Comparative evaluation and optimization of off-the-shelf cationic polymers for gene delivery purposes

Abstract

Cationic polymers are amongst the most utilized non-viral vectors for gene transfer owing to their ability to condense and protect the genetic material within polyplexes and to ferry it into cells. Quite a number of parameters, both related to the features of the vectors themselves (e.g. degree of branching, molecular weight, polydispersity) and to polyplexes (e.g. nitrogen to phosphate ratio (N/P), dose of complexes delivered, complexation buffer, etc.), are known to affect transfection behaviour. Consequently, some substantial discrepancy found in raw materials and in-home protocols across laboratories account for some disagreement and conflicting data about their performance. Hereinafter we provide a thorough chemical-physical and in vitro biochemical characterization, comparison, and optimization of the most widely used, commercially sourced polymers used in transfection, namely linear polyethylenimines (lPEIs), branched PEIs (bPEIs), linear poly-L-lysines (lPLLs), and polyamidoamine dendrimers (dPAMAMs). By means of a stepwise approach, we pinpointed the most effective molecular weight and complexation conditions specific to each of them and correlated the physicochemical features of polyplexes with their transfection effectiveness. Besides, taking separately into account the effects on transfection of the plasmid dose delivered to cells, the cell seeding density and the volume of the culture medium, we highlited a range of optimal conditions roughly specific to each studied polymer. Finally, we coped with the effect of the variation of these three parameters at once on the transfection effectiveness of lPEI and bPEI and pinpointed an array of settings specifically optimized to attain truly superior performances

Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation

“…Instead, lPEI-polyplexes in 150 mM NaCl behaved differently. Indeed, as already reported812, and irrespective of the mode of addition, this condition gave rise to a roughly heterogeneous population of big aggregates (PDI ≈ 1; D H  > 1 μm). Nonetheless, the ζ P was positive and similar to the other conditions studied, albeit lower when following the DROPPING mode.…”

“…For the sake of simplicity and comparison, polyplexes were invariably prepared at N/P 30, that was recently identified as the most effective for 25 kDa lPEI (from Polysciences)8. Each reagent was pre-warmed, added and incubated at 37 °C by means of a thermomixer in order to minimize temperature variations that might have some effect on complexation13.…”

Hydrophobe-substituted bPEI derivatives: boosting transfection on primary vascular cells

“…Therefore, we tested the transfection efficiency and the cytotoxicity of polyplexes prepared at different w/w with b PEI derivatives on primary VSMCs obtained from the human umbilical artery (HUASMCs) and from the porcine aorta (PAoSMCs), since pigs are commonly used in vascular research as a model of human disease [27]. The presence of serum in culture medium is necessary for long-term cell cultures and it is commonly employed to evaluate resistance to serum of gene delivery vectors prior to in vivo tests [28], consequently, transfection experiments were carried out in cDMEM, containing 10% FBS. Three w/w ratios (2.5, 5 and 10) were tested, given the recognised dependence of transfection activity on this parameter.…”

Hierarchical microchannel architecture in chitosan/bioactive glass scaffolds via electrophoretic deposition positive‐replica

“…Quantitative determination of DNA was performed after seeding SAOS‐2 cells on scaffolds in standard 96‐well plates using GEL method as described above and cultured for 14, 21, and 28 days. DNA was quantified using a SYBR‐Green protocol . Briefly, supernatants were diluted 1:10 in the assay buffer and TE solution (10 m M Tris/HCl, 1 m M EDTA) was added.…”

“…DNA was quantified using a SYBR-Green protocol. 38 Briefly, supernatants were diluted 1:10 in the assay buffer and TE solution (10 mM Tris/HCl, 1 mM EDTA) was added. DNA content was measured with SYBR-Green I (1:1250) by reading the absorbance at λ = 485 nm.…”