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Langmuir 1997, 13, 6516-6523
Kinetics of Protein-Induced Flocculation of Phosphatidylcholine Liposomes M. N. Dimitrova,†,‡ H. Matsumura,*,§ and V. Z. Neitchev† Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, and Electrotechnical Laboratory, AIST, MITI, Tsukuba 305, Japan Received April 14, 1997. In Final Form: August 4, 1997X The influence of protein adsorption on membrane-membrane interactions was studied regarding the kinetic stability of phosphatidylcholine (PC) liposome dispersions. The rates of flocculation were measured by the decrease in the transmitted light intensities in time. To elucidate the experimental data, we proposed an analytical method relating light transmittance to the average size of liposome flocs. The obtained relaxation time and maximum light absorbance were connected to the kinetic constant and the flocculation activation energy. These model parameters were calculated for the flocculation processes of PC liposomes in the presence of lysozyme, cytochrome c, and bovine serum albumin as a function (i) of the protein concentration and (ii) of the ionic strength. In addition to the generally accepted concept that the stability of PC liposomes is due to the action of hydration repulsive force, here we found that in the presence of soluble proteins the steric factors and electrostatics play essential roles for the colloidal stability of egg PC liposome dispersions.
1. Introduction The appropriate use of the phospholipid liposomes as a model of biomembranes and as a carrier in drug delivery systems1-3 requires control and prediction of the liposome dispersion stability. In order to explicate the relative stability of the disperse colloid system, it is necessary to determine the mechanisms of destabilization and their kinetics.4 Generally, in monographs and reviews more attention is paid to surface forces, surface interactions,5,6 and stable colloids, than to flocculation kinetics,7 which requires further fundamental knowledge. The necessity of theoretical and experimental studies on the kinetics of Brownian and gravitational coagulation of emulsions and liposome dispersions has been pointed out.4 It is generally known that phosphatidylcholine (PC) liposomes, in contrast with liposomes composed of other phospholipids, show relatively strong resistance to aggregation due to the action of hydration force at close contact.5,8 The PC liposomes are fairly stable upon alterations in pH, ionic strength, and addition of polyvalent ions,8 although PC aggregation caused by calcium and beryllium cation9 was recently reported. PC liposomes are aggregated by decreasing the temperature below the gel-to-liquid-crystalline phase transition temperature of vesicle membranes10-13 and by the addition of certain * To whom correspondence should be addressed. † Bulgarian Academy of Sciences. ‡ Present address: Tsukuba University, Department of Chemistry, Furusawa Laboratory, Tsukuba 305, Japan. § Electrotechnical Laboratory, AIST, MITI. X Abstract published in Advance ACS Abstracts, October 15, 1997. (1) Bentz, J.; Ellens, H. Colloids Surf. 1988, 30, 65. (2) Pozannsky, M. J.; Juliano, R. L. Pharmacol. Rev. 1984, 36, 277. (3) Yagi, K. Medical applications of liposomes; Japan Scientific Societies Press: Tokyo, 1986. (4) Dukhin, S. S.; Sjoblom, S. In Emulsions and Emulsion Stability, 1th ed.; Marcel Dekker, Inc.: New York, 1996. (5) Lis, L.; McAlister, M.; Fuler, N.; Rand, R.; Parsegian, V. Biophys. J. 1982, 37, 657. (6) Hunter, R. J. Foundations of Colloid Science; Oxford Science Publication; Clarenden Press: 1987; Vol. 1. (7) Sonntag, H.; Srenge, K. Coagulation Kinetics and Structure Formation; VEB Dentscher Verlag der Wissenschaften: Berlin 1987. (8) Matsumura, H.; Watanabe, K.; Furusawa, K. Colloids Surf., A 1995, 98, 175. (9) Minami, H.; Inoue, T.; Shimozawa, R. Langmuir 1996, 12, 3574. (10) Larrabee, A. L. Biochemistry 1979, 18, 33-21. (11) Schullery, S. E.; Schmidt, C. F.; Felgner, P.; Tillack, T. W.; Thompson, T. E. Biochemistry 1980, 19, 3919.
S0743-7463(97)00378-8 CCC: $14.00
polymers.14-17 Biochemical studies on protein-induced liposome aggregation have tended to focus on a possible fusion process in the presence of globular proteins,18 whereas the kinetics and thermodynamics of the proteininduced liposome flocculation require further studies. The main aim of this paper is to investigate the effect of globular protein adsorption on the colloidal stability of PC liposome dispersions and to determine the flocculation rate, measured through decrease in the transmitted light intensity in time. We also consider the flocculation activation energy and propose an analytical method for consideration of the kinetic velocity constant and floc’s stability by calculating the maximum absorbance values. The analytical method is well applied to the experimental data from the protein-induced phospholipid liposome flocculation under certain medium conditions. The stability of the liposome dispersion with constant volume fraction upon protein sorption is most likely to depend on a number of factors: protein species, amount of the adsorbed protein, mechanism of the sorption process, summation of the interacting forces, ionic strength, temperature, etc. Therefore, we studied the dependence of the flocculation rate and activation energy of the aggregation processes induced by typical globular proteins as a function of the concentration of added proteins. The variation of the ionic strength by addition of monovalent and bivalent electrolytes with different concentrations influences the flocculation kinetics and enables an understanding of how the particular electrostatic or hydrophobic interactions affect the liposome dispersion stability. 2. Experimental Section Materials. We used egg yolk PC, purchased from Sigma Chemical Co., Ltd., without further purification. The egg PC sample included a small amount of acidic impurities, which was (12) Wong, M.; Anthony, F. H.; Tillack, T. W.; Thompson, T. E. Biochemistry 1982, 21, 4126. (13) Wong, M.; Thompson, T. E. Biochemistry 1982, 21, 4133. (14) Sunamoto, J.; Iwamoto, K.; Kondo, H.; Shinkai, S. J. Biochemistry 1980, 88, 1219. (15) Massenburg, D.; Lentz, B. R. Biochemistry 1993, 32, 9172. (16) Viguera, A. R.; Mencia, M.; Goni, F. M. Biochemistry 1993, 32, 3708. (17) Viguera, A. R.; Alonso, A.; Goni, F. M. Colloids Surf., B 1995, 3, 263. (18) Schenkman, S.; Araudjo, P. S.; Dijkman, R.; Quina, F. H.; Chaimovich, H. Biochem. Biophys. Acta 1981, 649, 633.
© 1997 American Chemical Society
Kinetics of Protein-Induced Flocculation indicated by the negative electrophoretic mobility of its liposomes.19 Two types of globular proteins were used: (i) “soft”, which changes its conformation upon sorption on the liposome surface, bovine serum albumin (BSA) (isoelectric point at pH 4.9), and (ii) “hard” proteins, relatively stable to perturbations upon sorption, lysozyme (LSZ) (isoelectric point at pH 11.4) from hen’s egg and cytochrome c (CC) (isoelectric point at pH 10.1) from horse heart.20 All inorganic chemicals were analytical reagent grade. For the preparation of the solutions we used distilled and deionized water, obtained by an Autostill system (WG 240 Yamato Co.). PC liposomes (1-2 µm diameter) were prepared by using the vortex mixing method. On account of the size distribution importance for the studied flocculation, the liposome dispersion was dialyzed (1 µm pore size filter) to exclude the smaller liposomes and filtered (2 µm pore size filter) to remove the larger ones. Methods. The flocculation behavior of the PC liposomes was detected by a microscope (Olympus IMT-2) with a video-imageanalyzing system. The rates of the protein-induced flocculation processes, caused by the adsorption of 10-4, 10-3, 10-2, 10-1, and 1 mg/mL concentrations of the proteins under certain bulk conditions, were determined by measuring the decrease in the light transmittance (effective absorbance) at 1100 nm for a time period of 10 min. In these measurements we used a Jasco U best-30 double-beam spectrophotometer. The utilized spectrophotometer was modified with a jet device for rapid and controlled mixing of all the components (stopped-flow spectrophotometer).21 Solutions of protein or lipid alone were run concomitantly to provide suitable reference. For the interpretation of the PC liposomes’ surface properties, we measured their electrophoretic mobilities by a microelectrophoretic cell using a four-electrode configuration (Microtec Co., Ltd.). The consideration of the protein sorption effect on the membrane surface charge was examined by measuring the electrophoretic mobility of PC liposomes under different protein and electrolyte concentrations. All the measurements were performed at 24 °C.
3. Analytical Method In the stopped-flow experiments of the light absorbance with time, we measured the decrease in the light transmittance through the liposome dispersion due to the flocculation. During the process of flocculation, changes in the particle size distribution occur at constant total liposome volume; i.e., as the number of the particles decreases, their effective size increases. This leads to an increase in the scattered light intensity with an associated decrease in the transmitted light through the dispersion, which is effectively measured by the light absorbance. The intensity of scattered light from a particle with effective radius is described by21,22
I ∝ ar6 f(r) f(r) f 1
if
λ.r
f(r) f k
if
λ,r
where a is an effective constant, which depends on the particle refractive index, media refractive index, wavelength λ, etc., k is a constant, and f(r) is the correction factor for the Rayleigh equation, which depends on the r/λ ratio and the comparative refractive indexes of the dispersion phase with dispersion medium.23 (19) Matsumura, H.; Mori, F.; Kawahara, K.; Obata, C.; Furusawa, K. Colloids Surf., A 1994, 92, 87. (20) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (21) Furusawa, K.; Matsumoto, M. In Electrical Phenomena at Interfaces; Marcel Dekker, Inc.: New York and Basel, 1984; Chapter 8. (22) Heller, W.; Pangonist, W. J. Chem. Phys. 1957, 26, 498-506. (23) Nakagaki, M. In Experimental Methods of Light Scattering; Nankodo Publishing Co.: Tokyo/Kyoto, 1965;
Langmuir, Vol. 13, No. 24, 1997 6517
Total intensity of scattered light from unit volume of the suspension is
S ) nar6 where n is the particle number concentration. Assuming that scattered light does not fall onto the photodetector, S is equivalent to the effective light absorbance A. With this in mind, we write a zero-time absorbance (just before adding the protein)
A0 ) n0ar6 f(r)
(1)
where n0 is particle concentration of a uniform distribution of monomers. In the early stage of the flocculation after adding protein, we may consider only the formation of doublets is x; the monomer concentration will be n0 - 2x. The absorbance of the doublets is
Ad ) xard6 f(rd) where the effective doublet radius rd is defined by assuming a doublet-monomer volume ratio (rd/r)3 ) 2. We have for the effective light absorbance of the dispersion
A ) xard6 f(rd) + (n0 - 2x)ar6 f(r) In order to show more clearly the relation between doublet formation and the increase in effective light absorbance, we may write this in the form A ) A0 + ∆A, with
∆A ) 2xar6[2 f(rd) - f(r)]
(2)
Similar equations can be also derived for higher aggregation number flocs, but for simplicity we will restrict our attention to the framework of eq 2. The rate of effective encounters for doublet formation can be described by the following differential equation for the total particle concentration after the formation of x doublets, n ) n0 x24
n0 - n dn ) -Kn2 + Kb dt 2
(3)
where K is the flocculation kinetic constant of the forward reaction and Kb is the kinetic constant for the backward reaction. In the case of diffusional encounters of spherical particles25,26
K)
8kT -E*/kT e 3η
(4)
where kT is the Boltzmann factor, η is the suspension viscosity, and E* is the energy barrier of the flocculation. In the limit t f 0 we neglect the backward reaction term, obtaining by integration
1 1 + Kt ) n n0
(5)
Here, the total particle concentration n after forming x doublets is n0 - x. Then, n0 is expressed by using eq 1 and x by using eq 2 (24) Adamson, A. W. Physical Chemistry of Surfaces, 5th ed.; John Wiley & Sons, Inc.: New York, 1990. (25) Smoluchowski, M. Phys. Z. 1916, 17, 557-585. (26) Smoluchowski, M. Z. Phys. Chem. 1917, 92, 129.
6518 Langmuir, Vol. 13, No. 24, 1997
n0 )
A0 6
ar f(r)
and x )
Dimitrova et al.
∆A 2ar [2 f(rd) - f(r)] 6
Equation 5 then can be transformed to read
A0Kt ∆Af(r) ) A0[2f(rd) - f(r)] ar6 f(r)
(6)
And at very early times, t f 0, where also ∆A , A0 eq 6 can be expanded in series to the linear term:
∆A )
A02[2f(rd) - f(r)]Kt
(7)
ar6 f 2(r)
On the other hand, the experimental data for the increase in the effective light absorbance as a function of time seem to be well fitted by the following exponential function (see Figure 1)
∆A ) ∆A∞ (1 - e-t/τ)
(8)
where ∆A∞ is ∆A at t f ∞ and it is determined by the ratio of K/Kb; τ is the relaxation time of the process. From eq 2
∆A∞ ) A∞ - A0 ) 2x∞ar6 [2f(rd) - f(r)] where x∞ depends on the Kb/K ratio. When the kinetic constant of backward reaction Kb is small, the doublets are stable (x∞ f n0/2) and we obtain a large value of ∆A∞. At very early times eq 8 can be transformed in a linear form on the time t to read
∆A )
∆A∞ t τ
(9)
Comparing eqs 7 and 9 we can calculate the kinetic constant of the binary flocculation process
K)
∆A∞ar6 f 2(r)
(10)
A02τ[2f(rd) - f(r)]
where only the parameter a must be measured in a separate experiment and all other parameters are measured or easily calculated from our kinetic light absorbance data. The relaxation time τ is proportional to the inverse value of the flocculation kinetic constant K, and ∆A∞ relates to the stability of the formed flocs (the inability of redispersion back to monomers, that is small Kb). Substitution of eq 10 in eq 4 leads after simple transformations to the following expression for the activation energy of the flocculation process
{
E* ) -kT ln
3ηar6 f 2(r) ∆A∞
}
8kTA02τ[2f(rd) - f(r)]
(11)
When we observe the overall flocculation process, including flocculation beyond dimer formations, we can obtain (i) large flocs due to a small backward reaction or (ii) small flocs due to a certain large backward reaction. In some cases only monomers and doublets could be observed. 4. Results Dependence of the PC Flocculation Rate on Protein Concentration. The adsorption of LSZ at low bulk concentrations (10-4, 10-3, and 10-2 mg/mL) caused
Figure 1. A typical experimental plot for the effective light absorbance change as a function of time.
only a minor effect on the PC liposome dispersion stability (see Figure 2a). The LSZ adsorption at 10-1 mg/mL concentration led to the rapid formation of small flocs. Their size slowly increased over the next 2 h of the microscopic observations (see Figures 2b and 4). The adsorption of 1 mg/mL LSZ on the PC liposomes led to enhanced floc stability and to the formation of large flocs, which are shown on Figure 2c. The 1 mg/mL LSZ-induced flocs were relatively stable with respect to protein dilution (Figure 2d), which is an indirect indication for a bridging flocculation between protein-covered and protein-free PC liposomes. The LSZ-induced flocculation of PC liposomes was observed at the protein concentration for which surface charge of the PC membranes was negligible. Figure 3 shows the change of the electrophoretic mobility of PC liposomes versus the increase of the protein concentration. The electrophoretic mobility of PC liposomes at 10-1 mg/ mL LSZ was 0.20 (µm/s × cm/V) and had a zero value in the presence of 1 mg/mL LSZ. Figure 4 shows the change in the light transmittance in first and tenth minute after the rapid mixing of the components, as a function of the LSZ concentration. The adsorption of LSZ with concentrations below 10-2 mg/mL had a minimal effect on the effective light absorbance. By the protein concentration, the change in the effective light absorbance was enhanced and the flocculation rates were increasing. The adsorption of 4 × 10-1 mg/mL CC onto the PC membranes led to a formation of small flocs; with increasing the concentration of the added protein the formed flocs were enlarged. In contrast to LSZ and CC, the adsorption of BSA even at high protein concentrations did not influence the PC liposome dispersion stability. In the concentration range from 10-4 up to 1 mg/mL BSA, the microscopic observations and the effective light absorbance measurements did not show significant flocculation 2 h after adding the protein. Flocs were not formed and the liposome dispersion remained stable. The calculated values of the relaxation time τ, maximum light absorbance ∆A∞, the relative flocculation kinetic constant K′ ) K/a, and the relative activation energy E** ) E* + kT ln a for the PC liposome flocculation processes induced by different concentrations of LSZ and CC are shown in Table 1. The values of K′ are of the order of 10-40 m6/s clearly indicating slow flocculation. Values from (18 to 19) × 10-20 J for the activation energy E** per particle were obtained, implying that a relatively high energetic barrier in the potential curve should be overcome for flocculation to undergo. Dependence of the Protein-Induced PC Liposome Flocculation Rate on the Bulk Ionic Strength. To clarify the influence of electrostatic interactions on the dispersion stability, we examined the change in the kinetic
Kinetics of Protein-Induced Flocculation
Langmuir, Vol. 13, No. 24, 1997 6519
Figure 2. Microscope observations of (a) 10-2, (b) 10-1, and (c) 1 mg/mL LSZ-induced PC liposome flocs and (d) 1 mg/mL LSZinduced PC flocs after twice dilution of the bulk protein concentration.
rates and the activation energy of the protein (10-1 mg/ mL) induced flocculation of PC liposomes in the presence of monovalent and bivalent cations. The addition of CaCl2 to the LSZ (10-1 mg/mL) adsorbed PC liposomes led to the floc formations in the cation concentration range 10-4 up to 10-3 mol/L. In the presence of low electrolyte concentrations (up to 3 × 10-4 mol/L Ca2+) large flocs were formed (see Figure 9b); at 10-3 mol/L Ca2+ only small size flocs
were obtained; further increase of the bivalent cation completely prevented the flocculation of the liposomes. The addition of various concentrations of monovalent cation (Na) caused a different effect on the flocculation behavior of the LSZ (10-1 mg/mL) adsorbed PC liposomes. At low Na+ concentration (10-3 mol/L) the flocculation of LSZ/PC liposomes was prevented, while above 3 × 10-3 mol/L Na+ flocs were formed.
6520 Langmuir, Vol. 13, No. 24, 1997
Dimitrova et al.
Figure 3. Electrophoretic mobilities of 1 µm PC liposomes as a function of ([) BSA, (b) LSZ, or (2) CC concentration.
Figure 4. Flocculation rate dependencies on the LSZ concentration, measured in the ([) first and (2) tenth minute after the rapid mixing of the components. Table 1. τ, ∆A∞, K′, and E** Values for the Protein-Induced PC Liposome Flocculation protein
τ (s)
1 × 10-1 mg/mL LSZ 4 × 10-1 mg/mL LSZ 8 × 10-1 mg/mL LSZ 1 mg/mL LSZ 4 × 10-1 mg/mL CC 8 × 10-1 mg/mL CC 1 mg/mL CC
294 242 349 210 16 36 570
∆A∞ × K′ × 10-40 E** × 10-20 10-2 (m6/s) (J) E**/kT 2.2 5.6 8.3 5.5 5.3 2.8 8.3
6.05 7.58 8.44 7.38 4.73 13.41 13.37
18.337 18.243 18.199 18.256 18.438 18.009 18.013
44.557 44.244 44.223 44.359 44.803 44.760 43.771
The calculated values of τ, ∆A∞, K′, and E** of LSZ (10-1 mg/mL) induced PC liposome flocculation in the presence of Ca2+ and Na+ are shown in Table 2. With the increasing Na+ concentration E** gradually decreases, corresponding to an increase in K′. However, the stability of the formed flocs (∆A∞) decreases again at 10-1 mol/L Na+ showing dependence on the cation concentration. The calculated parameters for LSZ/PC flocculation in the presence of Ca2+ indicated that the flocs are most stable and K′ possesses the highest values at 3 × 10-4 mol/L concentration of the bivalent cation. With the addition of Ca2+ to BSA/PC or CC/PC dispersions we observed similar flocculation behavior of the protein-adsorbed PC liposomes. BSA/PC liposomes flocculated at the concentration of 3 × 10-4 mol/L Ca2+ (Figure 9a), where the rate is at the maximum in Figure 6a. Figure 7a shows the flocculation rate dependence of CC-adsorbed PC liposomes on the concentration of Ca2+. When Na+ was added to the dispersion medium, we observed flocs above 3 × 10-3 mol/L Na+ for BSA-absorbed PC liposomes and above 10-2 mol/L Na+ for CC-adsorbed PC liposomes. Figure 6b shows the flocculation rate of BSA-induced and Figure 7b presents the flocculation rate of CC-induced PC liposome aggregation as a function of the Na+
Figure 5. Electrophoretic mobilities of (9) PC liposomes, (b) BSA-absorbed PC liposomes, ([) LSZ-adsorbed PC liposomes, and (2) CC-adsorbed PC liposomes versus (a) CaCl2 and (b) NaCl concentration.
concentration. The flocculation rate is about zero at 10-3 mol/L Na+ and gradually increases with increasing the concentration of the electrolyte. The values of τ, ∆A∞, K′, and E** are given in Table 3 and show similar to LSZ/PC flocculation dependence on the concentration of the monovalent cation. Flocculation Rate at Zero Zeta Potential (ZZP). Figure 8 shows the calculated τ, K′, ∆A∞, and E** values of BSA-, LSZ-, or CC-induced PC flocs at cation concentration 3 × 10-4 mol/L Ca2+. A comparison between the three types of flocs at ZZP shows lower relaxation times and activation energies of the BSA- and LSZ-induced flocs than those for the CC flocculation. When all protein/PC liposomes had ZZP, BSA/PC- and LSZ/PC-type flocs showed similar stability and flocculation activation energy, as indicated by the approximately equal values of ∆A∞ and E**, while CC/PC-type flocs exhibited higher stability and activation energy (corresponding to higher ∆A∞ and slightly higher values of E**). Figure 9 shows the patterns formed in the three systems. The BSA/PC flocs developed compact structures (Figure 9a), whereas CC/PC flocs are more dendritic in appearance (Figure 9c), and LSZ/PC flocs combined both features (Figure 9b). 5. Discussion Our study clearly demonstrates the strong influence of LSZ and CC adsorption on the stability of 1 µm PC liposome dispersions without electrolyte. The effective protein concentration is above 10-1 mg/mL LSZ and 4 × 10-1 mg/ mL CC. The rates, as well as the E** values of the formation of LSZ- and CC-induced PC liposome flocs, show strong dependence on the amount of the adsorbed protein. As the concentration of the added protein is increased up to 8 × 10-1 mg/mL, the flocculation kinetic constant gradually increases and the activation energy gradually
Kinetics of Protein-Induced Flocculation
Langmuir, Vol. 13, No. 24, 1997 6521
Table 2. τ, ∆A∞, K′, and E** Values for 10-1 mg/mL LSZ-Induced PC Liposome Floc Formations in Different Ca2+ or Na+ Concentrations CaCl2 mol/L
τ (s)
∆A∞ × 10-2
10-4 3 × 10-4 10-3
370 502 314
2.56 4.98 2.08
NaCl
K′ × 10-40 (m6/s)
E** × 10-20 (J)
E**/kT
mol/l
3.09 4.63 2.98
18.609 18.445 18.628
45.221 44.819 45.264
3 × 10-3 10-2 3 × 10-2 10-1
τ (s)
∆A∞ × 10-2
K′ × 10-40 (m6/s)
E** × 10-20 (J)
E**/kT
365 241 130 45
7.38 9.13 10.74 7.90
6.25 11.13 23.06 32.88
18.329 18.087 17.779 17.639
44.538 43.950 43.203 42.863
Figure 6. Flocculation rates of BSA-induced PC liposomes aggregations as a function of (a) CaCl2 concentration in the (9) first and ([) tenth minute and (b) NaCl concentration in (9) first and (b) tenth minute.
Figure 7. Flocculation rates of CC-induced PC liposomes aggregations as a function of (a) CaCl2 concentration in (9) first and (b) tenth minute, and (b) NaCl concentration in (9) first and (2) tenth minute.
decreases, performing saturation phenomenon above 8 × 10-1 mg/mL protein concentration. These data correlate well with previously published research27 that adsorption does occur below a protein concentration 10-1 mg/mL but has only a minor effect, whereas the adsorption at a protein concentration above 10-1 mg/mL is more pronounced and has a significant effect on the properties of the PC membrane surface. The LSZ- and CC-induced flocculation of PC liposomes is observed when the protein adsorption reduces the surface charge on the PC membranes to a negligible value. The changes in the K′ and E** with the increase of the protein concentration up to 8 × 10-1 mg/ mL are clearly correlated with the electrophoretic mobility dependence of the protein/PC liposomes on the protein concentration (Figure 5). The activation energy is lowest, and the kinetic constant highest, for 8 × 10-1 mg/mL LSZor CC-induced PC liposome flocs. The electrophoretic mobility of such liposomes at 8 × 10-1 mg/mL concentration of the added protein is close to zero, indicating that the associated electrostatic surface potential is almost neutralized. Furthermore, LSZ and CC adsorb on mainly
account of electrostatic attraction (in the case of LSZ with concomitant hydrophobic dehydration interactions).28 Therefore, we consider the contribution of the electrostatic interactions to the overall flocculation processes as essential. For 1 mg/mL LSZ- and CC-induced PC liposome flocculation, the higher value of the activation energy and lower value of the kinetic constant are probably related to the protein surface coverage of the liposomes, which in the case of bridging flocculation plays an important role. It is well-known that a maximum flocculation occurs at θ ) 0.5 (i.e., at 50%) surface coverage and that with further increase of the protein surface coverage the flocculation rate decreases.29 If the surface of the liposomes is completely covered by protein molecules, the bridging flocculation between protein-covered and protein-free liposomes can hardly occur. When the density of the surface charge is not so high, only the electrostatic attraction limits the protein surface coverage θ. Thus, in
(27) Dimitrova, M.; Matsumura, H. Colloids Surf., B 1997, 8 (6), 287-293.
(28) Matsumura, H.; Dimitrova, M. Colloids Surf., B 1996, 6 (3), 165-172. (29) Wigsten, A. L.; Straton, R. A. Polymer adsorption and particle flocculation in turbulent flow. In Polymer adsorption and dispersion stability; ACS symposium Series; American Chemical Society: Washington, DC, 1984.
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Table 3. τ, ∆A∞, K′, and E** Values of 10-1 mg/mL BSA- or CC-Induced PC Liposome Flocculation in Presence of Different Na+ Concentrations BSA 10-1 mg/mL NaCl (mol/l) 3×
10-3
10-2 3 × 10-2 10-1
CC 10-1 mg/mL
τ (s)
∆A∞ × 10-2
K′ × 10-40 (m6/s)
E** × 10-20 (J)
τ (s)
∆A∞ × 10-2
275 377 135 44
4.27 9.08 9.06 7.6
5.66 10.03 28.74 70.82
18.363 18.129 17.695 17.326
263 147 47
10.40 10.59 8.82
K′ × 10-40 (m6/s) no flocculation 13.58 23.19 58.89
E** × 10-20 (J) 18.004 17.732 17.402
Figure 8. τ, ∆A∞, K′, and E** of 10-1 mg/mL BSA-, LSZ-, or CC-induced flocs in the presence of 3 × 10-4 mol/L Ca2+.
the case of adsorption caused mainly by the electrostatic attraction, bridging flocculation does not occur or is strongly obstructed in the adsorption saturation range, as in the case of 1 mg/mL LSZ- and CC-induced flocs. In contrast to LSZ and CC, without electrolyte the adsorption of BSA at the PC membranes had only a minor effect on the dispersion stability. The electrophoretic mobility of BSA-adsorbed PC liposomes decreases as the protein concentration increases (Figure 3) and reaches almost zero value. As the electrostatic repulsion is relatively weak, an auxiliary repulsive interaction between the BSA-absorbed PC liposomes must be acting. BSA absorption in the range of high-protein concentration leads to significant membrane destabilization due to dehydration hydrophobic force concomitant with conformation changes.27 This implies that the absorbed BSA molecules may cover almost completely the liposome surface, which may be the main cause for the repulsive interparticle interactions. Furthermore, BSA molecules are much larger compared to CC and LSZ molecules (size 11.6 × 2.7 × 2.7 nm30 rather than 2.8 × 3.0 × 3.4 nm31 and 4.6 × 3.0 (30) Norde, W. The adsorption of human plasma albumin and bovine pancreas ribonuclease on polustirene latices. PhD. Thesis of Wageningen Agricultural University, Wageningen, The Netherlands, 1976. (31) Bos, M. A. TIRF and its application to protein adsorption. Ph.D. Thesis of Wageningen Agricultural University, Wageningen, The Netherlands, 1994.
Figure 9. Microscope observations of (a) 10-1 mg/mL BSA-, (b) 10-1 mg/mL LSZ-, and (c) 10-1 mg/mL CC-induced PC liposome flocs in the presence of 3 × 10-4 mol/L Ca2+.
Kinetics of Protein-Induced Flocculation
× 3.0 nm,32 respectively). With respect to bridging flocculation, this indicates the possibility for steric obstructions between the BSA-absorbed PC liposomes while mutual approach. This appears to be an important topic for further investigation. The experimental data on electrophoretic mobilities versus Ca2+ concentration are very useful in interpreting the flocculation behavior of 10-1 mg/mL protein-adsorbed PC liposomes with additions of different concentrations of CaCl2 (Figure 5a). The electrophoretic mobilities of 10-1 mg/mL protein-adsorbed PC liposomes are essentially zero at 3 × 10-4 mol/L Ca2+. These medium conditions facilitate and strongly promote the protein/PC liposome flocculation, as confirmed by the minimum values of the activation energies and the maximum values of the kinetic constants. Above 3 × 10-4 mol/L Ca2+, the membrane surface charge changes its sign; a positive charge grows as the concentration of the cation increases. The associated increase in the electrostatic potential prevents the liposome flocculation. The addition of various concentrations of monovalent cation (Na) provides an additional knowledge for the protein-induced PC liposome flocculation. The calculated values of the relaxation time, K′, ∆A∞, and E** of 10-1 mg/mL protein-induced PC flocculation in the presence of Na+, shown in Table 2 and Table 3, are a clear confirmation of the essential role played by the electrostatic interactions in the overall flocculation processes. K′ increases with the increase in the Na+ concentration. This means that the reduction in the electrostatic interactions between the particles facilitates the flocculation process. However, the electrophoretic mobility of the 10-1 mg/mL adsorbed PC liposomes does not simply decrease with the increase in the cation concentration. The electrophoretic mobility of CC- or LSZ-adsorbed PC liposomes in the lower cation concentration range (10-5 up to 10-4 mol/L Na+) possess similar values to these at 10-2 mol/L concentration Na+ (Figure 5b). In the low salt concentration, however, we did not detect liposome dispersion instability. This suggests that the electrostatic potential is not the only important factor, but in addition the thickness of the double electric layer plays an essential role to the overall flocculation process. Hence, the decrease in the electrostatic potential and the screening of the electric double layer around the liposome membrane facilitate the proteininduced PC liposome flocculation. The flocculation rates and kinetic parameters of the obtained BSA-, LSZ-, or CC-induced PC liposome flocs can be compared on the basis of the influence of nonelectrostatic forces (including hydrophobic dehydration force), because at 3 × 10-4 mol/L Ca2+ and 10-1mol/L Na+ BSA, LSZ- or CC-adsorbed PC liposomes possess almost ZZP. K′ values in presence of Na+ are much larger compared (32) Norde, W.; Haynes, C. L. In Protein at Interfaces 2: Fundamentals and Applications; American Chemical Society: Washington, DC, 1995; Chapter 2.
Langmuir, Vol. 13, No. 24, 1997 6523
to the K′ values in Ca2+ for the protein-induced PC liposome flocculation. This indicates that Ca2+ impedes the rate of flocculation. The Ca2+ bonded on the PC liposomes probably obstructs to some extend the bridging flocculation between the protein-adsorbed liposomes. The kinetic parameters show strong dependence on the protein species, which indicates that the adsorption and flocculation mechanisms vary among the three proteins. Among the K′ values of the three proteins, these of BSA-induced PC liposome floc formations, in the presence of Na+ and Ca2+, are largest. This could be interpreted in terms of the adsorption mechanism regarding the short-range forces. BSA absorbs strongly on PC membranes due mainly to the action of hydrophobic dehydration force,28 which under these conditions is the most probable cause for the highest velocity constant among all three types of flocs. The main driving force for the adsorption of LSZ onto the PC membranes is electrostatic attraction, but with concomitant interaction between the hydrophobic residues of the protein and the membranes.28 The kinetic parameters of LSZ-induced PC flocs in the presence of Ca2+ are similar to these of BSA-induced ones, which supports our assertion for the importance of the hydrophobic dehydration force in the protein-induced flocculation process. The floc patterns formed by the absorption of the three proteins are rather different, thus suggesting different formation mechanisms. These data constitute clear evidence for the influence of the short-range forces on the dispersion stability. In protein-induced flocculation of the PC liposomes, short-range forces are manifested only after the electrostatic charge is negligibly small. Colloidal stability of the 1 µm PC liposomes, regarding the adsorption of globular proteins, thus appears to derive from electrostatic repulsion at far contact and hydration repulsion at close liposome contact with possible concomitant steric obstructions. 6. Conclusions The adsorption of the LSZ and CC influences the stability of the PC liposome dispersions. The effective protein concentrations, which caused PC liposome flocculation, are above 10-1 mg/mL LSZ and above 4 × 10-1 mg/mL CC. The values of the flocculation kinetic constants and the activation energies support the conclusion that the flocculation is slow and occurs only after relatively high energetic barrier in the potential curve is overcome. In the presence of monovalent and bivalent electrolytes around ZZP the flocculation caused by protein adsorption is strongly promoted due to the reduction of the repulsive force between the electric double layers of the adjacent PC membranes. Our study clearly shows how the proteininduced PC liposome bridging flocculation can be controlled and completely prevented by a minor modification of the medium salt conditions. LA970378J
