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Bio-ingénierie

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Published: November 17, 2017
doi:
10.3791/56625
, ,
1Department of Biomedical Engineering,University of Southern California

Summary

本文介绍了一种涉及单核细胞靶向肽 amphiphile 胶束的合成和表征方法, 并对胶束与单核细胞结合的生物相容性和能力进行了相应的测试。

Abstract

动脉粥样硬化是心血管疾病的主要贡献者, 是全世界死亡的首要原因, 每年有1730万人丧生。动脉粥样硬化也是导致猝死和心肌梗塞的主要原因, 由不稳定的斑块引起, 破裂并咬合血管, 无预警。目前的成像模式不能区分稳定和不稳定的斑块破裂。多肽分子胶束 (PAMs) 可以克服这一缺点, 因为它们可以修改的各种靶向基, 具体绑定到病变的组织。单核细胞已被证明是动脉粥样硬化的早期标记物, 而单核细胞的大量积累与易破裂的斑块有关。因此, 能够靶向单核细胞的纳米粒子可以被用来鉴别动脉粥样硬化的不同阶段。为此, 在这里, 我们描述了一个协议为单核细胞靶向 PAMs (核细胞趋化蛋白 protein-1 (MCP-1) PAMs) 的制备。MCP-1 PAMs 是通过合成在温和条件下形成纳米颗粒的 15 nm 直径与近中性表面电荷。在体外中, PAMs 被发现具有生物相容性, 对单核细胞具有较高的亲和力. 本文所描述的方法对动脉粥样硬化以及其他炎症性疾病的广泛应用有广阔的前景。

Introduction

心血管疾病仍然是全球死亡的主要原因, 全世界约有1730万人死亡,1。心血管疾病是由动脉粥样硬化造成的, 一种情况是斑块在动脉中形成, 从而抑制血液和氧气流向身体的细胞2,3。动脉粥样硬化的进展是由炎症反应、不规则脂代谢和斑块堆积引起的动脉增厚和硬化, 导致斑块破裂和心肌梗死4,5。内皮细胞表达细胞因子和黏附分子, 其中包括 MCP-1, 结合到 c-c 趋化因子受体 (CCR2) 的表面发现单核的6,7,8。氧化胆固醇在斑块形成的早期将单核细胞转化为巨噬生物, 从而放大了该地区的炎症反应, 导致组织损伤, 形成不稳定或易受伤害的斑块9,10。

传统上, 动脉粥样硬化是评估腔狭窄的解剖成像使用血管造影或超声11,12。然而, 这些方法只能确定严重的动脉壁狭窄, 而不是动脉粥样硬化的早期阶段, 因为最初的斑块生长导致动脉重塑, 以维持动脉大小和血流率12,13, 14。因此, 造影 underrepresent 动脉粥样硬化的患病率。此外, 非侵入性成像技术, 如单光子发射计算机断层扫描, 正电子发射断层扫描和磁共振成像最近被用来表征斑块形态, 因为它们可以提供初步的细节和斑块的特征。然而, 这些模式往往受限于缺乏灵敏度, 空间分辨率, 或要求使用电离辐射, 使成像斑块进展在不同阶段更具挑战性的15,16, 17。一个成像传递系统, 将专门确定斑块在不同阶段的动脉粥样硬化仍有待开发。

纳米粒子已显示为体内斑块靶和诊断的一个新兴平台18,19,20,21。特别是, PAMs 是有利的, 因为他们的化学多样性和能力, 以适应各种基, 组成, 大小, 形状和表面功能化22。多肽分子 (PAs) 由亲水性, 多肽 “headgroup” 附着在疏水的尾巴上, 通常是脂质;这两亲结构赋予自功能, 并允许在粒子的表面上的多肽的多显示22,23,24。肽 headgroups 可通过折叠和多肽25之间的氢键作用影响颗粒形状。通过β-片相互作用折叠的多肽已经被证明形成拉长的胶束, 而α-螺旋确认可以形成球形和拉长的胶束22,23,24, 25,26,27。聚乙二醇 (PEG) 连接, 屏蔽了肽的表面电荷可以放在亲水性肽和 PAMs 的疏水尾之间, 增强了纳米颗粒在系统循环中的可用性28,29,30,31. PAMs 也很有利, 因为它们具有生物相容性, 并被证明具有广泛的应用范围3233。胶束的水溶性比其他纳米系统的优势, 如某些高分子纳米颗粒, 不溶于水, 必须在增悬浮在注射剂34。此外, 创建 PAMs 的能力, 以响应特定的刺激, 使 PAMs 一个有吸引力的候选人, 控制细胞内药物传递35。

通过结合 CCR2 受体和积累在主动脉弓, PAMs 以前开发的单核细胞靶向监测不同阶段的动脉粥样硬化病变的主动脉9。在载脂蛋白-/-小鼠中, 单核细胞积累成正比地增加到斑块级数36。此外, 发现有易破裂的晚期斑块的患者有较高的单核细胞数量37。因此, 对 PAMs 的修改, 以纳入 MCP-1 是有用的, 因为它允许更大的目标特异性和分化的早期和晚期动脉粥样硬化病变。这些概念验证研究还证实了 PAMs 是足够安全的, 可以使用 pre-clinically 并清除 renally38。由于单核细胞和炎症的特点, 其他疾病, MCP-1 PAMs 有潜力用于治疗和诊断的应用以外的其他疾病的动脉粥样硬化8,39,40,41。

在此, 我们报告了高可伸缩性和自组装 MCP-1 PAMs 的制作, 证明了粒子的最佳大小, 表面电荷, 选择性靶向单核细胞的增强成像应用在动脉粥样硬化。

Protocol

注: 阅读试剂的 MSDS, 并按照当地机构的要求, 遵守所有化学安全措施。 1. MCP-1 PAMs 的制备 MCP-1 肽的制备 在反应容器 (RV) 中, 称 n-芴甲氧羰基-l-赖氨酸 (Boc)-摩尔0.25。用化学油烟机冲洗5毫升 dimethylforamide (DMF) 的 RV 侧面。 将 RV 加载到自动台式多肽合成器上。载入预包装的氨基酸瓶, n “-CYNFTINRKISVQRLASYRRITSS-c”, 从 c 到 N 末端。在最后的脱步骤中包…

Representative Results

MCP-1 PAM 的制备MCP-1 蛋白 [YNFTNRKISVQRLASYRRITSSK] 或炒多肽 [YNSLVFRIRNSTQRKYRASIST] 的 CCR2-binding 母题 (残留 13-35) 通过在 N 端部添加半胱氨酸残留物而改变。采用全自动肽合成器, 用甲氧羰基介导的固相法合成了 MCP-1 肽。用反相高效液相色谱法在50° c 的 C8 柱上用 0.1% TFA 在乙腈/水混合物中纯化了粗肽, 并以 MALDI 质谱 (图 1) 为特征。含半胱氨酸的…

Discussion

MCP-1 PAMs 是一个充满希望的分子成像平台, 包括一个亲水性靶向肽和疏水尾巴, 驱动自组装性质的纳米颗粒。这种单核细胞靶向胶束可以通过简单的合成和纯化步骤 MCP-1 肽和 DSPE-PEG (2000)-MCP-1。PAMs 有许多有益的特征为在体内分子成像, 如他们的自组装在温和的条件下, 内在生物降解性, 和结构和化学多样性允许纳入其他成像基或靶向多肽选择性地传递到特定的感兴趣的地点。它们的粒度、形状…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者要感谢南加州大学, 国家心脏, 肺和血液研究所 (aha), R00HL124279, Eli 和伊蒂丝广泛创新奖的财政支持, 以及力惠蒂尔基金会非癌症翻译研究奖授予 EJC。作者感谢中心的电子显微镜和微量分析, 卓越中心的 NanoBiophysics, 卓越中心的分子特性, 和翻译成像中心在南加州大学的援助仪器设置。

Materials

1,2-ethanedithiol VWR E0032 for peptide synthesis
10 mL disposable serological pipets VWR 89130-898 for cell culture
15 mL centrifuge tubes, polypropylene VWR 89401-566 for various applications
2,5-dihydroxybenzoic acid, 99% Fisher Scientific AC165200050 for MALDI
25 mL disposable serological pipets VWR 89130-900 for cell culture
2-Mercaptoethanol, 50 mM ThermoFisher Scientific 31350010 for cell culture
5 mL disposable serological pipets VWR 89130-896 for cell culture
50 mL centrifuge tubes VWR 89039-658 for various applications
75 cm2 culture flask Fisher Scientific 13-680-65 for cell culture
75 mL reaction vessel Protein Technologies 3000005 for peptide synthesis
96-wells cell culture plate VWR 40101-346 for MTS assay
Acetonitrile, HPLC grade Fisher Scientific A998SK-4 for HPLC purification
Borosilicate glass, 1 dram VWR 66011-041 for PAM synthesis
Borosillicate glass pipet, Long tips VWR 14673-043 for various applications
Coverslip, 0.16-0.19 mm, 22 x 22 mm Fisher Scientific 12-542B for confocal microscopy
Cy5 amine Abcam ab146463 for peptide conjugation
Diethyl ether, ACS grade Fisher Scientific E138-1 for peptide precipitation
Disposable syringes, 20 mL Fisher Scientific 14-817-54 for HPLC purification
Double neubauer ruled hemocytometer VWR 63510-13 for cell counting
DSPE-PEG(2000) amine Avanti 880128P for peptide conjugation
DSPE-PEG(2000) maleimide Avanti 880126P for peptide conjugation
DSPE-PEG(2000)-NHS ester  Nanocs PG2-DSNS-10K for conjugation to Cy5
Dulbecco's modified eagle medium-high glucose Sigma Aldrich D5796-500ML for cell culture
Fetal bovine serum, qualified, heat inactivated ThermoFisher Scientific 10438026 for cell culture
Fmoc-L-Ala-OH /HBTU Protein Technologies PS3-H5-A for peptide synthesis
Fmoc-L-Arg(Pbf)-OH /HBTU Protein Technologies PS3-H5-RBF for peptide synthesis
Fmoc-L-Asn(Trt)-OH /HBTU Protein Technologies PS3-H5-NT for peptide synthesis
Fmoc-L-Cys(Trt)-OH /HBTU Protein Technologies PS3-H5-CT for peptide synthesis
Fmoc-L-Gln(Trt)-OH /HBTU Protein Technologies PS3-H5-QT for peptide synthesis
Fmoc-L-Ile-OH /HBTU Protein Technologies PS3-H5-I for peptide synthesis
Fmoc-L-Leu-OH /HBTU Protein Technologies PS3-H5-L for peptide synthesis
Fmoc-L-Lys(Boc)-OH /HBTU Protein Technologies PS3-H5-KBC for peptide synthesis
Fmoc-L-Phe-OH /HBTU Protein Technologies PS3-H5-F for peptide synthesis
Fmoc-L-Ser(tBu)-OH /HBTU Protein Technologies PS3-H5-SB for peptide synthesis
Fmoc-L-Thr(tBu)-OH /HBTU Protein Technologies PS3-H5-TB for peptide synthesis
Fmoc-L-Tyr(tBu)-OH /HBTU Protein Technologies PS3-H5-YB for peptide synthesis
Fmoc-L-Val-OH /HBTU Protein Technologies PS3-H5-V for peptide synthesis
Fmoc-Lys(Boc)-wang resin, 100-200 mesh Novabiochem 856013 for peptide synthesis
Formic acid, optima LC/MS grade Fisher Scientific A117-50 for HPLC purification
Glycerol VWR M152-1L for confocal microscopy
Hand tally counter Fisher Scientific S90189 for cell counting
Magnetic stir bars, egg-shaped VWR 58949-006 for peptide conjugation
Methanol, ACS certified Fisher Scientific A412-4 for PAM synthesis
MTS cell proliferation colorimetric assay kit VWR 10191-104 for MTS assay
N,N-Dimethylformamide, sequencing grade Fisher Scientific BP1160-4 for peptide synthesis
N-Methylmorpholine Protein Technologies S-1L-NMM for peptide synthesis
Paraformaldehyde Fisher Scientific AC416780250 for fixing cells
PBS, pH 7.4 ThermoFisher Scientific 10010049 for various applications
Penicillin/streptomycin, 10,000 U/mL ThermoFisher Scientific 15140122 for cell culture
Peptide synthesis vessel, 25 mL  Fisher Scientific CG186011 for peptide synthesis
Phosphotungstic acid  Fisher Scientific A248-25 for TEM
Piperidine Spectrum P1146-2.5LTGL for peptide synthesis
Plain glass microscope slide 75 x 25 mm Fisher Scientific 12-550-A3 for confocal microscopy
Reagent reservoirs, sterile  VWR 95128093 for cell culture
Self-closing tweezer TedPella 515 for TEM
TEM support film TedPella 01814F for TEM
Trifluoroacetic acid  Fisher Scientific BP618-500 for peptide cleavage and HPLC purification
Triisopropylsilane VWR TCT1533-5ml for peptide cleavage
Trypan blue solution, 0.4% ThermoFisher Scientific 15250061 for cell counting
Tweezer, general purpose-serrated VWR 231-SA-SE for confocal microscopy
WEHI-274.1 ATCC ATCC CRL-1679 murine monocyte
automated benchtop peptide synthesizer Protein Technologies PS3 Benchtop Peptide Synthesizer
α- cyano- 4- hydroxycinnamic acid, 99% Sigma Aldrich 476870-2G for MALDI
DOWNLOAD MATERIALS LIST

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Tags

Keywords: Peptide AmphiphileMonocyte-targetingAtherosclerosisImagingSynthesisMCP-1PlaqueCardiovascularDiagnosisAutomated Peptide SynthesisCleavage
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Poon, C., Sarkar, M., Chung, E. J. Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis. J. Vis. Exp. (129), e56625, doi:10.3791/56625 (2017).
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