DNA binding chelates for nonviral gene delivery imaging.

Abstract

Noninvasive in vivo monitoring of gene delivery would provide a critically important information regarding the spatial distribution, local concentration, kinetics of removal and/or biodegradation of the expression vector. We developed a novel approach to noninvasive gene delivery imaging using heterobifunctional peptide-based chelates (PBC) bearing double-stranded DNA-binding groups and a technetium-binding amino acid motif. One of such chelates: Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys(4)-Lys-(N-epsilon-[4-(psoralen-8-yloxy)]butyrate)-NH(2) has been characterized and labeled with reduced (99m)Tc pertechnetate (oxotechnetate). The psoralen moiety (a DNA binding group of PBC) allowed linking to double-stranded DNA upon short-term irradiation with the near UV range light (>320 nm). Approximately 30-40% of added (99m)Tc-labeled PBC was nonextractable and co-eluted with a model pCMV-GFP vector during the gel-permeation chromatography. Nuclear imaging of "naked" DNA and DNA complexes with lipid-based transfection reagents ("lipoplexes") has been performed after systemic or local administration of (99m)Tc-PBC-labeled DNA in mice. Imaging results were corroborated with the biodistribution using (99m)Tc-PBC and (32)P-labeled DNA and lipoplexes. A markedly different biodistribution of (99m)Tc PBC-labeled DNA and lipoplexes was observed with the latter being rapidly trapped in the liver, spleen and lung. (99m)Tc PBC-DNA was used as an imaging tracer during in vivo transfection of B16 melanoma by local injection of "naked" (99m)Tc PBC-DNA and corresponding lipoplexes. As demonstrated by nuclear imaging, (99m)Tc PBC-DNA lipoplexes showed a slower elimination from the site of injection than (99m)Tc PBC-DNA alone. This result correlated with a higher expression of marker mRNA and green fluorescent protein as determined using RT-PCR and immunohistochemistry, respectively.