Preparation of cells for imaging

We used both darkfield and confocal imaging to determine the distribution of GNPs. For darkfield imaging, all cell lines were plated on coverslips placed on the bottom of 6 well dishes. The cells were treated with GNPPEG-RGD for 24 h to determine the extent of endocytosis. Upon completion of NP incubation, the cells were rinsed three times with PBS and fixed using 4% paraformaldehyde (PFA) for 20 min at 37 ͦC. The cover slips were washed with PBS, removed from each well, and mounted to a glass slide using Prolong Glass Antifade Mountant. Each sample was imaged using darkfield microscopy and HSI (CytoViva) under a 60X objective.

Live-cell imaging was performed using confocal microscopy (Zeiss LSM 980) using a 60X oil immersion lens. For confocal imaging, GNPPEG-RGD complexes had PEG-Cy5 (excitation 633 nm, emission filter 650 nm LP) conjugated as previously mentioned. To see general structure of the cell, microtubules (MTs) were stained with a viral transfection stain (CellLight Tubulin-GFP), which contains DNA coding for an α-tubulin/GFP construct. For live-cell confocal imaging, cells were plated on 3 cm coverslip-bottomed dishes in FluoroBrite media. For staining MTs, the cells were incubated in the viral stain for  > 24 h prior to treatment with fluorescent GNPPEG-RGD. After NP incubation, the cells were imaged after 24 h of endocytosis. To determine the retention, cells were first incubated with GNPs for 24 h, removed the media, added fresh media, and incubated for 24 h. All imaging parameters (acquisition settings) used between experiments was maintained constant.