An assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, its preparation method and applications

Abstract

The present invention relates to the field of biomedicine, and particularly to an assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, its preparation method and application. The assembled protein nanocarrier CABRi (CABRi series of siRNA protein delivery carriers) designed by the present invention can efficiently load siRNA without sequence selectivity, and can be mixed with one or more siRNAs in a certain ratio to form a carrier / small nucleic acid complex CABRi-siRNA, which self-assembles into a nanoparticle with a size of 10-100 nm. The present invention constructs a brand-new siRNA delivery carrier, breaking through three key technical bottlenecks in siRNA drug delivery, namely, extremely low in vivo delivery efficiency of siRNA, endosomal escape and extrahepatic delivery problems, and finally achieving the purposes of efficient delivery, endosomal escape, targeting 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 particularly to an assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, its preparation method and applications. Background Art

[0002] Since scientists discovered in 1998 that double-stranded RNA can specifically silence gene expression, RNA interference (RNAi) has shown great potential and application prospects in the treatment of various diseases as a post-transcriptional gene silencing method. At the same time, with the in-depth study of genomics and the rapid development of sequencing technology, gene therapy has become one of the revolutionary biotherapeutic solutions. In theory, siRNA can specifically and effectively inhibit any gene by directly targeting and reducing the expression of mRNA. Since the first siRNA drug Patisiran of Alnylam Pharmaceuticals was successfully launched in the United States on August 10, 2018, 6 siRNA drugs have been successfully launched one after another, but there is no fully self-developed siRNA drug approved for marketing in China.

[0003] The global R & D of siRNA drugs is still in the early stage of the industry's development, and there are still many technical bottlenecks in the development of siRNA drugs, especially the problem of low siRNA delivery efficiency. At present, the commonly used siRNA delivery systems include viral vectors and non-viral vectors. As we all know, 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 responses and liver toxicity. Therefore, the application of siRNA drugs with viral vectors is greatly limited. On the contrary, non-viral vectors include lipid nanoparticles (LNPs), polymeric nanoparticles, polypeptide complexes, and antibody oligonucleotide conjugate drugs (AOCs). Their characteristics are relatively high safety, but the siRNA targeting, delivery efficiency, endosomal escape, and safety need to be improved. The world's first approved siRNA drug, Patisiran, uses an LNP delivery system, which encapsulates the siRNA drug within the LNP and administers it by intravenous injection. The encapsulation of liposomes greatly improves the stability of the drug and its targeting to liver tissues, ensuring that siRNA is not filtered and cleared by the kidneys and is gradually taken up by target cells in liver tissues during blood circulation. This is the key to Patisiran's approval for marketing by overcoming delivery factors. The second small nucleic acid drug, Inclisiran, approved for marketing in December 2020, has a specially designed GalNAc-siRNA delivery system. The 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 marketed small nucleic acid drugs have been successfully marketed due to their special delivery methods.

[0004] Although the above-mentioned methods for administering siRNA in the market are relatively successful, there are still many problems. For example, in the current LNP-siRNA delivery system, PEG lipids, etc. are added during use, resulting in excessive immune responses and increased toxicity in the body, which is the main problem restricting clinical application. Therefore, before intravenous injection of LNP-mediated siRNA drugs (such as Patisiran), pretreatment with steroids and antihistamines is required to eliminate unnecessary allergic reactions. The GalNAc-modified siRNA drugs (such as Inclisiran) can only specifically target the liver and are not applicable to other organs, diseased tissue sites or tumors, etc., greatly limiting the application scenarios of siRNA drugs. Currently, there are mainly the following three key technical bottlenecks in siRNA drug delivery: 1) The problem of low in vivo delivery efficiency of siRNA. How to design siRNA and delivery vectors to form stable and effective complexes is the key point for drug development; 2) The problem of endosomal escape. For example, in the delivery technologies of marketed siRNA drugs, LNP or GalNAC, only less than 0.1-1% of siRNA can escape from endosomes, and how to improve siRNA endosomal escape is also the most crucial; 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 "bottleneck" technical difficulty that urgently needs to be solved currently.

[0005] Specifically designed artificial proteins have great potential as siRNA delivery vectors. Protein carriers are composed of natural amino acids and have the advantages of being non-toxic, easily degradable and not likely to cause immune responses. These carriers can be obtained through various methods, including prokaryotic expression, eukaryotic expression or cell-free expression in vitro. Through genetic recombination technology, tissue cell targeting ability can be imparted to protein carriers, and the functional domains binding to siRNA can be modified to enhance their siRNA delivery ability. Using mature recombinant protein technology, the complex structure of protein carriers can be precisely controlled, enabling them to protect siRNA from degradation by RNase, overcome the cell membrane barrier, enhance the uptake of siRNA by cells, promote endosomal escape, and ultimately exert the expected siRNA biological activity.

[0006] It should be noted that currently, protein-based siRNA delivery systems mostly remain in the stages of theoretical research and animal experiments. There are still many challenges to overcome in the clinical application of siRNA protein delivery carriers. First, the siRNA drugs delivered by proteins reported currently are usually administered locally or by intravenous injection, with limited oral bioavailability. Second, although the fusion expression of domains with different functions can be achieved through recombinant protein technology, how to ensure that the functions of individual domains within this recombinant protein are not affected remains an unsolved problem. Finally, the circulating half-life of protein-delivered siRNA drugs is relatively short, and special attention needs to be paid to how to improve the structural stability and extend the drug half-life.

[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 objective of the present invention is to provide an assembled protein nanocarrier CABRi for targeted delivery of small interfering nucleic acids, its preparation method and application, so as 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 of extrahepatic organs or tumors, and high safety.

[0009] To achieve the above objective, 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. The assembled protein nanocarrier CABRi includes an Assembled Functional Peptide (AFP), a linker, and a Small RNA Binding Protein / Peptide Segment (sRBP); in the present invention, the AFP refers to a functional property having a Cell-penetrating peptide (CPP) or simultaneously a pH-responsive endosome escape peptide (EEP) and a Specific targeting peptide (STP) for organs or tumor tissues.

[0011] The assembled functional peptide includes a polypeptide (CPP / STP) with transmembrane and pH-responsive functions and / or a specific targeting peptide (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 (Flexible Linker, FL) and / or a rigid linker (RigidLinker, RL); the number of amino acid residues of the flexible linker is 5 or 15; the amino acids constituting the flexible linker are selected from glycine and serine; this flexible linker connects CPP / EEP / STP to sRBP, or connects two sRBPs; the amino acid sequence of the flexible linker is as 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 constituting the rigid linker are selected from alanine, glutamic acid and lysine; this rigid linker connects CPP / EEP and STP; the amino acid sequence of the rigid linker is as 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 peptide of the present invention can achieve three functions: (1) As a cell-penetrating peptide (Cell-penetrating peptide, CPP), it can penetrate the membrane and enter the cell; (2) Some CPPs also serve as pH-responsive endosome escape peptides (pH-responsive endosome escape peptide, EEP) at the same time, realizing protonation or helix conversion in response to pH, and then escaping from the endosome, effectively releasing siRNA into the cytoplasm to play the function of gene silencing; (3) As a short peptide ligand, a specific targeting peptide (Specific targeting peptides, STP) for organs or tumor tissues, it can bind to specific receptors on the surface of organ tissues or tumor cells, and precisely target and deliver siRNA.

[0015] The sRBP described in the present invention is a protein that binds to siRNA molecules through a specific protein tertiary structure to form a stable protein-siRNA complex, enabling efficient delivery to cells within tissues. The sRBP described in the present invention may 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, ensuring the functional independence of each peptide domain and allowing the spontaneous formation of domain dimers or multimers within 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 carrier of the assembled protein nanocarrier CABRi includes prokaryotic expression vectors and eukaryotic expression vectors.

[0017] The assembled protein nanocarrier CABRi described in the present invention is a complete CABRi protein delivery carrier formed by arranging the assembled functional peptide, linker (flexible linker and / or rigid linker), and small RNA-binding protein in different ways, including but not limited to the integration of cell-penetrating peptides or polypeptides that also exhibit pH-responsive endosomal escape, organ- or tumor tissue-specific targeting peptides, flexible or rigid linkers, and small RNA-binding proteins / peptide segments, etc., thereby obtaining the recombinant protein carrier CABRi series capable of efficiently delivering siRNA. This assembled protein nanocarrier CABRi can self-assemble with siRNA into nanoparticles with a nanosize range of 10 - 100 nm, having a high deposition rate in the human respiratory system, and can cross capillaries or endothelial cells to achieve efficient and specific targeting of organ tissues (such as the 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 approximately 30 - 40 nm or 50 - 70 nm and have good deposition in the lungs upon nebulized inhalation.

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

[0019] Meanwhile, both unmodified or chemically modified siRNAs can be used in the present invention. Chemically modified siRNAs have better effects, enhancing the stability of siRNAs, strongly resisting nucleases, and having high delivery efficiency; at the same time, reducing the immunogenicity of small nucleic acids. These chemical modifications include 2ˋ-O-Methyl Base 2-methoxy modified base (2ˋ-ome), 2ˋ-Fluoro RNA 2ˋ-fluororibonucleic acid (2ˋ-FRNA), Phosphorothioate thiophosphoryl modification (PS), etc.

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

[0021] After connecting the assembled functional peptide, the linker, and the small RNA-binding protein by using seamless cloning technology, CABRi protein is obtained;

[0022] Cloning the CABRi protein onto 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; the basic vector includes prokaryotic system expression vectors and eukaryotic system expression vectors.

[0024] More preferably, the purification of the assembled protein nanocarrier CABRi is 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 application 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, and the siRNA drug delivery system includes the above-mentioned assembled protein nanocarrier CABRi.

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

[0028] The CABRi-siRNA nanocomplex provided by the present invention is a nanometer particle with a suitable size (10-100 nm) and strong stability, and can be used for in vivo delivery of siRNA. This complex has high safety and low toxicity. After releasing siRNA within 12-48 h of entering cells, the CABRi protein itself degrades rapidly. At the same time, this complex has the advantages of being easy to produce and having low cost.

[0029] The present invention provides the preparation method of the above siRNA nanocomplex, which includes the step of mixing the above assembled protein nanocarrier CABRi and siRNA, and incubating them to obtain the siRNA nanocomplex.

[0030] Further preferably, the molar ratio of the assembled protein nanocarrier CABRi protein to siRNA 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 min, and the temperature is room temperature.

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

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

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

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

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

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

[0038] The siRNA protein delivery vector CABRi series designed in the present invention can efficiently load siRNA without sequence selectivity. It can be mixed with one or more siRNAs in a certain proportion to form a vector / small nucleic acid complex CABRi-siRNA, which self-assembles into a nanoparticle with a size of 10 - 100 nm. AFP fused at the end of CABRi has three functional attributes: transmembrane penetration, ligand binding to specific cell surface receptors, and endosomal escape. It can achieve strong resistance to nuclease and high delivery efficiency in vivo; specifically target organ tissues (such as the lung and pancreas, etc.) or tumor tissues, etc.; pH acidic response protonation or α-helix conversion promotes the efficient escape of siRNA from endosomes, effectively releasing siRNA into the cytoplasm, and then exerting the 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 drug formulation of CABRi-siRNA designed in the present invention can meet intravenous injection, subcutaneous or oral administration methods, and effectively avoid glomerular clearance and circulatory system clearance. The present invention constructs a brand-new siRNA delivery vector, breaks through three key technical bottlenecks in siRNA drug delivery, and also overcomes the problems of extremely low in vivo delivery efficiency, endosomal escape, and extrahepatic delivery of siRNA, ultimately achieving the goals 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 and can be applied to the treatment fields of various diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (IF), lung cancer, pancreatic cancer, colorectal cancer and other malignant tumors. Brief Description of the Drawings

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

[0040] Figure 1 For CABRi-3 protein via Ni 2+Ni-NTA affinity chromatography Coomassie brilliant blue staining; where Marker is protein standard; Cell pellets is cell precipitate; Cell lysate is supernatant of cell lysate; Flow through is flow-through; Wash is wash buffer; Elution-first is the first eluate; Elution-second is the second eluate; Elution-third is the third eluate; Elution-fourth is the fourth eluate; Elution-fifth is the fifth eluate; Elution-last is the last eluate; Elution-total is the total eluate;

[0041] Figure 2 of CABRi-8 protein by Ni 2+ Ni-NTA affinity chromatography Coomassie brilliant blue staining; where Marker is protein standard; Cell lysate is supernatant of cell lysate; Flow through is flow-through; Wash is wash buffer; Elution-first is the first eluate; Elution-last is the last eluate; Elution-total is the total eluate;

[0042] Figure 3 of CABRi-12 protein by Ni 2+ Ni-NTA affinity chromatography Coomassie brilliant blue staining; where Marker is protein standard; Cell lysate is supernatant of cell lysate; Flow through is flow-through; Wash is wash buffer; Elution-first is the first eluate; Elution-last is the last eluate; Elution-total is the total eluate;

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

[0044] Figure 5 is the Coomassie brilliant blue staining of CABRi-8 separated and purified by cation exchange column; where Marker is protein standard; Purified sample is purified sample; Flow through is flow-through; Peak 1 is the first peak, A05 - A09 are the collection tubes of A05, A06, A07, A08 and A09 in sequence; Peak 2 is the second peak, A10 is the collection tube of A10, B05 - B10 are the collection tubes of B05, B06, B07, B08, B09 and B10 in sequence;

[0045] Figure 6 The particle size and potential of self - formed CABRi - 8 nanoparticles; among which, 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 self - formed CABRi - 12 nanoparticles; among which, 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; among which, 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] Figure 9 The particle size and potential of CABRi - 12 - siRNA nanoparticles; among which, 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] Figure 10 EMSA for detecting the binding efficiency of CABRi - 8 and siRNA;

[0050] Figure 11 EMSA for detecting the binding efficiency of CABRi - 12 and siRNA;

[0051] Figure 12 EMSA experiment for detecting the protection of siRNA by CABRi - 8 - siRNA nanoparticles;

[0052] Figure 13 Northern blotting for detecting the delivery of siRNA into cells by CABRi - 8 vector;

[0053] Figure 14 Confocal microscopy live - cell imaging technology for detecting the escape of CABRi - 8 - siRNA nanoparticles from endosomes / lysosomes and the release of siRNA into the cytoplasm;

[0054] Figure 15 Western Blotting experiment for detecting the reduction of the expression level of the target gene KRAS in cells by CABRi - 8 - siKRAS nanoparticles;

[0055] Figure 16 Plate cloning experiment for detecting the inhibition of the proliferation of H1975 cells by CABRi - 12 - siRNAs; among which, A is the culture diagram; B is the statistical graph of H1975 cells;

[0056] Figure 17 For fluorescence imaging experiments to detect the delivery of siRNA to tumor tissues by the CABRi-12 vector; where Lung is the lung; Heart is the heart; Tumor is the tumor; Liver is the liver, Spleen is the spleen; Kidney is the kidney;

[0057] Figure 18 For the growth inhibition of malignant tumors in mice by CABRi-12-siRNAs; where A is the statistical chart of tumor volume; B is the statistical chart of tumor mass;

[0058] Figure 19 For H&E staining to detect the toxic and side effects of the CABRi-12 delivery system on normal tissues and organs, where A is the H&E staining images of Lung (lung), Liver (liver), Brain (brain) and Intestine (intestine) under different treatments; B is the H&E staining images of Spleen (spleen), Kidney (kidney), Heart (heart) and Muscle (muscle) under different treatments. Detailed implementation manners

[0059] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0060] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0061] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0062] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0063] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0064] Some sequences related to the present invention are shown as follows:

[0065] The sequence of CPP / EEP is selected from one of those shown in 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 those shown in 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; <D

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

[0092] SEQ ID NO.25: GSSGG;

[0093] SEQ ID NO.26: GGGGSGGGGSGSSGG;

[0094] The amino acid sequence of RL is selected from one of those shown in SEQ ID NO: 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 NO.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 the CABRi series of siRNA targeted delivery protein nanocarriers

[0116] Overall construction plan of CABRi series vectors: The siRNA protein delivery vector CABRi series provided by the present invention uses seamless cloning technology and artificial gene synthesis technology to assemble the nucleotide sequences corresponding to FL / RL, sRBP, and AFP (CPP / EEP / STP) and insert them into prokaryotic expression vectors such as pET-28a and pGEX-4T-1, or they 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. Through a series of screenings such as the level of protein expression, the simplicity of purification, and the final yield, the optimal expression plasmid of the required siRNA protein delivery vector CABRi is finally determined.

[0117] In this example, taking CABRi-3 vector, CABRi-8 vector, and CABRi-12 vector as examples, the designed CABRi nucleotide sequence was cloned onto the prokaryotic expression vector pET-28a(+), and the insertion sites were 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 the identification and in vivo recognition of the protein vector; the middle segment contains a sRBP (SEQ ID NO.31); the C-terminus is AFP, 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 seamless connection of the following parts (no base insertion is required 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 and uses UltraOne Step Cloning Kit was used to insert another copy of the repetitive sRBP (SEQ ID NO.31)-FL (SEQ ID NO.26) into the middle region, thus forming two sRBPs. The amino acid sequence of the CABRi-8 protein in the CABRi-8 vector was obtained by seamless ligation of 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). The CABRi-12 vector was based on CABRi-8. Also by seamless cloning technology, STP (SEQ ID NO.18) was added after CPP / EEP (SEQ ID NO.6), and they were connected by RL (SEQ ID NO.27); the amino acid sequence of CABRi-12 in the CABRi-12 vector was obtained by seamless ligation of 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 (SEQ ID 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 examples, these expression plasmids were transferred into the Escherichia 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 h.

[0120] After collecting the bacterial solution, it was ultrasonically disrupted and lysed in sequence, and then the first round of Ni 2+ -NTA affinity chromatography column was used to purify the histidine-tagged protein, and SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining were carried out. The results of Coomassie Brilliant Blue staining are as Figures 1 - 3 shown Figures 1 - 3Coomassie brilliant blue staining results diagrams corresponding to CABRi-3 protein, CABRi-8 protein, and CABRi-12 protein respectively. Each component shown in the figure is as follows: Cell pellets are samples taken after collecting cells, adding lysis buffer to break them, centrifuging at high speed, and resuspending the precipitate with an equal volume of lysis buffer; Cell lysate is a sample taken from the supernatant after collecting cells, adding lysis buffer to break them, and centrifuging at high speed; Flow through is the flow-through liquid, which is the liquid that passes through after the supernatant of the cell lysate passes through Ni 2+ -NTA; Wash is the washing liquid, which is the washing liquid after washing Ni 2+ -NTA with Washbuffer; Elution-first is the first elution liquid, which is the elution liquid collected after eluting Ni 2+ -NTA with 6 ml of Elution buffer; Elution-second is the second elution liquid, which is the elution liquid collected after eluting Ni 2+ -NTA with 6 ml of Elution buffer; Elution-third is the third elution liquid, which is the elution liquid collected after eluting Ni 2+ -NTA with 8 ml of Elution buffer; Elution-fourth is the fourth elution liquid, which is the elution liquid collected after eluting Ni 2+ -NTA with 10 ml of Elution buffer; Elution-fifth is the fifth elution liquid, which is the elution liquid collected after eluting Ni 2+ -NTA with 10 ml of Elution buffer; Elution-last is the last elution liquid, which is the elution liquid collected after eluting Ni 2+ -NTA with 10 ml of Elution buffer; Elution-total is the total elution liquid, which is a sample taken by combining the above collected elution liquids.

[0121] It can be seen from Figures 1 - 3 that after the first-step separation by Ni 2+ -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 separation by Ni 2+ -NTA affinity chromatography, the second-round separation and purification are carried out using a protein purifier (model UEV-25M Yonglian Bio) and a cation exchange column (HiTrap TM SP / HP), and the separation and purification curve of the cation exchange column is as shown in Figure 4 It can be seen from Figure 4It can be seen that the CABRi-8 protein has two elution peaks (Peak 1 and Peak 2). At the same time, SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining were performed on the collection tubes corresponding to the elution peaks of the CABRi-8 protein. The results are as Figure 5 shown. Finally, the CABRi-8 protein with a purity greater than 95% was obtained and stored at -80 °C for later use.

[0123] Example 3 Preparation and Characterization of a Nanoscale Targeted Delivery Protein Carrier CABRi and siRNA Mixture

[0124] The CABRi series can form nanoparticles by itself. For example, the particle size and zeta potential of the nanoparticles formed by CABRi-8 and CABRi-8-siRNA are as Figure 6 and Figure 8 shown; the particle size and zeta potential of the nanoparticles formed by CABRi-12 and CABRi-12-siRNA are as Figure 7 and Figure 9 shown. The above values are summarized in Table 1.

[0125] Table 1 Particle Size and Zeta Potential of Nanoparticles Formed by the 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] Taking the synthesized siRNA, a specific siRNA targeting human KRAS (denoted as siKRAS), as an example, siRNA was dissolved in DEPC water to a solution with 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 sense strand: 5’-GUUGAUUACUUCUUAUUUUUC-3’, SEQ ID NO.36;

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

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

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

[0133] Mix the prepared CABRi protein with siRNA at different molar ratios (such as siRNA:CABRi = 1:4 or 1:8, etc.). In this example, the prepared CABRi protein and siRNA are mixed at a molar ratio of 4:1. The reaction system has a pH of 7.4 and reacts at room temperature for 30 min to obtain CABRi-siRNA mixture molecules (CABRi-8-siRNA and CABRi-12-siRNA), forming nanoparticles with appropriate size and strong stability. As Figure 8 , Figure 9 and 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 zeta potentials are about -7.258 and -19.31 respectively, which can be used for in vivo delivery of siRNA.

[0134] CABRi binding affinity for siRNA. Taking the CABRi-8 vector as an example, the proteins corresponding to the two peaks purified by cation exchange column are respectively prepared into CABRi-siRNA nanoparticles with biotin-labeled siKRAS at molar ratios of 1:1, 2:1, 4:1, and 8:1. The binding ability of CABRi to siRNA is detected by electrophoretic mobility shift assay (EMSA). The results are as Figure 10 shown. As Figure 10 shown, when the molar ratio of siKRAS and CABRi-8 protein is 1:2 or 1:4, the CABRi-8 protein can bind more than 90% of siKRAS. Similarly, as Figure 11 shown, the CABRi-12 protein can also efficiently bind siKRAS.

[0135] CABRi-siRNA nanoparticles are highly resistant to RNase degradation. Taking the CABRi-8 vector as an example, mix the CABRi-8 protein and siKRAS at a molar ratio of 4:1. The reaction system has a pH of 7.4 and reacts at room temperature for 30 min. Then add RNase at different concentrations (0 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL) to the reaction system and react at 37 °C for 40 min. The protective effect of CABRi on siKRAS is detected by electrophoretic mobility shift assay (EMSA). The results are as Figure 12 shown. As Figure 12 can be seen, under the protection of the CABRi-8 protein, siRNA can resist high concentrations of RNase without being degraded.

[0136] Under the condition that the total molar amount of siRNA remains unchanged, one or more siRNAs can be simultaneously encapsulated into the protein delivery vector CABRi. Either unmodified or chemically modified siRNAs are acceptable. Chemically modified siRNAs have better effects, enhancing the stability of siRNAs, strongly resisting nuclease, and having high delivery efficiency; meanwhile reducing the immunogenicity of small nucleic acids. These chemical modifications include 2ˋ-O-Methyl Base 2-methoxy modified base (2ˋ-ome), 2ˋ-Fluoro RNA 2ˋ-fluororibonucleic acid (2ˋ-F RNA), Phosphorothioate thiophosphoryl modification (PS), etc.

[0137] Example 4 Delivery efficiency of CABRi-siRNA and its effect on mediating endogenous target gene silencing

[0138] Delivery efficiency of the CABRi protein delivery vector for siRNA in cells. Taking the 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 the CABRi-8-siRNA nanoparticles were added to the cultured lung adenocarcinoma cells H1299 to be transfected, so that the final concentration of siKRAS was 60 nM. After transfection for 6 h, 12 h, and 24 h respectively, the transfected cancer cells were collected first, washed 3 times with PBS buffer, and RNA was extracted; Northern blotting analysis was performed, and the results are as Figure 13 shown. As Figure 13 can be seen, siKRAS could be detected at 6 h after transfection; the signal was enhanced at 12 h after transfection; and the signal became the strongest at 24 h after transfection. This result indicates that the CABRi-8 protein can efficiently deliver siRNA and gradually accumulate within a certain time range.

[0139] CABRi-siRNA nanoparticles escape from endosomes / lysosomes to release siRNA. Taking the CABRi-8 vector as an example, to detect the escape of siRNA from endosomes and avoid 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 the CABRi-8-siRNA nanoparticles were added to the cultured lung adenocarcinoma cells H1299 to be transfected, so that the final concentration of siKRAS was 60 nM. Lyso-Tracker Red was added half an hour before observing living cells under a confocal microscope, and the results are as Figure 14 shown. As Figure 14It can be seen that at 6 h after transfection, siKRAS can enter the cells and mostly co-localize in endosomes / lysosomes; at 16 h and 24 h after transfection, the pH-responsive endosomal escape peptide plays a role, resulting in the rupture of endosomes / lysosomes. At the same time, siKRAS dissociates from CABRi and releases siRNA from endosomes into the cytoplasm.

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

[0141] CABRi-12-siRNAs effectively inhibit the proliferation of tumor cells. Three effective siRNAs, including siKRAS, siSHP2, and siSOS1, were simultaneously packaged into the delivery protein vector CABRi-12 under the condition of constant total molar amount, that is, siRNA and CABRi-12 protein were mixed at a molar ratio of 1:4 to obtain CABRi-12-siRNA nanoparticles. The above CABRi-12-siRNA nanoparticles and CABRi-12 nanoparticles were used to treat the cells, and the same concentration of 240 nM of CABRi-12 nanoparticles was added; at the same time, an untreated group was set, that is, the malignant lung cancer cell line H1975 without any treatment was used as the untreated group. After treatment for 24 h, the treated malignant lung cancer cell line H1975 was inoculated at a density of 500 cells / well for plate clone growth culture. After 11 days of plate clone cell culture, crystal violet staining was performed for statistics, and the results are as Figure 16 shown. It can beFigure 16 It can be seen that, compared with the untreated group and the CABRi-12 treatment group, CABRi-12-siRNAs effectively inhibit the cell proliferation ability of malignant tumor cells.

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

[0143] Sense strand of siSHP2: 5’-GUUAGGAACGUCAAAGAAAGC-3’, SEQ ID NO.40;

[0144] Antisense strand of siSHP2: 5’-GCUUUCUUUGACGUUCCUAACAC-3’, SEQ ID NO.41.

[0145] Sense strand of siSOS1: 5’-CCCACAGUUGAGUGGCAUAUA-3’, SEQ ID NO.42;

[0146] Antisense strand of siSOS1: 5’-UAUAUGCCACUCAACUGUGGGAG-3’, SEQ ID NO.43.

[0147] Example 5 CABRi-12-siRNAs specifically target tumor tissues

[0148] First, PANC1 tumor cells of pancreatic cancer were subcutaneously inoculated into male nude mice (BALB / c-nu), and waited until the tumors 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 that of CABRi-8-siRNA nanoparticles in Example 4), and was injected by the tail vein injection method. The mice were sacrificed 24 h after injection, and the main organ tissues of the mice including the lung, heart, liver, kidney, spleen and tumor mass were separated, and the fluorescence intensity of siRNA-Cy5 in each organ was observed with a small animal fluorescence imaging instrument. The results are as Figure 17 shown. It can be Figure 17 seen that most of the siRNAs were mainly released in the tumor tissues. Although there was a little signal in the liver tissue, there was no signal in other organ tissues including the lung, heart, kidney and spleen. Therefore, it can be seen that the CABRi-12 vector successfully and efficiently specifically targets tumor tissues.

[0149] Example 6 CABRi-12-siRNAs effectively treat lung cancer cell xenografts

[0150] When the density of the lung adenocarcinoma A549 cell line reaches 80%-90% and the cell state is good, it can be used for experimental research. Before the experiment, the experimental cells in the logarithmic growth phase are washed once with PBS, 0.05% trypsin is added, and after digestion at 37°C for 1 min, the cells are dispersed and collected into a 15 mL centrifuge tube. After centrifugation at 800 g for 5 min, the supernatant is discarded, PBS is added for resuspension, and the cells are counted three times using a hemocytometer, and the average value is taken to prepare a single-cell suspension of 2×10 7 cells / mL (the cells must be fully dispersed to make a single-cell suspension).

[0151] According to the experimental needs, male BALB / c-nu nude mice at 6-7 weeks of age are used for the experiment. Without anesthesia, 0.1 mL of a single-cell suspension with a concentration of 2×10 7 / mL in the logarithmic growth phase is taken with a 1 mL syringe, and the needle is inserted subcutaneously about 0.5-1.0 cm on the back or under the armpit (to prevent leakage of the cell suspension). After raising a skin bleb subcutaneously, the needle is withdrawn, and the implantation is successful.

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

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

[0154] CABRi-12-siRNA group: 0.5 nmol siKRAS, 0.5 nmol siSHP2, 0.5 nmol siSOS1 and CABRi-12 protein are mixed at a molar ratio of 1:4, and after standing at room temperature for 30 min, CABRi-12-siRNA nanoparticles containing three siRNAs are obtained. Then, the CABRi-12-siRNA nanoparticles are injected into the experimental mice through the tail vein, and the treatment is carried out once every 4 days for a total of 3 times.

[0155] Continue to observe for 8 days after the treatment is over, then sacrifice the mice, dissect the tumors, measure the tumor volume, weigh them, and conduct statistics. The results are as Figure 18 shown. It can be seen from Figure 18 the records that after 3 treatments, compared with the control group, the tumor volume and weight of the treatment group are significantly reduced, indicating that CABRi-12-siRNA nanoparticles can significantly inhibit tumor growth in vivo.

[0156] Example 7 Safety evaluation of the 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. From aspects such as the food intake, body weight, and activity of the mice, they were all consistent with the control mice, and no toxic or side effects were found. After the treatment ended, the heart, liver, spleen, lung, kidney, brain tissue, intestinal tissue, muscle tissue, etc. of the mice were taken out and stained with H&E. The results are as Figure 19 shown. As can be Figure 19 seen, compared with the PBS control group, each tissue in the CABRi-12-siRNA treatment group was consistent with the normal tissue, and no side effects such as tissue lesions occurred. This indicates that the assembled protein nanocarrier CABRi has good in vivo safety and has no toxic or side effects on normal tissues and organs.

[0158] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention 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 CABRi-3 vector is composed of a flexible linker represented by SEQ ID NO. 25, a small RNA binding protein represented by SEQ ID NO. 31, a flexible linker represented by SEQ ID NO. 26, and a polypeptide represented by SEQ ID NO. 6, connected in sequence from the N-terminus to the C-terminus. The CABRi-8 vector is composed of a flexible linker represented by SEQ ID NO. 25, a small RNA binding protein represented by SEQ ID NO. 31, a flexible linker represented by SEQ ID NO. 26, a small RNA binding protein represented by SEQ ID NO. 31, a flexible linker represented by SEQ ID NO. 26, and a polypeptide represented by SEQ ID NO. 6, connected in sequence from the N-terminus to the C-terminus. From N-terminus to C-terminus, the CABRi-12 vector consists of a flexible linker shown in SEQ ID NO.25, a small RNA binding protein shown in SEQ ID NO.31, a flexible linker shown in SEQ ID NO.26, a small RNA binding protein shown in SEQ ID NO.31, a flexible linker shown in SEQ ID NO.26, a polypeptide shown in SEQ ID NO.6, a rigid linker shown in SEQ ID NO.27, and a specific targeting peptide shown in SEQ ID NO.

18.

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.

Images