Review Article
Advances in Liposomal Drug Delivery System in the Field of Chemotherapy
Ying-Jie Hu1#, Fan Zeng2#, Rui-Jun Ju3 and Wan-Liang Lu1*
1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug System, State Key Laboratory of Natural and Biomimetic Drugs, and School of Pharmaceutical Sciences, Peking University, China
2Beijing Neurosurgical Institute, China
3Department of Pharmaceutical Engineering, Beijing Institute of Petrochemical Technology, China
#Authors contribute to this paper equally
*Corresponding author: Wan-Liang Lu, School of Pharmaceutical Sciences, Peking University, Xueyuan Road 38, Beijing 100191, China
Published: 13 Sep, 2016
Cite this article as: Hu Y-J, Zeng F, Ju R-J, Lu W-L.
Advances in Liposomal Drug Delivery
System in the Field of Chemotherapy.
Clin Oncol. 2016; 1: 1092.
Abstract
Liposomal drug delivery system has experienced nearly 50 years from laboratory research to
clinical application. It has made evident breakthrough and innovation in the fields of anticancer,
anti-infection and pain management. The objectives of this study were to briefly summarize and
review the major fabrication approaches, progresses in the early studies, and clinical application of
drug liposomes for chemotherapy. The manufacturing technology of drug liposomes is becoming
mature now, consisting of film dispersion, reverse-phase evaporation, chemical gradient loading
and the other encapsulation methods. A number of liposomal strategies have been developed to
improve efficiency and function of drug delivery through passive, active and physicochemical
targeting approaches. Some new liposomal formulations are undertaking laboratory evaluations.
With regard to clinical application, tens of drug liposomes have been approved for clinical use
meanwhile a number of drug liposomes are undergoing clinical trial evaluations. During clinical
trials and uses, the liposomes have been evidenced having an optimal drug delivery efficiency and
better efficacy, despite the anticancer drug liposomes may lead to new side effects like hand-foot
syndrome. The drug liposomes can be enriched into the tumor site, hence demonstrating a better
efficacy and a reduced adverse reaction such as cardiotoxicity. Besides, the liposomal formulations
are capable of potentiating efficacy of anticancer drugs by circumventing multidrug resistance of
cancers and cancer stem cells, and by transferring drug across the blood-brain barrier (BBB). These
new functions have been evidenced in laboratory observations but await for clinical confirmation.
The review demonstrates that the liposomes are useful and promising drug delivery systems in the
field of chemotherapy.
Keywords: Liposomes; Drug delivery; Advance; Chemotherapy; Review
Introduction
During the latest decades, the comprehensive strategy which includes surgery, chemotherapy,
radiotherapy, and immunotherapy has been used in treatment of various cancers. The combination of
treatment plays an important role in enhancing the recovery rate and life quality of patients suffered
with this severe illness. However, there exist in scientific and technical issues during treatment. The
relapse and metastasis occur in the most of clinical cases. These are because the surgery is unable to
remove all cancer cells while the radiation treatment faces the same situation. In addition, in case
of the deteriorated health status, immunotherapy has a limited efficacy in improving the immune
system of patients in a short term.
As a fundamental approach, chemotherapy still plays a crucial role in eliminating cancer cells.
However, there have a number of obstacles to acquire a successful chemotherapy. On the first
hand, an anticancer drug has difficulty in maintaining an effective therapeutic concentration in the
tumor site but widely distributes in systemic organs and tissues, hence leading to severe damages
to healthy tissues and immune system. On the other hand, the multidrug resistance has been
experienced in clinical chemotherapy, and the resistance remains to a major problem in eliminating
cancer and cancer stem cells, resulting in a poor clinical prognosis. Furthermore, the physical and
chemical properties of anticancer drugs significantly affect the treatment outcomes. Consequently,
the development of suitable drug delivery systems is a pressing mission, and the significance for
studying new drug delivery strategies are no less favorable than that for hunting high performance
of new drug chemical entities.
As drug carriers, liposomes have been demonstrated to be the
useful delivery systems in improving unfavorable pharmacokinetics,
enhancing efficacy for removing cancer and cancer stem cells, and
reducing systemic side effects [1].
Liposomes are drug-loaded tiny capsules with bilayer membrane
structure, which are mainly made up of phospholipids and cholesterol
[2]. When amphipathic molecules phospholipids are dispersed in
water, the hydrophobic parts of molecules tend to gather together,
while the hydrophilic parts expose towards water, thereby forming
the round-shape vesicles with bilayer structure (Figure 1). This
construct can be used for encapsulation of pharmaceutical agents,
which a hydrophilic agent is entrapped into the aqueous vesicle
core of liposomes, while a hydrophobic agent inserted into the lipid
bilayers.
As drug delivery vehicles, liposomes are able to avoid the direct
exposure of drug, hence lowering drug degradation and blood
toxicity. Furthermore, the pegylated liposomes provide a prolonged
circulation in blood system through avoiding the rapid clearance
of reticuloendothelial system (RES) [3], and accumulate more into
cancer tissue by the enhanced permeability and retention (EPR)
effects [4,5]. Besides, a varied particle sizes of the liposomes change
the distribution of drug in tissues by a physical retention due to
the difference in compact degrees of each organ tissue. As a result,
liposomes are able to reduce the cardiotoxicity of doxorubicin
through this mechanism [6]. In addition to these favorable features,
more and more the modified liposomes have been fabricated to
reach unique actions for the purposes of transferring drug across the
physiological barrier, enhancing the uptake of drug-resistant cancer
cells, killing the “dormant” cancer stem cells, and interfering the
critical life signaling pathways or targets, thus not only eradicating
cancer cells themselves but also recovering the “soil” for growth of
health body cells.
Up to date, a dozen of liposomal drug formulations have been
applied to clinical treatments of cancer and other diseases, such as
Doxil for treatment of cancer [7], Depodur for relief of pain [8], and
AmBisome for treatment of fungal infection [9], etc. Moreover, the
laboratory studies show that the nanostructured liposomal drug
delivery system provides a promising strategy in cancer treatments.
In this review, the advances in drug liposomes are briefly summarized
and commented aiming at retrospection and prospection of the
development in this field.
Figure 1
Fabrication of Drug Liposomes
Film dispersion method
Film disperse method is also known as the Bangham method
or thin film hydration method and it is one of the most widely used
techniques for the formation of liposomes [10,11]. Film disperse
method is a fabrication process in which phospholipids with
lipophilic drugs are dissolved in appropriate amount of chloroform
or other solvents, and then the solvent is evaporated to form a lipid
film. A buffer solution containing water soluble drug is added to
the lipid film with shaking, yielding drug-loaded liposomes with a
particle size range of 1-5 μm. The liposome suspension needs to be
further treated by ultrasound or through the membrane extrusion
to make the particle size of liposomes uniform. To lower down and
homogenize the particle size of liposomes, several methods can be
selected, including ultrasonic method, film extrusion, and French
film extrusion method, etc.
Reverse-phase evaporation method
The reverse-phase evaporation process was first described by
Szoka and Papahadjopoulos, and it is based on the formation of
drops of water that are surrounded by lipid and dispersed in an
organic solvent, referred to as inverted micelles [12]. The method
is a preparation process in which phospholipid membrane material
is dissolved in an organic solvent (such as chloroform and ether),
and then aqueous drug solution is added to form W/O emulsions
under ultrasonic treatment. The organic solvent is removed vacuum
evaporation to yield liposomes. The liposomes prepared by reversephase
evaporation method are usually large unilamellar liposomes.
The problem of residual organic solvents can be solved by using
supercritical CO2, instead of organic solvents, known as supercritical
reverse evaporation method [13,14].
Chemical gradient methods
pH gradient method: pH gradient method is an active
encapsulation method. In this method, blank liposomes are firstly
prepared by film dispersion method, then the pH value of the
aqueous phase of liposome vesicles are adjusted to form a pH gradient
difference between internal and external vesicles, and the weak acid
or alkaline agents may be encapsulated in the internal phase of
liposomes in the form of ions by using the pH gradient (Figure 2).
This method makes it possible for preparing liposomes with high
drug entrapment efficiency. However, as the method is dependent
on drug structure, it cannot be applied to the drugs with arbitrary
structures. The active encapsulation methods are also known as
the remote loading methods, consisting of pH gradient method for
alkaline drugs, ammonium sulfate transmembrane gradient method
for alkaline drugs, and calcium acetate gradient method for weak
acidic drugs.
Ammonium sulfate gradient method: Ammonium sulfate
gradient method is designed according to the principle of chemical
equilibrium, and also an active encapsulation method. The
general process of the method could be described as the following
procedures (Figure 3). Firstly, blank liposomes are prepared by
using film dispersion method with ammonium sulfate solution as
the hydration solution. Secondly, the blank liposomes are dialyzed
in the dialysis tubing with phosphate buffered saline to remove the
ammonium sulfate outside, thus forming an ammonium sulfate
gradient between two sides of liposome vesicles, namely, the inside
has a high concentration of ammonium sulfate while the outside has
lower one. Finally, active encapsulation is achieved by incubation
with amphipathic weak bases solution in water bath at 60°C with
continually shaking for 20 min [15]. In this method, the stability
of the ammonium ion gradient is related to the low permeability
of its counterion, the sulfate, which also stabilizes anthracycline
accumulation for prolonged storage periods (> 6 months) due to the
aggregation and gelation of anthracycline sulfate salt [16].
Calcium acetate gradient method: The fabrication of calcium
acetate gradient method is briefly described as the following
procedures. Blank liposomes containing calcium acetate solution are
prepared by thin film dispersion method, and then a concentration
gradient is formed by removing the external calcium acetate of
liposomes. The calcium acetate concentration gradient that the
internal concentration is higher than the external one is produced
via the transmembrane movement of calcium acetate so that a large
number of protons transfer from the liposome interior to the outside,
forming a pH gradient. The method is suitable for encapsulating weak
acidic drugs. The drugs can bind with the liposome internal calcium
ion to form less soluble calcium salt, and prevent the drugs from
passing through the phospholipid bilayer, thereby improving the
entrapment efficiency and reducing drug leakage [17-19].
Additional methods
There are many other methods in the preparation of liposomes.
The ethanol and ether injection method can be used for dissolving
the lipids into an organic phase, followed by the injection of the
lipid solution into aqueous media, hence forming liposomes [20,21].
Besides, the heating method has been developed to produce blank
liposomes by hydration of phospholipids in an aqueous solution
containing 3% glycerol through raising the temperature to 60°C
or 120°C [22]. During preparation, the drug was incorporated into
blank liposomes using the heating method by addition of drug to
the solution at different temperature stages, including the beginning,
above the transition temperature of the lipids and the ambient
temperature. In addition, the freeze-drying of mono-phase solution
method for encapsulation of heat sensitive drugs such as DNA, and
the produced drug liposomes can be stored for a long time in a sealed
container [23].
Figure 2
Figure 3
Advance In Anticancer Drug Liposomes
Liposomes have demonstrated to be an excellent carrier system
for a variety of applications and are particularly ideal for anticancer
drug delivery because of the similarity to natural cells, and multiple
potentials in cancer chemotherapy.
Passive targeting drug liposomes
The passive targeting drug liposomes are the drug liposomes
with a mechanism by which drug liposomes can be preferentially
delivered to target cells in vivo, especially cells in cancer tissue.
Based on their physicochemical properties, such as suitable particle
size, drug liposomes can escape from nonspecific trapping by other
tissues but passively accumulate in target tissues with the circulation
of blood. Because cancer tissues are characterized by a high interstitial
pressure, enhanced vascular permeability and retention (EPR effect,
Figure 4), and the lack of functional lymphatic drainage, the passive
targeting has become a useful approach for drug delivery.
Regular drug liposomes: The regular drug liposomes are mainly
composed of phospholipid and cholesterol, without any modification.
After injection, the regular drug liposomes are mainly concentrated
in the liver, spleen, lung, lymph node, bone marrow and other
reticuloendothelial rich locations. In addition, they also aggregated
in the locations of inflammation, infection and vasculatures of solid
tumors, exhibiting passive targeting effect. Based on laboratory
evaluations, regular drug liposomes have shown multi-potentials in
the prevention and treatment of cancers.
Imanaka et al. [24] reported that the beta-sitosterol liposomes
exhibited the chemopreventive effect of tumor metastasis by oral
delivery. Their results showed that the amount of immune response
cytokines, such as IL-12 and IL-18, were increased in the small
intestine after the intake of liposomes. After administration of
the liposomes for 7 days, natural killer cell activity in the mice was
increased, suggesting that the immune surveillance activity of mice
was enhanced by the intake of beta-sitosterol liposomes. Furthermore,
daily intake of beta-sitosterol liposomes prevents the metastasis of
tumor.
Igarashi et al. [25] demonstrated that the liposomalization of
photosensitizer could be used to enhance the therapeutic efficacy
of photodynamic therapy for gastrointestinal tumors. The results
showed that the volume of necrotic tumor tissue was significantly
higher in the group of photofrin liposomes group than that in the
group of free photofrin. Moreover, the apoptotic index of the tumor
was also significantly higher after treatment with photofrin liposomes.
Their results revealed that the liposomalization of photosensitizer
were able to increase accumulation of the liposomes in tumor site,
with a resultant enhancement effect of photodynamic therapy.
Long-circulating drug liposomes: Long-circulating liposomes
can be prepared by coating liposome surface with a hydrophilic layer
of oligosaccharides, glycoproteins, polysaccharides and synthetic
polymers in order to achieve a continuous action on the tumor
tissue. Long circulating liposomes are also called stealth liposomes or
sterically stabilized liposomes [26]. When modified with ganglioside,
phosphatidylinositol or polyethylene glycol lipid derivatives, the
liposomes will be densely covered by the conformational clouds
around the surface of liposomes, hence playing a protective action
for liposomes. This steric protection is associated with the polymer
flexibility, steric hindrance and hydrophilicity, and it can prevent the
recognition of liposomes by opsonin and reduce the rapid clearance
of the liposomes by reticuloendothelial system [27,28]. Therefore,
the modified liposomes can last for a longer time in the circulation
system, and extend action time of drug during therapy.
Cogswell et al. [29] investigated that the long-circulating
econazole liposomes had a superior efficacy in treatment of breast
cancer by parenteral administration. In the study, long-circulating
econazole liposomes were prepared by a novel micelle exchange
technique in incorporating drug into the lipid bilayer of preformed
liposomes using a polyethylene glycol-linked phospholipid,
distearoylphosphatidyl ethanolamine (DSPE-PEG). This method
allowed for stable and efficient drug incorporation. Results showed
that the liposomes had a long-circulating effect and a better efficacy
but did not induce significant liver toxicity, renal toxicity or weight
loss in human breast cancer MCF-7 cells xenografted model in mice.
Furthermore, Fanciullino et al. [30] developed a kind of
pegylated liposomes of 2'-deoxyinosine (d-Ino), which was used
as a 5-fluorouracil (5-Fu) modulator, and evaluated its efficacy in
vitro and in tumor-bearing mice and its pharmacokinetics in rats.
The deoxyinosine liposomes exhibited a strong potentiation effect
in a combination use of 5-Fu in vitro, and displayed a 7-fold longcirculating
effect in animals. In tumor-bearing mice, the combination
of deoxyinosine liposomes with 5-fu led to 70 % of tumor reduction
with a doubling median survival time as compared to the control. In
addition to the long-circulating effect, the deoxyinosine liposomes
had demonstrated a capability to reverse the 5-Fu resistant colon
cancer SW620 cells.
In the latest ten years, the long-circulating liposomes have been
further assigned to be multifunctional long circulating liposomes,
such as pH sensitive or temperature sensitive long -circulating
liposomes, as discussed below.
Active targeting drug liposomes
Liposomes with passive targeting alone cannot reach the
selectivity of tumor tissues. Combining passive targeting with active
targeting brought a new strategy for chemotherapy, which could
lead to promoting tumor specificity as well as diminished systemic
adverse effects. Recently, efforts of scientists have been made on the
development of active targeting liposomes, which could target tumor
cells and cellular organelles. Enhanced intracellular uptake of the
active targeting liposomes is usually achieved by interaction of the
overexpressed receptors with ligands or the specific tumor antigens
with monoclonal antibodies.
Ligand-mediated liposomes: There are many receptors that
are overexpressed on the tumor cells but less expressed or nonexpressed
on the normal body cells, such as integrin receptors,
transferrin receptors, folate receptors, lectin receptors, and low
density lipoprotein receptors, etc. To selectively target tumor cell,
special ligands that could specifically bind to these receptors are
modified on the surface of liposomes. For instance, folate receptor is
overexpressed on many epithelial cancers, and it has been exploited
as the action target on cancer cells. As an example, folate receptor
is overexpressed on ovarian cancer cells, thus allowing the binding
of folate with folate receptor, and enhancing the internalization of
folate through folate receptor-mediated endocytosis. Accordingly,
liposomes modified with the folate demonstrated an active targeting
behavior on the ovarian cancer cells.
Zeng et al. [31] developed a kind of functional vincristine plus
dasatinib liposomes modified with a targeting molecule DSPEPEG2000-
c (RGDyK) for eradicating triple-negative breast cancer
(TNBC). C (RGDyK) is a cyclic peptide that has a specific affinity
with integrin receptor. It is found that the integrin receptor is
overexpressed on many malignant cancer cells, including TNBC
cells. Consequently, this cyclic peptide acts as a targeting molecule
in modifying drug liposomes for binding with integrin receptor on
the cancer cells.
Apart from the single modification, there were also multiple
modifications with more than one ligand, which could avoid the
heterogeneity of cancer cells. For example, Sriraman et al. [32]
developed a kind of pegylated doxorubicin liposomes modified
with folate (F), transferrin (Tf) or both (F+Tf). The dual-targeted
liposomes (F+Tf) showed a 7-fold increase in cell association
compared to either of the single-ligand targeted ones in human
cervical carcinoma (HeLa) cells.
Antibody-mediated liposomes: Monoclonal antibodies (mAbs)
and their derivatives are often used as the targeting molecules for
preparing the active targeting liposomes. The liposomes modified
with mAbs or their derivatives are defined as immunoliposomes,
which can be designed to improve pharmacological properties
of conventional anticancer drugs. The intracellular transport of
immunoliposomes could be increased by the antibody-antigen
interaction. To some extent, immunoliposomes can be described as a
kind of novel targeting liposomes with high active affinity to specific
cancer cells. A number of methods have been reported for modifying
antibodies onto the surface of the drug liposomes.
Huwyler et al. [33] developed a kind of daunorubicin
immunoliposomes by introducing anti-transferrin receptor OX26
antibody with PEG2000 on the liposomes, [34] which successfully
resulted in an enhanced accumulation at brain glioma sites.
Besides, Loureiro et al. [35] designed and prepared dual targeting
immounliposomes by modification with two antibodies, namely
the OX26 antibody and the anti-amyloid beta peptide antibody
(19B8MAb), as nanocarriers of drugs for Alzheimer's disease
therapy. Results showed that the established immounliposomes
could effectively cross the blood brain barrier (BBB) (Figure 5) and
concentrate at the Alzheimer area.
Physicochemical targeting liposomes
Although liposomal formulations of chemotherapy demonstrated
a significant reduction in systemic toxicity, the enhancement of
therapeutic efficacy has not fully reached. Many advanced drug
release strategies have been investigated ever since the liposomes
were introduced as drug delivery carriers. Such strategies include
utilization of pH, temperature, and enzyme sensitive systems, etc.
pH sensitive liposomes: In order to improve the anticancer efficacy
of liposomes, several passive targeting, active targeting methods, and
stimulus-responsive methods, have been developed [36]. Among
these stimulus-responsive systems, pH-sensitive liposomes have
attracted much interest [37]. Tumor microenvironment has been
confirmed to have mildly acidic (pH 6.0-7.0) condition due to the
glycolytic metabolism of glucose to lactate in tumor tissues, and this
lowered pH value from that of normal tissues (pH 7.4) has been used
for constructing pH sensitive drug liposomes [38].
Wang et al. [39] developed a pH sensitive drug delivery system,
octylamine-graft-PASP modified liposomes (OPLPs). The OPLPs
sustained a slow and steady release in the physiological pH 7.4
environment, while provided a fast release in sub-acid environment
(pH 6.0 of resembled tumor tissues). The in vitro tumor cytotoxicity
studies revealed that the tumor cells treated with OPLPs survived
only 35.0% after 48 h whereas normal cells survived 100 % in the
same condition. Júnior et al. [40] evaluated the tissue distribution of
stealth pH sensitive liposomes containing cisplatin (SpHL-CDDP),
compared with free cisplatin (CDDP), in solid Ehrlich tumor-bearing
mice. The longer circulation of SpHL-CDDP led to a higher blood
drug exposure and a higher accumulation of CDDP in tumor.
However, after internalization by tumor cells, the pH sensitive
liposomes would be captured by tumor cellular endosomes, in which
more acidic microenvironment in endosomes (pH 4.5-5.5) may lead
to degradation of the liposomes by endosomal enzyme and hydrolase.
For this reason, endosomal pH sensitive liposomes have been
formulated for endosomal escape. Moku et al. [41] reported a kind of
endosomal pH sensitive liposomes in which glutamic acid backbonebased
cationic amphiphiles play multifunctional roles in enhancing
cellular uptake by guanidine moiety and improving the endosomal
escape by histidine moiety. The endosomal pH-sensitive liposomal
drug carriers not only effectively deliver anti-cancer drugs to mouse
tumor, but also significantly contribute to enhancing anticancer
efficacy.
Temperature-sensitive liposomes: Tumor tissues are usually
exhibiting hyperthermia due to the rapid metabolism, and
accordingly, the fever is often occurred in the tumor sites similar to the
inflammatory response. This phenomenon inspired the development
of temperature-sensitive liposomes. In case of temperature rising,
the temperature-sensitive liposomes can release anticancer drug
at tumor sites under the condition of pathological hyperthermia
or external warming by which solid tumors can also be heated by a
controlled device with an external energy source, such as infrared
ray irradiation. This is because temperature-sensitive liposomes are
composed of lipids that could undergo a gel-to-liquid phase transition
at a critical temperature (transition temperature, Tm). Afterwards,
double molecular chain of phospholipids would gain a higher degree
of disorder and activity along with the increase of temperature, hence
resulting in the release of drug from the liposome vesicles.
Yatvin et al. [42] described the temperature-sensitive liposomes
which were able to release a hydrophilic drug when the temperature was
increased a few of degrees above physiological temperature. The lipid
materials for fabricating liposomes were based on 1,2-dipalmitoyl-snglycero-
3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-
phosphocholine (DSPC). Similarly, Kakinuma et al. [43] developed
a kind of temperature-sensitive liposomes containing cis-platinum
for treatment of brain glioma-bearing rats. And the results showed
a significantly increased concentration of cis-platinum in the brain
glioma sites.
Enzyme-sensitive liposomes: Recently, the over-expressed
enzyme systems in tumor microenvironment have been utilized to
trigger the release of anticancer drug from liposomes, such as matrix
metalloproteinases. Mura et al. [44] established enzyme-sensitive
liposomes were by coupling a monoclonal antibody 2C5 with the
polyethylene glycol chain via an MMP2-cleavable linker. Similarly,
the over-secretion of phospholipase A2 (sPLA2) has been found in
tumor site and can be used to initiate drug release of enzyme-sensitive
liposomes. Hansen et al. [45] evidenced that the activity of human
sPLA2 was highly sensitive to phospholipid acyl-chain length and
negative surface charge density of the liposomes, thereby triggering
drug release of enzyme-sensitive liposomes.
Physical adsorption-mediated liposomes: To adsorb onto the
membrane of cancer cells, a targeting effect can be achieved by
physical adsorption-mediated liposomes, which utilizes cationic
material to modify the surface of liposomes to produce a kind of
positively charged liposomes. The electropositive liposomes are able
to strongly adsorb onto the electronegative cell membrane of cancer
cells. Furthermore, after uptake by cancer cells, the cationic liposomes
can further accumulate into mitochondria of living cells in response
to mitochondrial membrane potential (Figure 6) [46-52].
Wang et al. developed mitochondrial targeting resveratrol
liposomes by modifying a conjugate of dequalinium (DQA) with
polyethylene glycol distearoylphosphatidylethanolamine (PEG2000-
DSPE). The results exhibited a significant antitumor efficacy in either
cancer cells or drug resistant cancer cells [53]. In addition, Ma et al.
developed mitochondrial targeting berberine liposomes by modifying
DQA-PEG2000-DSPE [54]. The mitochondrial targeting berberine
liposomes could transport across cancer stem cell membrane, and
selectively accumulate into the mitochondria of cancer cells. When
co-treatment with paclitaxel liposomes, mitochondrial targeting
berberine liposomes significantly potentiated the anticancer efficacy
in human breast cancer stem cells xenografts in nude mice.
Multifunctional liposomes: Multifunctional liposomes have
been developed in the latest years to achieve multiple purposes by
which one liposome formulation is able to reach a comprehensive
objective by combing passive, active and physicochemical targeting
effects. For example, Li et al. developed a kind of multifunctional
targeting paclitaxel plus artemether liposomes for treatment of brain
glioma [55]. In this construct, paclitaxel was used as the anticancer
drug and artemether used as the regulator of apoptosis and inhibitor
of vasculogenic mimicry channels. Two functional materials,
mannose-vitamin E derivative conjugate (MAN-TPGS1000) and
dequalinium-lipid derivative conjugate (DQA-PEG2000-DSPE), were
used to enhance the capabilities of liposomes in transferring drug
across blood-brain barrier (BBB), eliminating brain glioma stem
cells and destroying vasculogenic mimicry channels (Figure 7). The
transport mechanism of the liposomes across the BBB was associated
with receptor-mediated endocytosis by MAN-TPGS1000 conjugate via
glucose transporters, and adsorption-mediated endocytosis by DQAPEG
2000-DSPE conjugate via electric charge-based interaction.
Furthermore, Kono et al. [56] designed a kind of multifunctional
liposomes which combined the properties of active targeting and
temperature-sensitive liposomes, and were used for treatment and
diagnosis of human epidermal growth factor 2 (Her-2) positive
cancer by imaging, such as ovarian cancer and breast cancer. The
liposomes were functionalized with thermosensitive poly[2-(2-
ethoxy)ethoxyethyl vinyl ether] chains for triggering drug release
of liposomes (approximately 38°C), with conjugation of antibody
trastuzumab for targeting Her-2 positive cancer, and with entrapment
of indocyanine green for diagnosis by near-infrared fluorescence
imaging. The liposomes could retain drug under physiological
temperature while release drug immediately at a slightly higher
temperature in tumor, and exhibited significant ability in targeting
Her-2 positive cancer cells.
Figure 4
Figure 5
Figure 6
Figure 6
Covalently coupling conjugate for mitochondrial drug delivery [46-52].
Notes: A. The attachment of a lipophilic cation such as triphenyl phosphonium (TPP) improves the uptake of an attached molecule into mitochondria driven by
the large membrane potential across the mitochondrial membrane. B. The attachment of a mitochondrial targeting signal peptide (MTS) to ODN or PNA allows
mitochondrial delivery via TOM/TIM complex. C. A protein conjugated with protein transduction domain peptide (PTD) is delivered to mitochondria without passing
through the classical protein import pathway.
Figure 7
Figure 7
Characterization of targeting molecular materials and liposomes [55].
Notes: MALDI-TOF-MS spectra of TPGS1000 (A1) and MAN-TPGS1000 conjugate (A2); a schematic representation of the functional targeting paclitaxel plus
artemether liposomes (B); AFM images of paclitaxel liposomes (C1) and functional targeting paclitaxel plus artemether liposomes (C2).
Application of Drug Liposomes
Liposomes were first proposed by Bangham et al. [57] in 1965.
The study showed that the multilayer vesicles were spontaneously
formed with onion-like structure when phospholipids were dispersed
in water. This experiment makes people aware of the biodegradability
and biocompatibility of liposomes, and lays a foundation for the
liposomes to be used as drug carriers. In 1971, Gregoriadis et al.
[58] firstly encapsulated amyloglucosidase and 131I-albumin into
liposomes. After intravenous injection into rats, the liposomes were
mainly distributed in liver and spleen tissues. It indicated that the
liposomes could be used for loading bioactive substances, and had
a distinctive distribution in specific tissues, i.e., a targeting potential.
With laboratory study in-depth for five decades, a variety of liposomes
have been successfully used in the clinical practice, and there are still
many liposomes are undergoing clinical trial evaluations.
In 1995, doxorubicin liposomes were approved by Food and Drug
Administration (FDA) for treatment of various types of cancer [7].
It is a kind of decorated liposomes with hydrophilic polymer, which
is conjugated by polyethylene glycol with distearoylphosphatidyl
enthanolamine (PEG2000-DSPE). The use of PEG2000-DSPE conjugate
is used to prevent the adsorption of plasma proteins onto liposomes
or to prevent opsonization, thereby avoiding the rapid clearance of
liposomes by reticuloendothelial system. The pegylated liposomes
are able to extend the circulating time in blood circulation and to
accumulate more into the specific tissues such as solid tumor tissue.
Because of the decreased distribution in heart tissue, doxorubicin
liposomes could evidently lower the cardiotoxicity after uses.
Nevertheless, an unexpectedly hand-foot syndrome (HFS) has been
experienced during clinical application of doxorubicin liposomes.
Actually, the pegylated doxorubicin liposomes have become one of the
most common causes of HFS. The risk of developing HFS appears to be
doxorubicin dose-dependent. Drug formulations that prolong serum
drug levels or that concentrate drug at affected sites have higher rates.
This may be one reason why doxorubicin liposomes are associated
with a higher HFS incidence than the standard, nonencapsulated
formulation [59]. In spite of this, pegylated doxorubicin liposomes
are still considered as an efficient drug in tumor therapy.
The success of Doxil® has inspired the research and development
of liposomal drug delivery systems (Table 1) [7,60-77]. With the
gradual study of the liposomes in-depth, a number of drug liposomes
have been approved for clinical uses, such as daunorubicin liposomes
(DaunoXome®) [60], cytarabine liposome (Depocyt®) [61], vincristine
sulfate liposomes (Marqibo®) [64] and nonpegylated doxorubicin
liposomes (Myocet®) [65].
Meanwhile, several regular liposomes in clinical trials are
expected to be approved by drug administration authority soon. A
phase 2 clinical trial showed that the single-agent nanomolecular
liposomal annamycin appeared to be well tolerated, and exhibited
a significant clinical activity as a single agent in treatment of the
refractory adult acute lymphoblastic leukemia [73]. Another phase 2
study indicated that L-NDDP (Aroplatin), a liposomal formulation
of a structural analogue of oxaliplatin, was well tolerated in treatment
of the refractory patients with advanced colorectal cancer, and
demonstrated a positive anti-tumor activity. Further studies of
L-NDDP, preferably in combination with other agents such as
fluoropyrimidines, are warranted [70].
In addition to these, several active targeting liposomes are
undergoing clinical evaluation. MBP-426, a transferrin-mediated
liposomes containing oxaliplatin, is now in phase I trial as second
line treatment for gastric, gastroesophageal and esophageal
adenocarcinomas [72]. The other transferrin-mediated liposomes
that contain P53 plasmid DNA is now in phase Ib trial to treat solid
tumor [77]. Besides, immunoliposomes also have made a lot progress.
MCC-465, a type of pegylated doxorubicin liposomes functionalized
with the F(ab)2 of GAH antibody that show a significant anticancer
activity against GAH-positive colorectal and gastric cancer cells, is
progressed to phase I clinical trial [76].
However, clinical trials also revealed some unexpected adverse
events and results. According to early results of an ongoing phase
II trial, liposomal vincristine (Onco-TCS) was active and well
tolerated in this heavily pretreated population with relapsed non-
Hodgkin's lymphomas, but was neurotoxic in a fraction of patients
heavily exposed to prior neurotoxic agents [69]. The clinical study
of pegylated liposomal cisplatin (SPI-077) showed that SPI-077 was
essentially inactive against squamous cancers of head and neck and
only modestly active in patients with non-small-cell lung cancer
[67,68]. ThermoDox, a thermally sensitive liposomal doxorubicin,
was considered as an efficient agent against liver cancer. However it
was announced that ThermoDox failed in the phase 3 study, and it was
unable to demonstrate a significant improvement in progression-free
survival. Nonetheless, ThermoDox is not given up and a phase 3 study
(OPTIMA) is conducted now to determine whether ThermoDox is
effective in the treatment of non-resectable hepatocellular carcinoma
when used in conjunction with standardized radiofrequency ablation
(sRFA).
Table 1
Concluding Remarks
Up to date, the manufacturing methods of liposomes are becoming more and more mature, including film dispersion, reverse-phase evaporation, chemical gradient loading and the other encapsulation approaches. A variety of liposomal strategies have been developed to improve efficiency and functions of drug delivery through passive, active and physicochemical targeting methods. These new liposomal formulations are undertaking laboratory evaluations, exhibiting a promising and broad prospect, especially in the field of chemotherapy of cancers. In clinical application, tens of drug liposomes have been approved for clinical use meanwhile plenty of drug liposomes are undergoing clinical trial evaluations. During clinical trials and uses, the liposomes have been evidenced having an optimal drug delivery efficiency and a better efficacy, despite the anticancer drug liposomes may lead to new side effect like hand-foot syndrome induced by the pegylated doxorubicin liposomes. The drug liposomes can be effectively accumulated into the lesion or tumor site, demonstrate a better efficacy and a reduced adverse reaction such as cardiotoxicity of doxorubicin. Varying liposomal formulations are useful in improving the property of chemical drug itself. As a typical example, paclitaxel liposomes that are approved by State Food and Drug Administration of China (SFDA) in the 2004 are able to diminish the severe allergy as compared to the regular paclitaxel injection in which an allergic surfactant Cremophor is used as a solubilizer of insoluble paclitaxel. Besides, the liposomal formulations are capable of potentiating the efficacy of anticancer drugs by circumventing multidrug resistance of cancer and cancer stem cells, and by penetrating across biological barriers (the BBB barrier). These new functions have been evidenced in laboratory observations while await for clinical evaluations. It is believed that the liposomal drug delivery systems would have more applications in the field of chemotherapy with the progress of science and the development of pharmaceutical technology.
Acknowledgment
This study was supported by grants from the National Natural Science Foundation of China (No. 81373343), the National Basic Research Program of China (973 program, 2013CB932501) and the Key Grant of Beijing Natural Science Foundation (No. 7131009).
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