纳秒激光激活金纳米粒子透化细胞膜的重要因素
Important factors for cell-membrane permeabilization by gold nanoparticles activated by nanosecond-laser irradiation
摘要：
目的：脉冲激光照射附着在细胞上的吸光金纳米颗粒（AuNPs）瞬时增加细胞膜通透性，用于靶向分子递送。在这里，我们用AuNPs靶向卵巢癌细胞系OVCAR-3上的EGFR。为了优化膜渗透性并证明分子递送到贴壁OVCAR-3细胞中，我们系统地研究了不同的实验条件。材料和方法：通过缀合针对EGFR的抗体西妥昔单抗使AuNP（30 nm）官能化。通过银染、多光子成像和荧光寿命成像证明了颗粒的选择性结合。激光照射后，OVCAR-3细胞的膜通透性在不同条件下的金纳米粒子浓度，细胞孵育介质，和细胞-金纳米粒子孵育时间进行了研究。通过流式细胞术、测量碘化丙啶和异硫氰酸荧光素-葡聚糖摄取来评估膜渗透性和细胞活力。结果：EGFR-AuNP可有效靶向贴壁生长的OVCAR-3细胞。激光照射导致成功的透化，150 kDa的葡聚糖被成功地递送到细胞中，效率约为70%。结论：抗体靶向和激光照射的金纳米粒子可用于将分子递送到粘附细胞中。功效不仅取决于激光参数，而且取决于AuNP：细胞比率、细胞孵育介质和细胞- AuNP孵育时间。
英文摘要：
Purpose: Pulsed-laser irradiation of light-absorbing gold nanoparticles (AuNPs) attached to cells transiently increases cell membrane permeability for targeted molecule delivery. Here, we targeted EGFR on the ovarian carcinoma cell line OVCAR-3 with AuNPs. In order to optimize membrane permeability and to demonstrate molecule delivery into adherent OVCAR-3 cells, we systematically investigated different experimental conditions. Materials and methods: AuNPs (30 nm) were functionalized by conjugation of the antibody cetuximab against EGFR. Selective binding of the particles was demonstrated by silver staining, multiphoton imaging, and fluorescence-lifetime imaging. After laser irradiation, membrane permeability of OVCAR-3 cells was studied under different conditions of AuNP concentration, cell-incubation medium, and cell - AuNP incubation time. Membrane permeability and cell viability were evaluated by flow cytometry, measuring propidium iodide and fluorescein isothiocyanate - dextran uptake. Results: Adherently growing OVCAR-3 cells can be effectively targeted with EGFR-AuNP. Laser irradiation led to successful permeabilization, and 150 kDa dextran was successfully delivered into cells with about 70% efficiency. Conclusion: Antibody-targeted and laser-irradiated AuNPs can be used to deliver molecules into adherent cells. Efficacy depends not only on laser parameters but also on AuNP:cell ratio, cell-incubation medium, and cell - AuNP incubation time.
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