Liposomes: Applications

in Medicine

R. BANERJEE
Cardiovascular Research Institute, 3333 California Street,
Suite 150, University of California, San Francisco, CA 94118-1245

ABSTRACT: Liposomes are spherical lipid bilayers from 50 nm to 1000 nm in

diameter that serve as convenient delivery vehicles for biologically active com-
pounds. The field of liposome research has expanded considerably over the last
30 years. It is now possible to engineer a wide range of liposomes varying in size,
phospholipid composition and surface characteristics to suit the specific applica-
tion for which they are intended. This paper gives an overview of the main
advances in liposome research from a point of view of their applications in medi-
cine. Aqueous contrast enhancing agents entrapped in liposomal carriers can be
targeted to the liver and spleen and distinctions can be made between normal
and tumorous tissue using computed tomography. Topical application of
liposomes has great potential in dermatology. Liposomes have been used to
deliver anticancer agents in order to reduce the toxic effects of the drugs when
given alone or to increase the circulation time and effectiveness of the drugs.
From the original concept of encapsulating hemoglobin in an inert shell,
liposome-encapsulated hemoglobin (LEH) has evolved into a fluid proven to
carry oxygen, capable of surviving for reasonable periods in the circulation and
amenable to large-scale production. Liposomes may be used to target specific
cells by attaching amino acid fragments such as antibodies or proteins or appro-
priate fragments that target specific receptor sites. Liposomal DNA delivery vec-
tors and further enhancements in the forms of LPDI and LPDII are some of the
safest and potentially most versatile transfer vectors used to date. DNA vaccina-
tion and improved efficiency of gene therapy are just a few of the upcoming appli-
cations of liposomes.

E-mail: rban@itsa.ucsf.edu

JOURNAL OF BIOMATERIALS APPLICATIONS Volume 16 – July 2001 3

1530-8022/01/01 0003-19 $10.00/0 DOI: 10.1106/RA7U-1V9C-RV7C-8QXL
© 2001 Technomic Publishing Co., Inc.
Journal Online at http://techpub.metapress.com
4 R. BANERJEE

INTRODUCTION

The field of liposome research has expanded considerably over the

last 30 years. It is now possible to engineer a wide range of liposomes
varying in size, phospholipid composition and surface characteristics to
suit the specific application we are interested in. The surfaces of
liposomes can be modified by the choice of bilayer lipid as well as by the
incorporation and covalent linkage of glycoproteins and synthetic poly-
mers. In comparison with other drug carriers, liposomes have some
advantages, like biological degradability and relative toxicological and
immunological safety. It is impossible to describe the details of such a
vast field in a single review and do justice to all the relevant studies. This
paper gives an overview of the main advances in liposome research from
a point of view of their applications in medicine.
Liposomes are spherical lipid bilayers from 50 nm to 1000 nm in diam-
eter that serve as a convenient delivery vehicle for biologically active
compounds [1]. They are formed by the interaction of amphiphilic lipids
suspended in an aqueous phase, and they serve as osmotically sensitive
model membrane systems that are 105 times more permeable to anions
than cations [2].

CLASSIFICATION OF LIPOSOMES

Liposomes may be classified based on various characteristics, accord-

ing to size, lamellarity, lipid composition and applications, to name a
few. Table 1 summarises the common types of liposomes.

Based on the Circulation Time in vivo

Conventional Liposomes
Most of the early studies on the in vivo disposition of liposomes used
neutral phospholipids like phosphatidylcholine along with varying
amounts of cholesterol and sometimes small percentages of an acidic
phospholipid. Cholesterol was added to increase the stability of
liposomes in the presence of plasma, and the negative charge was added
to avoid aggregation and to increase encapsulation efficiency. These
compositions of liposomes are referred to as the conventional or classical
liposomes. They are recognised by the phagocytic cells of the
reticuloendothelial system and are removed from the circulation rap-
idly. Their half-life decreases with increasing diameter, negative surface
charge and fluidity [3,4].
 Liposomes: Applications in Medicine 5

Table 1. Classification of liposomes.

Types of Liposomes
According to size
Small unilamellar vesicles
Large unilamellar vesicles
Large multilamellar liposomes
According to circulation in vivo
Classical or conventional liposomes
Sterically stabilised liposomes
According to lamellarity
Unilamellar
Multilamellar
According to application
Diagnostic
Therapeutic
According to surface charge
Cationic/Liposomal DNA vector
Anionic
Neutral
Specialised liposomes
Targeted liposome
Immunoliposome
Transferosome
Liposomal DNA vector
LPDI
LPDII

Sterically Stabilised Liposomes

Addition of specific compounds to conventional liposomes caused an
enhancement of their stability in biological fluids and reduced reactivity
toward plasma proteins and cell surface receptors. Such conventional
liposomes that had been stabilised by additive means are referred to as
sterically stabilised liposomes. Most of the initial studies with sterically
stabilised liposomes used either GM1 ganglioside or phosphatidy
inositol (PI) as a stabilising component [5,6]. The introduction of poly-
ethylene glycol as the stabilising component was an important develop-
ment in steric stabilisation, which eventually superseded the use of GM1
or PI for achieving liposomes with long circulation [7,8]. The mechanism
whereby such a polyethylene glycol layer extends the liposome circula-
tion time is due to the ability of the polymer layer to prevent the associa-
tion and binding of opsonins in plasma, thereby inhibiting the body’s
6 R. BANERJEE

molecular recognition processes from labelling the liposomes as foreign

for subsequent uptake and removal by the cells of the reticuloendothelial
system, principally Kupffer cells in the liver. The polymer layer also
exerts a normal and lateral surface pressure that opposes close approach
by macromolecules preventing dissolution of liposomes by lipoprotein
particles in the bloodstream. This is called entropic exclusion [9].
Based on Surface Charge

Cationic Liposomes
As the name suggests, these liposomes have a net positive surface
charge. They are considered separately here due to their importance in
the genetic applications of liposomes. These are also referred to as
liposomal DNA delivery vectors and can also be considered as specialised
liposomes. Liposomes represent one of the safest and potentially most
versatile transfer vectors used to date. The most efficient lipid formula-
tions for in vitro delivery of DNA into cultured cells usually contain a
cationic lipid mixed in approximately equimolar ratios with the mem-
brane-destabilising lipid, dioleoyl phosphatidyl ethanolamine (DOPE).
DOPE is added with the assumption that the fusogenic properties at acid
pH will allow the release of DNA into the cytoplasm from the endosomal
compartment [10].
Specialised Liposomes

Targeted Liposomes
These are liposomes that are directed to recognise and bind to specific
cells in vivo. This can be achieved by incorporating a surface ligand if
there is no decrease in the half-life. When the surface ligand is an anti-
body, it is referred to as an immunoliposome. Attachment of whole anti-
bodies to the surface of classical or conventional liposomes increases
their already rapid clearance from circulation, resulting in circulation
times of only a few minutes [11].
Immunoliposomes that are formed from sterically stabilised
liposomes have circulation half-lives of several hours, which approach
those of sterically stabilised liposomes lacking targeting moieties and
are at least an order of magnitude higher than the half-lives of
immunoliposomes formed from classical liposomes [12].
Papahadjopoulos and Gabizon [13] found that the accumulation of
sterically stabilised liposomes in implanted mouse tumours could be
increased when tumour-recognising antibodies were conjugated to the
liposome surface. The possibility of targeting sterically stabilised
liposomes to specific tissues by the attachment of specific ligands was
 Liposomes: Applications in Medicine 7

demonstrated by Maruyama et al. [14] by using an antibody recognising

lung endothelial cells. Studies using sterically stabilised liposomes with
PEG-PE have indicated that such liposomes may be targeted to specific
cells without losing their property of a long half-life in blood and can
show anti-tumour effects in animal models if injected at an early stage in
tumour development [15].

PHARMACOKINETICS OF LIPOSOMES

Clearance of classical liposomes from the circulation has a biphasic

character—a rapid initial phase leading to 75% disappearance from the
blood in the first five minutes for large unsonicated liposomes and about
50% elimination for the smaller sonicated ones; this is followed by a con-
siderably slower rate of clearance after fifteen minutes for both types of
liposomes [16]. The rapid initial uptake by the liver may be ascribed to
endocytic activity of both Kupffer and endothelial cells of the liver. The
slow second phase of clearance from the blood probably reflects the
uptake of lecithin by hepatocytes mediated by lipoproteins [17,18].
The clearance of classical liposomes increases in direct proportion to
size [19] and presence of surface charge [20,21]. Increasing the dose of
classical liposomes decreases their clearance, presumably due to satura-
tion of the mononuclear phagocyte system uptake. Changes in the com-
position of classical liposomes can affect the clearance. Addition of
cholesterol [22] or the presence of high-phase transition phospholipids
[23] tighten the phospholipid bilayers and can be used to decrease the
clearance.
Circulation half-life is higher for sterically stabilised liposomes than
for classical liposomes at all doses. The circulation times of polyethylene
glycol containing liposomes are the same whether they contain
solid-phase phospholipids or added cholesterol [24,25]. Clearance of
sterically stabilised liposomes is less sensitive to liposome diameter than
is clearance of classical liposomes, with little size dependence between
sizes of 50–250 nm [26]. Due to their long circulation half-lives, there is
considerable uptake of sterically stabilised liposomes into tissues with
enhanced capillary permeability [27,28]. The clearance of
immunoliposomes formed from sterically stabilised liposomes appears
to be dependent on antibody density at the liposome surface. At low sur-
face densities of bound antibody (10–25 antibody molecules per 100 nm
liposome), the immunoliposomes are cleared at rates similar to that of
antibody-free liposomes [29]. At higher antibody densities, immuno-
liposomes are cleared rapidly, similar to those of immunoliposomes
formed from classical liposomes.
8 R. BANERJEE

FACTORS OF CLINICAL RELEVANCE FOR LIPOSOMES

Size

The size of liposomes plays an important role in the clearance of

liposomes—the extent of its importance differing for classical and
sterically stabilised liposomes. There is a strong size dependence in the
clearance of classical liposomes following intravenous administration,
as the liposome diameter increases the rate at which the liposomes are
removed from blood into the reticuloendothelial system also increases
[30].
In case of sterically stabilised liposomes, for liposomes up to 250 nm
diameter, uptake into the liver occurs slowly, and liposomes are found
primarily in liver Kupffer cells. Above 300 nm, increased uptake of
sterically stabilised liposomes into the spleen occurs, apparently by a
passive filtration mechanism with little or no increased liver uptake.
The ability to produce liposomes of controlled size distribution was ini-
tially obtained by sonication [31] and more recently by extrusion [32],
and it has provided control of circulation time and enhanced
extravasation.

Surface Charge

Positively or negatively charged liposomes are cleared more rapidly

than neutral classical liposomes. Jonah et al. [33] found the splenic
uptake of large negatively charged liposomes containing [14C] EDTA to
be approximately twofold greater than for positively charged liposomes.
Uptake by brain and lung tissue displayed a two- to fourfold preference
for positive as compared to negative liposomes. In contrast, Kimelberg
[34] found that small sonicated liposomes containing [3H] methotrexate
were preferentially present in the brain, spleen and bone marrow in case
of positive liposomes and in the lung for negative liposomes.
The formation of multilamellar liposomes and their ability to entrap
enzymes or solutes in the aqueous spaces between the lamellae depends
upon the capacity of the phospholipids to swell and form hydrated liquid
crystals consisting of a series of concentric bilayers that alternate with
aqueous compartments. The volume of these compartments, within
which enzymes may be entrapped, is determined by the net charge of the
lipids and the ionic strength of the swelling solution [35,36]. An increase
in the net anionic surface charge on the lamellae increases the capture of
enzyme as the like-sign repulsion of adjacent lipid layers causes an
increase in the volume of the aqueous compartments.
 Liposomes: Applications in Medicine 9

Cationic liposomes interact with DNA through charge interaction,

and there is an extensive lipid rearrangement during the complexation
of liposomes with DNA [37–40]. There are several advantages of using
cationic liposomes for gene delivery. First, unlike neutral or anionic
liposomes that require entrapment of DNA inside vesicles, cationic
liposomes form complexes with negatively charged DNA via charge
interaction. Second, liposome/DNA complexes are normally prepared in
such a way that the complexes are in slight excess in positive charge.
This allows an efficient interaction of the positively charged complexes
with the negatively charged cell membrane. Third, complexation of
cationic liposomes with DNA may help protect the DNA against physical
forces and enzymatic digestion. However, serum sensitivity is a major
drawback of cationic liposomes. Lipofection relies on a slight excess of
net positive charge of lipid/DNA complexes to efficiently interact with
negatively charged cell membranes. Interaction between liposome/DNA
complexes and anionic molecules in the serum would neutralise the posi-
tive charges and decrease the performance of liposome/DNA complexes.
Xu and Szoka [41] showed that water-soluble molecules with a high neg-
ative charge density such as dextran sulphate and heparin release DNA
from liposome/DNA complexes in the blood by a similar mechanism, ren-
dering DNA more susceptible to enzymatic digestion and decreasing the
efficacy of cationic liposome/DNA complexes.
Phase Transition or Fluidity of Membranes

Liposomes with high phase transition phospholipids (tighter bilayers)

are cleared more slowly than liposomes with fluid bilayers. IgG antibod-
ies shown to bind specifically to spin label lipid hapten III [42] were also
shown to fix complement in the presence of this hapten, presented either
in a DMPC or DPPC bilayer [43]. If complement fixation proceeded at
32°C, midway between the principal phase transition points of DMPC
(23°C) and DPPC (41°C), the amplitude of the peak of the biphasic
response was significantly greater for the case of fluid (DMPC)
liposomes than it was for the solid (DPPC) liposomes. These differences
in behaviour were almost totally obliterated if fixation proceeded at 4°C
rather than at 32°C, both types of liposomes were solid at 4°C and exhib-
ited virtually identical responses. Therefore, the inclusion of label III in
fluid rather than solid bilayers enhances its ability to induce comple-
ment fixation by IgG antibodies.
Lamellarity

Small unilamellar vesicles refer to sizes less than 50 nm having only

10 R. BANERJEE

one bilayer of lipid. Small unilamellar vesicles comprise a single internal

aqueous space enclosed in a single lipid bilayer. Large unilamellar vesi-
cles are produced by calcium-induced fusion of sonicated small
unilamellar vesicles to form large (0.2 to 2 microns in diameter) vesicles
with a single limiting membrane [44]. Multilamellar liposomes have
many bilayers of lipid enclosing the aqueous volume. Although the total
internal aqueous space of multilamellar liposomes is relatively large, the
individual compartments between each lipid lamella are too small to
accommodate large macromolecules. Multilamellar liposomes may be
used as informative model systems for studying entrapment of materials
and monitoring the properties of liposomal systems e.g., susceptibility to
hydrolysis by phospholipases [45]. However, it is relatively more diffi-
cult to produce a homogenous size distribution in multilamellar
liposomes. The trapping of material varies in multilamellar and
unilamellar liposomes. Small unilamellar vesicles are usually spherical
with a minimum surface/volume ratio, whereas multilamellar liposomes
are oblate cylinders and/or spheroids.

Captured Volume

The volume of a liposome is the sum of the volume occupied by the

hydrated lipid and the volume of the internal aqueous phase. However,
captured volume refers only to the amount of aqueous solution
entrapped per lipid. Although hydrating dried lipid films or powders pro-
duce large multilamellar liposomes, the high number of lamellae limits
the aqueous solution capturing efficiency of these liposomes. Another
problem is the phenomenon of solute exclusion [46]. Aqueous addition
allows hydration of surface lipid that organise into bilayers and because
water is more membrane permeable than most solutes, it permeates
through these outer layers to hydrate interior lipid with solute partially
excluded [47]. These problems are overcome by solvent removal tech-
niques like solvent injection method [48], in which lipid is first dissolved
in solvent that is then mixed with the aqueous phase in which the
liposomes are to be suspended at the time solvent is removed. Liposomes
of high captured volume have advantages in applications as slow-releas-
ing drug depositories, carriers of diagnostic imaging agents and as model
systems to study bilayer properties.

Permeability of Liposome Membrane

In diagnostic applications, liposomes with membranes impermeable

to an entrapped marker is advantageous, whereas in therapeutic appli-
 Liposomes: Applications in Medicine 11

cations, it is mandatory that the liposome delivers the drug at the target
site by release through its membranes. The rate of elution of an
entrapped marker from liposomes depends both on the nature of the
enclosed molecule and the lipid bilayer of the liposome. Molecular size
and charge are important characteristics of the entrapped substance in
this regard. Small molecules cross lipid bilayers more rapidly than large
ones, and anions cross more rapidly than cations. Vesicles made from
phosphatidylcholine alone are relatively permeable to 99mTcO4 [49].
Addition of cholesterol reduces this permeability to 99mTcO4, probably
by stiffening the membrane.

CLINICAL APPLICATIONS

Diagnostic

Large liposomes are cleared rapidly via uptake by resident phagocytic

cells of the reticuloendothelial system. This fact can be exploited to our
advantage in passively targeting large liposomes to the phagocytic cells
of the reticuloendothelial system for delivering diagnostic imaging
agents to the liver and spleen. Aqueous contrast-enhancing agents
entrapped in liposomal carriers can be targeted to these organs, and dis-
tinctions can be made between normal and tumorous tissue using com-
puted tomography [50,51]. For improved resolution in the case of
iodinating agents in CT imaging, liposomes with higher captured vol-
ume having higher iodine/lipid ratio are preferred. Various studies using
iodine/lipid ratios ranging from 1 [52] to 9 [53] have been described for
successful imaging of the liver and spleen.
Apart from aqueous contrast filled liposomes, liposomes which encap-
sulate gas are also of importance for diagnostic purposes. Such gas filled
liposomes are used for diagnostic ultrasound and magnetic resonance
imaging. The principle of the effectiveness of such liposomes is due to
their differing magnetic susceptibility and ability to reflect sound well.
The lipids chosen for such liposomes usually have surface active proper-
ties to help stabilise the liposome. DPPC/DSPC encapsulating gas have
been used with success for echocardiography by Adzamli et al. [54].
Simon et al. [55] have used similar air trapped liposomes for
neurosonography.

Therapeutic Applications

Localised/Regional Use
Topical application of liposomes has great potential in dermatology.
12 R. BANERJEE

An in vitro study of the percutaneous absorption of hydrocortisone

across human skin demonstrated that a liposomal formulation increased
drug delivery to the viable tissues by eightfold as compared to a commer-
cial pharmaceutical non-liposomal formulation [56]. The ratio of drug to
lipid recovered from the epidermis and dermis was higher than in the ini-
tial starting material suggesting that intact liposomes did not diffuse
through the stratum corneum. Masini et al. [57] found that
percutaneous absorption (quantity present in the epidermis, dermis and
receptor fluid) of retinoic acid in vitro using skin of hairless rats was
higher for a liposome formulation than for an alcoholic gel. The liposome
formulation decreased the lag time for release of retinoic acid from the
skin surface.
Wolf et al. [58] have studied the delivery of a liposome-encapsulated
DNA repair enzyme, T4 endonuclease V, in pH-sensitive liposomes com-
posed of phosphatidylcholine, phosphatidylethanolamine, oleic acid and
cholesteryl hemisuccinate (2:2:1:5 molar ratio). Liposome encapsulation
enhanced the delivery of T4 endonuclease across mouse skin in vivo lead-
ing to increased protection against UV radiation. This reduced the inci-
dence of skin cancer in a UV-induced mouse model and prevented
systemic immunosuppression of contact and delayed type of hypersensi-
tivity.
Clinical studies are being conducted to study the efficiency of use of
liposomes with interferon for viral lesions. Utility of a topical formula-
tion of interferon is being studied for the treatment of herpes simplex
infections [59] and warts [60]. Takeuchi et al. [61] studied the
antifibrogenic effects of liposomes-encapsulated interferons on skin
wounds.
Cevc and Blume [62] developed novel liposome delivery systems
termed transferosomes which are readily deformable and can penetrate
across the stratum corneum in response to osmotic gradients.
Liposomes can passively deliver bioactive compounds to hair follicles.
Topical delivery of plasmid DNA to keratinocytes would raise the possi-
bility of obtaining controlled gene expression in localised tissues. This
may be very beneficial for the design of vaccines and the treatment of
cancer [63]. Liposome-entrapped melanins, proteins, genes and small
molecules have been selectively targeted to the hair follicle and hair
shafts of mice. Liposomal delivery of these molecules is time dependent.
Negligible amounts of delivered molecules enter the dermis, epidermis
or bloodstream, thereby demonstrating selective follicle delivery. Naked
molecules are trapped in the stratum corneum and are unable to enter
the follicle. Liposomes thus have high potential in selective hair follicle
targeting of large and small molecules, including genes, opening the field
 Liposomes: Applications in Medicine 13

of gene therapy and other molecular therapy to restore hair growth,

physiologically restore or alter hair pigment and to prevent or accelerate
hair loss [64].
For ocular delivery, achieving greater adhesion is paramount because
of the constant washing process by tear flow. Drug corneal retention
time is dependent upon the chain length of phosphatidylcholine lipid
used with greater retention times for longer chain lipids. Corneal
allograft rejection in rats could be prevented by subconjunctival injec-
tions of liposomes containing dichloromethylene diphosphonate [65].

Systemic Applications

ANTICANCER THERAPY
Liposomes have been used to deliver anticancer agents in order to
reduce the toxic effects of the drugs when given alone or to increase the
circulation time and effectiveness of the drugs. Uziely et al. [66] utilised
a formulation of doxorubicin encapsulated in polyethylene glycol coated
liposomes (Doxil®, Liposome Technology Inc., Menlo Park, CA) which
improved the pharmacokinetics of free doxorubicin. This formulation
has been used as an alternative for standard therapy in Kaposi’s sarcoma
(the most common malignancy in patients infected with human immu-
nodeficiency virus). Northfelt et al. [67] treated fifty-three patients with
advanced Kaposi’s sarcoma refractory to standard therapy, with Doxil®.
One patient (2%) had a complete response whereas a partial response
was observed in 19 patients (36%).
Stage IV breast cancer patients also benefited from the treatment
with liposomal doxorubicin according to a study by Ranson et al. [68].
Complete and partial responses were obtained in 6% and 25% respec-
tively. Muggia et al. [69] found that liposomal doxorubicin had substan-
tial activity against refractory ovarian cancer.

ANTIMICROBIAL THERAPY
Liposomes have been used to administer drugs in infective conditions
like malaria and leishmaniasis. Alving et al. [70] found that liposomes
containing neutral glycolipids with a terminal glucose or galactose, on
intravenous injection, prevented the appearance of erythrocytic forms of
Plasmodium bergei in mice previously injected with sporozoites.
Liposome encapsulation of the aminoglycosides—amikacin and
gentamicin has significantly enhanced the activity of these antibiotics in
the treatment of intracellular mycobacterial infections [71].
14 R. BANERJEE

TREATMENT OF RESPIRATORY DISORDERS

Aerosolised liposomes can be used for inhalation therapy and is effec-
tive in relieving bronchial constriction in asthma. Treatment of adult
respiratory distress syndrome with liposomal prostaglandin E1
(Lip-PGE1) injected intravenously, decreased lung leak and lung lavage
neutrophil accumulation in rat models [72]. Acute lung injury in rats
was decreased by intratracheal administration of a liposomal
alpha-tochopherol [73].

ANTIARTHRITIS THERAPY
Shaw et al. [74] found that cortisol palmitate containing liposomes are
stable in rheumatoid synovial fluid at 37°C. The level of liposomal ste-
roid in the tissue was inversely related to the chronicity of inflammation.
Shinozawa et al. [75] compared the absorption and distribution of
liposome-entrapped prednisolone injected into the hip muscle of rats
with that of the free steroid. Liposomal prednisolone was found to be
retained by the injected tissue for longer periods of time.
Liposome-encapsulated aurothiomalate was found to reduce colla-
gen-induced arthritis in mice [76].

AS AN OXYGEN-CARRYING FLUID
From the original concept of encapsulating hemoglobin in an inert
shell, liposome-encapsulated hemoglobin (LEH) has evolved into a fluid
proven to carry oxygen, capable of surviving for reasonable periods in
the circulation and amenable to large-scale production. The formula for
the outer shell evolved from synthetic, non-lipid materials to egg-leci-
thin-based lipid mixtures to distearoyl-phosphatidylcholine-based
blends. In vivo studies have established that LEH has a circulation
half-life of 16–20 hours and can carry oxygen sufficient to sustain life.
Improvements of the LEH to increase its biocompatibility include carbo-
hydrate moiety addition as carbohydrates are expressed on the majority
of biological membranes including the red blood cell. It has been demon-
strated that inclusion of gangliosides into the liposomal bilayer results
in increased circulation times [77,78].

TARGETED LIPOSOMES
Liposomes are used to target specific cells by attaching amino acid
fragments such as antibodies or proteins or appropriate fragments that
target specific receptor sites. Targeted sterically stabilised liposomes
have an increased therapeutic efficiency against human breast cancer
xenographs in nude mice as compared to non-targeted liposomes [79].
On the basis of their characteristics of enhanced penetration, protec-
 Liposomes: Applications in Medicine 15

tion of associated agents and ability to transfer macromolecules across

the cell membrane, liposomes are being currently studied as delivery
vehicles for genes and oligonucleotides [80].

TOXICOLOGY

One of the well-established goals of encapsulating or complexing

drugs with lipids is to reduce acute toxicity. This has been demonstrated
for several classes of highly toxic drugs where lethality after a single dose
can be substantially reduced by lipid-based technology. The
toxicokinetic parameters of liposomal drugs differ from those of the
active drug substance, potentially leading to unique toxicity and efficacy
profiles. Liposomes are frequently taken up by the reticuloendothelial
system and may remain detectable in these tissues for prolonged periods
of time. New toxicities have been described with liposomal formulations
of doxorubicin-palmar-plantar erythrodysesthesia (hand-foot syn-
drome). This is similar to the effect of a continuous infusion of the con-
ventional drug, i.e., the slow release of doxorubicin from the liposomal
formulation mimics a continuous infusion [81].

Recent Developments and Future Perspectives

Cationic Liposome-Entrapped Polycation Condensed DNA (LPDI)

Polycations such as poly-L-lysine are known to efficiently condense
DNA. The binding of DNA to a polycation is stoichiometric, and the con-
densation process is highly cooperative. The LPDI particles have a
higher performance than the corresponding liposome/DNA complexes.
Unlike the liposome/DNA complexes, which are large and heteroge-
neous in size, LPDI particles are highly compact with a size ≤100 nm.
These particles would be more favorable for entering cells via an
endocytosis pathway. Also, LPDI particles offer better protection of
DNA from enzymatic digestion [82].

Anionic Liposome-Entrapped Polycation Condensed DNA (LPDII)

Structurally, these particles are similar to LPDI. Both contain a
highly condensed core of polylysine and DNA and a lipid shell. DNA is
first condensed with polylysine with the positive charge in excess. The
resulting cationic complex is then mixed with anionic liposomes carrying
a targeting ligand. Folate is commonly chosen as a targeting ligand
because many tumors, especially ovarian carcinoma, over-express folate
receptors. The advantages of LPDII are that besides being highly com-
pact, LPDII preparations do not require purification and are single vial
16 R. BANERJEE

formulations [83]. Compared with the traditional anionic and neutral

liposomal vectors, DNA is highly condensed in LPDII and is quantita-
tively encapsulated without the requirement of using excess amounts of
lipids. Finally, because anionic liposomes are relatively compatible with
biological fluids, LPDII can be potentially used for tissue-specific gene
delivery.
Liposomal research has come a long way since the initial discovery of
liposomes three decades ago. Liposomal pharmaceuticals have found
their place in clinical practice, and the number of their indications are
increasing by the day. Future applications will depend on the proper bio-
chemical and biophysical manipulation of liposomal lipids to modify
their interactions with biological fluids. DNA vaccination and improved
efficiency of gene therapy are just a few of the upcoming applications of
liposomes. It is clear that presently we have discovered only the tip of the
iceberg in the world of liposome research.

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