Evaluation and regulation of cellular functions through in-situ nucleic acid imaging

In situ-imaging of nucleic acids, such as messenger RNAs and microRNAs, could be a promising tool to understand a molecular basis of cellular functions. Both highly sensitive DNA probes and effective cytosolic delivery systems are required to construct the imaging tool.

Polycationic carriers with amino groups responsive to endosomal pH (between 7.4 to 5.5) provide efficient gene transfection, being a promising carrier for intra-cellular delivery. Polyethylenimine (PEI), chitosan, the polyamidoamine (PAMAM) dendrimer and poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) are examples of polycationic transfection reagents with weakly basic amino groups that are protonated at endosomal pH. The efficacy of these reagents is thought to be due to their lysosomotropic activity. Osmotic puncture of lysosomes through the proton buffer effect was proposed. An enhanced membrane disrupting effect through direct interaction with the protonated amino groups was recently proposed. To date, weakly basic amino groups, such as the imidazole group and ethylene diamine units, have been examined as endosomal pH-responsive groups. However, lack of readily incorporable basic groups with pKas around the endosomal pH has made polymeric carrier design difficult. Very few basic groups with pKa in the appropriate rage occur naturally, limiting our ability to design biocompatible gene carriers design.

We focused on the α-amino groups of naturally occurring α-amino acids as endosomal pH-responsive groups. The α-amino groups of the α-amino acids in an aqueous buffer have pKa > 9 and could not respond to endosomal pH. While basicity of the α-amino group is reduced by electron-withdrawing effect of the neighboring α-carboxyl group, its protonation is promoted by the neighboring α-carboxyl anion. It is, therefore, possible to reduce the pKa of the α-amino group to the neutral pH range by converting the carboxyl group to a nonionic ester or amide group. Further, the pKa of amino groups can be decreased when cationic groups are densely arranged along a polymer chain. Hence, polymers having α-amino acids as pendant groups are potential carriers with endosome-escaping function.

Amino groups of linear poly(allylamine) (PAA) or poly(L-lysine) (PLL) were coupled with α-carboxyl groups of α-amino acids (Gly, His, Lys, Arg, and Orn). Acid-base titration indicated that Lys-, Arg-, and Orn-pendant polymers had both strongly basic groups and endosomal pH-responsive α-amino groups. These polymers, like PAA and PLL, formed stable complexes with DNA. Lys-, Arg-, and Orn-pendant polymers were effective transfection reagents independent of the backbone polymers. The pH-responsive α-amino groups enhanced transfection activity as shown by the observation that acetylation of the α-amino group resulted in a considerable loss in transfection activity. These results strongly suggested a lysosomotropic activity of the α-amino groups. Among the α-amino acid-pendant polymers tested, the Orn-pendant polymer exhibited the highest transfection activity/toxicity index. Since PLL with α-amino acid-pendants is composed of naturally occurring amino acids, it is expected to be biodegradable, and these reagents have promise as cytosolic carriers.

主な論文

T. Wada, A. Kano, N. Shimada, A. Maruyama, α-Amino acid pendant polymers as endosomal pH-responsive gene carriers, Macromol. Res., in press.
H. Asanuma, T. Osawa, H. Kashida, T. Fujii, X. Liang, K. Niwa, Y.Yoshida, N. Shimada, A. Maruyama, Highly sensitive in-stem molecular beacon system chaperoned by a cationic copolymer, Chem. Commun, 48, 1760 – 1762, (2012), in press.
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