A polypeptide, peptide library, polypeptide-nucleic acid complex with high transfection efficiency and their applications

Abstract

The present invention relates to the field of biomedical technologies, and particularly to a polypeptide, a peptide library, a polypeptide-nucleic acid complex with high transfection efficiency and their applications. 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 by peptide bonds; the X peptide module is any one of the X<subgt;1< / subgt> peptide module and the X<subgt;2< / subgt> peptide module, and the Y peptide module is any one of the Y<subgt;1< / subgt> peptide module and the Y<subgt;2< / subgt> peptide module; the peptide library comprises a plurality of the above-mentioned polypeptides, and this peptide library helps to quickly find carrier formulations for different nucleic acid drugs, so as to better guide the construction of a drug delivery carrier platform; the polypeptide-nucleic acid complex comprises a polypeptide and a nucleic acid, and in the process of forming the polypeptide-nucleic acid complex, the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid is 15:1 to 30:1, and this polypeptide-nucleic acid complex is expected to be applied to drug preparation.

Description

Technical Field

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

[0002] Compared with traditional drug therapies, gene drugs can control diseases fundamentally by replacing pathogenic genes or changing the gene structure of diseased cells, etc., so they have broad clinical application prospects. However, due to the unsatisfactory pharmacokinetic properties of gene drugs and the difficulty in successfully delivering them into the cytoplasmic matrix of target cells, the progress of their clinical applications is limited. It is expected to overcome the pharmacokinetic defects through drug delivery carriers.

[0003] Currently, the commonly used nucleic acid drug delivery methods include biological methods such as liposome delivery, direct injection of DNA solution or receptor-mediated transfection of nucleic acids. However, these methods have disadvantages such as low biocompatibility, low transfection efficiency, and poor targeting. Although traditional polypeptide-based nucleic acid carriers can overcome problems such as poor liposome biocompatibility to a certain extent, there are still limitations such as low transfection efficiency and the ability to 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. For this reason, 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 the polypeptide-nucleic acid complex.

[0005] In order to achieve the first object, the technical solution adopted by the present invention is as follows:

[0006] A polypeptide with high transfection efficiency, the polypeptide includes five peptide modules A, X, B, C, and Y, and the five peptide modules A, X, B, C, and Y are sequentially connected by peptide bonds;

[0007] 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;

[0008] Wherein, 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;

[0009] 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.

[0010] Furthermore, it also includes a Z-peptide module, and the Z-peptide module is connected to the Y-peptide module through a peptide bond;

[0011] The amino acid sequence of the Z-peptide module is shown as SEQ NO.1;

[0012] SEQ NO.1 is as follows:

[0013] FLGLSSSSSSSSSSSSSSSS;

[0014] Among them, S is the abbreviation of serine.

[0015] In order to achieve the second object, the technical solution adopted by the present invention is:

[0016] A peptide library, the peptide library includes a plurality of polypeptides, and the polypeptides are the polypeptides with high transfection efficiency as described above.

[0017] In order to achieve the third object, the technical solution adopted by the present invention is:

[0018] A polypeptide-nucleic acid complex, including the polypeptide with high transfection efficiency as described above, and also including nucleic acid, and the polypeptide and the nucleic acid self-assemble to form a polypeptide-nucleic acid complex;

[0019] 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.

[0020] Furthermore, the polypeptide includes polypeptide I, polypeptide II and polypeptide III:

[0021] The polypeptide I is: AX1BCY1 or AX2BCY1, the amino acid sequence of AX1BCY1 is shown as SEQ NO.2, and the amino acid sequence list of AX2BCY1 is shown as SEQ NO.3;

[0022] The polypeptide II is: AX1BCY2 or AX2BCY2, the amino acid sequence of AX1BCY2 is shown as SEQ NO.4, and the amino acid sequence of AX2BCY2 is shown as SEQ NO.5;

[0023] The polypeptide III is: The amino acid sequence of AX2BCY2Z is shown as SEQ NO.6.

[0024] Furthermore, the molar ratio of the polypeptide I, the polypeptide II, and the polypeptide III is (10-30):(70-90):(0.1-1);

[0025] 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 - 25:1.

[0026] Furthermore, the molar ratio of the polypeptide I, the polypeptide II, and the polypeptide III is (15-25):(75-85):(0.1-1);

[0027] 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 - 20:1.

[0028] Furthermore, the nucleic acid includes at least one of random sequence DNA, plasmid DNA, mRNA, siRNA, and Cas9-mRNA / sgRNA.

[0029] To achieve the fourth object, the technical solution adopted by the present invention is:

[0030] An application of a polypeptide-nucleic acid complex, the polypeptide-nucleic acid complex being the polypeptide-nucleic acid complex as described in any one of the above, and the application including its application in the preparation of drugs.

[0031] Furthermore, the drug includes an anti-tumor drug.

[0032] One or more of the above technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0033] The present invention designs a polypeptide composed of peptide modules with different functions. The polypeptide is a polypeptide with high transfection efficiency. A library is formed by polypeptides composed of multiple different functional peptide modules, and a high-throughput screening platform for polypeptide carriers is established. Using this screening platform, the present invention has found an efficient delivery peptide library for siRNA to mRNA, plasmid, and even the CRISPR / Cas9 system, and has been successfully applied to in vivo administration. For the polypeptide with high transfection efficiency provided by the present invention, the peptide modules of its composition utilize the synergistic effects of enzyme response, lysosomal membrane targeting, and proton sponge effect to enhance the lysosomal escape ability of the polypeptide. Compared with traditional delivery carriers, the biocompatibility and delivery efficiency are significantly improved.

[0034] 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 enabling better guidance for the construction of drug delivery carrier platforms and more effective development of polypeptide-nucleic acid complexes with specific properties and functions. This polypeptide-nucleic acid complex is expected to play an important role in fields such as drug delivery and biomedical engineering.

[0035] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a transfection heat effect diagram of different polypeptide-nucleic acid complexes provided in Example 3 of the present invention.

[0037] Figure 2 is a transfection heat effect diagram of different polypeptide-nucleic acid complexes provided in Example 3 of the present invention.

[0038] Figure 3 is a transfection heat effect diagram of different polypeptide-nucleic acid complexes provided in Example 3 of the present invention.

[0039] Figure 4 is a circular dichroism spectrum diagram of different polypeptides and polypeptide-nucleic acid complexes provided in Example 4 of the present invention.

[0040] Figure 5 is a transmission electron microscopy morphology diagram of six different polypeptide-DNA complexes provided in Example 4 of the present invention.

[0041] Figure 6 is a ζ-potential column diagram of different polypeptides and polypeptide-DNA complexes provided in Example 4 of the present invention.

[0042] Figure 7 is a particle size distribution diagram of different polypeptide-DNA complexes provided in Example 4 of the present invention.

[0043] Figure 8 is an infrared spectrum diagram of different polypeptides provided in Example 4 of the present invention.

[0044] Figure 9 is an analysis diagram of the assembly process K of different polypeptide-nucleic acid complexes provided in Example 5 of the present invention; 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. 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.

[0045] Figure 10It is a confocal image of the cellular uptake process of different polypeptide-siRNA complexes provided in Example 6 of the present invention.

[0046] Figure 11 It is a microscopic image of Hela cells taking up the polypeptide (AX1BCY1)-siRNA complex provided in Example 6 of the present invention.

[0047] Figure 12 It is a microscopic image of Hela cells taking up the polypeptide (AX2BCY1)-siRNA complex provided in Example 6 of the present invention.

[0048] Figure 13 It is a bar graph of the flow cytometry efficiency of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention.

[0049] Figure 14 It is a fluorescence microscopic image of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention.

[0050] Figure 15 It is a flow cytometry result graph of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention; among them, A is the flow cytometry result graph of the polypeptide-nucleic acid complex of formulation R39, and B is the flow cytometry result graph of the polypeptide-nucleic acid complex of formulation R40.

[0051] Figure 16 It is a bar graph of the flow cytometry efficiency of different polypeptide-nucleic acid complexes provided in Example 6 of the present invention.

[0052] Figure 17 It is a statistical graph of the activity of Hela cells after treatment with different polypeptides provided in Example 7 of the present invention.

[0053] Figure 18 It is a fluorescence microscopic image of the polypeptide-siRNA complex for GFP gene silencing and gene editing of knocking out the GFP gene by the CRISPR / Cas9 system provided in Example 7 of the present invention.

[0054] Figure 19 It is a bar graph of the efficiency of the polypeptide-siRNA complex for GFP gene silencing and gene editing of knocking out the GFP gene by the CRISPR / Cas9 system provided in Example 7 of the present invention.

[0055] Figure 20 It is a graph of the hemolysis experiment results of different polypeptide-nucleic acid complexes provided in Example 8 of the present invention.

[0056] Figure 21 It is a bar graph of the hemolysis ratio of different polypeptide-nucleic acid complexes provided in Example 8 of the present invention.

[0057] Figure 22These are the typical whole-body fluorescence images of each group after 8 hours of intravenous injection of the polypeptide-Cy5 mRNA complex into mice provided in Example 8 of the present invention.

[0058] Figure 23 These are the typical whole-body bioluminescence images of each group after 24 hours of intravenous injection of the polypeptide-Fluc mRNA complex into mice provided in Example 8 of the present invention.

[0059] Figure 24 These are the ex vivo surface fluorescence images of each organ of mice after 48 hours of intravenous injection of the polypeptide-Fluc mRNA complex into mice provided in Example 8 of the present invention. Detailed implementation manners

[0060] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope 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.

[0061] In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0062] A polypeptide with high transfection efficiency, the polypeptide comprises five peptide modules A, X, B, C and Y, and the five peptide modules A, X, B, C and Y are sequentially connected by peptide bonds;

[0063] 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;

[0064] Wherein, 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;

[0065] Wherein, 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.

[0066] Preferably, it further comprises a Z peptide module, and the Z peptide module is connected to the Y peptide module by a peptide bond;

[0067] The amino acid sequence of the Z peptide module is shown in SEQ ID NO.1.

[0068] SEQ ID NO.1 is as follows:

[0069] FLGLSSSSSSSSSSSSSSSS;

[0070] Among them, S is the abbreviation of serine.

[0071] The peptide module composition, source and purity of different polypeptides are shown in Table 1, and the specific amino acid sequence compositions of different polypeptides are shown in Table 2.

[0072] Table 1 Structural composition, purity and source of different polypeptides

[0073]

[0074] Table 2 Amino acid sequence compositions of different polypeptides

[0075]

[0076] When preparing a single-component polypeptide sample, first dilute the DNA / RNA with water, and then add it to the polypeptide solution and vortex mix. When a multi-component polypeptide self-assembles with the DNA / RNA complex, first dilute the DNA / RNA with water, then add it to the premixed peptide solution, and vortex mix according to the calculated ratio. Before further structural characterization, the samples are usually incubated at room temperature for 30 minutes. If not specified, the DNA / RNA concentration is fixed at 50 μM, and the polypeptide-nucleic acid complex with the best N / P ratio is selected for related experiments.

[0077] Example 1 Preparation of a polypeptide with high transfection efficiency.

[0078] A polypeptide with high transfection efficiency, characterized by comprising the aforementioned six different functional peptide modules. In order to fully exert the effects of different peptide modules, the six peptide modules are sequentially connected in the order of their amino acid sequences. The A peptide module that binds nucleic acid through self-assembly, the X peptide module that realizes enzymatic cleavage and changes the polypeptide structure in response to lysosomal enzymes, the B peptide module that perturbs the lysosomal membrane and enhances lysosomal escape after enzymatic cleavage, the C peptide module that contains stimulus-responsive residues to trigger the dissociation of the polypeptide carrier in response to environmental pH changes, the targeting Y peptide module that targets specific cells or tissues, and the Z peptide module that resists the 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 sub-modules, coded as X1, X2 and Y1, Y2, and the Z module can be selectively added in the amino acid sequence.

[0079] The polypeptide was prepared by Tianjin Beiyang Kangtai Biotechnology Co., Ltd. through solid-phase synthesis. Taking the synthesis of 50 mg of AX2BCY1 polypeptide as an example, the Fmoc synthesis method was adopted. The specific preparation process is as follows:

[0080] Weigh 200 mg of dry 1% divinylbenzene (DVB)-crosslinked CTC resin (200 - 400 mesh) (SD: 1.211 mmol / g) into a solid-phase synthesis tube, add N,N-dimethylformamide (DMF), and swell it by passing nitrogen for 30 min. Take a small amount of resin for ninhydrin detection. In a test tube, add 2 - 3 drops of ninhydrin solution (solution A), phenol solution (solution B), and VC solution (solution C) in sequence. Take a small amount of resin into the test tube and heat it in an oil bath at 110 °C for 3 min, then observe the color change of the resin (purple / deep red / black - there are free amino acids on the resin and the Fmoc is successfully removed; yellow / blue - there are no free amino acids on the resin and the condensation is successful).

[0081] Amino acid coupling: Blow with nitrogen, pump dry with a vacuum pump. Then, under nitrogen protection, according to resin: Fmoc-amino acid: hydroxybenzotriazole (HXBT): N,N'-diisopropylcarbodiimide (DIC) = 1:3:4:4 (for the first amino acid, the reaction is carried out according to resin: Fmoc-amino: N,N-diisopropylethylamine = 1:2:8), dissolve in DMF, activate in an ice bath for 10 - 15 min, then pour it into the solid-phase synthesis tube, and carry out a dehydration condensation reaction at room temperature for 2 h. After the reaction, monitor the progress of the reaction by ninhydrin color development method. When the resin is colorless or yellow, it indicates that the condensation reaction is complete. Then add DMF to wash the resin 4 - 5 times and pump out the solvent.

[0082] Product capping: Add the capping solution dichloromethane (DCM): methanol: N,N-diisopropylethylamine = 17:2:1, 10 min each time, repeat 2 times, and then wash with DMF 5 times.

[0083] Remove the Fmoc protecting group: Add 20% piperidine-DMF solution to fully suspend the resin, blow with N2, pump dry, and remove the Fmoc protecting group. Then add DMF to wash the resin 4 - 5 times to remove the excess deprotection solution, pump out the solvent, and then carry out ninhydrin detection.

[0084] Repeat the processes of amino acid coupling and removing the Fmoc protecting group, and connect the amino acids in the operating sequence until the complete amino acid sequence is obtained.

[0085] Cleavage and excision of side chain protection: Wash the resin with DCM to remove DMF, repeat 2 times, 2 - 3 minutes each time. Then wash the resin with 100% methanol 3 times, 10 minutes each time, to fully shrink the resin. Filter by suction until the resin is dry. Add cleavage solution to the resin at a ratio of 10 - 15 mL / g (the amount can be appropriately increased according to the situation). (The composition of the cleavage solution is: water / triisopropylsilane (TIS) / trifluoroacetic acid (TFA) = 2.5 / 2.5 / 95, and it needs to be frozen at -20°C in advance before use.) Bubble nitrogen, and cleave for 2.5 hours (cleave at room temperature. If it is found that the cleavage solution volatilizes too fast during the process, new cleavage solution can be added). Then filter by suction, add a small amount of DCM to wash 2 times, 1 - 2 minutes each time, collect the filtrate. Rotavaporize the collected filtrate to remove most of the solvent. Drop the fully rotavaporized remaining solution into frozen ether at a volume ratio of 1:10 (operate in a centrifuge tube). Stir continuously with a glass rod during the dropping process to disperse the precipitated peptide as much as possible and avoid agglomeration. Then centrifuge at 3000 r / min for 3 minutes, discard the supernatant, and then filter by suction to remove ether. Discard the filtrate to obtain the crude peptide solid, and vacuum dry to obtain the dried crude peptide product.

[0086] Purification of polypeptide: Purify by preparative liquid chromatography (HPLC purification), concentrate and freeze-dry to obtain the pure polypeptide product.

[0087] Example 2 Preparation of polypeptide-nucleic acid complex.

[0088] 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.

[0089] A method for preparing a polypeptide-nucleic acid complex using a functional polypeptide with high transfection efficiency. The nucleic acids used include five common types of nucleic acid molecules, namely:

[0090] Random sequence DNA primer (length 100 bp, nonsense sequence);

[0091] Plasmid DNA (plasmid DNA capable of expressing GFP fluorescent protein);

[0092] mRNA (mRNA capable of expressing GFP fluorescent protein);

[0093] siRNA (siRNA for silencing the GFP fluorescent protein gene);

[0094] Cas9-mRNA / sgRNA (CRISPR / Cas9 nucleic acid drug capable of editing the GFP fluorescent protein gene).

[0095] The preparation process of the polypeptide-nucleic acid complex is as follows:

[0096] I. Preparation of polypeptide - mRNA complex.

[0097] Thaw the EGFP - mRNA purchased from APE×BIO at room temperature, centrifuge it at 3000 rpm for 10 s, slowly open it and then pipette to mix evenly to obtain the mRNA solution for standby.

[0098] Add the polypeptide prepared in Example 1 in different amounts into the buffer solution, pipette to mix thoroughly, and dissolve to obtain a polypeptide solution with a concentration of 1 mg / mL.

[0099] Quickly add the mRNA nucleic acid solution into 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) is 3, and incubate at room temperature for 30 minutes to obtain the polypeptide - mRNA complex.

[0100] Use N / P molar ratios of 3, 5, 10, 15, 20, 25, 30, 35, 40 respectively to replace the N / P molar ratio of 3 in this example. Additionally, use polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, AX2BCY2Z respectively for preparation, and the others are the same as this example to obtain different polypeptide - mRNA complexes respectively.

[0101] II. Preparation of polypeptide - DNA complex.

[0102] Centrifuge 1 OD of 100bp random - sequence DNA purchased from Ascent Scientific at 3000 rpm for 10 s, slowly open it and then add 33 ul of ultrapure water and oscillate to dissolve to obtain the solution for standby.

[0103] Add the polypeptide prepared in Example 1 in different amounts into the buffer solution, pipette to mix thoroughly, and dissolve to obtain a polypeptide solution with a concentration of 1 mg / mL.

[0104] Quickly add the DNA nucleic acid solution into 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) is 3, and incubate at room temperature for 30 minutes to obtain the polypeptide - DNA complex.

[0105] Use N / P molar ratios of 3, 5, 10, 15, 20, 25, 30, 35, 40 respectively to replace the N / P molar ratio of 3 in this example. Additionally, use polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, AX2BCY2Z respectively for preparation, and the others are the same as this example to obtain different polypeptide - DNA complexes respectively.

[0106] In addition, when preparing the polypeptide-plasmid DNA complex, take the EGFP-pDNA with a concentration of 1 mg / mL purchased from Yunzhou Biotech. After thawing it at room temperature, centrifuge it at 3000 rpm for 10 s to obtain the pDNA solution for standby, and carry out subsequent preparation according to the polypeptide-DNA complex preparation method.

[0107] III. Preparation of the polypeptide-siRNA complex.

[0108] Centrifuge 1 OD of siRNA purchased from Suzhou GenePharma Co., Ltd. at 3000 rpm for 10 s. Slowly open it and add 75 ul of ultrapure water, then oscillate to dissolve to obtain the solution for standby.

[0109] Add the polypeptide prepared in Example 1 in different amounts to the buffer solution, and pipette and blow to mix evenly to dissolve and obtain a polypeptide solution with a concentration of 1 mg / mL.

[0110] Quickly add the siRNA solution to the polypeptide solution so that the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid (N / P molar ratio) is 3, and incubate at room temperature for 30 minutes to obtain the polypeptide-siRNA complex.

[0111] Use N / P molar ratios of 3, 5, 10, 15, 20, 25, 30, 35, 40 respectively to replace the N / P molar ratio of 3 in this example. In addition, use polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, AX2BCY2Z respectively for preparation, and the others are the same as this example to obtain different polypeptide-siRNA complexes respectively.

[0112] IV. Preparation of the polypeptide-CRISPR / Cas9 complex.

[0113] Thaw the Cas9-mRNA purchased from APE×BIO and the sgRNA purchased from AnShengDa Technology at room temperature, and centrifuge at 3000 rpm for 10 s. Slowly open it and use a pipette to blow and mix evenly to obtain the Cas9-mRNA / sgRNA solution for standby.

[0114] Add the polypeptide prepared in Example 1 in different amounts to the buffer solution, and pipette and blow to mix evenly to dissolve and obtain a polypeptide solution with a concentration of 1 mg / mL.

[0115] Quickly add the Cas9-mRNA / sgRNA nucleic acid solution to the polypeptide solution so that the molar ratio of the protonated nitrogen of the polypeptide to the phosphate group of the nucleic acid (N / P molar ratio) is 3, and control the molar ratio of Cas9-mRNA to sgRNA in the mixed solution to be 3:1, and incubate at room temperature for 30 minutes to obtain the polypeptide-nucleic acid complex.

[0116] Respectively, the N / P molar ratios are 3, 5, 10, 15, 20, 25, 30, 35, 40; replacing the N / P molar ratio of 3 in this example, and respectively using polypeptides AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, AX2BCY2Z for preparation, with other conditions the same as this example, and different polypeptide-CRISPR / Cas9 complexes are obtained respectively.

[0117] Example 3 Formulation screening of polypeptide-nucleic acid complexes.

[0118] Based on the functional polypeptides obtained in Example 1 and the polypeptides formed by combining 5 different peptide modules therefrom: AX1BCY1, AX1BCY2, AX2BCY1, AX2BCY2, and AX2BCY2Z, the present invention designed a total of 101 combination methods for the polypeptide-complex, as shown in Tables 3 to 7, and screened these 101 combination methods by means of high-throughput screening.

[0119] Table 3 Composition of polypeptide-nucleic acid complexes

[0120]

[0121] Table 4 Composition of polypeptide-nucleic acid complexes

[0122]

[0123] Table 5 Composition of polypeptide-nucleic acid complexes

[0124]

[0125]

[0126] Table 6 Composition of polypeptide-nucleic acid complexes

[0127]

[0128] Table 7 Composition of polypeptide-nucleic acid complexes

[0129]

[0130] The present invention selected four commonly used nucleic acids for delivery, namely mRNA and pDNA capable of expressing GFP fluorescent protein, siRNA for silencing the GFP fluorescent protein gene, and CRISPR / Cas9 nucleic acid drug (Cas9-mRNA and sgRNA) capable of editing the GFP fluorescent protein gene, tested the transfection effects of polypeptides composed of different functional peptide modules on the four nucleic acids, and obtained a transfection effect heat map as Figure 1 、 Figure 2 、 Figure 3As shown, where ① represents polypeptide AX1BCY1, ② represents polypeptide AX2BCY1, ③ represents polypeptide AX1BCY2, and ④ represents polypeptide AX2BCY2. It can be seen from the heat map 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. As Figure 1 shown; when the molar ratio of the Y2 peptide module in the mixed polypeptide is 20%, the transfection effect is significantly improved, and this improvement is reflected to varying degrees when transfecting mRNA, pDNA, siRNA, and the Cas9 component. This indicates that the optimal molar ratio of Y2 is 20%. As Figure 2 shown; when the content of the Z peptide module reaches 20%, the delivery effect significantly decreases, only 85.88% of the original. However, when the proportion of the Z peptide module is less than 1%, the nucleic acid transfection effect is hardly affected, indicating that appropriate addition of the Z peptide module does not affect the delivery efficiency of the peptide carrier. As Figure 3 shown.

[0131] During the self-assembly process of the polypeptide and the nucleic acid molecule, the component composition of the polypeptide-nucleic acid complex with better transfection effect is shown in Table 8.

[0132] Table 8 Composition of the polypeptide-nucleic acid complex

[0133]

[0134] Example 4 Characterization of the polypeptide and the polypeptide-nucleic acid complex.

[0135] Characterization of a polypeptide-nucleic acid complex, taking the polypeptide-DNA complex as an example:

[0136] The circular dichroism spectra of the polypeptide and the polypeptide-DNA complex (referred to as the complex) were measured using a JASCO-810 circular dichroism spectrometer from JASCO Corporation. The optical path of the sample cell was 0.1 mm, the wavelength range was 185 - 350 nm, the scanning speed was 200 nm / min, the bandwidth was 1 nm, the test temperature was 25 °C, and the test was repeated three times and the average value was output in the device control software. As Figure 4 shown, it can be clearly seen from the figure that in a neutral environment, the results of the self-assembly of the two single polypeptides AX1BCY1 and AX1BCY2 mainly show the characteristics of β-sheets, and the formed polypeptide-DNA complex also shows the characteristics of β-sheets. While the two polypeptides AX2BCY1 and AX2BCY2 and the polypeptide-DNA complex show a secondary structure of α-helix.

[0137] The morphological structures of the complexes formed by the self-assembly of the following six polypeptides and nucleic acids were studied using cryogenic transmission electron microscopy:

[0138] AX2BCY1, AX2BCY1 + AX2BCY2, AX2BCY1 + AX2BCY2 + AX2BCY2Z, AX1BCY1, AX1BCY1 + AX1BCY2, and AX1BCY1 + AX1BCY2 + AX2BCY2Z.

[0139] The complex solution (10 μL) was deposited on the surface of a copper grid. After staying for 3 min, it was blotted with filter paper for 1 - 2 s. Subsequently, it was negatively stained twice with 10 μL of 1% phosphotungstic acid solution, staying for 30 s and 10 s respectively. During the test, the copper grid was installed in a cryo-sample holder and transferred to a JEM-1400 Flash cryo transmission electron microscope under cryogenic conditions. The results are as Figure 5 shown. From Figure 5 it can be seen that the polypeptide-nucleic acid complex presents a spherical nanoparticle structure. With the increase in the types of polypeptides, the particle size of the polypeptide-DNA complex nanoparticles increases, but the formed nanoparticles are more uniform, and it is less likely for the particles to aggregate, which is beneficial for the carrier to maintain a stable state in the in vivo environment.

[0140] Zeta potential and particle size analysis: Zeta potential and particle size were analyzed on a Zeta Plus (Brookhaven Instruments Corporation, USA). When preparing the samples, polypeptide carrier samples with different peptide modules were prepared according to different composition ratios. The electrode was immersed in the samples, and 1.7 mL of newly prepared samples with a concentration of 300 μM were directly analyzed. Each sample was run ten times, with ten cycles each time. The results obtained from the ten cycles were averaged and then statistically analyzed for the ζ potential. The results are as Figure 6 and Figure 7 shown. It can be seen from the figure that the assembled polypeptide-DNA complex has a positive potential, which is beneficial for the cell uptake of nucleic acid drugs, and the particle size of the polypeptide-DNA complex is about 200 nm, which is easily taken up by cells.

[0141] In addition, Fourier transform infrared spectroscopy (FTIR) was used to characterize the secondary structures of the polypeptides and the polypeptide-DNA complexes. The FTIR spectra of the samples were collected 16 times on average on a Bruker Vertex-70 Spectrometer (USA) with a resolution of 4 cm -1 and a range of 4000 - 400 cm -1 . When preparing the samples, the samples were fully frozen in liquid nitrogen and then freeze-dried in a vacuum freeze dryer for 48 h to obtain dry polypeptide powders. The dry polypeptide powders were mixed with KBr at a ratio of 1:100 (w / w) and ground into fine powders with a mortar. The samples were made into tablets by hydraulic pressing. The blank KBr tablet was scanned as the background, and the scanned sample spectra needed to subtract the blank KBr background. The scanning results are as Figure 8 shown. For the range of 1600 - 1800 cm in the figure-1 Analysis was performed on the peaks. The anti-β-sheet structure is dominant in the AX1BCY1 and AX2BCY2 polypeptides, accounting for 35% and 90.1% respectively. The α-helix structure is dominant in the AX2BCY1 and AX2BCY2 polypeptides, accounting for 83.5% and 60.1% respectively, which is basically consistent with the structure obtained by circular dichroism.

[0142] Example 5 Kinetic characteristics of the polypeptide-nucleic acid complex.

[0143] The process for determining the kinetic characteristics of a polypeptide-DNA complex is as follows:

[0144] All kinetic measurements were carried out in PBS at room temperature and under light-shielded conditions during the test. For each determination of k on experiment, first, the fluorescence emission of an 800 μL solution containing Atto488-labeled DNA (10 nM) was measured at 515 nM (excitation at 488 nM), and this value was used as the baseline at t = 0. Subsequently, a small amount of concentrated peptide solution was gradually added, controlling the final concentration of the polypeptide to reach 25 - 125 nM, and the decrease in its fluorescence emission intensity over time was monitored at 10-second intervals. Finally, the curve of the emission intensity versus time was fitted to a single-exponential function y = a*exp(-x / t) + y0 to calculate the k corresponding to each polypeptide concentration obs and then a curve of k obs versus the polypeptide concentration was plotted, and k was obtained from the slope of its linear fit on .

[0145] To determine k off , 800 μL of a solution containing Atto488-labeled DNA (10 nM) and a polypeptide solution (5 nM) were co-incubated until no further change in fluorescence was observed. Subsequently, the polypeptide-DNA complex was placed in an acidic buffer, and excess unlabeled ssDNA was used to displace 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 obtained data was fitted to a single-exponential function to calculate k off .

[0146] Dissociation constant K d was calculated using the association rate constant k on and the dissociation rate constant k off , and the results are as Figure 9 , and the results show that the polypeptide-DNA complex has nucleic acid binding-dissociation constants similar to those of viruses and has the ability to mimic viruses for nucleic acid delivery, which is consistent with its high transfection efficiency performance.

[0147] Example 6: Transfection of cells with polypeptides.

[0148] The polypeptide in this example is the polypeptide prepared in Example 2.

[0149] I. The treatment process of the cells used in the experiment is as follows:

[0150] Hela cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator with 5% CO2 at 37 °C until the logarithmic growth phase. When the cell state was good, the cells were resuspended with DMEM medium and seeded into a 12-well plate at a cell density of 5×10 4 cells / well, and incubated overnight in a cell culture incubator with 5% CO2 at 37 °C. When the cell density was about 70% - 80%, transfection was carried out. Before transfection, the medium was aspirated, and the cells were washed twice with PBS (1× phosphate buffered saline, pH 7.4), and then 500 μL of Opti-MEM (serum-free medium) was added to each well.

[0151] II. The process of testing the ability of the polypeptide-nucleic acid complex to escape from lysosomes is as follows:

[0152] The endocytosis and lysosomal escape process of the polypeptide-siRNA complex was observed by confocal microscopy. The siRNA labeled with fluorescein isothiocyanate (FITC) (green fluorescence);

[0153] siRNA sense strand nucleotide sequence: 3’-GCAUCGAGAAGUACGAGGATT-5’;

[0154] siRNA antisense strand nucleotide sequence: 3’-UCCUCGUACUUCUCGAUGCTT-5’;

[0155] The siRNA was co-cultured with Hela cells labeled with 4',6-diamidino-2-phenylindole (DAPI) for the nucleus and lysosomal red fluorescence probe (Lyso-Tracker Red) for lysosomes. At 4 hours, the positions of the polypeptide-siRNA complex and lysosomes in the cells were observed using a fluorescence microscope. The confocal microscopy images are as Figure 10As shown, it can be seen from the figure that after the polypeptide containing the X1 peptide module enters the cell, the level of green fluorescence produced is lower than that of the polypeptide containing the X2 peptide module. However, when the individual X1 peptide module or X2 peptide module enters the cell, it will be phagocytosed by lysosomes, and in the figure, the green fluorescence of the nucleic acid drug overlaps with the red fluorescence of the lysosomes. In the image of adding the polypeptide containing the Y2 peptide module, the composition of the polypeptide-nucleic acid complex is No. R23 in Table 6. It can be clearly seen that the green fluorescence label is released, more independent green labels appear in the image, and some of the red labels of the lysosomes become blurred, indicating that the lysosomes rupture; the polypeptide containing the Z peptide module hardly affects the lysosome escape ability, and the composition of the polypeptide-nucleic acid complex is No. R48 in Table 7.

[0156] III. The method for measuring the uptake rate of the polypeptide-siRNA complex is as follows:

[0157] 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 in the dark at room temperature for 30 min. Respectively take the complex of green fluorescence-labeled siRNA and polypeptide, dilute it with nuclease-free water to a final volume of 100 µL, add it to the culture well containing 500 µL of Opti-MEM medium, gently shake the cell culture plate to fully mix the complex of green fluorescence-labeled siRNA and polypeptide with the medium in the well. After 4 h, observe under an inverted fluorescence microscope (Model KQ-250E, Olympus Corporation, Japan), and the results are as Figure 11 and Figure 12 shown. The results show that the cell uptake efficiency of the polypeptide-siRNA complex is the highest when the nitrogen-to-phosphorus ratio is about 20.

[0158] IV. The process of measuring the uptake efficiency by flow cytometry is as follows:

[0159] Set a blank group and an experimental group. The cells in the blank group were not treated, and the cells in the experimental group were respectively taken up by the complexes with a molar ratio of protonated nitrogen to phosphate group (N / P) of 5, 10, 20, 30, and 40. The transfection method was the same as the treatment process in III above. After washing three times repeatedly with PBS, cell treatment and counting were carried out, and the cells were resuspended with the cell washing solution, which was PBS containing 2% BSA, to make the cell concentration 1×10 7 / mL. Take 200 μL of the cell resuspension and detect the fluorescence intensity inside the cells with a flow cytometer. The results are as Figure 11 , Figure 12 and Figure 13 shown. The results of the bar chart are basically consistent with those shown in the microscope images.

[0160] V. The transfection methods for mRNA and pDNA are as follows:

[0161] According to the preparation method in Example 2, the molar ratios of protonated nitrogen to phosphate groups (N / P molar ratios) are 15, 20, and 25 respectively. Assembly is carried out at room temperature for 30 min to obtain polypeptide-mRNA complexes and polypeptide-DNA complexes. The polypeptide-nucleic acid complexes are diluted with nuclease-free water to a final volume of 100 µL and added to the culture wells containing 500 µL of Opti-MEM medium. The cell culture plate is gently shaken to fully mix the polypeptide-mRNA complexes and polypeptide-DNA complexes with the medium in the wells. After 4 h, observation is carried out under an inverted fluorescence microscope, and the results are as Figure 14 shown. The microscopic image results show that the nucleic acid drugs are successfully delivered into the cells and expressed.

[0162] VI. The process for measuring transfection efficiency by flow cytometry is as follows:

[0163] A blank group and experimental groups are set up. The cells in the blank group are not treated, and the cells in the experimental groups are transfected with complexes with N / P = 15, 20, and 25. The method is the same as the treatment process in item III of this example. After washing repeatedly with PBS three times, cell counting is carried out. The cells are resuspended with cell washing solution (PBS containing 2% BSA) to make the cell concentration 1×10 7 / mL. Take 200 μL of the cell resuspension and detect the fluorescence intensity inside the cells with a flow cytometer. The results are as Figure 15 and Figure 16 shown. The illustrated results show that the compositions of the polypeptide-nucleic acid complexes are No. R39 and R40 in Table 6, which are the best composition methods for transfecting plasmid DNA and mRNA respectively.

[0164] Example 7 Effects of polypeptide-nucleic acid complexes on specific genes of cells and mice.

[0165] I. Cytotoxicity experiment.

[0166] Detection is carried out using a CCK-8 (Cell Counting Kit-8) kit. In the presence of an electron coupling reagent, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) can be reduced by dehydrogenases in mitochondria to generate a highly water-soluble orange-yellow formazan product. 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. The OD value is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, which can indirectly reflect the number of live cells.

[0167] Hela cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixture) in a cell culture incubator with 5% CO2 at 37°C. After the cells were in good condition, the cells in the logarithmic growth phase were resuspended in medium containing serum but no double antibody (penicillin-streptomycin mixture), and inoculated into a 96-well plate at a cell density of 5×10 4 cells / well, and incubated overnight in a cell culture incubator with 5% CO2 at 37°C. After the cells adhered, they were incubated with different concentrations of polypeptides 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. Each sample was measured six times in duplicate, and the results are as Figure 17 shown. The results showed that as the concentration of the polypeptide increased, the mortality rate of Hela cells gradually increased, but the toxicity of the polypeptide to Hela cells was generally small.

[0168] II. The antisense effect of the polypeptide-siRNA complex inhibits the expression of fluorescent protein.

[0169] The principle of the gene silencing effect of siRNA is as follows: Double-stranded siRNA molecules are assembled into RNA-induced silencing complexes (RISC) in the cytoplasm. Immediately afterwards, the sense strand in the siRNA is degraded, and the antisense strand (i.e., the guide strand) guides RISC to recognize and bind to the corresponding site of the mRNA complementary to its base sequence, thereby cleaving the target mRNA by ribonuclease II in the polypeptide-siRNA, so as to achieve the purpose of hindering the translation of specific genes and regulating the expression of target genes.

[0170] The specific operation process is as follows:

[0171] 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 with 5% CO2 at 37°C until the logarithmic growth phase. When the cells were in good condition, the cells were resuspended in DMEM medium and inoculated into a 12-well plate at a cell density of 5×10 4 cells / well, and incubated overnight in a cell culture incubator with 5% CO2 at 37°C. When the cell density was about 70%-80%, transfection was carried out. 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 aspirated, washed twice with PBS (1×, pH 7.4), and then the diluted polypeptide-siRNA complex was added to each well.

[0172] After 4 h of incubation, the polypeptide-nucleic acid complex was replaced with DMEM medium containing 10% fetal bovine serum, and incubation was continued for 48 h. Then, observations were made under an inverted fluorescence microscope. The results were as Figure 18 shown. Compared with the control group, the fluorescence in the experimental group was weakened to varying degrees.

[0173] qPCR quantitative detection of gene silencing efficiency: Total mRNA of Hela cells transfected with the green fluorescent protein (GFP) gene was extracted. An appropriate amount of cells was added with 1 mL of TRIzol reagent, shaken at room temperature for 3 - 5 min, and then left standing for 10 min. 250 μL of chloroform was added, shaken to form a powdery white mixture, and left standing at room temperature for 15 min. The supernatant was aspirated, and 500 μL of isopropanol was added to the supernatant to precipitate RNA. After shaking and leaving standing at room temperature for 15 min, centrifugation was carried out at 12,000 rpm at 4°C for 15 min. The supernatant was discarded, and the precipitate was left. The RNA precipitate was washed with 75% ethanol diluted with enzyme-free water, and then centrifuged at 10,000 rpm at 4°C for 10 min to obtain RNA. By mixing with Moloney murine leukemia virus reverse transcriptase, deoxynucleotide triphosphate (dNTP), and oligonucleotide (dT), an equal amount of RNA was reverse transcribed into complementary DNA (cDNA). Then, polymerase chain reaction was carried out by mixing primers, dNTP, cDNA, and DNA polymerase. The silencing efficiency of the GFP gene was quantitatively determined to be 85.6%. The results were as Figure 19 shown.

[0174] III. Experiment on knocking out the GFP gene by delivering the CRISPR / Cas9 gene editing component with the polypeptide-nucleic acid complex.

[0175] The principle of CRISPR / Cas9 gene editing in the form of sgRNA-mRNA is that the sgRNA and the mRNA capable of translating the Cas9 protein are co-delivered into target cells through the polypeptide-nucleic acid complex. Subsequently, the mRNA translates the Cas9 protein. Under the guiding action of the sgRNA, the gene editing component, namely the sgRNA-Cas9 protein complex, binds to the target gene in the genome. Subsequently, the gene editing component precisely cuts the target gene, and then the cell will attempt to repair the broken gene, thus achieving precise editing of the target gene.

[0176] The specific operation process is as follows:

[0177] 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 cell state was good, the cells were resuspended with DMEM medium and seeded at 5×10 5Cells were seeded at a density of cells / well into a 24-well plate and incubated overnight in a cell culture incubator with 5% CO2 at 37 °C. Transfection was performed when the cell density reached approximately 70%-80%. The polypeptide-CRISPR / Cas9 complex was prepared according to the method described in Example 2. Before transfection, the culture medium was aspirated, and the cells were washed twice with PBS (1×, pH 7.4). Then, 500 μL of Opti-MEM medium containing the vector-nucleic acid complex was added to each well.

[0178] After incubation for 4 h, the polypeptide-nucleic acid complex was replaced with DMEM medium containing 10% fetal bovine serum, and incubation was continued for 48 h. Observation was performed under an inverted fluorescence microscope, and the results are shown in Figure 18 Figure [X], indicating that the fluorescence in the experimental group was weakened to varying degrees compared with the control group.

[0179] qPCR quantification of gene silencing efficiency: Total mRNA was extracted from transfected Hela cells containing the green fluorescent protein (GFP) gene. An appropriate amount of cells was added to 1 mL of TRIzol reagent, shaken at room temperature for 3-5 min, and then allowed to stand for 10 min. 250 μL of chloroform was added, and the mixture was shaken until it became powdery white and then allowed to stand at room temperature for 15 min. The supernatant was aspirated, and 500 μL of isopropanol was added to the supernatant to precipitate RNA. The mixture was shaken and allowed to stand at room temperature for 15 min. Then, it was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was discarded, and the precipitate was retained. The RNA precipitate was washed with 75% ethanol diluted with enzyme-free water and then centrifuged at 10,000 rpm for 10 min at 4 °C to obtain RNA. An equal amount of RNA was reverse transcribed into complementary DNA (cDNA) by mixing with Moloney murine leukemia virus reverse transcriptase, deoxynucleotide triphosphate (dNTP), and oligonucleotide (dT). Subsequently, polymerase chain reaction was performed by mixing primers, dNTP, cDNA, and DNA polymerase. The editing efficiency of the GFP gene was quantitatively determined to be 43.7%, and the results are shown in Figure 19 Figure [X].

[0180] Example [X] Delivery of the polypeptide-nucleic acid complex in tumor model mice.

[0181] I. Evaluation of the in vivo hemolytic ability of the polypeptide-nucleic acid complex.

[0182] Take fresh mouse blood, centrifuge it at 1500 rpm for 10 min to obtain red blood cell precipitate, resuspend it with PBS buffer and centrifuge and wash it three times, and then dilute it to a final concentration of 5% (v / v). Take 500 μL of the red blood cell diluent, add TritonX-100 (control) and the polypeptide carrier respectively, and incubate at 37 °C for 1 hour. Then centrifuge the solution for 10 minutes, take 100 μL and add it to a 96-well plate, and read the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader, using the Triton X-100 well as the positive control and PBS as the negative control.

[0183] The results are as Figure 20 , Figure 21 shown. After adding Triton X-100 to the control group, obvious hemolysis occurred, that is, the red blood cells ruptured and the contents such as hemoglobin were released, resulting in the whole red blood cell suspension showing bright red. In contrast, the color of the red blood cell suspension added with the polypeptide sample was close to clear and transparent, and a clear boundary could be observed between the supernatant and the cell precipitate at the bottom of the test tube, indicating that almost no hemolysis occurred. 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 indicates that the polypeptide delivery system has good in vivo safety.

[0184] II. In vitro mouse tumor transplantation experiment.

[0185] The specific process is as follows:

[0186] The animal experiment was approved by the Ethics Committee of Tianjin International Joint Academy of Biomedicine and complied with the "Laboratory 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 1×10 7 HeLa cells were subcutaneously inoculated at the right abdominal area near the axilla of the mice. The specific steps were as follows: Put the fresh tumor tissue into HTK (histidine-tryptophan-ketoglutarate solution), and quickly take it into the laminar flow hood; Put the tumor tissue into a 6 cm culture dish filled with physiological saline, and rinse it 3-4 times repeatedly, each time with fresh water; Put the tumor tissue into a 6 cm culture dish filled with blank medium containing penicillin-streptomycin-amphotericin B, and rinse it 3-4 times repeatedly, each time with fresh medium; Press the tumor tissue in the blank medium with forceps and cut it into pieces with scissors, generally cut into 1 mm 3Just take the fragments on both sides; transfer the fragments to a new culture medium for the last wash, pick up the fragments with forceps and stuff them into the puncture needle, and continuously aspirate to make multiple fragments enter; wipe the whole body of the tumor-bearing mouse with an alcohol cotton ball, pick it up, expose the lateral abdomen, clip the hair in the middle of the lateral abdomen, and use scissors to cut a small 1-mm-long incision in the skin at the clipped hair area; insert the puncture needle subcutaneously through the small incision, probe forward towards the forelimb or hindlimb end, stop when the tip of the needle enters the root of the upper limb or lower limb, inject the tissue mass, and pull out the needle while rotating; use a cotton swab to gently rub along the injection direction close to the skin to make the tissue fragments as concentrated as possible at the injection site; dry the injection site with a sterilized cotton ball, put down the mouse, let it move freely, and measure the tumor growth status of the mouse every 2 days thereafter, and calculate the tumor volume according to the following formula:

[0187] V = L×W 2 ×0.5;

[0188] L and W are the longest and shortest diameters of the tumor respectively, and V is the volume of the tumor;

[0189] When the tumor volume reaches 50 - 100 mm 3 start treatment according to the subsequent part of this example.

[0190] III. In vivo distribution of the polypeptide - mRNA complex.

[0191] Use the screened polypeptide to deliver the untranslated Cy5-labeled mRNA to study its biodistribution in vivo. The results are as Figure 22 shown. Randomly divide 20 mice into 4 groups with 5 mice in each group. When the tumor volume reaches 50 - 100 mm 3 inject normal saline, formulation R5, formulation R23, formulation R40, and Lipofectamine 2000 intraperitoneally and subcutaneously respectively. 8 hours after administration, the luminescence intensity at the tumor site of the mice in the R40 group of the three components was observed to be the highest. Due to the lack of the Z peptide module, the stability of R5 and R23 decreased in vivo, so there was only weak fluorescence at the tumor site of these two groups of mice. Since Lipofectamine 2000 has no targeting property, its fluorescence was scattered more in the abdomen in addition to being enriched at the tumor site.

[0192] IV. Detection of in vivo delivery and expression of the polypeptide - mRNA complex.

[0193] After observing the accumulation of the polypeptide delivery system at the target tumor site, evaluate the in vivo delivery ability of the polypeptide delivery system to mRNA by using it to encapsulate and deliver FLuc mRNA. The results are as Figure 23 shown. Randomly divide 20 mice into 4 groups with 5 mice in each group. When the tumor volume reaches 50 - 100 mm 3At that time, normal saline, formulation R5, formulation R23, formulation R40 and Lipofectamine 2000 were respectively injected via the tail vein. After 24 hours of administration, the whole-body luminescence imaging results showed that the luciferase expression in the tumor sites of the mice in the R40 group was the highest, while the luciferase expression in the tumor sites of the mice in the R5 group and the R23 group was weak. At the same time, in the mice in the Lipofectamine 2000 group, luciferase expression was also detected in the abdomen in addition to the tumor sites, which was consistent with the aforementioned biodistribution results.

[0194] 48 hours after tail vein administration, the mice in the R40 group and the mice in the Lipofectamine 2000 group were dissected and their organs were excised. A fluorescence imager was used to observe the residual situation of the target protein with mRNA expression in each organ in vivo. The results are as Figure 24 shown. These results indicate that the mRNA translation protein in the R40 group was only expressed in the target tumor sites, while the mRNA translation protein in the Lipofectamine 2000 group was also expressed in organs such as the liver, spleen and kidney, indicating that the non-targeted delivery system would randomly deliver the drug to each organ, which might cause immunogenicity or kidney toxicity. These results indicate that the final formulation obtained through polypeptide screening can target the delivery of mRNA to the target site and has a strong ability to express the target protein.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate 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 is selected from any one of the amino acid sequences of SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5 and SEQ NO.

6.

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

3. A polypeptide-nucleic acid complex, characterized in that, It includes the polypeptide with high transfection efficiency as described in claim 1, and also includes nucleic acid. The polypeptide and the nucleic acid self-assemble to form a polypeptide-nucleic acid complex; During the self-assembly 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.

4. The polypeptide-nucleic acid complex according to claim 3, wherein The polypeptide is a mixture composed of three polypeptides with high transfection efficiency. The three polypeptides with high transfection efficiency are respectively selected from one of SEQ NO.2 and SEQ NO.3, one of SEQ NO.4 and SEQ NO.5, and SEQ NO.

6.

5. The polypeptide-nucleic acid complex according to claim 4, wherein The molar ratio of SEQ NO.2 or SEQ NO.3, SEQ NO.4 or SEQ NO.5, and SEQ NO.6 is (10 - 30):(70 - 90):(0.1 - 1); During the self-assembly 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.

6. The polypeptide-nucleic acid complex according to claim 5, wherein, The molar ratio of SEQ NO.2 or SEQ NO.3, SEQ NO.4 or SEQ NO.5, and SEQ NO.6 is (15 - 25):(75 - 85):(0.1 - 1); During the self-assembly 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.

7. The polypeptide-nucleic acid complex according to claim 3, wherein The nucleic acid includes at least one of random sequence DNA, plasmid DNA, mRNA, siRNA and Cas9-mRNA / sgRNA.

8. Use of a polypeptide-nucleic acid complex, characterized in that, The polypeptide-nucleic acid complex is the polypeptide-nucleic acid complex as described in any one of claims 3 to 7, and the application is its application in the preparation of drugs.

9. Use of the polypeptide-nucleic acid complex according to claim 8, characterized in that, The drug includes anti-tumor drugs.

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