A novel strategy for the preparation of liposomes: rapid solvent exchangefeigensonlab.mbg.cornell.edu/publications/Buboltz1999.pdf · A novel strategy for the preparation of liposomes:

A novel strategy for the preparation of liposomes:rapid solvent exchange

Je¡rey T. Buboltz, Gerald W. Feigenson *Section of Biochemistry, Molecular and Cell Biology, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA

Received 23 September 1998; received in revised form 21 December 1998; accepted 4 January 1999

Abstract

During the preparation of multi-component model membranes, a primary consideration is that compositionalhomogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipidmixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membranecomponents. A new preparative method has been developed which is specifically designed to avoid this intermediary state.This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organicsolvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapmentvolumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content,suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared byrapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional samplepreparation are discussed. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cholesterol ; Demixing; Homogeneity; Lipid mixture; Liposome preparation; Phase behavior

1. Introduction

Biological membranes are chemically complexmixtures, and any given biomembrane may containmore than a hundred di¡erent lipid species [1]. Bystudying these chemically well-de¢ned models of bio-membranes, researchers have sought physical-chem-ical insight into membrane biology.

Our laboratory has been studying phospholipid/cholesterol membranes in the regime of high choles-terol content. Speci¢cally, we have sought to deter-mine the maximum solubility of cholesterol in bi-layers composed of various phospholipids. Abovethis limit, excess cholesterol precipitates as crystalsof pure cholesterol monohydrate.

Initially, we expected that this solubility limitwould be straightforward to determine: simply pre-pare suspensions of increasing cholesterol contentand examine each sample for the presence of crystals.We found that several methods (e.g. 90³ light scatter-ing, X-ray di¡raction, ultra¢ltration) could be usedto detect cholesterol crystals, each with its own ad-vantages and disadvantages. But regardless of detec-tion method, one conclusion eventually became

0005-2736 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 9 9 ) 0 0 0 0 6 - 1

Abbreviations: FEP, £uorinated ethylene propylene; LUV,large unilamellar vesicle ; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl ; PIPES, piperazine-1,4-bis(2-ethanesulfonic acid); SUV, smallunilamellar vesicle

* Corresponding author. Fax: (607) 2552428;E-mail : [email protected]

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Biochimica et Biophysica Acta 1417 (1999) 232^245

clear: the reproducibility of these `straightforward'solubility limits was surprisingly poor. We were ledto conclude that the source of this variability wasinhomogeneity in the samples themselves and wefound that other workers had reported di¤culty inpreparing homogeneous samples of high cholesterolcontent [2]. Over the course of our research, we havelearned that artifactual crystal formation can occurduring conventional sample preparation [3]. Thismay help to explain the widely ranging values re-ported in the literature [3,4] for cholesterol solubilitylimits.

When preparing any model membrane, it is gener-ally important that the lipids remain well mixedthroughout the sample preparation procedure. Thisis especially true for physical-chemical studies of lip-id mixtures, such as those of phase behavior. But thepreparation methods which are most commonly usedin such studies entail an incubation of the lipid mix-ture in the form of a solvent-free ¢lm or powder,which are solids. It has long been known that sol-id-state mixtures are, in general, far more likely tophase separate (i.e. demix) than non-solid mixtures[5]. Therefore, we became concerned that the spuri-ous cholesterol crystal formation in our samplescould be traced to phase behavior in the solid-state¢lm or powder.

Here we describe the development of a novel meth-od for model membrane preparation which we callrapid solvent exchange (RSE). This method is specif-ically designed to form compositionally homogene-ous suspensions by the sudden precipitation of a lipidmixture in aqueous bu¡er. This is achieved by rap-idly exchanging the lipids between an organic solventenvironment and an aqueous environment. Accord-ingly, membrane structures are formed de novo inwater without passing the lipid mixture through anintermediary solid state. Phospholipid/cholesterolsuspensions have proven to be free of artifactualcrystals when prepared by rapid solvent exchange.

2. Materials and methods

2.1. Chemicals and materials

Phospholipids were purchased from Avanti PolarLipids (Alabaster, AL) and cholesterol was pur-

chased from Nu Chek Prep (Elysian, MN). Purity(s 99%) was con¢rmed by thin layer chromatogra-phy on washed, activated silica gel plates (AlltechAssociates, Deer¢eld, IL), developing with chlo-zroform/methanol/water = 65:25:4 for phospholipidanalysis or with petroleum ether/ethyl ether/chloro-form = 7:3:3 for cholesterol analysis. All solventsused were of HPLC grade. TLC plates were quanti-tated by charring and densitometry. (16:0,18:1)-NBD-N-PE was synthesized and puri¢ed accordingto a procedure already described [6,7]. Phospholipidstock solutions, as well as some suspensions, werequantitated by phosphate assay [8]. Aqueous bu¡er(pH 7.0, 5 mM piperazine-1,4-bis(2-ethanesulfonicacid) (PIPES), 200 mM KCl) was prepared from pu-ri¢ed water (Milli-Q system, Millipore) and ¢lteredthrough a 0.1 Wm ¢lter before use. Fluorinated ethyl-ene propylene (FEP) Te£on Oak Ridge centrifugetubes were purchased from Nalge. AnhydrousMgSO4 was of analytical reagent grade (Mallinck-rodt). Deuterated benzene (99.6% 2H) and chloro-form (99.8% 2H) were purchased from CambridgeIsotope Laboratories (Andover, MA). Dialysis mem-brane was Spectra/Por brand (Spectrum Medical In-dustries). 5(6)-Carboxy£uorescein (Eastman Labora-tory Chemicals, Rochester, NY) was used withoutfurther puri¢cation. Sodium hydrosul¢te was puri¢edgrade (Fisher Sci.). 1 mm, special glass X-ray capil-laries were purchased from Charles Supper (Natick,MA).

2.2. Rapid solvent exchange

The apparatus employed was developed throughtesting performed on a prototype. These deviceswere fabricated with the use of the Graduate Re-search Machine Shop facility supported by the Cor-nell Laboratory of Applied and Solid State Physics.The vacuum was maintained by a 1/3 h.p. mechan-ical pump (Gast, Benton Harbor, MI) with an ad-justable inlet valve for pressure regulation, and thepump outlet was vented to a laboratory hood. Either10 ml or 50 ml FEP Te£on Oak Ridge tubes servedas vessels for solvent exchange. During RSE, precip-itating lipids manifest transiently high surface activ-ity and FEP tubes were judged least likely to adsorblipid. A 100 Wl gas-tight, blunt-tipped syringe (point-style #3, Hamilton, Reno, NV) was used to intro-

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duce the lipid solution (10^100 Wl of 10^100 mMlipid) to the injection tubing; see Section 3 for adescription of the procedure. Standard vacuum pres-sure was 23 torr for sample preparation at roomtemperature (23³C). Samples containing di16:0-PCwere prepared by RSE into bu¡er which was main-tained at 50³C. For RSE at this temperature, thevacuum pressure was adjusted to 100 torr and thesample vessel was enclosed in a forced-air heatingjacket which was coupled to a heat gun.

2.3. Conventional sample preparations

2.3.1. Film deposition13U100 mm glass test tubes, each containing be-

tween 100 and 300 Wl of a lipid solution in chloro-form, were mounted on an analytical evaporator(Organomation Associates, South Berlin, MA) andbulk solvent was evaporated under a gentle streamof nitrogen gas. The test tubes were loaded into avacuum desiccator which was then coupled to a vac-uum line and the lipid ¢lms were incubated overnightunder a measured pressure of approx. 30 millitorr.The ¢lms were hydrated with aqueous bu¡er at roomtemperature and vortexed for 1 min to disperse thelipid. Samples containing di16:0-PC were hydratedand vortexed at 50³C, before being allowed to coolto room temperature.

2.3.2. Lyophilization13U100 mm glass test tubes, each containing be-

tween 100 and 300 Wl of a lipid solution in cyclo-hexane/methanol (99:1), were plunged into a bathof liquid nitrogen. The frozen samples were loadedinto a pre-cooled desiccator which was then coupledto a vacuum line. The desiccator was kept on waterice until the bulk solvent had been removed (2^3 h)after which the samples were allowed to warm toroom temperature. The vacuum incubation contin-ued overnight at approx. 30 millitorr. The volumi-nous, white lipid powders were hydrated and vor-texed in the same manner as the ¢lms.

2.3.3. Freeze-thawingSuspensions were subjected to ¢ve cycles of freeze-

thawing, between a bath of liquid nitrogen and aroom temperature water bath.

2.3.4. ExtrusionLarge unilamellar vesicles (LUVs) were prepared

by passing a suspension 10 times through a doublestack of 0.1 Wm Nucleopore polycarbonate mem-branes (Costar, Cambridge, MA), using a modi¢edextruder apparatus (Lipex Biomembranes, Vancou-ver, B.C.).

2.4. Solvent residue analysis

Liposomes were prepared by rapid solvent ex-change from 100 Wl of organic solvent (25 mM lipid)into 5 ml of aqueous bu¡er. In order to quantitatesolvent residue remaining at a given post-injectiontime-point, eight replicates were pooled and the sus-pension was centrifuged (20 000UgU5 min) to col-lect the vesicles in a pellet. The clear, lipid-free aque-ous supernatant was removed and the pellettransferred, along with about 50 Wl of associatedbu¡er, to a 13U100 mm glass tube and dispersedin 1.0 ml of deuterated solvent. The resulting lipid-stabilized emulsion (bu¡er in deuterated solvent) wasbroken by chemical dehydration: 100 mg of anhy-drous MgSO4 was added, followed by vortexing andlow-speed centrifugation. The desiccant pelleted,leaving a clear supernatant free of bulk water. Thisprocedure quantitatively recovers both lipid and sol-vent residue from the original aqueous suspension,while leaving little water in the extract. Without de-hydration, the extracts yielded NMR spectra con-taining large water peaks which interfered with theanalysis.

When the injection solvent was dichloromethane,deuterated chloroform was used for sample extrac-tion. In addition, samples for dichloromethane anal-ysis were prepared from lipids with fully saturatedacyl chains. This was to avoid interference from ole-¢nic 1H, whose resonance is near to that of dichloro-methane 1H. di12:0-PC was used to prepare samplesat room temperature and di16:0-PC for sample prep-arations at 50³C.

When the injection solvent was chloroform, deu-terated benzene was used for sample extraction.Since the chloroform 1H resonance is well resolvedfrom that of ole¢nic 1H, 16:0,18:1-PC was used toprepare suspensions for chloroform analysis, at bothroom temperature and 50³C.

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1H NMR spectra were collected on a Varian XL-200 NMR spectrometer with a 3 s pulse delay. Typ-ically, 160 transients were collected, though as manyas 1000 transients were taken when high sensitivitywas desired. When a solvent peak could be resolved(Fig. 1), it was integrated along with a nearby lipidproton peak, establishing a molar ratio between lipidand solvent residue. Control samples containing aknown quantity of solvent residue (0.1^1.0 mole%)were extracted and analyzed to validate the proce-dure.

2.5. X-Ray di¡raction

RSE liposomes (approx. 1 mg of lipid) were pre-pared and transferred to X-ray capillary tubes. Thesecapillaries were then ¢lled to the neck with bu¡erand placed in a buoyant support apparatus (Fig. 2)which allows the capillaries to withstand high speedcentrifugation in a swinging bucket rotor. Vesicleswere generally pelleted in the capillaries for 15 minat 20 000Ug, after which they were sealed with par-a¤n wax under argon gas.

In order to observe the e¡ects of centrifugation onthe di¡raction pro¢le of RSE vesicles, replicate sam-ples of 16:0,18:1-PC were prepared. Each of thesesuspensions was then exposed to centrifugation at adi¡erent level of centripetal force.

Di¡raction data were collected at the Macromo-lecular Di¡raction Facility of the Cornell High En-ergy Synchrotron Source (MacCHESS), on the F-1beamline. Samples were exposed to an intense syn-chrotron beam of wavelength 0.908 Aî , which waspassed through a 0.2 mm collimator. Data were re-corded either on Fuji imaging plates and then digi-tized by scanning, or with a Princeton 2K CCD de-tector containing 2048U2048 41-micron pixels [9,10].When desired, circular integration was performed to

transform the powder patterns into one-dimensionalpro¢les of di¡raction intensity.

2.6. External surface assay

Accessible external surface was assayed by a 7-ni-trobenz-2-oxa-1,3-diazol-4-yl (NBD)/dithionite assay[6]. In short, £uorescence is quantitated in samplescontaining a £uorescent surface marker (headgroup-labeled-NBD) both before and after chemical bleach-ing of the accessible external fraction by aqueousdithionite reagent. Subsequent addition of detergentmicellizes the liposomes, resulting in completebleaching of the remaining £uorophore.

For this assay, suspensions were prepared from0.25 Wmole of lipid (16:0,18:1-PC with 0.5 mole%16:0,18:1-NBD-N-PE) in 5 ml of bu¡er. Five repli-cate samples were prepared by each of three meth-ods: ¢lm deposition/freeze-thawing, ¢lm deposition/extrusion and rapid solvent exchange. Dithionite re-agent (1 M) was prepared as described by Gruberand Schindler [6].

To estimate the accessible external marker frac-tion, 2 ml of sample was placed in a quartz cuvettewith a masked stir bar, gently rotating at a set speed.After establishing the pre-dithionite signal, 5 Wl ofdithionite reagent was added to the cuvette. Whenthe rapid bleaching phase was complete and thepost-dithionite signal had been established, 50 Wl of20% (v/v) Triton X-100 was added to the cuvette todemonstrate complete bleaching.

Fluorescence time scans were collected on a Hita-chi Fluorescence Spectrometer (model F-3010) withexcitation at 470 nm (470 nm interference ¢lter;1.5 nm slit) and emission at 540 nm (480 nm long-pass ¢lter; 10 nm slit). With our samples (0.5 mole%NBD, 50 WM suspension), this arrangement yieldednegligible background signal (6 0.05%), even with

Table 1External surface fraction and entrapped volume of di¡erent liposome preparations

Preparative method External surface fraction (%) Entrapped volume (Wl/Wmole)

Film deposition n.d. 0.25 ( þ 0.04)Lyophilization n.d. 0.31 ( þ 0.04)Film deposition/extruded (LUV) 47.1 ( þ 3.9) n.d.Film deposition/frozen and thawed (FT-MLV) 13.4 ( þ 2.5) 4.07 ( þ 0.06)Rapid solvent exchange 33.1 ( þ 1.6) 4.50 ( þ 0.09)

Values presented are averaged from replicate samples (see Section 2); the range of values observed is shown in parentheses.

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turbid suspensions. This was demonstrated by con-trol samples prepared without £uorophore. For thisreason, no corrections were necessary for turbid sam-ples.

As observed by Gruber and Schindler, we foundthat turbid samples did yield higher £uorescence in-tensities (data not shown), but in our case this musthave been due to a greater e¡ective illuminated vol-ume caused by scattering of light within the sample.Since this signal enhancement was signi¢cant (ap-prox. 40%), we veri¢ed that the signal decreaseupon dithionite addition was not due to changes inthe light scattering properties of the suspension. Con-trol samples were observed by 90³ light scattering at470 nm, and subjected to both dithionite and Tritonaddition as in the £uorescence assay. Dithionite in-jection had no e¡ect on light scattering intensity(data not shown), so post-dithionite signal loss cansafely be attributed to bleaching only. Triton addi-tion decreased scattering intensity as expected, sincethis treatment micellizes the sample.

2.7. Entrapment assay

1 mM carboxy£uorescein (CF) was prepared inaqueous bu¡er and this was used to prepare threereplicate samples by each of four methods: ¢lm de-position, lyophilization, ¢lm deposition/freeze-thaw-ing and rapid solvent exchange. Samples contained5 mg of 16:0,18:1-PC in 5 ml of bu¡er.

After preparation, suspensions were transferred todialysis tubing (18 mm, 50 kDa MW cut-o¡) anddialyzed together against 600 ml CF-free bu¡er(changed every 3 h for 24 h) in order to removeunentrapped £uorophore. After dialysis, the CF re-maining in each sample was quanti¢ed by £uores-cence. In a quartz cuvette with a masked stir bar,100 Wl of the dialyzed sample was added to 3 ml ofbu¡er containing 2% Triton X-100, emulsifying theliposomes and relieving the self-quenching of en-trapped £uorophore. Each sample was also subjectedto phosphate analysis to verify the phospholipidconcentration in the suspension after dialysis,although no losses were ever observed. Control sam-ples (CF bu¡er without lipid) were included in eachexperiment to quantitate the very low level of unen-trapped, residual carboxy£uorescein remaining afterdialysis.

2.8. Di¡erential scanning calorimetry

Calorimetric measurements were made with a Mi-croCal MC-2 scanning calorimeter, at a scan rate of0.18³C/min. di16:0-PC RSE liposomes were preparedat 50³C and then transferred to a 50³C water bathwhich was cooled to room temperature overnight.Samples were degassed for 5 min and were keptunder nitrogen at 18 psi during data collection.

Fig. 1. Quantitation of solvent residue in aqueous lipid suspen-sions. 1H NMR spectra of suspension extracts. Insets show ex-panded view of solvent peak together with adjacent lipidpeak(s). (a) di12:0-PC with CH2Cl2 residue in CDCl3. Integra-tion of CH2Cl2 peak (5.28 ppm) together with lipid glycerolbackbone HCOCO peak (5.19 ppm broad) yield a solvent/lipidratio = 0.036 for this sample. (b) 16:0,18:1-PC with CHCl3 resi-due in C6D6. Integration of CHCl3 peak (6.14 ppm) togetherwith both nearby lipid peaks, CH = CH (5.60 ppm broad) andHCOCO (5.72 ppm broad), yield a solvent/lipid ratio = 0.026.

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3. Results

Sample preparation by rapid solvent exchange isaccomplished with the apparatus pictured in Fig. 3.This consists of a vacuum manifold, which couplesan FEP Te£on sample tube to the vacuum line, and alaboratory sample vortexer. The vacuum pressure isadjusted to be slightly higher than the vapor pressureof water; we routinely set it to approx. 23 torr forsample preparation at room temperature. The FEPtube, containing aqueous bu¡er, is mounted on thevortexer. The vortexer is actuated, forming the bu¡erinto a cylindrical shell, and the manifold is opened tothe vacuum. Next, a gas-tight, blunt-tipped syringecontaining the lipid solution in organic solvent ispositioned at the injection port. Finally, the syringeneedle is seated in the coupling sleeve and the syringe

plunger is quickly depressed, forcing the solutiondown through the stainless steel injection needleand out through a narrow side-ori¢ce in the tip(Fig. 4). As the organic solution passes throughthis ori¢ce it experiences a sudden drop in pressureso that vaporization of the solvent begins, along withsome evaporative cooling. The ori¢ce is only about acentimeter from the tube wall, so the solution drop-lets quickly contact the vortexing bu¡er which servesas a heat reservoir, transferring heat to the drop-lets so that vaporization can proceed to comple-tion. The cylindrical shell of bu¡er presents a largesurface area, and the vortexing action aids in thetransfer of solvent vapor to the vacuum. In thisway, bulk organic solvent is very rapidly and verye¤ciently vaporized. Solvent residue analysis showsthat s 99.99% of dichloromethane is removed withina second of injection (Figs. 5 and 6). In order toremove trace, residual solvent the sample continuesto vortex, maintaining maximal bu¡er surface areaunder reduced pressure for at least 20^30 s after in-jection. By this point no detectable solvent remains(Fig. 6), but our standard procedure is to leave each

Fig. 2. Buoyant support for centrifugation of samples in X-raycapillaries. A 28.5U105 mm polycarbonate tube is ¢lled withwater and a friction-¢t polypropylene closure, in which eight2 mm diameter holes have been drilled, is pressed into place.The water level is adjusted to be approx. 3^5 mm above thebottom of the closure. X-Ray capillaries, containing liposomesuspensions and ¢lled with bu¡er up into the neck, are loweredinto the holes. Supported in this way, the thin-walled capillariescan withstand centripetal forces in excess of 20 000Ug. The cen-trifuge must be started gently, so that capillaries are not brokenagainst the tube wall by the sudden torque on the rotor.

Fig. 3. Rapid solvent exchange apparatus.

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sample vortexing under vacuum for 1 min after in-jection.

The X-ray di¡raction pattern of 16:0,18:1-PCRSE liposomes is that of a £uid-phase bilayer withthe expected lamellar repeat spacing of about 65 Aî .The di¡raction is rather weak, though, unless thesample is subjected to centrifugation, as illustratedby the di¡raction pro¢les of samples exposed to in-creasing sedimentary force (Fig. 7). This is consistentwith images we obtained by thin-section electron mi-croscopy, which showed little multilamellar stackingin suspensions of RSE vesicles (data not shown).Likewise, the high fraction (approx. 33%) of accessi-ble external surface (Table 1) is consistent with a lowaverage lamellarity in RSE preparations.

RSE liposomes appear to be similar to frozen andthawed (FT) vesicles in aqueous entrapment volume,according to our entrapment assay (Table 1). No

Fig. 4. Cross-sectional schematic of the process of rapid solventexchange. A blunt-tipped, gas-tight syringe is seated in the in-jection sleeve and the plunger is depressed, forcing the lipid sol-ution down through the injection needle and out the side-ori-¢ce. Bulk solvent vaporizes and is removed within about asecond, while the lipid mixture precipitates in aqueous bu¡er.

Fig. 5. Removal of dichloromethane residue at 23³C. Expanded1H NMR spectra (as in Fig. 1a inset) of extracted di12:0-PCsamples prepared by rapid solvent exchange from a dichlorome-thane solution at room temperature. Shown are spectra for ex-tracts at 1, 3, 10 and 20 s post injection. Bulk solvent is quicklyvaporized; the solvent/lipid mole ratio (approx. 600 in the injec-tion solution) has been reduced to approx. 0.05 one secondafter injection. Residual solvent is then removed by continuedvortexing under vacuum so that by 20 s, no detectable solventpeak remains.

Fig. 6. Quantitative time courses of solvent residue removal.Zero time point (arrow) indicates solvent/lipid ratio in 25 mMinjection solution. b, dichloromethane removal at 23³C; eachpoint represents the averaged result of ¢ve independent experi-ments. a, dichloromethane removal at 50³C; results of three in-dependent experiments. F, chloroform removal at 23³C; threeexperiments. E, chloroform removal at 50³C; three experiments.The lines through the data represent exponential ¢ts. Error barsspan the range of solvent/lipid ratios obtained and the lowerlimit of the ordinate axis is the estimated detection limit of ourassay.

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attempt was made to account for the possible slowleakage of carboxy£uorescein, and since the time-scale of the dialysis assay was s 24 h the estimatedentrapment volumes should be regarded as lowerlimits.

Di¡erential scanning calorimetry of di16:0-PCRSE liposomes yielded a thermogram typical ofdi16:0-PC (data not shown), revealing both a pre-transition around 35³C and a sharp main transitioncentered at 41.3³C.

Fig. 8 shows X-ray di¡raction powder patterns ofa hydrated di16:0-PC/cholesterol mixture preparedin parallel by either ¢lm deposition or rapid solventexchange. Cholesterol crystals, which here indicateartifactual demixing (see Section 4), are present inthe sample prepared by ¢lm deposition (Fig. 8a).No crystals are evident in samples prepared byRSE (Fig. 8b).

4. Discussion

4.1. Properties of RSE liposomes

Rapid solvent exchange is a simple, convenientmethod for the preparation of liposomes, requiringno more than 1 min for each sample. RSE mem-branes form de novo within an aqueous environ-

Fig. 7. X-Ray di¡raction of liposomes prepared by rapid sol-vent exchange. X-Ray di¡raction pro¢les for 16:0,18:1-PC sus-pensions subjected to increasing centripetal force. Both beamintensity and exposure time were identical. The ¢rst-order re-peat is centered at about 65 Aî , and the second-order repeat atabout 32.5 Aî . Centrifugation improves the di¡raction intensity,though the positions and shape of the peaks are una¡ected.

Fig. 8. X-Ray di¡raction reveals artifactual cholesterol crystalsin a sample prepared by ¢lm deposition, but not in a sampleprepared by rapid solvent exchange. Powder patterns are shownfor aqueous suspensions of di16:0-PC/cholesterol at 50 mole%cholesterol, prepared by either ¢lm deposition or rapid solventexchange. This concentration of cholesterol is more than 15mole% below the true solubility limit in a hydrated bilayer ofdi16:0-PC [3]. Both preparations began with an aliquot takenfrom the same solution of phospholipid and cholesterol codis-solved in chloroform. (a) In the sample prepared by ¢lm depo-sition, the presence of crystalline cholesterol monohydrate is in-dicated by a characteristic pattern of lines at both low andwide angles (arrows). (b) No artifactual crystals appear in sam-ples prepared by RSE; only lamellar-phase lipid is evident.

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ment, so that larger entrapment volumes areachieved, and aqueous solutes should be uniformlydistributed across lamellae. The suspensions are oflow average lamellarity, with a high fraction of ex-ternal surface area. More to the point, rapid solventexchange does not pass the lipid mixture through anintermediary solid state.

4.2. RSE prevents artifactual cholesterol demixing

For some time, our laboratory has studied phos-pholipid/cholesterol bilayers in the regime of highcholesterol content. We have found [3] that choles-terol has a strong tendency to demix from such mix-tures during sample preparation by either of the twomethods most commonly used in physical-chemicalstudies of model membranes: ¢lm deposition andlyophilization. As discussed below, these preparativemethods entail an intermediary, solid-state lipid mix-ture. Cholesterol tends to precipitate from solid-statelamellae at concentrations which are well below itstrue solubility limit in hydrated lamellae [3]. When aphospholipid/cholesterol suspension is prepared inthis way, any demixed cholesterol can remain de-

mixed after hydration, becoming crystals of pure chol-esterol monohydrate. The presence of these crystalsis clearly revealed by X-ray di¡raction, as in Fig. 8.

The true solubility limit of cholesterol in a hy-drated bilayer is an important parameter for thephysical-chemical study of phospholipid-cholesterolinteractions. Our studies of several phospholipid/cho-lesterol mixtures were frustrated by experimentswhich were based on either of the two more conven-tional preparative methods. These yielded apparentsolubility limits whose reproducibility was poor, andwhich proved to be falsely low due to demixing ar-tifacts. In contrast, experiments based on RSE prep-arations have proven to yield reproducible solubilitylimits. For every phospholipid/cholesterol mixture,these solubility limits have been higher than was in-dicated by the conventional preparations [3], consis-tent with the elimination of a demixing artifact.

4.3. Two conventional methods of preparation:¢lm deposition and lyophilization

Liposome preparations are all aqueous lipid sus-pensions rather than solutions. On the other hand,

Fig. 9. Two conventional preparation methods which entail an intermediary solid-state lipid mixture. Film deposition (upper path-way): bulk solvent is removed by evaporation, depositing a solid-state lipid ¢lm on the wall of the sample vessel. After an extendedvacuum incubation to remove residual solvent, the ¢lm is hydrated to form an aqueous lipid suspension. Lyophilization (lower path-way): the sample is frozen and bulk solvent is removed by sublimation, leaving a voluminous lipid powder. After vacuum incubationto remove residual solvent, the powder is hydrated to form an aqueous lipid suspension.

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lipids are puri¢ed, quantitated and stored as solu-tions in organic solvent. As a consequence, essen-tially all methods for liposome preparation beginwith a lipid solution in organic solvent and endwith a lipid suspension in water (Fig. 9). The variouscomponents are typically combined by codissolvingthe lipids in an organic solvent and the organic sol-vent is then most often removed by either of twomethods: ¢lm deposition (Fig. 9 upper pathway) orlyophilization (Fig. 9 lower pathway). When all thesolvent has been removed, the solid lipid mixture ishydrated with aqueous bu¡er. Upon exposure towater, the lipids spontaneously swell to form lipo-somes [11,12]. At this point, many methods diverge,processing the suspensions in various ways to a¡ecttheir properties. Among these post-hydration treat-ments are vortexing, sonication [13], freeze-thawing[14], and high-pressure extrusion [15,16].

4.4. Intermediary solid-state lipid mixtures

Both ¢lm deposition and lyophilization entail aprolonged incubation in an intermediary state, nei-ther organic solution nor aqueous suspension: theintermediary solid-state lipid mixture (Fig. 9, ¢lm/powder). It is well known that solvent-free lipidsspontaneously form solid-state lamellae which exhib-

it an endothermic melting transition upon heating[12,17,18]. Likewise, X-ray di¡raction reveals thatthese solid-state lamellae are highly structured [19],far in excess of the order observed in the hydrated,£uid-state bilayer (Fig. 10).1 Our concern is thatsome apparent phase behavior observed in hydratedlipid mixtures may in fact be traced to the interme-diary solid state. More than 20 years ago, Gershfeldargued that this sort of e¡ect could explain disparatereports on phase behavior in phospholipid/cholester-ol mixtures [21].

It is the hydrated bilayer which is the biologicallyrelevant, physical-chemical state of interest, but both¢lm deposition and lyophilization take the lipid mix-ture from solution to aqueous suspension via a long-

Fig. 10. X-Ray di¡raction powder patterns contrasting the solid-state lamellar and hydrated, £uid lamellar states. Results are shownfor 16:0,18:1-PC/cholesterol mixtures at 10 mole% cholesterol, but similar results were observed for other lipid mixtures. (a) Solvent-free solid-state mixture prepared by ¢lm deposition from chloroform. Note the multiple orders of low-angle rings, produced by intensedi¡raction from stacked lamellae formed in the solid state. Moreover, the dense array of wide-angle rings indicates that the solid-statelipid is highly organized within the lamellae, revealing many di¡erent, characteristic structural spacings all on the order of a few ang-stroms. (b) Solvent-free solid-state mixture prepared by lyophilization from cyclohexane/methanol 99:1. The di¡raction pattern is simi-lar to that in (a). (c) Lipid mixture ¢lm, as in (a), after hydration in aqueous bu¡er. The solid-state lamellae have swollen and £uid-ized; the many wide-angle rings have been replaced by a single, gentle undulation which is characteristic of a £uid-state bilayer.

1 It is interesting to note that the lamellae which we seek tostudy in water are formed in a non-aqueous environment by thesepreparation methods. The process of hydration is really then aprocess of swelling [11,12], or the penetration of water acrossthese multilamellar structures. Of course, solutes permeate moreslowly and mechanical treatments are employed, after hydration,to achieve uniform distribution of solutes across lamellae [14].The lamellae themselves undergo transitions to new physicalstates associated with the shift to an aqueous environment[12,17,18,20,27], for example, the transition from an ordered, sol-id lamellar state to a £uid bilayer state upon hydration (compareFig. 10a with c).

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lived, intermediary solid state with its own phasediagram. The tendency of any mixture to remainstably mixed correlates with the phase state of thesystem: gas, liquid or solid. Gas-phase mixtures rep-resent one extreme, being miscible in all proportionsfor all known cases. Solid-phase systems representthe other extreme. While some multi-component sol-ids are known to be stably mixed, forming a single-phase `solid solution', it is generally true that solid-phase mixtures can manifest complex phase behavior[5], resulting in the demixing of components.

4.5. Demixing in solid-state mixtures

For example, solvent-free mixtures of n-alkanescan have complicated phase behavior in the solidstate. Binary mixtures of n-octane and n-decane arefully miscible in the £uid state but are largely immis-cible in the solid state, phase separating between 2%and 90% n-octane [22]. Similarly, mixtures of n-eico-sane (n-C20H42) and n-docosane (n-C22H46) are fullymiscible in the £uid-state but manifest complex phasebehavior in the solid state, with several di¡erent re-gimes of two-phase coexistence below the solidustemperature [23]. Recently, Dorset et al. have ob-served that while freshly solidi¢ed mixtures of n-C30H62 and n-C36H74 appear to be well-mixed, agedsamples reveal extensive demixing between 10% and90% n-C36H74 [24].

It has long been known that similar demixing canoccur in solid-state mixtures of amphiphiles. Alka-nols of di¡ering chain lengths can be largely immis-cible in the solid state, and the tendency to demix isenhanced when a primary alkanol is mixed with asecondary alkanol [25]. Binary mixtures of fatty acidswill phase separate in the solid state, if one compo-nent is saturated and the other is unsaturated, or ifthe di¡erence in chain length is signi¢cant [26]. An-hydrous soaps have shown similar tendencies in thesolid state [27]. While studying a natural lipid extractby X-ray di¡raction, Bear et al. concluded that theanhydrous lipid mixture showed evidence of threedistinct lipid phases [28]. While studying the e¡ectsof drying on myelin, Elkes and Finean [29] observedthe appearance of three new Bragg spacings whichthey attributed to lipid phase separation during thedrying process. Finally, as discussed above, choles-terol can demix from solid-state mixtures with phos-

pholipid, forming crystals of anhydrous cholesterol.Several groups have reported the presence of choles-terol crystals in solid-state phospholipid/cholesterolmixtures [3,28,30,31].

In all of these cases, components with signi¢cantstructural di¡erences seem to have a greater tendencyto demix from each other. This is not surprising,considering that solids are characterized by a highpropensity for organization and that space-¢lling,close-packing structures are favored in the solid state[32].

4.6. Possible manifestations of solid-state demixing inhydrated lipid mixtures

Of course, even if a solid-state mixture is unstable,demixing may be very slow to occur. On the time-scale of sample preparation, many solid-state lipidmixtures are likely to remain well mixed, even asmetastable solid solutions. However, in metastablesolid solutions of hydrocarbons, the rate of demixingis known to be accelerated by di¡erences in molec-ular structure [33], so it seems likely that some meta-stable solid-state lipid solutions could demix signi¢-cantly during conventional sample preparation. Ifthis occurs, then the resulting liposome suspensioncould be artifactually heterogeneous. This artifactualdemixing could be either reproducible or variable inextent, depending on both the particular mixture andthe timescale of sample preparation. In the case ofreproducible solid-state demixing, the apparent phasebehavior could be falsely assigned to the hydratedlipid mixture. On the other hand, variable (non-equi-librium) solid-state demixing might account for somelipid mixture formulations which are not well be-haved, manifesting poorly reproducible properties.

4.7. Strategy of rapid solvent exchange

The strategy behind RSE is simple. Consider, forexample, a chloroform solution of several lipid spe-cies. The interactions between chloroform and lipidmolecules are favorable, so the lipids are solvatedand can be considered to be well mixed. Now, imag-ine quickly exchanging the chloroform for water.Upon exposure to water, the lipids will begin toform lipidic colloids and the kinetics of this precip-itation process should be collision-limited, so that

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colloid formation is rapid and indiscriminate of spe-cies-speci¢c interactions. During this collision-limitedprecipitation, the various lipid species ought to formcompositionally homogeneous lipidic particles. Thesehomogeneous particles can then relax to form stablestructures de novo within the biologically relevant,aqueous environment.

4.8. Other methods compared with RSE

Of course, other sample preparation methods al-ready exist which do not entail an intermediary solid-state lipid mixture. Like RSE, they should producesuspensions free of solid-state demixing artifacts.These other methods fall into two categories, de-pending on the nature of the organic solvent.

Preparation from a miscible organic solvent: lip-osomes can be formed by precipitation from an or-ganic solvent which is miscible with water (see forexample [34^36]). Lipid is dissolved in a suitable sol-vent (methanol, etc.) which is then mixed with water,causing lipids to precipitate. Unfortunately, poor lip-id solubility is often an issue with such solvents.There is also the problem of residual solvent: sincethe solvent is miscible with water, removal requiresexchange of the bulk aqueous bu¡er (e.g. by dialysisor gel-¢ltration chromatography) and this can be in-convenient and time-consuming. A ¢nal, importantnote to these procedures is that they can produce ahigh proportion of small unilamellar vesicles (SUVs)[34].

Preparation from an immiscible organic solvent:several publications have described the preparationof vesicles by evaporation of water-immiscible sol-vent from an organic solution of lipids which is incontact with aqueous bu¡er (see for example [37^41]). It is somewhat surprising that the publicationsdescribing these methods often do not provide theresults of a quantitative assay for solvent residue.Moreover, we are not aware that any such publica-tion has quantitated the time course of solvent re-moval, so we cannot assess the timescale of eitherbulk or residual solvent removal. It seems clearthat the timescale of solvent removal has not beena controlling parameter in the design of the `vapor-ization' methods published to date. Rather, the ex-plicit goal has been to produce suspensions of lowlamellarity and/or to entrap solutes. In contrast to

these other methods, RSE has been speci¢cally de-signed for the rapid, e¤cient removal of solvent andthe timecourse of this process has been characterizedquantitatively.

If solvent is not quickly and e¤ciently removedupon exposure of the lipid mixture to water, at leasttwo concerns may be raised. First, some materials inthe bu¡er may be sensitive to prolonged solvent ex-posure. For example, globular proteins targeted forentrapment may be irreversibly denatured by signi¢-cant quantities of solvent [38], especially as the time-scale of exposure increases. Second, lipid structuresare likely to form in the presence of signi¢cant sol-vent, and this may create artifactual properties whichremain even after solvent has been removed. Consid-er the `ethanol injection' method, which tends toform a large proportion of SUV. The SUV structure,which is thermodynamically unstable, persists afterthe residual solvent has been removed or diluted [34].

We developed rapid solvent exchange in order tohave a method which would better approximate thecollision-limited precipitation strategy outlinedabove. RSE transfers the lipid mixture quickly be-tween an essentially pure solvent environment andan essentially pure aqueous environment. When pre-pared by RSE, lipid structures form de novo inwater, in the presence of insigni¢cant solvent residue.Our explicit goal was to preserve compositional ho-mogeneity throughout sample preparation, so thatany apparent phase behavior observed in the aque-ous suspension could be attributed only to the phys-ical chemistry of the hydrated lipid mixture. Becausethe method is speci¢cally designed for the fast, e¤-cient removal of organic solvent, it does not require ahighly volatile solvent. Rapid solvent exchangeworks with the most common, e¡ective lipid solvents(dichloromethane, chloroform) and allows the prep-aration of lipid mixtures which incorporate compo-nents of widely ranging solubility properties.

Acknowledgements

The authors are grateful to Dr. C.H. Spink forperforming the calorimetric measurements and toDr. J. Huang for invaluable help with the X-raydi¡raction experiments. This work was supportedby National Science Foundation Grant MCB-

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9722818. J.T.B. was supported, in part, by NationalInstitutes of Health Research Service Award I-T32-GM08267. This work is based upon research con-ducted at the Cornell High Energy SynchrotronSource (CHESS), which is supported by the NationalScience Foundation under award DMR-9311772, us-ing the Macromolecular Di¡raction at CHESS(MacCHESS) facility, which is supported by awardRR-01646 from the National Institutes of Health.

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