Multifunctional Thio-Stabilized Gold Nanoparticles for Near-Infrared Fluorescence Detection and Imaging of Activated Caspase-3.
Multifunctional Thio-Stabilized Gold Nanoparticles for Near-Infrared Fluorescence Detection and Imaging of Activated Caspase-3.
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DOI：
10.2174/1573411017999210112175743
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发表时间：
2021
影响因子：
1.8
通讯作者：
Achilefu S
中科院分区：
文献类型：
--
作者：
Fan J;Cheney PP;Bloch S;Xu B;Liang K;Odonkor CA;Edwards WB;Basak S;Mintz R;Biswas P;Achilefu S
金纳米粒子（AuNPs）由于其独特的光谱特性、化学和生物稳定性以及能够猝灭附着在其表面的有机染料的荧光而被广泛用于纳米医学。然而，球形AuNPs用于激活荧光传感分子过程的应用仅限于500 nm至600 nm光谱范围内的共振匹配荧光团，以最大限度地提高染料荧光猝灭效率。将荧光团系统扩展到发射>800 nm的近红外荧光方案，将有助于以高检测灵敏度分析多种生物事件。本研究的主要目的是确定球形aunp诱导的非共振近红外（NIR）荧光探针的辐射率抑制是否可以作为一种通用的纳米结构，用于水介质和癌细胞中活化的caspase-3的高灵敏度检测和成像。这需要开发可激活的caspase-3近红外荧光传感器，以克服水介质中表面涂层的非特异性降解和释放。我们利用球形aunp的荧光猝灭特性和多价性，开发了aunp模板的可激活近红外荧光探针，以检测活化的caspase-3，这是细胞内早期细胞死亡的报告因子。新鲜AuNPs包被一种多功能近红外荧光染料标记肽（LS422），该肽由RGD肽序列组成，靶向癌细胞表面αvβ3-整合素蛋白（αvβ3），介导肿瘤细胞对传感器的摄取和内化；一个用于报道通过caspase-3介导的近红外荧光增强诱导细胞死亡的DEVD肽序列；以及一个多齿正半胱氨酸序列，用于增强aunp上的自组装和稳定多功能结构。LS422的整合素结合亲和力和caspase-3动力学分别通过放射性寡配竞争结合和荧光肽测定。采用共聚焦近红外荧光光谱法和显微镜法检测肿瘤细胞内caspase-3、细胞活力和LS422内化水平。制备了窄尺寸AuNPs (13 nm)，并通过透射电镜和动态光散射对其进行了表征。在AuNPs上组装后，LS422对αvβ3的结合常数从13.2 nM提高到1.2 nM，提高了11倍。虽然LS422-AuNPs对caspase-3的催化转化率与参考荧光肽相似，但对该酶的结合亲和力增加了2倍。与αvβ3阳性而caspase-3阴性的乳腺癌MCF-7细胞不同，紫杉醇处理αvβ3和caspase-3阳性的肺癌A549细胞30分钟内荧光增强明显，这与caspase-3特异性激活LS422-AuNPs荧光有关。在荧光光谱仪中加入3.5 mW近红外激光源，检测灵敏度提高了一个数量级（检测极限~0.1 nM的cypate），并且相对于氙灯显著降低了信号噪声。这种灵敏度的增加使得在广泛的抑制剂浓度范围内检测底物水解而不光漂白cypate染料。多功能aunp展示了使用非共振猝灭策略来设计可激活的近红外荧光分子探针。纳米结构提供了一种选择性报告方法，用于检测活化的caspase-3，细胞活力成像，鉴定垂死细胞，以及可视化细胞内酶的功能状态。使用近红外荧光探针执行这些任务，创造了一个机会，将酶的体外和细胞分析转化为使用深层组织穿透近红外荧光分析方法对其功能状态的体内调查。
Gold nanoparticles (AuNPs) are commonly used in nanomedicine because of their unique spectral properties, chemical and biological stability, and ability to quench the fluorescence of organic dyes attached to their surfaces. However, the utility of spherical AuNPs for activatable fluorescence sensing of molecular processes have been confined to resonance-matched fluorophores in the 500 nm to 600 nm spectral range to maximize dye fluorescence quenching efficiency. Expanding the repertoire of fluorophore systems into the NIR fluorescence regimen with emission >800 nm will facilitate the analysis of multiple biological events with high detection sensitivity. The primary goal of this study is to determine if spherical AuNP-induced radiative rate suppression of non-resonant near-infrared (NIR) fluorescent probes can serve as a versatile nanoconstruct for highly sensitive detection and imaging of activated caspase-3 in aqueous media and cancer cells. This required the development of activatable NIR fluorescence sensors of caspase-3 designed to overcome the nonspecific degradation and release of the surface coatings in aqueous media. We harnessed the fluorescence-quenching properties and multivalency of spherical AuNPs to develop AuNP-templated activatable NIR fluorescent probes to detect activated caspase-3, an intracellular reporter of early cell death. Freshly AuNPs were coated with a multifunctional NIR fluorescent dye-labeled peptide (LS422) consisting of an RGD peptide sequence that targets αvβ3-integrin protein (αvβ3) on the surface of cancer cells to mediate the uptake and internalization of the sensors in tumor cells; a DEVD peptide sequence for reporting the induction of cell death through caspase-3 mediated NIR fluorescence enhancement; and a multidentate hexacysteine sequence for enhancing self-assembly and stabilizing the multifunctional construct on AuNPs. The integrin binding affinity of LS422 and caspase-3 kinetics were determined by a radioligand competitive binding and fluorogenic peptide assays, respectively. Detection of intracellular caspase-3, cell viability, and the internalization of LS422 in cancer cells were determined by confocal NIR fluorescence spectroscopy and microscopy. Narrow size AuNPs (13 nm) were prepared and characterized by transmission electron microscopy and dynamic light scattering. When assembled on the AuNPs, the binding constant of LS422 for αvβ3 improved 11-fold from 13.2 nM to 1.2 nM. Whereas the catalytic turnover of caspase-3 by LS422-AuNPs was similar to the reference fluorogenic peptide, the binding affinity for the enzyme increased by a factor of 2. Unlike the αvβ3 positive, but caspase-3 negative breast cancer MCF-7 cells, treatment of the αvβ3 and caspase-3 positive lung cancer A549 cells with Paclitaxel showed significant fluorescence enhancement within 30 minutes, which correlated with caspase-3 specific activation of LS422-AuNPs fluorescence. Incorporation of a 3.5 mW NIR laser source into our spectrofluorometer increased the detection sensitivity by an order of magnitude (limit of detection ~0.1 nM of cypate) and significantly decreased the signal noise relative to a xenon lamp. This gain in sensitivity enabled the detection of substrate hydrolysis at a broad range of inhibitor concentrations without photobleaching the cypate dye. The multifunctional AuNPs demonstrate the use of a non-resonant quenching strategy to design activatable NIR fluorescence molecular probes. The nanoconstruct offers a selective reporting method for detecting activated caspase-3, imaging of cell viability, identifying dying cells, and visualizing the functional status of intracellular enzymes. Performing these tasks with NIR fluorescent probes creates an opportunity to translate the in vitro and cellular analysis of enzymes into in vivo interrogation of their functional status using deep tissue penetrating NIR fluorescence analytical methods.
影响因子：
4.9
作者：
Zhang Z;Fan J;Cheney PP;Berezin MY;Edwards WB;Akers WJ;Shen D;Liang K;Culver JP;Achilefu S
通讯作者：
Achilefu S