Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI.
Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI.
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DOI：
10.1038/nprot.2017.081
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发表时间：
2017-10
期刊：
影响因子：
14.8
通讯作者：
Shapiro MG
中科院分区：
文献类型：
--
作者：
Lakshmanan A;Lu GJ;Farhadi A;Nety SP;Kunth M;Lee-Gosselin A;Maresca D;Bourdeau RW;Yin M;Yan J;Witte C;Malounda D;Foster FS;Schröder L;Shapiro MG
气体囊泡是一类独特的充气蛋白质纳米结构，其物理性质使其能够作为高灵敏度的成像剂用于超声和磁共振成像（MRI），可在亚纳摩尔浓度下检测。在这里，我们提供了一个协议，用于从本地和异源宿主生物体中分离气泡，这些纳米结构的功能与部分的目标和荧光，表征其生物物理特性和成像，使用超声和磁共振成像。气泡可以从天然的蓝藻和盐古菌宿主生物体或从E.表达异源气泡基因簇的大肠杆菌，并使用浮力辅助技术纯化。然后可以通过用工程化的异源表达变体替换表面结合的蛋白质或通过化学缀合来修饰它们，从而改变机械、表面和靶向性质。加压吸收光谱用于表征其机械性能，而动态光散射和透射电子显微镜用于确定纳米颗粒的尺寸和形态，分别。然后，可以在体外和体内使用针对其检测与背景进行优化的脉冲序列对气泡进行超声成像。它们也可以用超极化氙MRI成像，使用气泡结合和溶解氙之间的化学交换饱和转移-目前在体外实施的技术。这些可遗传编码的纳米结构需要3-8天的时间来制备，能够实现具有高灵敏度和分子靶向潜力的多模态、非侵入性生物成像。一类独特的蛋白质称为气体囊泡的物理特性使它们能够用作超声和磁共振成像（MRI）的造影剂。该方案描述了如何分离气体囊泡，用靶向和荧光部分使其功能化，并在体外和体内对其进行成像。
Gas vesicles are a unique class of gas-filled protein nanostructures whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and magnetic resonance imaging (MRI), detectable at sub-nanomolar concentrations. Here we provide a protocol for isolating gas vesicles from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and magnetic resonance imaging. Gas vesicles can be isolated from natural cyanobacterial and haloarchaeal host organisms or from E. coli expressing a heterologous gas vesicle gene cluster, and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants, or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, while dynamic light scattering and transmission electron microscopy are used to determine nanoparticle size and morphology, respectively. Gas vesicles can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between gas vesicle-bound and dissolved xenon – a technique currently implemented in vitro. Taking 3–8 days to prepare, these genetically encodable nanostructures enable multi-modal, noninvasive biological imaging with high sensitivity and potential for molecular targeting. The physical properties of a unique class of proteins called gas vesicles allow them to be used as contrast agents for ultrasound and magnetic resonance imaging (MRI). This protocol describes how to isolate gas vesicles, functionalize them with moieties for targeting and fluorescence, and image them in vitro and in vivo.
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影响因子：
2.9
作者：
Piedra, Mark;Allroggen, Achim;Lindner, Jonathan R.
通讯作者：
Lindner, Jonathan R.
影响因子：
17.1
作者：
Lakshmanan A;Farhadi A;Nety SP;Lee-Gosselin A;Bourdeau RW;Maresca D;Shapiro MG
通讯作者：
Shapiro MG
影响因子：
4.4
作者：
Witte, C.;Kunth, M.;Schroeder, L.
通讯作者：
Schroeder, L.
影响因子：
2.9
作者：
Kunth, Martin;Witte, Christopher;Schroeder, Leif
通讯作者：
Schroeder, Leif
体内检测cucultit [6]尿路，尿液是Xenon磁共振成像生物传感器的超极化XENON对比剂。
影响因子：
4.6
作者：
Hane FT;Li T;Smylie P;Pellizzari RM;Plata JA;DeBoef B;Albert MS
通讯作者：
Albert MS