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Due to charge interaction, cationic lipids spontaneously associate with nucleic acids, resulting in the formation of so-called lipoplexes. Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to expression of the genes (transfection). The mechanism involved in the cellular uptake of lipoplexes is most likely endocytosis, which occurs after nonspecific charge-mediated binding to cellular receptors. An important step in the transfection process following the actual internalization of lipoplexes is the release of the lipoplex and/or its DNA into the cytoplasm in order to evade lysosomal degradation. Here, the membranous nature of the lipoplex seems to be crucial in that it allows the exchange of lipids between the endosomal membrane and the lipoplex, which results in membrane perturbations that are a prerequisite in the endosomal escape of DNA. Interestingly, a hexagonal phase of the lipoplexes has been correlated with efficient transfection and it can be envisaged that such a phase could be instrumental in the creation of membrane perturbations. Subsequent to its release into the cytoplasm, the DNA has to be transferred into the nucleus. The nuclear import of DNA is most likely a protein-mediated process. In addition, the nuclear uptake of DNA may be facilitated at the time of nuclear envelope disassembly during mitosis. Currently, cationic liposomes are widely used as gene carrier system to deliver nucleic acids into cells in culture to study the cell-biological functioning of genes plus accompanying proteins. Ultimately, cationic lipids may be used in gene therapeutic protocols.

The utility of using a protammine/DNA complex coated with a lipid envelope made of cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) for transfecting CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryonal cells), and A17 (murine cancer cells) cells was examined. The widely used DOTAP/DNA lipoplex was employed as a reference. In all the tested cell lines lipid/protamine/DNA (LPD) nanoparticles were more efficient in transfecting cells than lipoplexes even though the lipid composition of the lipid envelope was the same in both devices. Physical-chemical properties were found to control the ability of nanocarriers to release DNA upon interaction with cellular membranes. LPD complexes easily release their DNA payload, while lipoplexes remain largely intact and accumulate at the cell nucleus. Collectively, these data explain why LPD nanoparticles often exhibit superior performances compared to lipoplexes in trasfecting cells and represent a promising class of nanocarriers for gene delivery. © 2011 American Chemical Society.

A combination of complementary analytical techniques has provided valuable information on the physicochemical properties of lipid-DNA complexes such as their size, shape, structure and surface charge. The experimental conditions for producing small and stable lipid-DNA complexes were determined. These complexes consist of spherical primary particles of approximately 60 nm in diameter connected to each other by DNA strands to form small clusters of approximately 70-200 nm in diameter. At high salt concentrations these complexes were not stable and aggregated. Several methods were used to stabilise the lipid-DNA complexes at physiological salt concentrations.

Although the properties of mesoscopic charged complexes, originating by the self-assembling of polyions onto oppositely charged particles, are been extensively investigated, both experimentally and theoretically, less attention has been addressed towards the existence of an equilibrium cluster phase in low-density colloidal suspensions, where particles maintain their integrity and polyions act as an electrostatic glue. The self-assembling of lipidic vesicles into mesoscopic aggregates induced by polyions is a hierarchical process where, at different basic levels, aggregates arrange themselves to form superstructures, giving rise to multi-lamellar complexes or honeycomb structures in which polyions are more or less intercalated between the lipid bilayers. This reorganization becomes relevant in many biological important processes such as drug delivery. However, besides these rather complex structural rearrangements, at a lower hierarchy level, clusters formed by intact vesicles, stuck together by oppositely charged polyions, may exist. These equilibrium clusters, which we call 'intermediate' aggregates, can be considered as a new class of colloids with a rich and not yet completely understood phenomenology. In this review, we will refer to a specific example and will present a well-documented experimental evidence of the formation of equilibrium clusters composed by positively charged liposomes built up by DOTAP, stuck together by a simple highly charged linear polyion (polyacrylate sodium salt). These structures, governed by a balance between long-range electrostatic repulsion and short-range attraction, give rise to the formation of relatively large, equilibrium clusters whose size and overall charge can be continuously tunable by simple environmental parameters. Under appropriate experimental conditions, in a long time limit, these liposome clusters may further evolve towards a different structural arrangement, giving account for the supramolecular structures of polyion-liposome complexes, such as those observed in DNA-lipid complexes. (C) 2006 Elsevier B.V. All rights reserved.

Charged colloidal particles whose interacting potential is governed by a short-range attraction and a long-range screened electrostatic repulsion contributions form aggregates whose shape, size, and overall charge are sensitively dependent on the balance between attraction and repulsion. In some cases, this class of colloidal systems shows an equilibrium cluster phase, where particles associate and dissociate reversibly into clusters. When the aggregation of the charged particles is induced by adding an oppositely charged polyion, very close to the isoelectric condition, the interaggregate interactions become very strong and a dynamical arrested state seems to occur. We provide some experimental evidences of this structural arrest in a colloid system composed by vesicles built up by a cationic lipid stuck together by an oppositely charged linear polyion, by means of the combined use of static and dynamic light scattering technique complemented by laser Doppler electrophoretic measurements. Our results show that the second virial coefficient, which is related to the potential of mean force between two adjacent aggregates, markedly increases in the vicinity of the isoelectric point. We interpret this increase as a print of strong interparticle interactions, yielding to a dynamical arrested state via cluster growth.

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 0.01M, 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.

A brief overview is given over recent theoretical and simulation work on complexes of oppositely charged macroions. The emphasis is on linear/ cylindrical macroions (flexible, semiflexible polyelectrolytes) and spherical/globular macroions (colloids, micelles, globular proteins). We discuss soluble complexes, dense complex phases, and micelles with cores consisting of dense complex phases, stabilized by neutral hydrophilic chains.

A detailed physicochemical study is presented on a new class of cationic amphiphiles, Sunfish amphiphiles, recently designed, synthesized, and tested for gene delivery. These materials have two hydrophobic tails, connected to the cationic pyridinium headgroup at the 1- and 4-positions. Two extreme morphologies can be visualized, i.e. one by back-folding involving association of both tails at one side of the pyridinium ring and one by independent unfolding of the tails, the two molecular geometries leading to considerable differences in the aggregate morphology. The behavior of six members of the Sunfish family in mixtures with DOPE, applying different conditions relevant for transfection, has been studied by a combination of techniques (DLS, DSC, NMR, SAXS, Cryo-TEM, fluorescence, etc.). The effects of structural parameters such as the presence of unsaturation in the tails and length of the alkyl chains on the properties of the aggregates have been assessed. A correlation of these structural data with cellular transfection efficiencies reveals that the highest transfection efficiency is obtained with those amphiphiles that are easily hydrated, form fluid aggregates, and undergo a transition to the inverted hexagonal phase in the presence of plasmid DNA (p-DNA) at physiological ionic strength.

Here we investigate the effect of membrane charge density on the protein corona that forms upon exposure of cationic liposomes to human plasma. We show that the protein corona around cationic liposomes is not uniquely defined by membrane charge density, but it is deeply affected by both the overall cationic charge and the available surface area. Remarkably, changes in protein composition are largely removed when a correcting quantity accounting for the ratio between the cationic charge and the surface area of different cationic liposome formulations is considered.