CRISPR-Cas9 near-infrared two-region precise light-operated editing system delivery carrier based on erythrocyte membrane as well as preparation method and application of CRISPR-Cas9 near-infrared two-region precise light-operated editing system delivery carrier

Abstract

The invention provides a CRISPR-Cas9 near-infrared two-region precise light-operated editing system delivery carrier based on an erythrocyte membrane, a preparation method and application, and particularly, the CRISPR-Cas9 gene editing system delivery carrier provided by the invention has the advantages of low cytotoxicity, efficient transfection, high yield, targeting property, space-time controllability, easiness in large-scale production and multifunctionality. Compared with a traditional liposome transfection reagent, the carrier shows lower cytotoxicity, protects the CRISPR / Cas9 compound from being influenced by the in-vivo environment, and ensures that the CRISPR / Cas9 compound is smoothly delivered to target cells. By embedding a specific targeting receptor, efficient targeting delivery is realized, and the pertinence and effectiveness of treatment are improved. And by combining a near-infrared two-region material, the time-space precise control of a CRISPR / Cas9 system is realized, and deeper tissue penetrating power and smaller biotoxicity are achieved. The preparation process is simple, the method is suitable for industrial production and clinical transformation, and new possibility is provided for gene therapy and personalized medical treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a CRISPR-Cas9 near-infrared second zone precise light-controlled editing system delivery vector based on red blood cell membrane, and a preparation method and application thereof. Background Art

[0002] As a revolutionary genome editing tool, the CRISPR-Cas9 system has become a key technology in biomedical research and treatment since its introduction. The system consists of a single guide RNA (sgRNA) and the Cas9 endonuclease, which can recognize and cut specific DNA sequences to achieve precise gene editing. CRISPR-Cas9 technology has a wide range of applications, including the treatment of genetic diseases, functional genomics research, and cancer treatment. However, the effective delivery of the CRISPR / Cas9 complex to target cells remains the focus and difficulty of current research.

[0003] At present, the delivery methods of CRISPR / Cas9 complexes mainly include physical methods, virus-mediated transfection, liposome-mediated transfection, and extracellular vesicle delivery. Although physical methods such as electroporation or microinjection are easy to operate, the delivery efficiency of exogenous genetic material is limited and there is some damage to cells. Although virus-mediated transfection has a high delivery efficiency, there are safety issues such as carcinogenicity, immunogenicity, and insertion mutagenesis. At the same time, it is costly and has a complex production process. Although liposome-mediated transfection has gained certain recognition as a non-viral delivery method, its transfection efficiency is relatively low and there are toxicity issues. Extracellular vesicle delivery has a high transfection efficiency, but the preparation process is cumbersome and the yield is low, which limits its wide application. In addition, the delivery of the CRISPR-Cas9 system is also faced with the problem of off-target effects, which may affect non-target genes and lead to unexpected biological effects.

[0004] In recent years, with the development of optogenetics, the activation of light-sensitive proteins has provided unprecedented control for CRISPR / Cas9-mediated genome manipulation. However, existing optogenetic systems are mainly based on blue light, which has limited penetration in complex human tissues and cannot be applied in deeper tumor tissues. In addition, the relatively large molecular size of CRISPR / Cas9 also makes it difficult to effectively deliver standard gene therapy vectors, and viral vectors are more likely to trigger unnecessary immunogenicity and carcinogenicity.

[0005] In order to solve the above problems, a biofilm-based CRISPR-Cas9 gene editing system delivery vector was developed with excellent biocompatibility, stability and active targeting ability, which can achieve efficient targeting and enrichment and retention effects on hypoxic HNSCC tumor tissues, and is of great significance for improving the delivery efficiency and safety of the CRISPR / Cas9 system. Summary of the invention

[0006] In view of this, this patent aims to provide a CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane, and its preparation method and application. Specifically, the present invention provides a CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane, which utilizes the unique properties of erythrocyte membrane, such as excellent biocompatibility and long circulation half-life, to improve the delivery efficiency and targeting of the CRISPR-Cas9 system. In addition, the present invention also provides a method for preparing the delivery vector and its application in cancer treatment, aiming to address the limitations of the prior art and provide a new strategy for gene editing therapy.

[0007] Specifically, the present invention provides a method for preparing a CRISPR-Cas9 near-infrared second zone precise light-controlled editing system delivery vector based on red blood cell membrane, comprising the following preparation steps:

[0008] (1) Preparation of Hsp-Cas9: Construction of the HSP70 promoter-driven Cas9 gene editing system;

[0009] (2) Preparation of red blood cell membranes: Separating a blood sample to obtain pure red blood cells, then using a lysis method to swell and eventually rupture the red blood cells, releasing the cell contents while maintaining the integrity of the cell membrane. After lysis, the cell fragments are separated by centrifugation or filtration to obtain free red blood cell membrane sheets, and then the collected red blood cell membrane sheets are further processed to disperse them into smaller red blood cell membrane fragments or liposomes, and finally a red blood cell membrane suspension is obtained;

[0010] (3) Synthesis of DSPE-PEG2000-αLDLR: DSPE-PEG2000-COOH was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, and then N-hydroxysuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide were added to react at room temperature. After the reaction, the pH was adjusted to 7.5 to obtain activated DSPE-PEG2000-COOH; then, the activated DSPE-PEG2000-COOH was incubated with anti-LDLR antibody, and then unbound COOH-PEG2000-DSPE and unbound anti-LDLR antibody were removed by dialysis, respectively, to finally prepare DSPE-PEG2000-αLDLR;

[0011] (4) Preparation of PT-Hsp-Cas9 complex: polylactic acid-co-glycolic acid copolymer is dissolved in ethyl acetate, PT is dissolved in THF, and Hsp-Cas9 is dissolved in RNase-free water to form a PT-Hsp-Cas9 (PC) core, and the PT solution and the Hsp-Cas9 solution are dropped into the polylactic acid-co-glycolic acid copolymer solution, followed by ultrasonication to obtain an initial emulsion; then, the initial emulsion is mixed with an aqueous solution of polyvinyl alcohol and ultrasonicated to form a multiple emulsion; finally, the multiple emulsion is mixed with an aqueous solution of polyvinyl alcohol and ultrasonicated to stabilize the emulsion to obtain a PT-Hsp-Cas9 complex; wherein the structural formula of the PT is shown in the following formula (I):

[0012] (5) Synthesis of ARPC nanoparticles: DSPE-PEG2000-αLDLR is added to the red blood cell membrane suspension to react to modify the red blood cell membrane, and then the DSPE-PEG2000-αLDLR modified red blood cell membrane is collected by centrifugation, and the prepared PT-Hsp-Cas9 complex is dissolved in the red blood cell membrane solution containing DSPE-PEG2000-αLDLR modified red blood cell membrane, sonicated on ice, and then incubated. After incubation, the excess red blood cell membrane is removed by centrifugation to obtain ARPC nanoparticles, i.e., the red blood cell membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector.

[0013] In some specific embodiments of the present invention, the preparation step of Hsp-Cas9 includes: obtaining the Hsp70 promoter fragment from the upstream region of the mouse HSP70 protein gene by PCR amplification.

[0014] In some specific embodiments of the present invention, the step of obtaining the Hsp70 promoter fragment from the upstream region of the mouse HSP70 protein gene by PCR amplification includes: first, genomic DNA needs to be extracted from the mouse sample. According to the operating instructions of the TaKaRaMini BEST genomic DNA extraction kit, genomic DNA is isolated from untreated mouse whole blood. A pair of groove primers for upstream amplification are designed in the 5'-UTR region of the Hsp70 gene, and the GC content and Tm value are as high as possible. Then, the designed primers and genomic DNA are used as templates for PCR amplification (PCR conditions: pre-denaturation to 94-96°C, denaturation to 94-96°C, annealing to 60-68°C, and extension to 72°C). Touch Down PCR is generally used, and two rounds of amplification are performed according to the groove primers, and the second round is based on the PCR product of the first round as a template. All the PCR reaction solution is added to the prepared agarose gel well, and a marker of appropriate size is added, and electrophoresis is performed to check the target band of the amplified product. Refer to the molecular weight of the Marker band to determine the target fragment band, cut the target band gel block on the UV luminometer with a scalpel, recover and purify the target gene fragment, and use NanoDrop1000 to detect the concentration of the gel recovery product. Finally, establish a connection system according to the instructions of the cloning vector. The above-amplified target gene PCR product is connected to the linearized eSpCas9-2A-GFP-(PX458) vector, and the connection product is transformed into DH5α competent cells by heat shock method. The transformed product coating rod is evenly coated on the LB plate medium containing ampicillin, and the plate is inverted in a 37°C incubator for overnight culture. Then, colony screening is performed. Use a sterile gun tip to pick 3-6 single colonies and place them in LB liquid medium containing ampicillin resistance for culture. After culture, add nucleic acid dyes for electrophoresis detection, select clones with bands consistent with the expected size, shake bacteria to extract plasmids, and then sequence. Finally, analyze and process the sequencing results. Compare the sequence with the expected design sequence to determine whether it is correct or not, and arrange the next experiment.

[0015] In some specific embodiments of the present invention, the steps of preparing red blood cell membranes include: first, centrifuging a blood sample to allow denser red blood cells to settle to the bottom, then removing the upper plasma and washing the red blood cells with PBS to remove plasma proteins and other impurities to ensure that only pure red blood cells remain; then using a mild lysis method to swell and eventually rupture the red blood cells to release the cell contents but maintain the integrity of the cell membrane; after lysis, separating the cell fragments by centrifugation or filtration to obtain free red blood cell membrane sheets; ultrasonically treating the collected membrane sheets or gently stirring them to disperse them into smaller red blood cell membrane fragments or liposomes to obtain a red blood cell membrane suspension.

[0016] In some specific embodiments of the present invention, the specific steps of synthesizing the DSPE-PEG2000-αLDLR include: dissolving DSPE-PEG2000-COOH in 2-(N-morpholino)ethanesulfonic acid buffer, then adding N-hydroxysuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, reacting at room temperature for 30 minutes, adjusting the pH to 7-7.5 to obtain activated DSPE-PEG2000-COOH; then incubating the anti-LDLR antibody with the activated DSPE-PEG2000-COOH at 4°C for 12-24 hours, then dialyzing through a 3500MW cutoff membrane for 4 hours to remove unbound COOH-PEG2000-DSPE, and then dialyzing through a 300k cutoff membrane for 4 hours to remove unbound antibodies, and finally preparing DSPE-PEG2000-αLDLR.

[0017] In some specific embodiments of the present invention, the specific steps of synthesizing the DSPE-PEG2000-αLDLR include: dissolving DSPE-PEG2000-COOH in 2-(N-morpholino)ethanesulfonic acid buffer, then adding N-hydroxysuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, reacting at room temperature for 30 minutes, adjusting the pH to 7.5 to obtain activated DSPE-PEG2000-COOH; then incubating the anti-LDLR antibody with the activated DSPE-PEG2000-COOH at 4°C for 12 hours, then dialyzing through a 3500MW cutoff membrane for 4 hours to remove unbound COOH-PEG2000-DSPE, and then dialyzing through a 300k cutoff membrane for 4 hours to remove unbound antibodies, and finally preparing DSPE-PEG2000-αLDLR.

[0018] In some specific embodiments of the present invention, the mass ratio of polylactic acid-glycolic acid copolymer and PT in the preparation of the PT-Hsp-Cas9 complex is 4:1-10:1.

[0019] In some specific embodiments of the present invention, the mass ratio of polylactic acid-glycolic acid copolymer and PT in the preparation of the PT-Hsp-Cas9 complex is 4:1.

[0020] In some specific embodiments of the present invention, the mass ratio of polylactic acid-glycolic acid copolymer and PT in the preparation of the PT-Hsp-Cas9 complex is 10:1.

[0021] In some specific embodiments of the present invention, the polymer of formula (I) is prepared according to the following reaction formula:

[0022]

[0023] In some specific embodiments of the present invention, the specific preparation steps of the polymer of formula (I) are as follows: 4-(2-ethylhexyl)-2,6-bis(trimethyltinyl)-4H-thieno[3,2-B:2',3'-D]pyrrole, 4,8-dibromo-6-(2-ethylhexyl)-[1,2,5]thiadiazole[3,4-f]benzotriazole, triphenylphosphine and 3 μmol of tri(dibenzylideneacetone)dipalladium are added to a Schlenk tube of chlorobenzene, and stirred at 110°C for 72 hours under nitrogen protection. The mixture is cooled to room temperature and precipitated in methanol. The precipitate is collected by filtration and dried to obtain a black solid, which is the polymer of formula (I).

[0024] In some specific embodiments of the present invention, the synthesis of the ARPC nanoparticles includes the following steps: adding DSPE-PEG2000-αLDLR to the red blood cell membrane suspension, reacting at 4°C overnight to modify the membrane, and then collecting the DSPE-PEG2000-αLDLR modified red blood cell membrane by centrifugation at 3500rpm at 4°C, dissolving the prepared PT-Hsp-Cas9 complex in the red blood cell membrane solution modified with DSPE-PEG2000-αLDLR, ultrasonicating in an ice bath for 20 minutes, incubating on a shaker at 4°C overnight, and then centrifuging to remove excess red blood cell membrane to obtain ARPC nanoparticles, i.e., the CRISPR-Cas9 near-infrared second zone precise light-controlled editing system delivery vector based on the red blood cell membrane.

[0025] The present invention also provides a method for preparing the above-mentioned erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector, and a erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector prepared therefrom.

[0026] The present invention also provides a method for preparing the above-mentioned erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector, and the use of the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector prepared therefrom or the above-mentioned erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector in the preparation of drugs for preventing and / or treating cancer.

[0027] The present invention also provides a method for preparing the above-mentioned CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane, and the use of the CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane or the above-mentioned CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane in cell transfection.

[0028] The present invention also provides a method for knocking out the PD-L1 gene in SCC7 cells using a CRISPR / Cas9 gene editing method, the specific steps comprising:

[0029] S1. Incubate SCC7 cells with the CRISPR-Cas9 gene editing system based on erythrocyte membrane prepared by the preparation method of the CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane or the CRISPR-Cas9 gene editing system based on erythrocyte membrane;

[0030] S2. The cells after incubation were exposed to 1060nm, 0.75W / cm 3 Incubate after laser stimulation.

[0031] In some specific embodiments of the present invention, S1. the incubation time is 12 to 24 hours.

[0032] In some specific embodiments of the present invention, S2. the laser irradiation time is 5-10 minutes.

[0033] In some specific embodiments of the present invention, S2. the incubation time after laser irradiation is 24-72 hours.

[0034] Compared with the prior art, the present invention has the following significant advantages and effects:

[0035] (1) Low cytotoxicity: Compared with traditional liposome transfection reagents, the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector provided by the present invention exhibits lower cytotoxicity, which helps to improve transfection efficiency while reducing damage to cells.

[0036] (2) Efficient transfection: The erythrocyte membrane has natural biocompatibility. The erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector provided by the present invention can effectively protect the CRISPR / Cas9 complex from the influence of the in vivo environment, ensuring its smooth delivery to the target cells and its function.

[0037] (3) High yield: Compared with extracellular vesicles, erythrocyte membranes are easier to obtain and have a higher yield, which means that the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector provided by the present invention can be mass-produced at a lower cost and is suitable for clinical applications.

[0038] (4) Targeting: By embedding specific targeting receptors (such as anti-LDLR antibodies) on the red blood cell membrane, efficient targeted delivery to specific cell types can be achieved, increasing the specificity and effectiveness of treatment.

[0039] (5) Spatiotemporal controllability: Combined with near-infrared zone II materials, precise spatiotemporal control of the CRISPR / Cas9 system can be achieved. Near-infrared zone II light has deeper tissue penetration and less biological toxicity, making it possible to remotely activate or inhibit CRISPR / Cas9, which is particularly valuable for in vivo applications.

[0040] (6) Easy to scale up production: Compared with viral vectors, the preparation process of the CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on red blood cell membrane provided by the present invention is relatively simple, does not require a complex biosafety laboratory, and is not prone to causing the risk of immune response or insertion mutation, making it more suitable for industrial production and clinical transformation.

[0041] (7) Multifunctionality: In addition to serving as a delivery vehicle for CRISPR / Cas9, red blood cell membrane-based nanoparticles can also carry other drugs or diagnostic agents to achieve multimodal treatment and monitoring.

[0042] In summary, the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector provided by the present invention not only overcomes some limitations of traditional delivery methods, but also shows obvious advantages in safety, effectiveness, cost-effectiveness and flexibility, providing new possibilities for future gene therapy and personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 Schematic diagram of the preparation process of ARPC nanoparticles.

[0045] Figure 2 ζ potential analysis data of PT, plasmid, PC, and ARPC, with mean and standard deviation, n=3.

[0046] Figure 3 is the hydrodynamic diameter of PC, RPC and ARPC.

[0047] Figure 4 The morphology detection results of nanoparticles, where (a) is the TEM image of PC; (b) is the element mapping transmission electron microscope image of PC; (c) is the transmission electron microscope image of RPC; (d) is the transmission electron microscope image of ARPC.

[0048] Figure 5 This is a picture of SDS-PAGE protein analysis.

[0049] Figure 6 Co-localization analysis of the red fluorescence of DID in DID-labeled RBCm and the yellow fluorescence of antibody-linked anti-LDLR IgGH&L (Alexa Fluor Plus 555) antibody observed by CLSM.

[0050] Figure 7 Agarose gel electrophoresis of plasmid, PC, RPC and ARPC.

[0051] Figure 8 Agarose gel electrophoresis of ARPC nanocomplexes at different plasmid / ARP ratios.

[0052] Fig. 9 UV-visible absorption spectra of PT, plasmid, RBCm, anti-LDLR, PC and ARPC.

[0053] Fig.10 (a) is the case with or without 0.75W / cm 2 GFP expression based on flow cytometry after SCC7 cells were transfected with PC, RPC and ARPC after laser; (b) Quantitative flow cytometry results of GFP expression, mean standard deviation, n = 3. *P < 0.05**P < 0.01***P < 0.001****P < 0.0001.

[0054] Fig.11 Fluorescence microscopic images of GFP in SCC7 cells, scale bar: 60m.

[0055] Fig.12 The expression levels of Hsp70 and PD-L1 proteins in SCC7 cells after treatment were assessed by Western blotting.

[0056] Fig.13 (a) The expression levels of Hsp70 and PD-L1 proteins in SCC7 cells after treatment, evaluated by CLSM, scale bar: 25 μm; (b) quantification of fluorescence from anti-Hsp70 and anti-PD-L1 staining using Image J, mean standard deviation, n=3.

[0057] Fig.14 (a) is the expression of PD-L1 based on flow cytometry after SCC7 cells were transfected with PC, RPC and ARPC after 0.75W / cm2 laser; (b) is the quantitative flow cytometry result of PD-L1 expression, mean standard deviation, n = 3. *P<0.05**P<0.01***P<0.001****P<0.0001.

[0058] Fig.15 This is the survival curve of tumor-bearing mice during the treatment process.

[0059] Fig.16 (a) is the tumor image at the end of treatment; (b) is the tumor weight change curve of mice after treatment and irradiation, all data are mean ± SEM (n = 5); (c) is the tumor volume change curve of mice after treatment and irradiation, all data are mean ± SEM (n = 5); (d) is the body weight change curve of mice after treatment and irradiation, all data are mean ± SEM (n = 5).

[0060] Fig.17 Flow cytometric analysis of PD-L1 expression in cancer cells at the end of anticancer treatment.

[0061] Fig.18 To detect the expression of PD-L1 and Hsp70 proteins in tumor tissues by Western blotting.

[0062] Fig.19 Representative immunofluorescence images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, PD-L1, and Hsp70 in tumor tissues. DETAILED DESCRIPTION

[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work belong to the protection scope of the present invention.

[0064] Protection scope.

[0065] Example 1 Preparation of CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector (ARPC) based on red blood cell membrane

[0066] Figure 1 The schematic diagram of the process for preparing ARPC nanoparticles is as follows:

[0067] (1) Preparation of Hsp-Cas9: The HSP70 promoter fragment was obtained from the upstream region of the mouse HSP70 protein gene by PCR amplification. Guide RNA, primers and pX458 plasmid were purchased from GenScript Biotechnology Co., Ltd. (Nanjing, China). First, the synthesis strategy was designed: the gene fragment analysis was not difficult, the mix synthesis sequence was directly synthesized, and the binding site was constructed on the vector. Then the primers were synthesized by an automatic synthesizer. After obtaining the target primers, PCR amplification was performed to obtain the PCR product and perform electrophoresis detection on it to obtain a PCR product with the expected size consistent with the target sequence, and the band with the expected size consistent with the target sequence was cut, and the product was recovered for connection and transformation. (PCR conditions: pre-denaturation at 94-96°C, denaturation at 94-96°C, annealing at 60-68°C, and extension at 72°C. Then perform the continuous transfer operation. The amplified target gene PCR product was connected to the linearized eSpCas9-2A-GFP-(PX458) vector, and the ligation product was transformed into DH5α competent cells by heat shock method. The transformed product was evenly coated on the LB plate culture medium containing ampicillin, and the plate was inverted in a 37°C incubator for overnight culture. Then, colony screening was performed. 3-6 single colonies were picked up with a sterile gun tip and placed in LB liquid culture medium containing ampicillin resistance for culture. After culture, nucleic acid dyes were added for electrophoresis detection, and clones with bands consistent with the expected size were selected for shaking bacteria to extract plasmids and then sequenced. Finally, the sequencing results were analyzed and processed. The sequence and file were compared with the expected designed sequence to determine whether it was correct or not, and the next experiment was arranged.

[0068] (2) Preparation of red blood cell membrane (RBCm): Whole blood was collected from the abdominal aorta of SD rats with a syringe, and then centrifuged at 2000 rpm for 10 minutes at 4°C. High-speed centrifugation can make the denser red blood cells precipitate to the bottom, then remove the upper plasma, and wash the red blood cells with PBS several times to remove plasma proteins and other impurities to ensure that only pure red blood cells are left. Then a mild lysis method (0.25X PBS) is used to swell and eventually rupture the red blood cells, releasing the cell contents but maintaining the integrity of the cell membrane. After lysis, the cell fragments are separated by centrifugation / filtration to obtain free red blood cell membrane sheets. The collected membrane sheets are further ultrasonically treated to disperse them into smaller membrane fragments or liposomes, which can be more easily coated on the surface of nanoparticles in subsequent steps. Finally, the RBCm suspension is obtained. The RBCm suspension needs to be stored at low temperature to maintain its biological activity and stability until it is ready for coating of nanocarriers.

[0069] (3) Synthesis of DSPE-PEG2000-αLDLR: First, DSPE-PEG2000-COOH was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer and reacted with N-hydroxysuccinimide (NHS) and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) at room temperature for 30 min, and then the pH was adjusted to 7.5 to obtain activated DSPE-PEG2000-COOH. The desired concentration of anti-LDLR antibody was incubated with activated DSPE-PEG2000-COOH at 4°C overnight (the molar ratio of αLDLR to DSPE-PEG2000-COOH was 2:1), and then unbound DSPE-PEG2000-COOH was removed by dialysis, first using a 3500MW cutoff membrane for 4 hours, then using a 300k cutoff membrane for 4 hours to remove unbound anti-LDLR antibody, and finally performing another 4-hour dialysis to completely replace and renew PBS. After dialysis, the obtained DSPE-PEG2000-αLDLR was stored at 4°C for future use.

[0070] (4) Preparation of PT-Hsp-Cas9 complex: Polylactic acid-co-glycolic acid (PLGA, 100 mg) was dissolved in ethyl acetate (5 mL), PT (10 mg) was dissolved in THF, and Hsp-Cas9 was dissolved in RNase-free water (DEPC 1 mL). To form the PT-Hsp-Cas9 (PC) core, the PT solution and the Hsp-Cas9 solution were dropped into the polylactic acid-co-glycolic acid solution, and ultrasonicated at 20% W for 5 min to obtain an initial emulsion. Then, the initial emulsion was mixed with 2% PVA solution and 20% W ultrasonically for 10 min to obtain a multiple emulsion. Finally, the multiple emulsion was mixed with an aqueous solution of 0.5% polyvinyl alcohol and ultrasonicated at 20% W for 10 min to stabilize the emulsion to obtain the PT-Hsp-Cas9 complex.

[0071] Wherein, the above PT is a polymer of formula (I), and its synthesis steps are as follows:

[0072] 0.1 mmol of 4-(2-ethylhexyl)-2,6-bis(trimethyltinyl)-4H-thieno[3,2-B:2',3'-D]pyrrole, 0.1 mmol of 4,8-dibromo-6-(2-ethylhexyl)-[1,2,5]thiadiazole[3,4-f]benzotriazole, 12 umol of triphenylphosphine and 3 umol of tris(dibenzylideneacetone)dipalladium were added to a Schlenk tube containing 5 mL of chlorobenzene. The mixture was stirred at 110° C. for 72 hours under nitrogen protection. The mixture was cooled to room temperature and precipitated in methanol. The precipitate was collected by filtration and dried to obtain a black solid, which is a polymer of formula (I). The reaction scheme is as follows:

[0073]

[0074] Synthesis of ARPC nanoparticles: DSPE-PEG2000-αLDLR was added to the above-mentioned red blood cell membrane suspension and reacted overnight at 4°C to modify the membrane. The red blood cell membrane modified with DSPE-PEG2000-αLDLR was collected by centrifugation at 3500 rpm for 10 min at 4°C. The prepared PT-Hsp-Cas9 complex was dissolved in the red blood cell membrane solution modified with DSPE-PEG2000-αLDLR and ultrasonicated on ice for 10 min (25 W / cm 2 ) for 20 min, incubate on a shaker at 4°C overnight, and the next day, remove excess erythrocyte membranes by centrifugation to obtain ARPC nanoparticles, which are the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vectors.

[0075] Example 2: Preparation of RPC

[0076] (1) Preparation of Hsp-Cas9: The HSP70 promoter fragment was obtained from the upstream region of the mouse HSP70 protein gene by PCR amplification. Guide RNA, primers and pX458 plasmid were purchased from GenScript Biotechnology Co., Ltd. (Nanjing, China).

[0077] (2) Preparation of red blood cell membrane (RBCm): Whole blood was collected from the abdominal aorta of SD rats with a syringe, and then centrifuged at 2000 rpm for 10 minutes at 4°C. High-speed centrifugation can make the denser red blood cells precipitate to the bottom, then remove the upper plasma, and wash the red blood cells with PBS several times to remove plasma proteins and other impurities to ensure that only pure red blood cells are left. Then a mild lysis method (0.25X PBS) is used to swell and eventually rupture the red blood cells, releasing the cell contents but maintaining the integrity of the cell membrane. After lysis, the cell fragments are separated by centrifugation / filtration to obtain free red blood cell membrane sheets. The collected membrane sheets are further ultrasonically treated to disperse them into smaller membrane fragments or liposomes, which can be more easily coated on the surface of nanoparticles in subsequent steps. Finally, the RBCm suspension is obtained. The RBCm suspension needs to be stored at low temperature to maintain its biological activity and stability until it is ready for coating of nanocarriers.

[0078] (3) Preparation of PT-Hsp-Cas9 complex (PC described below is prepared according to this step): polylactic acid-co-glycolic acid (PLGA, 100 mg) was dissolved in ethyl acetate (5 mL), PT (10 mg) was dissolved in THF, and Hsp-Cas9 was dissolved in RNase-free water (DEPC 1 mL). To form the PT-Hsp-Cas9 (PC) core, the PT solution and the Hsp-Cas9 solution were dropped into the polylactic acid-co-glycolic acid solution, and ultrasonicated at 20% W for 5 min to obtain an initial emulsion. Then, the initial emulsion was mixed with 2% PVA solution and 20% W ultrasonically for 10 min to obtain a multiple emulsion. Finally, the multiple emulsion was mixed with an aqueous solution of 0.5% polyvinyl alcohol and ultrasonicated at 20% W for 10 min to stabilize the emulsion to obtain the PT-Hsp-Cas9 complex, and finally lyophilized to remove the solution.

[0079] Wherein, the above PT is a polymer of formula (I), and its synthesis steps are as follows:

[0080] 0.1 mmol of 4-(2-ethylhexyl)-2,6-bis(trimethyltinyl)-4H-thieno[3,2-B:2',3'-D]pyrrole, 0.1 mmol of 4,8-dibromo-6-(2-ethylhexyl)-[1,2,5]thiadiazole[3,4-f]benzotriazole, 12 umol of triphenylphosphine and 3 umol of tris(dibenzylideneacetone)dipalladium were added to a Schlenk tube containing 5 mL of chlorobenzene. The mixture was stirred at 110°C for 72 hours under nitrogen protection. The mixture was cooled to room temperature and precipitated in methanol. The precipitate was collected by filtration and dried to obtain a black solid, which was the PT polymer. The reaction route is as follows:

[0081]

[0082] (4) Synthesis of RPC nanoparticles: The prepared PT-Hsp-Cas9 complex was dissolved in the red blood cell membrane solution and ultrasonicated in an ice bath for 10 min (25 W / cm 2 ) for 20 min, incubate on a shaker at 4°C overnight, and the next day, remove excess erythrocyte membranes by centrifugation to obtain RPC nanoparticles.

[0083] Example 3 Characterization of ARPC Nanoparticles Prepared in Example 1

[0084] The zeta potential of the ARPC nanoparticles, PC, Plasmid and PT prepared in Example 1 was measured. The measurement results are shown in the attached figure. Figure 2 As shown, after encapsulation of PCs with anti-LDLR-loaded RBCm, the zeta potential decreased from -24 mV to -12.9 mV, indicating a decrease in negative charge.

[0085] Dynamic light scattering (DLS) was used to characterize the particle sizes of PC, RPC, and ARPC. The results are shown in the attached figure. Figure 3 As shown by Figure 3 It can be seen that the initial average particle size of PC is 135 nm, and after RBCm encapsulation, the initial average particle size of RPC increases to 232 nm, and finally, the average particle size of ARPC is 313 nm.

[0086] To examine the morphology of the nanoparticles, we used transmission electron microscopy (TEM). First, we observed the morphology of RPCs. The results are shown in the attached figure. Figure 4 As shown in (a), the polycarbonate is approximately spherical with a particle size of about 150 nanometers, which is consistent with the particle size distribution data obtained previously. In order to verify the encapsulation of plasmid DNA and photothermal material PT, we performed scanning transmission electron microscopy (STEM) and energy dispersive spectroscopy (EDS) mapping. The test results are shown in the attached figure. Figure 4 As shown in (b), the presence of phosphorus (P) indicates plasmid DNA, while sulfur (S) is characteristic of PT. The overlap of these elements with the high-angle annular dark field (HAADF) image confirms the binding of plasmid DNA and the photothermal material PT in PC nanoparticles. We then coated the surface of PC nanoparticles with RBCm and incorporated anti-LDLR, and the detection results are shown in the attached figure. Figure 4 As shown in (c), RPC nanoparticles exhibited a membrane structure on their surface, and the particle size increased from 150nm to 233nm, which is still consistent with the previous particle size distribution data. Anti-LDLR antibodies were then embedded in the surface of RPC, and the test results are shown in the attached Figure 4 As shown in (d), the particle size increases further and a layer of flocculent material appears on the surface.

[0087] To investigate whether membrane proteins from RBCm were lost during the encapsulation process and to confirm the successful incorporation of anti-LDLR antibody, proteins were stained with Coomassie Brilliant Blue. Figure 5 As shown, the results showed that protein bands corresponding to RBCm and anti-LDLR were present in RPCs and ARPCs, indicating no protein loss.

[0088] To evaluate the presence of anti-LDLR antibodies on the surface of RBCm, DID staining and high-resolution confocal microscopy were used to observe the colocalization of RBCm and anti-LDLR antibodies. Figure 6 As shown, the results showed that anti-LDLR (yellow) was localized around RBCm (red), confirming that anti-LDLR was successfully incorporated as expected.

[0089] In addition, we evaluated whether the plasmid content in PC, RPC, and ARPC was affected by ultrasound. Figure 7 As shown, the results show that the band intensity remains consistent with nanoparticle formation, indicating minimal impact.

[0090] Next, the optimal loading amount of ARPC plasmid was studied. The results are shown in the attached Figure 8 As shown, the results showed that the maximum loading capacity was achieved when plasmid:ARP=4:1.

[0091] Finally, the UV absorbance of all components in ARPC was measured and the results are shown in the attached figure. Fig. 9 As shown, the results confirmed that ARPCs encapsulated all components, including PT, plasmid, RBCm and anti-LDLR.

[0092] Example 4: Transfection effect test of the red blood cell membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector (ARPC) prepared in Example 1

[0093] To verify the transfection effect of ARPC nanoparticles, two groups of blank, PC, RPC, and ARPC were co-cultured with SCC7 cells for 24 h, and then one of the groups was exposed to 1060 nm laser irradiation (0.75 W / cm 2 , 10 minutes), and the other group was not exposed to laser irradiation. After an additional 48 hours of incubation, the transfection efficacy of the ARPC system was evaluated using flow cytometry and CLSM. Fig.10 As shown, in the non-irradiated group (-laser), GFP expression was low, maintained at around 0.2%. However, after laser irradiation, ARPC showed a GFP expression level as high as 58.6%, which was significantly higher than PC (3.6%) and RPC (36%), indicating that the transfection efficiency using ARPC was significantly improved.

[0094] Fig.11 The fluorescence microscopic images of GFP in SCC7 cells also prove that ARPC treatment effectively enhances GFP expression in SCC7 cells.

[0095] Next, two groups of SCC7 cells were co-cultured with blank, PC, RPC or ARPC for 24 hours, and then one group was laser irradiated for 10 minutes, while the other group was not laser irradiated, and then cultured for another 72 hours. The expression levels of various proteins, including Cas9, Hsp70 and PD-L1, were then analyzed by WB and CLSM. The test results are shown in Figure 2. Fig.12 As shown in Figure 3, after laser irradiation, Hsp70 expression gradually increased, while PD-L1 expression was downregulated and Cas9 expression was upregulated. In the absence of laser irradiation, Cas9 expression was almost undetectable. To further confirm these findings, SCC7 cells were treated with anti-Hsp70, anti-PD-L1, and anti-Cas9 antibodies and imaged using CLSM. The detection results are shown in the attached figure. Fig.13As shown, with the upregulation of Hsp70 after laser irradiation, there was a trend of decreased PD-L1 expression. These test results collectively indicate that upregulation of Hsp70 leads to the expression of Cas9, which in turn leads to a decrease in PD-L1 expression.

[0096] Finally, to evaluate the efficiency of PD-L1 knockout, flow cytometry was used to detect the expression of PD-L1 in treated SCC7 cells. Fig.14 As shown, the proportion of PD-L1-negative cells in the ARPC (+ laser) group reached 45.3%, a significant increase compared with the 2.2% observed in the control group (- laser).

[0097] In summary, it can be confirmed that the CRISPR / Cas9 system mediated by the red blood cell membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector (ARPC) exhibits powerful editing capabilities.

[0098] Example 5: Testing the effect of Hsp70 controlling PD-L1 knockout in vivo

[0099] Female Balb / c mice, 6 weeks old, were purchased from GemPharmatech. 5×10 7 SCC7 cell suspension was injected into the cricothyroid region of the larynx. After 10 days, the mice were randomly divided into 5 groups (n=8) and injected with different formulations (PBS, ARP, ARP+L, ARPC, ARPC+L) peritumorally every other day. 24 hours after injection, the mice were treated with 1060nm laser (0.75W / cm 2 ) Tumors were irradiated for 9 minutes. The tumor volume and body weight of mice were recorded during the entire treatment period.

[0100] After in vivo treatment, the number of mice that died during the treatment was first recorded, and a survival curve was drawn, such as Fig.15 As shown in Figure 2, these curves indicate that there is little difference in survival before and after treatment. The tumor tissue was then dissected and photographs of the excised tumor tissue were taken for each group, as shown in Figure 2. Fig.16 As shown in (a); tumor weight was recorded as Fig.16 As shown in (b), the changes in tumor volume during the entire treatment process are recorded. Fig.16 As shown in (c); record the changes in mouse body weight, such as Fig.16 As shown in (d). It is clear that the presence or absence of laser treatment had a significant effect on tumor growth, with the smallest tumors observed in the ARPC+L group, suggesting that ARPC may have an enhanced immunostimulatory effect compared to ARP+L, and showing minimal changes in body weight, which remained stable at approximately 25g.

[0101] Next, the downregulation of PD-L1 in tumor tissues was analyzed. Flow cytometry was used for the first time to detect tumor tissues treated with ARPC. The results are shown in the attached figure. Fig.17 The results showed that the ARPC+L group exhibited the highest PD-L1 negativity rate, which was approximately four times that of the other groups.

[0102] To investigate whether the downregulation of PD-L1 is associated with Hsp70 activation, WB was performed on tumor tissues after treatment, and the results were as follows Fig.18 As shown, although both ARP+L and ARPC+L treatments induced Hsp70 upregulation due to photothermal therapy, PD-L1 expression was significantly lower in the ARPC+L group.

[0103] Tumor tissue sections were immunofluorescently stained with anti-Hsp70 and anti-PD-L1 antibodies, and the results were as follows: Fig.19 As shown, the above findings were confirmed, indicating the precise editing function of the ARPC nanoplatform in Hsp70 induction. In addition, HE and TUNEL staining were performed on tumor cells, and the detection results were shown in Fig.19 As shown, the results were consistent with expectations, with the ARP+L and ARPC+L groups showing stronger green fluorescence in TUNEL staining, which further elucidated the efficacy of tumor treatment.

[0104] Although the present invention is described using the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and these modifications and variations are all within the scope of protection of the present invention.

Claims

1. A method for preparing a CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on red blood cell membrane, characterized in that: The method comprises the following preparation steps: (1) Preparation of Hsp-Cas9: Construction of the HSP70 promoter-driven Cas9 gene editing system; (2) Preparation of red blood cell membranes: Separating a blood sample to obtain a red blood cell membrane suspension; (3) Synthesis of DSPE-PEG2000-αLDLR: DSPE-PEG2000-COOH was dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, and then N-hydroxysuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide were added to react at room temperature. After the reaction, the pH was adjusted to 7-7.5 to obtain activated DSPE-PEG2000-COOH; then, the activated DSPE-PEG2000-COOH was incubated with anti-LDLR antibody, and then unbound COOH-PEG2000-DSPE and unbound anti-LDLR antibody were removed by dialysis, and finally DSPE-PEG2000-αLDLR was prepared; (4) Preparation of PT-Hsp-Cas9 complex: polylactic acid-co-glycolic acid copolymer is dissolved in ethyl acetate, PT is dissolved in THF, and Hsp-Cas9 is dissolved in RNase-free water to form a PT-Hsp-Cas9 (PC) core, and the PT solution and the Hsp-Cas9 solution are dropped into the polylactic acid-co-glycolic acid copolymer solution, followed by ultrasonication to obtain an initial emulsion; then, the initial emulsion is mixed with an aqueous solution of polyvinyl alcohol and ultrasonicated to form a multiple emulsion; finally, the multiple emulsion is mixed with an aqueous solution of polyvinyl alcohol and ultrasonicated to stabilize the emulsion to obtain a PT-Hsp-Cas9 complex; wherein the structural formula of the PT is shown in the following formula (I): (5) Synthesis of ARPC nanoparticles: DSPE-PEG2000-αLDLR is added to the red blood cell membrane suspension to react to modify the red blood cell membrane, and then the DSPE-PEG2000-αLDLR modified red blood cell membrane is collected by centrifugation, and the prepared PT-Hsp-Cas9 complex is dissolved in the red blood cell membrane solution containing DSPE-PEG2000-αLDLR modified red blood cell membrane, sonicated on ice, and then incubated. After incubation, the excess red blood cell membrane is removed by centrifugation to obtain ARPC nanoparticles, i.e., the red blood cell membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector.

2. The method for preparing a CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on red blood cell membrane according to claim 1, characterized in that: The preparation step of Hsp-Cas9 includes: obtaining the HSP70 promoter fragment from the upstream region of the mouse HSP70 protein gene by PCR amplification.

3. The method for preparing a CRISPR-Cas9 near-infrared second zone precise light-controlled editing system delivery vector based on red blood cell membrane according to claim 1, characterized in that: The steps of preparing the red blood cell membrane include: firstly, centrifuging the blood sample to make the red blood cells with higher density settle to the bottom, then removing the upper plasma, and washing the red blood cells with PBS to remove plasma proteins and other impurities to ensure that only pure red blood cells are left; then using a mild lysis method to swell and eventually rupture the red blood cells to release the cell contents but maintain the integrity of the cell membrane; after lysis, separating the cell fragments by centrifugation or filtration to obtain free red blood cell membrane sheets; ultrasonically treating or gently stirring the collected membrane sheets to disperse them into smaller red blood cell membrane fragments or liposomes to obtain a red blood cell membrane suspension.

4. The method for preparing a CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on red blood cell membrane according to claim 1, characterized in that: The specific steps of synthesizing the DSPE-PEG2000-αLDLR include: dissolving DSPE-PEG2000-COOH in 2-(N-morpholino)ethanesulfonic acid buffer, then adding N-hydroxysuccinimide and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, reacting at room temperature for 30-60 minutes, adjusting the pH to 7.5, and obtaining activated DSPE-PEG2000-COOH; then incubating the anti-LDLR antibody with the activated DSPE-PEG2000-COOH at 4° C. for 12-24 hours, then dialyzing through a 3500MW cutoff membrane for 4 hours to remove unbound COOH-PEG2000-DSPE, and then dialyzing through a 300k cutoff membrane for 4 hours to remove unbound antibodies, and finally preparing DSPE-PEG2000-αLDLR.

5. The method for preparing a CRISPR-Cas9 near-infrared second zone precise light-controlled editing system delivery vector based on red blood cell membrane according to claim 1, characterized in that: The mass ratio of polylactic acid-glycolic acid copolymer and PT in the preparation of the PT-Hsp-Cas9 complex is 4:1-10:

1.

6. The method for preparing a CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on red blood cell membrane according to claim 1, characterized in that: The synthesis of the ARPC nanoparticles includes the following steps: adding DSPE-PEG2000-αLDLR to the erythrocyte membrane suspension, reacting at 4°C for 12-24 hours to modify the membrane, then collecting the DSPE-PEG2000-αLDLR modified erythrocyte membrane by centrifugation at 3500rpm at 4°C, dissolving the prepared PT-Hsp-Cas9 complex in the erythrocyte membrane solution modified with DSPE-PEG2000-αLDLR, ultrasonicating in an ice bath for 20 minutes, incubating on a shaker at 4°C for 12-24 hours, and then centrifuging to remove excess erythrocyte membrane to obtain ARPC nanoparticles, i.e., the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector.

7. A CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane, prepared by the method for preparing the CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector based on erythrocyte membrane as described in any one of claims 1 to 6.

8. Use of the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector prepared by the preparation method of the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector as described in any one of claims 1 to 6, or the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector as described in claim 7 in the preparation of drugs for preventing and / or treating cancer.

9. Use of the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector prepared by the preparation method of the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector as described in any one of claims 1 to 6, or the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector as described in claim 7 in cell transfection.

10. Use of the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector prepared by the preparation method of the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector as described in any one of claims 1 to 6, or the erythrocyte membrane-based CRISPR-Cas9 near-infrared zone II precise light-controlled editing system delivery vector as described in claim 7 in cell transfection.

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