Intracellular gene delivery is dependent on the type of non-viral carrier and defined by the cell surface glycosaminoglycans

Intracellular limiting steps and molecules involved in internalization and intracellular routing of non-viral gene delivery systems are still poorly understood. In this study, the intracellular kinetics of three different gene delivery systems calcium phosphate precipitates (CaP), polyethyleneimine (PEI) and N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride (DOTAP)) were quantified at cellular, nuclear, transcriptional and translational levels by using qRT-PCR. Additionally, a role of cell surface glycosaminoglycans (GAGs) was evaluated by performing the aforementioned studies in cells devoid of GAGs (pgsB-618) and cells lacking heparan sulphate (HS). The obtained data showed that the intracellular kinetics was dependent on the type of gene carrier and the weakest intracellular step varied between the carriers; rapid elimination of cell-associated pDNA in CaP, nuclear uptake in DOTAP and transcriptional and translational events in PEI mediated transfections. Overall, neither the amount of cell- nor nuclear associated pDNA correlated with transgene expression but the mRNA expression of the transgene correlated well with the expression at protein level. The nuclear uptake of pDNA in all cases was rapid and efficient thus indicating that the post-nuclear processes including transcription and translation steps have a critical role in defining the efficiency of non-viral gene delivery systems. Our study demonstrated that cell-surface GAGs are not essential for cell surface binding and internalization of gene delivery complexes, but they are able to define the intracellular routing of the complexes by leading them to pathways with high pDNA elimination.