alexa-fluor-546 and tetramethylrhodamine

Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. D-amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg9-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg9-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant.

Topics: Amino Acid Sequence; Animals; Biological Transport; Cell Membrane; Cell Survival; CHO Cells; Cricetinae; Cricetulus; Cytosol; Drug Delivery Systems; Endocytosis; Female; Fluorescent Dyes; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Peptides; Quinolinium Compounds; Reproducibility of Results; Rhodamines

The heterogeneity on photoinduced electron transfer (PET) kinetics between a labeled fluorophore and an amino acid residue has been extensively studied in biopolymers. However in aqueous solutions, the heterogeneity on PET kinetics between a fluorophore and a quencher has rarely been reported. Herein, we selected four commonly used fluorophores, such as tetramethylrhodamine (TMR), Rhodamine B (RhB), Alexa fluor 546 (Alexa546), and Atto655, and studied their respective PET kinetics in 50 mM tryptophan solutions with femtosecond transient absorption spectroscopy to explore the structural heterogeneity in their corresponding collision complexes. We measured the decay of the first excited electronic state of respective fluorophore with and without 50 mM tryptophan in aqueous solutions, and derived the charge separation rate in their corresponding collision complexes. We found that the PET process of all selected fluorophores in 50 mM tryptophan solutions has two charge separation rates, which indicates that the relevant states in the collision complex between respective fluorophore and tryptophan have strong structural heterogeneity. These femtosecond PET measurements are in agreement with Vaiana's molecular dynamics simulation (J. Am. Chem. Soc.2003, 125, 14564). In addition, with the obtained PET kinetic parameters, we derived the relative brightness of the collision complex between respective fluorophore and tryptophan, which are important parameters for the PET based fluorescence correlation spectroscopy study involving these fluorophores in biopolymers.

Topics: Fluorescent Dyes; Kinetics; Light; Quinolinium Compounds; Rhodamines; Spectrometry, Fluorescence; Tryptophan; Water