WO2024041526A1 - 递送核酸药物的多肽载体、***的核酸药物及其制备方法 - Google Patents

递送核酸药物的多肽载体、***的核酸药物及其制备方法 Download PDF

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WO2024041526A1
WO2024041526A1 PCT/CN2023/114224 CN2023114224W WO2024041526A1 WO 2024041526 A1 WO2024041526 A1 WO 2024041526A1 CN 2023114224 W CN2023114224 W CN 2023114224W WO 2024041526 A1 WO2024041526 A1 WO 2024041526A1
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nucleic acid
nanoparticles
tumor
drug
red blood
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梁璐
余细勇
张灵敏
岑慧裕
徐文艳
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广州医科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to an MMP2 enzyme-responsive polypeptide carrier for delivering nucleic acid drugs, a nucleic acid drug for treating tumors, and a preparation method thereof.
  • Lung cancer is one of the deadliest cancers in the world, and conventional treatments such as chemotherapy, radiotherapy and surgical resection have limited efficacy and severe side effects.
  • gene therapy has become an innovative and powerful means of treating challenging diseases such as cancer.
  • miR-126-3p has potential efficacy in the treatment of lung cancer.
  • the strong negative charge of microRNAs (miRNAs) hinders their internalization in the cell membrane, and rapid enzymatic digestion in physiological environments prevents the delivery of miRNAs to required sites in the body. How to safely and effectively deliver miRNAs to the cytoplasm and release them remains a major challenge in gene regulation and therapy.
  • vectors including viral and non-viral vectors, have been developed for the delivery of nucleic acids.
  • viral vectors have shortcomings such as low loading capacity, inflammatory/immunogenic responses, and difficulty in manufacturing on a large scale.
  • non-viral vectors have received increasing attention for their application in gene delivery due to their advantages such as convenient design, low cytotoxicity, and intelligent responsiveness.
  • drugs To effectively deliver miRNAs into cancer cells, drugs must overcome three transport barriers: (1) avoid capture by the immune system and reach the lesion, (2) be effectively taken up by cancer cells, and (3) protect and release therapeutic nucleic acids. Therefore, we designed an MMP2 enzyme-responsive biomimetic nanoparticle encapsulating microRNA to improve the delivery of nucleic acid drugs to the target site.
  • Natural cell membrane camouflage nanoparticles are a new type of biomimetic nanoparticles. Simple cell membrane engineering gives nanoparticles highly complex functions. They have both the unique functions of cell membranes and the versatility of nanomaterial synthesis, making nanoparticles wrapped in cell membranes have significant It has a series of advantages, such as prolonged circulation, specific targeting and immune evasion.
  • biofilms including tumor cells, stem cells, platelets, etc. have been widely studied in the biomedical field. Red blood cells are the oxygen transporters in our body, live about 4 months, and project many surface markers.
  • the cell membrane-penetrating peptide is rich in positively charged basic amino acid short peptides, which can self-assemble with negatively charged nucleic acid drugs through electrostatic interactions under physiological conditions to form non-covalently bound nanoparticles.
  • Matrix metalloproteinase 2 (MMP2) overexpressed in cancer cells can specifically recognize and cleave the peptide sequence Pro-Leu-Gly-Leu-Ala-Gly (PLGLAG).
  • Gene therapy is one of the important means of cancer treatment, but how to safely, efficiently and feasibly deliver miRNA into the body is an urgent problem that needs to be solved in current research.
  • many vectors including viral and non-viral vectors, have been developed for the delivery of nucleic acids.
  • viral vectors have shortcomings such as low loading capacity, inflammatory/immunogenic responses, and difficulty in manufacturing on a large scale.
  • non-viral vectors used for nucleic acid delivery mainly include organic polymers, inorganic nanoparticles, etc.
  • these nucleic acid delivery vectors have been widely studied, they still have many shortcomings, such as poor delivery targeting of organic polymers and inorganic nanoparticles. It has poor biodegradability and can easily accumulate in the body, causing biological toxicity.
  • the MMP2 enzyme-responsive peptide PLGLAG can be used to construct environmentally responsive nanoparticles to improve the release of miRNAs into lung cancer cells.
  • the stability, biological targeting, and in vivo activity of the PLGLAG sequence during delivery are not ideal.
  • the purpose of the present invention is to construct a system for efficient targeted delivery of miR-126-3p mimics using MMP2 enzyme-responsive biomimetic nanoparticles.
  • the first aspect of the present invention is to provide a polypeptide carrier for delivering nucleic acid drugs, the amino acid composition of which is shown in SEQ ID NO. 3.
  • a second aspect of the present invention provides the use of the above-mentioned polypeptide carrier in delivering MicroRNA drugs.
  • a second aspect of the present invention provides nanoparticles prepared by wrapping the above-mentioned polypeptide carrier and tumor-targeting MicroRNA drugs in red blood cell membranes.
  • the tumor is an MMP2 enzyme responsive tumor.
  • the tumor is lung cancer.
  • the MicroRNA drugs are miR-126-3p mimics.
  • a fourth aspect of the present invention provides a method for preparing the above-mentioned nucleic acid drug for treating tumors, which includes the following steps:
  • Nanoparticles are formed by self-assembly
  • the mass dosage ratio of the polypeptide carrier to the tumor-targeting MicroRNA drug is 20-40:1, preferably 28-32:1.
  • the mass ratio of the red blood cell membrane to the nanoparticles is 5:1 to 30:1, preferably 8 to 12:1.
  • the present invention constructs a system for efficient targeted delivery of miR-126-3p mimics using MMP2 enzyme-responsive biomimetic nanoparticles.
  • a cationic peptide with six arginine residues: 6R-PLGLAG-6R was synthesized at both ends of the MMP2 enzyme-cleavable peptide PLGLAG.
  • the cationic peptide can controllably load the miRNA and release the miRNA under environmentally responsive conditions, which is beneficial to the treatment of diseases.
  • the inventors of the present application screened out suitable preparation parameters based on accumulated experience.
  • the 6R-PLGLAG-6R was combined with miR-126-3p mimics through electrostatic adsorption and further modified with red blood cell membranes.
  • the obtained REMAIN has the smallest particle size. diameter and maximum transfection efficiency, which is conducive to the accumulation of nanoparticles into tumor tissue to and conducive to better delivery of miR-126-3p.
  • MMP2 enzyme-responsive biomimetic nanoparticles When the MMP2 enzyme-responsive biomimetic nanoparticles are absorbed by lung cancer cells, and MMP2 exists in large amounts in the lung cancer cells, 6R-PLGLAG-6R can be cleaved and decomposed by MMP2, and the miR-126-3p mimics are released, achieving the purpose of controlled release and effective Induces cancer cell apoptosis.
  • MMP2 enzyme-responsive biomimetic nanoparticles
  • Figure 3 RT-qPCR method determines the changes in the expression level of miR-126-3p in NCI-H299 cells after transfecting miR-126-3p with different peptide vectors and Lipo3000.
  • RBCM red blood cell membrane
  • MAIN refers to: MMP2 enzyme-responsive-PLGLAG-short peptide-loaded nanoparticles with miR-126-3p mimics.
  • REMAIN refers to: RBCM modified MAIN.
  • REMAIN-NC refers to: RBCM-modified MMP2 enzyme-responsive-PLGLA-short peptide-loaded nanoparticles with miRNA-NC mimics
  • PBS solution refers to: phosphate buffer solution.
  • the present invention designs polypeptides with good biocompatibility and degradability for delivering miRNA to treat tumors.
  • the present invention relates to a method and application of delivering miR-126-3p mimics based on red blood cell membrane-modified MMP2 enzyme-responsive nanoparticles. Modification of red blood cell membranes endows nanoparticles with cell-like functions, such as specific recognition, long-term blood circulation, and immune evasion. Based on specific adhesion molecules on the red blood cell membrane, the long blood circulation and cellular internalization of nanoparticles are significantly improved. Red blood cell membranes with long blood circulation can be used as biomimetic delivery systems for cancer-targeted gene therapy, inducing efficient apoptosis of cancer cells and leading to significant tumor suppression.
  • the present invention synthesizes a cationic peptide with 6 arginine residues (6R-PLGLAG-6R) at both ends of the MMP2 enzyme-cleavable peptide PLGLAG. Subsequently, 6R-PLGLAG-6R combined with miR-126-3p mimics (MMP2 stimulated peptide/miRNA-126-3p, MAIN) through electrostatic adsorption, and further modified MAIN (Red blood cell membrane, RBCM) with membrane/MMP2 stimulated peptide/miRNA-126-3p, REMAIN).
  • 6R-PLGLAG-6R 6R-PLGLAG-6R combined with miR-126-3p mimics (MMP2 stimulated peptide/miRNA-126-3p, MAIN) through electrostatic adsorption, and further modified MAIN (Red blood cell membrane, RBCM) with membrane/MMP2 stimulated peptide/miRNA-126-3p, REMAIN).
  • the MMP2 enzyme-responsive peptide thus obtained can effectively bind to miRNA mimics, and the camouflage of RBCM gives the nanoparticles a stealth function, thereby improving circulation life, immune evasion and biological safety.
  • miR-126-3p mimics are released, effectively inducing cancer cell apoptosis.
  • Example 1 Preparation of nanoparticles MAIN and REMAIN and optimization of polypeptide sequences and ratio parameters
  • the present invention designs three polypeptides with different amino acid sequences. There are 2, 4 and 6 arginines connected to both ends of the -PLGLA- peptide segment. Their amino acid sequences are respectively:
  • nucleic acid drug is prepared as follows:
  • the mass ratio of peptides to nucleic acids is 5:1 to 90:1.
  • the results of Malvern particle size analyzer measurement show that the particle size and PDI are smallest when the ratio is 30:1 ( Figure 1C). Therefore, 6R-PLGLAG-6R:miR-126 -3p is most preferably 30:1.
  • the mass ratio of red blood cell membrane and MAIN composite nanoparticles is 5:1 to 30:10.
  • the Marr particle sizer measurement results show that the particle size begins to become saturated when the ratio is 10:1 ( Figure 1D), so RBCM:MAIN is the best Preferably, it is 10:1.
  • a smaller particle size is beneficial to the accumulation of nanoparticles into tumor tissue.
  • all MAIN and REMAIN were prepared according to the optimal proportions in this example.
  • MAIN and REMAIN were synthesized as in Example 1.
  • Transmission electron microscopy (40K, 80K magnification) showed that the MAIN nanoparticles were approximately spherical and uniform in size (Figure 2A).
  • the results measured by a Malvern particle sizer showed that the average particle size of the MAIN nanoparticles was approximately 135.5nm, and the PDI was 0.222.
  • the size of the nanoparticles was similar to The transmission electron microscopy images showed consistent results ( Figure 2B).
  • Transmission electron microscopy (40K, 80K times) shows that REMAIN nanoparticles are approximately spherical, and the modification of the red blood cell membrane increases the size of REMAIN by about 20 nm compared with MAIN ( Figure 2C).
  • the average particle size is approximately 156.7nm, and the PDI is 0.239 (Figure 2D) .
  • the total protein of non-small cell lung cancer NCI-H1299 and normal cell lung epithelial cells BEAS-2B and human umbilical cord mesenchymal stem cells hUV-MSCs is 10%. After electrophoresis on SDS-PAGE (80V, 1.5 hours), transfer to PVDF membrane (250mA, 2 hours), incubate with primary antibody probe MMP2 overnight, then incubate with peroxidase-linked secondary antibody for 1 hour, and finally use Imaging with ultra-sensitive multifunctional imager. Western blotting results showed that MMP2 was overexpressed in non-small cell lung cancer NCI-H1299 ( Figure 2F).
  • MMP2 enzyme The environmental response characteristics of MMP2 enzyme are conducive to the controlled release of drugs.
  • the miR-126-3p release experiments were performed with or without MMP2 enzyme. 1 mL of dialysate was taken at different time points (1-72 h), and then 1 mL of fresh dialysate was added. The collected dialysate was measured using a fluorescent microplate reader at 488/425nm excitation and emission wavelengths, and its release was calculated. Cumulative release analysis showed that under the action of 300 nM MMP2 enzyme, MAIN showed about 75% release within 24 h, but only showed about 30% release in PBS (Figure 2G).
  • the environmental response characteristics of MMP2 enzyme promoted the release of nanomedicines in miR-126-3p (nucleotide sequence: UCGUACCGUGAGUAAAUAAUGCG SEQ ID NO. 4) is released in a specific tumor microenvironment and plays a role.
  • SDS-PAGE protein analysis for a series of membrane protein markers showed ( Figure 2E) that the characteristic proteins inherited from the erythrocyte membrane were well retained in the protein profile of REMAIN, and the erythrocyte membrane-modified nanoparticle REMAIN showed membrane protein markers, Endowing nanoparticles with cell-like functions.
  • Example 3 Determination of miR-126-3p expression in nanomedicines MAIN and REMAIN
  • NCI-H1299 cells were seeded into a 6-well plate at 1 ⁇ 10 5 cells per well and cultured in DMEM complete medium. After 24 hours, incubate with PBS, Lipo3000/miR-126-3p, MAIN, REMAIN-NC or free miR-126-3p (miR-126-3p is equivalent to 150nM) for 48h, and determine the miR by RT-qPCR after 48h. -126-3p expression level, RT-qPCR results showed that the expression level of miR-126-3p was significantly increased in the MAIN and REMAIN groups, and the effect was equivalent to the positive control group Lipo3000 ( Figure 3).
  • Example 4 Determination of expression levels of different types of microRNA in cells after being wrapped with polypeptides and cell membranes
  • NCI-H1299 cells were seeded into a 6-well plate at 1 ⁇ 10 5 cells per well and cultured in DMEM complete medium. After 24 hours, incubate with PBS, Lipo3000/microRNAs, 6R-PLGLAG-6R/microRNAs, RBCM/6R-PLGLAG-6R/miRNA-NC or free microRNAs (microRNAs equivalent to 150nM) for 48h, and then pass RT-qPCR after 48h. The expression levels of microRNAs were measured, and the RT-qPCR results showed that the expression level of microRNA loaded with red blood cell membrane-wrapped peptides was equivalent to the positive control group Lipo3000 (Table 1).
  • MAIN, REMAIN, and REMAIN-NC were synthesized according to the optimal method in Example 1.
  • Male BALB/c nude mice were implanted with 2 ⁇ 10 6 NCI-H1299 cells on the right side. After the tumor grows to about 200mm3 .
  • tail vein Mice treated with 200 ⁇ L of normal saline, REMAIN-NC and free miR-126-3p were injected every 3 days as the control group, gefitinib (20mg/kg) was used as the positive control drug, and REMAIN and MAIN were the experimental groups (every 20 ⁇ g of miRNA) for each mouse. On the 13th day after administration, the tumor tissue was taken, photographed, and weighed.

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Abstract

提供了一种递送核酸药物的多肽载体，其氨基酸构成如SEQ ID NO.3所示。还提供了上述多肽载体的应用以及相应的***的核酸药物，以及其制备方法。所述MMP2酶响应肽能够有效地结合miRNA mimics，而RBCM赋予纳米颗粒隐身功能，从而提高循环寿命、免疫逃逸和生物安全性。当MMP2酶响应的仿生纳米颗粒被肺癌细胞吸收后，在肺癌细胞中大量存在MMP2时，miR-126-3p mimics被释放，有效诱导癌细胞凋亡。所述的***的核酸药物提供了具有巨大应用前景的肺癌治疗新策略。

Description

递送核酸药物的多肽载体、***的核酸药物及其制备方法 技术领域
本发明属于生物医药领域，具体涉及一种MMP2酶响应型递送核酸药物的多肽载体、***的核酸药物及其制备方法。
背景技术
肺癌是世界上最致命的癌症之一，其常规治疗，如化疗、放疗和手术切除等治疗效果有限并且有严重的副作用。目前，基因疗法已成为治疗癌症等具有挑战性疾病的创新和有力手段。研究表明miR-126-3p在治疗肺癌方面具有潜在的疗效。然而，由于microRNAs(miRNAs)的强负电荷阻碍了其在细胞膜的内化，并且生理环境中的快速的酶消化会阻碍miRNAs传递到体内所需的位点。如何安全有效地将miRNAs输送至胞浆并释放仍然是基因调控和治疗的主要挑战。目前，许多载体，包括病毒和非病毒载体，已经被开发用于递送核酸。然而，病毒载体具有负载能力低，炎症/免疫原性反应，难以在大规模的中制造等缺点。近年来，非病毒载体因其设计方便、细胞毒性低、具有智能化响应性等优点使其在基因传递的应用受到越来越多的关注。为了有效将miRNAs递送到癌细胞中，药物必须克服三个运输障碍:(1)避免被免疫***俘获并到达病灶，(2)被癌细胞有效吸收，(3)保护并释放治疗性核酸。故我们设计了一种包裹microRNA的MMP2酶响应型仿生纳米粒子来改善核酸药物向靶部位的传递。
天然细胞膜伪装纳米粒子是一类新的仿生纳米粒子，简便的细胞膜工程化赋予纳米颗粒高度复杂的功能，它们同时具有细胞膜的独特功能和纳米材料合成的通用性，使包裹细胞膜的纳米颗粒具有显著的优势，如具有延长循环、特异性靶向和免疫逃逸等一系列优点。目前，根据不同的需要，包括肿瘤细胞、干细胞、血小板等的生物膜已在生物医学领域得到广泛地研究。红细胞是我们体内的氧气转运体，寿命约为4个月，并能投射出许多表面标记。由于定位在红细胞膜上的各种蛋白介导，如CD47，这些蛋白具有自我识别功能，可防止免疫细胞吞噬，从而延长了仿生纳米粒子的循环寿命。多肽类药物载体是重要的 非病毒载体之一，而细胞膜穿透肽富含带正电的碱性氨基酸短肽，可在生理条件下能够与带负电的核酸药物通过静电相互作用自组装形成非共价结合的纳米粒。而癌细胞中过表达基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)可特异性识别和切割肽序列Pro-Leu-Gly-Leu-Ala-Gly(PLGLAG)。
基因治疗是癌症治疗的重要手段之一，但如何安全高效可行地将miRNA递送至体内是目前研究中亟需解决的问题。目前，许多载体，包括病毒和非病毒载体，已经被开发用于递送核酸。然而，病毒载体具有负载能力低，炎症/免疫原性反应，难以在大规模的中制造等缺点。近年来，用于核酸递送的非病毒载体主要有有机聚合物、无机纳米颗粒等，虽然这些核酸递送载体被广泛研究，但其仍存在诸多缺点，例如有机聚合物递送靶向性差，无机纳米颗粒的生物可降解性差，容易在体内蓄积导致生物毒性等。
MMP2酶响应肽PLGLAG可以用于构建环境响应的纳米颗粒，从而改善向肺癌细胞释放miRNA。然而，PLGLAG序列在运载中，其稳定性和生物靶向性的以及体内活性都不理想。
发明内容
基于此，本发明的目的是构建一种MMP2酶响应型的仿生纳米颗粒高效靶向递送miR-126-3p mimics的体系。
实现上述目的的技术方案如下。
本发明的第一个方面，是提供一种递送核酸药物的多肽载体，其氨基酸构成如SEQ ID NO.3所示。
本发明的第二方面，是提供上述的多肽载体在递送MicroRNA药物中的应用。
本发明的第二方面，是提供由红细胞膜包裹上述的多肽载体和靶向肿瘤的MicroRNA药物形成的纳米粒制备而成。
在其中一些实施例中，所述肿瘤是MMP2酶响应型肿瘤。
在其中一些实施例中，所述肿瘤是肺癌。
在其中一些实施例中，所述MicroRNA药物为miR-126-3p mimics。
本发明的第四个方面，是提供上述***的核酸药物的制备方法，包括以下步骤：
(1)获得红细胞膜；
(2)将所述多肽载体溶解于PBS，得到多肽载体溶液；
(3)将靶向肿瘤的MicroRNA药物溶解于无核酶水，得到MicroRNA药物溶液；
(4)按照多肽载体与所述靶向肿瘤的MicroRNA药物的质量比例为20-40：1的比例，将多肽载体溶液与核酸药物溶液混合均匀，室温静置，使核酸与多肽通过正负电自组装的方式形成纳米粒；
(5)在所得纳米粒中加入所述红细胞膜，混合均匀，将制得的纳米颗粒通过梯度挤压得到粒径均一的纳米粒，获得核酸药物。
在其中一些实施例中，所述多肽载体与所述靶向肿瘤的MicroRNA药物的质量用量比例为20-40：1，优选为28-32：1。
在其中一些实施例中，所述红细胞膜与纳米粒的质量用量比为5:1～30:1，优选为8-12:1。
为了有效地将miRNAs有效递送到癌细胞中，药物必须克服三个运输障碍:(1)避免被免疫***俘获并到达病灶，(2)被癌细胞有效吸收，(3)保护并释放治疗性核酸。故本发明构建了一种MMP2酶响应型的仿生纳米颗粒高效靶向递送miR-126-3p mimics的体系。首先，在MMP2酶可切割肽PLGLAG两端合成了6个精氨酸残基的阳离子肽:6R-PLGLAG-6R。该阳离子肽可控地负载所述miRNA，并可在环境响应的条件下，释放所述miRNA，有利于疾病的治疗。其次，本申请发明人根据积累的经验，筛选到合适的制备参数，所述6R-PLGLAG-6R通过静电吸附结合miR-126-3p mimics，进一步用红细胞膜进行修饰，获得的REMAIN具有最小的粒径和最大转染效率，有利于纳米颗粒向肿瘤组织蓄积以 及有利于miR-126-3p更好的递送。当MMP2酶响应的仿生纳米颗粒被肺癌细胞吸收后，在肺癌细胞中大量存在MMP2时，6R-PLGLAG-6R可被MMP2切割分解，miR-126-3p mimics被释放，达到控释的目的，有效诱导癌细胞凋亡。然而，这类纳米颗粒用于肺癌基因药物的递送还未见报道。本发明探索和发展了一种具有巨大前景的肺癌治疗新策略。
附图说明
图1纳米药物MAIN、REMAIN的制备条件的对比。
图2纳米药物MAIN、REMAIN的表征。
图3 RT-qPCR法测定使用不同多肽载体及Lipo3000转染miR-126-3p后NCI-H299细胞中miR-126-3p表达水平变化。
图4纳米药物MAIN、REMAIN的体内抑瘤效果。
图5主要器官切片的HE染色结果。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是 为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
在阐述本发明的技术方案之前，定义本文中所使用的术语如下：
术语“RBCM”是指：红细胞膜。
术语“MAIN”是指：MMP2酶响应的-PLGLAG-短肽负载miR-126-3p mimics的纳米颗粒。
术语“REMAIN”是指：RBCM修饰的MAIN。
术语“REMAIN-NC”是指：RBCM修饰的MMP2酶响应的-PLGLA-短肽负载miRNA-NC mimics的纳米颗粒
术语“PBS溶液”是指：磷酸盐缓冲溶液。
本发明设计了生物生物相容性好、可降解的多肽，用于递送miRNA***。本发明涉及一种基于红细胞膜修饰的MMP2酶响应纳米颗粒递送miR-126-3p mimics的方法和应用。红细胞膜的修饰赋予纳米颗粒类似细胞的功能，例如特异性识别，长期血液循环和免疫逃逸。基于红细胞膜上的特异性粘附分子，纳米粒子的血液长循环和细胞内化明显改善。具有血液长循环的红细胞膜可以用作癌症靶向基因治疗的仿生递送***，诱导癌细胞的高效凋亡，并导致显着的肿瘤抑制。
本发明在MMP2酶可切割肽PLGLAG两端合成了6个精氨酸残基的阳离子肽(6R-PLGLAG-6R)。随后，6R-PLGLAG-6R通过静电吸附结合miR-126-3p mimics(MMP2 stimulated peptide/miRNA-126-3p,MAIN)，进一步用红细胞膜(Red blood cell membrane，RBCM)进行修饰MAIN(Red blood cell membrane/MMP2 stimulated peptide/miRNA-126-3p,REMAIN)。由此获得的MMP2酶响应肽能够有效地结合miRNA mimics，而RBCM的伪装赋予纳米颗粒隐身功能，从而提高循环寿命、免疫逃逸和生物安全性。当REMAIN被肺癌 细胞吸收后，在肺癌细胞中大量存在MMP2时，miR-126-3p mimics被释放，有效诱导癌细胞凋亡。
以下结合具体实施例对本发明作进一步详细的说明。
实施例1：纳米粒MAIN、REMAIN的制备及多肽序列及配比参数的条件优化
本发明设计了三条不同氨基酸序列的多肽，分别是在-PLGLA-肽段两端接有2个、4个和6个精氨酸，其氨基酸序列分别为：
2R-PLGLAG-2R：RRPLGLAGRR(SEQ ID NO.1)、
4R-PLGLAG-4R：RRRRPLGLAGRRRR(SEQ ID NO.2)、
6R-PLGLAG-6R：RRRRRRPLGLAGRRRRRR(SEQ ID NO.3)。
本实施例中，所述核酸药物的制备如下：
(1)红细胞膜的提取:通过2000rpm离心5分钟从全血中提取红细胞，并用红细胞裂解液进行裂解，在4℃进一步以14000rpm离心15分钟，将沉淀物重新溶解在含EDTA的去离子水中，超声处理3min，然后在4℃下以14000rpm离心15分钟。将该过程重复两次。最后，将获得的红细胞膜用含有EDTA的去离子水洗涤两次以除去血红蛋白。
(2)三种多肽载体分别溶解于PBS，制成1μg/μL的多肽溶液。将miR-126-3p mimics溶解于无核酶水，制成1μg/μL的核酸溶液。分别按照核酸:多肽＝1:5、1:10、1:20、1:30、1:40、1:50、1:60、1:90的质量比例将核酸溶液加入多肽溶液中，立刻进行涡旋震荡1min，使核酸与多肽充分混合均匀。室温静置15min，使核酸与多肽通过正负电自组装的方式形成纳米粒MAIN。
(3)将MAIN：RBCM＝1:5、1:10、1:20、1:30质量比将步骤(1)所得的RBCM分别加入步骤(2)所得的MAIN中，将混合物通过挤膜器梯度挤压得到粒径均一的REMAIN，即先将该混合物使用挤膜器挤压通过孔径为1μm碳酸脂膜，来回挤压过膜二十次，再以相同方法依次挤压通过孔径分别为400nm、200nm、100nm的碳酸脂膜。
(4)将制备的纳米药物进行适当稀释之后，使用马尔文粒径仪测定各组纳 米粒的粒径和多分散系数(polydispersity index,PDI)。
(5)分别将10μL各组纳米粒样品滴加至电镜铜网，静置5min后，使用吸水纸沿铜网边缘吸除多余液体，将铜网置于室温过夜晾干。按照透射电镜操作规程拍摄各组样品的电镜照片，以获知各样品纳米粒形态特征及粒径大小。
马尔为粒径仪测定结果显示6R-PLGLAG-6R整体上较其它肽序列粒径更小(图1A)，并且qRT-PCR结果表明6R-PLGLAG-6R转染miR-126-3p效率较其它多肽序列更高，并且与阳性对照组转染效率相当(图1B)，这是因为精氨酸数量的增加导致了多肽中氨基酸或亚胺的增加，增加了电荷密度，并改善了负电荷miR-126-3p的致密性，故多肽序列最优选为6R-PLGLAG-6R。更高的转染效率有利于miR-126-3p更好的递送。多肽与核酸的质量配比为5:1～90:1，马尔为粒径仪测定结果显示比例为30:1时粒径和PDI最小(图1C)，因此6R-PLGLAG-6R:miR-126-3p最优选为30:1。红细胞膜与MAIN复合纳米颗粒的质量配比为5:1～30:10，马尔为粒径仪测定结果显示比例为10:1时粒径开始趋于饱和(图1D)，故RBCM：MAIN最优选为10:1，更小的粒径有利于纳米颗粒向肿瘤组织蓄积。以下实施例中，制备的所有的MAIN、REMAIN都按照本实施例中最优配比的方式。
实施例2：纳米粒MAIN、REMAIN的表征
如实施例1方法合成MAIN、REMAIN。透射电镜(40K、80K倍)显示MAIN纳米粒近似于球形且大小均一(图2A)，马尔为粒径仪测定结果显示MAIN纳米粒平均粒径约为135.5nm，PDI为0.222，纳米粒大小与透射电镜图显示结果一致(图2B)。透射电镜(40K、80K倍)显示REMAIN纳米粒近似于球形，并且红细胞膜的修饰使REMAIN尺寸较MAIN增加了约20nm(图2C)，平均粒径约为156.7nm，PDI为0.239(图2D)。非小细胞肺癌NCI-H1299与正常细胞肺上皮细胞BEAS-2B、人脐带间充质干细胞hUV-MSCs等量的总蛋白在10％ SDS-PAGE上电泳(80V，1.5小时)后，转移到PVDF膜(250mA,2小时)，并与一抗探针MMP2孵育过夜后再与过氧化物酶连接的二抗孵育1小时，最后用超灵敏多功能成像仪成像。Western blotting结果表明MMP2在非小细胞肺癌NCI-H1299中过表达(图2F)，MMP2酶环境响应特性有利于药物的控释。首先制备含有FITC-miR-126-3p的REMAIN并装入透析膜(MW＝3500)中。miR-126-3p释放实验分别在有或没有MMP2酶的情况下进行，在不同时间点(1-72h)取1mL透析液，然后加入1mL新鲜透析液。采用荧光酶标仪在488/425nm激发和发射波长下测量收集的透析液，并计算其释放量。累积释放分析表明，在300nM MMP2酶的作用下，MAIN在24h内显示约75％的释放，但在PBS中仅显示约30％的释放(图2G)，MMP2酶环境响应特征促进了纳米药物在特定的肿瘤微环境内释放miR-126-3p(核苷酸序列为：UCGUACCGUGAGUAAUAAUGCG SEQ ID NO.4)并发挥作用。针对一系列膜蛋白标记物的SDS-PAGE蛋白分析表明(图2E)，从红细胞膜继承的特征蛋白在REMAIN的蛋白谱中具有良好的保留，红细胞膜修饰的纳米颗粒REMAIN显示了膜蛋白标记，赋予纳米颗粒具有细胞样功能。
实施例3：纳米药物MAIN、REMAIN中miR-126-3p表达量的测定
将NCI-H1299细胞接种到6孔板中，每孔1×105个细胞，在DMEM完全培养基中培养。24小时后，分别用PBS、Lipo3000/miR-126-3p、MAIN、REMAIN-NC或游离miR-126-3p(miR-126-3p等效于150nM)孵育48h，48h后通过RT-qPCR测定miR-126-3p表达水平，RT-qPCR结果表明MAIN及REMAIN组中miR-126-3p表达水平显著升高，并且与阳性对照组Lipo3000效果相当(图3)。
实施例4：多肽及细胞膜包裹后细胞中不同类型microRNA表达量的测定
将NCI-H1299细胞接种到6孔板中，每孔1×105个细胞，在DMEM完全培养基中培养。24小时后，分别用PBS、Lipo3000/microRNAs、6R-PLGLAG-6R/microRNAs、RBCM/6R-PLGLAG-6R/miRNA-NC或游离microRNAs(microRNAs等效于150nM)孵育48h，48h后通过RT-qPCR测定microRNAs表达水平，RT-qPCR结果表明红细胞膜包裹多肽负载microRNA表达水平与阳性对照组Lipo3000效果相当(表1)。
表1红细胞膜包裹多肽负载microRNA表达水平
所有组与RBCM/6R-PLGLAG-6R/microRNAs进行统计学分析比较，*P<0.05,**P<0.01,***P<0.001。实施例5：纳米颗粒在体内的抗肿瘤作用
如实施例1中最优方法合成MAIN、REMAIN、REMAIN-NC。雄性BALB/c裸鼠右侧植入2×106个NCI-H1299细胞。待肿瘤生长至约200mm3后。尾静脉 每隔3天分别注射200μL生理盐水、REMAIN-NC和游离miR-126-3p处理的小鼠作为对照组，吉非替尼(20mg/kg)作为阳性对照药，REMAIN、MAIN为实验组(每只小鼠20μg miRNA)，给药的第13天取肿瘤组织拍照、称重。从肿瘤图片(图4A)、重量(图4B)和肿瘤生长曲线(图4C)可以看出REMAIN-NC或游离miR-126-3p处理的小鼠较生理盐水组没有显著差异，而REMAIN、MAIN和吉非替尼均能显著抑制肿瘤生长。与MAIN组相比，REMAIN组表现出更好的抗肿瘤效果，类似于吉非替尼治疗组。上述结果表明了REMAIN通过向肿瘤组织递送miR-126-3p有效抑制肿瘤的生长。
并且苏木精-伊红染色结果表明，所有处理均未引起小鼠主要器官的明显组织学改变，对小鼠无明显毒性(图5)，说明本发明所述MMP2酶响应的仿生纳米颗粒载体具有良好的生物安全性。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (12)

  1. 一种递送核酸药物的多肽载体，其特征在于，其氨基酸构成如SEQ ID NO.3所示。
  2. 权利要求1所述的多肽载体在制备递送MicroRNA药物中的应用。
  3. 一种***的核酸药物，其特征在于，由红细胞膜包裹权利要求1所述的多肽载体和靶向肿瘤的MicroRNA药物形成的纳米粒制备而成。
  4. 根据权利要求3所述的核酸药物，其特征在于，所述肿瘤是MMP2酶响应型肿瘤。
  5. 根据权利要求4所述的核酸药物，其特征在于，所述肿瘤是肺癌。
  6. 根据权利要求3所述的核酸药物，其特征在于，所述MicroRNA药物为miR-126-3p mimics。
  7. 根据权利要求3所述的核酸药物，其特征在于，所述多肽载体与所述靶向肿瘤的MicroRNA药物的质量用量比例为20-40：1。
  8. 根据权利要求7所述的核酸药物，其特征在于，所述多肽载体与所述靶向肿瘤的MicroRNA药物的质量用量比例为28-32：1。
  9. 根据权利要求3-8任一项所述的核酸药物，其特征在于，所述红细胞膜与纳米粒的质量用量比为5:1～30:1。
  10. 根据权利要求9所述的核酸药物，其特征在于，所述红细胞膜与纳米粒的质量用量比为8-12:1。
  11. 权利要求3所述***的核酸药物的制备方法，其特征在于，包括以下步骤：
    (1)获得红细胞膜；
    (2)将所述多肽载体溶解于PBS，得到多肽载体溶液；
    (3)将靶向肿瘤的MicroRNA药物溶解于无核酶水，得到MicroRNA药物溶液；
    (4)按照多肽载体与所述靶向肿瘤的MicroRNA药物的质量比例为20-40：1的比例，将多肽载体溶液与核酸药物溶液混合均匀，室温静置，使核酸与多肽通过正负电自组装的方式形成纳米粒；
    (5)在所得纳米粒中加入所述红细胞膜，混合均匀，将制得的纳米颗粒通过 梯度挤压得到粒径均一的纳米粒，获得核酸药物。
  12. 根据权利要求11所述的制备方法，其特征在于，所述红细胞膜与纳米粒的质量用量比为5:1～30:1。
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