Journal of Colloid and Interface Science 304 (2006) 512–517www.elsevier.com/locate/jcis

Conductometric evidence for intact polyion-induced liposome clusters

F. Bordi a,b, C. Cametti a,b,∗, S. Sennato a,b, D. Viscomi a,b

a Dipartimento di Fisica, Universita’ di Roma “La Sapienza,” Piazzale A. Moro 5, I-00185 Rome, Italyb INFM-CRS SOFT, Universita’ di Roma “La Sapienza,” Rome, Italy

Received 9 June 2006; accepted 5 September 2006

Available online 9 September 2006

Abstract

In this note, we present a set of electrical conductivity measurements of polyion-induced liposome aggregate aqueous suspensions that supportsevidence for the existence of a cluster phase in low-density colloidal systems. Heavily NaCl-loaded liposomes, dispersed in a low-conductivityaqueous solution, are forced by electrostatic interactions with oppositely charged polyions to build up into individual aggregates, where the singlevesicles maintain their integrity and, upon an external force, are able to release their ionic content. The conductivity data, within the effectivemedium approximation theory for heterogeneous systems, are in agreement with the picture of a suspension built up by clusters of vesicles whichare able to preserve their content from the external medium. This finding opens new possibilities in multicompartment drug delivery techniques.© 2006 Elsevier Inc. All rights reserved.

Keywords: Liposome; Polyion-induced aggregation; Electrical conductivity; Liposome cluster

1. Introduction

The adsorption of polyelectrolytes onto oppositely chargedmesoscopic particles has been extensively investigated from ex-perimental and theoretical point of view, because of its impor-tance in a wide range of biological and technological processes[1–5]. This phenomenon is rather complex, depending on avariety of interconnected parameters which govern the finalspatial and electrical organization of the resulting complexesand, despite significant efforts and progresses, a complete un-derstanding of this phenomenology is still unsatisfactory.

In this note, we are going to focus on the complexationprocess of polyion–liposome systems at a low level of hier-archy, involving the formation of liposomal clusters of intactlipidic vesicles stuck together by polyion chains which act asan electrostatic glue [6].

At a higher hierarchical level, these aggregates can furtherevolve and a liposome aggregate restructuring is known to oc-cur, involving, as a consequence of the liposome fusion, therelease of the vesicle aqueous content and the formation ofelongated rod-like structures or internal multilamellar struc-

* Corresponding author.E-mail address: cesare.cametti@roma1.infn.it (C. Cametti).

0021-9797/$ – see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.jcis.2006.09.009

tures, where lipid bilayers alternate with ionized polyions [7–9].A variety of different experimental techniques, such as elec-tron microscopy [10,11], cryo-electron microscopy [12] andsmall-angle X-ray scattering [13], provides evidence for this re-structuring process.

On the same time, other techniques, such as dynamic lightscattering, transmission electron microscopy, electrophoreticand ζ -potential measurements [6,14–16] have shown that, inlow-density colloidal suspensions, an equilibrium, reversiblecluster phase exists, based on the balance between long-rangeelectrostatic repulsion (modeled by a Yukawa potential) andshort-range attraction. The short-range attraction potential ap-pears to be due to strong lateral correlation among adsorbedpolyions. The presence of a correlated adsorption [17] atthe surface of polyion-coated particles generates an orderedpatchwork-like structure [15], with excess negative charge andexcess positive charge domains, which impart the short-rangeattractive potential that governs the equilibrium cluster forma-tion.

In the last few years, we have extensively investigatedaggregates formed by unilamellar liposomes (built up bya cationic double chain lipid, dioleoyltrimethyl ammoniumpropane (DOTAP)) stuck together by a highly oppositelycharged linear polyion, polyacrylate sodium salt (NaPAA), that,

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thanks to its relatively simple chemical repeat unit, acts as a pro-totype of a polyion model [6,14–16]. The aggregate (cluster)formation involves two different processes. First, lipid mole-cules in aqueous solution self-assemble into charged liposomes,then, liposomes are assembled together into larger aggregatesby electrostatic interaction with oppositely charged polyions.We have provided experimental evidence that these aggregates,in low-density colloidal systems, i.e., at least in a range of lowenough volume fractions, maintain the integrity at the level ofthe single liposomes, without a lipid bilayer restructuring intomore complex arrangements. A clear evidence for the existenceof clusters composed by intact liposomes has been recently ob-tained by means of transmission electron microscopy (TEM)images, where the integrity of the single liposomes within thecluster, owing to the different concentrations of Cs+ ions intheir aqueous core, is clearly recognizable [16]. These com-partmentalized aggregates, which differ from vesosomes [18]because of the absence of an outer bilayer encapsulating mem-brane, could provide vehicles for multi-functional drug deliv-ery. Actually, the encapsulating membrane, if on one side mightfavor the overall biocompatibility, on the other hand it mightreduce the permeation properties. Moreover, this cluster phaserepresents a new and not yet completely understood colloid,with many unusual properties.

In order to provide a further evidence, among others, forthe existence, in NaPAA–DOTAP complexes, of this clusterphase, we have measured the low-frequency limit of the electri-cal conductivity of differently assembled liposome suspensions,from single liposomes, built up in high-conductivity salty aque-ous solution (0.3 M NaCl), to NaCl-loaded liposomes in low-conductivity aqueous suspension and finally to NaCl-loadedliposomes stuck together by an oppositely charged polyion,forming equilibrium, reversible isolated clusters of average sizeranging from 100 to 1000 nm. These aggregates were inducedto break down from an external stimulus, with the consequentrelease of their ionic content, whose amount can be monitoredby means of electrical conductivity measurements. Actually,d.c. electrical conductivity measurements probe primarily theconcentration of small ions in the bulk solution and conse-quently a change in the overall electrical conductivity shouldoccur if liposomes within the aggregate fuse, with the releaseof their ionic content. Our results confirm that liposomes withinthe single aggregate maintain their integrity, preserving theircontent from the external medium. In the conditions of the ex-periment, a further restructuring of the polyion–liposome com-plexes does not occur and this finding provides evidence fora new class of colloids with many fascinating and intriguingproperties [19], whose existence has been for quite a some timediscussed, originating a scientific dispute which, still today, hasnot been completely resolved.

2. Experimental

Unilamellar liposomes in aqueous solutions were obtainedfrom cationic double tailed lipids, dioleoyltrimethyl ammo-nium propane. Materials were purchased from Avanti PolarLipids (Alabaster, AL) and used without further purifica-

tion. The polyion-induced liposome aggregation has been in-duced by adding a highly charged linear polyion, polyacrylatesodium salt, purchased from Polysciences (Warrington, PA) as0.25 (wt/wt) aqueous solution, with a nominal molecular weightof 60 kD. In all the experiments, the liposome concentrationwas maintained constant to the value CL = 0.68 mg/ml, corre-sponding to a vesicle concentration of 4.2 × 1012 particle/ml.The mean hydrodynamic radius of the vesicles, measured bymeans of dynamic light scattering technique, was about 40 nm,with a log-normal size distribution (intensity averaged) with apolydispersity of the order of 0.20–0.25.

The polyion–liposome complex formation was induced byadding an appropriate amount of a polyion solution at the de-sired concentration to the liposome suspension in a single mix-ing step and gently shaking by hand. The size characterizationof the resulting complexes and the evaluation of their overallelectric charge during all the aggregation process was followedby means of dynamic light scattering and electrophoretic mea-surements.

Liposomes were initially prepared in 0.3 M NaCl electrolytesolution and subsequently an aliquot of this suspension was ex-tensively dialyzed against deionized water, by means of an AM-ICON 10 ml ultrafiltration cell, employing XM50-size mem-branes. The process removes the external salt concentration,leaving the liposome core content sufficiently unaffected. Thesame procedure was applied to the polyion-induced liposomeaggregates. After the initial check of the ion concentration bymeans of electrical conductivity measurements, the simple lipo-some suspensions and the polyion-induced aggregate suspen-sions were sonicated for 5 min in pulse power mode by meansof a Sonic Vibra Cell VC130. The process should induce theopening of transient pores, allowing the release of the liposomecore content in the external medium, whose ionic amount wasagain determined by means of electrical conductivity measure-ments.

2.1. Electrical conductivity measurements

The electrical conductivity of the liposome suspensions andpolyion-induced liposome aggregates was measured in the fre-quency range from 10 kHz to 2 GHz, at the temperature of25.0±0.2 ◦C, by means of two Radiofrequency Impedance An-alyzers Hewlett–Packard Models 4291A and 4294A. Detailsof the experimental procedure have been reported elsewhere[20,21]. Although the measurements extend over a wide fre-quency range, evidencing in the high frequency tail the begin-ning of the orientational dispersion of the aqueous phase, in thisnote, we consider exclusively the low-frequency value, neglect-ing the effects associated with the heterogeneity of the systeminvestigated and the surface ionic polarizations. Our aim is tocontrol the ion concentration in the aqueous phase, as a con-sequence of the state of aggregation of liposomes and of theirintegrity within the single aggregate. Low-frequency electricalconductivity, depending on the characteristic of the single ions(their concentrations and their mobility) should be an appropri-ate parameter, largely independent, to a first approximation, ofthe complex polarization mechanisms giving rise to dielectric

514 F. Bordi et al. / Journal of Colloid and Interface Science 304 (2006) 512–517

and conductivity dispersions in highly heterogeneous systems.However, in the following, for sake of completeness, we reportthe full spectra of the electrical conductivity, over the wholefrequency range investigated.

2.2. Dynamic light scattering (DLS) measurements

The size and size distribution of the polyion-induced li-posome aggregation have been measured by means of dy-namic light scattering technique, using an optical fiber probe(Brookhaven FOQELS) in conjunction with a Brookhaven9000 AT logarithmic correlator, to compute the scattered lightelectric field time autocorrelation function. The relaxationtimes that characterize the diffusing movement of the lipo-some aggregates were found using the standard data analysisprogram CONTIN [22], in terms of a continuous distribu-tion of exponential decay times. This analysis yields the av-erage diffusion coefficient 〈D〉 which can be translated into thehydrodynamic radius Rp by the Stokes–Einstein relationshipRp = KBT/(6πη〈D〉), where KBT is the thermal energy and η

the viscosity of the solvent.

2.3. Electrophoretic measurements

The electrophoretic measurements were carried out bymeans of the laser Doppler electrophoresis technique using aMALVERN Zetamaster apparatus equipped with a 5 mW HeNelaser. The mobility u of the diffusing aggregates was convertedinto a ζ -potential using the Smoluchowski relation ζ = uη/ε,where ε and η are the permittivity and the viscosity of the solu-tion, respectively.

3. Results

We have measured the electrical conductivity σ of a lipo-some suspension in three different aqueous environments, i.e.,(i) liposomes built up with a 0.3 M NaCl electrolyte solutionand dispersed in the same solution; (ii) liposomes with 0.3 MNaCl aqueous core dispersed in deionized water and, finally,(iii) this latter liposome suspension after a sonication cycle,where we expect the core content shared with the bulk solution.Fig. 1 compares the above stated d.c. electrical conductivitiesin the three different environments.

The excess NaCl salt was removed from the liposome sus-pension by extensive dialysis against deionized water and theconductivity of the overall suspension, starting from the valueσ = 2.88 mho/m, reduces to values of about 0.0173 mho/m.During this process, NaCl-loaded liposomes maintain their in-tegrity, preserving their salt concentration. It is well known thatunilamellar 100–200 nm sized liposomes are osmolitically in-ert [23–26] and that an osmotic shock does not produce a stablemembrane rupture.

The dialyzed suspension was then sonicated in order to pro-duce the opening and the following closure of the membranebilayer, with the consequent release of its ionic content. As aconsequence, the electrical conductivity, starting from the value

Fig. 1. The electrical conductivity σ as a function of frequency of liposomeaqueous suspensions, at the temperature of 25.0 ± 0.2 ◦C. The lipid concentra-tion is CL = 0.68 mg/ml. (a) Liposome suspension in 0.3 M NaCl electrolytesolution; (b) liposome suspension after exhaustive dialysis against deionizedwater; (c) liposome suspension after sonication (the electrolyte content in theliposome core has been released in the external medium, with a consequent in-crease of the conductivity).

Fig. 2. The normalized autocorrelation function g(1)(τ ) as a function of thecorrelation time τ for liposome suspensions in different aqueous environments.The inset shows the size distribution derived from the Laplace inversion of thecorrelation function using the constrained regularization algorithm CONTIN.(A) Liposomes built up in 0.3 M NaCl electrolyte solution; (B) liposomes afterdialysis against deionized water. The liposome aqueous core contains NaCl saltat a nominal concentration of 0.3 M; (C) liposomes in a dialyzed solution aftersonication.

of 0.0173 mho/m after the dialysis process, increases to thevalue of 0.0243 mho/m.

After the dialysis and the sonication treatments, the size andthe size distribution of liposomes do not vary too much, thesize distribution being monomodal, with a polydispersity of theorder of 0.20–0.25. This behavior was checked by means of dy-namic light scattering measurements, where the scattered lightintensity correlation function was analyzed by the CONTINalgorithm. The results are shown in Fig. 2, where the intensity–intensity autocorrelation function together with the size distrib-utions of liposomal particles, in the three different experimentalenvironments investigated, are compared.

For the systems investigated, we have also measured the ζ -potential, as a further check of the liposome integrity. We obtainvalues of 73 ± 3 mV for single liposomes after dialysis and avalue of 45 ± 5 mV for liposomes after sonication. These val-

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ues compare reasonably well with those measured for the samesystem [6].

Before proceeding any further, we want to justify the changein the conductivity observed in the liposome suspension afterthe dialysis and the sonication procedure. Within the effectivemedium approximation theory, the conductivity σ of a collec-tion of spheroidal particles of conductivity σp, uniformly dis-persed in a continuous medium of conductivity σm, is given by

(1)σ = σm2σm + σp − 2Φ(σm − σp)

2σm + σp + Φ(σm − σp),

where Φ is the volume fraction of the particles, given by

(2)Φ = 1

6a0RpN0CL

with a0 the area of the lipid head group, Rp the radius of theliposomes, CL the lipid concentration, and N0 the Avogadronumber. For typical values of a0 = 60 Å2, Rp = 70 nm, and thelipid concentration employed, CL = 0.68 mg/ml, the volumefraction is Φ = 4.7 × 10−3. Before the dialysis process, withliposomes dispersed in 0.3 M NaCl solution, since σp � σm,we expect that the conductivity of the suspension does not dif-fer appreciably from the conductivity of the external medium,σ � σm. After the dialysis process, σp � σm and Eq. (1) re-duces to σ/σm � (1 + 2Φ)/(1 − Φ) � 1.01. This means that,also in this case, in the above stated conditions, the presenceof the NaCl-loaded liposomes, at the concentration employed,causes only a very small increase of the overall conductivity ofthe system; in other words, the measured conductivity of thesuspension approaches the conductivity of the aqueous phase.

This picture deeply changes when the liposome suspensionis sonicated and the liposome aqueous core content is ex-changed with the external medium.

In vesicles containing high internal salt concentration,placed in a more dilute solution, the osmotic influx of solventinto the interior of a vesicle leads to its rupture, resulting in theformation of pores. Once a pore is formed, the internal con-tent of the vesicle begins to leak out, resulting in a decrease ofmembrane tension which favors the pore closure.

In this condition, the conductivity of the suspension, sincethe liposomes themselves go on not contributing because of thesmall value of the volume fraction, is expected to be

(3)σ = Φσp + (1 − Φ)σm.

By considering σp as the conductivity of 0.3 M NaCl elec-trolyte solution (σp = 3.79 mho/m at T = 25 ◦C) and σm as theconductivity of the dialyzed suspension (σm � 0.017 mho/m),Eq. (3) yields a value of 0.0250 mho/m, that is in verygood agreement with the one measured (Fig. 1c). This re-sult, enforced by the dynamic light scattering measurementand ζ -potential measurement, is a good evidence for intactNaCl-loaded liposomes dispersed in a much lower conductivitymedium, which can be stuck together by electrostatic interac-tions.

The aggregation process was induced by adding appropri-ate amount of NaPAA polyion to the liposome suspension. Asit is well known, the system experiences, depending on the

Table 1Characterization of DOTAP liposome and polyion-induced DOTAP liposomeaggregates by dynamic light scattering measurements

CL (mg/ml) Cp (mg/ml) 2Rp (nm) ζ -potential

0.3 M NaCl 0.68 140 ± 20Dialysis 0.68 160 ± 20 73 ± 6

Dialysis 0.68 0.035 315 ± 20 64 ± 4Sonication 0.68 0.035 120 ± 20

Dialysis 0.68 0.040 370 ± 20 58 ± 4Sonication 0.68 0.040 130 ± 20

Note. The mean diameters and standard deviations (SD) of particle size distrib-utions are averages of four different measurements.

polyion content, two peculiar phenomena known as reentrantcondensation and charge inversion, consisting in the formationof complexes whose average size, at the beginning, increasesuntil the isoelectric condition is reached, then decreases towardthe original size, when the polyion content is further increased.Concomitant to the reentrant condensation, the overall electriccharge of the aggregate changes, at the isoelectric condition,its sign, passing from a positive to a negative charge. Thisphenomenology has been investigated deeply in our laboratoryand a series of works dealing with the main features appeared[6,14–16].

We investigated the aggregation induced by two differentpolyion contents (Cp = 0.035 and 0.040 mg/ml, respectively),to which aggregates of average size of about 315 and 370 nmcorrespond. The characteristics of the systems investigated aresummarized in Table 1. The choice of these polyion concentra-tion values is due to a compromise between the need of a largepolyion concentration (in order to have a large aggregate) and,at the same time, the need of a small polyion content (in orderto have an overall moderate electrical conductivity, enough toevidentiate relatively small changes).

The whole evolution of the average size of the polyion-induced liposome aggregation has been investigated in our pre-vious work [27], where we have evidenced a rather sharp in-crease of the cluster size in the neighborhood of the isoelectriccondition. Moreover, in the presence of simple added salt, closeto the isoelectric condition, clusters are unstable and the sys-tem evolves toward a diffusion limited cluster aggregation [28].This complex phenomenology restricts the interval of polymerconcentration available for this experiment.

Figs. 3 and 4 show the main characteristic features ofthe electrical conductivity measurements for the two polyion-induced liposome aggregate suspensions. As far as Fig. 3 is con-cerned, the addition of a NaPAA solution (Cp = 0.035 mg/ml,electrical conductivity σ = 0.0040 mho/m) to a dialyzed li-posome suspension (CL = 0.68 mg/ml, electrical conductivityσ = 0.0172 mho/m) results in the polyion-induced liposomeaggregate suspension with a conductivity σ of the order of0.0691 mho/m. This increase is not surprising, since the drivingforce for the polyion–liposome self-assembly is the release ofcounterions into solution, which are one-dimensionally boundto NaPAA chains and two-dimensionally bound to the cationicsurface. This aspect has been considered in detail in an otherprevious paper of ours [29], where we followed the conductiv-

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Fig. 3. The electrical conductivity σ as a function of frequency of liposomeaqueous suspensions, at the temperature of 25.0 ± 0.2 ◦C. (a) Polyion solutionat a concentration Cp = 0.035 mg/ml; (b) liposome suspension after exhaustivedialysis against deionized water; (c) Polyion-induced liposome aggregates withan average size of 315 nm; (d) liposome aggregates after sonication (the elec-trolyte content of each liposome core has been released in the external medium).

Fig. 4. The electrical conductivity σ as a function of frequency of liposomeaqueous suspensions, at the temperature of 25.0 ± 0.2 ◦C. (a) Polyion solutionat a concentration Cp = 0.040 mg/ml; (b) liposome suspension after exhaustivedialysis against deionized water; (c) Polyion-induced liposome aggregates withan average size of 370 nm; (d) liposome aggregates after sonication (the elec-trolyte content in each liposome core has been released in the external medium).Note the linear scale to evidence small differences in curves d and c.

ity behavior of NaPAA polyion–DOTAP liposome system dur-ing the whole complexation process in an extended interval ofpolyion concentration, below and above the isoelectric condi-tion. It demonstrated that the polyion adsorption is concomitantwith the counterion release, whose magnitude is exactly the onepredicted by the lateral correlation adsorption theory [30–32].

Upon sonication, a further release of ions into solution oc-curs, exactly the ions in the aqueous core of the intact lipo-somes, which are a part of a single aggregate, and the valueof the conductivity increases from the one of the liposome ag-gregate suspension (σ = 0.0691 mho/m, Fig. 3c) to the valuemeasured after the sonication procedure (σ = 0.0776 mho/m,Fig. 3d). On the assumption that all the liposomes release theirionic content upon sonication, we are able to evaluate again theexpected conductivity increment on the basis of Eq. (3), Φ be-ing now the volume fraction of liposome aggregates into thesolution. Since the effective medium approximation theory [33]

Fig. 5. Size distribution of polyion induced liposome aggregates before (fullbars) and after (empty bars) sonication at two different polyion concentrations.Panel A: Cp = 0.035 mg/ml; Panel B: Cp = 0.040 mg/ml.

is insensitive to the geometry of the dispersed objects (in thelimit of spheroidal objects and for very dilute suspension), thefractional volume of dispersed aggregates equals the fractionalvolume of the single liposomes (Φ � 4.7 × 10−3). In this case,we obtain from Eq. (3) a value of σ = 0.0776 mho/m, that fitsvery well with what experimentally found. The complexation ata slightly higher polyion concentration (Cp = 0.040 mg/ml) be-haves similarly (Fig. 4), even if the difference before and afterthe sonication is smaller, since the ion release involves the sameamount of ions, now dispersed in a higher conductivity suspen-sion, because of the higher polyion concentration. Moreover,approaching the isoelectric condition, the counterion releasefrom the polyion chain (condensed counterion) and from theliposome surface increases, contributing to a further increase ofthe overall conductivity suspension.

Finally, we have to prove that liposome aggregates aftersonication continue to be formed by individual liposome clus-ters. Fig. 5 compares the size distribution of liposome sus-pension, before and after sonication. As can be seen, in bothcases, a monomodal distribution appears, lightly shifted towardsmaller average size immediately after sonication. The mea-surement of ζ -potential in the two aggregates before the soni-cation process yields a value of 64 ± 4 mV (in deionized water,Cp = 0.035 mg/ml) and a value of 58 ± 4 mV (in deionizedwater, Cp = 0.040 mg/ml). Also in this case, these values donot differ appreciably from those we observed in similar sys-tems [15].

These results furnish a further evidence, among others, basedon electrical conductivity measurements, for the existence of anequilibrium cluster phase in low-density liposome aqueous sus-pensions, where the complexation involves a low hierarchicallevel, different from the new kind of self-assembled multilayersobserved in lipidic systems at higher lipid content. Furthermore,

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the present results strengthen previous investigations based onTEM images of liposome aggregates to form isolated clusters,where the polyion acts as an electrostatic glue to join togetherdifferent intact liposomes. The possibility to control, by meansof simple environmental parameters, such as the ionic strength,the size and the electrical charge of clusters composed by lipo-somes which maintain their integrity offers new and interestingapplications in fields of chemistry, soft-matter physics and lifesciences.

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