Fluorophore:Dendrimer Ratio Impacts Cellular Uptake and Intracellular Fluorescence Lifetime

G5-NH₂-TAMRA_n (n = 1–4, 5+, and 1.5_avg) were prepared with n = 1–4 as a precise dye:dendrimer ratio, 5+ as a mixture of dendrimers with 5 or more dye per dendrimer, and 1.5_avg as a Poisson distribution of dye:dendrimer ratios with a mean of 1.5 dye per dendrimer. The absorption intensity increased sublinearly with n whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer. Flow cytometry was employed to quantify uptake into HEK293A cells. Dendrimers with 2–4 dyes were found to have greater uptake than dendrimer with a single dye. Fluorescence lifetime imaging microscopy (FLIM) showed that the different dye:dendrimer ratio alone was sufficient to change the fluorescence lifetime of the material observed inside cells. We also observed that the lifetime of G5-NH₂-TAMRA₅₊ increased when present in the cell as compared to solution. However, cells treated with G5-NH₂-TAMRA_1.5avg did not exhibit the high lifetime components present in G5-NH₂-TAMRA₁ and G5-NH₂-TAMRA₅₊. In general, the effects of the dye:dendrimer ratio on fluorescence lifetime were of similar magnitude to environmentally induced lifetime shifts.