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Bioingegneria

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

Published: February 01, 2019
doi:
10.3791/58444
, , , , , , ,
1Department of Thoracic and Cardiovascular Surgery, Clinical Research Laboratory,University Medical Center, 2Atherothrombosis and Vascular Biology,Baker Heart & Diabetes Institute, 3Department of Medicine,Monash University

Summary

在这里, 我们描述了一个方案的阳离子纳米脂质体的生成, 这是基于干膜法, 并可用于安全和有效的传递体外转录信使 rna。

Abstract

由于体外转录 (ivt) mrna 的积极特性, 基于信使 rna (mrna) 的治疗各种疾病的疗法的开发变得越来越重要。在 ivt mrna 的帮助下, 可以在不改变目标细胞生理状态的情况下诱导所需蛋白质的新合成。此外, 由于 ivt mrna 的瞬态效应, 可以精确地控制蛋白质的生物合成。

对于细胞的有效转染, 纳米脂质体 (nlps) 可能是治疗 mrna 的安全有效的载体。本研究描述了一种生成安全高效阳离子 nlps 的协议, 该方案由 dc-胆固醇和 1, 2-二醇-环甘油酯——————————————————————————————————————————————-nlps 具有定义的尺寸、均匀分布和较高的复用能力, 可使用干膜法生产。此外, 我们提出了不同的测试系统, 以分析他们的络合和转染效率使用合成增强绿色荧光蛋白 (transfection) mrna, 以及他们对细胞活力的影响。总体而言, 所提出的协议为 mrna 络合提供了一种有效和安全的方法, 可以促进和改进治疗性 mrna 的管理。

Introduction

在过去几年中, 改性 mrna 在治疗应用中的应用显示出巨大的潜力。在心血管、炎症和单基因疾病以及疫苗开发中, mrna 是一种很有前途的治疗药物1。

蛋白质替换疗法与 mrna 提供了几个好处比古典基因疗法, 根据脱氧核糖核酸转染入目标细胞2。mrna 功能直接在细胞溶胶中启动。尽管质粒 dna (pdna) 是一种双链结构, 含有启动子区域的圆形 dna 和编码治疗蛋白3的基因序列, 也在细胞溶胶中发挥作用, 但它只能被纳入正在进行有丝分裂的细胞中在转染的时候。这减少了组织中转染的细胞数量 1,4。具体来说, 有丝分裂活性弱的组织, 如心肌细胞的转染是困难的 5。与 pdna 不同的是, mrna 的转染和转化发生在组织1,6的有丝分裂和非有丝分裂细胞中。dna 与宿主基因组的病毒整合可能伴随着致突变效应或免疫反应7,8, 但在细胞转染后与蛋白质编码 mrna, 去重新合成所需的蛋白质自主开始 9,10。此外, 蛋白质合成可以通过单独剂量精确地调整到患者的需要, 而不会干扰基因组, 并有致突变效应11的危险.用伪尿素和 5 ‘-甲基胞苷代替尿素和胞苷12可显著降低合成生成的 mrna 的免疫激活潜力.假尿碱修饰的 mrna 也被证明具有更高的生物稳定性和显著提高的翻译能力13。

为了能够从基于 mrna 的治疗在临床应用中的有希望的特性中受益, 必须为 mrna 输送到细胞创造一个合适的工具。该飞行器应在体外和体内具有无毒特性, 保护 mrna 免受核酸酶降解的影响, 并提供足够的细胞吸收, 以便长时间地提供和翻译 mrna14。

在所有可能的体内药物传递载体类型中, 如碳纳米管、量子点和脂质体, 后者的研究最多 15、1 6.脂质体是由脂质双层10组成的囊泡。它们是具有疏水性和亲水性的两亲性, 通过这些分子的自排列, 形成了一个球形的双层。在脂质体内, 治疗药物或药物可以被封装, 从而防止酶降解 18.含有 n-[1, 2, 3-二烯二氧基) 丙基]-n 的脂质体, n、n-三甲基氯化铵 (dadma)19、[1, 2-bis (oley合 y)-3-(三甲基氨)丙烷] (dadap) 20 和二辛烷基氨基氨基氨基氨基氨基氨基胺胺 (dogs)21, 或dc-胆固醇22, 有很好的特征, 经常用于与 dna 或 rna 的细胞转染。

阳离子脂质体包括带正电荷的脂质和未带电的磷脂 23。通过阳离子脂质体转染是将核酸输送到细胞24,25的最常用方法之一。在核酸分子的主干中, 阳离子脂质颗粒与负电荷磷酸盐基团形成复合物26。这些所谓的脂质附着物附着在细胞膜表面, 并通过内吞或内皮样机制进入细胞 27。

1989年, 马龙等人。成功地描述了阳离子脂介导的mrna 转染28。然而, 使用 dotma 和 1, 2-二醇-sn-h神菌-3-磷乙醇胺 (dope) 的混合物, 该组发现 dotma 表现出细胞毒性作用28。此外, zohra等人还表明, dadap (1, 2-二烯二氧基 3-三甲基丙烷氯丙烷) 可用作 mrna 转染试剂29。然而, 为了有效转染细胞, dopap 应与其他试剂结合使用, 如纤维连接蛋白 29或 dope30。到目前为止, d同甲是市场上第一个用于基因传递的阳离子脂质 31.其他脂类被用作治疗载体或正在临床试验的不同阶段进行检测 (例如, 含有 dentap 作为脂质载体的 endatg-i) 目前正在第二阶段临床试验32中进行调查。

这项工作描述了一个协议, 用于生成含有 dc 胆固醇和 dope 的 nlps。此方法易于执行, 并允许生成不同大小的 nlps。使用干膜法生成 nlp 的总体目标是为 mrna 络合创建脂质体, 从而允许在体外高效和生物相容性的细胞转染 14,33。

Protocol

1. 阳离子纳米脂质体的产生 (图 1) 溶解脂质 dc-胆固醇 (3β-[n-(n ‘, n ‘-二甲基氨基乙烷)-氨基乙酰] 盐酸胆固醇) 和 dope (二烯醇磷脂酰乙醇胺), 以氯仿的形式提供, 最终浓度为 25 mg/ml。注: 将溶解的脂质存放在-20°c。 使用 25 mgml 两种脂类的库存解决方案。将溶解的 dc 胆固醇的40μl 和溶解的 dope 的80μl 混合在一个玻璃瓶中。注: 总脂量为3毫克。为避免氯仿快速…

Representative Results

利用该协议, 采用干膜法制备了由脂质 dc-胆固醇和 dope 组成的 nlps (图 1)。在制备过程中, 纳米脂质体溶液显示浊度的不同阶段 (图 2)。 然后, 通过使用 rna 定量试剂盒 (图3) 分析未封装的 mrna 的自由量, 可以分析 nlps 的封装效果。 <p class="jove_content" fo:keep-togeth…

Discussion

该协议描述了具有高封装效果的 nlps 的生成, 以合成改性 mrna, 以及细胞在体外的可靠转染。此外, nlps 保证 mrna 的释放, 而 mrna 又被转化为细胞内的功能蛋白。此外, 使用 nlps 的转染可以在常规细胞培养基中进行, 从而在转染过程中产生较高的细胞振动, 并在转染后最多持续三天。

使用 mrna 作为治疗, 自组装系统为其交付的首选。最常见的转染试剂包括阳离子脂质, 包括脂?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

没有

Materials

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) AppliChem, Darmstadt, Germany A2231
(3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol) Avanti, Alabama, USA 700001
4 ′,6-diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific, Darmstadt, Germany D1306
BD FACScan system BD Biosciences, Heidelberg, Germany
Cell Fix (10x) BD Biosciences, Heidelberg, Germany 340181
Chloroform Merck, Darmstadt, Germany 102445
Dimethyl sulfoxid (DMSO) Serva Electrophoresis GmbH, Heidelberg, Germany 20385.02
Dioleoyl phosphatidylethanolamine (DOPE) Avanti, Alabama, USA 850725
Fluorescence microscope Zeiss Axio, Oberkochen, Germany
Lipofectamine 2000 Thermo Fisher Scientific, Darmstadt, Germany 11668019
Mini extruder Avanti, Alabama, USA
Nuclease-free water Qiagen, Hilden, Germany 129114
Opti-Mem Thermo Fisher Scientific, Darmstadt, Germany 11058021
PBS buffer (w/o Ca2+/Mg2+) Thermo Fisher Scientific, Darmstadt, Germany 70011044
Quant-iT Ribo Green RNA reagent kit Thermo Fisher Scientific, Darmstadt, Germany Q33140
RPMI (w/o phenol red) Thermo Fisher Scientific, Darmstadt, Germany 11835030
Silica gel Carl Roth, Karlsruhe, Germany P077
Trypsin/EDTA (0.05%) Thermo Fisher Scientific, Darmstadt, Germany 25300054
HotStar HiFidelity Polymerase Kit Qiagen, Hilden, Germany 202602
QIAquick PCR Purification Kit Qiagen, Hilden, Germany 28104

Pseudouridine-5'-Triphosphate (Ψ-UTP)		 TriLink Biotechnologies, San Diego, USA N-1019
5-Methylcytidine-5'-Triphosphate (Methyl-CTP) TriLink Biotechnologies, San Diego, USA N-1014
Cyanine 3-CTP PerkinElmer, Baesweiler, Germany NEL580001EA
RNeasy Mini Kit Qiagen, Hilden, Germany 74104
MEGAscript T7 Transcription Kit Thermo Fisher Scientific, Darmstadt, Germany AM1333
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog New England Biolabs, Ipswich, USA S1411L
Antarctic Phosphatase New England Biolabs, Ipswich, USA M0289S
Agarose Thermo Fisher Scientific, Darmstadt, Germany 16500-500
GelRed Biotium, Fremont, USA 41003
peqGOLD DNA ladder mix VWR, Pennsylvania, USA 25-2040
Invitrogen 0.5-10kb RNA ladder Fisher Scientific, Göteborg,
Sweden		 11528766
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Tags

Keywords: Cationic NanoliposomesMRNA DeliveryTransfectionNanoparticleLipid FilmExtrusionNanolipoplexA549 Cells
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Michel, T., Link, A., Abraham, M., Schlensak, C., Peter, K., Wendel, H., Wang, X., Krajewski, S. Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA. J. Vis. Exp. (144), e58444, doi:10.3791/58444 (2019).
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