Polyelectrolyte Decorated Latex - Langmuir

Small DODAB BF (BF; refs 45−47 and references therein), 78−86-nm mean diameter and 38 ± 2 mV mean zeta potential (Table 1), were prepared by soni...
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Langmuir 2004, 20, 9535-9540

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Colloid Stability of Lipid/Polyelectrolyte Decorated Latex Felipe M. Correia, Denise F. S. Petri, and Ana M. Carmona-Ribeiro* Instituto de Quı´mica, Universidade de Sa˜ o Paulo, P.O. Box 26077, Sa˜ o Paulo, SP, 05513-970 Brazil Received April 28, 2004. In Final Form: August 2, 2004 The colloid stability of supramolecular assemblies composed of the synthetic cationic lipid dioctadecyldimethylammonium bromide (DODAB) on carboxymethyl cellulose (CMC) supported on polystyrene amidine (PSA) microspheres was evaluated via turbidimetry kinetics, dynamic light scattering for particle sizing, zeta-potential analysis, and determination of DODAB adsorption on CMC-covered particles. At 0.1 g L-1 CMC and 2 × 1011 PSA particles/mL, CMC did not induce significant particle flocculation, and a vast majority of CMC-covered single particles were present in the dispersion so that this was the condition chosen for determining DODAB concentration (C) effects on particle size and zeta potentials. At 0.35 mM DODAB, charge neutralization, maximal size, and visible precipitation indicated extensive flocculation and minimal colloid stability for the DODAB/CMC/PSA assembly. At 0.1 g L-1 CMC, isotherms of high affinity for DODAB adsorption on CMC-covered particles presented a plateau at a limiting adsorption of 700 × 1017 DODAB molecules adsorbed per square meter PSA which was well above bilayer deposition on a smooth particle surface. The polyelectrolyte layer on hydrophobic particles was swelled and fluffy (ca. 11-nm hydrodynamic thickness), and maximal adsorption of DODAB lipid onto this layer produced a compressed composite cationic film with 20 mV of zeta potential and about 10-nm mean thickness. The assembly of cationic lipid/CMC layer/polymeric particle was stable only well above charge neutralization of the polyelectrolyte by the cationic lipid, at relatively large lipid concentrations (at and above 1 mM DODAB) with charge neutralization leading to extensive particle aggregation.

Introduction Realization of practical and economic nanoscale structures is clearly going to depend on self-assembly as a necessary tool.1-4 A biomimetic approach to the selfassembly of nanostructures will probably involve bilayer membranes5-8 as either the nanostructures themselves or as templates or building blocks for more complex structures.9-12 Whether the vesicles are composed of surfactants, lipids, or polymers, their stability in various environments must be optimized to suit the particular task at hand. Stabilizing the bilayers has had a new surge of interest; they have been plated either by colloids13-16 or by polyelectrolytes,17-19 and these approaches have * Corresponding author. Telephone: 0055 11 3091 3810, extension 237. Fax: 0055 11 3818 5579. E-mail: [email protected]. (1) Tanford, C. The Hydrophobic Effect; Wiley: New York, 1980. (2) Bangham, A. D. Liposome Letters; Academic Press: San Diego, 1983. (3) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: San Diego, 1992. (4) Lehn, J.-M. Supramolecular Chemistry; VCH: Weinheim, 1995. (5) Fendler, J. H. Acc. Chem. Res. 1980, 13, 7. (6) Zasadzinski, J. A.; Kisak, E.; Evans, C. Curr. Opin. Colloid Interface Sci. 2001, 6, 85. (7) Carmona-Ribeiro, A. M. Chem. Soc. Rev. 2001, 30, 241. (8) Loidl-Stahlhofen, A.; Schmitt, J.; Noller, J.; Hartmann, T.; Brodowsky, H.; Schmitt, W.; Keldenich, J. Adv. Mater. 2001, 13, 1829. (9) Seddom, A. M.; Patel, H. M.; Burkett, S. L.; Mann, S. Angew. Chem., Int. Ed. 2002, 41, 2988. (10) Groves, J. T.; Boxer, S. G. Acc. Chem. Res. 2002, 35, 149. (11) Hubert, D. H. W.; Jung, M.; Frederik, P. M.; Bomans, P. H. H.; Meuldijk, J.; German, A. L. Langmuir 2000, 16, 8973. (12) Wong, M. S.; Cha, J. N.; Choi, K. S.; Deming, T. J.; Stucky, G. D. Nano Lett. 2002, 2, 583. (13) Carmona-Ribeiro, A. M.; Midmore, B. R. Langmuir 1992, 8, 801. (14) Carmona-Ribeiro, A. M.; Herrington, T. M. J. Colloid Interface Sci. 1993, 156, 19. (15) Sicchierolli, S. M.; Carmona-Ribeiro, A. M. J. Phys. Chem. 1996, 100, 16771. (16) Esumi, K.; Sugimuro, T.; Yamada, T.; Meguro, K. Colloids Surf., A 1992, 62, 249. (17) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Baumler, H.; Lichtenfeld, H.; Mo¨hwald, H. Macromolecules 2000, 33, 4538. (18) Fery, A.; Dubreuil, F.; Mo¨hwald, H. New J. Phys. 2004, 6, No. 18.

shown promise, both in stabilizing the bilayer structure and in creating novel nanostructures. On the other hand, in biology and medicine, cationization has been increasingly explored as a tool for targeting cells, tissues, and selected organs and delivering genes, drugs, and vaccines.20-26 In general, much effort has been devoted to the understanding of interactions between model mixtures of oppositely charged pairs.27-44 (19) Seitz, M.; Park, C. K.; Wong, J. Y.; Israelachvili, J. N. Langmuir 2001, 17, 4616. (20) Blau, S.; Jubeh, T. T.; Haupt, S. M.; Rubinstein, A. Crit. Rev. Ther. Drug Carrier Syst. 2000, 17, 425. (21) Carmona-Ribeiro, A. M. Curr. Med. Chem. 2003, 10, 2425. (22) Shiraishi, S.; Imai, T.; Otagiri, M. J. Controlled Release 1993, 25, 217. (23) Jameela, S.; Jayakrishnan, A. Biomaterials 1995, 16, 769. (24) Garcia-Chaumont, C.; Seksek, O.; Grzybowska, J.; Borowski, E.; Bolard, J. Pharmacol. Ther. 2000, 87, 255. (25) Uchegbu, I. F.; Schatzlein, A. G.; Tetley, L.; Gray, A. I.; Sludden, J.; Siddique, S.; Mosha, E. J. Pharm. Pharmacol. 1998, 50, 453. (26) Chen, J. P.; Chen, J. Y. J. Mol. Catal. B: Enzym. 1998, 5, 483. (27) Li, J.; Revol, J. F.; Marchessault, R. H. J. Colloid Interface Sci. 1996, 183, 365. (28) Liu, R. C. W.; Morishima, Y.; Winnik, F. M. Macromolecules 2001, 34, 9117. (29) Choi, U. S. Colloids Surf., A 1999, 157, 193. (30) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (31) Decher, G.; Lehr, B.; Lowack, K.; Lvov, Y.; Schmitt, J. Biosens. Bioelectron. 1994, 9, 677. (32) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Budde, A.; Mo¨hwald, H. Colloids Surf., A 1998, 137, 253. (33) Voight, A.; Lichtenfeld, H.; Sukhorukov, G. B.; Zastrow, H.; Baumler, H.; Mo¨hwald, H. Ind. Eng. Chem. Res. 1999, 38, 4037. (34) Shi, X. Y.; Cassagneau, T. T.; Caruso, F. Langmuir 2002, 18, 904. (35) Erbacher, P.; Zou, S. M.; Bettinger, T.; Steffan, A. M.; Remy, J. S. Pharm. Res. 1998, 15, 1332. (36) Divakaran, R.; Pillai, V. N. S.Water Res. 2001, 35, 3904. (37) Ashmore, M.; Hearn, J.; Karpowicz, F. Langmuir 2001, 17, 1069. (38) Huang, C. P.; Chen, Y. J. Chem. Technol. Biotechnol. 1996, 66, 227. (39) Watzke, H. J.; Dieschbourg, C. Adv. Colloid Interface Sci. 1994, 50, 1. (40) Safarik, I. Water Res. 1995, 29, 101. (41) Janes, K. A.; Calvo, P.; Alonso, M. J. Adv. Drug Deliverery Rev. 2001, 47, 83. (42) Woodley, J. Clin. Pharmacokinet. 2001, 40, 77.

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Despite these efforts, complex assemblies involving particles, polyelectrolytes, and bilayers remained challenging with respect to colloid stabilization of the whole assembly, knowledge of the assembled structure at a molecular level, and adequacy of the novel material to the required task. We report here determination of colloid stability for constructions via self-assembly of dioctadecyldimethylammonium bromide (DODAB) lipid from bilayer fragments (BF) on a carboxymethyl cellulose (CMC) layer supported on polystyrene amidine (PSA) microspheres. Our strategy involved cationic lipid adsorption from BF instead of closed vesicles.45-47 Thereby vesicle adhesion without vesicle disruption was circumvented and bilayer patches could possibly assemble onto the polyelectrolyte layer. The construction was characterized from turbidimetry kinetics, dynamic light scattering for particle sizing, zeta-potential analysis, quantitative analysis of lipid adsorption on polyelectrolyte-covered particles from adsorption isotherms, and visual observation of colloid stability, over a range of particle, polyelectrolyte, and lipid concentrations. Particles in the presence of oppositely charged polyelectrolytes can either flocculate or become stabilized as singlets or doublets depending on the polyelectrolyte molecular weight and concentration, particle number density, molar ratio for charges on particles and macromolecules, pH, and ionic strength.48-51 We have recently studied colloid stability of charged polymeric particles in the presence of oppositely charged polyelectrolytes so that experimental conditions for improved colloid stability, namely, occurrence of a vast majority of singlets and/or doublets in dispersion, could be established.50,51 In the present work the effect of adsorbing cationic lipids from BF on colloid stability of CMC-covered particles was evaluated. The production of colloidally stable particle singlets covered by a composite polyelectrolyte/lipid layer required relatively large concentrations of the cationic lipid, which massively adsorbed onto the polyelectrolyte layer and produced a compressed cationic film on particles with zeta potentials well below the one usually determined for DODAB cationic bilayers. Experimental Section Materials. CMC, sodium salt with a nominal degree of substitution of 0.7, molecular weight 89 300 g/mol, was purchased from Sigma. The pKa for carboxylate groups on CMC in pure water was 4.0.52-54 At pH 6.3, in pure water, practically all carboxylates will be dissociated. CMC is indeed expected to behave as a polyanion under our experimental conditions; that is, the degree of proton dissociation from the carboxylic moieties will be about 100% at pH 6.3. A 2 g L-1 CMC stock solution was prepared in pure water. This stock solution was diluted to yield final CMC concentrations in the range of 0.05-1.0 g L-1. DODAB (43) Ruckenstein, E.; Zeng, X. F. Biotechnol. Bioeng. 1997, 56, 610. (44) Strand, S. P.; Vandvik, M. S.; Varum, K. M.; Ostgaard, K. Biomacromolecules 2001, 2, 126. (45) Vieira, D. B.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 2001, 244, 427. (46) Moura, S. P.; Carmona-Ribeiro, A. M. Langmuir 2003, 19, 6664. (47) Pacheco, L. F.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 2003, 258, 146. (48) Stuart, M. A. C.; Fleer, G. J. Annu. Rev. Mater. Sci. 1996, 26, 463. (49) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319. (50) Vieira, D. B.; Lincopan, N.; Mamizuka, E. M.; Petri, D. F. S.; Carmona-Ribeiro, A. M. Langmuir 2003, 19, 924. (51) Reis, E. A. O.; Caraschi, J. C.; Carmona-Ribeiro, A. M.; Petri, D. F. S. J. Phys. Chem. B 2003, 107, 7993. (52) Hoogendam, C. W.; de Keizer, A.; Stuart, M. A. C.; Bijsterbosch, B. H.; Batelaan, J. G.; van der Horst, P. M. Langmuir 1998, 14, 3825. (53) Hoogendam, C. W.; de Keizer, A.; Stuart, M. A. C.; Bijsterbosch, B. H.; Smit, J. A. M.; Van Dijk, J. A. P. P.; van der Horst, P. M.; Batelaan, J. G. Macromolecules 1998, 31, 6297. (54) Fujimoto, J.; Petri, D. F. S. Langmuir 2001, 17, 56.

Correia et al. Table 1. Mean Diameter (D) and Zeta Potential (ζ) for Different Dispersions Such as PSA Microspheres, DODAB BF or LV, and Their Mixtures with CMC dispersion PSAa PSA in water PSA/CMC DODAB BF DODAB BF/CMC DODAB LV DODAB LV/CMC

DODAB, mM

CMC, g L-1

0.1 2 1 2 1

0.1 0.1

D, nm

ζ, mV

53 ( 8 63 ( 1 85 ( 1 77 ( 8 186 ( 2 529 ( 10 1083 ( 63

52 ( 1 -40 ( 1 37 ( 3 25 ( 1 55 ( 1 11 ( 2

a

PSA particle mean diameter from transmission electron microscopy as given by the supplier; the particle number density was 2 × 1011 particles/mL; the DODAB and CMC concentrations were 2 mM in water and 1 mM with CMC.

was purchased from Sigma. Small DODAB BF (BF; refs 45-47 and references therein), 78-86-nm mean diameter and 38 ( 2 mV mean zeta potential (Table 1), were prepared by sonication with the tip in Milli-Q water at 2.0 mM DODAB as previously described.55,56 DODAB large vesicles (LV), about 500-nm mean diameter and 55 ( 1 mV mean zeta potential, were obtained by vortexing the DODAB powder in pure water at 60 °C (Table 1). DODAB concentrations were analytically determined by microtitration57 or by a colorimetric method.58 Cationic PSA particles, nominal mean diameter of 53 nm, 1.1 × 106 cm2 g-1, specific surface area of 4.4 µC cm-2, and 52 ( 1 mV mean zeta potential (Table 1), were purchased from Interfacial Dynamics Corp. (Portland, OR, U.S.A.). NaCl and all other reagents were analytical grade. Water was Milli-Q quality. Determination of Mean Diameters, Size Distribution, and Zeta Potentials for the Mixtures or Mixture Components in Separate. The particle size (mean diameter Dz), size distribution, and zeta potential (ζ) in the presence or absence of CMC or CMC/DODAB were determined using the ZetaPlus zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY), which was equipped with a 677-nm laser and dynamic light scattering at 90° for particle sizing. ζ was determined from the electrophoretic mobility µ in pure water and from the Smoluchowski equation ζ ) µη/, where η is the medium viscosity and  the medium dielectric constant. Determination of Flocculation Kinetics for CMC/ DODAB or PSA/CMC Mixtures. The colloid stability of binary mixtures DODAB/CMC or PSA/CMC were evaluated by two different methods: (1) turbidity kinetics at 400 nm and (2) mean zeta-average diameter kinetics. Both methods gave essentially very similar results so that only the light scattering measurements will be shown in the results section. Equal volumes of DODAB BF or LV at 1.0 mM DODAB were rapidly (ca. 10 s) mixed in a cuvette with a CMC solution (0.2-2.0 g L-1 CMC), and mean diameters were recorded as a function of time for 10 min. Similarly, PSA particles (4 × 1011 particles mL-1) were mixed with a 0.2 g L -1 CMC solution to evaluate particle concentration effects or CMC concentration effects on the kinetics of the mean diameter at 25 °C. Details on interaction times, particle number densities, and DODAB and CMC concentrations are given in the figure captions. Determination of DODAB Adsorption Isotherms on PSA/ CMC Particles. After interaction (see details in Figure 7), mixtures were centrifuged (14 000 rpm/1 h/4 °C) to separate free from adsorbed DODAB on CMC-covered particles. The DODAB concentration was determined in each supernatant as previously described.58 The total surface area on particles was calculated from the mass fraction and the specific surface area. Adsorption was expressed as the number of DODAB molecules adsorbed per meter squared surface area of the CMC-covered particles. (55) Tran, C. D.; Klahn, P. L.; Romero, A.; Fendler, J. H. J. Am. Chem. Soc. 1978, 100, 1672. (56) Carmona-Ribeiro, A. M. Chem. Soc. Rev. 1992, 21, 209. (57) Schales, O.; Schales, S. S. J. Biol. Chem. 1941, 140, 879. (58) Stelmo, M.; Chaimovich, H.; Cuccovia, I. M. J. Colloid Interface Sci. 1987, 117, 200. (59) Carmona-Ribeiro, A. M.; Lessa, M. D. Colloids Surf., A 1999, 153, 355.

Colloid Stability of Decorated Latex

Figure 1. Effect of CMC concentration on colloid stability of 0.5 mM DODAB dispersions. The kinetics of the mean zetaaverage diameter were obtained upon adding CMC either to DODAB LV (A) or to BF (B) at a final CMC concentration of 0.01 (9), 0.02 (b), 0.05 (2), 0.10 (1), 0.20 ([), 0.50 (triangle pointing left), and 1.00 g L-1 (triangle pointing right).

Results 1. Colloid Stability of Cationic Bilayers or Particles in the Presence of CMC. Two types of DODAB cationic bilayers were evaluated regarding their stability in the presence of the anionic polyelectrolyte CMC in the water dispersion: cationic BF and LV. There was extensive aggregation and rapid increase in mean size as a function of time upon CMC addition to both BF and LV DODAB dispersions. Final sizes for LV/CMC (Figure 1A) were about 10 times larger than those for BF/CMC aggregates (Figure 1 B). For particles in the presence of 0.1 g L-1 CMC, the dependence of colloid stability on the particle number density was weak because at and above 2 × 1011 particles/ mL, final mean particle diameters were in the 66-80-nm range; that is, CMC-covered particles might have remained in the dispersion as singlets which exhibited a 3-17 nm increase in their diameters due to adsorption of a 1.5-8.5 nm CMC layer on the particles (Figure 2A; Table 1). Interestingly, this dispersion remained stable as such over long periods of time (not shown). However, taking into account the polydispersities of the scattering objects yielding a relatively broad particle size distribution, differences in diameters of 10 nm or less would be meaningless and it would not be possible from the present data to discriminate between singlets covered by one CMC layer or singlets covered by one CMC layer plus a certain extent of aggregated particles. At a fixed particle number density of 2 × 1011 particles/ mL, the effect of increasing the polyanion concentration was slightly increasing the mean particle size from 79 to 83 nm (over the CMC concentration range of 0.02-0.1 g L-1) or from 83 to 110 nm (over 0.1-0.2 g L-1 CMC; Figure 2B). However, above 0.2 g L-1 CMC, final sizes increased above about 120-nm mean diameter, suggesting significant particle aggregation. At the largest CMC concentration employed, 1 g L-1, further aggregation led to sizes around

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Figure 2. (A) the effect of 0.1 g L-1 CMC on the colloid stability of PSA microspheres over a range of particle number densities: 1 (0), 2 (O), 4 (4), 6 (3), 7 (]), 8 (triangle pointing left), 9 (triangle pointing right), and 10 × 1011 particles/mL (\). (B) Effect of CMC concentration on colloid stability of 2 × 1011 PSA particles/ mL: kinetics were obtained at a final CMC concentration of 0.01 (0), 0.02 (O), 0.05 (4), 0.10 (3), 0.20 (]), 0.50 (triangle pointing left), and 1.00 g L-1 (triangle pointing right).

200 nm compatible with three or more aggregated particles. The effect of polyelectrolyte concentration on colloid stability of the oppositely charged particulate (Figure 2B) was much less pronounced than its effect on colloid stability of the DODAB bilayer dispersions (Figure 1). Most final sizes ranged from 80 to 120 nm indicating at most a certain extent of aggregated particles in the dispersions (Figure 2B). The low colloid stability of DODAB BF and LV over a range of low ionic strengths has been well documented in the literature.60,61 Thus, it was necessary to investigate whether any electrolyte displaying the same ionic strength as the polyelectrolyte would yield a similar effect on the colloid stability of the bilayer dispersions. Over the 0-5.0 mM range of ionic strengths obtained by adding either the polyelectrolyte CMC or simply NaCl, Figure 3A,C,E,G shows the specificity of the CMC bridging effect that shifted size distribution of DODAB LV to higher values whereas a much smaller effect was obtained over the same range of ionic strength added as NaCl (Figure 3B,D,F,H). 2. Self-Assembly of DODAB from BF on CMCCovered Particles. Table 1 and Figure 4 present data on sizes and zeta potentials for dispersions of particles, bilayers, particles/CMC, bilayers/CMC, or particles/CMC/ bilayers. DODAB deposition from DODAB BF on CMCcovered particles reduced the thickness of the polyelectrolyte layer adsorbed on particles as shown from the comparison between parts A, C, and E. The final DODAB/ CMC/PSA assembly presented a mean diameter of 83 nm and a positive zeta potential of 20 mV (Figure 4E) in (60) Carmona-Ribeiro, A. M.; Yoshida, L. S.; Chaimovich, H. J. Phys. Chem. 1985, 89, 2928. (61) Carmona-Ribeiro, A. M. J. Phys. Chem. 1993, 97, 11843.

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Figure 3. Compared effect of CMC and NaCl on size distribution of 0.5 mM DODAB in the form of LV. In the left column are size distributions for dispersions of added CMC whereas in the right column are those mixed with NaCl instead of CMC solutions to yield the equivalent ionic strength. The mixtures interacted for 30 min before measurements. CMC final concentrations were 0 (A), 0.01 (C), 0.10 (E), and 1.00 g L-1 (G). NaCl final concentrations were 0 (B), 0.048 (D), 0.480 (F), and 4.800 mM (H).

contrast to 63 nm and 52 mV for the bare PSA particle (Table 1 and Figure 4A) and to 85 nm and -40 mV for the CMC-covered PSA (Figure 4C). The difference in diameter between PSA and DODAB/CMC/PSA was about 20 nm, yielding about 10 nm for the deposited layer thickness. At this point one should recall that the DODAB bilayer thickness is 4-5 nm. Thus, the CMC layer would be 5-6nm thick. The zeta-potential value of 20 mV for the DODAB/CMC/PSA particles (Figure 4E) was smaller than the 37 mV determined for DODAB BF alone (Table 1). In Figure 4C the size distribution showed a small peak at about 500 nm which might have been due to partially aggregated PSA/CMC particles. This would further prevent a clear interpretation of sizes measured as due to singlets or doublets as pointed out by a reviewer. The effect of the DODAB concentration on the sizes and zeta potentials of CMC-covered PSA is shown in Figure 5. There was a good correlation between colloid stability and electrostatics. Around 0 mV (region II), aggregation was at a maximum whereas over the range of negative (region I) or positive zeta potentials (region III) colloid stability was high and sizes were minimized. Size distributions for the DODAB/CMC/PSA system at DODAB concentrations belonging to regions I, slightly above II, and III are shown in Figure 6. In region I, where the DODAB concentration was not enough to cover all particles, one could imagine that that the two peaks in the size distribution would correspond to two types of particles: the stable ones covered with CMC and the aggregated ones covered with DODAB. At a DODAB concentration of 0.35 mM (region II in Figure 5A where

Figure 4. Size distributions for different dispersions and mixtures in pure water: only PSA particles at 2 × 1011 particles/ mL (A); only DODAB BF at 2 mM DODAB (B); a mixture of PSA particles and CMC (2 × 1011 particles/mL and 0.1 g L-1 CMC) at 0.5 h of interaction time (C); a mixture of DODAB BF and CMC (1 mM DODAB and 0.1 g L-1 CMC) at 0.5 h of interaction time (D); and a mixture of PSA particles first added to CMC, left to interact over 0.5 h, and then DODAB BF (2 × 1011 particles/mL, 0.1 g L-1 CMC and 1.0 mM DODAB BF as the final concentrations) added and left to interact again over 0.5 h before measurements (E). On the left in each part, the mean zeta potential and diameter are quoted.

charge neutralization occurs), a whitish precipitate was visible (not shown). For the other regions, where the mean zeta potential was either negative (region I) or positive (region III) precipitation was absent. Figure 7 shows an isotherm of high affinity for DODAB adsorption from DODAB BF onto CMC-covered particles. The limiting DODAB adsorption at about 700 × 1017 adsorbed DODAB molecules per square meter polystyrene was well above the amount expected for bilayer deposition (shown as the dashed line in Figure 7). Similar isotherms for DODAB adsorption were previously obtained for other hydrophilic adsorbents such as bacteria or fungus cell surfaces where biopolymers such as proteins and polysaccharides offered a very large surface area on a rough surface.21

Colloid Stability of Decorated Latex

Figure 5. Effect of DODAB concentration on mean z-average diameter (A) and zeta potential (B) for PSA particles first added to CMC and then added to DODAB BF over a range of final DODAB concentrations; final CMC concentration in the mixture was 0.1 g L-1. The three different moieties of the curves were named I, II, and III corresponding to negative, zero, and positive zeta potentials, respectively. Interactions of PSA/CMC took place over 0.5 h and between PSA/CMC and DODAB BF over 1 h before measurements.

Figure 6. Size distribution for PSA/CMC particles at three different DODAB BF concentrations belonging to regions I (A), II (B), and III (C) of the curve in Figure 5A. On the right, the mean zeta-potential and diameter values for each dispersion are quoted.

Discussion The present results have shown that the interaction between DODAB BF and CMC-covered PSA microspheres led to formation of a composite coverage on particles. This DODAB/CMC/PSA particulate required large concentrations of cationic lipid to produce colloidally stable particles as assessed by attainment of small mean particle sizes.

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Colloid stability was achieved well above charge neutralization of the polyelectrolyte layer in the dispersions over a restricted range of particle, lipid, and polyelectrolyte concentrations. At the particle number density of 2 × 1011 particles/mL, the colloid stability reported here as low mean sizes possibly due to prevalence of singlets contrasted with the extensive doublet formation for CMCcovered PSA51 and chitosan-covered poly(methyl methacrylate) particles under similar experimental conditions but in the absence of lipid.50 Apparently, adding the DODAB lipid well above charge neutralization of the CMC layer compressed the fluffy polyelectrolyte layer on the microspheres and produced a majority of colloidally stable PSA/CMC/DODAB singlets at the largest DODAB concentrations employed (Figure 5). The main features of the composite layer were essentially similar to those expected for a simple DODAB bilayer adsorbed onto a single polyelectrolyte layer, though the zeta potential of 20 mV for the composite layer was well below the usual zeta potential of 38-42 mV determined for the DODAB BF in pure water, and the DODAB adsorption was well above the amount expected for smooth deposition of a single bilayer on CMC-covered particles; indeed, CMC decoration on the polymer particle was not expected to generate a smooth particle surface for bilayer deposition. Possibly, ion pairs between DODAB polar heads and carboxyl moieties on the polyelectrolyte might have been extensively formed and a very dense and positively charged composite layer might have resulted around the polymeric particle which became electrostatically stabilized as shown from the effect of DODAB concentration on the zeta potential (Figure 5B), adsorption isotherm (Figure 7), microscopic colloid stability (Figure 5A), and CMC layer compression due to DODAB adsorption (Figure 4E). These three sets of data demonstrated internal consistency because a steep change in behavior took place above charge neutralization (at ca. 0.4 mM DODAB). Above 0.4 mM DODAB, adsorption and zeta potential remained remarkably constant at 700 × 1017 adsorbed DODAB molecules per meter squared of CMCcovered particle and 20 mV, respectively, and the dispersion was visibly stable with a mean diameter of 83 nm and a majority of particle singlets in dispersion despite the presence of some aggregates which increased polydispersity as seen from the size distribution (Figure 4E). The DODAB bilayer usually presents a zeta potential around 40 mV (Table 1); the low zeta potential of 20 mV for the DODAB/CMC/PSA assembly might be due to several reasons. The Smoluchowski approximation used for calculating potentials from electrophoretic mobilities valid for smooth and well-defined interfaces might not have been adequate to calculate the zeta potentials. The lipid-polyelectrolyte decoration around the particle might have diluted the outer DODAB charges within the thickness of the shell at the interface, and this might have reduced the charge density and, thus, the zeta potential. Some dissociated carboxylates from the CMC layer might have remained exposed to the water and contributed to the mean zeta potential despite the large amount of DODAB BF added to the dispersion. The particle might have been electrosterically stabilized, the DODAB bilayer contributing with the electrostatic repulsion and some protusions from the CMC layer eventually contributing with nonpaired and dissociated carboxylates plus steric stabilization. Furthermore, BF as the start material might have remained as adsorbed bilayer patches on the CMC layer, eventually generating a noncontinuous bilayer coverage with exposure of the CMC adsorbed layer.

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Figure 7. DODAB adsorption from DODAB BF onto CMC-covered PSA particles (0.1 g L-1 CMC; 2 × 1011 particles/mL) in pure water at 25 °C. The adsorption was expressed as a function of free DODAB concentration in the supernatant (A) or as the final DODAB concentration in each mixture (B). The dashed line corresponds to the adsorbed amount expected for adsorption of one single DODAB bilayer onto each CMC-covered PSA particle.

In the literature, mean thicknesses for one single layer of polyelectrolyte deposited on an hydrophilic colloidal template ranged from 1.5 up to 1.7 nm with a water content of 42%.62,63 For lipid membranes assembled on polyelectrolyte-coated colloidal particles, the assembly studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy revealed a homogeneous lipid coverage; freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support.64 Neutron reflectometry studied the preparation of polymer-cushioned lipid bilayers: a dimyristoylphosphatidylcholine bilayer onto a polyethylenimine-coated quartz substrate was examined.65 In the present work, taking into account extreme measurements for particle size, from dynamic light scattering, PSA particles had a mean diameter of 63 nm (Table 1) and this changed to 83 nm after CMC and DODAB adsorption on particles (Figure 4E). Thus, an about 20 nm increase in the mean diameter might have resulted from the deposition of a 5-nm layer of CMC on the particle and a 4.55-nm DODAB bilayer with a final zeta potential of 20 mV. Unfortunately, as emphasized by a reviewer, polydispersities for the samples did not allow stronger statements regarding thicknesses for layers on particles. Nevertheless, in agreement with the above interpretation, for the short-range forces and structures of softly supported phospholipid bilayers the force-distance profile measured with the surface force apparatus presented a (62) Estrela-Lopis, I.; Leporatti, S.; Moya, S.; Brandt, A.; Donath, E.; Mo¨hwald, H. Langmuir 2002, 18, 7861. (63) Lvov, Y.; Onda, M.; Ariga, K.; Kunitake, T. J. Biomater. Sci., Polym. Ed. 1998, 9, 345. (64) Moya, S.; Richter, W.; Leporatti, S.; Baumler, H. B.; Donath, E. Biomacromolecules 2003, 4, 808. (65) Wong, J. Y.; Majewski, J.; Seitz, M.; Park, C. K.; Israelachvili, J. N.; Smith, G. S. Biophys. J. 1999, 77, 1445.

long-range exponentially decaying repulsive force that had not been observed between rigidly supported bilayers on solid mica substrate surfaces.19 These repulsive forces in the intermediate distance regime (mica-mica separations from 15 to 40 nm) were shown to be due not to an electrostatic force between the bilayers but to compression (deswelling) of the underlying soft polyelectrolyte layer.19 Consistently, from our light-scattering measurements shown in Table 1 and Figure 4, mean diameters for CMC/ PSA and DODAB/CMC/PSA were 85 and 83 nm, respectively. Despite the large amount of adsorbed DODAB, the mean size did not increase; on the contrary, it became slightly smaller, possibly suggesting compression of the polyelectrolyte layer due to DODAB BF deposition. Conclusions The anionic polyelectrolyte CMC was adsorbed on hydrophobic cationic PSA microspheres via electrostatic multipoint attachment as a fluffy, highly charged and hydrated CMC layer which increased mean hydrodynamic diameter of the PSA particles by 22 nm. At limiting lipid adsorption onto these CMC-covered particles, the cationic lipid DODAB compressed the CMC layer and a composite DODAB/CMC film was formed on particles which exhibited a mean diameter about 20 nm above the one for bare PSA particles. Despite its polydispersity, at 1 mM DODAB (the largest lipid concentration employed), this particulate with a low mean particle size was positively charged (20 mV for the zeta potential) and colloidally stable. Below 1 mM DODAB, decorated particles were colloidally unstable. Acknowledgment. F.M.C. thanks FAPESP for a trainee technician fellowship. Financial support from FAPESP and CNPq is gratefully acknowledged. LA048938J