Fig. 2. In vitro uptake of 4-(N)-GemC18 in solution or in 4-(N)-GemC18-SLNs by TC-1-GR cells, and the effect of the neutralization of lysosomes with NH₄Cl on the intracellular degradation/hydrolysis of 4-(N)-GemC18 and the cytotoxicity of 4-(N)-GemC18 in solution or in 4-(N)-GemC18-SLNs in TC-1-GR cells.

(A) The level of 4-(N)-GemC18 in TC-1-GR cells after the cells were incubated with 40 μM of 4-(N)-GemC18 in solution or 4-(N)-GemC18-SLNs at either 4°C or 37°C for up to 6 h (n = 3). (B) The level of 4-(N)-GemC18 in TC-1-GR cells after the cells were incubated with 40 μM of 4-(N)-GemC18 in solution or 4-(N)-GemC18-SLNs for 2 h in the presence of various endocytosis inhibitors. Data shown are normalized to the levels of 4-(N)-GemC18 in TC-1-GR cells after 2 h of incubation in the absence of any inhibitors. ^aP < 0.01, 4-(N)-GemC18 vs. 4-(N)-GemC18-SLNs when chlorpromazine was used. (C) Co-localization of fluorescein-labeled SLNs (green) and lysosomes (red) at 0, 2 and 6 h after the removal of the nanoparticle-containing medium. Cell nuclei are in blue. (D) The level of 4-(N)-GemC18 detected in the lysosomal fraction of TC-1-GR cells after treatment with 40 μM of 4-(N)-GemC18 in solution or 4-(N)-GemC18-SLNs for 3 h (n = 3). Values reported are the % of 4-(N)-GemC18 detected in the lysosomal fraction divided by the total 4-(N)-GemC18 internalized by the cells within 3 h. ^bP < 0.05. (E) The percentage of 4-(N)-GemC18 remaining in TC-1-GR cells after it was delivered into the cells as 4-(N)-GemC18 alone or as 4-(N)-GemC18-SLNs and then incubated in the presence or absence of NH₄Cl for 16 h. ^cP < 0.001, 0 mM vs. 10 mM NH₄Cl for 4-(N)-GemC18-SLNs. (F) The IC₅₀ values of 4-(N)-GemC18 in solution and 4-(N)-GemC18-SLNs in TC-1-GR cells after 24 h of treatment in the presence or absence of 10 mM NH₄Cl (n = 5). ^dP < 0.01, 0 mM vs. 10 mM NH₄Cl for 4-(N)-GemC18-SLNs.