A targeted drug delivery carrier and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine technology and relates to a targeted drug delivery carrier and a preparation method thereof. The present invention provides a targeted drug delivery carrier comprising: a drug carrier and a targeting polypeptide; the drug carrier is a viral vector, and the nucleotides of the targeting polypeptide are operably connected to the nucleotides of the capsid protein of the viral vector; or, the drug carrier is a non-viral vector, and the targeting polypeptide is connected to the non-viral vector by surface modification, and the amino acid sequence of the targeting polypeptide is shown in any one of SEQ ID NO: 8-12 and SEQ ID NO: 14. The present invention designs and prepares an efficient drug delivery carrier by utilizing a targeting polypeptide that specifically recognizes a biomolecule, cell, tissue or organ.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a targeted drug delivery carrier and a preparation method thereof. Background Art

[0002] Currently, delivery vectors used for genetic diseases are divided into viral vectors and non-viral vectors according to whether they are based on viral modification. Viral vectors mainly include: lentivirus vectors (LV), adenovirus vectors (AdV) and adeno-associated virus vectors (AAV), etc. Non-viral vectors mainly include: lipid nanoparticles (LNP), virus-like particles (VLP), exosomes, etc.

[0003] AAV, a representative non-pathogenic and replication-defective viral vector, is widely used due to its low immunogenicity, resistance to host genome integration, and lack of pathogenicity. Currently, 90% of gene therapy clinical trials use AAV as a delivery vector. However, naturally occurring wild-type AAV has very limited organ targeting, limiting its therapeutic applications. Research indicates that AAV vectors have a high affinity for the liver and are naturally enriched in liver cells. Therefore, low doses of AAV vectors can be effective when targeting the liver alone. However, to achieve effective concentrations in other areas, the systemic AAV vector dose must be significantly increased. Existing research indicates that modifications to the AAV capsid can alter the biological properties of the vector. The AAV virus consists of an icosahedral protein capsid approximately 26 nm in diameter and a single-stranded DNA genome of approximately 4.7 kb. The genome is flanked by T-shaped inverted terminal repeats (ITRs), which contain the REP and CAP genes between the two ITRs. The REP gene encodes four proteins required for viral replication and regulation, while the CAP gene encodes the VP1, VP2, and VP3 proteins that comprise the capsid. The viral capsid is composed of 60 capsid protein monomers in a 1:1:10 ratio (VP1:VP2:VP3). Nine common variable regions (VRⅠ-VRIX) are found within the subunits that comprise the AAV capsid. These regions determine differences between AAV serotypes, including receptor recognition, gene transduction efficiency, and immune responses. This influences viral tissue tropism, transduction efficiency, antigenicity, and cross-immunogenicity between serotypes.

[0004] Non-viral vectors, such as lipid nanoparticles (LNPs), have become a key tool for nucleic acid vaccines and therapeutics due to their safety, tolerability, repeat dosing, and ability to carry large amounts of genetic cargo. Traditional LNPs contain four molecules: 1. Ionizable lipids: These bind nucleic acids and facilitate endosomal escape. 2. Amphiphilic phospholipids: These promote fusion with cell and endosomal membranes. 3. Cholesterol: These contribute to LNP stability. 4. Polyethylene glycol (PEG) lipids: These enhance colloidal stability and reduce clearance through the reticuloendothelial system. LNP technology stems from the study of cationic lipid complexes and ionizable cationic lipids, combining the physical properties and functional roles of lipids in membranes with their successful experience in delivering small molecule drugs, such as anticancer drugs.

[0005] Phage display technology (Phage-display) is a method for high-throughput screening of functional peptides. It can insert genes encoding exogenous peptides or proteins into the structural gene of the phage coat protein, so that the exogenous peptides or proteins form fusion proteins on the capsid protein of the phage. With the reassembly of the progeny phage, they are presented on the surface of the phage, which can maintain the relative spatial structure and biological activity. By inserting the peptide library into the structural gene of the phage capsid protein, a high-throughput phage library can be constructed. The phage library is used to screen specific targets. After repeated incubation, blocking, selection, elution and amplification steps, peptide sequences with high affinity to the target can be screened. All steps of phage display technology screening can be achieved in vitro. The phage used can be amplified in Escherichia coli. The steps are simple, and the peptide library throughput of the phage display peptide library is 10 9 The above meets the needs of high-throughput screening and has low experimental costs.

[0006] Wild-type AAV has limited organ targeting, and its therapeutic applications are also limited. In addition, AAV vectors have a high affinity for the liver and are naturally enriched in liver cells. Therefore, wild-type AAV will have some immune reactions and liver toxicity as a drug delivery vector. Traditional methods for improving the design of adeno-associated viruses mainly include directed evolution and rational design. Among them, directed evolution injects artificially prepared viral libraries into animals, combined with artificial pressurization and high-throughput sequencing methods. After multiple rounds of screening, new mutants are found in different tissues and organs. Its disadvantages are low screening efficiency, high time consumption and high cost. Rational design is based on prior knowledge to design and modify AAV capsid proteins. Although its efficiency is higher than directed evolution, its low throughput and reliance on original data seriously restrict the design and clinical application of viral vectors.

[0007] LNPs are primarily limited to intramuscular injection (e.g., the COVID-19 mRNA LNP vaccine) and intravenous injection targeting liver hepatocytes (e.g., Onpattro short interfering RNA LNPs). Their physicochemical properties are similar to those of very low-density lipoproteins (VLDLs), and they readily adsorb apolipoprotein E in plasma, limiting their use outside the liver.

[0008] Since the inherent characteristics of wild-type AAV and LNP vectors limit their scope of application, giving viral and non-viral vectors high targeting will expand their medical applications as drug delivery vectors. Summary of the Invention

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

[0010] To achieve the above object, the technical solution adopted by the present invention is:

[0011] In a first aspect, the present invention provides a targeted drug delivery carrier, comprising: a drug-carrying carrier and a targeting polypeptide; the drug-carrying carrier is a viral vector, and the nucleotides of the targeting polypeptide are operably linked to the nucleotides of the capsid protein of the viral vector; or, the drug-carrying 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 NOs: 8-12 and SEQ ID NO: 14.

[0012] The present invention screens out polypeptides with high affinity for targeting biological molecules, cells, tissues, and organs through phage display technology. The sequences of these polypeptides are shown in any one of SEQ ID NO: 8-12 and SEQ ID NO: 14. Such polypeptides are integrated into viral vectors or non-viral vectors, giving the viral vectors or non-viral vectors the ability to specifically recognize specific biological molecules, cells, tissues or organs, thereby improving the affinity of the vector for cells, tissues, organs, etc., and applying them to drug delivery vectors to improve the targeting of drug vectors and enhance therapeutic effects. The nucleotide sequence encoding the targeting polypeptide is operably connected to the nucleotide sequence of the capsid protein of the viral vector by genetic recombination, and the targeting polypeptide is expressed through the capsid protein of the viral vector. Specifically, these polypeptides can target muscle cells or muscles, and can improve the targeting of drug-loaded vectors to muscles.

[0013] As a preferred embodiment of the first aspect, the viral vector is any one of lentivirus, adenovirus, adeno-associated virus, retrovirus, and herpes virus; or / and, the non-viral vector is any one of lipid nanoparticles, virus-like particles, and exosomes.

[0014] As a preferred embodiment of the first aspect, the surface modification methods include: physical adsorption, surface deposition, coupling, esterification, and grafting. In the present invention, the targeting polypeptide can be fixed on the surface of the non-viral vector by a variety of surface modification methods, so that the non-viral vector has a certain targeting. For example: fixing the targeting polypeptide on the surface of the non-viral vector by the van der Waals force in the physical surface adsorption method; fixing the targeting polypeptide on the surface of the non-viral vector by deposition on the surface of the non-viral vector; fixing the targeting polypeptide on the surface of the non-viral vector by a coupling agent; fixing the targeting polypeptide on the surface of the non-viral vector by an esterification reaction between the targeting polypeptide and the surface of the non-viral vector; fixing the targeting polypeptide on the surface of the non-viral vector by a surface grafting method.‌

[0015] In a second aspect, the present invention provides a method for preparing the targeted drug delivery vector of the first aspect, comprising the following steps:

[0016] Step 1: Screening the targeted peptides of the target object by phage display method to obtain a phage library of the targeted peptides;

[0017] Step 2: performing multiple rounds of screening and sequencing on the phage library of step 1 to obtain a targeting polypeptide with high affinity to the target; the amino acid sequence of the targeting polypeptide is shown in any one of SEQ ID NOs: 8-12 and SEQ ID NO: 14;

[0018] Step 3: Connect the targeting polypeptide obtained in step 2 to the drug-carrying carrier.

[0019] The present invention utilizes phage display technology to screen various targeting peptides, which are then linked to drug-carrying carriers to enhance their targeting properties. The method of the present invention can yield a variety of targeted drug delivery vehicles. This method significantly enhances the affinity of drug carriers for specific targets, thereby increasing drug delivery efficiency and therapeutic efficacy. It also significantly reduces screening cycles and costs.

[0020] As a preferred embodiment of the second aspect, the target includes: biological molecules, cells, tissues, organs. The biological molecules include nucleic acid molecules and protein molecules.

[0021] The method of the present invention significantly improves the affinity of the drug carrier to biological molecules, cells, tissues or organs, thereby enhancing the delivery efficiency and therapeutic effect of the drug. At the same time, the method greatly shortens the screening cycle and screening costs.

[0022] As a preferred embodiment of the second aspect, the target object is a biological molecule or / and cell, and step 1 adopts the following method: after incubating the phage library with the target biological molecule or / and cell, washing, eluting and neutralizing, transfecting, culturing, and purifying; obtaining a phage library of targeted polypeptides.

[0023] As a preferred embodiment of the second aspect, the target object is a tissue and / or organ, and step 1 adopts the following method:

[0024] (1) Injecting the phage library into animals and collecting phages from tissues and / or organs;

[0025] (2) Transfecting, culturing, and purifying the phages collected in step (1) to obtain a phage library of targeted peptides.

[0026] The present invention directly injects the phage library into the animal body to obtain targeting polypeptides with high affinity to various tissues and / or organs. Connecting the polypeptides to drug carriers can improve the targeting of the carriers in various tissues and / or organs.

[0027] As a preferred embodiment of the second aspect, the method of linking the targeting polypeptide to the drug carrier in step 3 comprises the following steps:

[0028] (1) Inserting the nucleotide sequence of the targeting polypeptide into the capsid gene of a viral vector to construct an engineered viral vector;

[0029] (2) Expressing the targeting polypeptide by fusion with the capsid gene of the engineered viral vector to obtain a targeted viral vector drug delivery vehicle;

[0030] or,

[0031] The method for connecting the targeting polypeptide to the drug carrier in step 3 comprises the following steps:

[0032] The targeting polypeptide is fixed to the surface of a non-drug-carrying carrier to obtain a targeted non-viral drug-carrying carrier; the non-viral carrier is any one of lipid nanoparticles, virus-like particles, and exosomes.

[0033] Preferably, the non-viral vector is a lipid nanoparticle.

[0034] Preferably, the viral vector is an adeno-associated virus (AAV). The present invention inserts a targeting polypeptide nucleotide into the AAV capsid polynucleotide between the codons for any two adjacent amino acids at 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; the adeno-associated virus (AAV) includes: AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides a targeted drug delivery vector and its preparation method. Compared with traditional AAV vector screening methods, the preparation method of the present invention significantly shortens the screening cycle and screening costs. Through phage display technology, peptides with high affinity for target receptors or cells, tissues, and organs are screened. These peptides are then integrated into viral or non-viral vectors, resulting in high targeting of specific target receptors, cells, tissues, and organs, enabling targeted drug delivery.

[0037] For viral vectors, the present invention integrates the DNA sequence of the screened targeting polypeptide into the viral capsid protein sequence, enabling high targeting of target molecules or cells, tissues, and organs. For non-viral vectors, the present invention couples the screened polypeptide to the non-viral vector through chemical modification or physical coupling, and fixes the targeting polypeptide to the surface of the vector, enabling the non-viral vector to achieve high targeting of the target receptor or cell, tissue, or organ.

[0038] The above-mentioned targeted carriers significantly improve the affinity of drug carriers to specific biological molecules, cells, tissues or organs, thereby enhancing the delivery efficiency and therapeutic effect of drugs. At the same time, this method greatly shortens the screening cycle and screening costs.

[0039] Experiments have demonstrated that the functional polypeptides screened by the present invention (whose amino acid sequences are shown in any of SEQ ID NOs: 8-12 and SEQ ID NO: 14) can significantly improve the transfection efficiency of the Pint-series novel AAVs screened in representative muscle cells, C2C12, compared to wild-type AAV9. In C57 mice, the Ms-series novel AAVs screened can significantly improve the transduction efficiency of muscle tissue compared to wild-type AAV9. The new LNP formulation screened in C57 mice can significantly improve muscle tissue distribution and transduction efficiency compared to existing commercially available formulations, while reducing liver tissue distribution. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the present invention for summarizing the use of phage display technology to screen functional polypeptides;

[0041] Figure 2 Electrophoresis diagram of nucleic acid after phage genome library construction;

[0042] Figure 3 Plasmid maps constructed for the AAV capsid variants of the present invention;

[0043] Figure 4 This is a schematic diagram of AAV packaging and purification of the present invention;

[0044] Figure 5 Schematic diagram of the AAV virus titer amplification curve of the present invention;

[0045] Figure 6 Schematic diagram of the LNP-coupled polypeptide of the present invention;

[0046] Figure 7 A series of fluorescence images of PintAAV cells infected;

[0047] Figure 8 A series of PintAAV cell infection statistics

[0048] Figure 9 This is an image of Pint-E transfected mouse animals;

[0049] Figure 10 Schematic diagram of MsAAV obtained by phage mouse in vivo screening method;

[0050] Figure 11 This is the fluorescence intensity analysis diagram of LNP mouse in vivo imaging. DETAILED DESCRIPTION

[0051] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0052] The present invention constructs a targeted drug delivery carrier, comprising: a drug-carrying carrier and a targeting polypeptide;

[0053] The drug carrier is a viral vector, and the nucleotides of the targeting polypeptide are operably linked to the nucleotides of the capsid protein of the viral vector;

[0054] Alternatively, the drug carrier is a non-viral carrier, and the targeting polypeptide is connected to the non-viral carrier by surface modification.

[0055] Preferably, the viral vector is any one of lentivirus, adenovirus, adeno-associated virus, retrovirus, and herpes virus; or / and, the non-viral vector is any one of lipid nanoparticles, virus-like particles, and exosomes.

[0056] Preferably, the amino acid sequence of the targeting polypeptide is shown in any one of SEQ ID NOs: 8-12 and SEQ ID NO: 14.

[0057] Preferably, the surface modification methods include: surface adsorption, surface deposition, coupling, esterification, and grafting.

[0058] The preparation method of the above-mentioned targeted drug delivery carrier comprises the following steps:

[0059] Step 1: Screening the target-targeting peptides by phage display method to obtain a phage library of the targeted peptides; wherein the target can be any of biological molecules, cells, tissues, and organs.

[0060] Preferably, when the target is a biomolecule or / and a cell, the following method is used:

[0061] After incubating the phage library with target biological molecules and / or cells, the phage library is washed, eluted and neutralized, transfected, cultured and purified to obtain a phage library of targeted polypeptides.

[0062] Preferably, when the target is a tissue and / or organ, the following method is used:

[0063] (1) Injecting the phage library into animals and collecting phages from tissues and / or organs;

[0064] (2) Transfecting, culturing, and purifying the phages collected in step (1) to obtain a phage library of targeted peptides.

[0065] Step 2: The phage library of step 1 is subjected to multiple rounds of screening and sequencing to obtain a targeting polypeptide with high affinity to the target.

[0066] Step 3: Connecting the targeting polypeptide obtained in step 2 to a drug-carrying carrier; wherein the drug-carrying carrier includes a viral vector and a non-viral vector.

[0067] Preferably, when the drug carrier is a viral vector, the method of linking the targeting polypeptide to the drug carrier in step 3 comprises the following steps:

[0068] (1) Inserting the nucleotide sequence of the targeting polypeptide into the capsid gene of a viral vector to construct an engineered viral vector;

[0069] (2) Expressing the targeting polypeptide by fusion with the capsid gene of an engineered viral vector to obtain a targeted viral vector drug delivery vehicle.

[0070] Preferably, when the drug carrier is a non-viral carrier, the method for connecting the targeting polypeptide to the drug carrier in step 3 is: fixing the targeting polypeptide to the surface of the non-drug carrier to obtain a targeted non-viral carrier drug delivery system; wherein the non-viral carrier is any one of lipid nanoparticles, virus-like particles, and exosomes.

[0071] Example 1 Phage Screening for Integrin-Targeting Peptides

[0072] In this example, integrin is targeted and phage display technology is used to screen high-affinity integrin peptides. In subsequent experiments, the affinity integrin peptide sequences are integrated into viral vectors to improve the transduction efficiency and specificity of viral vectors to cells.

[0073] Main experimental materials: phage display kit (7-peptide NEB, #E8211S, 12-peptide NEB, #E8210S), integrin protein (SinoBiologic, #CT039).

[0074] Experimental steps:

[0075] 1. Integrin antigen coating: Dilute the integrin antigen protein to 0.5 μg / μL with PBS buffer, take 4 μg of protein into a flat-bottomed enzyme-linked microtiter plate, add 150 μL of antigen coating solution to each well, mix well; set up 4 replicate wells, and coat overnight at 4°C.

[0076] 2. Blocking: Discard the coating liquid in the ELISA plate and wash 6 times with PBST (phosphate buffered saline) on a plate washer; pat dry the residual liquid in the wells with a paper towel, add 200 μL of blocking solution to each well, and block at room temperature for 2 hours.

[0077] 3. Washing: After blocking, wash with PBST 6 times, 3 minutes each time.

[0078] 4. Binding or incubation: dilute the phage library to a titer of 5×10 11 pfu / mL, add 200uL of phage library dilution solution to each well of the ELISA plate and place it at room temperature for 2h.

[0079] 5. Washing: Discard excess diluent and wash 10 times with PBST.

[0080] 6. Elution and neutralization: After washing, add 600 μL of elution buffer to each well and shake at 400 rpm for 20 minutes at room temperature; then add 120 μL of neutralization buffer for neutralization and mix well.

[0081] 7. Transfection and culture: The neutralized solution obtained in step 6 was transferred into K12 Escherichia coli cultured to an OD600 of about 0.5. After mixed expansion culture for 4 hours, the supernatant was collected by centrifugation at 8000g for 5 minutes.

[0082] 8. Purification: Add 20% (w / v) phage precipitate to the supernatant in step 7, mix thoroughly, and incubate at 4°C for 4 h.

[0083] 9. Collection and re-purification: Centrifuge the phage precipitate from step 8 above at 13,000 g for 10 min, collect the precipitate, and repeat step 8 to purify the phage again.

[0084] 10. Calculate the titer of phage: Take 10 μL of the phage solution from step 9, perform a gradient dilution, and calculate the titer of phage using the blue-white staining method.

[0085] 11. Screening: Repeat steps 1-10 for three rounds of screening to obtain a phage peptide library with high affinity for integrin.

[0086] Example 2 Phage genome extraction and sequencing

[0087] The genome of the phage peptide library of Example 1 was extracted and subjected to high-throughput sequencing, and the polypeptide amino acid sequence was obtained by comparing the codons.

[0088] Experimental steps:

[0089] 1. Use phage DNA extraction kit to extract genome.

[0090] 2. Use Qubit nucleic acid quantification instrument to accurately quantify the genome.

[0091] 3. Use the extracted genomic DNA as a template to perform the first round of PCR amplification. The amplification primer sequences are shown in Table 1, the PCR reaction system is shown in Table 2, and the reaction procedure is shown in Table 3:

[0092] Table 1:

[0093]

[0094] Table 2:

[0095]

[0096] Table 3:

[0097]

[0098] 4. Add sequencing adapters to the PCR reaction products: Take 5 μL of the reaction solution for the second round of PCR amplification and add high-throughput sequencing adapters. The PCR reaction system is shown in Table 4 below, and the reaction procedure is shown in Table 5 below:

[0099] Table 4:

[0100]

[0101] Table 5:

[0102]

[0103] 5. Quantify the PCR product from step 4 using Qubit: Take 3 μL of the reaction solution and perform nucleic acid electrophoresis detection.

[0104] 6. Sequence the high-throughput library of PCR products using the Illumina sequencing platform.

[0105] The phage library was constructed, and the nucleic acid electrophoresis diagram after the library was constructed was as follows Figure 2 As shown, the upper 250bp are the successful addition of the sequencing adapter, and the lower 200bp are the original sequence. The peptide sequences obtained after sequencing are shown in Table 6. These sequences have high affinity with integrin antigens.

[0106] Table 6:

[0107]

[0108] Example 3 Phage display in mice

[0109] The phage peptide library is directly injected into mice. After 24 hours, the various organs of the mice are taken out for phage amplification, culture and purification. The purified phage is sequenced to obtain the corresponding polypeptide sequences. These sequences can reflect the preference of short peptides for various organs to a certain extent. By integrating the polypeptides into the surface of the viral vector, it is expected to obtain a viral vector with high affinity to the corresponding organs.

[0110] Experimental steps:

[0111] 1. Phage Screening in Vivo

[0112] 1. Experimental and control groups were set up. The experimental group was injected with 7 / 12 peptide phage library (phage display kit (7 peptides NEB, #E8211S, 12 peptides NEB, #E8210S), with three replicates for each peptide library. The control group was injected with normal saline.

[0113] 2. Phage library injection: dilute the peptide phage library 7 / 12 with PBS, and the injection dose is 1.0×10 11 Pfu, 200 μL was administered into the tail vein of mice.

[0114] 3. After 24 hours, the mice were bled thoroughly and the biceps, quadriceps, and diaphragm tissues were separated from the heart, liver, brain, spleen, lung, and kidney organs.

[0115] 4. Cut off some organ tissues and add them to PBST for tissue grinding.

[0116] 5. Collection and washing: Discard the supernatant and repeat the washing with PBST for 10 times.

[0117] 6. Elution and neutralization: Add 600 μL of elution buffer, shake and mix to fully elute the phage, and add 120 μL of neutralization buffer to terminate the reaction.

[0118] 7. Transfection and culture: The neutralized solution obtained in step 6 was transferred into K12 Escherichia coli cultured to an OD600 of about 0.5. After mixed expansion culture for 4 hours, the supernatant was collected by centrifugation at 8000g for 5 minutes.

[0119] 8. Purification: Add 20% (w / v) phage precipitate to the supernatant in step 7, mix thoroughly, and incubate at 4°C for 4 h.

[0120] 9. Collection and re-purification: Centrifuge the phage precipitate from step 8 above at 13,000 g for 10 min, collect the precipitate, and repeat step 8 to purify the phage again.

[0121] 10. Calculate the titer of phage: Take 10 μL of the phage solution from step 9, perform a gradient dilution, and calculate the titer of phage using the blue-white staining method.

[0122] 11. Screening: Repeat steps 1-10 for three rounds of screening to obtain a peptide library of high-affinity mouse muscle polypeptide sequences.

[0123] 2. Phage Genome Extraction and Sequencing

[0124] This step is consistent with the operation steps of Example 2.

[0125] Experimental results: After multiple rounds of screening and library construction and sequencing, a high-affinity mouse muscle peptide sequence was obtained. The peptides analyzed after sequencing are shown in Table 7:

[0126] Table 7:

[0127]

[0128] Example 4: AAV capsid variant plasmid construction

[0129] The nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 8-12 and 14 was integrated into the gene of the AAV virus capsid plasmid pAAV2 / 9n through genetic engineering methods; a BsmBI restriction site was introduced at the 588 / 589 site, and the gene sequence of the functional polypeptide was added to this site to give pAAV2 / 9n the corresponding transduction properties. The specific operation is as follows:

[0130] Experimental materials: capsid plasmid pAAV2 / 9n (Addgene, #112865), BsmBI restriction endonuclease (NEB, #R0580), BsiWI (NEB, #R0553V), and PmeI (NEB, #R0560V) restriction endonuclease, DH5α competent cells (Tsingke, #TSC-C14), and other reagents are common materials.

[0131] Experimental steps:

[0132] 1. Get the target fragment

[0133] 1. Nanjing GenScript was commissioned to synthesize the gene fragment sequence (SEQ ID NO: 3-7, SEQ ID NO: 13). The gene fragment was in the PUC57 plasmid glycerol strain and named pUC57-AAV588 / 589-BsmBI.

[0134] 2. Activate glycerol bacteria: Use glycerol bacteria to streak on LB resistance plates overnight. The next day, pick two monoclonal colonies and transfer them to 5 mL LB medium. Use a plasmid extraction kit to extract the bacteria. Each tube contains 1 tube of bacteria, 500 μL 50% glycerol + 500 μL bacterial solution.

[0135] 3. The plasmid was sequenced using Sanger sequencing to confirm the correctness of the sequence. The sequencing primer was the universal U6 primer.

[0136] 4. Use BsiWI and PmeI to double-digest the plasmid. The enzyme digestion system is shown in Table 8:

[0137] Table 8:

[0138]

[0139] React at 37°C for 1.5 h, add 6 μL of 10x DNA loading buffer and perform agarose gel electrophoresis.

[0140] 5. Obtain fragments: The vector fragment size is 2710 bp, and the target fragment size is 1230 bp. Use a gel recovery kit to recover the target fragment in the lower layer.

[0141]

[0142] The target fragments are: SEQ ID NO: 3-7, SEQ ID NO: 13.

[0143] 6. Determine the concentration of the target fragment using NanoDrop.

[0144] 2. Construction of vector fragments

[0145] 1. Glycerol bacteria containing the pAAV2-9n plasmid were purchased from Addgene (#112865). Lines were drawn on LB plates and cultured inverted at 37°C overnight.

[0146] 2. Pick two monoclonal clones into 5 mL of ampicillin-resistant LB medium, extract them using a plasmid miniprep kit, and verify the correctness of the plasmid by Sanger sequencing.

[0147] 3. Linearize the pAAV2-9n plasmid using BsiWI and PmeI enzymes. The enzyme digestion system is shown in Table 9:

[0148] Table 9:

[0149]

[0150] React at 37°C for 1.5 h, add 10 μL of 10× DNA loading buffer and perform agarose gel electrophoresis.

[0151] 4. Obtain linearized vector fragment: The linearized vector fragment is 6100 bp in size. Use a gel recovery kit to recover the upper vector fragment.

[0152] 5. Measure the concentration of recovered vector fragments using NanoDrop.

[0153] 3. Connecting the vector fragment to the target fragment

[0154] 1. Use DNA T4 ligase to carry out the reaction (the target fragment and linearized vector are used in an amount of 25 ng and 100 ng, respectively, and the number of fragments is 1:1). The ligation reaction is as shown in Table 10:

[0155] Table 10:

[0156]

[0157] The reaction was carried out at 25°C for 20 min to obtain the ligation product.

[0158] 2. Take 2 μL of the ligation product and transform it into DH5α E. coli competent cells. Spread on ampicillin-resistant plates. The next day, pick two monoclonal colonies and culture them in 5 mL of ampicillin-resistant LB medium for shake culture to extract the plasmid (one tube for each tube, 500 μL 50% glycerol + 500 μL bacterial solution).

[0159] 3. Sanger sequencing was used to confirm the correctness of the sequence. The correct plasmid was named: pAAV2 / 9n-588 / 589-BsmBI. The plasmid schematic is shown in Figure 3 .

[0160] A series of pAAV2 / 9n-588 / 589-BsmBI capsid plasmids were obtained, and their plasmid information is shown in Table 11:

[0161] Table 11:

[0162]

[0163] Example 5 AAV virus packaging

[0164] The capsid plasmid, target gene plasmid, and helper virus plasmid of the adeno-associated virus vector constructed in Example 4 are transfected into eukaryotic cells, and the targeted drug delivery vector is obtained through self-assembly. The specific operation is as follows:

[0165] Synthesis of AAV viral vectors requires three plasmids: capsid plasmid (Rep / Cap, i.e., the modified series of pAAV2 / 9n-588 / 589-BsmBI capsid plasmids of the present invention), target gene plasmid (GOI), and helper viral plasmid (Helper). These three plasmids are transfected into eukaryotic cells and self-assemble to form an AAV viral vector containing the target gene. After purification and quality testing, it can be used for drug delivery in vivo and in vitro. The overall flow chart is shown in FIG. Figure 4 .

[0166] Experimental steps ( Figure 4 ):

[0167] 1. Virus packaging and purification

[0168] 1. Plasmid extraction: The plasmids required for the three aforementioned AAVs were cultured overnight, and the endotoxin-free plasmids were extracted using a plasmid extraction kit (Promega #A2392).

[0169] 2. Plasmid detection: NanoDrop and Qubit were used to quantify the extracted plasmid, and nucleic acid electrophoresis was performed to verify the size and purity of the plasmid.

[0170] 3. Use 293T cells (ATCC#CRL-3216) to prepare viruses according to the tertiary seed bank.

[0171] 4. AAV virus packaging: PEI transient transfection was used to transfect 293T cells with a capsid plasmid: target gene plasmid: helper virus plasmid ratio of 1:1:1. 20 μg of total plasmid and 60 μL of PEI (DNA:PEI ratio of 1:3) were added to each 15 cm culture dish.

[0172] 5. Three days after transfection, cells were harvested for purification.

[0173] 6. Resuspend the cells and freeze-thaw repeatedly to lyse the cells and release the virus. Adjust the final nuclease concentration to 50 U / mL and incubate at 37°C for 60 min.

[0174] 7. Prepare samples before ultracentrifugation by preparing different density gradients of iodixanol. Finally, use a peristaltic pump to slowly and completely load all samples. After the heat-sealed tube is completely filled with liquid, heat seal it and seal it.

[0175] 8. Ultracentrifuge settings: 18°C, centrifugal speed 70,000 rpm, centrifugation time 1 hour 5 minutes, collect the virus.

[0176] 9. Sterile filtration: Use a 0.22 μm syringe filter to sterilize and filter the ultrafiltration recovered samples. Keep a sample to measure the physical titer and SDS-PAGE gel to measure the protein purity. Label the packaged samples and temporarily store them in an ultra-low temperature biological sample library.

[0177] 3. Virus Titer Detection

[0178] 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);

[0179] 2. The reaction system is shown in Table 12:

[0180] Table 12:

[0181]

[0182] At the same time, a nucleic acid gel was prepared for enzyme digestion verification. Using Qubit 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 dilution gradient of linearized plasmid 10 9 -10 4 The final volume was 200 μL, and a concentration gradient was formed with tenfold increments. qPCR reaction was performed with the reaction system shown in Table 13 and the reaction procedure shown in Table 14:

[0183] Table 13:

[0184]

[0185] Table 14:

[0186]

[0187] 3. Result analysis: From the amplification curve ( Figure 5 ) CT value and R 2 The value can show the accuracy of the standard curve;

[0188] 4. Adding 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, facilitating subsequent testing and comparison of the transfection ability of different viruses. The targeted virus is obtained, as shown in Table 15:

[0189] Table 15:

[0190]

[0191] Example 6 LNP preparation and polypeptide modification

[0192] The amino acid sequence of the polypeptides shown in SEQ ID NOs: 8-10 obtained in Example 2 is connected to a linker to obtain an amino acid sequence of a polypeptide shown in any one of SEQ ID NOs: 15-17;

[0193] A polypeptide with an amino acid sequence as shown in any one of SEQ ID NOs: 8-10 is coupled to a lipid nanoparticle to obtain a non-viral targeted drug delivery vector. The specific steps are as follows:

[0194] Lipid nanoparticles (LNP) are a nanoscale drug delivery system composed of lipids, usually composed of four components: phospholipids, cholesterol, cationic lipids, and PEG-lipids. Because their components are natural substances, they have low toxicity and immunogenicity to the human body and can be administered repeatedly.

[0195] In order to achieve targeted delivery of lipid nanoparticles to specific cells or tissues, in this embodiment, the amino acid sequence of the polypeptide shown in SEQ ID NO: 8-10 is connected to the surface of the lipid nanoparticles, as shown in the schematic diagram. Figure 6 When lipid nanoparticles enter the blood circulation, these peptide sequences can guide the lipid nanoparticles to bind precisely to receptors, thereby improving the delivery efficiency of drugs to target tissues / cells and reducing side effects on normal cells.

[0196] Experimental Materials:

[0197] 50mM sodium acetate (pH 5.5) buffer, SM102 (CAS: 2089251-47-6, molecular weight: 710.1653, molecular formula: C 44 H 87NO5), 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 luciferase). The peptide was commissioned to (Jiangsu Zhuanpeptide Biological) Company for synthesis, specifically:

[0198] Control peptide:

[0199] A2G80: VQLRNGFPYFSYGGC; M12-1: RRQPPRSISSHPGGGSC; The intergrin polypeptide sequence of the present invention is shown in Table 16:

[0200] Table 16:

[0201]

[0202] Among them, GGGS is the linker, and C is added by conjugation with -Mal (the linker of A2G80 polypeptide is GGC).

[0203] Experimental equipment:

[0204] Mainly microfluidic instruments are used, and the rest of the equipment is conventional equipment.

[0205] Experimental steps (the reagents and materials used in this example are all conventional materials for preparing LNPs and are all commercially available products):

[0206] 1. Preparation of lipid solution:

[0207] Weigh 15 mg of SM102 into a 1.5 mL clean EP tube, then add 200 μl of anhydrous ethanol to a concentration of 75 mg / mL.

[0208] Weigh 10 mg of DSPC into a 1.5 mL clean EP tube, then add 1.0 mL of anhydrous ethanol to a concentration of 15 mg / mL.

[0209] Weigh 10 mg of cholesterol into a 1.5 mL clean EP tube, then add 1.0 mL of anhydrous ethanol to a concentration of 75 mg / mL.

[0210] Weigh 10 mg of DMG-PEG2k (phospholipid polyethylene glycol) into a 1.5 mL clean EP tube, then add 1.0 mL of anhydrous ethanol to a concentration of 75 mg / mL.

[0211] 2. Preparation of DMG-PEG2k-Mal:

[0212] 2.1 Molar ratio of each lipid

[0213] SM102: DPPC: cholesterol: DMG-PEG2k: DSPE-PEG2k-Mal (phospholipid polyethylene glycol maleimide) = 50: 10: 38.5: 1.2: 0.3.

[0214] 2.2 Mass ratio

[0215] Total lipid:mRNA=40:1; total lipid concentration is 12.5mM.

[0216] 2.3 Lipid ethanol phase preparation

[0217] Taking 100ug of mRNA as an example, prepare 0.3% peptide-modified SM102 LNP: in the above-prepared lipid solution, take 46.3ul of SM102 solution, 77.3ul of DSPC, 145.7ul of cholesterol, 29.5ul of DMG-PEG2k and 8.6ul of DMG-PEG2k-Mal and place them in a 1.5mL clean EP tube. Then add 475.5ul of anhydrous ethanol to make the total lipid concentration 12.5mM and the total volume 782.9ul (1.5 times the required amount of lipid) and mix thoroughly until clear and transparent.

[0218] 2.4 mRNA aqueous phase preparation

[0219] Place 100 μg of Fluc mRNA (concentration 1 mg / mL) in a clean 5 mL tube, then add 1465.9 μl of 50 mM sodium acetate buffer (pH 5.5) and mix thoroughly.

[0220] 3. Microfluidic preparation:

[0221] Transfer the lipid-ethanol phase and the mRNA-water phase to 1 mL and 3 mL syringes, respectively. Remove any excess air bubbles. Insert the LNP chip into the device and insert two threaded syringes into the corresponding ports on the chip. Prepare two 15 mL collection tubes: one for sample collection and one for waste collection. Configure the software parameters to a total injection volume of 1.8 mL and a total flow rate of 12 mL / min (9 mL / min and a collection volume of 1.35 mL for the mRNA-water phase; 3 mL / min and a collection volume of 0.45 mL for the lipid-ethanol phase). Set the waste volume to 0.45 mL (0.4 mL initial waste volume and 0.05 mL final waste volume). Click "Start" to begin LNP preparation. Once preparation is complete, transfer the sample to a Pur-A-Lyzer Maxi 3500 dialysis tubing. The prepared sample should be dialyzed within 15 minutes. Dialyze against 1x PBS (Ca+ / Mg+-free) at 4°C for 4 hours, then dialyze overnight to remove ethanol. After dialysis, transfer the solution into a clean 5 mL tube and measure the volume. (If needed, concentrate the volume using a 30 kDa ultrafiltration tube at 3000 g at 4°C.) Note: The resulting solution can be stored at 4°C for several days before use. However, it is recommended to use the formulated LNPs as soon as possible to maintain consistent results and obtain the desired LNPs.

[0222] 4. Peptide conjugation modification of LNP:

[0223] 1) Using GGGS as a linker and C as a -Mal conjugate (GGC for the A2G80 peptide), the sulfhydryl group of the cysteine ​​residue reacts with the double bond of the maleimide to form a new chemical bond, thereby synthesizing peptides with amino acid sequences as shown in SEQ ID NOs: 15-17;

[0224] 2) Dissolve the synthesized peptide in PBS (pH 7.4) at a concentration of 4 mg / mL.

[0225] 3) Take out 90 μg of the LNP prepared in step 6, add the peptide solution (peptide:Mal molar ratio is 10:1), mix well and incubate at 4°C overnight.

[0226] 4) Use dialysis to remove unconjugated peptides. Use 1xPBS (without calcium and magnesium ions) to dialyze at 4°C for 4 hours, then change the medium. Then dialyze overnight. After dialysis, transfect into a clean 1.5mL EP tube and measure the volume (if needed, use a 30kDa ultrafiltration tube, 3000g, 4°C for volume concentration) to obtain peptide-modified LNPs.

[0227] Example 7 Targeted PintAAV virus cell infection experiment

[0228] Regarding the targeted peptide screened from integrins using phage display technology in Example 1, we believe that it can increase the infection efficiency of muscle cells. The polypeptide sequence was added to the AAV capsid using the construction method of Example 4, and the virus was packaged and purified using the method of Example 5. The packaged cargo contained the gene sequences of firefly luciferase (Fireflyluciferase) and red fluorescent protein (mCherry), and the infection efficiency of the virus was verified on muscle cells.

[0229] Experimental materials: C2C12 mouse myofibroblasts (ATCC#CRL-1772), C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), and other common materials.

[0230] Experimental equipment: carbon dioxide constant temperature incubator, small animal live imager, fluorescence microscope, laser confocal microscope, and other equipment are common equipment.

[0231] Experimental steps:

[0232] 1. Cell culture and induction: After thawing, C2C12 mouse myofibroblasts stabilized after more than three generations of culture and were plated. When the cell confluence reached approximately 60%, they were induced to form myotubes by adding 2% horse serum.

[0233] 2. Virus infection verification: After the purified virus passes the QPCR, SDS-PAGE, and endotoxin assay, the titer is determined using absolute quantitative PCR. The virus infection experiment is performed according to the infection titer per well MOI: 1E+4.

[0234] 3. Fluorescence Imaging and Analysis: 6 days after infection, cells were photographed using a Zeiss fluorescence microscope with a 10x objective lens. The mean fluorescence intensity (intDen) of the images was quantified using imageJ.

[0235] 4. Experimental results: Figure 7 、 8 The results showed that compared with the wild-type AAV9 virus, the screening method and viral vector of the present invention can have better transfection efficiency, indicating that the targeting polypeptide obtained by screening of the present invention has stronger muscle cell targeting and can improve the muscle targeting of the AAV vector.

[0236] Example 8 Targeted Pint-AAV virus transfection experiment in mice

[0237] For the new virus Pint-E with the highest fluorescence intensity, in order to verify the effect of the virus in mice, we injected the virus into the mice intramuscularly and observed the expression and distribution of the virus in the mice.

[0238] Experimental materials: C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), Pint-E virus, and other common materials.

[0239] Experimental equipment: small animal live imaging instrument, fluorescence microscope, laser confocal microscope, and other equipment are common equipment.

[0240] Experimental steps:

[0241] 1. Virus quality control: After the purified virus passes the QPCR, SDS-PAGE, and endotoxin assay, the titer is determined using absolute quantitative PCR.

[0242] 2. Determine the dosing volume. Weigh the mouse and calculate the injection volume according to the dose of 1.0×10^13 vg / kg. Use normal saline to dilute the virus during injection. The injection volume is 200 μL and the drug is administered through the tail vein.

[0243] 3. Live imaging of mice was performed on days 7, 14, 21, and 28 after administration to observe the distribution and expression of the delivered drug in the body. Organs were harvested from the mice on day 28, and the organs were cryopreserved and embedded for molecular-level testing.

[0244] Experimental results: The results show that Figure 9 The novel viral vector Pint-E screened based on this method has better transfection efficiency in vivo. This indicates that the targeted polypeptide screened by the present invention has stronger muscle targeting and can improve the muscle targeting of AAV vectors.

[0245] Example 9 Targeted MsAAV virus distribution experiment in mice

[0246] Regarding the targeted polypeptide screened from mice using phage display technology in Example 3, we believe that it can increase the distribution and infection efficiency of the corresponding target organs. The polypeptide sequence was added to the AAV capsid using the construction method of Example 4, and the virus was packaged and purified using the method of Example 5. The packaged product contained the gene sequences of firefly luciferase (Fireflyluciferase) and red fluorescent protein (MCherry), and the infection efficiency of the new virus was verified in mice.

[0247] Experimental materials: C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), MsAAV virus, and other materials are common materials.

[0248] 1. Virus quality control: After the purified virus passes the QPCR, SDS-PAGE, and endotoxin assay, the titer is determined using absolute quantitative PCR.

[0249] 2. Determine the dosing volume. Weigh the mouse and calculate the injection volume according to the dose of 1.0×10^13 vg / kg. Use normal saline to dilute the virus during injection. The injection volume is 200 μL and the drug is administered through the tail vein.

[0250] 3. Live imaging of mice was performed on days 7, 14, 21, and 28 after drug administration. Mice were weighed and intraperitoneally injected with 200 μL of anesthetic per 10 g of body weight and 100 μL of luminescent substrate (15 mg / mL) per 10 g of body weight. Ten minutes after substrate injection, live imaging was performed. The distribution and expression of the delivered drug in vivo were observed. On day 28, organs were harvested from the mice, cryopreserved, and embedded for molecular-level analysis.

[0251] The experimental results are as follows Figure 10 Compared with wild-type AAV, the novel viral vector MsAAV virus targets more muscle tissue and is less distributed in the liver, indicating that the targeting polypeptide screened by the present invention has stronger muscle targeting and can improve the muscle targeting of the AAV vector.

[0252] Example 10 In vivo administration of targeted LNP virus to mice

[0253] For the targeted LNP delivery vector constructed in Example 8, its targeting was verified by intravenous injection into mice, where 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 was detected using a small animal in vivo imaging instrument.

[0254] Experimental Materials:

[0255] The C57 mice used in this experiment were purchased from the Experimental Animal Center of Yunnan University. LNP was prepared according to Example 8, as were the anesthetic and luciferase substrate.

[0256] Experimental equipment:

[0257] Small animal in vivo imaging device, and the rest are common equipment.

[0258] Experimental groups:

[0259] (1) NC (PBS control group); (2) Non-pep (unmodified group); (3) A2G80; (4) M12-1; (5) Intergrin-1; (6) Intergrin-2; (7) Intergrin-3;

[0260] Experimental steps:

[0261] 1. Select 6-8 week old C57 mice of either sex, 3 per group, and inject the drug into the tail vein at a dose of 1.5 mg / kg in a total volume of 250 μL.

[0262] After 2 and 6 hours, mice were imaged using a small animal live imaging system. The mice were weighed and intraperitoneally injected with 200 μL of anesthetic per 10 g of body weight and 100 μL of luminescent substrate (15 mg / mL) per 10 g of body weight. Ten minutes after substrate injection, small animal live imaging was performed.

[0263] The experimental results are as follows Figure 11 Compared with the control group (unmodified LNP), intergrin-1 significantly increased the distribution and expression of LNP in muscle and decreased its distribution in the liver. This indicates that the targeted peptides screened in this invention have stronger muscle targeting and can improve the muscle targeting of AAV vectors.

[0264] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents 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-carrying carrier and a targeting polypeptide; The drug-carrying carrier is a viral vector, and the viral vector is adeno-associated virus AAV2; Inserting the nucleotide sequence of the targeting polypeptide into the gene of the adeno-associated virus capsid plasmid pAAV2 / 9n, introducing a BsmBI restriction site at the 588 / 589 site, and adding the nucleotide sequence of the targeting polypeptide at the site; The amino acid sequence of the targeting polypeptide is shown in SEQ ID NO: 8; the nucleotide sequence of the targeting polypeptide is shown in SEQ ID NO:

3.

2. A method for preparing a targeted drug delivery carrier according to claim 1, characterized in that: The preparation method comprises the following steps: Step 1: Screening the targeted peptides of the target object by phage display method to obtain a phage library of the targeted peptides; The target objects include: biological molecules, tissues, organs; The biomolecule is an integrin; The tissue is any one of biceps brachii, quadriceps femoris, and diaphragm tissue; The organ is any one of the heart, liver, brain, spleen, lung, and kidney; Step 2: The phage library from step 1 is subjected to multiple rounds of screening and sequencing to obtain a targeting polypeptide with high affinity to the target; the amino acid sequence of the targeting polypeptide is shown in SEQ ID NO: 8; Step 3: Connecting the targeting polypeptide obtained in step 2 to a drug-carrying carrier; the drug-carrying carrier is a viral vector.

3. The preparation method according to claim 2, wherein The target is a biomolecule, and step 1 adopts the following method: After the phage library is incubated with the target biological molecule, it is washed, eluted and neutralized, transfected, cultured and purified to obtain a phage library of targeted polypeptides.

4. The preparation method according to claim 2, wherein The target object is a tissue or / and organ, and step 1 adopts the following method: (1) Injecting the phage library into animals and collecting phages from tissues and / or organs; (2) Transfecting, culturing, and purifying the phages collected in step (1) to obtain a phage library of targeted peptides.

5. The preparation method according to claim 2, wherein The method for connecting the targeting polypeptide to the drug carrier in step 3 comprises the following steps: (1) inserting the nucleotide sequence of the targeting polypeptide into the gene of the capsid plasmid pAAV2 / 9n of adeno-associated virus AAV2, introducing a BsmBI restriction site at the 588 / 589 site thereof, and adding the nucleotide sequence of the targeting polypeptide at the site; (2) Expressing the targeting polypeptide by fusion with the capsid gene of an engineered viral vector to obtain a targeted viral vector drug delivery vehicle.

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