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Microcalorimetric and shear studies on the effects of cholesterol on the physical stability of lipid vesicles
Colloids and Surfaces A: Physicochemical and Engineering Aspects 172 (2000) 57 – 67 www.elsevier.nl/locate/colsurfa
Microcalorimetric and shear studies on the effects of cholesterol on the physical stability of lipid vesicles Der-Zen Liu, Wen-Yih Chen *, Li-Min Tasi, Shu-Ping Yang Department of Chemical Engineering, National Central Uni6ersity, Chung-Li, Taiwan, ROC Received 23 August 1999; accepted 16 March 2000
Abstract This work measured the dilution heat of phosphatidylcholine (PC) vesicles incorporated with various amounts of cholesterol using isothermal titration calorimetry (ITC). The interaction potential between these PC vesicles was analyzed and the changing size of the PC vesicles response to various shear force and the zeta potential of the PC vesicles were also measured and discussed along with the ITC results. Experimental data indicate that the repulsive surface potential and the repulsive electrostatic force increases as cholesterol is incorporated into the bilayer. This phenomenon can be attributed to the decrease in the binding ability between the PC vesicular surfaces and the cations in the buffer solution. Incorporating cholesterol into the bilayer promotes the repulsive surface potential (or energy barrier) between the PC vesicles from the surface potential viewpoint. Additionally, incorporating cholesterol into the bilayer makes the PC vesicles more rigid and sustains more severe shear stress in our shear forces studies. This work also reveals that temperature and PC vesicular stability are correlated with each other. The stability of PC vesicles are likely to decline with a rise in the temperature, and the elevated temperature effect is evidence of the rigidity or curvature of the PC vesicles. Furthermore, the repulsive surface potential (or energy barrier) between the PC vesicles declines with a rise in temperature. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Phosphatidylcholine bilayer vesicles; Cholesterol; Surface potential; ITC; Physical stability
1. Introduction Phospholipid bilayer vesicles (lipid vesicles, liposomes) have been widely utilized as a biomembrane model and as a carrier in drug delivery systems [1– 5]. The aqueous dispersion of lipid vesicles are metastable systems, therefore, control * Corresponding author. Tel.: +886-3-4227151, ext 4222; fax: +886-3-4225258. E-mail address: [email protected] (W.-Y. Chen)
and prediction of the lipid vesicles stability against aggregation or fusion are important throughout the various stages of the applications. Aggregation is a critical characteristic behavior of lipid vesicle systems as it depends on the overall interactive forces between lipid vesicles. Several investigations have demonstrated that lipid vesicles made of acidic phospholipids (PS, PG) possess negative electric charges on their dissociative − groups (such as PO− 4 and COO ) at neutral pH, and that the electrostatic repulsion forces are
0927-7757/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 0 ) 0 0 5 6 0 - 4
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the dominant contributing factor to the interaction between lipid vesicular surfaces. Nevertheless, these lipid vesicles are likely to be readily aggregated and sometimes fused by altering the pH or ionic compounds that are added to the solution, because this addition may reduce the repulsive electrostatic forces between the lipid vesicles surfaces [6–12]. In contrast, the lipid vesicles made of amphoteric phospholipid such as (PC or PE), exhibit considerably different properties than the acidic phospholipid bilayer vesicles. In fact, the structure of the PC head group is zwitterionic, and the z-potentials of PC vesicles are significantly smaller than those of the acidic phospholipid bilayer vesicles [13]. Hence, as generally assumed there is not a strong electrostatic repulsion force between the amphoteric phospholipid vesicles, and that the high stability of PC vesicles must be due to some other repulsive forces between lipid vesicles. These other repulsive forces include the repulsive hydration forces or the entropic forces [14 – 17] in a short distance range. The effect of cholesterol incorporation into phospholipid bilayer on the cation-induced lipid vesicular aggregation and fusion are complex and difficult to comprehend. Generally, when cholesterol is incorporated into the phospholipid bilayer it alters the nature of the lipid vesicle. Bental et al. [18] reported that cholesterol incorporated into bilayer attenuates the Ca2 + -induced vesicle aggregation of the large br-PS vesicle. Virden and Berg [19] discussed the behavior of the incorporation of varying amounts of cholesterol on DPPG small unilamellar vesicles on NaCl induced aggregation. Their studies demonstrated that at a low NaCl concentration (NaCl B0.8 M), increasing cholesterol amounts may alter ion binding or shear plane distance. The increasing stability of DPPG small unilamellar vesicles is caused by the expanded short-range repulsive hydration force While at high NaCl concentrations (NaCl \1.0 M). Rand et al. [20] studied the effects of interaction forces between lipid vesicles that include cholesterol indicating that cholesterol incorporated into the DPPC bilayer decreases the van der Waals attraction and increases the net repulsion forces between bilayers. Nevertheless, Eklund et al. [21]
observed enhancement of NaCl-induced aggregation of br-PS bilayer vesicles and attenuation of CaCl2-induced aggregation of DPPS bilayer vesicles by inclusion of cholesterol. According to their results, the phenomena is likely attributed to that incorporating the cholesterol into the bilayer slightly increases the lipid bilayer vesicle size and decreases the lipid bilayer vesicle curvature. This ultimately results in a diminished polar head group surface area and an increased charge density of the lipid vesicular surfaces. NaCl-induced aggregation of DPPS bilayer vesicular inclusion of cholesterol and CaCl2-induced aggregation of DMPG bilayer vesicular inclusion of cholesterol are distinct. Eklund et al. contended that the inclusion of cholesterol can enhance or attenuate the cation-induced lipid vesicle aggregation depending on the particular cation and the phospholipid fatty acid composition [21]. In light of the above discussion, incorporating cholesterol into the phospholipid bilayer can alter the phospholipid bilayer geometry (shear plane distance and bend curvature of lipid bilayer vesicle), and ultimately affect the cation-induced aggregation or fusion behavior. The mechanical properties of the lipid vesicular structure are heavily dependent on temperature. Several phases with temperature are identified in accordance with the state of lipid bilayer in which the acyl chain structure plays a decisive role [22]. Taylor et al. [23] and Dufourc et al. [24] proved that cholesterol are perpendicular to the phospholipid bilayer surface membrane with its small hydrophilic 3b-hydroxyl head group located in the vicinity of the phospholipid ester carbonyl groups after it is incorporated into the phospholipid bilayer. Simultaneously, the hydrophobic steroid ring of cholesterol orients itself parallel to the acyl chains of the phospholipid. Restated, the incorporation of cholesterol into the phospholipid bilayer will affect the movements of the acyl chains of the phospholipid bilayer. Previous investigations [25– 27] have demonstrated that incorporating cholesterol into bilayer will reduce the fluidity of phospholipid bilayer if the bilayer’s temperature is higher than the phase transition temperature of the acyl chain.
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The above discussion reveals that the effects of cholesterol are realized by considering the curvature of the lipid bilayer vesicle, the intravesicle interaction between lipids and the binding between the lipid and cation in the buffer. The changes of the interaction forces between the PC bilayers with added cholesterol present are still unclear, especially because it is impractical to directly measure the forces between PC vesicles. Therefore, the aim of this work is to measure the dilution heat of PC vesicles incorporated with various amounts of cholesterol by isothermal titration calorimetry (ITC). The parameter of the interaction forces and the surface potential of PC vesicles can then be calculated from the dilution heat empirical data coupled with a simple statistical thermodynamic model [28]. Furthermore, the shear modulus was established to examine the rigidity of the PC vesicle. In summary, the aggregation and fusion behaviors are explained using the shear forces studies and interaction forces (or surface potential) between PC vesicles with added various amount of cholesterol.
solvents. The PC bilayer film was hydrated at 323 K with a buffer solution composed of 20 mM Tris, 100 mM NaCl and 1 mM EDTA (pH being adjusted to 7.4 with HCl). Frozen and thawed (FT) PC vesicles were prepared from the MLVs to prepare small unilamellar vesicles and reduce the lamellarity [30]. These lipid vesicles were subjected to freezing (in boiling nitrogen) and thawing (in a water bath at 323 K) five times, and were then sonicated for 30 min using the Bandelin HD-200 sonication probe at a power setting of KE 76/D under nitrogen. The temperature of the lipid vesicles was maintained at around 323 K by circulating the water in the round-bottomed flask. Then, the final optically clear suspension was filtered through a 0.22-mm millipore. All of the vesicles were added the same amount of tocopherol with various quantities of cholesterol. Tocopherol was added to avoid oxidative damage caused by the interactions between free radicals and the PC lipid. Therefore, the disturbance of the chemical stability for the experimental measurements can be reduced.
2. Materials and methods
2.3. The lipid 6esicles calculation
2.1. Materials
The number of PC lipids within a PC bilayer vesicle can be estimated by calculating the ratio between the head -group surface areas of egg-PC (0.70 nm2) [31] and cholesterol (0.28 nm2) [32], then, making the total amount of egg-PC (3.75 mg − 1 ml − 1) divided by the quantity of PC lipids contained within one individual PC bilayer vesicles, thus the number of lipid vesicles per milliliter can be roughly calculated with this approach.
Egg-PC (:99%), cholesterol and a-tocopherol were purchased from Sigma (USA). Chloroform, methanol, Tris and sodium chloride were obtained from Merck (Germany). All chemicals were used as received without further purification.
2.2. Preparation of the lipid 6esicles suspension solution Multilamellar lipid vesicles (MLVs) were prepared according to the method proposed by Bangham et al. [29]. A solution containing egg-PC (37.5 mg − 1 10 ml − 1), cholesterol (0 or 9.66 or 19.32 mg − 1 10 ml − 1) (0 or 33 or 50 mol%) and 5.9 mg − 1 10 ml − 1 of a-tocopherol in a mixture of chloroform/methanol (2:1, v/v) was dried in a 25 ml round-bottomed flask. Nitrogen was applied to blow over the dried lipid film for approximately 30 min in order to remove the traces of organic
2.4. Determination of the particle size distribution of 6esicles by PCS Lipid vesicle size distribution was determined by a photon correlation spectroscopy employing an zetasize 3000 (Malvern Instruments, UK). A sample (lipid vesicles suspension solution) was diluted in a buffer solution until the measured size was independent of the particle concentrate. Then, the lipid vesicles suspension solution (contained in a 2 ml plastic cuvette) was placed in a
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sample holder at 298 K. The viscosity of the sample solution was 1 cp. A helium-neon laser of wavelength 633 nm and 5 mW acts as the light source. The laser was focused on the sample and scattered light was detected at 90° to the incident beam using a photomutiplier tube. The Malvern software controlled both the acquisition and the on-line analysis of the scattering data. Both the so-called z-average particle diameter and the polydispersity index were calculated by the comulant analysis method.
2.5. z-potential measurement The lipid vesicles suspension was prepared as described earlier. The electrophoretic mobility and the zeta potential of lipid vesicle suspension were determined by laser Doppler electrophoretic mobility measurements with the zetasizer 3000 (Malvern Instruments, UK).
2.6. The impact of high and low shear forces on the lipid 6esicle structure Different shear stresses and their impacts on the lipid vesicle structures were investigated via a cone and plate viscometer. A suspension solution of lipid vesicles prepared according to the previously mentioned instructions, was poured into a viscometer (Brookfield HVTD-II, CP40) cone. Various high shear stresses were applied to this lipid vesicles suspension solution for 3 min, and the changes on the particle size were measured afterwards. The low shear rate experiment was designed to understand the impact of shear rate on the structure of lipid vesicles. A suspension solution of lipid vesicles prepared as described in the previous section was put into a viscometer (Brookfield LVTDII, CP40) cone. The changes of particle size were observed under a continuous flow field generated by a 50 s − 1 shear rate. The temperatures of these shear forces and shear rate experiments were kept at 298 and 310 K using a water bath, and the number density of the lipid vesicles suspension solution in both the shear
stresses and shear liposome − 1 ml − 1.
rate
studies
was
1013
2.7. The isothermal titration calorimetry dilution heat and the surface potential of the lipid 6esicles The PC vesicles suspension solution dilution heat was determined by applying an isothermal titration calorimetry. The dilution heat of the PC vesicles with added 33 mol% cholesterol suspension solution as presented in Fig. 1. The thermal activity monitor (Thermometric AB, Sweden) controlled by digitam software performed the ITC. The ampoule was washed with water and acetone, and dried in the air prior to each experiment. A 2 ml suspension solution of lipid vesicles was placed in the ampoule in this experiment. A series of 150 ml buffer solution were titrated into the dispersion PC vesicles suspension solution using a Hamilton microliter syringe at 30 min intervals after the ampoule and the heat sink reached thermoequilibrium. The PC vesicles suspension solution was titrated five times per experiment. All experiments were performed at temperatures of 298 and 310 K. The dilution heats of the PC vesicles suspension solution with the number density of lipid vesicles suspension solution can be generally expressed as a function of the second order polynomial in Eq. (1): q E d d NkBT NkBT $ = b2 + b3r+ b4r 2 (1) dr dr For an open liquid system, the pressure change and the titration volume are neglected in the system total volume. The observed dilution heat of each titration is comparable to the internal energy change. Furthermore, the internal energy change can be expressed by the virial coefficient and the number density in Eq. (2): E 3 1 dBi + 1 i = −T % r NkBT 2 dT i=1 i
(2)
Comparing Eq. (1) with Eq. (2) reveals that the coefficient of the dilution heat polynomial
D.-Z. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 172 (2000) 57–67
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Fig. 1. The dilution heat of PC vesicles suspension solution. The PC vesicular composition is (PC:Chol:Vit E =4:2:1 mol ratio).
can be affiliated with the virial coefficients as in Eq. (3):
3.1. Zeta potential measurement
dB b2 = − T 2 dT
(3)
According to statistical mechanics, the relation between the second virial coefficient and the interaction potential energy function U(r) is: B2(T)= −2p
&
[exp −
0
U(r) −1]r 2dr kBT
(4)
and if a square-well energy potential function is picked and plugged into Eq. (4), then Eq. (3) can be rewritten as: b2 = − T
3. Results and discussion
dB2 = −B0(l 3 −1)e o/kBT(o/kBT) dT
b2/B0 = (l 3 −1)e o/kBT(o/kBT)
(5) (6)
where B0 =(16/3)pR , l= (2RHS +s)/2RHS and s, o denote the width and depth (or energy barrier) of the square-well energy potential function. 3 HS
This study measured the zeta potential (z-potential) of the PC vesicles incorporated with various amounts of cholesterol. Table 1 reveals that the PC vesicles made of egg-PC possess negative charges at pH 7.4. This finding is consistent with Carrion et al. [13] and McLaughlin et al. [33] indicating that there is a weak electrostatic repulTable 1 The values of zeta potential of PC vesicles at different temperatures with various amount of cholesterol incorporateda Composition PC:Vit E:Chol
Zeta potential (mv) (298 K)
Zeta potential (mv) (310 K)
4:1:0 4:1:2 4:1:4
−14.19 0.8 −17.991.1 −20.690.7
−4.790.7 −8.590.5 −11.491.4
a
Values are mean 9standard deviation, n = 5.
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sive force between the PC vesicles at pH 7.4. This experiment also demonstrated that the incorporation of cholesterol into the system can elevate the negative zeta potential. This phenomenon is possible because the cholesterol incorporated into the bilayer reduces the surface binding affinity among the cations in the buffer solution and its bilayer surface. Additionally, this study also observed that the zeta potential of the PC vesicles with or without cholesterol is higher at 310 than at 298 K because some hydrophilic headgroups become more hydrophobic at higher temperatures [34], increasing their ability to dehydrate and to bind with the cation in the buffer. Furthermore, the PC bilayer becomes more fluid as the temperature increases, disrupting the binding affinity with the cation in the buffer solution. The experimental data also indicates that the absolute zeta potential becomes more negative as the molar ratio of cholesterol changes from 0 to 33% in relation to its component contents within the lipid vesicle However, the decrease of zeta potential is not significantly altered as the molar ratio of cholesterol was added becomes 50% because of the cholesterol structural arrangements within the PC bilayer. The structural arrangements of the PC bilayer become irregular due to the incorporation of the cholesterol, thus affecting the binding ability between the PC vesicular surface and the cation as the molar ratio of cholesterol reaches 50% in the PC vesicle.
3.2. Interaction between PC lipid bilayer 6esicles The interaction forces between the lipid bilayers have been subjected to a significant amount of applied research as discussed in the introduction. Nir [35] suggested that incorporation of cholesterol into the bilayer increases the van der Waals forces among the lipid vesicles. However, Yeagle [36] contended that the electrostatic repulsive force decreases following the incorporation of cholesterol into the lipid vesicles. Mcintosh et al. [37] postulated that the annexation of cholesterol in the lipid vesicles affects the short distance repulsive force among the lipid vesicles. Therefore, we believe that incorporating cholesterol into the bilayer causes the lipid vesicles to change
their packing geometrical structures. These geometrical changes include lipid vesicle size, the curvatures of the surface bilayer, and surface lipid bilayer rigidity. We also believe that the Hamker constant, van der Waals forces, electrostatic repulsive forces, and hydration forces between the lipid bilayers or lipid vesicles will all be affected following the incorporation of cholesterol into the lipid vesicles. The interactive force and the effects of the forces between the PC vesicles are not directly measured when incorporating cholesterol into the PC bilayer. Therefore, a thermodynamic approach is utilized herein to examine the surface potential of the intervesicle interaction [28]. The dilution heat of the PC vesicle incorporated with various amounts of cholesterol by isothermal titration calorimetry was measured according to the derivation and discussion in the Section 2. The measurement was taken to obtain the energy barrier of the interaction potential between the PC vesicles. Namely, this study proposes a novel method of discussing the effect of cholesterol on the PC vesicle interaction potential. The system of PC vesicles has a positive b2 value with or without added cholesterol according to Table 2, indicating that the overall net interactive force is repulsive between the two PC vesicles. This net repulsive force poses a formidable barrier to aggregation and fusion. The value of b2/B0 (Table 2) is increased as the cholesterol is incorporated into the PC vesicles, indicating that the repulsive surface potential (energy barrier) of PC vesicles is also increased (higher values of o and s). Closely examining the interaction surface potential between the two PC vesicles, reveals that the higher interaction repulsive surface potential results in a system in which aggregation and fusion are both more difficult. A possible explanation is that the surface binding force between the PC vesicles and the cation in the buffer solution is declined as the cholesterol is incorporated into the PC bilayers, as observed in the zeta-potential experiments. Moreover, the hydration repulsive forces of PC vesicles may also be increased because the bounded water molecules are not displaced from the polar groups by cations.
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Table 2 The values of second order polynomial b2 and the b0 of virial coefficient of PC vesicles at different temperatures with various amount of cholesterol incorporated Reaction temperature (K)
Composition PC:Vit E:Chol (mol%)
b2 (nm3)
B0 virial coefficient (nm3)
b2/Bo
298
4:1:0 4:1:2 4:1:4
4.30×1011 7.54×1012 1.55×1013
7.88×105 1.66×106 2.47×106
5.46×105 4.54×106 6.27×106
310
4:1:0 4:1:2 4:1:4
3.84×1011 1.40×1012 2.45×1012
7.88×105 1.66×106 2.47×106
4.88×105 8.46×105 9.92×105
Fig. 2. The size of the PC vesicles of various amount of cholesterol incorporated with different high shear forces at different temperatures (each point value is average of three times). The particle size of liposome is the diameter.
The surface repulsive potential is diminished (lower values of o and s) or the net repulsive electrostatic force is decreased at a high temperature the b2/B0 value for 310 K is smaller than that of 298 K with or without cholesterol for a system of PC vesicles. The zeta potential experiment confirmed a less negative zeta potential at 310 than at 298 K. Therefore, the temperature effect on the total interaction potential between PC vesicles may be attributed to the temperature effect on the packing structure and the thermal motion of the PC bilayer. This temperature effect induces both the intervesicle and intravesicle interaction forces to change ultimately resulting in a total interaction potential.
3.3. Shear stress and its effect on particle size Fig. 2 illustrates how shear stress affects the PC vesicles system with or without cholesterol at 298 and 310 K. A larger threshold shear stress is necessary to destroy the PC vesicles with cholesterol added in bilayer, because adding cholesterol with the concentration range in this work can make the PC rearrange itself with higher order and rigidity. A high shear stress can impact the PC vesicular bilayer surface more effectively at higher temperatures according to our data. Nevertheless, the threshold shear stress needed to cause any changes in particle size at 310 K is smaller than that at 298 K because the disorder and
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flowing state of the PC bilayer vesicle increase at higher temperature. The shear stress can make the PC bilayer vesicle particle become smaller in size with or without incorporating cholesterol while the real cause for the reduction of the PC bilayer vesicle particle size is uncertain. Possible interpretations for this uncertainly include the notion that shear stress can increase the PC bilayer longitudinal interactions implying that the intravesicle may destroy the binding between PC lipids and may cause the PC vesicle to break into fragments or increase the bending energy of the PC lipid. Thus this would, in turn, increase the packing density of the PC lipids bilayer between the PC lipids.
by considering two mechanisms. First, the incorporation of cholesterol into the PC bilayer can make the PC vesicular bilayer surface reconstruct in a more rigid and ordered structure. Second, the PC vesicle will be unlikely to form a fusion state or become unstable following a collision. The shear rate can affect the PC bilayer surface more at higher temperature. Namely, the PC bilayer surface tends to form a fusion state or become unstable after a collision at a temperature higher than 298 K. The susceptibility of PC vesicle to temperature changes is related to its bilayer surface state as described in the previous section. The PC bilayer structure is more likely to become disordered and stay in a fluid state at higher temperature.
3.4. The effect of collision beha6ior on the lipid 6esicle
3.5. Particle size change
A lower shear rate (50 s − 1) is used to enhance the chance of collision to understand the PC vesicle collision behavior. Experimental results in Fig. 3 indicate that the threshold time needed to change the PC vesicular particle size is much longer following the incorporation of cholesterol, when accompanied by a fluid field of 50 s − 1. Conversely, the PC vesicle particle size is more susceptible to change and becomes bigger without incorporating cholesterol. This can be explained
Armengol and Estelrich [38] check the physical stability of PC vesicles by measuring their average size by a photon correlation spectroscopy. Figs. 4 and 5 display the changes of particle size of PC vesicle at 298 and 310 K, respectively. The PC vesicle particle size tends to become larger as time passes which is a sign that the fusion condition is irreversible. Experimental results suggest that originally, the PC vesicle was at a metastable state. In other words, the PC vesicle may con-
Fig. 3. The size of the PC vesicles of various amount of cholesterol incorporated with shear time at different temperatures. The lipid vesicles suspension solution was subjected to a continuous fluid field at a low shear rate of 50 s − 1 (each point value is average of three times). The particle size of liposome is the diameter.
D.-Z. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 172 (2000) 57–67
Fig. 4. The size of PC vesicles of various amount of cholesterol incorporated at 298 K with time. The number density of lipid vesicles suspension solution was 1013 liposome per ml (each point value is average of three times). The particle size of liposome is the diameter.
65
There are two continuous mechanisms for lipid vesicle fusion: the lipid vesicle has to aggregate; and intermediate structures such as inverted micelles or inverse hexagonal phase occur after the close juxtaposition and local perturbation in the lipid bilayer packing [39]. The incorporation of cholesterol into the bilayer will increase the repulsive forces and energy barrier between the PC vesicles according to the zeta potential and the b2/B0 value, and making it difficult for the PC vesicle to reach the primary minimum. Additionally, the shear stress and collision experiments indicated that the incorporation of cholesterol into the bilayer will make the PC vesicular bilayer surface more rigid and ordered. Obviously, the incorporation of cholesterol into the bilayer can decrease the state of fusion for the PC vesicle compared with the PC vesicle without cholesterol. The repulsive surface potential of the PC vesicle is lowered (lower values of o ) and the PC bilayer surface state become more fluid as the temperature increases from 298 to 310 K. Furthermore, the PC vesicles more easily reach the primary minimum, the so-called fusion state because the PC vesicular collision frequency is extended as the temperature rises.
4. Conclusion
Fig. 5. The size of PC vesicles of various amount of cholesterol incorporated at 310 K with time. The number density of lipid vesicles suspension solution was 1013 liposome per ml (each point value is average of three times). The particle size of liposome is the diameter.
strain the electrostatic repulsive forces to temporarily pause the second minimum state (or reversible aggregation). The PC vesicle will reach the first minimum state (or irreversible aggregation) may due to collision of PC vesicles or the conformational changes of PC vesicle with time. The incorporation of cholesterol into the bilayer will reduce the PC vesicle particle size changes since incorporating cholesterol can reduce the fusion between PC vesicles at either 298 or 310 K.
Incorporating cholesterol into small unilamellar vesicles of egg-PC increases the dispersion stability with respect to aggregation and fusion. Those can be explained with two aspects in our experiment. The experimental shear stress and shear rate demonstrate that adding cholesterol will make the composition of the PC bilayers more organized and rigid. The ITC and zeta potential experimental results qualitatively demonstrate that incorporating cholesterol into the PC bilayer can increase the electrical repulsion force and increase the repulsive interaction surface potential or energy barrier between the PC vesicles. Therefore, incorporating cholesterol into PC bilayer develop the physical stability. The raise of temperature accelerates the speed of PC vesicle fusion because the enhancement of temperature can increase the frequency of colli-
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sion between PC vesicles, and promote the thermal motion of the PC bilayer, resulting in a decreased order of PC bilayer structure. The ITC and zeta potential experiments also suggest a lower electrostatic repulsive and higher hydrophobic attractive surface potential between the PC at higher temperatures. Therefore, the raise of temperature will decline the physical stability.
Acknowledgements We would like to thank the National Science Council of the Republic of China for financially supporting this research under Grant No. NSC892214-E008-011.
Appendix A. Nomenclature PC PE PS PG q E N kB T r b2
B0 RHS s o
phosphatidylcholine lipid phosphatidylethanolamine lipid phosphatidylserine lipid phosphatidylglycerol lipid dilution heat of egg-PC liposome suspension solution (mJ) internal energy (mJ) number of lipid vesicles Boltzmann constant (J/K) absolute temperature (K) number density of lipid vesicles suspension solution (number per ml). second virial coefficient of the fitting polynomial of the dilution heat of a egg-PC liposome suspension solution virial coefficient the radius of lipid vesicle (nm) the width of the square-well potential function. the depth of the square-well potential function (energy barrier)
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Microcalorimetric and shear studies on the effects of cholesterol on the physical stability of lipid vesicles Der-Zen Liu, Wen-Yih Chen *, Li-Min Tasi, Shu-Ping Yang Department of Chemical Engineering, National Central Uni6ersity, Chung-Li, Taiwan, ROC Received 23 August 1999; accepted 16 March 2000
Abstract This work measured the dilution heat of phosphatidylcholine (PC) vesicles incorporated with various amounts of cholesterol using isothermal titration calorimetry (ITC). The interaction potential between these PC vesicles was analyzed and the changing size of the PC vesicles response to various shear force and the zeta potential of the PC vesicles were also measured and discussed along with the ITC results. Experimental data indicate that the repulsive surface potential and the repulsive electrostatic force increases as cholesterol is incorporated into the bilayer. This phenomenon can be attributed to the decrease in the binding ability between the PC vesicular surfaces and the cations in the buffer solution. Incorporating cholesterol into the bilayer promotes the repulsive surface potential (or energy barrier) between the PC vesicles from the surface potential viewpoint. Additionally, incorporating cholesterol into the bilayer makes the PC vesicles more rigid and sustains more severe shear stress in our shear forces studies. This work also reveals that temperature and PC vesicular stability are correlated with each other. The stability of PC vesicles are likely to decline with a rise in the temperature, and the elevated temperature effect is evidence of the rigidity or curvature of the PC vesicles. Furthermore, the repulsive surface potential (or energy barrier) between the PC vesicles declines with a rise in temperature. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Phosphatidylcholine bilayer vesicles; Cholesterol; Surface potential; ITC; Physical stability
1. Introduction Phospholipid bilayer vesicles (lipid vesicles, liposomes) have been widely utilized as a biomembrane model and as a carrier in drug delivery systems [1– 5]. The aqueous dispersion of lipid vesicles are metastable systems, therefore, control * Corresponding author. Tel.: +886-3-4227151, ext 4222; fax: +886-3-4225258. E-mail address: [email protected] (W.-Y. Chen)
and prediction of the lipid vesicles stability against aggregation or fusion are important throughout the various stages of the applications. Aggregation is a critical characteristic behavior of lipid vesicle systems as it depends on the overall interactive forces between lipid vesicles. Several investigations have demonstrated that lipid vesicles made of acidic phospholipids (PS, PG) possess negative electric charges on their dissociative − groups (such as PO− 4 and COO ) at neutral pH, and that the electrostatic repulsion forces are
0927-7757/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 0 ) 0 0 5 6 0 - 4
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the dominant contributing factor to the interaction between lipid vesicular surfaces. Nevertheless, these lipid vesicles are likely to be readily aggregated and sometimes fused by altering the pH or ionic compounds that are added to the solution, because this addition may reduce the repulsive electrostatic forces between the lipid vesicles surfaces [6–12]. In contrast, the lipid vesicles made of amphoteric phospholipid such as (PC or PE), exhibit considerably different properties than the acidic phospholipid bilayer vesicles. In fact, the structure of the PC head group is zwitterionic, and the z-potentials of PC vesicles are significantly smaller than those of the acidic phospholipid bilayer vesicles [13]. Hence, as generally assumed there is not a strong electrostatic repulsion force between the amphoteric phospholipid vesicles, and that the high stability of PC vesicles must be due to some other repulsive forces between lipid vesicles. These other repulsive forces include the repulsive hydration forces or the entropic forces [14 – 17] in a short distance range. The effect of cholesterol incorporation into phospholipid bilayer on the cation-induced lipid vesicular aggregation and fusion are complex and difficult to comprehend. Generally, when cholesterol is incorporated into the phospholipid bilayer it alters the nature of the lipid vesicle. Bental et al. [18] reported that cholesterol incorporated into bilayer attenuates the Ca2 + -induced vesicle aggregation of the large br-PS vesicle. Virden and Berg [19] discussed the behavior of the incorporation of varying amounts of cholesterol on DPPG small unilamellar vesicles on NaCl induced aggregation. Their studies demonstrated that at a low NaCl concentration (NaCl B0.8 M), increasing cholesterol amounts may alter ion binding or shear plane distance. The increasing stability of DPPG small unilamellar vesicles is caused by the expanded short-range repulsive hydration force While at high NaCl concentrations (NaCl \1.0 M). Rand et al. [20] studied the effects of interaction forces between lipid vesicles that include cholesterol indicating that cholesterol incorporated into the DPPC bilayer decreases the van der Waals attraction and increases the net repulsion forces between bilayers. Nevertheless, Eklund et al. [21]
observed enhancement of NaCl-induced aggregation of br-PS bilayer vesicles and attenuation of CaCl2-induced aggregation of DPPS bilayer vesicles by inclusion of cholesterol. According to their results, the phenomena is likely attributed to that incorporating the cholesterol into the bilayer slightly increases the lipid bilayer vesicle size and decreases the lipid bilayer vesicle curvature. This ultimately results in a diminished polar head group surface area and an increased charge density of the lipid vesicular surfaces. NaCl-induced aggregation of DPPS bilayer vesicular inclusion of cholesterol and CaCl2-induced aggregation of DMPG bilayer vesicular inclusion of cholesterol are distinct. Eklund et al. contended that the inclusion of cholesterol can enhance or attenuate the cation-induced lipid vesicle aggregation depending on the particular cation and the phospholipid fatty acid composition [21]. In light of the above discussion, incorporating cholesterol into the phospholipid bilayer can alter the phospholipid bilayer geometry (shear plane distance and bend curvature of lipid bilayer vesicle), and ultimately affect the cation-induced aggregation or fusion behavior. The mechanical properties of the lipid vesicular structure are heavily dependent on temperature. Several phases with temperature are identified in accordance with the state of lipid bilayer in which the acyl chain structure plays a decisive role [22]. Taylor et al. [23] and Dufourc et al. [24] proved that cholesterol are perpendicular to the phospholipid bilayer surface membrane with its small hydrophilic 3b-hydroxyl head group located in the vicinity of the phospholipid ester carbonyl groups after it is incorporated into the phospholipid bilayer. Simultaneously, the hydrophobic steroid ring of cholesterol orients itself parallel to the acyl chains of the phospholipid. Restated, the incorporation of cholesterol into the phospholipid bilayer will affect the movements of the acyl chains of the phospholipid bilayer. Previous investigations [25– 27] have demonstrated that incorporating cholesterol into bilayer will reduce the fluidity of phospholipid bilayer if the bilayer’s temperature is higher than the phase transition temperature of the acyl chain.
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59
The above discussion reveals that the effects of cholesterol are realized by considering the curvature of the lipid bilayer vesicle, the intravesicle interaction between lipids and the binding between the lipid and cation in the buffer. The changes of the interaction forces between the PC bilayers with added cholesterol present are still unclear, especially because it is impractical to directly measure the forces between PC vesicles. Therefore, the aim of this work is to measure the dilution heat of PC vesicles incorporated with various amounts of cholesterol by isothermal titration calorimetry (ITC). The parameter of the interaction forces and the surface potential of PC vesicles can then be calculated from the dilution heat empirical data coupled with a simple statistical thermodynamic model [28]. Furthermore, the shear modulus was established to examine the rigidity of the PC vesicle. In summary, the aggregation and fusion behaviors are explained using the shear forces studies and interaction forces (or surface potential) between PC vesicles with added various amount of cholesterol.
solvents. The PC bilayer film was hydrated at 323 K with a buffer solution composed of 20 mM Tris, 100 mM NaCl and 1 mM EDTA (pH being adjusted to 7.4 with HCl). Frozen and thawed (FT) PC vesicles were prepared from the MLVs to prepare small unilamellar vesicles and reduce the lamellarity [30]. These lipid vesicles were subjected to freezing (in boiling nitrogen) and thawing (in a water bath at 323 K) five times, and were then sonicated for 30 min using the Bandelin HD-200 sonication probe at a power setting of KE 76/D under nitrogen. The temperature of the lipid vesicles was maintained at around 323 K by circulating the water in the round-bottomed flask. Then, the final optically clear suspension was filtered through a 0.22-mm millipore. All of the vesicles were added the same amount of tocopherol with various quantities of cholesterol. Tocopherol was added to avoid oxidative damage caused by the interactions between free radicals and the PC lipid. Therefore, the disturbance of the chemical stability for the experimental measurements can be reduced.
2. Materials and methods
2.3. The lipid 6esicles calculation
2.1. Materials
The number of PC lipids within a PC bilayer vesicle can be estimated by calculating the ratio between the head -group surface areas of egg-PC (0.70 nm2) [31] and cholesterol (0.28 nm2) [32], then, making the total amount of egg-PC (3.75 mg − 1 ml − 1) divided by the quantity of PC lipids contained within one individual PC bilayer vesicles, thus the number of lipid vesicles per milliliter can be roughly calculated with this approach.
Egg-PC (:99%), cholesterol and a-tocopherol were purchased from Sigma (USA). Chloroform, methanol, Tris and sodium chloride were obtained from Merck (Germany). All chemicals were used as received without further purification.
2.2. Preparation of the lipid 6esicles suspension solution Multilamellar lipid vesicles (MLVs) were prepared according to the method proposed by Bangham et al. [29]. A solution containing egg-PC (37.5 mg − 1 10 ml − 1), cholesterol (0 or 9.66 or 19.32 mg − 1 10 ml − 1) (0 or 33 or 50 mol%) and 5.9 mg − 1 10 ml − 1 of a-tocopherol in a mixture of chloroform/methanol (2:1, v/v) was dried in a 25 ml round-bottomed flask. Nitrogen was applied to blow over the dried lipid film for approximately 30 min in order to remove the traces of organic
2.4. Determination of the particle size distribution of 6esicles by PCS Lipid vesicle size distribution was determined by a photon correlation spectroscopy employing an zetasize 3000 (Malvern Instruments, UK). A sample (lipid vesicles suspension solution) was diluted in a buffer solution until the measured size was independent of the particle concentrate. Then, the lipid vesicles suspension solution (contained in a 2 ml plastic cuvette) was placed in a
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sample holder at 298 K. The viscosity of the sample solution was 1 cp. A helium-neon laser of wavelength 633 nm and 5 mW acts as the light source. The laser was focused on the sample and scattered light was detected at 90° to the incident beam using a photomutiplier tube. The Malvern software controlled both the acquisition and the on-line analysis of the scattering data. Both the so-called z-average particle diameter and the polydispersity index were calculated by the comulant analysis method.
2.5. z-potential measurement The lipid vesicles suspension was prepared as described earlier. The electrophoretic mobility and the zeta potential of lipid vesicle suspension were determined by laser Doppler electrophoretic mobility measurements with the zetasizer 3000 (Malvern Instruments, UK).
2.6. The impact of high and low shear forces on the lipid 6esicle structure Different shear stresses and their impacts on the lipid vesicle structures were investigated via a cone and plate viscometer. A suspension solution of lipid vesicles prepared according to the previously mentioned instructions, was poured into a viscometer (Brookfield HVTD-II, CP40) cone. Various high shear stresses were applied to this lipid vesicles suspension solution for 3 min, and the changes on the particle size were measured afterwards. The low shear rate experiment was designed to understand the impact of shear rate on the structure of lipid vesicles. A suspension solution of lipid vesicles prepared as described in the previous section was put into a viscometer (Brookfield LVTDII, CP40) cone. The changes of particle size were observed under a continuous flow field generated by a 50 s − 1 shear rate. The temperatures of these shear forces and shear rate experiments were kept at 298 and 310 K using a water bath, and the number density of the lipid vesicles suspension solution in both the shear
stresses and shear liposome − 1 ml − 1.
rate
studies
was
1013
2.7. The isothermal titration calorimetry dilution heat and the surface potential of the lipid 6esicles The PC vesicles suspension solution dilution heat was determined by applying an isothermal titration calorimetry. The dilution heat of the PC vesicles with added 33 mol% cholesterol suspension solution as presented in Fig. 1. The thermal activity monitor (Thermometric AB, Sweden) controlled by digitam software performed the ITC. The ampoule was washed with water and acetone, and dried in the air prior to each experiment. A 2 ml suspension solution of lipid vesicles was placed in the ampoule in this experiment. A series of 150 ml buffer solution were titrated into the dispersion PC vesicles suspension solution using a Hamilton microliter syringe at 30 min intervals after the ampoule and the heat sink reached thermoequilibrium. The PC vesicles suspension solution was titrated five times per experiment. All experiments were performed at temperatures of 298 and 310 K. The dilution heats of the PC vesicles suspension solution with the number density of lipid vesicles suspension solution can be generally expressed as a function of the second order polynomial in Eq. (1): q E d d NkBT NkBT $ = b2 + b3r+ b4r 2 (1) dr dr For an open liquid system, the pressure change and the titration volume are neglected in the system total volume. The observed dilution heat of each titration is comparable to the internal energy change. Furthermore, the internal energy change can be expressed by the virial coefficient and the number density in Eq. (2): E 3 1 dBi + 1 i = −T % r NkBT 2 dT i=1 i
(2)
Comparing Eq. (1) with Eq. (2) reveals that the coefficient of the dilution heat polynomial
D.-Z. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 172 (2000) 57–67
61
Fig. 1. The dilution heat of PC vesicles suspension solution. The PC vesicular composition is (PC:Chol:Vit E =4:2:1 mol ratio).
can be affiliated with the virial coefficients as in Eq. (3):
3.1. Zeta potential measurement
dB b2 = − T 2 dT
(3)
According to statistical mechanics, the relation between the second virial coefficient and the interaction potential energy function U(r) is: B2(T)= −2p
&
[exp −
0
U(r) −1]r 2dr kBT
(4)
and if a square-well energy potential function is picked and plugged into Eq. (4), then Eq. (3) can be rewritten as: b2 = − T
3. Results and discussion
dB2 = −B0(l 3 −1)e o/kBT(o/kBT) dT
b2/B0 = (l 3 −1)e o/kBT(o/kBT)
(5) (6)
where B0 =(16/3)pR , l= (2RHS +s)/2RHS and s, o denote the width and depth (or energy barrier) of the square-well energy potential function. 3 HS
This study measured the zeta potential (z-potential) of the PC vesicles incorporated with various amounts of cholesterol. Table 1 reveals that the PC vesicles made of egg-PC possess negative charges at pH 7.4. This finding is consistent with Carrion et al. [13] and McLaughlin et al. [33] indicating that there is a weak electrostatic repulTable 1 The values of zeta potential of PC vesicles at different temperatures with various amount of cholesterol incorporateda Composition PC:Vit E:Chol
Zeta potential (mv) (298 K)
Zeta potential (mv) (310 K)
4:1:0 4:1:2 4:1:4
−14.19 0.8 −17.991.1 −20.690.7
−4.790.7 −8.590.5 −11.491.4
a
Values are mean 9standard deviation, n = 5.
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sive force between the PC vesicles at pH 7.4. This experiment also demonstrated that the incorporation of cholesterol into the system can elevate the negative zeta potential. This phenomenon is possible because the cholesterol incorporated into the bilayer reduces the surface binding affinity among the cations in the buffer solution and its bilayer surface. Additionally, this study also observed that the zeta potential of the PC vesicles with or without cholesterol is higher at 310 than at 298 K because some hydrophilic headgroups become more hydrophobic at higher temperatures [34], increasing their ability to dehydrate and to bind with the cation in the buffer. Furthermore, the PC bilayer becomes more fluid as the temperature increases, disrupting the binding affinity with the cation in the buffer solution. The experimental data also indicates that the absolute zeta potential becomes more negative as the molar ratio of cholesterol changes from 0 to 33% in relation to its component contents within the lipid vesicle However, the decrease of zeta potential is not significantly altered as the molar ratio of cholesterol was added becomes 50% because of the cholesterol structural arrangements within the PC bilayer. The structural arrangements of the PC bilayer become irregular due to the incorporation of the cholesterol, thus affecting the binding ability between the PC vesicular surface and the cation as the molar ratio of cholesterol reaches 50% in the PC vesicle.
3.2. Interaction between PC lipid bilayer 6esicles The interaction forces between the lipid bilayers have been subjected to a significant amount of applied research as discussed in the introduction. Nir [35] suggested that incorporation of cholesterol into the bilayer increases the van der Waals forces among the lipid vesicles. However, Yeagle [36] contended that the electrostatic repulsive force decreases following the incorporation of cholesterol into the lipid vesicles. Mcintosh et al. [37] postulated that the annexation of cholesterol in the lipid vesicles affects the short distance repulsive force among the lipid vesicles. Therefore, we believe that incorporating cholesterol into the bilayer causes the lipid vesicles to change
their packing geometrical structures. These geometrical changes include lipid vesicle size, the curvatures of the surface bilayer, and surface lipid bilayer rigidity. We also believe that the Hamker constant, van der Waals forces, electrostatic repulsive forces, and hydration forces between the lipid bilayers or lipid vesicles will all be affected following the incorporation of cholesterol into the lipid vesicles. The interactive force and the effects of the forces between the PC vesicles are not directly measured when incorporating cholesterol into the PC bilayer. Therefore, a thermodynamic approach is utilized herein to examine the surface potential of the intervesicle interaction [28]. The dilution heat of the PC vesicle incorporated with various amounts of cholesterol by isothermal titration calorimetry was measured according to the derivation and discussion in the Section 2. The measurement was taken to obtain the energy barrier of the interaction potential between the PC vesicles. Namely, this study proposes a novel method of discussing the effect of cholesterol on the PC vesicle interaction potential. The system of PC vesicles has a positive b2 value with or without added cholesterol according to Table 2, indicating that the overall net interactive force is repulsive between the two PC vesicles. This net repulsive force poses a formidable barrier to aggregation and fusion. The value of b2/B0 (Table 2) is increased as the cholesterol is incorporated into the PC vesicles, indicating that the repulsive surface potential (energy barrier) of PC vesicles is also increased (higher values of o and s). Closely examining the interaction surface potential between the two PC vesicles, reveals that the higher interaction repulsive surface potential results in a system in which aggregation and fusion are both more difficult. A possible explanation is that the surface binding force between the PC vesicles and the cation in the buffer solution is declined as the cholesterol is incorporated into the PC bilayers, as observed in the zeta-potential experiments. Moreover, the hydration repulsive forces of PC vesicles may also be increased because the bounded water molecules are not displaced from the polar groups by cations.
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Table 2 The values of second order polynomial b2 and the b0 of virial coefficient of PC vesicles at different temperatures with various amount of cholesterol incorporated Reaction temperature (K)
Composition PC:Vit E:Chol (mol%)
b2 (nm3)
B0 virial coefficient (nm3)
b2/Bo
298
4:1:0 4:1:2 4:1:4
4.30×1011 7.54×1012 1.55×1013
7.88×105 1.66×106 2.47×106
5.46×105 4.54×106 6.27×106
310
4:1:0 4:1:2 4:1:4
3.84×1011 1.40×1012 2.45×1012
7.88×105 1.66×106 2.47×106
4.88×105 8.46×105 9.92×105
Fig. 2. The size of the PC vesicles of various amount of cholesterol incorporated with different high shear forces at different temperatures (each point value is average of three times). The particle size of liposome is the diameter.
The surface repulsive potential is diminished (lower values of o and s) or the net repulsive electrostatic force is decreased at a high temperature the b2/B0 value for 310 K is smaller than that of 298 K with or without cholesterol for a system of PC vesicles. The zeta potential experiment confirmed a less negative zeta potential at 310 than at 298 K. Therefore, the temperature effect on the total interaction potential between PC vesicles may be attributed to the temperature effect on the packing structure and the thermal motion of the PC bilayer. This temperature effect induces both the intervesicle and intravesicle interaction forces to change ultimately resulting in a total interaction potential.
3.3. Shear stress and its effect on particle size Fig. 2 illustrates how shear stress affects the PC vesicles system with or without cholesterol at 298 and 310 K. A larger threshold shear stress is necessary to destroy the PC vesicles with cholesterol added in bilayer, because adding cholesterol with the concentration range in this work can make the PC rearrange itself with higher order and rigidity. A high shear stress can impact the PC vesicular bilayer surface more effectively at higher temperatures according to our data. Nevertheless, the threshold shear stress needed to cause any changes in particle size at 310 K is smaller than that at 298 K because the disorder and
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flowing state of the PC bilayer vesicle increase at higher temperature. The shear stress can make the PC bilayer vesicle particle become smaller in size with or without incorporating cholesterol while the real cause for the reduction of the PC bilayer vesicle particle size is uncertain. Possible interpretations for this uncertainly include the notion that shear stress can increase the PC bilayer longitudinal interactions implying that the intravesicle may destroy the binding between PC lipids and may cause the PC vesicle to break into fragments or increase the bending energy of the PC lipid. Thus this would, in turn, increase the packing density of the PC lipids bilayer between the PC lipids.
by considering two mechanisms. First, the incorporation of cholesterol into the PC bilayer can make the PC vesicular bilayer surface reconstruct in a more rigid and ordered structure. Second, the PC vesicle will be unlikely to form a fusion state or become unstable following a collision. The shear rate can affect the PC bilayer surface more at higher temperature. Namely, the PC bilayer surface tends to form a fusion state or become unstable after a collision at a temperature higher than 298 K. The susceptibility of PC vesicle to temperature changes is related to its bilayer surface state as described in the previous section. The PC bilayer structure is more likely to become disordered and stay in a fluid state at higher temperature.
3.4. The effect of collision beha6ior on the lipid 6esicle
3.5. Particle size change
A lower shear rate (50 s − 1) is used to enhance the chance of collision to understand the PC vesicle collision behavior. Experimental results in Fig. 3 indicate that the threshold time needed to change the PC vesicular particle size is much longer following the incorporation of cholesterol, when accompanied by a fluid field of 50 s − 1. Conversely, the PC vesicle particle size is more susceptible to change and becomes bigger without incorporating cholesterol. This can be explained
Armengol and Estelrich [38] check the physical stability of PC vesicles by measuring their average size by a photon correlation spectroscopy. Figs. 4 and 5 display the changes of particle size of PC vesicle at 298 and 310 K, respectively. The PC vesicle particle size tends to become larger as time passes which is a sign that the fusion condition is irreversible. Experimental results suggest that originally, the PC vesicle was at a metastable state. In other words, the PC vesicle may con-
Fig. 3. The size of the PC vesicles of various amount of cholesterol incorporated with shear time at different temperatures. The lipid vesicles suspension solution was subjected to a continuous fluid field at a low shear rate of 50 s − 1 (each point value is average of three times). The particle size of liposome is the diameter.
D.-Z. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 172 (2000) 57–67
Fig. 4. The size of PC vesicles of various amount of cholesterol incorporated at 298 K with time. The number density of lipid vesicles suspension solution was 1013 liposome per ml (each point value is average of three times). The particle size of liposome is the diameter.
65
There are two continuous mechanisms for lipid vesicle fusion: the lipid vesicle has to aggregate; and intermediate structures such as inverted micelles or inverse hexagonal phase occur after the close juxtaposition and local perturbation in the lipid bilayer packing [39]. The incorporation of cholesterol into the bilayer will increase the repulsive forces and energy barrier between the PC vesicles according to the zeta potential and the b2/B0 value, and making it difficult for the PC vesicle to reach the primary minimum. Additionally, the shear stress and collision experiments indicated that the incorporation of cholesterol into the bilayer will make the PC vesicular bilayer surface more rigid and ordered. Obviously, the incorporation of cholesterol into the bilayer can decrease the state of fusion for the PC vesicle compared with the PC vesicle without cholesterol. The repulsive surface potential of the PC vesicle is lowered (lower values of o ) and the PC bilayer surface state become more fluid as the temperature increases from 298 to 310 K. Furthermore, the PC vesicles more easily reach the primary minimum, the so-called fusion state because the PC vesicular collision frequency is extended as the temperature rises.
4. Conclusion
Fig. 5. The size of PC vesicles of various amount of cholesterol incorporated at 310 K with time. The number density of lipid vesicles suspension solution was 1013 liposome per ml (each point value is average of three times). The particle size of liposome is the diameter.
strain the electrostatic repulsive forces to temporarily pause the second minimum state (or reversible aggregation). The PC vesicle will reach the first minimum state (or irreversible aggregation) may due to collision of PC vesicles or the conformational changes of PC vesicle with time. The incorporation of cholesterol into the bilayer will reduce the PC vesicle particle size changes since incorporating cholesterol can reduce the fusion between PC vesicles at either 298 or 310 K.
Incorporating cholesterol into small unilamellar vesicles of egg-PC increases the dispersion stability with respect to aggregation and fusion. Those can be explained with two aspects in our experiment. The experimental shear stress and shear rate demonstrate that adding cholesterol will make the composition of the PC bilayers more organized and rigid. The ITC and zeta potential experimental results qualitatively demonstrate that incorporating cholesterol into the PC bilayer can increase the electrical repulsion force and increase the repulsive interaction surface potential or energy barrier between the PC vesicles. Therefore, incorporating cholesterol into PC bilayer develop the physical stability. The raise of temperature accelerates the speed of PC vesicle fusion because the enhancement of temperature can increase the frequency of colli-
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66
sion between PC vesicles, and promote the thermal motion of the PC bilayer, resulting in a decreased order of PC bilayer structure. The ITC and zeta potential experiments also suggest a lower electrostatic repulsive and higher hydrophobic attractive surface potential between the PC at higher temperatures. Therefore, the raise of temperature will decline the physical stability.
Acknowledgements We would like to thank the National Science Council of the Republic of China for financially supporting this research under Grant No. NSC892214-E008-011.
Appendix A. Nomenclature PC PE PS PG q E N kB T r b2
B0 RHS s o
phosphatidylcholine lipid phosphatidylethanolamine lipid phosphatidylserine lipid phosphatidylglycerol lipid dilution heat of egg-PC liposome suspension solution (mJ) internal energy (mJ) number of lipid vesicles Boltzmann constant (J/K) absolute temperature (K) number density of lipid vesicles suspension solution (number per ml). second virial coefficient of the fitting polynomial of the dilution heat of a egg-PC liposome suspension solution virial coefficient the radius of lipid vesicle (nm) the width of the square-well potential function. the depth of the square-well potential function (energy barrier)
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