designated treatment in C57 mice of different treatment groups bearing GL261-Luc cells orthotopically and the tumor distribution after HE staining of whole brain sections; Figure 2D It is a quantitative analysis of the tumor fluorescence levels of mice in different treatment groups during the specified treatment period. It can be seen that the tumor fluorescence values ​​of mice in the Cas9 / sgGDF15 nanocapsule treatment group are lower than those in the other two groups; Figure 2E This is the statistical result of the survival of mice in different treatment groups. It can be seen that the survival of mice was significantly prolonged after treatment with Cas9 / sgGDF15 nanocapsules; Figure 2F The editing status of GDF15 gene in tumor tissues of mice in different treatment groups. Cas9 / sgGDF15 nanocapsules successfully edited the GDF15 gene in the GL261 orthotopic glioma site; Figure 2G The editing efficiency of the GDF15 gene in mouse tumors after Cas9 / sgGDF1 nanocapsule treatment was detected at the DNA sequencing level, and the results showed that the mutation rate was 59.4%; Figure 2HImmunohistochemical analysis of GDF15, Caspase-3, and Ki67 expression in tumor tissues resected from mice after designated treatment showed a significant decrease in the presence of GDF15-positive tumor cells, and decreased Ki67 expression and increased Caspase-3 expression confirmed that Cas9 / sgGDF15 nanocapsules effectively inhibited tumor growth; Figure 2I-2K Flow cytometry was used to detect the CD206+TAM and CD86 in the tumors of mice in different treatment groups after treatment. + TAM and CD3 + CD8 + GZM + The content of T cells, as shown in the figure, after the treatment with Cas9 / sgGDF15 nanocapsules, the content of M1 macrophages in the tumor increased, the content of M2 macrophages decreased, and the content of cytotoxic T lymphocytes increased, which indicates that the nanocapsules targeting gliomas containing Cas9 / sgGDF15 complexes provided by the present invention successfully reversed the immunosuppressive microenvironment in tumor cells and restored the anti-tumor effect of immune cells, so as to achieve the purpose of treating gliomas; Figure 2L Representative multiple immunofluorescence staining of tumor tissues of mice in different treatment groups treated with designated drugs. The staining markers include DAPI (blue), PanCK (pink), CD8 (red), GZMB (yellow), CD86 (orange) and CD206 (green). The results further support the above conclusion that glioma-targeting nanocapsules containing Cas9 / sgGDF15 complexes can reshape the glioma immune microenvironment and effectively inhibit the progression of glioma.

[0052] Example 4: Verification of the tumor-suppressing effect of glioma-targeting nanocapsules containing Cas9 / sgGDF15 complexes in a virus-induced spontaneous glioma mouse model 1. Construction of virus-induced primary glioma model in mice a. Digest and count DF-1 cells infected with RCAS virus at 2×10^5 / cell, and resuspend in 4 μL of saline to prepare a single cell suspension; b. For G / tv-a;Arf - / - The mice were weighed and the dose required for anesthetizing the nude mice was calculated according to 0.07 mL / g. The anesthetic used in this experiment was 5% chloral hydrate. After intraperitoneal injection, the mice were completely anesthetized in about 5 minutes. The mice were fixed on a stereotaxic apparatus to prepare for surgery. c. Shave the hair of the mouse head, disinfect it, cut the midline skin of the head to expose the skull, use a stereotaxic instrument to locate the needle 1.2 mm to the right of the mouse's bregma and 0.5 mm from the front edge, and drill a hole to a depth of 2.5 mm; d. Single cell suspension was injected into the mouse brain at a rate of 1.5 μL / min.

[0053] e. After the injection stops, withdraw the needle by 0.5 mm every 2 minutes. f. After the needle is completely removed, use 75% alcohol cotton balls to disinfect the burr hole, suture the incision on the mouse's head with sterile sutures, and place the mouse in an incubator.

[0054] 2. Treatment of spontaneous glioma model mice On the 7th day after the glioma model was established, the three groups of mice were injected with targeted peptide nanocapsules, control nanocapsules and normal saline through the tail vein. The drugs were administered through the tail vein 4 times, namely on the 7th day, 14th day, 21st day and 28th day.

[0055] 3. Tumor tissue detection The same method as in Example 3 for detecting tumor tissue in the in situ glioma model mouse.

[0056] 4. Flow cytometry The same method as in Example 3 was used for the flow cytometry analysis of tumor cells.

[0057] The above results are shown in FIG3 , which shows the therapeutic effect of the nanocapsules of the present invention on mice with virus-induced spontaneous glioma. Figure 3A Schematic diagram of the construction of virus-induced spontaneous glioma model and the time point of drug administration; Figure 3B The tumor distribution of spontaneous glioma mice in different treatment groups after treatment was shown in HE staining of whole brain sections; Figure 3C It is a quantitative analysis of the tumor volume of mice in different treatment groups after the specified treatment. It can be seen that the tumor volume of mice treated with the Cas9 / sgGDF15 nanocapsules of the present invention is smaller than that of the other two groups; Figure 3D The growth curves of the relative volumes of tumors in mice in different treatment groups during the specified treatment period show that the tumor growth of mice in the Cas9 / sgGDF15 nanocapsule treatment group was significantly slowed down; Figure 3E This is the statistical result of the survival of mice in different treatment groups. The survival of mice treated with Cas9 / sgGDF15 nanocapsules was significantly prolonged. Figure 3F The editing status of GDF15 gene in tumor tissues of mice in different treatment groups. Cas9 / sgGDF15 nanocapsules successfully edited the GDF15 gene in spontaneous glioma sites; Figure 3G DNA sequencing was used to detect the editing efficiency of the GDF15 gene in mouse tumors after treatment with glioma-targeting nanocapsules containing the Cas9 / sgGDF15 complex, and the results showed that the mutation rate was 59.4%; Figure 3HImmunohistochemical analysis of GDF15, Caspase-3, and Ki67 expression in tumor tissues resected from mice after designated treatment showed a significant decrease in the presence of GDF15-positive tumor cells, and decreased Ki67 expression and increased Caspase-3 expression confirmed that Cas9 / sgGDF15 nanocapsules effectively inhibited tumor growth; Figure 3I-3K Flow cytometry was used to detect CD206 in tumors of mice in different treatment groups after treatment. + TAM, CD86 + TAM and CD3 + CD8 + GZM + The content of T cells is shown in the figure. After treatment with Cas9 / sgGDF15 nanocapsules, the content of M1 macrophages in the tumor increased, the content of M2 macrophages decreased, and the content of cytotoxic T lymphocytes increased, which indicates that the nanocapsules targeting gliomas containing the Cas9 / sgGDF15 complex provided by the present invention successfully reversed the immunosuppressive microenvironment in tumor cells and restored the anti-tumor effect of immune cells to achieve the purpose of treating gliomas; L is a representative multiple immunofluorescence staining of tumor tissues of mice treated with designated drugs in different treatment groups, and the staining markers include DAPI (blue), PanCK (pink), CD8 (red), GZMB (yellow), CD86 (orange) and CD206 (green). The results further confirmed the above conclusion that the nanocapsules targeting gliomas containing the Cas9 / sgGDF15 complex can effectively stimulate tumor immunity and thus curb the progression of spontaneous gliomas.

[0058] Example 5: Glioma-targeted nanocapsules containing Cas9 / sgGDF15 complexes can enhance the efficacy of PD-1 blockade therapy 1. Construction of mouse orthotopic glioma model The same method as in Example 3 for constructing the mouse orthotopic glioma model 2. Treatment of glioma model mice On the 7th day after the glioma model was established, the mice were injected with nanocapsules and PD-1 antibodies through the tail vein. The drugs were administered through the tail vein 4 times, namely on the 7th day, the 14th day, the 21st day, and the 28th day. The dosage of PD-1 monoclonal antibody was 6 mg / kg, and the dosage of nanocapsules was 2 mg / kg. Before each administration, the mice were imaged with a small animal imager, and the tumor fluorescence value of each mouse was recorded. The mice were euthanized 2 days after the last administration.

[0059] 3. Tumor tissue detection The same method as in Example 3 for detecting tumor tissue in the in situ glioma model mouse.

[0060] 4. Flow cytometry The same method as in Example 3 was used for the flow cytometry analysis of tumor cells.

[0061] The above results are shown in the attached Figure 4A-Figure 4I As shown, it shows the experimental results analysis of the effect diagram of the gene editing therapy mediated by the nanocapsule of the present invention combined with the anti-mouse PD1 monoclonal antibody treatment. Figure 4A The tumor fluorescence results after designated treatment in C57 mice of different treatment groups bearing GL261-Luc cells orthotopically and the tumor distribution after HE staining of whole brain sections; Figure 4B This is a quantitative analysis of the tumor fluorescence levels of four groups of mice treated differently during the specified treatment period. It can be seen that the Cas9 / sgGDF15 nanocapsule combined with PD-1 monoclonal antibody treatment group has the most significant effect in inhibiting mouse tumor growth; Figure 4C This is the statistical result of the survival period of four groups of mice with different treatments. It can be seen that the group treated with Cas9 / sgGDF15 nanocapsules combined with PD-1 monoclonal antibody has the longest survival period; Figure 4D DNA sequencing was used to detect the editing efficiency of the GDF15 gene in the tumors of two groups of mice treated with Cas9 / sgGDF15 nanocapsules alone and Cas9 / sgGDF15 nanocapsules combined with PD-1 monoclonal antibodies. The results showed that the mutation rates were 55.9% and 57.4%, respectively. Figure 4E Immunohistochemical analysis of GDF15, Caspase-3, and Ki67 expression in tumor tissues removed from mice after designated treatment. The decreased expression of Ki67 and the increased expression of Caspase-3 showed that the combination therapy of Cas9 / sgGDF15 nanocapsules and PD-1 monoclonal antibody had a significant inhibitory effect on tumor growth. Figure 4F-4H Flow cytometry was used to detect CD206 in tumors of mice in different treatment groups after treatment. + TAM, CD86 + TAM and CD3 + CD8 + GZM + T cell content; Fig. 4I Representative multiplex immunofluorescence staining of tumor tissues of mice treated with the indicated drugs in different treatment groups, e.g. Figure 4F-Figure 4I The results showed that compared with single treatment with Cas9 / sgGDF15 nanocapsules or PD-1 antibodies, combined treatment increased the expression of immune stimulatory cells (CD86 + TAM and CD3 + CD8 + GZM + T cells) and immunosuppressive cells (CD206 +The effect of reducing TAM content is the strongest, that is, the combined therapy has the most significant effect in reversing the immunosuppressive microenvironment of glioma. The GDF15 gene editing therapy presented in the present invention combined with PD-1 blocking therapy has a synergistic inhibitory effect on glioma, which provides a promising treatment option for GBM with a "cold" immune microenvironment.

[0062] In summary, the present invention discloses a nanocapsule targeting glioma containing a Cas9 / sgGDF15 complex. The glioma targeting of the nanocapsule is improved by cross-linking the target peptide Angiopep-2 (Ang) with strong brain penetration ability on the surface of the nanocapsule; adding a disulfide bond on the surface of the nanocapsule can protect the nanocapsule from degradation by enzymes in the blood and break in the high glutathione microenvironment of the tumor site, accurately releasing the encapsulated Cas9 / sgGDF15 complex therein, and then efficiently editing the GDF15 gene of glioma cells, specifically knocking out the GDF15 gene in glioma cells, reversing the immunosuppressive microenvironment of glioma, restoring the anti-tumor effect of immune cells, and achieving the purpose of treating glioma. It avoids the defects of low loading efficiency, non-specific targeting, the risk of causing neuroinflammation, and the lack of reactive drug release mechanism existing in traditional drug delivery carriers, thereby greatly improving the therapeutic effect. Therefore, delivering the Cas9 / sgGDF15 complex to the glioma area, knocking out the GDF15 gene, and reversing the suppressive immune microenvironment of glioma becomes a potential clinical treatment option.

Claims

1. A nanocapsule targeting glioma containing a Cas9 / sgGDF15 complex, characterized in that: The nanocapsule core is wrapped by the nanocapsule outer wall; wherein: The nanocapsule core contains a negatively charged Cas9 / sgGDF15 complex; The outer wall of the nanocapsule contains glioma targeting peptide Ang.

2. The glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex according to claim 1, characterized in that: The Cas9 / sgGDF15 complex is obtained by dissolving sgGDF15 and Cas 9 proteins in a molar ratio of 1:1 in HEPES buffer.

3. The glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex according to claim 2, characterized in that: The gene sequence of sgGDF15 is shown in SEQ ID NO:

1.

4. The glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex according to claim 1, characterized in that: The nanocapsule wall is polymerized by a disulfide bond crosslinking agent and polyethylene glycol, and the glioma targeting peptide Ang is connected to the disulfide bond group through an amidation reaction.

5. The glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex according to claim 1, characterized in that: The nanocapsule has a spherical shape and a size of 100~160nm.

6. The method for preparing a glioma-targeting nanocapsule containing a Cas9 / sgGDF15 complex according to any one of claims 1 to 5, characterized in that: include: Preparation of nanocapsule cores containing negatively charged Cas9 / sgGDF15 complexes; The outer wall of a nanocapsule containing the glioma targeting peptide Ang is prepared; the inner core of the nanocapsule is wrapped with the outer wall of the nanocapsule to prepare a glioma targeting nanocapsule containing a Cas9 / sgGDF15 complex.

7. The method for preparing a glioma-targeting nanocapsule containing a Cas9 / sgGDF15 complex according to claim 6, characterized in that: The method comprises the following steps: dissolving a positively charged Cas9 protein and a negatively charged sgGDF15 to obtain a negatively charged Cas9 / sgGDF15 complex; and then adding the negatively charged Cas9 / sgGDF15 complex to a positively charged citric acid buffer to obtain a nanocapsule core.

8. The method for preparing a glioma-targeting nanocapsule containing a Cas9 / sgGDF15 complex according to claim 6, characterized in that: The outer wall of the nanocapsule is prepared, including: polymerizing a disulfide bond crosslinking agent with a neutral molecule polyethylene glycol having an amide group and a succinic acid functional group, and connecting a glioma targeting peptide Ang with a disulfide bond group through an amidation reaction.

9. Use of the glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex according to any one of claims 1 to 5 in the preparation of a glioma-targeting drug.

10. Use of the glioma-targeting nanocapsule containing the Cas9 / sgGDF15 complex according to any one of claims 1 to 5 in the preparation of a PD1 antibody enhancer.

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