Assembly type protein nano-carrier CABRi for targeted delivery of small interfering nucleic acid as well as preparation method and application of assembly type protein nano-carrier CABRi

Abstract

The invention relates to the field of biological medicine, in particular to an assembled protein nano-carrier CABRi for targeted delivery of small interfering nucleic acid as well as a preparation method and application of the assembled protein nano-carrier CABRi. The assembled protein nano-carrier CABRi (siRNA protein delivery carrier CABRi series) designed by the invention can efficiently load siRNA without sequence selectivity, and can be mixed with one or more siRNAs according to a certain proportion to form a carrier / small nucleic acid compound CABRi-siRNA, and the carrier / small nucleic acid compound CABRi-siRNA is self-assembled into a nano particle with the size of 10-100nm. The invention constructs a brand new siRNA delivery carrier, breaks through three key technical bottlenecks in siRNA drug delivery, namely the problems of extremely low siRNA in-vivo delivery efficiency, endosome escape and extrahepatic delivery, and finally achieves the purposes of efficient delivery, endosome escape, targeting of extrahepatic organs or tumors, high safety and easy industrialization of siRNA drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, and a preparation method and application thereof. Background Art

[0002] Since scientists discovered in 1998 that double-stranded RNA can specifically silence gene expression, RNA interference (RNAi) as a post-transcriptional gene silencing method has shown great potential and application prospects in the treatment of various diseases. At the same time, with the in-depth study of genomes and the rapid development of sequencing technology, gene therapy has become one of the revolutionary biological treatment options. In theory, siRNA can specifically and effectively inhibit any gene by directly targeting and reducing the expression of mRNA. Since Alnylam's first siRNA drug Patisiran was successfully launched in the United States on August 10, 2018, 6 siRNA drugs have also been successfully launched in succession, but China has not yet approved the launch of fully independently developed siRNA drugs.

[0003] Global siRNA drug research and development is still in the early stages of industry development. There are still many technical bottlenecks in the development of siRNA drugs, especially the problem of low siRNA delivery efficiency. At present, commonly used siRNA delivery systems include viral vectors and non-viral vectors. It is well known that viral vectors have high siRNA delivery efficiency, but viral vectors will integrate into the genome, which may lead to many unpredictable consequences and cause many immune reactions and liver toxicity. Therefore, the application of siRNA drugs with viral vectors is greatly limited. In contrast, non-viral vectors include lipid nanoparticles (LNP), polymer nanoparticles, polypeptide complexes, and antibody oligonucleotide drug conjugates (AOCs). They are characterized by high safety, but siRNA targeting, delivery efficiency, endosomal escape, and safety need to be improved. Patisiran, the world's first siRNA drug approved for marketing, uses an LNP delivery system to encapsulate siRNA drugs in LNPs and administer them by intravenous injection. Liposome encapsulation greatly improves the stability of the drug and its targeting to liver tissue, ensuring that siRNA is not filtered and cleared by the kidneys, and is gradually taken up by target cells in liver tissue during blood circulation. This is the key to Patisiran's approval for marketing by overcoming delivery factors. Inclisiran, the second small nucleic acid drug approved for marketing in December 2020, has a specially designed GalNAc-siRNA delivery system. Its principle is that N-acetylgalactosamine (GalNAc) can specifically bind to the asialoglycoprotein receptor (ASGPR) on the liver cell membrane, thereby specifically targeting siRNA to the liver. These two small nucleic acid drugs on the market have been successfully marketed due to their special delivery methods.

[0004] Although the above-mentioned siRNA delivery methods on the market are relatively successful, there are still many problems. For example, the current LNP-siRNA delivery system will add PEG lipids when used, which will cause excessive immune response and increase toxicity in the body, which is the main problem limiting clinical application. Therefore, LNP-mediated siRNA drugs (such as Patisiran) need to be pretreated with steroids and antihistamines before intravenous injection to eliminate unnecessary allergic reactions. GalNAc-modified siRNA drugs (such as Inclisiran) can only specifically target the liver and are not suitable for other organs, tissue lesions or tumors, which greatly limits the application scenarios of siRNA drugs. At present, there are three key technical bottlenecks in siRNA drug delivery: 1) The problem of low efficiency of siRNA delivery in vivo. How to design siRNA and delivery carrier to form a stable and effective complex is the key to drug development; 2) The problem of endosomal escape. For example, with the siRNA drug delivery technology LNP or GalNAC on the market, only less than 0.1-1% of siRNA can escape from the endosome, and how to improve siRNA endosomal escape is also the most critical; 3) The problem of extrahepatic tissue delivery. For example, LNP and GalNAc currently only provide liver delivery effects, and how to solve the specific targeted delivery of siRNA in organs, tissues or tumors is also a technical difficulty that urgently needs to be solved.

[0005] Specific artificially designed proteins have great potential as siRNA delivery vectors. Protein vectors are composed of natural amino acids and have the advantages of being non-toxic, easily degradable, and not prone to causing immune responses. These vectors can be obtained by various methods, including prokaryotic expression, eukaryotic expression, or in vitro cell-free expression. Genetic recombination technology can be used to give protein vectors the ability to target tissue cells and modify the functional domains that bind to siRNA, thereby enhancing their siRNA delivery ability. Using mature recombinant protein technology, the complex structure of protein vectors can be precisely controlled, enabling them to protect siRNA from degradation by RNases, overcome cell membrane barriers, enhance cell uptake of siRNA, promote endosomal escape, and ultimately exert the expected siRNA biological activity.

[0006] It is worth noting that the current protein-based siRNA delivery system is still mostly in the stage of theoretical research and animal experiments, and there are still many challenges to overcome in the clinical application of siRNA protein delivery vectors. First, the currently reported protein-delivered siRNA drugs are usually administered locally or intravenously, and oral bioavailability is limited. Secondly, although the fusion expression of domains with different functions can be achieved through recombinant protein technology, how to ensure that the function of a single domain within the recombinant protein is not affected remains an unresolved issue. Finally, the circulation half-life of protein-delivered siRNA drugs is short, and how to improve structural stability and prolong drug half-life also requires special attention.

[0007] Based on the above technical obstacles, it is particularly important to develop a delivery system with efficient delivery, endosomal escape, targeting of extrahepatic organs or tumors, and high safety. Summary of the invention

[0008] The purpose of the present invention is to provide an assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids and its preparation method and application to solve the problems existing in the above-mentioned prior art. The assembled protein nanocarrier CABRi provided by the present invention has the advantages of efficient delivery, endosomal escape, targeting extrahepatic organs or tumors and high safety.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides an assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, wherein the assembled protein nanocarrier CABRi comprises an assembled functional peptide (Assembled Functional Peptide, AFP), a linker and a small RNA binding protein (Small RNA Binding Protein / Peptide Segment, sRBP); in the present invention, the AFP refers to a cell-penetrating peptide (CPP) or a pH-responsive endosome escape peptide (EEP), or a specific targeting peptide (STP) of an organ or tumor tissue having the functional properties;

[0011] The assembled functional peptides include polypeptides (CPP / STP) with membrane-penetrating and pH-responsive functions and / or specific targeting peptides (STP); the amino acid sequence of the polypeptide is any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.13; the amino acid sequence of the specific targeting peptide is any one of the sequences shown in SEQ ID NO.14-SEQ ID NO.24;

[0012] The linker includes a flexible linker (FL) and / or a rigid linker (RL); the number of amino acid residues of the flexible linker is 5 or 15; the amino acids of the flexible linker are selected from glycine and serine; the flexible linker connects CPP / EEP / STP with sRBP, or connects two sRBPs; the amino acid sequence of the flexible linker is shown in SEQ ID NO.25 or SEQ ID NO.26; the number of amino acid residues of the rigid linker is 12 or 15, and the amino acids of the rigid linker are selected from alanine, glutamic acid and lysine; the rigid linker connects CPP / EEP with STP; the amino acid sequence of the rigid linker is shown in SEQ ID NO.27 or SEQ ID NO.28;

[0013] The small RNA binding protein is derived from a double-stranded RNA binding domain (dsRNA-binding domain, DRBM or dsRBD); the amino acid sequence of the small RNA binding protein is any one of the sequences shown in SEQ ID NO.29-SEQ ID NO.35.

[0014] The assembled functional peptides described in the present invention can achieve three functions: (1) as a cell-penetrating peptide (CPP), it can penetrate the membrane into the cell; (2) some CPPs can also serve as pH-responsive endosome escape peptides (EEP), achieving pH-responsive protonation or helical conversion, thereby escaping the endosome and effectively releasing siRNA into the cytoplasm to exert gene silencing function; (3) as a short peptide ligand, specific targeting peptides (STP) of organs or tumor tissues, it can achieve binding to specific receptors on the surface of organ tissues or tumor cells, and precisely deliver siRNA in a targeted manner.

[0015] The sRBP of the present invention is a kind of protein-siRNA complex that is combined with siRNA molecules through a specific protein tertiary structure to form a stable protein-siRNA complex, which can achieve efficient delivery to cells in tissues. The sRBP of the present invention can be two or more repeated copies, which are connected by a flexible linker with an amino acid sequence of SEQ ID NO.25 or SEQ ID NO.26, which can ensure the functional independence of each peptide domain and spontaneously form domain dimers or polymers inside the protein.

[0016] Preferably, the copy numbers of the assembled functional peptide, the linker and the small RNA binding protein are all ≥1; the basic vector of the assembled protein nanocarrier CABRi includes a prokaryotic system expression vector and a eukaryotic system expression vector.

[0017] The assembled protein nanocarrier CABRi of the present invention is to form a complete CABRi protein delivery carrier by assembling functional peptides, linkers (flexible linkers and / or rigid linkers) and small RNA binding proteins in different assembly arrangements, including but not limited to cell-penetrating peptides or peptides that simultaneously respond to pH endosome escape, organ or tumor tissue-specific targeting peptides, flexible or rigid linkers and small RNA binding proteins / peptide segments, etc., so as to obtain a recombinant protein carrier CABRi series that can efficiently deliver siRNA. The assembled protein nanocarrier CABRi can self-assemble with siRNA into nanoparticles with a nanometer size in the range of 10-100nm, has a high deposition rate in the human respiratory system, can pass through capillaries or endothelial cells to achieve efficient and specific targeting of organ tissues (such as lungs and pancreas) or tumor tissues, and can also achieve targeted delivery of siRNA to organ tissues or tumors. Most CABRi-siRNA nanoparticles have an average diameter of about 30-40nm or 50-70nm, and atomized inhalation has good deposition in the lungs.

[0018] The assembled protein nanocarrier CABRi of the present invention can deliver one or more siRNAs into cells simultaneously by effectively loading various siRNAs into the protein carrier; siRNA can be efficiently released from the endosome, and further degrade the pathogenic mRNA or inhibit the protein translation of mRNA through the siRNA-induced silencing complex (siRISC), thereby exerting the effect of siRNA. Moreover, the assembled protein nanocarrier CABRi of the present invention can efficiently load siRNA without sequence selectivity, while protecting siRNA, and achieve efficient delivery.

[0019] At the same time, the siRNA of the present invention can be unmodified or chemically modified. Chemically modified siRNA has better effects, enhances siRNA stability, strong resistance to nucleases and high delivery efficiency; and reduces the immunogenicity of small nucleic acids. These chemical modifications include 2-O-Methyl Base2-methoxy modified base (2-ome), 2-Fluoro RNA2-fluoro RNA (2-FRNA), Phosphorothioate thio modification (PS), etc.

[0020] The present invention provides a method for preparing the above-mentioned assembled protein nanocarrier CABRi, comprising the following steps:

[0021] The assembled functional peptide, the linker and the small RNA binding protein are connected by seamless cloning technology to obtain a CABRi protein;

[0022] The CABRi protein is cloned into a basic vector to obtain the assembled protein nanocarrier CABRi.

[0023] Preferably, the copy numbers of the assembled functional peptide, the linker and the small RNA binding protein are all ≥1; and the basic vector includes a prokaryotic system expression vector and a eukaryotic system expression vector.

[0024] Further preferably, the purification of the assembled protein nanocarrier CABRi is carried out by a two-step method, including two of His affinity purification method, GST affinity purification method, ion exchange chromatography and gel filtration chromatography (molecular sieve).

[0025] The present invention provides the use of the above-mentioned assembled protein nanocarrier CABRi in the preparation of siRNA drug delivery systems, siRNA nanocomplexes and targeted drugs.

[0026] The present invention provides a siRNA drug delivery system, which comprises the above-mentioned assembled protein nanocarrier CABRi.

[0027] The present invention provides a siRNA nanocomplex, wherein the siRNA nanocomplex comprises the above-mentioned assembled protein nanocarrier CABRi and siRNA.

[0028] The CABRi-siRNA nanocomplex provided by the present invention is a nanoparticle of suitable size (10-100nm) and strong stability, which can be used for in vivo delivery of siRNA. The complex has high safety and low toxicity. After releasing siRNA within 12-48 hours after entering the cell, the CABRi protein itself is rapidly degraded. At the same time, the complex has the advantages of easy production and low cost.

[0029] The present invention provides a method for preparing the above-mentioned siRNA nanocomplex, comprising the steps of mixing the above-mentioned assembled protein nanocarrier CABRi and siRNA, and incubating them to obtain the siRNA nanocomplex.

[0030] Further preferably, the molar ratio of CABRi protein to siRNA in the assembled protein nanocarrier is 4:1.

[0031] Further preferably, the pH of the system obtained by mixing the assembled protein nanocarrier CABRi and siRNA is 7.4; the incubation time is 30 minutes, and the temperature is room temperature.

[0032] The present invention provides a targeted drug, which comprises the above-mentioned assembled protein nanocarrier CABRi and siRNA.

[0033] Preferably, the siRNA includes one or more of siKRAS, siSHP2 and siSOS1;

[0034] The sense chain sequence of the siKRAS is shown in SEQ ID NO.36, and the antisense chain sequence is shown in SEQ ID NO.37; the sense chain sequence of the siSHP2 is shown in SEQ ID NO.40, and the antisense chain sequence is shown in SEQ ID NO.41; the sense chain sequence of the siSOS1 is shown in SEQ ID NO.42, and the antisense chain sequence is shown in SEQ ID NO.43.

[0035] Further preferably, the drug comprises the above-mentioned assembled protein nanocarrier CABRi and an effective amount of siRNA.

[0036] Further preferably, the drug is administered by atomization, intravenous injection or subcutaneous administration.

[0037] The present invention discloses the following technical effects:

[0038] The CABRi series of siRNA protein delivery vectors designed by the present invention can efficiently load non-sequence selective siRNA, can be mixed with one or more siRNAs in a certain ratio to form a vector / small nucleic acid complex CABRi-siRNA, and self-assemble into a 10-100nm nanoparticle. The AFP fused to the end of CABRi has three functional properties: penetrating the membrane, binding to a ligand of a specific cell surface receptor, and escaping the endosome, which can achieve strong resistance to nucleases and high delivery efficiency in the body; achieve specific targeting of organ tissues (such as lungs and pancreas, etc.) or tumor tissues, etc.; pH acidic response protonation or α-helix conversion promotes siRNA to efficiently escape the endosome, effectively release siRNA into the cytoplasm, and then play a gene silencing function. Regarding the safety of CABRi-siRNA, the CABRi protein itself has low immunogenicity and can be rapidly degraded; siRNA is chemically modified to reduce immunogenicity. The pharmaceutical dosage form of CABRi-siRNA designed by the present invention can meet the intravenous, subcutaneous or oral administration methods, and effectively avoid glomerular clearance and circulatory system clearance. The present invention constructs a new siRNA delivery vector, breaks through the three key technical bottlenecks in siRNA drug delivery, and overcomes the extremely low efficiency of siRNA in vivo delivery, endosomal escape and extrahepatic delivery problems, and finally achieves the purpose of efficient delivery, endosomal escape, targeting extrahepatic organs or tumors, high safety and easy industrialization of siRNA drugs. At the same time, the present invention is a new method for siRNA drug delivery, which can be applied to the treatment of various diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (IF), lung cancer, pancreatic cancer and colorectal cancer and other malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 CABRi-3 protein was Ni 2+-NTA affinity chromatography Coomassie Brilliant Blue staining; Marker is a protein standard; Cell pellets is a cell pellet; Cell lysate is a cell lysate supernatant; Flow through is a flow-through solution; Wash is a rinse solution; Elution-first is the first elution solution; Elution-second is the second elution solution; Elution-third is the third elution solution; Elution-fourth is the fourth elution solution; Elution-fifth is the fifth elution solution; Elution-last is the last elution solution; Elution-total is the total elution solution;

[0041] Figure 2 CABRi-8 protein was Ni 2+ -NTA affinity chromatography Coomassie Brilliant Blue staining; Marker is a protein standard; Cell lysate is the supernatant of cell lysate; Flow through is the flow-through solution; Wash is the rinse solution; Elution-first is the first elution solution; Elution-last is the last elution solution; Elution-total is the total elution solution;

[0042] Figure 3 CABRi-12 protein was Ni 2+ -NTA affinity chromatography Coomassie Brilliant Blue staining; Marker is a protein standard; Cell lysate is the supernatant of cell lysate; Flow through is the flow-through solution; Wash is the rinse solution; Elution-first is the first elution solution; Elution-last is the last elution solution; Elution-total is the total elution solution;

[0043] Figure 4 This is the separation and purification curve of CABRi-8 by cation exchange column;

[0044] Figure 5 The CABRi-8 was separated and purified by a cation exchange column and then stained with Coomassie Brilliant Blue; Marker is a protein standard; Purified sample is a purified sample; Flow through is the flow-through liquid; Peak 1 is the first peak, A05-A09 are A05 collection tube, A06 collection tube, A07 collection tube, A08 collection tube and A09 collection tube respectively; Peak 2 is the second peak, A10 is A10 collection tube, B05-B10 are B05 collection tube, B06 collection tube, B07 collection tube, B08 collection tube, B09 collection tube and B10 collection tube respectively;

[0045] Figure 6 The particle size and potential of CABRi-8 self-formed nanoparticles; the green line is the first measurement curve, the orange line is the second measurement curve, and the blue line is the third measurement curve;

[0046] Figure 7 The particle size and potential of CABRi-12 self-formed nanoparticles; the green line is the first measurement curve, the orange line is the second measurement curve, and the blue line is the third measurement curve;

[0047] Figure 8 The particle size and potential of CABRi-8-siRNA nanoparticles; the green line is the first measurement curve, the orange line is the second measurement curve, and the blue line is the third measurement curve;

[0048] Fig. 9 The particle size and potential of CABRi-12-siRNA nanoparticles; the green line is the first measurement curve, the orange line is the second measurement curve, and the blue line is the third measurement curve;

[0049] Fig.10 EMSA was used to detect the binding efficiency of CABRi-8 and siRNA;

[0050] Fig.11 EMSA was used to detect the binding efficiency of CABRi-12 and siRNA;

[0051] Fig.12 To test the protection of siRNA by CABRi-8-siRNA nanoparticles for EMSA experiments;

[0052] Fig.13 Northern blotting was used to detect the delivery of siRNA into cells by CABRi-8 vector;

[0053] Fig.14 Confocal microscopy live cell imaging technology was used to detect the escape of CABRi-8-siRNA nanoparticles from endosomes / lysosomes and the release of siRNA into the cytoplasm;

[0054] Fig.15 Western Blotting experiments were used to detect whether CABRi-8-siKRAS nanoparticles reduced the expression level of the target gene KRAS in cells;

[0055] Fig.16 This is a plate cloning experiment to detect the inhibition of H1975 cell proliferation by CABRi-12-siRNAs; A is a culture graph; B is a H1975 cell statistics graph;

[0056] Fig.17 This is a fluorescence imaging experiment to detect the delivery of siRNA to tumor tissue by CABRi-12 vector; Lung is lung; Heart is heart; Tumor is tumor; Liver is liver; Spleen is spleen; Kidney is kidney;

[0057] Fig.18 The figure shows the effect of CABRi-12-siRNAs on inhibiting the growth of malignant tumors in mice; A is a statistical graph of tumor volume; B is a statistical graph of tumor mass;

[0058] Fig.19 H&E staining was used to detect the toxic and side effects of the CABRi-12 delivery system on normal tissues and organs, wherein A is the H&E staining image of the Lung, Liver, Brain and Intestine under different treatments; B is the H&E staining image of the Spleen, Kidney, Heart and Muscle under different treatments. DETAILED DESCRIPTION

[0059] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0060] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0061] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0062] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0063] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0064] The partial sequence involved in the present invention is as follows:

[0065] The sequence of CPP / EEP is selected from one of SEQ ID NO.1-13:

[0066] SEQ ID NO.1: VSRRRRGGRRRRRR;

[0067] SEQ ID NO.2:RRRRRRRRKKR;

[0068] SEQ ID NO.3: KVVVVKVVVVKVVVVKVVVVK;

[0069] SEQ ID NO.4: KLLLLKLLLLKLLLLKLLLLK;

[0070] SEQ ID NO.5: KLALKLALKALKAALKLA;

[0071] SEQ ID NO.6: KKALLAHALHLLALLALHLAHALKKA;

[0072] SEQ ID NO.7: KKALLALALHHLAHLALHLALALKKA;

[0073] SEQ ID NO.8: WEAKLAKALAKALAKHLAKALAKALKACEA;

[0074] SEQ ID NO.9: LIRLWSHLIHIWFQNRRLKWKKK;

[0075] SEQ ID NO.10: GLWRALWRLLRSLWRLLWRA;

[0076] SEQ ID NO.11: LLRLLRWWWRLLRLL;

[0077] SEQ ID NO.12: WEAALAEALAEALAEHLAEALAEALEALAA;

[0078] SEQ ID NO.13: HHEHHEHHEHHEHHEHHEHHEHHEHHE;

[0079] The sequence of STP is selected from one of SEQ ID NO.14-24:

[0080] SEQ ID NO.14: HLNILSTLWKYR;

[0081] SEQ ID NO.15: KLLLLKLLLLKLLLLKLLLLK;

[0082] SEQ ID NO.16: KVVVVKVVVVKVVVVKVVVVK;

[0083] SEQ ID NO.17: VVVVVVKKGRGDS;

[0084] SEQ ID NO.18: GRGDSGRGDS;

[0085] SEQ ID NO.19:CDCRGDCFC;

[0086] SEQ ID NO.20: SHSFSVGSGDHSPFT;

[0087] SEQ ID NO.21: GRFLTGGTGRLLRIS;

[0088] SEQ ID NO.22: KPVSLSYRSPSRFFESH;

[0089] SEQ ID NO.23: APWHLSSQYSRT;

[0090] SEQ ID NO.24:DMPGTVLP;

[0091] The sequence of FL is selected from one of SEQ ID NO.25-26:

[0092] SEQ ID NO.25: GSSGG;

[0093] SEQ ID NO.26: GGGGSGGGGGSSSGG;

[0094] The amino acid sequence of RL is selected from one of SEQ ID NOs: 27-28:

[0095] SEQ ID NO.27: AEAAAKEAAAKA;

[0096] SEQ ID NO.28: EAAAKEAAAKEAAAK;

[0097] The amino acid sequence of sRBP is selected from one of those shown in SEQ ID NOs. 29 - 35:

[0098] SEQ ID NO.29 (Human PKR / E2AK2 DRBM1 / 2):

[0099] MAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAK

[0100] LAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRLTVNYEQCASGVHGPEGFHYKCKMGQ KEYSIGTGSTKQEAKQLAAKLAYLQILSEETSV;

[0101] SEQ ID NO.30 (RNA silencing suppressor 1):

[0102] MERAIQGNDTREQANGERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKV

[0103] VFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLCEMAI RSKQELLQLTPVEVESNVSRGCPEGIETFKKESE;

[0104] SEQ ID NO.31 (RNA silencing suppressor2):

[0105] MERAIQGNDTREQAKRERWDGGSGGITSPFKLPDESPSWTEWRLYNDETNSNQDNPLGFKESWGFGKV

[0106] VFKRYLRYDRTEASLHRVLGSWTGDSVNYAASRFLGANQVGCTYSIRFRGVSVTISGGSRTLQHLSEMAI RSKQELLQLTPVEVESNVSRGAPEGIETFKKESE;

[0107] SEQ ID NO.32(Human TARBP2 DRBM1):

[0108] TPISLLQEYGTRIGKTPVYDLLKAEGQAHQPNFTFRVTVGDTSCTGQGPSKKAAKHKAAEVALKHLKG;

[0109] SEQ ID NO.33 (Human TARBP2 DRBM2):

[0110] NPVSPQQSECNPVGALQELVVQKGWRLPEYTVTQESGPAHRKEFTMTCRVERFIEIGSGTSKKLAKRNAA AKmLLRVHT;

[0111] SEQ ID NO.34 (Human Dicer dsRNA-binding fold):

[0112] AIGHINRYCARLPSDPFTHLAPKCRTRELLPDGTFYSTLYLPINSPLRASIVGPPMSCVRLAERVVALICCEKL HKIGELDDHLMPVGKETVKY;

[0113] SEQ ID NO.35(Human Dicer DRBM):

[0114] VPRSPVRELLEMEPETAKFSPAERTYDGKVRVTVEVVGKGKFKGVGRSYRIAKSAAARRALRSLKA.

[0115] Example 1 Construction of CABRi series of siRNA targeted delivery protein nanocarriers

[0116] The overall scheme for constructing the CABRi series of vectors: The CABRi series of siRNA protein delivery vectors provided by the present invention utilize seamless cloning technology and artificial gene synthesis technology to assemble the corresponding nucleotide sequences of FL / RL, sRBP, AFP (CPP / EEP / STP) together and insert them into prokaryotic expression vectors such as pET-28a and pGEX-4T-1, etc., and can also be inserted into eukaryotic expression vectors such as pcDNA3.1 V5-His A, pcDNA3.1-3xFlag and pcDNA3.1-3xHA. A series of vectors can be constructed from the above different combinations, named CABRi-1 vector, CABRi-2 vector, ... CABRi-99 vector, and the optimal expression plasmid of the desired siRNA protein delivery vector CABRi is finally determined through a series of screening such as the level of protein expression, the ease of purification and the final yield.

[0117] In this example, CABRi-3 vector, CABRi-8 vector and CABRi-12 vector are used as examples to clone the designed CABRi nucleotide sequence into the prokaryotic expression vector pET-28a(+), with the insertion sites being NheI and BamHI. The N-terminus of the CABRi-3 vector is 6×His / HA, the former is used for protein purification, and the latter is used for protein carrier identification and in vivo recognition; the middle segment contains an sRBP (SEQ ID NO.31); the C-terminus is AFP, which is composed of CPP / EEP (SEQ ID NO.6); the N-terminal region and the middle segment sRBP are connected by FL (SEQ ID NO.25), and the middle segment sRBP and the C-terminal CPP / EEP are connected by FL (SEQ ID NO.26). The amino acid sequence of the CABRi-3 protein in the CABRi-3 vector is obtained by seamlessly connecting the following parts (no bases are inserted between the sequences), specifically: FL (SEQ ID NO.25)-sRBP (SEQ ID NO.31)-FL (SEQ IDNO.26)-AFP (SEQ ID NO.6). The CABRi-8 vector is based on CABRi-3. UltraOne Step Cloning Kit, another duplicate copy of sRBP (SEQ ID NO.NO.31)-FL (SEQ ID NO.NO.26) was inserted into the middle region, thus forming two sRBPs, and the amino acid sequence of the CABRi-8 protein in the CABRi-8 vector was obtained by seamlessly connecting the following parts (no bases need to be inserted between the sequences), specifically: FL (SEQ ID NO.25)-sRBP (SEQ ID NO.31)-FL (SEQ ID NO.26)-sRBP (SEQ ID NO.31)-FL (SEQ ID NO.26)-AFP (SEQID NO.6). The CABRi-12 vector is based on CABRi-8. It also uses seamless cloning technology to add STP (SEQ ID NO.18) to CPP / EEP (SEQ ID NO.NO.6), and then connect them with RL (SEQ ID NO.27); the amino acid sequence of CABRi-12 in the CABRi-12 vector is obtained by seamlessly connecting the following parts (no bases need to be inserted between the sequences), specifically: FL (SEQ ID NO.25) -sRBP (SEQ ID NO.31) -FL (SEQ ID NO.26) -sRBP (SEQ ID NO.31) -FL (SEQ ID NO.26) -AFP (SEQ ID NO.6) -RL (SEQ ID NO.27) -STP (SEQID NO.18).

[0118] Example 2 Expression and purification of siRNA targeted delivery protein nanocarrier CABRi series

[0119] Taking the CABRi-3 vector, CABRi-8 vector and CABRi-12 vector constructed in Example 1 as an example, these expression plasmids were transferred into the E. coli expression strain BL21 (DE3) for culture. When the bacteria grew to an OD value of 0.6, IPTG with a final concentration of 0.2 mM was used for induction at 16°C for 16-20 hours.

[0120] After the bacterial solution was collected, it was ultrasonically disrupted and lysed in sequence, and then the first round of Ni 2+ -NTA affinity chromatography column was used to separate the purified histidine-tagged protein, and then SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining were performed. The results of Coomassie brilliant blue staining were as follows: Figure 1-Figure 3 As shown, Figure 1-Figure 3The Coomassie Brilliant Blue staining results for CABRi-3 protein, CABRi-8 protein, and CABRi-12 protein, respectively. The components shown in the figure are: Cell pellets are collected cells, broken by adding lysis buffer, and then centrifuged at high speed. The precipitate is fully resuspended in an equal amount of lysis buffer and then sampled; Cell lysate is collected cells, broken by adding lysis buffer, and then centrifuged at high speed and sampled; Flow through is the flow-through liquid, which is the supernatant of the cell lysate after Ni 2+ -The effluent after NTA; Wash is the rinsing solution, which is used to rinse Ni with Washbuffer 2+ -NTA post-rinsing buffer; Elution-first is the first elution buffer, which is 6 ml of Elution buffer to elute Ni 2+ -The eluate collected after NTA; Elution-second is the second eluate, which is eluted with 6 ml of Elution buffer. 2+ -The eluate collected after NTA; Elution-third is the third eluate, which is eluted with 8ml Elution buffer. 2+ -The eluate collected after NTA; Elution-fourth is the fourth eluate, which is eluted with 10ml Elution buffer. 2+ -The eluate collected after NTA; Elution-fifth is the fifth eluate, which is eluted with 10 ml Elution buffer. 2+ -The eluate collected after NTA; Elution-last is the last eluate, which is eluted with 10ml Elution buffer. 2+ -The eluate collected after NTA; Elution-total is the total eluate, which is sampled by combining the eluates collected above.

[0121] Depend on Figure 1-Figure 3 It can be seen that after the first step Ni 2+ After separation by -NTA affinity chromatography column, the target protein with a purity greater than 50% can be obtained.

[0122] Taking CABRi-8 as an example, after the first round of Ni 2+ -NTA affinity chromatography was used for separation and protein purification (model UEV-25M Yonglian Bio) and cation exchange column (HiTrap TM SP / HP) for the second round of separation and purification, the cation exchange column separation and purification curve is as follows Figure 4 As shown. Figure 4It can be seen that the CABRi-8 protein has two elution peaks (Peak 1 and Peak 2). At the same time, the collection tubes corresponding to the CABRi-8 protein elution peaks were subjected to SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. The results are as follows: Figure 5 As shown, the CABRi-8 protein with a purity greater than 95% was finally obtained and stored at -80°C for future use.

[0123] Example 3 Preparation and properties of a mixture of nanoscale targeted delivery protein carrier CABRi and siRNA

[0124] The CABRi series can form nanoparticles by themselves. For example, the particle sizes and potentials of the nanoparticles formed by CABRi-8 and CABRi-8-siRNA are as follows: Figure 6 and Figure 8 As shown; the particle size and potential of the nanoparticles formed by CABRi-12 and CABRi-12-siRNA are shown in Figure 7 and Fig. 9 The above values ​​are summarized in Table 1.

[0125] Table 1 Particle size and potential of nanoparticles formed by CABRi-8 carrier

[0126] Particle size (d, nm) Potential (mV) CABRi-8 Nanoparticles 42.3±0.1297 -6.124±0.9497 CABRi-12 Nanoparticles 79.24±26.55 -11.04±4.402 CABRi-8-siRNA nanoparticles 42.21±5.092 -7.258±1.673 CABRi-12-siRNA nanoparticles 54.03±1.968 -19.31±0.9709

[0127] The synthesized siRNA is a specific siRNA designed with human KRAS as the target (denoted as siKRAS) as an example. The siRNA is dissolved in DEPC water to a final concentration of 20 μM and stored at -20°C.

[0128] The sequences of the sense and antisense strands of siKRAS and siControl are as follows:

[0129] siKRAS positive strand: 5′-GUUGAUUACUUCUUAUUUUUC-3′, SEQ ID NO. 36;

[0130] siKRAS antisense strand: 5'-GAAAAAUAAGAAGUAAUCAACUG-3', SEQ ID NO.37;

[0131] siControl positive strand: 5'-CAUCAAGCUGGAGUGUCUCGC-3', SEQ ID NO.38;

[0132] siControl antisense strand: 5′-GCGAGACACUCCAGCUUGAUGUG-3′, SEQ ID NO.39.

[0133] The prepared CABRi protein and siRNA were mixed at different molar ratios (such as siRNA: CABRi = 1:4 or 1:8, etc.). In this embodiment, the prepared CABRi protein and siRNA were mixed at a molar ratio of 4:1, the reaction system was pH = 7.4, and the reaction was carried out at room temperature for 30 minutes to obtain CABRi-siRNA mixture molecules (CABRi-8-siRNA and CABRi-12-siRNA) to form nanoparticles of suitable size and strong stability. Figure 8 , Fig. 9 As shown in Table 1, the particle sizes of CABRi-8-siRNA nanoparticles and CABRi-12-siRNA nanoparticles are about 42.21 nm and 54.03 nm, respectively, and the potentials are about -7.258 and -19.31, respectively, which can be used for in vivo delivery of siRNA.

[0134] CABRi binding affinity to siRNA. Taking the CABRi-8 vector as an example, the proteins corresponding to the two peaks obtained by purification using a cation exchange column were mixed with biotin-labeled siKRAS at molar ratios of 1:1, 2:1, 4:1, and 8:1 to prepare CABRi-siRNA nanoparticles. The binding ability of CABRi to siRNA was detected by electrophoretic mobility shift assay (EMSA). The results are shown in Figure 2. Fig.10 As shown. Fig.10 As shown in Figure 2, when the molar ratio of siKRAS to CABRi-8 protein is 1:2 or 1:4, CABRi-8 protein can bind to more than 90% of siKRAS. Fig.11 As shown, CABRi-12 protein can also bind siKRAS with high efficiency.

[0135] CABRi-siRNA nanoparticles are highly resistant to RNAse degradation. Taking the CABRi-8 vector as an example, the CABRi-8 protein and siKRAS were mixed at a molar ratio of 4:1, the reaction system was pH = 7.4, and the reaction was carried out at room temperature for 30 minutes. Then, different concentrations of RNAse (0μg / mL, 10μg / mL, 15μg / mL, 20μg / mL) were added to the reaction system. After reacting at 37°C for 40 minutes, the protective effect of CABRi on siKRAS was detected by electrophoretic mobility shift assay (EMSA). The results are as follows Fig.12 As shown. Fig.12 It can be seen that under the protection of CABRi-8 protein, siRNA can resist high concentrations of RNases and not be degraded.

[0136] When the total molar number of siRNA remains unchanged, one or more siRNAs can be packaged into the protein delivery vector CABRi at the same time. Both unmodified and chemically modified siRNAs are acceptable. Chemically modified siRNAs have better effects, enhancing siRNA stability, strong resistance to nucleases and high delivery efficiency; while reducing the immunogenicity of small nucleic acids. These chemical modifications include 2-O-Methyl Base 2-methoxy modified base (2-ome), 2-Fluoro RNA 2-fluoro RNA (2-F RNA), Phosphorothioate thio modification (PS), etc.

[0137] Example 4 CABRi-siRNA delivery efficiency and endogenous target gene silencing

[0138] The delivery efficiency of CABRi protein delivery vector in siRNA in cells. Taking CABRi-8 vector as an example, first, biotin-labeled siKRAS and CABRi-8 protein were mixed at a molar ratio of 1:4 to prepare CABRi-8-siRNA nanoparticles; then, CABRi-8-siRNA nanoparticles were added to the cultured lung adenocarcinoma cells H1299 to be transfected, so that the final concentration of siKRAS was 60nM, and the transfected cancer cells were collected 6h, 12h and 24h after transfection, washed 3 times with PBS buffer, and RNA was extracted; Northern blotting analysis was performed, and the results are shown in the figure. Fig.13 As shown. Fig.13 It can be seen that siKRAS can be detected at 6h of transfection, the signal is enhanced at 12h of transfection, and the signal becomes the strongest at 24h of transfection. This result shows that CABRi-8 protein can efficiently deliver siRNA and gradually accumulate within a certain time range.

[0139] CABRi-siRNA nanoparticles escape endosomes / lysosomes to release siRNA. Taking the CABRi-8 vector as an example, in order to detect siRNA escaping endosomes and avoiding degradation in lysosomes, FAM'-labeled siKRAS and CABRi-8 protein were mixed at a molar ratio of 1:4 to prepare CABRi-8-siRNA nanoparticles. Then, CABRi-8-siRNA nanoparticles were added to the cultured lung adenocarcinoma cells H1299 to be transfected, so that the final concentration of siKRAS was 60nM. Lyso-Tracker Red was added half an hour before observing the living cells under a confocal microscope. The results are as follows Fig.14 As shown. Fig.14It can be seen that at 6h of transfection, siKRAS entered the cells and was mostly co-localized in endosomes / lysosomes; at 16h and 24h of transfection, the pH-responsive endosomal escape peptide exerted its effect, causing endosomal / lysosome rupture. At the same time, siKRAS dissociated from CABRi and released siRNA from the endosome into the cytoplasm.

[0140] The knockdown effect of CABRi-siRNA on target genes in cells. Taking CABRi-8 vector as an example, siKRAS and CABRi-8 proteins were mixed in a molar ratio of 1:4 to prepare CABRi-8-siRNA nanoparticles. A549 cells were cultured at a cell density of 1.5×10 5 / well were inoculated into 12-well plates; after overnight growth, cells were treated with CABRi-8-siKRAS nanoparticles or an equal amount of CABRi-8 nanoparticles alone, with a final concentration of 240 nM; after continued culture for 48 h or 72 h, cells were collected, washed with PBS, and lysate was added. The same amount of protein was taken for Western Blot detection, and the experimental results are shown in Fig.15 As shown. Fig.15 It can be seen that the protein delivery vector CABRi-8 can be detected to have a high efficiency of entering cells at 48h, and interestingly, most of the protein degradation of CABRi-8 has occurred at 72h; the protein expression level of the target gene KARS is significantly reduced, and the phosphorylation level of the downstream signaling molecule pERK1 / 2 is also significantly downregulated accordingly. This shows that CABRi-8-siKRAS nanoparticles can effectively reduce the expression level of the target gene KRAS in cells, and CABRi can be rapidly degraded within 72h.

[0141] CABRi-12-siRNAs effectively inhibit tumor cell proliferation. Three effective siRNAs including siKRAS, siSHP2 and siSOS1 were packaged into the delivery protein carrier CABRi-12 at the same time while keeping the total molar number unchanged, that is, siRNA and CABRi-12 protein were mixed at a molar ratio of 1:4 to obtain CABRi-12-siRNA nanoparticles. The cells were treated with the above-mentioned CABRi-12-siRNA nanoparticles and CABRi-12 nanoparticles, and the CABRi-12 nanoparticles were added at the same concentration of 240nM; at the same time, an untreated group was set up, that is, the malignant lung cancer cell line H1975 without any treatment was used as the untreated group. After 24 hours of treatment, the treated malignant lung cancer cell line H1975 was cultured for plate cloning growth at an inoculation size of 500 cells / well. After 11 days of plate cloning cell culture, crystal violet staining was performed and statistics were performed. The results are as follows Fig.16 As shown. Fig.16 It can be seen that CABRi-12-siRNAs effectively inhibited the cell proliferation ability of malignant tumor cells compared with the untreated group and the CABRi-12-treated group.

[0142] The sequences of siSHP2 and siSOS1 are as follows:

[0143] siSHP2 positive strand: 5′-GUUAGGAACGUCAAAGAAAGC-3′, SEQ ID NO.40;

[0144] siSHP2 antisense strand: 5′-GCUUUCUUUGACGUUCCUAACAC-3′, SEQ ID NO.41.

[0145] siSOS1 positive strand: 5'-CCCACAGUUGAGUGGCAUAUA-3', SEQ ID NO.42;

[0146] siSOS1 antisense strand: 5′-UAUAUGCCACUCAACUGUGGGAG-3′, SEQ ID NO.43.

[0147] Example 5 CABRi-12-siRNAs specifically targets tumor tissue

[0148] First, pancreatic cancer PANC1 tumor cells were subcutaneously inoculated into male nude mice (BALB / c-nu) until the tumor grew to a diameter of 8 mm. Cy5-labeled siKRAS was mixed with the delivery protein carrier CABRi-12 to prepare CABRi-12-siRNA nanoparticles (the preparation method was the same as the preparation method of CABRi-8-siRNA nanoparticles in Example 4) and injected via the tail vein. The mice were killed 24 hours after the injection, and the main organ tissues of the mice, including the lungs, heart, liver, kidneys, spleen and tumor masses, were separated. The fluorescence intensity of siRNA-Cy5 in each organ was observed using a small animal fluorescence imager. The results are as follows Fig.17 As shown. Fig.17 It can be seen that most siRNAs are mainly released in tumor tissues. Although there are a few signals in liver tissues, there are no signals in other organs and tissues including lungs, heart, kidneys, and spleen. Therefore, it can be seen that the CABRi-12 vector successfully and efficiently targets tumor tissues specifically.

[0149] Example 6 CABRi-12-siRNAs effectively treats lung cancer cell transplants

[0150] When the density of lung adenocarcinoma A549 cell line reaches 80%-90% and the cells are in good condition, they can be used for experimental research. Before the experiment, the experimental cells in the logarithmic growth phase were washed with PBS, 0.05% trypsin was added, and the cells were digested at 37°C for 1 min. After blowing the cells apart and collecting them in a 15mL centrifuge tube, centrifuged at 800g for 5 min, the supernatant was discarded, and PBS was added for resuspending. The cells were counted three times using a hemocytometer, and the average value was taken to prepare 2×10 7 Cells / mL single-cell suspension (be sure to fully disperse the cells to make a single-cell suspension).

[0151] According to the experimental requirements, 6-7 week-old male BALB / c-nu nude mice were used for the experiment. No anesthesia was required. 0.1 mL of 2×10 7 / mL of a single cell suspension in the logarithmic growth phase is inserted into the subcutaneous tissue of the back or armpit about 0.5-1.0cm (to prevent leakage of the cell suspension), and the needle is withdrawn after a skin mound is formed under the skin, indicating a successful implantation.

[0152] The experimental mice injected with tumor cells were kept for 2 weeks and the tumor growth was measured regularly. When the tumor diameter reached 5 mm, the mice were randomly divided into groups and drug treatment was started. The experiment was divided into the following 2 groups, with 10 mice in each group.

[0153] Control group: PBS tail vein injection / 4 days;

[0154] CABRi-12-siRNA group: 0.5 nmol siKRAS, 0.5 nmol siSHP2, 0.5 nmol siSOS1 and CABRi-12 protein were mixed at a molar ratio of 1:4 and allowed to stand at room temperature for 30 minutes to obtain CABRi-12-siRNA nanoparticles containing three siRNAs. The CABRi-12-siRNA nanoparticles were then injected into the experimental mice through the tail vein, once every 4 days, for a total of 3 treatments.

[0155] After treatment, the mice were observed for 8 days, and then the tumors were dissected, and the tumor volumes were measured, weighed, and statistically analyzed. Fig.18 As shown. Fig.18 It is recorded that after three treatments, the tumor volume and weight of the treatment group were significantly reduced compared with those of the control group, indicating that CABRi-12-siRNA nanoparticles can significantly inhibit tumor growth in vivo.

[0156] Example 7 Safety Evaluation of CABRi-siRNA Delivery System

[0157] In Example 6, mice treated with PBS were used as controls to compare the physiological toxicity of the CABRi-12 delivery system in vivo. The food intake, body weight, and activity of the mice were consistent with those of the control mice, and no toxic side effects were found. After the treatment, the heart, liver, spleen, lung, kidney, brain tissue, intestinal tissue, and muscle tissue of the mice were removed and stained with H&E. The results are as follows: Fig.19 As shown. Fig.19 It can be seen that compared with the PBS control group, the tissues in the CABRi-12-siRNA treatment group were consistent with normal tissues, and no side effects such as tissue lesions occurred. This shows that the assembled protein nanocarrier CABRi has good in vivo safety and has no toxic side effects on normal tissues and organs.

[0158] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, characterized in that: The assembled protein nanocarrier CABRi comprises an assembled functional peptide, a linker and a small RNA binding protein; The assembled functional peptides include polypeptides with membrane-penetrating and pH-responsive functions and / or specific targeting peptides; the amino acid sequence of the polypeptide is any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.13; the amino acid sequence of the specific targeting peptide is any one of the sequences shown in SEQ ID NO.14-SEQ ID NO.24; The linker includes a flexible linker and / or a rigid linker; the amino acid sequence of the flexible linker is shown in SEQ ID NO.25 or SEQ ID NO.26; the amino acid sequence of the rigid linker is shown in SEQ ID NO.27 or SEQ ID NO.28; The amino acid sequence of the small RNA binding protein is any one of the sequences shown in SEQ ID NO.29-SEQ ID NO.

35.

2. The assembled protein nanocarrier CABRi according to claim 1, characterized in that: The copy numbers of the assembled functional peptide, the linker and the small RNA binding protein are all ≥1; the basic vector of the assembled protein nanocarrier CABRi includes a prokaryotic system expression vector and a eukaryotic system expression vector.

3. The method for preparing the assembled protein nanocarrier CABRi according to claim 1, characterized in that: The following steps are involved: The assembled functional peptide, the linker and the small RNA binding protein are connected by seamless cloning technology to obtain a CABRi protein; The CABRi protein is cloned into a basic vector to obtain the assembled protein nanocarrier CABRi.

4. The preparation method according to claim 3, characterized in that: The copy numbers of the assembled functional peptide, the linker and the small RNA binding protein are all ≥1; and the basic vector includes a prokaryotic system expression vector and a eukaryotic system expression vector.

5. Use of the assembled protein nanocarrier CABRi according to claim 1 or 2 in the preparation of siRNA drug delivery systems, siRNA nanocomplexes and targeted drugs.

6. A siRNA drug delivery system, characterized in that: The siRNA drug delivery system comprises the assembled protein nanocarrier CABRi according to claim 1 or 2.

7. A siRNA nanocomplex, characterized in that: The siRNA nanocomplex comprises the assembled protein nanocarrier CABRi according to claim 1 or 2 and siRNA.

8. The method for preparing the siRNA nanocomplex according to claim 7, characterized in that: The method comprises the steps of mixing the assembled protein nanocarrier CABRi according to claim 1 and siRNA, and incubating the mixture to obtain the siRNA nanocomplex.

9. A targeted drug, characterized in that: The drug comprises the assembled protein nanocarrier CABRi according to claim 1 or 2 and siRNA.

10. The targeted drug according to claim 9, characterized in that: The siRNA includes one or more of siKRAS, siSHP2 and siSOS1; The sense chain sequence of the siKRAS is shown in SEQ ID NO.36, and the antisense chain sequence is shown in SEQ ID NO.37; the sense chain sequence of the siSHP2 is shown in SEQ ID NO.40, and the antisense chain sequence is shown in SEQ ID NO.41; the sense chain sequence of the siSOS1 is shown in SEQ ID NO.42, and the antisense chain sequence is shown in SEQ ID NO.43.

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