Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection.
Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection.
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DOI：
10.2147/ijn.s39658
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发表时间：
2013
影响因子：
8
通讯作者：
Rotomskis R
中科院分区：
文献类型：
--
作者：
Damalakiene L;Karabanovas V;Bagdonas S;Valius M;Rotomskis R
本研究的目的是通过关注非功能化量子点在不同细胞系中的时间依赖性积累和细胞内定位，与显微注射量子点相比，阐明非功能化量子点的自然摄取机制。应用共焦和稳态荧光光谱方法分析了非靶向 CdSe/ZnS 羧基涂层量子点（发射峰 625 nm）在 NIH3T3、MCF-7 和 HepG2 细胞中的累积动态。通过 Lysotracker® 染色研究量子点的细胞内共定位。在低温（4°C）下，细胞对量子点的吸收急剧减少，表明该过程依赖于能量。量子点细胞内定位的摄取动力学和成像揭示了37℃下羧基包被的量子点的三个积累阶段，即平台期、生长阶段和饱和阶段，其中包括四个形态阶段：粘附在细胞膜上；形成遍布整个细胞质的颗粒簇；粒状簇在核周区域的定位；多泡体样结构的形成及其在细胞质中的重新分布。在细胞质中观察到含有直径约0.5-8μm范围内的细胞内囊泡的多种量子点，但在细胞核中没有发现。含有量子点的囊泡在培养24小时后在NIH3T3细胞中形成多囊泡体样结构，在无血清培养基中Lysotracker阴性，在完全培养基中Lysotracker阳性。显微注射的量子点在细胞质中保持均匀分布至少 24 小时。细胞中量子点的自然吸收通过需要能量的机制经历三个积累阶段。与孵育相比，显微注射量子点后的细胞内分布的鲜明对比以及量子点从囊泡到细胞溶质的有限转移以及反之亦然，支持了量子点自然摄取的内吞起源。从培养基中吸附蛋白质的量子点在积累的最后阶段与不含蛋白质的量子点有不同的命运，这意味着不同的内化途径。
The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®. The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison with incubation as well as the limited transfer of quantum dots from vesicles into the cytosol and vice versa support the endocytotic origin of the natural uptake of quantum dots. Quantum dots with proteins adsorbed from the culture medium had a different fate in the final stage of accumulation from that of the protein-free quantum dots, implying different internalization pathways.