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DSPE-PEG2000 mixed monolayers on the water subphase at different temperatures
Colloids and Surfaces B: Biointerfaces 27 (2003) 333 /344 www.elsevier.com/locate/colsurfb
Thermodynamic characteristics of DSPC/DSPE-PEG2000 mixed monolayers on the water subphase at different temperatures Tzung-Han Chou, I.-Ming Chu * Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30043, Taiwan, ROC Received 15 April 2002; accepted 28 June 2002
Abstract Distearoylphosphatidylcholine (DSPC) spread at the air/water interface is used as a model membrane and to study the lateral interaction between DSPC and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPEPEG2000). DSPE-PEG2000 was found to be miscible with DSPC by our measurements of surface pressure /area per molecule (P /A ) isotherms at different temperatures. At different temperatures the nonideality and miscibility of mixed monolayer were determined by the analysis of excess area as a function of compositions, and the temperature effects on these deviations from ideality were evaluated. Furthermore, the interfacial thermodynamic characteristics of this mixed system including the change of entropies, the change of latent heats, and excess and mixing free energies during the compression process were calculated from the isotherms as a function of temperature in order to understand factors that affect the stability of mixed monolayer. It was found that increasing temperature and incorporation of DSPEPEG2000 both may make the mixed monolayer more compressible. # 2002 Elsevier Science B.V. All rights reserved. Keywords: DSPC; DSPE-PEG2000; Isotherm; Thermodynamic characteristics temperature effect; Mixed monolayers
1. Introduction The study of thermodynamic characteristics of artificial colloidal system, especially related to phospholipids and other amphipathic materials, may be very useful in getting better formulation for liposomes or emulsions products and can lead
* Corresponding author. Tel.: /886-3-571-3704; fax: /8863-571-5408 E-mail address: [email protected] (I.-M. Chu).
to fundamental understanding of the effect of each composition on system stability [1,2]. Such information on the physiochemical properties of liposomes is always needed in selecting appropriate materials in formulating a liposomal drug delivery system, and can lead to less trial and error steps involved. Distearoylphosphatidylcholine (DSPC), a zwitterionic lipid, is commonly used as a major component to prepare liposomes for an extensive range of practical applications [3 /5]. Other ingredients such as cholesterol and distearoylpho-
0927-7765/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 2 ) 0 0 0 9 6 - 6
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sphatidylethanolamine-poly ethylene glycol 2000 (DSPE-PEG2000) can help to increase liposome stability when included in the formulation. It is well established that adding certain amounts of cholesterol into liposomes is one of the effective methods to increase the stability of liposomes by its capability in condensing the lipid bilayer by lateral interaction with phospholipid [6]. As for DSPE-PEG2000, it has been found to eliminate the binding of plasma macromolecules including potential opsonizing factors such as immunoglobulins and complement proteins to liposomes and thus improve circulation life time as well as pharmacokinetics and antitumor therapeutic efficacy of liposomes [7 /10]. However, the basic mechanism of liposome stabilization, beyond that of protein adsorption prevention by lipid /poly ethylene glycol (PEG) is still not fully understood, despite intensive studies. Presently it was known that attachment of PEG molecules to the surface of liposomes increases the repulsive forces between liposomes to each other and to cells [11 /13]. Other works focused on studying the different forces between liposomes of surface-bound PEG layer [13 /16] and its effect on prolonging the circulation time in vivo [17 /21]. Besides, there are some reports about DSPE/DSPE-PEG monolayers for study using fluorescence microscope or neutron reflectometry [22,23]. They focus on the PEG / PEG interactions and how the structure of the phospholipid-solution interface is modified by the inclusion of bulk polymers /lipids. However, there has been very few research on detail thermodynamic characteristics, such as mixing, phase transition and compressibility, of mixed DSPC/DSPEPEG2000 monolayers at different temperatures and intermolecular interaction between DSPC and DSPE-PEG2000, which may be important in understanding the intrinsic membrane stability properties of these PEG-linked lipids. Although some efforts have been made to investigate the behavior of mixed DSPC/DSPEPEG2000 monolayers at room temperature in our previous report [24], it is necessary to further study this behavior at different temperatures. This study will provide us data on thermodynamic properties
of the mixed membranes and a more completely understanding of the system.
2. Materials and methods 2.1. Materials Synthetic L-a-DSPC was obtained from Sigma Aldrich (St. Louis, MO, USA) and used as received. DSPE-PEG2000 was purchased from Avanti Polar Lipids, Birmingham, AL, USA. The purity of both materials given by the manufacturer was /99%. All chemicals were used without further purification. The monolayer spreading solutions were made of HPLC grade chloroform and methanol, both obtained from Baker Inc., Paris. The desired molar ratios of DSPC/DSPE-PEG2000 mixtures were dissolved in chloroform/methanol (3:1, v/v) solution for monolayer spreading and stored at about 4 8C. The aqueous subphase for the experiments consisted of pure water purified by a Milli-Q plus purification system with a specific resistivity of 18.2 V-cm. All glassware in contact with sample should be exhaustively rinsed by pure water. 2.2. Measurement of lateral film pressure A NIMA Model 601M Langmuir /Blodgett trough coated with Teflon and the pressure sensor were both obtained from NIMA technology Ltd. UK. Before each experiment, the trough must be cleaned thoroughly. Then, it was filled with pure water and repeated blank compressions of the entire surface area were performed to ensure interfacial cleanliness. When the surface pressure fluctuation was less than 9/0.2 mN/m, a monolayer was then formed by dropping the spreading solution onto the water subphase with a microsyringe. Twenty minutes were allowed for solvent evaporation prior to compression. After allowed for solvent evaporation, the monolayer was compressed at the air/water interface with a rate of 5 cm2/min. The surface pressure /occupied area per molecule (P /A ) isotherms were measured by symmetric compression
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with two barriers. The surface pressure was monitored by the Wilhelmy plate method using a NIMA 9000 microbalance and the data were recorded continuously by a microcomputer. The temperature of subphase was controlled to 9/0.1 K. The P /A isotherms were carried out at 283, 297.5, and 310 K, respectively. Repeated measurements of compression isotherms resulted in a standard deviation no larger than 0.3 mN/m.
3. Results and discussion 3.1. P /A isotherms Monolayer characteristics at the air/water interface can be directly obtained through measurement of P /A isotherms. Fig. 1 shows P /A isotherms of DSPC/DSPE-PEG2000 mixed monolayers at different temperatures. The DSPC monolayer (Fig. 1(a)) exhibited a more condensed film than a pure DSPE-PEG2000 monolayer (Fig. 1(g)), which possesses an apparently larger occupied area per molecule. The isotherms of pure DSPEPEG2000 monolayer agree well with those found in the literatures [22,23] and shift toward to high position in P /A diagram with increasing temperature. Also, for pure DSPC monolayer, a sharp transition was observed when temperature was elevated to 310 K. Furthermore, one can find that the addition of DSPE-PEG2000 into DSPC monolayer dramatically affects the characteristics of P / A isotherms. At a constant temperature the plateau region of isotherm, the phase transition associated with a change in the film conformation, appeared after incorporation of DSPE-PEG2000 into the DSPC monolayer and became more apparent with increasing mole percentage of DSPE-PEG2000 (XDSPE-PEG2000). Change was also observed in the lifted-off values of mean molecular occupied area, which shift towards larger area with increasing XDSPE-PEG2000 at fixed subphase temperature. Besides, it was showed in Table 1 that the addition of DSPE-PEG2000 induces a rise larger ALE, the initial area of liquid-expanded phase. These phenomena indicate that the existence of DSPE-PEG2000 may make the mixed monolayer more expanded than the pure DSPC monolayer at
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the air/water interface at the same temperature because of random PEG side chains. In addition, the influence of temperature on the mixed monolayer behavior can also be found in Fig. 1. At a fixed composition all P /A isotherms when viewed as functions of temperature express similar behavior and shift towards larger areas with higher temperature. This implied that an increase in temperature indeed enhances the thermal mobility of the mixed films on the water subphase. This conclusion can also be supported from the data in Table 2, in which the lower collapse surface pressures always appeared at higher temperatures. The phase transition region in these compression isotherms was also affected by temperature. Fig. 2 illustrates the temperature dependence of the transition pressure pt, which corresponds to the beginning of liquid expanded / liquid condensed (LE /LC) phase transition region for DSPC/DSPE-PEG2000 mixed monolayers. It can be seen that transition pressure varies linearly with temperature, regardless of the composition in the mixed monolayers. Nevertheless, the slope of these lines dpt/dT , which was also listed in Table 1, seems to depend on XDSPE-PEG2000 in most cases although the difference between them was not obvious. In summary, these findings elucidate the effect of intermolecular interaction in determining the state of mixed monolayers. Both increasing temperature and incorporation of DSPE-PEG2000 into lipid domain make the mixed monolayers more disorder and expanded.
3.2. Entropy change and latent heat of mixed monolayer The main phase transition of the monolayer at several temperatures can be theoretically characterized by the two-dimensional Clausius /Clapeyron equation as follows [25]: dpt dT
and
Qt T(ALE AS )
(1)
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Fig. 1. Surface pressure-area per molecule isotherms of DSPC/DSPE-PEG2000 mixed monolayers with XDSPE-PEG /0 (a), 1 (b), 3 2000 (c) 5 (d), 7 (e), 10 (f), and 100% (g) at various temperatures. Curves are for T /283 ('), 297.5 (j), and 310 K (m).
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Fig. 1 (Continued)
DSt
Qt T
(2)
where pt is the surface pressure at the LE /LC phase transition commences, T is the absolute temperature, Qt is the latent heat of this phase change, and DSt is equal to the molar entropy change for an isothermal reversible phase transition process. ALE and AS are associated with the areas per molecule in the LE /LC phased, respectively. ALE is obtained at pt directly, while the value of AS is taken by extrapolating the liquidcondensed isotherm downward to pt. The accuracy of the experimental procedure may be questioned if the monolayer is labile owing to dissolution of some components into the subphase bulk. How-
ever, it has been well known for this binary system that DSPC and DSPE-PEG2000 are very insoluble in water and we did not find any obviously hysteresis after compression and cycles expansion of mixed monolayers (data not shown). Once the equilibrium has been reached, as is indicated by the reproducibility of the isotherms, the feasibility of thermodynamic analysis by means of Eqs. (1) and (2) can be assured. Table 1 gives corresponding thermodynamic parameters for the transition from LC to LE of mixed DSPC/DSPE-PEG2000 system that were determined from data at different temperatures. It shows that the values of the latent heats and entropy changes, defined for the transition from condensed to expanded monolayer, are overall
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Table 1 Thermodynamic characteristics of the main phase transition in the P /A isotherm for mixed DSPC/DSPE-PEG2000 monolayers with various mole fractions at different temperatures XDSPE-PEG
(%)
T (K)
Pt (mN/m)
ALE
dPt/dT
DSt (J/mol K)
Qt (J/mol)
283 297.5 310 283 297.5 310 283 297.5 310 283 297.5 310 283 297.5 310 283 297.5 310
6.9 7.6 8.6 5.8 6.6 7.6 5.5 6.0 7.4 5.9 6.9 7.4 5.94 7.6 8.6 9.2 10.7 11.6
65.9 68.4 82.8 72.8 78.1 90 81.3 85.9 96.3 88.1 93.1 99.4 105 108.1 111.3 350 380 416.7
0.065 0.065 0.065 0.065 0.065 0.065 0.069 0.069 0.069 0.054 0.054 0.054 0.101 0.101 0.101 0.089 0.089 0.089
7.03 8.01 13.64 9.15 11.24 15.901 11.89 13.83 18.09 12.63 14.26 16.30 31.83 33.72 35.62 134.23 150.36 170.05
1989.42 2382.83 4229.36 2589.52 3342.54 4927.89 3364.94 4114.09 5609.10 3573.15 4242.47 5054.07 9006.83 10031.90 11040.67 37991.62 44730.77 52714.48
2000
1
3
5
7
10
100
positive for all compositions studied. This means that transition from LC to LE structure of DSPC/ DSPE-PEG2000 mixed monolayer is characterized by entropy increase and endothermic process at different temperatures. The entropy for the transition from LC to LE film increases as expected from classical thermodynamic concept. Besides, the values of Qt and DSt are higher as temperature increases at the identical composition of the mixed monolayer. Thus it can be concluded that at higher
temperature, larger thermal motion results in a more random molecular configuration for DSPC/ DSPE-PEG2000 mixed monolayer at the air/water interface, thus it requires more energy for phase transition from LC to LE. Furthermore, it can be found from data in Table 1 that increasing the amount of DSPE-PEG2000 in the mixed monolayer has a dramatic effect on the entropy change and latent heat for the phase transition. When the temperature is fixed, the
Table 2 Isothermal compressibility (k , m/mN) and collapse pressures (pc, mN/m) of P /A isotherms of mixed DSPC/DSPE-PEG2000 monolayers with various molar percentages at different temperatures XDSPE-PEG
0%
1%
3%
5%
7%
10%
100%
ka kb kc pca pcb pcc
0.0028 0.0033 0.0046 59.9 59 45
0.0025 0.0055 0.0055 62 57 51
0.0033 0.0074 0.0062 55 55 52.5
0.0022 0.0062 0.0060 61 52 54
0.0040 0.0073 0.0083 53 58 51.5
0.0044 0.0080 0.0090 54 48 48.5
0.0057 0.0090 0.0105 64.8 64 60
2000
a b c
Indicated the parameters at 283 K. Indicated the parameters at 297.5 K. Indicated the parameters at 310 K.
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Fig. 2. Transition pressures as a function of temperature of DSPC/DSPE-PEG2000 mixed monolayers with XDSPE-PEG / 2000 1 ('), 3 (I), 5 (m), 7 (2), 10 (j), 100% (^).
entropy change of mixed monolayers and latent heat in the phase transition both increase with increasing XDSPE-PEG2000. It has been demonstrated before that polyethylene glycerol side chain can produce an expanded effect on the monolayer. Therefore, more DSPE-PEG2000 is believed to give more random molecular configuration during the LC /LE phase transition in the monolayer domain, thus results in larger energy requirement and entropy change for the phase transition. 3.3. Miscibility and excess area of mixed monolayers It is well established in previous literatures [26,27] that the miscibility of monolayers consisting of binary components can be determined simply and indirectly by means of the thermodynamically interfacial phase rule. If the two components in the monolayer at interface are miscible, the collapse surface pressures are dependent on the composition. In any event, a monolayer in which two components are immiscible will show two separate collapse surface pressures, corresponding to individual pure components. In Table 2, the collapse surface pressures of mixed DSPC/DSPE-PEG2000 monolayers at different
Fig. 3. Aex/Aid as a function of composition for DSPC/DSPEPEG2000 mixed monolayers at surface pressures of 5 ('), 10 (I), 15 (m), 20 (2), and 30 mN/m (j) on the water subphase at 283 (a), 297.5 (b), and 310 K (c).
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temperatures are listed. The collapse pressures of this mixed system did change with composition at a given temperature thought the differences between them are not all large. Consequently, it can be confidently concluded that DSPC and DSPEPEG2000 are miscible on the water subphase at temperatures investigated. Besides, the values of collapse pressures of mixed monolayer seem to be smaller at higher temperature. This phenomenon is consistent with the notion that enhanced mobility between molecules at higher temperatures causes easier collapse of mixed monolayers. More information on the behavior of intermolecular interaction in the mixed monolayer can be obtained by calculating the excess area (Aex). At a fixed surface pressure, the Aex can be estimated by the following equation [26,27], Aex A12 (X1 A1 X2 A2 )
(3)
where A12 is the actual occupied area per molecule of the mixed monolayer, X1 and X2 are the molar percentages of 1 and 2 components, respectively, and A1 as well as A2 imply the per molecular occupied areas of individual pure monolayers. Once two components form an ideally mixed monolayer or are immiscible, the Aex will be equal to zero at a given P . Otherwise, positive or negative values of Aex would indicate that miscible and nonideal mixed monolayers. In order to express clearly degrees of the deviation of excess area, the form of Aex/Aid should be used. Aex/Aid values versus the mole percentage of DSPE-PEG2000 in mixed monolayers at 283, 297.5, and 310 K, respectively, are shown in Fig. 3. Aex values of DSPC/DSPEPEG2000 mixed monolayers shown in Fig. 3 indicate that these values are not zero and are dependent on the subphase temperature and lateral surface pressure. It is clearly shown that DSPC and DSPE-PEG2000 are miscible and exhibit nonideal mixed behavior at the air/water interface at different temperatures. Nevertheless, it cannot be completely ruled out that DSPC and DSPE-PEG2000 might form ideal mixed monolayer or immiscible when XDSPE-PEG2000 is close to 1 or 0. There is a tendency to have more negative values of excess area as the temperature goes up, as shown in Fig. 3. It is believed that as temperature
increases, dehydration effect of PEG side chain would be more significantly affecting the monolayer. This would then intensify the negative trend of Aex/Aid as temperature goes up. The effect of lateral surface pressure on excess area was different at different temperatures. At 283 K the Aex/Aid decreased initially and then increased with increasing surface pressure (Fig. 3(a)). However when the temperature was higher, excess areas became significant more negative with the increase of surface pressure (Fig. 3(b and c)). This suggested that at higher temperatures the monolayers became more fluid and the mixed molecules are able to form more tightly packing than when molecular movement is restricted at lower temperature. This effect is more prominent at higher lateral surface pressure. Of course, the dehydration effect of PEG that accompanied by increasing temperature also plays a role. The dehydration is expected to decrease the area of PEG group and then enhances the packing directly. Moreover, composition effect for this mixed system on Aex/Aid at the different temperatures could be also identified from the data in Fig. 3. It is interesting to note that a maximum of Aex/Aid appeared at XDSPE-PEG200 /5 mol% either at high temperature or low temperature. This indicates that the intermolecular attractive interaction is weaker as well as special molecular packing pattern of the membrane at this composition. This finding lends support a notice in the research of DSPC/DSPE-PEG2000 mixed dispersions by others [28]. It was established that a phase transition occurs between mushroom to brush conformation for the PEG side chains at about XDSPEPEG200 /5 mol%. In our findings, the mixed membrane has special larger Aex at this composition, if the membranes are closely-packed enough, either under lower temperatures or under higher surface pressures. This seems to demonstrate the capability of the monolayer study in elucidating liposomal phenomena. 3.4. Monolayer stability The thermodynamic stability of forming a mixed monolayer consisted of two miscible mate-
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rials at the air/liquid interface can be determined
by the evaluation of free energy of mixing, DGmix,
Fig. 4. DGex vs. different mixed DSPC/DSPE-PEG2000 monolayers compositions at surface pressures of 5 ('), 10 (I), 15 (m), 20 (2), and 30 mN/m (j) on the water subphase at 283 (a), 297.5 (b), and 310 K (c).
Fig. 5. DGmix vs. different mixed DSPC/DSPE-PEG2000 monolayers compositions at surface pressures of 5 ('), 10 (I), 15 (m), 20 (2), and 30 mN/m (j) on the water subphase at 283 (a), 297.5 (b), and 310 K (c).
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or the excess free energy of mixing, DGex [26,27,29]. The DGex is defined as: II
g
DGex N0 [A12 (X1 A1 X2 A2 )]dP
(4)
0
where N0 is the Avogadro number and P is the surface pressure. Thus the value of DGex can be obtained directly by integration P /A isotherms. The DGmix, the partial molar thermodynamic properties, demonstrated the energetic tendency from a unmixed monolayer to a mixed monolayer and then was expressed as: DGmax DGex DGideal
(5)
where DGideal indicates the Gibbs energy of ideal mixing, which is given by: DGideal RT(X1 ln X1 X2 ln X2 )
(6)
where R is the universal gas constant and T is the absolute temperature. In Fig. 4, DGex is plotted as function of DSPEPEG2000 in mixed monolayers at different surface pressures and different temperatures. Obviously, the excess energy of this binary mixed system was significantly affected by temperature. The deviations from ideality are negative for the mixed monolayer with XDSPE-PEG2000 ‘/7% and the minimum of DGex appears at XDSPE-PEG2000 /7% at 283 and 297.5 K (Fig. 4(a and b)). This indicates that attractive interaction between molecules in a mixed monolayer became pronounced when XDSPE-PEG2000 ‘/7% and has a more significant influence on the mixed monolayer, at XDSPEPEG2000 /7% when mixed monolayers were at lower temperature (283 and 297.5 K). On the contrary, while XDSPE-PEG2000 B/7%, the positive deviations occurs, which means at this composition, repulsive interaction dominates. Nevertheless, when the temperature of subphase was elevated to 310 K, the values of DGex changes dramatically (Fig. 4(c)). Most of the values of DGex are negative and the degree of deviations became significant with increasing surface pressure especially as XDSPEPEG2000 ‘/5%. This indicates that at higher temperature the mixed monolayers are more stable comparing to ideal mixed monolayer than at lower temperature, and the intermolecular interaction
was stronger when the mixed monolayer was compressed to a more condensed state especially as mixed monolayers with XDSPE-PEG2000 ‘/5%. Plots of the DGmix versus XDSPE-PEG2000 for mixed DSPC/DSPE-PEG2000 monolayers on the water subphase at various surface pressures and different temperatures are illustrated in Fig. 5. The values of DGmix were changed from positive to negative as the compositions of mixed monolayers was ‘/5% at 283 and 297.5 K (Fig. 5(a and b)). According to the definition of DGmix, which can distinguish the difference between mixing and unmixing states in the monolayer system, one can say that the mixed monolayer with ‘/5% were energetically more favorable to form a mixed monolayer than a monolayer consisted of separated components at 283 and 297.5 K. However, all values of DGmix became negative and their magnitude increases when the surrounding temperature was raised to 310 K (Fig. 5(c)). This implied that the mixing tendency of this mixed system at the higher temperature was more advantageous than at lower temperature. Besides, it can be also found that the magnitude of DGmix seems to increase with increasing incorporation amounts of DSPE-PEG2000 and with increasing surface pressures at 310 K. In addition, one can see from results of either DGex or DGmix that mixed monolayer with XDSPEPEG2000 /5% stands at a point where DGex or DGmix values turns from positive to negative. It has been reported that bilayers containing from 1 to 9 mol% lipid /PEG2000 exhibited two different phase states characterized by two polymer conformation (mushroom and brush) [28,30]. The phase transition from mushroom to brush regimes also approximately occurred at the mixed lipid bilayer with 5 mol% of DSPE-PEG2000.
3.5. Isothermal compressibility of mixed monolayers In order to realize the equilibrium elasticity of a monolayer in a solid state at the interface, the 2-D isothermal compressibility, k , should be calculated and was defined as [31,32]:
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k
1 dA A dp
(7)
where A and p represented the mean molecular area and surface pressure, respectively. Hence, one can estimate k of a monolayer directly from the linear extrapolation of P /A isotherms. The evaluated range of compression isotherms for each monolayer defined as the difference in mean molecular areas from zero surface pressure to collapse surface pressure [32]. The isothermal compressibility of mixed DSPC/DSPE-PEG2000 monolayers at the air/water interface at 283, 297.5, and 310 K was listed in Table 2. The incorporation of DSPE-PEG2000 into DSPC monolayer affects compressibility for a mixed monolayer. At different temperatures compressibility of this binary mixed system increases with increasing the amounts of DSPE-PEG2000 or increasing environmental temperatures. Thus it can be concluded that the incorporation of DSPE-PEG2000 into DSPC monolayer or an increase in temperature would make the monolayer become more compressible although the differences in k of mixed monolayers are not obvious.
4. Conclusion The present study shows that mixed DSPC/ DSPE-PEG2000 monolayers, spread at the air/ water interface at different temperatures, are a useful model system for systematic studies of lateral interaction between them. From the analysis of P /A isotherms of this mixed system, DSPEPEG2000 is miscible with DSPC on water. The incorporation of DSPE-PEG2000 into DSPC monolayer or increasing temperature gives an expansion effect to the mixed monolayer. It is also an interesting system to study the thermodynamic properties of mixed monolayers. In this study, an increase in entropies for the transitions from LC to LE with increasing temperature or XDSPE-PEG2000, as well as an endothermic process of this transition were observed. The nonideal mixing behavior between DSPC and DSPE-PEG2000 and dehydration effect of PEG group with increasing
343
temperature were also found from the calculation of excess area. The stability of mixed system at different temperatures was judged by analysis of excess and mixing Gibbs free energies. The mixed monolayer with XDSPE-PEG2000 /5 mol% is an energy transition from positive to negative values of mixing energies. Moreover, influence of the subphase temperature, compositions, and lateral surface pressures on stability of mixed monolayers was discussed. Finally, the isothermal compressibility of mixed monolayers shows that the monolayer becomes more compressible as more DSPEPEG2000 added or as higher temperature increased.
Acknowledgements This research was supported by Grant NSC902214-E007-011 from the National Science Council of the Republic of China.
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[23] J. Majewski, T.L. Kuhl, M.C. Gerstenberg, J.N. Israelachvili, G.S. Smith, J. Phys. Chem. B 101 (1997) 3122. [24] T.-H. Chou, I.-M. Chu, Colloid Surf.: A, in press. [25] M.C. Phillips, D. Chapman, Biochim. Biophys. Acta 163 (1968) 301. [26] G.L. Gaines, Insoluble Monolayers at Liquid /Gas Interfaces (Chapter 6), Wiley-Interscience, New York, 1966. [27] K.S. Birdi, Lipid and Biopolymer Monolayers at Liquid Interfaces (Chapter 4), Plenum Press, New York, 1989. [28] A.K. Kenworthy, K. Hristova, D. Needham, T.J. McIntosh, Biophys. J. 68 (1995) 1921. [29] M.N. Jones, D. Chapman, Micelles, Monolayers, and Biomembranes (Chapter 2), Wiley-Liss, Inc, New York, 1995. [30] A.N. Nikolova, M.N. Jones, Biochim. Biophys. Acta 1372 (1998) 237. [31] K.S. Birdi, Lipid and Biopolymer Monolayers at Liquid Interfaces (Chapter 2), Plenum, New York, 1989. [32] R.A. Han, in: G. Roberts (Ed.), Langmuir /Blodgett Films (Chapter 2), Plenum Press, New York, 1990.
Thermodynamic characteristics of DSPC/DSPE-PEG2000 mixed monolayers on the water subphase at different temperatures Tzung-Han Chou, I.-Ming Chu * Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30043, Taiwan, ROC Received 15 April 2002; accepted 28 June 2002
Abstract Distearoylphosphatidylcholine (DSPC) spread at the air/water interface is used as a model membrane and to study the lateral interaction between DSPC and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPEPEG2000). DSPE-PEG2000 was found to be miscible with DSPC by our measurements of surface pressure /area per molecule (P /A ) isotherms at different temperatures. At different temperatures the nonideality and miscibility of mixed monolayer were determined by the analysis of excess area as a function of compositions, and the temperature effects on these deviations from ideality were evaluated. Furthermore, the interfacial thermodynamic characteristics of this mixed system including the change of entropies, the change of latent heats, and excess and mixing free energies during the compression process were calculated from the isotherms as a function of temperature in order to understand factors that affect the stability of mixed monolayer. It was found that increasing temperature and incorporation of DSPEPEG2000 both may make the mixed monolayer more compressible. # 2002 Elsevier Science B.V. All rights reserved. Keywords: DSPC; DSPE-PEG2000; Isotherm; Thermodynamic characteristics temperature effect; Mixed monolayers
1. Introduction The study of thermodynamic characteristics of artificial colloidal system, especially related to phospholipids and other amphipathic materials, may be very useful in getting better formulation for liposomes or emulsions products and can lead
* Corresponding author. Tel.: /886-3-571-3704; fax: /8863-571-5408 E-mail address: [email protected] (I.-M. Chu).
to fundamental understanding of the effect of each composition on system stability [1,2]. Such information on the physiochemical properties of liposomes is always needed in selecting appropriate materials in formulating a liposomal drug delivery system, and can lead to less trial and error steps involved. Distearoylphosphatidylcholine (DSPC), a zwitterionic lipid, is commonly used as a major component to prepare liposomes for an extensive range of practical applications [3 /5]. Other ingredients such as cholesterol and distearoylpho-
0927-7765/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 2 ) 0 0 0 9 6 - 6
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sphatidylethanolamine-poly ethylene glycol 2000 (DSPE-PEG2000) can help to increase liposome stability when included in the formulation. It is well established that adding certain amounts of cholesterol into liposomes is one of the effective methods to increase the stability of liposomes by its capability in condensing the lipid bilayer by lateral interaction with phospholipid [6]. As for DSPE-PEG2000, it has been found to eliminate the binding of plasma macromolecules including potential opsonizing factors such as immunoglobulins and complement proteins to liposomes and thus improve circulation life time as well as pharmacokinetics and antitumor therapeutic efficacy of liposomes [7 /10]. However, the basic mechanism of liposome stabilization, beyond that of protein adsorption prevention by lipid /poly ethylene glycol (PEG) is still not fully understood, despite intensive studies. Presently it was known that attachment of PEG molecules to the surface of liposomes increases the repulsive forces between liposomes to each other and to cells [11 /13]. Other works focused on studying the different forces between liposomes of surface-bound PEG layer [13 /16] and its effect on prolonging the circulation time in vivo [17 /21]. Besides, there are some reports about DSPE/DSPE-PEG monolayers for study using fluorescence microscope or neutron reflectometry [22,23]. They focus on the PEG / PEG interactions and how the structure of the phospholipid-solution interface is modified by the inclusion of bulk polymers /lipids. However, there has been very few research on detail thermodynamic characteristics, such as mixing, phase transition and compressibility, of mixed DSPC/DSPEPEG2000 monolayers at different temperatures and intermolecular interaction between DSPC and DSPE-PEG2000, which may be important in understanding the intrinsic membrane stability properties of these PEG-linked lipids. Although some efforts have been made to investigate the behavior of mixed DSPC/DSPEPEG2000 monolayers at room temperature in our previous report [24], it is necessary to further study this behavior at different temperatures. This study will provide us data on thermodynamic properties
of the mixed membranes and a more completely understanding of the system.
2. Materials and methods 2.1. Materials Synthetic L-a-DSPC was obtained from Sigma Aldrich (St. Louis, MO, USA) and used as received. DSPE-PEG2000 was purchased from Avanti Polar Lipids, Birmingham, AL, USA. The purity of both materials given by the manufacturer was /99%. All chemicals were used without further purification. The monolayer spreading solutions were made of HPLC grade chloroform and methanol, both obtained from Baker Inc., Paris. The desired molar ratios of DSPC/DSPE-PEG2000 mixtures were dissolved in chloroform/methanol (3:1, v/v) solution for monolayer spreading and stored at about 4 8C. The aqueous subphase for the experiments consisted of pure water purified by a Milli-Q plus purification system with a specific resistivity of 18.2 V-cm. All glassware in contact with sample should be exhaustively rinsed by pure water. 2.2. Measurement of lateral film pressure A NIMA Model 601M Langmuir /Blodgett trough coated with Teflon and the pressure sensor were both obtained from NIMA technology Ltd. UK. Before each experiment, the trough must be cleaned thoroughly. Then, it was filled with pure water and repeated blank compressions of the entire surface area were performed to ensure interfacial cleanliness. When the surface pressure fluctuation was less than 9/0.2 mN/m, a monolayer was then formed by dropping the spreading solution onto the water subphase with a microsyringe. Twenty minutes were allowed for solvent evaporation prior to compression. After allowed for solvent evaporation, the monolayer was compressed at the air/water interface with a rate of 5 cm2/min. The surface pressure /occupied area per molecule (P /A ) isotherms were measured by symmetric compression
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with two barriers. The surface pressure was monitored by the Wilhelmy plate method using a NIMA 9000 microbalance and the data were recorded continuously by a microcomputer. The temperature of subphase was controlled to 9/0.1 K. The P /A isotherms were carried out at 283, 297.5, and 310 K, respectively. Repeated measurements of compression isotherms resulted in a standard deviation no larger than 0.3 mN/m.
3. Results and discussion 3.1. P /A isotherms Monolayer characteristics at the air/water interface can be directly obtained through measurement of P /A isotherms. Fig. 1 shows P /A isotherms of DSPC/DSPE-PEG2000 mixed monolayers at different temperatures. The DSPC monolayer (Fig. 1(a)) exhibited a more condensed film than a pure DSPE-PEG2000 monolayer (Fig. 1(g)), which possesses an apparently larger occupied area per molecule. The isotherms of pure DSPEPEG2000 monolayer agree well with those found in the literatures [22,23] and shift toward to high position in P /A diagram with increasing temperature. Also, for pure DSPC monolayer, a sharp transition was observed when temperature was elevated to 310 K. Furthermore, one can find that the addition of DSPE-PEG2000 into DSPC monolayer dramatically affects the characteristics of P / A isotherms. At a constant temperature the plateau region of isotherm, the phase transition associated with a change in the film conformation, appeared after incorporation of DSPE-PEG2000 into the DSPC monolayer and became more apparent with increasing mole percentage of DSPE-PEG2000 (XDSPE-PEG2000). Change was also observed in the lifted-off values of mean molecular occupied area, which shift towards larger area with increasing XDSPE-PEG2000 at fixed subphase temperature. Besides, it was showed in Table 1 that the addition of DSPE-PEG2000 induces a rise larger ALE, the initial area of liquid-expanded phase. These phenomena indicate that the existence of DSPE-PEG2000 may make the mixed monolayer more expanded than the pure DSPC monolayer at
335
the air/water interface at the same temperature because of random PEG side chains. In addition, the influence of temperature on the mixed monolayer behavior can also be found in Fig. 1. At a fixed composition all P /A isotherms when viewed as functions of temperature express similar behavior and shift towards larger areas with higher temperature. This implied that an increase in temperature indeed enhances the thermal mobility of the mixed films on the water subphase. This conclusion can also be supported from the data in Table 2, in which the lower collapse surface pressures always appeared at higher temperatures. The phase transition region in these compression isotherms was also affected by temperature. Fig. 2 illustrates the temperature dependence of the transition pressure pt, which corresponds to the beginning of liquid expanded / liquid condensed (LE /LC) phase transition region for DSPC/DSPE-PEG2000 mixed monolayers. It can be seen that transition pressure varies linearly with temperature, regardless of the composition in the mixed monolayers. Nevertheless, the slope of these lines dpt/dT , which was also listed in Table 1, seems to depend on XDSPE-PEG2000 in most cases although the difference between them was not obvious. In summary, these findings elucidate the effect of intermolecular interaction in determining the state of mixed monolayers. Both increasing temperature and incorporation of DSPE-PEG2000 into lipid domain make the mixed monolayers more disorder and expanded.
3.2. Entropy change and latent heat of mixed monolayer The main phase transition of the monolayer at several temperatures can be theoretically characterized by the two-dimensional Clausius /Clapeyron equation as follows [25]: dpt dT
and
Qt T(ALE AS )
(1)
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Fig. 1. Surface pressure-area per molecule isotherms of DSPC/DSPE-PEG2000 mixed monolayers with XDSPE-PEG /0 (a), 1 (b), 3 2000 (c) 5 (d), 7 (e), 10 (f), and 100% (g) at various temperatures. Curves are for T /283 ('), 297.5 (j), and 310 K (m).
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Fig. 1 (Continued)
DSt
Qt T
(2)
where pt is the surface pressure at the LE /LC phase transition commences, T is the absolute temperature, Qt is the latent heat of this phase change, and DSt is equal to the molar entropy change for an isothermal reversible phase transition process. ALE and AS are associated with the areas per molecule in the LE /LC phased, respectively. ALE is obtained at pt directly, while the value of AS is taken by extrapolating the liquidcondensed isotherm downward to pt. The accuracy of the experimental procedure may be questioned if the monolayer is labile owing to dissolution of some components into the subphase bulk. How-
ever, it has been well known for this binary system that DSPC and DSPE-PEG2000 are very insoluble in water and we did not find any obviously hysteresis after compression and cycles expansion of mixed monolayers (data not shown). Once the equilibrium has been reached, as is indicated by the reproducibility of the isotherms, the feasibility of thermodynamic analysis by means of Eqs. (1) and (2) can be assured. Table 1 gives corresponding thermodynamic parameters for the transition from LC to LE of mixed DSPC/DSPE-PEG2000 system that were determined from data at different temperatures. It shows that the values of the latent heats and entropy changes, defined for the transition from condensed to expanded monolayer, are overall
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Table 1 Thermodynamic characteristics of the main phase transition in the P /A isotherm for mixed DSPC/DSPE-PEG2000 monolayers with various mole fractions at different temperatures XDSPE-PEG
(%)
T (K)
Pt (mN/m)
ALE
dPt/dT
DSt (J/mol K)
Qt (J/mol)
283 297.5 310 283 297.5 310 283 297.5 310 283 297.5 310 283 297.5 310 283 297.5 310
6.9 7.6 8.6 5.8 6.6 7.6 5.5 6.0 7.4 5.9 6.9 7.4 5.94 7.6 8.6 9.2 10.7 11.6
65.9 68.4 82.8 72.8 78.1 90 81.3 85.9 96.3 88.1 93.1 99.4 105 108.1 111.3 350 380 416.7
0.065 0.065 0.065 0.065 0.065 0.065 0.069 0.069 0.069 0.054 0.054 0.054 0.101 0.101 0.101 0.089 0.089 0.089
7.03 8.01 13.64 9.15 11.24 15.901 11.89 13.83 18.09 12.63 14.26 16.30 31.83 33.72 35.62 134.23 150.36 170.05
1989.42 2382.83 4229.36 2589.52 3342.54 4927.89 3364.94 4114.09 5609.10 3573.15 4242.47 5054.07 9006.83 10031.90 11040.67 37991.62 44730.77 52714.48
2000
1
3
5
7
10
100
positive for all compositions studied. This means that transition from LC to LE structure of DSPC/ DSPE-PEG2000 mixed monolayer is characterized by entropy increase and endothermic process at different temperatures. The entropy for the transition from LC to LE film increases as expected from classical thermodynamic concept. Besides, the values of Qt and DSt are higher as temperature increases at the identical composition of the mixed monolayer. Thus it can be concluded that at higher
temperature, larger thermal motion results in a more random molecular configuration for DSPC/ DSPE-PEG2000 mixed monolayer at the air/water interface, thus it requires more energy for phase transition from LC to LE. Furthermore, it can be found from data in Table 1 that increasing the amount of DSPE-PEG2000 in the mixed monolayer has a dramatic effect on the entropy change and latent heat for the phase transition. When the temperature is fixed, the
Table 2 Isothermal compressibility (k , m/mN) and collapse pressures (pc, mN/m) of P /A isotherms of mixed DSPC/DSPE-PEG2000 monolayers with various molar percentages at different temperatures XDSPE-PEG
0%
1%
3%
5%
7%
10%
100%
ka kb kc pca pcb pcc
0.0028 0.0033 0.0046 59.9 59 45
0.0025 0.0055 0.0055 62 57 51
0.0033 0.0074 0.0062 55 55 52.5
0.0022 0.0062 0.0060 61 52 54
0.0040 0.0073 0.0083 53 58 51.5
0.0044 0.0080 0.0090 54 48 48.5
0.0057 0.0090 0.0105 64.8 64 60
2000
a b c
Indicated the parameters at 283 K. Indicated the parameters at 297.5 K. Indicated the parameters at 310 K.
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339
Fig. 2. Transition pressures as a function of temperature of DSPC/DSPE-PEG2000 mixed monolayers with XDSPE-PEG / 2000 1 ('), 3 (I), 5 (m), 7 (2), 10 (j), 100% (^).
entropy change of mixed monolayers and latent heat in the phase transition both increase with increasing XDSPE-PEG2000. It has been demonstrated before that polyethylene glycerol side chain can produce an expanded effect on the monolayer. Therefore, more DSPE-PEG2000 is believed to give more random molecular configuration during the LC /LE phase transition in the monolayer domain, thus results in larger energy requirement and entropy change for the phase transition. 3.3. Miscibility and excess area of mixed monolayers It is well established in previous literatures [26,27] that the miscibility of monolayers consisting of binary components can be determined simply and indirectly by means of the thermodynamically interfacial phase rule. If the two components in the monolayer at interface are miscible, the collapse surface pressures are dependent on the composition. In any event, a monolayer in which two components are immiscible will show two separate collapse surface pressures, corresponding to individual pure components. In Table 2, the collapse surface pressures of mixed DSPC/DSPE-PEG2000 monolayers at different
Fig. 3. Aex/Aid as a function of composition for DSPC/DSPEPEG2000 mixed monolayers at surface pressures of 5 ('), 10 (I), 15 (m), 20 (2), and 30 mN/m (j) on the water subphase at 283 (a), 297.5 (b), and 310 K (c).
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temperatures are listed. The collapse pressures of this mixed system did change with composition at a given temperature thought the differences between them are not all large. Consequently, it can be confidently concluded that DSPC and DSPEPEG2000 are miscible on the water subphase at temperatures investigated. Besides, the values of collapse pressures of mixed monolayer seem to be smaller at higher temperature. This phenomenon is consistent with the notion that enhanced mobility between molecules at higher temperatures causes easier collapse of mixed monolayers. More information on the behavior of intermolecular interaction in the mixed monolayer can be obtained by calculating the excess area (Aex). At a fixed surface pressure, the Aex can be estimated by the following equation [26,27], Aex A12 (X1 A1 X2 A2 )
(3)
where A12 is the actual occupied area per molecule of the mixed monolayer, X1 and X2 are the molar percentages of 1 and 2 components, respectively, and A1 as well as A2 imply the per molecular occupied areas of individual pure monolayers. Once two components form an ideally mixed monolayer or are immiscible, the Aex will be equal to zero at a given P . Otherwise, positive or negative values of Aex would indicate that miscible and nonideal mixed monolayers. In order to express clearly degrees of the deviation of excess area, the form of Aex/Aid should be used. Aex/Aid values versus the mole percentage of DSPE-PEG2000 in mixed monolayers at 283, 297.5, and 310 K, respectively, are shown in Fig. 3. Aex values of DSPC/DSPEPEG2000 mixed monolayers shown in Fig. 3 indicate that these values are not zero and are dependent on the subphase temperature and lateral surface pressure. It is clearly shown that DSPC and DSPE-PEG2000 are miscible and exhibit nonideal mixed behavior at the air/water interface at different temperatures. Nevertheless, it cannot be completely ruled out that DSPC and DSPE-PEG2000 might form ideal mixed monolayer or immiscible when XDSPE-PEG2000 is close to 1 or 0. There is a tendency to have more negative values of excess area as the temperature goes up, as shown in Fig. 3. It is believed that as temperature
increases, dehydration effect of PEG side chain would be more significantly affecting the monolayer. This would then intensify the negative trend of Aex/Aid as temperature goes up. The effect of lateral surface pressure on excess area was different at different temperatures. At 283 K the Aex/Aid decreased initially and then increased with increasing surface pressure (Fig. 3(a)). However when the temperature was higher, excess areas became significant more negative with the increase of surface pressure (Fig. 3(b and c)). This suggested that at higher temperatures the monolayers became more fluid and the mixed molecules are able to form more tightly packing than when molecular movement is restricted at lower temperature. This effect is more prominent at higher lateral surface pressure. Of course, the dehydration effect of PEG that accompanied by increasing temperature also plays a role. The dehydration is expected to decrease the area of PEG group and then enhances the packing directly. Moreover, composition effect for this mixed system on Aex/Aid at the different temperatures could be also identified from the data in Fig. 3. It is interesting to note that a maximum of Aex/Aid appeared at XDSPE-PEG200 /5 mol% either at high temperature or low temperature. This indicates that the intermolecular attractive interaction is weaker as well as special molecular packing pattern of the membrane at this composition. This finding lends support a notice in the research of DSPC/DSPE-PEG2000 mixed dispersions by others [28]. It was established that a phase transition occurs between mushroom to brush conformation for the PEG side chains at about XDSPEPEG200 /5 mol%. In our findings, the mixed membrane has special larger Aex at this composition, if the membranes are closely-packed enough, either under lower temperatures or under higher surface pressures. This seems to demonstrate the capability of the monolayer study in elucidating liposomal phenomena. 3.4. Monolayer stability The thermodynamic stability of forming a mixed monolayer consisted of two miscible mate-
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341
rials at the air/liquid interface can be determined
by the evaluation of free energy of mixing, DGmix,
Fig. 4. DGex vs. different mixed DSPC/DSPE-PEG2000 monolayers compositions at surface pressures of 5 ('), 10 (I), 15 (m), 20 (2), and 30 mN/m (j) on the water subphase at 283 (a), 297.5 (b), and 310 K (c).
Fig. 5. DGmix vs. different mixed DSPC/DSPE-PEG2000 monolayers compositions at surface pressures of 5 ('), 10 (I), 15 (m), 20 (2), and 30 mN/m (j) on the water subphase at 283 (a), 297.5 (b), and 310 K (c).
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or the excess free energy of mixing, DGex [26,27,29]. The DGex is defined as: II
g
DGex N0 [A12 (X1 A1 X2 A2 )]dP
(4)
0
where N0 is the Avogadro number and P is the surface pressure. Thus the value of DGex can be obtained directly by integration P /A isotherms. The DGmix, the partial molar thermodynamic properties, demonstrated the energetic tendency from a unmixed monolayer to a mixed monolayer and then was expressed as: DGmax DGex DGideal
(5)
where DGideal indicates the Gibbs energy of ideal mixing, which is given by: DGideal RT(X1 ln X1 X2 ln X2 )
(6)
where R is the universal gas constant and T is the absolute temperature. In Fig. 4, DGex is plotted as function of DSPEPEG2000 in mixed monolayers at different surface pressures and different temperatures. Obviously, the excess energy of this binary mixed system was significantly affected by temperature. The deviations from ideality are negative for the mixed monolayer with XDSPE-PEG2000 ‘/7% and the minimum of DGex appears at XDSPE-PEG2000 /7% at 283 and 297.5 K (Fig. 4(a and b)). This indicates that attractive interaction between molecules in a mixed monolayer became pronounced when XDSPE-PEG2000 ‘/7% and has a more significant influence on the mixed monolayer, at XDSPEPEG2000 /7% when mixed monolayers were at lower temperature (283 and 297.5 K). On the contrary, while XDSPE-PEG2000 B/7%, the positive deviations occurs, which means at this composition, repulsive interaction dominates. Nevertheless, when the temperature of subphase was elevated to 310 K, the values of DGex changes dramatically (Fig. 4(c)). Most of the values of DGex are negative and the degree of deviations became significant with increasing surface pressure especially as XDSPEPEG2000 ‘/5%. This indicates that at higher temperature the mixed monolayers are more stable comparing to ideal mixed monolayer than at lower temperature, and the intermolecular interaction
was stronger when the mixed monolayer was compressed to a more condensed state especially as mixed monolayers with XDSPE-PEG2000 ‘/5%. Plots of the DGmix versus XDSPE-PEG2000 for mixed DSPC/DSPE-PEG2000 monolayers on the water subphase at various surface pressures and different temperatures are illustrated in Fig. 5. The values of DGmix were changed from positive to negative as the compositions of mixed monolayers was ‘/5% at 283 and 297.5 K (Fig. 5(a and b)). According to the definition of DGmix, which can distinguish the difference between mixing and unmixing states in the monolayer system, one can say that the mixed monolayer with ‘/5% were energetically more favorable to form a mixed monolayer than a monolayer consisted of separated components at 283 and 297.5 K. However, all values of DGmix became negative and their magnitude increases when the surrounding temperature was raised to 310 K (Fig. 5(c)). This implied that the mixing tendency of this mixed system at the higher temperature was more advantageous than at lower temperature. Besides, it can be also found that the magnitude of DGmix seems to increase with increasing incorporation amounts of DSPE-PEG2000 and with increasing surface pressures at 310 K. In addition, one can see from results of either DGex or DGmix that mixed monolayer with XDSPEPEG2000 /5% stands at a point where DGex or DGmix values turns from positive to negative. It has been reported that bilayers containing from 1 to 9 mol% lipid /PEG2000 exhibited two different phase states characterized by two polymer conformation (mushroom and brush) [28,30]. The phase transition from mushroom to brush regimes also approximately occurred at the mixed lipid bilayer with 5 mol% of DSPE-PEG2000.
3.5. Isothermal compressibility of mixed monolayers In order to realize the equilibrium elasticity of a monolayer in a solid state at the interface, the 2-D isothermal compressibility, k , should be calculated and was defined as [31,32]:
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k
1 dA A dp
(7)
where A and p represented the mean molecular area and surface pressure, respectively. Hence, one can estimate k of a monolayer directly from the linear extrapolation of P /A isotherms. The evaluated range of compression isotherms for each monolayer defined as the difference in mean molecular areas from zero surface pressure to collapse surface pressure [32]. The isothermal compressibility of mixed DSPC/DSPE-PEG2000 monolayers at the air/water interface at 283, 297.5, and 310 K was listed in Table 2. The incorporation of DSPE-PEG2000 into DSPC monolayer affects compressibility for a mixed monolayer. At different temperatures compressibility of this binary mixed system increases with increasing the amounts of DSPE-PEG2000 or increasing environmental temperatures. Thus it can be concluded that the incorporation of DSPE-PEG2000 into DSPC monolayer or an increase in temperature would make the monolayer become more compressible although the differences in k of mixed monolayers are not obvious.
4. Conclusion The present study shows that mixed DSPC/ DSPE-PEG2000 monolayers, spread at the air/ water interface at different temperatures, are a useful model system for systematic studies of lateral interaction between them. From the analysis of P /A isotherms of this mixed system, DSPEPEG2000 is miscible with DSPC on water. The incorporation of DSPE-PEG2000 into DSPC monolayer or increasing temperature gives an expansion effect to the mixed monolayer. It is also an interesting system to study the thermodynamic properties of mixed monolayers. In this study, an increase in entropies for the transitions from LC to LE with increasing temperature or XDSPE-PEG2000, as well as an endothermic process of this transition were observed. The nonideal mixing behavior between DSPC and DSPE-PEG2000 and dehydration effect of PEG group with increasing
343
temperature were also found from the calculation of excess area. The stability of mixed system at different temperatures was judged by analysis of excess and mixing Gibbs free energies. The mixed monolayer with XDSPE-PEG2000 /5 mol% is an energy transition from positive to negative values of mixing energies. Moreover, influence of the subphase temperature, compositions, and lateral surface pressures on stability of mixed monolayers was discussed. Finally, the isothermal compressibility of mixed monolayers shows that the monolayer becomes more compressible as more DSPEPEG2000 added or as higher temperature increased.
Acknowledgements This research was supported by Grant NSC902214-E007-011 from the National Science Council of the Republic of China.
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