Anal. Chem. 1996, 68, 3434-3440
Liposome Behavior in Capillary Electrophoresis Matthew A. Roberts,†,‡ Laurie Locascio-Brown,*,† William A. MacCrehan,† and Richard A. Durst‡
Chemical Sensing and Automation Technologies Group, Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001, and Bioanalytical Laboratory, Institute for Comparative and Environmental Toxicology, Cornell University, Geneva, New York 14456-0462
The behavior of liposomes in capillary electrophoresis is studied for the purpose of developing a potential method for characterizing liposomes prepared for use in industrial and analytical applications. This study characterizes the electrophoretic behavior of liposomes under various conditions to provide information about electrophoretic mobility and liposome-capillary surface interactions. The results of this method are compared with the results obtained using traditional laser light-scattering methods to obtain size information about liposome preparations. Additionally, reactions of liposomes and the surfactant n-octyl-β-D-glucopyranoside are performed off-line in bulk solution experiments and on-line in the capillary. Automated delivery of lysis agents by multiple electrokinetic injections is demonstrated as a general method for inducing on-capillary reactions between liposomes and other reagents. Furthermore, some preliminary evidence on the use of liposomes as a hydrophobic partitioning medium for analytical separations is presented. In aqueous solution, phospholipids self-assemble to form a lipid bilayer membrane which surrounds an aqueous cavity forming a spherical structure known as a liposome (also, lipid vesicles) as shown in Figure 1. Phospholipid molecules are composed of a polar head group and a double hydrophobic tail consisting of a diacyl chain backbone. The formation of liposomes, like the formation of micelles, is due to the energetically favorable association of the hydrophobic portion of the molecules to reduce interaction with the aqueous solvent. Liposomes have two distinct domains whose composition may be controlled: the liposome membrane itself and the interior aqueous cavity. Altering the constituents of the bilayer greatly affects liposome characteristics such as stability, surface charge, size, lamellarity, and reactivity.1 The interior aqueous cavity can be used for stable entrapment of water-soluble chemicals that may be released by lysis of the liposome. Control of the composition of the aqueous cavity may also be used to influence the size and stability of liposomes. It is important to characterize the properties of liposomes for several reasons: (1) liposomes are increasingly being used in commercial products, and the properties of these liposome preparations need to be monitored for quality control; (2) the liposome structure is a good model for cellular membrane behavior; and (3) liposomes have many characteristics which allow them to be used as sensitive reagents for analytical detection.2-4 †
National Institute of Standards and Technology. Institute for Comparative and Environmental Technology. (1) New, R. R. C. In Practical Approach Series; Rickwood, D., Hames, B. D., Eds.; Oxford University Press: New York, 1990, pp 301. ‡
3434 Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
Figure 1. Cross-sectional depiction of a liposome. Inset shows the phospholipid bilayer in some detail. Cholesterol is sequestered between phospholipid molecules in the bilayer. The head group of the cationic dye molecule, DiI-C18(C5), is represented as (9). Other phospholipid molecules in the bilayer are depicted as (b). The chemical structure of the dye molecule is also shown.
Because of their ability to entrap large quantities of the active ingredient either in the lipophilic bilayer or in the aqueous cavity, liposomes have found commercial use as encapsulants of cosmetic and pharmaceutical formulations. Such applications require rapid analytical methods, for the quality control of liposome preparations, that can characterize parameters such as vesicle size, charge, heterogeneity, and stability. Since liposomes are also a good model for cells,5,6 investigating their electrophoretic behavior will help to establish the capability of capillary electrophoresis (CE) for the more general characterization of biological vesicles. Recently several reports have focused on the use of CE for single-cell characterization. To date, all of the reports on single-cell analysis by CE have involved disruption of the cell membrane and quantitation of intracellular contents.7-11 Useful information might also be obtained from the migration characteristics of intact cells using CE. For example, interactions with cell surface receptors could potentially be measured by monitoring changes in the electrophoretic migration time following incubation with an appropriate ligand. (2) Plant, A. L.; Brizgys, M. V.; Locasio, L. E.; Durst, R. A. Anal. Biochem. 1989, 176, 420-426. (3) Locascio-Brown, L.; Plant, A. L.; Horvath, V.; Durst, R. A. Anal. Chem. 1990, 62, 2587-2593. (4) Reeves, S. G.; Rule, G. S.; Roberts, M. A.; Edwards, A. J.; Durst, R. A. Talanta 1994, 41, 1747-1753. (5) Kitano, H.; Kato, N.; Ise, N. Biotech. Appl. Biochem. 1991, 14, 192-201. (6) Hauser, H.; Gains, N. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1683-1687. (7) Hogan, H. L.; Yeung, E. S. Anal. Chem. 1992, 64, 2841-2845. (8) Chang, H. T.; Yeung, E. Anal. Chem. 1995, 67, 1079-1083. (9) Kristensen, H. K.; Lau, Y. Y.; Ewing, A. G. J. Neurosci. Methods 1994, 51, 183-188. (10) Schultz, N. M.; Huang, L.; Kennedy, R. T. Anal.Chem. 1995, 67, 924-929. (11) Xue, Q.; Yeung, E. S. J. Chromatogr. 1994, 661, 287-295. S0003-2700(96)00328-9 CCC: $12.00
© 1996 American Chemical Society
Finally, the use of liposomes makes new approaches to analysis possible. Liposomes have been modified with surface receptors for use as affinity chromatography matrices12 and as immunoassay reagents.3,4,13-15 In addition, we believe that liposomes could function as pseudostationary phases or active surfaces on which to perform analytical separations in CE. Liposomes may be engineered (1) to have a positive, negative, or zwitterionic surface charge to act as ion exchange membranes, (2) to incorporate different lipid combinations for retention of hydrophobic compounds, or (3) to include a surface-binding site for specific receptor-ligand interactions. Liposomes may be useful in CE immunoassays much like latex microspheres16 since the electrophoretically induced migration of the sample zone through an injected liposome zone can be used to evoke a chemical reaction. This potential can only be realized by first characterizing several key features of liposome behavior in CE. To our knowledge, this is the first effort to characterize the behavior of liposomes by CE. This study includes the evaluation of vesicle-wall adsorptive phenomena, bilayer stability in the presence of an electric field, and solvent- and surfactant-induced liposome lysis using CE. This report also evaluates the potential of CE for characterizing liposome populations as a function of pH. The use of liposomes as a pseudosolid phase for small molecule retention is explored using riboflavin as a model analyte. EXPERIMENTAL SECTION Reagents. Dimyristoylphosphatidylcholine (DMPC), dicetyl phosphate (DCP), and cholesterol were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). The 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine [DiI-C18(C5)] was obtained from Molecular Probes (Eugene, OR). Dibasic sodium phosphate (Na2HPO4‚7H2O), monobasic potassium phosphate (KH2PO4), and n-octyl-β-D-glucopyranoside (OGP) were obtained from Sigma Chemical Co. (St. Louis, MO). The two phosphate salts were used to prepare a 9.5 mmol L-1 solution with a pH of 7.4. This solution was used as the running buffer in the CE system and was used to dilute liposomes and all other reagents, unless otherwise specified. Preparation and Characterization of Liposomes. Liposomes were produced by a modified reverse-phase method similar to that previously described.15 A stock solution of 36 µmol of total lipids was prepared by dissolving DMPC, DCP, DiI-C18(C5), and cholesterol (molar ratio of 7:2:2:1) in 8 mL of chloroform-ethanol (5:3 v/v). The solution was then sonicated using an ultrasonic cleaner (125 W, 117 V, 50/60 Hz, Branson Cleaning Equipment Co., Shelton, CT) under nitrogen at 45 °C. While evaporation of the organic solvent proceeded, four 1-mL aliquots of buffer were added at 5-min intervals. The solution was then sonicated for an additional 5 min to ensure maximum solvation of the lipid components. During this process, there was a slight precipitation of the DiI-C18(C5), which was the least soluble component of the lipid mixture. The resulting liposome suspension was filtered using polycarbonate syringe filters (PCTE, Poretics Corp., Livermore, CA). Liposome preparations were passed once through 3and 0.4-µm filters, and then five times through a 0.2-µm filter. (12) Lundahl, P.; Yang, Q. J. Chromatogr. 1991, 544, 283-304. (13) Kim, C. K.; Lim, S. J. J. Immunol. Methods 1993, 159, 101-106. (14) Roberts, M. A.; Durst, R. A. Anal. Chem. 1995, 67, 482-491. (15) Siebert, T. A.; Reeves, S. G.; Durst, R. A. Anal. Chim. Acta 1993, 282, 297-305. (16) Rosenweig, Z.; Yeung, E. Anal. Chem. 1994, 66, 1771-1776.
Approximately 2.5 mL of solution was recovered and then dialyzed (Slide-A-Lysers, Pierce, Rockford, IL) three times in phosphate buffer. The dialysate absorbance was monitored at 650 nm, and no free dye was detected. After preparation, the liposomes were stored at 4 °C in the dark until needed. The mean diameter of liposomes was measured by laser diffraction particle size analysis (Coulter Model N4 submicron particle analyzer, Coulter Corp., Hialeah, FL). The amount of phospholipid present in the liposome preparation was determined by measuring the phosphorus content using inductively coupled plasma optical emission spectroscopy. Calculations for the liposome concentration based on phosphorus content were performed as previously described.15 Unless otherwise noted, all liposome solutions were diluted from stock to a concentration of 6 × 107 liposomes µL-1 or 0.6 nmol of liposomes L-1. Capillary Electrophoresis System and Capillary Conditioning. The CE system (Dionex, Sunnyvale, CA) was equipped with a variable-wavelength absorbance detector operating at 650 nm with a tungsten lamp. Samples were introduced using electrokinetic, pressure, or gravity injection. Data acquisition and handling were performed using the software supplied with the system. The 75-µm-i.d. polyimide-coated silica capillary tubing used for these experiments was underivatized and was cut to various lengths depending on experimental requirements. The polyimide coating at the detector window was removed by heating in a flame followed by a methanol wipe. New capillaries were conditioned by manually injecting a 0.5 mol L-1 sodium hydroxide solution for 10 min followed by a standard wash program. Great care was taken to ensure that no sodium hydroxide remained in the system after conditioning. The standard wash program consisted of 2-min pressure injections of distilled water, ethanol, distilled water, and phosphate buffer with a 1-min wait between each injection. After the wash program was complete, the capillaries were ready for liposome migration studies. On subsequent days, the capillary was cleaned using the standard wash program alone, i.e., without the sodium hydroxide conditioning. A conductivity of 1.82 mS was measured for the phosphate running buffer, and all subsequent buffer preparations were checked for similar conductivity as a measure of correct preparation. Detergent Lysis of Liposomes. Solutions of liposomes were made containing 0, 0.0001, 0.1, 2, 5, 10, 20, and 100 mg mL-1 OGP. These solutions were mixed, allowed to incubate for 20 min, and then gravity injected at a height of 3 cm for 3 s into a 75-cm capillary. A voltage of 30 kV was applied, and the absorbance was measured at 651 nm for 10 min. Capillaries were rinsed with running buffer for 3 min between each injection. Experiments were also performed to induce lysis of injected liposomes by on-capillary mixing with OGP. Liposomes were gravity injected at 3 cm for 3 s, followed by electrokinetic injection of buffer at 10 kV for 6 s (to ensure separation of zones at time zero) and then electrokinetic injection of OGP at 10 kV for various amounts of time. Solutions were injected with 0, 0.0001, 0.1, 2, 7.5, 10, 35, and 100 mg mL-1 OGP for 6 s to determine liposome stability as a function of OGP concentration. The 10 mg mL-1 solution was injected for 0, 12 , 24 , 48, and 96 s at 2 kV to elucidate the effects of contact time on subsequent liposome lysis. Measurement of Retention of Model Marker by Interaction with Liposomes. Riboflavin was used to observe the retention behavior as analyte interacts with the liposomes in the Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
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Table 1. Dil-Tagged Liposome Characteristicsa mean diameter ((1 SD)b,c volume liposome conc DiI-C18(C5) surface densityd (17 mol % of bilayer) diffusion coefficientb apparent electrophoretic mobility of mean (µa) Effective electrophoretic mobility (µe)c,e,f charge/sizeg liposome charge stability
355 ( 210 nm 2.34 × 10-11 µL-1 4.56 × 108 liposomes µL-1 2.75 × 106 molecules ip-1 1.5 × 10-8 cm2 s-1 4.97 × 10-4 cm2 s-1 -3.93 × 10-4 cm2 s-1 -7.41 × 10-5 C nm-1 -821 >5 months
a Measurements performed on liposomes after storage at 4 °C in PBS. b Measurements made by laser light-scattering particle size analysis. c Standard deviations correspond to the overall size distribution and not to uncertainty. Relative standard deviation of the mean is 1.5 kV/cm).18 Although present experiments were conducted at considerably lower field strengths, the field might still induce some lysis and produce lipidic precipitates that lead to spike formation in a small percentage of the liposome population especially when the bilayer is weakened by the presence of detergents or pH extremes. The formation of a few large particles may also have been caused by disruption of the bilayer membrane through loss of lipid material to the capillary wall. The loss of lipid from the membrane may have rendered it unstable favoring either agglomeration or disruption, leading to the formation of particulate material. Instead of an artifact produced in the CE experiment, another possible explanation for the appearance of spikes is that some precipitated lipidic particles or agglomerated liposomes form during storage of the liposomes that are not readily detectable in light-scattering experiments because they represent a very small subpopulation of particles in the solution. If this is true, then the CE method may be providing more information about the liposome mixture than is currently available by the light-scattering method. The particulate material detected by CE may be produced both under normal storage conditions and in the capillary. This notion is supported by the observation of spiked peaks in electropherograms of liposomes prepared under conditions known to cause agglomeration in bulk solution. When the solution pH is significantly lower than that of the stable liposome preparation, charge shielding of negative phospholipid head groups from buffer cations may occur. This has been shown in other studies to lead to both fusion and aggregation of sensitive bilayer vesicles.21-23 When our DiI-liposome preparations were (21) Allen,T. M.; Hong, K.; Papahadjopoulos, D. Biochemistry 1990, 29, 29762985.
Figure 5. Concentration of OGP versus the percent of liposome peak area. The percent liposome peak area is calculated as the (measured liposome area/original liposome area) × 100. The results obtained by incubation of liposomes in bulk solution with different concentrations of OGP are graphically represented by ([). Results obtained by electrokinetic injection of OGP are depicted by (~). The OGP critical micelle concentration (OGP cmc) is also shown. Inset shows the effect of varying electrokinetic injection time.
incubated in acidic buffer (pH
