Gene Editing Nanocapsule and Preparation Method and Use Thereof

Abstract

The present disclosure provides a gene editing nanocapsule and a preparation method and use thereof. The gene editing nanocapsule has a core-shell structure, wherein the inner core includes a Cas/sgRNA ribonucleoprotein complex, and the outer shell includes a polymer, the Cas/sgRNA ribonucleoprotein complex has a gene editing function, and the polymer acts as a carrier for the Cas/sgRNA ribonucleoprotein complex and protects it, because the polymer contains tumor microenvironment sensitive molecules, the nanocapsules can be efficiently released in tumor cells. Further, the surface of the outer shell can be modified with a targeting agent, so that the nanocapsule can specifically target tumor cells, which improves the endocytosis efficiency of the nanocapsule. The gene editing nanocapsule has good biocompatibility and biosafety, and is expected to become a safe and efficient gene therapy drug for tumors.

Claims

1. A gene editing nanocapsule, comprising an inner core and an outer shell encapsulating the inner core, wherein the inner core comprises a Cas/sgRNA ribonucleoprotein complex obtained by combining Cas nuclease and sgRNA; and the outer shell comprises a polymer polymerized by a monomer material, wherein the monomer material comprises a first monomer and a second monomer that can be polymerized with each other, wherein the first monomer is a molecule capable of electrostatically binding with the Cas/sgRNA ribonucleoprotein complex, and the second monomer is a tumor microenvironment sensitive molecule.

2. The gene editing nanocapsule according to claim 1, wherein the first monomer comprises at least one of guanidino acrylate, spermine acrylate, and N-(3-aminopropyl)methacrylamide; the tumor microenvironment sensitive molecule comprises at least one of a reduction-sensitive molecule, an acid-sensitive molecule and a ROS responsive molecule.

3. The gene editing nanocapsule according to claim 1, wherein the gene editing nanocapsule further comprises a targeting agent modified on an outer surface of the outer shell; the monomer material further comprises a third monomer that can be polymerized with the first monomer and/or the second monomer, wherein the third monomer is a molecule that can be connected to the targeting agent through a chemical bond.

4. The gene editing nanocapsule according to claim 1, wherein a target gene of the sgRNA is a tumor-targeted therapeutic gene.

5. The gene editing nanocapsule according to claim 3, wherein the molar ratio of the Cas nuclease and the sgRNA is 1:1˜1.5; the monomer material comprises acrylate polyethylene glycol succinimidyl formate, guanidino acrylate, and N,N′-bis(acryloyl)cystamine, wherein a molar ratio of the acrylate polyethylene glycol succinimidyl formate, the guanidino acrylate, and the N,N′-bis(acryloyl)cystamine is 1˜3:1˜3:1˜3; a molar ratio of the Cas nuclease and the guanidino acrylate is 1:200˜250; and the targeting agent is Angiopep-2, and a molar ratio of the Angiopep-2 and the acrylate polyethylene glycol succinimidyl formate is 1˜5:1.

6. A drug for treating a tumor, comprising the gene editing nanocapsule according to claim 1.

7. A preparation method for the gene editing nanocapsule according to claim 1, comprising following steps: step 1: incubating Cas nuclease and sgRNA in a buffer to form the Cas/sgRNA ribonucleoprotein complex; and step 2: adding the monomer material and an initiator to a system obtained in step 1, so that the monomer material undergoes a polymerization reaction to form the polymer that is coated on an outer surface of the Cas/sgRNA ribonucleoprotein complex.

8. The preparation method for the gene editing nanocapsule according to claim 7, wherein the preparation method further comprises: step 3: adding a targeting agent to a system obtained in step 2, wherein the targeting agent is connected to the polymer by a chemical bond; and the monomer material added in the step 2 further comprises a third monomer that can be polymerized with the first monomer and/or the second monomer, wherein the third monomer is a molecule that can be connected to the targeting agent through a chemical bond.

9. The preparation method for the gene editing nanocapsule according to claim 7, wherein the initiator comprises ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine, wherein a ratio of the ammonium persulfate and a reaction system is 1˜5 mg: 500 μL, a ratio of a N,N,N′,N′-tetramethylethylenediamine solution and the reaction system is 1˜5 μL: 500 μL, and a concentration of the N,N,N′,N′-tetramethylethylenediamine solution is 0.2%-0.8% w/v.

10. The gene editing nanocapsule according to claim 2, wherein the second monomer is the reduction-sensitive molecule.

11. The gene editing nanocapsule according to claim 2, wherein the reduction-sensitive molecule is a molecule containing disulfide bonds.

12. The gene editing nanocapsule according to claim 3, wherein the targeting agent comprises at least one of Angiopep-2, RGD peptide, apolipoprotein E, and transferrin.

13. The gene editing nanocapsule according to claim 4, wherein the tumor-targeted therapeutic gene comprises at least one of MTH1 gene and PLK1 gene.

14. The gene editing nanocapsule according to claim 4, wherein a sequence of a target site of the sgRNA on the PLK1 gene is shown in SEQ ID NO:1.

15. The drug for treating a tumor according to claim 6, wherein the tumor is glioma, non-small cell lung cancer or cervical cancer.

16. The preparation method for the gene editing nanocapsule according to claim 8, wherein in step 3, after adding the targeting agent, stirring is carried out for 1 to 3 hours.

17. The preparation method for the gene editing nanocapsule according to claim 8, wherein the third monomer comprises at least one of acrylate polyethylene glycol succinimidyl formate and acrylate polyethylene glycol maleimide.

18. The preparation method for the gene editing nanocapsule according to claim 9, wherein the step 1 is performed under a condition of 10° C.˜30° C., and an incubation time is 3 to 8 minutes; and the step 2 is performed at 0° C.˜5° C. in an oxygen-free environment with stirring, and a reaction time is 60 to 120 minutes.

19. The preparation method for the gene editing nanocapsule according to claim 9, wherein the preparation method for the gene editing nanocapsule further comprises: performing a step of removing impurities after the gene editing nanocapsules are prepared.

20. The preparation method for the gene editing nanocapsule according to claim 9, wherein an ultrafiltration centrifuge tube with a 10 kDa molecular weight cut-off is used for removing impurities.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] In order to illustrate the technical solutions of the embodiments of the present disclosure more clearly, the drawings need to be used in the embodiments will be briefly introduced below, it should be understood that the following drawings only show some embodiments of the present disclosure, and therefore should not be regarded as a limitation of the scope of the present disclosure.

[0057] FIG. 1A and FIG. 1B show the particle size distribution and morphological characteristics of ANC.sub.SS (Cas9/sgRNA) nanocapsules, wherein FIG. 1A is the particle size distribution diagram of ANC.sub.SS (Cas9/sgRNA) nanocapsules, FIG. 1B is a scanning electron micrograph of ANC.sub.SS (Cas9/sgRNA) nanocapsules.

[0058] FIG. 2A-FIG. 2E shows the experiment results of part of in vitro cells, wherein

[0059] FIG. 2A: a flow cytometry diagram of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA after incubating for 4 hours in U87MG-luc cells;

[0060] FIG. 2B: laser confocal micrograph of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA after incubating for 4 hours in U87MG-luc cells;

[0061] FIG. 2C: T7E1 experiment analyzes the editing efficiency in U87MG-luc cells, and the values are obtained by imageJ analysis;

[0062] FIG. 2D: WB analyzes the expression of PLK1 in U87MG-luc cells;

[0063] FIG. 2E: sequencing results of PLK1 gene editing in U87MG-luc cells treated with ANC.sub.SS (Cas9/sgRNA) nanocapsules.

[0064] FIG. 3A and FIG. 3B show the experiment results of another part of in vitro cells, wherein

[0065] FIG. 3A: T7E1 experiment analyzes the editing efficiency in CSC2-luc cells, and the values are obtained by imageJ analysis;

[0066] FIG. 3B: WB analyzes the expression of PLK1 in CSC2-luc cells.

[0067] FIG. 4A, FIG. 4B and FIG. 4C are in vivo experimental study results, wherein

[0068] FIG. 4A shows the pharmacokinetic study of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA;

[0069] FIG. 4B shows the qualitative distribution of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA in the heart, liver, spleen, lung, and kidney; the enlarged image on the right shows the tumor penetration condition of ANC.sub.SS (Cas9/sgRNA) and the control group observed through a confocal laser scanning microscope (CLSM);

[0070] FIG. 4C shows the quantitative distribution of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA in the heart, liver, spleen, lung, and kidney.

[0071] FIG. 5A-FIG. 5J shows the in vivo therapeutic effect of ANC.sub.SS (Cas9/sgRNA) nanocapsules on U87MG-luc tumor-bearing mice, wherein

[0072] FIG. 5A: schematic view showing in situ tumor study timetable;

[0073] FIG. 5B: relative photon quantity of different nanoparticles;

[0074] FIG. 5C: changes in body weight of mice during treatment;

[0075] FIG. 5D: bioluminescence of U87MG-luc;

[0076] FIG. 5E: survival rate of mice during treatment;

[0077] FIG. 5F: T7E1 experiment analyzes the editing efficiency for U87MG-luc tumor tissue, and the values are obtained through imageJ analysis;

[0078] FIG. 5G: H&E whole brain scanning to characterize tumor size;

[0079] FIG. 5H: WB analyzes the expression of PLK1 in U87MG-luc tumor tissue;

[0080] FIG. 5I: WB quantitative results;

[0081] FIG. 5J: gene editing and sequencing results of U87MG-luc tumor tissue.

[0082] FIG. 6A-FIG. 6J shows the in vivo therapeutic effect of ANC.sub.SS (Cas9/sgRNA) nanocapsules on CSC2-luc tumor-bearing mice, wherein

[0083] FIG. 6A: schematic view of establishment of the PDX derived GBM GSC orthotopic model;

[0084] FIG. 6B: biofluorescence of CSC2-luc cells;

[0085] FIG. 6C: H&E whole brain scanning to characterize tumor size;

[0086] FIG. 6D: relative photon quantity of different nanoparticles;

[0087] FIG. 6E: changes in body weight of mice during treatment;

[0088] FIG. 6F: survival rate of mice during treatment;

[0089] FIG. 6G: T7E1 experiment analyzes the editing efficiency for CSC2-Luc tumor tissue, and the values are obtained by imageJ analysis;

[0090] FIG. 6H: WB analyzes the expression of PLK1 in CSC2-Luc tumor tissues;

[0091] FIG. 6I: WB quantitative results;

[0092] FIG. 6J: gene editing and sequencing results of CSC2-Luc tumor tissue.

[0093] FIG. 7 shows the fluorescence intensity at different time points after ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA were injected into mice.

DETAILED DESCRIPTION OF EMBODIMENTS

[0094] Terms as used in the present disclosure:

[0095] “Prepared from” is synonymous with “comprising”. The terms “comprising”, “including”, “having”, “containing” or any other variations as used herein are intended to cover non-exclusive inclusion. For example, the composition, step, method, product or device comprising the listed elements is not necessarily only limited to those elements, but may include other elements not explicitly listed or elements inherent to such composition, step, method, product, or device.

[0096] The conjunction “consisting of” excludes any unspecified elements, steps or components. If used in a claim, this phrase will make the claim closed so that it does not include materials other than those described, except for the conventional impurities associated with it. When the phrase “consisting of” appears in a clause of the subject of a claim rather than immediately after the subject matter, it is only limited to the elements described in the clause; other elements are not excluded from the claims as a whole.

[0097] When amount, concentration, other value or parameter is expressed in ranges, preferred ranges, or ranges defined by a series of upper limit preferred values and lower limit preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of the upper limit or preferred value of any range and the lower limit or preferred value of any range, regardless of whether the ranges are separately disclosed. For example, when the range “1˜5” is disclosed, the described range should be interpreted as including the ranges “1˜4”, “1˜3”, “1˜2”, “1˜2 and 4˜5”, “1˜3 and 5” and the like. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0098] In these embodiments, unless otherwise specified, the parts and percentages mentioned are based on mass.

[0099] “Parts by mass” refers to the basic measurement unit that represents the mass ratio relationship of multiple components. 1 part can represent any unit mass, such as 1 g, or 2.689 g. If we say that the parts by mass of component A is a part, and the parts by mass of component B is b part, it means the ratio of the mass of component A to the mass of component B is a:b. Or, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number and represents a multiplying factor). It should not be misunderstood that, unlike the mass fraction, the sum of parts by mass of all components is not limited to 100 parts.

[0100] “And/or” is used to indicate that one or both of the stated conditions may occur, for example, A and/or B includes (A and B) and (A or B).

[0101] The implementation plan of the present disclosure will be described in detail below in conjunction with specific embodiments, but those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. If specific conditions are not indicated in the embodiments, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer are all conventional products that can be purchased on the market.

[0102] The sources of some reagents used in the embodiments of the present disclosure are as follows.

[0103] Guanidino acrylate was synthesized by using the method described in the literature of ROS-Responsive Polymeric siRNA Nanomedicine Stabilized by Triple Interactions for the Robust Glioblastoma Combinational RNAi Therapy;

[0104] dipropylene cystamine (sigma);

[0105] acrylate polyethylene glycol succinimidyl formate (Jenkem); and

[0106] Angiopep-2 (ChinaPeptides).

[0107] In the embodiments of the present disclosure, room temperature refers to a temperature condition of 10° C. to 30° C.

(1) Synthesis of Nanocapsules

Example 1 Preparation Method of ANC.SUB.SS .(Cas9/sgRNA) Nanocapsules

[0108] Step 1. Cas9 and sgRNA were added to 500 μl 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES buffer) (10 mM PH 7.4) at a molar ratio of 1:1.2, and incubated at room temperature for 5 minutes.

[0109] Specifically, the target gene of the sgRNA is the PLK1 gene, and the sequence of the target site of the sgRNA on the PLK1 gene is shown in SEQ ID NO:1.

[0110] Step 2. the above system was transferred to a 4° C. environment, acrylate polyethylene glycol succinimidyl formate was added and stirred for 10 minutes, then guanidino acrylate was added and stirred for 5 minutes, and then the degradable N,N′-bis(acryloyl)cystamine was added, wherein the molar ratio of acrylate polyethylene glycol succinimidyl formate, guanidino acrylate, and N,N′-bis(acryloyl)cystamine was 1:1:1.3 mg of ammonium persulfate and 3 μL of N,N,N′,N′-tetramethylethylenediamine solution were added to immediately initiate the polymerization reaction, wherein the polymerization reaction was carried out at 4° C. and under nitrogen protection for 90 minutes, the polymerization reaction process was always accompanied by mechanical stirring.

[0111] Specifically, the molar ratio of the Cas nuclease and the guanidino acrylate was 1:220.

[0112] Step 3. the amination-treated Angiopep-2 was added to the above system, stirred at room temperature for 2 hours, thereby finally forming the nanocapsules.

[0113] Specifically, in both step 2 and step 3, a magnetic stirrer was used for stirring, and the stirring speed was 250-350 rpm.

[0114] Step 4. a centrifugal filter tube with a 10 kDa molecular weight cut-off is used for impurity removal to remove unreacted monomers and initiators, PBS buffer (pH 7.4) is used to dilute the concentrated solution.

[0115] FIG. 1A and FIG. 1B are views of DLS test results of ANC.sub.SS (Cas9/sgRNA) nanocapsules prepared in Example 1. As shown in FIG. 1A and FIG. 1B, the average particle size of the nanocapsules prepared in Example 1 is 31 nm, and the particle size distribution is relatively uniform.

Example 2 Preparation Method of ANC.SUB.SS .(Cas9/sgRNA) Nanocapsules

[0116] The difference from Example 1 above is only that: the molar ratio of the Cas nuclease and the guanidino acrylate was 1:200.

Example 3 Preparation Method of ANC.SUB.SS .(Cas9/sgRNA) Nanocapsules

[0117] The difference from Example 1 above is only that: the molar ratio of the Cas nuclease and the guanidino acrylate was 1:250.

Comparative Example 1 Preparation Method of ANC (Cas9/sgRNA) Nanocapsules

[0118] The difference from Example 1 above is only that: in the step 2, hexamethylene diacrylate that cannot be degraded by GSH was used to replace N,N′-bis(acryloyl)cystamine that can be degraded by GSH, therefore, the polymers in the prepared ANC (Cas9/sgRNA) nanocapsules cannot be specifically degraded by tumor cells, thus the gene editing system (Cas9RNP) in the ANC (Cas9/sgRNA) nanocapsule cannot be efficiently released in tumor cells, the gene editing efficiency is lower.

Comparative Example 2 Preparation Method of NC.SUB.SS .(Cas9/sgRNA) Nanocapsules

[0119] The difference from Example 1 above is that: step 3 was not included (that is, Angiopep-2 was not added), and at the same time the acrylate polyethylene glycol succinimidyl formate was replaced with methoxy polyethylene glycol amine (mPEG-NH.sub.2) MW: 2000 (purchased from Ponsure), and the prepared NC.sub.SS (Cas9/sgRNA) nanocapsules do not have the ability of spanning BBB and cannot be efficiently swallowed by human glioma cells.

Comparative Example 3 Preparation Method of ANC.SUB.SS .(Cas9/sgScr) Nanocapsules

[0120] The difference from Example 1 above is only that: the sgRNA used in Comparative Example 3 was an invalid sgRNA, which does not have a targeting function.

Comparative Example 4 Preparation Method of Free Cas9/sgRNA

[0121] Cas9 and sgRNA were added to 500 μl HEPES buffer at a molar ratio of 1:1.2, and incubated for 5 minutes at room temperature.

[0122] It is worth mentioning that the ANC.sub.SS (Cas9/sgRNA) nanocapsules used in the following (2) cell experiment and (3) animal experiment were all the nanocapsules prepared in Example 1, ANC (Cas9/sgRNA) nanocapsule was prepared from Comparative Example 1, NC.sub.SS (Cas9/sgRNA) nanocapsule was prepared from Comparative Example 2, ANC.sub.SS (Cas9/sgScr) nanocapsule was prepared from Comparative Example 3, and Free Cas9/sgRNA was prepared from Comparative Example 4.

(2) Cell Experiment

[0123] {circle around (1)} Cell Endocytosis and Intracellular Release were Characterized by a Flow Cytometry and a Confocal Microscope

[0124] In the flow cytometry test, after U87MG-luc cells were inoculated in a 6-well cell culture plate (1×10.sup.6 cells/well) and cultured at 37° C. for 24 hours, 150 μL PBS solution (Cas9 concentration was 20 nM) of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA was added and incubated for 4 hours, the sample was sucked out, and the cells were digested with 500 μL trypsin. The obtained cell suspension was centrifuged at 1000×g for 3 minutes, washed twice with PBS buffer, and dispersed in 500 μL PBS buffer again, and tested by the flow cytometry (BD FACS Calibur, Becton Dickinson, USA) within 1 hour, Cell Quest software was used to circle 10,000 cells to obtain them.

[0125] The cell endocytosis and intracellular drug release behavior were observed and obtained through CLSM (Confocal laser scanning microscope) photos. After U87MG-luc cells were spread in a 24-well cell culture plate (1×10.sup.5 cells/well) containing microscope slides to culture for 24 hours, 50 μL PBS buffer (Cas9 concentration was 20 nM) of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA were added. After incubating for 4 hours, the culture medium was removed and the resultant was washed twice with PBS buffer. The cytoskeleton was stained with Phalloidin for 30 minutes and washed twice, and then the nucleus was stained with DAPI for 15 minutes and washed twice. The fluorescence picture was taken by CLSM (TCS SP5).

[0126] {circle around (2)} Editing Efficiency of Cells was Characterized by an In Vitro Gene Editing

[0127] U87MG-luc/CSC2-luc cells were inoculated in a 24-well plate (5×10.sup.4 cells/well) and cultured for 24 hours. 50 μL PBS solution (Cas9 concentration was 20 nM) of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC.sub.SS (Cas9/sgScr) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA was added and incubated overnight, and then culture medium was replaced, the cells were incubated again at 37° C. for 48 h. Universal genomic DNA kit (China, CWBIO) was used to extract genomic DNA. Then, the high-fidelity enzyme Kod-Plus-Neo (Japan, TOYOBO) was used to amplify the DNA fragment containing the sgRNA target site, and the PCR products were purified by the gel recovery kit (CWBIO, China). Finally, T7E1 enzyme (USA, NEB) detected insertion and deletion efficiency. The PCR products with T7E1 analysis indicating mutations were performed by DNA sequencing, and then subcloned into T cloning vector (Vazyme Biotech, China). Colonies were randomly selected and further analyzed by DNA Sanger sequencing using M13F as a primer (Sangon Biotech), the sequencing results of some clones are shown in FIG. 2E. Sanger sequencing results showed that 12 out of 21 clones had mutations in the target sequence, wherein the mutation type of 5 clones was T single base insertion, and the mutation type of 7 clones was base deletion.

[0128] {circle around (3)} Protein Expression Level in Cells was Characterized by In Vitro WB Experiment

[0129] U87MG-luc/CSC2-luc cells were inoculated in a 6-well plate (1×10.sup.5 cells/well) and cultured for 24 hours. 150 μL PBS solution (Cas9 concentration was 20 nM) of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC.sub.SS (Cas9/sgScr) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA was added and incubated overnight, and then culture medium was replaced, the cells were incubated again at 37° C. for 72 h. The cells were treated with lysis buffer (Beyotime, China), and the concentration of the obtained protein was quantified by the BCA kit (Beyotime, China). The lysate was separated by electrophoresis (SDS polyacrylamide gel) and transferred to PDVF membrane (Beyotime, China). The PDVF membrane and the anti-PLK1 primary antibody (mouse mAb 35-206, Abcam) were diluted at 1:1000 and incubated overnight at 4° C. The resultant was incubated for 1 h with ECL secondary antibody, showing protein bands (Licor, USA). Protein bands were analyzed by using ImageJ software.

(3) Animal Experiment

[0130] {circle around (1)} Pharmacokinetic Study

[0131] In the in vivo pharmacokinetic study, 6-8 weeks BALB/c mice were randomly divided into groups (3 mice in each group), 200 μL of ANC.sub.SS (Cas9/sgRNA) nanocapsules, ANC (Cas9/sgRNA) nanocapsules, NC.sub.SS (Cas9/sgRNA) nanocapsules, and Free Cas9/sgRNA (Cas9 dosage was 30 μg) were injected to the tail vein, blood was taken from the eye socket at a predetermined time point. The drug was extracted and separated from the blood sample by organic solvent, and quantified by a multimode reader. Pharmacokinetic parameters such as elimination half-life of drug in the body (t½), area under the drug concentration-time curve (AUC), clearance rate (CL) can be calculated by software fitting.

[0132] {circle around (2)} Anti-Tumor Effect

[0133] U87MG-Luc or CSC2-Luc glioma orthotopic model was established by transplanting tumor tissue into the brain of BALB/c nude mice (18-20 g, 6-8 weeks old). When the tumor volume was 20-30 mm.sup.3, it was used for treatment experiments; when the tumor volume was 100-150 mm.sup.3, it was used for biodistribution experiments.

[0134] Orthotopic model was established by luciferase-marked human glioma cell U87MG-Luc or human glioma stem cell CSC2-Luc, single-dose or multiple-dose administration was achieved through tail vein injection method, the tumor growth was tracked qualitatively and quantitatively by IVIS III. In the course of treatment, the systemic toxic and side effects and anti-tumor activity of nanomedicine were evaluated by the weight change and survival rate of mice. After the treatment was finished, the health status of normal organs and apoptosis condition of the tumor tissue in mice after nanomedicine treatment were analyzed through the histological staining methods such as H&E and TUNEL. Through treatment experiments, the systemic toxicity and the anti-tumor activity of the nanomedicine on U87MG-Luc or CSC2-Luc tumor-bearing nude mice can be determined.

[0135] {circle around (3)} Biodistribution

[0136] The nanomedicine was injected into the body of nude mice bearing orthotopic U87MG-luc/CSC2-Luc through the tail vein, and heart, liver, spleen, lung, kidney, brain and tumors and other major tissues of mice were collected at different time points, which were imaged in vitro by IVIS III. Subsequently, after each tissue was homogenized, extracted by the organic solvent and separated by centrifuging to obtain the supernatant, fluorescence spectrophotometer quantitatively analyzed the biodistribution of drug in the body at different time points. Through this experiment, the in vivo stability of nanomedicine, active targeting performance and the effect of enrichment, retention, and penetration of the released Cas9 drug on the tumor site can be known.

[0137] {circle around (4)} BBB Spanning Effect and Targeting

[0138] The nanomedicine was injected into the body of nude mice bearing orthotopic human glioma U87MG-luc through the tail vein, and the distribution of nanomedicine at different time points in the body was tracked by the small animal imager (IVIS III), the accumulation and retention of brain tumor sites were focused on, and by qualitative and quantitative comparison with the non-targeting control group (NC.sub.SS (Cas9/sgRNA) nanocapsules) and the non-sensitive control group (ANC (Cas9/sgRNA) nanocapsules), the BBB spanning efficiency and the tumor targeting ability of nanomedicine were investigated.

(4) Results and Discussion

[0139] (1) In Vitro Cell Experiment

[0140] Flow cytometry experiments (FIG. 2A) proved that ANC.sub.SS (Cas9/sgRNA) nanocapsules have very high cell endocytosis efficiency, and the cell endocytosis efficiency thereof is 2.5 times that of the non-targeting control group NC.sub.SS (Cas9/sgRNA), and 10.3 times that of the control group ANC (Cas9/sgRNA).

[0141] CLSM (confocal laser scanning microscope) (FIG. 2B) observed that after ANC.sub.SS (Cas9/sgRNA) nanocapsules were incubated for 4 hours, there was strong Cas9 fluorescence in the U87MG-luc nucleus, which confirmed that ANC.sub.SS (Cas9/sgRNA) nanocapsule can be quickly degraded in cell to release Cas9, which is transported to the nucleus to function.

[0142] In vitro gene editing experiments (FIG. 2C) proved that ANC.sub.SS (Cas9/sgRNA) nanocapsules have a very high gene editing efficiency for U87MG-luc cells, reaching 36.6%.

[0143] WB experiments (FIG. 2D) proved that ANC.sub.SS (Cas9/sgRNA) nanocapsules can significantly reduce the expression of PLK1 in U87MG-luc cells, thereby achieving the effect of inhibiting tumor growth.

[0144] (2) In Vivo Experimental Study on the Pharmacokinetics and Biodistribution of Targeting Nanomedicine ANC.sub.SS (Cas9/sgRNA) Nanocapsules

[0145] In vivo pharmacokinetics (FIG. 4A) study results showed that the targeting nanomedicine ANC.sub.SS (Cas9/sgRNA) has a longer circulation time in the body, which is equivalent to that of ANC (Cas9/sgRNA) nanocapsule and NC.sub.SS (Cas9/sgRNA) nanocapsule, and significantly longer than that of Free Cas9/sgRNA, which showed that the biocompatibility of nanocapsules is better.

[0146] In vivo biodistribution experiment (FIG. 4B, FIG. 4C) results of BALB/c mice bearing U87MG-luc glioma showed that the enrichment amount of ANC.sub.SS (Cas9/sgRNA) nanocapsules at the tumor site is significantly higher than that of Free Cas9/sgRNA, 4 hours after injection, the enrichment amount of Cas9 is 12% (the content of Cas9 in each gram of tissue accounts for the mass percentage of the total injection volume).

[0147] (3) Anti-Tumor Experiment and Histological Analysis of ANC.sub.SS (Cas9/sgRNA) Nanocapsules

[0148] The treatment experimental results of ANC.sub.SS (Cas9/sgRNA) nanocapsules in U87MG-luc tumor-bearing BALB/c nude mice are shown in FIG. 5A-FIG. 5J.

[0149] In FIG. 5A-FIG. 5J, ANC.sub.SS (Cas9/sgPLK1) indicates that the target gene of sgRNA is PLK1, ANC.sub.SS (Cas9/sgScr) indicates a negative control experiment, and PBS indicates PBS buffer (control experiment).

[0150] It can be seen from FIG. 5A-FIG. 5J that ANC.sub.SS (Cas9/sgRNA) nanocapsules can effectively inhibit tumor growth. The weight of the mice treated with ANC.sub.SS (Cas9/sgRNA) nanocapsules changed slightly, in comparison, the body weight of the PBS group decreased by 20% within 8 days, which shows that ANC.sub.SS (Cas9/sgRNA) nanocapsules have little toxic and side effects, in addition, those skilled in the art have known that the growth of brain tumors can cause weight loss, therefore, the experimental data of the present disclosure reflects that ANC.sub.SS (Cas9/sgRNA) nanocapsules have a better curative effect and can inhibit brain tumors.

[0151] The treatment experimental results of ANC.sub.SS (Cas9/sgRNA) nanocapsules in CSC2-luc tumor-bearing BALB/c nude mice are shown in FIG. 6A-FIG. 6J.

[0152] In FIG. 6A-FIG. 6J, ANC.sub.SS (Cas9/sgPLK1) indicates that the target gene of sgRNA is PLK1, ANC.sub.SS (Cas9/sgScr) indicates a negative control experiment, and PBS indicates PBS buffer (control experiment).

[0153] It can be seen from FIG. 6A-FIG. 6J that ANC.sub.SS (Cas9/sgRNA) nanocapsules can effectively inhibit tumor growth. The weight of the mice treated with ANC.sub.SS (Cas9/sgRNA) nanocapsules changed slightly, which shows that ANC.sub.SS (Cas9/sgRNA) nanocapsules have little toxic and side effects, in addition, those skilled in the art have known that the growth of brain tumors can cause weight loss, therefore, the experimental data of the present disclosure reflects that ANC.sub.SS (Cas9/sgRNA) nanocapsules have a better curative effect and can inhibit brain tumors.

[0154] What is striking is that after the mice were treated with an ANC.sub.SS (Cas9/sgRNA) nanocapsule dosage of 30 μg, the survival cycle was significantly prolonged. The histological analysis results by H&E staining proved that after treatment with ANC.sub.SS (Cas9/sgRNA) nanocapsules at a dosage of 30 μg, there is little harm to major organs including the heart, liver, spleen, lung, and kidney. The results once again show that ANC.sub.SS (Cas9/sgRNA) nanocapsules have extremely low systemic toxicity.

[0155] (4) Tumor Targeting Experiment of ANC.sub.SS (Cas9/sgRNA) Nanocapsules

[0156] The results of tracking the distribution (FIG. 7) of nanomedicine in the body at different time points by the small animal imager (IVIS III) showed that the fluorescence intensity of ANC.sub.SS (Cas9/sgRNA) nanocapsule is significantly higher than that of the control group, which indicates that it has a very good ability of targeting tumors.

[0157] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, not to limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, those ordinary skilled in the art should understand that: the technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features therein can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of technical solutions of the embodiments of the present disclosure.

[0158] In addition, those skilled in the art can understand that although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments means that they fall within the scope of the present disclosure and form different embodiments. For example, in the claims, any one of the claimed embodiments can be used in any combination. The information disclosed in the background section is only intended to deepen the understanding of the overall background of the present disclosure, and should not be regarded as an acknowledgement or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.