Langmuir 1993,9, 219-222
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Effect of Alkyl Chain Asymmetry on the Fusion and Crystallization Behavior of Vesicles Formed from Di-n-alkyl Phosphates Lisette Streefland,+Anno Wagenam,+Dick Hoekstra,* and Jan B. F. N. Engberta'J Department of Organic Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands, and Laboratory of Physiological Chemistry, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands Received June 5,1992. In Finul Form: September 9, 1992 Fusion of vesicles formed from synthetic, asymmetric (Le. mixed-chain) sodium di-n-alkyl phosphates (1-6) hae been studied with a resonance energy transfer w a y for lipid mixing and with transmieeion electron microscopy. Fusion was induced by Ca2+ions above the La LB phaee transition temperatures -+
of the different bilayers. We have found that two competing events take place simultaneously but independently from each other. These are fusion and formation of tubules, which have been characterized as anhydrouscrystals. Both processes are fast. The asymmetricdi-n-alkyl phosphates with alkyl chains that differ by only two methylenegroups (1and 2) showed initial fusion ratee comparableto that of vesicles formed from (symmetric)di-n-dodecylphosphate. With an increase in the difference in chain length (3, 4, and 51, the fusion rates increase,indicating that chain asymmetrydecreasesbilayer stability by reducing alkyl chain packing in the bilayer. However, when the differencein chain length becomes eight methylene group (61, fusion ie effectively suppressed. The sizes of the crystals formed from 1to 6 turned out to be rather different, but no simple relationship could be found with the extent of asymmetry of the alkyl Chains. fusion process.11J2 Especially a transition from the La to HII phase^ has received much attention and a mechanism has been proposed in which the transition proceeds via an inverted micellar intermediate (IMI).1413 Thism e d " was assumed to be appIicableto the synthetic di-n-dodecyl phosphate system as wells3Until now only the symmetric (e.g. equal chain) synthetic surfactants have been intigated in their fueogenicand polymorphic abilitiea. The aim of this study was to investigate the effect of chain asymmetry in di-n-alkyl phoephatee on the fusion and polymorphic behavior of vesicles formed from these surfactants. Chain asymmetry has been extensively studied in phospholipid bilayersl*Js but not in synthetic surfactant bilayers. It was anticipated that the bilayer packing will be significantlyinfluenced by thie structural change and therefore the vesicles might show a modified fusion behavior in the presence of Ca2+. Insight into their fusogenic ability would be important in planning a s p metric fusion experiments for these typea of surfactants.
Introduction Veaicles of chargedsyntheticsurfactants are increasingly used as membrane mimetic They show common features like phase transitions, osmotic sensitivity, reconstitution of membrane proteins, and fusion. Particularly the symmetric (e.g. between alike vesicles) fusion behavior of di-n-dadecyl phosphate has been studied in some detail.239Jo But ale0 asymmetricfusion,for example between synthetic surfactant vesicles and phospholipids, has been succesfully investigated and the results appear to suggest the potential use of these di-n-alkylphosphate veaiclea asdrugdeliverysyst%ms.~lIn them systems fusion was mually triggered by divalent cations, Cas+ being the one mostly used. The overall fusion event can be divided into two processes. First aggregation occurs in which the vesicles stiek together as a consequence of the reduction of repulsive electrostatic and hydration forces between the bilayers upon binding of Ca2+. In a second step the actual fusion process takes place accompanied by bilayer mixing and mixing of aqueous contents. Sometimes a lamellartononlamellar phase transition accompaniesthe
Experimental Section Materials. The asymmetric di-n-alkyl phoephatee were synthesized via the dihydrogen phosphate according to the method described previouely.10 N - ( 7 - N i t r 0 - 2 , 1 , 3 - ~ x a ~ l 4-y1)phoephatidylethanolamine (N-NBDPE)and N-(lirnamine RhodamineB sulphony1)phoephatidylethanolamine(N-Rh-PE) were obtained from Avanti Polar Lipids. HEPES (4-(2-hydro.yethyl)-l-piperazineethaneeulfonicacid) was from S i Chemical. Sodium acetate and calcium chloride were purchased h m
Organic Chemistry, University of Groningen. Laboratory of PhyeiologicalChemistry,University of Groningen. (1) (a) Rupert, L. A. M.;Hoehtra, D.; Engberta, J. B. F. N. J. Am. Chem.Soc. 1986,107,2628. (b)Rupert,L. A. M.; Hoekstra, D.; Engberta, + Department of t
J. B. F. N. J. Am. Chem. SOC.1986.108.3920. (2) Rupert, L. A. M.; Hoehtra, D.; E'ngberta, J. B. F. N. J. Colloid Interface Sci. 1987, 120, 126. (3) Rupert, L. A. M.; van Breemen, J. F. L.; van Bruggen, E. F. J.; Engberta,J. B. F. N.; Hoehtra, D. J. Membr. Biol. 1987,96,266. (4) Fendler,J. H. Membrane Mimetic Chemistry:Wilev-Intemience: New York, 1982. (6) Kunitake, T.;Okabata, Y . J. Am. Chem. Soc. 1977,99,3860. (6) Fontsijn, T.A. A; Engberta, J. B. F.N.; Hoehtra, D. J.Am. Chem.
Merck.
Vesicle Preparation. Vesicleswere prepared by the ethanolinjection method aa described by Rupert? Hereto, 10 mg of the amphiphilewaa diesolved in lWwL of ethanol. With a preheated Exmire syringe, 80 p L of thie solution waa injected into 2 mL of
Soc. 1990,112,8870.
(7) Fontsijn,T.A. A,; Engberta,J. B.F.N.; Hoehtra, D. Biochemistry
1991,30,6319. (8) For brief reviewe of
the hmgenic activity of synthetic bilayer membnnm,m (a)Mu"i,Y.;Kikuchi, J.4 InBioorganicChemistry Heidelberg, 1991;Vol. 2, p 73. (b)Fonbijn, Frontiers;Springer-Ver T.A. A;Wbrta,J. B. .;Hoekstra, D. In Cell and Model Membrane Intemctione; Ohki, S., Ed.; Plenum: New York, 1991; p 216. (9) Rupert, L.A. M.; Hoebtra, D.;Engberta, J. B. F.N. J.Phys. Chem.
(11) C u b , P. R.; De Kruijff, B. Biochim. Biophys. Acta 1WO,S69, 399. (12) Siegel, D. P.Biophys. J. 1986,49, 11%. (13) Siegel, D. P.Biophys. J. 1986,49, 1171. (14) Hui, X.; Huang, C. Biochemktry 1987,26,1036. (15) Hui, X.; Huang, C. Biochemktry 1987, I, 6448. (16) Wagenaar, A,; Rupert, L. A M.;Engberts, J. B. F. N.; Hoehtra, D. J. Org. Chem. 1989,64,2638.
A
1988,92,4416. (10) Rupert, L. A. M.; Engberta,J. B. F. N.;Hoebtra, D. Biochemistry 1988,8252.
(a
1993 American Chemical Society
220 Lagmuir, Vol. 9, No.1, 1993
CCaClpJ. mM
Streefland et al.
CCaClZ3, nM
Figure 1. (A) Initial rates of fusion for 1 (circles), 2 (squares), and 3 (triangles) at 42,69, and 48 OC, respectively. (B) Extenta of
fusion for 1,2, and 3 at the temperatures given above. Phosphate concentration waa 50 X 10-8 M, in a HEPES/NaAc buffer at pH 7.4.
an efficientlystirred 6 mM HEPES/6 mM NaAc buffer (pH 7.4) thermostated at 60 OC. Fumion Measurements. Vesicle fusion was monitored using a resonance energy transfer (RET) assay based on lipid mixing.'* Vesicles containing 0.8 mol % each of the fluorescent lipid analogues N-NBD-PE (donor) and N-Rh-PE (acceptor) were mixed with an equimolar amount of unlabeled vesicles. Fusion was induced by adding Ca2+ions. Upon bilayer mixing, dilution of the fluorescent probes takes place. The decrease in energy transfer efficiency, reflected as an increase of NBD fluorescence, was continuously monitored using a SLM-Aminco SPF-600 C spectrofluorometer,equippedwith a temperature-controlledcell holder, a magnetic stirriig device, and a chart recorder. The excitation wavelength was 466 nm whereas the emission waa followed at 630 nm. The fluorescence d e waa calibrated by adjusting the initial fluorescence of the nonfused vesicles at 0% fluorescence. The level of infinite dilution (100%fluorescence) was determined after disruption of the vesicles in a cetyltrimethylammonium bromide (CTAB, 1 % (w/v)) solution. Corrections were made for sample dilution and for effecta of the surfactants on the quantum yield of NBD. Initial fusion rates were calculated by taking the derivative at t = 0 and the extent of fusion was determined after the addition of an excess of the Ca*+binding agent EDTA (0.6 M solution of pH 8.0) when the fluorescence signal had just passed ita maximum. All fusion experiments were carried out in a 6 mM NaAc/HEPES buffer, pH 7.4, and occurred on a timescale at which the vesicles were (morphologically) stable in the absence of Caz+ions. Electron Miororcopy. Vesicles and crystals were vieualiied with transmiasion electron microscopy. Sampleswere prepared as described previ~usly.'~.~ The material was stained with a 1 % (w/v)solutionof uranyl acetate,after mounting on carbon-coated Fomvar grids. The sampleswere examined in a Philips EM300 electron microscope, operating at 80 kV.
Results and Discussion All di-n-alkyl phosphates investigated in this study (16) readily form unilamellar vesicles as revealed by transmission electron microecopy.le The fusion of theaevesicles, induced by Ca2+,was investigated with the RET assay (see Experimental Section), which allows the calculation (17)Hong, K.; Baldwin, P. A.; Allen, T. M.; Papahadjopoulos, D. Biochemistry 1988,27,3947. (18) Struck,D. K.; Hoeketra, D.; Pagano, R. E. Biochemistry 1981,20, 4093.
1: R1 = n-Ci~H25, R2 = nC14H29.4 R1 = nCloHnl, R2 = nC14H20 2 R1 = nC14H29, R2 = nCld33; 5: R1 = ~ C , O HR2 ~ ~= ,n-ClsH33 3: R1 = nC12H25, R2 = nClglj3; 6 R1 = nC1oH21, R2 nC18H37
of the extentsof fusion and initial fusion rates as a function of the Ca2+concentration. Since a change in fluorescence intensity may also result from a reversiblephase transition in the bilayer induced by Ca2+ the f i i extent of fluorescence increase was measured after addition of an excess of the Ca2+complexing agent EDTA. Usually the addition of EDTA was accompanied by only a negligible change in fluorescence intensity and the remaining fluorescence signal should be ascribed to (irreversible) merging of the bilayers. For 1-3 the rwulta are plotted in Figure 1. All measurementa were carried out at a temperature 10 OC above the main phase trausition temperature for the transition from the gel-like to liquidcrystalline phase of these vesicles. These T l s have been determined previously by fluorescence depolarization measurementa.le Below the Tis no fluorescenceincrease was observed. This indicates that a fluid membrane is a prerequisite for fusion to occur, consistent with previous resulta.23J Furthermore,there was no fusion activitybelow a Ca2+ threshold concentration, which is the minimum Ca2+concentration necessary for inducing a sufficiently close approach of the vesicles for the occurrence of fwion of the bilayers.l-a These threshold concentrations were 1.1, 1.6, and 2.0 mM Ca2+for 1, 2, and 3, respectively. Figure 1 shows that the initial rates of fusion are d e r for 1and 2, that have chainsthat differ only two methylene groups, than for 3 that has a differenceof four methylene groups between the two hydrocarbon tails. The initial rates of fusion for 1 and 2 are comparablewith that found for di-n-dodecylphosphate vesicles,the symmetricClaHaa analogue (maximum rate is 4.7% 8-9.2 Somewhat unexpectedly, the extent of fusion is higher for 1 and 2, than for 3 (Figure 1). Apparently the extent of fusion doea not parallel the initial rates of fluorescenceincrease. Previous work has shown that the maximum extent of fusion for
Effect of Alkyl Chain Asymmetry on Vesicle Behavior
Langmuir, Vol. 9, No. 1,1993 221
Table I. Maximum Extents of Fusion for the Ca2+-Induced Fusion of Vesicles Formed from 1 to 6 and of Di-m-dodecyl Phosphate Vesicles di-n-alkyl phosphate max. extent of fusion (% )a di-n-dodecyl phosphate 48b 1 36 i 4 2 38i2 3 28f2 4 36f2 5 14f2 6 0 a Corresponding with a maximum extent of fluorescence increase by 0.5 X value given. The data refer to measurements performed after addition of an excess of EDTA (see Experimental Section). b Reference 3.
DDP is 48%, indicating that this symmetric phosphate has slightly better fusogenic properties than 1-3. No quantitative fusion measurements could be performed with 4-6. Initial rates were toofast to be measured, from which one can conclude that bilayers formed from still more asymmetric surfactants are more easily destabilized. Differences in fusogenic activity between 3 and 4, which both have a difference of four methylene groups between the two alkylchains, suggestthat the total number of carbon atoms (28 and 24, respectively) is also a factor in determining bilayer stability. In the case of 4 and 5 the extent of fusion was 36 76 and 14% ,respectively(maximum values). This shows that an increasing asymmetry of the amphiphile is accompanied by a decrease in the extent of fluorescence increase, e.g. a decreased fusion activity, despite the increasinginitial rates of fluorescenceincrease. Theremight be a possibilitythat the crystallization process intervenes in the fluorescence assay. Surfactant 6, with a difference of eight methylene groups between the two hydrocarbon chains, confirms this assumption because after adding Ca2+a complete loss of the fluorescencesignal was observed upon addition of an EDTA solution. This suggests that the change in fluorescence intensity should be ascribed to an increase in fluorescence quantum yield resultingfrom a (reversible)Ca2+-inducedphase transition. Clearly no fusion had taken place at all. For the sake of convenience the maximum extents of fusion for 1-6 and di-n-dodecyl phosphate are summarized in Table I. Electron microscopic (EM) experiments were carried out using uranyl acetate as a stain and coloring material. For the different vesicular systems, the Ca2+-induced processes were followed by taking samples at several time periods after Ca2+addition and after EDTA addition. As an example, micrographs of vesicles formed from 4 are shown in Figure 2. Within a short time after Ca2+addition (ca.5 min) vesicleshad been transformed into long tubular structures that were quite uniform in length and width. This phenomenon was also observed for di-n-dodecyl phosphate3and the tubes were proposed to be an inverted hexagonal phase. However, we have now obtained strong X-ray evidencethat these structuresare, in fact, anhydrous crystals. The multidimensional alkyl chain packing in these crystals has been discussed in some detail.lg Unfortunately, no fused vesicles could be visualized by EM before the transformation of the vesicles into crystals took place. Essentially the same results were obtained for the other asymmetricphosphates, but there are differencesin vesicle sizes and crystal lengths. The relevant data for 1-6 are listed in Table 11. The most remarkable feature in this table is the variation in crystal lengths. Especially comparison of 4 (difference (19)Streefland, L.;Fang, Y.;Rand, R.P.; Hoekstra, D.; Engberta, J. B. F. N. hngmuir 1992,8,1715.
Figure2. Electron micrographs of (A) vesicles of 4 made by the ethanol injection method, (B)crystals formed upon Ca2+addition (2 mM),(C)vesicles formed after addition of EDTA, and (D) crystals formed from vesiclesof 6 upon addition of 2 mM of Ca2+. In A, B, and C, the bar represents 300 nm. In D the bar is 100 nm. All specimens were stained and colored with 1%uranyl acetate. Table 11. Vesicle Sizes and Tube Lengths for 1-6 Determined by EM in the Presence of Ca2+Ions at a Surfactant Concentration of 8 mM and a Final Cat+ Concentration of 2 mM average average average vesicle tube lengtha vesicle sizeb di-n-alkyl size before after Ca2+ after EDTA phosphate fusion (nm) addition (nm) addition (nm) 1 50 2000 140 2 100 1000 600 3 100 400 600 4 35 5000 100 5 65 1000 85 6 35 300 400 a A small increase in tube length has been observed in time. These values have been measured when no increase was observed anymore ( 1 / ~h3incubation). * Determined after the time the crystals needed to dissolve again (few min-*/2 h).
in chain lengthsof four methylene groups) and 6 (difference of eight methylene groups) highlights the large difference. Apparently, the amphiphile structure has a pronounced effect on the crystal formation. This may be a consequence of interdigitation of the alkyl chains in the bilayer before the crystal formation starts. In the anhydrous crystals the chains are noninterdigitated as shown by X-ray diffra~ti0n.l~ It is well-knownfor mixed-chain phosphatidylcholines that the chains can be interdigitated in the lamellar phase, particularly for the ones with a difference of eight methylene groups between the two ~ h a i n s . ~ ~ J ~ * ~ O - ~ ~ (20) Hui, S. W.;Mason, J. T.; Huang, C. Biochemistry 1984,223,5570. (21)McIntosh, T. J.; Simon, S. A.; Ellington, Jr.; Porter, N. A. Biochemistry 1984,223,4038.
222 Langmuir, Vol. 9, No. 1, 1993
Though there is no evidence for fusion from EM, some other intareetingobservationsemergefrom theEM resulta. Firstly, quite long cryetale are formed from 6 as compared to the initial vesicle s h , while the RET assay doee not indicate a substantial fusbn activity. Secondly, the shes of the veeiclae obtained after addition of EDTA are emall compared to the crystal sizes, particularly for 1 and 4. Apparently, there are two competing eventa, fusion and formation of crystab, which occur independently from each other. Most likely more than one vesicle can participate in the formation of a crystal upon addition of Ca2+ ions. Furthermore one crystal can form several vesicles upon addition of EDTA. This contrasta with the earlier interpretation for the DDP resulta which implied that tubes could only be formed from fused ve~icles.~ To obtain more evidence for these independently occurring proc-, we have carried out the eame EM experiment for DDP below the fusion and even aggregation Ca2+ threehold concentrations(1.38and 1.73 mM, respectively). At 1.0 mM Ca2+,crystals were indeed oberved which indica- that crystallizationoccurs even before extensive aggregation seta in. Still there seems to be some relationship between vesicle and crystal sizee. An experiment was carried out with vesicles formed from 4 that had been prepared by sonicationrather thanby the ethanol-inje&ion method. This method yields smaller unilnmellar vesicles, and now the crystals formed after addition of Ca2+were clearly shorter (about half the size). Apparently, c r p tallization is favored due to the preeenceof more nudeahon points. However, the data in Table I1 do not suggest a
Streefland et al. simple relation between chain asymmetzyand crystalsize. Due to the high rate of the pro", we cannot link the EM resulta with the kinetic resulta from the RET assay. Apparently the asymmetric di-n-alkylphosphate vesicles do exhibit fuaogenic activity, but increasing alkyl chain asymmetry does decrease the extent of fusion. Preeumably, the differently packed bilayers of the more asymmetric phosphates are more prone to form crystals, so that thisp r m becomesdominant over the fusion proteas. Conclusions We have investigatedthe effect of alkylchain asynunetry in synthetic di-n-alkyl phosphates on the fusogenic behavior of vesicles formed from these surfactante. Increasing asymmetry in the alkyl chains resulted in higher initial rates for Caa+-inducedfusion as revealed by a fluorescence technique based on lipid mixing. From this we conclude that the more asymmetric bilayers are more easilydestabilized. Apparently,the bilayer packing isless efficient than in the more symmetric ones. The procesees are so fast that no initial fusion producta could be observed with EM experiments. Extents of fusion decrease with increasing chain asymmetry. We contend, partially based on EM reaulta, that two competing events can take place; one being the fusion process and the other the formation of anhydrous crystals. Variation of surfactant structure and of the fusogenic agent may lead to lessrapidcrystallization, and studies along these lines are in momess. (22) Mattai, J.; Sripada, P.K.;Shipley, G.G.Biochemistry 1987,26, 3287.
