Journal of Antimicrobial Chemotherapy (1991) 28, Suppl.

B, 39-48

Overview of liposomes

Gregory Gregoriadis

Centre for Drug Delivery Research, The School of Pharmacy, University of London,
29-39 Brunswick Square, London WC1N 1AX, UK

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Many of the problems associated with conventional drug therapy may be circum-
vented by the use of delivery systems which, in a variety of ways, will optimize drug
action. Among the systems being investigated, liposomes hold considerable promise.
Versatility in both the structural characteristics of liposomes and their ability to
accommodate a wide range of drugs has contributed to the design of appropriate
formulations with optimal pharmacological actions. Areas in which such formula-
tions have been already applied successfully experimentally or clinically, include
cancer chemotherapy, antimicrobial therapy and vaccines.

Introduction
The last few decades have witnessed concerted efforts to enhance the effectiveness of
drugs used in therapeutic, diagnostic and preventive medicine by improving their
selectivity; with a few exceptions (e.g. antibiotics interfering with metabolic pathways in
bacteria that are not shared by the host), drug selectivity is generally poor. For
instance, in cancer chemotherapy cytostatic agents also damage normal, rapidly
dividing cells. Additional obstacles to effective drug action include inaccessibility of
targets (e.g. many antimicrobial agents fail to reach intra-cellular sites harbouring
micro-organisms), drug instability in the biological milieu, premature drug loss through
excretion and allergic reactions elicited by certain drugs (Gregoriadis, 1977).
Recently, accumulating information on the three-dimensional structure of drugs and
on quantitative structure activity relationships has contributed to better drug design
and optimization of the interaction of at least some drugs with their respective
molecular targets. However, it is more than likely that, in spite of progress in this area,
many of the aforementioned obstacles will remain, particularly those encountered
within the biological milieu interposed between the site of drug administration and the
target. On the other hand, developments in such unrelated areas as hybridoma and
recombinant DNA technology, the chemistry of polymers and colloids and the under-
standing of ligand receptor interactions and ensuing intracellular events have now
formed a realistic basis for the pursuit of an alternative approach to conferring
selectivity on drugs, namely targeted drug delivery. The concept was first aired early
this century and entails the use of carrier systems to deliver drugs to where they are
needed or facilitate their release there. Carriers can do so either because of an inherent
or acquired ability to interact selectively with certain cells or because they are
instructed to release drugs in a controlled fashion conducive to optimal pharmacolo-
gical action. In the former case for instance, antibodies bind with exquisite specificity to
39
0305-7453/91/28B039+10 J02.00/0 © 1991 The British Society for Antimicrobial Chemotherapy
40 G. Gregoriftdb

cell surface antigens, glycoproteins interact with receptors through terminal sugars and
colloid microspheres such as liposomes are endocytosed by a variety of cells. Following
injection, drug-loaded carriers may undergo changes which are detrimental to their
effective function. Changes include destabilization of the carrier by elements in the
biological milieu, its interception by non-target tissues and the development of immune
responses to the carrier. Over the years, such physiological considerations have led to
the study of carrier behaviour in vivo and, in many instances, its control (Gregoriadis,
1981). This certainly holds true for liposomes for which a wealth of relevant data has
already been amassed (Gregoriadis, 1988a).

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Structural characteristics of liposomes
Described in the mid-60s as a tool for the study of membrane biophysics, liposomes
(also known as vesicles) were, soon afterwards, to play a major role in drug delivery
(Gregoriadis, 1976). They consist of one or more concentric phospholipid bilayers
separated by aqueous compartments and their unique advantage over other carriers is
structural versatility (Figure 1). Thus, a wide spectrum of phospholipids with an array
of gel-liquid crystalline transition temperatures (TJ can, alone or in combination with
sterols, provide a range of membrane fluidity. This in turn controls the permeability of
membranes to entrapped drugs, bilayer stability and, as will become apparent later,
behaviour in vivo. A complement of amphiphiles incorporated into the lipid structure
will, when required, impose a surface charge. Very many drugs of varying solubility
and size (e.g. anti-tumour and antimicrobial drugs, enzymes, peptides, hormones,
vaccines and genetic material) have already been encapsulated in either the aqueous or
the lipid phase of liposomes. Drugs or ligands such as antibodies, certain glycoproteins
and neoglycoproteins and glycolipids can also be linked to the outer bilayer. Indeed, it
is now possible to design liposomes to satisfy particular needs in a variety of
applications ranging from biochemical and immunological assay kits and diagnostic
reagents to therapeutic preparations for enteral and parenteral uses and vaccines
(Gregoriadis, 1988a; Lopez-Berestein & Fidler, 1989). Major findings on the interaction
of liposomes with the biological milieu that have contributed towards the application
of the system both experimentally and clinically are summarized below.

Interaction of liposomes with the biological milieu

Since 1970 when liposomes were first proposed as a drug delivery system and tested in
animals (Gregoriadis & Ryman, 1972), a multitude of studies have shown that
behaviour in vivo will vary according to the type of vesicles used, the drug they carry,
the site of administration and the physiological state of the animals (Ryman & Tyrrell,
1980).
Soon after intravenous injection, drug-containing liposomes (especially those made
solely of unsaturated phospholipids), interact with at least two different groups of
plasma proteins (Gregoriadis, 1988A). One comprises the so-called opsonins, which
adsorb on to the surface of the vesicles to render them (and their contents) prey to the
phagocytic cells of the reticuloendothelial system (RES). The second group, high-
density lipoproteins (HDL), attack vesicles and with the help of a protein exhibiting
phosphatidylcholine transferase activity, remove phospholipid molecules from the
bilayers. This is followed by the disintegration of the vesicles or perhaps the formation
 Overview of Uposomes 41

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Figure 1. A section of an electron micrograph of a negatively-stained multilamellar liposome shows lipk)
bilaycn alternating with electron-opaque aqueous channels. Three of these bilayers are enlarged schematic-
ally to illustrate their bimolecular leaflet organization, in which polar heads of phospholipids face the water
phase and acyl chains form the hydrophobic regions of the leaflet. Open circles denote drugs entrapped in the
aqueous channels. Oblong shapes are cholesterol (filled) and membrane-soluble drugs (open) sandwiched
between phospholipid molecules. Liposomes can vary in size from about 20 run (small iinil»mH1nr vesicles) to
several microns in diameter. The latter can have one (large unilameilar vesicles) or more (multilamellar
vesicles) bilayers. Depending on the methodology and the type of drug, entrapment values can vary from less
than 1% to 100% of the drug used. (From Gregoriadis, 1990 with permission.)

of pores on their surface. In either case, encapsulated solutes are released into the
circulation; at the same time, vesicles at various stages of destabilization and with some
of their solute contents still entrapped, are rapidly (within minutes) removed by the
RES, probably through recognition by the cells of opsonins adsorbed on to the
liposomal surface. The rate of clearance of liposomes is influenced by vesicle size (e.g.
larger vesicles are eliminated more rapidly than smaller ones), surface charge and,
probably, by the extent of opsonin absorption (which is likely to depend on the extent
of bilayer destabilization; see later) (Gregoriadis, 19886).
Loss of structural integrity of liposomes in the circulating blood and the ensuing
premature release of their contents are obviously incompatible with the quantitative
delivery of drugs to tissues. This problem was resolved by packing the relatively loose
42 G. Gregortatts

100

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50

Figure 2. Permeability of DSPC liposomes in mouse plasma and clearance from the circulation. Small
imilamdlar liposomes containing quenched CF and composed of DSPC (O) or equimolar DSPC and
cholesterol ( • ) were incubated in the presence of mouse plasma at 37°C (a) or injected intravenously into
four mke (b). Values at time intervals are % latent CF of total CF present or % (mean±s.D.) latent CF of
injected latent CF per total mouse plasma (from Senior & Grtgoriadis, 1982, with permission).

phospholipid bilayers, usually made of egg phosphatidylcholine (PC), by the addition of

cholesterol or by substituting phosphatidylcholine, partly or wholly, with saturated
phospholipids that have T,s above 37°C and are therefore 'solid' at body temperature.
It has been shown that HDL is unable to remove phospholipids from such vesicles to a
significant extent. As a result, liposomes composed of, say, PC and increasing amounts
of cholesterol were found to lose correspondingly decreasing amounts of both their PC
component and entrapped water soluble markers on exposure to blood at 37°C in vitro
or in the circulation of injected animals. For liposomes made of equimolar amounts of
the saturated distearoyl phosphatidylcholine (DSPC) and cholesterol, complete bilayer
stability remained unchanged even after incubation with blood for over 48 h (Senior &
Gregoriadis, 1982) (Figure 2).
In the course of this work, an interesting relationship was observed between
liposomal stability and rate of vesicle clearance from the circulation; the more stable
the vesicles in the presence of serum in vitro, the longer their biological half-life. This
was again true for vesicles of all sizes; for small unilamellar vesicles (SUV) (diameter
40-80 nm) made of equimolar DSPC and cholesterol, a half-life of 20 h was measured
in mice. Such a relationship was explained as follows: vesicles with a loose bilayer
transfer some of their phospholipid to HDL. As a result, gaps are created in the bilayer
 Overview of Uposomes 43

•OPSONINS 1
y
• .llll)
LOOSE ^lU^V
^Jttfr-
BILAYERS ^fflfe —> ^ . V ^ ~"" ^> * ^ - • RES
# Raold
"^w^ ^/.}^ YV^" •
* clearance

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PACKED
BILAYERS

Figure 3. Correlation between stability of liposome* and clearance from blood circulation. In the proposed
scheme, the extent of bilayer porosity and leakage of solutes in blood is dependent on the facility with which
high-density lipoproteins (HDL) remove photpholipid molecules from the bilayer. Loose bilayen (top) are
attacked by HDL more effectively than packed bilayen (bottom). The greater the gaps after HDL attack, the
more extensive the opsonin adsorption on vesicles and uptake by the retkuloendothdial system (RES).
(From Grcgoriadis, 1988c, with permission.)

allowing the escape of trapped solutes and also the adsorption of opsonins which
promote vesicle uptake by the RES. In contrast, vesicles with packed bilayers are not
(or are only marginally) affected by HDL, solutes do not escape and opsonins are
unable to adsorb in amounts sufficient to mediate rapid vesicle removal by the RES
(Figure 3) (Gregoriadis, 1988*, c).
Progress in the control of liposomal stability and presence in the circulating blood
was instrumental in the effective targeting of the system to cells other than those that
normally intercept it (i.e. RES): liposomes coated with antibodies or other cell-specific
ligands must remain in the circulating blood for a period of time long enough to allow
their encounter and effective interaction with the target. It is generally advantageous to
employ large liposomes for the delivery of water soluble drugs since a large aqueous
phase will accommodate greater quantities of drugs, regardless of molecular size.
Unfortunately, however, even the most stable large liposomes exhibit brief half-lives.
On the other hand, it has been shown (Allen & Chonn, 1987; Gabizon &
Papahadjopoulos, 1988; Senior et al., 1991) that a highly hydrophilic bilayer surface
achieved by its enrichment with gangliosides or polyethylene glycol will substantially
extend the half-life of liposomes with a diameter of about 60 nm to a little over 100 nm.
One of the earliest observations on the fate of drug-containing liposomes in animals
was that uptake of the vesicles by the RES, namely the fixed macrophages and the
circulating monocytes, is carried out by endocytosis after which vesicles end up in the
lysosomal apparatus of the cells. Once in lysosomes, vesicles are disrupted by phospho-
lipases. Freed drug can then either act within the organdies or if small enough, diffuse
through the lysosomal membranes to reach other cell compartments (Gregoriadis,
1976). More recently, uposomes have been tailored to fuse in vitro with the cndocytic
vacuoles and release their contents into the cytoplasm thus avoiding passage through
the lysosomes (Connor & Huang, 1985). Because of their size (about 25 nm minimal
44 G. Gregoriadis

diameter),.intravenously injected liposomes escape only to a very small extent from the
intravascular space in normal animals. However, transcapillary passage has been
observed in inflamed areas in the body where vessels become leaky. Moreover,
although large liposomes preferentially localize in the macrophages of the liver and
spleen, small vesicles of an average diameter of about 100 nm can pass through the
fenestrae in the liver to reach the hepatic parenchymal cells by which they are
endocytosed. Small liposomes with long half-lives will also accumulate extensively in
the bone marrow macrophages (Gregoriadis, 1988ft).
Macrophages and other phagocytic cells will take up liposomes administered by
routes other than the intravenous. For example, although the majority of liposomes
injected intramuscularly or subcutaneously reach the blood circulation (and eventually

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the RES) via the lymphatics (small vesicles) or disintegrate locally (large vesicles), a
proportion end up in the lymph nodes draining the injected site. Similarly, circulation
through the lymphatic system appears to account for the recovery of a large proportion
of liposomes injected intraperitoneally, in the RES and the thoracic, renal, lumbar and
intestinal lymph nodes. Indeed, in terms of uptake per unit weight of tissue, lymph
nodes in all non-intravenous studies take up many more liposomes than do the liver
and spleen (Turner et al., 1983).
Numerous workers have attempted to resolve the question as to whether or not
orally administered liposomes protect entrapped agents in, and facilitate their uptake
by, the gut. Early experiments in several laboratories with insulin entrapped in
liposomes revealed a reduction in blood glucose levels in diabetic rats (Gregoriadis,
1988a). However, subsequent studies indicated that partial recovery of insulin and a
number of non-absorbable agents in the blood circulation of animals treated orally
with the Hposomal form of such agents, occurs through the absorption of complexes of
hposomal phospholipid, bile salt and agent rather than through absorption of intact
vesicles (Gregoriadis, 1988a).

Targeted liposomes
Association of drug-containing liposomes with cells that do not normally take up the
carrier has been achieved through the use of antibodies and other cell-specific ligands
linked to the outer bilayer of liposomes. Numerous in-vitro studies have shown
convincingly that polyclonal and monoclonal antibodies raised against a repertoire of
cell surface antigens mediate the association of the drug-containing liposomal moiety
(to which such antibodies are linked) with, and its introduction into, the relevant cells
(Machy, Barbet & Leserman, 1982). As expected, however, in-vivo targeting of
liposomes has proved a much more challenging proposition, especially when mediated
via antibodies, the Fc portion of which binds to its receptors on the macrophages. This
accelerates the removal of the carrier by the RES and diminishes its chances of contact
with the intended target cells. Avoidance of such RES interference has been achieved
by substituting for antibody its antigen-recognizing Fab portions or by the use of
antibody-coated small vesicles with long half-lives. In the latter case, Fc-mediated
reduction of the half-life of vesicles is modest enough to allow targeting to occur (Wolff
& Gregoriadis, 1984). Such complications, however, are absent when certain galactose-,
mannose-, or fucose-terminating glycoprotein and glyco-lipid ligands are used, as these
will associate exclusively with their receptors in vivo.
 Oeniew of liposomes 45

Medical applications of liposomes

Abundance of encouraging results with liposomal drugs in the treatment or prevention
of a wide spectrum of diseases in experimental nniTTinls and, to some extent, clinical
trials, has reinforced the prospect of clinical applications on a wide scale (Lopcz-
Berestein & Fidler, 1989). Progress towards clinical uses of liposomes has gained new
momentum thanks to the effects of liposome-based biotechnology companies which
sprang up in the early 80s. Some of the more promising developments, especially those
which have reached or are about to reach the stage of clinical investigation, are
discussed here briefly.
There is indirect evidence from animal work that certain tumour tissues take up

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drugs entrapped in small liposomes to a greater extent than neighbouring normal
tissues. It appears that this is due to one or more of the following factors: higher
endocytic activity of some tumour cells combined with increased local permeability of
adjacent capillaries, slow drug diffusion from vesicles either in circulation or lodged in
neighbouring tissues followed by preferential drug localization in the tumour.or indeed
as a result of migration of monocytes (together with engulfed liposomes) to tumours.
The case for using liposomal drugs in cancer chemotherapy appears stronger in work
aimed at reducing toxicity while maintaining the tumoricidal effect of the drug.
Experiments with liposomes containing anthracycline cytostatics have unequivocaUy
shown reduction of cardiotoxicity and dermal toxicity and prolonged survival of
tumour-bearing animals compared with controls receiving the free drug (Gregoriadis,
1988a). It is thought that liposomal drug taken up by the RES is released slowly to
penetrate neighbouring malignant cells and exert its effect Interestingly, there have
already been promising results in several related phase I and phase II clinical trails,
most of them carried out with daunomycin-containing formulations produced by
liposome-based companies which are responsible for the initiation of many of the
clinical trials. One of the companies has also produced an Indium Ill-containing SUV
preparation for cancer imaging. The product was effective with the majority of patients
studied, exhibiting specificity for malignant tissues and performing similarly for all
types of cancer and all locations (R. J. Crossley, personal communication). However,
criteria for imaging and therapy are different as therapeutic success in targeting is far
more stringent. This is because it not only requires selective localization and uptake of
the drug, but also release of the drug at an appropriate rate and position. In another
approach to cancer treatment, liposomes containing macrophage activation agents
were shown to transform the cells to a tumoricidal state and to eradicate metastases in
experimental animals. Macrophages (peripheral blood monocytes) have also been
activated to a tumoricidal state in clinical trials using muramyl tripeptide linked to
liposomal phospholipid.
Site-specific drug delivery in the treatment of microbial (including parasitic) disease
has been considered, mostly because of the inability of otherwise potent agents to enter
infected intracellular sites effectively. Since many microorganisms reside in the liver and
spleen, especially the RES component, liposomes with their propensity to localize in
these tissues are the obvious choice of carrier. A large body of evidence indicates that
liposomes are superior to the free agents both in terms of distribution to the relevant
intracellular sites and therapeutic efficiacy (Gregoriadis, 1988a). In the context of this
supplement, liposomes have also been applied in the treatment of fungal infections
often seen in immunosuppressed patients. Amphotericin B, used for the treatment of
46 G. Gregoriadis

such diseases, acts by binding to the ergosterol of fungal membranes, thus creating
channels through which vital molecules leak from the cells, which die as a result
Unfortunately, the drug also binds to the cholesterol of mammalian cells, hence its
toxicity. It has been demonstrated that disseminated candidosis in animals can be
treated successfully with amphotericin B incorporated into liposomes and similarly
enouraging observations have been made in patients suffering from fungal disease
(Lopez-Berestein et al., 1987) (see also other contributions in this supplement). It is
thought that the beneficial effect of liposomal amphotericin B is due to its considerably
lower toxicity to mammalian cells. Finally, work with liposome-borne agents for the
direct killing of microbes is now being supplemented with attempts to deliver (via
liposomes) immunostimulating agents such as muramyl dipeptide and its derivatives to

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activate macrophages to a microbicidal state (Lopez-Berestein & Fidler, 1989).
An application of liposomes which seems particularly realistic is in vaccines
(Gregoriadis, 1990). Developments in recombinant DNA technology and better under-
standing of the immunologica] structure of proteins and the ways by which cells and
mediators are involved in the induction of immune responses have led to a new
generation of recombinant subunit and synthetic peptide vaccines that mimic small
regions of viral, bacterial and protozoan proteins. These vaccines can elicit specific
immune responses, are denned at the molecular level and are, therefore, potentially
safe. However, subunit and peptide antigens are usually not immunogenic, or only
weakly so, in the absence of an immunological adjuvant. There is a diverse array of
unrelated substances that increase specific immune responses to antigens through a
variety of mechanisms. For instance, adjuvants can activate macrophages to release
mediators such as interleukin 1, which in turn stimulates the proliferation of
T-lymphocytes leading to humoral and cell-mediated immunity. Another mechanism
by which antigen-incorporating adjuvants may exert their action is slow degradation at
the site of injection, which facilitates uptake of the released antigen by the regional
lymph nodes. However, many of the available adjuvants induce side reactions and only
one (aluminium salts or alum) has been licensed for use in humans. Because alum is not
always effective and increases cell-mediated immunity only slightly if at all, a number of
alternative adjuvants are under investigation. It is perhaps not surprising that lipo-
somes, known to interact with macrophages avidly and to release their contents
gradually at the site of injection, potentiate strong immune responses to entrapped
antigens. This was originally established for diphtheria toxoid and later confirmed for a
large number of bacterial, viral and protozoan antigens relevant to human and
veterinary immunization (Gregoriadis, 1990). In several studies protection of animal
models was achieved by immunization with antigen-containing liposomes and at least
one of these preparations has been licensed for veterinary use. A further advantage of
liposomes over other adjuvants is their variability in structural characteristics, mode of
antigen accommodation and the type of immunity they promote, suggesting versatility
in immunoadjuvant action and vaccine design. For instance, targeted adjuvant activity
has been demonstrated with liposomes coated with a mannosylated ligand; this
facilitates binding to antigen-presenting cells that express the mannose receptor
(Garcon et al., 1988). Additional features include the possibility of using other
immunopotentiating agents in conjunction with liposomes to improve their
adjuvanticity further. Thus, with interleukin-2 co-entrapped with the antigen in the
same vesicles, responses to the antigen were increased above levels attained with the
liposomal antigen alone (Gregoriadis, 1990).
 Orerriew of liposome* 47

ConclusioD
Over 20 years of research have established liposomes as a promising drug delivery
system. Although liposomes are limited in tissue selectivity, their scope can be broad-
ened and their function optimized by a variety of structural manipulations, including
the use of cell-specific ligands anchored on their surface. A major disadvantage of
liposomes over other carriers is their size-imposed inability to cross most normal
membrane barriers. Nonetheless, evidence as presented in this supplement and else-
where indicates that several applications, notably in antimicrobial and cancer therapy
and vaccines, will soon reach clinical use.

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