Langmuir 1992,8, 2843-2845
2843
Notes Comparison of Barrier Properties of Bilayers Derived from an Ion-Paired Amphiphile with Those of a Phosphatidylcholine Analog' Yong-Chan Chung and Steven L. Regen'
both contain two hydrocarbon chains of identical length. Because we wanted to avoid ambiguities resulting from ionic interactions between an entrapped pemeant and the vesicle bilayer, an uncharged sugar molecule, ~ l ~ l s u c r o e e , was chosen for this study.'+
Department of Chemistry and Zettlemoyer Center for Surface Studies, Lehigh University, Bethlehem, Pennsylvania 18015
0
Hiroyuki Fukuda and Koji Hirano Nagoya Municipal Industrial Research Institute, 4-41 Rokuban 3-chome, Atsuta-ku, Nagoya 456,Japan Received June 2, 1992. In Fino1 Form: July 15, 1992
Introduction Single-chain cations that are paired with single-chain anions (i.e., ion-paired amphiphiles, IPA's) represent a novel class of bilayer-forming surfa~tants.~-~ In a sense, such molecules may be viewed as hybrids of micelle- and lamella-forming surfactants. Previously, it has been proposed that the ability of an IPA to form bilayers results from an overall reduction in head group area, due to electrostatic attraction between the anionic and cationic components? Exactly how tightly packed such surfactants can become in the gel and liquid-crystalline states, as a consequence of electrostatic and van der Waals forces, remains to be clarified. The issue of membrane packing is important not only from a fundamental standpoint, but also because it bears directly on the potential utility of IPA's for those applications that take advantage of their barrier properties, e.g., time-release carriers. In this paper we define the barrier properties and phase transition behavior of bilayers derived from one representative IPA, by use of high-sensitivity differential scanning calorimetry (ha-DSC) and by permeation measurements. Specifically, we have examined the gel to liquid-crystalline phase transition properties of bilayers made from cetyltrimethylammonium palmitate (CTAF'), and also their barrier properties toward [14Clsucrose. Analysis of the phase transition properties of a bilayer, via ha-DSC, defines the difference in intermolecularforces between its solidlike and liquidlike states. Permeation measurements provide a measure of membrane packing at a single temperature, and in any phase. In order to place our results with CTAP into perspective, we have compared them with those obtained from an analogous bilayer-formingphosphatidylcholine,1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC)? Our reason for c h m ing DPPC as a frame of reference stems from the fact that CTAF' and DPPC are both zwitterionic in character and
* Towhom correspondenceshould be addressedat the Department
of Chemistry, Lehigh University. (1) Supported by the National Science Foundation (Grant CHE9022581) and by the U.S. Army Research Office (Grant DAAL03-91-G0081). (2) Fukuda, H.; Kewata, K.; Okuda, H.; Regen, S. L. J. Am. Chem. SOC.1990,112, 1635. (3) Hirano, K.;Fukuda, H.; Regen, S. L. Langmuir 1991, 7, 1045. (4) Kaler. E. W.;Murthv, A. K.; Rodrirmez, . B. E.: Zaeadzineki, J. A. N. Science 1989,2&, 137i. ( 5 )Jokela, P. Jonsson, B.; Khan,A. J. Phys. Chem. 1987, 91, 3291. (6) Eibl, H. In Liposomes: From Physical Structure to Therapeutic Applications; Knight, C. G., Ed.; EleeviedNorth Holland Biomedical Press: Cambridge, 1981; p 19.
CTAP
DPPC
Materials and Methods GeneralMethods. Unlessstated otherwise,all chemicalsand reagents were obtained commercially and used without further purification. l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)was purchased from Avanti Polar Lipids ( B w h a m , AL). [W]Sucrose (250mCi/mmol, 20% ethanol solution) waa obtained from ICN Laboratories. Water was purified using a Milli-Q system consisting of one carbon, two ion-exchange, and one Organex-Q cartridges. Nitrogen analysis was determined using methodsthat were similar to those previouslydeacrihed,'OJ1 using DPPC and CTAP as standards for calibration curves. Dynamic light scattering was carried out by use of a Nicomp 270 submicrometer particle size analyzer? Specific methods used for liquid scintillation counting have been reported.? Cetyltrimethylammonium Palmitate (CTAP).An ionexchange resin (Bio-Rad, AGl-X8,hydroxide form, 3.2 mequiv/ g) was extractedwith methanol(Soxhlet)for 12h under nitrogen, and then packed into a column (2 X 7 cm). Cetyltrimethylamrecrystallid from monium bromide [0.500 g, 1.37 "01, methanoUether (Ul,v/v)l was dissolved in 20 mL of methanol and passed through the column; the column was then rinsedwith 60 mL of methanoL To the combined methanolic solution was of palmitic acid; subsequent removal added 0.351 g (1.37"01) of solventunder reduced pressure affordeda colorlesssolidwhich was recrystallized twice from methanoVethy1 acetate and then dried [12h, 23 OC (0.05 " H g ) ] to give 0.70 g (1.30mmol,96%) of CTAP: mp 149-153 OC; lH NMR (CDCb, 500M H Z ) b 0.90 (t, 6H, CHS),1.15-1.47 (s,50 H, CHd, 1.60 (m, 2 H, CH&H&=-O), 1.75 (m, 2 H,CHzCHzN+),2.18 (t, 2 H, CHzCO), 3.45 (e, 11 H, Anal. Calcd for CSSH~SNOT~HZO C, 75.33;H, CHZN(CHS)S). 13.54,N, 2.51. Found C, 75.30;H, 13.24;N, 2.61. Jhmination by IR (KBr disk) confirmed the complete disappearanceof the carboxylic acid moiety (1710cm-9 and the appearance of the ammonium carboxylate group (1560cm-'). DifferentialScanningCalorimetry(DSC).Multilamellar vesicleswere prepared by dispersing a thin lipid film (1.6mg) in 1.6 mL of water, and examined by high-sensitivity DSC using a Microcal MC-2instrument (Amherst, MA). Filma made from DPPC were cast from CHCls;those made from CTAP employed CHCWCHsOH Wl). Qpically, a thin film was heated in water (55 "C) for 0.5 h, followed by vortex mixing for 4 min. This procedure waa then repeated one time. Heating were recorded between 10 and 65 OC at a scan rate of 30 deg/h. T h e calorimetric data were analyzed to yield surfactant excess heat (7) Stefely,J.; Markowitz, M. A.; Regen, S. L. J. Am. Chem. SOC. 1988, 110, 7463. (8) Carmona-Ribeiio, A. M.; Chaimovich,H. Biochim. Biophys. Acta 1983, 733, 172. (9) Carmona-Ribeiio,A. M.; Yoehida, L. S.;Seeso, A,; Chaimovich,H. J. Colloid Interface Sci. 1984,100,433. (10) Miller, G.; Miller, E. Anal. Chem. 1948,20,481. (11) Stefely, J. Ph.D. Thesis, Marquette University, 1990.
Q743-7463/92/2408-2843$Q3.00/Q0 1992 American Chemical Society
2844 Langmuir, Vol. 8,No. 11, 1992
Notes
Table I. Comparison of Vesicles Made from CTAP and DPPC
W P b (cm/s) T m ("0 CTAP DPPC
41.6 41.6
(kcal mol-') 6.55 f 0.17 8.91 f 0.16
AH&
CU' (molecules) 210 f 5 310 25
*
captured volume (L/mol) 1.4 f 0.09 1.5 f 0.18
23 O C 4.3 f 1.7
50~"C ___ ~
20 5.2 2.6 0.27
*
Cooperativity units. Calculated by using a mean vesicle diameter of lo00 A. The observed halflifes for release from CTAP vescilea at 23 and 50 O C were 410 and 90min, respectively; the halflife for the release of sucroseout of DPPC vesicles at 50 O C was 680min. No detectable release of sucrose out of DPPC vesicles was observed over an &h period at 23 O C . Captured volumes and permeation coefficients that are reported are the average of three independent experiments (fl SD); calorimetricdata are averages of two independent experiments (flSD). a
z
P
c Z IW
'
E
capacities as a function of temperature, and the transition enthalpies were calculated by employing software supplied by Microcal. Captured Volume. Using procedures similar to those previously described? 3.0mg of CTAP (or DPPC) was dispersed in 1 mL of pure water, containing 1 pCi of [*%]sucrose. The resulting multilamellar dispersion was then subjected to five successive freeze-thaw cycles with vortex mixing, and extruded, sequentially, through two stacked0.4,0.2-, and 0.1-pmNuclepore polycarbonate membranes (four passes in each case) to give 1OOOA-diameter large unilamellar vesicles (dynamiclight scattering).1a After removal of nonentrappedsucrose by gel fiitration (Sephadex G-50), the vesicle dispersion was analyzed, simultaneously,for nitrogen and sucrose content. MembranePermeability. Aqueous vesicle dispersions (mean external diameter of 1OOO A, dynamic light scattering) were prepared from 3.0 mg of surfactant in 1mL of water containing [lWlsucrose, placed in a dialysis bag (Spectrapor 1 cellulose tubing, MW cutoff 8OOO) and then dialyzed against 300 mL of water for 15h. Periodic analysis for radioactivityinside the bag indicated that 15 h was sufficient to remove the nonentrapped sucrose, as judged by the complete disappearanceof a rapid fiist phase of the release kinetics. The bag was then placed in 300mL of pure water and the efflux rate monitored by periodic analysis for radioactivity.
Results and Discussion Thermotropic Phase Behavior. Dispersal of DPPC and CTAP into pure water, by use of standard vortex mixing procedures, afforded multilamellar vesicles having gel to liquid-crystalline phase transtion properties that are summarized in Table I. The temperatures a t which each of these bilayers is half-converted into the fluid phase, Tm, were found to be identical. The calorimetric enthalpy (AHcalcd) that was associated with chain melting for the CTAP membranes was, however, significantly lower than that of DPPC membranes. On the basis of their line widths a t half-maximum excess specific heat (ATlp), van't Hoff enthalpies were calculated by use of the following equa(12)Nayar, R.;Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1989, 986,200.
tion: AHVH= 6.9(Tm2/AT1/2).13J4Comparison of the experimentally determined calorimetric enthalpy with the van't Hoff enthalpy allows one to estimate the size of the molecular aggregate over which the motion of the molecules undergoing the phase transition is transmitted, i.e., the cooperativity of the melting process (CU = AHvH/AHcalcd). As seen from the data, the melting of CTAP membranes is somewhat less cooperative than the melting of DPPC bilayers; specificvalues of Tm, A H d , and CU, determined for DPPC membranes, are in good agreement with those previously reported.13J4 Captured Volume. Captured volumes were determined by dividing the fraction of entrapped [14Clsucrose by the surfactant concentration that was present in the di~persion.~Within experimental error, the capture efficiencies of CTAP and DPPC vesicles were identical.16 These captured volumes are in good agreement with those that have been previously reported for other 1000-Adiameter unilamellar vesicles.l6 Membrane Permeability. The rate of permeation of radioactive sucrose across vesicular bilayers can be readily determined via dialysis methods, provided that diffusion out of the vesicle is slower than diffusion acrose the dialysis m e m b r a ~ e . ~ JUnder ~ J ~ such conditione,the efflux kinetics obeys the relationship shown in eq 1. Here A1 = surface area of the bilayer, VI = internal volume of the vesicle, N1 = counts per minute (CPM) within the vesicle at time t = 0, VO= volume of dialysate, NO= counta in the dialysate beyond t = 0, and & I = the first-order rate constant which is defined by eq 2; in this latter equation, Prepresents the permeation coefficient expressed in unita of centimeters per second. When experimental conditions are chosen such that VO>> VI (as employed herein), then eq 1reduces to eq 3. Thus, the rate of disappearance of the radiolabel from within the vesicles (and the dialysis bag) d e f i e s a first-order plot whose slope, &I,depends on the size of the vesicles and the intrinsic permeability of the membrane, according to eq 2. Figure 1shows the efflux of [14Clsucroseout of CTAP vesicles at 50 OC. From this and related kinetic plota, estimated values for the permeation constant, P,have been obtained for CTAP and DPPC vesicles (Table I). In both their gel (23 "C) and fluid (50 OC) states,DPPC membranes are significantly less permeable toward sucrose than that of their CTAP analogs. These resulta indicate that the intermolecular associative interactions within DPPC bi(13)Mabrey-Gaud, S. In Liposomes: From Physical Structure to Therapeutic Applications; Knight, C. G., Ed.; Elnevier/North Holland Biomedical Press: Cambridge, 1981;p 105. (14)Mabrey, S.;Sturtavant, J. M. Methods Membr. Biol. 1978,9,237. (15)Control experimente that were carried out in which vesiclea of CTAP (and also DPPC) were incubated (0.5h) with externally-added [l%leucroee indicated an apparent adsorption that wm lew than 10% of that which was aemciated with the vesicles under capture conditions; the captured volumes have been corrected for this adsorption. (16)Bummer, P. M.; Zograf, G. Biophys. Chem. 1988,30,173. (17)Johneon, S. M.;Bangham, A. D. Biochim. Biophys. Acto 1969, 193. 82. (18)Bangham, A. D.; Hill, M. W.;Miller, N. G. A. Methods Membr. Biol. 1974,1, 1.
Langmuir, Vol. 8, No. 11, 1992 2845
Notes
In
[
Nl vo -
= (In N1)
[A]- 7vo 1 + Vl
(1) k1=
In [N+]
(A1/V1)P =-klt
(2)
(3)
layers are stronger than those within CTAP membranes, thereby providing a greater barrier for the passage of the
sucrose molecule. Thus, the CTAP bilayers are more loosely packed than those made from DPPC in both their gel and liquid-crystalline states. From a practical standpoint, these results also demonstrate the feasibilityof constructingtime-releasecarriers from an IPA. The relatively high rate at which sucrose is released from CTAP vesicles does, however, suggestthat IPA's may be more appropriate for the time release of larger molecules which diffuse across Surfactant membranes more slowly.
