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.

Folate Receptor Targeted Delivery of siRNA and Paclitaxel to Ovarian Cancer Cells via Folate Conjugated Triblock Copolymer to Overcome TLR4 Driven Chemotherapy Resistance

“…This was followed by coupling the previous product of acrylate-PCL- b -PEG-Fol (molecule C) with hy -PEI (25 kDa), as previously described. 36 A total of six different conjugates were synthesized consisting of two different PEG lengths (3.5 kDa or 5 kDa), varying grafting densities of PCL- b -PEG-Fol (10 or 30 µmol per 10 µmol of PEI), as well as one null folate conjugate (Table 1). Compounds synthesized were characterized by 1 HNMR, UV spectroscopy, and a folate composition assay.…”

Targeting receptor-mediated endocytotic pathways with nanoparticles: Rationale and advances

“…For instance, an amphiphilic peptide conjugated to folate can be mixed with polymers to form nanoparticles that are capable of encapsulating DNA for gene delivery [147, 148]. The uptake of folate-conjugated nanoparticles by cell lines overexpressing folate receptors can be inhibited by competition with free folate, indicating the specificity of folate-targeting [147, 148]. …”

From Stealthy Polymersomes and Filomicelles to “Self” Peptide-Nanoparticles for Cancer Therapy

“…Amphiphilic block copolymers that are suitable for biomedical application can be readily obtained by using hydrophilic polymers like PEG as macroinitiators. Furthermore, this synthetic strategy allows for the introduction of end-functional groups in the block copolymer that can be used for the incorporation of relevant molecules for application in drug delivery (23, 24). Alternatively, both polymers can be linked after being polymerized independently, provided that both possess functional end groups that can react with each other.…”