In Vitro and In Vivo Uncaging and Bioluminescence Imaging by Using Photocaged Upconversion Nanoparticles
In Vitro and In Vivo Uncaging and Bioluminescence Imaging by Using Photocaged Upconversion Nanoparticles
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DOI：
10.1002/anie.201107919
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Xing, Bengang
中科院分区：
文献类型：
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作者：
Yang, Yanmei;Shao, Qing;Xing, Bengang
细胞活动、生物学途径和基因表达的高度时空调控在复杂的生物学过程中至关重要。[1]一种能够实现这种控制的显着技术是使用光来操纵在各种生物系统中具有光活性（或光笼）的化合物。[2]以前，这种策略已被用于映射细胞功能，监测转基因的表达，并在体外和体内的细胞-细胞相互作用的动态过程的图像。[3，4]虽然所有这些尝试原则上都是成功的，但在光活化过程中使用高强度UV或可见光存在明显的局限性。过度暴露于紫外线可引起核酸的光反应并导致细胞损伤。此外，短波长UV或可见光不能穿透到组织中很远，这限制了其通过笼状化合物的光活化用于深层组织成像的效用。或者，具有长波长激发的多光子光解已用于实现深层组织成像和靶向基因表达[5]尽管其有用，但多光子光解过程通常需要复杂的实验装置，并且由于吸收截面窄而具有低转换效率。因此，非常需要开发一种简单的方法，该方法允许高深度地穿透到组织中并精确控制光笼系统以及限制细胞损伤。最近，镧系元素掺杂的上转换纳米粒子（UCNPs）的应用范围从生物标记到光学数据存储受到了相当大的关注。[6]这些纳米颗粒具有很高的光稳定性，并通过近红外（NIR）光照射实现深层组织渗透深度（高达10 mm），这使得它们对生物成像应用特别有吸引力。[7]在这里，我们展示了一种方法，用于uncaging photocaged分子在体外和体内，并进行生物发光成像研究相结合的通用photocaged化合物与UCNPs.Scheme 1说明了概念验证设计的光解笼d-胡萝卜素通过使用bioconjugated UCNPs。作为一种常用的生物发光探针，dexamin可以识别萤火虫荧光素酶（fLuc）报告基因，并在O2、Mg 2+离子和三磷酸腺苷（ATP）存在下产生生物发光。因此，d-胰蛋白酶提供了广泛应用于体外和体内分子成像的机会。[8]在我们的设计中，选择具有降低的表面淬灭效应的Tm/Yb共掺杂的NaYF 4核-壳纳米颗粒[9]作为用于d-胡萝卜素缀合的平台。核-壳纳米颗粒用硫醇化的硅烷分子包覆，随后偶联到用1-（2-硝基苯基）乙基笼住的d-氨基喹啉。由于光笼化的d-dCNPs的吸收带与纳米颗粒在UV区域（Tm 3+的1 I6 → 4F 3、1D 2 → 3 H6跃迁）中的上转换发射带重叠，所以用NIR光激发光笼化的UCNP可以触发d-dCNPs分子从纳米颗粒表面解离。重要的是，可以通过跟踪d-glucin的发光强度或通过使用fLuc酶报告基因来监测释放过程，从而提供了不需要UV或可见光的生物发光成像研究的可能性。
High temporal and spatial regulation of cellular activities, biological pathways, and gene expression is critical in complex biological processes.[1] One remarkable technique that enables such control is the use of light to manipulate compounds that are photoactive (or photocaged) in various biological systems.[2] Previously, this strategy has been used to map cellular functions, monitor the expression of transgenes, and image the dynamic processes of cell–cell interactions in vitro and in vivo.[3, 4] Although all of these attempts were successful in principle, there were significant limitations associated with the use of high-intensity UV or visible light in the photoactivation process. Excessive exposure to UV light can cause photoreactions in nucleic acids and result in cellular damage. Furthermore, short-wavelength UV or visible light does not penetrate into tissue very far, which limits its utility for deeptissue imaging by photoactivation of the caged compounds. Alternatively, multiphoton photolysis with long-wavelength excitation has been used to enable deep-tissue imaging and to target gene expression [5] Despite its usefulness, the multiphoton photolytic process typically requires a complex experimental set-up and has low conversion efficiency because of narrow absorption cross-sections. Therefore, the development of a simple approach that allows a high depth of penetration into tissue and precise control of photocaged systems as well as limiting cellular damage is highly desirable. Recently, lanthanide-doped upconversion nanoparticles (UCNPs) have received considerable attention for applications that range from biolabeling to optical data storage.[6] These nanoparticles offer high photostability and enable deep tissue-penetration depths (up to 10 mm) by irradiation with near-infrared (NIR) light, which makes them particularly attractive for bioimaging applications.[7] Herein, we demonstrate a method for uncaging photocaged molecules in vitro and in vivo and performing bioluminescence imaging studies by combining versatile photocaged compounds with the UCNPs.Scheme 1 illustrates the proof-of-concept design for the photolysis of caged d-luciferin by using bioconjugated UCNPs. As a commonly used bioluminescent probe, dluciferin can recognize firefly luciferase (fLuc) reporter genes and produce bioluminescence in the presence of O2, Mg2+ ions, and adenosine triphosphate (ATP). Therefore, d-luciferin provides opportunities for extensive applications in molecular imaging in vitro and in vivo.[8] In our design, Tm/Yb co-doped NaYF4 core-shell nanoparticles [9] that have a reduced surface-quenching effect were chosen as the platform for the conjugation of d-luciferin. The core-shell nanoparticles were coated with thiolated silane molecules and subsequently coupled to d-luciferin that was caged with a 1-(2-nitrophenyl) ethyl group. As the absorption band of the photocaged d-luciferin overlaps with the upconverted emission band of the nanoparticle in the UV region (1I6→ 4F3, 1D2→ 3H6 transitions of Tm3+), excitation of the photocaged UCNPs with NIR light can trigger disassociation of dluciferin molecules from the surface of the nanoparticle. Importantly, the uncaging process can be monitored either by tracking the luminescence intensity of d-luciferin or by using an fLuc enzyme reporter, thus providing the possibility of bioluminescence imaging studies without the need for UV or visible light.