Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

St&#233;phane Isabettini; Mirjam E. Baumgartner; Peter Fischer; Erich J. Windhab; Marianne Liebi; Simon Kuster

doi:10.3791/56812

JoVE Journal > Engineering

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Ingeniería

设计磁响应稀土离子螯合磷脂组件的制备方法及双折射测量

Published: January 03, 2018

doi:

10.3791/56812

Stéphane Isabettini¹, Mirjam E. Baumgartner¹, Peter Fischer¹, Erich J. Windhab¹, Marianne Liebi², Simon Kuster¹

¹Laboratory of Food Process Engineering,ETH Zurich, ²MAX IV Laboratory,Lund University

Summary

介绍了高磁响应稀土离子螯合多组件的制备方法。磁响应由装配尺寸决定, 它是通过纳米膜的挤压而定制的。采用双折射测量、核磁共振和小角度中子散射技术对组件的磁 alignability 和温度诱发的结构变化进行监测。

Abstract

Bicelles 是可调谐的圆盘状多组件, 由大量的脂质混合物组成。应用范围从膜蛋白结构研究的核磁共振 (NMR), 以纳米的发展, 包括形成光学活性和磁性可转换凝胶。这些技术需要对装配尺寸、磁响应和热阻进行高度控制。混合物 12-二-sn-glycero-3-phosphocholine (DMPC) 及其镧系离子 (Ln^{3 +}) 螯合磷脂共轭, 12-二-snglycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (甲醚-DTPA), 组装成高磁响应的组件, 如 DMPC/甲醚-DTPA/Ln^{3 +} (摩尔比率 4:1: 1) bicelles。在双层中引入胆固醇 (-OH) 和类固醇衍生物会导致另一组提供独特物理化学性质的组件。对于某一特定的脂质成分, 磁性 alignability 与 bicelle 大小成正比。在^{3 +}中的络合结果在大小和对准方向方面都产生了前所未有的磁响应。热可逆崩溃的圆盘状结构成泡后, 加热允许裁剪装配的尺寸, 通过挤出膜过滤器的定义孔径大小。磁性 alignable bicelles 通过冷却到5° c 再生, 导致装配尺寸由囊泡前体定义。在此, 解释了这个制造过程, 并通过在 5.5 T 磁场下的双折射测量来量化组件的磁性 alignability。双折射信号, 源自磷脂双层, 进一步使监测的多变化发生在双层。这种简单的技术是互补的核磁共振实验, 通常用于表征 bicelles。

Introduction

Bicelles 是从许多脂质混合物中获得的圆盘状多组件。¹^,²^,³^,⁴^,⁵广泛应用于膜生物分子的核磁共振波谱结构表征。⁶^,⁷然而, 最近的努力旨在扩大可能的应用领域。⁵^,⁸^,⁹最受研究的 bicelle 系统由 12-二-sn-glycero-3-phosphocholine (DMPC) 组成, 构成组件的平面部分, 12-己-snglycero-3-phosphocholine (DHPC)磷脂覆盖边缘。¹^,²^,³组成双层磷脂的分子几何决定了自组装多结构的结构。⁴^,⁵用甲醚-DTPA 替换 DHPC, 生成高磁响应和可调谐的 bicelle 系统。¹⁰^,¹¹ DMPC/甲醚-DTPA/Ln^{3 +} (摩尔比 4:1: 1) bicelles 与更多的顺磁性镧系离子 (Ln^{3 +}) 在双层表面上相关联, 从而增强了磁响应。¹⁰此外, 用甲醚-DTPA/Ln^{3 +}取代水溶性 DHPC 分子, 可以形成耐稀释的 bicelles。¹¹

平面多组件的磁性 alignability 是由它们的整体磁场能量决定的,

(1)

其中 B 是磁场强度, 磁常数, n 的聚合数和分子磁性的易感性各向异性组成双层的脂质. 因此, DMPC/甲醚-DTPA/Ln^{3 +} bicelles 对磁场的响应是根据它们的大小 (集合数 n) 和分子磁性磁化率各向异性Δχ来调整的。后者很容易通过改变螯合物的性质在^{3 +}中实现。¹²^,¹³^,¹⁴^,¹⁵在双层中引入胆固醇 (铁-OH) 或其他甾体衍生物, 可以调节聚合数 n 和Δχ的磁化率。¹¹^,¹⁶^,¹⁷^,¹⁸^,¹⁹对于给定的脂质组合, 较大的组件含有更多的脂类, 能够对 E_mag (更大的聚合数 n) 做出贡献, 从而导致更多的 alignable 物种。DMPC/DHPC bicelles 的大小, 例如, 是常规控制通过优化的组成脂质比或总浓度。²⁰^,²¹^,²²虽然这在 DMPC/甲醚-DTPA/Ln^{3 +} bicelles 中是可能的, 但在加热时, 它们从 bicelle 到泡的热可逆转换提供了更多的剪裁选项。机械手段, 如挤压通过膜过滤器, 可以形成的囊泡。磁性 alignable bicelles 在冷却到5° c 时再生, 并且他们的维度从泡前体被口授。¹¹在这里, 我们专注于机械制造过程的潜力, DMPC/甲醚-DTPA/tm^{3 +} (摩尔比率 4:1: 1) 或 DMPC/, OH/甲醚-DTPA/tm^{3 +} (摩尔比 16:4: 5:5) 作为参考系统。在使用其他 Ln^{3 +}而不是 Tm^{3 +}时, 该进程工作类似。这些技术提供的各种可能性在图 1中得到了突出显示, 并在其他地方进行了广泛的讨论。²³

图 1: 可能的制造过程的示意图概述.所研究的磁性 alignable 在^{3 +}螯合多组件是由 DMPC/甲醚-DTPA/Tm^{3 +} (摩尔比 4:1: 1) 或 DMPC/, OH/甲醚-DTPA/Tm^{3 +} (摩尔比 16:4: 5:5) 组成。干脂膜是水合的50毫米磷酸缓冲液 pH 值为 7.4, 总脂质浓度为15毫米。有效的脂质膜水化需要的是冻融循环 (FT) 或加热和冷却循环 (H & #38; C)。H 和 #38; C 周期是必要的, 以再生样品后, 最后冻结解冻步骤, 或再生样品保持长期冻结超过一段时间, 如果他们要使用不进一步挤压。Isabettini et al广泛讨论了这些步骤。²³最大的 alignable 多组件实现, 根据脂质组成提供不同的组件体系结构。通过纳米膜过滤器, 挤出 (Ext) 可调节 bicelle 尺寸和磁性 alignability。所呈现的对齐系数_f是从2D 小角中子散射 (san) 模式 (DMPC/甲醚/DTPA/Tm^{3 +} (摩尔比率 16:4: 5:5) 中计算的, 通过800、400、200或 100 nm 孔隙进行拉伸。san 度量是量化 bicelle 对齐的补充方法, 此处不会详细介绍。¹¹^,¹⁶一个_f范围从-1 (平行中子散射或垂直对准的 bicelles 与磁场方向) 到0的各向同性散射。请点击这里查看更大版本的这个数字。

bicelles 的结构在广泛的表征技术中得到了广泛的研究。¹³使用核磁共振波谱法或小角度中子散射 (san) 实验对暴露在磁场中的 bicelles 的对准进行了量化。⁵^,¹⁰^,¹¹^,¹²^,¹³^,¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁴^,²⁵但是, 在 Ln^{3 +}的情况下发生的核磁共振峰值的移位和展宽是对该方法的严重限制。¹⁵^,²⁶^,²⁷^,²⁸尽管 san 实验不受此限制的影响, 但在解决方案中对组件的磁致对准的常规定量化方面, 还是可取的替代方法。双折射测量是一个可行的和比较简单的选择。类似核磁共振实验, 双折射测量揭示了有价值的信息, 脂质重和脂质相发生在双层。此外, 多组件中发生的几何变换, 如温度等变化的环境条件进行了监测。¹¹^,¹²^,¹³^,¹⁶磁致双折射Δn′用于研究各种类型的磷脂系统。¹³^,²⁹^,³⁰基于磁场相位调制技术的双折射测量是检测 bicelles 方位的可行方法。¹²^,¹⁶^,¹⁸^,²⁹^,³¹^,³²在高磁场中 bicelles 的双折射率高达 35 T 的可能性也由 m. Liebi et al.显示¹³

当偏振光进入一个各向异性物质时, 它会在一个普通的和特殊的波中折射。¹¹两个波具有不同的速度, 并在相位上由一个迟滞δ转移。延迟δ的程度被测量并且转换成双折射信号来量化材料中各向异性的程度

(2)

其中λ是激光的波长, d 是样品的厚度。磷脂是光学的各向异性, 它们的光轴与它们的长分子轴重合, 与烃尾平行。¹¹^,¹²如果磷脂在溶液中是随机定向的, 则不测量延迟。当磷脂相互平行时, 就会测量迟钝。磁致双折射可根据磁场中分子的取向, 产生正负符号; 请参见图 2。与 x 轴平行的磷脂将导致一个负的, 而那些沿 z-axis 对齐结果为正值的. 当与 y-axis 平行时, 当光学轴与光传播的方向重合时, 不观察到双折射。

图 2: 磷脂的对准和磁致双折射的对应符号 .测量的的符号取决于在磁场中磷脂的方向. 虚线表示分子的光轴。光在45°上极化, 并在 y 方向传播。磁场 B 在 z 方向。这一数字已从 m Liebi 中修改。¹¹ 请单击此处查看此图的较大版本.

在 bicelles 的各向同性胶体悬浮的情况下, 磷脂在双层中的排列所诱导的取向将会丢失, 使迟滞δ零化。bicelles 还必须对准, 以便在它们的双层中定位光学活性磷脂, 导致偏振光δ。因此, 双折射是一个敏感的工具, 量化的磁性 alignability 的多组件。垂直于磁场的 Bicelles 将产生一个正的, 而那些对齐的平行将产生一个负的. 该符号取决于设置的对齐方式, 并且可以使用引用示例进行检查。

Protocol

1. DMPC/甲醚-DTPA/tm3 + (摩尔比 4:1: 1) 和 DMPC/-OH/甲醚-DTPA/tm3 + (摩尔比 16:4: 5:5) 多组件的制作程序初步准备用乙醇稳定的氯仿 (#62; 99% 氯仿) 冲洗所有的玻璃器皿, 用压缩空气烘干。在乙醇稳定氯仿 (#62 中生产2种不同的10毫克/毫升的 DMPC 和甲醚-DTPA 的库存溶液; 99% 氯仿), 在乙醇稳定的氯仿 (和 #62; 99% 氯仿) 和10毫米的 TmCl 的库存溶液中, 有一个10毫米的电液.3…

Representative Results

non-extruded DMPC/甲醚-DTPA/Tm3 +的双折射信号 (摩尔比 4:1: 1) 在一个 5.5 T 磁场的加热和冷却循环期间, 从5到40° c 和背部以1° c/分钟 (图 6) 的速度监测样品。双折射结果在5° c 时确认了高磁对齐, 其值为 1.5 x 10-5, 为所报告的挤出系统的两倍强。6,7,23 DMPC 在24° c 以上的 T<s…

Discussion

详细介绍了双折射测量如何与 san 结合使用, 以评估产生高磁响应的方法在^{3 +}螯合磷脂组件中的 Isabettini et al.²³所建议的制造协议也适用于由较长的联邦和 DPPE-DTPA 磷脂组成的组件, 或用于含有其双层内化学工程类固醇衍生物的部件。¹¹^,¹²^,¹⁷^,¹⁸^,¹⁹…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

作者承认瑞士国家科学基金会资助 SMhardBi (项目编号 200021_150088/1)。在瑞士 Villigen 谢瑞 Instute 的瑞士散裂中子源 SINQ 进行了无 san 实验。作者热烈感谢 Dr. Kohlbrecher 的指导和无实验。在高磁场下的双折射测量装置是从荷兰奈梅亨型磁性实验室 HFML 的现有设置中得到启发的。我们感谢布鲁诺菲斯特的帮助, 开发的电子双折射设置, 1月 Corsano 和丹尼尔 Kiechl 为构建框架允许精细和轻便的激光对准, 和 Dr. 伯纳德科勒为持续的技术支持。

Materials

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)	Avanti Polar Lipids	850345P	>99%
1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate acid hexammonium salt (DMPE-DTPA)	Avanti Polar Lipids	790535P	>99%
Thulium(III) chloride	Sigma-Aldrich	439649	anhydrous, powder, 99.9% trace metals basis
Dysprosium(III) chloride	Sigma-Aldrich	325546	anhydrous, powder, 99.9% trace metals basis
Ytterbium(III) chloride	Sigma-Aldrich	439614	anhydrous, powder, 99.9% trace metals basis
Chloroform	Sigma-Aldrich	319988	contains ethanol as stabilizer, ACS reagent, ≥99.8%
Methanol	Sigma-Aldrich	34860	≥99.9%
Cholesterol	Amresco	433	Ultra pure grade
D2O	ARMAR chemicals	1410	99.8 atom % D
Ultrapure water	Millipore		Synergy pak2 (SYPK0SIX2), Millipack GP (MPGP02001)
electronic pH meter	Metrohm	17440010
Whatmann Nuclepore 25 mm 100nm membrane filter	VWR	515-2028
Whatmann Nuclepore 25 mm 200nm membrane filter	VWR	515-2029
Whatmann Nuclepore 25 mm 400nm membrane filter	VWR	515-2030
Whatmann Nuclepore 25 mm 800nm membrane filter	VWR	515-2032
Whatmann Filter paper	VWR	230600
25 ml round bottom flask	VWR	201-1352	14/23 NS
3 ml glass snap-cup	VWR	548-0554	ND18, 18x30mm
2.5 ml glass syringe	Hamilton
Sodium dihydrogen phosphate dihydrate	Merk	1.06342	Salt used to make phosphate buffer
di-Sodium hydrogen phosphate	Merk	1.06586	Salt used to make phosphate buffer
Liquid Nitrogen	Carbagas	–
Pressurized Nitrogen gas	Carbagas	–	200 bar bottle
Lipid Extruder 10 ml	Lipex	–	Fully equipped with thermobarrel
High-pressure PVC tube	GR NETUM	–	must resist more than 4 MPa
Serto adaptors	Sertot	–
Nitrile gloves	VWR	–
2 ml glass pipettes	VWR	612-1702	230 mm long
Diode Laser	Newport	LPM635-25C
DSP Dual Phase Lock-in Amplifier	SRS	SR830
Photodiode Detector	Silonex Inc.	SLSD-71N5	5mm2, Silicon, photo-conductive
5.5 T Cryogenic Magnetic	Cryogenic/Oerlikon AG	–	12 bar He-cooled. RW4000/6000 compressor, RGD 5/100 TA cryo-head
Second order low pass filter	home-built	–	Linear power supply 24V DC, second order, Sallen Key, cut-off frequency 360 Hz, +/- 12V, max 10 mA
Photoelastic modulator	Hinds instruments	PEM-90
Glan-Thompson Calcite Polarizer	Newport	10GT04	25.4mm diameter
Quartz sample cuvette	Hellma	165-10-40	temperature controlled cell, 0.8 ml, 10mm path length
Temperature probe	Thermocontrol	–	Type K, 0.5mm diameter, Thermocoax
Non-polarizing mirrors	Newport	50326-1002	25.4mm
RS 232 cables	National Instruments	189284-02	For Connecting to the RS-232 Port on the front of Compact FieldPoint Controllers
BNC 50 Ω cable and connectors	National Instruments	763389-01
cFP-AI-110	National Instruments	777318-110	8-Channel Analog Voltage and Current Input Module for Compact FieldPoint
cFP-CB-1	National Instruments	778618-01	Integrated Connector Block for Wiring to Compact FieldPoint I/O
cFP-CB-3	National Instruments	778618-03	Integrated Isothermal Connector Block for Wiring Thermocouples to the cFP-TC-120 Module
cFP-TC-120	National Instruments	777318-120	8-Channel Thermocouple Input Module for Compact FieldPoint
cFP-1804	National Instruments	779490-01	Ethernet/Serial Interface for NI Compact FieldPoint
LabView 2010	National Instruments	–
Industrial power supply	Traco Power	TCL 060-124	100-240V AC
Waterbath	Julabo	FP40-HE	refrigerated/Heating Circulator

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Isabettini, S., Baumgartner, M. E., Fischer, P., Windhab, E. J., Liebi, M., Kuster, S. Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies. J. Vis. Exp. (131), e56812, doi:10.3791/56812 (2018).