The stability and targeting ability of nanocarrier gene delivery systems are necessary conditions to ensure the good therapeutic effect and low nonspecific toxicity of cancer treatment. Poly(ethylene glycol) (PEG) has been widely applied for improving stability and as a spacer for linking ligands and nanocarriers to improve targetability. However, the cellular uptake and endosomal escape capacity of nanocarriers has been seriously harmed due to the introduction of PEG. In the present study, we synthesized a new gene delivery vector by coupling divalent folate-PEG (PEG3.4k-FA2) onto polyamidoamine-polyethylenimine (PME) copolymer (PME-(PEG3.4k-FA2)1.72). Both PEG and monovalent folate-PEG (PEG3.4k-FA1) modified PME were prepared as control polymers, which were named as PME-(PEG3.5k)1.69 and PME-(PEG3.4k-FA1)1.66, respectively. PME-(PEG3.4k-FA2)1.72 exhibited strong DNA condensation capacity like parent polymer PME which was not significantly influenced by PEG. PME-(PEG3.4k-FA2)1.72/DNA complexes at N/P = 10 had a diameter ∼143 nm and zeta potential ∼13 mV and showed the lowest cytotoxicity and hemolysis and the highest transfection efficiency among all tested polymers. In folate receptor positive (FR-positive) cells, the cellular uptake and transfection efficiency were increased with the increase in the number of folates coupled on PEG; the order was PME-(PEG3.4k-FA2)1.72 > PME-(PEG3.4k-FA1)1.66 > PME-(PEG3.5k)1.69. Folate competition assays showed that PME-(PEG3.4k-FA2)1.72 complexes had stronger targeting ability than PME-(PEG3.5k)1.69 and PME-(PEG3.4k-FA1)1.66 complexes due to their higher folate density per PEG molecule. Cellular uptake mechanism study showed that the folate density on PEG could change the endocytosis pathway of PME-(PEG3.5k)1.69 from clathrin-mediated endocytosis to caveolae-mediated endocytosis, leading to less lysosomal degradation. Distribution and uptake in 3D multicellular spheroid assays showed that divalent folate could offer PME-(PEG3.4k-FA2)1.72 complexes stronger penetrating ability and higher cellular uptake. With these advantages, PME-(PEG3.4k-FA2)1.72 may be a promising nonviral vector candidate for efficient gene delivery. This study also indicates that divalent folate modification on PEG can serve as an efficient strategy to improve the cellular uptake and targeting ability of PEGylated cationic polymers for gene delivery.
Cationic polymers have been regarded as promising non-viral gene carriers because of their advantages over viral gene vectors, such as low cost, a high level of safety and easy manipulation. However, their poor transfection efficiency in the presence of serum and high toxicity are still limiting issues for clinical applications. In addition, the lack of adequate understanding of the gene delivery mechanism hinders their development to some extent. In this study, new polycations (PAPEs) consisting of a low generation polyamidoamine (PAMAM) core and branched polyethyleneimine (PEI-1.8k) outer layers were synthesized and their transfection activity and mechanism were studied. PAPEs were characterized by FTIR, (1)H NMR and gel permeation chromatography. PAPEs were able to self-assemble with pDNA and form spherical nanoparticles with sizes of 70-204 nm and zeta potentials of 13-33 mV. Importantly, the PAPE-pDNA complexes displayed lower cytotoxicity and higher transfection activity than PEI 25k in various cell lines, specifically in the presence of serum. The transfection mechanism was evaluated by endocytosis inhibition with specific inhibitors, time-dependent transfection, and intracellular trafficking inspection by CLSM. The high levels of transgene expression mediated by PAPEs were attributed to caveolae-mediated cellular uptake, the reduced entry into lysosomes and the entry into the nucleus through mitosis.
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