Use of EPR and FTIR to detect biological effects of ultrasound and microbubbles on a fibroblast cell line.

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.

Radiofrequency dielectric loss relaxation in polyion-induced liposome aggregates.

In this note, we present a set of dielectric loss relaxation measurements of aqueous charged liposome suspensions during the whole aggregation process induced by oppositely charged adsorbing polyions. The system experiences two concomitant effects known as "reentrant condensation" and "charge inversion," resulting in the formation of liposome aggregates whose average size reaches a maximum in the vicinity of the electroneutrality condition, accompanied to a progressive reduction of their overall electrical charge. Far from the neutrality, from both sides, polyion-coated liposomes exist with a charge of opposite sign. The dielectric loss relaxation in these complex aggregating systems has never been measured so far and we report here, for the first time, the dielectric loss behavior of liposomes built up by a cationic lipid and stuck together by poly(acrylate), which is a flexible oppositely charged polyion. The data are analyzed in the framework of standard electrokinetic model theory. The evolution of the aggregation process as a function of the polyion content is mainly characterized by a counterion polarization effect, governed by the surface charge density of the aggregates and hence by the zeta-potential.

Polyion-induced liposomal vesicle aggregation: a radiowave dielectric relaxation study.

The radiowave dielectric properties of aqueous heterogeneous systems during the complexation of charged polyions and oppositely charged liposomal particles have been measured in a wide frequency range, between 100 Hz and 2 GHz. The formation of a polyion-liposome complex driven by the correlated polyion adsorption at the particle surface implies two concomitant effects referred to as reentrant condensation and charge inversion. Both of them are governed by electrostatic interactions and there is now strong evidence, based on experiments and simulations, that counterion release is the driving force of the aggregation process. From this point of view, dielectric technique may offer a suitable tool in the investigation of the structural properties of these aggregates. In spite of the fact that interaction of polyions with oppositely charged surfaces was extensively experimentally investigated, there are no papers concerning the dielectric properties during the polyion-induced aggregation. To get an insight into this important topic, the authors present here an extensive set of radiowave dielectric measurements of liposomal vesicle aqueous suspensions where the liposome aggregation was induced by an oppositely charged polyion. The aggregation was followed from the beginning, when most of the isolated liposomes predominate, up to the formation of polyion-coated liposomes of inverted charge, crossing the isoelectric condition, where large, almost neutral, aggregates appear. The authors describe the observed dielectric dispersions as due to counterion polarization in the adjacency of the liposome and liposome aggregate surface, primarily governed by the zeta potential, according to the standard electrokinetic model.

Conductometric evidence for intact polyion-induced liposome clusters.

In this note, we present a set of electrical conductivity measurements of polyion-induced liposome aggregate aqueous suspensions that supports evidence for the existence of a cluster phase in low-density colloidal systems. Heavily NaCl-loaded liposomes, dispersed in a low-conductivity aqueous solution, are forced by electrostatic interactions with oppositely charged polyions to build up into individual aggregates, where the single vesicles maintain their integrity and, upon an external force, are able to release their ionic content. The conductivity data, within the effective medium approximation theory for heterogeneous systems, are in agreement with the picture of a suspension built up by clusters of vesicles which are able to preserve their content from the external medium. This finding opens new possibilities in multicompartment drug delivery techniques.

Counterion release in overcharging of polyion-liposome complexes.

We present a set of electrical conductivity measurements of a mesoscopic equilibrium cluster phase in the aggregation process of charged particles induced by oppositely charged polyions. These measurements supply strong experimental evidence that correlated adsorption of polyions is driven by the counterion release. This phenomenon, known to occur in DNA-liposome mixtures in lamellar phase, i.e., when liposomes fuse together to form a sandwichlike structure encompassing DNA chains, was not previously observed in aqueous suspension of clusters of intact liposomes stuck together by polyions to form reversible aggregates. A simple statistical model of the lateral correlation of polyions at the particle surface justifies quantitatively the observed behavior of the counterion release, as shown by electrical conductivity measurements.