Liposomes Review

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Liposomes, or (phospho)lipid vesicles, are self-

assembled colloidal particles that occur naturallyand can be prepared artificially1, as shown by

Bangham and his students in the mid-1960s2. At first,they were used to study biological membranes; severalpractical applications, most notably in drug delivery,emerged in the 1970s2. Today, they are a very usefulmodel, reagent and tool in various scientific disciplines,including mathematics and theoretical physics (topologyof two-dimensional surfaces floating in a three-dimensional continuum), biophysics (properties of cellmembranes and channels), chemistry (catalysis, energyconversion, photosynthesis), colloid science (stability,thermodynamics of finite systems), biochemistry (func-tion of membrane proteins) and biology (excretion, cell

function, trafficking and signalling, gene delivery andfunction)1. On the applied side, several products rely ontheir colloidal, chemical, microencapsulating and surfaceproperties; these products range from drug-dosage forms(antifungals, anticancer agents, vaccines) and cosmeticformulations (skin-care products, shampoos) to diagnos-tics and various uses in the food industry. However, itseems that drug-delivery applications are now the mostwidely investigated area of their practical applications1.

Liposomes were introduced as drug-delivery vehiclesin the 1970s. Early results were, however, rather disap-pointing, owing mainly to their colloidal and biologi-cal instability, and their inefficient and unstable encap-sulation of drug molecules. Their utility was improved

following basic research that increased our understand-ing of their stability and interaction characteristics3. Inparallel, several pharmaceutical enterprises werefounded that survived the decline in the commercialappreciation of liposomes in the 1980s and early 1990sand, eventually, put several commercial products on themarket. At present, these include antifungal and anti-cancer preparations that compare favourably to exist-ing treatments, but a recent renaissance in liposomeresearch is promising many more products to come.

Amphiphiles and self assemblyMolecules can be roughly divided into the polar and

the nonpolar according to the symmetry and distri-

bution of their electronic clouds. Polar molecules are

soluble in polar solvents and insoluble in nonpolar sol-vents, and vice versa; for example, it is well known thatoil and water do not mix. Some molecules, however,posses a polar and a nonpolar group on the same mol-ecule; these molecules are called amphiphiles and,owing to hydrophilic and lipophilic interactions, theycan self-organize and form ordered structures in sol-vents. In these self-assembled structures, molecules areoriented in such a way that the polar portion of themolecule is in contact with the polar environment andshields the nonpolar part, and vice versa. Well-knownamphiphiles include soaps, detergents and polar lipids(lecithins, kephalins) and, in aqueous mixtures, thesemolecules are able to form several different phases. At

high concentrations, they form liquid-crystalline phases(characterized by long-range order) that can, upondilution in excess water, be dispersed into relativelystable colloidal particles1. The most frequently formedliquid-crystalline phases are the lamellar and, to a lesserextent, hexagonal and cubic phases (Fig. 1). Thesemacroscopic phases can be dispersed into colloidal par-ticles known as liposomes, hexasomes and cubosomes,respectively; the particles retain the short-rangesymmetry of their original parent phase.

The symmetry of lipid self assembly and liquid-crystalline-phase formation shows strong dependenceon the molecular shape of the amphiphile. In the firstapproximation, one can define the cross-sectional areas

of the hydrophobic (Anp) and polar (Ap) parts. ForAp Anp, structures with high curvature (such asmicelles) are formed. When the areas are comparable,a bilayered configuration is the most stable form, while,forAnp Ap, inverse micelles (with negative surfacecurvature) are formed. Furthermore, in self-assembledmulticomponent systems, one can define an averagevalue for the two parameters, Anp and Ap,which are typically a linear combination of individualAs multiplied by the mole fraction of a particular mol-ecule. Figure 2 shows the influence of molecular geom-etry on the symmetry of the phases formed4. BecauseAnp and Ap, as well as Anp and Ap, can bechanged [by temperature or ionization(pH), and by

change of composition forAs], phase transitionsbetween various phases can be induced, as will beshown below.

TIBTECH JULY 1998 (VOL 16) Copyright 1998, Elsevier Science Ltd. All rights reserved. 0167 7799/98/$19.00. PII: S0167-7799(98)01220-7 307

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Novel applications of liposomesDan D. Lasic

Opinions of the usefulness of liposomes in various biotechnological applications range from unsubstantiated optimism to

undeserved pessimism. This article reviews the background and development of liposomes, describes products that are

commercially available and speculates optimistically about some future applications. The current deepening and widening

of interest in liposomes in many scientific disciplines, and their application in medicine, immunology, diagnostics, cosmetics,

ecology, cleansing and the food industry are promising novel breakthroughs and products.

D. D. Lasic is at Liposome Consultations, 7512 Birkdale Drive,Newark, CA 94560, USA.


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This molecular-shape analysis and the concept of theshape parameter (Anp/Ap), which were introduced (onthe basis of similar concepts in emulsion technologyand in Tanfords treatment of the shapes of micelles) byIsraelachvili and co-workers, is very useful for a quali-tative understanding of the topology of lipid aggregateswith different lipid compositions. However, one mustbe careful not to attempt rigorous thermodynamicanalyses of liposome models based on these concepts,

because such an analysis can be applied only to systemsat thermodynamic equilibrium1. If applied to lipo-somes containing lipids with a given shape parameter,such an analysis would yield a very narrow size distri-bution, which is never observed in practice. Suchthermodynamic models also predict the spontaneousformation of liposomes, but a high-energy process istypically needed to produce liposomes (see spontaneousvesiculation, below).

Current industrial applications of liposomes are basedon their colloidal properties (size, microencapsulation,membrane mechanical strength and surface properties)as they are defined during their preparation. Novel sys-tems may incorporate some time-dependent or other

specific inducible changes in the liposome membraneor its coating to produce intelligent liposomes that willchange their properties (e.g. leakage rate, fusogenic

activity or interaction with particular cells) upon a spe-cific trigger following their application. Examples ofsuch sytems are temperature- and pH-sensitive lipo-somes, which will be described briefly below. In moregeneral terms, such preprogrammed changes can bedescribed by phase transitions within the bilayer, itscoating or the encapsulated agents.

Lipid bilayers and liposomes exhibit various phasetransitions that might be used to trigger drug release or

liposome fusion upon an appropriate trigger. Lipidbilayers can exist in a low-temperature solidorderedphase and, above a certain temperature, in a fluiddisordered phase; the temperature of this phase transi-tion, Tc, can be tailored by selecting the proper lipids.The transition can be exploited to control liposomeleakage: for example, the leakage is maximal at tem-peratures around Tc, owing to the coexistence of twophases and numerous defects on the boundariesbetween the domains5. Such liposomes can be used todeliver encapsulated drugs, possibly with external heat-ing, more effeciently into target tissues. Bilayers can alsoundergo transitions into a different liquid-crystallinephase: by increasing either the total or average area

of polar heads Ap on the outer monolayer byhydrolysis or protonation of lipids containing weakbasic groups, a lamellarmicellar phase transition can

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Inverse-prolate micelleProlate micelleInverse micelleMicelle

Hexagonal phaseInverseHexagonal phaseNormal

Oblate micelle,bilayered fragments Lamellar phase

Largeunilamellar

vesicleSmall

unilamellarvesicle

Large multilamellar vesicle

Liposomes

Figure 1Self aggregation of polar lipid molecules. Depending on the temperature, the molecular shape of the lipids and the conditions in the lipidwater

mixture (concentration and ionic strength), lipid molecules may self assemble into different colloidal particles. At higher concentrations,

these particles can organize into ordered liquid-crystalline phases. Several different types of liposome are shown in the centre of the figure.


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be triggered. This results in rapid leakage of the encap-sulated molecules followed by liposome dissolution.Analogously, by decreasing Ap, lamellarinverse-hexagonal phase transition can occur. These transitionscan induce liposome fusion and the release of their con-tents. For membranes containing a lipid with a proton-atable weak-acid group (pH-sensitive liposomes6), thiscan be accomplished by lowering the pH; alternatively,

the transition can be induced by depleting lipids withlarge Ap, such as poly(ethylene glycol)-linked lipids(PEG-lipids) [typically, a diacyl lipid with attachedpoly(ethylene glycol) polymer], either by faster disso-ciation kinetics of these more-hydrophilic moleculesfrom the bilayer [by tailoring the hydrocarbon chain(s)and/or the size of the PEG polymer] or by designinga biodegradable linkage of PEG to the lipid710. Suchshedding of a polymer coating can also induce lipo-some destabilization (a stealthnonstealth phase tran-sition, resulting in enhanced cell uptake), as will be dis-cussed below10. At this point, we should note thatsuccessful operation in the test tube is a necessary, butnot a sufficient, condition for the system to work in

vivo. The presence of various plasma proteins can sta-bilize liposomes and prevent liposome disintegration orfusion. Table 1 shows some of phase transitions in

liposomes and the effect they have on the parentliposome.

Liposomes and sterically stabilized liposomesLiposomes are microscopic spherical particles in

which membranes, consisting of one or more lipidbilayers, encapsulate a fraction of the solvent in whichthey are suspended into their interior1. Studies during

the 1970s and 1980s typically used ill-defined largemultilamellar vesicles in the micrometre size range,while more recent investigations mostly use homo-geneous unilamellar vesicles in the size range50150 nm. This size range is a compromise betweenloading efficiency of liposomes (increases with increas-ing size), liposome stability (decreases with increasingsize above an optimal 80200 nm range) and ability toextravasate (decreases with increasing size). The thick-ness of the membrane is around 4 nm, and it can con-tain a polymer coating and/or ligands with definedfunctions, such as specific binding or fusogenic activ-ity. Although, in the early studies, liposome stabilitypresented a big problem, very stable liposomes can now

be produced in large quantities.Liposome stability can be subdivided into physical,chemical and biological stabilities, which are all

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Species Shape Organization Phase

SoapsDetergents

Lysophospholipids

PhosphatidylcholinePhosphatidylserinePhosphatidylinositolSphingomyelinDicetylphosphateDODAC

PhosphatidylethanolaminePhosphatidic acidCholesterolCardiolipinLipid A

Inverted cone

Micelles

Cylinder

Bilayer

Cone

Reverse micelles

IsotropicHexagonal 1

Lamellar(Cubic)

Hexagonal 2

Figure 2The effect of molecular shape on the structure of the amphiphilic aggregate. Cone-like molecules tend to pack into structures with high radii

of curvature and inverse-cone-like molecules form structures with large negative curvatures; cylindrical molecules organize into flat bilay-

ers. Combining cone- and inverse-cone-like molecules (which individually pack into structures with high curvature) can result in the formation

of a flat bilayer. Such a bilayer can, however, be unstable and undergo a phase transition into a normal or inverse-micellar structure.


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inter-related. The shelf-life stability of pharmaceuticaland cosmetic products is determined by the physicaland chemical stability of the liposomes (uniformity ofsize distribution and encapsulation efficiency, and mini-mal degradation of all compounds, respectively). Byoptimizing the size distribution, pH and ionic strength,as well as the addition of antioxidants and chelatingagents, liquid liposome formulations can be stable foryears. Furthermore, the addition of appropriate cryo-

protectants allows their storage in frozen or lyophilizedform. Biological stability, however, depends on thepresence of agents that interact with liposomes uponapplication to the subject. Forin vivo cases, it thereforealso depends on the administration route1.

Problems with the colloidal instability of liposomesin the test tube were solved simply by increasing thecharge on the liposomes and/or decreasing the ionicstrength of the medium. Alternatively, the repulsionbetween liposomes can be increased sterically. Stericstabilization is more universal because it is salt inde-pendent, more effective at higher liposome (colloidalparticle, in general) concentrations and more reversibleupon particle aggregation by freezing. Additionally, the

presence of inert surface groups also reduces liposomeinteractions with macromolecules and thus stabilizesthese particles in media that are deleterious to liposomeintegrity. A major breakthrough in liposomal drugdelivery was the realization that the electrostatic stabi-lization of liposomes cannot provide adequate stabilityto liposomes in the presence of disintegrating sub-stances such as the proteins and enzymes encounteredin in vivo applications1. Consequently, the colloidal andbiological stability of liposomes was drastically increasedby coating liposomes with inert hydrophilic poly-mers1,1117. Because prolonged liposome circulation inblood and stability in general are due to steric stabi-lization by surface-grafted polymers, which reduce

interactions with blood proteins, the polymer-coated,long-circulating liposomes were called sterically stabi-lized liposomes11,16,17 but, owing to their ability to

evade the defenses of the immune systems of the body,they are sometimes also referred to as stealth lipo-somes16 (Stealth liposome is a trademark of SequusPharmaceuticals). The majority of sterically stabilizedliposomes are ultimately cleared by macrophages, albeitwith a lower rate than other formulations, but they arecleared by macrophages in tissues other than the liverand spleen, which take up the majority of conventionalliposomes. This altered biodistribution opens up the

possibility of targeting these sites, which may harbourmany infectious diseases (HIV, mycobacteria) and arealmost inaccessible to regular drug-delivery systems.

Experiments have shown that optimal stability isobtained at around 5 mol% of PEG-lipid [PEG mole-cular weight 2000 Da (2000PEG)]17. The fundamentalquestion becomes liposome stability as a function of thepolymer-chain density (D, the distance between poly-mer-attachment points on the bilayer) and degreeof polymerization (N). Following scaling analyses18,19,we can express the thickness of polymer coating (hc) asEqn 1,

hc DN(d/D)35 (1)

where dis the size of the monomer. Comparing repul-sive force with van der Waals attraction [which can, atthe distance hc from a particle with diameter b, beapproximated as Ab/hc (where A is the Hamakerconstant), and should be smaller than thermal energykT to prevent binding (where k is the Boltzmannconstant, Tthe temperature)], one gets hc/b A/kT.The latter quotient is typically 0.1 and it follows that,forhc 0.1b, one gets sufficient steric stabilization toovercome attractive interactions with other macro-molecules or liposomes. In other words, the polymercoating should have a thickness of around 10% of theparticle diameter17. Experiments have shown nice

agreement between the model and measured forcedistance profiles by the osmotic-stress technique20 andwith surface-force apparatus21; sterically stabilized


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Table 1. Phase transitions in liposomes

Phase transition Trigger Result

Gelliquid-crystal Temperature Liposome leakage

Ionic strength Liposome leakageLamellarmicellar Hydrolysis Leakage, disintegrationIonization of weak amphiphilic bases (pH) Leakage, liposome disintegrationProtonation of surface-attached polymer Leakage, liposome disintegration

Lamellarinverse-hexagonal Protonation of weak amphiphilic acids Liposome fusion, leakage(pH change)

PEGPE-linkage break in an unstable bilayer Liposome fusion, leakagePEGPE dissociation from the bilayer Liposome fusion, leakage

Steric destabilization (stealthnonstealth) Removal of PEG from a stable bilayer Liposome destabilization,macrophage uptake

Collapse of a polymer brush Temperature, presence of specific ions Liposome destabilization, fusion(random-coilhelix )

Precipitation or gelation of liposome- Gradient loading Stable encapsulation, shapeentrapped agent change

Solubilization of encapsulated gel, crystals pH change, addition of ionophores Leakage of encapsulatedmolecules

Abbreviations: PE, phosphatidyl ethanolamine; PEG, poly(ethylene glycol).


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micelles and emulsions are a logical variation of theseliposomes. This work has triggered considerable theo-retical and experimental efforts to study the origin ofsteric stabilization, polymers at interfaces and nonfoul-ing and nonadsorbing surfaces in the systems based onPEGylated bilayers. This system offers well-controlled

and -defined conditions compared with adsorbed(block co)polymers, such as PEGpolyoxypropyleneoxide. However, it seems that, owing to some biolog-ical constraints (possibly some filtering mechanism inthe spleen), we still cannot prepare larger long-circulating liposomes (200500 nm).

The generality of steric stabilization was shown whenother polymers that conveyed similar protection werediscovered22,23. In parallel, chemical methods weredeveloped to synthesize various polymer lipids16,22,23

and conjugate them with antibodies or their fragments,proteins, peptides and oligosaccharides24. Because theycan evade nonspecific uptake, sterically stabilized lipo-somes with surface-attached ligands also revived hopes

of achieving the elusive goal of site-specific drug tar-geting by using liposomes2528. Additionally, other thera-peutic benefits (such as passive targeting of tumours)were discovered, as will be described below29,30.

Spontaneous formation of liposomesNumerous papers claim erroneously that liposomes

form spontaneously upon hydration of lipids. Withoutagitation, however, only few liposomes would form,caused by crystal defects and not to the spontaneity ofliposome formation upon swelling of lipids. However,below, we shall show that this is exactly the feature thatmakes liposomes useful for drug encapsulation anddelivery.

A few simple observations indicate that liposomes arenot, in general, a thermodynamically stable state and socannot form spontaneously. First, in order to produceliposomes, some energy (e.g. sonication, extrusion, orhomogenization) must be dissipated into the system.Also, after liposomes are formed, they are, in general,not colloidally stable and slowly aggregate and fuse intolarge and more lamellar structures with greater radii ofcurvature that eventually settle out of the liquid. Ther-modynamically stable systems, such as micelles, wouldform simply by mixing surfactant into water andwould, disregarding chemical degradation, stay in thisphase forever. A long-term goal of liposome manu-facture is therefore the preparation of spontaneous

liposomes that is, vesicles that would form just bymixing lipids with an aqueous phase.Following the theoretical work of Franks31 and, espe-

cially, Helfrich32, this instability was explained by bend-ing elasticity. Symmetric membranes prefer to be flat(spontaneous curvature, C0, 0) and energy is requiredto curve them. The bending elasticity per unit area (Eb)for small distortions with both principal curvaturesbeing equal is approximated by Eqn 21,32,

Eb 12Kb (2CC0)

2 kC2 (2)

where Kb is the bending elastic modulus, k is themodulus of Gaussian curvature and Cis the curvature

( 1/radius, R). Examining the equation above andneglecting Gaussian curvature (kK), we can see thatliposomes can form spontaneously only in the case of

extremely soft bilayers in which the excess energy iscomparable to the thermal energy kT; such liposomesare entropically stabilized (i.e. Eb kT). Alternatively,for non-zero values of spontaneous curvature, thecurved state also has lower free energy and liposomescan form spontaneously (i.e. Eb 0 for 2C C0). An

example of forming a C00 system is increasing thearea of one monolayer with respect to the opposed one,for instance by asymmetrically changing Ap of theouter monolayer by ionization (pH) or inserting mol-ecules1. In practice, mixing charged and uncharged sur-factants3335 or detergents with opposite charges atunbalanced ratios36 gives rise to a heterogeneous popu-lation of vesicles. Spontaneous formation indicates athermodynamically controlled process, but the lack ofa narrow size distribution indicates that the energyminimum is very shallow.

Obviously, a liposome with an asymmetric distribu-tion of surface-attached and interacting polymersbetween the two monolayers is also characterized by a

non-zero spontaneous curvature35. Experimentally,spontaneous vesiculation has been achieved in a mix-ture of an ionic surfactant (such as sodium dodecyl sul-fate or cetyl-trimethyl-ammonium chloride), PEG-ylated surfactant (5000PEGstearate) and octanol bysimply increasing the ionic strength of this mixedmicellar solution. Micelles are formed in pure water.Typically, 10 g l1 of sodium dodecyl sulfate, 614 g l1

octanol and 620 g l1 of 5000PEGstearate are dis-persed in water and diluted with an equivalent volumeof 4% NaCl. The translucent mixture progressivelybecomes more turbid and vesicles of extremely uniformsize distribution are formed; heating accelerates thevesicle formation. Although the final state a stable,

homogeneous population of vesicles can be explainedby the natural curvature of asymmetric membranes, thevesiculation process that starts in an isotropic micellarsolution is more difficult to comprehend. When thesalinity increases, micelles start to grow and change toa disc-like conformation; the presence of octanol maystabilize the disc-like conformation over the rodlike.While these micelles fluctuate (similar to the shapechanges and inversions of an umbrella in the wind),PEG-surfactant distributes to each surface according toits curvature via rapid diffusion in-the-bilayer and overthe edges of fluctuating bilayer fragments. The micellesgrow by fusion and, when a disc-like micelles is largeenough, it self-closes into a vesicle. This elimination of

edges locks the asymmetric distribution of PEG poly-mer in and sets C0 0. The process is possible if thediffusion rate exceeds the fluctuation frequency of themicelle37.

Although the dream of any formulator, such lipo-somes may only be effective for the delivery ofhydrophobic drug molecules, because soft bilayers can-not encapsulate hydrophilic drugs stably. Such lipo-somes may be important in cosmetics, where they cansimplify the preparation of various topical formulationsand potentially improve the percutaneous absorptionof agents because their flexible and soft bilayers maymix effectively with the skin lipids. This may soften thelipid filling between keratinized cells in the stratum

corneum, and possibly their cell membranes, and, inparallel, enhance drug permeation into the skin. Addi-tionally, these surfactants are strong skin-penetration


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enhancers and it is difficult to separate the variouseffects responsible for the penetration of the drug intothe skin. On the theoretical side, spontaneous vesicu-lation is very important for our understanding ofvesicles and their thermodynamics.

In drug delivery, however, spontaneous vesicles rep-

resent a curious paradox: it is the thermodynamic insta-bility of normal liposomes that makes them stable as adrug carrier! Contrary to general belief, for an effec-tive drug-delivery system, self-assembled particles mustbe in a kinetically trapped, and not a thermodynami-cally stable, state. The systems that are thermodynami-cally stable simply change phase upon a change in theenvironment (e.g. upon administration to the patient),while systems or particles that are kinetically trappedstates are much more stable and can maintain theirproperties for extended times after administration. Asimple example from the test tube can illustrate thispoint: upon dilution, micelles and microemulsions,which are thermodynamically stable phases, disinte-

grate or aggregate, respectively, while liposomes re-main essentially unchanged1,38. Similarly, spontaneousvesicles alter their phase, depending on final concen-trations (i.e. move to a different position in a phasediagram).

Solving Eqn 2 for a closed vesicle with zero sponta-neous curvature produces Eqn 3,

e Eb Area 12Kb (2C)

2 4R2

8Kb

(3)

and we can see that, with each vesicle, there is an excessof free energy (e) of 8Kb (when the vesicle closes, the

area becomes 4R2 and C1

R). In this (Hookean)approximation, the stretching elasticity of the bilayer isnot taken into account and the excess energy of vesi-cle is size independent. Therefore, the fusion of twovesicles halves the total excess energy of the system.Although this process is not entropically favoured, thiscontribution can typically be neglected with respect toelastic energy (e kT).

In living cells, billions of vesicles are being generated(by pinching off from lipid tubules, exocytosis andendocytosis) and readsorbed (fusion with plasma mem-branes) constantly. The energy for this process comesfrom various proteins and their conformationalchanges. It has been speculated39 that this excess energy

can be conserved in living cells, where the fusion ofvesicles is constantly occurring. The excess energy, e,associated with each vesicle is around 1050 kT, whichcorresponds approximately to the energy provided bythe hydrolysis of several adenosine triphosphate (ATP)molecules (~15 kTper reaction)40. This analysis pointsto the possibility that some of the energy in the cell canbe stored in the curvature of various vesicles and mayeventually become bioavailable upon vesicle fusionwith the membrane39.

Industrial manufacture of liposomesAs for any new high-technology biotechnological

discipline, the transfer from the academic bench to an

industrial enterprise was crucial for liposomes.Although the first experiments in humans wereperformed with liposomes prepared fresh daily, any

commercial product must have well-defined stabilitycharacteristics and a shelflife of over a year.

In contrast to the many pessimistic forecasts duringthe 1980s, the reproducible preparation of large vol-umes (in batches of hundreds of litres) of stable lipo-somes no longer presents a problem. They are prepared

from well-characterized raw mater ials with establishedsafety profiles1,10,41. Furthermore, in most cases, long-term (shelf-life) stability problems have been success-fully solved as well.

The first larger-scale liposome manufacture wasperformed in the cosmetics industry, with more-demanding pharmaceutical formulations followingquickly. A characteristic liposome formulation consistsof several different lipids that have to be mixed beforehydration; normal practice is to lyophilize the lipidmixture from tert-butanol or spray-dried lipid powder,or to use a thoroughly dried thin film (although, inpractice, it can be very thick and supported on variousinert beads, which increase the surface area). Adding

organic solvents such as chloroform or methylene chlor-ide to solubilize and mix lipids is not recommended(US regulations allow 50 ppm of chloroform and500 ppm of methylene chloride in the formulation).Alternatively, lipid solutions in water-miscible (ethanol,propylene glycol) or -immiscible (ether, freon) organicsolvents can be injected into an aqueous phase followedby organic-solvent removal (and possibly recycling) byevaporation, filtration or dialysis. For cosmetic andsome nutritional products, this may not be neededbecause the remaining solvent (propylene glycol orethanol) may not be harmful and, additionally, preventsmicrobial growth. At higher lipid concentrations, allthese hydration methods give rise to large multilamel-

lar liposomes, which are not suitable for most appli-cations owing to their large size and low encapsulationvolumes for water-soluble compounds. These largeliposomes can be converted into smaller, unilamellarliposomes by several methods but, for large-scale appli-cations, extrusion and homogenization are basically theonly applicable techniques.

Stable, efficient drug encapsulation may be the great-est challenge for liposome manufacture. The hydro-philic drugs are typically introduced in the hydrationsolution or can be, in the case of weak bases or acids,loaded into preformed liposomes by a HendersohnHasselbalch distribution, because liposomes can main-tain various gradients across their membranes. The

most efficient loading is achieved by the entrapment ofweak bases by a pH (acidic inside)42,43 or ammonium-sulfate-concentration44 gradient. Analogously, weakacids can be loaded by a pH (alkaline inside) or sodium-bicarbonate-concentration gradient45. The stability ofthe entrapment of such molecules is, in general, muchlower than of the weak bases that can form precipi-tates with encapsulated anions because weak acids donot normally form precipitates with encapsulatedcations1,45. Electrostatic potential, osmotic pressure andother gradients are not very effective for drug loading.To ensure high trapping efficiencies of drugs that donot respond to gradients, however, work at high drugand, especially, lipid concentrations (100250 mM) is

still required. Theoretical encapsulation efficiencies ofabove 70% are never matched experimentally, whereencapsulation efficiencies around 50% are considered


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to be high and generally 2035% encapsulation effi-ciencies are all that can be obtained.

Hydrophobic drugs are added in the organic lipidsolution, but we must be aware that membrane load-ing can rarely incorporate more than 5 mol% of thehydrophobic drug into the bilayer (compared to the

lipid) and typically only 12%, which may be an orderof magnitude less in terms of (mg drug) (mg lipid)1

than in liposomes loaded by a gradient method. Fur-thermore, these bilayers are less stable because they donot contain cholesterol; its presence typically decreasesdrug loading.

Figure 3 shows a general way to prepare pharmaceu-tical liposomal formulations. Various short cuts are pos-sible in this scheme, for instance, skipping free-drugremoval in some remote loading methods or lyophiliza-tion for liquid or frozen products. If the liposomes arelarger than ~150 nm, sterile filtering is probably notpossible and so the whole process must be performedaseptically, which is not very practical from the engi-

neering or economical viewpoints. Other approaches,such as irradiation and autoclaving, in general causelipid or drug degradation, although liposomes can, insome cases, survive autoclaving.

In addition to liquid, frozen or lyophilized formula-tions, liposomes can be delivered as proliposomes,either in an organic solution (such as propylene glycol)or as a finely dispersed powder that transform into lipo-somes upon hydration during application. Self-swellingmixtures can be encapsulated into capsules for oraladministration from which liposomes can be formedupon capsule disintegration in the intestine.

Liposomes as drug-delivery vehicles

Liposomes resemble cell membranes in their struc-ture and composition. They are typically made fromnatural, biodegradable, nontoxic and nonimmunogeniclipid molecules and can encapsulate or bind a varietyof drug molecules into or onto their membranes. Con-sequently, all these properties make them attractive can-didates for use as drug-delivery vehicles, as advocatedby Schneider [his patent application for liposomesas drug-delivery systems in the mid-1960s wasrejected because of a pre-existing patent from IGIFarbenIndustrie (1934), which claimed that aqueoussuspensions of lecithin and cholesterol can be drugcarriers], Bangham, Ryman and, in particular,Gregoriadis in the early 1970s1,4648. After more than a

decade of work, it turned out, however, that thephysicochemical and biological properties of liposomesused at that time (e.g. leakage of drug molecules andshort residence in blood) limited their utility in drugdelivery and, especially, in cancer therapy1,48.

Liposome applications in drug delivery depend, andare based on, physicochemical and colloidal character-istics such as composition, size, loading efficiency andthe stability of the carrier, as well as their biologicalinteractions with the cells. There are four major inter-actions between liposomes and cells1,46. Lipid exchangeis a long-range interaction that involves the exchangeof liposomal lipids for the lipids of various cell mem-branes; it depends on the mechanical stability of the

bilayer and can be reduced by alloying the membranewith cholesterol (which gives rise to greatly improvedmechanical properties, such as an increased stretching

elastic modulus, resulting in stronger membranes and

reduced permeability). The second major interactionis adsorption onto cells, which occurs when the attrac-tive forces (electrostatic, electrodynamic, van der Waals,


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Clean and depyrogenize all equipment,prefilter and ultrafilter all solutions

Mix lipids in organic solvent

Solventrecovery

Dry Inject

Hydrate Diafilter

MLV

DownsizeExchange of

external buffer

LoadingRemove free drug

Prefilter

Sterile filter

Fill vials

Lyophilize

Seal

Label and package

Asepticconditions

Figure 3

A schematic representation of the manufacture of a liposomal product. Lipids are

mixed in an organic solvent; in the case of chloroform, methylene chloride, methanol,

tertiary butanol and some other solvents, these solvents are removed by evapora-

tion and vacuum drying or lyophilization (for t-butanol). The dry lipid film, paste or cake

is then hydrated. Alternatively, one can inject an ethanol or propylene-glycol lipid solu-

tion into the aqueous phase and remove the solvent by chromatography, filtration or

dialysis. Upon hydration of lipids, large multilamellar vesicles are typically formed.Their size can be reduced by extrusion, homogenization or sonication. Hydrophilic

drugs are normally added in the hydrating medium, while hydrophobic ones are codis-

solved in the organic phase. In the case of weak bases and acids, these molecules

can be loaded into a preformed liposome by a pH or ammonium-sulfate gradient; a

pH gradient can be established by exchanging the external solution by dialysis or gel

filtration, or by simple titration of the suspension. Free (i.e. nonencapsulated) drug

can be removed by dialysis, chromatography, filtration or centrifugation. For clinical

applications, these liposomes must be sterile; if they are smaller than 100150 nm

this can be achieved by sterile filtration (0.2 m filters; in many cases, ny lon filters

are the least likely to clog) but, for larger liposomes, the whole process must be

performed aseptically although, in some cases, heat sterilization can be applied.


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hydrophobic insertion, hydrogen bonding, specificlock-and-key etc.) exceed the repulsive forces (elec-trostatic, steric, hydration, ondulation, protrusionetc.)17. Obviously, this depends on the surface charac-teristics of liposomes and can be specific or nonspecific.Adsorption onto phagocytic cells is normally followed

by endocytosis or, rarely, by fusion. Endocytosis deliv-ers the liposome and its contents into the cytoplasmindirectly via a lysosomal vacuole in which low pH andenzymes may inactivate the encapsulated agent. Dur-ing fusion, however, the liposomes contents are deliv-ered directly into the cell and the liposomal lipidsmerge into the plasma membrane. Therefore, a sub-stantial effort is being undertaken to utilize this modeof drug entry. Efforts range from the incorporation offusogenic proteins into the bilayer to the preparation ofmetastable bilayers and pH-sensitive polymer coatings.As has already been mentioned, however, the presenceof plasma and the consequent adsorption of plasma pro-tein can stabilize liposomes mechanically and inactivate

these liposome-degradation mechanisms.For drug delivery, liposomes can be formulated as a

suspension, as an aerosol or in a (semi)solid form suchas a gel, cream or dry powder; in vivo, they can beadministered topically or parenterally. After systemic(usually intravenous) administration, which seems to bethe most promising route for this carrier system, lipo-

somes are typically recognized as foreign particles andconsequently endocytosed by cells of the mononuclearphagocytic system (MPS), mostly fixed Kupffer cells inthe liver and spleen16,46. This fate is very useful fordelivering drugs to these cells but, in general, excludesother applications, including site-specific drug delivery

by using ligands expressed on the liposome surface inorder to bind to receptors (over)expressed on the dis-eased cells1. For this reason, a search for liposomes thatcould evade rapid uptake by the MPS started and a fewlipid compositions that prolonged liposome blood-circulation times were discovered49,50, culminating inthe development of PEG-coated, sterically stabilizedliposomes1117.

Based on the liposome properties introduced above,several modes of drug delivery can be envisaged: themajor ones are enhanced drug solubilization (e.g.amphotericin B, minoxidil), protection of sensitivedrug molecules (e.g. cytosine arabinose, DNA, RNA,antisense oligonucleotides, ribozymes), enhanced

intracellular uptake (all agents, including antineoplasticagents, antibiotics and antivirals) and altered pharmaco-kinetics and biodistribution of the encapsulated drug.The latter accounts for the decreased toxicity of lipo-somal formulations because liposome-associated drugmolecules cannot normally spill to organs such as theheart, brain and kidneys. However, we must be aware


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Table 2. Liposomal products on the market or in advanced clinical studiesa

Company Product Status

Sequus, Menlo Park, CA, USA Doxil: dox in stealth liposomes On the market since 1995 (USA) and

1996 (Europe)Spy 07: cisPt in stealth liposomes Phase I

NeXstar, Boulder, CO, USA Ambisome: AmpB in liposomes On the market since 1990 (Europe) and1997 (USA)

DaunoXome: dauno in liposomes On the market since 1996 (USA andEurope)

MiKasome: liposomal amikacin Phase ILiposome Co., Princeton, NJ, USA DC99: liposomal dox Phase III

Ventus: liposomal PGE1 Phase III not successfulAsta Medica, Frankfurt, Germany Topical anticancer cream On German marketAronex,The Woodlands, TX, USA Nyotran: liposomal nystatin Phase III

Liposomal annamycin Phase IIAtragen: liposomal retinoic acid Phase II

Inex, Vancouver, BC, Canada Liposomal vincristine Phase ISwiss Serum Institute, Bern, Switzerland Epaxal: hepatitis-A vaccine On Swiss market since 1994

Trivalent influenza vaccine Phase IIIHepatitis-A and -B vaccine Phase IDiphtheria, tetanus and hepatitis-A Phase Ivaccines

Diphtheria, tetanus, influenza and hepatitis-A Phase Ivaccine

Novavax, Rockville, MD, USA Escherichia colivaccine in synthetic Phase Iliposomes

Shigella flexnerivaccine in synth. liposomes Phase IIGI, Vineland, NJ, USA (veterinary) Newcastle-disease vaccine (chicken) On the market

Avian-rheovirus vaccine On the marketBiozone Labs, Pittsburgh, CA, USA ELA-Max: liposomal lidocaine On the US market since 1998

aOnly formulations that contain liposomes are shown. There are several lipid-based commercially available drug formulations whose structure ismicelar, mixed micellar, a liquid-crystalline suspension or a lipid-stabilized emulsion. Several liposomeDNA complexes are also in various phases of

clinical trials for cancer and cystic-fibrosis gene therapy.Abbreviations: AmpB, amphotericin B; dauno, daunorubicin; dox, doxorubicin HCl; cisPT, cisplatin; PGE1, prostaglandin E1


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that, in some cases, liposomes can be used only as a sus-pending vehicle and not as a drug carrier. Examplesinclude taxol, cyclosporin B, prostaglandins and somelow molecular weight non-steroid anti-inflammatorydrugs. Often, such molecules are neither hydrophilicnor hydrophobic and, if they can be suspended, tend

to stick to the surface of liposomes. Obviously, uponparenteral administration, the drug is washed away inseconds and its pharmacokinetics and biodistributionresemble formulations that solubilize the drug (such aswater, 400PEG, Cremophor or tetraglycol solutions ofthe drug). As discussed above, conventional liposomesare taken up by macrophages and can therefore serve asexcellent drug-delivery vehicles to these cells. How-ever, sterically stabilized liposomes, which are notavidly taken up by MPS cells, have different biodistri-bution properties and have shown enhanced accumu-lation in sites of trauma, such as tumours, infections andinflammations29,30,50, which are characterized by leakycapillaries51. This accumulation is due simply to their

prolonged circulation and small size (100 nm), whichenable them to extravasate52. Very small neutral andmechanically stabilized liposomes (produced by prepar-ing a bilayer with high mechanical strength, measuredby the stretching elasticity of the membrane53) alsoexhibit prolonged circulation times and may also accu-mulate in sites of trauma16,46,5456. In the first approxi-mation, we can simply view this as a statistical prob-lem: the longer a liposome can circulate (by avoidinguptake in the liver), the greater the possibility that itextravasates (leaves the vascular system) at the siteswhere the blood vessel are porous52. Whether non-sticky PEGylated liposomes extravasate more effi-ciently than conventional liposomes is not yet clear.

Measurements of extravasation (permeability coeffi-cients of liposomes in tumours) have shown, however,that the microvascular permeability of stealth liposomesin tumours is approximately twice that of conventionalliposomes, while, in nontumuor tissues, permeabilitiesare similar and lower57. We must also mention that thelong circulation times of liposomes in several studieswere artefacts of macrophage saturation or even theirimpairment by the toxic drug (as with doxorubicin);only recently, a long-overdue study has shown that theadministration of doxorubicin in conventional lipo-somes can severely deplete macrophages in the liver andpose a health hasard58.

Medical applications of liposomes in humansThere are more than ten thousand papers on numer-ous medical applications of liposomes in various pre-clinical models. However, we shall concentrate only onliposome applications in humans.

In many cases, effective chemotherapy is severely lim-ited by the toxic side effects of the drugs. Liposomeencapsulation can alter the spatial and temporal distri-bution of the encapsulated drug molecules in the body,which may significantly reduce unwanted toxic sideeffects and increase the efficacy of the treatment.Although many drugs have been studied in preclinicalsettings in numerous animal disease models1,4648, forhuman therapy, liposomal therapeutics are at present

used only in systemic fungal infections and cancer ther-apy47. However, in preventative medicine, liposome-based vaccines show great promise. Table 2 lists liposo-

mal products on the market and in advanced phases ofclinical development47.

Targeting the drug to macrophages (where the infec-tious agents often reside) and the reduced toxicity ofthe formulation (resulting from the limited spillage ofdrug to other tissues) increase the therapeutic efficiency

of treatment. The drug of choice in the treatment ofsystemic fungal infections is amphotericin B, which is,owing to its aqueous insolubility, typically formulatedinto detergent micelles. However, micelles are unstableupon systemic administration, and severe neuro- andnephrotoxicity limit the dose that can be administered.If the drug is formulated in a stable colloidal particle,it is delivered much more efficiently to macrophagesand, additionally, toxicity can be significantly reduced.Following this rationale, three lipid-based ampho-tericin-B formulations are commercially available:AmBisome (NeXstar, Boulder, CO, USA) contains thedrug formulated into small, negatively charged lipo-somes59; Amphotec (Sequus, Menlo Park, CA, USA)

is a stable mixed micelle of drug complexed withcholesterol sulfate60; and Abelcet (The LiposomeCo., Princeton, NJ, USA) is a homogenized liquid-crystalline suspension of drug and lipids61.

Although these products are getting an increasedshare of the market, a number of possible improvementscan be envisaged, including encapsulation of more-potent antifungals (which are too toxic for systemic usein a free form) and sterically stabilized liposomal anti-fungals and antibiotics for the targeting of targets awayfrom the MPS system. Additionally, such liposomes canact as long-circulating platforms to bind and neutralizepathogens or reduce inflammation by blocking appro-priate receptors overexpressed at the site of trauma.

Liposomal expression of oligosaccharides or antibodiesincreases the circulation times of ligands and, addition-ally, the multivalency of binding. For instance, sialyl-Lewis-x liposomes (liposomes with attached oligosac-charides to target inflammation receptors) were shownto be 750-times more effective at inhibiting selectin-E-mediated cellular adhesion than the free ligand62,while antibodies expressed on stealth liposomes tar-geted to the same receptors on cultured endothelialcells have shown 275-times higher selectivity and canbe used to deliver drugs to these cells effectively withincreased specificity63.

In general, the saturation of various receptors may bean important new modality in liposome applications

and such systems may become useful for treatinginflammatory, autoimmune and cardiovascular diseases.Owing to their systemic accessibility, these applicationsof targeted liposomes may be more important than incancer therapy, in which application the tumours arerarely directly accessible.

Liposomes in anticancer therapyMost of the medical applications of liposomes that

have reached the preclinical stage are in cancer treat-ment4648. Very early studies mostly showed reducedtoxicity of the liposome-encapsulated drug but, in mostof the cases, the drug molecules were not bioavailable,resulting not only in reduced toxicity but also in

severely compromised efficacy. Unfortunately, this wasalso found to be true for primary and secondary livertumours. Despite the fact that most of the drug ended


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up in the liver, it did not diffuse into malignantcells64.

Several clinical studies did not support the use ofconventional liposomes in cancer treatment48,64,65.Although it is still not clear whether such liposomes(especially the ones with remotely loaded anthracy-

clines) can be beneficial in cancer therapy, it has beendemonstrated that small, stable liposomes can passivelytarget several different tumours16,29,30,57 because theycan (owing to their biological stability) circulatefor prolonged times and (owing to their smallsize, 50150 nm) extravasate in tissues with enhancedvascular permeability51, which is often the case intumours51,57.

Two liposomal formulations have been approved bythe US Food and Drug Administration (FDA) andare commercially available in the USA, Europe andJapan for the treatment of AIDS-related Kaposissarcoma. Doxil (Sequus) is a formulation of doxo-rubicin precipitated in sterically stabilized liposomes66,

while DaunoXome (NeXstar) is daunorubicin encap-sulated in small liposomes with very strong and cohe-sive bilayers, which can be referred to as mechanicalstabilization53.

DaunoXome is a small liposome (small unilamellarvesicles consisting of distearoylphosphatidylcholinecholesterol (2:1), total lipid concentration ~27 mM)with daunorubicin (1 mg ml1) loaded by a pH gradi-ent48. These liposomes are relatively stable in the cir-

culation because they are small and their membrane iselectrically neutral and mechanically very strong53. Thisreduces the charge-induced and hydrophobic bindingof plasma components but does not protect againstvan der Waals adsorption. Also, uncharged liposomeformulations are less colloidally stable than charged

ones.Doxil is a liquid suspension of 80100 nm liposomes

(2000PEGdistearoylphosphatidylethanolaminehydro-genated-soya-bean phosphatidylcholinecholesterol,20 mM) with doxorubicin HCl at 2 mg ml1. The drugis encapsulated into preformed liposomes by an ammo-nium-sulfate-gradient technique and is, additionally,precipitated with encapsulated sulfate anions. Theseliposomes circulate in patients for several days, whichincreases their chances of extravasating at sites with aleaky vascular system. Their stability is due to their sur-face PEG coating as well as to their mechanically verystable bilayers16,20,21,53,66. Doxil was the first liposomaldrug approved by the FDA and has been on the mar-

ket since 1995, while DaunoXome was approvedapproximately six months later. Currently, both for-mulations are used in the treatment of Kaposis sarcoma,while some studies in solid tumours promise that theiruses may soon expand55,67.

DaunoXome was shown to be as effective as con-ventional therapies, with reduced drug toxicity andimproved quality-of-life scores of the patients55. Theuse of Doxil in Kaposis sarcoma has shown a high


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Box 1

Formulation Distribution Elimination time Clearance Volume of distributiona Ref.tg

(h) tg(h) (ml min1) (l)

Free Dox Minutes 27 48 000 1764 101PCPGdox 28 44 000 1400 102PCCLdox 7 8200 81 103PCChdox 17 19 000 160 104PCChdox 12 10 000 234 105Doxil 46b 90 5 106Doxil 39b 100 3.5 107Doxil 5.8,5.6c 51,56c 42,57c 2.8 68DaunoXome 4.4b 17.3 6.4 55DaunoXome 48b 400700 34 108

aVolume of steady-state distribution.bIn many experiments, it is difficult to distinguish between the distribution and elimination times; the reported t

gis simply calculated as

tg ln2 volume of distribution clearance rate.

cFor 10 and 20 mg m2 dose, respectively.Abbreviations: CL, cardiolipin; Ch, cholesterol; dauno, daunorubicin; dox, doxorubicin; PC, phosphatidyl choline; PG, phosphatidyl glycerol.

This table shows the pharmacokinetic parameters of various anthracycline formulations in patients. The free drug and drug weaklybound to the liposomes (phosphatidylcholinephosphatidylglycerol) show large volumes of distribution and consequently high clearancerates. The phosphatidylcholinecardiolipin formulation exhibits stronger doxorubicin binding but lower elimination time, indicating liveruptake. The phosphatidylcholinecholesterol formulation reflects a mixed pattern of liposome leakage and uptake by the mononuclearphagocytic system (MPS). DaunoXome exhibits very stable drug encapsulation, but a low drug-circulation half-life t15 points to MPS uptake.Doxil shows very low leakage and prolonged presence in the plasma; consequently, drug concentrations were shown to be 2.521times higher in Kaposis-sarcoma lesions than in normal skin107, as well as 11.4-, 5.2- and 9.9-fold higher than the free drug107 inpatients at doses of 10, 20 and 40 mg m2, respectively108.It should be noted that several of these formulations were fairly effective in mice tumour models, indicating the importance of stabledrug encapsulation. The difference in therapeutic activity between mice and humans can be explained by the thermodynamics of theliposome drug system and associated drug leakage. The situation is completely different in mice (where a four- to fivefold dilution

occurs upon bolus injection) and humans (where the dilution upon infusion can be up to 5000-fold). The same was true for manyformulations containing hydrophobic derivatives of various drugs.


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response rate in comparison with standard treatments68.Owing to the altered biodistribution of Doxil, its tox-icity profile is quite different from those of the free drugor conventional liposomes. Although the dose-limitingtoxic effect is handfoot syndrome and stomatitis (lipo-somes that are not taken by the MPS or do not

extravasate eventually end up in the skin, especially atthe areas where vascular system is constantly underpressure and slightly damaged)69, but nausea, vomitingand alopecia, which are usual after conventional ther-apy, are notably mild or absent after liposomal (Doxil)treatment70. Doxil has also been shown to have a 4.5-times-lower medium-pathology score for doxorubicin-induced cardiotoxicity than the free drug71, while thelevel of neutropenia is similar to that of the free drug.Despite these convincing results, however, it seems that,in the long run, the potential of this formulation isgreater for the treatment of solid tumours. For instance,in salvage therapy of ovarian carcinoma in patientsrefractory to platinum and paclitaxel, a 20% overall

response was recorded following treatment withDoxil72.

The difference between the various formulations canbe explained by the pharmacokinetic data shown inBox 1 and biodistribution reports, which have shownsignificant drug accumulation in tumours in the case ofsmall and stable liposomes16,48,50,57,68; free drug distrib-utes to a large volume (causing systemic toxicity), isquickly washed away and, because no specific tumourtargeting occurs, the activity is relatively low. Conven-tional liposomes are distributed to a smaller volume,depending on the drug leakage from liposomes, whichis a function of lipid composition and the method ofdrug encapsulation; this decreases in the following

order: phosphatidyl-cholinephosphatidyl-glycerol(PCPG) PCcholesterol PCcardiolipin69. Cor-respondingly, with improved liposome encapsulation,the volume of distribution decreases and the clearancerates are smaller. Because such liposomes typically donot target tumours (low blood-circulation times andlarger size) and end up in the MPS, the drug activityin most tumour models is compromised and, generally,the reduction in drug toxicity does not justify the useof such formulations. When the drug is encapsulatedin small, stable vesicles, the volume of distribution isvery small (mostly blood, some in liver and in tissueswith leaky vasculature), reducing toxicity and, ifaccompanied by liposome accumulation in tumours,

increased activity. Clearance rates are low because thedrug is stably encapsulated. The elimination times ofthe drug are not a good indicator of their activitybecause, typically, they represent the second phase, inwhich smaller amounts of the drug are involved. Theelimination times are calculated from the clearance rateand volume of distribution and do not correlate to lipo-some stability. It is actually the blood-circulation timethat, in the case of small, stable liposomes, determinestheir accumulation in tumours.

From this simple pharmacokinetic analysis, we canconclude that high clearance times, which are associ-ated with low volumes of distribution and smallchanges in biodistribution toward tumours, character-

ize good liposomal anticancer formulations. Of course,for a drug to remain active, it must not be encapsulatedtoo stably. When liposomes accumulate and become

trapped in the tissue, they start to disintegrate and drugmolecules leak into the surroundings, as can be inferredfrom observed drug-degradation products, fluorescent(confocal) microscopy and their anticancer activity. Theorigin of liposome disintegration is one of the leastunderstood processes, but it is very likely that various

lipases degrade the lipids and the softer bilayers start todisintegrate. PEG-lipids are also lost slowly, owingto their greater hydrophilicity, which allows enzymesto approach closely to the liposomes and cells to inter-nalize them. In parallel, gradients can dissipate and thedrugs can leak from topologically intact liposomes;mechanical damage and lipid phase transitions can alsooccur in regions with reduced liquid concentration.Although the real situation is far more complex, thissimplified explanation of pharmacokinetic and biodis-tribution data may explain the differences betweendifferent formulations.

Because of the complexity of systemic drug delivery,the potency of the immune system and the anatomy of

tumours, it is difficult to predict whether it will be pos-sible to prepare formulations that provide much moreeffective drug delivery. The next developments maythus rely instead on combinational therapy, which hasbeen found to be so effective in the treatment of AIDSand other diseases. Consequently, Sequus is developingcisplatin in sterically stabilized liposomes (phase I),while liposomal vincristine (phase II) and annamycin(phase II) are being developed by Inex (Vancouver, BC,Canada) and Aronex (The Woodlands, TX, USA),respectively. A second liposomal agent in the combi-national therapy may, however, additionally includevarious cytokines, such as interferons, interleukins andtumour necrosis factor (TNF), which are very active

(but also very toxic) anticancer agents. Preliminary dataon combinational therapy of TNF in sterically stabi-lized liposomes with Doxil have shown arrested tumourgrowth, whereas either agent alone only decreased thegrowth rate73.

Since the early days of liposome applications, sci-entists have been trying to develop liposomes thatcan target specific cells via surface-attached lig-ands1,2528,46,48,74. Promising in vitro experiments were,however, not reproduced in vivo because of nonspecificliposome clearance by the MPS. Obviously, stealthliposomes have revived the concept of targeted liposo-mal drug delivery. The immunogenicity of current for-mulations can be reduced by using fragments of

humanized antibodies, so the remaining problems arethe accessibility of tumours and cellular uptake of theliposomes. Solid tumours are rather inaccessible and thetherapeutic benefit can be expected mostly within thevascular endothelium or in other body fluids, especiallyin the case of targeting internalizing epitopes48,7476. Ifthe therapy proves to be effective, we must also be awarethat the large-scale manufacture of targeted liposomesis technically considerably more demanding and theeconomics more critical than for nontargeted liposo-mal formulations. The future research will thereforeprobably concentrate on small liposomes with human-ized antibody fragments that will target internalizingreceptors. In addition to cancer therapy, such liposomes

could be used to target endothelial cells and circulat-ing cells and components in the blood circulationsystem for other (e.g. cardiovascular) diseases.


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Immunogenicity can be further reduced byusing smaller ligands. One example of this is folate,because its receptor (which also mediates endocytosis)is upregulated in many tumours. Its lipid, and especiallyPEG-lipid, conjugates can be easily synthesized andpurified, thus simplifiying liposome manufacture and

preservation, and reducing costs27. Next-generationligands may include targeting peptides from variouslibraries.

Because of their accumulation in tumours and sitesof infection and inflammation, sterically stabilized lipo-somes can be used to deliver other agents to these sites:antisense oligonucleotides and ribozymes are verypotent agents, but the major problem is their deliveryto and into the appropriate cells10. It is very likely thatencapsulation of these agents in stealth liposomes maynot only dramatically improve the therapeutic indexbut also be the optimal way to deliver these molecules10.

Other applications: vaccination, gene therapy and

diagnosticsAs a result of their special properties, liposomes are

being studied for the treatment of other diseases andfor vaccination applications. Examples are liposomalantibiotics, antivirals, prostaglandins, steroid and non-steroid anti-inflammatory drugs, insulin, and manyothers. Administration routes being investigatedinclude the pulmonary, topical and other parenteralroutes. Although several approaches look promising, alot more basic research will have to be performedbefore meaningful clinical studies can be initiated.Some promising concepts are described below.

Infections and inflammations are characterized by aleaky vascular system in a similar fashion to tumours.

Sterically stabilized liposomes may therefore be aneffective delivery vehicle for the delivery of appropri-ate drugs in such circumstances. Many infectious agentsreside in cells that are practically inaccessible tochemotherapy, such as deep-tissue macrophages. Asalready mentioned, a large fraction of stealth liposomesend up in the skin, where many are ultimately takenup by macrophages. It is also believed that these cellsharbour many bacterial and viral infections and sothe potential of long-circulating liposomes should bethoroughly exploited.

Recombinant-DNA technology and studies of genefunction and gene therapy all depend on the deliveryof nucleic acids into cells in vitro and in vivo. Although

in vitro techniques can rely on a number of physical andchemical methods, in vivo delivery is more demanding.DNAcarrier systems include several colloidal particles:cationic liposomes have been shown to complex (nega-tively charged) DNA, and such complexes were able totransfect cells in vitro74, resulting in the expression ofthe protein encoded in the DNA plasmid in the targetcells. Obviously, for gene therapy (the treatment of dis-eases on the molecular level by switching genes on oroff ), in vivo delivery is preferred. It was discovered thatcationic lipid-based DNA complexes can transfect cer-tain cells in vivo upon localized (mostly lung-epithelialcells upon intratracheal instillation) or systemic(endothelial cells in the lung) administration. However,

the first series ofin vivo studies yielded rather low lev-els of gene expression. Improvements were sought bothon the molecular level, where many novel lipids were

synthesized and many different combinations of lipidstried7881, and the colloidal level, where the struc-tureactivity relationships of the DNAlipid complexwere studied10,8285. Despite the fact that the QSAR(quantitative structureactivity relationships) are stillnot understood, developments in DNA-plasmid

design, the synthesis of novel cationic lipids and chol-esterol stabilization of complexes81,85, and improve-ments in the colloidal structures85 have resulted in morethan a thousandfold increase in gene expression overthe initial experiments10. Although these developmentsare impressive, the absolute expression levels are stillrather low, the tissue specificity of expression uponin vivo administration rather uncontrollable and theduration of expression short; before initiating realisticclinical trials, more research on the delivery vehiclesand the DNA vector will be required (for example, thelatter may include DNA sequences that would give riseto nuclear localization10). Improvements in the lipid-based carr iers may include coating reactive cationic sur-

faces with negatively charged bilayers or by stericallystabilizing coatings. It has been shown that PEG-lipidscan coat conventional liposomes by transfer from PEG-lipid micelles into liposomes in the incubationmedium86, and a similar approach may be used to coatDNAlipid complexes. Such liposomes may containtargeting ligands and, perhaps, incorporate somefusogenic function or component. Alternatively, othercondensing agents can be used and the condensedDNA entrapped into neutral or negatively chargedliposomes.

Because site-specific, efficient, long-term geneexpression is still an elusive goal, researchers should notconcentrate on demanding targets, such as cystic fibro-

sis, but instead try to treat diseases where a low level ofshort-term expression might be sufficient. Therapeu-tic genes may include various cytokines and other regu-latory molecules for the treatment of inflammations andinfections. Gene expression in the lung epithelium,upon pulmonary administration, or by the endothelialcells or hepatocytes, upon systemic administration, canlead to therapeutic activity. Hepatocyte targeting bysmall, flexible liposomes is well known. Larger, stable(sterically stabilized) liposomes are taken up by thespleen. The massive, rapid targeting of endothelial cellsin the liver was recently reported by the small liposomesderivatized with cis-aconitic hydride; with increasingvesicle size, the distribution shifted towards liver

macrophages (Kupffer cells)87

. Taking into account allthese data and designing similar particles with encap-sulated or associated/complexed/coated nucleic acids,some realistic therapeutic targets and goals may well beestablished.

Lipid-based gene delivery is the focus of several spe-cialized high-technology companies, of which Vical(San Diego, CA, USA), Genzyme (Farmington, MA,USA), GeneMedicine (The Woodlands, TX, USA)and Megabios (Burlingame, CA, USA) have productsin clinical trials.

A liposomal vaccine against hepatitis A has beensuccessfully launched by the Swiss Serum Institute(Bern, Switzerland)88. Purified hepatitis-A-virion cap-

sule, viral phospholipids and envelope glycoproteinfrom influenza virus are mixed with PC andphosphatidylethanolamine in the presence of excess


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detergent. Upon detergent removal, liposomes con-taining antigen and some viral lipids and proteins[including fusogenic glycoprotein (influenza haemag-glutinin)] are formed. The liposomes act as an adjuvantand carrier of coadjuvants (viral glycolipids and glyco-proteins), and potentiate an immune response to the

vaccine antigen. The same company is developing vac-cines for influenza, hepatitis B, diphtheria and tetanus.Other companies, such as Orasomal Technology(Chicago, IL, USA) are working on the developmentof other vaccines, including oral vaccination, and drugdelivery by polymerized liposomes.

A significant barrier to the development of liposomesfor oral or mucosal delivery of macromolecules hasbeen the susceptibility of liposomes formed from com-monly available lipids to enzymatic degradation and tothe low pH in the stomach and bile-salt dissolution inthe intestine. Common liposomes thus fail to protectmacromolecules against enzymatic degradation in thegastrointestinal tract. Mechanically and sterically stabi-

lized liposomes can survive these conditions, but areconsequently too stable in the intestine to deliver theencapsulated drug via the normal absorption process.A possible solution to this problem would be to designliposomes with time-dependent stability. Direct uptakeby M cells in Peyers patches, which sample the sur-roundings for antigens, is too low for substantialabsorption of drugs but may be effective for vaccina-tion, when low concentration of antigens may be ade-quate to stimulate an immune response. Recently, thepotential of utilizing polymerized liposomes as oraldrug and vaccine vehicles has been explored89,90. Suchliposomes survive intestinal transit intact and can betaken up rapidly by M cells and, subsequently, by

macrophages. Providing M-cell-targeting molecules bythe covalent attachment of lectins and other ligandsprovides further increases in the uptake of liposomes bythe intestine90. Very stable liposomes and polymerized-liposome vehicles may provide a route for the com-mercial development of liposome technology for oralvaccination and, eventually, for a wide variety ofmacromolecular drugs.

Alternatively, liposomes (or their precoursors) can bedelivered directly into intestine in various capsules. Itis possible that the mere presence of lipids increasesabsorption in the gut, as it is well known that variousmicelles, mixed micelles and vesicles help to transporthydrophobic molecules across the epithelial membranes

in the intestine. Additionally, lipids may inactivate someenzymes that would digest or pump out drugs.

Future prospectsConsidering future medical applications of lipo-

somes, we can expect several novel anticancer agents,cytokines, antifungals, antibiotics and antivirals in con-ventional and long-circulating liposomes. Their usemay also spread into cardiovascular area, possibly bydelivering prostaglandins, and the delivery of antisenseoligonucleotides and ribozymes is also very promising.Novel modes of action may include: the developmentof liposomes as a long-circulating platform to bind andinactivate various blood-borne toxins; to form local-

ized liposomal depots for various drugs [including alipid foam with programmable release of cytosine arabi-nose, morphine or other drugs that is being developed

by DepoTech (San Diego, CA, USA)]; to saturatevarious receptors with liposome-bound ligands; and totarget macrophages in lymph nodes and skin. Theinhalation of drug-laden liposomal aerosols may alsofind applications. Liposome-based artificial blood andencapsulated allergens or their extracts may be further

candidates for future development as desensitizers in thetreatment of allergies46.

Immunotherapy and targeted delivery are two otherareas in which many researchers expect progress. Muchsimpler, however, would be developments of formula-tions for the treatment of tropical antiparasitic infec-tions, such as malaria and leishmaniasis91,92. Despite thefact that there are hundreds of millions of peopleaffected, there does not seem to be enough commer-cial interest to develop such formulations. Eibl and col-leagues developed synthetic amphiphilic lipids that canform liposomes or micelles as a treatment for cancer(lipids are by themselves anticancer agents)93; such for-mulations were also very effective, however, in the

treatment of tropical parasitic infections94.Another very promising use of liposomes is in diag-

nostic assays95. For instance, several blood-clottingassays rely on the colloidal instability of various cells ordifferent lipid extracts from, for example, rabbit brains.It is only natural to expect that synthetic liposomes hav-ing similar but optimized and always identical lipidcomposition will give rise to more reproducibleresults, as liposomal kits for measuring prothrombintimes have shown (Innovin and Actin by DADEInternational, Miami, FL, USA).

Annexins are membrane-binding proteins and ahighly sensitive and precise assay for their determi-nation was developed on the basis of liposome aggre-

gation as monitored by turbidimetry96. Several otherdiagnostic assays are based on the agglutination of lipo-somes or on their ability to amplify responses. Forinstance, in ELISA, one antibody typically carries onelabelling group; however, if the ligand is associated witha liposome, it could carry thousands of markers thatenhance the signal proportionally. As another example,receptorligand interactions can be accompanied bychromatic transitions and thus used to detect viruses.For instance, a system consisting of polyacetylene bilay-ers functionalized by a sialic-acid analogue, which is asite-specific ligand for influenza-virus haemagglutinin,undergoes a colour change upon binding that is observ-able to the naked eye (bluered)97.

Less sophisticated but better known to the generalpublic are liposome applications in the cosmetics andfood industries, as well as in the oral delivery of nutri-ents [e.g. liposomal vitamins, minerals and herb extractsfor oral administration (Biozone Labs, Pittsburgh, CA,USA]. These are based on the ability of liposomes tosolubilize hydrophobic molecules in natural lipidwatersystems, act as slow-release capsules and, perhaps, enhancepenetration through mucosal membranes. Althoughthese claims are difficult to prove, one can safely say thatthe presence of lecithin does have other beneficialeffects, from reducing irritation in the stomach toreducing blood-cholesterol levels47. The explosivegrowth of liposomal cosmetic products, which started

in 1987 by Diors Captureand LOreals Niosomes andNactosomes (skin-rejuvenating creams), seems to havestalled, because other novel technologies and agents


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have captured the imagination of customers. Despitethat, there are several hundred products on the market,including skin creams, sunscreens, dentifrices, skin-whitening lotions and perfumes, which generate wellover a billion US dollars in annual sales1,95.

In parallel, the applications of liposomes will spread

to several other areas, such as the food and nutritionindustry, diagnostics, and the coating industry.Although the first liposome applications were high-technology formulations made from expensive rawmaterials for the pharmaceutical industry (and slightlyless expensive ones in cosmetics), inexpensive lipo-somes will soon come of age. Low-purity lipid extractsin the price range US$ 150 kg1 and new processingmethods (such as direct hydration during mixing andchurning or via propylene-glycol injection, followedperhaps by a large-scale homogenization) will allow thepreparation of large quantities of very cheap liposomes,which will find applications in the food and nutritionalindustry, and in cleansing formulations. On a large

scale, such liposomes may have an impact on agr icul-tural and ecological applications, for example, in metaldetoxification or for cleaning up reactor spills; it is pos-sible that the metal ions can be bound to liposomeswith surface-attached chelators to simplify the sep-aration. After metal binding, the liposomes can beprecipitated and the decontaminated water safelydecanted. Several other scenarios, including the decon-tamination of oil spills on land and water, have beendiscussed95.

ConclusionThe synergistic input from colloid science, physics,

chemistry, biology, pharmacology and medicine has

resulted in the successful development of liposomaldrug delivery in less than 30 years, and the solid theo-retical and experimental basis that have been developedpromise new improvements and products1,88,98100.Only time will tell which of the above applications andspeculations will prove to be successful. However, basedon the already available products, we can say that lipo-somes have definitely established their position inmodern technology.

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