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Real‐Time Intracellular Transport of Gene Nanocarriers Studied by Multiple Particle Tracking

We used real‐time multiple particle tracking to quantitatively characterize the type and rates of transport of gene nanocarriers within live cells. The heterogeneous cytoplasmic transport of polyethylenimine (PEI)/DNA gene carriers was quantified by tracking their mean‐square displacements over time and classified into active and nonactive transport populations on the basis of their effective diffusivities versus time. Nonactive gene carriers frequently displayed hop‐diffusion trajectories, suggesting a porous cytoplasmic network of flexible biopolymers or sequential attachment and detachment events. Microtubule‐dependent active transport of gene carriers resulted in an effective diffusivity 30‐fold greater than that of nonactive carriers (at a time scale of 3 s). Compared to nonactive carriers in control cells with intact microtubules, microtubule depolymerization enhanced short‐range motion of gene carriers but resulted in similar long‐range transport. Multiple particle tracking characterizes gene carrier transport in complex biological environments and, therefore, may be a useful tool in quantifying rate‐limiting steps in gene delivery within cells and other biological media.