Anal. Chem. 1903, 65, 178-180
170
The Use of Tetraalkylammonium Ion-Sensitive Electrodes for the Liposome Marker Release Assay Takashi Katsu Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan
A u r k s of tetraalkylammonlum Ion-sensltlve electrodes was constructed wlth the a h of flndlng new Ionic markers wltable for use In the permeablllty away of I l p o m a l membranes. The wries comprbedthe tetramethyl-, tetraethyl-, tetrapropyl-, tetrabutyl-, and tetraamylammonlumIon homologues, and they were compared wlth tetraphenylphorphonlum and K+ lonunrttlve electrodes. Tetraalkylammonlum Ions wlth alkyl chaln lengths shorter than that of a propyl group and octanolwater partltlon coefflclents of lew than 5 X lo-* dld not dgnlflcantly petmeate llporomalmembranes composed of egg phosphatldylchdlne and were trapped stably In the Inner Ilporomal aquoous phase. We selected the tetraethylammonium Ion for further study and verlfled Its valldlty for use as a marker by comluctlng an experiment to determbw antlbody and complement actlvltles by measurlng the lmmunoreactlonlrtduced llporomal membrane permeablllty changes. I n addltlon to being sultable for use In such a conventlonal permeablllty assay, the tetraalkylammonlum Ion-unrltlvo electrodes were wed succeutully to determlno the slzes of channels formed In Ilpowmal membranes.
INTRODUCTION Liposome permeability measurements are fundamental to investigations of the interactions between biologically active substances and membranes. Rapid changes in liposomal permeability can be brought about by calcium-mediated fusion, interaction with drugs, antibody/complement-mediated lysis of antigen-bearing liposomes, and passage through the phase-transition temperature. When measuring such permeability changes, it is important to choose a marker moleculewhich neither passes through intact membranes nor associates with membranes in any way that destabilizes or aggregates them and can be separated easily from liposomes by conventional methods.’ Various markers satisfying these criteria have been developed, for example, glucose, arsenazo111,and carboxyfluorescein, which can be detected by various spectrometric methods.’ Ionic substances also are convenient markers and are detected potentiometrically with the corresponding ionselectiveelectrode~.Z-~ Potentiometric measurement has some inherent advantages over spectrometric methods, Le., simpler procedure, low cost, and ease of continuous monitoring of marker ion release from liposomes. Umezawa and coworkers3~4prepared liposomes loaded with tetraamylammonium ions (TAA+)and monitored the immune lysis-induced release of the TAA+marker with a TAA+-sensitiveelectrode. However, the liposomes had to be freshly prepared for each (1) New, R. R.C. In Liposomes: a Practical Approach; New, R. R. C., Ed.; IRL Press: Oxford, 1990; pp 105-161. (2) DOrazio, P.; Rechnitz, G. A. Anal. Chem. 1977, 49, 2083-2086. (3) Shiba, K.; Umezawa, Y.; Watanabe, T.; Ogawa, S.; Fujiwara, S. Anal. Chem. 1980,52, 161C-1613. (4) Umezawa, Y.; Sofue, S.; Takamoto, Y. Talanta 1984,31,375-378. (5)Katsu, T.; Tanaka, A.; Fujita, Y. Chem. Pharm. Bull. 1982, 30,
1504-1507. 0003-2700/93/0365-0176$04.00/0
experiment, because storage for more than about 12h resulted in scattered data.3 It is likely that, as a result of ita high lipophilicity, TAA+ is released outside the liposomes spontaneously. Such lipophilic cations are particularly useful for membrane potential measurements6 which require establishment of a rapid ionic equilibrium across membranes, and the lipophilic tetraphenylphosphonium ion (TPP+)has been used extensively for such ~tudies.~*8 The prime aim of our study was to find a less leaky ionic marker that could be used for liposomal membrane permeability assays. We chose a series of tetraalkylammonium ions, R4N+,where R = methyl, ethyl, propyl, butyl, and amyl, abbreviated to TMA+, TEA+, TPA+, TBA+, and TAA+, respectively, and compared them with TPP+7 3 and K+5 as marker ions. First, we investigated the relationship between the liposomal permeability to and the octanol-water partition coefficient of each marker ion. We found that permeation of tetraalkylammonium ions with alkyl chain lengths shorter than that of a propyl group through a liposomal membrane composed of egg phosphatidylcholine (PC) was insignificant, which corresponded with their octanol-water partition coefficients of less than 5 x 10-2. Next, we entrapped TEA+in liposomes and proved that it was loaded stably in the liposomes and was superior in this respect to the marker cation, TAA+,used in a previous study.3~4These TEA+-loaded liposomes were used to determine antibody and complement activities. In addition to the conventional permeability assay, tetraalkylammonium ion markers were used to determine the sizes of channels that formed in the liposomal membranes by utilizing their characteristic subtle size increases with increasing alkyl chain lengths. We determined the size of the channel formed by amphotericin B.9@ This channel rendered the membrane permeable to TMA+,whereas it was impermeable to TEA+. Therefore, the diameter of the amphotericin B channel was between that of TMA+ (0.7 nm) and TEA+ (0.8 nm). This method was useful for the precise determination of the sizes of channels formed in liposomal membranes.
EXPERIMENTAL SECTION Reagents. The sources of the reagents used were as follows: tetramethylammoniumchloride(TMACI),tetraethylammonium chloride (TEACl),tetrabutylammonium chloride (TBACI), din-octyl phthalate and 1-octanol from Tokyo Kasei Kogyo, Japan; tetrapropylammonium chloride (TPACl) from Alfa Products, USA; tetraamylammonium chloride (TAACl) from Kanto Kagaku, Japan; valinomyin from Boehringer Mannheim, Germany; tetraphenylphosphoniumchloride (TPPCl),sodium tetrakis[3,5bis(trifluoromethyl)phenyl]borate, and 2-fluoro-2’-nitrodiphenyl ether from Dojindo Laboratories, Japan; poly(viny1 chloride) (6) Rottenberg, H. Methods Enzymol. 1979,55,547-569. (7) Kamo, N.; Murataugu, M.; Hongoh, R.; Kobatake, Y .J.Membrane Biol. 1979, 49, 105-121. (8)Hosoi, S.; Mochizuki, N.; Hayashi, S.; Kasai, M. Biochim. Biophys. Acta 1980,600, 844-852. (9)De Kruijff, B.; Demel, R. A. Biochim. Biophys. Acta 1974, 339, 57-70. (10) Bolard, J. Biochim. Biophys. Acta 1986,864, 257-304.
0 1993 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65,
(PVC) (degree of polymerization, 1020) from Nacalai Tesque, Japan; dipalmitoylphosphatidylcholine (DPPC), dicetyl phosphate (DCP), cholesterol, amphotericin B, and melittin from Sigma,USA;egg phosphatidylcholine (PC)from The Green Cross Corporation, Japan; preserved sheep blood was purchased from The Japan Lamb Corporation and rabbit anti-sheep hemolysin and guinea pig complement (titer, 182 CHa units/mL) were obtained from Kyokuto Seiyaku Kogyo, Japan. All the other chemicals used were of analytical reagent grade. Electrode System. Tetraalkylammonium ion-sensitiveelectrodes were constructed using a PVC-based membrane with the following composition:ll 0.5 mg of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, 60 pL of 2-fluoro-2'-nitrodiphenyl ether, and 30 mg of PVC. The materials were dissolved in about 1mL of tetrahydrofuran and poured into a flat Petri dish (30-mmdiameter), and the solvent was allowed to evaporate at room temperature. The resulting membrane was cut and stuck to a PVC tube (4-mm outer diameter, 3-mm inner diameter) with tetrahydrofuran. A TPP+-sensitiveelectrode was similarly constructed. A PVC-sensor membrane of a K+-sensitiveelectrode contained valinomycin as a neutral carrier and di-n-octyl phthalate as a membrane solvent.12 The electrochemical cell used in this study is represented as follows:11J2Ag,AgCl/internal solution/PVC-sensor membrane/sample solution/l M ",NO3 (salt bridge)/lO mM KCl/AgCl,Ag. Each internal solution comprised a 10 mM solution of the chloride salt of the required marker cation. This electrode system, including the reference electrode,12is compact, and therefore, assay sample volumes were decreased down to 0.3-1 mL. The electromotive force between the pair of Ag/AgClelectrodes was measured with an appropriate field-effect transistor operational amplifier (input resistane > 10l2 Q) and recorded. The selectivity coefficients of the ionsensitive electrodes were determined using the separate solution method with the respective chloride salts (10 rnM).l3 Partition Coefficients. The octanol-water partition coefficients of the tetraalkylammonium chloride series, TPPC1, and KC1 were determined as follows. Each marker substance was dissolvedin water (2 mL), an equal volume of 1-octanolwas added, the mixtures were equilibrated by shaking in a capped test tube for 1h at 37 "C, and the partition coefficientwas determined by measuring the concentration of marker ion in the aqueous phase before and after the addition of octanol using the corresponding ion-sensitive electrode. However, the decreases in the marker ion concentrations, except that of T U + in the aqueous phase were too small to determine, which indicates that the tetraalkylammonium ions, except T U + , distributed almost exclusively in water. In order to determine the concentrations of the small amounts of marker dissolved in octanol, we took 1mL of octanol phase, reextracted it with 1 mL of water, and measured the concentration in the water using the appropriate ion-sensitive electrode and used this value as the concentration in 1-octanol. In addition, Gran's was used for quantification in some cases to reduce the slight experimental error arising from differencesbetween the electrode calibration curves of pure water and octanol-contaminated water. Membrane Permeabilities to Marker Ions. The membrane permeability to each marker ion was evaluated by measuring the ion uptake rate into the inner liposomal aqueous phase induced by the membrane potential. It is well-known that lipophilic cations are drawn inside membranes when the inner membrane potential is negative.'sB In this study, a negative membrane potential was created by adding valinomycin to K+-loaded liposomes suspended in a K+-free medium as follows. Egg PC liposomes were prepared by the reversed-phase evaporation rnethod.l2 Egg PC (10 pmol, 7.7 mg) was dissolved in 1.5 mL of diethyl ether, 1mL of aqueous solution containing 150mM KC1/5 m M 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid (HEPES)-NaOH (pH 7.4) was added, and the mixture was sonicated (Ohtake Works, Japan; Type 5201) for 2 min at 0 "C to obtain (11) Katsu, T.; Sanchika, K.; Okazaki, H.; Kondo, T.;Kayamoto, T.; Fujita, Y. Chern. Pharrn. Bull. 1991,39,1651-1654. (12) Katsu, T.;Kobayaehi, H.;Fujita,Y. Biochirn. Biophys. Acta 1986,
860,608-619. (13) Katsu, T.;Kayamoto, T.;Fujita, Y. Anal. Chirn. Acta 1990,239, 23-27. (14) Gran, G. Analyst 1952, 77, 661-671.
NO. 2, JANUARY
15, 1993
177
a homogeneous emulsion. Then, the diethyl ether solvent was removed completely using a conventionalrotary evaporator under reduced pressure (by water aspirator) at 25 "C, after which a homogeneoussuspension of liposomesremained. The liposomes were centrifuged (105000g, 30 min) and washed twice with 150 mM NaC1/5 mM HEPES-NaOH (pH 7.4) to remove the untrapped K+and the final pellet was suspended in 1mL of 150 mM NaC1/5 mM HEPES-NaOH (pH 7.4). The experiments to determine the marker uptake were carried out at 25 "C as follows. An aliquot of the liposome suspension (0.1 mL) was diluted with 0.2 mL of 150 mM NaC1/5 mM HEPES-NaOH (pH 7.4), and an aqueous solution of the required marker cation (3 pL, final concentration 0.1 mM) was added, followed by addition of an ethanolic solution of valinomycin (0.3 pL, final concentration0.1 pM), which generated a membrane potential due to the difference between the K+ concentrations across the liposomal membrane and induced uptake of the lipophilicmarker cation into the inner liposomal compartment. The addition of an aqueous solution of melittinlSJ6 (3 pL, final concentration 20 pM) abolished this membrane potential and brought about effluxof the accumulated lipophilic cations to the external medium. Complement-MediatedImmune Lysis. Forssman antigen, incorporated into liposomes, was used as a lipid antigen and a lipid mixture containing it was isolated from sheep erythrocytes, as described by Rose and Oklander." About 50 mg of lipid was extracted from 50 mL of preserved sheep blood, which was stabilized and diluted with Alsever's solution (50%, v/v). Multilamellar TEA+-loaded liposomes were prepared, using procedures similar to those described previ~usly.~ The dried thin film of lipid mixture, which contained DPPC (20 pmol, 14.7 mg), cholesterol (15 pmol, 5.8 mg), DCP (2 pmol, 1.1mg), and the above-described lipid extract (2.0 mg), was swollen in 2 mL of 150mM TEACl at 55 "C, centrifuged, and washed three times to remove any untrapped marker. The final lipid mixture pellet was dispersed in 2 mL of 0.15 M NaCl solution. Immunoreaction was carried out at 37 "C as follows. An aliquot (0.1 mL) of the liposome suspension was diluted with an appropriate volume of modified veronal buffer, which comprised 3.13 mM barbital, 1.82 mM sodium barbital, 0.15 mM CaCL0.5 mM MgC12, and 145 mM NaC1.5 The final reaction volume, including the antiserum and complement that were added later, was 500 rL. In order to determine the antibody activities, the liposomes were sensitized initially with various dilutions of rabbit anti-sheep antiserum for 5 min, guinea pig complement (final dilution 150)was added, and the potential changeresultingfrom TEA+efflux was recorded 15 min later in a similar manner conducted previou~ly.~ Similar experiments were carried out to determine the complement activities, in which the final dilution of the antiserum remained constant (1500) and the dilution of complement varied. Channel Size Determination. Both TEA+ and K+ were incorporated simultaneouslyinto liposomesfor the determination of the sizes of the channels formed by amphotericin B. Liposomes were prepared using the reversed-phase evaporation method. Egg PC (10 pmol, 7.7 mg) and cholesterol (2 pmol, 0.77 mg) were dissolved in 1.5 mL of diethyl ether, and 1 mL of an aqueous solutioncontaining 50 mM KCV50mM TEACVl00mM sucrose/5 mM HEPES-NaOH (pH 7.4) was added. Sucrose was added to the inner aqueous liposomal phase, as the reversed-phase liposomes without sucrose floated after the centrifugation procedure, because the density of the TEA+-containing solution was lower than that of water and the addition of a substance, such as sucrose, with a much greater density was necessary to sedimentthe liposomes. The final pellet was suspended in 1mL of 150mM NaC1/5mM HEPES-NaOH (pH 7.4),an aliquot (100 pL) of this liposomalsuspension was diluted with 400 pL required assay mixture solution at 25 "C to produce final suspension media of either (a) 50 mM NaC1/200mMsucrose/6 mM HEPES-NaOH (pH 7.4) or (b) 150 mM NaC1/5 mM HEPES-NaOH (pH 7.4), to which amphotericin B dimethyl sulfoxide solution (10 pL, final concentration 200 pg/mL) was added, and the amounts of effluxed markers were recorded 15min later. The total amounts of marker ions were determined by disrupting liposomes by addition of an aqueous solution of melittinl5Je ( 5 pL, final (15) Katsu, T.;Kuroko, M.; Morikawa, T.;Sanchika, K.; Fujita, Y.; Yamamura, H.; Uda, M. Biochirn. Biophys. Acta 1989,983, 135-141.
178
ANALYTICAL CHEMISTRY, VOL. 65, NO. 2, JANUARY 15, 1993 IogCoctanol ( M
-4
-3
-2
1 -1
02 0
% h
-1
z Y
-2 -7
-6 -5 -4
-3
-2
log ITEA*] or log IK+l ( M )
Flgurr 1. Callbratlon curves of TEA+ and K+ electrodes in 150 mM NaC115 rnM HEPES-NaOH (pH 7.4) buffer at 25 'C.
-3 Flgurr 3. Octanol-water panition experiments with marker ions.
10 min _ I
Melittin
TPP'
TAA'
TBA'
T PA'
Flgurr 2. Valinomycin-induced uptake of marker ions into reversedphase egg PC liposomes at 25 O C . At the time indicated by the fhst
arrow, valinomycin was added: the second arrow Indicates the time when melittin was added. The concentration of marker ion before addition of vallnomycln was expressed as 100% . concentration 20 wM).The following diameters of tetraalkylammonium ions were used for estimationof channelsize:l*TMA+, 0.7 nm; TEA+,0.8 nm; TPA+,0.9 nm.
RESULTS AND DISCUSSION Electrode Response Characteristics. The calibration curves of the TEA+ and K+ electrodes, in a buffer solution comprising 150 mM NaC1/5 mM HEPES-NaOH (pH 7.4), are shown in Figure 1. The detection limits of the TEA+and K+ electrodes were 0.5 and 5 fiM, respectively, which were determined by extrapolating the linear regions and baseline of the calibration curve and the intersection point was defined as the detection limit.13 The slopes in the linear range were 58 mvldecade for the TEA+ and 56 mV/decade for the K+ electrode. The selectivity coefficients of the electrodes are shown in Table I. The K+electrode was not subject to serious interference from tetraalkylammonium ions with alkyl chain lengths shorter than that of a propyl group. The TEA+ electrode was not subject to interference from K+, although interference from ions with alkyl chain lengths greater than an ethyl group was marked. Membrane Permeability and Partition Coefficient. The membrane permeability of each marker ion was evaluated by measuring the membrane potential-induced uptake of the (16)Ohhashi, T.; Katsu, T.; Ikeda, M. Biochirn. Biophya. Acta 1992, 1106,165-170. (17)Rose, H. G.;Oklandar, M. J. Lipid Rea. 1966,6,428-431. (18)Fujishrro, R.; Wada, G.; Tamamushi, R. Gendai Butauri-Kagaku Koza; Tokyo Kagaku Dojin: Tokyo, 1B68;Vol. 8,Chapter 4.
ion into the inner liposomal aqueous phase. Valinomycin is known to evoke a membrane potential when there is a K+ gradient across the liposomal membrane. In our study, liposomes containingKC1solution were suspended in a NaClcontainingmedium. When a lipophilic ion, which permeated the membrane freely, was added to this liposome suspension, movement of the ion inside the liposomes occurred immediately. The uptake rates of the various marker cations are shown in Figure 2. The TAA+ was taken up more rapidly than TPP+, which is used widely for membrane potential measurementa.7~8 The uptake rate decreased markedly as the alkyl chain length of the quaternary ions became shorter and virtually no TPA+ was taken up. In order to confiim that the uptake has been induced by the membrane potential, a membrane disrupting agent, melittin,16J6 was added to the suspension, which abolished the membrane potential and caused efflux of the accumulated lipophilic ions. The octanol-water partition coefficients of each marker cation, at various concentrations,were determined, and their equilibrium distributions are shown in Figure 3. The slopes of all the lines were about 0.5, which indicates that all the marker cations bound strongly to their counter anion C1- in octanol, as discussed below. The partition coefficient is defined as
P = CJC,
(1)
where Gois the marker ion concentration in octanol and C, is that in water. When the marker substance (RX)does not dissociate in octanol, but does so in water (R+ + X-), we can obtain
P = CJC,2 (2) because the concentrations [R+l and [X-I are equal to [RXI. Therefore, in these circumstances the slope of the plot of log C, vs log C, is 0.5. Our results were very close to this value, and the partition coefficients are summarized in Table 11. The membrane permeability experimentashowed that TPA+ did not permeate egg PC liposomal membranes appreciably and ita octanol-water partition coefficient, expressed as the logarithm, was -1.3. Therefore, it is reasonable to assume that the phospholipid membrane was less permeable to substances with partition coefficients below this value. Use of Tetraethylammonium Ions for the Marker Release Assay. We selected TEA+as a marker ion and used it for the determination of antibody and complement activities. In previous studies,3-6 T U +and K+were used for this assay, but we found that TAA+was not particulary euitable
ANALYTICAL CHEMISTRY, VOL. 65, NO. 2, JANUARY 15, 1993
Table I. Selectivity Coefficients of TEA+ and K+ Electrodesa Na+ K+ TMA+ TEA+ -1.8 TEA+ electrode -4.0 -3.9 -3.0 -3.0 K+electrode -2.9 a
Expressed as the logarithm of the selectivity coefficient, log
179
TPA+
TBA+
TAA+
TPP+
1.7 -1.9
3.3 -0.2
5.0 1.9
4.6 1.0
or log
-
I
il
I
-*ooc 1
1
1
104 103 Ant iserurn d i l u t i o n
105
Co rnpl erne nt d i Iu t i o n
Figure 4. Potential vs concentratlon graphs of (a)antiserum and (b)
complement. Table 11. Octanol-Water Partition Coefficients. K+ TMA+ TEA+ TPA+ TBA+ TAA+ -3.4 a
-2.7
-2.1
-1.3
0.4
1.7
TPP+ 0.4
Expressed as the logarithm of the partition coefficient, log P.
because liposomes were higly permeable to it. As for K+, although it was trapped stably in liposomes, the level of K+ in the antiserum and complement had to be reduced beforehand by dialysis5 to avoid it contaminating the assay system. These disadvantages could be overcome by using a new marker, TEA+, which did not leak appreciably, even after storage in a refrigerator at 4 OC for 2 weeks. The TEA+sensitive electrode did not suffer serious interference from Na+, K+, or other cations present in biological fluids, which is evident from the selectivity coefficients of the electrode presented in Table I. The antibody and complement activities, based on the potential changes caused by TEA+ efflux from liposomes as a result of immunoreaction, determined are shown in Figure 4. The amount of TEA+ effluxed was dependent on the dilution of both antiserum and complement; in our system, the immune response could be observed with dilutions as low as 1:5 X 105 of antiserum and l:lO3 of complement. These results demonstrated that the combination of TEA+-loaded liposomes and a TEA+-sensitive electrode is suitable for measuring the activities of immune reagents. Channel Size Determination. Liposomes loaded with both K+and TEA+ were prepared and used to determine the sizes of channels formed in the liposomes by monitoring the difference between the rates of K+ and TEA+ effluxes. No cross-interference of the K+and TEA+ electrodes during the determination process was observed,as discussed above. The amphotericin B-induced K+ and TEA+ effluxes from liposomes suspended in the medium containing 50 mM NaC1/ 200 mM sucrose/5 mM HEPES-NaOH (pH 7.4) are shown in Figure 5a. The medium contained a high concentration of sucrose to protect the liposomes against osmotic lysis,l9920 which leads to TEA+ efflux despite formation of small-sized channels. Such osmotic lysis was actually observed in the next experiment (Figure 5b). The amphotericin molecules, (19) Katsu, T:;Ninomiya, C.; Kuroko, M.; Kobayashi, H.; Hirota, T.; Fujita, Y.Btochtrn. Biophys. Acta 1988,939, 57-63. (20) Oku, N.; Nojima, S.; Inoue, K.Biochirn. Biophys. Acta 1980,595, 277-290.
s
L
I v
I
0.7
0.8
Marker diameter
0.9 (nm)
Figure 6. Relationship between the percent efflux Induced by amphotericin B and the tetraaikyiammonium ion diameters.
which are known to form channels in cholesterol-containing liposomes?JO induced efflux of K+, but not of TEA+. When the liposomal membrane was disrupted by adding melittin,16Js a large potential change due to TEA+ efflux was observed, which indicated that TEA+was trapped in the liposomes and verified that amphotericin B only induced K+ efflux from liposomes. Liposomes were also suspended in medium containing 150 mM NaC1/5 mM HEPES-NaOH (pH 7.4). Under these conditions, if the ionophore can open a channel which is large enough to permit the permeation of Na+ present in the external medium, the osmotic pressure of the internal aqueous phase becomes higher than that of outer one and the liposomes burst,19320 resulting in TEA+ efflux. Amphotericin induced TEA+efflux, which was conducted by Na+ influx (Figure Sb). These two experiments with and without sucrose in the medium afforded information that the size of the channels opened by the amphotericin molecules was between that of Na+ and TEA+. In order to determine the channel size more precisely, we prepared liposomes loaded with TMA+instead of TEA+and performed the same experiment under conditions of osmotic protection. Simultaneous efflux of TMA+and K+ was observed clearly. We also prepared liposomes loaded with TPA+, the ionic size of which was larger than that of TEA+, but this ion was not released as we expected. The
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 2, JANUARY 15, 1993
relationship between the percent efflux and marker ion sizes are shown in Figure 6. It was clear that the diameter of the channels made by the amphotericin molecules was between that of TMA+ (0.7 nm) and TEA+ (0.8 nm), which accorded well with the size reported previ~usly.~ Therefore, this assay can provide detailed information about the sizes of channels formed in liposomal membranes. Concluding Remarks. In this study, we have shown that the use of a series of tetraalkylammonium ions is suitable for the assay of liposomalmembrane permeability, and combining marker ions of various sizes enabled us to determine the precise size of channels formed in liposomal membranes.
ACKNOWLEDGMENT We wish to thank Ms.Kyoko Ito, who carried out some of the preliminary experiments that were extended in this study, and Ms. Tokiko Kawakami and Mr. Takashi Kayamoto for their technical assistance. This work was supported by a Grant-in-Aid from the Shimadzu Science Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. RECEIVED for review July 1, 1992. Accepted October 22, 1992.
