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Langmuir 2006, 22, 4910-4913
Polymeric Vesicle Permeability: A Facile Chemical Assay Giuseppe Battaglia,*,† Anthony J. Ryan,‡ and Salvador Tomas*,‡ Department of Engineering Materials, The Kroto Research Institute, UniVersity of Sheffield, Broad Lane, Sheffield, U.K., S3 7HQ, and Department of Chemistry, The UniVersity of Sheffield, Sheffield, U.K., S3 7HF ReceiVed February 6, 2006. In Final Form: April 6, 2006 We present a simple method to characterize vesicles and determine, at the same time, the membrane permeability to specific molecules. The method is based on encapsulating highly hydrophilic 3,3′,3′′-phosphinidynetris-benzenesulfonic acid (PH) into vesicles and subsequently monitoring its reaction with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). We tested the method by measuring the membrane permeability of vesicles formed from a series of poly(ethylene oxide)co-polybutylene oxide (EB) copolymers and egg yolk phosphatidylcholine. We found that the experimental data are in good agreement with calculations based on Fick’s first law. We therefore quantified the DTNB permeability across vesicle membranes, finding that polymeric EB membranes have a more selective permeability toward polar molecules compared to phospholipids membranes.
Natural phospholipids1 are able to form supramolecular membranes that play a central role in both the structure and function of all animal and plant cells. They define the sub- and extracellular compartments determining the nature of communication between the inside and the outside of the cell. This communication may take the form of the actual passage of ions and molecules between the different compartments (passive and active diffusion) or it may be in the form of information transmitted through conformational changes that drive the formation of hollow structures made of an enclosed aqueous volume: vesicles. New advances in polymer chemistry have recently allowed the design of a new class of amphiphilic membranes based on amphiphilic block copolymers.2 The molecular weight of these “superamphiphiles” is much higher than that of biological amphiphiles; therefore, they generate highly entangled3 membranes providing the final structure with improved mechanical properties.4 Polymeric membranes have been shown to form structures whose geometry can be as simple as spherical vesicles, with size ranging from tens of nanometers to hundreds of micrometers3,4 or more complex multilamellar tubules, with lengths of many millimeters.5 The most important property of amphiphilic membranes is their ability to partition aqueous volumes with different compositions and concentrations, which is due to their selective permeability to hydrophobic and hydrophilic molecules. Assessing the permeability of specific molecules is therefore one of the most important measurements to characterize amphiphilic membranes fully. Water and ion permabilities have been extensively measured by different techniques such as fluorescence quenching methods,6-8 membrane potential measurements,9 micropipet aspiration techniques,10 and anti-Stokes Raman * Corresponding authors. (G.B.) E-mail:
[email protected]. Fax: +44(0)114 222 5962. Tel: +44(0)114 222 5945. (S.T.) E-mail:
[email protected]. Fax: +44(0)114 222 9346. Tel: +44(0)144222455. † Department of Engineering Materials, University of Sheffield. ‡ Department of Chemistry, University of Sheffield. (1) Cevc, G.; Marsh, D. Phospholipid Bilayers: Physical Principles and Models; John Wiley & Sons: New York, 1987; Vol. 5. (2) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973. (3) Battaglia, G.; Ryan, A. J. J. Am. Chem. Soc. 2005, 127, 8757-8764. (4) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143-1146. (5) Battaglia, G.; Ryan, A. J. Angew. Chem., Int. Ed. 2006, 45, 2052-2056. (6) Mathai, J. C.; Sprott, G. D.; Zeidel, M. L. J. Biol. Chem. 2001, 276, 2726627271. (7) Garcia, A. M. Methods Enzymol. 1992, 207, 501-510.
scattering.11 The permeability of more complex and nonionic molecules has been measured only by NMR techniques.12,13 However, molecular exchange through amphiphilic membranes always takes place in an aqueous environment, and the permeating molecules undergo no great variation in their individual properties. Consequently, only the slow release of encapsulated molecules can be detected under nonequilibrium conditions in dialysis experiments. Any rapid exchange is not observable under such circumstances. Here we present a method based on simple chemistry and dynamic light scattering measurements (DLS) that has allowed the real-time measurements of the permeability of membranes formed by amphiphilic block copolymers poly(ethylene oxide)co-poly(butylene oxide) (EB). Copolymers have been chosen covering a range of molecular weights and three different architectures: EmBn diblocks and two triblocks BnEmBn and EmBnEm (where E stands for poly(ethylene oxide) and B stands for poly(butylene oxide), and m and n indicate the degrees of polymerization, respectively). The block copolymer results have been compared with the permeability of vesicles formed by egg yolk phosphatidylcholine (PC). Specific molecule permeability can be measured by monitoring the reaction between two chemicals separated by amphiphilic membranes when one of the two chemicals permeates much more slowly than the other. Under such conditions, the reaction rate is directly correlated to the permeability of the permeating molecule. We have accordingly chosen to encapsulate a highly hydrophilic molecule, 3,3′,3′′-phosphinidynetris-(benzenesulfonic acid) (i.e., PH), inside vesicles. This choice ensures, with great certainty, the localization of any PH-involving reaction inside the vesicle aqueous core because its solubility in the hydrophobic inner membrane is negligible. In particular, we have monitored the reaction of PH with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, also known as Ellman’s reagent14 (Scheme 1). PH reduces the DTNB disulfide bond, leading to the formation of thionitroben(8) Chen, P.-Y.; Pearce, D.; Verban, A. S. Biochemistry 1988, 27, 57135718. (9) Ward, J. M. Plant Physiol. 1997, 114, 1151-1159. (10) Olbrich, K.; Rawicz, W.; Needham, D.; Evans, E. Biophys. J. 2000, 79. (11) Potma, E. O.; Boeij, W. P. d.; Haastert, P. J. M. V.; Wiersma, D. A. Proc. Natl. Acad. Sci. 2001, 98, 1577-1582. (12) Johnson, S. M.; Bangham, A. D. Biochim. Biophys. Acta 1969, 193, 82-91. (13) Rumplecker, A.; Fo¨rster, S.; Za¨hres, M.; Mayer, C. J. Chem. Phys. 2004, 120, 8740-8747.
10.1021/la060354p CCC: $33.50 © 2006 American Chemical Society Published on Web 04/25/2006
Letters
Langmuir, Vol. 22, No. 11, 2006 4911
Scheme 1. Reaction from 3,3′,3′′-Phosphinidynetris-(benzenesulfonic acid) (PH) and 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) to form Thionitrobenzoate (TNB) and Tris(3-sulfonatophenyl)Phosphine Oxidea
a
The TNB dianion absorbs at λ ) 412 nm.
Figure 1. (a) Optical micrograph of PH-loaded microvesicles of E16B22 following the addition of DTNB. (b) UV/vis spectra from PH-loaded vesicles as a function of time after DTNB addition. (c) Increase in the intensity of a band centered at 412 nm caused by the reaction of DTNB with PH encapsulated in E16B22 vesicles. (d) Initial absorbance at 410 nm following the addition of DTNB to E16B22 vesicles encapsulating PH after storage for extended periods of time.
zoate, TNB. The formation of TNB is reflected in the increase of a band in the UV spectra centered at 412 nm. DTNB is significantly less hydrophilic than PH and can easily permeate the inner hydrophobic membranes.15 This assay can be applied to measure DTNB permeability through amphiphilic membranes and, at the same time, to give a rapid and clear answer to the fundamental question of whether a given amphiphile forms vesicles: because only enclosed aqueous volumes can encapsulate highly hydrophilic molecules such as PH. The assay comprises three steps. (1) Vesicles are formed in the presence of the PH molecules. (2) The PH-loaded vesicles are separated from the original solution. (3) DTNB is added to the loaded vesicle dispersion, and the DTNB + PH reaction kinetics are monitored. The localization of the PH + DTNB reaction in the vesicle aqueous core can be visualized for micrometer-sized PH-loaded vesicles formed by electroformation.16 The micrograph in Figure 1a shows that the inner vesicle volume becomes yellow as a (14) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77. (15) Cline, D. J.; Redding, S. E.; Brohawn, S. G.; Psathas, J. N.; Schneider, J. P.; Thorpe, C. Biochemistry 2004, 43, 15195-15203.
consequence of the formation of TNB. The reaction kinetics and consequently the DTNB permeability can be quantified by applying the method to low-polydispersity nanosized vesicles formed by rehydration and extrusion. In Figure 1b and c, the kinetics of TNB formation is monitored by UV/vis spectroscopy. The reaction of DTNB with free PH is very fast under the experimental conditions15 (k > 10 M-1 s-1). Even in the presence of empty vesicles, the reaction is not affected (data not presented). When PH is encapsulated inside vesicles, the PH + DTNB kinetics have been found to be on the order of hours, even in the thinnest membranes of E16B22 vesicles (Figure 1c). This indicates that DTNB first has to cross the membrane and then react with PH. Therefore, the kinetic data, plotted in Figure 1c, are dependent on DTNB membrane permeation. The PH confinement inside the vesicles has also been tested by measuring the absorbance at 410 nm immediately after the DTNB addition (Figure 1d). Because the reaction between PH and DTNB is almost instantaneous, this value relates to the presence of free PH in solution. As can be seen in Figure 1d, even 24 h after encapsulation (16) Angelova, M. I.; Dimitrov, D. S. Faraday Discuss. 1986, 81, 303.
4912 Langmuir, Vol. 22, No. 11, 2006
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Table 1. Permeation Rates Calculated from Different Amphiphiles amphiphile
membrane thickness/nm
encapsulation [PH]i/[PH]f%
hydrodynamic diameter/nm
permeability/ n ms-1
191.5 ( 11
196.4 ( 12 195.1 ( 14 143.2 ( 7
0.93 ( 0.06 0.25 ( 0.06 3.56 ( 0.06 19.30 ( 0.06 0.43 ( 0.08 0.49 ( 0.12 0.25 ( 0.10
E16B22 (pH 7.2) E16B22 (pH 8) E16B22 (pH 7) E16B22 (pH 6) E50B70 (pH 7.2) E68B65 (pH 7.2) E115B103 (pH 7.2)
2300
2.40 ( 0.26a
7300 7700 12500
4.53 ( 0.44a 4.07 ( 0.41a 7.56 ( 0.56a
0.156 0.180 0.127 0.161 0.064 8.7 × 10-4 0.040
B37E77B37 (pH 7.2) B46E99B46 (pH 7.2)
8700 11000
3.15 ( 0.29a 3.42 ( 0.33a
0.022 2.8 × 10-4
171.3 ( 9 188.2 ( 10
0.73 ( 0.14 0.63 ( 0.12
E31B54E31 (pH 7.2) E34B75E34 (pH 7.2) E40B100E40 (pH 7.2)
6600 8400 11400
4.16 ( 0.32a 4.62 ( 0.41a 6.37 ( 0.43a
4.8 × 10-4 0.010 0.033
118.4 ( 10 160.2 ( 13 147.1 ( 9
0.47 ( 0.06 0.45 ( 0.07 0.34 ( 0.07
3.4b
0.109 0.067 0.104 0.078
197.6 ( 10
0.06 ( 0.01 0.08 ( 0.01 0.12 ( 0.05 0.67 ( 0.11
PC (pH 7.2) PC (pH 8) PC (pH 7) PC (pH 6) a
Mw/ g mol-1
750
Data from ref 3. b Data from ref 1.
no significant changes have been detected, indicating that the PH remains fully encapsulated. Assuming that the diffusion of DTNB through vesicle membranes is rate-limiting and reversible, Fick’s first law can be applied to define the DTNB flux, JDTNB, as a function of the diffusion coefficient, DDTNB, the gradient of concentration from inside, Ci(t), and outside the membrane, Ce(t), and the membrane thickness, d:
JDTNB ) DDTNB
dC(t) = p(Ce(t) - Ci(t)) dx
(1) p)
The permeability p can be defined as the ratio between the diffusion coefficient DDTNB and the membrane thickness d
DDTNB p) d
(2)
dC(t) ) NvesiclesJDTNBAvesicle dt
(3)
where V is the volume of solution where C(t) applies, Avesicle ) 4π(D/2)2 is the surface area at the middle plane of the membrane, D is the vesicle diameter measured by DLS, and Nvesicles is the number of vesicles calculated from encapsulation e as follows
Nvesicles )
Venc eV ) Vvesicle Vvesicle
(4)
where the encapsulation, e, is calculated as
e)
[PH]f [PH]i
Vvesicle dC(t) dC(t) D ) e[DTNB]Avesicle dt 6e[DTNB] dt
(5)
where Vvesicle ) (4/3)π(D/2)3 is the volume of the single vesicle and Venc is the encapsulated volume that is calculated as a product of the total volume, V, and encapsulation, e, defined as ratio between the final and initial PH concentrations (Supporting Information). Because the reaction of DTNB with PH is very fast under these conditions,15 we can assume that all of the DTNB that permeates reacts immediately with the encapsulated PH. DTNB is always in large excess compared to the encapsulated PH, the
(6)
Alternatively, the process can be thought of as a reaction between DTNB and the vesicle membrane, governed by the following kinetic equation
dC(t) ) k[DTNB][A] dt
According to the conservation of mass
V
concentration gradient Ce(t)-Ci(t) can thus be assumed to be constant and equal to the added DTNB concentration, [DTNB], until the PH is nearly completely consumed. This is reflected in a linear increase of the absorbance in the earlier stages of the permeation kinetics (Figure 1c). During this time, dC(t)/dt is constant and can easily be derived from the absorbance gradient using the Beer-Lambert law. Accordingly, by combining eqs 1, 3, and 4, the permeability can be calculated as a function of experimental values as follows:
(7)
where [A] is the concentration of the vesicle membrane expressed as the vesicle membrane area per unit volume:
[A] )
NvesicleAvesicle V
(8)
Combining eqs 4, 7 and 8, eq 6 is derived where the rate constant k is equal to the permeability, p. Equation 6 has been used to calculate the DTNB permeability at pH 7.2 for PC vesicles and for the different EB molecular weights and architectures. Permeability measurements were also made at pH 8, 7, and 6 but only for vesicles formed from E16B22 and PC (Table 1). The permeability, measured at pH 7.2, is plotted as a function of the membrane thickness in Figure 2. EB membranes have been found to be almost 1 order of magnitude more permeable to DTNB than phospholipids. This is due to the more hydrophilic nature of the polyether membrane compared to the aliphatic lipid membranes. Moreover, diblocks and triblocks fall on the same master curve, indicating that the butylene oxide membrane is interpenetrated and not a classic bilayer, confirming previously reported results for the same system.3 The data can be fitted by eq 2, allowing the calculation of the diffusion coefficient for DTNB in the EB membranes: DDTNB ) 2.5 ( 0.3 nm2 s-1, the
Letters
Figure 2. Permeability as function of the EB and PC membrane thicknesses. The data have been fitted by eq 2, and the DTNB diffusion coefficient has been calculated to be 2.50 ( 0.3 nm2 s-1.
consistent diffusion coefficient providing an internal validation of the method used. Further insights into the molecular properties of EB membranes can be gained by measuring the pH dependence of the permeability. At pH 6, DTNB permeability is 20 times larger for the E16B22 diblock copolymer than for PC vesicles, whereas at pH 8 the ratio is less than 3-fold larger (Figure 3). DTNB is a weak diacid (with both pKa values being virtually identical17 at around 2.5); therefore, as a function of pH the relative proportions of neutral and charged DTNB species change exponentially. According to the graph in Figure 3, EB membranes are more permeable than lipid membranes to neutral molecules, as indicated by the high permeability difference at acidic pH. Conversely, EB membranes are almost as permeable as lipid membranes toward charged species, as evidenced by the fact that the two permabilities seem to converge at higher pH. This behavior relates, ultimately, to the chemical structure of the membrane and gives a good estimation of the relative hydrophobicities of two the membranes. In conclusion, we have developed a simple chemical assay to characterize vesicles and measure the membrane permeability. (17) Danehy, J. P.; Elia, V. J.; Lavelle, C. J. J. Org. Chem. 1971, 36, 10031005.
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Figure 3. Permeability of EB and PC membranes as a function of pH.
This has allowed us to determine the permabilities of different polymeric membranes, finding that the permeability depends on the thickness of the membrane as predicted by Fick’s first law. Our data also suggest that EB membranes show an improved selectivity between charged and uncharged molecules, when compared with PC membranes. This leads to the conclusion that the vesicle permeability can be tuned by choosing the appropriate composition of the amphiphilic membrane, opening new possibilities in controlled release applications. Finally, this assay can be used for assessing, both qualitatively and quantitatively, vesicle formation because highly hydrophilic molecules can be encapsulated only by a membrane-confined aqueous volume (i.e., vesicle). Acknowledgment. We thank the ICI Strategic Technology Group for financial support. We also acknowledge the contributions of Dr. Shao-Min Mai, who synthesized the block copolymers used in the present work. Supporting Information Available: Experimental method and DLS size distribution of nanovesicles. This material is available free of charge via the Internet at http://pubs.acs.org. LA060354P
