Targeting drug delivery carrier and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to a targeting drug delivery carrier and a preparation method thereof. The invention provides a targeting drug delivery carrier. The targeting drug delivery carrier comprises a drug carrier and targeting polypeptide, the drug carrier is a virus carrier, and the nucleotide of the targeting polypeptide is operably connected with the nucleotide of the capsid protein of the virus carrier; or the drug carrier is a non-viral carrier, the targeting polypeptide is connected with the non-viral carrier in a surface modification mode, and the amino acid sequence of the targeting polypeptide is shown as any one of SEQ ID NO: 8-12 and SEQ ID NO: 14. The efficient drug delivery carrier is designed and prepared by utilizing targeting polypeptide for specifically recognizing biomolecules, cells, tissues or organs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a targeted drug delivery carrier and a preparation method thereof. Background Art

[0002] Currently, the delivery carriers for genetic diseases are classified into viral carriers and non-viral carriers according to whether they are based on virus modification. Among them, the main viral carriers are: lentiviral vectors (Lentivirus, LV), adenoviral vectors (Adenovirus, AdV), adeno-associated viral vectors (Adeno-associated virus, AAV), etc. The main non-viral carriers are: lipid nanoparticles (Lipid nanoparticle, LNP), virus-like particles (Virus-like particle, VLP), exosomes (Exosome), etc.

[0003] As a representative of non-pathogenic and replication-defective viral vectors, AAV is widely used because of its characteristics such as low immunogenicity, genetic material not integrating into the host genome, and non-pathogenicity. Currently, 90% of gene therapy clinical trials use AAV as a delivery carrier. However, the organ targeting of naturally occurring wild-type AAV is very limited, and its therapeutic applications are also restricted. Research has pointed out that AAV vectors have a high affinity for the liver and will naturally accumulate in liver cells. Simply targeting the liver, a low dose of AAV vectors is effective. But if targeting other parts, to reach an effective concentration, the dose of AAV vectors in systemic administration must be greatly increased. Existing research shows that the modification of the AAV capsid can change the biological characteristics of the vector. The AAV virus consists of an icosahedral protein capsid with a diameter of about 26 nm and a single-stranded DNA genome of about 4.7 kb. There are T-shaped inverted terminal repeats (Inverted terminal repeat, ITR) at both ends of the genome. The REP and CAP genes are contained between the two ITRs. The REP gene is responsible for encoding four proteins required for virus replication and regulation, and the CAP gene is responsible for encoding the VP1, VP2, and VP3 proteins that make up the capsid. The virus capsid is polymerized by 60 capsid protein monomers, with a ratio of 1:1:10 (VP1:VP2:VP3). There are 9 common variable regions (VRⅠ-VRⅨ) on the subunits that make up the AAV capsid. These regions determine the differences between different AAV serotypes, including receptor recognition, gene transduction efficiency, and immune response. Thus, they affect the tissue tropism, transduction efficiency, antigenicity, and cross-reactivity of immunogenicity between serotypes of the virus.

[0004] The non-viral vector LNP (Lipid Nanoparticle), namely lipid nanoparticle, has become a key tool for nucleic acid vaccines and therapies due to its advantages such as safety, tolerance, repeat dosing ability, and the ability to carry a large amount of gene cargo. Traditional LNP contains four molecules: 1. Ionizable lipids: used to bind nucleic acids and assist endosomal escape. 2. Amphiphilic phospholipids: promote fusion with cell and endosomal membranes. 3. Cholesterol: helps with the stability of LNP. 4. Polyethylene glycol (PEG) lipids: improve colloidal stability and reduce reticuloendothelial system clearance. LNP technology originated from the research on cationic lipid complexes and ionizable cationic lipids, combining the physical properties and functional roles of lipids in membranes, as well as their successful experience in the delivery of small molecule drugs (such as anti-cancer drugs).

[0005] Phage-display is a method for high-throughput screening of functional polypeptides. It can insert the gene encoding an exogenous polypeptide or protein into the structural gene of the phage coat protein, enabling the exogenous polypeptide or protein to form a fusion protein on the capsid protein of the phage and be presented on the surface of the phage along with the reassembly of the progeny phages, while maintaining the relative spatial structure and biological activity. By inserting a polypeptide library into the structural gene of the phage capsid protein, a high-throughput phage library can be constructed. Using the phage library to screen for specific targets, after repeating steps such as incubation, blocking, panning, elution, and amplification several times, a polypeptide sequence with high affinity for the target can be screened out. All steps of phage display screening can be achieved in vitro, and the phages used can be amplified in Escherichia coli, with simple steps. And the peptide library flux of the phage display peptide library is above 10 9 which meets the requirements of high-throughput screening and has low experimental costs.

[0006] Wild-type AAV has limited organ tropism and its therapeutic applications are also restricted. Moreover, the AAV vector has a high affinity for the liver and will naturally accumulate in liver cells. Therefore, wild-type AAV as a drug delivery vector will exhibit some immune responses and liver toxicity. Traditional methods for improving the design of adeno-associated viruses mainly include methods such as directed evolution and rational design. Among them, directed evolution injects an artificially prepared virus library into an animal body, combined with artificial pressure and high-throughput sequencing methods. After multiple rounds of screening, novel mutants are found in different tissues and organs. Its disadvantages are low screening efficiency, high time and cost consumption. Rational design is based on prior knowledge to design and modify the AAV capsid protein. Although it is more efficient than directed evolution, due to its low throughput and dependence on original data, it severely restricts the design and clinical application of viral vectors.

[0007] LNPs are mainly limited to intramuscular injection (such as COVID-19 mRNA LNPs vaccine) and intravenous injection targeting liver hepatocytes (such as Onpattro short interfering RNA LNPs). Its physicochemical properties are similar to very low density lipoproteins, and it is easy to adsorb apolipoprotein E in plasma, resulting in limited application outside the liver.

[0008] Due to the limitations of the characteristics of wild-type AAV and LNP vectors themselves, endowing high targeting properties to viral and non-viral vectors will expand their applications in medicine as drug delivery vectors. Summary of the Invention

[0009] The present invention provides a targeted drug delivery vector and a preparation method thereof. By screening targeted polypeptides with high affinity and specificity and integrating them into viral vectors or non-viral vectors, the present invention effectively solves the problems of insufficient targeting and low delivery efficiency in existing drug delivery vectors.

[0010] To achieve the above object, the technical solutions adopted by the present invention are as follows: In a first aspect, the present invention provides a targeted drug delivery vector, comprising: a drug-carrying carrier and a targeted polypeptide; the drug-carrying carrier is a viral vector, and the nucleotide of the targeted polypeptide is operably linked to the nucleotide of the capsid protein of the viral vector; or, the drug-carrying carrier is a non-viral vector, and the targeted polypeptide is linked to the non-viral vector by surface modification; the amino acid sequence of the targeted polypeptide is as shown in any one of SEQ ID NO: 8-12 and SEQ ID NO: 14.

[0011] The present invention screens out polypeptides with high affinity for targeted biomolecules, cells, tissues, and organs through phage display technology. The sequences of these polypeptides are as shown in any one of SEQ ID NO: 8-12 and SEQ ID NO: 14. And integrating such polypeptides into viral vectors or non-viral vectors endows viral vectors or non-viral vectors with the ability to specifically recognize specific biomolecules, cells, tissues, or organs, enhances the affinity of the vectors for cells, tissues, organs, etc., and applying them to drug delivery vectors can improve the targeting of drug carriers and enhance the therapeutic effect. Operably linked means inserting the nucleotide sequence encoding the targeted polypeptide into the nucleotide sequence of the capsid protein of the viral vector by genetic recombination, and expressing the targeted polypeptide through the capsid protein of the viral vector. Specifically, these polypeptides can target muscle cells or muscle, and can improve the targeting of the drug-carrying carrier to muscle.

[0012] As a preferred embodiment of the first aspect, the viral vector is any one of lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes virus; or / and, the non-viral vector is any one of lipid nanoparticles, virus-like particles, exosomes.

[0013] As a preferred embodiment of the first aspect, the surface modification methods include: physical adsorption, surface deposition, coupling, esterification, grafting. In the present invention, various surface modification methods can be used to immobilize the targeting polypeptide on the surface of the non-viral vector, so that the non-viral vector has a certain targeting property. For example: immobilize the targeting polypeptide on the surface of the non-viral vector through the van der Waals force in the physical surface adsorption method; immobilize the targeting polypeptide on the surface of the non-viral vector by depositing on the surface of the non-viral vector; immobilize the targeting polypeptide on the surface of the non-viral vector through a coupling agent; immobilize the targeting polypeptide on the surface of the non-viral vector by esterifying the targeting polypeptide with the surface of the non-viral vector; immobilize the targeting polypeptide on the surface of the non-viral vector through the surface grafting method.

[0014] In the second aspect, the present invention provides a preparation method of the targeting drug delivery carrier of the first aspect, including the following steps: Step 1: Screen the targeting polypeptide of the target through the phage display method to obtain the phage library of the targeting polypeptide; Step 2: Perform multiple rounds of screening and sequencing on the phage library in Step 1 to obtain the targeting polypeptide with high affinity for the target; the amino acid sequence of the targeting polypeptide is shown as any one of SEQ ID NO: 8-12, SEQ ID NO: 14; Step 3: Connect the targeting polypeptide obtained in Step 2 to the drug-loading carrier.

[0015] The various targeting polypeptides screened by the phage display technology of the present invention and connecting these various targeting polypeptides to the drug-loading carrier can improve the targeting property of the drug-loading carrier. Through the method of the present invention, various targeting drug delivery carriers can be obtained. The method of the present invention significantly improves the affinity of the drug carrier for specific targets, thereby enhancing the drug delivery efficiency and therapeutic effect. At the same time, the method greatly shortens the screening cycle and screening cost.

[0016] As a preferred embodiment of the second aspect, the target includes: biomolecules, cells, tissues, organs. The biomolecules include nucleic acid molecules and protein molecules.

[0017] The method of the present invention significantly improves the affinity of the drug carrier for biomolecules, cells, tissues or organs, thereby enhancing the drug delivery efficiency and therapeutic effect. At the same time, the method greatly shortens the screening cycle and screening cost.

[0018] As a preferred embodiment of the second aspect, the target is a biomolecule or / and a cell, and the method of step 1 is as follows: After incubating the phage library with the target biomolecule or / and cell, wash, elute and neutralize, transfect, culture, and purify; to obtain a phage library of the targeting polypeptide.

[0019] As a preferred embodiment of the second aspect, the target is a tissue or / and an organ, and the method of step 1 is as follows: (1) Inject the phage library into an animal, and collect phages from the tissue or / and organ; (2) Transfect, culture, and purify the phages collected in step (1); to obtain a phage library of the targeting polypeptide.

[0020] In the present invention, the phage library is directly injected into an animal, and a targeting polypeptide with high affinity for each tissue or / and organ can be obtained. Connecting this polypeptide with a drug carrier can improve the targeting of the carrier in each tissue or / and organ.

[0021] As a preferred embodiment of the second aspect, the method for connecting the targeting polypeptide with the drug-loaded carrier in step 3 includes the following steps: (1) Insert the nucleotide sequence of the targeting polypeptide into the capsid gene of the viral vector to construct an engineered viral vector; (2) Express the targeting polypeptide by fusion of the capsid gene of the engineered viral vector to obtain a targeting viral vector drug delivery carrier; Or, The method for connecting the targeting polypeptide with the drug-loaded carrier in step 3 includes the following steps: Fix the targeting polypeptide to the surface of the non-drug-loaded carrier to obtain a targeting non-viral vector drug-loaded carrier; the non-viral vector is any one of lipid nanoparticles, virus-like particles, and exosomes.

[0022] Preferably, the non-viral vector is lipid nanoparticles.

[0023] Preferably, the viral vector is an adeno-associated virus vector (AAV). In the present invention, the nucleotide of the targeting polypeptide is inserted between the codons of any two adjacent amino acids among positions 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, or 704-714 of the amino acid sequence in the AAV capsid polynucleotide; the adeno-associated virus (AAV) includes: AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a targeted drug delivery carrier and a preparation method thereof. The preparation method of the present invention greatly shortens the screening period and screening cost compared with the traditional AAV vector screening method. Through phage display technology, polypeptides with high affinity for target receptors or cells, tissues, and organs are screened, and the polypeptides are integrated into viral vectors or non-viral vectors to endow them with high targeting to specific target receptors or cells, tissues, and organs, and targetedly deliver drugs.

[0025] For viral vectors, the present invention integrates the DNA sequence of the screened targeting polypeptide into the viral capsid protein sequence to endow it with high targeting to target molecules or cells, tissues, and organs. For non-viral vectors, the present invention couples the screened polypeptide to the non-viral vector by chemical modification or physical coupling, and immobilizes the targeting polypeptide on the surface of the vector to endow the non-viral vector with high targeting to target receptors or cells, tissues, and organs.

[0026] The above-mentioned targeting vector significantly improves the affinity of the drug carrier for specific biomolecules, cells, tissues, or organs, thereby enhancing the drug delivery efficiency and therapeutic effect. At the same time, this method greatly shortens the screening period and screening cost.

[0027] Experimental results show that the functional polypeptides screened by the present invention (the amino acid sequences of which are shown in any one of SEQ ID NO: 8-12 and SEQ ID NO: 14) can significantly improve the transfection efficiency of the cells in the novel AAV of the Pint series screened from typical muscle cells C2C12 compared with wild-type AAV9. In C57 mice, in the novel AAV of the Ms series screened, the transduction efficiency of muscle tissue can be significantly improved compared with wild-type AAV9. In the new LNP formulation screened in C57 mice, the distribution and transduction efficiency of muscle tissue can be significantly improved compared with the original formulation on the market, and the distribution in liver tissue is reduced. Brief Description of the Drawings

[0028] Figure 1 It is a schematic overview diagram of screening functional polypeptides by phage display technology of the present invention; Figure 2 It is a nucleic acid electrophoresis diagram after phage genome library construction; Figure 3 It is a plasmid diagram of AAV capsid variant construction of the present invention; Figure 4 It is a schematic diagram of AAV packaging and purification of the present invention; Figure 5 It is a schematic diagram of AAV virus titer amplification curve of the present invention; Figure 6 It is a schematic diagram of LNP coupled polypeptide of the present invention; Figure 7 It is a fluorescence image of a series of PintAAV cell infections; Figure 8 It is a statistical chart of a series of PintAAV cell infections Figure 9 It is an imaging diagram of Pint-E transfected mice; Figure 10 It is a schematic diagram of MsAAV obtained by the phage screening method in mice; Figure 11 It is an analysis diagram of the fluorescence intensity of LNP in vivo imaging of mice. Detailed implementation manners

[0029] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] The present invention constructs a targeted drug delivery vector, including: a drug-loading carrier and a targeting polypeptide; The drug-loading carrier is a viral vector, and the nucleotide of the targeting polypeptide is operably linked to the nucleotide of the capsid protein of the viral vector; Or, the drug-loading carrier is a non-viral vector, and the targeting polypeptide is linked to the non-viral vector by surface modification.

[0031] Preferably, the viral vector is any one of lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes virus; and / or, the non-viral vector is any one of lipid nanoparticles, virus-like particles, exosomes.

[0032] Preferably, the amino acid sequence of the targeting polypeptide is shown in any one of SEQ ID NO: 8-12, SEQ ID NO: 14.

[0033] Preferably, the surface modification methods include: surface adsorption, surface deposition, coupling, esterification, grafting.

[0034] The preparation method of the above-mentioned targeted drug delivery vector includes the following steps: Step 1: Screen the targeting polypeptide of the target by phage display method to obtain a phage library of the targeting polypeptide; wherein, the target can be any one of biomolecules, cells, tissues, organs.

[0035] Preferably, when the target is a biomolecule and / or a cell, the following method is adopted: After incubating the phage library with the target biomolecule and / or cell, wash, elute and neutralize, transfect, culture, and purify; obtain a phage library of the targeting polypeptide.

[0036] Preferably, when the target is a tissue or / and an organ, the following method is adopted: (1) Inject the phage library into an animal body and collect phages from the tissue or / and organ; (2) Transfect, culture, and purify the phages collected in step (1) to obtain a phage library of targeting polypeptides.

[0037] Step 2: Perform multiple rounds of screening and sequencing on the phage library in step 1 to obtain a targeting polypeptide with high affinity for the target.

[0038] Step 3: Connect the targeting polypeptide obtained in step 2 with a drug-loading carrier; wherein, the drug-loading carrier includes a viral carrier and a non-viral carrier.

[0039] Preferably, when the drug-loading carrier is a viral carrier, the method for connecting the targeting polypeptide with the drug-loading carrier in step 3 includes the following steps: (1) Insert the nucleotide sequence of the targeting polypeptide into the capsid gene of the viral carrier to construct an engineered viral carrier; (2) Fuse and express the targeting polypeptide through the capsid gene of the engineered viral carrier to obtain a targeting viral vector drug delivery carrier.

[0040] Preferably, when the drug-loading carrier is a non-viral carrier, the method for connecting the targeting polypeptide with the drug-loading carrier in step 3 is: fix the targeting polypeptide to the surface of the non-drug-loading carrier to obtain a targeting non-viral carrier drug delivery system; wherein, the non-viral carrier is any one of lipid nanoparticles, virus-like particles, and exosomes.

[0041] Example 1 Screening of Targeting Polypeptides for Integrin by Phage In this example, integrin is used as the target, and phage display technology is utilized to screen polypeptides with high affinity for integrin. Subsequently, the polypeptide sequence with affinity for integrin is integrated into a viral carrier in subsequent experiments to improve the transduction efficiency and specificity of the viral carrier for cells.

[0042] Main experimental materials: Phage display kits (7-peptide NEB, #E8211S; 12-peptide NEB, #E8210S), integrin protein (SinoBiologic, #CT039).

[0043] Experimental steps: 1. Coating of integrin antigen: Dilute the integrin antigen protein to 0.5 μg / μL with PBS buffer, take 4 μg of the protein into a flat-bottom enzyme-linked well plate, add 150 μL of antigen coating solution to each well, and mix well; set 4 replicates and coat overnight at 4°C.

[0044] 2. Blocking: Discard the coating liquid in the ELISA plate and wash it 6 times with PBST (phosphate buffer solution) on a plate washer; Pat dry the residual liquid in the wells with a tissue paper, add 200 μL of blocking solution to each well, and block at room temperature for 2 h.

[0045] 3. Washing: After blocking, wash 6 times with PBST, 3 min each time.

[0046] 4. Binding or Incubation: Dilute the phage library titer to 5×10 11 pfu / mL with the blocking solution, add 200 μL of the phage library dilution to each well of the ELISA plate, and place it at room temperature for 2 h.

[0047] 5. Washing: Discard the excess dilution, and wash 10 times with PBST.

[0048] 6. Elution and Neutralization: After washing, add 600 μL of elution solution to each well, shake and elute at 400 rpm at room temperature for 20 min; Then add 120 μL of neutralization solution for neutralization and mixing.

[0049] 7. Transfection and Cultivation: Transfer the neutralization solution obtained in step 6 into K12 Escherichia coli cultured to an OD600 of about 0.5, mix and expand the culture for 4 h, and then centrifuge at 8000 g for 5 min to collect the supernatant.

[0050] 8. Purification: Add 20% (w / v) phage precipitation solution to the supernatant in step 7, mix well and let it stand at 4 °C for 4 h.

[0051] 9. Collection and Re-purification: Centrifuge the phage precipitation solution in step 8 above at 13000 g for 10 min, collect the precipitate, and repeat step 8 to purify the phage again.

[0052] 10. Calculate the phage titer: Take 10 μL of the phage solution in step 9, dilute it stepwise, and calculate the phage titer by the blue-white spot color development method.

[0053] 11. Screening: Repeat steps 1-10, and after three rounds of screening, obtain a phage peptide library with high affinity for integrin.

[0054] Example 2 Phage Genome Extraction and Sequencing Extract the genome of the phage peptide library in Example 1 for high-throughput sequencing, and obtain the polypeptide amino acid sequence by referring to the codons.

[0055] Experimental steps: 1. Extract the genome using a phage DNA extraction kit.

[0056] 2. Accurately quantify the genome using a Qubit nucleic acid quantifier.

[0057] 3. Use the extracted genomic DNA as a template for the first round of PCR amplification. The primer sequences for amplification are shown in Table 1 below, the PCR reaction system is shown in Table 2 below, and the reaction program is shown in Table 3 below: Table 1: Table 2: Table 3: 4. Add sequencing adapters to the PCR reaction products: Take 5 μL of the reaction solution for the second round of PCR amplification, add high-throughput sequencing adapters. The PCR reaction system is shown in Table 4 below, and the reaction program is shown in Table 5 below: Table 4: Table 5: 5. Quantify the PCR products in step 4 using Qubit: Take 3 μL of the reaction solution for nucleic acid electrophoresis detection.

[0058] 6. Sequence the high-throughput library of the PCR products. The sequencing platform is Illumina.

[0059] Construct a library for the phage. The nucleic acid electrophoresis pattern after library construction is as shown in Figure 2 shown. The 250 bp band above indicates successful addition of the sequencing adapter, and the arrow at 200 bp below indicates the original sequence. The polypeptide sequences obtained after sequencing are shown in Table 6. These sequences have a high affinity for integrin antigens.

[0060] Table 6: Example 3 Phage Display in Mice Inject the phage polypeptide library directly into mice. After 24 h, take various organs of the mice for phage amplification culture and purification. Sequence the purified phage to obtain the corresponding polypeptide sequences. To a certain extent, these sequences can reflect the preference of the short peptides for various organs. Integrate the polypeptides onto the surface of the viral vector, and it is expected to obtain a viral vector with high affinity for the corresponding organs.

[0061] Experimental steps: I. In vivo screening of phage 1. Set up experimental groups and control groups. The experimental group is the injection group of the 7 / 12-mer phage library (phage display kit (7-mer NEB, #E8211S, 12-mer NEB, #E8210S)), with 3 replicates for each peptide library. The control group is the injection of normal saline.

[0062] 2. Phage library injection: Dilute the 7 / 12 peptide phage library with PBS, and the injection dose is 1.0×10 11 Pfu, and administer 200 μL via the tail vein of mice.

[0063] 3. After 24 h, harvest the mice. When harvesting, fully bleed the mice, and separate the biceps brachii, quadriceps femoris, diaphragm tissues and the materials of heart, liver, brain, spleen, lung, and kidney organs.

[0064] 4. Cut off the tissues of some organs and add them to PBST for tissue grinding instrument.

[0065] 5. Collection and washing: Discard the supernatant, and repeat the washing with PBST for 10 times.

[0066] 6. Elution and neutralization: Add 600 μL of eluent, shake well to fully elute the phage, and add 120 μL of neutralizing solution to terminate.

[0067] 7. Transfection and culture: Transfer the neutralizing solution obtained in step 6 into K12 Escherichia coli cultured to an OD600 of about 0.5, mix and expand the culture for 4 h, and then centrifuge at 8000 g for 5 min to collect the supernatant.

[0068] 8. Purification: Add 20% (w / v) phage precipitation solution to the supernatant in step 7, mix well and let it stand at 4°C for 4 h.

[0069] 9. Collection and re-purification: Centrifuge the phage precipitation solution in step 8 above at 13000 g for 10 min, collect the precipitate, and repeat step 8 to re-purify the phage.

[0070] 10. Calculate the phage titer: Take 10 μL of the phage solution in step 9, dilute it in gradient, and calculate the phage titer by the blue-white plaque color development method.

[0071] 11. Screening: Repeat steps 1-10, and after three rounds of screening, obtain a polypeptide sequence library with high affinity for mouse muscle.

[0072] II. Phage genome extraction and sequencing This step is the same as the operation steps in Example 2.

[0073] Experimental results: After multiple rounds of screening and library construction and sequencing, a polypeptide sequence with high affinity for mouse muscle was obtained. The polypeptides after analysis after sequencing are shown in Table 7: Table 7: Example 4: Construction of AAV capsid variant plasmid The nucleotide sequences encoding the amino acid sequences shown in any one of SEQ ID NOs: 8-12, 14 were integrated into the gene of the AAV viral capsid plasmid pAAV2 / 9n by genetic engineering methods; and a BsmBI restriction site was introduced at the 588 / 589 site, and the gene sequence of the functional polypeptide was added at this site to endow pAAV2 / 9n with corresponding transduction characteristics. The specific operations are as follows: Experimental materials: Capsid plasmid pAAV2 / 9n (addgene, #112865), BsmBI restriction endonuclease (NEB, #R0580), BsiWI (NEB, #R0553V) and PmeI (NEB, #R0560V) restriction endonucleases, DH5α competent cells (Tsingke, #TSC-C14), and the rest of the reagents are common materials.

[0074] Experimental procedures: I. Obtaining the target fragment 1. Entrust GenScript Nanjing to synthesize the gene fragment sequences (SEQ ID NOs: 3-7, SEQ ID NO: 13). The gene fragments are in the PUC57 plasmid glycerol bacteria, which are named pUC57-AAV588 / 589-BsmBI.

[0075] 2. Activate the glycerol bacteria: Streak the glycerol bacteria on an LB resistant plate overnight. The next day, pick 2 monoclonal colonies into 5 mL of LB medium, extract using a plasmid miniprep kit, and preserve 1 tube for each tube, with 500 μL of 50% glycerol + 500 μL of the bacterial solution.

[0076] 3. Determine the correctness of the sequence of the plasmid by Sanger sequencing, and the sequencing primer is the universal U6 primer for full-length sequencing; 4. Double-digest the plasmid with BsiWI and PmeI. The digestion system is shown in Table 8: Table 8: React at 37 °C for 1.5 h, and add 6 μL of 10x DNA loading Buffer for agarose gel electrophoresis.

[0077] 5. Obtain the fragment: The size of the vector fragment is 2710 bp, and the size of the target fragment is 1230 bp. Use a gel extraction kit to recover the lower-layer target fragment.

[0078]

[0079] The target fragments are: SEQ ID NO: 3 - 7, SEQ ID NO: 13.

[0080] 6. Measure the concentration of the target fragments by NanoDrop.

[0081] II. Construct the vector fragment 1. The glycerol bacteria containing the pAAV2 - 9n plasmid were purchased from (Addgene #112865), and streaked on an LB plate and incubated overnight at 37°C in an inverted position.

[0082] 2. Pick 2 single colonies into 5 mL of LB medium with ampicillin resistance, extract using a plasmid miniprep kit, and verify the correctness of the plasmid by Sanger sequencing.

[0083] 3. Linearize the pAAV2 - 9n plasmid using double digestion with BsiWI and PmeI. The digestion system is shown in Table 9: Table 9: React at 37°C for 1.5 h, and add 10 μL of 10×DNA loading Buffer for agarose gel electrophoresis.

[0084] 4. Obtain the linearized vector fragment: The size of the linearized vector fragment is 6100 bp, and recover the upper - layer vector fragment using a gel extraction kit.

[0085] 5. Measure the concentration of the recovered vector fragment by NanoDrop.

[0086] III. Ligate the target fragment to the vector fragment 1. Use DNA T4 ligase for the reaction (the amounts of the target fragment and the linearized vector are 25 ng and 100 ng respectively, and the fragment ratio is 1:1). The ligation reaction is as follows in Table 10: Table 10: React at 25°C for 20 min to obtain the ligation product.

[0087] 2. Take 2 μL of the ligation product and transform it into DH5α Escherichia coli competent cells, spread on an ampicillin - resistant plate. The next day, pick 2 single - colony colonies into 5 mL of LB medium with ampicillin resistance for shaking culture to extract plasmids (preserve 1 tube for each tube, 500 μL of 50% glycerol + 500 μL of bacterial solution).

[0088] 3. Determine the correctness of the sequence by Sanger sequencing, and name the correct plasmid as: pAAV2 / 9n - 588 / 589 - BsmBI. The plasmid schematic diagram is shown in Figure 3 .

[0089] A series of pAAV2 / 9n-588 / 589-BsmBI capsid plasmids were obtained, and the information of the series of plasmids is shown in Table 11: Table 11: Example 5 Packaging of AAV Viruses The capsid plasmid, target gene plasmid, and helper virus plasmid of the adeno-associated virus vector constructed in Example 4 were transfected into eukaryotic cells, and a targeted drug delivery vector was obtained through self-assembly; the specific operations are as follows: Synthesizing an AAV virus vector requires three plasmids, a capsid plasmid (Rep / Cap, that is, the series of pAAV2 / 9n-588 / 589-BsmBI capsid plasmids modified in the present invention), a target gene plasmid (GOI), and a helper virus plasmid (Helper). The three plasmids were transfected into eukaryotic cells, and an AAV virus vector containing the target gene could be formed through self-assembly. After purification and passing the quality inspection, it can be used for drug delivery in vivo and in vitro. The overall flow chart is shown in Figure 4 .

[0090] Experimental steps ( Figure 4 ): I. Virus Packaging and Purification 1. Plasmid extraction: The three plasmids required for the aforementioned AAV were subjected to overnight shaking culture, and an endotoxin-free plasmid extraction was performed using a plasmid large-scale extraction kit (Promega #A2392).

[0091] 2. Plasmid detection: The extracted plasmids were quantified using NanoDrop and Qubit, and nucleic acid electrophoresis was performed to verify the size and purity of the plasmids.

[0092] 3. Use 293T cells (ATCC #CRL-3216) to prepare viruses according to the three-level seed bank.

[0093] 4. Packaging of AAV viruses: Adopt the PEI transient transfection method, and transfect 293T cells in the ratio of capsid plasmid: target gene plasmid: helper virus plasmid of 1:1:1. Add a total of 20 μg of plasmid and 60 μL of PEI (DNA:PEI ratio is 1:3) to each 15 cm culture dish.

[0094] 5. Three days after transfection, collect the cells for purification.

[0095] 6. Resuspend the cells, and repeatedly freeze-thaw the cells to lyse the cells and release the viruses. Adjust the final concentration of nuclease to 50 U / mL and incubate at 37 °C for 60 min.

[0096] 7. Treat the samples before ultracentrifugation, prepare different density gradients of iodixanol, and finally use a peristaltic pump to slowly and completely load all the samples. After the heat-sealed tube is completely filled with liquid, heat-seal it and seal it.

[0097] 8. Ultracentrifugation settings: 18°C, centrifugal speed 70,000 rpm, centrifugation time 1 hour and 5 minutes, collect the virus.

[0098] 9. Sterile filtration: sterilize and filter the ultrafiltration recovered samples with a 0.22um syringe filter, retain samples to measure physical titer and SDS-PAGE gel to measure protein purity, label the packaged samples and temporarily store them in an ultra-low temperature biological sample library.

[0099] 3. Virus titer detection 1. Linearize the pAAV_IRES-hrEGFP plasmid and digest it with SacⅠ-HF enzyme (select the restriction site according to the specific plasmid) (37℃, 30min); 2. The reaction system is shown in Table 12: Table 12: At the same time, nucleic acid gel was prepared for enzyme digestion verification. Using Qubit for quantification, the concentration of the reaction solution was measured to be 16.5 ng / μL. The copy number per μL was calculated to be 2.68×10 9 vg, converted to a linearized plasmid dilution gradient of 10 9 -10 4 , the final volume is 200 μL, and ten times is one gradient to form a concentration gradient. qPCR reaction is performed, the reaction system is shown in Table 13, and the reaction procedure is shown in Table 14: Table 13: Table 14: 3. Result analysis: From the amplification curve ( Figure 5 ) CT value and R 2 The value can show the accuracy of the standard curve; 4. The addition of a commercially available standard virus product (wild-type AVV9) can verify the accuracy of the test, and from then on, the titer of the virus can be accurately calibrated, thereby quantifying the titer of each virus, which is convenient for subsequent testing and comparison of the transfection ability of different viruses. The targeted virus is obtained, as shown in Table 15: Table 15: Example 6 LNP preparation and polypeptide modification The amino acid sequence of the polypeptide with the amino acid sequences shown in SEQ ID NO: 8-10 obtained in Example 2 was linked to a linker to obtain a polypeptide amino acid sequence shown in any one of SEQ ID NO: 15-17; The polypeptide with the amino acid sequence shown in any one of SEQ ID NO: 8-10 was linked to lipid nanoparticles by coupling to obtain a non-viral targeted drug delivery carrier. The specific operation is as follows: Lipid Nanoparticles (LNP) is a nanoscale drug delivery system composed of lipids, usually composed of four components: phospholipids, cholesterol, cationic lipids, and polyethylene glycolated lipids (PEG-lipids). Because its components are natural substances, it has low toxicity and immunogenicity to the human body, and can be administered repeatedly.

[0100] To achieve the targeted delivery of lipid nanoparticles to specific cells or tissues, in this example, the amino acid sequence of the polypeptide with the amino acid sequences shown in SEQ ID NO: 8-10 was linked to the surface of lipid nanoparticles, and its schematic diagram is as Figure 6 . When the lipid nanoparticles enter the blood circulation, these polypeptide sequences can guide the lipid nanoparticles to precisely bind to the receptors, thereby improving the delivery efficiency of the drug to the target tissue / cells and reducing the side effects on normal cells.

[0101] Experimental materials: 50 mM sodium acetate (pH 5.5) buffer, SM102 (CAS: 2089251-47-6, molecular weight: 710.1653, molecular formula: C 44 H 87 NO5), DSPC (CAS: 816-94-4, molecular weight: 790.15, molecular formula: C44H 88 NO8P), cholesterol (CAS: 57-88-5, molecular weight: 386.65, molecular formula: C 27 H 46 O), DMG-PEG2k (CAS: 160743-62-4, molecular weight: 2509.2, molecular formula: C 34 H 66 O: (C2H4O)n), DMG-PEG2k-mal (phospholipid polyethylene glycol), Fluc mRNA (messenger RNA encoding firefly luciferase). The polypeptide was synthesized by (Jiangsu Zhuanpeptide Biotechnology) company. Specifically: Control polypeptide: A2G80: VQLRNGFPYFSYGGC; M12-1: RRQPPRSISSHPGGGSC; The Intergrin polypeptide sequence of the present invention is shown in Table 16: Table 16: Among them, GGGS is the linker, and C is added for conjugation synthesis with -Mal (the linker of the A2G80 polypeptide is GGC).

[0102] Experimental equipment: Mainly use microfluidic instruments, and the rest of the equipment are conventional equipment.

[0103] Experimental steps (the reagents and materials in this example are all conventional materials for preparing LNP and are all commercially available products): 1. Preparation of lipid solution: Weigh 15 mg of SM102 and place it in a 1.5 mL clean EP tube, then add 200 μL of absolute ethanol to a concentration of 75 mg / mL.

[0104] Weigh 10 mg of DSPC and place it in a 1.5 mL clean EP tube, then add 1.0 mL of absolute ethanol to a concentration of 15 mg / mL.

[0105] Weigh 10 mg of cholesterol and place it in a 1.5 mL clean EP tube, then add 1.0 mL of absolute ethanol to a concentration of 75 mg / mL.

[0106] Weigh 10 mg of DMG-PEG2k (phospholipid polyethylene glycol) and place it in a 1.5 mL clean EP tube, then add 1.0 mL of absolute ethanol to a concentration of 75 mg / mL.

[0107] 2. Preparation of DMG-PEG2k-Mal: 2.1 Molar ratio of each lipid SM102: DPPC: cholesterol: DMG-PEG2k: DSPE-PEG2k-Mal (phospholipid polyethylene glycol maleimide) = 50:10:38.5:1.2:0.3.

[0108] 2.2 Mass ratio Total lipid: mRNA = 40:1; The total lipid concentration is 12.5 mM.

[0109] 2.3 Preparation of lipid ethanol phase Taking 100 μg of mRNA as an example, prepare SM102 LNP modified with 0.3% polypeptide: In the lipid solution prepared above, take 46.3 μL of SM102 solution, 77.3 μL of DSPC, 145.7 μL of cholesterol, 29.5 μL of DMG-PEG2k and 8.6 μL of DMG-PEG2k-Mal and place them in a clean 1.5 mL EP tube. Then add 475.5 μL of absolute ethanol to make the total lipid concentration 12.5 mM and the total volume 782.9 μL (1.5 times the required amount of lipids), and mix well until clear and transparent.

[0110] 2.4 Preparation of mRNA aqueous phase Take 100 μg of Fluc mRNA (concentration 1 mg / mL) and place it in a clean 5 mL tube. Then add 1465.9 μL of 50 mM sodium acetate buffer (pH 5.5) and mix well.

[0111] 3. Preparation by microfluidics: Transfer the lipid ethanol phase and the mRNA aqueous phase to 1 mL and 3 mL syringes respectively. Remove the excess air bubbles. Insert the LNP chip into the device, insert the two syringes with screw threads into the corresponding interfaces of the chip, and prepare two 15 mL collection tubes, one for sample collection and one for waste liquid collection. Design the parameters on the software, set the total injection volume to 1.8 mL and the total flow rate to 12 mL / min (the flow rate of the mRNA aqueous phase is 9 mL / min and the collection volume is 1.35 mL; the flow rate of the lipid ethanol phase is 3 mL / min and the collection volume is 0.45 mL). Design the waste liquid volume to 0.45 mL (the initial waste liquid volume is 0.4 mL and the final waste liquid volume is 0.05 mL). Click "Start" to prepare the LNP. After preparation, transfer it to a Pur-A-Lyzer Maxi 3500 dialysis tube. The prepared sample should be dialyzed within 15 minutes. Dialyze with 1xPBS (without calcium and magnesium ions) at 4 °C for 4 hours and then change the solution, and then dialyze overnight (to remove ethanol). After dialysis, transfer it to a clean 5 mL tube and measure the volume. (If necessary, a 30 kDa ultrafiltration tube can be used, centrifuged at 3000 g at 4 °C for volume concentration) Note: The generated solution can be stored at 4 °C for several days before use. However, it is recommended to use the prepared LNPs as soon as possible to obtain consistent results and get the LNP.

[0112] 4. Polypeptide conjugation modification of LNP: 1) Using GGGS as the linker, C was conjugated and added with -Mal (the A2G80 polypeptide was GGC). The sulfhydryl group of the cysteine residue underwent a thiol - double bond addition reaction with the double bond of maleimide to form a new chemical bond, synthesizing polypeptides with amino acid sequences shown in SEQ ID NO: 15 - 17; 2) Dissolve the synthesized polypeptide with PBS (pH 7.4) at a concentration of 4 mg / mL.

[0113] 3) Take 90 μg from the LNP prepared in step 6, add the polypeptide solution (the molar ratio of polypeptide:Mal is 10:1), mix well and incubate overnight at 4°C.

[0114] 4) Use dialysis to remove the non - conjugated polypeptides. Dialyze with 1xPBS (without calcium and magnesium ions) at 4°C for 4 hours and then change the solution, followed by overnight dialysis. After dialysis, transfer to a clean 1.5 mL EP tube, measure the volume (if necessary, a 30 kDa ultrafiltration tube can be used, 3000 g, 4°C for volume concentration) to obtain polypeptide - modified LNP.

[0115] Example 7 Targeted PintAAV Virus Cell Infection Experiment Regarding the targeted polypeptide screened from integrins using phage display technology in Example 1, we believe that it can increase the infection efficiency of muscle cells. Add the polypeptide sequence to the AAV capsid using the construction method of Example 4, and use the method of Example 5 for virus packaging and purification. The packaged cargo contains the gene sequences of Firefly luciferase and mCherry, and verify the virus infection efficiency on muscle cells.

[0116] Experimental materials: C2C12 mouse myoblasts (ATCC#CRL - 1772), C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), and the rest of the materials are common materials.

[0117] Experimental equipment: Carbon dioxide constant temperature incubator, small animal in - vivo imager, fluorescence microscope, laser confocal microscope, and the rest of the equipment are common equipment.

[0118] Experimental steps: 1. Cell culture and induction: The C2C12 mouse myoblasts after resuscitation become stable after more than three generations of culture and are plated. When the cell confluence is about 60%, induce by adding 2% horse serum to form myotubes.

[0119] 2. Virus infection verification: After the purified virus passed the tests of QPCR, SDS-PAGE, and endotoxin determination, absolute quantitative PCR was used to measure the titer, and a virus infection experiment was carried out according to the infection titer MOI of 1E+4 per well.

[0120] 3. Fluorescence imaging and analysis: A Zeiss fluorescence microscope was used to take pictures of the fluorescence field of the cells 6 days after infection, with the background value unified, and the pictures were taken under a 10x objective lens. The average fluorescence intensity (intDen) of the pictures was quantified by Image J.

[0121] 4. Experimental results: As Figure 7 , 8 The results showed that compared with the wild-type AAV9 virus, the screening method and virus vector of the present invention had better transfection efficiency, indicating that the targeting polypeptide obtained by screening in the present invention had stronger muscle cell targeting ability and could improve the muscle targeting of the AAV vector.

[0122] Example 8 In vivo transfection experiment of targeted Pint-AAV virus in mice For the novel virus Pint-E with the highest fluorescence intensity, in order to verify the effect of the virus in mice, we injected the virus into mice by intramuscular injection and observed the expression and distribution of the virus in mice.

[0123] Experimental materials: C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), Pint-E virus, and the rest of the materials were common materials.

[0124] Experimental equipment: Small animal in vivo imager, fluorescence microscope, laser confocal microscope, and the rest of the equipment were common equipment.

[0125] Experimental steps: 1. Virus quality control: After the purified virus passed the tests of QPCR, SDS-PAGE, and endotoxin determination, absolute quantitative PCR was used to measure the titer.

[0126] 2. Determine the administration volume, weigh the mice, calculate the injection volume according to the dose of 1.0×10^13 vg / kg, dilute the virus with normal saline during injection, and the injection volume was 200 μL. The tail vein administration method was used.

[0127] 3. Perform in vivo imaging on the mice on the 7th, 14th, 21st, and 28th days after administration to observe the distribution and expression of the delivered drug in the body. On the 28th day, take organ samples from the mice, perform cryopreservation and embedding, and conduct molecular level detection.

[0128] Experimental results: The results showed that as Figure 9, the novel viral vector Pint-E screened based on this method has better transfection efficiency in vivo. It shows that the targeting polypeptide obtained by screening in the present invention has stronger muscle targeting ability and can improve the muscle targeting of AAV vectors.

[0129] Example 9 Distribution Experiment of Targeted MsAAV Virus in Mice For the targeting polypeptide screened from mice using phage display technology in Example 3, we believe it can increase the distribution and infection efficiency in the corresponding target organs. The polypeptide sequence was added to the AAV capsid using the construction method of Example 4, and virus packaging and purification were carried out using the method of Example 5. The packaged cargo carried the gene sequences of Firefly luciferase and mCherry. The infection efficiency of the novel virus was verified in mice.

[0130] Experimental materials: C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), MsAAV virus, and the rest of the materials are common materials.

[0131] 1. Virus quality control: After purification, the virus was qualified by QPCR, SDS-PAGE, and endotoxin determination, and then absolute quantitative PCR was used for titer determination.

[0132] 2. Determine the dosing volume: Weigh the mice. The dose was calculated as 1.0×10^13 vg / kg, and the injection volume was calculated. When injecting, the virus was diluted with normal saline, and the injection volume was 200 μL. The tail vein injection method was used.

[0133] 3. Perform in vivo imaging on the mice on the 7th, 14th, 21st, and 28th days after dosing. Weigh the mice, inject 200 μL of anesthetic per 10 g of body weight intraperitoneally, and inject 100 μL of the luminescent substrate (15 mg / mL) per 10 g of body weight. Perform small animal in vivo imaging ten minutes after the substrate injection. Observe the distribution and expression of the delivered drug in vivo. On the 28th day, take organs from the mice for cryopreservation and embedding, and perform molecular level detection.

[0134] The experimental results are as Figure 10 , compared with wild-type AAV, the novel viral vector MsAAV virus targets more muscle tissues and distributes less to the liver, indicating that the targeting polypeptide obtained by screening in the present invention has stronger muscle targeting ability and can improve the muscle targeting of AAV vectors.

[0135] Example 10 Administration of Targeted LNP Virus in Mice For the targeted LNP delivery vector constructed in Example 8, the intravenous injection of mice was used to verify its targeting. The control sequence was a sequence that has been reported to change the targeting of LNP. Fluc-mRNA (firefly luciferase mRNA) was packaged using LNP, and the distribution and expression of the cargo in mice were detected by a small animal in vivo imaging instrument.

[0136] Experimental materials: All C57 mice used in this experiment were purchased from the Experimental Animal Center of Yunnan University. The LNP was prepared in Example 8, and anesthetic and luciferase substrate were used.

[0137] Experimental equipment: Small animal in vivo imager, and the rest are common equipment.

[0138] Experimental grouping: (1)NC (PBS control group); (2) Non-pep (unmodified group); (3) A2G80; (4) M12-1; (5) Intergrin-1; (6) Intergrin-2; (7) Intergrin-3; Experimental steps: 1. Select C57 mice aged 6 - 8 weeks, regardless of gender, with 3 mice in each group. Intravenous injection was performed via the tail vein at a dose of 1.5 mg / kg, and the total volume was 250 μL.

[0139] 2. After 6 hours, the mice were imaged using a small animal in vivo imager. The mice were weighed, and 200 μL of anesthetic was intraperitoneally injected per 10 g of body weight, and 100 μL of luminescent substrate (15 mg / mL) was injected per 10 g of body weight. Small animal in vivo imaging was performed ten minutes after the substrate injection.

[0140] The experimental results are as Figure 11 , compared with the control group (unmodified LNP), intergrin-1 significantly improved the distribution and expression of LNP in muscle and reduced the distribution of LNP in the liver. This indicates that the targeted polypeptide screened in the present invention has stronger muscle targeting and can improve the muscle targeting of the AAV vector.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A targeted drug delivery carrier, characterized in that, The drug delivery carrier includes: a drug-loaded carrier and a targeting polypeptide; The drug-loaded carrier is a viral vector, and the nucleotide of the targeting polypeptide is operably linked to the nucleotide of the capsid protein of the viral vector; Or, the drug-loaded carrier is a non-viral vector, and the targeting polypeptide is linked to the non-viral vector by surface modification; The amino acid sequence of the targeting polypeptide is shown in any one of SEQ ID NO: 8-12 and SEQ ID NO:

14.

2. The drug delivery carrier according to claim 1, wherein The viral vector is any one of lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes virus; and / or, the non-viral vector is any one of lipid nanoparticles, virus-like particles, exosomes.

3. The drug delivery carrier according to claim 1, wherein The surface modification methods include: surface adsorption, surface deposition, coupling, esterification, grafting.

4. A method for preparing a targeted drug delivery carrier according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: Step (1): Screen the targeting polypeptide of the target by phage display method to obtain a phage library of the targeting polypeptide; Step (2): Perform multiple rounds of screening and sequencing on the phage library in Step 1 to obtain a targeting polypeptide with high affinity for the target; the amino acid sequence of the targeting polypeptide is shown in any one of SEQ ID NO: 8-12 and SEQ ID NO: 14; Step (3): Link the targeting polypeptide obtained in Step 2 to the drug-loaded carrier; the drug-loaded carrier includes a viral vector and a non-viral vector.

5. The preparation method according to claim 4, characterized in that, The target includes: biomolecules, cells, tissues, organs.

6. The preparation method according to claim 5, characterized in that, When the target is a biomolecule or / and a cell, the following method is adopted in Step 1: After incubating the phage library with the target biomolecule or / and cell, wash, elute and neutralize, transfect, culture, and purify; obtain a phage library of the targeting polypeptide.

7. The preparation method according to claim 5, characterized in that, When the target is a tissue or / and an organ, the following method is adopted in Step 1: (1) Inject the phage library into an animal body, and collect phages from the tissue or / and organ; (2) Transfect, culture, and purify the phages collected in Step (1); obtain a phage library of the targeting polypeptide.

8. The preparation method according to claim 4, characterized in that, The method for linking the targeting polypeptide to the drug-loaded carrier in Step 3 includes the following steps: (1) Insert the nucleotide sequence of the targeting polypeptide into the capsid gene of the viral vector to construct an engineered viral vector; (2) Fusion express the targeting polypeptide through the capsid gene of the engineered viral vector to obtain a targeted viral vector drug delivery carrier; Or, The method for linking the targeting polypeptide to the drug-loaded carrier in Step 3 includes the following steps: Fix the targeting polypeptide to the surface of the non-drug-loaded carrier to obtain a targeted non-viral vector drug-loaded carrier; the non-viral vector is any one of lipid nanoparticles, virus-like particles, exosomes.

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