[9] LIPOSOME PREPARATION 193

[9] L i p o s o m e Preparation and Size Characterization

By MARTIN C. WOOVLE and DEMETRIOS PAPAHADJOPOULOS

Introduction
Liposomes are rapidly becoming accepted as pharmaceutical agents
which m a y improve the therapeutic activity of a wide variety of com-
pounds. Consequently, there is an increasing need for easily reproducible
and efficient preparation methods at both a laboratory and an industrial
scale. A number of reviews have described studies of liposome production
and properties, their use as carders for drugs, and their interaction with a
variety of cell types, t-v M a n y studies have shown that liposome behavior
can vary dramatically, depending on several properties, including particle-
size distribution, number of lamellae, chemical composition, and a m o u n t
of trapped volume. Therefore, it is essential to use methods producing
well-defined liposomes, with respect to these parameters, in order to un-
derstand the impact of each property on liposome function. Reh'able char-
acterization of liposomes with respect to these parameters is critical and
especially so for particle-size distribution.
Recent reviews have focused on the therapeutic properties of liposomes
without consideration of the differences produced by current production
methods, which m a y greatly affect the behavior of liposomes. 6,a-11 In this
review we will present a scheme categorizing all liposome preparation
methodologies along with a detailed description of the most useful
methods. Principal concerns and problems for producing well-defined

1F. C. Szoka and D. Papahadjopoulos,Annu. Rev. Biophys. Bioeng. 9, 467 (1980).

2 D. Dearaer and P. S. Uster, in "The Liposoraes"(M. J. Ostro, ed.), p. 27. Dekker, New
York, 1983.
3G. Gregoriadis(ed.), "Liposorae Technology,"Vols. 1-3. CRC Press, Boca Raton, Flor-
ida, 1984.
4 p. R. Cullis, M. J. Hope, M. B. Bally, T. D. Madden, L. D. Mayer, and A. S. Janoff, in
"Liposoraes from Biophysicsto Therapeutics" (M. J. Ostro, ed.), p. 39. Dekker, New
York, 1987.
5 D. Lichtenbergand Y. Barenholz,Methods Biochem. Anal. 33, 337 (1988).
6 j. H. Senior, Crit. Rev. Ther. Drug Carrier Syst. 3, 123 (1987).
P. Machy and L. Leserman (eds.), "Liposora~in Cell Biologyand Pharmacology,"p. 6.
Libbey, London, 1987.
8j. N. Weinstein,Cancer Treat. Rep. 68, 127 (1984).
9 G. Lopez-Berestein,Antimicrob. Agents Chemother. 31, 675 (1987).
10R. Kirsh and G. Poste,Adv. Exp. Med. Biol. 202, 171 (1986).
i1 j. Sunamoto and K. Iwaraoto, Crit. Rev. Ther. Drug Carrier Syst. 2, 117 (1986).

Copyrisht 1989 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 171 All rights of~'l~roduction in any form reserved.
 194 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

liposomes will also be discussed. Finally, the existing methods for size
characterization will be reviewed. The usefulness of these methods as
applied to liposomes will be described.

Conceptual Mapping of Liposome Preparation Methods

The procedures for liposome preparation can be generalized and di-
vided into three stages: first, preparation of the aqueous and lipid
phases; second, primary processing involving lipid hydration; and third,
secondary processing steps essential in some cases and optional in others.
The secondary processing steps can be used separately or combined, in
some cases. Based on these three stages it is possible to categorize all
existing preparation methodologies. Such a conceptual mapping helps to
clarify the relationship between methods and thus the choices that must be
made when choosing one. A categorization is presented below with notes
on the critical elements involved in each step and the specific method to
which it relates.

Preparation of Molecular Mixtures of Components

Preparation of Aqueous Phase. Aqueous buffer, drug, chelator, osmo-
larity, ionic strength, pH, and concentration of material to be encapsulated
have to be adjusted in relation to the medium in which the liposomes will
be suspended after formation. For most detergent solubilization methods,
detergent is added to the aqueous phase.
Preparation of Molecular Mixture of Lipids. The lipid material is dis-
solved in a suitable (organic) solvent or mixture which can be either
miscible or immiscible with the aqueous phase, depending on the method
to be used.
For many methods, a dry lipid mixture is produced by removing the
solvent. The form of the dry lipid (powder, film, etc.) affects the liposomes
formed and is determined by both the solvent removal process and the
surface used for drying the lipid. The solvent can be removed either by
solvent vaporization [classic multilamellar vesicle (MLV)], by solvent sub-
limation (freeze drying), or by spray drying. 12-14

,2 F. J. T. Fildes, in "Research Monographs in Cell and Tissue Physiology. Liposomes: From

Physical Structure to Therapeutic Applications" (C. G. Knight, ed.), Vol. 7, p. 465.
Elsevier, Amsterdam, 1981.
,3 T. E. Thompson, B. R. Lentz, and Y. Barenholz, FEBSSymp. 42, 47 (1985).
,4 G. Redziniak and A. Meybeck, U.S. Patent 4,508,703 (1985).
[9] LIPOSOME PREPARATION 195

Primary Processing (Hydration of Lipids: Liposome Formation)

Hydration of Dry Powder or Film with Aqueous Phase. After removing
the solvent, dry lipid mixtures can be dispersed into the aqueous phase by
shaking or vortexing, forming the now classic MLV (see Hydration of Dry
Lipid Films). Large (LUV) or small (SUV) unilamellar vesicles can be
formed spontaneously by controlling the lipid components and/or the
form of the dry lipid mixture. This is discussed in detail below (see Sponta-
neous Vesiculation). Detergent solub'flization of lipid, either dry mixture or
previously hydrated, produces mixed micclles of detergent and lipids.
Controlled selective removal of the detergent results in lipid rcorganiTation
into unilameUar vesicles. Details of the controlling parameters arc dis-
cussed below (see Detergent Solubilization and Removal).
Replacement of Immiscible Organic Solvent with Aqueous Phase. When
lipids are dissolved in aqueous immiscible solvents they can form an
emulsion in the aqueous phase. Removal of the organic solvent under the
proper conditions forms LUVs. This is called the reverse phase evapora-
tion (REV) procedure which is discussed below with several modified
versions (see Reverse Phase Evaporation and Solvent Vaporization; see also
Large Unilamellar Vesicles, Reverse Phase Evaporation).
Hydration by injection of the lipid solution into the aqueous phase,
maintained under a reduced pressure to vaporize the solvent, produces
oligolamellar vesicles (OLVs), LUVs, or MLVs, depending on the parame-
term This technique will be described in more detail below (see Reverse
Phase Evaporation and Solvent Vaporization; see also Volatile Solvent
Injection).
Replacement of Miscible Organic Solvent with Aqueous Phase. Injec-
tion of aqueous miscible lipid solutions, typically ethanol, into the aqueous
phase under the proper conditions will form SUVs. Strategies for removal
of the solvent include dialysis, ultrafiltration, and gel permeation chroma-
tography. This method will be discussed in greater detail [see Solvent
(Ethanol) Injection].

Secondary Processing (Size Changes after Liposome Formation)

Mechanical Fragmentation. Many methods exist for fragmentation of
MLVs into particles that are smaller and/or unilamellar. Ultrasonication
of an MLV dispersion converts the lipid into minimally sized SUVs.
Passage through the French Press cell also converts MLVs into SUVs.
Homogenization of MLVs can form either SLrVs or OLVs, depending on
conditions and number of passes used. Two types of equipment, micro-
fluidizers available from BDC or Microiluidics and traditional homogen-
 196 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

izers from Gaulin, have been used. Extrusion of MLV dispersions through
filters with defined pores can be used to reduce the particle size to approxi-
mately that of the pore and to reduce the number oflamellae. LUVs can be
formed in some cases. [See Description of Specific Methods for detailed
descriptions of these processes (pp. 203 and 208).]
Physicochemically Induced Fragmentation. By temporarily altering the
chemical nature of the aqueous phase, the physical properties of the head
group are changed, dramatically altering the organization of the lipid
dispersions. One manifestation of such a method is based on hydration
changes occurring on protonation or deprotonation of the lipid head
group. Addition of sufficient base to alter the protonation of head groups
followed by a return to neutral pH will convert MLVs containing an acidic
lipid into LUVs. The details of this procedure are described below [see
Description of Specific Methods (p. 207)].
Fusion-Mediated Size Increase of SUVs. SUVs are unstable, spontane-
ously increasing in size during storage. This results in LUVs in some cases
and large aggregates in others. Freezing and thawing SUVs (or LUVs) will
induce the release of entrapped aqueous contents. Simultaneously, larger
particles are formed which are typically multilamellar. Addition of ions or
other materials can induce aggregation and fusion, resulting in LUVs in
some cases. Most commonly, divalent cations are added to formulations
with acidic lipids, resulting in cochleated cylinders which convert to LUVs
upon addition of chelators. Addition of detergents and subsequent removal
can convert SUV to LUV. [See Description of Specific Methods for details
(p. 208).]
Dehydration- Rehydration- Induced Size Increases (AlL Vs). Dehydra-
tion of the lipid head groups can be achieved by evaporation, lyophiliza-
tion, or even freezing. Reconstitution (rehydration of the dry lipid in the
case of evaporation, and lyophilization and thawing in the case of freezing)
results in substantial changes in the particle structure. Typically, SUVs
increase in size, forming OLVs or MLVs. Freeze-thaw cycling of MLVs
increases the aqueous entrapment, but with only minimal changes in
overall size distribution. [See Description of Specific Methods for details
(pp. 199 and 200).]

Guide for Choice of M e t h o d

The methodology for liposome preparation has evolved rapidly during
the last few years as a response to the need for preparing well-defined
liposomes for specific applications. As a consequence, the value of each
method or each combination of methods depends entirely on the intent of
the investigator and the requirements of their intended use. It follows that,
depending on the specification at hand, some methods are much more
[9] LIPOSOME PREPARATION 197

suitable than others. The critical appraisal that follows is based on a broad
categorization of various types of liposomes and some specific usages.
Specific details and references for each method will be given in the follow-
ing section. As we discuss the methods of choice, we will also define
whether they are particularly suitable for laboratory scale (1.0-100 ml of
10- 100 mg/ml), for the relatively larger scale required for clinical testing
(up to 10 liters of 100-300 mg/ml), or for industrial-scale production
(more than 10 liters of up to 400 mg/ml).

Multilamellar Liposomes
If there is no restriction or specific preference concerning geometry or
size, the MLV form of liposomes is especially advantageous when lipophi-
lic material is to be encapsulated, or if there is no concern with the
efficiency of aqueous encapsulation. For laboratory-scale production, the
classic "thin film hydration" method is adequate. Its usefulness can be
extended by subsequent "extrusion" through defined pore membranes,
which produces a reasonably narrow size distribution. For a larger scale,
other strategies should be considered: alternative drying methods, such as
lyophilization, spray drying, or solvent injection and vaporization, all of
which can be optimized for large-scale production.
If the efficiency of encapsulation is a concern, and there is a desire for
high encapsulation of water-soluble solutes, the following methods should
be considered: (1) reverse phase evaporation; (2) one of the freeze-thaw or
dehydration-rehydration methods followed by extrusion; or (3) extrusion,
homogenization, or solvent injection and vaporization under conditions
giving high final lipid concentrations. The latter methods (3) are particu-
larly well-suited for preparations of relatively large amounts of liposomes,
such as 0.1- to 1.0-liter volumes, while the former methods (1 and 2) are
best for laboratory scale.

Small Unilamellar Liposomes

The small size, relatively narrow size distribution, and high surface-to-
volume ratio of SUVs makes them a good choice for some applications,
especially if there is no need for efficient encapsulation of water-soluble
solutes. For laboratory-scale preparation, sonication, French Press, or eth-
anol injection methods should be considered. The choice between these
depends on the importance of the specific limitations of each, such as
exposure to ultrasonic irradiation, residual ethanol, or limited access to
equipment (such as a French Press). For larger scale, up to a few liters,
homogenization procedures produce fairly small liposomes after repeated
passage. These may not be unilamellar, but can be made at high enough
198 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

lipid concentrations to achieve reasonable capture of water-soluble solutes.

Alternatively, ethanol injection on a large scale should be considered when
an ethanol content of up to 10% is acceptable. In fact, this level of ethanol
may be of interest as a preservative in some situations where the resulting
liposome properties, such as increased bilayer fluidity and aqueous leak-
age, are not a problem.

Large Unilamellar Liposomes

These liposomes are particularly well suited for efficient encapsulation
of water-soluble solutes because of their favorable aqueous volume-to-sur-
face ratio. They are also most desirable for membrane protein reconstitu-
tion and other studies of membrane permeability and fusion. The deter-
gent-removal methods are most commonly used for membrane protein
reconstitution, while the reverse phase evaporation is the most efficient
procedure for high degrees of encapsulation of water-soluble solutes and
macromolecules. Convenient commercial apparatus for laboratory-scale
preparations has recently been introduced for the detergent dialysis
method (Dupont, Wilmington, Delaware). Extrusion is recommended for
secondary processing to achieve a narrower size distribution. When extru-
sion is used under high pressure, with high lipid concentration and small
pore-size membranes, it produces liposomes with both high capture and a
very homogeneous size distribution.
Although good methods for large-scale production of LUVs particu-
larly at the industrial scale, are currently lacking, all the possibilities have
not been exhausted and solutions are expected, perhaps even in the near
future. For example, modification of existing methods, such as reverse
phase evaporation, into continuous processes, rather than simply increas-
ing the batch size, have yet to be fully explored. Other strategies for
modifying existing methods or development of entirely new methods may
provide larger scale LUV production.

Description of Specific Methods

Multilamellar Vesicles
Hydration of Dry Lipid Films. Liposomes prepared by shaking or
vortexing dried lipids with the aqueous phase were the first to be character-
ized as closed vesicles composed of multiple concentric lipid bilayers
(MLVs). ~5-~7These liposomes are very heterogeneous in size 18 and have a

~sA. D. Bangham, M. M. Standish, and J. C. Watkins, J. Mol. Biol. 13, 238 (1965).
16A. D. Bangham, J. de Crier, and G. D. Greville, Chem. Phys. Lipids 1, 225 (1967).
17A. D. Ballgham, M. W. Hill, and N. G. A. Miller, Methods Membr. Biol. 1, I (1974).
[9] LIPOSOME PREPARATION 199

low level of aqueous encapsulation. However, because the method is ex-

ceptionally simple and the MLVs form spontaneously, it is used exten-
sively as the hydration step, frequently followed by one or more secondary
processing steps such as extrusion or sonication.
Many studies have demonstrated that lipid composition and concen-
tration as well as the mode of hydration affect the final product. The mode
of hydration may be the most important factor. ~7,1s The usual approach
has been to use a large surface area to dry the lipid and hence to reduce the
thickness of the lipid film. The method for drying the lipid, such as spray
drying, also has shown that the exact form of the dried lipid can influence
the resulting liposomes?2-~4 Nevertheless, the full extent of the role played
by the form of the ~ lipid has yet to be determined. Many other factors
are also important, such as initial moisture content, aqueous phase used,
final lipid concentration, lipid composition, the actual hydration process,
and time for hydration. Is The principal problem in producing MLVs by
shaking or vortexing is reproducible control of all the parameters. Often
the form of dry lipid and the hydration process vary uncontrollably, mak-
ing it difficult to achieve a well-defined preparation.
Dehydration-Rehydration. An approach which improves entrapment
of aqueous solutes by MLV preparations is to dry the lipid in the presence
of the aqueous solute to be entrapped, thus forming a mixed lipid film with
solute trapped between layers. ~9.2This mixed film is then hydrated gradu-
ally with a minimum of aqueous phase. Interestingly, the mixed film
hydrates differently when certain aqueous solutes are used.2 The result is a
convenient method for producing MLVs with a higher percentage of en-
trapped aqueous phase. In the usual procedure, which is denoted
dehydration-rehydration, preformed liposomes are added to an aqueous
solution of the solute and the mixture is either lyophilized19 or evapo-
rated2 with similar results. SUVs may be preferable to MLVs, but both
have been used. Subsequent rehydration forms MLVs with a high level of
entrapment of the aqueous solutes added. Consequently, this method is
useful for entrapping a wide variety of aqueous components, especially
proteins which can be freeze-dried, without the need for exposure of
aqueous solutes to organic solvents or detergents. Another manifestation of
this technique is to add the aqueous solution to an ethanolic solution of the
lipid, which is then dried. 2~

is F. Olson, C. A. Hunt, F. C. Szoka, W. J. Vail, and D. Papahadjopoulos, Biochim. Biophys.

Acta 557, 9 (1979).
t9 C. J. Kirby and G. Gregoriadis, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. 1,
p. 19. CRC Press, Boca Raton, Florida, 1984.
20R. L. Shew and D. W. Dcamcr, Biochim. Biophys. Acta 816, 1 (1985).
21 S. M. Gruner, R. P. Lenk, A. S. Janoff, and M. J. Ostro, Biochemistry 24, 2833 (1985).
200 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

In most cases, gradual controlled rehydration of the resulting material

with a minimal aqueous volume gives a heterogeneous population of
MLVs but with a higher percentage of entrapped aqueous solutes. Subse-
quent processing, such as extrusion, ~sis recommended to reduce the size of
the particles and improve the homogeneity.
Freezing alone may essentially dehydrate the lipid head g r o u p s , 22 with
rehydration occurring upon thawing. Therefore, freeze-thaw cycles are
effectively a form of dehydration- rehydration. This method is discussed in
the next section.
Freeze-Thaw Processing of MLVs. Repeated freezing followed by
thawing is a convenient method for increasing the trapped volume of MLV
preparations or inducing leakage of SUVs and L U V s . 23-27 The bilayer
structure is dramatically altered as determined by freeze-fracture electron
microscopy (EM) and nuclear magnetic resonance (NMR) studies.26 A
likely explanation may involve a combination of both head group dehy-
dration and physical disruption of the multilamellar structure by ice crys-
tals formed during freezing. These result in improved hydration of the lipid
upon thawing. However, despite the appearance of an isotropic component
in NMR spectra attributed to small liposomes, the overall size distribution
is not significantly decreased.27 Both the lipid and aqueous compositions
influence the process such that the compositions which reduce membrane
damage on freezing also diminish the effects of the freeze-thaw procedure.
The presence of cryoprotectants and nonionic aqueous solutes reduce
membrane damage. 2s,29 Interestingly, entrapped glycoprotein from serum
also exhibits protection during freezing. Low salt concentrations and cho-
lesterol are also stabilizing.29 Therefore, each formulation may behave
differently and this must be considered and tested.
Reverse Phase Evaporation and Solvent Vaporization. Both of these
general procedures were developed to produce LUVs, a.3~ but under many

22S. W. Hui, T. P. Stewart, L. T. Boni, and P. L. Yeagle, Science 212, 921 (1981).
23 R. Rendi, Biochim. Biophys. Acta 135, 333 (1967).
24 D. Siminovitch and D. Chapman, FEBS Lett. 16, 207 (1971).
25 U. Pick, Arch. Biochem. Biophys. 212, 186 (1981).
2~ L. D. Mayer, M. J. Hope, P. R. Cullis, and A. S. Janoff, Biochim. Biophys. Acta 817, 193
(1985).
27 M. J. Hope, M. B. Bally, L. D. Mayer, A. S. Janoff, and P. R. Cullis, Chem. Phys. Lipids
40, 89 (1986).
2s L. M. Crowe, C. Womersley, J. H. Crowe, D. Reid, L. Appell, and A. Rudolph, Biochim.
Biophys. Acta 861, 131 (1986).
29 G. Strauss, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. 1, p. 197. CRC Press,
Boca Raton, Florida, 1984.
3o D. Deamer and A. D. Bangham, Biochim. Biophys. Acta 443, 629 (1976).
3~F. Szoka and D. Papahadjopoulos, Proc. Natl. Acad. Sci. U.S.A. 75, 4194 (1978).
[9] LIPOSOME PREPARATION 201

conditions MLVs are actually foITned.21'32 The specific conditions under

which these methods can be used to prepare LUVs are discussed below (see
Large Unilamellar Vesicles). Nevertheless, these methods are very useful
for MLV production, either as the hydration step to be followed by second-
ary processing or for situations where heterogeneous MLVs are adequate.
Solvent injection and vaporization are especially good methods for
situations requiring large-scale hydration of lipid and a relatively large
percentage of aqueous entrapment, even though OLVs or MLVs may be
formed.33-~ The reverse phase evaporation method is most useful on the
laboratory scale. 3~ Claims of unique stability have been made21 for MLVs
produced by a specific modification of the REV technique, and to indicate
this they have been given a special name, stable plurilameUar vesicles
(SPLVs). The SPLV method consists of bath sonicating an emulsion of
aqueous phase in an ether solution of lipid while evaporating the ether. 2~
Another method using phase reversal produces multivesicular lipo-
somes.37, 38

Unilamellar Vesicles
Unilamellar vesicles are liposomes with a tingle bilayer surrounding the
entrapped aqueous phase. The term small unilamellar vesicles is used for
minimally sized liposomes. The minimum size (200-250 A diameter),
usually attributed to a maximum of curvature allowed by the packing of
lipids in the bilayer, results in a low amount of entrapped aqueous volume
per lipid. As the size increases, the amount of entrapped aqueous phase
per mole of lipid increases,a9 Stability of size also increases with size,
usually attributed to a decrease in the amount of curvature and resulting
s t r a i n . 39-42 Unilamellar liposomes with diameters of 100 nm and larger are
generally referred to as large unilamellar vesicles. In practice, however,

32C. Pidgeon, F. McNeely, T. Schmidt, and J. Johnson, Biochemistry 26, 17 (1987).

33 D. Deamer, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. l, p. 29. CRC Press,
Boca Raton, Florida, 1984.
34 D. S. Caliso, H. R. Petty, and H. M. McConnell, Biochim. Biophys. Acta 649, 129 (1981),
3s H. Sehieren, S. Rudolph, M. Finkelstein, P. Coleman, and G. Weissmann, Biochim.
Biophys. Acta 542, 137 (1978).
3~F. J. Martin and G. West, U.S. Patent Appl. Serial No. 908765 (1986).
37 S. Kim and S. B. Howell, Cancer Treat. Rep. 71, 705 (1987).
as S. Kim, M. S. Turker, E. Y. Chi, S. Sela, and G. M. Martin, Biochim. Biophys. Acta 728,
339 (1983).
39 D. Lichtenberg, E. Freire, C. F. Schmidt, Y. Barenholz, P. L. Feigner, and T. E. Thomp-
son, Biochemistry 20, 3462 (1981).
40 M. P. Sheetz and S. I. Chan Biochemistry 11, 4573 (1972).
4t C. Huang and J. T. Mason, Proc. Natl. Acad. Sci. U.S.A. 75, 308 (1978).
42 B. R. Lentz, T. J. Carpenter, and D. R. Alford, Biochemistry 26, 5389 (1987).
202 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

oligolamellar vesicles, which have a large entrapped volume per lipid but
with several bilayers widely separated, are often described as unilamellar
due to a difficulty in distinguishing between the two. In some proce-
dures, 43,44 unilamellar vesicles with diameters as large as 10/zm have been
made and are called giant unilamellar vesicles (GUVs). Despite variation
in terminology in the literature, the distinctions between SUVs, LUVs,
OLVs, and G U V s are becoming standardized. 5,7,45

S m a l l Unilamellar Vesicles
Sonication. Sonication o f MLVs or other aqueous dispersions o f phos-
pholipids produces small particles '~,47 that have been shown to be SUVs
that entrap an aqueous space. 4s Extensive characterization o f these mini-
mally sized SUVs 49 has greatly enhanced the understanding o f lipid hi-
layers, liposomes, and biological membranes in general. 1~,5 Because o f
their minimal size, these liposomes are a relatively homogeneous popula-
tion o f unilamellar vesicles, but they are strained 4'4~,5~ and are likely to
undergo spontaneous slow transformations to larger vesicles. 4,44,52-55
High-speed centrifugation has been used as a simple but effective proce-
dure to remove any larger liposomes. ~6 The principal disadvantages o f
SUVs in general are a relatively low percentage o f aqueous entrapment and
an incompatibility for sonication with m a n y compounds, including some
lipidsY Sonication under an inert atmosphere has been used to reduce
lipid damage, 49 although this is not always successful, especially with probe
sonicators. 57 Milder sonication using lower power bath sonicators m a y

43S. Kim and G. M. Martin, Biochim. Biophys. Acta 646, 1 (1981).

N. Oku and R. C. MacDonald, Biochemistry 22, 855 (1983).
45D. Papahadjopoulosand W. J. Vail, Ann. N.Y. Acad. Sci. 308, 259 (1978).
L. Saunders, J. Perdn, and D. B. Gammack, J. Pharm. Pharmacol. 14, 567 (1962).
47M. B. Abramson, R. Katzman, and H. P. G-regor,J. Biol. Chem. 239, 70 (1964).
D. Papahadjopoulosand N. Miller, Biochim. Biophys. Acta 135, 624 (1967).
49C. Huang, Biochemistry 8, 344 (1969).
50L. B. Margolis, Biochim. Biophys. Acta 779, 161 (1984).
5mT. E. Thompson, C. Huang, and B. J. Litman, in "The Cell Surface in Development"
(A. A. Moscona, ed.), p. 1. Wiley, New York, 1974.
52j. Suurkuusk, B. R. Lentz, Y. Barenholz, R. L. Biltonen, and T. E. Thompson, Biochem-
istry 15, 1393 (1976).
53A. L. Larrabee, Biochemistry 18, 3321 (1979).
M. Wong, F. H. Anthony, T. W. Tillack, and T. E. Thompson, Biochemistry 21, 4126
(1982).
55R. A. Parente and B. R. Lentz, Biochemistry 23, 2353 (1984).
56y. Barenholz, D. Gibbes, B. J. Litman, J. Goll, T. E. Thompson, and E. D. Carlson,
Biochemistry 16, 2806 (1977).
5~H. Hauser, Biochem. Biophys. Res. Commun. 45, 1049 (197l).
[9] LIPOSOME PR~-PARATIOrq 203

have an advantage of reducing lipid damage. 5s,s9 However, the bath soni-
cators have several disadvantages: they require much longer times; sample
sizes, containers, and placement in the bath are often critical so that
reproducibility is hard to achieve; and the residual concentration of large
particles is greater. ~ The model Gll2SP1T bath sonicator (Laboratory
Supplies Co., Hicksville, New York) has an excellent reputation, but in our
experience the actual bath component has a short lifetime. Finally, for
large-scale production, sonication is severely limited by ditiieulties in
achieving uniform results and in scale up.
French Press. Extrusion of MLV dispersions through a French Press is a
gentle method for producing SUVs.6-63 The average size of the resulting
particles is slightly larger and the heterogeneity of the preparations with
respect to size is greater than that obtained by sonication. Thus the lipo-
somes may well be more stable but the reproducibility of the method has
yet to be established.
Homogenization. Homogenization of MLVs or other lipid dispersions
also produces SUVs in some circumstances. Several different types of
equipment have been used: microfluidizers such as those supplied by
Microfluidics and Biotechnology Development Corporation64 and homo-
genizers such as those supplied by Gaulin. 6s Unfortunately, little data have
been published and much of that is only limited data in support of a
patent. The principal consideration is the shear force generated, tempera-
ture of operation, and lipid fluidity at that temperature,t~,ss The resulting
SUVs are larger than the minimal size produced by sonication and thus
may be more stable. However, significant amounts of larger particles are
also present.
In the case of the microfluidizer developed by Biotechnology Develop-
ment Corporation, the instrument is based on a high-pressure collision of
two feed streams containing MLVs. Despite only limited data, the resulting

ss D. Papahadjopoulos and H. K. Kimelberg, in "Progress in Surface" (S. G. Davidson, ed.),

Vol. 4, Part 2, p. 141. Pergamon, Oxford, 1973.
59E. Racker, Biochem. Biophys. Res. Commun. 55, 224 (1973).
6oy. Barenholz, S. Amselem, and D. Lichtenberg FEBS Left. 99, 210 (1979).
61 R. U Hamilton, J. Goerke, L. S. S. Guo, M. C. Williams, and R. J. Havel, J. Lipid Res.
21,981 (1980).
62R. L. Hamilton and U S. S. Guo, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. 1,
p. 37. CRC Press, Boca Raton, Florida, 1984.
63p. I. Lelkes, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. 1, p. 51. CRC Press,
Boca Raton, Florida, 1984.
E. Mayhew, R. Lazo, W. J. Vail, J. King, and A. M. Green, Biochim. Biophys. Acta 775,
169 (1984).
6s R. J. Klimchak and R. P. Lenk, Biopharmacology 1, 18 (1988).
R. C. Gamble, Eur. Patent Appl. 0-190-050 (1986).
204 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

size clearly depends on the pressure used and the number of passes through
the device.64 However, even after 40 recycles at the maximum pressure, the
resulting liposomes contained a large quantity of larger particles. Whether
the size distribution was bimodal or a single population skewed toward
large particles was not determined. 5 The microfluidizer can be easily scaled
up to an industrial scale, but it is only applicable to situations where SUVs
are desirable and even then a second processing step may be necessary if
the presence of larger particles cannot be tolerated.
Solvent (Ethanol) Injection. Injection of lipid solutions in a solvent
miscible with the aqueous phase is a simple method for preparing SUVs.
However, it is limited by the need for subsequent processing to remove the
solvent, the inevitability of residual solvent, and the low solubility of some
lipid components in aqueous miscible solvents such as ethanol. Ethanol
was the first solvent used for this method and is still the most common. 67,68
The size of the resulting liposomes is dependent on several parameters:
relatively homogeneous SUVs can be produced by keeping a low lipid
concentration, a fast rate of injection, and keeping the aqueous phase
above the Tc phase transition of the lipid. 6s Removal of ethanol from the
resulting liposomes by gel permeation chromatography may be more ef-
fective than dialysis, but still does not remove it completely.69 For some
applications, the residual solvent may not be a limitation, as, for example,
when using ethanol with very low levels of impurities.
Spontaneous Vesiculation. Small unilamellar vesicles can also be
formed spontaneously during the hydration of certain lipids, such as a
mixture of short- and long-chain lecithins7,7~ or lecithin with lysoleci-
thin. 72 A recent report also indicates that the surface on which the phos-
pholipids are deposited during dehydration can contribute to spontaneous
formation of SUVs upon hydration, without sonication or application of
shearing forces.73 In a related procedure, micelles are homogeneously con-
verted to SUVs by acyl transfer to lysophosphatidylcholine (lyso-PC). TM

67 S. Batzri and E. D. Korn, Biochim. Biophys. Acta 298, 1015 (1973).

J. M. H. Kxemer, M. W. J. Esker, C. Pathmamanoharan, and P. H. Wiersema, Biochemis-
try 16, 3932 (1977).
s9 j. R. Nodund, C. F. Schmidt, S. N. Dicken, and T. E. Thompson, Biochemistry 20, 3237
(1981).
7oN. E. Gabriel and M. F. Roberts, Biochemistry 23, 4011 (1984).
7~ N. E. Gabriel and M. F. Roberts, Biochemistry 25, 2812 (1986).
~2 H. Hauscr, Chem. Phys. Lipids 43, 283 (1987).
73 D. D. Lasic, J. Kidric, and S. Zagorc, Biochim. Biophys. Acta 896, 117 (1987).
74 V. Gavino and D. Deamor, J. Bioenerg. Biomembr. 14, 513 (1982).
[9] LIPOSOME PREPARATION 205

Small or Large Unilamellar Vesicles

Detergent Solubilization and Removal. The method of detergent solu-
bilization and removal was originally developed for purification and re-
constitution of membrane proteins giving rise to particles or vesicles that
were eventually described as liposomes. 75-~s With this procedure, either
dried lipid mixtures or preformed liposomes are solubilized with the deter-
gent-containing aqueous phase. In either case the critical step is the deter-
gent removal from the mixed micelles. By controlling the rate of detergent
removal, the size and structure of the resulting liposomes can be con-
trolled, but the actual dependence on lipids, detergent(s), and conditions
are not clear. Despite advances in development of the theory, 5 the current
approach is largely empirical. 79-s2 Nevertheless, by carefully controlling
the detergent removal, SUVs as small as 60 n m or LUVs of 100 n m can be
produced. 79's3 Recent introduction of a commercial apparatus by Dupont
may facilitate use of this method on the laboratory scale.
One of the principal problems with this method is removal of residual
detergent, zs4 Several methods have been explored based on differences
between lipids and detergents. Detergent dialysis is based on a substantial
concentration of detergent monomers, approximately the CMC, com-
pared to a very low concentration of lipid monomers. Therefore, dialysis of
the detergent is considerably faster than that of the lipid. 77-79,s5 The resid-
ual detergent is determined by the dialysis conditions used as well as the
detergent and lipids. Dilution is based on the same difference between
detergent and lipid monomer concentration. When mixed micelles are
diluted below the CMC for the detergent, all the detergent leaves the
micelles, forcing the lipid to reorganize into bilayers. However, the deter-
gent monomer still must be removed. A couple of removal strategies are

7~S. Fleischer and H. Klouwen, Biochem. Biophys. Res. Commun. 5, 378 (1961).
7~M. Kasahara and P. Hinlde, Proc. Natl. Acad. Sci. U.S.A. 73, 396 (1976).
77y. Ka~awa and E. Racker, J. Biol. Chem. 246, 5477 (1971).
7s S. RazJn, Biochim. Biophys. Acta 265, 241 (1972).
79H. G. Weder and O. Zumbuehl, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. 1,
p. 79. CRC Press, Boca Raton, Florida, 1984.
so R. E. Stark, G. J. Grosselin, J. M. Donovan, M. C. Carey, and M. F. Roberts, Biochemis-
try 24, 5599 (1985).
s~ S. Almog, T. Kushnir, S. Nir, and D. Lichtenberg, Biochemistry 25, 2597 (1986).
s2 p. Schurntenbergvr, R. Bertani, and W. Kan~g, J. Colloid Interface Sci. 114, 82 (1986).
s3 M. H. Milsmann, R. A. Schwendener, and H. G. Weder, Biochim. Biophys. Acta 512, 147
(1978).
T. M. Allen, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. 1, p. 109. CRC Press,
Boca Raton, Florida, 1984.
ss V. Rhodcn and S. Goldin, Biochemistry 18, 4173 (1979).
206 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

based on selective detergent removal. In one application, the mixed micelle

solution is passed over a gel permeation column. As the particles pass
down the column, the detergent monomers are retained, depleting the
detergent and eventually forming liposomes, which are eluted, s6-ss In
another method, specific absorption of detergent to heads is used.ss-9~
Recently, a different strategy, using detergents, was reported92 for prep-
aration of homogeneous LUVs containing membrane proteins. Well-de-
fined preformed liposomes are added to solutions of detergent-solubilized
protein, but without an excess of detergent. Presumably, the liposomes are
not completely solubilized and their homogeneity is maintained. The pre-
liminary data reported for bacteriorhodopsin indicate that not only were
the resulting liposomes very homogeneous, but also a very high degree of
specificity for orientation was found.

Large Unilamellar Vesicles

Reverse Phase Evaporation. The reverse phase evaporation technique3~
has become a widely accepted method for producing LUVs with a high
percentage of aqueous phase entrapment on a laboratory scale) The actual
type and size of liposomes produced depends on a number of factors,
such as choice of lipids (percentage cholesterol and charged lipids), lipid
concentration used in the organic solvent, choice of solvent, ratio of or-
ganic to aqueous volumes in the emulsion, rate of evaporation, and ionic
strength of the aqueous phase.t By controlling these parameters it is possi-
ble to produce LUVs or MLVs (or SPLVs) reproducibly. 32,93-96 By com-
bining the procedure with extrusion, the final size can be selected over a
wide range. 96,97However, scale up of the method for LUVs is limited due
86j. Bruner, P. Skrabal, and H. Hauser, Biochim. Biophys. Acta 455, 322 (1976).
8~H. G. Enoch and P. Strittmatter, Proc. Natl. Acad. Sci. U.S.A. 76, 145 (1979).
88 T. M. Allen, A. Y. Romans, H. Kereret, and J. P. Segrest, Biochim. Biophys. Acta 601, 328
(1980).
89 p. W. Holloway, Anal. Biochem. 53, 304 (1973).
9o W. J. Gerritsen, A. J. Verldeij, R. F. Zwaal, and L. L. M. Van Deenen, Eur. J. Biochem.
85, 255 (1978).
91 p. S. J. Cheetam, Anal. Biochem. 92, 447 (1979).
92 M. Paternostre and J. L. Rigaud, Biophys. J. 53, 504a (1988).
93j. N. Weinstein, R. L. Magin, M. B. Yatvin, and D. S. Zaharko, Science 204, 188 (1979).
94C. G. Knight (ed.), "Research Monographs in Cell and Tissue Physiology. Liposomes:
From Physical Structure to Therapeutic Appfications," Vol. 7. Elsevier, Amsterdan, 1981.
9s F. C. Szoka and D. Papahadjopoulos, in "Research Monographs in Cell and Tissue
Physiology. Liposomes: From Physical Structure to Therapeutic Applications" (C. G.
Knight, ed.), Vol. 7, p. 51. Elsevier, Amsterdan, 1981.
96 N. Duzgunes, J. Wilschut, K. Hone,, R. Fraley, C. Perry, D. S. Friend, T. L. James, and D.
Papahadjopoulos, Biochim. Biophys. Acta 732, 289 (1983).
97 F. Szoka~ F. OIson, T. Heath, W. Vail, E. Mayhew, and D. Papahadjopoulos, Biochim.
Biophys. Acta 601, 559 (1980).
[9] LIPOSOME PREPARATION 207

to a requirement for a small surface-area-to-volume ratio. Other limita-

tions of the method include sonication of lipids during emulsion forma-
tion, limited solubility of lipids and instability of some proteins in the
solvent, and a need to eliminate the peroxide impurities in ether that may
degrade the lipids. These factors are not necessarily limiting for every
situation and can be circumvented in some cases. For example, sonication
to form the emulsion has been replaced with other m e t h o d s . 9s
Volatile Solvent Injection. Injection of solutions of lipid in volatile
solvents into an aqueous phase under reduced pressure is a very convenient
method for controlled hydration of lipids even on larger scales. 3,34-~,99,1
In general, the aqueous phase is maintained above the phase transition of
the lipids (To) and at a reduced pressure to vaporize the lipid solvent during
the injection. This method can produce LUVs with a relatively high per-
centage of entrapped aqueous volume per mole of lipid.27,33 However,
OLVs and MLVs can also form, depending on the conditions. A slow rate
of injection combined with rapid solvent removal and low lipid concentra-
tion in the solvent favors LUV formation. The total percentage of aqueous
volume entrapped can be as high as 65% when the final aqueous lipid
concentration is high.35 Unfortunately, a complete understanding of the
relationship between process conditions and the lameUarity of the resulting
liposomes is lacking. Solubility of the lipids in the solvent can be a severe
limitation for some lipids. The solvent used originally, ethyl ether, is also
limited by its tendency to decompose into peroxides, which are not volatile
and induce lipid damage. For these reasons use of other volatile solvents
has been explored?4-36 When an appropriate solvent is found the method
has many advantages. Even when mostly OLVs or MLVs are formed, the
preparation can be extruded at high lipid concentration to produce lipo-
somes with a high aqueous entrapment efficiency and with the size con-
trolled over a wide range.27,97
Transient pH .lump. A recently developed method for reorganizing
MLVs into LUVs takes advantage of lipid hydration and structural
changes following deprotonation of acidic lipids. 1~-~4 Currently this

98p. Machy and L. D. Leserman, in "Liposome Technology" (G. Gregoriadis, ed.), Vol. 1, p.
221. CRC Press, Boca Raton, Florida, 1984.
99 M. J. Ostro, D. Giacomoni, D. Lavelle, W. Paxton, and S. Dray, Nature (London) 274,
921 (1978).
~ooM. J. Ostro, D. Lavelle, W. Paxton, B. Matthews, and D. Giacomoni, Arch. Biochem.
Biophys. 201, 392 (1980).
iol H. Hauser and N. Gains, Proc. Natl. Acad. Sci. U.S.A. 79, 1683 (1982).
~o2N. Gains and H. Hauser, Biochim. Biophys. Acta 731, 31 (1983).
to3 T. S. Aurora, W. Li, H. Z. Cummins, and T. H. Haines, Biochim. Biophys. Acta 020, 250
(1985).
~o4W. Li and T. H. Haines, Biochemistry 25, 7447 (1986).
208 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

method has been demonstrated to work with only a few acidic lipids, but
this restriction may decrease with further studies. 72 Characterization of the
dependence of size and distribution on m a n y parameters has yet to be
completed, but some control m a y be possible.
Fusion. A number of fusion processes can alter the size of liposomes
but most give rise to poorly defined particles and little control is possible.
SUV are relatively unstable, especially when produced by sonication, and
can spontaneously, but slowly, grow in size, at best becoming LUV 42 and at
worst forming large aggregates. Several techniques to fuse SUV are avail-
able such as addition of specific m o n o or divalent ions to acidic
liposomes, 15-~9 incubation at temperatures at or below the Tc of the
lipids, 54 or addition and subsequent removal of detergents, sl'sT,H How-
ever, with some exceptions 54,~7,~t~ these methods are difficult to control
and often the resulting liposomes are poorly defined. Another related
method uses freeze thaw techniques similar to those applied to M L V
(p. 200) to fuse SUV, forming LUV and G U V up to 10/zm in diameter) n

Unilamellar or Oligolamellar Vesicles

Extrusion through Filters with Well-Defined Pores. Extrusion of M L V
dispersions through polycarbonate membranes with defined diameter
pores results in a decrease in particle size and polydispersity. ~s The charac-
teristics of the final liposomes depend on m a n y factors, including lipid
composition and charge, solvent composition, filter pore diameter, the
pressure used for the extrusion, temperature, and number of passes
used. 1s,~3 The influence of all the parameters is not clearly understood but
the following conclusions can be made. With a pore size of 0.2/zm and
larger, the resulting liposomes are not unilamellar nor is the population
very homogeneous. However, by repeatedly extruding the sample the num-

~o5D. Papahadjopoulos,W. J. Vail, K. Jacobson, and G. Poste, Biochim. Biophys. Acta 394,
483 (1975).
to~ D. Papahadjopoulos,W. J. Vail, C. Newton, S. Nit, K. Jacobson, G. Poste, and R. Lazo,
Biochim. Biophys. Acta 465, 579 (1977).
t07j. Wilschut,N. Duzgunes,and D. Papahadjopoulos,Biochemistry 20, 3126 (1981).
~0sN. Duzgunes and D. Papahadjopoulos,in "Membrane Fluidity in Biology"(R. I. Aloia,
ed.), Vol. 2, p. 187. AcademicPress, New York, 1983.
to9 N. Duzgunes, R. M. Straubingex,P. A. Baldwin, D. S. Friend, and D. Papahadjopoulos,
Biochemistry 24, 3091 (1985).
Hop. Schurntenberger,N. Mazer, and W. Kan~g,J. Phys. Chem. 89, 1042 (1985).
m S. Gould-Fogeriteand R. J. Mannino, Anal. Biochem. 148, 15 (1985).
H2R. I. MacDonaldand R. C. MacDonald,Biochim. Biophys. Acta 735, 243 (1983).
,t3 L. D. Mayer, M. J. Hope, and P. R. Cullis,Biochim. Biophys. Acta 858, 161 (1986).
[9] LIPOSOME PREPARATION 209

ber of lameUae can be reduced to less than five. H4,11s With repeatedextru-
sion the heterogeneity of the size distribution also decreases, but this effect
is diminished as the falter size increases. By using high pressure, up to
15 arm, MLV samples can be extruded directly through 0.1- or 0.05-gin
filters to form LUVs. Interestingly, combining this technique with two
stacked filters may improve the size homogeneity.27 An onionskin model
has been proposed to explain the results of the extrusion method. H6
The use of extrusion as a secondary processing technique has many
advantages. MLVs can be converted to well-defined oligolamellar vesicles
over a large range of sizes (0.2 to 0.5 #m) controlled by the pore size of the
filter used, or to LUVs from 0.1 to 0.05 gin. Extrusion can be performed
on a large scale, limited primarily by the size of filters and the availability
of apparatus which can withstand high pressures. Identifying filter failure
often presents a difficulty, especially during the final passes when only a
small percentage of large particles remain.

Critical Issues and Problems to Avoid

Aqueous Phase
The choice of aqueous phase must take into consideration several
factors that depend on the desired final product: osmolarity and ionic
strength, pH, choice of buffer and concentration, any aqueous component
to be entrapped, and the maximum levels of contaminants such as metal
ions and dust. Liposomes are osmotically active; they shrink or swell with
changes in osmolarity. 1~ In extreme cases this can result in loss of en-
trapped aqueous markers and large changes in size distribution. Minimally
sized SUVs seem to be an exception to this in that they are not sensitive to
osmolarity changes. Many ions, and most notably divalent cations, can
interact specifically with the head group of negatively charged lipids, re-
suiting in changes in zeta potential, which can lead to aggregation, fusion,
or affect the interaction between liposomes and other surfaces, such as
cells. Likewise, the pH can alter the ionization of lipids, giving rise to
similar effects or even completely destabilizing the bilayer structure in
some cases, such as is observed for pure phosphatidylethanolamine

~4 D. Lichtenberg and T. Markello, 3. Pharrn. Sci. 73, 122 (1984).

Hs H. Talsma, H. Jousma, K. Nicolay, and D. J. A. Crommelin, Int. J. Pharm. 37, 171
(1987).
tt6 H. Jousma, H. Talsma, F. Spies, J. G. H. Joosten, H. E. Junginger, and D. J. A.
Crommelin, Int. J. Pharm. 35, 263 (1987).
210 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

(PE). 48,~17-H9 Furthermore, most lipids are subject to acid- or base-cata-

lyzed hydrolysis. Lipid oxidation is also enhanced by aqueous components,
especially some metal ions. Consequently, it is essential to control the
osmolarity, ionic strength, and pH of the aqueous phase. In addition,
antioxidants such as tocopherol, aqueous antibiotics or bacterial inhibitors,
and metal chelators are often added to the aqueous phase to improve the
overall stability of liposomes.

Organic Solvent
The choice of an organic solvent for solubilizing the lipids is based on
two overriding considerations. First, all the lipid components must be
soluble at the desired concentration so that a molecular mixture can be
formed. Second, removal of the solvent from the lipids must be uniform so
that the lipid components are uniformly distributed throughout the
particles. ~2-'22 For example, many lipid components crystallize before
others during the drying process. When this happens the film will be
heterogeneous and in some cases the liposome preparations will contain
heterogeneities or even residual crystals. In addition, c o m m o n impurities
or degradation products of some solvents are concentrated upon solvent
removal and can lead to lipid degradation. This is especially true of perox-
ides formed by many ethers.

Lipid Choice
Although the choice for lipids depends on the particular application,
the following guidelines generally relate to the factors that enter in choos-
ing a particular lipid over another. Although liposomes can be made with
any phospholipid, most work on liposomes has been done with phosphati-
dylcholine (PC) derived from hen egg yolks. This material is available in
both large quantifies and high purity and is probably the best characterized
phospholipid. It is usually mixed with cholesterol at a 2:1 or 1 : I mole

~7 R. J. Y. Ho, B. T. Rouse, and L. Huang, Biochem. Biophys. Res. Commun. 138, 931
(1986).
,,s H. Ellens, J. Bentz, and F. C. Szoka, Biochemistry25, 285 (1986).
H9 H. EUens, J. Bentz, and F. C. Szoka, Biochemistry 25, 4141 (1986).
~2oT. M. Estep, D. B. Mountcastle, R. L. Biltonen, and T. E. Thompson, Biochemistry 17,
1984 (1978).
~2~T. M. Estep, D. B. Mountcastle, Y. Barenholz, R. U Biltonen, and T. E. Thompson,
Biochemistry 18, 2112 (1979).
t22 D. Bach, Y. Barenholz, T. E. Thompson, and I. R. Miller, Thermochim. Acta, in press
(1989).
[9] LIPOSOME PREPARATION 21 1

ratio for increasing the stability and decreasing the permeability of lipo-
somes both in buffer and in the presence of plasma proteins.~,n3-~25
A negatively charged phospholipid is added in many preparations in
the form of phosphatidylglycerol (PG) or phosphatidylserine (PS) at a
10-30% mole ratio to PC. The inclusion of the acidic lipid decreases the
tendency of PC liposomes to aggregate and can increase the efficiency of
encapsulation of certain drugs either by improving the aqueous entrap-
ment 1~ or by specific charge interactions.~ The negative charge also in-
creases the efficiency of the uptake ofliposomes by ceils in vitro/26,127 and
in vivo decreases their half-life (hrz) in blood following iv injection. 12s The
specific lipid used to impart the negative charge is important for some
applications. Recent work indicates that when either ganglioside Gu~ or
phosphatidylinositol (PI) is added for the negative charge, the resulting
liposomes tend to have long hi2 in blood. ~29,~3
The fluidity of the liposome bilayer can be controlled by the choice of
lipid, in terms of the number of double bonds and the length of the acyl
chains. Synthetic PCs with long, saturated acyl chains, such as dipalmityl-
phosphocholine (DPPC) and distearylphosphocholine (DSPC) (with and
without cholesterol) have been used to produce liposomes which are
"rigid" at ambient temperatures. These show much lower permeability to
various solutes, and increased stability and circulation time in
blood. ~24,'2g,~3~However, such liposomes have to be prepared at a tempera-
ture above the solid-to-fluid phase transition (To) for each lipid (42 and
54 , respectively, for DPPC and DSPC). Once formed and annealed at
high temperature (including during extrusion and other secondary process-
ing), they can then be used at low temperature. Sphingomyelin is a natural
phospholipid that has a high transition temperature m and also forms very
stable liposomes.~3a

123D. Papahadjopoulos, M. Cowden, and H. K. Kimelberg, Biochim. Biophys. Acta 330, 8

(1973).
124C. J. Kirby, J. Clarke, and G. Gregoriadis, Biochem. J. 186, 591 (1980).
125L. S. S. Guo, R. L. Hamilton, J. Goerke, J. N. Weinstein, and R. J. Havel, J. Lipid Res.
21,993 (1980).
~26j. Damen, J. Regts, and G. Scherphof, Biochim. Biophys. Acta 665, 538 (1981).
t27 R. Fraley, R. M. Straubinger, G. Rule, L. Springer, and D. Papahadjopoulos, Biochemis-
try 20, 6978 (1981).
i~ j. Senior, G. Gregoriadis, and K. A. Mitropoulos, Biochim. Biophys. Acta 760, 111
(1983).
,29 T. M. Allen and A. Chonn, FEBSLett. 223, 42 (1987).
13oA. Gabizon and D. Papahadjopoulos, Proc. Natl. Acad. Sci. U.S.A. 85, 6949 (1988).
131j. Senior and G. Gregoriadis, Life Sci. 30, 2123 (1982).
m M. Shinitzky and Y. Barenholz, J. Biol. Chem. 249, 2652 (1974).
133T. M. Allen, Biochim. Biophys. Acta 640, 385 (1981).
212 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

Liposomes become very permeable at temperatures close to the Tc134

and this has been used to create temperature-sensitive liposomes for drug
delivery to hyperthermic tumors, s4 The presence of cholesterol at high
mole ratio abolishes the Tc and also inhibits the formation of a highly
permeable state.l~- ~36
For most phospholipids, liposomes can be formed at a wide pH range
of 6 - 9 in the presence of 0.1 M monovalent ions. When liposomes are
made of acidic phospholipids such as PS, PG, PI, or phosphatidic acid
(PA), care should be taken to avoid divalent or polyvalent metals, which
tend to bind strongly to negatively charged groups on the lipid, thus
producing aggregation and fusion. ~8 Addition of metal chelators can re-
duce this problem.
PE, when pure, is the only phospholipid that does not form closed
vesicles at neutral pH and physiological ionic strength. ~37 It will form
liposomes however, at low ionic strength and high pH 137or in combination
with another component.~3s-~4oThis property has been utilized to produce
pH-sensitive liposomes, by incorporating PE or other amphiphilic mole-
cules that have a carboxyl group that can be protonated as the pH drops
from 7.0 to 6.0, ~9,Hs'H9 or to produce target-sensitive liposomes. H7
Although most of the work with liposomes has been done with pure
lipids or mixtures of pure lipids, certain impurities can be tolerated while
still maintaining acceptable stability. For example, the small percentage of
lyso-PC or fatty acids that accumulates during hydrolysis would be accept-
able if there were no other interference with any of the specific functions
contemplated. However, storage conditions at either low (< 6.5) or high pH
(->8.5) may generate enough hydrolytic products to destabilize liposomes,
depending on the time and temperature. Oxidation is another difficult
problem that results in unstable liposomes. ~4~-~ Since oxidation is an

,34 D. Papahadjopoulos, K. Jacobson, S. Hir, and T. Isac, Biochim. Biophys. Acta 311, 330
(1973).
,35 E. Oldtield and D. Chapman, FEBSLett. 23, 285 (1972).
~ M. K. Jain, Curt. Top. Membr. Transp. 6, 1 (1975).
,3~ D. Papahadjopoulos and J. C. Watkins, Biochim. Biophys. Acta 135, 639 (1967).
' n P. R. Cullis and B. DeKruijff, Biochim. Biophys. Acta 559, 399 (1979).
,39 R. J. Y. Ho and L. Huang, J. Immunol. 134, 4035 (1985).
,4o T. F. Tarashi, T. M. Van Der Steen, B. DeKruijff, C. Tellier, and A. J. Verkeij, Biochemis-
try 21, 5756 (1982).
~4~T. Nakazawa and S. Nagatsuka, Int. J. Radiat. Biol. 38, 537 (1980).
,42 D. A. Barrow and B. R. Lentz, Biochim. Biophys. Acta 645, 17 (1981).
,43 T. Nakazawa, S. Nagatsuka, and T. Sakurai, Int. J. Radiat. Biol. 40, 365 (1981).
~ F. Ianzini, L. Guidoni, P. L. Indovina, V. Viti, G. Erriu, S. Onnis, and P. l~mdaeeio,
Radiat. Res. 98, 154 (1984).
[9] LIPO$OME PREPARATION 213

autocatalytic process accelerated by metal ions, both the purity of the

starting material and the storage conditions (including antioxidants, chela-
tion, and nitrogen or argon atmosphere) are to be monitored and con-
trolled. This is particularly important for polyunsaturated fatty acids
present in both egg yolk and soybean PC. In this respect, synthetic palmi-
tyloleylphosphatidylcholine (POPC), DPPC, and DSPC arc much more
stable to oxidative degradation and, if this is a crucial parameter, their use
is recommended. In terms of stability to hydrolysis, sphingomyelins and
ether analogs of PC (instead of the diacyl-PC) are much more stable than
egg or soybean PC.
Liposomes can also be made of single-chain amphophiles, such as
mixtures of long-chain fatty acids and alcohols~4~ or nonphospholipid
synthetic ether compounds, ~ which may have some advantages for cer-
tain applicationsthat requireincreasedstabilityto hydrolysis.
Finally,when liposomes arc made to encapsulatelipophilicdrugs that
tend to associatewith the bilayer,the stabilityand physicochcmical char-
acteristicsof the system may be altered depending on the interaction
between lipidand the drug.94

Methods for Particle-SizeDetermination

Particle-size distribution is an especially important parameter for lipo-
somes. For example, it clearly influences the distribution of the liposomes
within the body when they are administered parenterally. 147-15~However,
despite the existence of many methods, there is no single method capable
of accurate and reliable size distribution analysis of liposome samples.
Except for electron microscopy, none of the methods is capable of resolv-
ing the distribution over the entire range of particle size required for
liposomes, from l0 nm to 50/zm. In fact, many methods only determine
the average size and provide little or no indication of the distribution.
Particle shape can also be important, especially for larger particles, those
greater than 1 gin. Therefore, comparison and compilations of results from

,4s W. R. Hargreaves and D. Deamer, Biochemistry 17, 3759 (1978).

,46 A. J. Bailli, A. T. Florence, L. R. Hum, G. T. Muirhead, and A. Rogerson, J. Pharm.
Pharmacol. 37, 863 (1985).
,4~ R. Juliano and D. Stamp, Biochem. Biophys. Res. Commun. 63, 651 (1975).
,48 R. M. Abra and C. A. Hunt, Biochim. Biophys. Acta 666, 493 (1981).
,49 T. M. Allen and J. M. Everest, J. Pharmacol. Exp. Ther. 226, 539 (1983).
,so y. Sato, H. Kiwada, and Y. Kato, Chem. Pharm. Bull, 34, 4244 (1986).
,5, F. C. Szoka, D. Milholland, and M. Barza, Antimicrob. Agents Chemother. 31,421 (1987).
214 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

many methods are necessary to fully characterize the sizes of liposomes

within a particular preparation.
When choosing size measurement methods, several factors must be
taken into consideration. Because liposomes are populations of particles,
there is a distribution of size, rather than a single value, which can be
expressed in a variety of ways. For instance, the percentage of mass,
volume, or even surface area in each resolved size class can be determined.
However, each of these may have a very different shape and calculated
mean, even for the same distribution. |52
While a single, absolute liposome size-distribution measurement does
not exist, a variety of useful and reliable sets of information can be ob-
tained. When a more specific question is asked, then the method or
methods can be chosen more effectively. A few generalized situations have
been addressed in the following discussion. Large liposomes, those greater
than 1/tin, can be adequately measured by light microscopy and the
Coulter counter. For very small liposomes, those smaller than 0.2/tm,
several methods are very useful: gel permeation chromatography (GPC),
dynamic light scattering (DLS), and EM. The major difficulty is in the
range between these two extremes. Apart from EM, which is cumbersome,
modifications of one or more methods, such as centrifugation, sedimenta-
tion field flow fractionation, or flow cytometry, may improve size charac-
terization of liposomes in this intermediate range. Alternatively, new
methods may be developed to fall this need.
So far, this discussion has assumed that absolute size distributions are
to be measured. Nevertheless, a number of situations can be satisfied by
relative measurements or indications of change. For example, control of
process parameters that may affect size distribution or identification of
component failures can be accomplished by measurements of change
without absolute measures. In such cases, measurements limited to the
mean size, such as DLS and turbidity, can be quite useful.

Single-Particle Properties
Microscopic methods have been used extensively to characterize lipo-
somes, not only for size but also for shape and structure. However, the
advantage of measurements on individual particles is offset by a need to
count a large number of particles for a good representation of the entire
sample. In addition, quantitation of images is difficult and tedious, espe-
cially for a large number of particles. Image-processing techniques can be

1s2 C. Orr and E. Y. H. Keng, in "Handbook on Aerosols" (R. L. Dennis, ed.), p. 114. Energy
Res. Dev. Admin., G. C. A. Corp., Bedford, M~_~chusetts, 1976.
[9] LIPOSOME PREPARATION 215

applied to facilitate the data collection and analysis but have not been
standardized.
Light microscopy has the advantage of determining particle shape and
to some extent the amount of multilameUarity. The principal limitation is
the lack of resolution below 0.5/zm with the best of optics, although the use
of lasers may extend the range even further. 153 Another problem is that
visualization by light microscopy of truly unilamellar liposomes of any size
is difficult, if not impossible, without the use of encapsulated fluorescent
markers.
Electron microscopy has a much smaller minimum resolution but the
samples must be fixed. The fixation can introduce artifacts, especially with
negative staining. Consequently, quantitation of images from negative
stain are of very limited use. Freeze-fracture has less of a tendency for
artifacts but must be corrected for deviations of the fracture plane from the
midplane of the particle. ~54 Freeze etching minimizes this problem.l,~s,~55
The Coulter counter is very useful for determination of the distribution
by particle volume but only for particles of 1/zm and larger. It is also
dependent to a lesser extent on particle asymmetry. Because of the range
limitation it is best for determination of the number of particles present
above 1 #m or for use in combination with another method.

Hydrodynamic Radius Using Gel Permeation Chromatography

This method fractionates the sample by hydrodynamic radius, allowing
a determination of the amount of phospholipid in each size class resolved
(fraction), but with some limit to the actual resolution.49 When the column
is calibrated the amount of lipid (or drug) mass versus size can be deter-
mined. 156,t5~However, the currently available columns are only capable of
resolving particle sizes smaller than 0.2/zm. All the larger particles are
lumped together in the void volume fraction. Thus, use of GPC in combi-
nation with the Coulter counter can provide size characterization from
10 nm to more than 1 #m, but with a gap from 0.2 to 1/zm. Recent
procedures using high-performance liquid chromatography (HPLC) col-
umns can be automated for routine analyses. ~Ss

153W. Jiskoot, T. Teedink, E. C. Beuvery, and D. J. A. Crommelin, Pharm. Weekbl. Sci. Ed.
8, 259 (1986).
154R. Van Venetie, J. Leunissen-Bijvelt, A. J. Vcrkleij, and P. H. Vcrvcrgaext, J. Microsc.
118, 401 (1980).
155p. Guiot and P. Baudhuin, in "Liposom Technology" (G. Gregoriadis, ed.), Vol. 1,
p. 163. CRC Press, Boca Raton, Florida, 1984.
i~ y. Nozaki, D. D. Lasic, C. Tanford, and J. A. Reynolds, Science 217, 366 (1982).
157j. A. Reynolds, Y. Nozaki, and C. Tanford, Anal. Biochem. 130, 471 (1983).
lss M. Ollivon, A. Walter, and R. Blumenthal, Anal. Biochem. 1S2, 262 (1986).
2 16 MODEL MEMBRANES AND THEIR CHARACTERISTICS [9]

Light Scattering
The turbidity of liposome samples depends on the particle-size distri-
bution, among other parameters. Turbidity has been used to characterize
the average size but it is severely limited by the many other factors which
also affect turbidity. ~59 The primary usefulness of turbidity is in detecting
formation of particles, or a change in particle size, as processing takes
place.
Dynamic light scattering [also referred to as photocorrelation spectrom-
etry (PCS) and quasielastic light scattering (QELS)] has been used exten-
sively to characterize liposome size distribution but is reliable only for
homogeneous samples. The reliability and accuracy deteriorates rapidly
with increasing heterogeneity. 16 In addition, the method is limited to sizes
smaller than 3/zm and its ability to resolve 1-/zm particles in the presence
of smaller ones is questionable. Nevertheless, DLS can be very useful to
characterize liposome samples, especially SUVs and small LUVs, giving an
average diameter and polydispersity. These values can be used to detect
relative differences or changes occurring on processing or swelling. 16~-~6a
The mathematical techniques required to calculate the size distribution
operate by fitting a summation of exponentials to the autocorrelation
function. The effect is that the distribution analyses can often be mislead-
ing. By use of multiple mathematical procedures on a single autocorrela-
tion function this problem can be minimized but not eliminated. ~6

Density
Centrifugation can be used to fractionate liposome preparations by
differences in sedimentation rate; the resultant separation may be due to
either differences in size or some other factor affecting liposome density,
such as number of lamellae.~64 In order for the method to be applicable,
there must be sufficient density differences between the particles and the
solvent. For liposomes, ultracentrifugation or alteration of the buffer den-
sity may be required, especially for smaller particles. By use of analytical
ultracentrifuge techniques the distribution of scattered intensity versus
density can be calculated. ~65 If the particle density is homogeneous, and

159D. A. Barrow and B. R. Lentz, Biochim. Biophys. Acta 597, 92 (1980).

16oT. Prouder, ACSMonogr., 332 (1987).
161 E. Hantz, A. Cao, J. Escaig, and E. Taillandicr, Biochim. Biophys. Acta 862, 379 (1986).
le2 W. Li, T. S. Aurora, T. H. Haines, and H. Z. Cummins, Biochemistry 25, 8220 (1986).
J63 M. S. McCracken and M. C. Sammons, J. Pharm. Sci. 76, 56 (1987).
164E. Goormaghtigh and G. A. Scarborough, Anal. Biochem. 1.$9, 122 (1986).
165 H. Coil and C. G. Seades, J. Colloid Interface Sci. 115, 121 (1987).
[ 10] MICROCAPSULE PREPARATION FROM ERYTHROCYTES 217

known, the distribution with size can be calculated. Since the density of
liposomes may vary, this presents a limitation of the method's accuracy.
Sedimentation field flow fractionation (SFFF) suffers from the same
limitations as centrifugation, because the determination is also based on
density differences between the particles and the solvent. Initial testing of
SFFF for liposomes was promising but no further reports have appeared, tu

Others
A variety of other methods have been used for characterization of
liposome size based on parameters influenced by size, in addition to other
properties: NMR, DSC, zeta potential, flow cytometry, etc. In most cases,
the dependence of the method on size varies and the extent of the influence
of other parameters makes the results uncertain. Nevertheless, the results
from each of these methods can provide important alternative information
for a complete characterization of the liposomes. In the case of flow
cytometry, it is conceivable that fluorescent measurements of liposomes
may be possible down to 0.1/tm by optimization for much smaller particle
size. By a combination of lipid-soluble dyes and aqueous entrapped dyes,
simultaneous measurements of both lipid and aqueous mass per particle
are conceivable, allowing determination of the lamellarity of each particle.
Unfortunately, none of the commercial instruments has been designed for
this purpose.
i~ j. j. Kirkland, W. W. Yau, and F. C. Szoka, Science 215, 296 (1982).

[10] P r e p a r a t i o n o f M i c r o c a p s u l e s f r o m H u m a n
Erythrocytes: Use in Transport Experiments of
G l u t a t h i o n e a n d Its S - C o n j u g a t e
B y TAKAHITO KONDO

Sealed inside-out microvesicles have been used in the characterization

of transport systems in erythrocyte membranes.~-s The method for prepar-
ing the microvesicles was originally reported by Steck and Kant, 6,7 using
H. Sze and A. K. Solomon, Biochim. Biophys. Acta 550, 393 (1979).
2 j. E. Mollman and D. E. Pleasure, J. Biol. Chem. 255, 569 (1980).
3j. M. Gimbel, J. Biol. Chem. 257, 10781 (1982).
4 A. J. Caride, A. F. Rega, and P. J. Garahan, Biochim. Biophys. Acta 734, 363 (1983).
s j. S. Lolkema and G. T. Robillard, Eur. J. Biochem. 147, 69 (1985).
6 T. L. Steck, R. S. Weinstein, J. H. Straus, and D. F. H. Wallach, Science 168, 255 (1970).
T. L. Steck and J. A. Kant, this series, Vol. 31, p. 172.

Copyright 1989 by AcKlemic ~ Inc.

METHODS IN ENZYMOLOGY, VOL. I 71 All rights of ~roducfion in any form ra~rv~L