Fig. 5: Mechanisms of cellular uptake of ^intraArg-TRM.

A Competition experiment using arginine. The cells were incubated with DiD-loaded ^intraArg-TRM (400 µg/mL) at 42 °C for 30 min in the culture medium containing 10% FBS supplemented with various concentrations of arginine hydrochloride (mean ± SD, n = 4, Dunnett’s test; versus 0 mM). The culture medium originally contained 1.15 mM arginine hydrochloride according to the supplier. B Confocal fluorescence microscopic images of colon-26 cells incubated with ^intraArg-TRM (400 µg/mL) at 42 °C for 30 min and further incubated at 37 °C for 240 min. Red: DiD-loaded micelles, green: LysoTracker Red; scale bars: 20 μm. C Effect of endocytosis inhibitors on the cellular uptake of DiD-loaded ^intraArg-TRM. The cells were preincubated with various endocytosis inhibitors in the culture medium containing 10% FBS for 60 min at 37 °C, followed by incubation with ^intraArg-TRM (400 µg/mL) at 42 °C for 30 min in the presence of inhibitors (mean ± SD, n = 4). The concentration of each inhibitor for this experiment was set based on the cytotoxicity determined by WST-8 assay (Table S5) and morphological observation. ^‡p < 0.001 versus without inhibitor (Dunnett’s test). D Cellular uptake of a macropinocytosis marker molecule. The cells were co-incubated with TRITC-labeled 70 kDa dextran (100 µg/mL) and DiD-loaded micelles (400 µg/mL) for 30 min at 37 or 42 °C (mean ± SD, n = 4). ^†p < 0.005 versus 37 °C (Student’s t test). E Schematic illustration of the proposed mechanism and pathways of cellular internalization of ^intraArg-TRM upon heating.