Liposomes Part B

502 Pages·2003·4.491 MB·English

by  Colowick S.P., Kaplan N.O.

METHODS IN ENZYMOLOGY
EDITORS-IN-CHIEF
John N. Abelson Melvin I. Simon
DIVISIONOFBIOLOGY
CALIFORNIAINSTITUTEOFTECHNOLOGY
PASADENA,CALIFORNIA
FOUNDING EDITORS
Sidney P. Colowick and Nathan O. Kaplan
Preface
The origins of liposome research can be traced to the contributions by Alec
Bangham and colleagues in the mid 1960s. The description of lecithin
dispersions as containing ‘‘spherulites composed of concentric lamellae’’
(A. D. Bangham and R. W. Horne, J. Mol. Biol. 8, 660, 1964) was followed
by the observation that ‘‘the diffusion of univalent cations and anions out of
spontaneously formed liquid crystals of lecithin is remarkably similar to
the diffusion of such ions across biological membranes (A. D. Bangham,
M.M.StandishandJ.C.Watkins,J.Mol.Biol.13,238,1965).Followingearly
studies on the biophysical characterization of multilamellar and unilamellar
liposomes,investigators began to utilize liposomes as awell-defined model to
understand the structure and function of biological membranes. It was also
recognized by pioneers including Gregory Gregoriadis and Demetrios Papa-
hadjopoulos that liposomes could be used as drug delivery vehicles. It is
gratifying that their efforts and the work of those inspired by them have lead
to the development of liposomal formulations of doxorubicin, daunorubicin
and amphotericin B now utilized in the clinic. Other medical applications of
liposomes include their use as vaccine adjuvants and gene delivery vehicles,
whicharebeingexploredinthelaboratoryaswellasinclinicaltrials.Thefield
hasprogressedenormouslyinthe38yearssince1965.
This volume includes applications of liposomes in biochemistry, molecular
cellbiologyandmolecularvirology.Ihopethatthesechapterswillfacilitatethe
work of graduate students, post-doctoral fellows, and established scientists
entering liposome research. Subsequent volumes in this series will cover add-
itionalsubdisciplinesinliposomology.
The areas represented in this volume are by no means exhaustive. I have
tried to identify the experts in each area of liposome research, particularly
thosewhohavecontributedtothefieldoversometime.Itisunfortunatethat
I was unable to convince some prominent investigators to contribute to the
volume. Some invited contributors were not able to prepare their chapters,
despite generous extensions of time. In some cases I may have inadvertently
overlookedsomeexpertsinaparticulararea,andtotheseindividualsIextend
myapologies.Theirprimarycontributionstothefieldwill,nevertheless,notgo
unnoticed,inthecitationsinthesevolumesandintheheartsandmindsofthe
manyinvestigatorsinliposomeresearch.
xiii
xiv preface
I would like to express my gratitude to all the colleagues who graciously
contributedtothesevolumes.IwouldliketothankShirleyLightofAcademic
Pressforherencouragementforthisproject,andNoelleGracyofElsevierInc.
forherhelpatthelaterstagesoftheproject.
I am especially thankful to my wife Diana Flasher for her understanding,
supportandloveduringtheendlesseditingprocess,andmychildrenAveryand
Maxinefortheiruniquecuriosity,creativity,cheer,andlove.Iwishtodedicate
thisvolumetoDiana,AveryandMaxine.
Nejat Du¨zgu¨nes
Contributors to Volume 372
Articlenumbersareinparenthesesandfollowingthenamesofcontributors.
Affiliationslistedarecurrent.
Alicia Alonso (3), Unidad de Biofisica Sue E. Delos (428), Department of
and Departamento de Bioqu´ımica, Uni- Cell Biology, UVA Health System,
versidad Del Pa´ıs Vasco, Aptdo. 644, School of Medicine, P.O. Box 800732,
48080Bilbao,Spain Charlottesville,Virginia22908
Bruno Antonny (151), CNRS-Institut de Arnold J. M. Driessen (86), University
PharmacologieMoleculaireetCellulaire, of Groningen, Department of Microbiol-
660 Route des Lucioles, 06560 Sophia ogy, P. O. Box 14, Haren 9750AA, The
Antipolis-Valbonne,France Netherlands
John D. Bell (19), Department of Physi- Nejat Du¨zgu¨nes, (260), Department of
ology and Developmental Biology, Brig- Microbiology, School of Dentistry,
ham Young University, Provo, Utah University of the Pacific, 2155
84602 WebsterStreet,SanFrancisco,California
Robert Bittman (374), Department of 94115
Medical Microbiology, Molecular Vir- Laurie J. Earp (428), Department of
ology Section, University of Groningen, Cell Biology, UVA Health System,
Ant.Deusinglaan1,9713AVGroningen,
School of Medicine, P.O. Box 800732,
TheNetherlands
Charlottesville,Virginia22908
Pierre Bonnafous (408), Crucell Holland Raquel F. Epand (124), Department of
BV, Archimedesweg 4, P.O. Box 2048,
Biochemistry,McMasterHealthSciences
Leiden,TheNetherlands
Center, Hamilton, Ontario L8N 3Z5,
Mauro Dalla Serra (99), CMR-ITC In- Canada
stitute of Biophysics, Section at Trento,
Richard M. Epand (124), Department of
Via Sommarive 18, Povo, Trento 38050,
Biochemistry,McMasterHealthSciences
Italy
Center, Hamilton, Ontario L8N 3Z5,
David W. Deamer (133), Department of Canada
Chemistry and Biochemistry, University
of Californi-Santa Cruz, Santa Cruz, Shiroh Futaki (349), Faculty of Pharma-
California95064 ceutical Sciences, The University of To-
kushima, Shomachi 1-78-1, 770–8505
Pietro De Camilli (248), Department of Tokushima,Japan
Cell Biology, Howard Hughes Medical
Institute,YaleUniversitySchoolofMedi- YvesGaudin(392),LaboratoiredeGenet-
cine, 295 Congress Avenue, New Haven, iquiedesVirusduCNRS, GifsurYvette
Connecticut06510 Cedex91198,France
Jeanine De Keyzer (86), University of Re´my Gibrat (166), Plant Biochemistry
Groningen, Department of Microbiology, and Molecular Biology, Agro-M/CNRS/
P. O. Box 14, Haren 9750AA, The ONRA/UMII,ENSA-INRA,Montpellier,
Netherlands 34060Cedex1,France
ix
x contributors to volume 372
Fe´lixM.Gon˜i(3),UnidaddeBiofisicaand Daniel Le´vy (65), Institut Curie, UMR-
Departamento de Bioqu´ımica, Universi- CNRS 168 and LRC-CEA 34V, 11 Rue
dad Del Pa´ıs Vasco, Aptdo. 644, 48080 PierreetMarieCurie,75231ParisCedex
Bilbao,Spain 05,France
Ckayde Grignon (166), Plant Biochemis- SongLiu(274),DepartmentofBiochemis-
try and Molecular Biology, Agro-M/ try and Cell Biology, Rice University,
CNRS/ONRA/UMII, ENSA-INRA, Houston,Texas77005
Montpellier,34060Cedex1,France
Manas Mandal (319), Department of
Hideyoshi Harashima (349), Faculty of PharmaceuticalSciences,CollegeofPhar-
Pharmaceutical Sciences, The University macy,UniversityofMichigan,428Church
of Tokushima, Shomachi 1-78-1, 770– Street,AnnArbor,Michigan48109
8505Tokushima,Japan
Elizabeth Mathew (319), Department of
Theodore L. Hazlett (19), Laboratory Pharmaceutical Sciences, College of
forFluorescenceDynamics,Universityof Pharmacy, University of Michigan, 428
Illinois at Urbana-Champaign, Urbana, Church Street, Ann Arbor, Michigan
Illinois61801 48109
Lorraine D. Hernandez (428), Depart- James A. McNew (274), Department
ment of Cell Biology, UVA Health of Biochemistry and Cell Biology, Rice
System, School of Medicine, P.O. University,Houston,Texas77005
Box 800732, Charlottesville, Virginia
22908 ThomasJ.Melia(274),CellularBiochem-
istry and Biophysics Program, Memorial
AndreasHoffman(186),Macromolecular Sloan-Kettering Cancer Center, New
Crystallography Laboratory, NCI at
York,NewYork10021
Frederick, 539 Boyles Street, Frederick,
Maryland21702 GianfrancoMenestrina(99),CMR-ITC
InstituteofBiophysics,SectionatTrento,
RobertHuber(186),InstituteofCelland Via Sommarive 18, Povo, Trento 38050,
Molecular Biology, University of Edin- Italy
burgh, Michael Swann Building, The
King’s Building Mayfield Road, EH9 Pierre-Alain Monnard (133), Depart-
3JREdinburgh,Scotland ment of Chemistry and Biochemistry,
UniversityofCaliforni-SantaCruz,Santa
HiroshiKiwada(349),FacultyofPharma- Cruz,California95064
ceutical Sciences, The University of
Tokushima, Shomachi 1-78-1, 770–8505 Jose´ L.Nieva(3,235),UnidaddeBiofisica
Tokushima,Japan and Departamento de Bioqu´ımica, Uni-
versidad Del Pa´ıs Vasco, Aptdo, 644,
Kyung-Dall Lee (319), Department of 48080Bilbao,Spain
Pharmaceutical Sciences, College of
Pharmacy, University of Michigan, 428 ShlomoNir(235),SeagramCenterforSoil
Church Street, Ann Arbor, Michigan and Water Sciences, Faculty of Agricul-
48109 tural, Food and Environmental Quality
Sciences,Rehovot76100,Israel
TatianaS.Levchenko(339),Department
ofPharmaceuticalSciences,Northeastern Olivier Nosjean (216), Pharmacology
University, 360 Huntington Avenue, Moleculaire et Cellulaire, Institut de Re-
Boston,Massachusetts02115 cherchesServier,Crossy-sur-Seine,France
contributors to volume372 xi
ChristianOker-Blom(418),Universityof Brenton L. Scott (274), Department of
Jvaskyla, Department of Biological and BiochemistryandCellBiology,RiceUni-
Environmental Sciences, P.O. Box 35, versity,Houston,Texas77005
FIN40351Jyvaskyla,Finland
Yechiel Shai (361), Department of Bio-
Frank Opitz (48), University of Leipzig, logicalChemistry,WeigmannInstituteof
Institute for Medical Physics and Bio- Science,Rehovot76100,Israel
physics, Liebigstrasse 27, Leipzig D-
04103,Germany Jolanda M. Smit (374), Department of
Medical Microbiology, Molecular Vir-
Sergio Gerardo Peisajovich (361), De- ology Section, University of Groningen,
partment of Biological Chemistry, Weig- Ant.Deusinglaan1,9713AVGroningen,
mannInstituteofScience,Rehovot76100, TheNetherlands
Israel
James E. Smolen (300), Department of
Jens Pittler (48), University of Leipzig,
Pediatrics, Baylor College of Medicine,
Institute for Medical Physics and Bio-
1100Bates,Room6014,Houston,Texas
physics, Liebigstrasse 27, Leipzig D-
77030
04103,Germany
Chester Provoda (319), Department of Toon Stegmann (408), Crucell Holland
BV, Archimedesweg 4, P.O. Box 2048,
Pharmaceutical Sciences, College of
Leiden,TheNetherlands
Pharmacy, University of Michigan, 428
Church Street, Ann Arbor, Michigan Reiko Tachibani (349), Faculty of
48109
Pharmaceutical Sciences, The University
Ram Rammohan (339), Department of of Tokushima, Shomachi 1-78-1, 770-
Pharmaceutical Sciences, Northeastern 8505Tokushima,Japan
University, 360 Huntington Avenue,
VladimirP.Torchilin(339),Department
Boston,Massachusetts02115
ofPharmaceuticalSciences,Northeastern
Jean-Louis Rigaud (65), Institut Curie, University, 360 Huntington Avenue,
UMR-CNRS 168 and LRC-CEA 34V, Boston,Massachusetts02115
11 Rue Pierre et Marie Curie, 75231
ParisCedex05,France Chris Van der Does (86), University of
Groningen, Department of Microbiology,
Karine Robbe (151), CNRS-Institut de P. O. Box 14, Haren 9750AA, The
PharmacologieMoleculaireetCellulaire, Netherlands
660 Route des Lucioles, 06560 Sophia
Antipolis-Valbonne,France MartinVanderLaan(86),Universityof
Groningen, Department of Microbiology,
Ste´phane Roche (392), Laboratoire de
P.O. Box 14, Haren 9750AA, The
Genetiquie des Virus du CNRS, Gif sur
Netherlands
YvetteCedex91198,France
Bernard Roux (216), Physico-Chemie JeffreyS.VanKomen(274),Department
of Biochemistry and Cell Biology, Rice
Biologique,UniversiteCBernard-Lyon1,
University,Houston,Texas77005
Villeurbanne,France
Susana A. Sanchez (19), Laboratory for AnaV.Villar(3),UnidaddeBiofisicaand
Fluorescence Dynamics, University of Departamento de Bioqu´ımica, Universi-
Illinois at Urbana-Champaign, Urbana, dad Del Pa´ıs Vasco, Aptdo. 644, 48080
Illinois61801 Bilbao,Spain
xii contributors to volume 372
Natalia Volodina (339), Department of Markus R. Wenk (248), Department of
Pharmaceutical Sciences, Northeastern Cell Biology, Howard Hughes Medical
University, 360 Huntington Avenue, Institute,YaleUniversitySchoolofMedi-
Boston,Massachusetts02115 cine, 295 Congress Avenue, New Haven,
Connecticut06510
MattiVuento(418),UniversityofJvasky-
la,DepartmentofBiologicalandEnviron- Judith M. White (428), Department of
mentalSciences,P.O.Box35,FIN40351
Cell Biology, UVA Health System,
Jyvaskyla,Finland
School of Medicine, P.O. Box 800732,
Barry-Lee Waarts (374), Department of Charlottesville,Virginia22908
Medical Microbiology, Molecular Vir-
JanWilschut(374),DepartmentofMed-
ology Section, University of Groningen,
ical Microbiology, Molecular Virology
Ant.Deusinglaan1,9713AVGroningen,
Section, University of Groningen, Ant.
TheNetherlands
Deusinglaan 1, 9713 AV Groningen, The
ThomasWeber(274),DepartmentofMo- Netherlands
lecular, Cell, and Developmental Biology
andCarlC.IcahnInstituteforGeneTher- Olaf Zscho¨rnig (48), University of Leip-
apy and Molecular Medicine, Mount zig, Institute for Medical Physics and
Sinai School of Medicine, New York, Biophysics, Liebigstrasse 27, Leipzig D-
NewYork10029 04103,Germany
[1] phospholipase–liposome interactions 3
[1] Interaction of Phospholipases C and
Sphingomyelinase with Liposomes
By Fe´lix M. Gon˜i, Ana V. Villar, Jose´ L. Nieva, and Alicia Alonso
Introduction
Theconventionalclassificationofmembraneproteinsasintrinsicorin-
tegral,andasextrinsicorperipheral,hasonthewholebeensupersededby
amorecomplexpatterninwhichacontinuumofpossibilitiesisconsidered,
fromtheintegralproteinfirmlyembeddedinthebilayertothesolublepro-
tein that contacts the membrane only transiently for a specific function.
Phospholipasesstandinaclassoftheirownasmembraneproteinsbecause,
irrespective of their more or less ‘‘peripheral’’ location, they perturb the
physical properties of the membrane through chemical modification of its
lipid components. Thus it is not their mere binding and/or insertion into
the bilayer, but the chemical reactions they catalyze, that determines
ultimately the nature oftheirinteraction with the membrane.
In this laboratory we have examined the membrane interactions of
phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC), and of
the sphingomyelin-specific phospholipase C usually known as sphingomy-
elinase. Morerecently,we haveexploredtheeffectsofaphosphatidylino-
sitol(PI)-specificphospholipaseC(PI-PLC).Theeffectsoftheseenzymes
occur essentially through their lipid end-products, diacylglycerol or cera-
mide. Depending on the enzyme, and on the bilayer lipid compositions, a
variety of effects can be observed. Enzyme activity is commonly followed
by vesicle–vesicle aggregation, and, under certain conditions, by interve-
sicularlipidmixing,andbymixingofvesicularaqueouscontents.Observa-
tion of intervesicular contents mixing is always accompanied by detection
of mixing of lipid inner monolayers, indicative of vesicle–vesicle fusion.
Moreover,effluxofvesiclecontents,whetherornotaccompaniedbyother
effects, is observed often as a result of phospholipase C treatment. All of
the above-described phenomena can be monitored conveniently through
the use of fluorescence spectroscopy techniques, as detailed below. A
summary of the results obtained by these methods in our laboratory is
presented in a review.1
1F.M.Gon˜iandA.Alonso,Biosci.Rep.20,443(2000).
Copyright2003,ElsevierInc.
Allrightsreserved.
METHODSINENZYMOLOGY,VOL.372 0076-6879/03$35.00
4 liposomes in biochemistry [1]
Materials
Enzymes
Phospholipase C (EC 3.1.4.3) from Bacillus cereus (MW, (cid:1)23,000) is
usually obtained from Roche Molecular Biochemicals (Indianapolis, IN)
and used without further purification. Routine sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS–PAGE) controls reveal that the
enzyme preparations supplied by this company are (cid:2)90% pure. The
enzyme shows broad specificity (see below) and is active on glycero-
phospholipidsinavarietyofaggregationalstates,forexample,monomeric
in solution, dispersed in detergent-mixed micelles, and in model bilayers.
Roche Molecular Biochemicals has discontinued the sale of this enzyme.
Othersuppliersprovideequivalentenzymes,buttheyhavenotbeentested
thoroughlyinour laboratory.
Phosphatidylinositol-specific phospholipase C (EC 4.6.1.13) from
B.cereusissuppliedbyMolecularProbes(Eugene,OR)andusedwithout
furtherpurification.Sphingomyelinase(EC3.1.4.12)fromB.cereusispur-
chased from Sigma (St. Louis, MO). As indicated by the manufacturer,
preparationsofthisenzymeoftencontainsignificantphospholipaseCcon-
tamination, in amounts that vary from batch to batch. We have been
unable to separate the PC-PLC impurity from sphingomyelinase, using a
variety of chromatographic methods. In our case, and with the exception
of those experiments in which the simultaneous activities of PC-PLC and
sphingomyelinase are required, the PC-PLC inhibitor o-phenanthroline is
used routinely in sphingomyelinase assays (see below). In the absence of
PLC activity, sphingomyelinase is found to cleave specifically sphingomy-
elin, and not any glycerophospholipid. Activity on sphingophospholipids
otherthansphingomyelin,forexample,ceramidephosphorylethanolamine,
hasnotbeentested.
Substrates
Egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE),
andwheatgermphosphatidylinositol(PI)aregradeIfromLipidProducts
(South Nutfield, Surrey, UK). Egg sphingomyelin (SM) is from Avanti
Polar Lipids (Alabaster, AL). The purity of the above-described lipids is
checked by running 0.1 mg of lipid on a thin-layer chromatography plate
thatislaterrevealedbycharringinanovenunderconditionsthatallowde-
tection of1 (cid:1)goflipid.Dihexanoylphosphatidylcholine (DHPC)and cho-
lesterol are supplied by Sigma. All these lipids are used without further
purification. Glycosylphosphatidylinositol (GPI) is purified from rat liver
according to Varela-Nieto et al.2 GPI is stored at (cid:3)20(cid:4) and used within
[1] phospholipase–liposome interactions 5
the following 2 weeks. Oxidation or other forms of degradation are
detected after long-term storage. DHPC is used below its critical micellar
concentration (i.e., below 10 mM) to obtain dispersions of monomeric
phospholipid. However, enzyme assays on defined substrates are usually
carried outwith phospholipid vesicles(liposomes).
Forliposomeproduction,phospholipiddispersionsarepreparedbyre-
hydrating lipid films dried from organic solvents. Solvents are evaporated
thoroughlyundera current of N , and then leftfor atleast 2 h underhigh
2
vacuum to remove solvent traces.
Small unilamellar vesicles are prepared by sonication3 from aqueous
phospholipid dispersions, consisting mainly of multilamellar vesicles
(MLVs). Samples on ice are treated in a Soniprep 150 probe sonicator
(MSE,Crawley,Surrey,UK)with10-to12-(cid:1)mpulsesfor30 min,alternat-
ing on and off periods every 10 s. Probe debris and MLV remains are
pelleted by centrifugation at6000 g and 4(cid:4) for 10 min.
Large unilamellar vesicles (LUVs) are prepared by the extrusion
method.4Toobtainthesevesiclesaqueouslipidsuspensions(MLVs)areex-
truded10timesthroughtwostackedNuclepore(Pleasanton,CA)polycar-
bonatefilters(porediameter,0.1 (cid:1)m).TheextruderissuppliedbyNorthern
Lipids(Vancouver, BC,Canada).Extrusiontakes place atroom tempera-
ture,exceptforLUVsconsistingofpureSM,inwhichcasetheextruderis
equilibrated at 42(cid:4) with a temperature regulation accessory. Average ves-
iclediametersaremeasuredbyquasi-elasticlightscattering(QELS),using
a Zetasizer instrument (Malvern Instruments, Malvern, Worcestershire,
UK).LUVmeandiametersare(cid:1)100–115and(cid:1)160–190 nmforPC-based
liposomesandSM-basedliposomes,respectively.
Toascertainthat theextrusion proceduredoesnotalterthelipidcom-
position of the systems under study, the lipid mixtures are quantitated oc-
casionally after the extrusion treatment. For that purpose, the resulting
LUV suspensions are extracted with chloroform–methanol (2:1, v/v). The
organicphaseisconcentratedandseparatedonthin-layerchromatography
(TLC) Silica Gel 60 plates, using successively in the same direction the
solvents chloroform–methanol–water (60:30:5, v/v/v) for the first 10 cm
and petroleum ether-ethyl ether-acetic acid (60:40:1, v/v/v) for the whole
plate. After charring with a sulfuric acid reagent, the spot intensities are
quantified with a dual-wavelength TLC scanner (CS-930; Shimadzu,
Tokyo, Japan). The results of these studies have shown that, under our
2I. Varela-Nieto, L. Alvarez, and J. M. Mato, ‘‘Handbook of Endocrine Research
Techniques,’’p.391.AcademicPress,SanDiego,CA,1993.
3A.Alonso,R.Sa´ez,A.Villena,andF.M.Gon˜i,J.Membr.Biol.67,55(1982).
4L.D.Mayer,M.H.Hope,andP.R.Cullis,Biochim.Biophys.Acta858,161(1986).