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.
Highlights
Branched and linear polyethylenimines 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
In order to investigate the influence of differences in the complexation protocol on the physicochemical and biological properties of the resulting polyplexes, two distinct modes of addition of reagents in the preparation of poly(ethylene imine) (PEI)/plasmid DNA (pDNA) polyplexes were compared in different experimental setups
Even though lPEI and bPEI have been found effective at different molecular sizes, PEIs with a MW ≈ 25 kDa are by far the most studied and employed in transfection[11,12], and were investigated in the present study
Summary
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. The development of non-viral vectors capable of efficiently delivering nucleic acids to cells and tissues (i.e. transfection) is an inherently interdisciplinary and a rapidly advancing area of research Since their introduction in 19871, a number of cationic polymer-based vectors have been designed and developed[2,3]. Despite extensive research in this area, some conflicting results about the performances of non-viral gene delivery vectors in general, and PEIs have been reported. Such a wide range of outcomes arises, at least in part, from the large variability in experimental conditions employed in transfection assays[7,8].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
