含纳米片生物活性剂的配制与表征
Summary
在这里，我们描述了含有纳米盘的生物活性剂的生产和表征。以两性霉素B纳米盘为例，逐步描述该方案。
Abstract
术语纳米盘是指由双层形成脂质、支架蛋白和集成生物活性剂组成的离散类型的纳米颗粒。纳米盘被组织成圆盘状的脂质双层，其周边由支架蛋白限制，支架蛋白通常是可交换载脂蛋白家族的成员。许多疏水性生物活性剂通过集成到颗粒脂质双层的疏水环境中，在纳米盘中有效地溶解，产生了直径为10-20nm的颗粒群。纳米盘的配制需要单个组分的精确比例，每种组分的适当顺序添加，然后对配方混合物进行浴超声处理。两亲性支架蛋白自发接触并重组分散的双层形成脂质/生物活性剂混合物，形成离散、均匀的纳米盘颗粒群。在此过程中，反应混合物从不透明、浑浊的外观转变为澄清的样品，当完全优化时，离心时不会产生沉淀。表征研究涉及生物活性剂增溶效率的测定、电子显微镜、凝胶过滤色谱、紫外可见（UV/Vis）吸光光谱和/或荧光光谱。随后通常使用培养的细胞或小鼠研究生物活性。在含有抗生素（即大环内酯类多烯类抗生素两性霉素B）的纳米盘的情况下，可以测量它们作为浓度或时间函数抑制酵母或真菌生长的能力。相对容易配制、相对于组分的多功能性、纳米级粒径、固有稳定性和水溶性允许纳米盘技术的无数体 外 和 体内 应用。在本文中，我们描述了一种配制和表征含有两性霉素B作为疏水生物活性剂的纳米盘的一般方法。
Introduction
新生的盘状高密度脂蛋白（HDL）是人体循环系统中更丰富的球形高密度脂蛋白的天然祖先。这些新生颗粒，也称为pre-ß HDL，具有独特而独特的结构特性1。事实上，新生的HDL不是作为球状粒子存在的，而是圆盘状的。对天然和重组盘状高密度脂蛋白的广泛结构表征研究表明，它们由磷脂双层组成，其周长由两亲性可交换载脂蛋白（apo）限制，例如apoA-I。在人脂蛋白代谢中，循环的新生高密度脂蛋白从外周细胞中积累脂质，并在依赖于关键蛋白质介质的过程中成熟为球形高密度脂蛋白，包括ATP结合盒转运蛋白A1和卵磷脂：胆固醇酰基转移酶2。这个过程代表了反向胆固醇转运途径的关键组成部分，被认为可以预防心脏病。有了这些知识和重建盘状HDL的能力，研究人员将这些颗粒用作治疗动脉粥样硬化的治疗干预措施3。从本质上讲，将重组高密度脂蛋白（rHDL）输注到患者中会促进胆固醇从斑块沉积物中排出，并将其返回到肝脏以转化为胆汁酸并从体内排泄。一些生物技术/制药公司正在推行这种治疗策略4。
与此同时，在实验室中产生这些粒子的能力引发了一系列研究活动，导致了新的应用和新技术。一个突出的应用涉及使用rHDL颗粒作为微型膜，在天然环境中容纳跨膜蛋白5。迄今为止，已有数百种蛋白质成功掺入盘状rHDL，研究表明，这些蛋白质作为受体、酶、转运蛋白等保留了天然构象和生物活性。这些被称为“纳米盘”的颗粒也被证明适合结构表征，通常以高分辨率6。这种跨膜蛋白研究方法被认为优于洗涤剂胶束或脂质体的研究，因此正在迅速发展。重要的是要认识到，已经报道了两种能够形成rHDL的不同方法。“胆酸盐透析”方法13在与在rHDL双层5中掺入跨膜蛋白相关的应用中很常用。基本上，该配制方法涉及在含有洗涤剂胆酸钠（或脱氧胆酸钠;胶束分子量[MW]为4，200Da）的缓冲液中混合形成磷脂的双层，支架蛋白和目标跨膜蛋白。去垢剂可有效溶解不同的反应组分，使样品能够针对缺乏去垢剂的缓冲液进行透析。在透析步骤中，当洗涤剂从样品中取出时，会自发形成rHDL。当这种方法用于捕获感兴趣的跨膜蛋白时，产物颗粒被称为纳米盘5。然而，尝试使用这种方法掺入小分子疏水生物活性剂（MW <1，000 Da）在很大程度上是不成功的。与跨膜蛋白不同，小分子生物活性剂能够与洗涤剂一起从透析袋中逸出，大大降低了它们在rHDL中的掺入效率。通过从配方混合物14中省略洗涤剂解决了这个问题。相反，组分依次加入到水性缓冲液中，从双层形成脂质开始，形成含有rHDL的稳定生物活性剂，称为纳米盘。其他人则使用rHDL掺入和运输体内成像剂7。最近，由载脂蛋白支架和阴离子甘油磷脂心磷脂组成的专用rHDL已被用于配体结合研究。这些颗粒为研究心磷脂与各种水溶性配体（包括钙、细胞色素c和抗癌剂阿霉素8）的相互作用提供了一个平台。
本研究的重点是配制具有稳定掺入疏水性生物活性剂（即纳米盘）的rHDL。这些试剂整合到盘状rHDL颗粒的脂质环境中的能力有效地赋予它们水溶性。因此，纳米盘具有 体内 治疗应用的潜力。在配制纳米盘时，需要特定的孵育/反应条件才能成功地将离散疏水性生物活性剂掺入产品颗粒中，本报告的目的是提供详细的实用信息，可用作为特定应用创建新型纳米盘颗粒的基础模板。因此，在本手稿的上下文中，术语nanodisc和nanodisk是不可互换的。纳米盘是指配制为含有嵌入其脂质双层5中的跨膜蛋白的rHDL，而术语纳米盘是指配制为掺入低分子量（<1，000 Da）疏水性生物活性剂（例如两性霉素B14）的rHDL。
有多种方法可用于获取合适的支架蛋白。可以从制造商处购买支架蛋白[例如apoA-I（SRP4693）或apoE4（A3234）]，但是，成本可能是一个限制因素。首选方法是在大肠杆菌中表达重组支架蛋白。针对人类apoA-I9，apoE410以及昆虫血淋巴蛋白apolipophorin-III11的协议已发布。为了本文描述的实验的目的，使用重组人apoE4的N端（NT）结构域（氨基酸1-183）。合成编码人apoE4-NT的核苷酸序列并将其插入到与载体编码的pelB前导序列直接相邻的pET-22b（+）表达载体中。该构建体导致pelB领导序列-apoE4-NT融合蛋白的表达。蛋白质合成后，细菌pelB领导序列将新合成的蛋白质引导到周质空间，其中前导肽酶切割pelB序列。所得的apoE4-NT蛋白没有序列标签或尾巴，随后逃逸细菌并积聚在培养基11，12中，简化了下游加工。
Protocol
Representative Results
Discussion
含有纳米盘的生物活性剂的配制提供了一种溶解否则不溶的疏水化合物的便捷方法。由于产品生物活性剂纳米盘完全溶于水介质，因此它们为各种疏水分子提供了一种有用的递送方法（表1）。这些包括小分子、天然和合成药物、植物营养素、激素等。配方策略通常遵循标准方案，该方案必须考虑生物活性剂在有机溶剂中的溶解度特性。除了选择合适的有机溶剂来溶解生物活性剂外，?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（R37 HL-64159）的资助。
Materials
| Amphotericin B | Cayman Chemical Company | 11636 | ND Formulation & Standard Preparation |
| Ampicillin | Fisher Scientific | BP17925 | Transformation & Expansion |
| ApoE4-NT Plasmid | GenScript | N/A | Transformation |
| Baffled Flask | New Brunswick Scientific | N/A | Expansion & Expression |
| BL21 competent E coli | New England Biolabs | C2527I | Transformation |
| Centrifuge bottles | Nalgene | 3140-0250 | Expression |
| Chloroform | Fisher Scientific | G607-4 | ND Formulation |
| DMSO | Sigma Aldrich | 472301 | Standard Prepartation |
| Dymyristoylphosphatidylcholine | Avanti Lipids | 850345P | ND Formulation |
| Erlenmeyer flask | Bellco Biotechnology | N/A | Expansion & Expression |
| Falcon Tubes | Sarstedt Ag & Co | D51588 | Yeast Viability Assay |
| Glass borosilicate tubes | VWR | 47729-570 | ND Formulation |
| GraphPad (Software) | Dotmatics | N/A | Yeast Viability Assay |
| Heated Sonication Bath | VWR | N/A | ND Formulaton |
| Heating and Nitrogen module | Thermo Scientific | TS-18822 | ND Formulation |
| HiTrap Heparin HP (5 mL) | GE Healthcare | 17-0407-03 | Purification |
| Isopropyl β-D-1-thiogalactopyranoside | Fisher Scientific | BP1755 | Expression |
| J-25 Centrifuge | Beckman Coulter | J325-IM-2 | Expression |
| JA-14 Rotor | Beckman Coulter | 339247 | Expression |
| Lyophilizer | Labconco | 7755030 | ND Formulation |
| Methanol | Fisher Scientific | A452-4 | ND Formulation |
| Nitrogen gas | Praxair | UN1066 | ND Formulation |
| NZCYM media | RPI Research Products | N7200-1000.0 | Expansion & Expression |
| Pet-22B vector | GenScript | N/A | Transformation |
| Petri dish | Fisher Scientific | FB0875718 | Transformation & Expansion |
| Quartz Cuvettes | Fisher Brand | 14385 928A | Spectral Analysis |
| Shaking Incubator | New Brunswick Scientific | M1344-0004 | Transformation, Expansion, & Expression |
| Slide-A-Lyzer Buoys | Thermo Scientific | 66430 | Purification |
| SnakeSkin Dialysis Tubing | Thermo Scientific | 68100 | Purification |
| SnakeSkin Dialysis Tubing | Thermo Scientific | 88243 | Purification |
| Sodium Chloride | Fisher Scientific | S271 | Purification |
| Sodium Phosphate dibasic | Fisher Scientific | S374-500 | Purification |
| Sodium Phosphate monobasic | Fisher Scientific | BP329-500 | Purification |
| Spectra/POR Weighted Closures | Spectrum Medical Industries | 132736 | Purification |
| Spectrophotometer | Shimadzu UV-1800 | 220-92961-01 | spectral analysis |
| Tabletop Centrifuge | Beckman Coulter | 366816 | ND Formulation |
| UVProbe 2.61 (Software) | Shimadzu | N/A | Spectral Analysis |
| Vacuum filter | Millipore | 9004-70-0 | Expression & Purification |
| Vacuum pump | GAST Manufacturing Inc | DOA-P704-AA | Expression & Purification |
| Vortex | Fisher Scientific | 12-812 | ND Formulation |
| Yeast | N/A | BY4741 | Yeast Viability Assay |
| Yeast Extract-Peptone-Dextrose | BD | 242820 | Yeast Viability Assay |
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