Direct evidence of multicompartment aggregates in polyelectrolyte-charged liposome complexes

Abstract

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.

FIGURE 1

Average hydrodynamic diameter 〈2R〉 (open symbols), and ζ-potential (solid symbols), of NaPA-DOTAP complexes as a function of the polyion/DOTAP charge ratio parameter ξ, for the two different molar concentrations of CsCl employed in liposome preparation: (a) CsCl 0.1 M and (b) CsCl 0.01 M. Lines are to guide the eye only.

FIGURE 2

ESI-TEM image of a typical aggregate of heavily (0.1 M) and lightly (0.01 M) Cs-loaded liposomes. In panel a, the aggregate appears built up by globular particles and darker and lighter individual globules are clearly recognized. In the absence of any staining, the contrast observed is to be ascribed to differences in the elastic scattering of electrons due to local changes in the Cs density. Panel b shows the Cs map of the same aggregate as in panel a, obtained applying the two-windows method (50) around the M_{4, 5}-Cs edge (see Fig. 3). In this image, red-gold levels correspond to variations in Cs concentration. Comparing this topographic map and the real image in panel a makes evident that Cs is contained within the aggregate, and that the liposomes in the aggregate, loaded with different Cs concentrations (0.1 M and 0.01 M) maintain their individuality. In panel c, the Cs map, but with a threshold set at 50% intensity, and the high-contrast reference image of panel a, are superimposed. Bars represent 100 nm.

FIGURE 3

Electron energy-loss spectroscopy spectrum, collected from a zone containing lipoplexes, showing the characteristic M_{4, 5} edge of Cs.

FIGURE 4

At a lower magnification, the smaller aggregates appear to have a more compact and approximately roundish shape, while larger aggregates are more elongated. Bar represents 400 nm.

FIGURE 5

In some cases, large fractal-like aggregates appear. Darker and lighter liposomes (high Cs and low Cs concentration, respectively) are clearly distinguishable. Bar is 200 nm.

FIGURE 6

Larger liposomes, when deposited on the support, tend to assume a doughnut shape (as the cartoon in the inset illustrates), probably due to the combined effects of the finite minimum radius of curvature that the vesicle double layer can bend, and of the surface tension of the filling aqueous medium, which forces the electrolyte to withdraw from the central region of the vesicles when they collapse in the microscope vacuum chamber. Bar is 100 nm.

FIGURE 7

(a) A typical aggregate induced by DNA polyion in a mixture of water-filled and Cs-loaded liposomes. Water-filled liposomes are not visible and only appear as empty space between the Cs-loaded liposomes. Bar is 200 nm. (b) Negative staining allows the water-filled liposome contour to be recognizable. Bar is 50 nm.

FIGURE 8

Number-weighted and intensity-weighted size distribution. (a, open bar) Number-averaged distribution of the liposome size from the analysis of TEM images; (shaded bar) intensity-averaged distribution of liposomes considering an r⁶ weight. (b) The corresponding size distribution of liposome size from light-scattering measurements. (Open bar) Number-averaged distribution; (shaded bar) intensity-averaged distribution.