title: Modular synthesis of folate conjugated ternary copolymers: polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate for targeted gene delivery.
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.
authors.affiliation: Department of Pharmaceutics and Biopharmacy, Philipps-University Marburg , Ketzerbach 63, D-35032 Marburg, Germany.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Mengyao
authors.initials: M
authors.lastname: Zheng
authors.forename: Thomas
authors.initials: T
authors.lastname: Renette
authors.forename: Thomas
authors.initials: T
authors.lastname: Kissel
mesh_terms: D045744:Cell Line, Tumor; D057930:Click Chemistry; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics; D004705:Endocytosis; D005492:Folic Acid / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D011091:Polyesters / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011094:Polyethyleneimine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D011091:Polyesters; C576039:polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol); D011094:Polyethyleneimine; D004247:DNA; D005492:Folic Acid