Spatiotemporal Tracing of the Cellular Internalization Process of Rod- Shaped Nanostructures

YF Wang and QR Zhang and FL Tian and HD Wang and YF Wang and XW Ma and QQ Huang and MJ Cai and YL Ji and XC Wu and YL Gan and Y Yan and KA Dawson and ST Guo and JC Zhang and XH Shi and YP Shan and XJ Liang, ACS NANO, 16, 4059-4071 (2022).

DOI: 10.1021/acsnano.1c09684

Endocytosis, as one of the main ways for nanostructures enter cells, is affected by several aspects, and shape is an especially critical aspect during the endocytosis of nanostructures. However, it has remained challenging to capture the dynamic internalization behaviors of rod- shaped nanostructures while also probing the mechanical aspects of the internalization. Here, using the atomic force microscopy-based force tracing technique, transmission electron microscopy, and molecular dynamic simulation, we mapped the detailed internalization behaviors of rod-shaped nanostructures with different aspect ratios at the single- particle level. We found that the gold nanorod is endocytosed in a noncontinuous and force-rebound rotation manner, herein named "intermittent rotation". The force tracing test indicated that the internalization force (similar to 81 pN, similar to 108 pN, and similar to 157 pN) and time (similar to 0.56 s, similar to 0.66 s, and, similar to 1.14 s for a 12.10 nm x 11.96 nm gold nanosphere and 26.15 nm x 13.05 nm and 48.71 nm x 12.45 nm gold nanorods, respectively) are positively correlated with the aspect ratios. However, internalization speed is negatively correlated with internalization time, irrespective of the aspect ratio. Further, the energy analysis suggested that intermittent rotation from the horizontal to vertical direction can reduce energy dissipation during the internalization process. Thus, to overcome the energy barrier of internalization, the number and angle of rotation increases with aspect ratios. Our findings provide critical missing evidence of rod-shaped nanostructure's internalization, which is essential for fundamentally understanding the internalization mechanism in living cells.

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