REVIEWS

RECENT ADVANCES WITH

LIPOSOMES AS PHARMACEUTICAL
CARRIERS
Vladimir P. Torchilin
Abstract | Liposomes — microscopic phospholipid bubbles with a bilayered membrane
structure — have received a lot of attention during the past 30 years as pharmaceutical
carriers of great potential. More recently, many new developments have been seen in the area
of liposomal drugs — from clinically approved products to new experimental applications, with
gene delivery and cancer therapy still being the principal areas of interest. For further successful
development of this field, promising trends must be identified and exploited, albeit with a clear
understanding of the limitations of these approaches.

RETICULO-ENDOTHELIAL
Since the pioneering observation of Alec Bangham improvement in survival1–3. The same set of indications
SYSTEM roughly 40 years ago that phospholipids in aqueous was targeted by a combination therapy comprising
The physiological system systems can form closed bilayered structures, lipo- liposomal doxorubicin and paclitaxel4 or Caelyx
responsible for the elimination somes have moved a long way from being just another (Schering-Plough)(doxorubicin in PEG liposomes)
of foreign macromolecules and
particles from the body; macro-
exotic object of biophysical research to become a and carboplatin5. Caelyx is also in Phase II clinical trials
phages of liver, spleen and pharmaceutical carrier of choice for numerous prac- for patients with squamous cell cancer of the head and
lymphatic system play a key role tical applications (FIG. 1; BOX 1). The real breakthrough neck6 and ovarian cancer7. Clinical research showed the
in this elimination. developments in the area during the past 15 years impressive effect of doxorubicin in PEG liposomes
have resulted in the approval of several liposomal against unresectable hepatocellular carcinoma8, cuta-
drugs, and the appearance of many unique biomedical neous T-cell lymphoma9 and sarcoma10. Liposomal
products and technologies involving liposomes. The lurtotecan was found to be effective in patients with
interest in the field remains high — almost 2,000 topotecan-resistant ovarian cancer11. Other indications
papers and more than 150 reviews on various aspects targeted by liposomal formulations include ampho-
of liposomology were published in 2003 alone. tericin B for the treatment of visceral leishmaniasis12
Clearly, within the frame of a single review paper it is and long-acting analgesia with liposomal bupivacaine
impossible to address all the of the pertinent issues, in healthy volunteers13. It would seem that liposomal
but I will attempt to identify the most important drugs have a very promising future.
trends in liposomology, as well as the most significant
achievements and challenges. Liposomes in drug delivery: evolution
The clinical applications of liposomes are well One of the drawbacks of the use of liposomes is the fast
Department of known (TABLE 1). The initial success achieved with elimination from the blood and capture of the liposomal
Pharmaceutical Sciences, many liposome-based drugs has fuelled further clinical preparations by the cells of the RETICULO-ENDOTHELIAL
Northeastern University, investigations. Recently, we have seen the selective SYSTEM, primarily in the liver. A number of developments
360 Huntington Avenue, delivery of the anticancer agent doxorubicin in poly- have aimed to reduce this problem.
Boston, Massachusetts
02115, USA.
ethylene gylcol (PEG) liposomes for the treatment of
e-mail: v.torchilin@neu.edu solid tumours in patients with breast-carcinoma Immunoliposomes. To increase liposomal drug accu-
doi:10.1038/nrd1632 metastases, which have resulted in a subsequent mulation in the desired tissues and organs, the use of

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REVIEWS

targeted liposomes with surface-attached ligands capa- Current research on PEG liposomes focuses on
ble of recognizing and binding to cells of interest has attaching PEG in a removable fashion to facilitate lipo-
been suggested (FIG. 1B). Immunoglobulins (Ig) of the some capture by cells. After PEG-liposomes accumulate
IgG class and their fragments are the most widely used at the target site, through the enhanced permeability and
targeting moieties for liposomes, which can be retention (EPR) effect22, the PEG coating is detached
attached to liposomes, without affecting liposomal under the action of local pathological conditions
integrity or the antibody properties, by covalent binding (decreased pH in tumours). New, detachable PEG con-
to the liposome surface or by hydrophobic insertion jugates have been described23, in which the detachment
into the liposomal membrane after modification with process is based on the mild thiolysis of the dithiobenzy-
hydrophobic residues14. Still, despite improvements in lurethane linkage between PEG and an amino-containing
targeting efficacy, the majority of immunoliposomes substrate (such as PE).
accumulate in the liver as a consequence of insufficient Although, PEG remains the gold standard for the steric
time for the interaction between the target and targeted protection of liposomes, attempts continue to identify
liposome. Better target accumulation can be expected other polymers that could be used to prepare long-circu-
if liposomes can be made to remain in the circulation lating liposomes. Earlier studies with various water-soluble
long enough. flexible polymers have been summarized elsewhere20,24.
More recent papers describe long-circulating liposomes
Long-circulating liposomes. Different methods have that were prepared using poly[N-(2-hydroxypropyl)
been suggested to achieve long circulation of liposomes methacrylamide)] 25, poly-N-vinylpyrrolidones26,
in vivo, including coating the liposome surface with L -amino-acid-based biodegradable polymer–lipid
inert, biocompatible polymers, such as PEG, which conjugates27 and polyvinyl alcohol28. Investigation of
form a protective layer over the liposome surface and the relative roles of the liposome charge and protective
slow down liposome recognition by opsonins and polymer molecular mass revealed that opsonins with
therefore subsequent clearance of liposomes15,16 (FIG. 1C). different molecular masses might be involved in the
Long-circulating liposomes are now being investigated clearance of liposomes containing differently charged
in detail and are widely used in biomedical in vitro and lipids29.
in vivo studies; they have also found their way into Continuing interest in using long-circulating lipo-
clinical practice17,18. An important feature of protective somes in cancer chemotherapy30,31 is supplemented by
polymers is their flexibility, which allows a relatively their potential use for other purposes, such as carrying
small number of surface-grafted polymer molecules to imaging agents and the treatment of infection32. Inter-
create an impermeable layer over the liposome estingly, recent evidence showed that PEG liposomes,
surface19,20. Long-circulating liposomes demonstrate previously considered to be biologically inert, could still
dose-independent, non-saturable, log-linear kinetics induce certain side reactions through activation of the
and increased bioavailability21. complement system33.

A B C D E
h j
f i
d e
q
c g k

b s
a
p r
+ l
+
+
–
o n m

Figure 1 | Evolution of liposomes. A | Early traditional phospholipids ‘plain’ liposomes with water soluble drug (a) entrapped into
the aqueous liposome interior, and water-insoluble drug (b) incorporated into the liposomal membrane (these designations are not
repeated on other figures). B | Antibody-targeted immunoliposome with antibody covalently coupled (c) to the reactive
phospholipids in the membrane, or hydrophobically anchored (d) into the liposomal membrane after preliminary modification with a
hydrophobic moiety. C | Long-circulating liposome grafted with a protective polymer (e) such as PEG, which shields the liposome
surface from the interaction with opsonizing proteins (f). D | Long-circulating immunoliposome simultaneously bearing both
protective polymer and antibody, which can be attached to the liposome surface (g) or, preferably, to the distal end of the grafted
polymeric chain (h). E | New-generation liposome, the surface of which can be modified (separately or simultaneously) by different
ways. Among these modifications are: the attachment of protective polymer (i) or protective polymer and targeting ligand, such as
antibody (j); the attachment/incorporation of the diagnostic label (k); the incorporation of positively charged lipids (l) allowing for the
complexation with DNA (m); the incorporation of stimuli-sensitive lipids (n); the attachment of stimuli-sensitive polymer (o); the
attachment of cell-penetrating peptide (p); the incorporation of viral components (q). In addition to a drug, liposome can loaded
with magnetic particles (r) for magnetic targeting and/or with colloidal gold or silver particles (s) for electron microscopy.

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 REVIEWS

derivatives and thiols, which yields thioether bonds.

Box 1 | Liposomes Many lipid derivatives used in these techniques are com-
Liposomes are spherical, self-closed structures formed by one or several concentric lipid mercially available38. Other approaches also exist, such as
bilayers with an aqueous phase inside and between the lipid bilayers. There are a number those that yield the carbamate bond via the reaction of
of different types of liposomal vesicle: p-nitrophenylcarbonyl- and amino-group37; this topic
• Multilamellar vesicles: these range in size from 500 to 5,000 nm and consist of several
is reviewed in REFS 39,40. (See BOX 2 and some popular
concentric bilayers. coupling schemes in FIG. 2).
• Small unilamellar vesicles: around 100 nm in size and formed by a single bilayer.
New ligands for targeting liposomes
• Large unilamellar vesicles: range in size from 200 to 800 nm. Antibody-mediated liposome targeting. Although various
• Long-circulating liposomes: liposomes modified in such a way (usually surface-grafted monoclonal antibodies have been shown to deliver lipo-
with certain polymers) that they can stay in the blood much longer (for hours) than somes to many targets, the optimization of properties of
non-modified liposomes. immunoliposomes is an ongoing concern. The majority
• Immunoliposomes: liposomes carrying antibodies attached to their surfaces and able of research in this area relates to cancer targeting, which
to accumulate in the area within the body where an attached antibody recognizes and utilizes a variety of antibodies. Internalizing antibodies
binds its antigen. are required to achieve a much-improved therapeutic
efficacy with antibody-targeted liposomal drugs, as
Attractive biological properties of liposomes: shown with B-lymphoma cells and internalizable epi-
• Liposomes are biocompatible. topes (CD19), for example41. An interesting concept was
• Liposomes can entrap water-soluble (hydrophilic) pharmaceutical agents in their developed to target HER2-overexpressing tumours
internal water compartment and water-insoluble (hydrophobic) pharmaceuticals into using anti-HER2 liposomes42. The antibody CC52,
the membrane. which is directed against rat colon adenocarcinoma
• Liposome-incorporated pharmaceuticals are protected from the inactivating effect of CC531 lines, was attached to PEGylated liposomes and
external conditions, yet do not cause undesirable side reactions. resulted in the specific accumulation of liposomes in a rat
model of metastatic CC531 (REF. 43). NUCLEOSOME-specific
• Liposomes provide a unique opportunity to deliver pharmaceuticals into cells or even
antibodies capable of recognizing various tumour cells
inside individual cellular compartments.
through tumour-cell-surface-bound nucleosomes
• Size, charge and surface properties of liposomes can be easily changed simply by improved Doxil (Alza) targeting to tumour cells and
adding new ingredients to the lipid mixture before liposome preparation and/or by increased its cytotoxicity44. Immunoliposomes contain-
variation of preparation methods.
ing the novel antitumoral drug fenretinide, and targeting
For key monographs on biomedical application of liposomes, see REFS 213–218. the GANGLIOSIDE GD2, induced apoptosis in neuroblas-
toma and melanoma cell lines, and demonstrated strong
antineuroblastoma activity both in vitro and in vivo in
Long-circulating immunoliposomes. The further devel- mice45. A combination of immunoliposome and endo-
opment of liposomal carriers involved the attempt to some-disruptive peptide improves the cytosolic delivery
combine the properties of long-circulating liposomes and of the liposomal drug, increases cytotoxicity and opens
immunoliposomes in one preparation34–36. Early experi- up new avenues for constructing targeted liposomal sys-
ments have been performed by simple co-immobilization tems; this was shown with the diphtheria toxin A chain,
of an antibody and PEG on the surface of the same lipo- which was combined with pH-dependent fusogenic
some, although the protective polymer can create steric peptide diINF-7 into integrated liposomes specifically
hindrances for target recognition with the targeting targeted to ovarian carcinoma46.
moiety34. To achieve better selectivity of PEG-coated
liposomes, it is advantageous to attach the targeting lig- Folate-mediated liposome targeting. Targeting tumours
and via a PEG spacer arm, so that the ligand is extended with folate-modified liposomes represents a popular
outside of the dense PEG brush, which reduces steric approach, because folate receptors (FR) are frequently
hindrance of binding to the target. Currently, various overexpressed in a range of tumour cells. After early
advanced technologies are used, and the targeting moiety studies established the possibility of delivering macro-
is usually attached above the protecting polymer layer, by molecules47 and then liposomes48 into living cells using
coupling it with the distal water-exposed terminus of FR endocytosis, which could bypass multidrug resis-
activated liposome-grafted polymer molecule35,37 (FIG. 1D). tance, interest in folate-targeted drug delivery by lipo-
somes grew rapidly (for important reviews see REFS 49,50).
NUCLEOSOME Liposome surface-modification chemistry Liposomal daunorubicin51 as well as doxorubicin52
Subunit of chromatin (the The preparation of modified liposomes with controlled have been delivered into various tumour cells through
complex of DNA plus specialized
properties requires the chemical conjugation of proteins, FR and demonstrated increased cytotoxicity. Recently,
proteins — histones — in
eukaryotic cells) composed of a peptides, polymers and other molecules to the liposome the application of folate-modified doxorubicin-loaded
short length of DNA wrapped surface. In general, the conjugation methodology is liposomes to the treatment of acute myelogenous
around a core of histone proteins. based on three main reactions, which are quite efficient leukaemia was combined with the induction of FR
and selective: reaction between activated carboxyl using all-trans retinoic acid53. Folate-targeted lipo-
GANGLIOSIDES
Glycolipids with large size
groups and amino groups, which yields an amide bond; somes have been proposed as delivery vehicles for
molecules; usually present on the reaction between pyridyldithiols and thiols, which yields boron neutron capture therapy54 and also used for target-
outer surface of cell membranes. disulphide bonds; and reaction between maleimide ing tumours with haptens for tumour immunotherapy55.

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Table 1 | Liposomal drugs approved for clinical application or undergoing clinical evaluation*
Active drug Product name‡ Indications
Daunorubicin DaunoXome Kaposi’s sarcoma
Doxurubicin Mycet Combinational therapy of recurrent breast cancer
Doxorubicin in PEG-liposomes Doxil/Caelyx Refractory Kaposi’s sarcoma; ovarian cancer; recurrent breast cancer
Amphotericin B AmBisome Fungal infections
Cytarabine DepoCyt Lymphomatous meningitis
Vincristine Onco TCS Non-Hodgkin’s lymphoma
Lurtotecan NX211 Ovarian cancer
Nystatin Nyotran Topical antifungal agent
All-trans retinoic acid Altragen Acute promyelocytic leukaemia; non-Hodgkin’s lymphoma; renal-
cell carcinoma; Kaposi’s sarcoma
Platinum compounds Platar Solid tumours
Annamycin Doxorubicin-resistant tumours
E1A gene Various tumours
DNA plasmid encoding HLA-B7 Allovectin-7 Metastatic melanoma
and α2 microglobulin
Liposomes for various drugs and Broad applications
diagnostic agents (lipoMASC)
*In different countries the same drug could be approved for different indications or be at different phases of clinical trials). ‡Name of
liposome formulation.

Within the field of gene therapy, folate-targeted lipo- target PEG liposomes with radionuclides to VIP receptors
somes have been used for both gene targeting to on the surface of tumour cells, and resulted in enhanced
tumour cells56 and for targeting tumours with antisense breast cancer inhibition in rats70. In addition, PEG lipo-
oligonucleotides57. somes loaded with doxorubicin have been targeted by
RGD peptides to the integrins of tumour vasculature and
Transferrin-mediated liposome targeting. Transferrin demonstrated increased efficiency against C26 colon car-
(Tf) receptors (TfR) are overexpressed on the surface of cinoma in murine model71. RGD peptides were also used
many tumour cells, and so antibodies against TfR, as well for targeting liposomes to integrins on activated platelets,
as Tf itself, are popular ligands for liposome targeting to and could therefore be used to specifically target the car-
tumours and inside tumour cells58. Recent studies involve diovascular system72. Similarly, an angiogenic homing
the coupling of Tf to PEG on PEGylated liposomes to peptide was used for targeted delivery of drug-loaded
combine longevity and targetability for drug delivery into liposomes to vascular endothelium in the experimental
solid tumours59. A similar approach was applied to the treatment of tumours in mice73. Epidermal growth factor
delivery of agents for photo-dynamic therapy, including receptor (EGFR)-targeted immunoliposomes have been
hypericin, into tumours60,61, and for intracellular delivery specifically delivered to variety of tumour cells that over-
of cisplatin into gastric cancer62. Tf-coupled doxoru- express EGFR74. The incorporation of mitomycin C into
bicin-loaded liposomes demonstrate increased binding long-circulating hyaluronan-targeted liposomes increases
and toxicity against C6 glioma cells63. Interestingly, the its activity against tumours that overexpress hyaluronan
increase in the expression of TfR was also discovered in receptors75. Research also continues on the use of galacto-
post-ischaemic cerebral endothelium, which was used to sylated liposomes to target drugs to the liver for the treat-
deliver Tf-modified PEG liposomes to post-ischaemic ment of liver tumours or metastases76. The ability of
brain in rats64. Tf65 as well as anti-TfR antibodies66,67 have galactosylated liposomes to accumulate specifically in
also been used to facilitate gene delivery into cells by parenchymal cells led to their application in gene delivery
cationic liposomes. Tf-mediated liposome delivery was to these cells (see REF. 77 for a review). Cisplatin-loaded
also successfully used for brain targeting. Immuno- liposomes that specifically bind to chondroitin sulphate,
liposomes incorporating the OX26 monoclonal antibody, which is overexpressed in many tumour cells, have been
which is directed to rat TfR, were found to concentrate on used for the successful suppression of tumour growth and
brain microvascular endothelium68. metastases in vivo78. Another new approach to liposome
targeting involves the functionalization of liposomes with
Other ligands. The search for new ligands for liposome peptide-amphiphiles79.
targeting is focused on specific receptors that are over-
expressed on target cells (particularly cancer cells) and pH-sensitive liposomes
certain specific components of pathological cells. For To achieve the pH-sensitive release of liposome content,
example, liposome targeting to tumours has been liposomes are constructed from pH-sensitive compo-
achieved by using vitamin and growth factor receptors69. nents; after being endocytosed in the intact form, these
Vasoactive intestinal peptide (VIP) has been used to fuse with the endovacuolar membrane as a result of the

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Liposomes as carriers of protein and peptides

Box 2 | Liposome surface modification: liposomal proteins and peptides
During the past two decades native, biologically active
Liposome surface modification serves various goals and involves various compounds of protein/peptide origin, such as enzymes,
modifiers: peptide hormones, cytokines and so on, have become
drugs of choice for the treatment of various diseases
• Attachment of specific ligands serves to prepare targeted liposomes.
(BOX 2).
• Attachment of certain hydrophilic polymers with highly flexible main chain results in
long-circulating liposomes. Liposomal proteins. From a clinical point of view, the
• Attachment of various labels or label carriers allows for monitoring the fate of potential ability of liposome-encapsulated enzymes to
liposomes in a body or cell and for the preparation of contrast liposomes for enhanced enter the cytoplasm or lysosomes of live cells is of crucial
diagnostic imaging. importance for the treatment of inherited diseases
• Incorporation of positively charged lipid derivatives or positively charged polymers caused by the abnormal functioning of some intra-
allows for DNA binding and efficient cell transfection. cellular enzymes (that is, the lysosomal storage diseases)
• Attachment of either antibody or antigen onto the surface of liposomes leads to a and cancer88.
liposome-based immunoassay system. The use of the liposome-encapsulated asparaginase
improves the survival of animals with asparagine-
Liposomes serve as carriers of proteins and peptides to be used in various areas: dependent P1534 tumours compared with free enzyme89.
• Incorporation of protein and peptide drugs into liposomes to improve their therapeutic It is also an important feature of this approach that the
activity (in a broad sense) and to diminish various drawbacks and side effects frequently encapsulation of asparaginase into liposomes prevents
characteristic of such drugs. the production of anti-asparaginase antibodies. Super-
• Incorporation of various proteins and peptides into liposomes to modulate the immune oxide dismutase (SOD), which protects against the
response towards these proteins and peptides or to other antigens (for example, by cytotoxic effects of superoxide anions, has been encap-
protein- or peptide-modulated activation of certain components of the immune system). sulated into liposomes and reduced ischaemia-repefusion
• Attachment of certain proteins and peptides (usually monoclonal antibodies or their
oxidative stress in gerbil brain when injected as an
Fab fragments) to the liposome surface to target liposomes (drug- or diagnostic-agent- intreaperitoneal bolus by increasing enzyme activity
loaded) to certain pathological areas in the body or even inside cells (using so-called (longevity) and decreasing membrane peroxidation in
transduction proteins and peptides). various regions of the brain90. Spray-dried powder for-
mulations of the active SOD in liposomes mixed with
• Liposomal delivery of DNA into certain cells to initiate the in situ production of a
disaccharides have also been described91. Experimental
therapeutically active protein (usually enzyme) to treat a local or systemic disease.
thrombolytic therapy with the liposomal tissue plas-
• Reconstitution of various membrane proteins into liposome to investigate the fine minogen activator in rabbits with jugular vein thrombo-
details of functioning of these proteins in vivo. sis demonstrated the benefits of the liposomal enzyme
over the native enzymes: fourfold higher dose of the
native enzyme was required to provide the same degree
lower pH inside the endosome, and release their contents of the lysis as the liposomal enzyme92.
into the cytoplasm (FIGS 3,4). Studies of pH-sensitive A very interesting approach to the use of liposomal
liposomes focus on the development of new lipid enzymes is their application to antibody-directed enzyme
compositions that confer pH-sensitivity to liposomes; prodrug therapy (ADEPT) based on the on-site acti-
liposome modification with various pH-sensitive poly- vation of chemically modified inactive phospholipid
mers; and combining liposomal pH-sensitivity with derivatives of various anticancer and antiviral agents.
longevity and ligand-mediated targeting. Long-circu- The application of phospholipid prodrugs incorporated
lating PEGylated pH-sensitive liposomes, although into liposome membranes brings several benefits93: the
having a decreased pH-sensitivity, still effectively deliver efficiency of prodrug incorporation is high; prodrugs
their contents into the cytoplasm (for a recent review do not leak from the liposome into the aqueous phase;
see REF. 80). Antisense oligonucleotides can be delivered drugs are protected against metabolic degradation; and
into cells by anionic pH-sensitive phosphatidylethanol- long-lasting therapeutic drug levels can be achieved.
amine (PE)-containing liposomes that are stable in the The achieve the specific generation of active cytotoxic
blood, but which, however, undergo phase transition at molecules from inactive prodrugs in the vicinity of
acidic endosomal pH. This facilitates oligo release into tumour cells, a conjugate of a tumour-specific antibody
cell cytoplasm (reviewed in REF. 81). New pH-sensitive with an enzyme responsible for the conversion of a pro-
liposomal additives have recently been described that drug into the active drug is targeted towards tumour. To
include oleyl alcohol82 and a mono-stearoyl derivative increase the efficiency of the required enzyme in the
of morpholine83. Serum-stable, long-circulating tumour, rather than just ‘straight’ antibody-enzyme
PEGylated pH-sensitive liposomes were also prepared conjugates, immunoliposomes have been loaded with
using a combination of PEG and a pH-sensitive, ter- the required enzyme (immuno-enzymosomes)94.
minally alkylated co-polymer of N-isopropylacryl-
amide and methacrylic acid84. The combination of Liposomal peptides. The incorporation of insulin into
liposome pH-sensitivity and specificity of ligand tar- liposomes purposely exploits delivery to the liver (the
geting for cytosolic drug delivery using decreased natural target organ for liposomes), prolongs insulin
endosomal pH values has been described for both action in the body and enhances the oral absorption of
folate and Tf-targeted liposomes85–87. insulin. In animal experiments, the attempt to improve

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O
a O EDC b
O
NGPE C OH R1 N C N R2
N O C (CH2)2 S S SPDP
EDC pH 5.5, R1 = C2H5 N
room temperature R2 = (CH3)2NC3H6 O

O HNR1 Sulpho-NHS PE NH2 + SPDP Ligand NH2 + SPDP

NGPE C O C O –
SO 3
NR2
Sulpho-NHS pH 5.5, HO N
room temperature MeOH
TEA Pyr S S CH2 N Ligand
O O
SO–3
O O
DTT
NGPE C O N + R1 NH C NH R2

O PE N CH2 S S Pyr HS CH2 N Ligand

H2N-ligand
pH 7.5, 0–4°C

O
NGPE C NH Ligand PE N CH2 S S CH2 N Ligand

c O pH 8.0
O

PE NH C (CH2)2 S S PE NH C (CH2)2 S S Fab′ + S

N + Fab-S-H N
H
PDP-PE-vesicle Fab′-vesicle 2-Thriopyridone

O O SATA
d
O O O
PE NH2 + N O C CH2Ι Ligand + N O C CH2 S C C3H

O O
NHSΙA
O O
Ligand – NH C CH2 S C CH3 + NH2OH

O O O
PE NH C CH2Ι PE NH C CH2 S Ligand Ligand – NH C CH2 SH

e
CH3(CH2)m–O–CH2
CH3(CH2)m–O–CH O O
CH2 O P O CH2CH2NH O (CH2CH2O)n C NO2 + NH2 – Ligand
OH

CH3(CH2)m–O–CH2 Aqueous buffer, pH >8.5

CH3(CH2)m–O–CH O
CH2 O P O CH2CH2NH O (CH2CH2O)n NH Ligand
OH
Figure 2 | Chemical reactions to attach various ligands (antibodies) to the liposome surface (all these reactions can
be used to directly attach ligands to the liposome surface or to attach ligands to liposomes via the PEG spacer).
a | Attachment of amino-group-containing ligands to liposomes via the liposome-incorporated reactive phospholipid N-glutaryl-
phosphatidylethanolamine (NGPE). Water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy-
sulphosuccinimide (sulpho-NHS) are used to activate NGPE for the interaction with amino-group of the ligand. b | Attachment of
amino-group-containing ligands to amino-group-containing phospholipid (such as phosphatidylethanolamine (PE)) incorporated into
the liposomal membrane using a heterobifunctional reagent, N-succinimidyl pyridyl dithio propionate (SPDP). Both amino-containing
liposomal phospholipid and ligand are activated with SPDP, producing pyridyl dithio propionate (PDP) derivatives; the PDP group in
the ligand is reduced with dithiothreitol (DTT) into the HS-group. As a result of the interaction of activated liposome with activated
ligand aromatic pyridyl sulphydryl (-S-Pyr) group of PDP-lipid is displaced with the aliphatic -SH group yielding a disulphide bridge
between the liposome and the ligand. c | In a particular case of SPDP-mediated coupling, SH-group-containing Fab fragment (Fab-SH)
of immunoglobulin G antibody can be directly attached to a PDP-containing liposome with the formation of a disulphide bridge.
d | Attachment of thioacetate-activated ligand to the iodoacetate-activated liposome. The thioacetate group can be introduced into
the amino-group-containing ligand (protein) by activation with succinimidyl-S-acetyl thioacetate (SATA) and converted into a free
sulphydryl group by the subsequent treatment with hydroxylamine. The iodoacetate group can be introduced onto the liposome
surface by the activation of amino-group-containing phospholipid with N-hydroxysuccinimido-iodoacetate (NHSIA). SH-containing
ligand then interacts with iodoacetate-liposome. e | Attachment of amino-group-containing ligands to PEGylated liposomes via
p-nitrophenylcarbonyl (p-NP) group on the distant terminus of the lipid-conjugated polyethylene glycol (PEG) chain. Direct interaction
of the ligand amino-group with the liposomal pNP group yields carbamate (urethan) bond.

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the bioavailability of oral liposomal insulin by coating efficiency of transfection. (The assembly of liposome–
insulin-containing liposomes with PEG or mucin95 has DNA complexes is discussed in REF. 106.) To combine
successfully resulted in long-lasting lowering of glu- the longevity of liposomal preparations with efficient
cose levels. This is explained by the better interaction DNA delivery, pre-condensed DNA has been encapsu-
of polymer-coated liposomes with the mucus layer lated into PEGylated cationic liposomes107. Recently,
and better retention of insulin under the aggressive the use of polycationic liposomes for gene delivery
conditions of the stomach and gastro-intestinal tract. has been proposed — that is, liposomes modified by
The efficiency of oral administration of liposomal cetylated polyethylene imine, which anchors in the
insulin in liposomes of different phospholipid compo- membrane via cetyl residues and binds DNA via positive
sition was also confirmed in rats96. However, the high charges. Such liposomes demonstrate good loading
variability of effects caused by oral liposomal insulin with DNA and high transfection efficacy108. The in vivo
still represents a challenge. Buccal delivery of the lipo- results achieved with cationic liposome-mediated
somal insulin generated encouraging results in rabbit gene delivery and the future prospects of this technology
experiments97 and might represent an interesting alter- have been discussed in detail109,110. Liposomes are also
native. The pharmacodynamics of insulin in PEG- used for the targeting of antisense oligonucleotides to
coated liposomes after intravenous administration has specific tissues, in particular for neuroblastoma treat-
been studied in rats98. PEGylated liposomes provided ment, an approach that is exemplified by the use of
the strongest and the longest decrease in the glucose coated cationic liposomes made of a central core of a
level, which supports the hypothesis that the hormone cationic phospholipid bound to oligonucleotide, and
is slowly released from PEG liposomes and maintains an outer shell of neutral lipid. Such liposomes are
a constant concentration in the blood for a long time. additionally modified with a monoclonal antibody
The interest in the liposomal insulin for both oral and against neuroectoderma antigen and target antigen-
PARENTERAL applications remains high. positive cells both in vitro and in vivo111. The first
Cytokines are frequently candidates for liposomal papers on cationic liposome-mediated delivery of small
dosage. The incorporation of recombinant interleukin-2 interfering RNA are now being published, and have par-
into liposomes increased its blood-circulation time by ticularly compared intravenous and intraperitoneal
eightfold99. Mannosylated liposomes with muramyl administration routes in adult mice112.
dipeptide significantly inhibited liver metastases in
tumour-bearing mice100. PEG liposomes have also been Administration
proposed for the oral delivery of recombinant human Liposomes for oral administration. The oral adminis-
EGF101. Liposomal recombinant human tumour- tration of liposomes requires that they are highly stabile
necrosis factor strongly suppressed parasitaemia and and that the liposomes move from the gut to the blood,
protected against Plasmodium berghei k173-induced and only begin to release the drug when in the blood113.
experimental cerebral malaria in mice102. Liposomal Early attempts to use polymerized liposomes as potential
systems have also been considered as a cytokine supple- oral vaccine carriers114 were only partially successful.
ment in tumour cell vaccines, because they could pro- Many alternative schemes are currently under develop-
vide a cytokine reservoir at the antigen-presentation ment. For example, chitosan-coated insulin liposomes
site103; and the benefits of the liposomal interferon-γ in have been shown to produce hypoglycaemic effects in
the generation of systemic immune responses in B16 mice after oral administration115. Liposomes made
melanoma model have been clearly demonstrated by with the addition of gangliosides GM1 and GM type
these authors. III are stable in different biological media and can
The incorporation into liposomes of the antimicro- survive the gastro-intestinal tract 116. PEG-coated
bial and antiendotoxin cationic peptide CM3 was liposomes were used for oral delivery of recombinant
suggested for aerosol delivery, and corresponding human epidermal growth factor for gastric ulcer
models describing its potential distribution in lungs healing 117. Hypocalcaemic effects of liposomal
of patients with different breathing patterns have been salmon calcitonin after oral administration has also
developed104. been demonstrated118. PEG liposomes have also been
considered for use as oral vaccines — for example,
Liposomes in gene delivery ovalbumin in PEG-coated liposomes induces the best
The use of liposomes for gene delivery applications is mucosal immune response of all carriers tested119. To
a huge area that will be only briefly addressed here. improve protein and peptide bioavailability via the
Although viral systems are currently the most com- oral route, an oral colon-specific drug delivery system
mon means for DNA delivery, non-viral systems have for bee venom peptide was developed that was based
also been developed. Cationic lipid-based liposomes105 on coated alginate gel beads entrapped in liposomes120.
are easy to prepare, reasonably cheap and non-
immunogenic. Many of the finer features of these Liposomal aerosols. After liposome drying methods
delivery systems and mechanisms remain insuffi- were developed121, aerosolized liposomal preparations
PARENTERAL ciently understood, and so recent studies in this popular became a feasible route for delivering drugs to the lung.
Administered by means other
than through the alimentary
area have tended to concentrate on structure, function, Among the examples in this area, a few deserve special
tract (such as intramuscular or structure–activity relationships, detailed mechanisms attention.A combined aerosol of liposomal paclitaxel and
intravenous injection). of liposome-mediated gene delivery, and improved cyclosporin A achieves better results in the treatment of

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A Liposome B Liposome

a Viral components

Drug
a Drug b b

Lysosome

Endosome

c
f Endosome g
Nucleus Nucleus
c

h Drug efflux

e d

Figure 3 | Liposome-cell interaction. A | Drug-loaded liposomes can specifically (a) or nonspecifically (b) adsorb onto the cell
surface. Liposomes can also fuse with the cell membrane (c), and release their contents into the cell cytoplasm, or can be
destabilized by certain cell membrane components when adsorbed on the surface (d) so that the released drug can enter cell via
micropinocytosis. Liposome can undergo the direct or transfer-protein-mediated exchange of lipid components with the cell
membrane (e) or be subjected to a specific or nonspecific endocytosis (f). In the case of endocytosis, a liposome can be delivered
by the endosome into the lysosome (g) or, en route to the lysosome, the liposome can provoke endosome destabilization (h), which
results in drug liberation into the cell cytoplasm. B | Liposome modified with specific viral components (a) and loaded with a drug
can specifically interact with cells (b), provoke endocytosis, and, via the interaction of viral components with the inner membrane of
the endosome (c), allow for drug efflux into the cell cytoplasm (d).

pulmonary metastases of renal-cell carcinoma in mice liposomes is supported by the results of transdermal
than each alone122. Spray-dried powder formulations delivery of pergolide in liposomes, in which elastic vesi-
of liposomes and disaccharides have also been used as cles have been shown to be more efficient131. The combi-
carriers for SOD123. Improved delivery of rifampicin nation of liposomes and IONTOPHORESIS for transdermal
by aerosolized liposomes to alveolar macrophages delivery yielded promising results132.
might become significant in the treatment of tubercu-
losis124. Aerosolized liposomal budesonide is effective Lymphatic targeting with liposomes. Because subcuta-
against experimental asthma in mice125. Aerosol formu- neous administration of liposomes results in their
lations of liposomal 9-nitrocamptothecin are non-toxic uptake by draining lymphatic capillaries at the injection
and efficiently treated melanoma and osteosarcoma site, and active capture of liposomes by macrophages in
lung metastases in mice126. Liposomal paclitaxel in regional lymph nodes, plain and ligand-targeted lipo-
aerosol effectively treated pulmonary metastases in a somes have been proposed as a potentially useful
murine renal carcinoma model127. Nebulization was approach to targeting lymphatics for therapeutic and
recently proposed as a means to deliver liposomal diagnostic applications after subcutaneous adminis-
aerosols128. In this particular case, a dispersion of the tration133. Liposome uptake by lymph nodes might be
physical mixture of drugs and phospholipid in saline increased by using biotin-bearing liposomes for pre-
was used that spontaneously formed liposomes with liminary injection, and avidin for subsequent adminis-
the drug inside. tration that aggregates biotin liposomes and increases
their uptake by lymph-node macrophages134. Lipo-
Topical application of liposomes. During recent years, somes have been used for lymphatic delivery of metho-
the topical delivery of liposomes has been applied to trexate135 and for magnetic resonance imaging (MRI)
different applications and in different disease models129. with gadolinium (Gd)-loaded liposomes136.
Current efforts in this area concentrate around opti-
mization procedures and new compositions. New, Combination therapy with liposomes. An interesting
IONTOPHORESIS highly flexible liposomes called transferosomes that example of a new approach is to combine radio-fre-
A means of enhancing the flux follow the trans-epidermal water activity gradient in the quency tumour ablation with intravenous liposomal
of ionic compounds across a
membrane (such as the skin) by
skin have been proposed. Diclofenac in transferosomes doxorubicin, which resulted in better tumour accu-
the application of an electric was effective when tested in mice, rats and pigs130. The mulation of liposomes and increased necrosis in
current across it. concept of increased deformability of transdermal tumours137.

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A Liposome membrane destabilization Drug efflux and improved immune response (activation of CD8+
T cells), evidently because of protection of the antigen
a Liposome membrane by liposomes142.
Oral delivery of antigens in liposomes (ovalbumin
was used as a model antigen) effectively induces ORAL
143
TOLERANCE . Various approaches for delivering lipo-
somal proteins to the cytoplasm and Golgi of antigen-
b
presenting cells were recently reviewed144. In recent
developments, liposomes have been successfully used
B Destabilization of endosomal membrane Drug efflux
for the delivery of peptide vaccines and CTL epitopes
Endosome membrane to dendritic cells (DCs) to improve the immune
response to antigenic peptides145,146. Liposomal formu-
lations of peptide vaccines load and activate DCs,
which leads to protective antiviral and antitumour
a immune responses147. Liposomes have successfully
b delivered CTL epitopes to DCs148. A hybrid CTL epitope
delivery system has also been proposed that consists of
Lipososme
Antennapedia homeodomain peptide vector in lipo-
Figure 4 | Fusogenic and stimuli-sensitive liposomes. A | Liposome membrane somes149. Antigen in mannosylated liposomes enhances
destabilization. After accumulation in required sites in the body, liposomes containing stimuli- the uptake and activation of DCs, and increases their
sensitive components, such as lipids (a) in the membrane and drug (b) inside, and after being ability to induce primed T-cell proliferation 150. In
subjected to local action of the corresponding stimulus (such as pH or temperature), undergo
general, the future of the liposomal vaccines seems to
local membrane destabilization (transfer from left to right of panel A) that allows for drug efflux
from the liposome into surroundings. B | Destabilization of endosomal membrane. After being
be promising.
endocytosed by the cell and taken inside the endosome, the liposome containing stimuli (pH)-
sensitive components, such as lipids (a) in the membrane and drug (b) inside, can undergo pH- Liposomes in diagnostic imaging
dependent membrane destabilization and initiate the destabilization of the lysosomal membrane The use of liposomes for the delivery of imaging agents
(transfer from left to right of panel B) that allows for drug efflux into the cell cytoplasm. for all imaging modalities (BOX 3) has a long history151.
FIGURE 5 demonstrates the principal scheme of liposome
loading with various reporter moieties, which might be
Liposomal vaccines covalently or non-covalently incorporated into different
Liposomes were long ago been shown to be effective compartments of these particulate carriers. The relative
immunological adjuvants for protein and peptide anti- efficacy of entrapment of contrast materials into differ-
gens138,139. They are capable of inducing both humoral ent liposomes, as well as the advantages and disadvan-
and cellular immune responses towards the liposomal tages of various liposome types, have been discussed by
antigens. Liposomes with encapsulated protein or pep- Tilcock152. Liposomal contrast agents have been used for
tide antigen are phagocytosed by macrophages and experimental diagnostic imaging of liver, spleen, brain,
eventually accumulate in lysosomes. Once in the lyso- cardio-vascular system, tumours, inflammation and
somes, degraded peptides are presented to the major infections151,153.
histocompatibility complex class II (MHCII) complex GAMMA-SCINTIGRAPHY and MRI both require a suffi-
on the macrophage surface. This results in the stimula- cient quantity of radionuclide or paramagnetic metal
tion of specific T-helper cells, and, ultimately, stimulation to be associated with the liposome. There are two pos-
of specific B cells, which results in the subsequent sible routes to improve the efficacy of liposomes as
secretion of antibodies139. A fraction of the liposomal contrast mediums for gamma-scintigraphy and MRI:
antigen can escape from endosomes into the cytoplasm increasing the quantity of carrier-associated reporter
(for example, when pH-sensitive liposomes are used) metal (such as 111In or Gd), and/or enhancing the signal
and in this case the liberated antigen is processed and intensity. To increase the load of liposomes with
presented in association with the MHCI complex, which reporter metals, amphiphilic chelating polymers, such
induces a cytotoxic T-lymphocyte (CTL) response; this as N,α-(DTPA-polylysyl)glutaryl phosphatidyl ethanol-
provides liposomes with certain benefits over traditional amine, were introduced154. These polymers easily
ORAL TOLERANCE
The acquisition of a specific
adjuvants (such as Freund’s adjuvant) that do not incorporate into the liposomal membrane and
nonresponsiveness, via oral induce any significant CTL response. markedly increase the number of chelated Gd or In
administration, to a Synthetic human MUC1 peptides, which are con- atoms attached to a single lipid anchor. In the case of
molecule recognized by the sidered candidates for therapeutic cancer vaccines, MRI, metal atoms chelated into these groups are
immune system.
have been incorporated into liposomes or attached to directly exposed to the water environment, which
GAMMA-SCINTIGRAPHY
the surface of liposomes, and in both cases elicited enhances the signal intensity of the paramagnetic ions
Medical diagnostic imaging strong antigen-specific T-responses140. Formaldehyde- and leads to corresponding enhancement of the vesicle
modality based on the inactivated ricin toxoid in liposomes for intra-pulmonary contrast properties. The overall performance of chel-
application of γ-emitting vaccination generated protection against inhaled ricin141. ating polymer-bearing liposomes might be further
radioactive materials, such as
99m-Tc, 111-In, 125- and 131-I,
Liposomal composition incorporating Antennapedia improved by additional incorporation of amphiphilic
67-Ga, and some other isotopes homeodomain fused to a poorly immunogenic CTL PEG into the liposome membrane, because of the pres-
with variable decay times. epitope increased the immunogenicity of the construct ence of the increased concentration of PEG-associated

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carbon dioxide. Gas bubbles stabilized inside the phos-

Box 3 | Liposome in diagnostic imaging
pholipid membrane demonstrate good performance
Diagnostic imaging requires that appropriate intensity of signal from an area of interest and low toxicity of these contrast agents in rabbit and
is achieved to differentiate certain structures from surrounding tissues, regardless of the porcine models. Recently, liposomal applications for
modality used. Currently used imaging modalities include gamma-scintigraphy (involving ultrasound and gamma-scintigraphic imaging have
the application of γ-emitting radioactive materials); magnetic resonance (based on the been discussed162.
transition between different energy levels of atomic nuclei under the action of a
radiofrequency signal); computed tomography (which utilizes ionizing radiation with New-generation liposomes
the aid of computers to acquire cross-images of the body and three-dimensional images of The following few examples illustrate the variety of cur-
areas of interest); and ultra-sonography (the modality using irradiation with ultrasound rent approaches to using liposomes as pharmaceutical
and based on the different rate at which ultrasound passes through various tissues). carriers.
To facilitate the accumulation of contrast in the required zone, various
microparticulates, including liposomes, have been suggested as carriers for contrast agents.
Virosomes. VIROSOMES (FIG. 3) represent one more devel-
Possible methods of liposome labelling with reporter group (label): opmental avenue for liposomes. In these applications,
• Label is added during the manufacturing process to liposomes (label is incorporated which aim to enhance tissue targeting, the liposome sur-
into the aqueous interior of liposome or into the liposome membrane). face was modified with fusogenic viral envelope
• Label is adsorbed onto the surface of preformed liposomes. proteins163. Initially, virosomes were intended for the
intracellular delivery of drugs and DNA164,165. Later, viro-
• Label is incorporated into the lipid bilayer of preformed liposomes.
somes became a cornerstone for the development of
• Label is loaded into preformed liposomes using membrane-incorporated transporters new vaccines. The delivery of protein antigens to the
or ion channels.
immune system by fusion-acting virosomes was found
Clinically acceptable diagnostic liposomes will have to meet the following to be very effective166, in particular when targeting
requirements: DCs167. As a result, a whole set of virosome-based vac-
• The labelling procedure should be simple and efficient. cines have been developed for use in humans and ani-
mals. Special attention has been paid to the delivery of
• The reporter group should be affordable, stable and safe/easy to handle.
influenza vaccine using virosomes containing the spike
• Liposomes should be stable in vivo stability with no release of free label. proteins of influenza virus168, used because it elicits high
• Liposomes need to be stable on storage — within acceptable limits. titres of influenza-specific antibodies. Trials of virosome
influenza vaccine in children showed that it is highly
immunogenic and well tolerated169. A similar approach
water protons in the close vicinity of chelated Gd ions was used to prepare virosomal hepatitis A vaccine that
located on the liposomal membrane. In addition to the elicited high antibody titres after primary and booster
enhanced RELAXIVITY, the coating of liposome surface vaccination of infants and young children170; these data
with PEG polymer can help in preventing the contrast have been confirmed in healthy adults171 and in elderly
agent being taken up at the site of injection by resident patients172. A combination of influenza protein-based
phagocytic cells. This approach results in efficient lipo- virosomes with other antigens might be used to prepare
somal contrast agents for MRI of the blood pool155 other vaccines173. In general, virosomes can provide an
(FIG. 5). MRI using pH-responsive contrast liposomes excellent opportunity for the efficient delivery of both
allows for the visualization of pathological areas with various antigens and many drugs (including nucleic
decreased pH values156. Liposomes loaded with contrast acids, cytotoxic drugs and toxoids)173,174, although they
agent have also used for the in vivo monitoring of tissue might present certain problems associated with their
pharmacokinetics of liposomal drugs in mice157. stability/leakiness and immunogenicity.
Because of its short half-life and ideal radiation
energy, 99mTc is the most clinically attractive isotope for Magnetic liposomes. An interesting approach for tar-
gamma-scintigraphy. Recently, new methods for geted drug delivery under the action of magnetic field
labelling preformed glutathione-containing liposomes is the use of liposomes loaded with a drug and a ferro-
with various 99mTc and 186Re complexes have been devel- magnetic material. In one example, magnetic lipo-
oped158,159; these are extremely effective and result in a somes containing doxorubicin were intravenously
very stable product. administered to osteosarcoma-bearing hamsters.
Computerized tomograhphy contrast agents (pri- When the tumour-implanted limb was placed between
marily heavily iodinated organic compounds) can be two poles of a 0.4 Tesla magnet, the application of the
included in the inner water compartment of liposomes field for 60 minutes resulted in a fourfold increase in
RELAXIVITY or incorporated into the liposome membrane. For drug concentration in the tumour175. In the same
The property of certain metal
example, iopromide has been incorporated into osteosarcoma model in which the magnet was
ions to increase proton
relaxation rate (relates to plain160 and PEGylated liposomes161 and demonstrated implanted into the tumour, magnetic liposomes
magnetic resonance imaging). favourable biodistribution and imaging potential in loaded with adriamycin demonstrated better accumu-
rats and rabbits. Liposomes for sonography are pre- lation in tumour vasculature and resulted in enhanced
VIROSOMES pared by incorporating gas bubbles (which are efficient tumour-growth inhibition176. Intravenous injection in
Liposomes with surface-
attached or membrane-
reflectors of sound) into the liposome, or by forming the rats of liposomes loaded with 99mTc-albumin and mag-
incorporated fragments of the bubble directly inside the liposome as a result of a chemi- netite resulted in a 25-fold increase in accumulated
viral protein coat. cal reaction, such as bicarbonate hydrolysis yielding radioactivity in the right kidney, near which a SmCo

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A d B 50 C
* 45
900
40
e b 800
* 35
* * 700

1/T1 (1/sec)
30
600

T1 (msec)
* c * 25
a * * 20
500
400
b 15
a 300
10 200
5 100
f 0 0
0 20 40 60 80 0 20 40 60 80
Liposomal lipid (mM) Time post-injection (min)
Figure 5 | Liposomes in diagnostic imaging. A | Schematics of liposome loading with a contrast agent. A liposome consisting of
a bilayered membrane (a) and internal aqueous compartment (b) can entrap a contrast agent (asterisk) into the inner water space of
liposome as a soluble entity (c), or it can be incorporated into the liposome membrane as a part of monomeric (d) or polymeric (e)
amphiphilic unit. Additionally, liposomes can be sterically protected by amphiphilic derivative of polyethylene glycol (PEG) or PEG-like
polymer (f). B | Molecular relaxivities (1/T1) of PEG-liposomes containing the same molar fraction of Gd-loaded monomeric (a) or
polymeric (b) DTPA chelate. Because the use of the polymeric chelate allows for loading much larger number of Gd atoms per single
lipid anchor, the relaxivity (that is, contrast properties) of such liposome is much higher. C | Effect of Gd-polychelate-containing PEG
liposomes on T1 parameter of the blood in vivo in dog. Such long-circulating contrast liposomes allow for strong and prolonged
blood opacification required for diagnostically significant magnetic resonance imaging of the blood pool. Gd, gadolinium.

magnet was implanted, compared with the control left helped to treat haemorrhagic shock in rats188. However,
kidney177. This might become a promising way of drug some side effects were found for PEG haemosomes. For
targeting by liposomes. example, they were shown to be phagocytosed by human
peripheral blood monocytes and macrophages via the
Cytoskeleton-specific immunoliposomes. Specific anti- opsonin-independent pathway189. In addition, some
cardiac myosin monoclonal antibodies have an excellent studies show complement activation after administration
capacity to recognize and bind hypoxic cells with of PEGylated haemosomes190.
damaged plasma membranes when intracellular myosin
is exposed into extracellular space178. This property of ATP liposomes. There is interest in liposomal forms of
the antimyosin antibody has been successfully used ‘bioenergic’ substrates, such as ATP, and some encour-
for the delivery of antibody-bearing liposomes in the aging results with ATP-loaded liposomes in various in
field of experimental myocardial infarction34. In addition, vitro and in vivo models have been reported. ATP lipo-
immunoliposomes specifically targeting ischaemically somes were shown to protect human endothelial cells
damaged cardiomyocytes (cytoskeleton-specific immuno- from energy failure in a cell culture model of sepsis191. In
liposomes) seal membrane damage and decrease the a brain ischaemia model, the use of the liposomal ATP
level of cell death both in vitro179 and in the isolated rat increased the number of ischaemic episodes tolerated
heart model180. A similar approach was used for decreas- before brain electrical silence and death192. In a HYPO-
ing haemorrhage after focal embolic stroke by anti- VOLEMIC shock-reperfusion model in rats, the adminis-
actin-targeted liposomes in rats181. Cytoskeleton-specific tration of ATP liposomes provided effective protection
immunoliposomes can fuse with damaged cells, and so to the liver193. ATP liposomes also improved the rat liver
they were used as carriers for successful gene delivery energy state and metabolism during the cold storage
into hypoxic cells182 (FIG. 6). preservation194. Similar properties were also demon-
strated for the liposomal coenzyme Q10 (REF. 195).
Liposomal haemoglobin. Active research continues in the Interestingly, biodistribution studies with the ATP lipo-
area of liposomal haemoglobin (haemosomes) as a blood somes demonstrated significant accumulation in the
substitute. To make long-circulating haemosomes, tech- damaged myocardium196. Recently, ATP-loaded lipo-
nology for PEG post-insertion was developed, in which somes were shown to effectively preserve mechanical
the resulting liposomes do not lose any haemoglobin and properties of the heart under ischaemic conditions in an
circulate longer in rabbits183. PEGylated liposomal isolated rat heart model197. ATP-loaded immunolipo-
haemoglobin was found to be stable at storage for 1 year somes have also been prepared that possess specific
even at room temperature184 and to circulate longer in affinity towards myosin — that is, which are capable of
rabbits when labelled with 99mTc (half-clearance time of specifically recognizing hypoxic cells198.
48 h)185. Further optimization of vesicle composition led
to the conclusion that the use of saturated lipids is prefer- Liposomes in photo-dynamic therapy. Photo-dynamic
able because it allows escaping lipid peroxidation186. therapy (PDT) is a rapidly developing modality for
HYPOVOLEMIC
Good microvascular perfusion was achieved with lipo- the treatment of superficial tumours, in which photo-
A decrease in the volume of somal haemoglobin in hamsters187. Haemoglobin vesicles sensitizing agents are used for the photo-chemical erad-
circulating blood. suspended in recombinant human serum albumin ication of malignant cells. In PDT, liposomes are used

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A Normal cell B Sarcolemmal disruption

Cytoskeleton Cytoskeleton

b
a

c
d

D Intracellular delivery of drug or DNA C Interaction

Cytoskeleton Cytoskeleton

Figure 6 | Cytoskeleton-specific immunoliposomes for drug and DNA delivery. Liposome (a) modified with cytoskeleton-
specific monoclonal antibody (b) and loaded with DNA (c) or drug molecules (d) can specifically recognize the sites of sarcolemmal
disruption (B), attach to these sites via the antibody-specific interaction with the exposed cytoskeletal antigen (C), and, on fusion
with the cell, deliver drugs or DNA into the cell cytoplasm (D).

both as drug carriers and enhancers, and a review on the active against tumours in sarcoma-bearing mice201. PDT
use of liposomes in PDT has recently been published199. with liposomal photofrin provides better results against
Targeting as well as the controlled release of photo- human gastric cancer in mice than is achieved with free
sensitizing agent in tumours might still further enhance drug202. Another porphyrin derivative (SIM01) in
the outcome of the liposome-mediated PDT. A benzo- dimyristoylphosphatidylcholine iposomes also produces
porphyrin derivative encapsulated in polycation lipo- better results in PDT, mainly due to better accumulation
somes modified with cetyl-polyethyleneimine was used in the tumour (human adenocarcinoma in nude
for antiangiogenic PDT. This drug, encapsulated in such mice)203. Liposomal meso-tetrakis-phenylporphyrin is
liposomes, was better internalized by human umbilical effective in PDT of human amelanotic melanoma in
vein endothelial cells and was found in the intranuclear nude mice204. The interest in this area of liposomology is
region and associated with mitochondria200. The com- still growing.
mercial liposomal preparation of the benzoporphyrin
derivative monoacid ring A (Visudyne; Novartis) is Liposomes modified with cell-penetrating peptides. A
new approach to drug delivery has recently emerged,
which is based on the use of certain viral proteins that
Box 4 | Liposomal formulations for clinical application have the ability to penetrate into cells (the so-called
‘protein transduction’ phenomenon). The transactivating
According to Crommelin and Storm212, the following quality-control assays should be
transcriptional activator (TAT) protein from HIV-1
applied to liposomal formulations:
enters various cells when added to the surrounding
• Basic characterization assays: pH; osmolarity; trapped volume; phospholipid media205. Recent data indicate that there is more than
concentration; phospholipid composition; phospholipid acyl chain composition; one mechanism used by cell-penetrating peptides and
cholesterol concentration; active compound concentration; residual organic solvents proteins (CPP) and CPP-mediated intracellular delivery
and heavy metals; active compound/phospholipid ratio; proton or ion gradient of various molecules and particles. TAT-mediated
before and after remote loading.
intracellular delivery of large molecules and nanoparticles
• Chemical stability assays: phospholipid hydrolysis; non-esterified fatty acid occurs through energy-dependent macropinocytosis,
concentration; phospholipid acyl chain auto-oxidation; cholesterol autoxidation; active with subsequent enhanced escape from endosome into
compound degradation. the cell cytoplasm206, whereas individual CPPs or CPP-
• Physical characterization assays: appearance; vesicle size distribution; sub-micron conjugated small molecules penetrate cells via electro-
range; micron range; electrical surface potential and surface pH; zeta potential; static interactions and hydrogen bonding and and the
thermotropic behaviour, phase transition, and phase separation; percentage of free drug. penetration does not seem to be associated with meta-
• Microbiological assays: sterility; pyrogenicity (endotoxin level). bolic energy (that is, it is a purely physical, not biological,

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process)207. Traversing cellular membranes represents a covalent coupling of TAT-peptides to microparticu-

major barrier for the efficient delivery of macromole- late drug carriers could provide an efficient tool for
cules into cells, and therefore CPPs, whatever their the cytosolic delivery of various drugs and DNA in
mechanism of action, could serve to transport various vitro and even in vivo in certain protocols of local
drugs and even drug-loaded pharmaceutical carriers treatment.
into mammalian cells in vitro and in vivo. It has been
demonstrated that relatively large particles, such as Conclusion
liposomes, can be delivered into various cells by several The development of ‘pharmaceutical’ liposomes is
TAT-peptide or other CPP molecules attached to the currently a growth area. The increasing variety of sug-
liposome surface208–210. Complexes of TAT-peptide gested applications, and encouraging results from early
liposomes with a plasmid (plasmid pEGFP-N1, which clinical applications and clinical trials of different
encodes the green fluorescent protein) were used for liposomal drugs (TABLE 1), need to be augmented by
successful in vitro transfection of various tumour and simple production processes and a variety of quality-
normal cells, as well as for in vivo transfection of control assays for liposomal formulations (BOX 4).
tumour cells in mice bearing Lewis lung carcinoma211. With these requirements met, we are surely likely to
Whatever mechanisms underlie the TAT-mediated see more liposomal pharmaceuticals on the market in
delivery of large cargo such as liposomes into cells, the the foreseeable future.

1. Symon, Z. et al. Selective delivery of doxorubicin to patients 20. Torchilin, V. P. & Trubetskoy, V. S. Which polymers can make of polymeric chains combines effective target binding with
with breast carcinoma metastases by stealth liposomes. nanoparticulate drug carriers long-circulating? Adv. Drug long circulation times. Biochim. Biophys. Acta 1149,
Cancer 86, 72–78 (1999). Deliv. Rev. 16, 141–155 (1995). 180–184 (1993).
2. Perez, A. T., Domenech, G. H., Frankel, C. & Vogel, C. L. 21. Allen, T. M. & Hansen, C. Pharmacokinetics of stealth versus A good paper on long-circulating immunoliposomes
Pegylated liposomal doxorubicin (Doxil) for metastatic breast conventional liposomes: effect of dose. Biochim. Biophys. with specific ligands attached to distant termini of
cancer: the Cancer Research Network, Inc., experience. Acta 1068, 133–141 (1991). liposome-grafted polymeric chains.
Cancer Invest. 20 (Suppl. 2), 22–29 (2002). 22. Maeda, H., Sawa, T. & Konno, T. Mechanism of tumor- 36. Abra, R. M. et al. The next generation of liposome delivery
3. O’Shaughnessy, J. A. Pegylated liposomal doxorubicin in targeted delivery of macromolecular drugs, including the systems: recent experience with tumor-targeted, sterically-
the treatment of breast cancer. Clin. Breast Cancer 4, EPR effect in solid tumor and clinical overview of the stabilized immunoliposomes and active-loading gradients.
318–328 (2003). prototype polymeric drug SMANCS. J. Control. Release 74, J. Liposome Res. 12, 1–3 (2002).
4. Schwonzen, M., Kurbacher, C. M. & Mallmann, P. Liposomal 47–61 (2001). 37. Torchilin, V. P. et al. p-Nitrophenylcarbonyl-PEG-PE-
doxorubicin and weekly paclitaxel in the treatment of One of the key publications on the mechanism and liposomes: fast and simple attachment of specific ligands,
metastatic breast cancer. Anticancer Drugs 11, 681–685 significance of the enhanced permeability and including monoclonal antibodies, to distal ends of PEG
(2000). retention (EPR) effect. chains via p-nitrophenylcarbonyl groups. Biochim. Biophys.
5. Goncalves, A. et al. Phase I study of pegylated liposomal 23. Zalipsky, S., Qazen, M., Walker, J. A., Mullah, N., Quinn, Y. P. Acta 1511, 397–411 (2001).
doxorubicin (Caelyx) in combination with carboplatin in & Huang, S. K. New detachable poly(ethylene glycol) 38. Torchilin, V. P. & Klibanov, A. L. in Phospholipid Handbook
patients with advanced solid tumors. Anticancer Res. 23, conjugates: cysteine-cleavable lipopolymers regenerating (ed. Cevc, G.) 293–321 (Marcel Dekker, New York, 1993).
3543–3548 (2003). natural phospholipid, diacyl phosphatidylethanolamine. 39. Torchilin, V. P., Weissig, V., Martin, F. J. & Heath, T. D. in
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