Liposomes Part A

320 Pages·2003·4.209 MB·English

by  Colowick S.P., Kaplan N.O.

Preface
The origins of liposome research can be traced to the contributions of Alec
Bangham and colleagues in the mid 1960s. The description of lecithin disper-
sions as containing ‘‘spherulites composed of concentric lamellae’’ (A. D.
Bangham and R. W. Horne, J. Mol. Biol. 8, 660, 1964) was followed by the
observationthat‘‘thediffusionofunivalentcationsandanionsoutofspontan-
eouslyformedliquidcrystalsoflecithinisremarkablysimilartothediffusionof
such ions across biological membranes (A. D. Bangham, M. M. Standish and
J. C. Watkins, J. Mol. Biol. 13, 238, 1965). Following early studies on the
biophysicalcharacterizationofmultilamellarandunilamellarliposomes,inves-
tigators began to utilize liposomes as a well-defined model to understand the
structure and function of biological membranes. It was also recognized by
pioneers,includingGregoryGregoriadisandDemetriosPapahadjopoulos,that
liposomes could be used as drug delivery vehicles. It is gratifying that their
effortsandtheworkofthoseinspiredbythemhaveledtothedevelopmentof
liposomalformulationsofdoxorubicin,daunorubicin,andamphotericinB,now
utilizedintheclinic.Othermedicalapplicationsofliposomesincludetheiruse
as vaccine adjuvants and gene delivery vehicles, which are being explored in
thelaboratoryaswellasinclinicaltrials.Thefieldhasprogressedenormously
since1965.
This volume describes methods of liposome preparation, and the physico-
chemicalcharacterizationofliposomes.Ihopethatthesechapterswillfacilitate
theworkofgraduatestudents,post-doctoralfellows,andestablishedscientists
entering liposome research. Subsequent volumes in this series will cover
additionalsubdisciplinesinliposomology.
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.ItisunfortunatethatI
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.
ix
x preface
Inthelastfiveyears,theliposomefieldhaslostsomeofitsmajormembers.
DemetriosPapahadjopoulos(oneofAlec Bangham’sprotegesandoneof my
mentors)wasasignificantmoverofthefieldandaninspirationtomanyyoung
scientists.Heorganizedthefirstconferenceonliposomesin1977inNewYork.
Hewasalsoaco-founderofacompanytoattempttocommercializeliposomes
for medical purposes. Danilo Lasic brought in his sophisticated biophysics
background to help understand liposome behavior, wrote and co-edited nu-
merousvolumesonvariousaspectsofliposomes,andhelpedtheirwidespread
appreciation with short reviews. David O’Brien was a pioneer in the field of
photoactivatable liposomes, most likely inspired by his earlier work on rhod-
opsin.Hewastohavecontributedachaptertothelastvolumeof‘‘Liposomes’’
inthisseries.Foralltheircontributionstothefield,thisvolumeisdedicatedto
thememoriesofDrs.Papahadjopoulos,LasicandO’Brien.
I would like to express my gratitude to all the colleagues who graciously
contributedtothesevolumes.IwouldliketothankShirleyLightofAcademic
Press for her encouragement for this project, and Noelle Gracy of Elsevier
Scienceforherhelpatthelaterstagesoftheproject.Iamespeciallythankfulto
my wife Diana Flasher for her understanding, support and love during the
endless editing process, and my children Avery and Maxine for their unique
curiosity,creativity,cheer,andlove.
Nejat Du¨zgu¨nes
Mill Valley
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
Contributors to Volume 367
Articlenumbersareinparenthesesandfollowingthenamesofcontributors.
Affiliationslistedarecurrent.
Patrick Ahl (80), Bio Delivery Sciences Wilson Capparo´s-Wanderley (70),
International, Inc., UMDNJ-New Jersey School of Pharmacy, Lipoxen Technolo-
Medical School, 185 South Orange gies Ltd., University of London, 29-39
Avenue, ADMC4, Newark, New Jersey Brunswick Square, London WC1N 1AX,
07103 England
Juha-Matti Alakoskela (129), Institute Laurie Chow (3), Inex Pharmaceutical
ofBiomedicine,P.O.Box63,Biomedcum Copre, Glenlyon Business Park1, 100-
Haartmaninkatu 8, University of 8900GlenlyonParkway,Burnaby,British
Helsinki,Helsinki,FIN00014,Finland Columbia,CanadaV5J5J8
MiglenaAngelova(15),InstituteofBio- Joel A. Cohen (148), Department of
medicine, P.O. Box 63, Biomedicum Physiology, University of the Pacific
Haartmaninkatu 8, University of SchoolofDentistry,2155WebsterStreet,
Helsinki,Helsinki,FIN00014,Finland SanFrancisco,California94115
KlausArnold(253),InstituteforMedical Rivka Cohen (270), Laboratory of
Physics and Biophysics, Faculty of
Membrane and Liposome Research,
Medicine, University of Leipzig,
Hebrew Univeristy-Hadassah Medical
D-04103Leipzig,Germany School,Jerusalem91120,Israel
Jesus Arroyo (213), Facultad Farmacia y
E.AnibalDisalvo(213),FacultadFarm-
Bioqu´ımica, Universidad de Buenos
aciayBioqu´ımica,UniversidaddeBuenos
Aires,Junin9562P,BuenosAires11113,
Aires,Junin9562P,BuenosAires11113,
Argentina
Argentina
AndrewBacon(70),SchoolofPharmacy,
Lipoxen Technologies Ltd., University Nejat Du¨zgu¨nes (23), Department of
of London, 29-39 Brunswick Square, Microbiology, University of the Pacific
LondonWC1N1AX,England SchoolofDentistry,2155WebsterStreet,
SanFrancisco,California94115
Luis A. Bagatolli (233), MEMPHYS-
Center for Biomembrane Physics, Simcha Even-Chen (270), Laboratory of
DepartmentofBiochemistryandMolecu- Membrane and Liposome Research,
lar Biology, Campusvej 55, DK-5230 Hebrew Univeristy-Hadassah Medical
OdenseM,Denmark School,Jerusalem91120,Israel
YechezkelBarenholz(270),Laboratory Gregory Gregoriadias (70), School of
of Membrane and Liposome Research, Pharmacy, Lipoxen Technologies
Hebrew Univeristy-Hadassah Medical Ltd.,UniversityofLondon,29-39Bruns-
School,Jerusalem91120,Israel wick Square, London WC1N 1AX,
England
DeliaL.Bernik(213),FacultadFarmacia
y Bioqu´ımica, Universidad de Buenos SadaoHirota(177),TokyoDenkiUniver-
Aires, Junin 956 2P, Buenos Aires sity, 6-6-18 Higashikaigan-Minami,
11113,Argentina Chigasaki-Shi253-0054,Japan
vii
viii contributors to volume 367
Juha M. Holopainen (15), Institute of WalterR.Perkins(80),Transave,Inc.,11
Biomedicine, P.O. Box 63, Biomedicum Deerpark Drive, Suite 117, Monmouth
Haartmaninkatu 8, University of Junction,NewJersey08552
Helsinki,Helsinki,FIN00014,Finland
Gertrud Puu (199), Swedish Defense Re-
ReumaHonen(270),LaboratoryofMem- search Agency, NBC Defence, SE 90182
brane and Liposome Research, Hebrew Umei,Sweden
Univeristy-Hadassah Medical School,
Jerusalem91120,Israel RamonBarnadasiRodrı´guez(28),Uni-
tat de Biofisica, Facultat de Medicine,
Michael Hope (3), Inex Pharmaceutical Universitat Auto`noma de Barcelona,
Copre, Glenlyon Business Park1, 100- Catalonia, 08193 Cerdanolya del Valle`s,
8900 Glenlyon Parkway, Burnaby, Spain
BritishColumbia,CanadaV5J5J8
Rolf Schubert (46), Pharmazeutisches
Jana Jass (199), The Lawson Health Re- Institut, Lehrstuhl fu¨r Pharmazeutische
search Institute, 268 Grosvenor Street, Technologie,Albert-Ludwigs-Universita¨t-
London,Ontario,CanadaN6A4U2 Freiburg, Hermann-herder Strasse 9,
Andrea Kasˇna´ (111), Veterinary D-79104Freiburg,Germany
Research Institute, Department of Im- Hilary Shmeeda (270), Shaare Zedek
munology, Hudcova 70, 62132 Brno,
Medical Center, Department of
CzechRepublic
Experimental Oncology, POB 3235,
PaavoK.J.Kinnunen(15,129),Institute Jerusalem91031,Israel
of Biomedicine, P.O. Box 63, Torbjo¨rn Tja¨rnhage (199), Swedish De-
Biomedicum Haartmaninkatu 8, Univer-
fense Research Agency, NBC Defence,
sity of Helsinki, Helsinki, FIN 00014,
SE90182Umei,Sweden
Finland
Peter Laggner (129), Institute of Bio- Jaroslav Tura´nek (111), Veterinary Re-
search Institute, Department of Immun-
physics and X-Ray Structure Research,
ology, Hudcova 70, 62132 Brno, Czech
AustrianAcademyofSciences,Schmiedl-
Republic
strasse6,A-8042Graz,Austria
BrendaMcCormack(70),SchoolofPhar- Carmela Weintraub (270), Laboratory
of Membrane and Liposome Research,-
macy, Lipoxen Technologies Ltd.,
Hebrew Univeristy-Hadassah Medical-
University of London, 29-39 Brunswick
Square,LondonWC1N1AX,England School,Jerusalem91120,Israel
Barbara Mui (3), Inex Pharmaceutical EwoudC.A.VanWinden(99),Regulon
Copre, Glenlyon Business Park1, Gene Pharmaceuticals A.E.B.E.,
100-8900 Glenlyon Parkway, Burnaby, Auxentiou Grigoriou 7, Alimos, 17455
BritishColumbia,CanadaV5J5J8 Athens,Greece
Jirˇı´Necˇa(111),VeterinaryResearchInsti- Manuel Sabe´s i Xaman´ı (28), Unitat de
tute, Department of Immunology, Biofisica, Facultat de Medicine,
Hudcova70,62132Brno,CzechRepublic Universitat Auto`noma de Barcelona,
Catalonia, 08193 Cerdanolya del Valle`s,
Shinpei Ohki (253), Department of Physi- Spain
ology and Biophysics, School of
Medicine and Biomedical Sciences, DanaZa´luska´ (111),VeterinaryResearch
StateUniversityofNewYorkatBuffalo, Institute, Department of Immunology,
Buffalo,NewYork14214 Hudcova70,62132Brno,CzechRepublic
[1] extrusion technique 3
[1] Extrusion Technique to Generate Liposomes of
Defined Size
By Barbara Mui, Laurie Chow and Michael J. Hope
Introduction
Liposome extrusion is a widely used process in which liposomes are
forced under pressure through filters with defined pore sizes to generate
a homogeneous population of smaller vesicles with a mean diameter that
reflects that of the filter pore.1 This technique has grown in popularity
andhasbecomethemostcommonmethodofreducingmultilamellarlipo-
somes, usually called multilamellar vesicles (MLVs), to large unilamellar
vesicles (LUVs) formodelmembraneanddrugdelivery research.
TheextrusionconceptwasinitiallyintroducedbyOlsonetal.,2whode-
scribed the sequential passage of a dilute liposome preparation through
polycarbonate filters of decreasing pore size, using a hand-held syringe
and filter holder attachment, in order to produce a homogeneous size dis-
tribution. This procedure was further developed and made more practical
by the construction of a robust, metal extrusion device that employed
medium pressures (800 lb=in2) to rapidly extrude MLV suspensions dir-
ectly through polycarbonate filters with pore diameters in the range of 50
to200 nmtogenerateLUVs.1Atthetimethisprocessrepresentedamajor
advance for those routinely preparing LUVs. Other size reduction
methods, such as the use of ultrasound or microfluidization techniques,
tendtogeneratesignificantpopulationsof‘‘limitsize’’vesiclesthataresub-
ject to lipid-packing constraints3 and also suffer from lipid degradation,
heavy metal contamination, and limited trapping efficiencies. Reversed
phase evaporation (REV) methods were also common in the 1980s and
usually involved the formation of aqueous–organic emulsions followed by
solvent evaporation to produce liposome populations with large trapped
volumesandimprovedtrappingefficiencies.4However,thesemethodsare
restricted by lipid solubility in solvent or solvent mixtures; moreover,
1M.J.Hope,M.B.Bally,G.Webb,andP.R.Cullis,Biochim.Biophys.Acta812,55(1985).
2F.Olson,C.A.Hunt,F.C.Szoka,W.J.Vail,andD.Papahadjopoulos,Biochim.Biophys.
Acta557,9(1979).
3M.J.Hope,M.B.Bally,L.D.Mayer,A.S.Janoff,andP.R.Cullis,Chem.Phys.Lipids40,
89(1986).
4F. Szoka, F. Olson, T. Heath, W. Vail, E. Mayhew, and D. Papahadjopoulos, Biochim.
Biophys.Acta601,559(1980).
Copyright2003,ElsevierInc.
Allrightsreserved.
METHODSINENZYMOLOGY,VOL.367 0076-6879/03$35.00
4 methods of liposome preparation [1]
removal of residual solvent can be tedious. Detergent dialysis tech-
niques are also subject to similar practical difficulties associated with lipid
solubilityand completeremovalof detergent.
Consequently,theconvenienceandspeedofextrusionbecameamajor
advantageoverothertechniques.Extrusioncanbeappliedtoawidevariety
oflipidspeciesandmixtures,itworksdirectlyfromMLVswithouttheneed
forsequentialsizereduction,processtimesareontheorderofminutes,and
it is only marginally limited by lipid concentration compared with other
methods.Manufacturingissuesrelatedtoremovaloforganicsolventsorde-
tergentsfromfinalpreparationsareeliminatedandtheequipmentavailable
for extrusion scales well from bench volumes (0.1 to 10 mL) through pre-
clinical(10 mLto1liter)toclinical(>1liter)volumesemployingrelatively
low-costequipment,especiallyattheresearchandpreclinicallevels.
Extrusion and Extrusion Devices
MLVsformspontaneouslywhenbilayer-forminglipidmixturesarehy-
drated in excess water, but they exhibit a broad size distribution ranging
from 0.5 to 10 (cid:3)m in diameter and the degree of lamellarity varies
dependingonthemethodofhydrationandlipidcomposition.Thesefactors
restrictseverelythepracticalapplicationofMLVsformembraneanddrug
delivery research, as discussed in detail elsewhere.3 In general, <10% of
the total lipid present in a normal multilamellar liposome is present in
the outer monolayer of the externally exposed bilayer compared with
50%intheoutermonolayerofalargeunilamellarsystem.1Consequently,
the LUV better reflects the bilayer structure of a typical plasma or large
organellemembrane.OtherlimitationsofMLVsincludetheirlargediam-
eter,sizeheterogeneity,multipleinternalcompartments,lowtrapvolumes,
and inconsistencies from preparation to preparation. Therefore, sizing
MLV preparations by extrusion is an effective way to overcome some of
these problems and to generate reproducible model membrane systems
forbasic research, applied research, and clinicalapplications.
Only moderate pressures (typically 200–800 lb=in2) are required to
force liquid crystalline MLVs through polycarbonate filters with defined
pore sizes. The majority of laboratories specializing in liposome research,
particularly as applied to drug delivery, use a heavy-duty device com-
mercially available from Northern Lipids (Vancouver, BC, Canada;
www.northernlipids.com). The Lipex extruder is an easy-to-use, robust
stainless steel unit, which can operate up to pressures of 800 lb=in2
(Fig.1). Aquick-fitsampleport assembly allows forrapid and convenient
cycling of preparations through the filter holder. The sequential use of
large to small pore filters2 to reduce back pressure is not necessary for
[1] extrusion technique 5
Fig. 1. A research-scale extrusion device (Lipex extruder) manufactured by Northern
Lipids(Vancouver,BC,Canada)hasa10-mLcapacityandcanbeoperatedoverawiderange
oftemperatureswhenusedincombinationwithacirculatingwaterbath.Thequick-release
sampleportatthetopoftheunitallowsforrapidcyclingofsamplethroughthefilters.
6 methods of liposome preparation [1]
the majority of lipid samples, and large multilamellar systems can be ex-
truded directly through filter pore sizes as small as 30 nm. The equipment
is also fitted with a water-jacketed, sample-holding barrel that enables the
extrusion of lipids with gel–liquid crystalline phase transitions above room
temperature, an important feature as gel-state lipids will not extrude (see
Effect of Lipid Composition on Extrusion, later).
Extrusion can also be performed with a hand-held syringe fitted with
a standard sterilization filter holder or purpose-built hand-held units, such
as those supplied by Avanti Polar Lipids (Alabaster, AL; www.avanti-
lipids.com) and Avestin (Ottawa, ON, Canada; www.avestin.com). These
devicesare suitable only forsmall-volumeapplications (typically <1 mL);
oneexampleconsistsoftwoHamiltonsyringesconnectedbyafilterholder,
allowingforback-and-forthpassageofthesample.5Usingthistechnique,a
dilutesuspensionofliposomes(composedofliquidcrystallinelipid)canbe
passed through the filters to reduce vesicle size. This method, however, is
limitedbythebackpressurethatcanbetoleratedbythesyringeandfilter
holder, as well as the pressure that can be applied manually. Gener-
ally, phospholipid concentrations must be less than 30 mM in order to
comfortably extrude liposomesmanually.
Avarietyoffilterssuitableforreducingthemeandiameterofliposome
preparations are available from scientific suppliers. The most commonly
used are standard polycarbonate filters (with straight-through pores).
Other filter materials can be used, but the polycarbonate type has proved
to be reliable, inert, durable, and easy to apply to filter supports without
damage. Pore density influences extrusion pressure. In our experience
thereisusuallylittlevariationbetweenfiltersfromthesamemanufacturer.
However, on occasion users may notice changes in vesicle diameter pre-
pared when using filters from different batches from the same supplier or
when using filters in which the pores are created by different manufactur-
ing processes. Tortuous path type filters do not have well-defined pore
diameters like the straight-through type, and back pressure tends to be
higherwhenusingthesefiltersforliposomeextrusion.However,adequate
size reduction canstill be achieved.
Mechanism of Extrusion and Vesicle Morphology
As the concentric layers of a typical MLV squeeze into the filter pore
under pressure during extrusion, a process of membrane rupture and
resealingoccurs.Thepracticalconsequenceofthisisthatanysolutetrapped
5R. C. MacDonald, R. I. MacDonald, B. P. Menco, K. Takeshita, N. K. Subbarao, and
L.R.Hu,Biochim.Biophys.Acta1061,297(1991).
[1] extrusion technique 7
insideanMLVorlargeliposomebeforesizereductionwillleakoutduring
theextrusioncycle.Therefore,whenspecificsolutesaretobeencapsulated,
extrusionisnearlyalwaysperformedinthepresenceofmediumcontaining
thedesiredfinalsoluteconcentrationandexternal(unencapsulated)solute
is removed only when sizing is complete. In a study on the mechanism of
liposome size reduction by extrusion, Hunter and Frisken6 demonstrated
thatthe pressureneeded toreduce theparticlesize ofvesiclesduring pas-
sage through a 100-nm pore correlated with the force needed to rupture
thelipidmembraneandnottheforcerequiredsimplytodeformthebilayer.
Interestingly,theseauthorsalsonotedthatasflowratethroughthefilterin-
creasedthemeanvesiclesizedecreased.Thisisattributedtothethicknessof
the lubricating layer formed by fluid associated with the sides of the pore
from which particles are excluded. As the velocity of the fluid increases
the thickness of the lubricating layer also increases, effectively reducing
thepore diameter experienced by vesiclestraversing the membrane.6,7
Theruptureandresealingprocesscanalsogiverisetoovalorsausage-
shapedvesicles,andMui et al.8showedthat this shapedeformationis dic-
tatedlargelybyosmoticforce.Asvesiclesaresqueezedthrough thepores
they elongate and lose internal volume through transient membrane
rupturetoaccommodatetheincreaseinsurfacearea-to-volumeratioasso-
ciated with the nonspherical morphology. On exiting the pore the mem-
brane wants to adopt a spherical shape, thermodynamically the lowest
energy state for the bilayer, but the required increase in trapped volume
is opposed by osmotic force. Therefore, in the presence of impermeable
or semipermeable solutes (e.g., common buffers and salts) oval or saus-
age-shaped vesicles are produced, whereas vesicles made in pure water
arespherical (Fig.2A and B).
Sausage-like and dimpled vesicle morphology is observed when extru-
sion occurs even in solutions of relatively low osmolarity, such as 10 mM
NaCl. It should be noted that these vesicle morphologies have been ob-
servedonlywhenemployingcryoelectronmicroscopytechniques,inwhich
vesicles are visualized through thin films of ice in the absence of cryopro-
tectants. Freeze–fracture methods do not reveal sausage-like morphology
under the same conditions, which may be due to the high concentrations
of membrane-permeable glycerol (25%, v=v), used as a cryoprotectant,
affectingtheosmoticgradient.Roundingupofvesiclesisreadilyachieved
bysimplylowering theionicstrength ofthe externalmedium.8
6D.G.HunterandB.J.Frisken,Biophys.J.74,2996(1998).
7G.GompperandD.M.Kroll,Phys.Rev.EStat.Phys.PlasmasFluidsRelat.Interdiscip.
Topics52,4198(1995).
8B.L.Mui,P.R.Cullis,E.A.Evans,andT.D.Madden,Biophys.J.64,443(1993).