A polypeptide and its application

Abstract

The present invention discloses a polypeptide and its application. The polypeptide is the KW polypeptide with the sequence shown in SEQ ID No.1 or the KHW polypeptide with the sequence shown in SEQ ID No.2. In the polypeptide delivery system provided by the present invention, the polypeptide is composed of natural amino acids, can be biodegradable in cells, will not cause damage to target cells, and ensures the safety of drug administration; it can be wound with RNA through electrostatic and hydrogen bond interactions, so as to assemble into nanoparticles encapsulating RNA, with a high encapsulation rate; and it can carry multiple siRNAs with different targets at the same time, so as to produce a synergistic gene silencing effect in the same targeted cells and improve the drug efficacy of RNAi; it has a wide range of applications and can be used to encapsulate various drugs such as siRNA, mRNA, shRNA, microRNA, ASO or DNA.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide delivery systems, and particularly to a polypeptide and its applications. Background Art

[0002] The characteristics of branched polypeptides mainly include the following aspects: (1) They can wind with RNA through electrostatic and hydrogen bond interactions, thus assembling into nanoparticles encapsulating RNA, with a high encapsulation rate and a preference for the cell membrane interface; (2) They can selectively target a variety of tissues and cell types and can be used for local or systemic delivery; (3) They can carry multiple siRNAs with different targets simultaneously, enabling a synergistic gene silencing effect in the same targeted cells and improving the drug efficacy of RNAi; (4) They have a wide range of applications and can be used to encapsulate various drugs such as siRNA, miRNA, and mRNA; (5) They are composed of natural amino acids, can be biodegradable inside cells, and have lower toxicity; (6) They have good water solubility. Due to these characteristics, such polypeptide macromolecules have extensive potential applications in the biological and medical fields.

[0003] However, there are the following problems in the synthesis of branched polypeptides at present:

[0004] (1) There are few reported methods for the synthesis of branched polypeptides in China.

[0005] (2) In the reported solid-phase synthesis methods of polypeptides, when connecting the next amino acid during the synthesis process, the amino acid and the condensing agent are directly added to the polypeptide reaction tube, resulting in incomplete reactions and the generation of by-products.

[0006] (3) In the reported solid-phase synthesis methods of polypeptides, the purity is not high, and there is a problem of trifluoroacetic acid residue. If purified by HPLC, the purification efficiency is low and the yield loss is large.

[0007] (4) In the reported solid-phase synthesis methods of polypeptides, the finally obtained polypeptide contains water and needs to be dehydrated by freeze-drying technology, which is time-consuming.

[0008] A drug delivery system is a system that delivers drugs to the drug action target. Naked nucleic acid drugs are easily degraded by nucleases in plasma and tissues, or are rapidly filtered and cleared by the kidneys and excreted from the body, resulting in a very short circulation time of nucleic acid drugs in the body. A drug delivery system can usually improve the stability of drugs, reduce drug degradation, improve the bioavailability of drugs, and increase the drug concentration in the target area. Therefore, the delivery of nucleic acid drugs requires a suitable carrier to improve the delivery efficiency.

[0009] According to different delivery technologies, nucleic acid drug delivery platforms can be classified into: viral vector delivery systems, non-viral vector delivery systems (including lipid-based nanoparticles, GalNAc conjugate delivery systems, and peptide-based delivery systems), and other delivery systems including magnetic nanoparticle delivery, protoplast delivery, and physical methods based on ultrasound, electroporation, or magnetism, etc.

[0010] Peptide-based gene delivery systems have become a new type of non-viral vector. Compared with polymer and lipid carriers, they have advantages such as better biocompatibility, diverse design, easy synthesis, and lower risk of immune response. However, peptide delivery systems have become a research hotspot in the pharmaceutical field in recent years and are expected to provide new ideas for the precise treatment of various major diseases. A suitable peptide delivery system can encapsulate nucleic acids, protect nucleic acid drugs from degradation by nucleases, and at the same time deliver nucleic acid drugs into cells. The present invention aims to provide a safe and effective peptide nanoparticle system for the delivery of nucleic acid drugs. Summary of the Invention

[0011] In view of the deficiencies of the prior art, the present invention provides a peptide delivery system for encapsulating nucleic acid drugs and its application. The peptide delivery system is safe and effective, can effectively protect nucleic acid drugs, improve the stability of nucleic acid drugs, and at the same time has good transfection efficiency for cells and can normally release nucleic acid drugs in cells, having a wide application prospect in the field of nucleic acid drug delivery.

[0012] To solve the above technical problems, the technical solution of the present invention is as follows: A peptide delivery system for encapsulating nucleic acid drugs, wherein the peptide structure in the peptide delivery system is KW peptide, KHW peptide, KKK-KW peptide, or KKK-KHW peptide; the structural formula of the KKK-KW peptide or KKK-KHW peptide is as follows:

[0013]

[0014] In the formula, R is KW peptide or KHW peptide; the sequence of the KW peptide is KWHHHKWHHHKWHHHKWHHHK; the sequence of the KHW peptide is KHWHKHWHKHWWHKHWHK.

[0015] The present invention also provides a peptide, which is KW peptide or KHW peptide; the sequence of the KW peptide is as shown in SEQ ID No.1; the sequence of the KHW peptide is as shown in SEQ ID No.2.

[0016] Furthermore, the positively charged lysine and histidine in the peptide bind to the negatively charged nucleic acid drug through electrostatic interaction to form peptide nanoparticles; tryptophan in the peptide plays an anchoring role on the cell membrane.

[0017] Preferably, the nucleic acid drug is selected from siRNA, mRNA, shRNA, microRNA, ASO, DNA or plasmid. More preferably, the nucleic acid drug is selected from siRNA or mRNA. The polypeptide and siRNA form relatively non-smooth nanoparticles in a coiled manner, where lysine and histidine are positively charged and bind to the nucleic acid drug through electrostatic interaction, and tryptophan plays an anchoring role on the cell membrane.

[0018] Preferably, the molar ratio of the KW polypeptide to siRNA is 1-60:1-10; the molar ratio of the KKK-KW polypeptide to siRNA is 1-20:1-10; the mass ratio of the KKK-KW polypeptide to mRNA is 0.5-8:1.

[0019] Preferably, the molar ratio of the KHW polypeptide to siRNA is 1-20:20-1; the molar ratio of the KKK-KHW polypeptide to siRNA is 1-20:20-1; the mass ratio of the KKK-KHW polypeptide to mRNA is 0.5-8:1.

[0020] The present invention also provides a method for preparing the polypeptide delivery system for encapsulating a nucleic acid drug, and the method is operated as follows: first dissolve the polypeptide in water with a pH of 2-5 to prepare a polypeptide solution with a concentration of 0.5-5 mg / mL, mix the nucleic acid drug and the polypeptide according to the molar ratio and let it stand, and then add water or DMEM medium and let it stand for 5-25 min to obtain a polypeptide complex solution encapsulating the nucleic acid.

[0021] The present invention also provides the application of the polypeptide delivery system in siRNA transfection, siRNA gene silencing, mRNA vaccine or CRISPR / Cas9 gene editing. Preferably, it is applied in siRNA transfection and siRNA gene silencing, and the cells for transfection and gene silencing are selected from any one of Vero cells, HEK293 cells, 293T cells, 293A cells, MDCK cells, W2 cells, RPE cells, etc., and the tumor cells are selected from any one of breast cancer Hela cells, pancreatic cancer PANC-1 cells, liver cancer HepG2 cells, lung cancer A549 cells, etc.

[0022] The present invention also provides the application of the polypeptide delivery system in the preparation of drugs for preventing or treating infectious diseases, cancers or metabolic diseases.

[0023] In the polypeptide delivery system provided by the present invention, the polypeptide is composed of natural amino acids, can be biodegradable within cells, and will not cause unintentional damage to target cells, ensuring the safety of drug administration. There are two main domains in this polypeptide delivery system: one is the amino acid pairing domain responsible for polypeptide self-assembly, and the other is the cell-penetrating domain, which is a cell-penetrating functional group or a group of cationic amino acids. Lysine (K): It is a positively charged basic amino acid, and the amino group on the side chain can undergo dehydration condensation with the carboxyl group of other amino acids to form branched polypeptides. Tryptophan (W), due to its aromaticity, flat rigid shape and π-electron structure, has a strong preference at the membrane interface and serves as an anchor on the cell membrane, thereby enhancing peptide-membrane interaction. Histidine (H) has been proven to enhance the disruption of endosomal membranes through the proton sponge effect.

[0024] The polypeptide delivery system delivers siRNA and mRNA to numerous target organs except the liver through local or systemic administration, overcoming the targeting and stability obstacles of drugs; and can carry multiple siRNAs with different targets simultaneously, enabling them to produce a synergistic gene silencing effect in the same target cells and improving the drug efficacy of RNAi; it has a wide range of applications and can be used to encapsulate various drugs such as siRNA, mRNA, microRNA, ASO or DNA. Due to these characteristics, the KKK-KW and KKK-KHW polypeptide delivery systems have broad application prospects in the field of nucleic acid drugs.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The polypeptide delivery system provided by the present invention can deliver nucleic acid drugs into cells, and has better delivery efficiency and transfection effect than commercially available liposome reagents, with a high encapsulation rate and low toxicity.

[0027] The present invention proves through knockdown experiment results that the polypeptide delivery system can normally release siRNA in cells, and the silencing effect of siRNA is better than that of commercially available liposome reagents. This experiment can be completed by simply standing at room temperature for 30 minutes. The present invention discovers through Malvern particle size analyzer that the diameter of the above-mentioned nanoparticles after encapsulating nucleic acids is about 100 - 200 nm and the encapsulation rate is greater than 90%.

[0028] The nucleic acid-KKK-KW complex disclosed by the present invention can encapsulate siRNA and mRNA; can effectively transfect HEK293 cells and Vero cells, and the transfection effect is better than that of commercial transfection reagents RNAiMAX and Lipo2000; the in vitro transfection siRNA gene silencing efficiency is better than that of commercial reagent Lipo2000 and is comparable to RNAiMAX; the particle size is uniform and the potential is positive; the encapsulation rate is higher than 90%; the cytotoxicity is small and far lower than that of Lipo2000.

[0029] Through cytotoxicity experiments, it was found that the cytotoxicity of KKK-KHW is comparable to that of Lipo2000 and RNAiMAX, and it has relatively low cytotoxicity to cells. Through serum enzyme degradation experiments, it was found that KKK-KHW can effectively protect siRNA and mRNA, and it is hardly degraded by serum enzymes after 6 hours of incubation.

[0030] In the present invention, KKK-KHW can completely encapsulate siRNA when the molar ratio of KKK-KHW to siRNA is 5:1, and can completely encapsulate mRNA when the ratio of KKK-KHW to mRNA is 2:1. The transfection efficiency of KKK-KHW is higher than that of Lipo2000 and RNAiMAX in both low-concentration and high-concentration cases of siRNA. KKK-KHW delivers siGFP into cells, which can knockdown EGFP with an inhibition rate of about 40%. Brief Description of the Drawings

[0031] Figure 1 Reaction formula of Fmoc4-KKK-OH;

[0032] Figure 2 Synthesis steps of Fmoc-KKK-OH;

[0033] Figure 3 Synthesis step diagram of R4-KKK-CONH2;

[0034] Figure 4 It is an agarose gel electrophoresis diagram of siRNA-KW complexes formed by mixing siRNA and KW polypeptide at different molar ratios;

[0035] Figure 5 It is the transfection efficiency of siRNA-KW complexes at different siRNA concentrations and in different cells;

[0036] Figure 6 It is an agarose gel electrophoresis diagram of siRNA-KKK-KW complexes formed by mixing siRNA and KKK-KW polypeptide at different molar ratios;

[0037] Figure 7 It is the transfection efficiency of siRNA-KKK-KW complexes at different molar ratios in HEK293 cells;

[0038] Figure 8 It is the transfection efficiency of siRNA-KKK-KW complexes at different siRNA concentrations and in different cells;

[0039] Figure 9 It is the in vitro transfection gene silencing efficiency of siRNA-KKK-KW complexes;

[0040] Figure 10 It is the standard curve of the encapsulation rate of siRNA-KKK-KW complexes;

[0041] Figure 11 is the cytotoxicity and IC 50 value of the siRNA-KKK-KW complex;

[0042] Figure 12 is the agarose gel electrophoresis pattern of the mRNA-KKK-KW complex formed by mixing mRNA and KKK-KW polypeptide at different molar ratios;

[0043] Figure 13 is the standard curve of the encapsulation efficiency of the mRNA-KKK-KW complex;

[0044] Figure 14 is the electrophoresis pattern for detecting the ability of KKK-KHW to encapsulate siRNA;

[0045] Figure 15 is the electrophoresis pattern for detecting the ability of KKK-KHW to encapsulate mRNA;

[0046] Figure 16 is the delivery efficiency of KKK-KHW delivering siRNA in Vero cells;

[0047] Figure 17 is the delivery efficiency of KKK-KHW delivering siRNA in Vero cells;

[0048] Figure 18 is the knockdown efficiency of KKK-KHW delivering siRNA;

[0049] Figure 19 is the serum enzyme degradation level of KKK-KHW-siRNA;

[0050] Figure 20 is the cytotoxicity and IC50 detection of KKK-KHW;

[0051] Figure 21 is the in vitro biological activity detection of KKK-KW and KKK-KHW delivering mRNA. Specific Embodiments

[0052] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions. The meanings of the English abbreviations involved in the following embodiments are shown in Table 1.

[0053] Table 1

[0054]

[0055] Example 1

[0056] A preparation method of a four-chain polypeptide R4-KKK-CONH2, the structure of which is shown as follows:

[0057]

[0058] In the formula, K is lysine, and R is any linear polypeptide sequence of different permutations and combinations of 1 to 20 kinds of amino acids, including but not limited to the KW sequence (KWHHHKWHHHKWHHHKWHHHK) and the KHW sequence (KHWHKHWHKHWWHKHWHK).

[0059] Step 1: Synthesize the polypeptide Fmoc-KKK-OH

[0060] Three Fmoc-Lys(Boc)-OH are connected to dichloride resin: After the dichloride resin is swollen and washed with DMF, the first Fmoc-Lys(Boc)-OH is introduced; after the reaction is completed, it is washed, capped, and washed in sequence; then the protection is removed and then washed again. Prepare a lys condensation solution, connect a Fmoc-Lys(Boc)-OH, repeat the second lys step to connect the third Fmoc-Lys(Boc)-OH, and after washing, carry out cleavage. After the cleavage reaction, precipitation is carried out; after the precipitate is dissolved, TFA is removed, and then precipitation and water removal are carried out to obtain the polypeptide Fmoc-KKK-OH.

[0061] In step 1, the amount of each amino acid added is 4eq of the amount of dichloride resin;

[0062] The capping method involved in step 1 is: the amount of AcOH added is 4eq of the amount of dichloride resin, and then 8eq of TEA is added; the capping method involved in step 3 is: the amount of AcOH added is 4eq of the amount of AM resin. AcOH reacts with HBTU for 10 min first, and then DIPEA is added.

[0063] The LC-MS detection of Fmoc-KKK-OH is as Figure 3 shown: Select the positive ion mode, m / z range: 200 - 800, Frag = 15.0V.

[0064] LC-MS result analysis: The exact molecular weight of Fmoc-KKK-OH is: 624.4. The detected molecular weight is: 625.2 [M+H] + , 313.2 [M+2H] 2+ , 209.0 [M+3H] 3+ , and the synthesized Fmoc-KKK-OH has the correct structure.

[0065] Step 2: Synthesize the main chain Fmoc4-KKK-OH

[0066] The reaction of Fmoc-KKK-OH with Fmoc-OSu under alkaline conditions to produce Fmoc4-KKK-OH is shown by the reaction formula as Figure 1 shown below.

[0067] (1) Dissolve Fmoc-KKK-OH in 10 mL of THF + 10 mL of MeOH, add 455 μl of TEA, and place it in a round-bottom flask; add Fmoc-OSu to 10 mL of THF and place it in a constant-pressure dropping funnel on top of the round-bottom flask. Slowly drip it for 20 min, and after dripping, stir the reaction at room temperature for 2 h.

[0068] (2) Rotate and evaporate the solvent to concentrate it to 5 ml, add 30 mL of EA and 10 mL of PE, mix well, and place it in a -20 °C refrigerator overnight to wait for precipitation.

[0069] (3) Centrifuge at 12000 r / min for 2 min, discard the supernatant, dissolve the solid by ultrasonic wave with 3 mL of DCM + 1 mL of MeOH, add 30 mL of EA, mix well and then centrifuge, discard the supernatant. Repeat this step 3 - 4 times, and check by TLC to see if the impurities are removed completely.

[0070] (4) The LC-MS detection of Fmoc4-KKK-OH is as Figure 5 shown below: Select the negative and positive ion modes, m / z range: 900 - 1400, Frag = 15.0 V.

[0071] LC-MS result analysis: The exact molecular weight of Fmoc4-KKK-OH is: 1290.57. The detected molecular weight is: 1289.4 [M-H] - , 1325.4 [M+Cl] - , and the synthesized Fmoc4-KKK-OH has the correct structure.

[0072] Step 3: Synthesize the four-chain polypeptide R4-KKK-CONH2

[0073] After the AM resin is swollen, washed with DMF, deprotected, and then washed again, prepare the condensation solution and introduce Fmoc4-KKK-OH. After the reaction is completed, wash, cap, and wash in sequence, then deprotect and wash again. Prepare the condensation solution of the first side-chain amino acid and introduce the first side-chain amino acid. After washing, repeat the above steps to synthesize 4 R side-chains simultaneously. After the reaction is completed, wash and then cleave; after the cleavage reaction, precipitate; after the precipitate is dissolved, remove TFA, and then precipitate and remove water to obtain the four-chain polypeptide R4-KKK-CONH2;

[0074] The washing methods involved in Step 1 and Step 3 are: wash with DMF twice, swell with DCM twice, shrink with EtOH twice, swell with DCM and wash with DMF; the usage amount only needs to cover the resin.

[0075] When connecting the next amino acid in Step 1 and Step 3, it is necessary to prepare the amino acid condensation solution in advance. Dissolve the amino acid and HBTU in DMF at a molar ratio of 1:1 and react alone for 10 min, then pour it into the polypeptide reaction tube, and then add DIPEA. The preparation process of this DIPEA is as follows: the volume ratio of DIPEA to DMF is 174:826, and the added amount is 0.6 ml per 0.1 mmol of amino acid.

[0076] Precipitation is carried out after the cleavage reaction in Step 1 and Step 3; after the precipitate is dissolved, TFA is removed, and then precipitation and water removal are carried out again. The specific operations are as follows: after cleavage, ice-cold diethyl ether is used for precipitation, and the precipitate is precipitated at 1 - 5 °C, and the precipitate is obtained by centrifugal separation;

[0077] Removing TFA from the precipitate: The precipitate after centrifugation is ultrasonically dissolved in a small amount of MeOH, a trace amount of TEA is added dropwise to react with TFA to form a salt, and then it is added dropwise to 30 times the volume of EA to precipitate, centrifuged at 12000 r / min for 2 min. The precipitate after centrifugation is dissolved in MeOH, TEA is added dropwise, and then it is added dropwise to EA to precipitate and then centrifuged. Repeat 2 - 3 times until no white smoke is emitted when adding TEA. At this time, the TFA residue in the polypeptide is completely removed, and the precipitate after centrifugation is collected.

[0078] The precipitate collected after removing TFA from the precipitate is ultrasonically dissolved in a small amount of MeOH, then rotary evaporated to a transparent film, and then PE and EA are added to precipitate a white turbidity, and rotary dried to obtain a white powdery polypeptide. This step can remove the water in the product by dissolving the precipitate in organic solvents multiple times and rotary drying, achieving the purpose of water removal, and the product does not need to be freeze-dried subsequently.

[0079] The synthesis steps of Fmoc-KKK-OH are as Figure 2 shown, and the synthesis steps of R4-KKK-CONH2 are as Figure 3 shown.

[0080] Preparation of KKK-KW polypeptide in Example 2

[0081] The synthesis method of the four-chain polypeptide KW4-KKK-CONH2, the structure is as shown below, and the preparation method includes the following steps:

[0082]

[0083] Step 1: Synthesize the polypeptide Fmoc-KKK-OH;

[0084] Step 2: Synthesize the main chain Fmoc4-KKK-OH;

[0085] Step 3: Synthesize the four-chain polypeptide KW4-KKK-CONH2;

[0086] LC-MS detection: Select the positive ion mode, m / z range: 100 - 2200, Frag = 100.0V.

[0087] LC-MS result analysis: The exact molecular weight of KW4-KKK-CONH2 is: 12524.32. The detected molecular weight is: 118.1 [M+107H] 107+ , 235.3 [M+59Na] 59+ , and the synthesized KW4-KKK-CONH2 has the correct structure.

[0088] HPLC detection conditions and results:

[0089] Column: C8 (10*250mm, 5μm); Column temperature: 35°C; Flow rate: 1ml / min; Detection wavelength: 220nm; Mobile phase: A 0.05% phosphoric acid aqueous solution, B HPLC methanol; Method: 0→20min, 65% A→40% A, 20→30min, 40% A→0% A; Retention time: 7.328min; Purity: 99.1%.

[0090] Preparation of Example 3 KKK-KHW

[0091] Synthesis method of the four-chain polypeptide KHW4-KKK-CONH2, the structure is as shown below, and the preparation method includes the following steps:

[0092]

[0093] Step 1: Synthesize the polypeptide Fmoc-KKK-OH

[0094] Step 2: Synthesize the main chain Fmoc4-KKK-OH

[0095] Step 3: Synthesize the four-chain polypeptide KHW4-KKK-CONH2

[0096] LC-MS detection: Select the positive ion mode, m / z range: 200 - 1400, Frag = 50.0V.

[0097] LC-MS result analysis: The exact molecular weight of KHW4-KKK-CONH2 is: 11963.97. The detected molecular weight is: 227.2 [M+53H] 53+ , 268.1 [M+52K] 52+ , 317.1 [M+43K] 43+ , 380.8 [M+35K] 35+ , 449.3 [M+28Na] 28+ , 542.4 [M+23Na] 23+ , 704.2 [M+17H]17+ The synthesized KHW4-KKK-CONH2 has the correct structure.

[0098] HPLC detection conditions and results:

[0099] Column: C8 (10*250mm, 5μm); Column temperature: 35°C; Flow rate: 1 ml / min; Detection wavelength: 220 nm; Mobile phase: A 0.05% phosphoric acid aqueous solution, B HPLC methanol; Method: 0→20 min, 65% A→40% A, 20→30 min, 40% A→0% A; Retention time: 7.548 min; Purity: 99.1%.

[0100] Preparation of nucleic acid-polypeptide complex in Example 4

[0101] First, dissolve the polypeptide (KW, KHW, KKK-KHW or KKK-KW) in water with a pH of 2-4 to prepare a 1 mg / mL polypeptide solution. Mix mRNA, siRNA or siRNA-FAM (200 nM) with the polypeptide solution according to the molar ratio and let it stand for 5 min. Then add a certain amount of water or DMEM medium and let it stand for 5-25 min to obtain the nucleic acid-KKK-KW complex solution.

[0102] The gene name of the siRNA used in this example is GFP-1, and the base sequence of its sense strand is as follows. The siRNA is synthesized by Tsingke Biotechnology.

[0103] Sense strand 5’-3’GGCUACGUCCAGGAGCGCA

[0104] Antisense strand 5’-3’UGCGCUCCUGGACGUAGCC

[0105] Example 5 Study on the binding ability of siRNA with KW polypeptide and KKK-KW polypeptide

[0106] Using agarose gel electrophoresis technology (1.2% w / v, 1×TBE, 55V, 1h), the binding ability of siRNA with KW polypeptide and KKK-KW polypeptide at a series of different molar ratios was studied respectively. The principle of the gel retardation experiment is as follows: Alone siRNA is small in size and negatively charged, so it can easily migrate through the gel; When siRNA does not bind or binds incompletely with the polypeptide, due to its small size and negative charge, it can easily migrate through the gel, showing a strong fluorescence intensity; When the cationic siRNA-polypeptide complex is positively charged and large in size, their migration through the gel is hindered. Therefore, the free siRNA detected in the agarose gel will decrease, showing a weakened fluorescence intensity; When the siRNA-polypeptide complex is completely bound, no free siRNA can be detected in the agarose gel and no fluorescence is shown.

[0107] As Figure 4 shown in the first lane, due to its small size and negative charge, siRNA can easily migrate through the gel. As the molar ratio increases, due to the larger size and positive charge of the cationic siRNA-KW complex, their migration in the gel is hindered, and the fluorescence intensity gradually weakens. When the siRNA:KW molar ratio is 1:30, the siRNA molecules are completely bound to the KW polypeptide, so free siRNA is not detected in the agarose gel. Therefore, when the siRNA:KW molar ratio is greater than 1:30, all siRNA molecules can be completely bound to the KW polypeptide.

[0108] As Figure 6 shown, the first lane is siRNA. As the molar ratio increases, when the siRNA:KKK-KW molar ratio is 1:10, the siRNA molecules are completely bound to the KKK-KW polypeptide, so free siRNA is not detected in the agarose gel. Therefore, when the siRNA:KKK-KW molar ratio is greater than 1:10, all siRNA molecules can be completely bound to the KKK-KW polypeptide.

[0109] Example 6 Investigation of the transfection efficiency of siRNA-KW complexes at different siRNA concentrations and in different cells

[0110] At the same time, the transfection efficiencies of two types of cells, HEK293 and Vero, were investigated. siRNA-FAM with a fluorescent label and KW polypeptide were prepared into siRNA-KW complexes according to a molar ratio of 1:30, and were transfected into the cells seeded in 24-well plates respectively. After culturing at 37°C for 24 h, the medium was removed, the cells were washed three times with PBS, the cells were digested with trypsin, 150 μL of PBS was added to each well to resuspend the cells evenly, 20 μL of the cell suspension was taken and added to the cell counting chamber, and the transfection rate was detected using the "GFP mode" of the cell counter Count Star S2.

[0111] As Figure 5 shown, the siRNA-KW complex can perform cell transfection. The transfection efficiency in Vero cells is lower than that of the commercial transfection reagent RNAiMAX, and the transfection efficiency in HEK293 cells is higher than that of the commercial transfection reagent RNAiMAX.

[0112] Example 7 Investigation of the transfection efficiency of siRNA-KKK-KW complexes at different molar ratios in HEK293 cells

[0113] The siRNA-FAM (200 nM) with fluorescent label was formulated with the KKK-KW polypeptide into siRNA-KKK-KW complexes according to a series of molar ratios of 1:10, 1:12, 1:14, and 1:16, and added to HEK293 cells in a 24-well plate for transfection. After culturing at 37 °C for 24 h, the medium was removed, the cells were washed three times with PBS, the cells were digested with trypsin, 150 μL of PBS was added to each well to resuspend the cells, 20 μL of the cell suspension was taken and added to a cell counting chamber, and the transfection efficiency was detected using the "GFP mode" of the cell counter Count Star S2. When the siRNA-KKK-KW complex was transfected into the cells, the cells in the bright field and the fluorescence in the dark field could be seen corresponding to each other under an exposure intensity of 4000, and the transfection efficiency was shown by detecting the fluorescence value.

[0114] As Figure 7 shown, in HEK293 cells, the transfection efficiency of the KKK-KW polypeptide on siRNA was much higher than that of the commercial transfection reagents RNAiMAX and Lipo2000, and the highest transfection efficiency of the KKK-KW polypeptide was 80.2%.

[0115] Example 8 Investigation of the transfection efficiency of the siRNA-KKK-KW complex at different siRNA concentrations and in different cells

[0116] The siRNA-FAM (50 nM, 100 nM, 200 nM) with fluorescent label was respectively formulated with the KKK-KW polypeptide into siRNA-KKK-KW complexes according to a molar ratio of 1:16, and were respectively inoculated into HEK293 cells and Vero cells in a 24-well plate for transfection. After culturing at 37 °C for 24 h, the medium was removed, the cells were washed three times with PBS, the cells were digested with trypsin, 150 μL of PBS was added to each well to resuspend the cells, 20 μL of the cell suspension was taken and added to a cell counting chamber, and the transfection efficiency was detected using the "GFP mode" of the cell counter Count Star S2. When the siRNA-KKK-KW complex was transfected into the cells, the cells in the bright field and the fluorescence in the dark field could be seen corresponding to each other under an exposure intensity of 4000, and the transfection efficiency was shown by detecting the fluorescence value.

[0117] As Figure 8 shown, in HEK293 cells and Vero cells, the transfection efficiency of the KKK-KW polypeptide on siRNA was higher than that of the commercial transfection reagents RNAiMAX and Lipo2000.

[0118] Example 9 Investigation of the in vitro transfection gene silencing efficiency of the siRNA-KKK-KW complex

[0119] Vero cells were seeded at 1.5 * 10 per well 5The cells were seeded at a density of

[0120] per well in a 24-well plate and incubated for 24 h. Next, the commercial reagent EL was used to transfect the PEGFP-N3 plasmid into Vero cells. After 4 - 6 h, the cells were washed and the medium was changed. siGFP (100 nM, 200 nM) was transfected into the cells using different delivery systems, Lipo2000, KKK-KW, and RNAiMAX, respectively. After culturing at 37 °C for 24 h, the medium was removed, and the cells were washed three times with PBS. The cells were then digested with trypsin, and 150 μL of PBS was added to each well to resuspend the cells. 20 μL of the cell suspension was taken and added to a cell counting chamber, and the transfection efficiency was detected using the "GFP mode" of the cell counter Count Star S2. When the siRNA-KKK-KW complex was transfected into the cells, the cells in the bright field and the fluorescence in the dark field corresponded to each other at an exposure intensity of 500, and the transfection efficiency was shown by detecting the fluorescence value. Figure 9 In vitro transfection was used to study the efficiency of the siRNA-KKK-KW complex in silencing the EGFP gene at the siRNA level. RNAiMAX and Lipo2000 were used as positive controls. As

[0121] shown, in Vero cells, the siRNA-KKK-KW complex induced a 53% EGFP gene silencing efficiency at a molar ratio of 1:16, the siRNA-RNAiMAX complex induced a 65% EGFP gene silencing rate, and the siRNA-Lipo2000 complex induced a 51% EGFP gene silencing rate. The results indicate that the KKK-KW polypeptide can effectively deliver siRNA into the cytoplasm and induce specific gene silencing, and the gene silencing efficiency of the KKK-KW polypeptide is better than that of the commercial reagent Lipo2000 and comparable to RNAiMAX.

[0121] Example 10 Detection of the particle size, PDI, and zeta potential of the siRNA-KKK-KW complex

[0122] siRNA (200 nM) and the KKK-KW polypeptide were prepared into the siRNA-KKK-KW complex at a series of molar ratios of 1:10, 1:12, 1:14, and 1:16. After standing for 5 min, it was added to enzyme-free and sterile water, and after mixing, the particle size, PDI, and zeta potential were detected.

[0123] As shown in Table 2, at the optimal molar ratio of 1:16, the particle size of the siRNA-KKK-KW complex in water was 231.4 nm, the PDI was 0.3877, and the zeta potential was 35.65 mV.

[0124] Table 2

[0125]

[0126] Example 11 Detection of the Encapsulation Efficiency of the siRNA-KKK-KW Complex

[0127] The Quant-it kit method was used to detect the encapsulation efficiency of the siRNA-KKK-KW complex. The Quant-it reagent solution was added to 1×TE buffer to prepare a reaction solution. The siRNA-KKK-KW complexes at different molar ratios were mixed with the reaction solution and allowed to stand for 5 min, then added to an enzyme-linked immunosorbent assay (ELISA) plate. The fluorescence value was detected using an ELISA reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

[0128] Calculation formula: Encapsulation efficiency = 1 - (fluorescence value of free RNA measured - blank / fluorescence value of total RNA - blank) × 100%.

[0129] As shown in Table 3 and Figure 10 it can be seen that the encapsulation efficiency of the siRNA-KKK-KW complex is greater than 90%. At a molar ratio of 1:16, the encapsulation efficiency is as high as 94.6%.

[0130] Table 3

[0131]

[0132] Example 12 Detection of the Cytotoxicity and IC 50 value of the siRNA-KKK-KW Complex

[0133] Two 96-well plates were seeded with Vero cells, with 8000 cells per well. One column of each plate was left blank without cells. After culturing at 37 °C for 24 h, the drugs were administered. For the detection of cytotoxicity, 8 experimental groups were required, namely the drug administration groups siRNA-KKK-KW, siRNA-RNAiMAX, siRNA-Lipo2000, KKK-KW, Lipo2000, RNAiMAX, the control group Cell, and the blank group Blank. For the detection experiment of IC 50 , the drug administration groups were set at 0.1 μM, 0.5 μM, 1 μM, 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM, the control group Cell, and the blank group Blank. After culturing for another 24 h, the cell plates were taken out, 10 μL of CCK-8 was added to each well, incubated at 37 °C for 1 h, and then detected using an ELISA reader with a wavelength of 450 nm.

[0134] Cell survival rate = (absorbance of the drug administration group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group) × 100%

[0135] As Figure 11As shown, the cytotoxicity of the siRNA-KKK-KW complex in Vero cells was lower than that of siRNA-Lipo2000 and comparable to that of siRNA-RNAiMAX. As Figure 11 shown, the IC 50 of the siRNA-KKK-KW complex was 56.38 uM.

[0136] Example 13 Study on the binding ability of mRNA to KKK-KW polypeptide

[0137] Using agarose gel electrophoresis technology (1.2% w / v, 1×TBE, 55V, 1h), the binding ability of an mRNA encoding the S protein of the novel coronavirus to the KKK-KW polypeptide at a series of different mass ratios was studied. The mRNA was an mRNA expressing the RBD region of the S protein of the original strain of the novel coronavirus.

[0138] As Figure 12 shown, when the mRNA:KKK-KW mass ratio was greater than 1:2, all mRNA molecules could completely bind to the KKK-KW polypeptide.

[0139] Example 14 Detection of the particle size, PDI and zeta potential of the mRNA-KKK-KW complex

[0140] mRNA (200 nM) and the KKK-KW polypeptide were respectively prepared into mRNA-KKK-KW complexes at mass ratios of 1:2 and 1:4. After standing for 5 min, they were added to enzyme-free and sterile water, and after mixing, the particle size, PDI and zeta potential were detected.

[0141] As shown in Table 4, at a mass ratio of 1:2, the particle size of the mRNA-KKK-KW complex in water was 180.8 nm, the PDI was 0.1655, and the zeta potential was 15.63 mV; at a mass ratio of 1:4, the particle size of the mRNA-KKK-KW complex in water was 211 nm, the PDI was 0.258, and the zeta potential was 19.89 mV.

[0142] Table 4

[0143]

[0144] Example 15 Detection of the encapsulation efficiency of the mRNA-KKK-KW complex

[0145] The encapsulation efficiency of the mRNA-KKK-KW complex was detected by the method of the Quant-it kit.

[0146] Calculation formula: Encapsulation efficiency = 1 - (fluorescence value of free RNA measured - blank / fluorescence value of total RNA - blank) × 100%.

[0147] As shown in Table 5 andFigure 13 As shown, when the mass ratio of the mRNA-KKK-KW complex is 1:2, the encapsulation efficiency is 91.27%, and when the mass ratio is 1:4, the encapsulation efficiency is 81.86%.

[0148] Table 5

[0149]

[0150] Example 16 Detection of the encapsulation ability of KKK-KHW for siRNA

[0151] The agarose gel electrophoresis experiment was used to study the encapsulation ability of KKK-KHW and the KHW side chain for siRNA at different molar ratios. The molar ratios of siRNA:KKK-KHW were selected as 1:5, 1:6, 1:7, 1:8, 1:9, 1:10; the molar ratios of siRNA:KHW side chain were selected as 1:5, 1:10, 1:16, 1:20, 1:30, 1:40, 1:50, 1:60. A 1.2% agarose gel was prepared, and the polypeptide powder was dissolved to 1 μg / μL. KKK-KHW and siRNA were mixed according to the above molar ratios, and after standing at room temperature for 30 min, DNA loading buffer was added, and the samples were added to the wells. The voltage was 80 V, and after running the gel for 1 h, the gel was placed under a gel imager for observation. Since the complex completely encapsulated by KKK-KHW has a particle size larger than the pore size of the agarose gel, it will not run out of the gel wells. It was determined that siRNA:KKK-KHW was completely encapsulated at a molar ratio of 1:5, and siRNA:KHW side chain was completely encapsulated at a molar ratio of 1:30. The results are as Figure 14 shown.

[0152] Example 17 Detection of the encapsulation ability of KKK-KHW for mRNA

[0153] The agarose gel electrophoresis experiment was used to study the encapsulation ability of KKK-KHW for mRNA at different mass ratios. The mass ratios of mRNA:KKK-KHW were selected as 2:1, 1:1, 1:2, 1:4, 1:6, 1:8. A 1.2% agarose gel was prepared, and the polypeptide powder was dissolved to 1 μg / μL. KKK-KHW and mRNA were mixed according to the above mass ratios, and after standing at room temperature for 30 min, DNA loading buffer was added, and the samples were added to the wells. The voltage was 80 V, and after running the gel for 1 h, the gel was placed under a gel imager for observation. Since the complex completely encapsulated by KKK-KHW has a particle size larger than the pore size of the agarose gel, it will not run out of the gel wells. It was determined that mRNA:KKK-KHW was completely encapsulated at a mass ratio of 1:2, as Figure 15 shown.

[0154] Example 18: Detection of the particle size, PDI and zeta potential of nanoparticles encapsulating siRNA and mRNA with KKK-KHW

[0155] siRNA: KKK-KHW was formulated at molar ratios of 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. After standing for 10 min, 1 mL of water was added, and then it was left standing for another 10 min. The test solution was added to a disposable folding capillary cuvette with a syringe, and the particle size and zeta potential were detected using a Malvern particle size analyzer. When setting the parameters, the material was selected as protein. The dispersion system was water, the temperature was 25 °C, and the equilibration time was 60 s. The particle size was 267.3 nm when the molar ratio of KKK-KHW to siRNA was 1:5. mRNA: KKK-KHW was formulated at mass ratios of 1:2 and 1:4, and the solvent was water or DMEM. The nanoparticles composed of siRNA, mRNA, and KKK-KHW had uniform particles and a small particle size in water, as shown in Tables 6 and 7.

[0156] Table 6

[0157]

[0158] Table 7

[0159]

[0160]

[0161] Example 19: Determination of the encapsulation efficiency of siRNA and mRNA encapsulated by KKK-KHW

[0162] RiboGreen dye exhibits fluorescence characteristics when binding to RNA, and the fluorescence signal intensity is positively correlated with the RNA concentration. The RNA concentration is reflected by the fluorescence signal intensity. The detection kit was taken out of the refrigerator and placed at room temperature for 30 min. 20×TE buffer was diluted to 1×TE buffer, and Quant-it reagent was diluted with 1×TE buffer. After KKK-KHW was mixed with siRNA and mRNA and left standing for 20 min, a black 96-well microplate was taken, and 198 μL of the diluted Quant-it reagent solution was added to each well, and then 2 μL of the test sample was added. Six replicates were made for each sample, and at the same time, a naked RNA group and a control group with only the polypeptide were set up. After incubation for 5 min, the fluorescence of the samples was measured on a fluorescence microplate reader. The fluorescence excitation wavelength was 485 nm, and the emission wavelength was 520 nm. The results showed that the encapsulation efficiency of siRNA encapsulated by KKK-KHW was greater than 95%, and the encapsulation efficiency of mRNA encapsulated by KKK-KHW was about 90%. As shown in Table 8.

[0163] Table 8

[0164]

[0165] Example 2 Detection of the delivery efficiency of KKK-KHW delivering siRNA in Vero cells

[0166] Weigh 1 mg of KKK-KHW into a centrifuge tube using a ten-thousandth balance, dissolve KKK-KHW with 1 mL of sterilized water (pH = 3), and vortex to mix evenly. Dissolve the siRNA-FAM dry powder in DEPC water according to the label to a concentration of 20 μM. Plate Vero cells at a cell density of 1.5×10 5 cells / well, and culture for 18 - 24 h after plating. The final concentrations of siRNA in the 24-well plate are 50 nM, 100 nM, and 200 nM. Mix KKK-KHW with siRNA at a molar ratio of 1:7 and 1:10, let it stand for 5 min, then add 100 μL of opti-MEM medium after standing. After standing for another 15 min, add the mixture to the 24-well plate. The control groups are the Lipo2000 and RNAiMAX groups. The final concentrations of siRNA in the 24-well plate are 50 nM, 100 nM, and 200 nM. When the concentration is 50 nM, the usage amounts of Lipo2000 and RNAiMAX are 1 μL / well; when the concentration is 100 nM, the usage amounts of Lipo2000 and RNAiMAX are 2 μL / well; when the concentration is 200 nM, the usage amounts of Lipo2000 and RNAiMAX are 3 μL / well. The transfection method follows the instructions. 24 h after transfection, discard the medium in the 24-well plate, wash the cells 3 times with PBS, discard the PBS, then add 100 μL of trypsin to each well, rinse and discard the trypsin, and digest for 3 min. Add 150 μL of PBS to each well to resuspend the cells, take 20 μL of the cell suspension and add it to a cell counting chamber, and analyze it using the GFP module of the Countstar cell analyzer. The results are as Figure 16 and Figure 17 shown. The transfection efficiencies of KKK-KHW and KKK-KW are better than those of Lipo2000 and RNAiMAX.

[0167] Example 21 Detection of the knockdown efficiency of KKK-KHW delivering siRNA

[0168] Plate Vero cells at a cell density of 1.5×10 5Cells were seeded at a density of Figure 18 per well, and cultured for 18 - 24 h after plating. pEGFP-N3 was transfected into the cells using the transfection reagent EL(TRANS) according to the instructions, with 1 μg of pEGFP-N3 per well. siRNA transfection was performed 6 h after plasmid transfection. Before transfection, the plates were washed 3 times with 1*PBS to exclude the influence of the EL transfection reagent. The final concentrations of siGFP in the 24-well plates were 100 nM and 200 nM. KKK-KHW was mixed with siRNA at a molar ratio of 1:10 and allowed to stand for 5 min. After standing, 100 μL of opti-MEM medium was added, and the mixture was added to the 24-well plates after standing for another 15 min. The control groups were Lipo2000 and RNAiMAX groups. The medium in the 24-well plates was discarded, and the cells were washed 3 times with PBS. The siRNA mixture was added to the wells. After 24 h, analysis was performed using the GFP module of the Countstar cell analyzer. The results showed that the siRNA delivered by KKK-KHW could knockdown EGFP and inhibit the expression of its fluorescent protein. As Figure 18 shown.

[0169] Example 22 Detection of the Serum Enzyme Degradation Level of KKK-KHW-siRNA

[0170] For the KKK-KHW-siRNA experimental group (10% FBS), 12 μL of KKK-KHW + 3 μL of siRNA were taken and mixed for 5 min. 15 μL of the mixture was added to 15 μL of DMEM (50% FBS) in a 200 μL EP tube and mixed. Incubation was carried out at 37 °C for 0 h, 2 h, and 6 h. For the siRNA control group, 3 μL of siRNA was added to 12 μL of DEPC water. 15 μL of the mixture was added to 15 μL of DMEM (50% FBS) in a 200 μL EP tube and mixed. Incubation was carried out at 37 °C for 0 h, 2 h, and 6 h. For the control group, 12 μL of KKK-KHW was added to 3 μL of DEPC water. 15 μL of the mixture was added to 15 μL of DMEM (50% FBS) in a 200 μL EP tube and mixed. Incubation was carried out at 37 °C for 0 h, 2 h, and 6 h. Agarose gel with a concentration of 1% was prepared. Before loading, 10 μL of the sample from the experimental group was taken and added to 2 μL of 1% SDS for depolymerization, and mixed well and incubated at room temperature for 2 min. 10 μL of the sample was added to each well, and agarose gel electrophoresis was carried out for 30 min. KKK-KHW could effectively protect siRNA from degradation by serum enzymes, as Figure 19 shown.

[0171] Example 23 Detection of the Cytotoxicity and IC50 of KKK-KHW

[0172] Vero cells were seeded in a 96-well plate at 8,000 cells per well. The four sides of the 96-well plate were filled with 100 μL of PBS, leaving one column as a blank group without cells seeded. After 24 h, the drug was administered with a final concentration of 100 nM of siRNA, and there were 6 replicates for each sample. After 24 h, the cytotoxicity was detected. The cell plate was taken out, 10 μL of CCK-8 was added to each well, and incubated at 37 °C for 1 h. After 1 h, it was detected using a microplate reader with a wavelength of 450 nm. The cell survival rate was calculated as follows: (absorbance of the drug-treated group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group) * 100%, and the results are as Figure 20 shown.

[0173] Example 24. In vitro biological activity detection of KKK-KW and KKK-KHW in delivering mRNA

[0174] Using KKK-KHW, KKK-KW and Lipo2000 transfection reagent, mRNA (receptor binding domain of the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) was introduced into 293A cells. After culturing, the supernatant was taken, and the expression of RBD protein was detected by direct ELISA method. First, HEK293 cells were seeded at 3×10 5The cells at a density of [number] / well were seeded in a 12-well plate and cultured at 37°C in 5% CO2 for 24 hours, then reserved. Dissolve KKK-KW and KKK-KHW to 1 μg / μL. The mass ratio of mRNA (3 μg / well) to KKK-KHW (12 μg / well) and KKK-KW (12 μg / well) is 1:4. Mix them in Opti-MEM and let stand at room temperature for 20 min, then add to the cell plate. The mass ratio of Lipo2000 to mRNA is 1:1. Mix according to the Lipo2000 instruction manual and let stand at room temperature for 20 min, then add to the cell plate. After culturing at 37°C for 24 h, centrifuge to collect the cell supernatant and detect the expression of RBD protein by ELISA. Prepare the standard curve according to the instruction manual, prepare the test samples, dilute the cell supernatant to be tested to a suitable concentration with 1× dilution buffer, and record the dilution factor. Detection sample treatment: Plate washing: Wash the plate three times with 1× washing buffer. Sample incubation: Add 100 μL of control, test sample, Lipo2000 Only sample, and standard solutions of each concentration of the standard curve to each well. Add 100 μL of 1× dilution buffer to the ELISA blank control. Add them to the enzyme-linked immunosorbent assay (ELISA) plate in duplicate and seal with a sealing film, then incubate at 25°C for 2 hours. Plate washing: Wash the plate three times with 1× washing buffer. Incubation of enzyme-labeled detection antibody: Except for the ELISA blank control well, add 100 μL of the detection antibody pre-prepared to the working concentration to each well. Add 100 μL of 1× dilution buffer to the ELISA blank control, seal with a sealing film, and incubate at 25°C for 1 hour. Plate washing: Wash the plate three times with 1× washing buffer. Color development: Add 200 μL of the pre-prepared substrate solution to each well of the enzyme-linked immunosorbent assay (ELISA) plate and incubate at 25°C in the dark for 20 minutes. Termination: Add 50 μL of the termination solution to each well of the enzyme-linked immunosorbent assay (ELISA) plate. Set the detection wavelength to 450 nm on a multifunctional microplate reader and measure its absorbance. As Figure 21 The results showed that both KKK-KHW and KKK-KW could deliver mRNA into cells and express normally, and the effect of KKK-KHW was better than that of KKK-KW.

[0175] In summary, the polypeptide delivery system provided by the present invention can deliver siRNA and mRNA to many target organs except the liver through local or systemic administration, overcoming the targeting and stability obstacles of drugs; and can carry multiple siRNAs with different targets at the same time, enabling them to produce a synergistic gene silencing effect in the same target cells and improving the drug efficacy of RNAi; it has a wide range of applications and can be used to encapsulate various drugs such as siRNA, mRNA, microRNA, ASO, or DNA. Due to these characteristics, the KKK-KW and KKK-KHW polypeptide delivery systems have broad application prospects in the field of nucleic acid drugs.

[0176] It should be noted that the above embodiments are only preferred embodiments of the present invention, and do not limit the protection scope of the present invention. Any equivalent replacement or substitution made on this basis falls within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide is KW polypeptide; the sequence of the KW polypeptide is as shown in SEQ ID No.

1.

2. Use of the polypeptide according to claim 1 in the preparation of a nucleic acid drug delivery system, characterized in that, The nucleic acid drug is siRNA.

3. Use of the polypeptide according to claim 2 in the preparation of a nucleic acid drug delivery system, characterized in that, The molar ratio of the KW polypeptide to siRNA is 20 to 60:

1.

4. Use of the polypeptide according to claim 2 or 3 in the preparation of a nucleic acid drug delivery system, characterized in that, The application operation is as follows: first dissolve the polypeptide in water with a pH of 2 - 5 to prepare a polypeptide solution with a concentration of 0.5 - 5 mg / mL, mix the nucleic acid drug with the polypeptide according to the molar ratio or mass ratio and let it stand still, then add it to water or a culture medium and let it stand still for 5 - 25 min to obtain a polypeptide complex solution encapsulating the nucleic acid.

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