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Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be overexpressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse-like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized and evaluated for targeted siRNA delivery via folate-FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI-PCL-PEG-Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI-PCL-PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV, which was lower than that of micelleplexes without folate-conjugation (+13-16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI-PCL-PEG-Fol/siRNA micelleplexes were observed in SKOV-3 cells, an FR overexpressing cell line, in comparison with the nonfolate-conjugated ones. Moreover, PEI-PCL-PEG-Fol/siRNA micelleplexes exhibited excellent stability in vivo during the analysis of 120 min and a longer circulation half life than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per gram in the tumor after 24 h due to the effective folate targeting and the prolonged circulation.

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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