AFM and TEM characterization of siRNAs lipoplexes: A combinatory tools to predict the efficacy of complexation

This work aims to evaluate the effects of two different surface modification strategies: PEG conventional coupling (PEG-Lpx) and postpegylation technique (postPEG-Lpx), on lipoplexes obtained between liposomes and siRNAs. Photon correlation spectroscopy (PCS) and gel electrophoreses (as conventional techniques), and atomic force microscopy (AFM) and transmission electron microscopy (TEM) (proposed to complete the assessments of lipoplexes) were employed to investigate reorganization, structure, qualitative-quantitative stabilization of siRNAs, and PEG covering of lipoplexes. The results suggested that postPEG-Lpx exhibited high level of homogeneity with a mean diameter (Z-Average) of about 320 nm, low tendency to aggregation (a polydispersion index, PDI, close to 0.06) and high loading efficiency (E.E. 82%). Otherwise, PEG-Lpx showed a Z-Average greater than 1 mu m, high aggregation rate (PDI >0.3) and a low E.E. (10%). The definition of the architecture by using optimized microscopical procedure allows to suggest postpegylation technique as a promising technology for the preparation of applicable complexes. This formulation strategy lead to a stable siRNA condensation and full compaction of gene material, moreover the PEG coverage generated a homogeneous hydrated surface, well described by the "phase" AFM approach. The microscopical techniques can provide a predictive and useful tool to use in the preformulative technological studies of complicated gene complexes. (C) 2013 Elsevier B.V. All rights reserved.