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Langmuir 1998, 14, 5802-5805
Electroporation of Unilamellar Vesicles Studied by Using a Pore-Mediated Electron-Transfer Reaction N. Mariano Correa and Z. A. Schelly* Center for Colloidal and Interfacial Dynamics, Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas 76019-0065 Received June 5, 1998. In Final Form: July 30, 1998 The dynamics of electric field-induced pore formation (electroporation) is investigated in 186 nm diameter, unilamellar bilayer vesicles (liposomes) prepared from the phospholipid surfactant dioleoylphosphatidylcholine (DOPC). Formation of reversible transient pores is detected via the spectral changes associated with the electron-transfer reaction between Fe(CN)64- ions entrapped in the interior compartment of the vesicle and Ir(Cl)62- ions placed in the bulk medium. Upon application of a high-voltage square pulse (E ) 8 kV/cm) to the suspension, poration occurs only if the pulse length is g200 µs. The field-induced elongation of the time average spherical vesicles to prolate ellipsoid in the direction of the applied field E is detected through the transient birefringence of the system. The onset of elongation precedes the availability of pores large enough for Fe(CN)64- to pass through, with an estimated radius of ∼9.6 Å. The average rate of radius growth of the reversible pores (4 × 10-6 m s-1) is found to be 5 orders of magnitude smaller than that of irreversible electropores in planar black lipid membranes of similar phospholipids.
Introduction Electroporation is the fully reversible transient pore formation in surfactant bilayers such as cell membranes, liposomes, or, in general, vesicles, induced by an externally applied electric field pulse to the suspension.1 The method is commonly used for the insertion of biologically active materials into cells and cell membranes for purposes of transfection, in vivo gene therapy, cloning, etc.2 The species to be inserted reaches its target destination through diffusional transport during the limited lifetime (typically microseconds to seconds) of the open pores. A main issue in applications of electroporation has been finding the proper experimental conditions (e.g., applied field strength E and the number, shape, and length of pulses) which result in pores of suitable size and lifetime, without permanent destruction of the membrane or fusion of the cells. Due to the wide diversity in composition and structure of biological membranes, only general patterns of the requirements and their effects have been established. In successful procedures, provided the induced membrane potential is above particular threshold values and the pulse length falls within certain limits, the size of the pores created grow with the length of the pulse. However, it is fair to say that the detailed mechanism of the field-induced pore opening is not known.1,3 The problem associated with the complexity of natural cell membranes can be avoided by investigating synthetic liposomes or vesicles4 which mimic the geometry, topology, and skeletal structure of cell membranes, but are void of ion channels and the multitude of other embedded components. In previous studies of the effects of electric field on organized assemblies, our laboratory focused on the dynamics of field-induced phase separation in water-in(1) Neumann, E., Sowers, A. E., Jordan, C. A., Eds. Electroporation and Electrofusion in Cell Biology; Plenum: New York, 1989. (2) Chang, D. C., Chassy, B. M., Saunders, J. A., Sowers, A. E., Eds. Guide to Electroporation and Electrofusion; Academic Press: San Diego, CA, 1992. (3) Wilhelm, C.; Winterhalter, M.; Zimmermann, U.; Benz, R. Biophys. J. 1993, 64, 121. (4) Teissie, J.; Tsong, T. Y. Biochemistry 1981, 20, 1548.
Figure 1. Schematic representation of the DOPC liposome system studied, with [Fe(II), inside] ) 2 × 10-3 M and [Ir(IV), outside] ) 5.6 × 10-5 M. The amount of compartmentalized Fe(II) present corresponds to a global Fe(II) concentration of 3 × 10-5 M.
oil microemulsions,5 and structural changes in reverse micelles6-8 and synthetic vesicles.9,10 In recent papers, we have reported the preparation of quantum dots via the electroporation of vesicles11 and studied the timing of pore opening relative to structural changes of the bilayer shell.12 In the present paper, the dynamics of electroporation of unilamellar bilayer vesicles, prepared from the phospholipid dioleoylphosphatidylcholine (DOPC), is investigated by the use of a pore-mediated indicator reaction
Fe(CN)64- + Ir(Cl)62- f Fe(CN)63- + Ir(Cl)63- (1) By entrapping Fe(II) in the interior compartment of the vesicle and placing Ir(IV) in the bulk medium (Figure 1), we can use the spectral changes associated with the electron-transfer Fe(II) + Ir(IV) f Fe(III) + Ir(III) to signal the opening of pores in the separating bilayer. Reaction 1 is a fast (k ) 5 × 108 M-1 s-1),13 essentially irreversible process that had been used previously for monitoring (5) Tekle, E.; Ueda, M.; Schelly, Z. A. J. Phys. Chem. 1989, 93, 5966. (6) Chen, H. M.; Schelly, Z. A. Langmuir 1995, 11, 758. (7) Feng, K.-I; Schelly, Z. A. J. Phys. Chem. 1995, 99, 17212. (8) Gu, J.; Schelly, Z. A. Langmuir 1997, 13, 4256. (9) Asgharian, N.; Wu, X.; Meline, R. L.; Derecskei, B.; Cheng, H.; Schelly, Z. A. J. Mol. Liq. 1997, 72, 315. (10) Asgharian, N.; Meline, R. L.; Schelly, Z. A. in Materials Science of the Cell, Mulder, B.; Vogel, V.; Schmidt, C., Eds.; Materials Research Society: Warrendale, PA, 1998. (11) Correa, N. M.; Zhang, H.; Schelly, Z. A. J. Am. Chem. Soc., submitted for publication. (12) Correa, N. M.; Schelly, Z. A. J. Phys. Chem., submitted for publication.
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Electroporation of Unilamellar Vesicles
Langmuir, Vol. 14, No. 20, 1998 5803
solubilizate exchange between reverse micellar aggregates.14 In contrast, in the present study the mere occurrence rather than the rate of the reaction is relevant since the timing of pore opening is established post facto from the length of the shortest successful pulse applied. Experimental Section Preparation of Liposomes and Passive Entrapment of Fe(II). The phospholipid surfactant dioleoylphospahatidylcholine (DOPC, from Avanti Polar Lipids) was used without further purification. After the chloroform solvent was evaporated off and the film was dried under reduced pressure, large multilamellar vesicles (MLV) were prepared by hydrating the dry lipid film with a 2 × 10-3 M aqueous solution of the molecule to be encapsulated (K4(Fe(CN)6‚3 H2O, Aldrich, 99%) through mixing (Vortex-2 Genie) for about 5 min at room temperature. The resulting solution of MLV, with Fe(II) both entrapped and in the bulk medium, had a lipid concentration of 1 mg/mL. To prepare unilamellar vesicles, the MLV suspension was extruded five times (Extruder, Lipex Biomembranes) through two stacked polycarbonate filters of pore size 200 nm under nitrogen pressure of up to 3.4 atm. The unilamellar nature of pure DOPC vesicles prepared using the extrusion method15 had been confirmed previously9 through measuring the extent of quenching by Mn2+ of their 31P NMR signals.16 The water used in solution preparations was double-deionized, distilled, and filtered (0.2 µm). Gel Chromatography. The replacement of K4Fe(CN)6 in the bulk with (NH4)2Ir(Cl)6 was carried out under balanced ionic strength conditions, using gel filtration17 in a 1.1 cm i.d. × 30 cm glass column (Aldrich) and Sephadex G-25 (Medium, Sigma) as the gel medium. Due to the light-sensitivity of the iridium compound, this and all subsequent manipulations were carried out under exclusion of light, except for inevitable brief illuminations during spectral and birefringence measurements. Prior to use, the gel (6 g) was swelled in 40 mL of the eluent solution for 24 h at room temperature, and degassed under reduced pressure (aspirator) for 2 h with periodic swirling. The composition of the eluent (8 × 10-5 M (NH4)2Ir(Cl)6 plus 0.019 M NaCl (Matheson Coleman & Bell)) was chosen such as to avoid excessive absorption by the iridium compound in subsequent spectral measurements. After the swelled gel was filled into the column, it was saturated with pure, empty vesicles (1 mg/mL lipid with 0.02 M NaCl inside/ outside) by four passages of 5 mL of fresh solution each. With the column thus prepared, 1 mL of the sample (vesicles with Fe(II) inside/outside) was loaded, and 0.5 mL fractions were collected at an elution flow rate of 1 mL/min. The desired vesicles (Fe(II) inside, Ir(IV) outside) were eluted as a sharp peak (2 mL) at the excluded volume V0 ) 8-10 mL, and the extravesicular solute (including Fe(II)) was eluted at the internal volume Vi ) 20-25 mL. The Ir(IV) concentration in the resulting solution to be electroporated was 5.6 × 10-5 M. The chromatography was followed by light scattering measurements to identify the fractions containing the vesicles. Dynamic Light Scattering. The mean hydrodynamic diameter 〈Dh〉 of the vesicles was determined at 25 °C by quasielastic light scattering (QELS) using a Brookhaven BI-200SM multiangle goniometer in conjunction with an argon ion laser (Lexel 85-1, 514.5 nm, 20-80 mW) as light source and a 72channel BI-2030 digital correlator. The performance of the instrument was evaluated by calibration using a polystyrene latex standard of 200 nm diameter. All measurements were carried out at a 90° scattering angle. The QELS data were analyzed by the nonnegative least-squares method. Although gel filtration slightly altered the size distribution of the vesicles (Figure 2), their mean hydrodynamic diameter 〈Dh〉 ) 186 nm (13) Bruhn, H.; Nigam, S.; Holzwarth, J. F. Faraday Discuss. Chem. Soc. 1982, 74, 129. (14) Fletcher, P. D. I.; Howe, A. M.; Robinson, B. R. Faraday Trans. 1 1987, 83, 985. (15) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161. (16) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55. (17) Gel Filtration Principles and Methods, 6th ed.; Pharmacia Biotech: Sweden.
Figure 2. Size distribution of DOPC vesicles prior to (a) and after (b) gel chromatography. In both cases 〈Dh〉 ) 186 nm.
Figure 3. Absorption spectra of DOPC vesicles prior to (multiband spectrum), and after (E), electroporation. The bands A (487.5 nm, ) 4207 cm-1 M-1), B (425 nm), and C (233.5 nm) correspond to Ir(IV) in the bulk, and band D (215 nm) corresponds to Fe(II) entrapped in the vesicular compartments. After electroporation (using nine 200 µs pulses), bands A-D are absent, and a single band (E) due to Ir(III) and Fe(III) emerges. If the vesicles are lysed instead, ∼47% of the absorbance of Ir(IV) in the visible range remains (see text). remained unchanged. On the basis of QELS measurements, electroporation affects neither. Absorption Spectra. The occurrence of the indicator reaction, eq 1, mediated by field-induced pores was detected spectrophotometrically after the electroporation experiments. The UV-vis absorption spectra were recorded at 0.2 nm increment setting on a Gilford Response II computerized spectrophotometer at 25 ( 0.1 °C, using 1 cm path length and stoppered quartz cuvettes. Prior to electroporation, the spectrum of the system is simply the superposition of the spectra of the individual reactants (separated by the bilayer) in water (Figure 3A-D). This solution is free of spontaneous transmembrane reaction for at least 5 h tested. If the barrier between the reactants is removed by lysing the vesicles with Triton X-100 (Fluka), which forms mixed micelles with phospholipids,18 all the entrapped Fe(II) is released and reacts with Ir(IV). Since Fe(II) is present in limiting amount, the bands of Ir(IV) in the visible are reduced only by (18) Jakubowski, H. V.; Penas, M.; Saunders: K. J. Chem. Educ. 1994, 71, 347.
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Figure 4. Oscilloscope trace of the transmitted light intensity (I) due to transient electric birefringence of DOPC vesicles (with Fe(CN)64- encapsulated and Ir(CN)62- in the bulk), relative to an inducing electric field (E) square pulse (insert). Electroporation (EP) occurs at the directional intersection of the arrows. The extra long pulse is chosen only for illustration of the full course of the birefringence signal. ∼53%. From this, the global concentration of the originally entrapped 2 × 10-3 M Fe(II) can be estimated to be ∼3 × 10-5 M. Electroporation Experiments and Transient Electric Birefringence Measurements. The experimental setup and details of the operation of the instrument were described previously.5 The sample solution is placed between a pair of gold-plated stainless steel electrodes (2.5 mm apart) of the thermostated (25 ( 0.5 °C) Kerr cell with an optical path length of 5 cm. The rise and fall times of the high-voltage (up to 2.1 kV) square pulse delivered to the electrodes are
