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.

Polyethylenimine-based micro/nanoparticles as vaccine adjuvants

“…NPs and successfully transferred DNA into nerve stem cells. 2 Subsequently, PEI has drawn more attention in biomedical field, especially as drug carriers, [13][14][15] biological labels 16,17 and vaccine adjuvants (Table 1). 18 Among all the delivered drugs, the most common ones are nucleic acids.…”

“…20,21 PEI also works as a biological label when conjugated with imaging agents (such as the Fe 3 O 4 and fluorescent NPs), and reflects the specific cell or sub-cell information, such as migrations and other behaviors, under certain conditions. 16,22,23 The adjuvant effect of PEI is an emerging area. In a series of recent studies, vaccines composed of PEI as the immunostimulants are quite competent in treating infections or tumors.…”

Two‐Photon Absorption Properties and Structures of BODIPY and Its Dyad, Triad and Tetrad

“…Two‐photon absorption cross‐sections : The two‐photon excitation spectra were obtained by upconverted fluorescence measurements using a Ti:sapphire femtosecond laser Insight DS with pulse width <120 fs and a repetition rate of 80 MHz (Spectra‐Physics) as described previously . The excitation beam was collimated over the cell length (10 mm) and the fluorescence, collected at 90° of the excitation beam, was focused into an optical fiber connected to a spectrometer.…”

Biophysical characterization of quaternary pyridinium functionalized polynorbornenes for DNA complexation and their cellular interactions

“…The size and the zeta potential measurements of the complexes were made by Zetamaster 3000 (Malvern Instruments, Paris, FRANCE) with the following specifications: sampling time, 30 s; medium viscosity, 1.054 cP; refractive index (RI) medium, 1.34; RI particle, 1.45; scattering angle, 90°; temperature, 25°C. Data were analyzed using the multimodal number distribution software of the instrument …”