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Langmuir 1998, 14, 5438-5445
Temperature Dependence of Protein-Induced Flocculation of Phosphatidylcholine Liposomes Mariana N. Dimitrova,†,‡ Hideo Matsumura,*,§ Vassil Z. Neitchev,‡ and Kunio Furusawa† Department of Chemistry, The University of Tsukuba, Tsukuba 305, Japan, Electrotechnical Laboratory, AIST, MITI, Tsukuba 305, Japan, and Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Received February 3, 1998. In Final Form: July 20, 1998 We studied the influence of the ambient temperature on the lysozyme- and cytochrome c-induced flocculation of phosphatidylcholine liposomes and its relation to the adsorption of the proteins at phospholipid membrane surfaces. The temperature dependence of two kinetic parameters, flocculation rate and activation energy, was experimentally determined. From 10 to 45 °C, the maximum increase in the activation energy was on the order (4-5) × 10-20 J/particle (about 8-10 kT). The influence of the temperature was more emphasized on the values for the flocculation rate, which was the primary calculated parameter, rather than for the activation energy. Within the studied temperature range a 5-fold increase in the relative flocculation rate was observed in the case of 1.0 mg/mL cytochrome c-induced liposome flocculation. The temperature dependencies of the activation energy and the flocculation rate were considered in terms of Brownian motion of the phosphatidylcholine liposomes, protein adsorption mechanism, and bridging mechanism of the protein-induced liposome flocculation.
1. Introduction Liposomes have long been regarded as therapeutic agents and drug delivery systems for a growing range of medical applications, including the treatment of lung disorders,1 cancer therapy,2 and gene delivery.3,4 Contact of blood with implanted liposomes often triggers a dynamic, sequential protein adsorption process, which promotes significant changes in the surface properties of the liposomes. For the appropriate use of phospholipid liposomes as a model of biomembranes, and a carrier in drug delivery,5-7 systematic knowledge for protein/liposome membrane interactions, as well as for proteininduced fusion and aggregation processes is necessary. Relative to liposomes composed of acidic phospholipids, large phosphatidylcholine (PC) liposomes strongly resist aggregation and fusion mainly due to the action of the hydration force at close liposome contact.8 Although PC liposome aggregation caused by calcium and beryllium cations was recently reported,9 these liposomes are fairly stable upon alterations in pH, ionic strength, and the addition of polyvalent ions.10 The PC liposome aggregation has been observed by decreasing the temperature below
the gel-to-liquid-crystalline phase transition point for the vesicle membranes11-14 and by the addition of certain polymers.15-18 Previous reports have shown that soluble proteins such as bovine serum albumin (BSA), lysozyme (LSZ), and cytochrome c (CC) adsorb on the PC liposome membrane (in the case of BSA absorb into the PC membrane) and, above certain effective protein concentrations (e.g., at 10-1 mg/mL), can strongly change the membrane surface properties.19 The adsorption of LSZ and BSA molecules on the phospholipid liposomes causes membrane destabilization and leakage of the inner liposome content to the outside medium.20 In addition, LSZ and CC, which adsorb mainly due to electrostatic attraction (in the case of LSZ with concomitant hydrophobic interactions), induce PC liposome bridging flocculation.21,22 The flocculation rate and the activation energy of the protein-induced liposome flocculation (PILF) strongly depend on the bulk protein concentration.22 On the other hand, PILF can be controlled: enhanced or hampered, even totally blocked, by slight modification of the bulk ionic strength. The influence of the ambient temperature on the overall PILF, presented here, is a further natural development
†
The University of Tsukuba. Bulgarian Academy of Sciences. § Electrotechnical Laboratory. ‡
(1) Perkins, W. R.; Dause, R. B.; Parente, R. A.; Minchey, S. R.; Neumen, K. C.; Gruner, S. M.; Taraschi, T. F.; Janoff, A. S. Science 1996, 273, 330. (2) Lassic, D. D.; Martin, F. Stealth Liposomes; CRS Press: Boca Raton, FL, 1995. (3) Lassic, D. D.; Strey, H.; Stuart, M. C. A.; Podgornic, R.; Frederic, P. M. J. Am. Chem. Soc. 1997, 119, 832. (4) Ra¨dler, J. O.; Koltover, I.; Slditt, T.; Safiniya, C. R. Science 1997, 275, 810. (5) Bentz, J.; Ellens, H. Colloids Surf. 1988, 30, 65. (6) Pozannsky, M. J.; Juliano, R. L. Pharmacol. Rev. 1984, 36, 277. (7) Yagi, K. Jpn. Sci. Soc. Press: Tokyo 1986. (8) Lis, L.; McAlister, M.; Fuler, N.; Rand, R.; Parsegian, V. Biophys. J. 1982, 37, 657. (9) Minami, H.; Inoue, T.; Shimozawa, R. Langmuir 1996, 12, 3574. (10) Matsumura, H.; Watanabe, K.; Furusawa, K. Colloids Surf. A: Physicochem. Eng. Aspects 1995, 98, 175. (11) Larrabee, A. L. Biochemistry 1979, 15, 3321.
(12) Schullery, S. E.; Schmidt, C. F.; Felgner, P.; Tillack, T. W.; Thompson, T. E. Biochemistry 1980, 19, 3919. (13) Wong, M.; Anthony, F. H.; Tillack, T. W.; Thompson, T. E. Biochemistry 1982, 21, 4126. (14) Wong, M.; Thompson, T. E. Biochemistry 1982, 21, 4133. (15) Sunamoto, J.; Iwamoto, K.; Kondo, H.; Shinkai, S. J. Biochem. (Tokyo) 1980, 88, 1219. (16) Massenburg, D.; Lentz, B. R. Biochemistry 1993, 32, 9172. (17) Viguera, A. R.; Mencia, M.; Goni, F. M. Biochemistry 1993, 32, 3708. (18) Viguera, A. R.; Alonso, A.; Goni, F. M. Colloids Surf. B: Biointerfaces 1995, 3, 263. (19) Matsumura, H.; Dimitrova, M. Colloids Surf. B: Biointerfaces 1996, 6 (3), 165. (20) Dimitrova, M.; Matsumura, H. Colloids Surf. B: Biointerfaces 1997, 8 (6), 287. (21) Meulenaer, B.; van der Meeren, P.; De Cuyper, M.; Vanderdeelen, J.; Baert, L. J. Colloid Interface Sci. 1997, 189, 254. (22) Dimitrova, M. N.; Matsumura, H.; Neitchev, V. Z. Langmuir 1997, 13 (24), 6516.
S0743-7463(98)00127-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/15/1998
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of the above studies. The changes in the rate and the activation energy of protein-induced liposome flocculation have been experimentally determined for temperatures ranging from 10 to 45 °C. From the measured light absorbance change in time, applying a previously reported analytical model,22 we have calculated the rate and activation energy of LSZ- or CC-induced liposome flocculation at different ambient temperatures. Variations in the bulk temperature affect the stability of protein/ liposome dispersion, with volume fraction constant, most likely through influence on a number of factors: Brownian motion, flocculation mechanism, protein adsorption, protein solubility, rearrangements of the protein molecules in the adsorbed layer, adsorption/desorption of the water molecules from the hydration layer, etc. We discuss the observed temperature dependencies of the PILF rate and activation energy on the basis of the relative importance of the above-mentioned factors, emphasizing the liposomes’ Brownian motion, the protein adsorption mechanism, and the bridging mechanism of the liposome flocculation as the major factors in the overall PILF. 2. Experimental Section Materials. The lipid, which was used for the formation of the liposomes, was egg yolk PC, purchased from Sigma Chemical Co., Ltd. The PC liposomes possessed negative electrophoretic mobility of the order of -1 (µm/s × cm/V), indicating that the natural egg PC lipid includes a small amount of acidic phospholipids.23 Three types of globular proteins were used: LSZ (isoelectric point at pH 11.4) from hen’s egg, CC (isoelectric point at pH 10.1) from horse heart, and BSA (isoelectric point at pH 4.9). All inorganic chemicals were analytical reagent grade purchased from WAKO Chemicals, Japan. For the preparation of the solutions we used distilled and deionized water, produced by the Autostill system (WG 240 Yamato Co.). Liposomes’ Preparation. Large-sized PC liposomes (mean diameter 1000 nm) were prepared by the vortex mixing method. On account of the importance of size distribution for the flocculation study, the liposome dispersion was dialyzed through a 1000 nm pore size filter to exclude the smaller liposomes and filtered through a 2000 nm pore size filter to remove the larger ones. For the preparation of 400 nm size PC liposomes, we used the extrusion method. The size distribution of the PC liposome dispersions was monitored using a dynamic light scattering size meter (Photal Otsuka electronics DLS 800, Japan, Co. Ltd.). Methods. The effective light absorbance was measured using 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).24 For continuous and precise temperature control, the spectrophotometer was improved with a Lauda RCS 6 compact low-temperature thermostat (temperature control at 10 °C is (0.03 °C). The rates of PILF caused by the adsorption of 10-1, 4 × 10-1, 8 × 10-1, and 1.0 mg/mL protein solutions were measured by the decrease in light transmittance (effective absorbance) within 10 min after the rapid mixing of all components. For the 400 and 1000 nm diameter PC liposomes, the respective utilized wavelengths were 800 and 1100 nm. Solutions of protein or liposome dispersions alone were run simultaneously to provide suitable references. The average values for the flocculation rate and the activation energy were calculated22 on the basis of three (or more) independent experimental measurements of the effective light absorbance change with time (10 min) under certain bulk temperature. Applying the curve fitting by the method of the least squares, we have determined τ and ∆A∞, and knowing A0 from the experimental data, we have calculated the flocculation rate and activation energy and have determined their average values. (23) Matsumura, H.; Mori, F.; Kawahara, K.; Obata, C.; Furusawa, K. Colloids Surf. A: Physicochem. Eng. Aspects 1994, 92, 87. (24) Furusawa, K.; Matsumoto, M. In Electrical Phenomena at Interfaces; Kitahara, A., Watanabe, A., Eds.; Marcel Dekker: New York and Basel, 1984; pp 225-266.
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Figure 1. Microscopic image of PC liposome dispersion (1000 nm liposome diameter) in the presence of 1 mg/mL BSA at bulk temperature 10 °C. The bridging flocculation of PC liposomes was directly observed using an Olympus IMT-2 optical microscope. The microscope stage was modified with a micro cool plate Kitazato MD-10 F (temperature control at 22 °C is (1 °C). The liposome electrophoretic mobility was measured using a microelectrophoretic cell with a four-electrode configuration (Microtec Co. Ltd).
3. Results The results were classified in three subsections according to the protein that caused PC liposome flocculation: BSA, CC, or LSZ. One supplementary subsection, which represents a comparison between the rates of CCor LSZ-induced PILF at different protein concentrations and bulk temperatures, was also added. Generally, BSA caused only a minor effect on the PC liposome dispersion stability, excluding some reversible doublets and triplets, which were formed at low bulk temperature (10 °C). The highest flocculation rates were induced by using CC, added at relatively high concentrations of about 1.0 mg/mL to a 1000 nm liposome suspension at 45 °C. Bovine Serum Albumin. Figure 1 presents direct microscopic observation of a 1000 nm PC liposome dispersion in the presence of 1 mg/mL BSA at ambient temperature 10 °C. At relatively low bulk temperature only unstable, small-sized flocs were formed. The average floc size corresponded to mainly that of doublets and triplets of liposomes. Above 10 °C, the absorption of BSA on the PC liposomes had only a minor effect on the dispersion stability and the microscopic observations (not shown here) were very similar to those obtained by using a pure liposome dispersion without protein. Generally, single liposomes can be seen, but in rare occasions, some of the liposomes adhered to each other, thus forming doublets, triplets, and quadruplets. Cytochrome C-Induced Flocculation. Microscopic observations on the flocculation behavior of CC-induced PC liposome flocs showed that an increase in the temperature changed both the flocs’ pattern, and the average size of the aggregates. Figure 2 shows the resultant flocs’ pattern induced by 1 mg/mL CC at 15, 25, 35, and 45 °C. The flocs formed at 15 °C appeared denser, and more compact, than those formed at 25 °C. By close observation of Figure 2, it can be seen that at higher temperatures the rarer flocs with a larger size and a more dendrite-like structure were formed. In the analytical method, which we have described in detail in ref 22, we have shown how the activation energy of PILF can be calculated using experimentally attainable
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Figure 2. Microscopic images of CC-induced PILF at ambient temperatures (a) 15 °C, (b) 25 °C, (c) 35 °C, and (d) 45 °C. The main diameter of the liposomes is 1000 nm, and the concentration of the protein is 1 mg/mL.
parameters from the effective light absorbance measurements. From the applied analytical method (see eq 11 in ref 22), we can obtain the relative activation energy, E** ) E* + kTlna, where the first term, E*, is the flocculation activation energy and the second term is a product of the Boltzman factor, kT, and asan effective experimental constant that depends on the particle refractive index, media refractive index, wavelength, etc. Here, however, the relative activation energy, E**, is equal to the real activation energy, E*, shifted with a temperature including constant kTlna. Without knowing the effective experimental constant, a, we can evaluate the temperature dependence of the activation energy, E*, using the following equation: E**/T ) E*/T + klna. Therefore, we plotted the obtained experimental results as E**/T versus 1/T, which according to the above written simple equation, gives the desired temperature dependence of the flocculation activation energy. A precise theoretical consideration of a would probably show a temperature dependence of the constant itself. In the experiments, however, the effective light absorbance of a pure liposome
dispersion varied less than 1% over the studied temperature range (data are not shown here). On the basis of these experiments, presently we can neglect thorough consideration of a possible temperature dependence of a, which would not change markedly our data. For the CCinduced flocculation of 1000 nm PC liposomes, the inverse temperature dependence of E**/T was plotted at four different protein concentrations and was produced in Figure 3. At the lowest studied CC concentration, 10-1 mg/mL, E**/T increased from 6.02 × 10-22 to 6.32 × 10-22 J/particle, which gave a 5% increase in the initial value at 15 °C. We can see a minimum in the curves of 4 × 10-1 and 8 × 10-1 mg/mL CC-induced PILF at the ambient temperature of 25 °C. It must be noted, however, that the concentration dependence of the activation energy increase was not a simple uniform one. E**/T of the CC-induced flocculation at 8 × 10-1 mg/mL almost coincided with that for 1.0 mg/mL at 15 and 25 °C and with that for 4 × 10-1 mg/mL at 35 and 45 °C. For the 1 mg/mL CC-induced PC liposome flocculation, we noticed a linear dependence, with a reverse slope to that of 10-1 mg/mL CC-induced PILF.
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Figure 3. E**/T versus the inverse temperature of CC-induced PILF for (b) 10-1 mg/mL, (2) 4 × 10-1 mg/mL, ([) 8 × 10-1 mg/mL, and (1) 1 mg/mL concentration of the added protein in the temperature range 15-45 °C.
Interestingly, in this case, as the temperature increased from 15 to 45 °C, the E**/T values decreased from 6.07 × 10-22 to 5.92 × 10-22 J/particle, which gave a 2.5% decrease with respect to the horizontal X-axis. The temperature dependence of E**/T was enhanced at the lower CC concentrations. Lysozyme-Induced Flocculation. The E**/T values of the LSZ-induced PC liposome flocculation were plotted in Figure 4a,b as a function of the inverse temperature and in Figure 5 as a function of the LSZ concentration. For clarity, the curves in Figure 4 were plotted on two separate graphs. Figure 4a shows the E**/T values of LSZ-induced flocculation at low protein concentration in the temperature range 10-45 °C, and Figure 4b shows the LSZ-induced flocculation for four different protein concentrations (10-1, 4 × 10-1, 8 × 10-1, and 1.0 mg/mL LSZ) in the temperature range 15-45 °C. The curves in Figure 4a revealed a shallow minimum between 15 and 25 °C. In Figure 4b, E**/T values increased as the temperature was raised from 15 to 45 °C; whereas for 10-1 mg/mL LSZ, E**/T increased 4.32% compared to the value at 15 °C, for 4 × 10-1 mg/mL LSZ the increase was 1.29%, almost 0% for 8 × 10-1 mg/mL LSZ, and a 1% increase for 1 mg/mL LSZ. Both parts a and b of Figure 4 clearly indicate that the temperature dependence of the activation energy of LSZ-induced flocculation alters with the concentration of the adsorbed protein. Figure 5 shows the E**/T dependence of the LSZinduced flocculation for 1000 nm PC liposomes versus the added protein concentration at isothermal conditions. The plots revealed the following tendencies: (1) there was a minimum in the E**/T curves around 8 × 10-1 mg/mL LSZ, as the E**/T values at this protein concentration were very close in magnitude for all the different ambient temperatures; (2) at the same protein concentration (in the case of 10-1 and 4 × 10-1 mg/mL LSZ), the E**/T increased with temperature (see also Figure 4); (3) at isothermal conditions the influence of the ambient temperature on the flocculation parameter E**/T was more accented at the lower protein concentrations (lower protein surface coverage). Rates of Cytochrome C- and Lysozyme-Induced Flocculation. In Figure 6 we have shown the temperature dependence of the relative kinetic constant, K′ ) K/a,22 where K is the flocculation rate constant. At 10-1 mg/mL concentration of the added LSZ, a 2-fold decrease in K′ was observed as the temperature increased from 15 to 45 °C, where the fastest change in K′ was noticed
Figure 4. (a) E**/T versus the inverse temperature of LSZinduced PILF at low protein concentration, ([) 10-1 mg/mL and (2) 4 × 10-1 mg/mL, in the temperature range 10-45 °C. (b) E**/T versus the inverse temperature of LSZ-induced PILF for (b) 10-1 mg/mL, (2) 4 × 10-1 mg/mL, ([) 8 × 10-1 mg/mL, and (1) 1 mg/mL concentration of the added protein in the temperature range 15-45 °C.
Figure 5. E**/T of LSZ-induced PILF versus the concentration of the added protein at isothermal conditions: (b) 15 °C; (2) 25 °C; ([) 35 °C; (1) 45 °C.
between 25 and 35 °C. Alternative temperature dependencies were obtained upon addition of 10 times more concentrated proteinss1.0 mg/mL LSZ or CC. In such an environment, one can perceive a 5-fold increase in K′ from 15 to 45 °C for the 1.0 mg/mL CC-induced flocculation, while for the LSZ-induced flocculation there was only a 2-fold increase from 15 to 25 °C, and then K′ decreased with the further temperature increase up to 45 °C. Figure 7 represents a histogram comparison between the rates of CC- or LSZ-induced flocculation processes at
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4. Discussion
Figure 6. Relative flocculation rates of PILF, induced by (1) 10-1 mg/mL CC, (2) 1 mg/mL CC, ([) 10-1 mg/mL LSZ, and (b) 1 mg/mL LSZ versus the ambient temperature in the range 15-45 °C (main diameter of the liposomes 1000 nm).
Figure 7. Relative flocculation rates of PILF, induced by 10-1 mg/mL LSZ, 1 mg/mL LSZ, 10-1 mg/mL CC, and 1 mg/mL CC, estimated for the ambient temperatures 15, 25, and 35 °C (main diameter of the liposomes 400 nm).
15, 25, and 35 °C. In this case the main diameter of the flocs-forming liposomes was 400 nm, and the protein concentrations were 10-1 and 1.0 mg/mL CC or LSZ. The relative flocculation rate, K′, of LSZ-induced liposome flocculation exhibited a nontypical maximum at 25 °C, while for the CC-induced PILF it gradually rose as the temperature increased from 15 to 35 °C. A very similar tendency was observed in the case of 1 mg/mL LSZ-induced flocculation (see Figure 6), where the liposome diameter was 1000 nm. Images of flocs, formed from 400 nm in diameter liposomes in 1 mg/mL LSZ solutions for 2 h after adding the protein, are shown in Figure 8. Figure 8a represents flocculation at 15 °C, and parts b and c show PILF at 25 °C, Figure 8d, at 35 °C, and Figure 8e, at 45 °C. The largest flocs were formed at 25 °C (Figure 8b,c). Slightly lower density flocs were formed at 15 °C (Figure 8a). At 35 and 45 °C, the floc density decreased with the temperature increase. The photographs in Figure 8 coincide qualitatively with the histogram in Figure 7, confirming fair agreement between direct microscopic observations and the kinetic parameters obtained by applying the analytical method for the effective light absorbance data interpretation. The quantitative differences most probably relate to the time of the flocculations 10 min for the histogram in Figure 7 and 2 h for the images in Figure 8. (25) Israelachvili, J. N. Intermolecular and Surfaces Forces, 2nd ed.; Academic Press Inc.: San Diego, CA, 1991; p 154.
This study clearly reveals that the increase in the ambient temperature has a strong influence on the flocculation rate and the activation energy of PILF. The alteration in the temperature affects the stability of protein-adsorbed liposome dispersions through influence on a number of factors, among which Brownian motion, the bridging mechanism of flocculation, protein adsorption, rearrangements of the protein molecules in the adsorbed layer, etc., manifest major importance for the overall PILF. The increase in the ambient temperature leads to an increase of the Brownian energy of each particle with 0.5 k∆T for each degree of freedom.25 Thus, the temperature increase must lead to an increase in the rate of the effective collisions between the liposomes in the suspension, i.e., to an increase in the flocculation rate. On the other hand, regarding the liposomes within the already formed flocs, the temperature increase may lead to two controversial processes, redispersion or coagulation and eventual fusion, depending on the interaction potential between proteinadsorbed liposomes. In our real systems, however, the added proteins play a major significant role. BSA, LSZ, and CC are water soluble, globular proteins that adsorb on the PC liposome surfaces (in the case of BSA deeply absorb into the phospholipid bilayer). Moreover, these relatively large protein molecules under certain bulk conditions can simultaneously adsorb onto the surfaces of two liposomes, thus causing bridging flocculation between protein-covered and protein-free liposome surfaces.21,22 A mosaic mechanism of PILF is less probable, since the adsorption of the positively charged CC and LSZ on the negatively charged liposome surface neutralizes the surface charge but does not cause surface charge overcompensation even at very high protein concentration.19 The depletion mechanism is also unlikely to cause the liposome flocculation, since the bulk protein concentrations, here studied, are far below the concentrations at which depletion aggregation induced by polymers has been observed.26,27 In addition, previously reported experimental data for the influence of the bulk protein concentration (in the studied range 10-4 to 1 mg/mL) on the overall PILF showed no indication for depletion effect.22 For the most probable bridging mechanism of the PILF, a maximum rate of the formed flocs is expected at a protein adsorption equal to 50% surface coverage28 (or more precisely to an optimum protein surface coverage). The surface coverage of the liposomes by protein molecules depends on the bulk protein concentration and temperature, as in accordance with the Gibbs equation (see eq III-83 in ref 29): increasing the temperature decreases the adsorption. Structural rearrangements of the protein molecule in the adsorbed layer are unfavorable since the bridge formation is most effective when a liposome collides with another liposome just after a protein molecule has been adsorbed.30 In the frames of the present work, the thermal unfolding of the proteins, protein conformation, and solubility changes in the overall temperature range are not thoroughly considered. At neutral pH, the thermal unfolding (26) Hirtzel, C. S.; Rajagopalan, R. Colloidal Phenomena Advanced Topics; Noyes Publishers: Park Ridge, NJ, 1985; p 95. (27) Hunter, R. J. Foundations of Colloid Science; Clarendon Press: Oxford, U.K., 1993; Vol. 1, p 492. (28) Wigsten, A. L.; Stratton, R. A. Polymer adsorption and dispersion stability; ACS Symposium Series; American Chemical Society: Washington, DC, 1984. (29) Adamson, A. W. Physical chemistry of surfaces, 5th ed.; Jonh Wiley & Sons: New York, 1990. (30) Gregory, J. Colloids Surf. 1988, 31, 231.
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Figure 8. Microscopic images of LSZ-induced PILF at ambient temperatures (a) 15 °C, (b) 25 °C, (c) 25 °C, (d) 35 °C, and (e) 45 °C. The main diameter of liposomes is 400 nm and the concentration of the protein is 1 mg/mL.
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of the proteins used is above 80 °C.31 The temperature increase in the range 10-45 °C has only a minor influence on the protein conformation and solubility in solution.32 The conformational stability of proteins upon and after adsorption on the interface, however, may vary over the studied temperature range. This appears to be an interesting and important topic for further investigation. The role of the hydration forces, which are generally considered as one of the major factors for the stability of PC liposome dispersions, was also not clearly seen as very significant in our experiments. Taking into account all the above regarded factors, we discuss our experimental data in terms of Brownian motion and the bridging flocculation mechanism, which have contradictory influence on the overall PILF with the temperature increase in the range 10-45 °C. For the experimentally observed high stability of PC liposome dispersion in the presence of BSA, the contribution of structural rearrangements of the protein molecules in the adsorbed layer should also be taken into account in addition to the previously considered steric stabilization and electrostatic repulsion.22 BSA is classified as “soft”, easy to perturb upon protein sorption,33 whose large and flexible molecules most probably rearrange in the adsorbed layer. These changes are unfavorable and obstruct or even fully prevent the formation of “bridges” between protein-covered and protein-free liposome surfaces. The microscopic observation in Figure 1 is fairly consistent with this hypothesis, showing that with a decrease in the ambient temperature, and therefore a decrease in the unfavorable conditions for the bridging flocculation such as rearrangements of the protein molecules in the adsorbed layer and the thermal undulations of the phospholipid membranes, the BSA-absorbed PC liposome dispersion becomes less stable. The activation energy of the LSZ-induced liposome flocculation under different bulk conditions (see Figures 4 and 5) shows the tendency of increasing in the temperature range from 15 to 45 °C. The increase in E** most probably relates to conformational changes of the adsorbed protein, which affect the protein adsorption and furthermore the flocculation mechanism. Structural rearrangements and mobility of the protein molecules in the adsorbed layer as well as thermal undulations of the liposome membrane also contribute to the noticeable decrease in the probability for a bridging formation. The temperature increase in this case has a greater influence on the less saturated surfaces (10-1 and 4 × 10-1 mg/mL LSZ), and completely covered ones, where bridging forces should be of low strength. The influence of the ambient temperature on the activation energy of 8 × 10-1 mg/mL LSZ-induced liposome flocs is the least pronounced. The two experimental facts, (i) the minima in the three curves for the 25, 35, and 45 °C LSZ-induced PILF (Figure 5) at around 8 × 10-1 mg/mL added protein and (ii) the fair similarity of all four E**/T values around this protein concentration, clearly indicate that at this protein concentration the activation energy possessed the lowest value, and the bridging PILF was the most facilitated. Regarding the bridging mechanism of PILF, the maximum flocculation should occur at the optimum surface coverage of the liposome membranes with LSZ molecules. Therefore, 8 × 10-1 mg/mL LSZ appears to be the bulk protein concentration that leads to the optimum surface coverage. A comparison between both graphs infers an important (31) Privalov, P. L.; Khechinashvili, N. M. J. Mol. Biol. 1974, 86, 665. (32) Ataka, M.; Asai, M. J. Cryst. Growth 1988, 90, 86. (33) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87.
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conclusion that at conditions at which PILF is maximum, the influence of the ambient temperature is less pronounced. This means that the structural rearrangements of the protein molecules in the adsorbed layer, as well as possible temperature movements of the peptide chain, have probably a less inhibitory effect on the “bridge” formations between the protein-covered and protein-free surfaces of the approaching liposomes. The structure and the adsorbed amount of LSZ and the configuration of the molecules in the adsorbed layer at this certain protein concentration may alter only slightly over the temperature range 15-45 °C and thus be fairly conducive to the bridging PILF. The two minima in the activation energy curves for the LSZ-induced flocculation between 15 and 25 °C in Figure 4a can be explained in terms of Brownian motion. Below 15 °C the increase in temperature heightened the energy of the liposomes, thus increasing the probability of the effective collisions. In other words, the temperature change leads to energizing the basic liposome suspension but does not influence the absolute energy level of the transition state that corresponds to the energy barrier (activation energy, E*). A further increase in the temperature may raise the energy level of the transition state because of the enhanced oscillation amplitudes of the individual liposomes. Thus, the energy of the joined oscillators in the transition states grows more quickly than the energy of the independent liposomes in the suspension. From the experimental results (Figure 4a,b), we may conclude that PILF diminishes the energy of the transition state at lower temperatures. Above the minimum in the experimental graphs (Figure 4,b), a further increase in the ambient temperature heightens the activation energy of the process, most probably due to the unfavorable conditions for the bridging mechanism such as rearrangements of the protein in the adsorbed layer, thermal movements of the peptide chain, and thermal undulations of the liposome membrane, which prevail over Brownian motion and hampered the flocculation. The increase in the protein concentration shifts the minimum in the E**/T versus 1/T for 10-1, 4 × 10-1, and 8 × 10-1 mg/mL LSZ (Figure 4b) toward higher temperatures due to the bridging mechanism of PILF, for which the amount of the adsorbed protein plays an essential role. The temperature dependence of the relative flocculation rate for 0.1 mg/mL LSZ-induced flocculation (see Figure 6), can be pertinently explained in terms of the bridging mechanism of PILF. As has already been discussed, the temperature change from 15 to 45 °C increases the activation energy of the bridging flocculation (Figures 4a,b and 5), and hence lowers the number of effective collisions per unit time. A low collision rate lowers the flocculation rate, which was experimentally observed by a 2-fold decrease of the relative flocculation rate with the temperature increase in the studied range. Changing the protein and its concentration revealed the significant influence of the protein adsorption mechanism, and the amount of adsorbed molecules on the PILF rate. The maximum flocculation rate in the K′/T curve for 1.0 mg/ mL LSZ at 25 °C corresponds to a minimum activation energy shown in Figures 4b and 5, which is in agreement with the hypothesis for the bridging flocculation as the main factor and the Brownian energy increase in the liposome flocs as the main complementary factor. The microscopic images in Figure 2 and the rapid increase in the flocculation rate of 1.0 mg/mL CC-induced PILF, indicate flocculation behavior different from that of LSZ. The microscopic observations of CC-induced flocs at four different temperatures (see Figure 2) show that
Flocculation of Phosphatidylcholine Liposomes
at higher ambient temperature more dendrite and large flocs were formed. These changes in the floc’s size and pattern are fairly consistent with the rapid increase of K′ (Figure 6). The calculated 5-fold increase in the flocculation rate was accompanied by a 2.5% decrease in the E**/T versus 1/T in respect to the X-axis (see Figure 3). In fact more detailed consideration of the estimated opposite linear slope indicates that the activation energy in this case also increased, but the increase was less pronounced. Supposing that the flocculation activation energy is constant when the bulk temperature increases from 15 to 45 °C, then a 9.56% decrease must be observed in the E**/T versus inverse temperature plots (from the E*/288 value to the E*/318 one). The experimentally determined 2.5% decrease indicates that with the bulk temperature increase, an increase in the activation energy for the 1.0 mg/mL CC-induced liposome flocculation was detected. The increase, however, was distinctively small. We think that the difference in the temperature dependence of the flocculation rate, and activation energy of the CC- and LSZ-induced PC liposome flocculation, which is most pronounced at 1.0 mg/mL concentrations of these two proteins, closely relates to their different mechanisms of adsorption on the phospholipid membrane. In the electrophoretic measurements,19,20 for example, we have seen that the adsorption of CC more strongly depends on the bulk cation concentration compare to the LSZ one. This is a clear indication that the electrostatic attraction is the main driving force for the CC adsorption. The adsorption of LSZ on the phospholipid membranes, on the other hand, causes local membrane destabilization and leakage of inner liposome content to the outer medium, which was experimentally observed in the fluorescent measurements by using dye/quencher fusion assay.20 This confirms a very close approach and penetration of the LSZ molecules into the PC membrane, which relates to the additional hydrophobic dehydration interactions
Langmuir, Vol. 14, No. 19, 1998 5445
between the hydrophobic domains of LSZ molecules and PC membranes. On the basis of the previously reported studies about the adsorption of these two proteins on the phospholipid membranes,19,20 we think that the contribution of the total entropy change in both cases over the investigated temperature range is essential for the different temperature dependence of CC- and LSZ-induced PILF. In the case of LSZ, which adsorbs strongly into the phospholipid membranes, the hydrodynamic movements of the lipid hydrocarbon chains are most probably greatly obstructed, which causes an entropy loss. As the bulk temperature increases, the entropy loss increases. This is unfavorable and hampers the adsorption of the protein molecules on the liposome membranes. Furthermore, it leads to a smaller probability for bridge formations and smaller relative flocculation rate. In the case of CC, which adsorbs only due to the long-range electrostatic attraction, the hydrodynamic movement and thermal undulations of the lipid hydrocarbon chains are not so strongly influenced by the adsorbed proteins. There is not such a great loss of entropy as the ambient temperature increases. Therefore, as the temperature increased, the flocculation rate and the activation energy of CC-induced PC liposome flocculation simultaneously increased. In conclusion, the complex temperature dependence of the overall PILF confirms the hypothesis for a complementary action of the bridging and Brownian forces during the flocculation process, with a strong parallel influence of the protein adsorption mechanism. Acknowledgment. The work was partially supported by a research fellowship N 962129 awarded to M.N.D. by the Japanese Ministry of Education, Monbusho. The authors are grateful to Dr. P. Reay for the challenging discussions and critical reading of the manuscript. LA980127Q
