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 non-viral carrier, and the non-viral carrier is any one of lipid nanoparticles, virus-like particles and exosomes; the amino acid sequence of the targeting polypeptide is as shown in SEQ ID NO: 8; the targeting polypeptide is connected with the non-viral vector in a surface modification manner. The efficient drug delivery carrier is designed and prepared by utilizing targeting polypeptide for specifically recognizing biomolecules, tissues or organs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a targeted drug delivery carrier and its preparation method. Background Technology

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

[0003] As a representative of non-pathogenic and replication-defective viral vectors, aeroids (AAV) are widely used due to their low immunogenicity, lack of genetic material integration into the host genome, and non-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, thus limiting its therapeutic applications. Studies indicate that AAV vectors have a high affinity for the liver and naturally accumulate in liver cells; targeting the liver alone, low doses of AAV vectors are effective. However, to achieve effective concentrations when targeting other parts of the body, the dose of AAV vector administered systemically must be significantly increased. Existing research shows that modifying the AAV capsid can alter the biological characteristics 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 has T-shaped inverted terminal repeats (ITRs) at both ends, with the REP and CAP genes located 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 make up the viral capsid. The viral capsid is composed of 60 capsid protein monomers in a 1:1:10 ratio (VP1:VP2:VP3). There are nine 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. This affects the virus's tissue susceptibility, transduction efficiency, antigenicity, and cross-reactivity of immunogenicity between serotypes.

[0004] Lipid nanoparticles (LNPs), also known as non-viral vectors, have become a key tool for nucleic acid vaccines and therapies due to their advantages in safety, tolerability, repeatability, and ability to carry large amounts of genetic material. Traditional LNPs contain four molecules: 1. Ionizable lipids: used to bind nucleic acids and facilitate endosome escape. 2. Amphiphilic phospholipids: promote fusion with cell and endosome membranes. 3. Cholesterol: contributes to LNP stability. 4. Polyethylene glycol (PEG) lipids: improve colloidal stability and reduce reticuloendothelial system clearance. LNP technology originates from research on cationic lipid complexes and ionizable cationic lipids, combining the physical properties and functional roles of lipids in membranes with successful experience in the delivery of small molecule drugs (such as anticancer drugs).

[0005] Phage display is a high-throughput method for screening functional peptides. It involves inserting the gene encoding a foreign peptide or protein into the structural gene of a phage capsid protein, causing the foreign peptide or protein to form a fusion protein on the capsid protein. This fusion protein is then presented on the phage surface during the reassembly of progeny phages, maintaining its relative spatial structure and biological activity. By inserting a peptide library into the structural gene of the phage capsid protein, a high-throughput phage library can be constructed. Using this library, specific targets can be screened. Through repeated steps such as incubation, blocking, selection, elution, and amplification, peptide sequences with high affinity for the target can be identified. All steps of phage display screening can be performed in vitro, and the phages used can be amplified in *E. coli*. The process is simple, and the throughput of a phage-displayed peptide library is over 10^6 times. 9 The above meets the requirements for high-throughput screening with low experimental costs.

[0006] Wild-type AAV has limited organ targeting, thus restricting its therapeutic applications. Furthermore, AAV vectors have a high affinity for the liver and naturally accumulate in liver cells, leading to immune responses and liver toxicity when used as drug delivery vectors. Traditional adeno-associated virus (AAV) improvement design methods include directed evolution and rational design. Directed evolution involves injecting artificially prepared viral libraries into animals, combined with artificial pressure and high-throughput sequencing. After multiple rounds of screening, novel mutants are discovered in different tissues and organs. Its disadvantages include low screening efficiency and high time and cost. Rational design, based on prior knowledge, designs and modifies the AAV capsid protein. While its efficacy is higher than directed evolution, its lower throughput and reliance on existing data severely limit the design and clinical application of viral vectors.

[0007] LNPs are mainly limited to intramuscular injection (such as the COVID-19 mRNA LNP vaccine) and intravenous injection targeting liver cells (such as Onpattro short interfering RNA LNPs). Their physicochemical properties are similar to very low-density lipoproteins, and they readily adsorb apolipoprotein E in plasma, which limits their application outside the liver.

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

[0009] This invention provides a targeted drug delivery carrier and its preparation method. By screening for targeted peptides with high affinity and specificity and integrating them into a non-viral carrier, this invention effectively solves the problems of insufficient targeting and low delivery efficiency in existing drug delivery carriers.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a targeted drug delivery carrier, the drug delivery carrier comprising: a drug-carrying carrier and a targeting peptide; the drug-carrying carrier is a non-viral carrier, the non-viral carrier being any one of lipid nanoparticles, virus-like particles, and exosomes; the amino acid sequence of the targeting peptide is shown in SEQ ID NO: 8; the targeting peptide is linked to the non-viral carrier by surface modification.

[0011] This invention utilizes phage display technology to screen for peptides with high affinity for targeting biomolecules, tissues, and organs. The sequences of these peptides are shown in SEQ ID NO: 8. These peptides are then integrated into non-viral vectors, endowing the non-viral vectors with the ability to specifically recognize specific biomolecules, tissues, or organs, thereby enhancing the vector's affinity for tissues and organs. When applied to drug delivery carriers, this improves the targeting specificity of the drug delivery vehicle and enhances therapeutic efficacy. Specifically, these peptides can target muscle, improving the targeting specificity of drug delivery vehicles to muscle.

[0012] As a preferred embodiment of the first aspect, the surface modification methods include: physical adsorption, surface deposition, coupling, esterification, and grafting. In this invention, targeted peptides can be immobilized on the surface of a non-viral carrier through various surface modification methods, thereby giving the non-viral carrier a certain degree of targeting. For example: targeted peptides can be immobilized on the surface of a non-viral carrier through van der Waals forces in physical surface adsorption; targeted peptides can be immobilized on the surface of a non-viral carrier through intermolecular self-assembly hydrophilic-hydrophobic interactions and electrostatic interactions; and targeted peptides can be immobilized on the surface of a non-viral carrier through chemical coupling methods such as aminoamide condensation, esterification, reductive amination, thiol-maleimide reaction, and Click chemistry.

[0013] In a second aspect, the present invention provides a method for preparing a targeted drug delivery carrier as described in the first aspect, comprising the following steps: Step 1: Screen for targeting peptides of the target analyte using phage display to obtain a phage library of the targeting peptides; Step 2: Perform multiple rounds of screening and sequencing on the phage library obtained in Step 1 to obtain a targeting peptide with high affinity for the target; the amino acid sequence of the targeting peptide is shown in SEQ ID NO: 8; Step 3: Connect the targeted peptide obtained in Step 2 to the drug delivery carrier; the drug delivery carrier is a non-viral carrier; The target objects include: biomolecules, tissues, and organs; the biomolecules are integrins. The tissue is any one of the biceps brachii muscle tissue, quadriceps femoris muscle tissue, and diaphragm tissue; the organ is any one of the heart, liver, brain, spleen, lung, and kidney.

[0014] This invention utilizes phage display technology to screen targeted peptides, which are then linked to drug delivery carriers, thereby enhancing the targeting specificity of the carriers. The method of this invention provides targeted drug delivery carriers. This method significantly improves the affinity of drug carriers for specific targets (biomolecules, tissues, or organs), thereby enhancing drug delivery efficiency and therapeutic efficacy. Simultaneously, this method greatly shortens the screening cycle and reduces screening costs.

[0015] As a preferred embodiment of the second aspect, the target substance is a biomolecule, and step 1 is carried out by the following method: after incubating the phage library with the target biomolecule, the phage library is washed, eluted and neutralized, transfected, cultured and purified to obtain a phage library of the targeted peptide.

[0016] In a preferred embodiment of the second aspect, the target object is a tissue and / or organ, and step 1 is performed using the following method: (1) Inject the phage library into the animal and collect the phage from the tissues and / or organs; (2) Transfect, culture and purify the phages collected in step (1) to obtain a phage library of targeted peptides.

[0017] This invention involves directly injecting a phage library into an animal to obtain targeting peptides with high affinity for various tissues and / or organs. Linking these peptides to a drug carrier can improve the targeting of the carrier in various tissues and / or organs.

[0018] As a preferred embodiment of the second aspect, the method for linking the targeted peptide to the drug delivery carrier in step 3 includes the following steps: First, the targeting peptide is linked to the linker amino acid sequence and introduced into the reaction site. Then, it is coupled to a non-viral vector through the linker amino acid sequence. The non-viral vector is any one of lipid nanoparticles, virus-like particles, or exosomes.

[0019] Preferably, the non-viral carrier is a lipid nanoparticle.

[0020] In a preferred embodiment of the second aspect, the linker is an amino acid sequence; the amino acid sequence is (GGGGS). n C, (GGGS) n C, (GGS) n C, (GS) n One of the types of C.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a targeted drug delivery carrier and its preparation method. The preparation method of this invention significantly shortens the screening cycle and reduces screening costs compared to traditional AAV carrier screening methods. By using phage display technology, peptides with high affinity for target receptors, tissues, and organs are screened and integrated into a non-viral vector, giving it high targeting specific receptors, tissues, and organs. This enhances the affinity of the drug carrier for specific biomolecules, tissues, or organs, thereby improving drug delivery efficiency and therapeutic efficacy. Simultaneously, this method significantly shortens the screening cycle and reduces screening costs.

[0022] Experiments have shown that modifying the surface of an LNP vector with the selected functional peptides (whose amino acid sequences are shown in SEQ ID NO: 8) can significantly improve the distribution and transduction efficiency of muscle tissue and reduce the distribution of liver tissue. Attached Figure Description

[0023] Figure 1 This is a schematic diagram outlining the use of phage display technology to screen functional peptides in this invention. Figure 2 This is an electrophoresis image of nucleic acids after bacteriophage genome library construction; Figure 3 This is a plasmid diagram of the AAV capsid variant constructed according to the present invention; Figure 4 This is a schematic diagram of the AAV packaging and purification process of the present invention; Figure 5 This is a schematic diagram of the AAV virus titer amplification curve of the present invention; Figure 6 This is a schematic diagram of the LNP-coupled polypeptide of the present invention; Figure 7 Here are fluorescence images of a series of PintAAV cells infected; Figure 8Statistical graph of PintAAV cell infection series Figure 9 Image of a mouse transfected with Pint-E; Figure 10 A schematic diagram of MsAAV obtained by the in vivo screening method for bacteriophage mice; Figure 11 This is a fluorescence intensity analysis diagram of LNP mice in vivo imaging. Detailed Implementation

[0024] 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.

[0025] This invention constructs a targeted drug delivery carrier, comprising: a drug-loaded carrier and a targeted peptide; The drug delivery carrier is a non-viral carrier, which is any one of lipid nanoparticles, virus-like particles, and exosomes. The amino acid sequence of the targeted polypeptide is shown in SEQ ID NO: 8; The targeted polypeptide is linked to the non-viral vector in a manner that allows it to be attached.

[0026] Preferably, the surface modification method includes: physical adsorption, surface deposition, coupling, esterification, and grafting.

[0027] The preparation method of the above-mentioned targeted drug delivery carrier includes the following steps: Step 1: Screen for targeting peptides of the target analyte using phage display to obtain a phage library of the targeting peptides; Step 2: Perform multiple rounds of screening and sequencing on the phage library obtained in Step 1 to obtain a targeting peptide with high affinity for the target; the amino acid sequence of the targeting peptide is shown in SEQ ID NO: 8; Step 3: Connect the targeted peptide obtained in Step 2 to the drug delivery carrier; the drug delivery carrier is a non-viral carrier; The target objects include: biomolecules, tissues, and organs; The biomolecule integrin; The tissue is any one of the biceps brachii tissue, quadriceps femoris tissue, and diaphragm tissue; The organ in question is any one of the following: heart, liver, brain, spleen, lungs, and kidneys.

[0028] Preferably, when the target substance is a biomolecule, step 1 is performed using the following method: After incubating the phage library with the target biomolecule, the phage library was washed, eluted and neutralized, transfected, cultured and purified to obtain the phage library containing the targeted peptide.

[0029] Preferably, when the target object is a tissue and / or organ, step 1 is performed using the following method: (1) Inject the phage library into the animal and collect the phage from the tissues and / or organs; (2) Transfect, culture and purify the phages collected in step (1) to obtain a phage library of targeted peptides.

[0030] Example 1: Phage screening of integrin-targeting peptides This embodiment uses integrin as the target and employs phage display technology to screen for peptides with high affinity for integrin. In subsequent experiments, the peptide sequences of affinity integrin are integrated into viral vectors to improve the transduction efficiency and specificity of viral vectors to cells.

[0031] Main experimental materials: Phage display kit (7-peptide NEB, #E8211S, 12-peptide NEB, #E8210S), integrin protein (SinoBiologic, #CT039).

[0032] Experimental steps: 1. Integrin antigen coating: Dilute the integrin antigen protein to 0.5 μg / μL with PBS buffer, take 4 μg of protein and put it into a flat-bottomed ELISA plate, add 150 μL of antigen coating solution to each well and mix well; set up 4 replicates and coat overnight at 4℃.

[0033] 2. Blocking: Discard the coating liquid in the ELISA plate, wash 6 times with PBST (phosphate buffer) on a plate washer; pat dry the remaining 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.

[0034] 3. Washing: After sealing, wash with PBST 6 times, 3 minutes each time.

[0035] 4. Binding or incubation: Dilute the phage library titer to 5 × 10⁻⁶ using blocking buffer. 11 Add 200 μL of phage library dilution to each well of the microplate at pfu / mL and incubate at room temperature for 2 hours.

[0036] 5. Washing: Discard excess diluent and wash 10 times with PBST.

[0037] 6. Elution and neutralization: After washing, add 600 μL of elution buffer to each well and elute by shaking at 400 rpm at room temperature for 20 min; then add 120 μL of neutralization buffer to neutralize and mix.

[0038] 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 mixing and expanding culture for 4 h, the supernatant was collected by centrifugation at 8000g for 5 min.

[0039] 8. Purification: Add 20% (w / v) phage precipitate to the supernatant from step 7, mix thoroughly, and let stand at 4°C for 4 hours.

[0040] 9. Collection and re-purification: Centrifuge the phage precipitate from step 8 above at 13000g for 10 min, collect the precipitate, and repeat step 8 to purify the phage again.

[0041] 10. Calculate the phage titer: Take 10 μL of the phage solution from step 9, dilute it serially, and calculate the phage titer using the blue-white staining method.

[0042] 11. Screening: Repeat steps 1-10 for three rounds of screening to obtain a phage peptide library with high affinity for integrins.

[0043] Example 2: Phage Genome Extraction and Sequencing The genome of the phage peptide library from Example 1 was extracted and subjected to high-throughput sequencing. The amino acid sequence of the peptide was obtained by comparing the codons.

[0044] Experimental steps: 1. Genomic DNA extraction was performed using a phage DNA extraction kit.

[0045] 2. The genome was accurately quantified using the Qubit nucleic acid quantification instrument.

[0046] 3. Using the extracted genomic DNA as a template, the first round of PCR amplification was performed. The amplification primer sequences are shown in Table 1 below, the PCR reaction system is shown in Table 2 below, and the reaction procedure 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 reaction solution for a 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 product from step 4 using Qubit: Take 3 μL of the reaction solution for nucleic acid electrophoresis detection.

[0047] 6. Sequencing of the high-throughput library of PCR products was performed using the Illumina sequencing platform.

[0048] Phages were used to construct a library, and the resulting nucleic acid electrophoresis image is shown below. Figure 2 As shown, the top 250bp represents the successfully added sequencing adapter, and the bottom 200bp (arrow) represents the original sequence. The resulting polypeptide sequences after sequencing are shown in Table 6. These sequences have a high affinity for integrin antigens.

[0049] Table 6: Example 3: Phage display in mice Phage peptide libraries were directly injected into mice. After 24 hours, various organs of the mice were collected for phage amplification culture and purification. Sequencing of the purified phages yielded corresponding peptide sequences. These sequences can, to some extent, reflect the preference of short peptides for various organs. Integrating the peptides into the surface of viral vectors may yield viral vectors with high affinity for the corresponding organs.

[0050] Experimental steps: I. In vivo screening of bacteriophages 1. The experiment was set up with an experimental group and a control group. The experimental group was the injection group of 7 / 12 peptide phage library (phage display kit (7 peptide NEB, #E8211S, 12 peptide NEB, #E8210S) with 3 replicates for each peptide library. The control group was injected with physiological saline.

[0051] 2. Phage library injection: The 7 / 12 peptide phage library was diluted with PBS, and the injection dose was 1.0 × 10⁻⁶. 11 Pfu, 200 μL, was administered to mice via the tail vein.

[0052] 3. Tissue samples were collected from the mice 24 hours later. During the sampling process, the mice were thoroughly bled, and the biceps brachii, quadriceps femoris, diaphragm tissue, and organs such as the heart, liver, brain, spleen, lungs, and kidneys were separated.

[0053] 4. Cut off a portion of the organ's tissue and add it to a PBST grinding instrument.

[0054] 5. Collection and washing: Discard the supernatant and wash repeatedly with PBST, 10 times.

[0055] 6. Elution and neutralization: Add 600 μL of elution buffer, shake to mix thoroughly and elute the phage, then add 120 μL of neutralization buffer to terminate the elution.

[0056] 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 mixing and expanding culture for 4 h, the supernatant was collected by centrifugation at 8000g for 5 min.

[0057] 8. Purification: Add 20% (w / v) phage precipitate to the supernatant from step 7, mix thoroughly, and let stand at 4°C for 4 hours.

[0058] 9. Collection and re-purification: Centrifuge the phage precipitate from step 8 above at 13000g for 10 min, collect the precipitate, and repeat step 8 to purify the phage again.

[0059] 10. Calculate the phage titer: Take 10 μL of the phage solution from step 9, dilute it serially, and calculate the phage titer using the blue-white staining method.

[0060] 11. Screening: Repeat steps 1-10 and perform three rounds of screening to obtain a peptide library of high-affinity mouse muscle peptide sequences.

[0061] II. Phage Genome Extraction and Sequencing This step is the same as the operation steps in Example 2.

[0062] Experimental Results: After multiple rounds of screening and library construction and sequencing, peptide sequences from high-affinity mouse muscle were obtained. The peptides analyzed after sequencing are shown in Table 7. Table 7: Example 4: Construction of AAV capsid variant plasmid The nucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NO: 8-12, 14 was integrated into the gene of the AAV virus capsid plasmid pAAV2 / 9n using genetic engineering methods; and a BsmBI restriction site was introduced at position 588 / 589, where the gene sequence of the functional polypeptide was added to the site, giving pAAV2 / 9n the corresponding transduction properties. The specific operation is 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 other reagents were common materials.

[0063] Experimental steps: I. Obtaining the target segment 1. The gene fragment sequence (SEQ ID NO: 3-7, SEQ ID NO: 13) was synthesized by Nanjing GenScript Corporation. The gene fragment was found in PUC57 plasmid glycerol bacteria and was named pUC57-AAV588 / 589-BsmBI.

[0064] 2. Activation of glycerol bacteria: Streaking of glycerol bacteria on LB resistant plates overnight, picking 2 single colonies the next day and transferring them to 5 mL of LB medium. Plasmids were extracted using a plasmid mini-prep kit. One tube was used for preservation, with 500 μL of 50% glycerol and 500 μL of bacterial culture.

[0065] 3. The plasmid was sequenced using Sanger sequencing to confirm the correctness of the sequence. The sequencing primers were universal U6 primers. 4. The plasmid was digested with BsiWI and PmeI double enzymes. The enzyme digestion system is shown in Table 8: Table 8: The reaction was carried out at 37°C for 1.5 h, and then 6 μL of 10x DNA loading buffer was added for agarose gel electrophoresis.

[0066] 5. Fragment obtained: The vector fragment size is 2710 bp, and the target fragment size is 1230 bp. The target fragment in the lower layer is recovered using a gel extraction kit.

[0067]

[0068] The target fragments are: SEQ ID NO: 3-7, SEQ ID NO: 13.

[0069] 6. NanoDrop determines the concentration of the target fragment.

[0070] II. Constructing Carrier Fragments 1. Glycerol bacteria containing pAAV2-9n plasmid were purchased from Addgene (112865), streaked on LB plates, and incubated overnight at 37°C with the plates inverted.

[0071] 2. Pick two single clones and put them into 5 mL of ampicillin-resistant LB medium. Extract the plasmids using a plasmid mini-extraction kit and verify the correctness of the plasmids using Sanger sequencing.

[0072] 3. Linearize the pAAV2-9n plasmid using BsiWI and PmeI double digestion. The digestion system is shown in Table 9: Table 9: The reaction was carried out at 37℃ for 1.5 h, and then 10 μL of 10× DNA loading buffer was added for agarose gel electrophoresis.

[0073] 4. Obtaining the linearized vector fragment: The linearized vector fragment size is 6100bp. The upper vector fragment was recovered using a gel extraction kit.

[0074] 5. NanoDrop was used to determine the concentration of the recovered carrier fragments.

[0075] III. Connecting the carrier fragment to the target fragment 1. The reaction was performed using DNA T4 ligase (the amount of the target fragment and the linearized vector were 25 ng and 100 ng, respectively, with a fragment ratio of 1:1). The ligation reaction is shown in Table 10 below: Table 10: The reaction was carried out at 25°C for 20 minutes to obtain the ligation product.

[0076] 2. Take 2 μL of the ligation product and transform it into DH5α Escherichia coli competent cells. Spread the mixture on an ampicillin-resistant plate. On the second day, pick two single colonies and culture them in 5 mL of ampicillin-resistant LB medium to extract plasmids (one tube per culture, 500 μL 50% glycerol + 500 μL bacterial solution).

[0077] 3. Sanger sequencing confirmed the sequence's correctness, and the correct plasmid was named pAAV2 / 9n-588 / 589-BsmBI. A schematic diagram of the plasmid is shown below. Figure 3 .

[0078] A series of pAAV2 / 9n-588 / 589-BsmBI capsid plasmids were obtained, and their information is shown in Table 11: Table 11: Example 5: AAV virus packaging 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 the targeted drug delivery vector was obtained through self-assembly. The specific operation is as follows: Synthesizing AAV viral vectors requires three plasmids: a capsid plasmid (Rep / Cap, specifically the modified pAAV2 / 9n-588 / 589-BsmBI capsid plasmid of this invention), a target gene plasmid (GOI), and a helper viral plasmid. These three plasmids are transfected into eukaryotic cells, where they self-assemble to form an AAV viral vector containing the target gene. After purification and quality testing, the vector can be used for in vitro and in vivo drug delivery. The overall flowchart is shown below. Figure 4 .

[0079] Experimental steps ( Figure 4 ): I. Virus Packaging and Purification 1. Plasmid extraction: The three types of plasmids required for AAV were cultured overnight and endotoxin-free plasmids were extracted using a plasmid large-scale extraction kit (Promega#A2392).

[0080] 2. Plasmid detection: NanoDrop and Qubit were used to quantify the extracted plasmids, and nucleic acid electrophoresis was performed to verify the size and purity of the plasmids.

[0081] 3. Virus preparation was carried out using 293T cells (ATCC#CRL-3216) according to the three-level seed bank.

[0082] 4. Packaging of AAV virus: 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.

[0083] 5. Collect cells for purification three days after transfection.

[0084] 6. Resuspend the cells and repeatedly freeze and thaw them 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.

[0085] 7. Sample preparation before ultracentrifugation: Prepare iodixanol with different density gradients. Finally, use a peristaltic pump to slowly and completely load all samples into the tube. After the tube is completely filled with liquid, heat seal it.

[0086] 8. Use the following settings for high-speed centrifugation: 18℃, centrifugation speed 70,000 rpm, centrifugation time 1 hour and 5 minutes to collect the virus.

[0087] 9. Sterilization filtration: Use a 0.22µm syringe filter to sterilize the ultrafiltration recovered sample. The sample is retained for physical titer measurement and SDS-PAGE gel chromatography to determine protein purity. After aliquoting, the sample is labeled and temporarily stored in the cryogenic biobank.

[0088] III. 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: Simultaneously, nucleic acid gels were prepared for enzyme digestion verification. Quantification was performed using Qubit, and the concentration of the reaction solution was determined to be 16.5 ng / μL. The copy number per μL was calculated to be 2.68 × 10⁻⁶. 9 vg, the dilution gradient of the linearized plasmid (10). 9 -10 4 The final volume was 200 μL, with a concentration gradient of 10-fold increments. The qPCR reaction was performed, and the reaction system is shown in Table 13, while the reaction procedure is shown in Table 14. Table 13: Table 14: 3. Results Analysis: From the amplification curve ( Figure 5 CT value and R 2 The value indicates the accuracy of the standard curve; 4. Adding commercially available standard virus products (wild-type AVV9) can corroborate the accuracy of the detection, allowing for precise titer determination of the virus and quantification of the titer of each virus. This facilitates subsequent testing and comparison of the transfection capabilities of different viruses. The resulting targeted viruses are shown in Table 15. Table 15: Example 6: LNP preparation and peptide modification The amino acid sequences of the polypeptides with amino acid sequences as shown in SEQ ID NO: 8-10 obtained in Example 2 are linked with linkers to obtain polypeptide amino acid sequences as shown in any one of SEQ ID NO: 15-17. By conjugating a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO: 8-10 to a lipid nanoparticle, a non-viral targeted drug delivery carrier is obtained. The specific procedure is as follows: Lipid nanoparticles (LNPs) are nanoscale drug delivery systems composed of lipids. They are typically composed of four components: phospholipids, cholesterol, cationic lipids, and polyethylene glycol-modified lipids (PEG-lipids). Because their components are natural substances, they have low toxicity and immunogenicity to the human body and can be repeatedly administered.

[0089] To achieve targeted delivery of lipid nanoparticles to specific cells or tissues, in this embodiment, the amino acid sequence of a peptide with the amino acid sequence shown in SEQ ID NO: 8-10 is attached to the surface of the lipid nanoparticles, as illustrated in the schematic diagram below. Figure 6 Once lipid nanoparticles enter the bloodstream, these polypeptide sequences can guide them to bind precisely to receptors, thereby improving drug delivery efficiency to target tissues / cells and reducing side effects on normal cells.

[0090] Experimental materials: 50mM 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), DSPC-PEG2000-Mal (maleimide-phospholipid polyethylene glycol), and Fluc mRNA (messenger RNA encoding firefly luciferase). The peptides were synthesized by Jiangsu Zhuntai Biotechnology Co., Ltd., specifically: Control peptide: A2G80: VQLRNGFPYFSYGGC; M12-1: RRQPPRSISSHPGGGSC; The intermediate peptide sequences of this invention are shown in Table 16: Table 16: GGGSC is the linker sub-linker.

[0091] Experimental equipment: The main equipment used is microfluidic instruments, while the rest of the equipment is conventional.

[0092] Experimental Procedure (The reagents and materials used in this example are all standard materials for preparing LNPs and are all commercially available products): 1. Preparation of lipid solutions: Weigh 15 mg of SM102 into a clean 1.5 mL EP tube, and then add 200 μL of anhydrous ethanol to a concentration of 75 mg / mL.

[0093] Weigh 10 mg of DSPC into a clean 1.5 mL EP tube, and then add 1.0 mL of anhydrous ethanol to a concentration of 15 mg / mL.

[0094] Weigh 10 mg of cholesterol into a clean 1.5 mL EP tube, and then add 1.0 mL of anhydrous ethanol to a concentration of 75 mg / mL.

[0095] Weigh 10 mg of DMG-PEG2k (phospholipid polyethylene glycol) into a clean 1.5 mL EP tube, and then add 1.0 mL of anhydrous ethanol to a concentration of 75 mg / mL.

[0096] Weigh 1 mg of DSPE-PEG2k-Mal (maleimide-phospholipid polyethylene glycol) into a clean 1.5 mL EP tube, and then add 0.5 mL of anhydrous ethanol to a concentration of 2 mg / mL.

[0097] 2. Preparation of targeted peptide-modified lipid nanoparticles: 2.1 Molar ratio of each lipid SM102:DPPC:cholesterol:DMG-PEG2k:DSPE-PEG2k-Mal (maleimide-phospholipid polyethylene glycol) = 50:10:38.5:1.2:0.3.

[0098] 2.2 mass ratio Total lipids:mRNA = 40:1; total lipid concentration was 12.5 mM.

[0099] 2.3 Preparation of lipid-ethanol phase Taking 100 μg of mRNA as an example, prepare 0.3% peptide-modified SM102 LNP: 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 DSPC-PEG2000-Mal and place them in a 1.5 mL clean EP tube. Then add 475.5 μl of anhydrous ethanol to make the total lipid concentration 12.5 mM and the total volume 782.9 μl (1.5 times the required amount of lipid). Mix thoroughly until clear and transparent.

[0100] 2.4 mRNA aqueous phase preparation Take 100 μg of Fluc mRNA (concentration of 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 thoroughly.

[0101] 3. Microfluidic control equipment: Transfer the lipid ethanol phase and mRNA aqueous phase to 1 mL and 3 mL syringes, respectively. Remove any excess air bubbles. Insert the LNP chip into the device, and insert the two threaded syringes into the corresponding interfaces on the chip. Prepare two 15 mL collection tubes, one for sample collection and one for waste collection. Configure the parameters in the software, setting the total injection volume to 1.8 mL and the total flow rate to 12 mL / min (9 mL / min flow rate and 1.35 mL collection volume for the mRNA aqueous phase; 3 mL / min flow rate and 0.45 mL collection volume for the lipid ethanol phase). Design the waste volume to 0.45 mL (initial waste volume 0.4 mL, final waste volume 0.05 mL). Click "Start" to prepare the LNP. After preparation, transfer the samples to Pur-A-Lyzer Maxi 3500 dialysis tubes. Dialyze the prepared samples within 15 minutes. Dialyze with 1xPBS (calcium and magnesium ion-free) at 4°C for 4 hours, then change the medium and dialyze overnight (to remove ethanol). After dialysis, transfer to a clean 5mL tube and measure the volume. (If necessary, the volume can be concentrated using a 30kDa ultrafiltration tube, 3000g, 4°C.) Note: The resulting solution can be stored at 4°C for several days before use. However, it is recommended to use prepared LNPs as soon as possible to obtain consistent results.

[0102] 4. LNP peptide conjugation modification: 1) Using GGGSC as the linker, the sulfur amino group of the cysteine ​​residue undergoes a thiol-double bond addition reaction with the double bond of maleimide to form a new chemical bond, thus synthesizing a polypeptide with an amino acid sequence as shown in SEQ ID NO: 15-17; 2) Dissolve the synthesized peptide in PBS (pH 7.4) at a concentration of 4 mg / mL.

[0103] 3) Take 90 μg of the LNP prepared in step 6, add it to the peptide solution (the molar ratio of peptide to Mal is 10:1), mix well and incubate at 4°C overnight.

[0104] 4) Use dialysis to remove peptides without conjugation. Dialyze with 1xPBS (without calcium and magnesium ions) at 4°C for 4 hours, then change the medium and dialyze overnight. After dialysis, transfer the peptides to a clean 1.5mL EP tube and measure the volume (if necessary, a 30kDa ultrafiltration tube can be used to concentrate the volume at 3000g and 4°C) to obtain peptide-modified LNPs.

[0105] Example 7: Targeted PintAAV Virus Cell Infection Experiment For the targeted peptides screened from integrins using phage display technology in Example 1, we believe they can increase the infection efficiency of muscle cells. Using the construction method of Example 4, the peptide sequence was added to the AAV capsid, and the virus was packaged and purified using the method of Example 5. The packaged goods contained the gene sequences of firefly luciferase and red fluorescent protein (mCherry). The infection efficiency of the virus was verified on muscle cells.

[0106] Experimental materials: C2C12 mouse myofibroblasts (ATCC#CRL-1772), C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), and other common materials.

[0107] Experimental equipment: carbon dioxide incubator, small animal in vivo imaging system, fluorescence microscope, laser confocal microscope, and other common equipment.

[0108] Experimental steps: 1. Cell culture and induction: After resuscitation, C2C12 mouse myofibroblasts tend to be stable after three or more generations of culture. They are then plated and induced when the cell confluence is about 60%. Myotubes are induced by adding 2% horse serum.

[0109] 2. Virus infection verification: After the purified virus passed the qPCR, SDS-PAGE and endotoxin test, the titer was determined by absolute quantitative PCR. Virus infection experiments were conducted according to the infection titer MOI of 1E+4 per well.

[0110] 3. Fluorescence Imaging and Analysis: Fluorescence field images of cells 6 days after infection were taken using a Zeiss fluorescence microscope. Background values ​​were standardized, and images were taken under a 10x objective lens. The average fluorescence intensity (intDen) of the images was quantified using Imgae J.

[0111] 4. Experimental results: such as Figure 7 , 8 The results show that the screening method and viral vector of the present invention have better transfection efficiency than wild-type AAV9 virus, indicating that the targeted peptides obtained by the present invention have stronger muscle cell targeting and can improve the muscle targeting of AAV vector.

[0112] Example 8: In vivo transfection experiment of targeted Pint-AAV virus in mice To verify the effects of the novel virus Pint-E, which exhibits the highest fluorescence intensity, we injected the virus intramuscularly into mice and observed its expression and distribution within the mice.

[0113] Experimental materials: C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), Pint-E virus, and other common materials.

[0114] Experimental equipment: small animal in vivo imaging system, fluorescence microscope, laser confocal microscope, and other common equipment.

[0115] Experimental steps: 1. Virus quality control: After the purified virus passes the tests of qPCR, SDS-PAGE and endotoxin assay, the titer is determined by absolute quantitative PCR.

[0116] 2. Determine the administration volume: Weigh the mice, and calculate the injection volume according to the dosage of 1.0×10^13 vg / kg. Dilute the virus with physiological saline before injection. The injection volume is 200μL, and the administration is carried out via tail vein.

[0117] 3. On days 7, 14, 21 and 28 after drug administration, mice were subjected to in vivo imaging to observe the distribution and expression of the delivered drug in the body. On day 28, organs were taken from the mice, cryopreserved and embedded, and molecular-level detection was performed.

[0118] Experimental results: The results show that... Figure 9 The novel viral vector Pint-E screened using this method exhibits better transfection efficiency in vivo. This indicates that the targeting peptides obtained by screening in this invention have stronger muscle targeting, which can improve the muscle targeting of AAV vectors.

[0119] Example 9: In vivo distribution experiment of targeted MsAAV virus in mice For the targeted peptides screened from mice using phage display technology in Example 3, we believe that they can increase the distribution and infection efficiency of the corresponding target organs. Using the construction method of Example 4, the peptide sequence was added to the AAV capsid, and the virus was packaged and purified using the method of Example 5. The packaged goods contained the gene sequences of firefly luciferase and red fluorescent protein (MCherry). The infection efficiency of the novel virus was verified in mice.

[0120] Experimental materials: C57 / BL mice (purchased from the Experimental Animal Center of Yunnan University), MsAAV virus, and other common materials.

[0121] 1. Virus quality control: After the purified virus passes the tests of qPCR, SDS-PAGE and endotoxin assay, the titer is determined by absolute quantitative PCR.

[0122] 2. Determine the administration volume: Weigh the mice, and calculate the injection volume according to the dosage of 1.0×10^13 vg / kg. Dilute the virus with physiological saline before injection. The injection volume is 200μL, and the administration is carried out via tail vein.

[0123] 3. In vivo imaging of mice was performed on days 7, 14, 21, and 28 after drug administration. Mice were weighed and injected intraperitoneally 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. In vivo imaging of small animals was performed 10 minutes after substrate injection. The distribution and expression of the delivered drug in vivo were observed. Organs were harvested from mice on day 28, cryopreserved, and embedded for molecular-level detection.

[0124] Experimental results are as follows Figure 10 Compared to wild-type AAV, the novel viral vector MsAAV targets more muscle tissue and distributes less to the liver, indicating that the targeted peptides obtained by screening in this invention have stronger muscle targeting and can improve the muscle targeting of AAV vectors.

[0125] Example 10: In vivo administration of targeted LNP virus to mice For the targeted LNP delivery vector constructed in Example 8, its targeting ability was verified by intravenous injection into mice. The control sequence was a sequence that has been reported to alter LNP targeting. The LNP was used to package Fluc-mRNA (firefly luciferase mRNA), and the distribution and expression of the cargo in mice were detected by a small animal in vivo imaging instrument.

[0126] Experimental materials: C57 mice used in this experiment were all purchased from the Experimental Animal Center of Yunnan University. LNP was prepared as described in Example 8. Anesthetic and luciferase substrate were also used.

[0127] Experimental equipment: The small animal live imaging device is used; the rest are common equipment.

[0128] Experimental Groups: (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 6-8 week old C57 mice, regardless of sex, with 3 mice in each group, and administer a tail vein injection at a dose of 1.5 mg / kg, with a total volume of 250 μL.

[0129] 2. Six hours later, the mice were imaged using a small animal in vivo imaging system. The mice were weighed and injected intraperitoneally with 200 μL of anesthetic per 10g of body weight, and 100 μL of luminescent substrate (15 mg / mL) per 10g of body weight. Small animal in vivo imaging was performed 10 minutes after the substrate injection.

[0130] Experimental results are as follows Figure 11 Compared with the control group (unmodified LNP), intergrin-1 (SEQ ID NO: 15) significantly increased the distribution and expression of LNP in muscle and decreased the distribution of LNP in liver. This indicates that the targeted peptides obtained by screening in this invention have stronger muscle targeting and can improve the muscle targeting of drug carriers.

[0131] 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 modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A targetable drug delivery vehicle, comprising: a biocompatible polymer matrix; a drug; and a targeting agent covalently bonded to the polymer matrix. The drug delivery carrier comprises a drug-loaded carrier and a targeting polypeptide; The drug-loaded carrier is a non-viral carrier, and the non-viral carrier is any one of a lipid nanoparticle, a virus-like particle, and an exosome; The amino acid sequence of the targeting polypeptide is shown in SEQ ID NO: 8; The targeting polypeptide is connected to the non-viral carrier by surface modification.

2. The drug delivery vehicle of claim 1, wherein, The surface modification includes physical adsorption, surface deposition, coupling, esterification, and grafting.

3. A method for preparing the targeting drug delivery vehicle as claimed in claim 1 or 2, characterized by, The preparation method comprises the following steps: Step 1: screening a targeting polypeptide for a target by a phage display method to obtain a phage library of the targeting polypeptide; 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 SEQ ID NO: 8; Step 3: assembling or coupling the targeting polypeptide obtained in step 2 with a drug-loaded carrier; the drug-loaded carrier is a non-viral carrier; The target includes a biomolecule, a tissue, and an organ; The biomolecule is an integrin; The tissue is any one of a biceps tissue, a quadriceps tissue, and a diaphragm tissue; The organ is any one of a heart, a liver, a brain, a spleen, a lung, and a kidney.

4. The production method according to claim 3, wherein When the target is a biomolecule, step 1 adopts the following method: After incubating the phage library with the target biomolecule, the phage library is washed, eluted and neutralized, transfected, cultured, and purified to obtain a phage library of the targeting polypeptide.

5. The production method according to claim 3, wherein When the target is a tissue or an organ, step 1 adopts the following method: (1) injecting the phage library into an animal body to collect phages from the tissue or the organ; (2) transfecting, culturing, and purifying the collected phages of step (1) to obtain a phage library of the targeting polypeptide.

6. The production method according to claim 3, wherein The method for connecting the targeting polypeptide to the drug-loaded carrier in step 3 comprises the following steps: First, connect the targeting polypeptide to a linker to introduce a reaction site, and then couple the linker to the non-viral carrier; The non-viral carrier is any one of a lipid nanoparticle, a virus-like particle, and an exosome.

7. The production method according to claim 6, wherein The linker is an amino acid sequence. the amino acid sequence is (GGGGS) n C, (GGGS) n C, (GGS) n C, (GS) n C.

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