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Pharmaceutical Nanotechnology, 2015, 3, 000-000 1
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Recent Advances in Liposomal Drug Delivery: A Review
Vijaykumar Nekkanti1*and Sandeep Kalepu2
1College of Pharmaceutical Sciences, Western University of Health Sciences,
Pomona, CA, USA; 2Shri Vishnu College of Pharmacy, Bhimavaram, Andhra
Abstract: Since the discovery of liposomes, the drug delivery technology has made a
tremendous advancement in the field of medicine. Owing to their biocompatibility, efficacy,
targeting ability and improved in vivo performance, liposomes has become popular as
versatile drug carrier systems. Controlled and sustained drug release, lowered systemic
toxicity and improved pharmacokinetic and pharmacodynamic properties of drug are the potential
applications of liposomal drug delivery. Drugs encapsulated in liposomes can be targeted actively and
passively to the tumor specific site with improved efficacy and reduced off-target effects. Development of
multifunctional liposomes for targeting cell organelles, long acting (PEGylated) and with combination of
drugs is a hot-topic of current research. In recent years, the focus of liposomal technology has been on the
combined applications of diagnostics and therapeutics. The present work reviews the liposomal drug delivery
field, summarizes the success of liposomal technology in translation from concept to clinical acceptance and
recent developments in the delivery of anti-fungal, antibiotic, anti-inflammatory and anti-cancer drugs.
Keywords: Liposomes, immunoliposomes, proteins and peptides, vaccines, gene delivery, active
The availability of advanced techniques in drug
discovery and research has led to the identification
of a wide number of therapeutic molecules.
However, most of them are unsuccessful in the
developmental process due to poor correlation
between in vitro and in vivo results [1, 2]. Potent
drugs with a narrow therapeutic index generally
require a targeted or site specific delivery. This
can be achieved by using a carrier mediated drug
delivery system which includes liposomes,
microparticles, nanoparticles, etc. [3-7]. Liposomes
as a drug carrier have gained significant importance
owing to its well established properties contributing
to the delivery of drugs . Liposomes are closed
concentric vesicles of phospholipids in the colloidal
size range of 0.01-5.0 µm. Phospholipids are
amphiphilic molecules, containing hydrophilic and
*Address correspondence to this author at the College of
Pharmaceutical Sciences, Western University of Health
Sciences, 309 E. Second Street, Pomona, CA, 91766, USA;
Tel: (909) 469-6476; Fax: (909) 469-5600;
hydrophobic parts which when dispersed in water,
form into bilayered vesicles, where non polar part
is shielded with polar part [9-11]. Fig. (1) shows
the self-assembled phospholipid bilayer structure
of a liposome.
Liposomes were first reported by A. D.
Bangham in early 1960s, who investigated the
formation of spherical structures when phospholipids
come into contact with water. This discovery has
led to encapsulation of a wide category of drugs in
the phospholipid bilayers for improving therapeutic
performance . Aqueous drugs can be entrapped
in aqueous space; whereas poorly soluble drugs can
be accommodated within the phospholipid bilayers
. Hence, the application of an effective and
safer phospholipids for transport of drugs to a
specific site has showed a path to the development
of liposomal drug delivery system. Literature
reveals various types of liposomes, that are
classified based on number of bilayers, size,
composition and methods of preparation. But most
of them are classified in relation to their size and
2 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
Fig. (1). Self-assembled phospholipid bilayer structure of a liposome.
Fig. (2). Various types of liposomes classified based on the lamellae.
number of lamellae, which include multilamellar
vesicle (MLV) and unilamellar vesicles (ULV).
MLVs consists of two or more bilayers and have a
size range of 0.5-5 µm. These vesicles have a
moderate trapped volume (aqueous volume: lipid)
and are stable on long term storage. On the other
side, ULVs having single lamellae can be grouped
into large unilamellar vesicles (LUV) and small
unilamellar vesicles (SUV). LUVs are greater than
100 nm in size and have a high trapped volume,
Advances in Liposomal Drug Delivery Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 3
whereas SUVs are less than 100 nm in size and
have low trapped volume. SUVs are also
characterized based on their long circulation half-
lives [14, 15]. Various types of liposomes classified
based on the lamellae are depicted in Fig. (2).
MATERIALS USED IN LIPOSOME
A wide range of phospholipids are available for
preparation of liposomes. Phosphatidylcholines can
be extracted from natural sources or prepared by
synthetic and semisynthetic methods .
Phosphatidylcholines differ markedly from other
phospholipids in the orientation of bilayer sheets
with respect to the micellar structures. A variety of
natural and semisynthetic phospholipids are used
in the preparation of liposomes. Phospholipids
tend to undergo a distinctive gel-liquid crystalline
phase transition depending on the presence of acyl
or branched chains at a specific temperature range
[17-19]. A comprehensive list of commonly used
phospholipids and their phase transition temperatures
(Tc) are summarized in Table 1.
The most commonly used natural phospholipids
are egg and soya lecithin, obtained from egg and
soya, respectively. These are frequently used as
principal components in the liposomes for various
applications, due to their low cost, lack of net charge
and chemical inertness. Lipid composition and
surface charge affects the tissue distribution and in
vivo clearance of drug loaded liposomes. Neutral
lipids consist of sphingomyelin or alkyl ether
lecithin analogs in addition to phosphatidylcholine.
Increased resistance of lipids to hydrolysis without
affecting the physical properties of the corresponding
membrane can be achieved by replacing the ester
with ether linkages. A variety of phospholipid
structures can be obtained by combining the polar
head groups (e.g., phosphatidylcholilne (PC),
phsophatidylethanolamine (PE), phosphatidylserine
(PS), phosphatidylglycerol (PG) and phosphatidic
acid (PA)) with various fatty acids chains like
lauric, myristic, palmitic, stearic and oleic acids.
Similarly, cardiolipin (CL) and sphingomyelin
(SM) combined with these fatty acid chains
provide additional lipids for specialized drug
Table 1. List of commonly used phospholipids and
their phase transition temperatures (Tc).
Soybean phosphatidylcholine (SPC)
Hydrogenated soybean phosphatidylcholine (HSPC)
Egg sphingomyelin (ESM)
Egg phosphatidylcholine (EPC)
Dimyristoyl phosphatidylcholine (DMPC)
Dipalmitoyl phosphatidylcholine (DPPC)
Dioleoyl phosphatidylcholine (DOPC)
Distearoyl phosphatidylcholine (DSPC)
Dimyristoyl phosphatidylglycerol (DMPG)
Dipalmitoyl phosphatidylglycerol (DPPG)
Dioleoyl phosphatidylglycerol (DOPG)
Distearoyl phosphatidylglycerol (DSPG)
Dimyristoyl phosphatidylethanolamine (DMPE)
Dipalmitoyl phosphatidylethanolamine (DPPE)
Dioleoyl phosphatidylethanolamine (DOPE)
Dimyristoyl phosphatidylserine (DMPS)
Dipalmitoyl phosphatidylserine (DPPS)
Dioleoyl phosphatidylserine (DOPS)
Liposomes prepared utilizing only phospholipids
are usually not rigid enough, mainly due to low
phase transition temperature, and/or unsaturation
in the fatty alkyl chains, which causes imperfections
in the bilayer packing. Such liposomes leak the
encapsulated drug during storage. In order to
prevent such leakage, one or more bilayer additives
are usually included in the formula composition.
The most commonly used additives are cholesterol
and α-tocopherol . Encapsulation efficiency of
liposomes varies with changes in the phospholipid
bilayer composition. Cholesterol is a major
constituent of natural membranes and its
incorporation into liposome bilayers causes major
changes in their properties. Cholesterol does not,
by itself, form bilayer structures, but it can be
incorporated into phospholipid membranes in high
concentrations. Due to tight packing of the
bilayers, rigidity is increased and permeability to
water soluble molecules is decreased. Thus,
4 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
cholesterol improves retention of hydrophilic
drugs by decreasing the bilayer permeability.
Above the phase transition temperature (Tc),
cholesterol decreases the fluidity for making the
bilayer more ordered . Cholesterol molecule
accommodates/orients itself among the
phospholipid molecules with its tricyclic ring
sandwiched between the first few carbons of the
fatty acyl chains and its hydroxyl group facing
towards water phase . Blood proteins such as
albumin, macroglobulin and m-transferrin have a
propensity to react more easily with cholesterol
devoid liposomes, and tend to destabilize the
vesicle leading to its reduced application as a drug
delivery system .
Preparation of Liposomes
In 1965, Bangham and coworkers have
prepared the MLVs (first type of liposomes) by
thin film hydration technique . In this method,
the phospholipids were dissolved in an organic
solvent in a round bottom flask. The solvent is
then removed using a rotary evaporator to obtain a
thin film of lipids, which is subsequently hydrated
by slow addition of an aqueous buffer with
simultaneous shaking or vortexing. This hydration
results in the formation of liposomes. The amount of
drug being encapsulated in the liposome depends
on various factors like film thickness, lipid
composition, temperature, rate of hydration and
duration of shaking. Based on its solubility, the
drug can either be incorporated in the organic
solvent or in the aqueous buffer [25, 26].
Small unilamellar liposomes (SUV) are
optically clear preparations consisting of single
phospholipid bilayers surrounding aqueous spaces.
SUVs are spherical with a minimum radius of
about 20 nm and have a size up to 100 nm .
The most important feature of SUV is that they are
homogeneous population of liposomes. SUVs are
mostly prepared by sonication (bath/probe) of the
MLVs . By using high energy transfer
capability of ultrasonic waves, MLVs of any size
can be converted to SUVs . Alternatively, they
can also be prepared by solvent injection method
[30, 31]. However, the use of SUVs as drug
carriers is limited due to their low trapped volume,
low encapsulation efficiency and possibility of
spontaneous fusion to form larger vesicles below
the phase transition temperature of the lipid .
Large unilamellar vesicles (LUV) encapsulate a
higher percentage of the initial aqueous phase, and
hence higher aqueous volume to lipid ratios. LUVs
provide several advantages compared to MLVs
and SUVs and therefore are commonly employed
as drug carriers. LUVs have higher encapsulation
efficiency owing to the large volume of their
cavity . LUVs may have sizes more than 100
nm up to few micrometers (µm) . Various
methods like detergent dialysis, ether injection,
reverse phase evaporation techniques and even
extrusion can be used to prepare LUVs. Extrusion
is related to a downsizing technique because in
this process, significantly small sized LUVs are
produced by extruding MLVs through small pore
filters (made up of ceramic or polycarbonate). On
the other side, clogging of the polycarbonate
membrane filters is a major drawback while using
a high concentration of lipid/ cholesterol for
extruding large sized MLVs .
In all these methods, the drug is added to the
organic solvent along with the lipids or to the
aqueous hydrating fluid, depending on its
solubility. These methods entrap the drug
passively due to which large proportion of the
drug remain un-entrapped, and their removal from
the suspension is tedious. Also, after complete
removal of non-entrapped drug, due to osmotic
imbalance (concentration gradient), the entrapped
drug leaks out during storage. Such a problem can
be resolved by active loading of drugs, which
ensures one way passage of drug molecules (from
exterior to interior) by creating a differential
environment in the liposomal dispersion. This
difference in environment helps in achieving a
maximum encapsulation and entrapment efficiency
of almost 100% independent of type and
composition of lipid and size of the bilayered
Many drugs are weakly basic and lipophilic.
Most of these drugs are unionized at neutral pH,
but become charged at acidic pH, and therefore
cannot cross the lipid barrier. This principle was
employed in pH gradient loading of liposomes
. In this method, liposomes are prepared using
buffers of acidic pH. Once liposomes are prepared,
the external medium is changed to high pH buffers
and the drug to be encapsulated is added. Because
of this pH gradient, the un-protonated form of the
drug crosses the liposome membrane and
accumulates inside the liposome. The drug entered
Advances in Liposomal Drug Delivery Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 5
into the liposome becomes protonated, and the
resulting charged species are trapped inside the
liposomes since they are unable to cross the bilayer
[36, 37]. Doxorubicin has been encapsulated up to
95% using the pH-gradient principle. However,
sometimes the gradients are not very stable and the
drug molecules may leak out. Also, if the outside
pH is lowered, re-equilibrium takes place. To
avoid these problems, Lasic and coworkers (1993)
have described a method which causes gelation of
the interior of liposomes. In this method instead of
a pH gradient, an ammonium sulfate gradient is
used. Unlike the pH-gradient technique, the
ammonium sulphate gradient utilizes the
permeability coefficient across the lipid bilayer of
ammonium and sulphate ions . The sulphate
ions aggregate inside the liposome due to its low
solubility leading to a gel like precipitation of
doxorubicin and stabilization of the vesicle. Labile
drugs can be loaded into liposomes using these
gradient loading techniques. Some of the
advantages of liposomal drug delivery include:
improved pharmacokinetics such as increased
circulation half-life and reduced elimination,
increased safety and efficacy, increased stability,
flexibility of active and passive targeting etc .
Various applications of liposomes are summarized
in Table 2.
Characterization of Liposomes
Liposomes differ in their physicochemical
characteristics owing to different methods of
preparation. Therefore, a precise and reproducible
quality control tests are required in order to
characterize and predict the in vitro as well as in
vivo behavior of liposomes [8, 22, 39]. The
parameters for characterizing liposomes can be
grouped into physical (size, morphology, lamellarity
and shape), chemical (potency and purity) and
biological parameters. Some of the important
parameters of liposomes in the product development
process which require a strong quality control
check are discussed below [40, 41].
The size of the vesicle is of primary
consideration, since it determines the in vivo fate
of the drug loaded into liposome [42-46]. Various
methods for determining size and size distribution
can be grouped into (1) microscopy techniques
(optical microscopy, scanning electron microscopy,
negative stain transmission electron microscopy
and cryo-transmission electron microscopy), (2)
hydrodynamic technique (field flow fractionation,
ultracentrifugation and gel permeation), (3)
diffraction and scattering techniques (laser light
scattering, quasi-elastic light scattering and photon
correlation spectroscopy) [47-49]. The size of uni-
modal systems of mean diameter less than 1
micron can be determined using these techniques.
An important factor that governs the
encapsulation efficiency of liposomes is trapped
volume and is usually expressed in µl/mg of total
lipids. SUVs have a trapped volume of 0.5 µl/mg
and LUVs has around 30 µl/mg. Experimental
trapped volume can be determined using 22Na or
14C-inulin (non-permeable radioactive solute
molecules) . Initially the lipid is dispersed in
an aqueous medium containing the solute. This
leads to the formation of bilayered vesicle trapping
the non-permeable radioactive solutes which are
Table 2. Applications of liposomes.
Passive targeting to the cells of the immune system
Vaccines, immunomodulators, antimonials, porphyrins
Systemic or local sustained release
Doxorubicin, cytosine arabinoside, cortisones, vasopressin
Site avoidance mechanism
Doxorubicin and amphotericin B
Site specific targeting
Anti-inflammatory, anti-cancer and anti-infectives
Improved penetration into the target tissues
Corticosteroids, anesthetics and insulin
Improved efficacy and therapeutic index
Taxol and amphotericin B
6 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
then separated by centrifugation, gel filtration or
dialysis. Then the amount of residual radioactivity
per lipid is determined. Alternatively, trapped
volume can also be determined using water soluble
markers like sucrose, 14C or 3H-glucose or 6-
carboxyfluorescein . The encapsulation
efficiency depends on physicochemical properties
of the drug, lipid composition, and charge on the
lipid. Prior studies have indicated that lipid charge
will have significant influence on the drug
entrapment efficiencies. It was found that
positively or negatively charged liposomes
entrapped more drug than neutral liposomes under
the same study conditions . Packing of lipid in
the liposome membranes has a major role on the
physical membrane properties such as
permeability, membrane elasticity, surface charge
and binding properties of proteins . The
presence of cholesterol in liposome formulations
may change the packing of the phospholipids to a
more ordered and rigid membrane and may
stabilize to avoid drug leakage . Several
methods such as centrifree filtration, gel filtration,
mini-column centrifugation, Ficoll density
gradient, protamine aggregation and dialysis were
reported to measure drug encapsulation [54-58].
The choice of method depends on the number of
samples, purpose of separation and cost of
analysis. The centrifree filtration was reported to
be the best method for measuring the encapsulated
drug at concentrations < 5mg/ml. Density gradient
method was ideal for rapid separation of non-
encapsulated drug. In this method, there is no
requirement of sample dilution and accurate
estimation of encapsulation can be obtained. Mini-
column, gel filtration and dialysis techniques are
tedious methods and are not suitable for routine
analysis. Protamine aggregation is economical but
requires prolonged incubation times (16-24 h) and
the test sample cannot be recovered due to
protamine contamination .
Zeta potential is considered to be one of the
important factors affecting cellular uptake and
drug delivery . Neutral-charged liposomes
with tightly packed membranes tend to remain
longer in the circulation and exhibit increased drug
retention, compared to charged systems. Certain
plasma proteins have an affinity for liposomes,
and the affinity is enhanced if the liposome is
charged. In particular, cationic systems are expected
to quickly interact with various components in
systemic circulation and thus having shorter half-
life in vivo . It is also known that anionic
liposomes containing negatively charged lipids such
as phospatidylserine (PS), phosphatidicacid (PA)
and phosphatidylglycerol (PG) are taken up by
macrophages and thus disappear from circulation
in short time [60, 61].
Determination of Lamellarity
The lamellarity determination is essential to
define the structure of liposome and its in vivo
performance. The encapsulation efficiency and
drug release kinetics are significantly influenced
by the number of lipid bilayers of the liposome.
The liposome uptake and intercellular fate are
affected by the lamellarity. The liposomal
lamellarity widely varies based on the choice of
lipids and preparation methods. Liposomal
lamellarity can be measured using 31P NMR,
Cryo-electron microscopy and small angle X-ray
scattering (SAXS) techniques .
In vitro Release
A reproducible in vitro release method with
suitable simulated physiological medium or human
plasma should be established for evaluating the
release of drug from the liposomes. This method is
primarily important for measuring the (a)
liposomal drug product quality, (b) suitability of
the process controls, (c) drug release from the
product over time and (d) effect of minor changes
in the manufacturing process or facility.
Stability of Liposomal Formulations
Stability is an important parameter to be
considered in the application of liposomal drug
delivery. The physical stability and chemical
integrity of a drug molecule depends on the
process of manufacturing and conditions of
storage. The liposomal stability governs the
therapeutic activity of the drug molecules. The
physical stability of liposomes is connected with
size, size distribution, morphology and drug
retention, whereas chemical stability is concerned
with oxidation and hydrolysis of phospholipids, as
well as drug degradation. A stable liposomal drug
product has a qualified physical, chemical and
Advances in Liposomal Drug Delivery Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 7
microbial stability, which provides assurance
concerning the products integrity during its storage
period. Hence, a defined protocol for stability
studies marks its importance in ascertaining the
product’s physical and chemical integrity having a
well-established manufacturing, characterization,
efficacy and stability testing methods.
Phospholipids are vulnerable to hydrolysis (due
to the formation of lysophospholipid) leading to
chemical instability. Moreover, phospholipids
containing unsaturated fatty acids are prone to
oxidative reactions. In some instances, the bilayer
lipids and drug itself might also play a role in the
stability of liposomal drug product. For example
unprotected cholesterol is prone to rapid oxidation
in an aqueous dispersion. Free (un-entrapped) drug
has a different stability profile, when compared to
its encapsulated/entrapped state. One of the
strategies to protect the drug from biological
environment is to encapsulate within a liposome.
One such example is the encapsulation of insulin
in the liposomes so as to protect from the
proteolytic enzymes of gastrointestinal tract.
Liposomes of different sizes are formed when
phospholipids are hydrated in water. In order to
attain a thermodynamically stable state, these
vesicles tend to aggregate leading to an increase in
their size. The physical stability of liposomal drug
product also gets affected due to fusion and
breakup of vesicles. Light scattering and electron
microscopic techniques can be used to monitor the
size, shape and morphology of the vesicles having
a control on stability of the drug product . The
most commonly encountered problem in evaluating
the stability of disperse systems is to estimate the
shelf life of a drug product. However, there are no
standardized tests available for stability determination
and in many instances the investigation is carried
out on case-by-case basis using stability testing
protocol . Various parameters relating to
particle size profiles, rheological profiles, drug
leakage, chemical stability, etc. are monitored to
ascertain the stability of drug product.
The behavior of drug loaded liposomes in vivo
is mainly governed by various pharmacokinetic
parameters like absorption, distribution and
elimination of the vesicles. Fixed tissue macrophages
in the liver, spleen and bone marrow are the major
sites of potential access by liposomes after
intravenous administration [7, 64]. Large
liposomes (> 0.5 µm diameter) are taken up by
phagocytosis of fixed tissue macrophages and
blood monocytes. For small liposomes (< 0.1 µm),
the pathway of phagocytosis by phagocytic cells
and the uptake by liver parenchymal cells are
involved in the elimination of these liposomes
from blood . Liposomal pharmacokinetic
studies performed by intravenous administration
revealed their rapid clearance from blood mainly
by liver and spleen. The lipid composition also has
a role in the tissue/bio-distribution and blood
clearance. The fate of liposomes is governed by
the surface charge, presence of specific ligand on
the surface, binding properties of proteins  and
liposomal membrane permeability to entrapped
markers. Protein opsonization on to the surface of
neutrally charged liposomes is minimum due to
their tightly packed and rigid membrane, which
facilitate in retention of the drug . Anionic
liposomes composed of negatively charged lipids
like phosphatidylglycerol, phosphatidylserine and
phosphatidic acid have a reduced circulation time
due to their uptake by macrophages [13, 61].
Negatively charged small liposomes are cleared
more rapidly than its counter parts neutral and
positively charged liposomes . Furthermore, a
biphasic pattern of clearance is observed with the
negative small liposomes. On the other hand,
blood monocytes and lungs play a major role in
the uptake of negatively charged large liposomes
compared to neutral and positively charged
liposomes. Surface modified liposomes (carrying
ligand) are more prone to clearance than that of the
native liposomes. However, liposome uptake by
the liver can be reduced to some extent by
incorporating cholesterol, which may change the
phospholipids packing to a more rigid and ordered
membrane. Moreover, this may improve the
stability of liposomes by avoiding drug leakage
and increase the retention of liposomes in vivo
. Site-specific or targeted delivery of
liposomes can be more advantageous in terms of
systemic availability of the drug. Using targeted
delivery, a substantial amount of drug can be made
available at the specific site when compared to the
drug concentration in other tissues. The amount
and velocity of liposomal entrapped drug available
to the target tissue directs the ultimate bioavailability
of the drug. Hence the onset, duration and degree
8 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
of action depend on the rate and extent of drug
release from liposomes at the target site (tissue)
Limitations of Liposomes
Liposomes have shown a great potential as
carrier based drug delivery system. However,
some of the limitations associated with the
development of liposomes are product stability,
low drug entrapment, short circulation half-life of
liposomes, vesicle size and high production costs
etc. The stability of the liposomes is majorly
governed by the final composition of the liposomal
formulation. Physical instability occurs predominantly
due to oxidation of unsaturated acyl chains or
hydrolysis of ester bonds, drug leaking and
aggregation of vesicles resulting in bigger
liposomes which are rapidly cleared by Reticulo-
endothelial system (RES). Some of these stability
issues can be addressed by freeze drying the
liposomal suspension in to powder formulation
using cryoprotectant (ex., AmBisomeTM). During
long term storage, the liposomal products may
undergo chemical hydrolysis of ester
glycerophospholipids to free fatty acids,
lysophopholipids and glycerophospho compounds
[68-70]. These hydrolysis products may result in
increased particle size  and increased
permeability from the liposomal bilayers . The
extent of encapsulation efficiency is generally
higher for non-polar drugs as compared to polar
drugs. Non-polar drug such as daunomycin has
shown higher encapsulation efficiency compared
to polar drug cytosine arabinoside [73, 74]. The
pH sensitive liposomes requisites stability of
vesicles in systemic circulation following parenteral
administration. However, various components of
blood act as potential destabilizers including
lipoproteins [75, 76]. Albumin was reported to
destabilize dioleoyl phophotidyl ethanolamine/oleic
acid liposomes due to extraction of oleic acid from
the membrane resulting in leakage of its contents
. Stability of the liposomes can be improved
by preventing oxidation, aggregation, and fusion
by preparing and storing the liposomes in an inert
atmosphere and by using antioxidants .
Liposomes for Fungal Treatment
A polyene antibiotic, Amphotericin B (AmpB)
is used to treat fungal infections. It binds to sterols
in fungal and mammalian membranes resulting in
the formation of transmembrane pores which
allow the leakage of vital intracellular components
leading to cell death. AmpB has a degree of
selectivity for fungal membranes due to their
ergosterol content, however, binding to the
cholesterol-containing mammalian membranes
results in toxicity. Nephrotoxicity was reported in
the systemic use of AmpB and often resulted in
central nervous system side effects on chronic use.
Studies revealed the effectiveness of liposomal
Amphotericin B (L-AmpB) in experimental fungi
and parasitic diseases [79, 80]. Moreover, L-
AmpB is less toxic to mammalian cells when
compared to free AmpB and hence established to
be superior for the treatment of systemic fungal
infections. A reduced nephrotoxicity due to
minimized accumulation in the kidney and a
biphasic pattern of clearance was observed with L-
AmpB on intravenous administration .
Liposomes for Cancer Treatment
Liposomes are the first pharmaceutical drug
products based on nanotechnology applications
that are approved for cancer and other therapeutic
applications. Increased levels of protein binding is
observed with positively and negatively charged
liposome as well as those containing unsaturated
lipids [82, 83]. Literature revealed that coating of
liposomes with polyethylene glycol could prolong
their circulation time in the systemic circulation
and can be considered as a major breakthrough in
drug delivery research. The coated poly ethylene
glycol (PEG) acts as a steric barrier by forming a
protective layer over the liposomes due to which
the vesicle escapes its recognition by the opsonins
and phagocytic cells of RES and hence enjoy a
comfortable period in the systemic circulation
before they are being cleared [84-87]. Currently,
these long circulating liposomes are being
explored extensively and their wide use in
biomedical in vitro and in vivo studies has paved a
path into clinical investigation. These PEG coated
liposomes can be referred to as Stealth® or
sterically stabilized liposomes, which has a
circulation half-life of greater than 24 h in rats and
even longer in humans. The clearance kinetics of
such type of liposomes is dose dependent, log-
linear and non-saturable. It is worth to note that,
Stealth® technology was used for the formulation
of the anti-cancer drug, doxorubicin. Doxil® was
Advances in Liposomal Drug Delivery Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 9
the first commercial liposomal formulation that is
loaded with the anti-cancer drug, doxorubicin and
was the foremost to be approved by FDA in the
The fate of liposomes in the body is dependent
on size of the vesicles . Literature suggests a
vesicle size of less than 200 nm can escape from
the physical liver screening process. However,
depending on the size of liver sinusoids, a vesicle
of size less than 150 nm is required for penetrating
into the malignant tissue through highly permeable
tumor blood vessels. It is therefore governed by
the enhanced permeation rate (EPR) effect, which
helps the liposome to accumulate in the tumor by
passive targeting. Vasculature differences between
healthy and tumor tissue enables the EPR effect.
Blood vessels in turn have large gaps in the cells
due to less perfect cellular packing leading to more
leaky nature as depicted in Fig. (3). Hence,
liposomes accumulate in the tumor via passive
targeting effect by escaping vasculature [83, 89,
90]. Passive targeting to several different tumors is
directed by the size and stability of liposomes in
vivo. This can be attributed to their prolonged
circulation times and extravasation in tissues
owing to their small size. Hence, considering the
reported data from various pharmacological
studies of liposomes, it can be concluded that
smaller liposomes have more chances of escaping
from the non-specific uptake by RES system .
Doxorubicin hydrochloride (C27H29NO11 HCl,
molecular weight 579.99) is a cytotoxic
anthracycline antibiotic. At cumulative doses of
550 mg/m2, doxorubicin had shown a 7.5 %
incidence of clinical cardiomyopathy, indicating
dose dependent cardio toxicity. When a close
clinical follow-up including serial determination of
left ventricular ejection fractions was made, the
incidence rose to 20%. Hence, a general dose of
450-500 mg/m2 was set up for treatments
employing free doxorubicin that is given by bolus
every three weeks. Therefore, to reduce the
toxicity and improve pharmacokinetics, doxorubicin
was encapsulated into the liposomes. Doxil® is a
formulation containing doxorubicin in a
precipitated form with in the bilayers of sterically
stabilized liposomes. The pharmacokinetics and
mechanism of drug delivery to tumors by Doxil®
(doxorubicin hydrochloride liposome injection)
and other stealth liposome preparations are
significantly different from conventional
chemotherapy. The encapsulated drug demonstrated
1000 fold increase in AUC with profound decrease
in clearance and distribution . The half-life of
the encapsulated drug was also increased
remarkably i.e., to 60-80 h. The important aspect
enabling Doxil® to reach the target tissues with
prolonged circulation can be attributed to the
stable drug retention and circulation half-life of
doxorubicin in the vesicle [92, 93]. The stability of
Stealth® liposomes in plasma helps in transporting
a major fraction of the administered dose in
reaching the tissues, whereas the remaining minimal
amount (5%) of drug leaks from liposomes and
gets distributed in the tissue as a free drug during
circulation. Clinical studies have revealed that, in
patients suffering with breast carcinoma, the
concentration of doxorubicin (Doxil®) in tumor
tissue was 10 fold higher, when compared to
Fig. (3). Passive targeting by enhanced permeation rate (EPR) effect.
10 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
adjacent muscle tissue, signifying an enhanced
drug delivery to these tumors .
Apart from these advantages, Doxil® also
suffers from some disadvantages, which include its
unique toxicity due to accumulation of drug in
large amounts on healthy tissues and mucosa. This
results in a reduced maximal tolerated dose in
comparison with conventionally administered
doxorubicin. Currently, Doxil® is approved for
patients suffering with ovarian cancer and AIDS-
related Kaposi’s sarcoma [94, 95].
Sustained Release Liposomes
Anticancer drugs can be delivered to the
systemic circulation in a sustained release mode by
encapsulating in the liposomes [15, 96]. DepoCyt®
consisting of Cytarabine is first sustained-release
injectable product to reach the market in the year
1999. DepoFoam™, a sustained release injectable
technology of Skye Pharma is applied in
DepoCyt® that is used in the treatment of
lymphoma, i.e. lymphomatous meningitis. Though
Cytarabine can be used for controlling such type
of lymphoma, its short plasma half-life of around
20 min necessitates spinal injection of the drug on
frequent basis, which incurs patient incompliance,
distress and high treatment costs. On the contrary,
the frequency of injection could be reduced to
every 2nd week with the use of DepoCyt®, which
consists of drug encapsulated within the
nonconcentric internal aqueous chambers of
spherical particles. The bilayer lipid membranes
separating the internal chambers are composed of
synthetic analogs of naturally occurring lipids. In
comparison to unentrapped Cytarabine, DepoCyt®
administered through intrathecal route maximized
the therapeutic potential of cytotoxic agents that
are specific to S-phase of cell cycle. In addition,
the dosing frequency could be reduced due to the
prolonged CSF t1/2 of Cytarabine [97-99].
Typically either a purified antigen or an
attenuated pathogen is used as immunogen in a
known vaccine. However, a long term immune
response may not be induced by purified antigens
and even sometimes doesn’t induce a response at
all. On the other hand, attenuated vaccines can
produce a response in patient under immunization.
However, delivering the antigen encapsulated within
a liposome can induce a long term response, which
is not observed in direct immunization with certain
antigens [100, 101]. Studies revealed that cell
membrane of malignant cells can form liposomes
encapsulating potential antigens.
Literature was reported on the ability of
peptides encapsulated in liposomes for their
therapeutic application as cancer vaccine . In
this study, BLP25 (a 25 amino acid sequence
containing synthetic human MUC1 peptide) was
evaluated for its ability to act as a cancer vaccine
. Monophosphoryl lipid A (1% w/w) contained
in distearoylphosphatidylcholine, cholesterol and
dimyristoylphosphatidylglycerol (3:1:0.25 molar
ratios) were used for the preparation of liposomes,
which were then incorporated with lipid
conjugated and non-conjugated MUC1 peptides.
Immunization of C57BL/6 mice was done with
peptide associated liposomes, peptide mixed with
peptide free liposomes and lipopeptide alone. The
results indicated a profound effect of liposomal
formulation on the immune response. A strong
immune response (antigen specific T-cell cell
responses) was observed with physically
associated liposome but not with peptide mixed
with peptide free liposome or lipopeptide alone.
Humoral immune responses were significantly
affected by the nature of association, which could
be justified by the inducing of MUC1-specific
antibodies by the surface exposed peptide
liposomes. Hence, either a preferential cellular
response could be induced by tailoring the
liposomal drug delivery system that gives out a
hypothesis that, different liposomal formulations
stimulate different immune pathways .
Among the various types of liposomes,
immunoliposomes have gained wide attention due
to their targeting capabilities. Due to the presence
of antibodies attached on to their surface, these
liposomes exhibit immunologic response [95,
105]. The preparation of immunoliposomes, i.e.
conjugation of antibodies to liposomes, is not that
straightforward and even can pose a challenge
during their formulation. Protein molecules and
monoclonal antibodies can be conjugated directly
on to the liposome, PEGylated liposome or PEG
chain of the PEGylated liposomes. Similar to other
liposomes, the RES can scavenge and clear the
immunoliposome from systemic circulation
Advances in Liposomal Drug Delivery Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 11
rapidly [106, 107]. Therefore, to prevent the
uptake and increase the circulation half-life,
liposomes are PEGylated (coated with PEGs).
Similarly, conjugation of antibodies on to the
PEGylated liposomes has been reported. However,
the drawback of this delivery system is that it is
hard to conjugate the antibody on to the
PEGylated liposome, where in the high molecular
weight PEG chain cause steric hindrance to the
antibodies to be bound onto the liposomes. In
addition, the targeting capacity of the bound
antibodies is also reduced due to the presence of
the PEG chain. To overcome such problems and to
achieve the desired targeting objective using
antibodies have been conjugated on to the PEG
chain of the PEGylated liposomes. Thus, the dual
benefit of targeting and long circulation could be
achieved. Fig. (4) represents various types of
Immunoliposomes are prepared using various
types of linkage between the antibodies or their
fragments and the lipids/liposomes. Depending
upon the method of preparation, the linkage can be
carried out on the lipid which can then be used to
make the liposome or can be made on to the
liposomes. The commonly used types of linkages
are the covalent and non-covalent conjugation
between the antibodies and lipids. In covalent
conjugation, the amino group (amide bond
formation) or the sulfhydryl groups (maleimide
reaction) are the major active sites for the
conjugation process. Whereas, in non-covalent
conjugation, liposomes prepared with biotin
modified lipids onto which the targeting protein
molecules are attached. Increased circulation half-
life, targeting specificity and minimized drug loss
and degradation are the major advantages of
immunoliposomes. Apart from the promising
applications, immunoliposomes suffer from a
major drawback, i.e. immunogenicity and increased
rate of clearance from circulation can be observed
due to repeated injections. Immunoliposomes of less
than 80 nm (as required for an effective delivery)
may get eliminated rapidly from the tumor site.
Liposomes for Gene Delivery
Various discoveries related to human genomes
and their use in disease treatment has become
more approachable with the advances of science
and technology. In spite of these developments,
choosing a right carrier for the delivery of the gene
to the target is of paramount importance. One such
important carrier is liposomes, which can deliver
DNA, anti-sense oligonucleotides, siRNA and
other potential agents into the nucleus. Specially
engineered liposomes like cationic liposomes, pH
sensitive liposomes, fusogenic liposomes and
genosomes are explored for gene delivery .
Since, a strong negative charge is imparted by
DNA, its transfection of cells becomes a difficult
task. Delivery of DNA into the cell nucleus can be
performed using different methods. They can be
broadly categorized into physical, chemical,
Fig. (4). Various types of immunoliposomes.
12 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
biological and mechanical. Delivery of DNA using
liposomes falls under the chemical category.
Cationic liposomes have been showing promising
results as a carrier for DNA transfection .
However, best transfection can be observed with
the lipids having three major components: a
positively charge head group with which the
negatively charged DNA interacts, a lipid solubility
determining linker group and a hydrophobic group
which helps in anchoring the lipid on to the bilayer
[18, 21]. Lipid DNA complexation is formed by
the electrostatic adsorption of DNA on the lipid
surface owing to the cationic charge .
Delivery of contents might be attributed due to
membrane fusion of the cationic liposomes with
simultaneous avoidance of nucleolous and
lysosomal degradation of DNA . The size of
the liposomes is an important determinant for
clearance by the RES. In this respect, the passage
of the endothelium is the first barrier when
liposomes are used for gene delivery. The most
important target organ for gene transfer is the
liver. The size of the sinusoidal fenestrae is an
important determinant of the efficiency of
different gene transfer vectors. In essence, the
diameter of liposomes should be lower than 80-
100 nm to have a potential for gene transfer into
Some of the most commonly used cationic
liposomal formulations include, N-[1-dioleyloxy)
and dioleoylphophati-dylethanolamine (DOPE) in
a 1:1 phospholipid mixture (Lipofectin®) and 2,3-
(DOSPA or LipofectAMINE®) from Invitrogen
Corporation, USA. 1,2-bis(oleoyloxy)-3-
(trimethylammonio) propane (DOTAP), 1,2-
ammonium bromide (DMRE), 3β[N-(N',N'-
dimethylaminoethane) -carbamoyl] cholesterol
(DC-CHOL), and dioctadecylamino-glycyl-spermine
(DOGS or Transfectam®, Promega Corporation,
Liposomes protect the DNA from degradation
during circulation and promote the delivery of
DNA at the desired site bypassing the cell
Table 3. Examples of liposomal based protein and peptide d rug d elivery and preparation methods.
Dry lipid hydration
Dry lipid hydration
Basic fibroblast growth factor
Bovine serum albumin
Dry lipid hydration
Epidermal growth factor receptor
Dry lipid hydration extrusion
Reverse phase evaporation
Dry lipid hydration
Nerve growth factor
Reverse phase evaporation
Dry lipid hydration
Advances in Liposomal Drug Delivery Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 13
membrane. On the contrary, rapid clearance due to
their cationic charge is the major drawback of
cationic liposomes. Even when compared to
neutral lipids, these are larger in size and pose
toxicity with repeated administration. PEGylation
can be done to these lipids so as to improve the
circulation half-life, however, this may result in a
decreased transfection efficiency . Apart
from these, aggregation and nuclear degradation
can be prevented by the tight condensation of
DNA, which can be achieved by incorporating
cationic polymers (polylysine and protamine) into
cationic liposome-DNA complex. The delivery of
plasmid DNA to tumors, skin, brain and lungs can
be successfully achieved using cationic lipids as
carriers. In vitro transfection mainly uses DNA
entrapped cationic liposomes. The future scope of
this technology along with a detailed discussion
can be available concerning the in vivo behavior of
the cationic liposomal gene delivery in the
literature [114, 115].
Liposomal Antibiotics for Pulmonary Delivery
The systemic delivery of liposomal antibiotics
for the treatment of pulmonary infections has
several limitations such as low accumulation of
drug in the lungs, and possible adverse side effects
to other tissues and organs. Apart from the
invasive intravenous route, the inhalation route is
becoming a more promising for the administration
of antibiotics because of the patient’s convenience
and the possibility of administering the antibiotics
directly into the lungs. Delivery of liposomal
antibiotics directly to lungs enables high
concentrations of drug at the site of infection
potentially overcoming adaptive resistance and
preventing their leakage into the blood stream and
distribution to other healthy tissues or organs
circumventing the potential for cumulative systemic
toxicities . The efficiency of liposomal
antibiotics (aminoglycosides, fluoroquinolones) as
dry powder inhaler, or nebulized aerosol form in
animals with lung infections is certainly
revolutionizing the treatment of pseudomonas
aeruginosa patients [117-119]. These studies have
indicated a valuable path to study the safety,
efficacy or pulmonary deposition of nebulized
liposomal amikacin and inhaled liposomal
ciprofloxacin in clinical trials for the treatment of
cystic fibrosis (CF) and non CF bronchiectasis
Liposomes for Protein and Peptide Delivery
Proteins and peptides are potent therapeutic
agents used in the treatment of various diseases.
However because of their unstable nature and
degradation at physiological conditions the
delivery of these drugs at the targeted site is
extremely complicated . Most of the protein
and peptide drugs produce their mechanism of
action extracellularly by interacting with the
receptors. Encapsulation of proteins and peptides
in to lipid vesicles for improving the therapeutic
properties has been extensively investigated. The
ability of liposomal encapsulated enzymes to
penetrate into the cytoplasm or lysosomes of living
cells is utmost important for treating abnormal
functioning of the intracellular enzymes. The
phospholipids utilized in the liposomal
formulations are generally regarded as safe
(GRAS), biocompatible and offer protection to the
encapsulated drug from inactivation due to fusion
during the storage and handling. A list of protein
and peptide drugs investigated using liposomal
based drug delivery is summarized in Table 3. The
bio distribution of liposomes containing β-
Glucuronidase showed that 50% of the administered
enzyme was found within an hour and 25% in the
liver after 48h . When liposomal based
asparginase was administered to P1534 tumor
induced animals; the survival of animals was
improved by a factor of 2 following the treatment
and generation of antibodies in response to the
enzyme was also found reduced when compared to
free asparginase . Recombinant human factor
FVIII (rFVIII) when formulated in to PEGylated
liposome, demonstrated prolonged circulation time
and enhanced clotting in hemophilic and non-
hemophilic mice .
Liposomes for Diagnostics and Therapeutic
Liposomes are extensively utilized in
diagnostic and therapeutic applications. Liposomes
offer potential advantages in the areas of X-ray
and magnetic resonance imaging, ultrasound and
nuclear medicine because of their high loading
capacity, tolerability and tissue selectivity and are
more preferred when compared to other forms of
tissue-selective contrast media . Medical
imaging entails an appropriate intensity of signal
in order to distinguish certain structures from
14 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
Table 4. List of approved (marketed) liposo mal drug products.
Aronex Pharmaceuticals Inc.
Acute promyelocytic leukemia
Sequus Pharmaceuticals Inc.
Fungal infections leishmaniasis
NeXstar Pharmaceuticals Inc.
Serious fungal infections
Sequus Pharmaceuticals Inc.
Serious fungal infections
The Liposome Company.
Serious fungal infections
Dry protein free powder of DPPC-
Britannia Pharm, UK
Expanding lung diseases in infants
Avian retrovirus vaccine
Killed avian retrovirus
Vineland lab, USA
NeXstar Pharmaceuticals Inc., USA
Kaposi sarcoma in AIDS
Pacira Pharmaceuticals Inc
Post-surgical pain reliever
Kaposi sarcoma in AIDS
Pacira Pharmaceuticals Inc
Treatment of lymphomatous
Sequus Pharmaceuticals Inc.
Kaposi sarcoma in AIDS
The liposome company, USA
Metastatic breast cancer
Epaxal –Berna Vaccine
Inactivated hepatitis-A Virions
Swiss serum & vaccine institute,
Bristol-Myers Squibb, Netherland
Serious fungal infections
NeXstar Pharmaceuticals Inc.
Aronex Pharmaceuticals Inc.
Systemic fungal infections
Ozone Pharmaceuticals Ltd.
The liposome company
Systemic inflammatory disease
NeXstar Pharmaceuticals Inc.
neighboring tissues. Superparamagnetic liposomes
are reported as highly efficient magnetic resonance
imaging (MRI) contrast agents. Example,
maghemite encapsulated liposomes synthesized
from egg PC (phosphatidylcholine) and DSPE-
A number of liposome-based pharmaceutical
products have been approved (Table 4) and many
more are in clinical development (Table 5). The
success of liposome technology has spawned the
growth of a “support industry” including
equipment and excipient suppliers, as well as a
number of biotechnology companies that focus
specifically on the development of liposome-based
New Generation of Liposomes
Liposomal drug delivery has created an
opportunity to formulate a wide variety of difficult
to deliver therapeutic agents. In spite of many
products in the market and several others in the
clinical trials, the instability of a drug during its
transfer to the targeted site is still a problem.
Therefore, to improve the drug stability, efficacy
and to reduce the adverse effects by targeting the
site of action, a new generation of liposomes have
been explored using various phospholipids and
their derivatives . The new generation
liposomes demonstrated considerable advantages
with potential therapeutic benefits (Table 6).
However, still further investigation is needed to
Advances in Liposomal Drug Delivery Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 15
Table 5. List of liposomal drug products in clinical development .
Acute myeloid leukemia
Gastric and pancreatic cancer
Glioma and colon cancer
ErbB2-positive breast cancer
Gastric cancer and gastro-esophageal junction
Relapsed solid tumors
Newly diagnosed or relapsed solid tumors
Non-small cell lung cancer
Adult relapsed ALL
Primary hepatocellular carcinoma
Refractory chest wall breast cancer
Colorectal liver metastases
Cationic liposomal paclitaxel
Triple negative breast cancer
siRNA targeting transthyretin (TTR)
siRNA targeting PCSK9 RNAi targeting
Liver cancer and liver metastases
TKM-PLK1 TKM-ApoB (Tekmira)
RNAi targeting polo-like kinase 1
(POLO) RNAi targeting apoB
High levels of LDL cholesterol
Anti-MUC1 cancer vaccine
Non-small cell lung cancer
Pseudomonas aeruginosa lung infection and
Lipoquin Pulmaquin® (Aradigm)
Non-cystic fibrosis bronchiectasis
Table 6. New generation liposomes and their advantages.
One or more lipids containing diether linkages
Highly stable liposomes
Non-ionic surfactant and cholesterol
Less susceptible to bile salts
Monoester of polyoxyethylene fatty acids, cholesterol and free fatty acids.
Two to seven bilayer shells
High drug loading
Lipid supramolecular aggregates
Highly flexible suitable for transdermal delivery
Phospholipids and alcohol in relatively high concentration
More disruptive in the skin lipid bilayer organization
suitable for transdermal delivery
16 Pharmaceutical Nanotechnology, 2015, Vol. 3, No. 1 Nekkanti and Kalepu
(Table 6) Contd….
Lipids surface modified with fusogenic viral envelope proteins
Intracellular delivery of antigens and DNA
Phospholipids and polaxamers or PEG
Internal solid fat core surrounded by phospholipid bilayer
Suitable for encapsulation of hydrophobic drugs
Multidrug formulations are possible
Complex of cationic phospholipids and a functional gene or DNA
Suitable for gene delivery
overcome the limitations encountered in terms of
long term stability, entrapment efficiency and
Liposomes have been used in many
pharmaceutical applications. Numerous formulation
and characterization techniques were developed to
evaluate liposomes of different sizes, lamellarities,
charge and entrapped volumes. Encapsulation of
drugs into liposomes demonstrated improved
therapeutic performance with concomitant reduction
in toxicity. Decreased volume of distribution, long
circulation half-life and slower drug clearance was
observed with PEGylated liposomes. High
specificity of immunoliposomes has shown
promising applications in targeted drug delivery.
Clinical benefits of using liposomes in conjunction
with drug molecules clearly proved their ability in
drug delivery applications. Moreover, the
integration of liposomal drug delivery with
nanotechnology has an added advantage in the
field of theranostics i.e., therapeutics and
diagnostics. In conclusion, this work summarizes
the potential applications of liposomes and
discusses, in brief the recent advances in the drug
delivery technology. A promising sign is the
growing interest in the development of liposomal
based formulations. Further advancements in
liposomal drug delivery technology will spur the
complete evolution of liposomes as drug carriers.
CONFLICT OF INTEREST
The author(s) confirm that this article content
has no conflict of interest.
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Received: May 05, 2015 Revised: July 04, 2015 Accepted: July 08, 2015