title: Fluorophore:dendrimer ratio impacts cellular uptake and intracellular fluorescence lifetime.
abstract: G5-NH2-TAMRAn (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-TAMRA1.5(avg) 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.
authors.affiliation: Department of Chemistry, ‡Department of Biomedical Engineering, and §Department of Physics, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors.forename: Casey A
authors.initials: CA
authors.lastname: Dougherty
authors.forename: Sriram
authors.initials: S
authors.lastname: Vaidyanathan
authors.forename: Bradford G
authors.initials: BG
authors.lastname: Orr
authors.forename: Mark M
authors.initials: MM
authors.lastname: Banaszak Holl
mesh_terms: D003593:Cytoplasm / Q000378:metabolism*; D050091:Dendrimers / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D061848:Optical Imaging; D012235:Rhodamines / Q000032:analysis / Q000378:metabolism*
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D050091:Dendrimers; D005456:Fluorescent Dyes; C104700:PAMAM Starburst; D012235:Rhodamines