Modular Synthesis of Folate Conjugated Ternary Copolymers: Polyethylenimine-<i>graft</i>-Polycaprolactone-<i>block</i>-Poly(ethylene glycol)-Folate for Targeted Gene Delivery

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-<i>g</i>-PCL-<i>b</i>-PEG-Fol) as targeted gene delivery system using a modular synthesis approach including “click” conjugation of folate moieties with heterobifunctional PEG-<i>b</i>-PCL at PEG terminus and subsequently the introduction of PEI by a Michael addition between folate-PEG-<i>b</i>-PCL and PEI via active PCL terminus. This well-controlled synthetic procedure avoids tedious separation of byproduct. The structure of PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol was confirmed by ¹H NMR and UV spectra. DNA condensation of PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol was tested using a SYBR Gold quenching assay and agarose gel electrophoresis upon heparin competition assay. Although PEI-<i>g</i>-PCL-<i>b</i>-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-<i>g</i>-PCL-<i>b</i>-PEG-Fol/pDNA polyplexes was observed in FR overexpressing KB cells in comparison to unmodified PEI-<i>g</i>-PCL-<i>b</i>-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-<i>g</i>-PCL-<i>b</i>-PEG-Fol/pDNA polyplexes was increased approximately 14-fold in comparison to folate-negative polyplexes. Therefore, the PEI-<i>g</i>-PCL-<i>b</i>-PEG-Fol merits further investigation under in vivo conditions for targeting FR overexpressing tumors