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Recent Advances in Liposomal Drug Delivery: A Review

July 2015
Pharmaceutical Nanotechnology 3(1):35-55

Authors:

Shri Vishnu College of Pharmacy

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.

Self-assembled phospholipid bilayer structure of a liposome.

…

. List of approved (marketed) liposomal drug products.

…

. List of liposomal drug products in clinical development.

…

Examples of liposomal based protein and peptide drug delivery and preparation methods.

…

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Pharmaceutical Nanotechnology, 2015, 3, 000-000 1

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

Pradesh, India

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

targeting.

INTRODUCTION

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 [8]. 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;

E-mail: vnekkanti@westernu.edu

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 [12]. Aqueous drugs can be entrapped

in aqueous space; whereas poorly soluble drugs can

be accommodated within the phospholipid bilayers

[13]. 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

V. Nekkanti

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

PREPARATION

Phospholipids

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 [16].

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

delivery [20].

Table 1. List of commonly used phospholipids and

their phase transition temperatures (Tc).

Phospholipids

Tc (°C)

Soybean phosphatidylcholine (SPC)

-20 to-30

Hydrogenated soybean phosphatidylcholine (HSPC)

Egg sphingomyelin (ESM)

Egg phosphatidylcholine (EPC)

-5 to-15

Dimyristoyl phosphatidylcholine (DMPC)

Dipalmitoyl phosphatidylcholine (DPPC)

Dioleoyl phosphatidylcholine (DOPC)

-22

Distearoyl phosphatidylcholine (DSPC)

Dimyristoyl phosphatidylglycerol (DMPG)

Dipalmitoyl phosphatidylglycerol (DPPG)

Dioleoyl phosphatidylglycerol (DOPG)

- 18

Distearoyl phosphatidylglycerol (DSPG)

Dimyristoyl phosphatidylethanolamine (DMPE)

Dipalmitoyl phosphatidylethanolamine (DPPE)

Dioleoyl phosphatidylethanolamine (DOPE)

- 16

Dimyristoyl phosphatidylserine (DMPS)

Dipalmitoyl phosphatidylserine (DPPS)

Dioleoyl phosphatidylserine (DOPS)

-10

Bilayer Additives

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 [21]. 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 [22]. 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 [23]. 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 [24].

Preparation of Liposomes

In 1965, Bangham and coworkers have

prepared the MLVs (first type of liposomes) by

thin film hydration technique [12]. 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 [27].

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 [28]. By using high energy transfer

capability of ultrasonic waves, MLVs of any size

can be converted to SUVs [29]. 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 [32].

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 [4]. LUVs may have sizes more than 100

nm up to few micrometers (µm) [33]. 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 [34].

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

vesicle [23].

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

[35]. 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 [38]. 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 [23].

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].

Vesicle Size

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.

Encapsulation Efficiency

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) [50]. 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.

Application

Examples

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

Actinomycin-D

Reduced toxicity

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 [41]. 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 [51]. 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 [52]. 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 [53]. 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 [58].

Zeta Potential

Zeta potential is considered to be one of the

important factors affecting cellular uptake and

drug delivery [59]. 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 [60]. 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 [62].

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.

Chemical Stability

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 [63]. 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 [39]. Various parameters relating to

particle size profiles, rheological profiles, drug

leakage, chemical stability, etc. are monitored to

ascertain the stability of drug product.

Pharmacokinetics

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 [65]. 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 [66] 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 [53]. 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 [67]. 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

[53]. 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)

[67].

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 [71] and increased

permeability from the liposomal bilayers [72]. 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

[77]. 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 [78].

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 [81].

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

year 1995.

The fate of liposomes in the body is dependent

on size of the vesicles [88]. 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 [88].

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 [91]. 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 [92].

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].

Liposome Vaccine

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 [102]. 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

[103]. 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 [104].

Immunoliposomes

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.

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 [108].

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 [109].

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 [110].

Delivery of contents might be attributed due to

membrane fusion of the cationic liposomes with

simultaneous avoidance of nucleolous and

lysosomal degradation of DNA [111]. 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

hepatocytes [112].

Some of the most commonly used cationic

liposomal formulations include, N-[1-dioleyloxy)

propyl]-N,N,N-trimethylammonium (DOTMA)

and dioleoylphophati-dylethanolamine (DOPE) in

a 1:1 phospholipid mixture (Lipofectin®) and 2,3-

dioleyloxy-N-[2(sperminecarboxamido) ethyl]-N,N-

dimethyl-1-propanammonium trifluoroacetate

(DOSPA or LipofectAMINE®) from Invitrogen

Corporation, USA. 1,2-bis(oleoyloxy)-3-

(trimethylammonio) propane (DOTAP), 1,2-

dimystyloxypropyl-3-dimethylhydroxyethyl

ammonium bromide (DMRE), 3β[N-(N',N'-

dimethylaminoethane) -carbamoyl] cholesterol

(DC-CHOL), and dioctadecylamino-glycyl-spermine

(DOGS or Transfectam®, Promega Corporation,

USA) [23].

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.

Protein/pepti de

Preparation method

Reference

Adamantyltripeptides

Dry lipid hydration

[129]

Anti-ovalbumin antibodies

Dry lipid hydration

[130]

Basic fibroblast growth factor

Freeze-thawing extrusion

[131]

Bovine serum albumin

Double emulsification

[132]

Calcitonin

Dry lipid hydration

[133]

Enkephalin

Double emulsification

[134]

Epidermal growth factor receptor

Freeze-thawing extrusion

[135]

Hemoglobin

Dry lipid hydration extrusion

[136]

Horseadish peroxidase

Extrusion

[137]

Human gamma-globulin

Dehydration-rehydration

[138]

Insulin

Reverse phase evaporation

[139]

Leishmania antigen

Freeze-thawing extrusion

[140]

Leridistim

Double emulsification

[141]

Leuprolide

Dry lipid hydration

[142]

Nerve growth factor

Reverse phase evaporation

[143]

Octreotide

Double emulsification

[134]

Progenipoietin

Double emulsification

[144]

Superoxide dismutase

Dry lipid hydration

[51]

TATa peptide

Extrusion

[145]

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 [113]. 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 [116]. 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

[120-122].

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 [123]. 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 [123]. 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 [124]. Recombinant human factor

FVIII (rFVIII) when formulated in to PEGylated

liposome, demonstrated prolonged circulation time

and enhanced clotting in hemophilic and non-

hemophilic mice [125].

Liposomes for Diagnostics and Therapeutic

Applications

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 [126]. 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.

Product

Drug

Company

Indication target

AtragenTM

Tretinoin

Aronex Pharmaceuticals Inc.

Acute promyelocytic leukemia

Amphotec

Amphotericin B

Sequus Pharmaceuticals Inc.

Fungal infections leishmaniasis

AmbisomeTM

Amphotericin B

NeXstar Pharmaceuticals Inc.

Serious fungal infections

AmphocilTM

Amphotericin B

Sequus Pharmaceuticals Inc.

Serious fungal infections

AbelcetTM

Amphotericin B

The Liposome Company.

Serious fungal infections

ALECTM

Dry protein free powder of DPPC-

Britannia Pharm, UK

Expanding lung diseases in infants

Avian retrovirus vaccine

Killed avian retrovirus

Vineland lab, USA

Chicken pox

DaunoXomeTM

Daunorubicin citrate

NeXstar Pharmaceuticals Inc., USA

Kaposi sarcoma in AIDS

DepoDur

Morphine

Pacira Pharmaceuticals Inc

Post-surgical pain reliever

Daunoxome

Daunorubicin citrate

Galen Ltd

Kaposi sarcoma in AIDS

Depocyt

Cytarabine

Pacira Pharmaceuticals Inc

Treatment of lymphomatous

meningitis

Doxil

Doxorubicin

Sequus Pharmaceuticals Inc.

Kaposi sarcoma in AIDS

Estrasorb

estradiol

Novavax

Menopausal Therapy

EvacetTM

Doxorubicin

The liposome company, USA

Metastatic breast cancer

Epaxal –Berna Vaccine

Inactivated hepatitis-A Virions

Swiss serum & vaccine institute,

Switzerland

Hepatitis A

Fungizone

Amphotericin B

Bristol-Myers Squibb, Netherland

Serious fungal infections

Mikasome®

Amikacin

NeXstar Pharmaceuticals Inc.

Bacterial infection

NyotranTM

Nystatin

Aronex Pharmaceuticals Inc.

Systemic fungal infections

Topex Br

Terbutaline sulphate

Ozone Pharmaceuticals Ltd.

Asthma

Ventus

Prostoglandin-E1

The liposome company

Systemic inflammatory disease

VincaXome

Vincristine

NeXstar Pharmaceuticals Inc.

Solid Tumors

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-

PEG(2000) [127].

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

pharmaceuticals.

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 [128]. 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 .

Product

Drug

Indications

Current Status

CPX-351 (Celator)

Cytarabine:daunorubicin

Acute myeloid leukemia

Phase II

CPX-1 (Celator)

Irinotecan HCI:floxuridine

Colorectal cancer

Phase II

Gastric and pancreatic cancer

Phase II

MM-398 (Merrimack)

CPT-11

Glioma and colon cancer

Phase I

MM-302 (Merrimack)

ErbB2/ErbB3-targeted doxorubicin

ErbB2-positive breast cancer

Phase I

MBP-436 (Mebiopharm)

Transferrin-targeted oxaliplatin

Gastric cancer and gastro-esophageal junction

Phase II

Brakiva (Talon)

Topotecan

Relapsed solid tumors

Phase I

Alocrest (Talon)

Vinorelbine

Newly diagnosed or relapsed solid tumors

Phase I

Lipoplatin (Regulon)

Cisplatin

Non-small cell lung cancer

Phase III

L-annamycin (Callisto)

Annamycin

Adult relapsed ALL

Phase I

Primary hepatocellular carcinoma

Phase III

Refractory chest wall breast cancer

Phase II

ThermoDox (Celsion)

Thermosensitive doxorubicin

Colorectal liver metastases

Phase II

Pancreatic cancer

Phase II

Endo-Tag-1 (Medigene)

Cationic liposomal paclitaxel

Triple negative breast cancer

Phase II

ALN-TTR ALN-PCS

siRNA targeting transthyretin (TTR)

TTR amyloidosis

Phase I

Hypercholesterolemia

Phase I

ALN-VSP (Alnylam)

siRNA targeting PCSK9 RNAi targeting

liver cancer

Liver cancer and liver metastases

Phase I

TKM-PLK1 TKM-ApoB (Tekmira)

RNAi targeting polo-like kinase 1

Liver tumors

Phase I

(POLO) RNAi targeting apoB

High levels of LDL cholesterol

Phase I

Stimuvax (Oncothyreon/Merck)

Anti-MUC1 cancer vaccine

Non-small cell lung cancer

Phase III

Nerve block

Phase II

Exparel (Pacira)

Bupivacaine

Epidural

Phase I

Arikace™ (Insmed)

Amikacin

Pseudomonas aeruginosa lung infection and

nontuberculous mycobacterian

Phase III

Lipoquin Pulmaquin® (Aradigm)

Liposomal ciprofloxacin

Non-cystic fibrosis bronchiectasis

Phase III

Table 6. New generation liposomes and their advantages.

Type

Modification

Advantages

Archaeosomes

One or more lipids containing diether linkages

Highly stable liposomes

Niosomes

Non-ionic surfactant and cholesterol

Less susceptible to bile salts

Novasomes

Monoester of polyoxyethylene fatty acids, cholesterol and free fatty acids.

Two to seven bilayer shells

High drug loading

Transfersomes

Lipid supramolecular aggregates

Highly flexible suitable for transdermal delivery

Ethosomes

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….

Type

Modification

Advantages

Virosomes

Lipids surface modified with fusogenic viral envelope proteins

Intracellular delivery of antigens and DNA

Cryptosomes

Phospholipids and polaxamers or PEG

Improved stability

Emulsomes

Internal solid fat core surrounded by phospholipid bilayer

Suitable for encapsulation of hydrophobic drugs

Vesosomes

Multilamellar liposomes

Multidrug formulations are possible

Genosomes

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

active targeting.

CONCLUSION

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.

ACKNOWLEDGEMENTS

Declared none.

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Received: May 05, 2015 Revised: July 04, 2015 Accepted: July 08, 2015

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy

Article

Full-text available

Oct 2022
INT J MOL SCI

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy

Preprint

Full-text available

Sep 2022

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg PE, DSPC, cholesterol, and DSPE-PEG-2000, and the mean size of the RTL was in the range of 114 to 133 nm, as measured by NTA. The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three i.v. injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Aliskiren Hemifumarate Proliposomes for Improved Oral Drug Delivery: Formulation Development, In Vitro and In Vivo Permeability Testing

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Full-text available

Jul 2022
MOLECULES

The objective of this study was to develop proliposomal formulations for a poorly bioavailable drug, aliskiren hemifumarate (AKH). A solvent evaporation method was used to prepare proliposomes using different lipids. The lipids of selection were soy phosphatidylcholine (SPC), dimyristoylphosphatidylcholine (DMPC), and dimyristoylphosphatidylglycerol sodium (DMPG Na), stearylamine, and cholesterol in various ratios. Proliposomes were evaluated for particle size, zeta potential, in vitro drug release, in vitro permeability, and in vivo pharmacokinetics upon hydration with aqueous phase. In vitro drug release studies were conducted in 0.01 N hydrochloric acid using USP type II dissolution apparatus. Parallel artificial membrane permeation assay (PAMPA) and Caco-2 cell line models were used to study the in vitro drug permeation. Male Sprague-Dawley (SD) rats were used to conduct in vivo pharmacokinetic studies. Among different formulations, proliposomes with drug/DMPC/cholesterol/stearylamine in the ratio of 1:5:0.025:0.050 (w/w/w/w) demonstrated the desired particle size, higher zeta potential, and higher encapsulation efficiency. The PAMPA and Caco-2 cell line experiments showed a significantly higher permeability of AKH with proliposomes as compared to pure AKH. In animal studies, the optimized formulation of proliposomes showed significant improvement in the rate and extent of absorption of AKH. Specifically, following a single oral administration, the relative bioavailability of AKH proliposome formulation was 230% when compared to pure AKH suspension.

Pediatrik Rehabilitasyonda Duyu Bütünleme Teorisi

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Mar 2022

Liposomal Dry Powder: Future Prospects In Pulmonary Drug Delivery System

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Apr 2022

Fundamental Aspects of Lipid-Based Excipients in Lipid-Based Product Development

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Apr 2022

Poor aqueous solubility of drugs is still a foremost challenge in pharmaceutical product development. The use of lipids in designing formulations provides an opportunity to enhance the aqueous solubility and consequently bioavailability of drugs. Pre-dissolution of drugs in lipids, surfactants, or mixtures of lipid excipients and surfactants eliminate the dissolution/dissolving step, which is likely to be the rate-limiting factor for oral absorption of poorly water-soluble drugs. In this review, we exhaustively summarize the lipids excipients in relation to their classification, absorption mechanisms, and lipid-based product development. Methodologies utilized for the preparation of solid and semi-solid lipid formulations, applications, phase behaviour, and regulatory perspective of lipid excipients are discussed.

Epigenetic Alterations of Preimplantation Embryo

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Mar 2022

Immunoliposomes: A Targeted Drug Delivery System for Cancer Therapeutics and Vaccination

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Mar 2022

Cancer has become one of the world's most lethal and life-threatening disorders, resulting in many deaths. Drug targeting and managing drug delivery are concepts that are implemented to increase a drug's therapeutic index by enhancing its specificity to particular cells, tissues, or organs, and reducing its action and harmful side effects. Liposomes have proven to be one of the most innovative drug delivery systems in medicine. Immunoliposomes, also known as antibody-coupled liposomes, have gained a lot of attention as a homing device for targeted therapies. Monoclonal antibodies or antibody fragments that combine with liposomes to create immunoliposomes have been considered a leading technique for targeted delivery. Various functionalization strategies are adopted for the non-covalent and covalent binding of monoclonal antibodies and their components to liposomal surfaces, such as thiolation, amide bonds, hydrazone bonds, and electrostatic interactions, hydrophobic interactions, hydrogen bonding, etc. for cancer-specific targeting. This review emphasized an overview of various stimulus-responsive immunoliposomes capable of regulating drug release in response to an exogenous magnetic field, changes in response stimulation.temperature or pH, enzyme concentration, endogenous stimuli, and applications of immunoliposomes in vaccination and cancer therapeutics and endogenous immune

Liposomes as a skin delivery system for cosmeceuticals

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Aug 2022

This chapter explains the science of liposomes, which are closed concentric phospholipid vesicles, of colloidal range, with an aqueous core surrounded by a lipophilic membrane. The mechanism of dermal penetration is discussed. Human skin, due to its large surface area, is suitable for transdermal administration of potent drugs and an alternative to overcome the limitations of conventional drug delivery systems. The transport of drugs occurs mainly through the diffusion process, and hence, the lipid solubility influencing the concentration gradient for the transport plays a critical role. The chapter also focuses on newer technologies for liposomal preparation, such as supercritical fluid technology, dual asymmetric centrifugation, and membrane contactor technology, all of which are gaining more importance for industrial manufacturing. The regulatory requirements for liposomal drug products address the chemistry, manufacturing, and controls (CMC), pharmacokinetics and bioavailability studies, and labeling requirements for submission of NDA and ANDA. The major applications of liposomes include skin and hair care products containing liposomes. These skin care products include moisturizers, sunscreen and UV protection, antiaging lotions, whitening and freckle-removing cosmetics, and antiacne cream. Hair care products include those treating alopecia.

Tailoring the multi-functional properties of phospholipids for simple to complex self-assemblies

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Jul 2022

The unique interfacial properties, huge diversity, and biocompatible nature of phospholipids make them an attractive pharmaceutical excipient. The amphiphilic nature of these molecules offers them the property to self-assemble into distinct structures. The solubility, chemical and structural properties, surface charge, and critical packing parameters of phospholipids play an essential role during formulation design. This review focuses on the relationship between the structural features of a phospholipid molecule and the formation of different lipid-based nanocarrier drug delivery systems. This provides a rationale and guideline for the selection of appropriate phospholipids while designing a drug delivery system. Finally, we refer to relevant recent case studies covering different types of phospholipid-based systems including simple to complex assemblies. Different carriers in the size range of 50 nm to a few microns can be prepared using phospholipids. The carriers can be delivered through oral, intravenous, nasal, dermal, transmucosal, and subcutaneous routes. A wide range of applicability can be achieved by incorporating various hydrophilic and lipophilic additives in the phospholipid bilayer. Advanced research has led to the discovery of phospholipid complexes and cell membrane mimicking lipids. Overall, phospholipids remain a versatile pharmaceutical excipient for drug delivery. They play multiple roles as solubilizer, emulsifier, surfactant, permeation enhancer, coating agent, release modifier, and liposome former.