Polypeptide and application thereof

Abstract

The invention discloses a polypeptide and application thereof. The polypeptide is a KW polypeptide with a sequence as shown in SEQ ID No. 1 or a KHW polypeptide with a sequence as shown in SEQ ID No. 2. The polypeptide in the polypeptide delivery system provided by the invention is composed of natural amino acids, can be biodegraded in cells, does not cause damage to target cells, and ensures the safety of administration; the nano-particles can be wound with RNA through the interaction of static electricity and hydrogen bonds, so that the nano-particles wrapping the RNA are assembled, and the encapsulation efficiency is high; a plurality of different targeted siRNAs can be carried at the same time, so that a synergistic gene silencing effect is generated in the same targeted cell, and the drug curative effect of RNAi is improved; the application range is relatively wide, and the compound can be used for wrapping a plurality of medicines such as siRNA, mRNA, shRNA, microRNA, ASO or DNA (Deoxyribose Nucleic Acid) and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide delivery systems, and in particular to a polypeptide and an application thereof. Background Art

[0002] The characteristics of branched polypeptide macromolecules mainly include the following aspects: (1) they can be entangled with RNA through electrostatic and hydrogen bond interactions to assemble into RNA-encapsulated nanoparticles with high encapsulation efficiency and 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 at the same time, so that they produce a synergistic gene silencing effect in the same targeted cell, thereby improving the drug efficacy of RNAi; (4) they have a wide range of applications and can be used to encapsulate a variety of drugs such as siRNA, miRNA and mRNA; (5) they are composed of natural amino acids, can be biodegraded in cells, and have lower toxicity; (6) they have good water solubility. Due to these characteristics, this type of polypeptide macromolecule has a wide range of potential applications in the biological and medical fields.

[0003] However, the current synthesis of branched peptides has the following problems:

[0004] (1) There are relatively few methods for synthesizing branched polypeptides reported in China.

[0005] (2) In the reported solid-phase peptide synthesis method, when connecting the next amino acid during the synthesis process, the amino acid and the condensation agent are directly added to the peptide reaction tube, which results in incomplete reaction and the generation of by-products.

[0006] (3) The reported solid-phase synthesis method of peptides has low purity and has the problem of trifluoroacetic acid residue. If HPLC is used for purification, the purification efficiency is low and the yield loss is large.

[0007] (4) The reported solid-phase synthesis methods of peptides finally obtain peptides containing water, which need to be removed by freeze-drying technology, which is time-consuming.

[0008] The drug delivery system is a system that delivers drugs to the drug target. Naked nucleic acid drugs are easily degraded by nucleases in plasma and tissues, or are quickly filtered and excreted by the kidneys, which makes the nucleic acid drugs circulate in the body for a short time. Drug delivery systems can usually improve the stability of drugs, reduce drug degradation, increase drug bioavailability, and increase drug concentration in the target area. Therefore, the delivery of nucleic acid drugs requires a suitable carrier to improve delivery efficiency.

[0009] According to different delivery technologies, nucleic acid drug delivery platforms can be divided into: viral vector delivery systems, non-viral vector delivery systems (including lipid-based nanoparticles, GalNAc conjugated 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.

[0010] Peptide-based gene delivery systems have become a new type of non-viral vectors, which have the advantages of better biocompatibility, design diversity, easy synthesis and lower risk of immune response compared with polymer and lipid carriers. However, peptide delivery systems have become a research hotspot in the medical field in recent years, and are expected to provide new ideas for the precision treatment of various major diseases. Suitable peptide delivery systems can encapsulate nucleic acids, protect nucleic acid drugs from degradation by nucleases, and deliver nucleic acid drugs into cells. The present invention aims to provide a safe and effective polypeptide nanoparticle system for the delivery of nucleic acid drugs. Summary of the invention

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

[0012] In order to solve the above technical problems, the technical solution of the present invention is as follows: a polypeptide delivery system encapsulating a nucleic acid drug, wherein the polypeptide structure in the polypeptide delivery system is a KW polypeptide, a KHW polypeptide, a KKK-KW polypeptide or a KKK-KHW polypeptide; wherein the general structural formula of the KKK-KW polypeptide or the KKK-KHW polypeptide is as follows:

[0013]

[0014] Wherein R is a KW polypeptide or a KHW polypeptide; the KW polypeptide sequence is KWHHHKWHHHKWHHHKWHHHK; the KHW polypeptide sequence is KHWHKHWHKHWWHKHWHK.

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

[0016] Furthermore, the positively charged lysine and histidine in the polypeptide are combined with the negatively charged nucleic acid drug through electrostatic interaction to form polypeptide nanoparticles; and the tryptophan in the polypeptide 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. Further preferably, the nucleic acid drug is selected from siRNA or mRNA. The polypeptide and siRNA are entangled with each other to form relatively rough nanoparticles, wherein lysine and histidine are positively charged and bind to the nucleic acid drug through electrostatic action, and tryptophan plays an anchoring role on the cell membrane.

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

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

[0020] The present invention also provides a method for preparing the polypeptide delivery system encapsulating nucleic acid drugs, and the method is operated as follows: firstly, the polypeptide is dissolved in water with a pH of 2-5 to prepare a 0.5-5 mg / mL polypeptide solution, the nucleic acid drug and the polypeptide are mixed according to a molar ratio and allowed to stand, and then water or DMEM culture medium is added and allowed to stand for 5-25 minutes to obtain a polypeptide complex solution encapsulating 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, the application in siRNA transfection and siRNA gene silencing, wherein 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 use of the polypeptide delivery system in preparing medicines for preventing or treating infectious diseases, cancers or metabolic diseases.

[0023] The polypeptides in the polypeptide delivery system provided by the present invention are composed of natural amino acids, can be biodegraded in cells, will not cause unintentional damage to target cells, and ensure the safety of drug administration. There are two main domains in the present polypeptide delivery system: one is the amino acid pairing domain responsible for the self-assembly of the polypeptide, and the other is the cell penetration domain, which is a cell penetration functional group or a group of cationic amino acids. Lysine (K): It is a positively charged alkaline amino acid, and the amino group on the side chain can be dehydrated and condensed with the carboxyl groups of other amino acids to form branched polypeptides. Tryptophan (W) has a strong preference for the membrane interface due to its aromaticity, flat rigid shape and π electron structure, and acts as an anchor point on the cell membrane, thereby enhancing peptide-membrane interactions. Histidine (H) has been shown to enhance the destruction of endosomal membranes through the proton sponge effect.

[0024] The peptide delivery system delivers siRNA and mRNA to many target organs except the liver through local or systemic administration, overcoming the obstacles of drug targeting and stability; and can carry multiple siRNAs with different targets at the same time, so that they produce synergistic gene silencing effects in the same target cell, improving the drug efficacy of RNAi; it has a wide range of applications and can be used to encapsulate a variety of drugs such as siRNA, mRNA, microRNA, ASO or DNA. Due to these characteristics, the KKK-KW and KKK-KHW peptide 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, high encapsulation rate and low toxicity.

[0027] The present invention proves through the 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. The experiment can be completed within 30 minutes at room temperature. The present invention finds through the Malvern particle sizer test that the diameter of the above nanoparticles after encapsulating nucleic acid is about 100-200nm and the encapsulation rate is greater than 90%.

[0028] The nucleic acid-KKK-KW complex disclosed in 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 the commercial reagent Lipo2000, and is equivalent to that of RNAiMAX; the particle size is uniform and the potential is positive; the encapsulation rate is higher than 90%; the cytotoxicity is small, which is much lower than that of Lipo2000.

[0029] The present invention found through cytotoxicity experiments that the cytotoxicity of KKK-KHW is comparable to that of Lipo2000 and RNAiMAX, and has less cytotoxicity. The serum enzyme degradation experiment found that KKK-KHW can effectively protect siRNA and mRNA, and is almost not degraded by serum enzymes after incubation for 6 hours.

[0030] The KKK-KHW of the present invention 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 at low and high siRNA concentrations is better than that of Lipo2000 and RNAiMAX. KKK-KHW delivers siGFP into cells and can knock down EGFP with an inhibition rate of about 40%. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0036] Figure 6 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 is the transfection efficiency of siRNA-KKK-KW complexes at different molar ratios in HEK293 cells;

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

[0039] Fig. 9 is the gene silencing efficiency of in vitro transfection of siRNA-KKK-KW complex;

[0040] Fig.10 is the standard curve of the encapsulation efficiency of siRNA-KKK-KW complex;

[0041] Fig.11 Cytotoxicity and IC of siRNA-KKK-KW complex 50 value;

[0042] Fig.12 It is an agarose gel electrophoresis diagram of mRNA-KKK-KW complex formed by mixing mRNA and KKK-KW polypeptide at different molar ratios;

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

[0044] Fig.14 This is the electrophoresis diagram of the siRNA packaging ability test of KKK-KHW;

[0045] Fig.15 This is the electrophoresis diagram of the mRNA packaging ability test of KKK-KHW;

[0046] Fig.16 is the delivery efficiency of siRNA delivered by KKK-KHW in Vero cells;

[0047] Fig.17 is the delivery efficiency of siRNA delivered by KKK-KHW in Vero cells;

[0048] Fig.18 is the knockdown efficiency of siRNA delivered by KKK-KHW;

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

[0050] Fig. 20 It is the cytotoxicity and IC50 test of KKK-KHW;

[0051] Fig.21 It is an in vitro biological activity test of mRNA delivered by KKK-KW and KKK-KHW. DETAILED DESCRIPTION

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

[0053] Table 1

[0054]

[0055] Example 1

[0056] A method for preparing a four-branched polypeptide R4-KKK-CONH2, the structure of which is shown below:

[0057]

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

[0059] Step 1: Synthesize peptide Fmoc-KKK-OH

[0060] Connect three Fmoc-Lys(Boc)-OHs on dichloro resin: the dichloro resin is swollen, washed with DMF and then connected with the first Fmoc-Lys(Boc)-OH; after the reaction is completed, it is washed, capped and washed in sequence; then it is deprotected and washed again, a Lys condensation solution is prepared, and the first Fmoc-Lys(Boc)-OH is connected; the second Lys step is repeated to connect the third Fmoc-Lys(Boc)-OH, which is washed and cleaved, and precipitated after the cleavage reaction; after the precipitate is dissolved, TFA is removed, and then precipitation and water removal are performed to obtain the polypeptide Fmoc-KKK-OH.

[0061] The amount of each amino acid added in step 1 is 4 eq of dichlororesin;

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

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

[0064] LC-MS result analysis: The precise 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+ , the synthesized Fmoc-KKK-OH structure is correct.

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

[0066] Fmoc-KKK-OH reacts with Fmoc-OSu under alkaline conditions to produce Fmoc4-KKK-OH. Figure 1 shown.

[0067] (1) Dissolve Fmoc-KKK-OH in 10 mL THF + 10 mL MeOH, add 455 μl TEA, and place in a round-bottom flask; add Fmoc-OSu to 10 mL THF and place in a constant pressure dropping funnel, place on the round-bottom flask, and slowly add dropwise for 20 min. After the addition is complete, stir and react at room temperature for 2 h.

[0068] (2) Concentrate the solvent to 5 ml by rotary evaporation, add 30 mL EA and 10 mL PE, mix well, and place in a -20 ° C refrigerator overnight to wait for precipitation.

[0069] (3) Centrifuge at 12000 r / min for 2 min, remove the supernatant, dissolve the solid with 3 mL DCM + 1 mL MeOH by ultrasonic, add 30 mL EA, mix well and centrifuge, remove the supernatant, repeat this step 3 to 4 times, and climb the plate to see whether the impurities have been removed.

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

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

[0072] Step 3: Synthesis of four-branched polypeptide R4-KKK-CONH2

[0073] AM resin is swollen, washed with DMF, deprotected, and washed again, and then a condensation solution is prepared and connected to Fmoc4-KKK-OH. After the reaction is completed, it is washed, capped, and washed in sequence, and then deprotected and washed again. The first amino acid condensation solution of the side chain is prepared and connected to the first side chain amino acid. After washing, the above steps are repeated to synthesize 4 R side chains at the same time. After the reaction is completed, it is washed and cleaved; after the cleavage reaction, precipitation is performed; after the precipitation is dissolved, TFA is removed, and then precipitation and water removal are performed to obtain a four-branched polypeptide R4-KKK-CONH2;

[0074] The cleaning method involved in step 1 and step 3 is: 2 times of DMF cleaning, 2 times of DCM swelling, 2 times of EtOH shrinkage, DCM swelling and DMF cleaning; the amount used should be enough 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 for 10 minutes, then pour into the peptide reaction tube and add DIPEA. The DIPEA preparation process is as follows: DIPEA:DMF volume ratio 174:826, the amount added is 0.6ml for every 0.1mmol amino acid.

[0076] After the cleavage reaction in step 1 and step 3, precipitation is performed; after the precipitate is dissolved, TFA is removed, and then precipitation and water are removed. The specific operation is as follows: after cleavage, precipitation is performed using ice ether, the precipitate is precipitated at 1-5°C, and the precipitate is obtained by centrifugation;

[0077] Removal of TFA from precipitate: Use a small amount of MeOH to ultrasonically dissolve the precipitate after centrifugation, add a trace amount of TEA to react with TFA to form salt, then add it dropwise to 30 times the volume of EA to precipitate, centrifuge at 12000r / min for 2min, dissolve the precipitate after centrifugation with MeOH, add TEA dropwise, then add it dropwise to EA to precipitate and centrifuge, repeat 2 to 3 times until white smoke is no longer emitted when TEA is added, at this time, the TFA residue in the polypeptide is completely removed, and the precipitate after centrifugation is collected.

[0078] After removing TFA from the precipitate, the collected precipitate is ultrasonically dissolved with a small amount of MeOH, then rotary evaporated to a transparent film, and then PE and EA are added to precipitate white turbidity, and the white powdery polypeptide is obtained by spin drying. In this step, the water in the product can be removed by dissolving the precipitate with an organic solvent multiple times and spin drying, thereby achieving the purpose of removing water, and the product does not need to be freeze-dried later.

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

[0080] Example 2 Preparation of KKK-KW polypeptide

[0081] The synthesis method of the four-branched polypeptide KW4-KKK-CONH2, the structure of which is shown below, comprises the following steps:

[0082]

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

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

[0085] Step 3: Synthesis of four-branched polypeptide KW4-KKK-CONH2;

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

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

[0088] HPLC test conditions and results:

[0089] Column: C8 (10*250mm, 5μm); column temperature: 35℃; 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] Example 3 Preparation of KKK-KHW

[0091] The synthesis method of the four-branched polypeptide KHW4-KKK-CONH2, the structure of which is shown below, comprises the following steps:

[0092]

[0093] Step 1: Synthesize peptide Fmoc-KKK-OH

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

[0095] Step 3: Synthesis of four-branched polypeptide KHW4-KKK-CONH2

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

[0097] LC-MS result analysis: The precise 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 structure is correct.

[0098] HPLC test conditions and results:

[0099] Column: C8 (10*250mm, 5μm); column temperature: 35℃; 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: 7.548min; purity: 99.1%.

[0100] Example 4 Preparation of Nucleic Acid Polypeptide Complex

[0101] First, the polypeptide (KW, KHW, KKK-KHW or KKK-KW) is dissolved in water with a pH of 2-4 to prepare a 1 mg / mL polypeptide solution, mRNA, siRNA or siRNA-FAM (200 nM) is mixed with the polypeptide solution according to the molar ratio and allowed to stand for 5 minutes, and then a certain amount of water or DMEM culture medium is added and allowed to stand for 5 to 25 minutes to obtain a 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 positive strand is as follows. The siRNA is synthesized by Qingke Biotechnology.

[0103] Positive strand 5'-3'GGCUACGUCCAGGAGCGCA

[0104] Anti-strand 5'-3'UGCGCUCCUGGACGUAGCC

[0105] Example 5 Study on the Binding Ability of siRNA to KW Peptide and KKK-KW Peptide

[0106] Using agarose gel electrophoresis (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. The principle of gel retardation experiment is: single siRNA is easy to migrate through the gel due to its small size and negative charge; when siRNA and polypeptide are not bound or the binding is incomplete, it is easy to migrate through the gel due to its small size and negative charge, and the fluorescence intensity displayed is strong; when cationic siRNA-peptide complexes contain positive charge and large size, their migration through the gel is hindered, so the free siRNA detected in the agarose gel will be reduced, and the fluorescence intensity displayed will be weakened; when the siRNA-peptide complexes are fully bound, no free siRNA can be detected in the agarose gel, and no fluorescence is displayed.

[0107] like Figure 4 As shown, in lane 1, siRNA easily migrates through the gel due to its small size and negative charge. As the molar ratio increases, the migration of cationic siRNA-KW complexes in the gel is hindered due to their larger size and positive charge, and the fluorescence intensity gradually decreases. When the siRNA:KW molar ratio is 1:30, the siRNA molecules are completely bound to the KW polypeptide, so no free siRNA is detected in the agarose gel. Therefore, when the siRNA:KW molar ratio is greater than 1:30, the siRNA molecules can be completely bound to the KW polypeptide.

[0108] like Figure 6 As shown, the first lane is siRNA. As the molar ratio increases, when the molar ratio of siRNA:KKK-KW is 1:10, the siRNA molecules are completely bound to the KKK-KW polypeptide, so no free siRNA is detected in the agarose gel. Therefore, when the molar ratio of siRNA:KKK-KW is greater than 1:10, the siRNA molecules can completely bind to the KKK-KW polypeptide.

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

[0110] The transfection efficiency of HEK293 and Vero cells was investigated at the same time. Fluorescently labeled siRNA-FAM and KW polypeptide were prepared into siRNA-KW complex at a molar ratio of 1:30, and inoculated into cells in 24-well plates for transfection. After culturing at 37°C for 24 hours, the culture medium was removed, the cells were washed three times with PBS, and the cells were digested with trypsin. 150uL of PBS was added to each well to blow the cells evenly, 20uL of cell suspension was taken and added to the cell counting plate, and the transfection rate was detected using the "GFP mode" of the cell counter Count Star S2.

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

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

[0113] Fluorescently labeled siRNA-FAM (200nM) and KKK-KW polypeptide were prepared 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 hours, the culture medium was removed, the cells were washed three times with PBS, and the cells were digested with trypsin. 150uL of PBS was added to each well to blow the cells evenly, 20uL of the cell suspension was added to a cell counting plate, and the transfection rate 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 4000, and the transfection efficiency was displayed by detecting the fluorescence value.

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

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

[0116] Fluorescently labeled siRNA-FAM (50nM, 100nM, 200nM) was respectively mixed with KKK-KW polypeptide at a molar ratio of 1:16 to prepare siRNA-KKK-KW complexes, which were inoculated into HEK293 cells and Vero cells in 24-well plates for transfection. After culturing at 37°C for 24 hours, the culture medium was removed, the cells were washed three times with PBS, and the cells were digested with trypsin. 150uL of PBS was added to each well to blow the cells evenly, 20uL of the cell suspension was added to a cell counting plate, and the transfection rate 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 4000, and the transfection efficiency was displayed by detecting the fluorescence value.

[0117] like Figure 8 As shown, in HEK293 cells and Vero cells, the transfection efficiency of KKK-KW peptide for siRNA is higher than that of commercial transfection reagents RNAiMAX and lipo2000.

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

[0119] Vero cells were plated at 1.5*10 5The cells / well were inoculated in a 24-well plate and incubated for 24 hours. Next, the PEGFP-N3 plasmid was transfected into Vero cells using the commercial reagent EL, and the cells were washed and replaced after 4 to 6 hours. siGFP (100nM, 200nM) was transfected using different delivery systems Lipo2000, KKK-KW, and RNAiMAX, respectively. After culturing at 37°C for 24 hours, the culture medium was removed, the cells were washed three times with PBS, the cells were digested with trypsin, 150uL of PBS was added to each well to blow the cells evenly, 20uL of cell suspension was taken and added to the cell counting plate, and the transfection rate 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 displayed by detecting the fluorescence value.

[0120] The efficiency of siRNA-KKK-KW complex in silencing EGFP gene at siRNA level was studied by in vitro transfection. RNAiMAX and Lipo2000 were used as positive controls. Fig. 9 As shown, in Vero cells, the siRNA-KKK-KW complex induced EGFP gene silencing efficiency of 53% at a molar ratio of 1:16, the siRNA-RNAiMAX complex induced EGFP gene silencing efficiency of 65%, and the siRNA-Lipo2000 complex induced EGFP gene silencing efficiency of 51%. The results showed that the KKK-KW polypeptide can effectively deliver siRNA to the cytoplasm and induce specific gene silencing, and the gene silencing efficiency of the KKK-KW polypeptide is better than the commercial reagent Lipo2000 and comparable to RNAiMAX.

[0121] Example 10 Particle size, PDI and potential detection of siRNA-KKK-KW complex

[0122] siRNA (200 nM) and KKK-KW polypeptide were prepared into siRNA-KKK-KW complexes according to a series of molar ratios of 1:10, 1:12, 1:14, and 1:16. After standing for 5 min, they were added into enzyme-free sterile water, mixed, and then the particle size, PDI, and 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 potential was 35.65 mV.

[0124] Table 2

[0125]

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

[0127] The encapsulation efficiency of siRNA-KKK-KW complex was detected by Quant-it kit method. Quant-it reagent solution was added to 1×TE buffer to prepare reaction solution, siRNA-KKK-KW complex at different molar ratios was mixed with reaction solution and allowed to stand for 5 min, then added to ELISA plate, and fluorescence value was detected by ELISA instrument at excitation wavelength of 485 nm and emission wavelength of 520 nm.

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

[0129] As shown in Table 3 and Fig.10 As shown, the encapsulation efficiency of the siRNA-KKK-KW complex was greater than 90%, and at a molar ratio of 1:16, the encapsulation efficiency was as high as 94.6%.

[0130] Table 3

[0131]

[0132] Example 12 Cytotoxicity and IC of siRNA-KKK-KW complex 50 Value detection

[0133] Two 96-well plates were plated with Vero cells, 8,000 cells were required per well, and one blank column was left on each plate without cells. The cells were cultured at 37°C for 24 hours before administration. The cytotoxicity test required eight experimental groups, 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. IC 50 The detection experiment was set up with 0.1uM, 0.5uM, 1uM, 10uM, 25uM, 50uM, 75uM, 100uM for the drug group, Cell for the control group, and Blank for the blank group. After culturing for another 24h, the cell plate was taken out, 10uCCK-8 was added to each well, incubated at 37℃ for 1h, and then detected using a 450nm wavelength microplate reader.

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

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

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

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

[0138] like Fig.12 As shown, when the mass ratio of mRNA:KKK-KW is greater than 1:2, the mRNA molecules can completely bind to the KKK-KW polypeptide.

[0139] Example 14 Particle size, PDI and potential detection of mRNA-KKK-KW complex

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

[0141] As shown in Table 4, at the 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 potential was 15.63 mV; at the 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 potential was 19.89 mV.

[0142] Table 4

[0143]

[0144] Example 15 Detection of Encapsulation Efficiency of mRNA-KKK-KW Complex

[0145] The Quant-it kit was used to detect the encapsulation efficiency of the mRNA-KKK-KW complex.

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

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

[0148] Table 5

[0149]

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

[0151] Agarose gel electrophoresis was used to study the ability of KKK-KHW and KHW side chains to encapsulate siRNA at different molar ratios. The molar ratio of siRNA: KKK-KHW was selected as 1:5, 1:6, 1:7, 1:8, 1:9, 1:10; the molar ratio of siRNA: KHW side chain was selected as 1:5, 1:10, 1:16, 1:20, 1:30, 1:40, 1:50, 1:60. Prepare 1.2% agarose gel, dissolve the polypeptide powder into 1ug / uL, mix KKK-KHW and siRNA according to the above molar ratio, let it stand at room temperature for 30min, then add DNA loading buffer, and add the sample to the well. The voltage was 80V, and the gel was placed under a gel imager for observation after running for 1h. The complex completely encapsulated by KKK-KHW would not run out of the gel hole because its particle size was larger than the pore size of the agarose gel. It was determined that siRNA:KKK-KHW was completely wrapped at a molar ratio of 1:5, and siRNA:KHW side chains were completely wrapped at a molar ratio of 1:30. Fig.14 shown.

[0152] Example 17 Detection of mRNA packaging ability by KKK-KHW

[0153] Agarose gel electrophoresis was used to study the mRNA encapsulation ability of KKK-KHW at different mass ratios. The mass ratios of mRNA:KKK-KHW were selected as 2:1, 1:1, 1:2, 1:4, 1:6, and 1:8. Prepare 1.2% agarose gel, dissolve the polypeptide powder into 1ug / uL, mix KKK-KHW and mRNA according to the above mass ratio, let it stand at room temperature for 30min, then add DNA loading buffer and add the sample to the well. The voltage was 80V, and after running the gel for 1h, the gel was placed under a gel imager for observation. The complex completely encapsulated by KKK-KHW will not run out of the gel hole because its particle size is larger than the pore size of the agarose gel. It was determined that mRNA:KKK-KHW was completely encapsulated at a mass ratio of 1:2, such as Fig.15 shown.

[0154] Example 18 Particle size, PDI and potential detection of nanoparticles encapsulating siRNA and mRNA in KKK-KHW

[0155] siRNA: KKK-KHW was prepared in a molar ratio of 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. After standing for 10 minutes, 1 mL of water was added and the mixture was allowed to stand for another 10 minutes. The test solution was added to a disposable folding capillary cuvette using a syringe, and the particle size and potential were detected using a Malvern particle size analyzer. When setting the parameters, protein was selected as the material. The dispersion system was water, the temperature was 25°C, and the equilibrium time was 60 seconds. The particle size of KKK-KHW and siRNA was measured to be 267.3 nm when the molar ratio was 1:5. mRNA: KKK-KHW was prepared in a mass ratio of 1:2 and 1:4, and the solvent was water or DMEM. The nanoparticles composed of siRNA, mRNA, and KKK-KHW were uniform in water and had a small particle size, 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 fluorescent properties when combined with RNA, and the intensity of the fluorescent signal is positively correlated with the RNA concentration, and the concentration of RNA is reflected by the intensity of the fluorescent signal. Take the test kit out of the refrigerator and place it at room temperature for 30 minutes. Dilute 20*TE buffer to 1*TE buffer, and dilute Quant-itreagent with 1*TE buffer. After mixing KKK-KHW with siRNA and mRNA, let it stand for 20 minutes. Take a black 96-well ELISA plate, add 198uL of diluted Quant-itreagentsolution to each well, and then add 2uL of the sample to be tested. Make 6 duplicate wells for each sample, and set up a naked RNA group and a control group of only polypeptides. After incubation for 5 minutes, measure the fluorescence of the sample on a fluorescent ELISA instrument, with a fluorescence excitation wavelength of 485nm and an emission wavelength of 520nm. The results show that the encapsulation efficiency of KKK-KHW-encapsulated siRNA is greater than 95%, and the encapsulation efficiency of KKK-KHW-encapsulated mRNA is about 90%. As shown in Table 8.

[0163] Table 8

[0164]

[0165] Example 20 Detection of the delivery efficiency of siRNA delivered by KKK-KHW in Vero cells

[0166] Use a 1 / 10,000 balance to weigh 1 mg of KKK-KHW into a centrifuge tube, dissolve KKK-KHW with 1 mL of sterile water (pH = 3), and vortex to mix. Add DEPC water to the siRNA-FAM powder according to the label and dissolve to 20uM. Vero cells were plated, with a cell volume of 1.5*10 5 / well, and cultured for 18-24h after plating. The final concentration of siRNA in 24-well plates was 50nM, 100nM, and 200nM. KKK-KHW was mixed with siRNA at a molar ratio of 1:7 and 1:10 and allowed to stand for 5min. After standing, 100uL of opti-MEM medium was added, and the mixture was added to the 24-well plate after standing for another 15min. The control group was Lipo2000 and RNAiMAX groups. The final concentration of siRNA in 24-well plates was 50nM, 100nM, and 200nM. The amount of Lipo2000 and RNAiMAX used at 50nM was 1uL / well, the amount of Lipo2000 and RNAiMAX used at 100nM was 2uL / well, and the amount of Lipo2000 and RNAiMAX used at 200nM was 3uL / well. The transfection method was referred to the instructions. 24h after transfection, discard the culture medium in the 24-well plate, wash the cells 3 times with PBS, discard the PBS, add 100uL of trypsin to each well, rinse and discard the trypsin, digest for 3min. Add 150uL PBS to each well to resuspend the cells, take 20uL of cell solution and add it to the cell counting plate, and analyze it using the GFP module of the Countstar cell analyzer. The results are shown in Fig.16 and Fig.17 As shown, the transfection efficiency of KKK-KHW and KKK-KW was better than that of Lipo2000 and RNAiMAX.

[0167] Example 21 Knockdown efficiency detection of siRNA delivered by KKK-KHW

[0168] Vero cells were plated, with a cell count of 1.5*10 5 / well, cultured for 18-24h after plating. Use transfection reagent EL (TRANS) to transfect pEGFP-N3 into cells according to the instructions, and pEGFP-N3 is 1ug per well. siRNA transfection was performed 6h after plasmid transfection. Wash the plate 3 times with 1*PBS before transfection to eliminate the influence of EL transfection reagent. The final concentration of siGFP in the 24-well plate is 100nM and 200nM. KKK-KHW is mixed with siRNA at a molar ratio of 1:10 and allowed to stand for 5min. After standing, 100uL opti-MEM culture medium is added, and the mixture is added to the 24-well plate after standing for 15min. The control group is Lipo2000 and RNAiMAX group. The culture medium in the 24-well plate is discarded, the cells are washed 3 times with PBS, and the siRNA mixture is added to the well plate. After 24h, the GFP module of the Countstar cell analyzer is used for analysis. The results showed that the siRNA delivered by KKK-KHW could knock down EGFP and inhibit its fluorescent protein expression. Fig.18 shown.

[0169] Example 22 Detection of serum enzyme degradation level of KKK-KHW-siRNA

[0170] KKK-KHW-siRNA experimental group (10% FBS), take 12μL KKK-KHW + 3μL siRNA, mix for 5min. Take 15μL and add 15μL DMEM (50% FBS) to a 200μL EP tube, mix, and incubate at 37℃ for 0h, 2h, and 6h. siRNA control group Take 3μL siRNA and add 12uL DEPC water, take 15μL and add 15μL DMEM (50% FBS) to a 200μL EP tube, mix, and incubate at 37℃ for 0h, 2h, and 6h. Control group Take 12μL KKK-KHW and add 3uL DEPC water, take 15μL and add 15μL DMEM (50% FBS) to a 200μL EP tube, mix, and incubate at 37℃ for 0h, 2h, and 6h. Prepare agarose gel with a concentration of 1%. Before loading, 10uL of sample was taken out of the experimental group and 2uL of 1% SDS was added for depolymerization, mixed and incubated at room temperature for 2min. 10uL of sample was added to each well and agarose gel electrophoresis was performed for 30min. KKK-KHW can effectively protect siRNA from degradation by serum enzymes, such as Fig.19 shown.

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

[0172] Vero cells were plated in 96-well plates, with 8,000 cells per well. The four sides of the 96-well plate were filled with 100uL PBS, leaving one column blank group without cells. Drugs were administered after 24 hours, the final concentration of siRNA was 100nM, and 6 replicates were used for each sample. Cytotoxicity was tested after 24 hours, the cell plate was taken out, 10uCCK-8 was added to each well, and incubated at 37°C for 1 hour; after 1 hour, the plate was tested using a 450nm wavelength microplate reader; the cell survival rate was calculated = (absorbance of the drug group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group) * 100%, and the results are as follows Fig. 20 shown.

[0173] Example 24 In vitro biological activity detection of mRNA delivered by KKK-KW and KKK-KHW

[0174] KKK-KHW, KKK-KW and Lipo2000 transfection reagents were used to introduce mRNA (SARS-CoV-2 Spike protein receptor binding region) into 293A cells. After culture, the supernatant was collected and the expression of RBD protein was detected by direct ELISA. First, HEK293 cells were cultured at 3*10 5The amount of cells / well was inoculated in a 12-well plate, and cultured at 37°C, 5% CO2 for 24 hours before use. KKK-KW and KKK-KHW were dissolved to 1ug / uL, and the mass ratio of mRNA (3ug / well) to KKK-KHW (12ug / well) and KKK-KW (12ug / well) was 1:4. They were mixed in Opti-MEM and allowed to stand at room temperature for 20 minutes before being added to the cell plate. The mass ratio of Lipo2000 to mRNA was 1:1. After mixing according to the instructions of lipo2000, they were allowed to stand at room temperature for 20 minutes before being added to the cell plate. After culturing at 37°C for 24 hours, the cell supernatant was collected by centrifugation, and the expression of RBD protein was detected by ELISA. The standard curve was prepared according to the instructions, and the test sample was prepared. The supernatant of the cells to be tested was diluted to a suitable concentration with 1× dilution buffer, and the dilution multiple was recorded. Test sample processing: plate washing: Wash the plate three times with 1× washing buffer. Sample incubation: add 100μl of control substance, test sample, Lipo2000 Only sample and standard solution of each concentration of standard curve to each well, add 100μl of 1× dilution buffer to ELISA blank control, add to the ELISA plate in duplicate, seal with sealing film, and incubate at 25℃ for 2 hours. Washing: wash the plate three times with 1× washing buffer. Enzyme-labeled detection antibody incubation: except for the ELISA blank control well, add 100μl of detection antibody prepared in advance to the working concentration to each well, add 100μl of 1× dilution buffer to the ELISA blank control, seal with sealing film, and incubate at 25℃ for 1 hour. Washing: wash the plate three times with 1× washing buffer. Color development: add 200μl of pre-prepared substrate solution to each well of the ELISA plate and incubate at 25℃ in the dark for 20 minutes. Termination: add 50μl of stop solution to each well of the ELISA plate. Set the detection wavelength to 450nm on the multifunctional microplate reader and measure its absorbance. Fig.21 The results showed that both KKK-KHW and KKK-KW could deliver mRNA into cells and express it normally, among which KKK-KHW was more effective than 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 by local or systemic administration, overcoming the obstacles of drug targeting and stability; and can carry multiple siRNAs with different targets at the same time, so that they produce a synergistic gene silencing effect in the same target cell, improving the drug efficacy of RNAi; the scope of application is wide, and can be used to encapsulate siRNA, mRNA, microRNA, ASO or DNA and other drugs. 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 are not intended to limit the protection scope of the present invention. Equivalent replacements or substitutions made on the basis of the above embodiments all fall within the protection scope of the present invention.

Claims

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

2.

2. The use of the polypeptide according to claim 1 in delivering nucleic acid drugs, characterized in that: The nucleic acid drug is selected from siRNA, mRNA, shRNA, microRNA, ASO, DNA or plasmid.

3. Use of the polypeptide according to claim 2 in delivering nucleic acid drugs, characterized in that: The nucleic acid drug is selected from siRNA or mRNA.

4. Use of the polypeptide according to claim 3 in delivering nucleic acid drugs, characterized in that: The molar ratio of the KW polypeptide:siRNA is 1-60:1-10.

5. Use of the polypeptide according to claim 3 in delivering nucleic acid drugs, characterized in that: The molar ratio of the KHW polypeptide:siRNA is 1-60:

1.

6. Use of the polypeptide according to any one of claims 1 to 5 in delivering nucleic acid drugs, characterized in that: The method is operated as follows: firstly, the polypeptide is dissolved in water with a pH of 2-5 to prepare a 0.5-5 mg / mL polypeptide solution, the nucleic acid drug and the polypeptide are mixed according to a molar ratio or a mass ratio and allowed to stand, and then added to water or a culture medium and allowed to stand for 5-25 minutes to obtain a polypeptide complex solution encapsulating the nucleic acid.

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