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Complexation of Anionic Polyelectrolytes with Cationic Liposomes: Evidence of Reentrant Condensation and Lipoplex Formation F. Bordi,† C. Cametti,*,† M. Diociaiuti,‡ D. Gaudino,† T. Gili,† and S. Sennato† Dipartimento di Fisica, Universita´ di Roma “La Sapienza”, Piazzale A. Moro 5, I-00185 Rome, Italy, and INFM-CRS SOFT, Unita´ di Roma 1, Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanita´ , I-00185 Rome, Italy Received October 27, 2003. In Final Form: January 26, 2004
We have studied the complexation process taking place in cationic liposomes in the presence of anionic polyelectrolytes, in the polyion concentration range from the dilute to the concentrated regime, by combining dynamic light scattering and transmission electron microscopy techniques. We employed as the cationic lipid a two-chained amphiphile (Dioleoyltrimethylammoniumpropane) and sodium polyacrylate salt as the flexible anionic polyelectrolyte. The results evidence a variety of different structures, mainly depending on the liposome-polyion charge ratio, whose peculiar dynamical and structural features are briefly described. In particular, three different polyion concentration regions are found, within which a monomodal or bimodal distribution of aggregates, with a well-defined time evolution, is present. At low polyion content, close to the isoelectric point, large aggregates are formed, deriving from the collapse of the liposomal bilayers into extended charged surfaces, where adsorbed polyions form a two-dimensional strongly correlated array and organize into a two-dimensional Wigner liquid. At high polyion content, above a critical concentration, the size distributions of the complexes are clearly bimodal and a large-component aggregate, continuously increasing with time, coexists with a population of smaller-size aggregates. At an intermediate polyion concentration, spherical, small-size vesicular structures are reformed, connected in a network by polymer chains. A brief discussion tries to summarize our results into a consistent picture.
Introduction The complexation between a flexible polyelectrolyte and a charged colloidal particle of opposite sign has been recently studied both experimentally1-7 and theoretically8-12 by various authors, owing to its interest in different fields, ranging from biological processes, such as purification of proteins and immobilization of enzymes in polyelectrolyte complexes, to commercial processes that include enhanced oil recovery,13 water treatment by colloidal flocculation,14 and stabilization of preceramic suspensions.13 * To whom correspondence should be addressed. † Universita ´ di Roma “La Sapienza”. ‡ Istituto Superiore di Sanita ´. (1) Wagner, K.; Harries, D.; May, S.; Kahl, V.; Ra¨dler, J. O.; BenShaul, A. Langmuir 2000, 16, 303-306. (2) de Meijere, K.; Brezesinski, G.; Mo¨hwald, H. Macromolecules 1997, 30, 2337-2342. (3) McQuigg, D. W.; Kaplan, J. I.; Dublin, P. L. J. Phys. Chem. 1992, 96, 1973-1981. (4) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science 1998, 282, 11111114. (5) Caruso, F.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 1998, 102, 2011-2016. (6) Engelking, J.; Wittemann, M.; Rehahn, M.; Menzel, H. Langmuir 2000, 16, 3407-3413. (7) Ge, L.; Mo¨hwald, H.; Li, J. Biochem. Biophys. Res. Commun. 2003, 303, 653-659. (8) Dobrynin, A. V.; Deshkovski, A.; Rubinstein, M. Phys. Rev. Lett. 2000, 84, 3101-3104. (9) Dobrynin, A. V.; Deshkovski, A.; Rubinstein, M. Macromolecules 2001, 34, 3421-3436. (10) Nguyen, T. T.; Grosberg, A. Y.; Shklovskii, B. I. Phys. Rev. Lett. 2000, 85, 1568-1571. (11) Nguyen, T. T.; Shklovskii, B. I. J. Chem. Phys. 2001, 114, 59055916. (12) Nguyen, T. T.; Shklovskii, B. I. J. Chem. Phys. 2001, 115, 72987308. (13) Gregoriadis, G. Liposome Technol. 1993, 1, 1-63. (14) Schwoyer, W. Polyelectrolytes for water and wastewater treatment; CRC Press: Boca Raton, FL, 1981.
The phenomenology underlying this complexation process is rather complex, depending on many parameters such as the charge and the size of the particles, the charge density on the polyion chain, the flexibility of the polyelectrolyte backbone, and the ionic strength of the medium. In aqueous solutions, charged mesoscopic particles are surrounded by a diffuse layer of spatially confined counterions that, at least partially, screens long-range electrostatic interactions. Nevertheless, polyelectrolyte chains strongly interact with these mesoscopic particles, in particular when they bear a charge of opposite sign, and form a large variety of structures. The strong correlation among polyions adsorbed onto a charged colloidal particle results in two important effects, that is, the “charge inversion” and the presence of a short-range attractive potential between polyion-coated particles. Charge inversion15,16 occurs when, in the adsorption at the charged surface, more polyions collapse than are necessary to neutralize it. The resulting complex can, therefore, display an overall charge of opposite sign to the one the particle originally bears. Charge inversion is a phenomenon due to the strong lateral correlation between adsorbed polyelectrolytes.9,10 Also, the short-range attractive potential, which close to the isoelectric point causes the aggregation of lipoplexes, stems from the lateral correlation of adsorbed polyelectrolytes. Avoiding each other and residing as far away as it is possible to minimize their electrostatic interactions, polyelectrolytes leave the particle surface partially uncovered. At the isoelectric point, electrostatic repulsion between complexes that are almost neutral is small. They, therefore, can now get so close to each other that the local electrostatic interaction (15) Nguyen, T. T.; Shklovskii, B. I. Physica A 2001, 293, 324-338. (16) Mateescu, E.; Jeppepsen, C.; Pincus, R. Europhys. Lett. 1999, 46, 493-498.
10.1021/la036006u CCC: $27.50 © 2004 American Chemical Society Published on Web 05/25/2004
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between a polyion on one complex and the bare oppositely charged particle surface on the other complex can bind them together. This short-range attractive potential arises from a nonhomogeneous charge distribution. The purpose of this paper is to present a comprehensive scenario of the complexation between a charged liposome and an oppositely charged linear polyelectrolyte, combining dynamic light scattering measurements and transmission electron microscopy (TEM) measurements, to explore both the hydrodynamic and structural properties of the resulting aggregates. This strategy is intended to provide further information on the complexation process of liposomes and to reconcile the somewhat diverging pictures emerging from spectroscopic and structural techniques. The study described here focuses on the assembly process of cationic liposomes built up by dioleoyltrimethylammoniumpropane (DOTAP) lipids that have long been used as models for biological membranes and have proved to be advantageous as carriers for drug-delivery systems.17 Liposomes, vesicles whose typical sizes range from 20 nm to micrometers in diameter, are closed shells of self-assembled lipid bilayers that encompasse an aqueous core.18 Complexes of cationic liposomes with DNA have recently received much interest as nonviral gene delivery vehicles19 for a variety of biomedical applications.20 However, as a result of the complexity of the physical transformation occurring during the polyionliposome self-assembly, much remains unknown, in particular the physical characterization of the resulting aggregates that form a class of new colloids yet poorly defined. Most experimental studies on liposome-DNA interaction are focused on supramolecular complex structural details21-24 with little attention to the kinetics of complex formation. Although several authors observed that different preparation procedures might influence the structure of the resulting lipoplexes and their transfection efficiency, no systematic attempt has been devoted to investigating the aggregation kinetics and to clarifying the basic mechanisms that cause the aggregation. To some extent, this lack of basic knowledge has to be ascribed to the fact that, despite the wide activity in this field, lipoplexes with an optimal transfection efficiency are not still available to be employed in gene therapy. For example, only recently, the role of the overall charge density of lipoplexes in determining their transfection efficiency has been pointed out,25 while the effects of condensed counterions around a polyelectrolyte on the lipoplexes formation are still unclear. In particular, the counterion release has been claimed as the driving force that leads to DNA-liposome aggregation and, eventually, to the formation of lipoplexes. From an experimental point of view, the evaluation of the counterion condensation in systems, at finite polyion (17) Barenholz, Y. Curr. Opin. Colloid Interface Sci. 2001, 6, 66-77. (18) Lasic, D. Liposomes in gene delivery; CRC Press: Boca Raton, FL, 1997. (19) Woodle, M. C.; Scaria, P. Curr. Opin. Colloid Interface Sci. 2001, 6, 77-86. (20) Pedroso De Lima, M.; Simoes, S.; Pires, P.; Faneca, H.; Duzgunes, N. Adv. Drug Delivery Rev. 2001, 47, 277-294. (21) Stenberg, B.; Sorgi, F.; Huang, L. FEBS Lett. 1994, 356, 361366. (22) Lasic, D. D.; Strey, H.; Stuart, M. C. A.; Podgornik, R.; Fredick, P. M. J. Am. Chem. Soc. 1997, 119, 832-833. (23) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810-814. (24) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 281, 78-81. (25) Lin, A.; Slack, N.; Ahmad, A.; George, C.; Samuel, C.; Safinya, C. Biophys. J. 2003, 84, 3307-3316.
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concentration, is generally a difficult task, especially for a rigid polyelectrolyte such as DNA. On the other hand, for a flexible polyelectrolyte, it is well documented that the extent of counterion condensation strongly affects the local chain conformation. In this case, owing to the balance between electrostatic and polymer-solvent interactions, the polyion chain can be described as an extended sequence of partially coiled domains (blobs), where electrostatic interactions between charged groups are screened by condensed counterions and each blob bears an effective renormalized charge. On the basis of these considerations, it is possible, in principle, to evaluate the influence of counterion condensation during the polyelectrolyteliposome interactions through the polyion chain conformation. To this end, we employed sodium polyacrylate salt (NaPAA), a highly flexible polyion, whose electrical behavior has been extensively investigated and the effect of counterion condensation, depending on the polymer concentration, well-established.26,27 Moreover, as pointed out by Raspaud et al.,28 the aggregation is not a consequence of the intrinsic structure and flexibility of the polyion, and different experimental results have clearly demonstrated that the mechanism leading to aggregation is qualitatively independent of the polymer flexibility, and, rather, the common feature is the electrostatic interaction due to the highly charged character of the system. In the present study, the size distribution and the morphology of cationic lipid DOTAP-polyelectrolyte (NaPAA) complexes have been investigated in a wide polyion concentration range, evidencing at least three distinct steps in the mechanism of complex formation. Polyion-coated liposomes, at low polyion concentration, evolve toward a liposome aggregation resulting, close to the isoelectric point, in large charged surfaces where adsorbed polyions form an ordered array, resulting in a two-dimensional Wigner liquid. At an appropriate polyion concentration, well above the isoelectric condition, a component whose size increases with time coexists with single small complexes that do not show a tendency to aggregate further. The knowledge of these peculiar behaviors and the characterization of the different structures resulting in the overall complexation process is crucial to obtaining insights into the properties that modulate the biological activity of these systems. 2. Experimental Section 2.1. Materials. Three different samples of NaPAAs, [-CH2CH(CO2Na)-]n,, with nominal molecular weights of 5.1, 60, and 225 kD, were purchased from Polysciences, Inc. (Warrington, PA), as a 0.25 (w/w) solution in water and were used as anionic polyelectrolytes. DOTAP was purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. DOTAP, one of the most popular cationic lipids, is a two-chained amphiphile whose acyl chains are linked to the propylammonium group through an ester bond. 2.2. Liposome Preparation. An appropriate amount of DOTAP (17.0 mg) was dissolved in 10 mL of a methanolchloroform solution (1:1 v/v), and the organic solvent was subsequently removed by overnight vacuum desiccation. The resulting dried lipid film was resuspended in water at a temperature of 25 °C well above the phase transition temperature of this lipid (Tf ) 0°), for 1 h. The resulting aqueous lipid mixture was sonicated at a temperature of 25 °C for 1 h at a pulsed power (26) Bordi, F.; Cametti, C.; Gili, T. Phys. Rev. E 2002, 66, 021803021811. (27) Bordi, F.; Cametti, C.; Gili, T. Phys. Rev. E 2003, 68, 011805011812. (28) Raspaud, E.; Olvera de la Cruz, M.; Sikorav, J.; Livolant, F. Biophys. J. 1998, 74, 381-393.
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mode, until the solution appeared optically transparent in white light. A homogeneous liposomal suspension of approximately uniformly sized unilamellar vesicles with an average diameter of (90 ( 5 nm) was obtained. The final liposome concentration was 1.5 × 1013 particles/mL. Complex formation was initiated by adding the NaPAA aqueous solution (prepared at an appropriate concentration) by a single mixing step to an existing DOTAP liposome suspension to a final volume of 1 mL, with a final concentration of 7.5 × 1012 particles/mL. Under these experimental conditions for complex formation, the order of addition of polyion and liposomes has essentially no effect on the resultant aggregate size.29 The solution was gently mixed to avoid bubble formation prior to measurements. The pH values of the solutions were around pH ) 6.2. All experiments were carried out at a temperature of 25.0 ( 0.2 °C and were repeated several times to check reproducibility. 2.3. Dynamic Light Scattering Measurements. The size and size distribution of liposomes and liposome-polyelectrolyte complexes were characterized by dynamic light scattering measurements. This technique measures the normalized (secondorder) time autocorrelation function g(2)(τ) of the intensity of the scattered light, which is related to the normalized (first-order) autocorrelation function g(1)(τ) by the Siegert relationship
g(2)(τ) ) 1 + β| g(1)(τ)|2
(1)
where β is a spatial coherence factor dependent on the geometry of the detection system. For a dilute suspension of monodisperse particles, g(1)(τ) decays exponentially with a decay rate Γ ) q2D, where q is the magnitude of the scattering wavevector and D the translational diffusion coefficient, which is related to the hydrodynamic radius RH through the Stokes-Einstein relationship
RH )
KBT 6πηD
(2)
where KBT is the thermal energy and η is the viscosity of the medium. For a polydisperse system, g(1)(τ) can be expressed as
g(1)(τ) )
∫ G(Γ) exp(-Γt) dΓ ∞
0
(3)
where the distribution function G(Γ) was found using the standard data analysis program CONTIN,30 a fit routine that starts from a preliminary unsmoothed solution in a form of equally spaced ln(Γ) and employs the constrained regularization method, according to statistical criteria. 2.4. TEM Measurements. TEM measurements were carried out by means of a ZEISS 902 microscope operating at 80 kV, equipped with an electron energy loss filter. To enhance contrast, the electron spectroscopy imaging (ESI) mode was used. A droplet of the suspension containing liposome-polyelectrolyte complexes was deposited onto 300-mesh copper grids (for electron microscopy) and covered with a very thin amorphous carbon film (about 20 nm). The liquid excess was removed by placing the grid on the appropriate filter paper. The staining solution, filtered by polycarbonate 0.2-µm filters, consists of 2 % (w/v) of phosphotungstate acid (PTA) in a buffered aqueous solution at pH ) 7.3 (NaOH). In a few cases, grids have been made hydrophilic, by using the glow-discharge method.31 No difference has been observed in the morphology of the samples deposited on hydrophilized or nonhydrophilized grids. The image acquisition was performed by a digital chargecoupled device camera model PROSCAN HSC2 (1K × 1K pixels) thermostated by a Peltier cooler. Image analysis was carried out by a digital analyzer SIS 3.0, which allows the contrast and sharpness of the acquired images to be enhanced and morphological quantification and statistics to be performed. The overall (29) Kennedy, M.; Pozharski, E.; Rakhmanova, V.; MacDonald, R. Biophys. J. 2000, 78, 1620-1633. (30) Provencher, S. Comptut. Phys. Commun. 1982, 27, 213-242. (31) Gustafssonn, J.; Arvidson, G.; Karlsson, G.; Almgrem, M. Biochim. Biophys. Acta 1995, 1235, 305-312.
attainable resolution can be evaluated on the order of 2 nm. Moreover, the digital fast Fourier transform technique was used to study the symmetry and dimensions of the two-dimensionalmolecular periodic arrangements observed.
3. Results and Discussion The formation of polyion-coated liposomes or, more generally, polyion-liposome complexes, has been directly monitored in terms of their apparent hydrodynamic radius RH, as determined by dynamic light scattering measurements. Complex formation was studied as a function of the polyion content in a wide concentration range, from a very low concentration, in the dilute regime, to a high concentration, in the concentrated-entangled regime. We systematically varied the composition of the NaPAADOTAP mixture by varying the polyion concentration from 0.01 to 15 mg/mL. We introduce a polyion-to-DOTAP charge ratio defined as
ξ)
2NfMp MwD Mwp MD
(4)
where Mp and MD and Mwp and MwD are the weights per unit volume of the solution and the molecular weights of the polyion and DOTAP, respectively, N is the degree of polymerization of the polyion, and f is the fraction of free counterions (the fraction of ionized groups on the polyion backbone, after counterion condensation occurred). According to eq 4, the factor 2 comes from considering only the liposome charge distributed on the outer leaflet of the vesicle (1/2 of the total charge present in the liposomal component, assuming for vesicles of 100 nm in diameter approximately the same radius for the outer and inner leaflet of the bilayer). We varied the parameter ξ from 0.09 to 130, crossing the neutralization region. In all the experiments, the liposome concentration was MD ) 0.85 mg/mL, corresponding to a vesicle concentration of 7.5 × 1012 particles/mL. Moreover, three different polyion molecular weights (5.1, 60, and 225 kD) were investigated. Among the others, the polyion-liposome charge ratio parameter plays a crucial role, resulting in an important effect known as “charge inversion”. In the presence of excess liposomes, the complexes exhibit a positive charge, and conversely, in the presence of excess NaPAA, negatively charged structures are formed. As pointed out by Ra¨dler et al.,23,32 the charge inversion produces a structural change in the complex arrangement. In the course of the polyion addition to the DOTAP suspension, the system presents a very complex phenomenology, whose main features are shown in Figure 1, which summarizes the different regimes occurring in the polyion concentration range we have investigated. The size of complexes of DOTAP with NaPAA remains fairly constant (on the order of 200 nm) until a charge ratio of 0.5 is reached, at which point a gradual increase in size is observed up to the charge neutralization. At this point, aggregation of complexes occurs, resulting in structures greater than 1-2 µm in diameter. In all the three polyion molecular weights studied, this growing regime appears. Because the interaction between cationic liposomes and negative polyion is thought to be largely electrostatic, the presence of large structures near the neutral charge condition suggests aggregation governed by diffusion(32) Ra¨dler, J. O. Koltover, I.; Jamieson, A.; Salditt, T.; Safinya, C. R. Langmuir 1998, 14, 4272-4283.
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Figure 2. Hydrodynamic diameter 〈2RH〉 of NaPAA-DOTAP structures in the high polyion concentration regime. The polyion molecular weight is Mw ) 60 kD. Two different concentration regimes are evidenced. In the range lower than 4 mg/mL, only a single component is present as shown in the inset on the left. In the range from 4 to 12 mg/mL (marked by two vertical dotted lines), two distinct populations are present, whose time evolution is shown in the inset on the right. The arrows mark the two particular polyion concentrations at which the size time evolutions are shown.
Figure 1. Average hydrodynamic diameter 〈2RH〉 of NaPAADOTAP structures as a function of the NaPAA concentration, for different molecular weights: (A) Mw ) 5.1 kD; (B) Mw ) 60 kD; and (C) Mw ) 225 kD. The vertical arrows mark the polyion concentration interval, where two distinct populations are simultaneously present in the system, the first with a timeindependent average size in the range 200-250 nm and the second with a time-dependent size in the range 500 nm to 5 µm. In panel B, the letters a-d indicate the polyion concentration at which the TEM measurements have been carried out (see Figures 5-8).
limited cluster aggregation (DLCA), in which two approaching nearly neutral complexes tend to stick to each other due to a short-range attractive potential. Anionic polyions serve generally as a bridge between positively charged liposomes, bringing them to close proximity, leading to their aggregation or fusion. Remarkably, the size of the complexes at the neutral condition appears to be dependent on the polyion molecular weight, that is, on the length of the polyion. As can be seen in Figure 1, with the increase of the molecular weight from 5.1 to 225 kD, the average size of the aggregates spans from 600 to 1300 nm. This behavior can be partially expected because longer polyions can simultaneously contact more liposomes. As the charge ratio is further increased (at moderate excess NaPAA charge), a sharp transition toward smallersize particles is observed, where the increased presence of polyion provides again a different pathway for the formation of smaller-size lipoplexes. As the charge ratio is further progressively increased to values significantly higher than unity (corresponding to the polyion concentration in the range from 0.2 ÷ 0.3 mg/mL to 1 ÷ 3 mg/mL), the hydrodynamic radius does not vary anymore significantly from the value following the initial mixing, indicating that, in these conditions,
the resulting structures are stable against any further aggregation. For the polyions of higher molecular weights (60 and 225 kD), a polyion concentration near 2-4 mg/mL up to 10 mg/mL (corresponding to a charge ratio in the range from about 15 to 80) leads to a different growing kinetic regime in which two different populations appear with two distinct evolution times. It must be noted that the observed phenomenology is directly dependent on the way the experiment was carried out. In this case, the formation of lipoplexes has been obtained by a single addition of the appropriate amount of the polyelectrolyte solution. As a consequence, each sample is independently prepared and the data reported in Figure 1 represent a steady-state regime. The complexity of the system is confirmed by the marked deviation from a single-exponential decay of the electric field autocorrelation function. The analysis by the CONTIN method allows one to obtain the distribution of the relaxation times. For the systems investigated, in this polyion concentration range, we obtain two distinct decay modes, corresponding to two different typical populations, whose size increase appears to occur in two phases. First, for each polyion concentration within the above stated interval, the initially formed lipoplexes undergo a further aggregation forming even larger structures whose average size continuously increases in time. The size distribution of these structures exhibits a unimodal log-normal distribution. We have measured complexes with an estimated size larger than 1-5 µm at time on the order of 103 min from the beginning. Beyond this point, complexes have a size that is impossible to accurately determine by dynamic light scattering. Second, an essentially static regime, in which the lipoplexes are stable and do not undergo further aggregation, is present in the system. Also in this case, this component displays a unimodal log-normal distribution. Figure 2 shows this peculiar behavior in the case of polyion of molecular weight Mw ) 60 kD, where the time dependence of the hydrodynamic radius is shown at two selected polyion concentrations, in the single population region, for a charge ratio of ξ ) 15, and in the two populations region, for a charge ratio of ξ ) 80. It must be noted that the average characteristic times and the amplitude of the two decay modes vary consistently, suggesting that the presence of two different populations
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cannot be attributed to an artifact in the CONTIN analysis. Similar findings have been previously reported by refs 33 and 34 for DNA-dimethyldioctadecyl ammonium bromide/ cholesterol cationic liposomes. However, whereas in that system the time-dependent component rapidly reaches (on the order of 1-5 min) steady-state values, in the NaPAA-DOTAP complexes, the growing kinetics extends over large time intervals (on the order of 103 min or more). A bimodal distribution, in which both the two components are stable upon time, has been observed in zwitterionic liposome-surfactant mixtures, where small closed particles and open structures with formation of tubular aggregates coexist.35 The simultaneous presence of two well-separated components represents a significative novel feature for these systems where aggregation properties differ from those of more conventional charged-stabilized colloidal systems. In fact, the kinetics of the aggregation of chargestabilized colloidal particle suspensions, after a transient regime, proceeds according to the DLCA regime, which results, in the long time limit, in the formation of infinitesize clusters. On the contrary, as can be seen in Figure 2, in the present case, the liposome complexation results in the formation of clusters coexisting with stable, smallsize aggregates. The kinetics of aggregation is described by dynamical scaling36 with a time-dependent average hydrodynamic radius given by the power law
RH(t) ) RH(0)(t/t0)β
(5)
where β ) z/df with df being the fractal dimension of the cluster and z the kinetic exponent. This cluster increase is not justified by the progressive disappearance of the small component, as suggested by the application of the mass action law. Finally, at a slightly higher polymer concentration (about 10 mg/mL), a new regime appears, where gel-like homogeneous samples are found. However, because the amplitude of the correlation function of the scattered light does not decrease, as generally occurs, in viscoelastic gels where the degrees of freedom are partially frozen, this gel-like appearance is probably due to an increase in viscosity.37 It is striking to observe that, in this high polymer concentration, spherical, small-size vesicular structures are reformed, probably connected by polyions into networks. This region is similar to the one suggested by Johner et al.,38 who postulated the possibility of a gel phase induced by a further bridging between partially coated “multiplet” structures, before a complete saturation is reached. The peculiar feature of the aggregation process in the moderate polyion concentration range is the presence of large structures (size on the order of 1 µm), close to the isoelectric condition. The presence of large equilibrium clusters in the system of aggregating particles has been recently suggested by Groenewold and Kegel39 on the basis of a stabilization mechanism that involves a small amount (33) Lai, E.; van Zanten, J. H. J. Controlled Release 2002, 82, 149158. (34) Lai, E.; van Zanten, J. H. J. Pharm. Sci. 2002, 91, 1225-1232. (35) De la Maza, A.; Coderch, L.; Lopez, O.; Parra, J. Microsc. Res. Tech. 1998, 40, 63-71. (36) Bordi, F.; Cametti, C.; Gili, T.; Gaudino, D.; Sennato, S. Bioelectrochemistry 2003, 59, 99-106. (37) Barretta, P.; Bordi, F.; Rinaldi, C.; Paradossi, G. J. Phys. Chem. B 2000, 104, 11019-11026. (38) Johner, A.; Joanny, J.; Diez Orrite, S.; Bonet Avalos, J. Europhys. Lett. 2001, 56, 549-555. (39) Groenewold, J.; Kegel, W. K. J. Phys. Chem. B 2001, 105, 1170211709.
of charge on each aggregating particle. Although these authors explicitly restrict their model to colloidal particles with very low surface charge and, moreover, that are dispersed in a nonpolar solvent, the phenomenology that is expected is exactly the one we observe in our system. The stabilization mechanism for finite-size clusters arises from the repulsion due to a small amount of charge on each aggregating particle. Up to a given size of the aggregate, the electrostatic repulsion is not sufficient to counterbalance the short-range attractive potential that allows the particles to stick together. However, as the size of the cluster increases, a sufficient charge is built up, until an equilibrium between repulsion and attraction between aggregating particles is attained. Consistently with this picture, the aggregation of polyion-coated liposomes is observed in the region around the isoelectric condition, where the adsorbed polyions nearly exactly counterbalance the external surface charge of the lipoplexes. Within this scheme,39 by treating the driving force of clustering in the capillary approximation, Groenewold and Kegel have shown that even a small charge is sufficient to stabilize clusters with a large aggregation number. Close to the isoelectric point, each polyion-coated liposome bears a vanishingly small charge owing to the polyion coverage of the liposome surface, and the mechanism suggested yields aggregates on the order of some micrometers. By minimizing the free energy per particle in a cluster, these authors found that the equilibrium aggregation number n0 is given by
n0 )
2γv0 (z0e)2
(6)
where γ is the negative excess energy per unit surface associated to the particle at the cluster surface, v0 is the volume of the “elementary” aggregating particle that contributes to the cluster formation, and z0e the charge per particle. Assuming γ ) 0.3 erg/cm2, which roughly corresponds to 50-100 hydrogen bonds per liposome, the permittivity of the aqueous phase ) 80, the volume of a single polyion-coated particle v0 ) 2.5 × 10-16 cm3, and a charge z0e ) 10 elementary charges result in an aggregation number n0 on the order of 5 × 102, which yields a cluster of a size on the order of RH ) 0.35-0.40 µm. It is noteworthy that this value is in markedly good agreement with the value derived from light scattering measurements (the maximum observed in Figure 1, at the isoelectric point). Moreover, within the same scheme, the average size of the aggregate, below and above the isoelectric point, scales with the “effective” charge of the polyion-coated liposome as
RH ∝ (qs)-1/3
(7)
where qs is the number of dissociable sites on the liposome surface, after polyion adsorption. The presence of the adsorbed polyion has the effect of renormalizing the effective number density of the charged sites available for interacting with counterions. Assuming the normalized surface charge on the aggregating particles qs ∼ (QL kQP)/QP, where QL are charges of the external surface of the liposome and QP those of the polyion and k is an adjustable parameter, the size of the resulting aggregates, for all three of the molecular weights investigated, scales with qs as shown in Figure 3, with the scaling exponent given by eq 7. The values of the parameter k we used in the scaling law are 0.40, 0.44, and 0.40 for the three
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According to the scaling theory of polyelectrolyte solutions,8,9 in the range of polyion concentrations investigated and in the case of the good solvent condition (T > Θ), the polyion chain is modeled as a random walk of Nξ0 correlation blobs of size ξ0, each of them containing g monomers. The scaling laws yield
Figure 3. Scaling behavior of the average diameter 〈2RH〉 of the aggregates as a function of the normalized charge on the aggregating polyion-coated liposomal particles. Closed symbols: above the isoelectric point; (2) 5.1 kD, (9) 60 kD, and (b) 225 kD. Open symbols: below the isoelectric point; (4) 5.1 kD, (0) 60 kD, and (O) 225 kD. The full lines are the dependences predicted by eq 7 with the expected exponent (-1/3).
molecular weights (5.1, 60, and 225 kD) investigated, respectively. As can be seen, the experimental data are in good agreement with the scaling law (eq 7) over an extended range, around the peak where the aggregates reach their maximum size and their overall charge is practically zero. On the other hand, at the isoelectric point, QL ) fQP and, hence, the parameter k must represent the fraction of the ionized groups on the polyion chain and consequently the fraction of free counterions. For this polyion, in the range of concentrations we studied, a value on the order of f ) 0.4 is in very good agreement with values derived from low-frequency electrical conductivity measurements.40 As we have noted above, the formation of clusters of finite size is determined by the balance between electrostatic repulsion due to the small effective charge of the polyion-coated liposomes and short-range attractive interactions. This attractive potential depends on the inhomogeneous distribution of polyions adsorbed at the liposome surface. The role of the strong lateral correlation between polyions adsorbed at a charged surface in determining the phenomenon of “charge inversion” and the short-range attractive potential has been recently demonstrated by Nguyen et al.10-12 As far as the polyion structural organization on the charged liposome surface is concerned, Dobrynin et al.8,9 have proposed a scaling analysis based on a balance between electrostatic interactions of charged polyion blobs at the charged surface and short-range repulsion between adjacent blobs. This process gives rise to different regimes, depending on the surface charge density. For a flexible polyion chain, the key parameters that govern the adsorption process and the resulting structural organization are the monomer size b, the degree of polymerization N, the fraction f of free counterions (derived from the ionization of the polyion charged groups) and the Bjerrum length lB (lB ∼ 7 Å in water at 25 °C). According to Dobrynin et al.,8,9 when the surface charge is higher than a value given by
σ ∼ [b2f1/3N(lB/b)2/3]-1
(8)
the strong electrostatic repulsion between chains forces them to get organized, at the charged surface, in a twodimensional strongly correlated Wigner liquid crystal. The cell size becomes on the order of the electrostatic blob size when the surface charge σ reaches the value σ ∼ f/b2. (40) Bordi, F.; Colby, R.; Cametti, C.; De Lorenzo, L.; Gili, T. J. Phys. Chem. B 2002, 106, 6887-6893.
Nξ0 ∼ Nb3/2c1/2(lB/b)3/7
(9)
g ∼ (B/b)3/2c-1/2
(10)
Here, B ) (b/f 2lB)2/7 is the ratio of the chain contour length Nb and the actual extended size L ) Nξ01/2 and c is the polyion concentration expressed as momomers per unit volume. Each blob bears an electric charge qξ0 ) (ze)fg. If the overall chain conformation in the surface adsorption process is not altered, at the neutralization condition, the charge of each blob must be balanced by the charge of a corresponding Wigner cell of size a
qξ0 ) σa2
(11)
Within this scheme, the polymer adsorption results in an array of correlation blobs whose characteristic length is the size a. In the case of NaPAA of 60-kD molecular weight, we have a blob concentration of about 2.7 × 1015 blobs/mL, and for a surface charge density of σ ∼ 1/0.65 nm-2, where condition (8) applies, eq 11 leads to a cell size of about a ∼ 4 nm. As we will discuss in what follows, this value compares reasonably well with the evidence of a hexagonal Wigner cell with a size of the same order of magnitude, observed by TEM measurements. The semiquantitative agreement of our findings with the Dobrynin et al. model gives support to the view that the polyion maintains its blob configuration and, hence, a substantial fraction of condensed counterions remains unchanged. The ionic character of the solvent strongly influences the lipoplex formation process.41 Lipoplexes induced by NaCl electrolyte solution exhibit smaller size aggregates compared to the values found for lipoplexes prepared in the presence of NaPAA solution, at the same [NaCl]/ [DOTAP] molar charge ratio. It appears that, to induce aggregation or fusion, the “inducer” should possess a high density of negative charge; that is, a polyion is much more effective than small ions bearing the same overall charge. Although small ions act in a similar way, namely, by lowering the surface charge density of the liposome, they are unable to promote membrane fusion as confirmed by the morphology observed by TEM, where the aggregates clearly appear as formed by flattened individual liposomes (data not shown). Figure 4 compares the hydrodynamic diameters 〈2RH〉 of the resulting aggregates formed in NaCl and NaPAA aqueous solutions. As can be seen, although the behavior is, to a first approximation, similar, in the high salt concentration regime, where the aggregation can be attributed to the usual destabilization mechanism, the formation of aggregates due to the charge neutralization mechanism in the case of NaCl solutions is completely absent, as expected. However, it is important to underline that, also in this case, a two populations regime appears for the NaCl concentration higher than a critical value (on the order of 70 mg/mL, corresponding to a charge ratio of ξ ) 1970), at a very large NaCl excess. (41) Bordi, F.; Cametti, C. Colloids Surf., B 2002, 26, 341-350.
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Figure 5. TEM image (negative staining) of NaPAA-DOTAP complexes in the very low polyion content region (concentration marked by the letter a) in panel B of Figure 1. Liposomes appear isolated, with the adsorbed polyions that form small bumps on the liposome surface. In samples prepared from the same liposome stock suspension, in the absence of added polyions, these bumps are never observed (images not shown).
Figure 4. (A) Hydrodynamic diameter 〈2RH〉 of NaCl-DOTAP structures as a function of electrolyte NaCl concentration. The inset shows the time dependence of the two populations present in the suspension at the NaCl concentration marked by the arrow. (B) The dependence of 〈2RH〉 on the NaPAA polyion concentration (Panel B) of Figure 1, shown by comparison. The polyion (NaPAA) and small ion (NaCl) concentrations, expressed as monomers per unit volume, span the same interval. Note the different scales on the ordinate of panels A and B.
To properly interpret the morphology of the various complexes formed between NaPAA and cationic liposomes and to reveal their structural features, we employed TEM measurements. The TEM technique applied to negatively stained liposome structures has been shown35,42 to be appropriate to study the formation and the morphology of liposomal aggregates (complexes) of different sizes, formed during the interactions with different surfactants and charged micelles. Micrographs from TEM shown here were chosen from some 10th of the negatives and are the result of a series of subjective decisions. Images are taken at different representative polyion concentrations (marked by the letters a-d in panel B of Figure 1). The polyion molecular weight is Mw ) 60 kD. Figure 5 shows a negatively stained TEM micrograph where rather polydisperse polyion-coated liposomal particles are visible. The figure shows the representative structures formed in the system in the range of small to moderate polyion concentrations (less than 0.04 mg/mL, corresponding to a molar charge ratio ξ ) 0.35). Liposomes appear isolated, with adsorbed polyions that form small bumps. Also apparent in the figure is the spherical shape of the vesicles and the smooth integral appearance of the layer where the polymer chains are in a random coil conformation (low-grafting concentration). At higher polyion concentration, but below the isoelectric point, liposomes begin to aggregate in larger structures (42) Ge, L.; Mo¨hwald, H.; Li, J. Colloids Surf., A 2003, 221, 49-53.
Figure 6. TEM images of NaPAA-DOTAP complexes at moderate polyion concentration, below the isoelectric point (concentration marked by the letter b in panel B of Figure 1). Panel A: by using TEM in the ESI mode, aggregates can be imaged without any staining. Liposomes within the aggregate maintain their approximately spherical shape. Panel B: with negative staining, liposomes appear interwoven in a complicated network of elongated ribbons or threads. This network (panel C) results as composed of individual threads with an apparent thickness of about 4 nm.
maintaining their approximately spherical shape and their individuality. This situation is depicted in Figure 6, panel A), where an image of NaPAA-DOTAP complexes at a polyion concentration of 0.07 mg/mL is shown, by using TEM in the ESI mode. This image is obtained without staining. With negative staining (panel B), new details appear and liposomes, whose outlines are now barely visible, appear interwoven in a complex network of elongated ribbons and threads. This network at a higher magnification (panel C) results, composed of individual threads with an apparent thickness of about 4 nm. This value is above the resolution limit of the technique (≈2 nm) and is of the right order of magnitude for the effective size of a blob, although there could be some pending
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Figure 8. TEM images of NaPAA-DOTAP liposome complexes formed at a very large excess of polyion (concentration marked by the letter d in panel B) of Figure 1). Liposomes now appear isolated again but surrounded by a diffuse halo of adsorbed polyions. Also, clearly visible are bundles of “free” polyion chains in excess. Again, from the apparent “polyion thickness”, a blob size of about 4 nm can be evaluated.
Figure 7. TEM images of NaPAA-DOTAP liposome complexes very close to the isoelectric point (concentration marked by the letter c in panel B of Figure 1). The sharp, straight contours of aggregates, which have lost any resemblance to the rounded outlines that appeared in Figure 6, suggest an extended planar sheet structure. At higher magnification (panel B), a welldefined texture (negative staining) appears. A Fourier transform of the image (see inset) evidences a hexagonal Wigner cell with a size of about 4 nm.
questions regarding the interpretation of the TEM images. The full stretched appearance of the blob chain can be attributed to the stress due to the chain being partially squeezed between adjacent liposomes. The dye used in the negative staining (PTA) is in acidic form and is repelled by the PAA- polyion, so that the thickness of the “polyelectrolyte domain” has to be measured as the thickness of the “white” domain outlined by the stainer. The diameter of the bare polyion chain can be estimated lower than 1 nm, well below the resolution limit of the microscope. At the isoelectric point, huge aggregates form, where single (individual) liposomes can no longer be recognized. Many deformed, partially ruptured, and faceted large vesicles were present together with a large fraction of disklike structures. Figure 7 shows a TEM image of NaPAA-DOTAP complexes at a polyion concentration of 0.15 mg/mL. In panel A, the sharp, straight contours of the aggregates suggest that they are now composed of extended planar sheets. This finding suggests that neutralization of the DOTAP positive charges is required to allow destabilization of the lipid bilayer and promotion of the liposome-liposome fusion. On some of the aggregates, weak patterns (striations) are seen. These striations may represent multilamellar structures in which NaPAA is entrapped between the lamellae, whose formation must be regarded as a breakdown or a collapse of the liposome structure in the presence of polyions. Similar structures have been observed by Gustafssonn et al.31 in DNA-cationic liposome structures visualized by cryo-transmission electron microscopy measurements. At a high magnification (panel B), with negative staining, a well-defined texture appears on the sheets. A Fourier transform of the image, shown in the inset, evidences a hexagonal Wigner cell with a size of about 4 nm. This texture is attributed to the adsorbed polyelectrolyte that forms a strongly correlated two-dimensional Wigner liquid between phospholipid lamellae. Also in this case, our findings confirm the presence of a blob conformation and that the electrostatic interactions with lamel-
Figure 9. Distribution of the hexagonal Wigner cell size measured in different images of NaPAA-DOTAP aggregates formed very close to the isoelectric point.
lae take place through the renormalized charge, including the counterion condensation effect. Finally, in a NaPAA excess, at a polyion concentration higher than 0.3 mg/mL, isolated polyion-coated liposomes appear again. Typical sizes are around 200-250 nm. Figure 8 shows a TEM image of liposome complexes where bundles of “free” polyelectrolyte chains in excess are clearly visible. A final comment is in order. The distribution of the hexagonal Wigner cell size measured in different images of lipoplex sheets at the isoelectric point obtained with negative PTA staining is shown in Figure 9. At the surface charge density that characterizes the DOTAP double layer (an area of 0.65 nm2 per lipid molecule), the surface should be covered by hard-packed PAA blobs that, to minimize their electrostatic interactions, arrange themselves in a hexagonal lattice. PTA interacts preferentially with the liposome surface, avoiding the polyelectrolyte domain, and contributes in this way to the definition of the hexagonal lattice of the correlation blobs. As can be seen, as we have previously pointed out, the size of 4 nm compares well with the cell size in the correlation blob. Furthermore, it must be pointed out that in the images obtained without staining (data not shown), the cell size appears much less precisely defined. The broad resulting distribution, extending up to a d spacing of 20 nm, can be attributed to the fact that the contrast of the image is now due to the
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inhomogeneous distribution of the electron density, that is, to the inhomogeneous distribution of the polyelectrolyte mass. To conclude, our dynamic light scattering and TEM experiments give strong evidence for a variety of different continuously evolving structures in the complexation of anionic polyelectrolytes and cationic liposomes and offer support to the coexistence of structures with different sizes in the range of concentrations covering the dilute to the concentrated-entangled regime. At least three different polyion concentration regimes are evidenced, where the morphology of the resulting complexes is deeply different, ranging from an isolated polyion-coated liposome to the formation of hexagonally ordered polyion blobs at the
Bordi et al.
surface of the lipid bilayer, resulting from the rupture or the fusion of different approaching liposomes, up to a region where polyion re-organization is preferred and isolated polyion-covered liposomes newly appear. In particular, characteristic polyion concentrations have been evidenced, beyond which the aggregates start to grow significantly and coexist with smaller aggregates whose sizes do not change with time. The presence of a blob structure in the different conditions we have investigated suggests that the release of counterions cannot be assumed as the driving force in the polyelectrolyte-liposome complexation. LA036006U
