WO2021027425A1 - 一种抗炎靶向递送系统及其制备方法 - Google Patents

一种抗炎靶向递送系统及其制备方法 Download PDF

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WO2021027425A1
WO2021027425A1 PCT/CN2020/099625 CN2020099625W WO2021027425A1 WO 2021027425 A1 WO2021027425 A1 WO 2021027425A1 CN 2020099625 W CN2020099625 W CN 2020099625W WO 2021027425 A1 WO2021027425 A1 WO 2021027425A1
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platelet
inflammatory
delivery system
targeted delivery
platelets
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汪超
麻庆乐
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苏州大学
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the invention relates to an anti-inflammatory targeted delivery system and a preparation method thereof, and belongs to the technical field of pharmaceutical carrier preparation.
  • Atherosclerosis is the pathological basis of cardiovascular disease and is a chronic inflammatory disease characterized by local immune response dysfunction.
  • the pathogenesis of atherosclerosis is still controversial and unknown.
  • traditional treatments for atherosclerotic diseases include drugs to lower cholesterol and blood clotting, or surgical procedures such as percutaneous coronary intervention.
  • these medical treatments have adverse effects, and they mainly focus on relieving symptoms rather than reducing the underlying atherosclerosis. Therefore, more effective anti-atherosclerotic strategies need to be developed.
  • vascular inflammation plays an important role in atherosclerosis.
  • Vascular inflammation has been confirmed to be closely related to the acceleration of atherosclerotic complications.
  • downstream biomarkers of pro-inflammatory signaling pathways such as signaling pathway protein 3 (NLRP3) containing pyrin domain, high-sensitivity C-reactive protein and interleukin-1 ⁇ (IL-1 ⁇ ) are closely related to increased cardiovascular risk, and Cholesterol level is irrelevant.
  • NLRP3 signaling pathway protein 3
  • IL-1 ⁇ interleukin-1 ⁇
  • Canakinumab is a human anti-IL-1 ⁇ monoclonal antibody that can neutralize IL-1 ⁇ signaling. It has shown low cardiovascular (CV) events in clinical trials, opening a new era of anti-inflammatory treatment for atherosclerosis .
  • the disadvantages of the existing method for platelet membrane encapsulation of PLGA include cumbersome extraction and separation steps, the need to extract and separate the platelet membrane and then modify the surface of the polymer PLGA nanoparticles, the cumbersome and complicated steps, and the high cost, which limit its further clinical application .
  • the present invention provides an anti-inflammatory targeted delivery system, which is prepared from platelets, does not contain any synthetic polymers or other materials, and has high biological safety.
  • Platelet-derived vesicles can be obtained by a one-step method of activated platelets, with simple separation process, simple operation steps, and wide sources.
  • platelets have an inherent affinity for inflammatory sites, including atherosclerotic plaques, and platelet-derived targeted delivery systems can selectively bind to multiple cell types, such as macrophages and atherosclerotic plaques. Relevant endothelial cells, therefore, using this targeted delivery system can deliver anti-inflammatory drugs to the site of inflammation.
  • the first object of the present invention is to provide an anti-inflammatory targeted delivery system.
  • the anti-inflammatory targeted delivery system includes platelet-derived vesicles and loaded in the platelet-derived vesicles or attached to the platelets. Derive anti-inflammatory drugs on the surface of vesicles, and the platelet-derived vesicles are obtained by ultracentrifugation activated platelets.
  • the particle size of the platelet-derived vesicles is 100-10000 nm.
  • the anti-inflammatory drug is a hydrophilic or hydrophobic small molecule anti-inflammatory drug.
  • small molecule compounds are active small molecule drugs.
  • the anti-inflammatory drug is one or a combination of MCC950, auranofin, TAK-242, bromoxone, and curcumin.
  • the platelets are derived from autologous or donors with the same blood type.
  • the second object of the present invention is to provide a method for preparing the anti-inflammatory targeted delivery system, which includes the following steps:
  • step (2) Mix the anti-inflammatory drugs with the platelets obtained in step (1) uniformly, and then perform platelet activation;
  • step (3) Centrifuge the activated platelets in step (2) at 600-1000g for 15-25 minutes, and collect the supernatant;
  • step (3) The supernatant collected in step (3) is subjected to ultracentrifugation at a rotation speed of at least 2 500 g, and the precipitate is collected to obtain the anti-inflammatory drug targeted delivery system.
  • step (1) it also includes the step of purifying the platelets: centrifuge the supernatant collected in step (1) at 600-1000g, collect the platelet pellet, and add a buffer solution containing a platelet anticoagulant inhibitor to wash After washing, centrifuge at 600-1000g to collect the precipitate to purify platelets. When in use, use buffer to resuspend.
  • the platelet activation is activated by adding a platelet activation inducer.
  • the platelet activation inducer includes one or a combination of thrombin, collagen, thrombin sensitive protein, ADP, and thromboxane A2.
  • step (4) the time of ultracentrifugation is at least 30 min.
  • one or more platelet inhibitors are provided to the sample, and at least one of the platelet inhibitors is prostaglandin H1.
  • step (3) the anti-inflammatory drug and purified platelets are mixed in a ratio higher than 1:1.
  • the buffer is PBS buffer.
  • the anti-inflammatory targeted delivery system of the present invention is prepared from platelets. Since platelets are derived from organisms, platelets can be completely biodegraded, participate in the metabolism of the body, and have no toxic side effects. Therefore, platelet-derived vesicles derived from platelets also have good biocompatibility. The obtained platelet-derived vesicles do not contain any artificially synthesized polymers or other materials, and the anti-inflammatory targeted delivery system of the present invention does not require the use of heterologous or artificial synthetic materials such as polylactic glycolic acid (PLGA) as anti-inflammatory Targeted delivery vehicle with high biological safety.
  • PLGA polylactic glycolic acid
  • the process of the present invention is simple, without the need to extract platelet membranes, platelet-derived vesicles are very easy to obtain, platelets can be derived from the patient itself or from donors with the same blood type, platelet-derived vesicles can be obtained by a one-step method of activated platelets, and the separation process is simple , The operation steps are simple.
  • platelets have an inherent affinity for inflammatory sites, including cardiovascular diseases such as atherosclerotic plaques, and platelet-derived targeted delivery systems can selectively bind a variety of inflammatory-activated cell types, such as macrophages and Atherosclerotic plaques form related endothelial cells, so this targeted delivery system can deliver anti-inflammatory drugs to the site of inflammation.
  • Figure 1 is a microscopic view of the platelet-derived vesicle of the present invention
  • Figure 2 is a particle size distribution diagram of platelet-derived vesicles of the present invention.
  • Figure 3 is a graph showing the particle size changes of platelet-derived vesicles of the present invention in different solutions
  • Figure 4 is an electrophoretic analysis diagram of platelet-derived vesicles and platelets of the present invention.
  • Figure 5 is a Western Blot result diagram of platelet-derived vesicles and platelets of the present invention.
  • Figure 6 is a diagram of the inflammatory factors secreted by platelet-derived vesicles and platelets of the present invention.
  • Figure 7 is a diagram of platelet-derived vesicles targeting macrophages of the present invention.
  • Figure 8 is a diagram of platelet-derived vesicles targeting endothelial cells of the present invention.
  • Figure 9 is an imaging diagram of the platelet-derived vesicles of the present invention targeting small animals in vivo;
  • Fig. 10 is a confocal view of a tissue section of the present invention.
  • Figure 11 is an ultraviolet absorption graph of the anti-inflammatory composition MCC950 of the present invention.
  • Figure 12 is the sustained release curve of the anti-inflammatory composition MCC950 of the present invention.
  • Figure 13 is a graph showing the expression of IL-1 ⁇ on macrophages by the anti-inflammatory composition of the present invention.
  • Figure 14 is a graph showing the expression of IL-1 ⁇ on endothelial cells by the anti-inflammatory composition of the present invention.
  • Figure 15 is a diagram showing the expression of NLRP3 on macrophages by the anti-inflammatory composition of the present invention.
  • Figure 16 is a diagram showing the expression of NLRP3 on endothelial cells by the anti-inflammatory composition of the present invention.
  • Figure 17 is an HE staining diagram showing the effect of the anti-inflammatory composition of the present invention on the size of mouse aortic arch plaque;
  • Figure 18 is an immunohistochemical staining diagram of IL-1 ⁇ of aortic arch plaque by the anti-inflammatory composition of the present invention.
  • Figure 19 is an immunohistochemical staining diagram of Mac3 of aortic arch plaque by the anti-inflammatory composition of the present invention.
  • Figure 20 is a graph showing the expression of blood inflammatory factors by the anti-inflammatory composition of the present invention.
  • mice and ApoE-KO mice aged 6-8 weeks were purchased from Changzhou Cavins Laboratory Animal Co., Ltd. The mice were processed in accordance with the guidelines of the Laboratory Animal Management Committee (IACUC) of the Institute of Biochemistry and Cell, Chinese Academy of Sciences.
  • IACUC Laboratory Animal Management Committee
  • Mouse macrophages RAW264.7 and endothelial cells HUVECs were purchased from the American Type Culture Collection (ATCC). RAW264.7 and HUVECs were stored in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100U/ml streptomycin. The third or fourth generation subcultured cells were used in each embodiment of the present invention.
  • Blood was taken from the orbit of C57BL/6 mice and stored in PBS containing 5mM EDTA and 1 ⁇ M PGE1. Centrifuge at 100g for 15 minutes to remove red blood cells, repeat the steps until the supernatant is clear, collect the supernatant and centrifuge at 800g for 20 minutes; store the centrifuged pellet at room temperature for further use after resuspension.
  • TEM transmission electron microscopy
  • Figure 1 The results are shown in Figure 1, with a diameter of about 100-150 nm.
  • LDS dynamic light scattering
  • Figure 2 and Figure 3 Stored in various buffers at 4°C for at least 4 days .
  • PGE1 the size of PVs did not change significantly, indicating the high stability of PVs.
  • SDS-PAGE gel electrophoresis was used to analyze the platelets and platelet-derived vesicle proteins.
  • Figure 4 From this, it can be concluded that platelet-derived vesicles contain platelet-derived vesicles. Some proteins, however, lost high molecular weight cytoplasmic proteins such as actin and myosin.
  • IL-1 ⁇ and IL-6 in the supernatant activated by thrombin was detected by enzyme-linked immunosorbent assay. The results are shown in Fig. 6 that platelet-derived vesicles do not secrete pro-inflammatory factors, indicating that platelet-derived vesicles do not produce side effects on the body when used as anti-inflammatory targeted delivery vehicles, and have good development prospects as drug carriers for inflammation treatment.
  • Example 4 Targeting analysis of platelet-derived vesicles
  • the platelets were stained with DiD, and DiD-labeled platelet-derived vesicles were collected after activation.
  • the macrophages were treated with oxidized LDL to convert them into foam cells, and then the foam cells were incubated with DiD-labeled platelets or platelet-derived vesicles PVs.
  • the results are shown in Figure 7. It was observed by fluorescence imaging that platelet-derived vesicle PVs showed a higher affinity for foam cells compared with non-activated macrophages.
  • DiD-labeled platelets or platelet-derived vesicles PVs were incubated with LPS-treated human umbilical vein endothelial cells. The results are shown in Fig. 8.
  • Free DiD or DiD-labeled platelet-derived vesicle PVs were injected intravenously into ApoE-KO mice. Six hours after the injection, the mice were euthanized. The heart and aorta are removed for ex vivo imaging. The results are shown in FIG. 9, DiD-labeled platelet-derived vesicles significantly accumulated in mouse atherosclerotic lesions in ApoE-KO mice. In contrast, little signal was detected in ApoE-KO mice treated with free dye. In addition, the accumulation of PVs in the aorta was not shown in wild-type mice, indicating that the targeting ability is caused by the affinity between PVs and activated macrophages or endothelium.
  • the anti-inflammatory drug MCC950 was mixed with purified platelets and incubated for 24 hours.
  • the precipitate was obtained by centrifugation at 800g for 20 minutes.
  • the precipitate was platelets loaded with MCC950. Resuspend the precipitate and add 2U/mL thrombin to activate it at 37°C. After 30 minutes, centrifuge at 800g for 20 minutes, collect the supernatant and perform ultracentrifugation to obtain platelet-derived vesicles loaded with MCC950, and concentrate them into particles at a rate of 100,000 rpm (2 hours), which is an anti-inflammatory targeted delivery system.
  • the ultraviolet absorption peak of the anti-inflammatory composition was measured by an ultraviolet absorber. As shown in FIG. 11, the characteristic absorption peak of MCC950 in the absorption spectrum indicated that the anti-inflammatory drug MCC950 was successfully loaded on the platelet-derived vesicles.
  • the loading and release curves of MCC950 were measured by high performance liquid chromatography (HPLC). As shown in Figure 12, the drug loading percentage was about 15.3%, and the drug release curve showed sustained release.
  • Example 7 Analysis of the anti-inflammatory effect of the anti-inflammatory composition
  • ApoE-KO mice were fed high-fat diet for twelve weeks, and mice fed with normal diet were used as untreated controls.
  • Mice fed with high-fat diet received 9 intravenous infusions of PBS, anti-inflammatory drug MCC950 or anti-inflammatory composition MCC950-PVs within 21 days, and the dose of MCC950 administered for each infusion was 5 mg/kg. All mice were euthanized 24 hours after the final infusion.
  • the mouse aortic arch was dissected, and the plaques in the mouse aortic arch after various treatments were analyzed quantitatively.
  • the cross-section of the atherosclerotic plaque was immunostained with hematoxylin and eosin (H&E), and IL-1 ⁇ , Mac3 (macrophages).

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Abstract

一种抗炎靶向递送系统及其制备方法。该抗炎靶向递送系统包括血小板衍生囊泡和装载在血小板衍生囊泡内或附着于血小板衍生囊泡表面的抗炎药物，血小板衍生囊泡通过超速离心活化后的血小板获得。该抗炎靶向递送系统由血小板制备得到，不包含任何人工合成高分子或其他材料，生物安全性高。血小板衍生囊泡可通过活化血小板一步法获得，分离工艺简单，操作步骤简单，并且来源广泛。此外，血小板对炎症部位具有内在的亲和力，包括动脉粥样硬化斑块，而血小板来源的靶向递送系统可以选择性地结合多种细胞类型，如巨噬细胞和与动脉粥样硬化斑块形成相关的内皮细胞，因而利用此靶向递送系统能够将抗炎药物递送至炎症部位。

Description

一种抗炎靶向递送系统及其制备方法 技术领域
本发明涉及一种抗炎靶向递送系统及其制备方法，属于药物载体制备技术领域。
背景技术
心血管疾病(Cardiovascular disease，CVD)仍然是死亡率和发病率的主要原因。动脉粥样硬化(Atherosclerosis，AS)是心血管疾病的病理基础，是一种慢性炎症性疾病，其特征是局部免疫反应功能障碍。动脉粥样硬化的发病机制仍存在争议和未知因素。在临床实践中，动脉粥样硬化疾病的传统治疗包括降低胆固醇和凝血的药物，或经皮冠状动脉介入治疗等外科手术。然而，这些医学治疗存在不良反应，且主要集中在缓解症状而不是减少潜在的动脉粥样硬化，因此，需要开发更有效的抗动脉粥样硬化策略。
最近的研究表明，炎症在动脉粥样硬化中起重要作用。血管炎症已被证实与动脉粥样硬化并发症的加速密切相关。例如，促炎信号通路的下游生物标志物，如含有pyrin结构域的信号通路蛋白质3(NLRP3)，高敏C反应蛋白和白细胞介素-1β(IL-1β)与心血管风险增加密切相关，与胆固醇水平无关。2017年，Canakinumab是人类抗IL-1β单克隆抗体可中和IL-1β信号，在临床试验中已显示出较低的心血管(CV)事件，开创了抗炎治疗动脉粥样硬化的新时代。尽管有前景，但仍需改善疗效，同时减少副作用。针对炎症进行治疗仍然是一大挑战，仍需要进一步开发用于调节动脉粥样硬化局部炎性微环境的靶向递送系统。
目前已有多个用于动物模型的抗炎靶向递送系统的报道，如Thijs J.Beldman等人用透明质酸作为抗炎靶向递送系统，通过使用透明质酸纳米粒来 探讨动脉粥样硬化相关的炎症，改善了动脉粥样硬化。Diana Dehaini等人用红细胞膜和血小板膜包PLGA来作为抗炎靶向递送系统。由此可见，目前绝大多数的抗炎靶向递送系统采用合成材料，或需要进行化学修饰，工艺相对复杂，在体内参与的生物化学反应和降解产物往往不够明确，这些都是临床转化面临的挑战。因此，对人体健康的潜在风险较大。因此，将利用化学物质改性或缔合的抗炎靶向系统用于人体时，其使用的安全性是最大的问题。
此外，现有的血小板膜包PLGA的方法的缺陷包括提取分离步骤繁琐，需要提取、分离血小板膜之后再修饰到高分子PLGA纳米颗粒表面，步骤繁琐复杂，成本高昂，限制了其进一步的临床应用。
发明内容
为解决上述技术问题，本发明提供一种抗炎靶向递送系统，其由血小板制备得到，不包含任何人工合成高分子或其他材料，生物安全性高。血小板衍生囊泡可通过活化血小板一步法获得，分离工艺简单，操作步骤简单，并且来源广泛。此外，血小板对炎症部位具有内在的亲和力，包括动脉粥样硬化斑块，而血小板来源的靶向递送系统可以选择性地结合多种细胞类型，如巨噬细胞和与动脉粥样硬化斑块形成相关的内皮细胞，因而利用此靶向递送系统能够将抗炎药物递送至炎症部位。
本发明的第一个目的是提供一种抗炎靶向递送系统，所述的抗炎靶向递送系统包括血小板衍生囊泡和装载在所述的血小板衍生囊泡内或附着于所述的血小板衍生囊泡表面的抗炎药物，所述的血小板衍生囊泡通过超速离心活化后的血小板获得。
进一步地，所述的血小板衍生囊泡的粒径为100-10000nm。
进一步地，所述的抗炎药物为亲水性或疏水性的小分子抗炎药物。在一些情况下，小分子化合物为活性小分子药物。
进一步地，所述的抗炎药物为MCC950、auranofin、TAK-242、bromoxone、姜黄素中的一种或一种以上组合。
进一步地，所述的血小板来源于自体或血型一致的捐献者。
本发明的第二个目的是提供所述的抗炎靶向递送系统的制备方法，包括如下步骤：
(1)将含有血小板的血液样品分散于含有血小板凝集抑制剂的缓冲液中，在50-150g离心至上清液澄清，去除红细胞和白细胞，获得只含有血小板的上清液；
(2)将抗炎药物与步骤(1)得到的血小板混合均匀，然后进行血小板活化；
(3)将步骤(2)活化后的血小板在600-1000g离心15-25min，收集上清液；
(4)将步骤(3)收集的上清液以至少2 500g转速进行超速离心，收集沉淀，得到所述的抗炎药物靶向递送系统。
进一步地，在步骤(1)之后还包括对血小板进行纯化的步骤：将步骤(1)收集的上清液在600-1000g离心，收集血小板沉淀，并加入含有血小板抗凝抑制剂的缓冲液洗涤，洗涤后在600-1000g离心，收集沉淀，为纯化血小板。在使用时，采用缓冲液进行重悬。
进一步地，所述的血小板活化通过添加血小板活化诱导剂活化。
进一步地，所述的血小板活化诱导剂包括凝血酶、胶原、凝血酶敏感蛋白、ADP、血栓素A2中的一种或一种以上组合。
进一步地，在步骤(4)中，超速离心的时间为至少30min。
进一步地，向所述样品中提供一种或多种血小板抑制剂，所述血小板抑制剂中的至少一种为前列腺素H1。
进一步地，在步骤(3)中，所述的抗炎药物与纯化血小板按照高于1:1比例进行混合。
进一步地，在步骤(1)中，所述的缓冲液为PBS缓冲液。
本发明的有益效果是：
本发明的抗炎靶向递送系统，其由血小板制备得到。由于血小板来自于生物体，因此血小板是可以完全生物降解，参与了体内的新陈代谢，没有毒副作 用，因而来源于血小板的血小板衍生囊泡也具有良好的生物相容性。获得的血小板衍生囊泡不包含任何人工合成高分子或其他材料，利用本发明的抗炎靶向递送系统无需使用异源或是聚乳酸甘醇酸(PLGA)等人工合成材料类物质作为抗炎靶向递送载体，生物安全性高。此外，本发明工艺简单，无需提取血小板膜，血小板衍生囊泡非常容易获得，血小板可来源于病人本身或者来源于血型一致的捐献者，血小板衍生囊泡可通过活化血小板一步法获得，分离工艺简单，操作步骤简单。此外，血小板对炎症部位具有内在的亲和力，包括动脉粥样硬化斑块等心血管疾病，而血小板来源的靶向递送系统可以选择性地结合多种炎症活化的细胞类型，如巨噬细胞和与动脉粥样硬化斑块形成相关的内皮细胞，因而利用此靶向递送系统能够将抗炎药物递送至炎症部位。
附图说明
图1为为本发明的血小板衍生囊泡的显微镜检图；
图2为本发明的血小板衍生囊泡的粒径分布图；
图3为本发明的血小板衍生囊泡在不同溶液里的粒径变化图；
图4为本发明的血小板衍生囊泡、血小板的电泳分析图；
图5为本发明的血小板衍生囊泡、血小板的Western Blot结果图；
图6为本发明的血小板衍生囊泡、血小板分泌的炎性因子图；
图7为本发明的血小板衍生囊泡靶向巨噬细胞图；
图8为本发明的血小板衍生囊泡靶向内皮细胞图；
图9为本发明的血小板衍生囊泡体内靶向小动物成像图；
图10为本发明的组织切片共聚焦图；
图11为本发明的抗炎组合物MCC950紫外吸收图；
图12为本发明的抗炎组合物MCC950缓释曲线；
图13为本发明的抗炎组合物对巨噬细胞IL-1β表达图；
图14为本发明的抗炎组合物对内皮细胞IL-1β表达图；
图15为本发明的抗炎组合物对巨噬细胞NLRP3表达情况图；
图16为本发明的抗炎组合物对内皮细胞NLRP3表达情况图；
图17为本发明的抗炎组合物对小鼠主动脉弓斑块大小影响的HE染色图；
图18为本发明的抗炎组合物对主动脉弓斑块IL-1β免疫组化染色图；
图19为本发明的抗炎组合物对主动脉弓斑块Mac3免疫组化染色图；
图20为本发明的抗炎组合物对血液炎性因子表达图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
6-8周龄的C57BL/6小鼠和ApoE-KO小鼠购自常州卡文斯实验动物有限公司。小鼠按照中国科学院生化与细胞所实验动物管理委员会(IACUC)的指导操作方法进行处理。
小鼠巨噬细胞RAW264.7,内皮细胞HUVECs购自美国模式菌种保藏中心(简称ATCC)。RAW264.7和HUVECs于添加有10％胎牛血清、100U/ml青霉素和100U/ml的链霉素的Dulbecco改良Eagle培养基中保藏。使用第三代或第四代传代培养的细胞用于本发明的各实施例。
实施例1：血小板的提取
从C57BL/6小鼠眼眶取出血液，储存在含有5mM EDTA和1μM PGE1的PBS中。100g离心15分钟去除红细胞，重复步骤直到上清液澄清，收集上清液并以800g离心20分钟；将离心沉淀物在室温下储存，以在重新悬浮后进一步使用。
实施例2：血小板衍生囊泡的提取
将血小板沉淀物重新悬浮后，加入2U/mL的凝血酶，于37℃活化30分钟，再以800g离心20分钟，收集上清液并进行超速离心获取血小板衍生囊泡，以2 500g(2小时)的速率浓缩成颗粒，即为血小板衍生囊泡。
实施例3：血小板衍生囊泡的定性分析
通过透射电镜(TEM)检测血小板衍生囊泡的表征，结果如图1所示，直径约100-150nm。通过动态光散射(DLS)测量粒径以及血小板衍生囊泡在不同溶液里随时间的粒径变化，结果如图2，图3所示，在4℃下储存在各种缓冲 液中至少4天。在没有血小板聚集抑制剂PGE1的情况下，PVs的大小没有显着变化，表明PVs的高稳定性。提取血小板及血小板衍生囊泡蛋白后，利用SDS-PAGE凝胶电泳对血小板及血小板衍生囊泡蛋白质进行蛋白质分析，结果如图4所示，由此，可得出血小板衍生囊泡含有来自血小板的部分蛋白质，然而，失去了高分子量的胞质蛋白质，如肌动蛋白和肌球蛋白。
之后利用Western Blot检测技术，采用10％的SDS-PAGE凝胶电泳分离血小板和血小板衍生囊泡蛋白，转膜至二氟化树脂膜(PVDF膜)上后，进行一抗二抗孵育。然后用AMERSHAM IMAGER 600超灵敏多功能成像仪对CD41的含量进行显影分析。结果如图5所示，CD41的存在进一步证实了血小板衍生囊泡确实来自血小板。
为了测定血小板衍生囊泡在激活后是否释放促炎细胞因子，通过酶联免疫吸附试验检测凝血酶激活的上清液中的IL-1β和IL-6表达。结果如图6所示，血小板衍生囊泡不分泌促炎因子，表明血小板衍生囊泡在作为抗炎靶向递送载体时，对机体不产生副作用，作为炎症治疗的药物载体具有良好的发展前景。
实施例4：血小板衍生囊泡的靶向性分析
(1)体外靶向分析
首先将血小板用DiD染色，经活化后收集得到DiD标记的血小板衍生囊泡。将巨噬细胞用氧化LDL处理使之转化为泡沫细胞，然后将泡沫细胞与用DiD标记的血小板或血小板衍生囊泡PVs孵育。结果如图7所示，通过荧光成像观察到，与未活化的巨噬细胞相比，血小板衍生囊泡PVs对泡沫细胞显示出更高的亲和力。DiD标记的血小板或血小板衍生囊泡PVs与经LPS处理人脐静脉内皮细胞共孵育，结果如图8所示，同样的，血小板衍生囊泡靶向活化后的内皮细胞。总之，这些结果在表明靶向递送系统与动脉粥样硬化的主要组分具有强亲和力，其可用于药物靶向递送至动脉粥样硬化斑块炎性微环境。
(2)体内靶向分析
将游离DiD或DiD标记的血小板衍生囊泡PVs，静脉内注射到ApoE-KO小鼠中。注射后6小时，对小鼠实施安乐死。切除心脏和主动脉用于离体成像。 结果如图9所示，DiD标记的血小板衍生囊泡在ApoE-KO小鼠中的小鼠动脉粥样硬化病变中的显着积累。相反，在游离染料处理的ApoE-KO小鼠中检测到很少的信号。此外，在野生型小鼠中未显示主动脉中PVs的积累，表明靶向能力是由PVs与活化的巨噬细胞或内皮之间的亲和力引起的。在共聚焦荧光显微镜下进一步检查动脉粥样硬化病变的切片，结果如图10所示。与游离DiD处理或野生型小鼠相比，动脉粥样硬化斑块中血小板衍生囊泡的富集进一步证实了血小板衍生囊泡的靶向能力。
实施例5：抗炎组合物的制备
将抗炎药物MCC950与纯化血小板混合进行共孵育24小时，800g离心20分钟获取沉淀物，沉淀物为MCC950装载的血小板，将沉淀物重新重悬，加入2U/mL的凝血酶，于37℃活化30分钟，再以800g离心20分钟，收集上清液并进行超速离心获取MCC950装载的血小板衍生囊泡，以100,000rpm(2小时)的速率浓缩成颗粒，即为抗炎靶向递送系统。
实施例6：抗炎组合物的定性分析
利用紫外吸收仪测定抗炎组合物的紫外吸收峰，结果如图11所示，MCC950在吸收光谱中的特征吸收峰表明抗炎药物MCC950成功加载到血小板衍生囊泡上。
利用高效液相色谱(HPLC)测定MCC950的加载和释放曲线，结果如图12所示，药物负载百分比约为15.3％，药物的释放曲线呈现缓释性。
实施例7：抗炎组合物的抗炎效果分析
(1)在体外减轻炎症的治疗效果
巨噬细胞和内皮细胞铺板培养后，待细胞均匀长至60％时，弃去原培养液，加入含100ng/mL LPS的新培养液，1小时后再分别加入同样剂量的抗炎药物MCC950，靶向递送系统血小板衍生囊泡PVs，抗炎组合物MCC950-PVs。24小时后收集细胞上清液使用ELISA检测炎性因子IL-1β的表达。同样的在24小时后提取细胞蛋白，利用Western Blot检测技术检测NLRP3的表达情况。结果分别如图13-16所示，由图13-16可看出用LPS处理的巨噬细胞和内皮细胞 分泌大量IL-1β，以及上调NLRP3表达。相反的，抗炎药或抗炎组合物处理后显著抑制IL-1β和NLRP3表达，说明本发明的抗炎组合物具有良好的抗炎效果。
(2)在体内减轻炎症的治疗效果
ApoE-KO小鼠高脂饲料喂养十二周，另普通饲料喂养的小鼠作未处理的对照。高脂饲料喂养的小鼠在21天内接受9次静脉输注PBS，抗炎药物MCC950或抗炎组合物MCC950-PVs，每次输注施用的MCC950剂量为5mg/kg。最终输注后24小时对所有小鼠实施安乐死。解剖取小鼠主动脉弓，对经各种治疗的小鼠主动脉弓中的斑块进行了定量组织学分析。动脉粥样硬化斑块的横截面用苏木精和伊红(H&E)，并对IL-1β，Mac3(巨噬细胞)进行免疫染色。结果如图17-19所示，组织学检查证实高脂饲料喂养小鼠未经治疗组中存在较大的斑块。在治疗组中，我们发现接受抗炎组合物MCC950-PVs治疗的小鼠内膜增厚与高胆固醇组和纯药物治疗相比显着降低，斑块明显减少，并且病变也明显减少。值得注意的是，抗炎组合物MCC950-PVs的治疗效果更好，可能是由于通过靶向递送系统靶向递送在斑块部位具有更高的MCC950浓度。
通过ELISA测试了外周血中炎性因子IL-1β，IL-6和TNF-α的表达。结果如图20所示，促炎细胞因子的表达在高脂肪组中上调，相反，在抗炎药物MCC950或抗炎组合物MCC950-PVs处理组中，促炎因子的分泌显着下降。这些结果表明本发明的抗炎组合物也缓解了全身性炎症。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一种抗炎靶向递送系统，其特征在于，所述的抗炎靶向递送系统包括血小板衍生囊泡和装载在所述的血小板衍生囊泡内或附着于所述的血小板衍生囊泡表面的抗炎药物，所述的血小板衍生囊泡通过超速离心活化后的血小板获得。
  2. 根据权利要求1所述的一种抗炎靶向递送系统，其特征在于，所述的血小板衍生囊泡的粒径为100-10000nm。
  3. 根据权利要求1所述的一种抗炎靶向递送系统，其特征在于，所述的抗炎药物为亲水性或疏水性的小分子抗炎药物。
  4. 根据权利要求3所述的一种抗炎靶向递送系统，其特征在于，所述的抗炎药物为MCC950、auranofin、TAK-242、bromoxone、姜黄素中的一种或一种以上组合。
  5. 一种权利要求1-4任一项所述的抗炎靶向递送系统的制备方法，其特征在于，包括如下步骤：
    (1)将含有血小板的血液样品分散于含有血小板凝集抑制剂的缓冲液中，在50-150g离心至上清液澄清，去除红细胞和白细胞，获得只含有血小板的上清液；
    (2)将抗炎药物与步骤(1)得到的血小板混合均匀，然后进行血小板活化；
    (3)将步骤(2)活化后的血小板在600-1000g离心15-25min，收集上清液；
    (4)将步骤(3)收集的上清液以至少2 500g转速进行超速离心，收集沉淀，得到所述的抗炎药物靶向递送系统。
  6. 根据权利要求5所述的方法，其特征在于，在步骤(2)中，所述的血小板活化通过添加血小板活化诱导剂活化。
  7. 根据权利要求6所述的方法，其特征在于，所述的血小板活化诱导剂包 括凝血酶、胶原、凝血酶敏感蛋白、ADP、血栓素A2中的一种或一种以上组合。
  8. 根据权利要求5所述的方法，其特征在于，在步骤(4)中，超速离心的时间为至少30min。
  9. 根据权利要求5所述的方法，其特征在于，所述血小板凝集抑制剂中的至少一种为前列腺素H1。
  10. 根据权利要求5所述的方法，其特征在于，在步骤(3)中，所述的抗炎药物与纯化血小板按照高于1:1比例进行混合。
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