Water permeation through block-copolymer vesicle membranes

Water permeation through block-copolymer vesicle membranes

Chemical Physics Letters 444 (2007) 268–272 www.elsevier.com/locate/cplett Water permeation through block-copolymer vesicle membranes Alina Leson a, ...

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Chemical Physics Letters 444 (2007) 268–272 www.elsevier.com/locate/cplett

Water permeation through block-copolymer vesicle membranes Alina Leson a, Volkan Filiz b, Stephan Fo¨rster b, Christian Mayer a b

a,*

University Duisburg-Essen, Physical Chemistry, D-45141 Essen, Germany University of Hamburg, Physical Chemistry, D-20146 Hamburg, Germany Received 27 March 2007; in final form 2 July 2007 Available online 12 July 2007

Abstract The molecular exchange of water molecules through membranes of dispersed vesicles prepared from different block copolymers was studied using nuclear magnetic resonance spectroscopy combined with pulsed field gradients. Vesicles from four different block copolymers with different hydrophobic sections (poly-2-vinylpyridine, polyisoprene, polybutadiene and polylactid) were used for the determination of average residence times for water molecules in the encapsulated state. The activation energies for the water transfer through the different membranes were obtained in a systematical variation of the dispersion temperature.  2007 Elsevier B.V. All rights reserved.

1. Introduction Vesicles are hollow, spherical structures with potential applications as drug carriers [1]. Their size depends on the chemical constitution and the molecular mass of the polymer as well as on the preparation method and environmental factors [2,3]. Nuclear magnetic resonance echo experiments with pulsed field gradients (PFG-NMR) have proven to be a powerful approach for the analysis of the molecular exchange through vesicle membranes [4–7]. PFG-NMR studies give access to the translational mobility of any system component as a function of an adjustable observation time D. Based on these data, a distinction between the molecules of the continuous external phase and the encapsulated ones is possible. At the same time, it allows one to observe an exchange process between both domains in the equilibrium state [8–15]. A characteristic parameter for the exchange process is the activation energy for the membrane permeation. In the given case, it characterizes the energy barrier which an individual water molecule has to overcome when it penetrates through the hydrophobic region of the vesicle membrane. In many applications of vesicle dispersions, this number may be cru*

Corresponding author. Fax: +49 201 183 2570. E-mail address: [email protected] (C. Mayer).

0009-2614/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2007.07.023

cial for the evaluation of the encapsulation properties as it is indicative of the permeability of the membrane for polar molecules. In the following, we present studies on vesicles formed by four different amphiphilic block copolymers (poly(2-vinylpyridine-block-ethylene oxide) (P2VP-PEO), polyisoprene-block-ethylene oxide (PI-PEO), polybutadiene-block-ethylene oxide (PB-PEO) and polylactid-blockethylene oxide (PLA-PEO)). 2. Theory The hindered diffusion inside the encapsulated volume of small vesicles has little or no effect on the echo decay. In contrast, the Brownian motion of the vesicles is a very efficient mechanism of translational dislocation and dominates the echo decay for the confined molecules. Hence, it is a good approximation to consider the echo decay process as an exchange between two molecular reservoirs with two different exponential relaxation times as it has been treated in detail by Woessner [16] for a general type of relaxation and has been adapted to the PFG-NMR measurement by Ka¨rger [17]. This approach has been successfully applied to the problem of molecular exchange through vesicle or capsule walls in the past (see [18] or [19] for examples). For our experiments, we will assume that the spin–spin and the spin-lattice relaxation do not differ for the internal

A. Leson et al. / Chemical Physics Letters 444 (2007) 268–272

and the external domain. This condition is justified as long as the solvent and the concentrations of all constituents are identical for both domains of the vesicles. Further, we neglect the influence of spin diffusion, a process which could principally contribute to the magnetization exchange for stimulated echo experiments with consequences similar to molecular exchange, an assumption which has been justified by earlier studies [13]. With the approximation D @ (D  d/3) which is valid for short gradient pulses and long separations, the echo decay is described by [16,17]: I=I 0 ¼ I rel ¼ exp½c2 d2 G2 DðD  d=3Þ

ð1Þ

I rel ¼ P 0a expðk 0a DÞ þ P 0b expðk 0b DÞ

ð2Þ

with: k 0a ¼ C 1  C 2

ð3Þ

k 0b

ð4Þ

¼ C1 þ C2 h i 1 1 1 ðP  P Þðk  k Þ þ þ b a a b sa sb 1 4 P 0a ¼ þ 2 C2 h i 1 ðP b  P a Þðk a  k b Þ þ s1a þ s1b 4 1 P 0b ¼  2 C2   1 1 1 ka þ kb þ þ C1 ¼ 2 sa sb " #1=2 2 1 1 1 4 ka  kb þ  þ C2 ¼ 2 sa sb sa sb k a ¼ c2 G2 D0a d2 and k b ¼ c2 G2 D0b d2

ð5Þ

ð6Þ ð7Þ ð8Þ ð9Þ ð10Þ

with Pb and Pa being the relative populations of the encapsulated and the free phase, respectively (with Pa + Pb = 1). The apparent diffusion constants D 0 a and D 0 b determine the initial and the final slope of an individual plot Irel vs. G2, while the average residence times sa and sb take influence on the dependence of the final plateau level on the diffusion time D (with sa/sb = Pa/Pb). This analytical approach has been further improved by introducing an apparent diffusion coefficient that accounts for the internal diffusion [20]. The reciprocal average residence time s1 b is proportional to the rate constant for the membrane transfer. Therefore, the activation energy for the transfer of water through the vesicle mebranes can be obtained in an Arrhenius-like plot 1 of ln s1 b vs. T . Similar to a conventional Arrhenius-plot, its slope is equivalent to the term Ea/R which allows for the determination of the activation energy Ea. 3. Experimental methods The block copolymers that were used were prepared by sequential living anionic polymerization of the different hydrophobic parts and ethylene oxide. The degrees of the polymerization are summarized in Table 1. For vesicle preparation, water is added to a solution of the polymer in chloroform and the two-phase mixture is stirred vigor-

269

Table 1 Parameters of the used block-copolymers Polymer

P2VP-PEO 10 PI-PEO 18.2 PB-PEO 15 PLA-PEO 53

Block lengths

M [g/mol]

Hydrophobic part

PEO

54 32 40 100

34 27 25 50

7350 3350 3340 5760

ously for three days. After evaporation of the organic solvent, the aqueous phase contains spherical vesicles at a concentration of 3 mg/ml. Mean hydrodynamic radii were determined as Rh = 86 nm (PI-PEO), Rh = 80 nm (PBPEO), and Rh = 140 nm (PLA-PEO). All PFG-NMR measurements were performed on a Bruker Avance 400 spectrometer (Bruker AG, Karlsruhe, Germany) equipped with a BAFPA 40 gradient amplifier and a Bruker DIFF30 probe. The instrument was tuned to 400 MHz proton frequency, gradient pulses were adjusted to gradient strengths between 0 and 4 T/m with 1.2 ms duration. For all measurements, the stimulated echo sequence (90–s1–90–s2– 90–s1–echo) was used in combination with gradient pulses during each waiting period s1. The spacing D between the two gradient pulses (and, correspondingly, the waiting period s2) was varied between 100 and 300 ms. The desired temperature is adjusted on the integrated temperature unit and kept stable for at least 30 min to allow for complete equilibration of the sample. Three PFG-NMR experiments were run on each of four temperature settings in the range of 25–37 C, the results were averaged. 4. Results and discussion 4.1. Water diffusion and exchange Fig. 1 shows a set of echo decay curves observed on the corresponding water signal of vesicle dispersions of P2VPPEO and PI-PEO under variation of the field gradient strength G for different spacings D between the two gradient pulses. Logarithmic relative echo amplitudes Irel are plotted vs. the parameter c2G2d2(D  d/3) such that a linear dependence with the slope equal to the negative self diffusion constant (D) is obtained for free diffusion according to Eq. (1). In the case of P2VP-PEO vesicles the exchange of water molecules through a vesicle membrane is very rapid and only free diffusion can be observed as shown in Fig. 1. This is only partially a consequence of the limitations of the experiment, but rather due to the fact that some vesicle membranes do not represent a significant barrier for the self diffusion. In the case of the vesicles from PI-PEO18.2 as well as for PB-PEO and PLA-PEO, the NMR signals of the free and the encapsulated fractions could be well separated by the action of the pulsed field gradients. For all three systems, the free water is represented by an initial rapid decay of

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A. Leson et al. / Chemical Physics Letters 444 (2007) 268–272 0.0 100 ms PI-PEO

-1.0

200 ms PI-PEO

ln I/I0

-2.0

300 ms PI-PEO

-3.0

100 ms P2VP-PEO

-4.0

200 ms P2VP-PEO 300 ms P2VP-PEO

-5.0 -6.0 -7.0 -8.0 -9.0 -10.0 0.00

0.25

0.50

0.75 2

2 2

1.00

γ G δ (Δ-δ/3) [10

1.25 10

1.50

1.75

2.00

s/m²]

Fig. 1. Examples of echo decay curves observed on the water signal of dispersed vesicles from P2VP-PEO (full symbols) and PI-PEO (open symbols) for T = 298 K. The data refer to pulse spacings of D = 100 ms (triangles), D = 200 ms (squares), and D = 300 ms (diamonds), respectively. The corresponding solid lines show the best fit obtained by Eqs. (2)–(10) using a single data set: Da = 2.0 · 1009 m2/s ± 0.02 · 1009 m2/s, Db = 1.8 · 1012 m2/ s ± 0.09 · 1012 m2/s, sb = 120 ms ± 0.2 ms, Pb = 0.6% ± 0.01%.

the echo intensity, while the encapsulated water gives rise for a relatively stable plateau value for larger gradients (see Fig. 1 for PI-PEO18.2). The average residence time in the encapsulated state can be derived from the dependence of the plateau level on the separation D of the gradient pulses. The echo decay curves for each vesicle type at each temperature have been fitted based on Eqs. (2)–(10) with a single parameter set. Fig. 1 shows an example for PI-PEO vesicles at T = 298 K. With a single parameter set (see caption in Fig. 1), the curves for three different pulse spacings (100 ms, 200 ms, 300 ms) are well reproduced. The initial very steep slope down to ln(I/I0) = 5, which is very similar to the one for P2VP-PEO10, corresponds to the nearly free diffusion of the water in the continuous phase, which here contributes to (98.87 ± 0.016)% of the overall water content. The common slope of all three curves reflects a self diffusion constant of Da = (2.0 · 109 ± 0.02 · 109) m2/s which is close to the value for free self diffusion of pure water with 2.26 · 109 m2/s for 298 K. The final slope, again shared by all three echo decay curves, is related to the Brownian motion of the vesicles which, according to the overall best fit, amounts to a diffusion coefficient of Db = (1.8 · 1012 ± 0.09 · 1012) m2/s. The average residence time of water molecules in the encapsulated state is reflected by the decreasing plateau level of I/I0 for high gradients. For water in PI-PEO18.2 vesicles, it amounts to sb = 120 ms at T = 298 K. The resulting residence times for all vesicle types at temperatures between 25 and 37 C are summarized in Table 2. At 25 C, the values for sb vary between very short periods for P2VP-PEO10, 96 ms for PB-PEO15, 120 ms for PIPEO18.2 and 250 ms for PLA-PEO53. The variation of

Table 2 Measured average residence times and calculated activation energies Polymer

sb (25 C) [ms]

P2VP-PEO 10 PI-PEO 18.2 PB-PEO 15 PLA-PEO 53

Not detectable 120 100 96 83 250 150

sb (30 C) [ms]

sb (35 C) [ms]

sb (37 C) [ms]

Ea [kJ/mol]

90 75 90

80 68 80

34.3 22.4 83.1

the water permeability for the different vesicle types can be partially attributed to the different polarities of the hydrophobic sections of the block-copolymers. Poly(isoprene) in PI-PEO, as an example, is much less polar than poly(2-vinylpyridine) in P2VP-PEO, which explains for the higher permeability of P2VP-PEO membranes. The low permeability of the PLA-PEO membranes as compared to P2VP-PEO may be explained by the larger thickness of its hydrophobic barrier which amounts to 100 monomer units instead of 54 (Fig. 3). In general, the differences in permeability seem to decrease with increasing temperature: at 37 C, the water residence times for PIPEO and PLA-PEO are practically identical (sb = 80 ms); with sb = 68 ms, the one for PB-PEO is only slightly smaller. 4.2. Activation energy of the membrane transfer The average residence times sb have been determined with low experimental uncertainty (below 3%), therefore, they are expected to reflect the temperature dependence

A. Leson et al. / Chemical Physics Letters 444 (2007) 268–272

271

3.0

2.5 PB-PEO PI-PEO

ln τb

-1

2.0

1.5 PLA-PEO

1.0

0.5

0.0 0.00320

0.00325

0.00330 1/T [1/K]

0.00335

0.00340

Fig. 2. Arrhenius-plots for the exchange of water molecules through membranes of PB-PEO (diamonds), PLA-PEO (triangles), and PI-PEO (squares). The dashed lines indicate the individual best fits for the Arrhenius plots (the corresponding activation energies are listed in Table 2).

PEO 50

PEO 34

PEO 25

PEO 27

P2VP 54

PEO

PLA 100

PI PB 40 PEO 25

32 PEO 27

34

PEO 50

Fig. 3. Schematic representation of the relation between the hydrophilic (light-grey) and the hydrophobic parts (dark-grey) of the vesicle membranes used in the experiments. Numbers refer to the number of monomer units in the polymer blocks, the relative sizes of the blocks are indicated by the relative widths of the shaded sections.

of the exchange process even though the temperature window is restricted to only 12 K. The activation energy for the water transfer through the vesicle membranes is obtained from a plot of the natural logarithm of s1 vs. b the reciprocal temperature (see Fig. 2). For vesicles from PB-PEO, PLA-PEO and PI-PEO, three plots are obtained

which can be well approximated by three linear functions. From their slopes m, activation energies are calculated according to the equation Ea = mR, the resulting data are listed in Table 2. On a most simple level of interpretation, one may assume that the obtained activation energies mark the

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A. Leson et al. / Chemical Physics Letters 444 (2007) 268–272

energy barriers which a mole of individual water molecules has to overcome in order to cross the hydrophobic section of the given membrane. In this first approximation, one may neglect the temperature-induced variation of the molecular packing of the hydrophobic chains which may contribute to reduced or enhanced water permeation through the inner layer of the membrane. This given, the obvious similarity of the activation energies for membranes of PB-PEO (Ea = 22 kJ/mol) and PI-PEO (Ea = 33 kJ/mol) is quite easily understood: in both cases, we deal with a layer of aliphatic hydrocarbon which only weakly interacts with water molecules. Hereby, the principal portion of the activation energy derives from the fact that water molecules have to be separated from the bulk water phase and brought into an environment with little residual interaction. In case of a hypothetically vacuum layer, this energy would correspond to the evaporation enthalpy of water which amounts to 44 kJ/mol. The differences of 22 kJ/ mol for PB-PEO and 11 kJ/mol for PI-PEO could therefore correspond to the residual interactions of water with the PB or PI sections, respectively. With 83.1 kJ/mol, the activation energy for the water permeation through PLA-PEO membranes is significantly higher. At this point of time, we can only give a more speculative interpretation for this observation: in contrast to PB-PEO and PI-PEO, this block-copolymer carries carbonyl groups in its hydrophobic part and hence is capable of forming hydrogen bonds in the inner layer of the vesicle membrane. This would reduce the energy barrier connected to the separation from the bulk water and the intrusion into the hydrophobic layer. However, it may result in a temporary localization of water molecules inside this hydrophobic section, since the hydrogen bonds have to be broken in order to allow for water diffusion towards the other side of the membrane. In addition, the PLA chains may undergo more significant structural changes in the temperature region between 25 and 37 C as compared to PB or PI. In this case, the higher activation energy for PLA-PEO membranes would include the influence of a

decrease in packing density and an increase in molecular mobility in its hydrophobic layer. Acknowledgements We gratefully acknowledge financial support from the Volkswagen-Stiftung in connection with the project titled ‘Block-copolymer vesicles with controlled uptake/release functions for drugs and genes’. We also thank M. Stolzenburg from the University of Hamburg for the used block copolymers. References [1] F. Najafi, M.N. Sarbolouk, Biomaterials 24 (2003) 1175. [2] M. Antonietti, S. Fo¨rster, Adv. Mater. 15 (2003) 1323. [3] M. Regenbrecht, S. Akari, S. Fo¨rster, H. Mo¨hwald, Surf. Interf. Anal. 27 (1999) 418. [4] E.O. Stejskal, J.E. Tanner, J. Chem. Phys. 42 (1965) 288. [5] E.O. Stejskal, J. Chem. Phys. 43 (1965) 3597. [6] J.E. Tanner, E.O. Stejskal, J. Chem. Phys. 49 (1968) 1768. [7] R. Kimmich, NMR tomography diffusometry, relaxometry, Springer, Berlin, 1997. [8] P.W. Kuchel, C.J. Durrant, J. Magn. Reson. 139 (1999) 258. [9] K.I. Momot, P.W. Kuchel, B.E. Chapman, D. Whittaker, Langmuir 19 (2003) 2088. [10] C. Mayer, D. Hoffmann, M. Wohlgemuth, Int. J. Pharm. 242 (2002) 37. [11] A. Rumplecker, S. Fo¨rster, M. Za¨hres, C. Mayer, J. Chem. Phys. 120 (2004) 8740. [12] A. Bauer, S. Hauschild, M. Stolzenburg, S. Fo¨rster, C. Mayer, Chem. Phys. Lett. 419 (2006) 430. [13] C. Mayer, A. Bauer, Prog. Coll. Polym. Sci. 133 (2006) 22. [14] T. Adalsteinsson, W.F. Dong, M. Scho¨nhoff, J. Phys. Chem. B 108 (2004) 20056. [15] P.T. Callaghan, I. Furo, J. Chem. Phys. 120 (2004) 4032. [16] D.E. Woessner, Relaxation effects of chemical exchange, in: D.M. Grant, R.K. Harris (Eds.), Encyclopedia of NMR, Wiley, Chichester, 1995. [17] J. Ka¨rger, J. Ann. Phys. 7 (1971) 107. [18] M. Scho¨nhoff, O. So¨dermann, J. Phys. Chem. 101 (1997) 8237. [19] M. Wohlgemuth, C. Mayer, J. Colloid Interface Sci. 260 (2003) 324. [20] J. Pfeuffer, U. Flo¨gel, W. Dreher, D. Leibfritz, NMR Biomed. 11 (1998) 19.