A preparation method of CRISPR / Cas9 gene delivery system

Abstract

The present invention discloses a method for preparing a CRISPR / Cas9 gene delivery system. The particle size of the gene delivery system is about 180-200 nm, the carrier is a two-dimensional iron sulfide nanosheet, CRISPR / Cas9 is used for gene therapy, and the two-dimensional iron sulfide nanosheet is used to induce ferroptosis and enhance photothermal therapy. The preparation method includes chelation, reactant mixing, solvent thermal reaction, FeS / CRISPR / Cas9 complex preparation, red blood cell membrane extraction, tumor cell membrane extraction, and hybrid membrane modification steps. The present invention prepares a CRISPR / Cas9 gene delivery system, and the synthesized product has stability and repeatability, which is conducive to biomedical research on gene delivery and synergistic tumor therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for preparing a CRISPR / Cas9 gene delivery system. Background Art

[0002] As a cutting-edge medical technology, gene therapy can precisely intervene and regulate gene expression, and has great application potential in tumor treatment. However, it faces many challenges in practical application. Among them, the most prominent problem is the lack of an effective vector system. Existing vector systems often have problems such as low efficiency and high toxicity. Therefore, the development of efficient and low-toxic vector systems has become a key issue that needs to be urgently solved in the field of gene therapy.

[0003] Among the existing vector systems, cationic vectors have attracted much attention due to their high gene loading rate and high transfection efficiency, and have unique advantages in cancer treatment. Cationic vectors can interact electrostatically with negatively charged cell membranes, thereby achieving efficient gene delivery. However, cationic vectors also have obvious toxicity problems. Their high positive charge characteristics easily bind to negatively charged substances in cells, destroying cell structures and inducing adverse reactions such as cell apoptosis. In addition, the poor degradability of the vector causes the vector to accumulate in cells, increasing its potential toxicity.

[0004] In order to solve the limitations of existing carriers, acid-degradable carriers have attracted much attention due to their unique properties. This type of carrier can be rapidly degraded in an acidic environment, thereby reducing its residence time and toxicity in cells. At the same time, the good degradability of the carrier can also achieve targeted release of genes and improve the effect of gene therapy. Two-dimensional nanomaterials have significant advantages in the loading of gene drugs, mainly due to their extremely high specific surface area, which provides more attachment points for the loading of DNA, RNA, etc. However, due to the heterogeneity of tumors, single gene therapy models face problems such as drug resistance and poor prognosis. Multimodal synergistic therapy has become an important development direction for tumor treatment.

[0005] Iron, as an essential element for the human body, can be removed from the body through typical iron metabolic pathways and has good safety. Iron-based nanomaterials with photothermal properties can play a good synergistic effect as a carrier for gene delivery. The free radicals produced during ferroptosis may also directly destroy the structure of heat shock proteins (HSPs), or affect their transcription and translation processes, causing cancer cells to lose their heat stress protection mechanism, making the heat generated by PTT more effective in destroying cancer cells, thereby improving the therapeutic effect. Therefore, the synthesis of a biodegradable, biocompatible, high gene delivery efficiency and synergistic therapeutic function iron-based two-dimensional nanomaterial has good application potential in the field of tumor treatment. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) provide the material basis for the realization of this concept. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a CRISPR / Cas9 gene delivery system and a preparation method thereof.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Technical solution 1: A method for preparing a CRISPR / Cas9 gene delivery system, comprising the following steps:

[0009] Step 1: Chelation: Dissolve 0.3-0.6 mmol of ammonium ferrous sulfate hexahydrate and 0.1-0.2 mmol of trisodium citrate in 5-10 mL of ethylene glycol to obtain solution A; dissolve 250-500 mg of PEI in 5-10 mL of ethylene glycol to obtain solution B; mix equal volumes of solution A and solution B, and stir at room temperature to obtain solution C;

[0010] Step 2: Mixing reactants: dissolving 0.25-0.5 mmol of thioacetamide in 5-10 mL of ethylene glycol to obtain solution D; mixing solution D and solution C at a volume ratio of 1:2, stirring at room temperature to obtain solution E; dropping 1-2 mL of triethanolamine into solution E, stirring at room temperature to obtain solution F;

[0011] Step 3: Solvothermal reaction: subject solution F to a solvothermal reaction, centrifuge after the reaction is complete, wash with ethanol, collect the precipitate, resuspend it with ethanol, obtain a FeS ethanol solution, and store it for later use;

[0012] Step 4: Preparation of FeS / CRISPR / Cas9 complex: centrifuge the FeS ethanol solution, remove the ethanol, resuspend it in water to 0.4-0.8 mg / mL, prepare the CRISPR / Cas9 plasmid into an aqueous solution of 0.1-0.2 mg / mL, add an equal volume of the CRISPR / Cas9 plasmid solution to the FeS aqueous solution, and incubate it to obtain solution G;

[0013] Step 5: Extraction of erythrocyte membrane: add mouse peripheral blood erythrocytes into deionized water, place in an ice bath under hypotonic conditions, centrifuge, repeat hypotonic and centrifuge treatments, resuspend in PBS, and obtain erythrocyte membrane solution H. Determine protein content by BCA method, and calculate cell membrane mass based on membrane mass being twice protein mass.

[0014] Step 6: Extraction of tumor cell membrane: Add tumor cells to membrane protein extraction reagent, freeze and thaw repeatedly 3-5 times, centrifuge to remove cell nuclei and organelles, take supernatant and centrifuge, resuspend in PBS to obtain tumor cell membrane solution I, determine protein content by BCA method, and calculate cell membrane mass based on membrane mass being twice protein mass;

[0015] Step 7: Hybrid membrane modification: Solution G was mixed with solutions H and I in a mass ratio of 1:1:1 to 1:2:2, and the gene delivery system was obtained by probe ultrasound in an ice bath.

[0016] Preferably, in step 1, in the solution A, the content of ammonium ferrous sulfate is 0.6 mmol, the content of trisodium citrate is 0.2 mmol, and the volume of ethylene glycol is 10 mL; in the solution B, the content of PEI is 500 mg, and the volume of ethylene glycol is 10 mL; the stirring in the solution C is magnetic stirring at a speed of 400 rpm; and the stirring time at room temperature is 120 min.

[0017] Preferably, in step 2, in the solution E, the amount of thioacetamide is 0.5 mmol, and the volume of ethylene glycol is 10 mL; in the solution F, the amount of triethanolamine added is 2 mL; the stirring is magnetic stirring, and the speed is 400 rpm; and the stirring time is 5 min.

[0018] Preferably, in step 3, the solvent thermal reaction is carried out in a high pressure reactor at a temperature of 200°C for 24 h; the centrifugal speed is 14,000 rpm for 10 min; the washing is carried out with anhydrous ethanol until the supernatant is colorless; and the storage is carried out by dispersing the product in ethanol and storing it at -20°C.

[0019] Preferably, in step 4, the centrifugal speed of the centrifugal treatment is 14000 rpm, and the time is 10 min; the concentration of the FeS aqueous solution is 0.4 mg / mL, and the concentration of the CRISPR / Cas9 plasmid solution is 0.1 mg / mL, and the two are mixed in equal volumes, the incubation temperature is room temperature, and the incubation time is 15 min.

[0020] Preferably, in step 5, the ice bath placement time is 1 h, the centrifugation condition is 20000 g, the time is 15 min, and the number of hypotonic times is 2 times.

[0021] Preferably, in step 6, the centrifugation conditions for the first centrifugation are 780 g for 10 min, and the centrifugation conditions for the supernatant centrifugation are 20,000 g for 15 min.

[0022] Preferably, in step 7, the mixing mass ratio is G:H:I = 1:1:1, and the ultrasonic conditions are 100 W, on 3 s / off 3s, 10 min.

[0023] By adopting the above technical effects, the present invention achieves the following beneficial effects:

[0024] (1) The synthetic method for preparing the CRISPR / Cas9 delivery system of the present invention is simple and has strong operability; the synthesized product is stable and reproducible.

[0025] (2) The CRISPR / Cas9 delivery system synthesized by the present invention has a high gene delivery capability.

[0026] (3) The CRISPR / Cas9 delivery system synthesized by the present invention has low systemic toxicity.

[0027] (4) The CRISPR / Cas9 delivery system synthesized in the present invention has the ability to induce ferroptosis of tumor cells.

[0028] (5) The CRISPR / Cas9 delivery system synthesized in the present invention has the ability to enhance PTT through ferroptosis.

[0029] The vector of the CRISPR / Cas9 delivery system prepared by the present invention has a simple synthesis method and strong operability. The synthesized product is stable and repeatable, which is conducive to the efficient delivery of genes and the reduction of the toxicity of the system, and can play a synergistic tumor treatment function. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a transmission electron microscope image of the FeS ultrathin nanosheet prepared in Example 1 of the present invention.

[0031] Figure 21 is the photothermal temperature rise curve of FeS aqueous dispersion with different concentrations in the present invention.

[0032] Figure 3 is the light-to-heat conversion efficiency of FeS in the present invention.

[0033] Figure 4 Agarose gel electrophoresis diagram of FC formed by FeS nanosheets loaded with CRISPR / Cas9 at different mass ratios in the present invention.

[0034] Figure 5 This is the transmission electron microscopy elemental analysis of the FCRM formed by FeS nanosheets loaded with CRISPR / Cas9 and coated with hybrid membranes in the present invention.

[0035] Figure 6 is the average particle size of FeS nanosheets, FC and FCRM in the present invention.

[0036] Figure 7 is the potential of FeS nanosheets, FC and FCRM in the present invention.

[0037] Figure 8 The membrane proteins stained with Coomassie brilliant blue in the present invention are retained. Group I is erythrocyte membrane, Group II is tumor cell membrane, Group III is hybrid membrane, and Group IV is FCRM.

[0038] Fig. 9 These are the uptake images of MGC 803 cells treated with different concentrations of FCRM and FCR obtained by fluorescence microscopy in the present invention.

[0039] Fig.10 It is the survival rate of MGC 803 cells transfected with FCRM in the present invention.

[0040] Fig.11 The figure shows the biocompatibility of FCRM at different concentrations on HUVEC cells in the present invention.

[0041] Fig.12 The free radicals produced in MGC 803 cells after being treated with different drugs in the present invention.

[0042] Fig.13 It is the lipid peroxidation level of MGC 803 cells after being treated with different drugs in the present invention.

[0043] Fig.14 The expression of Survivin protein was determined by immunoblotting after MGC 803 cells were treated with different drugs in the present invention.

[0044] Fig.15 The GPX4 protein expression was determined by immunoblotting after MGC 803 cells were treated with different drugs in the present invention.

[0045] Fig.16 The HSP70 protein expression was determined by immunoblotting after MGC 803 cells were treated with different drugs in the present invention. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0047] As introduced in the background technology section, the current cationic gene delivery system has problems of low transfection efficiency and high toxicity, which limits the development of gene therapy. In addition, the effect of single gene therapy is limited. Ferroptosis-enhanced PTT can cooperate with gene therapy to achieve a more efficient tumor suppression effect.

[0048] Based on this, the purpose of the present invention is to provide a preparation method of a CRISPR / Cas9 delivery system and its application in synergistic tumor therapy. The present invention uses ammonium ferrous sulfate and thioacetamide as raw materials, and obtains ultrathin FeS nanosheets through a solvothermal reaction driven by PEI. The FeS aqueous solution is mixed with the CRISPR / Cas9 aqueous solution and allowed to stand to obtain a carrier / gene complex, and red blood cell membranes and tumor cell membranes are added to make hybrid membrane-modified FCRM under probe ultrasound. The gene delivery system prepared by the present invention is simple and has strong repeatability, good degradability, good long blood circulation ability and tumor targeting, which can overcome the problems of low efficiency and high toxicity faced by the current cationic gene delivery system, and has good application potential.

[0049] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0050] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels. Embodiment 1:

[0051] A method for preparing the CRISPR / Cas9 gene delivery system described in Example 1 comprises the following steps:

[0052] Step 1: Chelation: Dissolve 0.6 mmol of ammonium ferrous sulfate hexahydrate and 0.2 mmol of trisodium citrate in 10 mL of ethylene glycol to obtain solution A; dissolve 500 mg of PEI in 10 mL of ethylene glycol to obtain solution B; mix equal volumes of solution A and solution B, and stir at room temperature to obtain solution C;

[0053] Step 2: Mixing reactants: Dissolve 0.5 mmol of thioacetamide in 10 mL of ethylene glycol to obtain solution D; mix solution D and solution C at a volume ratio of 1:2, stir at room temperature to obtain solution E; add 2 mL of triethanolamine dropwise to solution E, stir at room temperature to obtain solution F;

[0054] Step 3: Solvothermal reaction: subject solution F to a solvothermal reaction, centrifuge after the reaction is complete, wash with ethanol, collect the precipitate, resuspend it with ethanol, obtain a FeS ethanol solution, and store it for later use;

[0055] Step 4: Preparation of FeS / CRISPR / Cas9 complex: centrifuge the FeS ethanol solution, remove the ethanol, resuspend it in water to 0.4 mg / mL, prepare the CRISPR / Cas9 plasmid into a 0.1 mg / mL aqueous solution, add an equal volume of the CRISPR / Cas9 plasmid solution to the FeS aqueous solution, and incubate it to obtain solution G;

[0056] Step 5: Extraction of erythrocyte membrane: add mouse peripheral blood erythrocytes into deionized water, place in an ice bath under hypotonic conditions, centrifuge, repeat hypotonic and centrifuge treatments, resuspend in PBS, and obtain erythrocyte membrane solution H. Determine protein content by BCA method, and calculate cell membrane mass based on membrane mass being twice protein mass.

[0057] Step 6: Extraction of tumor cell membrane: Add tumor cells to membrane protein extraction reagent, freeze and thaw repeatedly for 5 times, centrifuge to remove cell nuclei and organelles, take supernatant and centrifuge, resuspend in PBS to obtain tumor cell membrane solution I, determine protein content by BCA method, and calculate cell membrane mass based on membrane mass being twice protein mass;

[0058] Step 7: Hybrid membrane modification: Solution G was mixed with solutions H and I in a mass ratio of 1:1:1, and probe ultrasound was performed under ice bath to obtain the gene delivery system.

[0059] In this embodiment, 235.3 mg of ammonium ferrous sulfate and 5.8 mg of trisodium citrate were dissolved in 10 mL of ethylene glycol, 500 mg of PEI was dissolved in 10 mL of ethylene glycol, and the mixture was uniformly reacted at room temperature and 400 rpm under magnetic stirring for 120 min. Subsequently, 56.4 mg of thioacetamide was dissolved in 20 mL of ethylene glycol, added to the above solution, reacted at room temperature and 400 rpm under magnetic stirring for 5 min, 2 mL of triethanolamine was added dropwise, and reacted at room temperature and 400 rpm under magnetic stirring for 5 min. After the reaction, all the liquids were transferred to a stainless steel autoclave lined with Teflon, and heated at 200 ° C for 24 h. Finally, the mixture was collected by centrifugation at 14000 rpm for 10 min, and washed with anhydrous ethanol until the supernatant was clear to obtain the product FeS nanosheets. The product was dispersed in ethanol and stored at -20 ° C.

[0060] The FeS ethanol solution was centrifuged, the ethanol was removed, and the solution was resuspended into a 0.4 mg / mL aqueous solution. An equal volume of 0.1 mg / mL CRISPR / Cas9 plasmid solution and 0.4 mg / mL FeS aqueous solution were mixed and incubated at room temperature for 15 min to obtain FC.

[0061] Peripheral blood of mice was collected, centrifuged at 700 g to collect red blood cells in the lower layer, deionized water was added, and the cells were placed in an ice bath for 1 h for hypotonic treatment. The upper hemoglobin was removed by centrifugation at 20,000 g for 15 min, and the precipitate was collected and repeated hypotonic and centrifugation operations were repeated until the lower precipitate was white, and the precipitate was resuspended in PBS to obtain the red blood cell membrane solution. Tumor cells were also collected, membrane protein extraction reagent was added, and the cells were repeatedly frozen and thawed 5 times, centrifuged at 780 g for 10 min, the nucleus and organelles were removed, the supernatant was centrifuged at 20,000 g for 15 min, and the PBS was resuspended to obtain the tumor cell membrane solution, which was stored at -80 °C. FC was mixed with red blood cell membrane and tumor cell membrane in a mass ratio of 1:1:1, and the gene delivery system was obtained by probe ultrasound under ice bath at parameters of 100 W, on 3 s / off 3 s, 10 min.

[0062] like Figure 1 As shown, 500 μg / mL FeS ethanol solution was added dropwise onto the ultra-thin copper mesh, with a total of 40-50 μL added, dried, and observed under a transmission electron microscope. The prepared FeS was in the shape of a flake with a particle size of about 80-100 nm.

[0063] like Figure 2As shown in the figure, FeS was made into aqueous dispersions of different concentrations and irradiated with NIR near-infrared light. The irradiation parameters were 808nm, 5 min, and 1.5 W. The photothermal heating process was recorded and the photothermal heating curve was drawn. The results showed that as the concentration of FeS aqueous dispersion increased, the photothermal heating effect increased. When the Fe concentration was 40 μg / mL, the temperature increased from 33.1 ℃ to 65.4 ℃: Figure 2 The horizontal axis is time (S) and the vertical axis is temperature (℃).

[0064] like Figure 3 As shown in the figure, the FeS aqueous dispersion with an Fe concentration of 20 μg / mL was irradiated with NIR near-infrared light, and the irradiation parameters were 808 nm, 5 min, and 1.5 W. The photothermal heating and cooling processes were recorded, and the photothermal conversion efficiency was calculated. The results showed that the photothermal conversion efficiency η of FeS was as high as 38.96%, indicating that it has high photothermal performance, which is conducive to the synergistic application of gene therapy and PTT.

[0065] like Figure 4 As shown in the figure, FeS was made into water dispersions of different concentrations, mixed with equal volumes of CRISPR / Cas9 plasmid solution in a certain ratio, incubated at room temperature for 15 min to obtain FC, loaded on 1% agarose gel, and photographed on a gel imager after electrophoresis. The results showed that when the mass ratio of Fe to plasmid was greater than 1:1, no plasmid band emerged, indicating that FeS had a good loading capacity for plasmids.

[0066] like Figure 5 As shown, 500 μg / mL of FeS / CRISPR / Cas9 / hybrid membrane aqueous solution, i.e., FCRM, was added dropwise onto an ultra-thin copper mesh, a total of 40-50 μL, and dried. Transmission electron microscopy results showed that the prepared FCRM showed phosphorus elements belonging to the plasmid and cell membrane, indicating that the FCRM was successfully prepared.

[0067] like Figure 6 As shown, the aqueous solutions of FeS, FC, and FCRM were 50 μg / mL each. The average particle size of FC measured by dynamic light scattering was larger than that of FeS, and the particle size of FCRM was larger after coating, indicating the gradual and successful construction of FCRM, among which, FeS: iron sulfide nanosheets, FC: FeS / CRLISPR / Cas9, FCRM: FeS / CRISPR / Cas9 / hybrid membrane.

[0068] like Figure 7 As shown, the FeS, FC, and FCRM aqueous solutions were 50 μg / mL each. The potential measured by dynamic light scattering decreased gradually, and after coating the cell membrane, the potential reversed to -17.9 mV, indicating the gradual and successful construction of FCRM.

[0069] like Figure 8As shown in the figure, erythrocyte membrane, tumor cell membrane, hybrid membrane and FCRM with a membrane mass of 15 μg were loaded on sodium dodecyl sulfate polyacrylamide gel, and protein bands were displayed by Coomassie brilliant blue staining after electrophoresis. The membrane protein retention results showed that the protein bands of hybrid membrane Ⅲ and FCRMⅣ were similar to those of erythrocyte membrane Ⅰ and tumor cell membrane Ⅱ, indicating that the membrane protein remained stable after ultrasound, centrifugation and working temperature related to membrane extraction process. The membrane protein can be effectively transferred to FCRM together with the lipid membrane during the coating process to ensure the subsequent cell membrane function; among them, Ⅰ: erythrocyte membrane; Ⅱ tumor cell membrane; Ⅲ: hybrid membrane; Ⅳ: FCRM.

[0070] like Fig. 9 As shown, MGC 803 cells were seeded in laser confocal microplates at a density of 2×105 / well. After overnight culture, FCR labeled with Cy5.5 dye was used, where the mass ratio of FC to erythrocyte membrane R was 1:2, the Fe concentration in FCR was 40 μg / mL, and the Fe concentration of FCRM was 10, 20, and 40 μg / mL. The cells were treated for 4 h and photographed under a fluorescence microscope. The results showed that FCRM showed a concentration-dependent increase in uptake, and FCRM had a stronger uptake than FCR, indicating that hybrid membrane modification had better targeting than single erythrocyte membrane modification, where FCRM: FeS / CRLISPR / Cas9 / hybrid membrane; FCR: FeS / CRLISPR / Cas9 / erythrocyte membrane; Cy5.5: cyanine fluorescent dye.

[0071] like Fig.10 As shown in the figure, MGC 803 cells were seeded in 96-well plates at a density of 1×104 / well, and the firefly luciferase plasmid was used as the control group. The gene delivery system constructed was FLRM. The CRISPR / Cas9 plasmid carrying the Survivin target gene was the experimental group. MGC 803 cells were transfected with different concentrations of FLRM and FCRM for 48 h. The cell survival rate was determined by the MTT method. The results showed that FCRM could cause a certain degree of cell apoptosis due to the Survivin gene, indicating that it has a high gene transfection efficiency and can achieve the effect of gene therapy. FCRM+NIR showed the lowest cell survival rate. The therapeutic effect was the synergistic effect of ferroptosis, gene therapy and photothermal therapy, among which FLRM: FeS / luciferase plasmid / hybrid membrane; NIR: near-infrared light irradiation.

[0072] like Fig.11As shown in the figure, HUVEC cells were seeded in a 96-well plate at a density of 1×104 / well, and the cells were treated with FCRM at different concentrations of 0, 90, 180, and 360 μg / mL and incubated for 48 h. The cell viability was determined by the MTT method. The results showed that when the FCRM concentration reached 360 μg / mL, the viability of HUVEC cells remained above 80%. This fully demonstrates that FCRM has good biocompatibility, laying a solid foundation for subsequent in-depth research and application.

[0073] like Fig.12 As shown, MGC 803 cells were seeded at a density of 2×105 / well in laser confocal microplates, and the cells were treated with different drugs and incubated for 4 h. The reactive oxygen species level was stained with DCFH-DA fluorescent probe and photographed under a fluorescence microscope. The results showed that MGC 803 cells had a concentration-dependent increase in the production of hydroxyl free radicals ·OH after FCRM treatment. The FCR group had a lower intracellular ·OH level due to its lower cellular efficiency. After the addition of ferroptosis inhibitors, the ·OH level decreased; among them, Fer-1: ferroptosis inhibitor; DCFH-DA: 2.7-dichlorofluorescein diacetate (reactive oxygen probe).

[0074] like Fig.13 As shown in the figure, MGC 803 cells were seeded in laser confocal microplates at a density of 2×105 / well, and the cells were treated with different drugs and incubated for 6 h. The lipid peroxidation level in the cells was stained with C11-BODIPY fluorescent probe and photographed under a fluorescence microscope. The results showed that the fluorescence of the oxidized form of MGC 803 cells increased after FCRM treatment, while the fluorescence of the reduced form decreased, and this phenomenon was alleviated after the addition of ferroptosis inhibitors, indicating that FCRM can effectively induce ferroptosis and cause an increase in the lipid peroxidation level of tumor cells; among them, C11-BODIPY OX: undecanoic acid oxidized form; C11-BODIPY Non-OX: undecanoic acid non-oxidized form.

[0075] like Fig.14 As shown in the figure, MGC 803 cells were seeded in 6-well plates at a density of 1×105 / well. Cells were treated with different drugs. Near-infrared light irradiation (NIR) was performed 1 h after drug addition. The ferroptosis inhibitor Fer-1 was added together with the drug. After 4 h, the drug solution was discarded and replaced with new culture medium, and cultured for 48 h. RIPA lysis buffer was added to extract cell proteins, and the concentration of each target protein was determined by immunoblotting. The results showed that the FCRM group carrying the Survivin target gene showed a significant downregulation of Survivin protein compared with the FLRM group carrying the luciferase reporter gene, and it was concentration-dependent, indicating its efficient gene transfection efficiency; Survivin: apoptosis inhibitor protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase (internal reference).

[0076] like Fig.15 As shown, the cell transfection treatment was the same as above. The results showed that FCRM could cause a decrease in GPX4 protein expression, and GPX4 expression was restored after the addition of ferroptosis inhibitors, indicating that it can effectively induce ferroptosis of tumor cells; where: GPX4: glutathione peroxidase 4.

[0077] like Fig.16 As shown, the cell transfection treatment was the same as above. The results showed that the expression of HSP 70 protein increased significantly after 45 ℃ heat treatment, but decreased after FCRM treatment, and HSP did not increase significantly after NIR irradiation in the presence of FCRM, while HSP increased after NIR irradiation in the presence of FCRM and Fer-1. This result indicates that FCRM can effectively reduce HSP70 expression through ferroptosis, thereby reducing the photothermal resistance behavior of tumor cells during the PTT process, which is conducive to more effective photothermal therapy; HSP70: heat shock protein 70. Embodiment 2:

[0078] The difference from Example 1 is that: in step 1, in the solution A, the amount of ammonium ferrous sulfate is 0.6 mmol, the amount of trisodium citrate is 0.2 mmol, and the volume of ethylene glycol is 5 mL; in the solution B, the amount of PEI is 500 mg, and the volume of ethylene glycol is 5 mL; in the solution C, the stirring is magnetic stirring, and the speed is 400 rpm; and the stirring time is 120 min.

[0079] In step 2, in the solution E, the amount of thioacetamide is 0.5 mmol, and the volume of ethylene glycol is 5 mL; in the solution F, the amount of triethanolamine added is 2 mL; the stirring is magnetic stirring, and the speed is 400 rpm; the stirring time is 5 min.

[0080] In step 3, the solvent thermal reaction is carried out in a high pressure reactor at a temperature of 200°C for 24 h; the centrifugal speed is 14,000 rpm for 10 min; the washing is carried out with anhydrous ethanol until the supernatant is colorless; and the storage is carried out by dispersing the product in ethanol and storing it at -20°C.

[0081] In step 4, the centrifugal speed is 14000 rpm and the time is 10 min; the concentration of the FeS aqueous solution is 0.8 mg / mL, the concentration of the CRISPR / Cas9 plasmid solution is 0.2 mg / mL, and the two are mixed in equal volumes. The incubation temperature is room temperature and the incubation time is 15 min.

[0082] In step 5, the ice bath time is 1 h, the centrifugation condition is 20000 g, the time is 15 min, and the number of hypotonic times is 2 times.

[0083] In step 6, the first centrifugation condition is 780 g for 10 min, and the supernatant is centrifuged at 20,000 g for 15 min.

[0084] In step 7, the mixing mass ratio is G:H:I = 1:1:2, and the ultrasonic conditions are 100 W, on 3 s / off 3 s, 10 min. Embodiment three:

[0085] The difference from Example 1 is that: Step 1: 0.6 mmol of ammonium ferrous sulfate hexahydrate and 0.2 mmol of trisodium citrate are dissolved in 8 mL of ethylene glycol to obtain solution A; 500 mg of PEI is dissolved in 8 mL of ethylene glycol to obtain solution B; solution A and solution B are mixed in equal volumes and stirred at room temperature to obtain solution C;

[0086] Step 2: Dissolve 0.5 mmol of thioacetamide in 8 mL of ethylene glycol to obtain solution D; mix solution D and solution C in a volume ratio of 1:2, stir at room temperature to obtain solution E; add 2 mL of triethanolamine dropwise to solution E, stir at room temperature to obtain solution F;

[0087] Step 3: Solvent thermal reaction of solution F at 180 °C for 24 h;

[0088] Step 4: Centrifuge the FeS ethanol solution, remove the ethanol, resuspend it in water to 0.6 mg / mL, prepare the CRISPR / Cas9 plasmid into a 0.15 mg / mL aqueous solution, add an equal volume of the CRISPR / Cas9 plasmid solution into the FeS aqueous solution, and incubate it to obtain solution G;

[0089] Step 5: Add mouse peripheral blood red blood cells into deionized water, place in an ice bath under hypotonic conditions, centrifuge, repeat hypotonic and centrifuge treatments for 4 times, resuspend in PBS, and obtain red blood cell membrane solution H;

[0090] Step 6: Add the tumor cells to the membrane protein extraction reagent, freeze and thaw repeatedly for 4 times, centrifuge to remove the cell nucleus and organelles, take the supernatant and centrifuge, resuspend in PBS to obtain tumor cell membrane solution I;

[0091] Step 7: Mix solution G with solutions H and I in a mass ratio of 1:2:2, and obtain the gene delivery system by probe ultrasound in an ice bath.

Claims

1. A method for preparing a CRISPR / Cas9 gene delivery system, characterized in that: The following steps are involved: Step 1: Chelation: Dissolve 0.3-0.6 mmol of ammonium ferrous sulfate hexahydrate and 0.1-0.2 mmol of trisodium citrate in 5-10 mL of ethylene glycol to obtain solution A; dissolve 250-500 mg of PEI in 5-10 mL of ethylene glycol to obtain solution B; mix equal volumes of solution A and solution B, and stir at room temperature to obtain solution C; Step 2: Mixing reactants: dissolving 0.25-0.5 mmol of thioacetamide in 5-10 mL of ethylene glycol to obtain solution D; mixing solution D and solution C at a volume ratio of 1:2, stirring at room temperature to obtain solution E; dropping 1-2 mL of triethanolamine into solution E, stirring at room temperature to obtain solution F; Step 3: Solvothermal reaction: subject solution F to a solvothermal reaction, centrifuge after the reaction is complete, wash with ethanol, collect the precipitate, resuspend it with ethanol, obtain a FeS ethanol solution, and store it for later use; Step 4: Preparation of FeS / CRISPR / Cas9 complex: centrifuge the FeS ethanol solution, remove the ethanol, resuspend it in water to 0.4-0.8 mg / mL, prepare the CRISPR / Cas9 plasmid into an aqueous solution of 0.1-0.2 mg / mL, add an equal volume of the CRISPR / Cas9 plasmid solution to the FeS aqueous solution, and incubate it to obtain solution G; Step 5: Extraction of erythrocyte membrane: add mouse peripheral blood erythrocytes into deionized water, place in an ice bath under hypotonic conditions, centrifuge, repeat hypotonic and centrifuge treatments, resuspend in PBS, and obtain erythrocyte membrane solution H. Determine protein content by BCA method, and calculate cell membrane mass based on membrane mass being twice protein mass. Step 6: Extraction of tumor cell membrane: Add tumor cells to membrane protein extraction reagent, freeze and thaw repeatedly 3-5 times, centrifuge to remove cell nuclei and organelles, take supernatant and centrifuge, resuspend in PBS to obtain tumor cell membrane solution I, determine protein content by BCA method, and calculate cell membrane mass based on membrane mass being twice protein mass; Step 7: Hybrid membrane modification: Solution G was mixed with solutions H and I in a mass ratio of 1:1:1 to 1:2:2, and probe sonication was performed under ice bath to obtain the gene delivery system; In the step 1, in the solution A, the amount of ammonium ferrous sulfate is 0.6 mmol, the amount of trisodium citrate is 0.2 mmol, and the volume of ethylene glycol is 10 mL; in the solution B, the amount of PEI is 500 mg, and the volume of ethylene glycol is 10 mL; the stirring in the solution C is magnetic stirring, and the speed is 400 rpm; the stirring time at room temperature is 120 min; In the step 2, in the solution E, the amount of thioacetamide is 0.5 mmol, and the volume of ethylene glycol is 10 mL; in the solution F, the amount of triethanolamine added is 2 mL; the stirring is magnetic stirring, and the speed is 400 rpm; the stirring time is 5 min; In the step 3, the solvent thermal reaction is carried out in a high pressure reactor at a temperature of 200°C for 24 hours; the centrifugal speed of the centrifugal treatment is 14,000 rpm for 10 minutes; the washing is carried out with anhydrous ethanol until the supernatant is colorless; the storage is carried out by dispersing the product in ethanol and storing it at -20°C; In step 4, the centrifugal speed of the centrifugal treatment is 14000 rpm, and the time is 10 min; the concentration of the FeS aqueous solution is 0.4 mg / mL, and the concentration of the CRISPR / Cas9 plasmid solution is 0.1 mg / mL, and the two are mixed in equal volumes; the incubation temperature is room temperature, and the incubation time is 15 min; In step 5, the ice bath time is 1 h, the centrifugation condition is 20000 g, the time is 15 min, and the number of hypotonic times is 2 times; In step 6, the parameters of the first centrifugation are set to 780 g for 10 min, and the parameters of the supernatant centrifugation are set to 20,000 g for 15 min; In the step 7, the mass ratio of solution G to solution H and solution I is G:H:I = 1:1:1, and the ultrasonic condition is 100 W, on 3 s / off 3 s, 10 min.

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