Biopolymer-Vescicle InteractionsLarge interest is devoted to clarify the behavior ofcolloid systems composed by proteins (or DNA) andsurfactant-based vesicles. [1] Catanionic ones (anacronym derived from the term [cationic + anionic])are stu<strong>di</strong>ed in more detail. [2] Such vesicles, VES, areeasily prepared and are a valid alternative toliposomes as carriers in gene therapy. They arethermodynamically and kinetically stable. Their layeris similar to biological membranes and allowsencapsulating hydrophilic and hydrophobicmolecules. Many properties of catanionic vesicles,such as their surface charge density, double layerflui<strong>di</strong>ty, etc., can be properly tuned. Suchpossibilities ensure their use in bio-chemicallyrelevant methods.It is important to define which driving forcesinfluences the interaction process between VES andDNA, or proteins. Object of this research line is tounderstand the mechanisms responsible for theformation of vesicle-protein complexes, termed lipoplexes.Stu<strong>di</strong>es reported so far are based on TEM[3](Cryo-TEM) or fluorescence microscopy. Noinformation on the parameters controlling theinteraction processes, i.e. the charge density and theiso-electric point of the complexes, can be obtainedfrom the above techniques. The driving forcecontrolling biopolymer-vesicle interactions iselectrostatic, and the thickness of the electricaldouble layer around biopolymers, vesicles and theircomplexes is significantly affected from theinteractions. The surface charge density and somerelated quantities may be significantly mo<strong>di</strong>fied. Todetect polarization effects and mo<strong>di</strong>fications in theelectrical double layer around such charged particles,CONTIN <strong>di</strong>stribution function of the vesicles size,expressed as normalized intensity (in arbitraryunits) vs. the particle <strong>di</strong>ameter, R H (nm) in 6.0mmol l -1 , mole ratio [1.7/1.0], SDS-CTAB catanionicmixtures, up, in the same system with 0.14 mmoll -1 added LYS, me<strong>di</strong>um, and with 1.4 mmol l -1 LYS,down, at 20.0 °C.Dielectric relaxation spectra, reported as ε vs.applied frequency in the SDS-CTAB vesicular<strong>di</strong>spersion [6.0 mmol l -1 and 1.7/1.0 mole ratio], ○,for 0.42, ◊, 0.82, □, and 1.40, ∆, mmol l -1 LYS in thevesicular pseudo-solvent, at 20.0 °C.Dielectric Relaxation Spectroscopy (DS),Electrophoretic mobility and ζ-potential (ζ-P) areused.They allow to characterize in detail the interactionprocesses and give information on the charge<strong>di</strong>stribution around vesicles, proteins, DNA and theirlipo-plexes. Dynamic Light Scattering (DLS), andCircular Dichroism (CD) are also used. The firstmethod estimates the average size of the vesiclesand lipo-plexes formed upon interaction withbiopolymers, the second gives information on theconformation of the biomacromolecules bound ontovesicles.Vesicles formed by SDS and CTAB are used. They arenegatively charged, i.e. the SDS content is in excesswith respect to CTAB. Hence, interactions withproteins having high iso-electric point, such aslysozyme, LYS, may be experienced. The behavior ofmixtures containing SDS-CTAB based negativelychargedvesicles in the presence of increasingamounts of LYS is under study. The interactionprocess continues up to complete saturation of theavailable bin<strong>di</strong>ng sites onto the vesicles and theprotein release from vesicles is demonstrated.References1- Zuber, G., Dauty, E., Nothisen, M., Belguise, P.,Behr, J.P. Adv. Drug Delivery Rev., 52, 245, (2001).2- Marques, E.F., Regev, O., Khan, A., Lindman, B.Adv. Colloid Interface Sci.,100-102, 83 (2003).3- Mel’nikova, Y.S., Lindman, B. Langmuir, 16, 5871(2000).AuthorsAdalberto Bonincontro 1 and Camillo La Mesa 21<strong>Dipartimento</strong> <strong>di</strong> <strong>Fisica</strong>; 2 <strong>Dipartimento</strong> <strong>di</strong> ChimicaUniversità <strong>di</strong> Roma “La <strong>Sapienza</strong>”85SOFT Scientific <strong>Report</strong> 2004-06
Scientific <strong>Report</strong> – Self Assembly, Clustering, Structural arrestPolyelectrolyte-Charged Liposome Complexes: Evidence ofEquilibrium Multi-Compartment AggregatesAggregation of colloidal particles induced byoppositely charged polyions has been extensivelyinvestigated both theoretically and experimentally,owing to its importance in many field of physicalchemistry. Recently, these phenomena attractedrenewed interest because the properties of theresulting aggregates, combined with thecharacteristics of self-organi-zation of the process,possess high potential for applications in materialresearch and bio-nanotechnologies. Among thesestructures, cationic lipid-DNA complexes havewitnessed of an increasing acceptance as preferentialDNA delivery vehicles in gene therapy [1], since theirFig. 1. Top: The average ra<strong>di</strong>us of the aggregatesfrom DLS measurements (o) reaches its maximumat the point of charged inversion, as measured by ζ-potential (o), i.e. at ζ=0.Bottom: a): TEM image of a typical pd-liposomeaggregate. Contrast is obtained mixing liposomesprepared loa<strong>di</strong>ng their acqueous core with two<strong>di</strong>fferent concentrations of CsCl. “Heavily” and“lightly” loaded globules are clearly <strong>di</strong>stinguished.b): Cs elemental map for the same frame, obtainedby filtering the transmitted electrons in an energywindow characte-ristic of the Cs-M 4,5 edge. c): theoriginal image a) and the map b) with a threshold at≈ 50% of the peak value are superimposed.enhanced permeability through the membranebilayer favors the delivery of DNA into the cells.“Columnar” (stacks of alternate planar layers of lipidand polymer) or “hexagonal” (lipids in the hexagonalphase H II with a DNA chain filling each honey-combcell) structures that form in these systems close tothe isoelectric point (where the stoichiometric chargeof DNA counterbalances the stoichiometric charge ofthe lipid phase) have been described [2].However, by means of the combined use of DynamicLight Scattering (DLS) and Transmission ElectronMicroscopy (TEM) measurements, we pro-vide astrong and <strong>di</strong>rect evidence for the existence, in thepolyion-induced liposome aggregation, of anequilibrium cluster phase where liposomes maintaintheir integrity, with the ability of preserving theaqueous content of their core from the externalme<strong>di</strong>um. Oppositly charged polyelectrolytes stick tothe liposome surface and, with the increasingpolymer/lipid charge concentration ratio, ξ, theoverall electric charge of these “polyelectrolytedecoratedliposomes” (pd-liposomes) decreases,passing through the zero (isoelectric point) andreaching large negative values when polyions are inexcess (“overcharging” or charge inversion).Maximum aggregation occurs close to the point ofcharge inversion. As we have recently shown, theseclusters are equilibrium aggregates [3,4].Aggregation is due to a balance of long range electrostaticrepulsion and of short-range attraction,arising from the correlated adsorption of polyions atthe liposome surface ("charge patch" attraction).This cluster phase shows very interesting features inview of possible biome<strong>di</strong>cal applications since thestructure of the aggregates, with liposomesreversibly "glued together" by a non-uniform<strong>di</strong>stribution of adsorbed polyelectrolytes, could fin<strong>di</strong>nteresting applications as "multi-compartmentliposomal aggregates" for controlled-release drugdelivery.References[1] D. Ferber, Science, 294, 1638 (2001).[2] I. Koltover, T. Sal<strong>di</strong>tt, J. O. Rädler, C. R. Safnya,Science, 281, 78 (1998).[3] F. Bor<strong>di</strong>, C. Cametti, M. Diociaiuti, S. Sennato,Phys. Rev. E, 71, 050401(Rd) (2005).[4] F.Bor<strong>di</strong>, C.Cametti, M.Diociaiuti, S.Sennato,Biophys. J. 2006, 91,1513-1520AuthorsF. Bor<strong>di</strong> § , C. Cametti § , S. Sennato § , M. Diociaiuti #§<strong>Dipartimento</strong> <strong>di</strong> <strong>Fisica</strong>, Università <strong>di</strong> Roma ″La<strong>Sapienza</strong>″, Roma and INFM CRS-SOFT#<strong>Dipartimento</strong> <strong>di</strong> Tecnologie e Salute, IstitutoSuperiore <strong>di</strong> Sanità, Roma.SOFT Scientific <strong>Report</strong> 2004-0686
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