Polypeptide with high transfection efficiency, peptide library, polypeptide-nucleic acid compound and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a polypeptide with high transfection efficiency, a peptide library, a polypeptide-nucleic acid compound and application thereof, the polypeptide with high transfection efficiency comprises five peptide modules A, X, B, C and Y, and the five peptide modules A, X, B, C and Y are sequentially connected through peptide bonds to form the polypeptide; the X peptide module is any one of an X1 peptide module and an X2 peptide module, and the Y peptide module is any one of a Y1 peptide module and a Y2 peptide module; the peptide library comprises a plurality of polypeptides, and the peptide library is helpful for quickly finding carrier formulas for different nucleic acid drugs, so that the construction of a drug delivery carrier platform can be better guided; the polypeptide-nucleic acid compound comprises polypeptide and nucleic acid, in the assembling and forming process of the polypeptide-nucleic acid compound, the molar ratio of protonated nitrogen of the polypeptide to phosphate groups of the nucleic acid is 15: 1-30: 1, and the polypeptide-nucleic acid compound is expected to be applied to medicine preparation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a polypeptide with high transfection efficiency, a peptide library, a polypeptide-nucleic acid complex and applications thereof. Background Art

[0002] Compared with traditional drug therapy, gene drugs have broad clinical application prospects because they can fundamentally control diseases by replacing pathogenic genes or changing the genetic structure of diseased cells. However, due to the unsatisfactory pharmacokinetic properties of gene drugs, it is difficult to successfully deliver them to the cytoplasm of target cells, which limits the progress of their clinical application. Drug delivery vectors are expected to overcome their pharmacokinetic defects.

[0003] Currently, the more commonly used methods for delivering nucleic acid drugs include liposome delivery, direct injection of DNA solution or receptor-mediated transfection of nucleic acid and other biological methods. However, these methods have disadvantages such as low biocompatibility, low transfection efficiency and poor targeting. Although traditional polypeptide nucleic acid carriers can overcome the problems of poor biocompatibility of liposomes to a certain extent, they still have the limitations of low transfection efficiency and can only transfect a certain type of nucleic acid. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a polypeptide with high transfection efficiency, the second object of the present invention is to provide a peptide library, the third object of the present invention is to provide a polypeptide-nucleic acid complex, and the fourth object of the present invention is to provide an application of a polypeptide-nucleic acid complex.

[0005] In order to achieve the first purpose, the technical solution adopted by the present invention is: A polypeptide with high transfection efficiency, the polypeptide comprising five peptide modules A, X, B, C and Y, wherein the five peptide modules A, X, B, C and Y are sequentially connected by peptide bonds; The X peptide module is any one of the X1 peptide module and the X2 peptide module, and the Y peptide module is any one of the Y1 peptide module and the Y2 peptide module; Among them, the amino acid sequence of the A peptide module is KKK, the amino acid sequence of the X1 peptide module is GFLG, the amino acid sequence of the X2 peptide module is RRGK, the amino acid sequence of the B peptide module is LLL, the amino acid sequence of the C peptide module is HHH, the amino acid sequence of the Y1 peptide module is KKK, and the amino acid sequence of the Y2 peptide module is GYQTI; Among them, K is the abbreviation of lysine, G is the abbreviation of glycine, F is the abbreviation of phenylalanine, L is the abbreviation of leucine, R is the abbreviation of arginine, H is the abbreviation of histidine, Y is the abbreviation of tyrosine, Q is the abbreviation of glutamine, T is the abbreviation of threonine, and I is the abbreviation of isoleucine.

[0006] Furthermore, it also includes a Z peptide module, and the Z peptide module is connected to the Y peptide module via a peptide bond; The amino acid sequence of the Z peptide module is shown in SEQ NO.1; SEQ NO.1 is as follows: FLGLSSSSSSSSSSSSSSSS; Here, S is the abbreviation for serine.

[0007] In order to achieve the second purpose, the technical solution adopted by the present invention is: A peptide library comprising a plurality of polypeptides, wherein the polypeptides are the polypeptides with high transfection efficiency as described above.

[0008] In order to achieve the third purpose, the technical solution adopted by the present invention is: A polypeptide-nucleic acid complex, comprising the polypeptide with high transfection efficiency as described above, and also comprising a nucleic acid, wherein the polypeptide and the nucleic acid self-assemble to form the polypeptide-nucleic acid complex; During the self-assembly process of the polypeptide and the nucleic acid, the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid is 15:1 to 30:1.

[0009] Furthermore, the polypeptide includes polypeptide I, polypeptide II and polypeptide III: The polypeptide I is: AX1BCY1 or AX2BCY1, the amino acid sequence of AX1BCY1 is shown in SEQ NO.2, and the amino acid sequence of AX2BCY1 is shown in SEQ NO.3; The polypeptide II is: AX1BCY2 or AX2BCY2, the amino acid sequence of AX1BCY2 is shown in SEQ NO.4, and the amino acid sequence of AX2BCY2 is shown in SEQ NO.5; The polypeptide III is: AX2BCY2Z amino acid sequence is shown in SEQ NO.6.

[0010] Furthermore, the molar ratio of the polypeptide I, the polypeptide II and the polypeptide III is (10-30): (70-90): (0.1-1); During the self-assembly process of the polypeptide and the nucleic acid, the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid is 15:1 to 25:1.

[0011] Furthermore, the molar ratio of the polypeptide I, the polypeptide II and the polypeptide III is (15-25): (75-85): (0.1-1); During the self-assembly process of the polypeptide and the nucleic acid, the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid is 15:1 to 20:1.

[0012] Furthermore, the nucleic acid includes at least one of random sequence DNA, plasmid DNA, mRNA, siRNA and Cas9-mRNA / sgRNA.

[0013] In order to achieve the fourth purpose, the technical solution adopted by the present invention is: An application of a polypeptide-nucleic acid complex, wherein the polypeptide-nucleic acid complex is any of the polypeptide-nucleic acid complexes described above, and the application includes its application in preparing a drug.

[0014] Furthermore, the drug includes an anti-tumor drug.

[0015] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention designs a polypeptide composed of peptide modules containing different functions, which is a polypeptide with high transfection efficiency. A polypeptide library formed by multiple peptide modules with different functions is established to establish a high-throughput screening platform for polypeptide carriers. The present invention uses this screening platform to find an efficient delivery peptide library for siRNA to mRNA, plasmids and even CRISPR / Cas9 systems, and successfully applies it to in vivo administration. The polypeptide with high transfection efficiency provided by the present invention, the constituent peptide modules of which utilize the synergistic effects of enzyme response, lysosomal membrane targeting and proton sponge effect, enhance the lysosomal escape ability of the polypeptide, and significantly improve the biocompatibility and delivery efficiency compared with traditional delivery carriers.

[0016] Compared with the prior art, the peptide library provided by the present invention helps to quickly find carrier formulations for different nucleic acid drugs, thereby better guiding the construction of drug delivery carrier platforms and more effectively developing polypeptide-nucleic acid complexes with specific properties and functions. The polypeptide-nucleic acid complexes are expected to play an important role in the fields of drug delivery, biomedical engineering, etc.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the transfection thermal effects of different polypeptide-nucleic acid complexes provided in Example 3 of the present invention.

[0019] Figure 2 This is a diagram of the transfection thermal effects of different polypeptide-nucleic acid complexes provided in Example 3 of the present invention.

[0020] Figure 3 This is a diagram of the transfection thermal effects of different polypeptide-nucleic acid complexes provided in Example 3 of the present invention.

[0021] Figure 4 It is the circular dichroism spectra of different polypeptides and polypeptide-nucleic acid complexes provided in Example 4 of the present invention.

[0022] Figure 5 It is a transmission electron microscopy morphology image of six different polypeptide-DNA complexes provided in Example 4 of the present invention.

[0023] Figure 6 This is a zeta potential bar graph of different polypeptides and polypeptide-DNA complexes provided in Example 4 of the present invention.

[0024] Figure 7 This is a particle size distribution diagram of different polypeptide-DNA complexes provided in Example 4 of the present invention.

[0025] Figure 8 It is the infrared spectra of different polypeptides provided in Example 4 of the present invention.

[0026] Fig. 9 The different polypeptide-nucleic acid complex assembly process K provided in Example 5 of the present invention d Analysis diagram; wherein, the polypeptide contained in the complex in Figure A is AX2BCY1, the polypeptide contained in the complex in Figure B is AX2BCY1+AX2BCY2, the polypeptide contained in the complex in Figure C is AX1BCY1, and the polypeptide contained in the complex in Figure D is AX2BCY1+AX2BCY1.

[0027] Fig.10 It is a confocal image of the cellular uptake process of different polypeptide-siRNA complexes provided in Example 6 of the present invention.

[0028] Fig.11 This is a microscope image of the Hela uptake polypeptide (AX1BCY1)-siRNA complex provided in Example 6 of the present invention.

[0029] Fig.12 This is a microscopic image of the Hela uptake polypeptide (AX2BCY1)-siRNA complex provided in Example 6 of the present invention.

[0030] Fig.13 It is a flow efficiency bar graph of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention.

[0031] Fig.14It is a fluorescence microscope image of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention.

[0032] Fig.15 It is a flow cytometry result diagram of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention; wherein A is a flow cytometry result diagram of the polypeptide-nucleic acid complex of formulation R39, and B is a flow cytometry result diagram of the polypeptide-nucleic acid complex of formulation R40.

[0033] Fig.16 It is a flow efficiency bar graph of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention.

[0034] Fig.17 This is a statistical graph of Hela cell activity after treatment with different polypeptides provided in Example 7 of the present invention.

[0035] Fig.18 This is a fluorescence microscope image of the polypeptide-siRNA complex provided in Example 7 of the present invention for GFP gene silencing and CRISPR / Cas9 system gene editing to knock out the GFP gene.

[0036] Fig.19 This is a bar graph showing the efficiency of GFP gene silencing by the polypeptide-siRNA complex provided in Example 7 of the present invention and GFP gene knockout by gene editing using the CRISPR / Cas9 system.

[0037] Fig. 20 This is a graph showing the results of a hemolysis experiment of different polypeptide-nucleic acid complexes provided in Example 8 of the present invention.

[0038] Fig.21 It is a bar graph of the hemolysis ratios of different polypeptide-nucleic acid complexes provided in Example 8 of the present invention.

[0039] Fig. 22 These are typical whole-body fluorescence images of mice provided in Example 8 of the present invention after intravenous injection of polypeptide-Cy5 mRNA complex for 8 hours.

[0040] Fig.23 These are typical whole-body bioluminescence images of mice injected intravenously with polypeptide-Fluc mRNA complex for 24 hours provided in Example 8 of the present invention.

[0041] Fig.24 This is an in vitro surface fluorescence image of various organs of mice 48 hours after intravenous injection of the polypeptide-Fluc mRNA complex provided in Example 8 of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0043] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0044] A polypeptide with high transfection efficiency, the polypeptide comprising five peptide modules A, X, B, C and Y, wherein the five peptide modules A, X, B, C and Y are sequentially connected by peptide bonds; The X peptide module is any one of the X1 peptide module and the X2 peptide module, and the Y peptide module is any one of the Y1 peptide module and the Y2 peptide module; Among them, the amino acid sequence of the A peptide module is KKK, the amino acid sequence of the X1 peptide module is GFLG, the amino acid sequence of the X2 peptide module is RRGK, the amino acid sequence of the B peptide module is LLL, the amino acid sequence of the C peptide module is HHH, the amino acid sequence of the Y1 peptide module is KKK, and the amino acid sequence of the Y2 peptide module is GYQTI; Among them, K is the abbreviation of lysine, G is the abbreviation of glycine, F is the abbreviation of phenylalanine, L is the abbreviation of leucine, R is the abbreviation of arginine, H is the abbreviation of histidine, Y is the abbreviation of tyrosine, Q is the abbreviation of glutamine, T is the abbreviation of threonine, and I is the abbreviation of isoleucine.

[0045] Preferably, it further comprises a Z peptide module, wherein the Z peptide module is connected to the Y peptide module via a peptide bond; The amino acid sequence of the Z peptide module is shown in SEQ NO.1, SEQ NO.1 is as follows: FLGLSSSSSSSSSSSSSSSS; Here, S is the abbreviation for serine.

[0046] The peptide module composition, source and purity of different polypeptides are shown in Table 1 , and the specific amino acid sequence composition of different polypeptides is shown in Table 2 .

[0047] Table 1 Structural composition, purity and source of different peptides Table 2 Amino acid sequence composition of different peptides For single-component peptide sample preparation, first dilute the DNA / RNA with water, then add to the peptide solution and vortex mix. For multi-component peptide-DNA / RNA self-assembled complexes, first dilute the DNA / RNA with water, then add to the premixed peptide solution and vortex mix according to the calculated ratio. Samples are usually incubated at room temperature for 30 minutes before further structural characterization. If not specified, the DNA / RNA concentration is fixed at 50 μM, and the peptide-nucleic acid complex with the best N / P ratio is selected for the relevant experiments.

[0048] Example 1 Preparation of a polypeptide with high transfection efficiency.

[0049] A polypeptide with high transfection efficiency, characterized in that it contains the aforementioned six peptide modules with different functions. In order to give full play to the effects of different peptide modules, the six peptide modules are connected in sequence according to their order in the amino acid sequence. A peptide module that binds nucleic acid through self-assembly, an X peptide module that responds to lysosomal enzymes to achieve enzymatic cleavage and change the structure of the polypeptide, a B peptide module that disturbs the lysosomal membrane after enzymatic cleavage and enhances lysosomal escape, a C peptide module containing stimulus-responsive residues that responds to environmental pH changes to trigger the dissociation of the polypeptide carrier, a Y peptide module that targets specific cells or tissues, and a Z peptide module that resists adhesion between the polypeptide and biological tissues. Among them, the A, B, and C peptide modules are the basic skeletons of the polypeptide, the X peptide module and the Y peptide module each have two different submodules, codenamed X1, X2 and Y1, Y2, and the Z module can be selectively added in the amino acid sequence.

[0050] The peptide was prepared by Tianjin Beiyang Kangpeptide Biotechnology Co., Ltd. through solid phase synthesis. Taking the synthesis of 50mg AX2BCY1 peptide as an example, the Fmoc synthesis method was used. The specific preparation process is as follows: Weigh 200 mg of dry 1% divinylbenzene (DVB) cross-linked CTC resin (200-400mesh) (SD: 1.211mmol / g) in a solid phase synthesis tube, add N,N-dimethylformamide (DMF), and swell with nitrogen for 30 min. Take a small amount of resin for ninhydrin detection, add 2-3 drops of ninhydrin solution (liquid A), phenol solution (liquid B) and VC solution (liquid C) in the test tube, take a small amount of resin in the test tube, heat it in an oil bath at 110℃ for 3min, and observe the color change of the resin (purple / dark red / black-there are free amino acids on the resin, Fmoc removal is successful; yellow / cyan-there are no free amino acids on the resin, condensation is successful).

[0051] Amino acid coupling: nitrogen was blown through, vacuum pump was used to dry, and then under nitrogen protection, resin: Fmoc-amino acid: hydroxybenzotriazole (HXBT): N, N'-diisopropylcarbodiimide (DIC) = 1:3:4:4 (the first amino acid was reacted according to resin: Fmoc-amino: N, N-diisopropylethylamine = 1:2:8), dissolved in DMF, activated in an ice bath for 10-15 minutes, and then poured into a solid phase synthesis tube, and dehydrated and condensed for 2 hours at room temperature. After the reaction, the reaction was monitored by the ninhydrin colorimetric method. When the resin was colorless or yellow, it indicated that the condensation reaction was complete. Then, DMF was added to wash the resin 4-5 times, and the solvent was removed.

[0052] Product blocking: Add blocking solution dichloromethane (DCM): methanol: N,N-diisopropylethylamine = 17:2:1, 10 min / time, repeat 2 times, and then wash with DMF 5 times.

[0053] Removal of Fmoc protecting group: Add 20% piperidine-DMF solution to fully suspend the resin, blow through N2, drain and remove the Fmoc protecting group. Add DMF to wash the resin 4-5 times, remove the excess deprotection solution, remove the solvent, and then perform ninhydrin detection.

[0054] The amino acid coupling and Fmoc protection group removal process is repeated, and amino acids are connected according to the operation sequence until a complete amino acid sequence is obtained.

[0055] Cutting and removal of side chain protection: Use DCM to wash away DMF, repeat 2 times, 2-3 min each time, then wash the resin 3 times with 100% methanol, 10 min each time, to make the resin fully shrink, filter until the resin is dry, add cutting liquid at 10-15 mL / g (the amount can be increased appropriately according to the situation) to the resin, (the cutting liquid composition is: water / triisopropylsilane (TIS) / trifluoroacetic acid (TFA) = 2.5 / 2.5 / 95, and needs to be frozen at -20°C before use) blow nitrogen, cut for 2.5 h (cut at room temperature, if the cutting liquid is found to evaporate too quickly in the middle, new cutting liquid can be added), then filter, then add a small amount of DCM to wash 2 times, 1-2 min each time, collect the filtrate, and remove most of the solvent by rotary evaporation. The remaining solution after sufficient rotary evaporation is 1: 10% was added dropwise to the frozen ether (operated in a centrifuge tube), and a glass rod was used to stir continuously during the addition process to disperse the precipitated peptide as much as possible to avoid agglomeration. Then, the mixture was centrifuged at 3000 r / min for 3 min, the supernatant was discarded, and the ether was removed by suction filtration. The filtrate was discarded to obtain a crude peptide solid, and vacuum dried to obtain a dried crude peptide product.

[0056] Peptide purification: Purify by preparative liquid chromatography (HPLC purification), concentrate and freeze-dry to obtain pure peptide.

[0057] Example 2 Preparation of polypeptide-nucleic acid complex.

[0058] The polypeptide-nucleic acid complex is self-assembled from a polypeptide and a nucleic acid, the polypeptide is a polypeptide with high transfection efficiency, and the polypeptide-nucleic acid complex includes one or more of the polypeptides.

[0059] A method for preparing a polypeptide-nucleic acid complex using a functional polypeptide with high transfection efficiency, wherein the nucleic acid used includes five commonly used nucleic acid molecules, namely: Random sequence DNA primer (length 100 bp, nonsense sequence); Plasmid DNA (plasmid DNA capable of expressing GFP fluorescent protein); mRNA (mRNA capable of expressing GFP fluorescent protein); siRNA (siRNA that silences the GFP fluorescent protein gene); Cas9-mRNA / sgRNA (CRISPR / Cas9 nucleic acid drug capable of editing the GFP fluorescent protein gene).

[0060] The preparation process of polypeptide-nucleic acid complex is as follows: 1. Preparation of polypeptide-mRNA complex.

[0061] Thaw the EGFP-mRNA purchased from APE×BIO at room temperature, centrifuge at 3000 rpm for 10 s, open it slowly and pipette it evenly to obtain the mRNA solution for use; The polypeptide prepared in Example 1 was added to the buffer solution in different amounts, and mixed by pipetting to obtain a polypeptide solution with a concentration of 1 mg / mL; The mRNA nucleic acid solution was quickly added to the polypeptide solution so that the molar ratio of protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid (N / P molar ratio) was 3, and incubated at room temperature for 30 minutes to obtain a polypeptide-mRNA complex.

[0062] The N / P molar ratios were 3, 5, 10, 15, 20, 25, 30, 35, and 40 respectively; instead of the N / P molar ratio of 3 in this example, polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, and AX2BCY2Z were used for preparation, and the other aspects were the same as in this example, to obtain different polypeptide-mRNA complexes.

[0063] 2. Preparation of polypeptide-DNA complex.

[0064] Centrifuge 1 OD of 100 bp random sequence DNA purchased from Anshengda Technology at 3000 rpm for 10 s, open it slowly, add 33 ul of ultrapure water and shake to dissolve, and obtain a solution for use; The polypeptide prepared in Example 1 was added to the buffer solution in different amounts, and mixed by pipetting to obtain a polypeptide solution with a concentration of 1 mg / mL; The DNA nucleic acid solution was quickly added to the polypeptide solution so that the molar ratio of protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid (N / P molar ratio) was 3, and the mixture was incubated at room temperature for 30 minutes to obtain a polypeptide-DNA complex.

[0065] The N / P molar ratios were 3, 5, 10, 15, 20, 25, 30, 35, and 40 respectively; instead of using the N / P molar ratio of 3 in this example, polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, and AX2BCY2Z were used for preparation, and the other aspects were the same as in this example, to obtain different polypeptide-DNA complexes.

[0066] In addition, when preparing the polypeptide-plasmid DNA complex, EGFP-pDNA with a concentration of 1 mg / mL purchased from Yunzhou Biotechnology was taken, thawed at room temperature, and centrifuged at 3000 rpm for 10 seconds to obtain a pDNA solution for use, and then subsequent preparation was carried out according to the polypeptide-DNA complex preparation method.

[0067] 3. Preparation of peptide-siRNA complex.

[0068] Centrifuge 1 OD of siRNA purchased from Suzhou Jima Biotechnology at 3000 rpm for 10 s, open it slowly, add 75 ul of ultrapure water and shake to dissolve, and obtain a solution for use; The polypeptide prepared in Example 1 was added to the buffer solution in different amounts, and mixed by pipetting to obtain a polypeptide solution with a concentration of 1 mg / mL; The siRNA solution was quickly added to the polypeptide solution so that the molar ratio of protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid (N / P molar ratio) was 3, and the mixture was incubated at room temperature for 30 minutes to obtain a polypeptide-siRNA complex.

[0069] The N / P molar ratios were 3, 5, 10, 15, 20, 25, 30, 35, and 40 respectively; instead of using the N / P molar ratio of 3 in this example, polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, and AX2BCY2Z were used for preparation, and the other aspects were the same as in this example, to obtain different polypeptide-siRNA complexes.

[0070] 4. Preparation of peptide-CRISPR / Cas9 complex.

[0071] Thaw the Cas9-mRNA purchased from APE×BIO and the sgRNA purchased from Anshengda Technology at room temperature, centrifuge at 3000 rpm for 10 s, open slowly and pipette to obtain the Cas9-mRNA / sgRNA solution for use; The polypeptide prepared in Example 1 was added to the buffer solution in different amounts, and mixed by pipetting to obtain a polypeptide solution with a concentration of 1 mg / mL; The Cas9-mRNA / sgRNA nucleic acid solution was quickly added to the polypeptide solution to make the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid (N / P molar ratio) be 3, and the molar ratio of Cas9-mRNA and sgRNA in the mixed solution was controlled to be 3:1. The mixture was incubated at room temperature for 30 minutes to obtain a polypeptide-nucleic acid complex.

[0072] The N / P molar ratios were 3, 5, 10, 15, 20, 25, 30, 35, and 40 respectively; instead of the N / P molar ratio of this example being 3, the polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, and AX2BCY2Z were used for preparation respectively, and the other aspects were the same as this example, to obtain different polypeptide-CRISPR / Cas9 complexes.

[0073] Example 3 Screening of formulations of polypeptide-nucleic acid complexes.

[0074] According to the functional polypeptide obtained in Example 1 and the polypeptides formed by combining the five different peptide modules obtained therefrom: AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2 and AX2BCY2Z, the present invention designed a total of 101 combinations for the polypeptide-complex, as shown in Tables 3 to 7, and screened these 101 combinations using a high-throughput screening strategy.

[0075] Table 3 Composition of polypeptide-nucleic acid complexes Table 4 Composition of polypeptide-nucleic acid complexes Table 5 Composition of polypeptide-nucleic acid complexes Table 6 Composition of polypeptide-nucleic acid complexes Table 7 Composition of polypeptide-nucleic acid complexes The present invention selected four commonly used nucleic acids for delivery, namely mRNA and pDNA that can express GFP fluorescent protein, siRNA that silences the GFP fluorescent protein gene, and CRISPR / Cas9 nucleic acid drugs (Cas9-mRNA and sgRNA) that can edit the GFP fluorescent protein gene. The transfection effect of polypeptides composed of peptide modules with different functions on the four nucleic acids was tested, and the transfection effect heat map was obtained as shown in the figure. Figure 1 , Figure 2 , Figure 3 As shown in the figure, ① represents polypeptide AX1BCY1, ② represents polypeptide AX2BCY1, ③ represents polypeptide AX1BCY2, and ④ represents polypeptide AX2BCY2. According to the heat map, it can be seen that the transfection ability of the polypeptide containing the X2 peptide module is slightly stronger than that of the polypeptide containing the X1 peptide module. Figure 1 As shown in the figure; when the molar ratio of the Y2 peptide module in the mixed polypeptide is 20%, the transfection effect is significantly improved. This improvement is reflected to varying degrees when transfecting mRNA, pDNA, siRNA and Cas9 components, which shows that the optimal molar ratio of Y2 is 20%. Figure 2 As shown in the figure, when the content of Z peptide module reaches 20%, the delivery effect decreases significantly, only 85.88% of the original. However, when the proportion of Z peptide module is less than 1%, the effect of nucleic acid transfection is almost unaffected, indicating that the appropriate addition of Z peptide module will not affect the delivery efficiency of peptide carriers. Figure 3 shown.

[0076] In the process of self-assembly of the polypeptide and the nucleic acid molecule, the composition of the polypeptide-nucleic acid complex with better transfection effect is shown in Table 8.

[0077] Table 8 Composition of polypeptide-nucleic acid complex Example 4 Characterization of polypeptides and polypeptide-nucleic acid complexes.

[0078] Characterization of a polypeptide-nucleic acid complex, taking a polypeptide-DNA complex as an example: The circular dichroism spectra of peptides and peptide-DNA complexes (referred to as complexes) were determined using a JASCO jasCO-810 circular dichroism spectrometer from JASCO. The sample cell had an optical path of 0.1 mm, a wavelength range of 185-350 nm, a scanning speed of 200 nm / min, a bandwidth of 1 nm, and a test temperature of 25°C. The test was repeated three times and the average value was output in the equipment control software, as shown in Figure 4As shown in the figure, it can be clearly seen that in a neutral environment, the assembly results of the two single polypeptides AX1BCY1 and AX1BCY2 mainly show the characteristics of β-folding, and the polypeptide-DNA complex formed also shows the characteristics of β-folding. However, the two polypeptides AX2BCY1 and AX2BCY2 and the polypeptide-DNA complex show the secondary structure of α-helix.

[0079] The morphological structures of the following six peptide-nucleic acid self-assembled complexes were studied by cryogenic transmission electron microscopy: AX2BCY1, AX2BCY1+AX2BCY2, AX2BCY1+AX2BCY2+AX2BCY2Z, AX1BCY1, AX1BCY1+AX1BCY2 and AX1BCY1+AX1BCY2+AX2BCY2Z.

[0080] The complex solution (10 μL) was deposited on the surface of the copper mesh, and after staying for 3 minutes, it was absorbed with filter paper for 1-2 seconds, and then negatively stained twice with 10 μL 1% phosphotungstic acid solution, staying for 30 seconds and 10 seconds respectively. During the test, the copper mesh was installed in a cryogenic sample holder and transferred to a JEM-1400Flash cryogenic transmission electron microscope under low temperature conditions. The results are shown in Figure 5 As shown, from Figure 5 It can be seen that the polypeptide-nucleic acid complex presents a spherical nanoparticle structure. As the number of polypeptide types increases, the particle size of the polypeptide-DNA complex nanoparticles increases, but the formed nanoparticles are more uniform and less likely to aggregate, which is beneficial for the carrier to maintain a stable state in the in vivo environment.

[0081] Zeta potential and particle size analysis: Zeta potential and particle size were analyzed on a Zeta Plus (Brookhaven Instruments, USA). When preparing samples, peptide carrier samples with different peptide modules were prepared according to different composition ratios. The electrodes were immersed in the samples and 1.7 mL of freshly prepared samples with a concentration of 300 μM were directly analyzed. Each sample was run ten times, ten cycles each time, and the results of the ten cycles were averaged and statistically analyzed for the zeta potential. The results are shown in Figure 2. Figure 6 and Figure 7 As shown in the figure, it can be seen that the potential of the assembled polypeptide-DNA complex is positive, which is conducive to the cell's uptake of nucleic acid drugs, and the particle size of the polypeptide-DNA complex is about 200nm, which is easily taken up by cells.

[0082] In addition, Fourier transform infrared spectroscopy (FTIR) was used to characterize the secondary structures of the peptides and peptide-DNA complexes. An average of 16 FTIR spectra were collected on a Bruker Vertex-70 Spectrometer (USA) with a resolution of 4 cm-1 , ranging from 4000-400 cm -1 When preparing the sample, the sample was fully frozen in liquid nitrogen and then freeze-dried in a vacuum freeze dryer for 48 hours to obtain a peptide dry powder. The dried peptide powder was mixed with KBr at a ratio of 1:100 (w / w) and ground into fine powder with a mortar and pestle. The blank KBr tablet was scanned as the background. The blank KBr background needs to be subtracted from the sample spectrum obtained by scanning. The scanning results are as follows: Figure 8 As shown in the figure, for 1600~1800 cm -1 The peaks at the left and right sides of the chromatograms were analyzed. The anti-β-folded structure was dominant in the AX1BCY1 and AX1BCY2 polypeptides, accounting for 35% and 90.1% respectively. The α-helical structure was dominant in the AX2BCY1 and AX2BCY2 polypeptides, accounting for 83.5% and 60.1% respectively, which was basically consistent with the structure obtained by circular dichroism.

[0083] Example 5 Kinetic characteristics of polypeptide-nucleic acid complexes.

[0084] The kinetic characteristics of a peptide-DNA complex are determined as follows: All kinetic measurements were performed in PBS at room temperature and protected from light. on In the experiment, the fluorescence emission of 800 μL of Atto488-labeled DNA (10 nM) solution was first measured at 515 nM (488 nM excitation), and this value was used as the benchmark for t = 0. Subsequently, a small amount of concentrated peptide solution was gradually added to control the final concentration of the peptide to 25~125 nM, and the decrease in its fluorescence emission intensity over time was monitored at intervals of 10 seconds. Finally, the relationship between emission intensity and time was fitted to a single exponential function y=a*exp(-x / t)+y0, so as to calculate the k corresponding to each peptide concentration. obs , then draw k obs The curve of peptide concentration was plotted and k was obtained from the slope of its linear fit. on .

[0085] To determine k off 800 μL of Atto488-labeled DNA (10 nM) and peptide solution (5 nM) were incubated until no further changes in fluorescence were observed. The peptide-DNA complex was then placed in an acidic buffer and an excess of unlabeled ssDNA was used to replace the encapsulated Atto488-labeled DNA. After adding 1 μM unlabeled ssDNA, the change in fluorescence emission at 515 nm was monitored every 10 seconds. The resulting data were fitted to a single exponential function to calculate k off .

[0086] Dissociation constant K d Using the association rate constant k on and the dissociation rate constant k off Calculation, the result is as follows Fig. 9 The results showed that the polypeptide-DNA complex has a nucleic acid binding-dissociation constant similar to that of viruses and has the ability to mimic viruses in nucleic acid delivery, which is consistent with its high transfection efficiency performance.

[0087] Example 6 Transfection of cells with polypeptides.

[0088] The polypeptide in this example is the polypeptide prepared in Example 2.

[0089] 1. The processing process of cells used in the experiment is as follows: Hela cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator at 5% CO2 and 37°C until the logarithmic growth phase. When the cells were in good condition, the cells were resuspended in DMEM medium and incubated at 5×10 4 The cells were inoculated into a 12-well plate at a density of 100 cells / well and incubated overnight in a cell culture incubator at 5% CO2 and 37°C. Transfection was performed when the cell density was about 70% to 80%. Before transfection, the culture medium was removed, the cells were washed twice with PBS (1× phosphate buffered saline, pH 7.4), and then 500 µL of Opti-MEM (reduced serum medium) was added to each well.

[0090] 2. The process of testing the ability of polypeptide-nucleic acid complexes to escape lysosomes is as follows: The endocytosis and lysosomal escape process of the peptide-siRNA complex was observed by confocal microscopy. siRNA labeled with fluorescein isothiocyanate (FITC) (green fluorescence); siRNA sense strand nucleotide sequence: 3′-GCAUCGAGAAGUACGAGGATT-5′; siRNA antisense strand nucleotide sequence: 3′-UCCUCGUACUUCUCGAUGCTT-5′; Hela cells were co-cultured with siRNA and 4',6-diamidino-2-phenylindole (DAPI) to label the nucleus and Lyso-Tracker Red to label the lysosomes. The location of the peptide-siRNA complex and lysosomes in the cells was observed using a fluorescence microscope at 4 hours. The confocal microscope images are shown in the figure. Fig.10As shown in the figure, it can be seen that after the polypeptide containing the X1 peptide module enters the cell, the green fluorescence level generated is lower than that of the polypeptide containing the X2 peptide module. However, after the single X1 peptide module or X2 peptide module enters the cell, it will be engulfed by the lysosome. The figure shows that the green fluorescence of the nucleic acid drug overlaps with the red fluorescence of the lysosome. In the image of the polypeptide containing the Y2 peptide module, the composition of the polypeptide-nucleic acid complex is numbered R23 in Table 6. It can be clearly seen that the green fluorescent label is released, more independent green labels appear in the image, and some lysosomal red labels become blurred, indicating that the lysosome is ruptured; the polypeptide containing the Z peptide module has almost no effect on the lysosomal escape ability, and the composition of the polypeptide-nucleic acid complex is numbered R48 in Table 7.

[0091] 3. The method for determining the peptide-siRNA complex uptake rate is as follows: One end of the delivered siRNA was labeled with FITC green fluorescence, and the complex was prepared according to the preparation method of the polypeptide-siRNA complex in Example 2, so that the molar ratio of protonated nitrogen to phosphate group (N / P molar ratio) was 5, 10, 20, 30 and 40, and assembled at room temperature in the dark for 30 min. The green fluorescence-labeled siRNA and polypeptide complexes were taken separately, diluted to a final volume of 100 μL with nuclease-free water, and added to the culture wells containing 500 μL Opti-MEM culture medium, and the cell culture plate was gently shaken to fully mix the green fluorescence-labeled siRNA and polypeptide complexes with the culture medium in the wells. After 4 hours, the complexes were observed under an inverted fluorescence microscope (KQ-250E model of Olympus, Japan). The results are as follows: Fig.11 and Fig.12 As shown, the results indicate that the cell uptake efficiency of the polypeptide-siRNA complex is highest when the nitrogen-phosphorus ratio is around 20.

[0092] 4. The process of measuring uptake efficiency by flow cytometry is as follows: A blank group and an experimental group were set up. The cells in the blank group were not treated. The cells in the experimental group were taken up with complexes with a molar ratio (N / P) of protonated nitrogen to phosphate groups of 5, 10, 20, 30, and 40, respectively. The transfection method was the same as the above three treatment processes. After repeated washing with PBS three times, the cells were processed and counted, and the cells were resuspended with a cell washing solution containing 2% BSA in PBS to a cell concentration of 1×10 7 / mL, take 200 μL of cell resuspension, and use flow cytometry to detect the intracellular fluorescence intensity. The results are as follows Fig.11 , Fig.12 and Fig.13 As shown, the bar graph results are basically consistent with the microscope image results.

[0093] 5. The transfection methods of mRNA and pDNA are as follows: According to the preparation method in Example 2, the molar ratio of protonated nitrogen to phosphate groups (N / P molar ratio) was 15, 20 and 25 respectively, and assembled at room temperature for 30 min to obtain polypeptide-mRNA complexes and polypeptide-DNA complexes. The polypeptide-nucleic acid complex was diluted with nuclease-free water to a final volume of 100 μL, added to the culture well containing 500 μL Opti-MEM culture medium, and the cell culture plate was gently shaken to fully mix the polypeptide-mRNA complex and polypeptide-DNA complex with the culture medium in the well. After 4 hours, the results were observed under an inverted fluorescence microscope. Fig.14 As shown, the microscopy images showed that the nucleic acid drug was successfully delivered into the cells and expressed.

[0094] 6. The process of determining transfection efficiency by flow cytometry is as follows: A blank group and an experimental group were set up. The cells in the blank group were not treated. The cells in the experimental group were transfected with complexes with N / P=15, 20, and 25. The method was the same as the treatment process of the above three examples. After repeated washing with PBS for three times, the cells were processed and counted, and the cells were resuspended with cell washing solution (PBS containing 2% BSA) to a cell concentration of 1×10 7 / mL. Take 200 μL of cell resuspension and use flow cytometry to detect the intracellular fluorescence intensity. The results are as follows Fig.15 and Fig.16 As shown, the graphical results indicate that the compositions of the polypeptide-nucleic acid complexes are numbered R39 and R40 in Table 6, which are the compositions with the best transfection effects for plasmid DNA and mRNA, respectively.

[0095] Example 7 Effects of polypeptide-nucleic acid complexes on specific genes in cells and mice.

[0096] 1. Cytotoxicity assay.

[0097] Using CCK-8 (Cell Counting Kit-8) kit for detection, in the presence of electron coupling reagent, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) can be reduced by dehydrogenase in mitochondria to generate highly water-soluble orange-yellow formazan products. The depth of its color is positively correlated with cell proliferation and negatively correlated with cytotoxicity. For the same cells, the depth of color is linearly related to the number of cells. Using a microplate reader to measure the OD value at a wavelength of 450 nm can indirectly reflect the number of living cells.

[0098] Hela cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator at 5% CO2 and 37°C. When the cells were in good condition, the cells in the logarithmic growth phase were resuspended in medium containing serum but not double antibody (penicillin-streptomycin mixture) and incubated at 5×10 4 Cells were seeded into 96-well plates at a density of 100 cells / well and incubated overnight in a cell culture incubator at 5% CO2 and 37°C. After the cells adhered, they were incubated with different concentrations of peptides at 37°C for 48 hours. Then 10 μl of Cell Counting Kit-8 solution was added to each well. After incubation for 2 hours, the absorbance at 450 nm was measured at 25°C using a microplate reader. Six replicates were performed for each sample, and the results are shown in Table 1. Fig.17 As shown, the results showed that with the increase of polypeptide concentration, the death rate of Hela cells gradually increased, but the toxicity of the polypeptide to Hela cells was generally small.

[0099] 2. The antisense effect of the peptide-siRNA complex inhibits the expression of fluorescent protein.

[0100] The principle of siRNA's gene silencing effect is as follows: double-stranded siRNA molecules are assembled into RNA-induced silencing complexes (RISC) in the cytoplasm, followed by degradation of the sense strand in the siRNA, and the antisense strand (i.e., guide strand) guides RISC to recognize and bind to the corresponding site of the mRNA that is complementary to its base, thereby cleaving the target mRNA through the ribonuclease II in the polypeptide-siRNA, thereby achieving the purpose of hindering the translation of specific genes and regulating the expression of target genes.

[0101] The specific operation process is as follows: Hela cells containing the green fluorescent protein (GFP) gene were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator at 5% CO2 and 37°C until the logarithmic growth phase. When the cells were in good condition, the cells were resuspended in DMEM medium and incubated at 5×10 4 The cells were inoculated into a 12-well plate at a density of 100 μL / well and incubated overnight in a cell culture incubator at 5% CO2 and 37°C. Transfection was performed when the cell density was about 70%-80%. Before transfection, the polypeptide-siRNA complex was obtained according to the method in Example 2, and diluted with Opti-MEM medium at 500 μL / well, the medium was removed, and the cells were washed twice with PBS (1×, pH 7.4), and then the diluted polypeptide-siRNA complex was added to each well.

[0102] After 4 h of incubation, the polypeptide-nucleic acid complex was replaced with DMEM medium containing 10% fetal bovine serum and incubated for another 48 h. The culture was observed under an inverted fluorescence microscope. Fig.18 As shown, compared with the control group, the fluorescence of the experimental groups was weakened to varying degrees.

[0103] qPCR quantitative detection of gene silencing efficiency: Extract total mRNA from Hela cells containing the green fluorescent protein (GFP) gene after transfection. Take an appropriate amount of cells and add 1mL TRIZOL reagent, shake at room temperature for 3-5min and let stand for 10min. Add 250μL chloroform (trichloromethane), shake until it turns pink, and let stand at room temperature for 15min. Aspirate the supernatant, add 500μL isopropanol to the supernatant to precipitate RNA, shake and let stand at room temperature for 15min, and then centrifuge at 12000rpm at 4℃ for 15min. Discard the supernatant, keep the precipitate, add 75% ethanol diluted with enzyme-killing water to wash the RNA precipitate, and then centrifuge at 4℃ 10000rpm for 10min to obtain RNA. By mixing with Moloney murine leukemia virus reverse transcriptase, deoxynucleotide triphosphate (dNTP) and oligonucleotide (dT), equal amounts of RNA are reverse transcribed into complementary DNA (cDNA). Then, the silencing efficiency of the GFP gene was quantitatively obtained to be 85.6% by mixing primers, dNTPs, cDNA and DNA polymerase for polymerase chain reaction. Fig.19 shown.

[0104] 3. Experiment on knocking out GFP gene by delivering CRISPR / Cas9 gene editing components using peptide-nucleic acid complexes.

[0105] The principle of CRISPR / Cas9 gene editing using the sgRNA-mRNA format is to co-deliver sgRNA and mRNA that can translate Cas9 protein into target cells through a polypeptide-nucleic acid complex. The mRNA then translates the Cas9 protein. Under the guidance of the sgRNA, the gene editing component, i.e. the sgRNA-Cas9 protein complex, binds to the target gene in the genome. The gene editing component then precisely cuts the target gene. The cell then tries to repair the broken gene, thereby achieving precise editing of the target gene.

[0106] The specific operation process is as follows: Hela cells containing the green fluorescent protein (GFP) gene were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator at 5% CO2 and 37°C until the logarithmic growth phase. When the cells were in good condition, the cells were resuspended in DMEM medium and incubated at 5×10 5Cells were inoculated into 24-well plates at a density of 100 cells / well and incubated overnight in a cell culture incubator at 5% CO2 and 37°C. Transfection was performed when the cell density was about 70%-80%. Preparation of polypeptide-CRISPR / Cas9 complexes was prepared according to the method in Example 2. Before transfection, the culture medium was removed, washed twice with PBS (1×, pH 7.4), and then 500 μL of Opti-MEM culture medium containing the vector-nucleic acid complex was added to each well.

[0107] After 4 h of incubation, the polypeptide-nucleic acid complex was replaced with DMEM medium containing 10% fetal bovine serum and incubated for another 48 h. The culture was observed under an inverted fluorescence microscope. Fig.18 As shown, compared with the control group, the fluorescence of the experimental groups was weakened to varying degrees.

[0108] qPCR quantitative detection of gene silencing efficiency: Extract total mRNA from Hela cells containing the green fluorescent protein (GFP) gene after transfection. Take an appropriate amount of cells and add 1mL TRIZOL reagent, shake at room temperature for 3-5min and let stand for 10min. Add 250μL chloroform (trichloromethane), shake until it turns pink, and let stand at room temperature for 15min. Aspirate the supernatant, add 500μL isopropanol to the supernatant to precipitate RNA, shake and let stand at room temperature for 15min. Then centrifuge at 12000rpm for 15min at 4℃. Discard the supernatant, keep the precipitate, add 75% ethanol diluted with enzyme-killing water to wash the RNA precipitate, and then centrifuge at 4℃ 10000rpm for 10min to obtain RNA. Equal amounts of RNA are reverse transcribed into complementary DNA (cDNA) by mixing with Moloney murine leukemia virus reverse transcriptase, deoxynucleotide triphosphate (dNTP) and oligonucleotide (dT). Then, the polymerase chain reaction was performed by mixing primers, dNTPs, cDNA and DNA polymerase. The editing efficiency of the GFP gene was quantitatively determined to be 43.7%. Fig.19 shown.

[0109] Example 8 Delivery of polypeptide-nucleic acid complexes in tumor model mice.

[0110] 1. Evaluation of the in vivo hemolytic ability of peptide-nucleic acid complexes.

[0111] Fresh blood from mice was centrifuged at 1500rpm for 10min to obtain red blood cell pellets, which were resuspended and washed three times with PBS buffer and then diluted to a final concentration of 5% (v / v). 500μL of red blood cell dilution was added to TritonX-100 (control) and polypeptide carrier, respectively, and incubated at 37°C for 1 hour. The solution was then centrifuged for 10 minutes, 100μL was added to a 96-well plate, and the absorbance at 450nm was read using an ELISA reader, with the Triton X-100 well as the positive control and PBS as the negative control.

[0112] The results are as follows Fig. 20 , Fig.21 As shown in the figure, after adding Triton X-100 to the control group, obvious hemolysis occurred, that is, red blood cells ruptured and hemoglobin and other contents were released, causing the red blood cell suspension to appear bright red as a whole. In contrast, the color of the red blood cell suspension with the addition of the polypeptide sample was close to clear and transparent, and a clear boundary could be observed between the supernatant and the cell sediment at the bottom of the test tube, indicating that hemolysis almost did not occur. The results showed that the hemolysis rate caused by the polypeptide was less than 1%, while the hemolysis rate of the control group reached about 95%. This result shows that the polypeptide delivery system has good in vivo safety.

[0113] 2. In vitro mouse tumor transplantation experiment.

[0114] The specific process is as follows: Animal experiments were approved by the Ethics Committee of Tianjin International Joint Institute of Biomedicine and were in accordance with the Experimental Animal Law. BALB / c female nude mice (6 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). Forty mice were randomly divided into 8 groups and inoculated subcutaneously with 1×10 7 HeLa cells. The specific steps are: put the fresh tumor tissue into HTK (histidine-tryptophan-ketoglutarate solution) solution and quickly take it to the clean bench; put the tumor tissue into a 6cm culture dish filled with physiological saline and rinse it repeatedly 3-4 times, and change the water each time; put the tumor tissue into a 6cm culture dish filled with blank culture medium containing penicillin-streptomycin-amphotericin B, and rinse it repeatedly 3-4 times, and change the culture medium each time; press the tumor tissue into the blank culture medium with tweezers and cut it into pieces with scissors, generally cut into 1mm 3The fragments are moved to new culture medium for washing for the last time, and the fragments are picked up with tweezers and inserted into the puncture needle, and suction is continuously applied to allow multiple fragments to enter. The tumor-bearing mouse is wiped with alcohol cotton balls all over the body, picked up, and the flank is exposed. The hair is cut in the middle of the flank, and a small incision of 1 mm in length is made in the skin at the hair-cut site with scissors. The puncture needle is inserted into the subcutaneous tissue through the small incision, and the needle tip is moved forward to the forelimb or hind limb end, and the needle tip stops at the root of the upper limb or lower limb, and the tissue block is injected, and the needle is rotated to pull out. A cotton swab is used to push and rub the skin in the injection direction to concentrate the tissue fragments as much as possible at the injection site. The injection site is wiped dry with a sterilized cotton ball, and the mouse is put down to allow it to move freely. The tumor growth of the mouse is measured every 2 days thereafter, and the tumor volume is calculated according to the following formula: V=L×W 2 ×0.5; L and W are the longest and shortest diameters of the tumor, respectively, and V is the volume of the tumor; When the tumor volume reaches 50-100 mm 3 Then, treatment is started according to the subsequent part of this embodiment.

[0115] 3. Distribution of polypeptide-mRNA complex in vivo.

[0116] The screened peptides were used to deliver non-translated Cy5-labeled mRNA to study its biodistribution in vivo. Fig. 22 As shown, 20 mice were randomly divided into 4 groups, with 5 mice in each group. When the tumor volume reached 50-100 mm 3 At the same time, physiological saline, formula R5, formula R23, formula R40 and liposome 2000 were subcutaneously injected into the abdominal cavity. Eight hours after administration, it was observed that the luminescence intensity of the tumor site of mice in the three-component R40 group was the highest. R5 and R23 lacked the Z peptide module, and their stability in the body decreased, so these two groups of mice had only weak fluorescence at the tumor site. Since Lipofectamine2000 has no targeting, its fluorescence is not only enriched in the tumor site, but also scattered in the abdomen.

[0117] 4. Detection of in vivo delivery and expression of polypeptide-mRNA complexes.

[0118] After observing the accumulation of the peptide delivery system at the target tumor site, the ability of the peptide delivery system to deliver mRNA in vivo was evaluated by using it to encapsulate FLuc mRNA and deliver it. Fig.23 As shown. 20 mice were randomly divided into 4 groups, 5 mice in each group. When the tumor volume reached 50-100 mm 3At the same time, normal saline, formula R5, formula R23, formula R40 and Lipofectamine2000 were injected through the tail vein respectively. 24 hours after administration, the results of whole-body luminescence imaging showed that the expression of luciferase in the tumor site of mice in the R40 group was the highest, while the expression of luciferase in the tumor site of mice in the R5 and R23 groups was weaker. At the same time, in addition to the tumor site, luciferase was also expressed in the abdomen of mice in the Lipofectamine2000 group, which was consistent with the above-mentioned biodistribution results.

[0119] 48 hours after tail vein administration, the mice in the R40 group and the Lipofectamine2000 group were dissected and their organs were removed. A fluorescent imager was used to observe the residual status of the target protein expressed by mRNA in various organs in the body. The results are as follows Fig.24 As shown, the results show that the mRNA translation protein of the R40 group is only expressed in the target tumor site, while the mRNA translation protein of the Lipofectamine2000 group is also expressed in organs such as the liver, spleen, and kidney, indicating that the non-targeted delivery system will randomly deliver the drug to various organs, which may cause immunogenicity or renal toxicity. These results show that the final formula obtained through peptide screening can target the delivery of mRNA to the target site and has a strong ability to express the target protein.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide with high transfection efficiency, characterized in that The polypeptide comprises five peptide modules, A, X, B, C and Y, wherein the five peptide modules A, X, B, C and Y are connected in sequence through peptide bonds; The X peptide module is any one of the X1 peptide module and the X2 peptide module, and the Y peptide module is any one of the Y1 peptide module and the Y2 peptide module; Among them, the amino acid sequence of the A peptide module is KKK, the amino acid sequence of the X1 peptide module is GFLG, the amino acid sequence of the X2 peptide module is RRGK, the amino acid sequence of the B peptide module is LLL, the amino acid sequence of the C peptide module is HHH, the amino acid sequence of the Y1 peptide module is KKK, and the amino acid sequence of the Y2 peptide module is GYQTI; Among them, K is the abbreviation of lysine, G is the abbreviation of glycine, F is the abbreviation of phenylalanine, L is the abbreviation of leucine, R is the abbreviation of arginine, H is the abbreviation of histidine, Y is the abbreviation of tyrosine, Q is the abbreviation of glutamine, T is the abbreviation of threonine, and I is the abbreviation of isoleucine.

2. The polypeptide with high transfection efficiency according to claim 1, characterized in that Also included is a Z peptide module, wherein the Z peptide module is connected to the Y peptide module via a peptide bond; The amino acid sequence of the Z peptide module is shown in SEQ NO.

1.

3. A peptide library, characterized in that The peptide library comprises a plurality of polypeptides, and the polypeptides are the polypeptides with high transfection efficiency as described in claim 1 or 2.

4. A polypeptide-nucleic acid complex, characterized in that: A method comprising: comprising: comprising: providing a polypeptide with high transfection efficiency as claimed in claim 1 or 2, and further comprising a nucleic acid, wherein the polypeptide and the nucleic acid self-assemble to form a polypeptide-nucleic acid complex; During the self-assembly process of the polypeptide and the nucleic acid, the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid is 15:1 to 30:

1.

5. The polypeptide-nucleic acid complex according to claim 4, characterized in that The polypeptides include polypeptide I, polypeptide II and polypeptide III: The polypeptide I is: AX1BCY1 or AX2BCY1, the amino acid sequence of AX1BCY1 is shown in SEQ NO.2, and the amino acid sequence of AX2BCY1 is shown in SEQ NO.3; The polypeptide II is: AX1BCY2 or AX2BCY2, the amino acid sequence of AX1BCY2 is shown in SEQ NO.4, and the amino acid sequence of AX2BCY2 is shown in SEQ NO.5; The polypeptide III is: AX2BCY2Z amino acid sequence is shown in SEQ NO.

6.

6. The polypeptide-nucleic acid complex according to claim 5, characterized in that The molar ratio of the polypeptide I, the polypeptide II and the polypeptide III is (10-30): (70-90): (0.1-1); During the self-assembly process of the polypeptide and the nucleic acid, the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid is 15:1 to 25:

1.

7. The polypeptide-nucleic acid complex according to claim 5, characterized in that The molar ratio of the polypeptide I, the polypeptide II and the polypeptide III is (15-25): (75-85): (0.1-1); During the self-assembly process of the polypeptide and the nucleic acid, the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid is 15:1 to 20:

1.

8. The polypeptide-nucleic acid complex according to claim 4, characterized in that The nucleic acid includes at least one of random sequence DNA, plasmid DNA, mRNA, siRNA and Cas9-mRNA / sgRNA.

9. An application of a polypeptide-nucleic acid complex, characterized in that: The polypeptide-nucleic acid complex is the polypeptide-nucleic acid complex according to any one of claims 4 to 8, and the use includes use thereof in preparing a drug.

10. The use of the polypeptide-nucleic acid complex according to claim 9, characterized in that: The drugs include anti-tumor drugs.

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