title: Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging.
abstract: We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.
authors.affiliation: Groupe des Matériaux Organiques, Institut de Physique et Chimie des Matériaux de Strasbourg, Université Louis Pasteur (CNRS, UMR 7504), BP 43 23 rue du Loess, F-67034 Strasbourg, France.
authors.forename: Ali
authors.initials: A
authors.lastname: Hayek
authors.forename: Sebnem
authors.initials: S
authors.lastname: Ercelen
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bolze
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Nicoud
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Schaub
authors.forename: Patrice L
authors.initials: PL
authors.lastname: Baldeck
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
mesh_terms: D004247:DNA / Q000737:chemistry; D004705:Endocytosis; D005449:Fluorenes / Q000138:chemical synthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010647:Phenyl Ethers / Q000138:chemical synthesis / Q000737:chemistry*; D017785:Photons; D011094:Polyethyleneimine / Q000737:chemistry*; D013057:Spectrum Analysis; D014162:Transfection
chemical_list: C522261:4-(2-(4-(4-isothiocyanatobutyloxy)-3,5-dimethoxy)styryl)-4'-(2-(3,4,5-tris(1,4,7,10,13-pentaoxotetradecyl))styryl)-9,9'-di-n-propylfluorene; D005449:Fluorenes; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D010647:Phenyl Ethers; C037152:isothiocyanic acid; D011094:Polyethyleneimine; D004247:DNA