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

Abstract

Although branched and linear polyethylenimines (bPEIs and lPEIs) are gold standard transfectants, a systematic analysis of the effects of the preparation protocol of polyplexes and the composition of the transfection medium on their physicochemical behaviour and effectiveness in vitro have been much neglected, undermining in some way the identification of precise structure-function relationships. This work aimed to address these issues. bPEI/DNA and lPEI/DNA, prepared using two different modes of addition of reagents, gave rise to polyplexes with exactly the same chemical composition but differing in dimensions. Upon dilution in serum-free medium, the size of any kind of polyplex promptly rose over time while remained invariably stable in complete DMEM. Of note, the bigger the dimension of polyplexes (in the nano- to micrometer range), the greater their efficiency in vitro. Besides, centrifugal sedimentation of polyplexes displaying different dimensions to speed up and enhance their settling onto cells boosted transfection efficiencies. Conversely, transgene expression was significantly blunted in cells held upside-down and transfected, definitively pointing out the impact of gravitational sedimentation of polyplexes on their transfection efficiency. Overall, much more attention must be paid to the actual polyplex size that relies on the complexation conditions and the transfection medium.

Comparison of random and gradient amino functionalized poly(2‐oxazoline)s: Can the transfection efficiency be tuned by the macromolecular structure?

“…Interestingly, polyplex formations in 150 mM NaCl showed small polyplexes with a low PDI for P(M n A m ) whereas for P(E n A m ) mostly aggregates larger than 700 nm with a high PDI could be detected (Table ). The influence of N/P ratio and transfection medium for l ‐PEI was recently shown and are in good accordance to our results …”

High cell density transient transfection of CHO cells for TGF‐β1 expression

“…The results also showed that with the addition of fresh CHOMACS CD medium up to 5 mL at 5 hpt, the unknown PEI inhibitory components of CHOMACS CD medium had a negligible effect on transfection efficiency. Consistent with this finding, most transfection events occurred within the first hour after transfection, when 80% of the pDNA/PEI polyplex is taken up by the cells [16,33]. Therefore, volumetric upscaling of the high cell density transfected culture through medium addition at 5 hpt decreased its cell density to extend the protein production time as showed in Supporting Information Figure S2.…”

Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

“…To guarantee appropriate biological performance and a successful transfection with the nanocomplexes, it is essential to evaluate their colloidal stability in the transfection medium. In this study, we used Opti-MEM™, as it is commonly used for transfection purposes in various cell lines [43,44]. Furthermore, we supplemented the medium with HEPES and mannitol, based on a recommendation for transfection with the commercially available CS-based transfection reagent, Novafect, from NovaMatrix ® (Sandvika, N).…”