Opposites attract: influence of the molar mass of branched poly(ethylene imine) on biophysical characteristics of siRNA-based polyplexese

Abstract

Polymer-based carriers, in particular polycations, represent an interesting alternative to viral vectors, as they form so-called polyplexes with nucleic acids by entropic driven, electrostatic interactions. In this study, we investigate in detail polyplexes based on small interfering RNA (siRNA), the delivery of which into eukaryotic cells represents an attractive route for treating genetic diseases by inhibition of harmful gene expression. Although plasmid DNA (pDNA) based polyplexes are well characterized, we show that not all knowledge can be adopted from pDNA, as siRNA is around 250 times smaller and shows a higher rigidity. The used polymer component is the polycation branched poly(ethylene imine) (B-PEI) of a high range of molar masses (0.6, 1.8, 10, 25 kDa), which are further analyzed by potentiometric titration and cytotoxicity tests. The formation, size, and net-charge of the polyplexes are examined at different ratios of nitrogen of the different polymers and phosphates of the RNA (N/P). Moreover, the stability of siRNA polyplexes against heparin and time was investigated. The obtained physicochemical parameters were then correlated to the cellular internalization of polyplexes. A strong dependency of the molar mass on the polyplex characteristics of the used B-PEI was found. Thereby, high molar mass B-PEI ≥ 10 kDa forms smaller polyplexes of around 50 nm radius with zeta potentials > 25 mV, increased long-term stability, and enhanced cellular uptake compared to low molar mass ones. To gain deeper insight into the differences and characteristics of siRNA based polyplexes, the characterization by analytical and preparative ultracentrifugation (AUC, PUC) is applied on siRNA polyplexes for the first time and referred to conventional characterization methods such as DLS. AUC was also used to identify non-complexed PEI in the polyplex solutions. A virtual N/P of 1.3 for siRNA was measured, independent of the used molar masses of B-PEI. Additionally, differences in cellular uptakes of siRNA and pDNA based polyplexes were found. The results of this study will help to understand the properties of siRNA-based polyplexes and could lead to more efficient polymer design.