Quantitative Assessment of Endosomal Escape of Various Endocytosed Polymer‐Encapsulated Molecular Cargos upon Photothermal Heating

Abstract Encapsulated molecular cargos are efficiently endocytosed by cells. For cytosolic delivery, understanding the dynamic process of cargos release from the carrier vehicles used for encapsulation and the lysosomes where the carrier vehicles are trapped (which in general is the bottleneck), followed by diffusion in the cytosol is important for improving drug/gene delivery strategies. A methodology is reported to image this process on a millisecond scale and to quantitatively analyze the data. Polyelectrolyte capsules with embedded gold nanostars to encapsulate 43 fluorescent molecular cargos with diverse properties, ranging from small fluorophores to fluorescently labeled proteins, siRNA, etc., are used. By short laser irradiation intracellular release of the molecular cargos from endocytosed capsules into the cytosol is triggered, and their intracellular spreading is imaged. Most of the released molecular cargos evenly distribute inside the entire cell, while others are enriched in certain cell compartments. The time the different molecular cargos take to distribute within cells, i.e., the spreading time, is used as a quantifier. Quantitative analysis reveals that intracellular spread cannot be described by free diffusion, but is determined by interaction of the molecular cargo with intracellular components.