Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains To Overcome Intracellular Barriers

Abstract

The prospective use of the block copolymers poly(ethylene oxide)-b-poly[2-(diethylamino)ethyl methacrylate] (PEO-b-PDEA) and poly[oligo(ethylene glycol)methyl ether methacrylate]-b-poly[oligo(ethylene glycol)methyl ether methacrylate-co-2-(diethylamino)ethyl methacrylate-co-2-(diisopropylamino)ethyl methacrylate] (POEGMA-b-P(OEGMA-co-DEA-co-DPA)) as nonviral gene vectors was evaluated. The polymers are able to properly condense DNA into nanosized particles (R ≈ 75 nm), which are marginally cytotoxic and can be uptaken by cells. However, the green fluorescent protein (GFP) expression assays evidenced that DNA delivery is essentially negligible meaning that intracellular trafficking hampers efficient gene release. Subsequently, we demonstrate that cellular uptake and particularly the quantity of GFP-positive cells are substantially enhanced when the block copolymer polyplexes are produced and further supplemented by BPEI chains (branched polyethylenimine). The dynamic light scattering/electrophoretic light scattering/isothermal titration calorimetry data suggest that such a strategy allows the adsorption of BPEI onto the surface of the polyplexes, and this phenomenon is responsible for increasing the size and surface charge of the assemblies. Nevertheless, most of the BPEI chains remain freely diffusing in the systems. The biological assays confirmed that cellular uptake is enhanced in the presence of BPEI and principally, the free highly charged polymer chains play the central role in intracellular trafficking and gene transfection. These investigations pointed out that the transfection efficiency versus cytotoxicity issue can be balanced by a mixture of BPEI and less cytotoxic agents such as for instance the proposed block copolymers.

Diethylaminoethyl- chitosan as an efficient carrier for siRNA delivery: Improving the condensation process and the nanoparticles properties

“…However, for DEAE-CH 50 nanoparticles, the presence of excess polycations did not affect the efficiency. On the contrary, at an N/P ratio of 10, the best silencing was achieved, agreeing with previous reports by other groups that indicated that an excess of free or weakly-associated polycation chains can improve the lysosomal release [12,13].…”

“…nanoparticles encapsulating siRNA prepared with chitosan 470 kDa, in the presence of polyguluronate mediated silencing efficiencies near 50% with HEK 293FT and HeLa cells [10]. However, transfection efficiencies of low Mw chitosan nanoparticles may be adjusted by increasing the amino to phosphate N/P ratios and recent studies have indicated that an excess of free or weakly associated polycation chains plays an important role on the uptake process and transfection efficiency [11][12][13]. Chitosans having low and high Mws, (2 kDa to 500 kDa) are used in large charge excess to siRNA, and N/P ratios in the range of 10 to 200 are commonly found in the literature [14][15][16].…”

Sweet Vector for Gene Delivery: the Sugar Decoration of Polyplexes Reduces Cytotoxicity with a Balanced Effect on Gene Expression

“…The respective flow cytometry dot plots are given in the Supporting Information (Figures S5 and S6, Supporting Information). Indeed, the obtained values are consistent with a recent study we conducted . The cellular uptake, subcellular localization, and gene expression of the BPEI and BPEI‐Lac polyplexes were finally examined qualitatively via fluorescence microscopy.…”

Hyperbranched poly(ethylenimine-co-oxazoline) by thiol–yne chemistry for non-viral gene delivery: investigating the role of polymer architecture

“…It has more recently be shown that free polymer chains can have an impact gene transfection. [51][52][53] Whereby the mechanism of cationic polymer mediated plasmid transfection, could be explained by free polymer interaction with the interior endosomal membrane and creation of pores, allowing an amount of polyplex into the cytoplasm. Any small change in the transfection process could have disproportionate effect on observed expression due to endosome escape being very inefficient.…”