DNA–polycation complexation and polyplex stability in the presence of competing polyanions

Abstract

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.

DNA Release Dynamics from Reducible Polyplexes by Atomic Force Microscopy

“…AFM results further indicate that the condensates are highly compact and approximately half of them contain only one DNA plasmid. Destabilization experiments by a polyanion exchange reaction found that compact DNA condensates are more stable against polyanion exchange 15,16. While all the polycations bind equally well to DNA they show selectivity toward dissociation.…”

Enhancement of chitosan-mediated gene delivery through combination with phiC31 integrase

“…1) were used (1) pCMVeGFP-attB, expressing the reporter gene green fluorescent protein (GFP); (2) pCMVCEP290attB, expressing the CEP290 gene (gene responsible for LCA 10), both driven by the cytomegalovirus (CMV) promoter and containing the attachment sites for integrase (attB); and (3) pCMVINT, an integrase expression plasmid (all kindly provided by Dr. Jean Bennett, University of Pennsylvania, USA) [8,26]. Plasmids were amplified in Top10 bacteria and purified using a Plasmid Maxi kit (Qiagen, USA) following manufacturer guidelines.…”

“…Cells were cultured at 37°C, under a 5% CO 2 For selection studies, HEK293 cells were transfected as described above and 72 h post-transfection selection with Geneticin Ò (Gibco, USA) was initiated. Selection of transfected cells was carried out for 2 weeks using 300-500 lg/mL of Geneticin Ò in complete media.…”

“…Gene therapy shows great promise regarding treatment of several inherited diseases and, in recent years, gene therapy has been featured in numerous clinical trials with encouraging outcomes [1][2][3][4]. Currently, there are two general methodologies used to deliver transgenes: a viral mediated and a non-viral mediated approach.…”

Co-delivery of small interfering RNA and plasmid DNA using a polymeric vector incorporating endosomolytic oligomeric sulfonamide

“…This is the result of the siRNA chain being short and rigid [14], unlike long and flexible pDNA. In general, the electrostatic strength between polycations and polyanions (here, nucleic acids) increases with higher molecular weights (MW) [14, 15]. These short siRNAs (usually duplexes of 21–23 nucleotides) may result in one hundredth of the electrostatic strength of pDNA (more than 2000 base pairs) in an association with a given polycation [16].…”