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Among polymeric polycations, chitosan has emerged as a powerful carrier for gene delivery. Only a few studies have focused on the stability of the chitosan/DNA complex under storage, although this is imperative for nanomedicinal applications. Here, we synthesized polyelectrolyte complexes at a charge ratio of 10 using 50 kDa chitosan and plasmid (p)DNA that encodes a GFP reporter. These preparations were stable up to 3 months at 4 °C and showed reproducible transfection efficiencies in vitro in HEK293 cells. In addition, we developed a methodology that increases the in vitro transfection efficiency of chitosan/pDNA complexes by 150% with respect to standard procedures. Notably, intracellular pDNA release and transfected cells peaked 5 days following transection of mitotically active cells. These new developments in formulation technology enhance the potential for polymeric nanoparticle-mediated gene therapy.

The design of biocompatible polyelectrolyte complexes is a promising strategy for in vivo delivery of biologically active macromolecules. Particularly, the condensation of DNA by polycations received considerable attention for its potential in gene delivery applications, where the development of safe and effective non-viral vectors remains a central challenge. Among polymeric polycations, Chitosan has recently emerged as a very interesting material for these applications. In this study, we compare the observed aggregation behavior of Chitosan-DNA complexes with the predictions of existing models for the complexation of oppositely charged polyelectrolytes. By using different and complementary microscopy approaches (AFM, FESEM and TEM), light scattering and electrophoretic mobility techniques, we characterized the structures of the complexes formed at different charge ratios and Chitosan molecular weight. In good agreement with theoretical predictions, a reentrant condensation, accompanied by charge inversion, is clearly observed as the polycation/DNA charge ratio is increased. In fact, the aggregates reach their maximum size in correspondence of a value of the charge ratio where their measured net charge inverts its sign. This value does not correspond to the stoichiometric 1:1 charge ratio, but is inversely correlated with the polycation length. Distinctive "tadpole-like" aggregates are observed in excess polycation, while only globular aggregates are found in excess DNA. Close to the isoelectric point, elongated fiber-like structures appear. Within the framework of the models discussed, different apparently uncorrelated observations reported in the literature find a systematic interpretation. These results suggest that these models are useful tools to guide the design of new and more efficient polycation-based vectors for a more effective delivery of genetic material., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Interaction of DNA with oppositely charged objects, such as multivalent ions, cationic surfactants, cationic liposomes, basic proteins, and alcohols, up to nano- or mesoscopic particles, gives rise to a very interesting and fascinating phenomenology, where the shape, size, and stability of the resulting aggregates depend on a delicate balance between different driving forces, mainly of electrostatic origin. We have studied the cationic liposome-DNA complexes during the whole complexation process, below, close to, and above the isoelectric condition, where the number of cationic lipids equals the number of phosphate groups on the DNA chain. We took advantage of the combined use of dynamic light scattering, laser Doppler electrophoretic mobility, and radio-wave dielectric relaxation measurements in order to characterize both the structural parameters (hydrodynamic radius) and the electrical parameters (charge and counterion concentration) of the resulting structures. These structures are fundamentally of two types, clusters of liposomes stuck together by DNA chains (cluster phase in low-density colloidal suspension) and coexisting DNA coils and DNA globules, according to the procedure through which interactions occur (liposomes in excess DNA solution or DNA in excess liposome suspension).

By means of the combined use of dynamic light scattering and transmission electron microscopy measurements, we provide a direct evidence for the existence of an equilibrium cluster phase in the polyion-induced liposome aggregation, where the liposomes maintain their integrity, with the ability of preserving the aqueous core content from the external medium. We prepared single liposomes containing, in their interior, different CsCl electrolyte solutions at different concentrations (0.1 and 0.01 M, respectively). During the polyion-induced complexation of a mixture of these two differently loaded liposomes, reversible aggregates form, whose multicompartmental structure reveals the simultaneous presence of nonfused liposomes. Clusters composed by mesoscopic-sized vesicles and realized by charged lipids coupled to oppositely charged polyions are playing an increasingly important role as model systems in a variety of phenomena in soft matter and for their potential use in biomedical applications as drug delivery systems. Aggregates of liposomes such as those described in this article, where the electrostatic interactions are the primary driving forces promoting aggregation, may represent a new and interesting class of colloids which give rise to a rich phenomenology with several unusual colloidal behaviors that deserve to be further investigated.

We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion-liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion-liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion-liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases ("reentrant" condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.