Liposomes in Drug Delivery

Julia Nofadini (1306370972), Teknologi Bioproses

1.1 Liposomes: Brief Overview of Preparation and Properties

Liposomes, phospholipid bubbles with a bilayered membrane structure, are considered as
promising pharmaceutical carriers for different applications. Currently, liposomes are used experimentally
and clinically to deliver various pharmaceuticals including drugs and diagnostic agents as well as genes
and related products. Research in the field of liposomes aims now at the development of various
liposome-based multifunctional nanopreparations for therapy and diagnostics or for both simultaneously
(theranostics).
Liposomes are artificial phospholipid vesicles, which can be obtained by a variety of methods
from lipid dispersions in water. The most frequently used methods for liposome preparation include
ultrasonication, reverse phase evaporation, detergent removal from mixed lipid-detergent micelles by
dialysis or gel filtration, freezethawing, extrusion, and lipid film hydration. Various amounts of
cholesterol are incorporated into the liposomal membrane to increase liposome membrane stability and
slow down their disintegration and release of incorporated drugs. Liposome size depends on their
composition, preparation method, and intended use, and can vary from around 80 nm or even less to
greater than 1 m in diameter.
The release rate of different compounds from liposomes in vitro is usually under 1% per hour,
assuming that the incubation temperature sufficiently differs from the phase transition temperature of a
given phospholipid, since the maximal permeability of liposomes is observed at temperatures close to the
phase transition temperature of the liposomal phospholipid. In vivo, the release rate can vary from
minutes to hours and depends on the liposome composition and location in the body. Liposomes are in
general biocompatible, cause no or very little antigenic, pyrogenic, allergic, and toxic reactions (unless
contain impurities or contaminations), easily undergo biodegradation, protect the host from any
undesirable effects of the encapsulated drug, and protect an entrapped drug from premature inactivation
by the physiological medium. Different methods of liposomal content delivery into the cytoplasm have
been elaborated. (The principal mechanisms of liposomecell interaction are presented in Fig. 1.)

Fig 1. Liposomecell interaction. Drug-loaded liposomes can adsorb on the cell surface specifically (1) or nonspecifically (2).
Liposome can also fuse with the cell membrane releasing its contents inside cell cytoplasm (3). It can also be destabilized by
certain cell membrane components when adsorbed on the surface so that the released drug can enter cell via
micropinocytosis (4). Liposome can undergo the direct or transfer protein-mediated exchange of lipid components with the
cell membrane (5). It can also be subjected to a specific or nonspecific endocytosis (6). In this case, a liposome can be
delivered by the endosome into the lysosome (6a) or, en route to lysosome, liposome can provoke endosome destabilization,
which results in drug liberation into the cell cytoplasm (6b). Drug-loaded liposome modified with certain viral components can
specifically interact with cells, provoke endocytosis, and, via the interaction of viral components with the inner membrane of
the endosome, allow for the drug efflux into the cell cytoplasm (7).

Liposomes loaded with drugs can incorporate these drugs in a variety of fashions: water-soluble
drugs are entrapped in the liposomal inner aqueous space (and, in case of multilammellar liposomes, into
the aqueous space between bilayers), while less soluble drugs may be incorporated in the phospholipid
membrane (see Fig. 2).

Fig. 2 Attachment of various modifiers to the liposome surface. 1 liposome; 2 soluble drug in inner
aqueous space; 3 insoluble drug in the membrane; 4 targeting ligand attached to a distal end of
protective polymer chain; 5 stimuli-sensitive or cell-penetrating function on the surface of liposomes; 6

contrast moiety attached to the liposome surface for liposome visualization in the body

1.2 Liposomes In Vivo: Achievements and Problems

Liposomes are eliminated from the blood by the cells of the mononuclear phagocyte system
(MPS), primarily in the liver. To increase liposomal drug accumulation in the desired areas, the use of
targeted liposomes with surface-attached ligands capable of recognition and binding to cells of interest
was attempted (see Fig. 2). It has been clearly demonstrated that liposomes, similar to macromolecules
and other nanoparticulates, are capable of accumulating in various pathological areas with compromised
vasculature (such as tumor, infarcts, and inflammations) via the so-called enhanced permeability and
retention (EPR) effect. Thus, the longer circulation of liposomes should enhance this means of target
accumulation, allowing for more passages through the target region and longer time for accumulation.
Different methods have been suggested to achieve long circulation of liposomes in vivo, including
coating the liposome surface with inert, biocompatible polymers, such as PEG. Alternatively, PEG may
prevent liposome-cell interaction and thus slow down the capture of liposomes by cells. Long circulating
liposomes demonstrate dose independent, nonsaturable, log linear kinetics, and increased bioavailability.
Further development of liposomal carriers has involved attempts to combine the properties of
long-circulating liposomes and targeted liposomes in one preparation, since longevity should also allow
for the better interaction of targeted liposomes with the target. Currently, various advanced technologies
for the preparation of targeted long-circulating liposomes are used, and the targeting moiety is usually
attached above the protecting polymer layer, to minimize the steric hindrances for the interaction with the
target, by coupling it with the distal water-exposed terminus of a liposome-grafted polymer molecule.
The combination of a targeting antibody and an endosome disruptive peptide on the same liposome has
been shown to improve the cytosolic delivery of liposomal drug and its cytotoxicity. Early studies
demonstrated the possibility of delivery of macromolecules and liposomes into living cells utilizing folate
receptor (FR) endocytosis, which could by pass multidrug resistance.
Recently, attempts have been made to target liposomes and other pharmaceutical nanocarriers to
cancer cells by residues of ascorbic acid attached to the liposome surface. Another recent approach
suggests the use of phage coat fusion proteins as targeting ligands for liposomes, purified from phage
preparations specifically selected against target cells and incorporated into the liposome membrane via
the hydrophobic fragment of the protein. If successful, such an approach can substitute expensive and
unstable monoclonal antibodies with cheap, stable, and easy to prepare phage proteins. In general, the
conjugation methodology for attaching specific ligands to the liposome surface is based on several
chemical reactions, which are efficient and selective: reaction between activated carboxyl groups and
amino groups yielding an amide bond; reaction between pyridyldithiols and thiols yielding disulfide
bonds; and reaction between maleimide derivatives and thiols yielding thioether bonds.
In many cases, drug loaded liposomes should bring the drug inside cells and allow for its release
from the endosomes into cell cytoplasmto escape the lysosomal degradation. To achieve this, the

liposome could be made pH-sensitive, i.e. made of pH-sensitive components and, after being
endocytosed, it interacts with the endovacuolar membrane under the action of lowered pH inside the
endosome, releasing its content into the cytoplasm. Antisense oligos could be delivered into cells by
anionic pH-sensitive PE-containing liposomes, which are stable in blood, but undergo a phase transition
at acidic endosomal pH and facilitate release of the oligo into cell cytoplasm. Liposomes that can carry on
their surface multiple functionalities, such as a targeting ligand and a residue of a cell penetrating peptide
allowing for an effective intracellular delivery, and that demonstrate different properties depending on the
specific conditions of surrounding tissues (e.g. lowered pH in tumors). Another approach to intracellular
drug delivery is based on the use of certain viral proteins demonstrating a unique ability to penetrate into
cells (protein transduction phenomenon).
1.3 Administration Routes for Liposome-Based Preparations
Liposomes as a dosage form allow for a broad variety of administration routes, each having its
own limitations. Oral administration requires high liposome stability and drug loaded liposome delivery
from the gut to the blood with subsequent drug release. PEG coated liposomes were used for oral delivery
of recombinant human epidermal growth factor for gastric ulcer healing. Nebulization was recently
suggested to deliver liposomal aerosols. In this particular case, a dispersion of a physical mixture of drugs
and phospholipids in saline, which spontaneously formed liposomes with the drug inside, was used. In
general, liposomes were found to increase skin penetration of many hydrophilic substances. Highly
flexible liposomes, Transferosomes that follow the transepidermal water activity gradient in the skin have
been proposed. Liposomes loaded with various drugs and decorated with various targeting moieties, such
as sugar residues, have also been utilized for nasal and ocular drug delivery. According to Crommelin
(2003), the following quality control assays should be applied to liposomal formulations for use in
humans:
Basic characterization assays, such as pH; osmolarity; trapped volume; phospholipid concentration;
phospholipid composition; phospholipid acyl chain composition; cholesterol concentration; active
compound concentration; residual organic solvents and heavy metals; active
compound/phospholipid ratio; and proton or ion gradient before and after remote loading.
Physical characterization assays, such as appearance; vesicle size distribution; submicron range;
micron range; electrical surface potential and surface pH; zeta potential; thermotropic behavior,
phase transition, and phase separation; and percentage of free drug.
Chemical stability assays, such as phospholipid hydrolysis; nonesterified fatty acid concentration;
phospholipid acyl chain autoxidation; cholesterol autoxidation; and active compound degradation.
Microbiological assays, such as sterility; and pyrogenicity (endotoxin level).
1.4 A Special Case of Liposomal Peptide and Protein Drugs
Proteins and peptides intended as therapeutic agents often demonstrate low biological stability.
One of the technologies to improve pharmacological properties of proteins and peptides is their
incorporation into liposomes (see schematics in Fig. 3). From the clinical point of view, the potential
ability of liposome encapsulated enzymes to enter the cytoplasm or lysosomes of live cells is of primary
importance for the treatment of inherited diseases caused by abnormal functioning of some intracellular
enzymes, especially in the liver and CNS cells. The use of liposomes for the transfer of therapeutic
enzymes through the bloodbrain barrier, which permits delivery of these enzymes into cells of the
central nervous system, also seems very attractive. Eventually, it was found that encapsulation into
liposomes could affect certain properties of enzymes or even cause certain undesirable side effects.

Fig. 3 Liposomes as carriers for proteins and peptides. Native proteins and peptides easily lose their
activity in vivo via unfolding or proteolytic degradation (cased by body temperature, oxidation or reduction,
influence of various ions, body enzymes and inhibitors, etc.). Immobilization of proteins and peptides into
liposomes stabilizes them significantly slowing down many of the inactivation processes

1.5 Liposomes as Vehicles for Delivery of DNA and Related Materials

The use of liposomes for gene delivery is a big and well-elaborated area. Although viral systems
are currently the most common means for DNA delivery, nonviral systems have also been developed.
Cationic lipid-based liposomes are easy to prepare, reasonably cheap, and nonimmunogenic. To combine
the longevity of liposomal preparations with efficient DNA delivery, precondensed DNA was
encapsulated into PEGylated cationic liposomes. Polycationic liposomes for gene delivery have been
suggested, i.e. liposomes modified by cetylated polyethylene imine, which anchors in the membrane via
cetyl residues and binds DNA via positive charges. Such liposomes demonstrate good loading with DNA
and high transfection efficacy. Liposomes are also used for targeting of antisense oligonucleotides, in
particular for neuroblastoma treatment, exemplified by coated cationic liposomes composed of a central
core of a cationic phospholipid bound to oligonucleotide, and an outer shell of neutral lipid. Such
liposomes are additionally modified with a monoclonal antibody against neuroectodermal antigens and
target antigen positive cells both in vitro and in vivo. Liposomes have also been used for intraperitoneal
delivery of siRNA for therapy of advanced ovarian cancer. Liposomes decorated with cell penetrating
peptides appear to be a promising mean to effectively deliver siRNA inside cells and silence the target
gene.
1.6 Liposomes as Immunological Adjuvants
Liposomes are known to be effective immunological adjuvant for various antigens since they are
capable of inducing both humoral and cellular immune responses toward liposomal antigens. The ability
to induce the cytotoxic T lymphocytes (CTL) response provides liposomes with certain benefits when
compared to traditional adjuvants, such as Freunds adjuvant, which do not induce significant CTL
response. Liposomal formulations of peptide vaccines loaded and activated dendritic cells (DCs), leading
to protective antiviral and antitumor immune responses.
1.7 Liposomes in Medical Imaging
There exist several different methods to label/load the liposome with a contrast/reporter group:
(a) Label is added to liposomes during the manufacturing process, either into the aqueous interior of
liposome or into the liposome membrane; (b) Label is adsorbed onto the surface of preformed liposomes;
(c) Label is incorporated into the lipid bilayer of preformed liposomes; (d) Label is loaded into preformed
liposomes using membrane-incorporated transporters, ion channels, or concentration gradients. In any
case, the labeling procedure should be simple and efficient, the reporter group should be affordable,
stable, and safe/easy to handle, and the liposomes should be stable during storage and in vivo, with no
release of free label. In addition to the enhanced relaxivity, the coating of liposome surface with PEG
polymer can help in avoiding contrast agent uptake in the site of injection by resident phagocytic cells.
Furthermore liposomes for sonography are prepared by incorporating gas bubbles (which are efficient
reflectors of sound) into the liposome, or by forming the bubble directly inside the liposome as a result of
a chemical reaction, such as bicarbonate hydrolysis yielding carbon dioxide. Gas bubbles stabilized inside
the phospholipid membrane demonstrate good performance and low toxicity of these contrast agents in
rabbit and porcine models.

Conclusion :

Liposomes, phospholipid bubbles with a bilayered membrane structure, are considered as

promising pharmaceutical carriers for different applications.
Liposomes, similar to macromolecules and other nanoparticulates, are capable of accumulating in
various pathological areas with compromised vasculature (such as tumor, infarcts, and
inflammations) via the so-called enhanced permeability and retention (EPR) effect.
Liposomes are known to be effective immunological adjuvant for various antigens since they are
capable of inducing both humoral and cellular immune responses toward liposomal antigens.

Reference :
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The next generation of liposome delivery systems: recent experience with tumor-targeted,
sterically-stabilized immunoliposomes and active-loading gradients. J Liposome Res 12:13.
Crommelin DJ, Storm G (2003) Liposomes: from the bench to the bed. J Liposome Res 13:3336.
Juergen Siepmann et al. (ed.) Fundamental and Applications of Controlled Release Drug Delivery,
Springer.