J. Phys. Chem. B 1998, 102, 9319-9322
9319
Dynamics of Electroporation of Synthetic Liposomes Studied Using a Pore-Mediated Reaction, Ag+ + Br- f AgBr 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: May 26, 1998; In Final Form: August 11, 1998
The dynamics of electric field-induced transient pore formation (electroporation) is studied in 178 nm diameter synthetic unilamellar bilayer liposomes, prepared from the surfactant dioleoylphosphatidylcholine (DOPC). With Ag+ ions entrapped in the interior compartment of the vesicle and Br- ions placed in the bulk medium, details of the poration process are revealed through the pore-mediated production of (AgBr)n clusters which are detected spectrophotometrically in the UV. The field-induced elongation of the spherical bilayer shells to prolate ellipsoids is monitored through the transient birefringence of the system. Effects of the field strength E, pulse length, and number of the high-voltage square pulses applied are investigated, and threshold values of these parameters necessary for electroporation are established. It is found that polarization and elongation of the liposomes precede pore opening by 30 µs, and that 75% of the entrapped Ag+ ions are ejected during a single pulse into the bulk solution, where the indicator reaction Ag+ + Br- f AgBr takes place.
Introduction Electroporation is the field-induced formation of reversible, transient holes in surfactant bilayers such as cell membranes and liposomes or vesicles.1 Since the temporary pores allow the alteration of the interior content of closed bilayer shells, electroporation techniques have been routinely used for gene transfection and insertion of biologically active molecules into cells, cell membranes, and vesicles (for drug delivery). The homogeneous electric field E externally applied to the suspension is typically of the form of a high-voltage dc square pulse or exponentially decaying2 pulse, and radio frequency (RF ac)3 or bursts of high-frequency bipolar oscillations.4,5 A crucial issue in practical applications is finding the proper conditions (concentrations, applied field strength E, pulse length, etc.) which result in pores of suitable size and lifetime, without permanently damaging the bilayer and fusing neighboring membrane shells.6 Main sources of the challenge to identifying the proper conditions are the complex and diverse composition and structure of various biological cell membranes, and the incomplete understanding of the events involved in the fieldinduced poration process.7 The situation, of course, is much simpler in the case of pure synthetic vesicles which can be prepared with a uniform composition, and are void of structural complications such as ion channels and embedded proteins. Prior to perturbation by the applied field, vesicles undergo thermal shape fluctuations with a time average spherical shape of the bilayer shell. The extent of fluctuations are mainly governed by the bending elasticity8 κ of the membrane. Upon application of the field at time t ) 0, its primary effects are the appearance of a potential difference ∆V (on top of the natural membrane potential) between the exterior (Ve) and interior (Vi) boundaries of the bilayer, and the global polarization of the system. For a spherical shell with radius a much greater than the membrane thickness, the buildup of ∆V at polar coordinate * Author to whom correspondence should be addressed.
θ (with respect to -E) due to a homogeneous, constant applied field is given by9
∆V(θ,t) ) Ve - Vi ) 1.5 aE cosθ [1 - exp(-t/τp)] f
(1)
τp ≈ aCm(ri + re/2)f
(2)
f ≈ [1 + aGm(ri + re/2)]-1
(3)
where Cm is the membrane capacitance per unit area, ri and re are the specific resistances of the interior and exterior media, and Gm the membrane conductance per unit area (assumed to be uniform over the entire shell surface). Prior to opening of pores Gm is usually negligible (f ≈ 1), and ∆V is greatest in the polar regions of the shell facing the electrodes, where eventually the pores are formed. The relaxation time of polarization τp refers to a specific polarization process in the limit of small perturbation (E f 0). The overall polarization, however, entails electronic and interfacial (Maxwell-Wagner) polarization as well as that resulting from the partial alignment parallel to E of the permanent and induced-dipolar moieties of the constituent surfactant molecules. These processes occur over different time scales, typically in the picosecond to microsecond range. In addition, the spherical shell is elongated to a prolate ellipsoid with its major axis parallel to E, and the induced-dipolar and instantaneously dipolar shells are also partially aligned in the direction of the field. The resulting structural anisotropy of the solution is manifested in a transient birefringence which can be monitored. The underlying electromechanical deformation of vesicles and the rotational relaxation of the dipoles typically occur on the microsecond time scale.10,11 Not surprisingly, the nature and dynamics of the overall polarization and deformation processes are similar to those observed in other spheroid organized assemblies such as reverse micelles and water-in-oil microemulsions.12 Although it is generally known that the experimental parameters must be within certain limits for electroporation to occur
10.1021/jp9823648 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/27/1998
9320 J. Phys. Chem. B, Vol. 102, No. 46, 1998
Correa and Schelly
Figure 1. Schematic representation of the DOPC liposome system studied, with [Ag+, inside] ) [Br-, outside] ) 5 × 10-3 M.
without bursting different cell membranes1,6 or planar black lipid membranes (BLM),7 the aim of the present study is to establish the timing of pore opening relative to the above-mentioned polarization and deformation events in synthetic liposomes. Poration is detected via the formation of product of the diffusioncontrolled reaction
Ag+ + Br- f AgBr
(4)
with the reactants originally segregated by the membrane. It is shown that the onset of vesicle elongation precedes the formation of pores of a size that permits the passage of Ag+ ions. Experimental Section Preparation of Liposomes and Entrapment of Ag+ Ions. The phospholipid surfactant dioleoylphospahatidylcholine (DOPC, from Avanti Polar Lipids) was used without further purification. After evaporating the chloroform solvent and drying under reduced pressure, large multilamellar vesicles (MLV) were prepared by hydrating the dry lipid film with a 5 × 10-3 M aqueous solution of the molecule to be encapsulated (AgClO4, Fluka, 99.9%) through mixing (Vortex-2 Genie) for about 5 min at room temperature. The resulting solution of MLV with Ag+ both entrapped and in the bulk medium had a lipid concentration of 4 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 method13 had been confirmed previously10 through measuring the extent of quenching by Mn2+ of their 31P NMR signals.14 To replace the Ag+ ions outside the vesicles by Br-, equal volumes of the vesicular solution and a 0.02 M aqueous solution of KBr (Fluka, 99.9%) were mixed. The AgBr precipitate was separated by centrifugation (Hettich EBA 12R) for 90 min at 23 900 g. The resulting system (Figure 1), with 5 × 10-3 M Ag+ inside and 5 × 10-3 M Br- outside the liposomes, was free of spontaneous transmembrane reaction for up to a week tested. Notwithstanding, fresh solutions were used in the electroporation experiments. Due to the zwitterionic polar headgroup of DOPC, the surfactant contributes no free ions to the solution. All manipulations were carried out under the exclusion of light, except for inevitable brief illuminations during birefringence and spectral measurements. The water used in solution preparations was double-deionized, distilled, and filtered (0.2 µm). Size of the Liposomes. The mean hydrodynamic diameter of the vesicles was determined at 25 °C by quasi-elastic light scattering (QELS) using a Brookhaven BI-200SM multiangle goniometer in conjunction with an argon ion laser light source (514.5 nm, 20-80 mW) and a 72-channel BI-2030 digital correlator. The performance of the instrument was evaluated
Figure 2. Size distribution of DOPC vesicles (〈Dh〉 ) 178 nm) containing entrapped Ag+ ions, and Br- ions in the bulk, at 25 °C.
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. Neither centrifugation nor electroporation was found to affect the monodisperse size distribution and the mean hydrodynamic diameter ) 178 nm determined (Figure 2). Electroporation Experiments and Transient Electric Birefringence Measurements. The experimental setup and details of the operation of the instrument were described previously.15 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
