Abstract
Folate receptor (FR) is overexpressed in a variety of human cancers. Gene delivery vectors conjugated with folate as a ligand could possibly deliver gene materials into target tumor cells via FR-mediated endocytosis. This study addresses novel folate-conjugated ternary copolymers based on polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (PEI-g-PCL-b-PEG-Fol) as targeted gene delivery system using a modular synthesis approach including "click" conjugation of folate moieties with heterobifunctional PEG-b-PCL at PEG terminus and subsequently the introduction of PEI by a Michael addition between folate-PEG-b-PCL and PEI via active PCL terminus. This well-controlled synthetic procedure avoids tedious separation of byproduct. The structure of PEI-g-PCL-b-PEG-Fol was confirmed by (1)H NMR and UV spectra. DNA condensation of PEI-g-PCL-b-PEG-Fol was tested using a SYBR Gold quenching assay and agarose gel electrophoresis upon heparin competition assay. Although PEI-g-PCL-b-PEG-Fol could condense DNA completely at N/P ratio >2, polyplexes of N/P ratio 10 with sizes of about 120 nm and positive zeta potentials were selected for further biological evaluations due to polyplex stability. An enhancement of cellular uptake of PEI-g-PCL-b-PEG-Fol/pDNA polyplexes was observed in FR overexpressing KB cells in comparison to unmodified PEI-g-PCL-b-PEG, through flow cytometry analysis and confocal laser scanning imaging. Importantly, this enhanced cellular uptake could be inhibited by free folic acid and did not occur in FR-negative A549 cells, demonstrating specific cell uptake by FR-mediated endocytosis. Furthermore, the transfection efficiency of PEI-g-PCL-b-PEG-Fol/pDNA polyplexes was increased approximately 14-fold in comparison to folate-negative polyplexes. Therefore, the PEI-g-PCL-b-PEG-Fol merits further investigation under in vivo conditions for targeting FR overexpressing tumors.
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