Use of dendritic polypeptides in the preparation of small nucleic acid drug delivery systems
The dendritic delivery system, formed by co-incubating dendritic peptide DP7 with small nucleic acid drugs, solves the problems of small nucleic acid drugs' inability to cross cell membranes and lack of extrahepatic targeting, achieving efficient intracellular delivery and liver targeting, and improving the delivery efficiency and targeting of drugs in vivo.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SICHUAN UNIV
- Filing Date
- 2024-02-23
- Publication Date
- 2026-07-03
AI Technical Summary
Existing technologies make it difficult for small nucleic acid drugs to be efficiently delivered across cell membranes, and there is a lack of effective extrahepatic targeted delivery carriers, resulting in low drug delivery efficiency in vivo.
Using dendritic peptide DP7 as a carrier, a dendritic structure formed by lysine linkage is co-incubated with a small nucleic acid drug to form a dendritic peptide delivery system, achieving efficient intracellular delivery and liver targeting.
It improves the intracellular delivery efficiency and lymph node targeting efficiency of small nucleic acid drugs, significantly enhances liver targeting effects, simplifies the preparation process, and reduces costs.
Smart Images
Figure CN118542944B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to the use of dendritic peptides in the preparation of small nucleic acid drug delivery systems. Background Technology
[0002] Small nucleic acid technology, as one of the disruptive innovative therapies in recent years, offers numerous advantages over traditional drugs, including rapid target screening, high development success rate, low likelihood of drug resistance, broad indications, and long-lasting therapeutic effects. It can effectively supplement or replace existing therapies, fill gaps in indications, and demonstrate enormous market potential. Broadly defined, small nucleic acid drugs refer to oligonucleotide sequences less than 30 nt in length, including antisense oligonucleotides (ASO), small interfering RNA (siRNA), miRNA, oligonucleotide aptamers, and CpG oligonucleotides. The development of small nucleic acid drug delivery vectors is recognized as one of the core barriers and technical challenges in the industry. Because small nucleic acids are exogenous drugs, they may be degraded by nucleases in the bloodstream. Furthermore, due to their large molecular weight and negative charge, they cannot cross cell membranes to exert their therapeutic effects. In vitro experiments show that only 1% of nucleic acid drugs can enter the cytoplasm, while in vivo experiments show this proportion to be less than 0.1%. Therefore, developing small nucleic acid delivery systems that can overcome natural biological barriers and achieve efficient transmembrane and in vivo transport has become a critical problem that urgently needs to be solved.
[0003] DP7 (VQWRIRVAVIRK) is a novel cationic hydrophilic antimicrobial peptide developed by the applicant based on previous research on amino acid activity prediction methods. After modification with cholesterol, Chol-DP7 (DP7-C) was found to function as a small nucleic acid drug delivery carrier. It can efficiently deliver small nucleic acid drugs to various cells (including normal cells, tumor cells, and immune cells) via caveolin- and clathrin-dependent pathways. Although previous experiments have demonstrated that DP7-C can enhance the intracellular delivery efficiency of small nucleic acid drugs, subcutaneous injection after direct incubation of DP7-C and small nucleic acid drugs lacks significant organ targeting, thus failing to meet the requirement of tissue-specific targeting of small nucleic acid drugs during translational applications to reduce drug administration toxicity.
[0004] Liver-targeted drug delivery, especially of nucleic acid drugs, is a hot research topic in this field. Currently, mature and well-developed liver-targeted technologies for nucleic acid drugs with extensive clinical pipelines mainly rely on GalNAc modification, for which China lacks independent intellectual property rights. Finding new and effective extrahepatic targeted delivery methods is also a critical issue that urgently needs to be addressed in this field. Summary of the Invention
[0005] The technical problem to be solved by this invention is to improve the intracellular delivery efficiency, lymph node drainage efficiency, and liver targeting efficiency of small nucleic acid drugs, as extrahepatic targeting delivery carriers are scarce.
[0006] One of the technical solutions of the present invention to solve the above-mentioned technical problems is to provide the use of dendritic polypeptide DP7 in the preparation of small nucleic acid drug delivery vectors.
[0007] The aforementioned polypeptide is a DP7 polypeptide with the amino acid sequence VQWRIRVAVIRK (SEQ ID No. 1); the dendritic polypeptide formed by the coupling of DP7 polypeptides is composed of 2 to 16 DP7 polypeptide molecules.
[0008] Preferably, the dendritic polypeptide is composed of 2, 4, or 8 DP7 polypeptide molecules coupled together.
[0009] In the above-mentioned uses, the DP7 polypeptide molecules are coupled together by lysine as a linking medium.
[0010] In the above-mentioned uses, the DP7 polypeptide molecule participates in coupling at the carbon-terminal and / or nitrogen-terminal amino acid residues.
[0011] Furthermore, the dendritic polypeptide involved in the above-mentioned uses has at least one of the following structural formulas:
[0012]
[0013] or
[0014]
[0015] Among them, the small nucleic acid drug mentioned in the above uses is at least one of DNA drug or RNA drug.
[0016] The small nucleic acid drug mentioned in the above-mentioned uses is at least one of a single-stranded nucleic acid drug or a double-stranded nucleic acid drug.
[0017] The size of the small nucleic acid drug described in the above-mentioned uses is 19nt to 29nt when it is a single-stranded small nucleic acid drug; and 19bp to 29bp when it is a double-stranded small nucleic acid drug. Preferably, when it is a single-stranded small nucleic acid drug, the size is 19nt to 23nt; and when it is a double-stranded small nucleic acid drug, the size is 19bp to 23bp.
[0018] Among them, the small nucleic acid drugs mentioned in the above uses are one or more of antisense oligonucleotide (ASO) drugs, small interfering RNA (siRNA) drugs, miRNA drugs, oligonucleotide aptamers, and CpG oligonucleotides.
[0019] Furthermore, the small nucleic acid drug delivery carrier described in the above-mentioned uses is a subcutaneous drug delivery carrier.
[0020] Furthermore, the aforementioned subcutaneous administration refers to subcutaneous injections or subcutaneous embedding preparations.
[0021] The present invention also provides a small nucleic acid drug delivery system, which is prepared from dendritic peptides and small nucleic acids as raw materials. The peptides are dendritic peptides formed by coupling DP7 peptides with the amino acid sequence VQWRIRVAVIRK to each other, and are composed of 2 to 16 DP7 peptide molecules coupled together.
[0022] Furthermore, the aforementioned small nucleic acid drug delivery system is prepared from dendritic peptides and small nucleic acids at a mass ratio of 0.1 to 20:1. Preferably, it is prepared from dendritic peptides and small nucleic acids at a mass ratio of 1 to 4:1.
[0023] Furthermore, the aforementioned small nucleic acid drug delivery system is prepared by co-incubating a dendritic polypeptide with a small nucleic acid drug. Preferably, it is prepared by co-incubating the small nucleic acid drug with the dendritic polypeptide in water or liquid culture medium for 5–15 min.
[0024] In the aforementioned small nucleic acid drug delivery system, the dendritic polypeptide is composed of 2, 4, or 8 DP7 polypeptide molecules coupled together.
[0025] In the aforementioned small nucleic acid drug delivery system, the DP7 polypeptide molecules are coupled together using lysine as a linking medium.
[0026] In the aforementioned small nucleic acid drug delivery system, the DP7 involved in coupling is an amino acid residue at the carbon terminus and / or nitrogen terminus.
[0027] The dendritic polypeptide described in the above-mentioned small nucleic acid drug delivery system has at least one of the following structural formulas:
[0028]
[0029]
[0030] The small nucleic acid drug in the above-mentioned small nucleic acid drug delivery system is at least one of DNA drug or RNA drug.
[0031] Furthermore, the small nucleic acid drug is at least one of a single-stranded nucleic acid drug or a double-stranded nucleic acid drug.
[0032] The size of the small nucleic acid drug in the above-mentioned small nucleic acid drug delivery system is as follows: when it is a single-stranded small nucleic acid drug, the size is 19nt to 29nt; when it is a double-stranded small nucleic acid drug, the size is 19bp to 29bp; preferably, when it is a single-stranded small nucleic acid drug, the size is 19nt to 23nt; when it is a double-stranded small nucleic acid drug, the size is 19bp to 23bp.
[0033] Among them, the small nucleic acid drug in the above-mentioned small nucleic acid drug delivery system is one or more of the following: antisense oligonucleotide (ASO) drug, small interfering RNA (siRNA) drug, miRNA drug, oligonucleotide aptamer, and CpG oligonucleotide.
[0034] Among them, the small nucleic acid drugs mentioned in the above-mentioned small nucleic acid drug delivery system are small nucleic acid drugs targeting immune checkpoint molecules and cancer therapeutic targets.
[0035] The immune checkpoint molecules in the aforementioned small nucleic acid drug delivery system are at least one of CTLA-4, PD-1, PD-L1, LAG3, TIGIT, TIM3, B7H3, CD39, CD73, adenosine A2A receptor, SIGLEC10, or CD47; the cancer therapeutic targets are at least one of CYP1A1, p53, NQO1, ALDH2, EPHX1, STAT3, VEGF, Pin1, FGFR, and UCK2.
[0036] The small nucleic acid drug in the aforementioned small nucleic acid drug delivery system is an siRNA targeting the immune checkpoint molecule PD-L1, and its nucleotide sequence is as follows:
[0037] sense(5'-3')CUCCAAAGGACUUGUACGUTT
[0038] antisense(5'-3')ACGUACAAGUCCUUUGGAGTT.
[0039] Based on the above technical solutions, the present invention also provides a small nucleic acid drug formulation prepared by adding pharmaceutically acceptable excipients or auxiliary components to the above-mentioned small nucleic acid drug delivery system as the main component.
[0040] Furthermore, the aforementioned small nucleic acid drugs also contain immune adjuvants.
[0041] Among them, the dosage form of the aforementioned small nucleic acid drugs is injection.
[0042] Among them, the dosage form of the aforementioned small nucleic acid drugs is a subcutaneous administration formulation.
[0043] Furthermore, the aforementioned subcutaneous drug delivery formulation is a subcutaneous injection formulation or a subcutaneous embedding formulation.
[0044] Furthermore, the present invention also provides the use of dendritic polypeptides in the preparation of liver-targeted drug delivery carriers, wherein the dendritic polypeptide is a DP7 polypeptide with the amino acid sequence VQWRIRVAVIRK; and it is formed by the coupling of 2 to 16 DP7 polypeptide molecules to form a dendritic polypeptide.
[0045] The dendritic polypeptide described in the above-mentioned uses is composed of 2, 4, or 8 DP7 polypeptide molecules coupled together.
[0046] In the above-mentioned uses, the DP7 polypeptide molecules in the dendritic polypeptides are coupled together by lysine as a linking medium.
[0047] In the above-mentioned uses, the DP7 in the dendritic polypeptide participates in coupling at the carbon-terminal and / or nitrogen-terminal amino acid residues.
[0048] Wherein, the structural formula of the dendritic polypeptide described in the above-mentioned uses is at least one of the following:
[0049]
[0050]
[0051] The drug delivered in the above-mentioned uses is at least one of peptide drugs, protein drugs, or nucleic acid drugs. The nucleic acid drugs mentioned in the above-mentioned uses are small nucleic acid drugs.
[0052] The small nucleic acid drug mentioned in the above-mentioned uses is at least one of antisense oligonucleotide (ASO), small interfering RNA (siRNA), miRNA, oligonucleotide aptamer, and CpG oligonucleotide.
[0053] The administration route for the liver-targeted drug delivery carrier described in the above-mentioned uses is injection.
[0054] The administration route of the liver-targeted drug delivery carrier described in the above-mentioned uses is subcutaneous administration.
[0055] Furthermore, the subcutaneous administration described in the above uses refers to subcutaneous injection or subcutaneous embedding.
[0056] The beneficial effects of this invention are as follows: Based on the inventive synthesis of dendritic DP7, this invention further discovers its unique and superior properties. First, the dendritic DP7 peptide can serve as a small nucleic acid drug delivery carrier. Experimental results show that the dendritic DP7 peptide can efficiently transfect target drugs, especially siRNA, into normal cells, tumor cells, and dendritic cells (DCs). For example, the transfection efficiency of the dendritic DP7 peptide KK2DP7 into siRNA in normal cells, tumor cells, and DCs can all reach over 85%. Second, subcutaneous para-lymph node injection after co-incubation of the dendritic DP7 peptide with the target drug can improve the lymph node targeting efficiency of small nucleic acid drugs and can also serve as a lymph node-specific targeted delivery carrier. In addition, the dendritic DP7 peptide, after loading the drug, also exhibits a liver-targeting effect, significantly improving the liver-targeting efficacy of the drug. In particular, it can effectively improve the liver-targeting efficacy of drugs after subcutaneous administration. For the art, using subcutaneous administration formulations can improve patient compliance with medication and provide liver targeting, offering multiple superior benefits. Meanwhile, the dendritic DP7 peptide is simple and low-cost to prepare for drug formulations, which is conducive to its subsequent promotion and use and has a good application prospect. Attached Figure Description
[0057] Figure 1 HPLC and MS data of KK2DP7.
[0058] Figure 2 The efficiency of KDP7, KK2DP7, KK2K4DP7 and siRNA uptake by normal cells, tumor cells and dendritic cells (DCs) after incubation.
[0059] Figure 3 Interference efficiency of KK2DP7 / siPD-L1 in B16 tumor cells.
[0060] Figure 4 Interference efficiency of KK2DP7 / siPD-L1 in DCs.
[0061] Figure 5 Interference efficiency of KK2DP7 / siPD-L1 in Hep1-6 tumor cells.
[0062] Figure 6 Interference efficiency of KK2DP7 / siVEGF in Hep1-6 tumor cells.
[0063] Figure 7 Statistical results of the efficiency of KK2DP7 / Cy5-siRNA targeting lymph nodes after para-lymph node injection.
[0064] Figure 8Fluorescence intensities in the liver at different time points after subcutaneous administration of KDP7, KK2DP7, KK2K4DP7, and Cy5-siRNA following incubation. (***P<0.001)
[0065] Figure 9 Therapeutic effects of KK2DP7 / siPD-L1 and KK2DP7 / siVEGF in mice and statistical analysis of the number of liver tumor nodules. (***P<0.001, *P<0.05) Detailed Implementation
[0066] The present invention will be specifically described below through the introduction of specific embodiments.
[0067] In the preliminary research of this invention, a peptide with antibacterial activity was obtained, with the sequence VQWRIRVAVIRK (SEQ ID No. 1), and named DP7. Further research revealed that hydrophobic modification of the DP7 peptide resulted in an amphiphilic compound, DP7-C, capable of self-assembling into micelles. This compound can reduce the cytotoxicity of the DP7 peptide while maintaining its antibacterial activity; furthermore, when assembled into nanoparticles, it can serve as a delivery carrier for some drugs. Although previous experiments have demonstrated that DP7-C can enhance the intracellular delivery efficiency of small nucleic acid drugs, subcutaneous injection after direct incubation of DP7-C and small nucleic acid drugs showed no significant targeting.
[0068] To reduce the tissue-specific targeting effect and drug-induced toxicity of small nucleic acid drugs during conversion and application, this invention creatively modifies DP7 to obtain dendritic DP7 peptides. The DP7 peptide molecules in the dendritic DP7 peptides are coupled together using lysine residues as linkers. The coupling sites of the DP7 peptides can be amino acid residues at the C-terminus or N-terminus. Coupling can be achieved by linking multiple DP7 peptide molecules, typically 2 to 16 molecules. In the embodiments of this invention, dendritic DP7 peptides composed of 2, 4, and 8 DP7 peptide molecules were prepared and investigated, namely, bibranched DP7 (hereinafter abbreviated as KDP7, structure as shown in Formula I), tetrabranched DP7 (hereinafter abbreviated as KK2DP7, structure as shown in Formula II), and octagonal DP7 (hereinafter abbreviated as KK2K4DP7, structure as shown in Formula III).
[0069] Further research revealed that these dendritic peptides, when incubated with small nucleic acid drugs, can efficiently deliver the drugs into cells.
[0070] This invention also discovered that these dendritic peptides, after incubation with small nucleic acid drugs, possess lymph node-targeting properties. This enables the preparation of lymph node-targeting small nucleic acid drug formulations.
[0071] Furthermore, during subcutaneous administration experiments, it was unexpectedly discovered that subcutaneous administration can specifically and efficiently target the liver. In one embodiment, the targeting effect was optimal after incubation of the dendritic DP7 peptide KK2DP7 with a small nucleic acid drug. Therefore, it is believed that the subcutaneous administration technique after direct incubation of the dendritic DP7 peptide with the drug does not require reliance on the commonly used N-acetylgalactosamine modification method in the art, thus solving the technical problem of the scarcity of extrahepatic targeted delivery carriers. The preparation process only requires co-incubating the dendritic peptide with at least one of the target drugs, such as peptide drugs, protein drugs, or nucleic acid drugs, for an appropriate time. For example, in one embodiment of the present invention, it is prepared by co-incubation with a small nucleic acid drug for 5 minutes; for use, it can be administered via subcutaneous injection. The preparation method is simple, low-cost, and conducive to its subsequent widespread use.
[0072] The aforementioned small nucleic acid drugs can be at least one of DNA drugs or RNA drugs. Generally, the size of the small nucleic acid drugs selected is 19nt to 29nt for single-stranded small nucleic acid drugs and 19bp to 29bp for double-stranded small nucleic acid drugs. Preferably, the size of the small nucleic acid drug is 19nt to 23nt for single-stranded small nucleic acid drugs and 19bp to 23bp for double-stranded small nucleic acid drugs.
[0073] The small nucleic acid drug delivered by the dendritic DP7 peptide in this invention can be at least one of antisense oligonucleotide (ASO) drugs, small interfering RNA (siRNA) drugs, miRNA drugs, oligonucleotide aptamers, and CpG oligonucleotides.
[0074] For example, it can be used to deliver small nucleic acid drugs that target immune checkpoint molecules. Common immune checkpoint molecules include CTLA-4, PD-1, PD-L1, LAG3, TIGIT, TIM3, B7H3, CD39, CD73, adenosine A2A receptor, SIGLEC10, or CD47.
[0075] For example, it can also deliver small nucleic acid drugs targeting cancer therapeutic targets. Common cancer therapeutic targets include at least one of CYP1A1, p53, NQO1, ALDH2, EPHX1, STAT3, VEGF, Pin1, FGFR, and UCK2.
[0076] In one embodiment of the present invention, siRNA targeting the immune checkpoint molecule PD-L1 was used and a good targeted anti-tumor effect was obtained. Its nucleotide sequence is as follows:
[0077] sense(5'-3')CUCCAAAGGACUUGUACGUTT
[0078] antisense(5'-3')ACGUACAAGUCCUUUGGAGTT.
[0079] Of course, the aforementioned drugs may also contain various pharmaceutically acceptable adjuvants and excipients. Furthermore, when the aforementioned drugs are vaccines, they may also contain immune adjuvants.
[0080] The drug of this invention can be administered via subcutaneous administration, intravenous injection, or intraperitoneal injection.
[0081] Furthermore, the aforementioned subcutaneous administration route can be subcutaneous injection or subcutaneous implantation.
[0082] The present invention will be further described in detail below through embodiments. The main experimental materials and equipment used in the embodiments are as follows:
[0083] 1. Experimental cell lines and experimental animals
[0084] LO2, B16, and HEP-1-6 cell lines were purchased from the American Type Culture Collection (ATCC). They were cultured on RPMI-1640 medium (Gibico) containing 10% fetal bovine serum (FBS, Gibico). Six- to eight-week-old female C57 / BL6J mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd., and housed in an SPF-grade environment.
[0085] 2. Main Reagents, Materials, and Kits
[0086] The cell culture media used in the experiment: RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco, USA. siVEGF and siPD-L1 were synthesized by Shanghai Gemma Gene Technology Co., Ltd. Cy3 and Cy5-labeled siNC were also synthesized by Shanghai Gemma Gene Technology Co., Ltd.
[0087] 3. Main instruments and equipment
[0088] Flow cytometer: FACSCalibur. Western blotting system: e-Blot. Small animal in vivo imaging system: PerkinElmerIVIS Lumina.
[0089] Example 1: Synthesis of dendritic peptides KDP7, KK2DP7, and KK2K4DP7
[0090] The dendritic DP7 peptide was synthesized using a standard solid-phase peptide synthesis method. A brief description is as follows: Fmoc-Lys(Fmoc)-OH was attached to a resin, two Fmoc atoms were removed, exposing two NH2 atoms. Following the peptide sequence VQWRIRVAVIRK, condensation was performed from right to left, thus obtaining a two-branched peptide.
[0091] By attaching Fmoc-Lys(Fmoc)-OH to the resin, removing two Fmoc molecules, and then exposing two NH2 molecules, two more Fmoc-Lys(Fmoc)-OH molecules are attached to the two exposed NH2 molecules. After removing all Fmoc molecules, four amino groups are exposed. Following the polypeptide sequence VQWRIRVAVIRK, the polypeptide is condensed from right to left to obtain a four-branched polypeptide.
[0092] By attaching Fmoc-Lys(Fmoc)-OH to the resin, removing two Fmoc atoms, two NH2 atoms are exposed. These two exposed NH2 atoms are then attached with two more Fmoc-Lys(Fmoc)-OH atoms. After removing all Fmoc atoms, four amino groups are exposed, and then four more Fmoc-Lys(Fmoc)-OH atoms are attached. After removing all Fmoc atoms, eight amino groups are exposed. Following the peptide sequence VQWRIRVAVIRK, condensation is performed from right to left, thus obtaining an eight-branched peptide.
[0093] The synthesized dendritic peptides were analyzed by HPLC and MS to verify the final products. We synthesized dendritic peptides KDP7, KK2DP7, and KK2K4DP7. For example, the HPLC and MS results for KK2DP7 are shown below. Figure 1 .
[0094] Example 2: Determination of the uptake efficiency of KDP7 / siRNA, KK2DP7 / siRNA, and KK2K4DP7 / siRNA complexes by normal cells, tumor cells, and dendritic cells (DCs).
[0095] 1. Preparation of KDP7, KK2DP7, KK2K4DP7 and siRNA complexes
[0096] KDP7(VQWRIRVAVIRKK), KK2DP7((VQWRIRVAVIRK)2KK), and KK2K4DP7(((VQWRIRVAVIRK)2K)2KK) were synthesized by Shanghai ChuTai Biotechnology Co., Ltd. using a solid-phase peptide synthesis method. The final products were purified by HPLC and identified by mass spectrometry.
[0097] KDP7, KK2DP7, and KK2K4DP7 lyophilized powders can be dissolved directly in deionized water, aliquoted, and stored at -20°C. To prepare their complexes with siRNA, simply incubate them with an aqueous siRNA solution in deionized water and culture medium for 5 minutes.
[0098] 2. Acquisition and culture of DC cells
[0099] ① Take the tibia and fibula of adult female C57BL / 6J mice at about 6 weeks of age, soak them in 75% ethanol for 5 minutes to kill bacteria, then remove the muscle tissue, soak the leg bones in RPMI 1640 + 1% PS medium; cut off both ends of the leg bones with sterile scissors, and then use a syringe to draw fresh RPMI 1640 + 1% PS medium to blow out bone marrow cells until all bone marrow cells are blown out;
[0100] ② Filter the collected culture medium containing bone marrow cells through a 70μm sieve, centrifuge at 1200rpm for 3min, discard the supernatant, and resuspend the cells in erythrocyte lysis buffer (weigh 1.3g Tris-base and 3.74g NH4Cl, dissolve in 490ml ultrapure water, adjust the pH of the solution to 7.2-7.4 with concentrated hydrochloric acid, add ultrapure water to make up to 500ml, remove bacteria with a 0.22μm filter, store at 4℃, prepare fresh before use) and let stand at room temperature for 3min, centrifuge at 1200rpm for 3min, and finally wash away the erythrocyte lysis buffer with RPMI 1640 + 10% FBS + 1% PS medium and resuspend the cells.
[0101] ③ Transfer the resuspended cells to culture dishes, 2 × 10⁶ cells per dish. 6 -3×10 6 For each cell culture dish, add 10 ml of RPMI 1640 + 10% FBS + 1% PS medium and 20 ng / ml GM-CSF cytokine. Incubate the dishes at 37°C. On the third day of culture, add fresh RPMI 1640 + 10% FBS + 1% PS medium containing 20 ng / ml GM-CSF until day 8 to obtain immature DCs (imDCs). Take the DCs cultured to day 8, wash off the medium with 1 ml PBS, resuspend the cells in 100 μl PBS, add 1 μl of APC Hamster Anti-Mouse CD11c flow cytometry antibody, gently mix, and incubate at 4°C in the dark for 40 min. After incubation, wash off excess antibody with PBS, resuspend the cells in 200 μl PBS, and detect CD11c using flow cytometry. + The proportion of DCs. When the proportion of CD11c is greater than 80%, it indicates that DC induction was successful.
[0102] 3. Efficiency of KK2DP7 / siRNA complex transfection of normal cells, tumor cells, and dendritic cells (DCs)
[0103] Normal liver cells (LO2), tumor cells (Hep-1-6), and immature dendritic cells (DCs) cultured for 8 days were seeded into 24-well plates, with 5 × 10⁶ cells per well. 5 Cells were incubated for 5 min with 30 μl of KK2DP7 / siNC complex in 1640 complete medium (0.24 μg siNC and 0.48 μg KK2DP7). After 24 h of incubation, cells were harvested, excess medium was washed off, and cells were resuspended in 200 μl PBS. The proportion of fluorescent cells was then determined by flow cytometry.
[0104] The results show that the transfection efficiency of KK2DP7 in transfecting siRNA into normal cells, tumor cells, and dendritic cells can all reach over 85%. Figure 2 ).
[0105] Example 3: Detection of interference efficiency of KK2DP7 / siPD-L1 complex after uptake by tumor cells and dendritic cells (DCs)
[0106] 1. Cell processing and protein sample preparation
[0107] Tumor cells B16, Hep-1-6, and immature DCs cultured for 8 days were seeded into 6-well plates, 2 × 10⁶ cells per well. 6 Cells were cultured for 48 hours. 120 μl of either the KK2DP7 / siPD-L1 or KK2DP7 / siVEGF complex was added and incubated for 5 min in 1640 complete medium. The concentrations of siPD-L1 and siVEGF in the KK2DP7 / siPD-L1 or KK2DP7 / siVEGF complex were 0.96 μg / ml, and the concentration of KK2DP7 was 1.92 μg / ml. After culturing for another 48 h, excess medium was washed away with PBS. Cells were collected using a cell scraper and lysed on ice for 30 min using RIPA lysis buffer.
[0108] 2. BCA method for determining protein concentration
[0109] Mix 50 parts of solution A with 1 part of solution B and add 200 μl to each well of a 96-well plate. Dilute the protein standard solution serially twofold, and add 20 μl to the corresponding well for the standard curve. Add 20 μl of the diluted sample to the sample assay well. Incubate at 37°C for 30 min. Measure the OD570 value using a microplate reader. Calculate the sample protein concentration based on the standard curve.
[0110] 3. Preparation of protein samples for SDS-PAGE electrophoresis
[0111] Add an appropriate amount of SDS-PAGE loading buffer to 30 μg of protein sample, mix well, boil for 10 min, centrifuge at 13000 rpm for 10 min, and take the supernatant for later use.
[0112] 4. SDS-PAGE electrophoresis
[0113] Add the protein sample and protein marker to the corresponding wells. At room temperature and with a voltage of 80V, the protein sample moves from the stacking gel to the separating gel interface. Once the protein sample has moved from the stacking gel to the separating gel interface, the voltage is increased to 120V, and the protein sample moves to the bottom of the separating gel, at which point the electrophoresis step is complete.
[0114] 5. Transfer membrane
[0115] After electrophoresis, the separating gel and a methanol-activated PVDF membrane of similar size were arranged in the following order: blackboard-sponge-3 sheets of filter paper-gel-PVDF membrane-3 sheets of filter paper-sponge-whiteboard to form a transfer sandwich structure. The membrane was transferred at a constant voltage of 100V for 1.5 hours.
[0116] 6. Enclosed
[0117] After the transfer was completed, the PVDF membrane was placed in 5% skim milk powder prepared with PBST and incubated at 37°C for 1 hour.
[0118] 7. Incubation of primary antibody
[0119] Depending on the size of the protein being detected, place the PVDF membrane in the appropriate primary antibody dilution buffer and incubate overnight at 4°C with gentle shaking; 8. Incubate with secondary antibody.
[0120] Wash away the primary antibody with PBST three times, 15 min each time. Place in the secondary antibody dilution buffer corresponding to the primary antibody and incubate at 37°C for 1 h.
[0121] 9. Exposure
[0122] The secondary antibody was washed away with PBST three times, 15 min each time. The PVDF membrane was then wetted with a 1:1 mixture of developing and fixing solutions, and exposed and photographed using a chemical imaging system.
[0123] The results show that KK2DP7 / si-PD-L1 can significantly reduce the expression level of PD-L1 in B16. Figure 3 KK2DP7 / siPD-L1 can significantly reduce PD-L1 expression levels in DCs. Figure 4 KK2DP7 / siPD-L1 can significantly reduce the expression level of PD-L1 in Hep1-6. Figure 5 KK2DP7 / siVEGF can significantly reduce the expression level of VEGF in Hep1-6. Figure 6 ).
[0124] Example 4: Detection of lymph node targeting efficiency of the KK2DP7 / Cy5-siRNA complex
[0125] 40 μg of KK2DP7 and 20 μg of Cy5-siNC were incubated in 100 μl of PBS for 5 min and then injected into the subcutaneous lymph nodes of mice. After 24 h, proximal and distal lymph nodes of the mice were collected for fluorescence imaging, and the fluorescence intensity in the lymph nodes was detected and statistically analyzed.
[0126] The results showed that, compared with the siRNA group alone, the fluorescence intensity in both proximal and distal lymph nodes was significantly increased in the KK2DP7 / Cy5-siRNA group. Figure 7 This indicates that KK2DP7 / Cy5-siRNA can target lymph nodes.
[0127] Example 5: Detection of liver targeting efficiency of the KK2DP7 / Cy5-siRNA complex
[0128] 120 μg of DP7, DP7-C, KDP7, KK2DP7, KK2K4DP7 and 60 μg of Cy5-siRNA were incubated in 100 μl PBS for 5 min and then injected subcutaneously into the right back of mice. Major organs of the mice were collected at different time points (24 h, 9 days, 15 days, and 30 days) to observe the fluorescence intensity in each organ.
[0129] The results showed that compared with the siRNA groups alone, the fluorescence intensity in the liver was significantly increased in the KDP7 / Cy5-siRNA, KK2DP7 / Cy5-siRNA, and KK2K4DP7 / Cy5-siRNA groups, with the KK2DP7 / Cy5-siRNA group showing the greatest increase in fluorescence intensity. Figure 8 This indicates that KK2DP7 / Cy5-siRNA has the best liver-targeting effect.
[0130] Example 6: Efficacy of subcutaneous administration of KK2DP7 / siVEGF and KK2DP7 / siPD-L1 in the treatment of in situ hepatocellular carcinoma
[0131] Hep1-6 was in situ injected into the liver of 8-week-old C57 mice on day 0, with a dose of 1×10⁻⁶ per mouse. 6 On day 3, mice were subcutaneously injected with KK2DP7 (120ug) / siRNA (60ug). Mice were sacrificed on day 25, and their livers were photographed. The therapeutic effect of KK2DP7 / siRNA was evaluated. Results showed that both KK2DP7 / siVEGF and KK2DP7 / siPD-L1 inhibited the growth of orthotopic hepatocellular carcinoma in mice. Figure 9As shown in the figure, almost no tumor growth was observed in the livers of the KK2DP7 / siVEGF and KK2DP7 / siPD-L1 groups, while significant tumor growth was observed in the livers of the NS and KK2DP7 / siNC groups (white areas represent in situ liver tumors; the less white area, the fewer liver tumors). Compared to the NS and KK2DP7 / siNC groups, the number of tumor nodules in the livers of mice in the KK2DP7 / siVEGF and KK2DP7 / siPD-L1 groups was significantly reduced. Figure 9 ).
[0132] This invention provides a small nucleic acid drug delivery system based on dendritic peptides KDP7, KK2DP7, and KK2K4DP7, exhibiting good lymph node and liver targeting capabilities in the above examples. The delivery system based on these dendritic peptides can efficiently deliver small nucleic acid drugs to tumor cells, liver cells, and immune cells. KK2DP7, in particular, demonstrates superior overall performance. This delivery system successfully improves the targeting of small nucleic acid drugs to lymph nodes and the liver. In the preparation process, the effect can be achieved simply by co-incubating the dendritic peptides with the small nucleic acid drug or other drugs for an appropriate time followed by subcutaneous injection. The preparation method is simple, low-cost, and conducive to subsequent widespread use, showing great application potential.
Claims
1. The use of dendritic polypeptides in the preparation of small nucleic acid drug delivery carriers, characterized in that: The polypeptide is a DP7 polypeptide with the amino acid sequence VQWRIRVAVIRK; it is a dendritic polypeptide formed by coupling 2, 4, or 8 DP7 polypeptide molecules; the structural formula of the dendritic polypeptide is at least one of the following: 、 、 。 2. The use according to claim 1, characterized in that: The small nucleic acid is at least one of DNA or RNA; or the small nucleic acid is at least one of single-stranded or double-stranded nucleic acid.
3. The use according to claim 2, characterized in that... The size of the small nucleic acid is as follows: when it is a single-stranded small nucleic acid, the size is 19 nt to 29 nt; when it is a double-stranded small nucleic acid, the size is 19 bp to 29 bp.
4. The use according to claim 3, characterized in that: When the small nucleic acid is a single-stranded small nucleic acid, the size of the small nucleic acid is 19nt to 23nt; when the small nucleic acid is a double-stranded small nucleic acid, the size of the small nucleic acid is 19bp to 23bp.
5. The use according to claim 1, characterized in that: The small nucleic acid is one or more of antisense oligonucleotides (ASO), small interfering RNA (siRNA), miRNA, oligonucleotide aptamers, or CpG oligonucleotides.
6. The use according to any one of claims 1 to 5, characterized in that: The small nucleic acid drug delivery carrier is a lymph node-targeting or liver-targeting small nucleic acid drug delivery carrier; the structural formula of the dendritic polypeptide is as follows: 。 7. Small nucleic acid drugs, characterized in that... The product is prepared from dendritic polypeptides and small nucleic acids, wherein the polypeptide is a DP7 polypeptide with the amino acid sequence VQWRIRVAVIRK; dendritic polypeptides are formed by coupling 2, 4, or 8 DP7 polypeptide molecules; and the structural formula of the dendritic polypeptide is at least one of the following: 、 、 。 8. The small nucleic acid drug according to claim 7, characterized in that: It is prepared from dendritic polypeptides and small nucleic acids in a mass ratio of 0.1 to 20:
1.
9. The small nucleic acid drug according to claim 8, characterized in that: It is prepared from dendritic polypeptides and small nucleic acids in a mass ratio of 1 to 4:
1.
10. The small nucleic acid drug according to claim 7, characterized in that: It is prepared by co-incubating dendritic polypeptides with small nucleic acids.
11. The small nucleic acid drug according to claim 10, characterized in that: It is obtained by incubating small nucleic acids and dendritic polypeptides together in water or liquid culture medium for 5 to 15 minutes.
12. The small nucleic acid drug according to claim 7, wherein the small nucleic acid is at least one of DNA or RNA; or, the small nucleic acid is at least one of single-stranded nucleic acid or double-stranded nucleic acid.
13. The small nucleic acid drug according to claim 12, characterized in that: The size of the small nucleic acid is 19 nt to 29 nt when it is a single-stranded small nucleic acid; and 19 bp to 29 bp when it is a double-stranded small nucleic acid.
14. The small nucleic acid drug according to claim 13, characterized in that: When the small nucleic acid is a single-stranded small nucleic acid, the size of the small nucleic acid is 19nt to 23nt; when the small nucleic acid is a double-stranded small nucleic acid, the size of the small nucleic acid is 19bp to 23bp.
15. The small nucleic acid drug according to any one of claims 7 to 14, characterized in that: The small nucleic acid mentioned is one or more of antisense oligonucleotides (ASO), small interfering RNA (siRNA), miRNA, oligonucleotide aptamers, and CpG oligonucleotides.
16. The small nucleic acid drug according to claim 15, characterized in that: The small nucleic acid drugs mentioned are small nucleic acid drugs that target immune checkpoint molecules and / or cancer therapeutic targets.
17. The small nucleic acid drug according to claim 16, characterized in that: The immune checkpoint molecules are at least one of CTLA-4, PD-1, PD-L1, LAG3, TIGIT, TIM3, B7H3, CD39, CD73, adenosine A2A receptor, SIGLEC10, or CD47; or the cancer therapeutic targets are at least one of CYP1A1, p53, NQO1, ALDH2, EPHX1, STAT3, VEGF, Pin1, FGFR, or UCK2.
18. The small nucleic acid drug according to claim 16, characterized in that: The small nucleic acid drug described is an siRNA targeting the immune checkpoint molecule PD-L1, and its nucleotide sequence is as follows: sense 5'-3' CUCCAAAGGACUUGUACGUT; antisense 5'-3' ACGUACAAGUCCUUUGGAGTT.
19. The small nucleic acid drug according to any one of claims 7 to 14, characterized in that... It also contains pharmaceutically acceptable excipients or auxiliary ingredients.
20. The small nucleic acid drug according to claim 19, characterized in that... It also includes immune adjuvants.
21. The small nucleic acid drug according to claim 19, characterized in that... The small nucleic acid drug is in the form of an injection.
22. The small nucleic acid drug according to claim 19, characterized in that... The small nucleic acid drug is a subcutaneous formulation.
23. The small nucleic acid drug according to claim 22, characterized in that... The subcutaneous drug delivery formulation is a subcutaneous injection formulation or a subcutaneous embedding formulation.
24. The use of dendritic peptides in the preparation of liver-targeted small nucleic acid drug delivery carriers, characterized in that: The polypeptide is a DP7 polypeptide with the amino acid sequence VQWRIRVAVIRK; it is a dendritic polypeptide formed by coupling 2, 4, or 8 DP7 polypeptide molecules; the structural formula of the dendritic polypeptide is at least one of the following: 、 、 。 25. The use according to claim 24, characterized in that: The small nucleic acid mentioned is one or more of antisense oligonucleotides (ASO), small interfering RNA (siRNA), miRNA, oligonucleotide aptamers, and CpG oligonucleotides.
26. The use according to any one of claims 24 or 25, characterized in that: The administration route of the liver-targeted drug delivery vehicle is injection.
27. The use according to claim 26, characterized in that: The administration route of the liver-targeted drug delivery carrier is subcutaneous.
28. The use according to claim 27, characterized in that: The subcutaneous administration mentioned refers to subcutaneous injection or subcutaneous embedding.