Department of Engineering Design, Indian Institute of Technology Madras, Chennai 600036, India.
Department of Electrical Engineering, University of Cambridge, Cambridge CB3 0FA, UK.
Analyst. 2021 Aug 7;146(15):4756-4766. doi: 10.1039/d0an02432e. Epub 2021 Jul 9.
Targeted intracellular delivery of biomolecules and therapeutic cargo enables the controlled manipulation of cellular processes. Laser-based optoporation has emerged as a versatile, non-invasive technique that employs light-based transient physical disruption of the cell membrane and achieves high transfection efficiency with low cell damage. Testing of the delivery efficiency of optoporation-based techniques has been conducted on single cells in monolayers, but its applicability in three-dimensional (3D) cell clusters/spheroids has not been explored. Cancer cells grown as 3D tumor spheroids are widely used in anti-cancer drug screening and can be potentially employed for testing delivery efficiency. Towards this goal, we demonstrated the optoporation-based high-throughput intracellular delivery of a model fluorescent cargo (propidium iodide, PI) within 3D SiHa human cervical cancer spheroids. To enable this technique, nano-spiked core-shell gold-coated polystyrene nanoparticles (ns-AuNPs) with a high surface-to-volume ratio were fabricated. ns-AuNPs exhibited high electric field enhancement and highly localized heating at an excitation wavelength of 680 nm. ns-AuNPs were co-incubated with cancer cells within hanging droplets to enable the rapid aggregation and assembly of spheroids. Nanosecond pulsed-laser excitation at the optimized values of laser fluence (45 mJ cm), pulse frequency (10 Hz), laser exposure time (30 s), and ns-AuNP concentration (5 × 10 particles per ml) resulted in the successful delivery of PI dye into cancer cells. This technique ensured high delivery efficiency (89.6 ± 2.8%) while maintaining high cellular viability (97.4 ± 0.4%), thereby validating the applicability of this technique for intracellular delivery. The optoporation-based strategy can enable high-throughput single cell manipulation, is scalable towards larger 3D tissue constructs, and may provide translational benefits for the delivery of anti-cancer therapeutics to tumors.
靶向细胞内生物分子和治疗 cargo 的输送使细胞过程的控制操纵成为可能。基于激光的光穿孔已成为一种多功能、非侵入性技术，它利用基于光的细胞膜瞬时物理破坏，并实现高转染效率和低细胞损伤。基于光穿孔的技术的输送效率测试已经在单层中的单细胞上进行，但尚未探索其在三维 (3D) 细胞簇/球体中的适用性。作为 3D 肿瘤球体生长的癌细胞广泛用于抗癌药物筛选，并可潜在用于测试输送效率。为此，我们在 3D SiHa 人宫颈癌球体中演示了基于光穿孔的模型荧光 cargo（碘化丙啶，PI）的高通量细胞内输送。为了实现这一技术，制造了具有高表面积与体积比的纳米刺核壳金涂聚苯乙烯纳米颗粒（ns-AuNPs）。ns-AuNPs 在 680nm 的激发波长下表现出高电场增强和高度局域加热。ns-AuNPs 与癌细胞一起在悬滴中孵育，以实现球体的快速聚集和组装。在优化的激光能量密度（45 mJ cm）、脉冲频率（10 Hz）、激光暴露时间（30 s）和 ns-AuNP 浓度（5×10 个颗粒/ml）下进行纳秒脉冲激光激发，导致 PI 染料成功输送到癌细胞中。该技术确保了高输送效率（89.6±2.8%），同时保持高细胞活力（97.4±0.4%），从而验证了该技术在细胞内输送中的适用性。基于光穿孔的策略可以实现高通量单细胞操作，可扩展至更大的 3D 组织构建体，并可能为抗癌治疗剂输送到肿瘤提供转化益处。
