Accepted Manuscript

Composition design and medical application of liposomes

Mingyuan Li, Chunyang Du, Na Guo, Yuou Teng, Xin Meng, Hua Sun, Shuangshuang
Li, Peng Yu, Hervé Galons

PII: S0223-5234(19)30007-8
DOI: https://doi.org/10.1016/j.ejmech.2019.01.007
Reference: EJMECH 11014

To appear in: European Journal of Medicinal Chemistry

Received Date: 7 December 2018

Revised Date: 4 January 2019
Accepted Date: 4 January 2019

Please cite this article as: M. Li, C. Du, N. Guo, Y. Teng, X. Meng, H. Sun, S. Li, P. Yu, Hervé. Galons,
Composition design and medical application of liposomes, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.007.

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Composition design and medical application of liposomes

Mingyuan Li, Chunyang Du, Na Guo, Yuou Teng, Xin Meng, Hua Sun, Shuangshuang Li, Peng
Yu*, Hervé Galons*
(China International Science and Technology Cooperation Base of Food Nutrition/Safety and
Medicinal Chemistry, Tianjin International Cooperation Research Centre of Food Nutrition/

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Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science &
Technology, Tianjin, China, 300457)

Abstract

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Liposomes, which possess the properties of nano-scale, biofilm similar structure, excellent
biocompatibility, become more and more useful in the drug development as the delivery system.

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Liposomes are relatively stable, their aqueous phase could contain the hydrophilic drugs and
their phospholipid bilayer should localize the lipophilic drugs. Moreover, their surface-
modifiable characteristics have really extended the liposomes’ application to targeting and

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environmental sensitive delivery system. In order to make the common liposome more fit the
human and animal body’s complex environment, the structural variation strategy in the head, tail
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and bond of lipid molecules have been employed to develop the different functionalized
liposomes-based drug delivery system for the localizable relieve and organ/tissue targeting
relieve. In this paper, we would like to summarize the recent development on the design and
optimization of liposomes, including Long-circulation liposomes, Specific active targeting
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liposomes, Environmental sensitive liposomes, Multifunctional liposomes, and so on. And the
liposome content selection and current status of clinical application are systematically discussed.
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Keywords Liposome; Drug delivery; Composition design; Functional modification; Medical

application
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*Corresponding author.
E-mail address: yupeng@tust.edu.cn (P. Yu), hervegalons@yahoo.com (H. Galons)
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1. Introduction

In 1965, Bangham A [1] first discovered that the phospholipid molecules could
spontaneously form the closed bilayer vesicles in water. Soon after that, liposomes, with the size
of range from 5 to 200 nm [2], were reported to encapsulate hydrophilic or lipophilic drugs in the
aqueous phase or bilayer membrane phase by using the affinity of different parts of vesicles, and
the liposomes were introduced into the field of drug delivery system. Conventional

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chemotherapy to cancer is based on low molecular weight drugs (generally <1000 Da) [3, 4].
Due to their small size, chemotherapeutic agents, such as doxorubicin, vincristine, gemcitabine
or cisplatin, have unfavorable pharmacokinetics and a suboptimal biodistribution, as exemplified

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by a short blood half-life and prominent off-target accumulation in multiple healthy organs (Fig.
1A). This, together with the unspecific mechanism of action of chemotherapeutic drugs and their
large volume of distribution, causes severe side effects, such as myelosuppression, mucositis,

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neurotoxicity, nausea, vomiting and alopecia [5].

Fig 1. was inserted here.

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In the case of liposome encapsulating drugs, kidney excretion can be reduced, and the
blood half-life prolonged (Fig. 1B). Moreover the drug loaded liposomes could be accumulated
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in tumors via the enhanced permeability and retention (EPR) effect, which was first described by
Matsumura and Maeda in 1986 [7]. EPR relies on different specific pathophysiological
characteristics between tumors and healthy tissues. In healthy tissues, low-molecular-weight
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drugs can easily extravasate out of blood vessels, while drug loaded liposomes are unable to do
so due to their size (Fig. 1A). Conversely, the tumors possess the abnormally wide fenestrations
in the blood vessels which allow the extravasation of liposomes. This, together with the absence
of lymphatic drainage, leads to a relatively more effective and selective accumulation of
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liposomes in tumors [8-11].

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Liposomes, as a carrier, have many advantages, such as cell-like membrane structure,

high biocompatibility, low immunogenicity, protection of the drugs or active groups,
prolongation of drug half-life, reducing toxicity and increasing efficiency, and so on. In order to
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obtain better liposome delivery systems, structural modification and surface modification based
on classical lipid molecules have been carried out to generate novel liposomes with specific
biological effects, which greatly expand the application of liposomes in biomedicine [12]. Since
the first liposomes encapsulating drug have entered the clinical trail stage in 1985 [13], more
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than 40 liposome loaded drugs have been successfully marketed or are in different clinical
research stages. In this paper, we would like to summarize the recent development on the
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design and optimization of liposomes, including long-circulation liposomes, Specific active

targeting liposomes, Environmental sensitive liposomes, Multifunctional liposomes, and so on.

2. Design of lipid molecules

Liposomes consist of classical lipid molecules and novel lipid molecules, both of which
belong to amphiphilic molecules, and are generally composed of hydrophilic head, hydrophobic
tail and bonds. The classical lipid molecules generally use glycerol-like molecules as the
backbone, attaching hydrocarbon chains to the two of three branches the glycerol backbone as
the tail and an N-containing group to the other branch as the head.
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2.1. Hydrophilic head of lipid molecules

The hydrophilic heads of lipid molecules can be divided into three types according to
their charges: cationic, neutral and anionic head. Firstly, the cationic head helps to attract the
liposome to the negatively charged cell membrane, thus increasing the cell incorporation rate.
And the cationic head containing primary and secondary amines can escape the lysosome
through the "proton sponge effect" to prevent the lysosome from degrading the therapeutic agent.

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In addition, cationic liposomes loaded with drugs or molecular probes can also absorb negatively
charged biological macromolecules, thus achieving the goal of double loading. DOTAP ((2,3-
dioleoyl-propyl)-trimethylamine), DDAB (dimethyl dioctadecyl ammonium bromide) and CTAB

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(trimethylhexadecyl ammonium bromide) were all changes based on DOTMA (2,3-dioleoyl-
propyl)-trimethylamine bromide), which was invented by Felgner and his colleagues in 1987
(The shape and some steroid hydrophobic domain of cationic lipids were showed as Fig 2 and

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Fig 3) [14-17]. In addition, derivatives with similar physical and chemical properties can be
obtained by subtle modification of the head of monoammonium group. Wolff et al. obtained
DOKIR or DMRIE by substituting hydroxyethyl group for methyl group on ammonium group.
These lipid molecules showed better membrane penetration ability than DOTAP in vitro and in

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vivo [18].
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Fig 2. was inserted here.
Fig 3. was inserted here.
Cationic lipids not only have stronger gene transfer ability, but also have cytotoxicity
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which cannot be ignored. In order to design low toxic and high efficient lipid molecules, it is an
excellent choice to construct lipid molecules with endogenous amino acids. Jiang et al. [19]
chose a second-generation dendrimer as the head consisting of three amino acids, arginine, lysine
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and histidine. Confocal and flow cytometry experiments showed that the lipid molecules headed
by the second-generation arginine showed the best transfection efficiency, thanks to the
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membrane-penetrating peptide-like and antiserum abilities of histidine. It can significantly

enhance the entry of cells, the escape of lysosomes and the ability to enter the nucleus. And it
also showed good transfection effect during in vivo experiment. Through further optimization,
this kind of liposome is expected to have broader application prospects. Different from cationic
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lipids, commonly used nontoxic neutral lipids mainly include phosphatidylethanolamine (PE),
neutral phosphatidylcholine (PC) and cholesterol, such as EPC, DPPC (1,2-Dipalmitoyl-sn-
Glycero-3-Phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DOPE
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(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and so on. While phosphatidylserine (PS) is

the representative of anionic lipids. In particular, the transformation of DOPE is beneficial to
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gene escape from lysosomes and increase the success rate of transfection. The addition of
adjuvant lipids can help stabilize the structure of lipid membranes, resist the damage of serum
proteins to membranes, and adjust membrane transition temperature especially for
thermosensitive liposome.

2.2. hydrophobic tail of lipid molecules

Studies have shown that the hydrophobic tail can affect the volume of hydrophobic cavity
and the ratio of hydrophobic part to hydrophobic part of liposomes, so it also plays an important
role in the structure of liposomes (as shown in Fig 4). Generally speaking, the hydrophobic tail
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part is two kinds of fatty hydrocarbons and cholesterol. In gene delivery, single-tailed lipid
molecules are more toxic and less efficient than double-tailed lipid molecules for most classical
lipid molecules with aliphatic hydrocarbon tails. Tang and Hughes [20] has reported that hexyl
6-(2,5-diamino-valeroxy) esters with single tail have higher transfection efficiency and lower
toxicity than DOTAP with double tail. Unlike other double carbon chain phospholipids, MSPC
(Monostearoylphosphatidylcholine) has only one carbon chain tail. This structure enables the
phospholipid membranes containing MSPC to rapidly change phase and release the encapsulated

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drugs immediately when they reach a certain phase transition temperature. MSPC, also known as
lysophospholipids, is a necessary component of the thermosensitive liposome membrane [21]. In
addition to the number of hydrocarbon chains affecting liposome properties, the tail length and

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saturation are also important factors. Koynova et al. [22] synthesized a series of lipid molecules
with different length and saturation of hydrocarbon chains and evaluated their biological effects.
It was found that the transfection efficiency of the liposome increased with the increase of tail

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length and unsaturation.
Li et al. [23] compared the transfection efficiency of three cholesterol derivatives
(lysinylated cholesterol, histidinylated cholesterol, and argininylated cholesterol), and found that

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the toxicity of cholesterol-tailed liposomes was dozens of times higher than DOTAP. However,
most of the lipid molecules of cholesterol derivatives are inhibitors of protein kinase C (PKC),
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which is a cytotoxic kinase. Therefore, cholesterol derivatives are generally highly cytotoxic.
Kaddah et al. [24] prepared GRs-loaded (GRs: cortisol, cortisone, fludrocortisone acetate,
methylprednisolone, prednisolone and prednisone) liposomes consisting of DPPC and
cholesterol. Fluidity and permeability studies showed that the cholesterol content, GR
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concentration, period of incubation and the experimental protocol design are factors determining
the outcomes of the research.
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Fig 4. was inserted here.

2.3. Bond of lipid molecules

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Usually, the linkage between the hydrophobic and hydrophobic ends of lipid molecules
are ester bonds, ether bonds and amide bonds. Liposomes with ether bonds as linking bonds have
higher transfection efficiency, but the stability and the non-degradation of ether bonds increases
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the cytotoxicity of liposomes. In comparison, liposomes with ester and amide bonds have better
biodegradability and lower cytotoxicity [25-27].
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In order to ensure the stability of liposomes in vivo and further reduce the cytotoxicity of
liposomes, human environment-responsive bonds were added into the structure of liposomes.
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These liposomes are stable in normal tissue environments, but targeted release therapeutic agents
and biodegraded by response to environmentally sensitive keys. As early as 1998, Tang and
Hughes [28] tried to add disulfide bond to lipid molecules. The liposomes made up of these lipid
molecules could degrade and release gene directionally in tumor cells with high glutathione
concentration. The transfection efficiency of the liposomes is 50 times that of the liposomes
made up of DOTAP/DOPE. This structure is still widely used in recent years, liposomes
designed by Wang. et al. [29], for example, also use disulfide bond breaks in cells to release
loaded drugs. There were numerous liposomes designed by using pH gradient change between
normal tissues, tumor tissues and intracellular lysosomes. The commonly used pH-sensitive
bonds are ortho-ester bonds [30], carbamate bonds [31], hydrazone bonds [32] and so on.
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3. Functional modification of liposomes

The tumor tissue is different from other normal tissues because of its too fast growth, the
generation rate of normal vessels is difficult to meet the demand, and the tumor cells themselves
often constitute tubular structure, transporting nutrients, which are called "vasculogenic
mimicry", or generating substandard blood tube rapidly. There are many fissures on both
vascular walls of the tumor tissue, which can reach about 10~780nm. When the liposomes flow

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through the tumor tissue in the blood circulation, they are easy to leak out from the fissure of the
vascular wall to the tumor tissue, then accumulating there. The pathophysiological characteristics
of tumors and their microenvironment can be harnessed for passive targeting of liposomes [33].

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The EPR effect is mainly based on leaky blood vessels within tumors, and is unique to the tumor
tissues [33]. However, liposomes have non-specific adsorption with plasma proteins when
circulating in vivo, and they are easy to be degraded, easily scavenged by macrophages, and have

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low targeting. Therefore, many liposomes have shown good results in cell experiments, but it is
difficult to cope with complex in vivo environment. In recent years, in order to obtain more
effective liposomes in vivo, people began to further optimize the function of liposomes.

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3.1. Long-circulation liposomes
Liposomes are easily ingested by the Mononuclear Phagocyte System (MPS), resulting in a
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rapid decrease in blood drug concentration and eventually accumulation in liver, spleen and other
organs. Obviously, this phenomenon not only reduces the drug distribution in target tissues (non-
MPS-related tissues), but also causes some damage to accumulated organs. In order to solve this
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problem, people first attempted to pre-inject a large number of blank liposomes to block MPS,
and then inject drug-loaded liposomes to extend the circulation time of liposomes. By examining
the distribution of Tc-labeled liposomes in vivo, Kao et al. [34] found that the tissue in the MPS-
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blocked group was 1.5 times more radioactive than that in the non-blocked group, and that the
radiation intensity per gram of tumors was twice as strong as that in other tissues.
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However, for clinical treatment, blocking MPS is not the best solution. Long-circulation
liposomes is a kind of modified liposomes by coating their surface with inert polymeric
molecules, such as oligosaccharides, glycoproteins, polysaccharides, and synthetic polymers [35].
Among these hydrophilic molecules, polyethylene glycol (PEG) polymer is a successful material
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which has been used to stabilize the liposomes. Blume [36] and Klibanov [37] first used
polyethylene glycol (PEG) to modify liposomes. The improved surface properties are associated
with the steric hindrance effect offered by hydrophilic polymer, which could prevent the
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modified liposomes from being rapidly eliminated by the RES [38]. Tang et al. [39] designed
Gambogenic acid-loaded PEGylated liposomes (GNA-PEG-LPs), which was used to reduce the
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toxicity of Gambogenic acid, prolong the half-life and enhance anticancer efficacy. The
pharmacokinetic study showed that the t1/2 and the AUC of GNA-PEG-LPs were higher than
GNA solution approximately 2.84-fold and 2.97-fold, respectively. As successful examples,
several formulations of pegylated doxorubicin liposomes have been approved for treatment of
cancers, such as Caelyx, Myocet, and Doxil [40].
Although great achievements have been made, the long-circulation liposomes exhibit some
drawbacks as well, such as "accelerated blood clearance" (ABC) phenomenon, low cellular
uptake, and poor selectivity in cancer cells. Dams et al. [41] found that PEG liposomes have
ABC phenomenon, which occurred in rats after the first intravenous injection of PEG liposomes,
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and the pharmacokinetic behavior of the liposomes changed abnormally after a certain interval of
time. Lai et al. [42] studied the correlation between Kupffer cells (KCs) and ABC phenomenon.
Effect of Kupffer depletion on ABC phenomenon induced by PEGylated liposomes (PL) and
Kupffer cells-targeted liposomes (MFPL) respectively was evaluated by pharmacokinetics and
biodistribution of liposomes, which demonstrated that the clearance function of KCs to PL and
KCs could participate in the happening of ABC phenomenon.

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3.2. Specific active targeting
Tumor cells and normal cells have many differences in gene and gene expression, this make
cell surface or blood vessel surface of tumor tissues have a series of specific and over expressed

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receptors (such as folate receptor [43], HER2 receptor [44], and transferrin receptor [45], et al.).
And these receptors are closely related with tumor growth and proliferation, such as
incontrollable malignant tumor proliferation, insensitivity of negative growth regulation,

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angiogenesis, tissue invasion and metastasis, apoptosis escape, and other physiological
characteristics [46]. Based on this, people designed highly efficient targets to bind with highly
expressed receptors in tumor tissues, such as antibodies, small-molecule drug, polysaccharides,

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proteins, polypeptides, and oligonucleotide aptamers are described below. When we attach these
specific targets to liposomes, they become active-targeting nanocarriers to tumor tissues [47].
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Antibodies were first used in the design of active targeting liposomes. In 1975, it was
reported that the efficiency of IgM modified liposomes was 100 times higher than that of non
modified liposomes [48]. Rodallec et al. [49] developed an immunoliposome in breast cancer,
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combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab.

Immunoliposomes made of natural (ANC-1) and synthetic lipids (ANC-2) were synthesized
using standard thin film method and the antiproliferative activity was tested on human breast
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cancer models ranging from almost negative (MDA-MB-231), positive (MDA-MB-453) to

overexpressing HER2. Both ANC showed similar or higher in vitro antiproliferative activities
than standard treatment. The IC50s for docetaxel combined to free trastuzumab were 8.7±4, 2±0.7
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and 6±2 nM, the IC50s for ANC-1 were 2.5±1, 1.8±0.6 and 3.4±0.8 nM and for ANC-2 were
1.8±0.3 nM, 2.8±0.8 nM and 6.8±1.8 nM with MDA-MB-231, MDA-MB-453 and SKBR3,
respectively.
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Folic acid is a typical representative of small molecule drug target, it has high affinity
between folate receptor and folate binding protein expressed on cell membrane [50]. Folate
receptors were overexpressed in ovarian cancer, uterine cancer, osteosarcoma, meningioma and
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other cancer cells, but not in normal tissues. Moghimipour et al. [51] prepared folic acid-
modified liposomes for tumor targeting of 5-fluorouracil. Folate targeted liposomes showed
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higher cellular uptake, lower IC50 (12.02 µM compared to 39.81 µM for liposomal 5FU and
39.81 µM for free 5FU) and higher ROS production than free drug (62,271.28 vs 2369.55
fluorescence intensity) on cancer cells. Results of hemolysis assay confirmed the blood
biocompatibility of the liposomes. Moreover, folate targeted liposomes showed better tumor
inhibition than free drug (88.75 mm3 tumor volume vs 210.00 mm3) and no tissue abnormalities
were found in histological examination.
Because a large number of receptors specifically binding to glycosyls are expressed on the
membrane of some human tissues and cells, glycosyl-liposomes can be prepared by
incorporating polysaccharides into the bilayer of liposomes. Adulterable polysaccharides include
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galactose, hyaluronic acid, mannose derivatives, branched-chain amylose, dextran, etc. Ravar et
al. [52] successfully enhanced the endocytosis of paclitaxel liposomes with liposomes coated
with hyaluronic acid, and in vivo tests in mice found that the accumulation of targeted liposomes
in tumor tissue was higher than that of drugs alone or non-targeted groups.
Transferrin (TfR), the most representative protein-targeting molecule, is a membrane
glycoprotein that is highly expressed in metabolically active tissues requiring large amounts of

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iron, such as tumor cells and blood-brain barrier cells [53]. Jhaveri et al. [54] designed
resveratrol-loaded liposomes with transferrin modified on the surface (Tf-RES-Ls). The Tf-RES-
Ls were significantly more cytotoxic and induced higher levels of apoptosis accompanied by

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activation of caspases 3/7 in GBM cells when compared to free RES or RES-L. As ligand
molecules, polypeptides have the characteristics of easy preparation, low immunogenicity and
high stability compared with large molecular weight proteins [55]. Integrin αvβ3 is a common

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receptor that is highly expressed in tumor cells involving in vascular regeneration, and the
corresponding target is arginine-glycine-aspartate (RGD) polypeptide [56]. Jiang et al. [57]
prepared RGD-modified paclitaxel and curcumin co-loaded liposomes were developed to
evaluate their antitumor activity in vitro and in vivo. The results showed RGD-modified co-

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loaded liposomes had superior antiproliferative effect on A549 cells than free drug or
unmodified liposomes (the in vitro cytotoxicity was shown in Fig 5.). Dasa et al. [58] designed
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plectin targeted peptide (PTP) liposomes encapsulated with a PARP inhibitor (AZ7379). The in
vivo tumor growth studies were performed to determine the efficacy of PTP liposomes in
preventing PARP activity in mice bearing OVCAR8 tumors. PTP liposomal AZ7379 delivery
not only enhanced PARP inhibition but also resulted in decelerated tumor growth in mice
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bearing subcutaneous and intraperitoneal OVCAR8 tumors. In mice bearing subcutaneous or

intraperitoneal tumors, treatment with PTP liposomes resulted in a 3- and 1.7-fold decrease in
tumor volume, respectively, compared to systemic drug treatment.
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Fig 5. was inserted here.

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Oligonucleotide aptamer is an oligonucleotide short chain derived from RNA or DNA

(~15kD). Because of the advantages of oligonucleotide aptamers, such as small molecular weight,
lack of immunogenicity, easier tumor penetration and targeting, and no change in affinity by
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chemical modification, they have great potential in the design of targeted liposomes [59].
Peddada et al. [60] delivered antisense oligonucleotides using poly (alkylene oxide)-poly
(propylacrylic acid) graft copolymers in conjunction with cationic liposomes. The data supported
a strong role for the hydrophilic-lipophilic balance of the graft copolymer in achieving serum
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stability and cellular uptake, and this novel nanoparticle delivery system can be extended to the
delivery of plasmid DNA, siRNA, or aptamers for preclinical and clinical development.
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3.3. Environmental sensitivity

During the delivery of therapeutic agents by liposomes in vivo, the dynamic
microenvironmental changes (such as pH changes, glutathione, enzymes, etc.) can make some
new liposomes release drugs in specific lesion tissues, and some new liposomes can be
controlled release at targeting tissues artificially (such as heating, ultrasound, light, magnetic
effect, etc.). Both modes of drug release from liposome are known as "trigger release".
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The pH of normal tissue is about 7.4, while tumor tissue pH is lower than 6.5, and the pH
inside cell inclusions is less than 5.5 [61]. Based on the these characteristics, pH sensitive
liposome is a widely used environmental sensitive liposome. The earliest pH-sensitive liposomes
were composed of DOPE and its derivatives. These liposomes can undergo layered to hexagonal
phase transitions in the inclusion environment, then destroy the inclusion membrane and increase
the probability of liposomes escaping from lysosomes [62, 63]. Jiang et al. [64] used two
methylmaleic anhydride (DMA) modified DSPE to prepare paclitaxel loaded liposomes (DSPE-

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Lys-DMA, DLD), which could be adsorbed to negatively charged cell membranes at pH 6.8 to
increase cell uptaken. Yuba et al. [65] designed a pH-sensitive delivery system for bleomycin
(BLM) was developed using egg yolk phosphatidylcholine liposomes modified with PEG (PEG-

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PE) for long circulation in the bloodstream and 2-carboxycyclohexane-1-carboxylated
polyglycidol-having distearoyl phosphatidylethanolamine (CHexPG-PE) for pH sensitization.
And the results showed that the PEG-PE/CHexPG-PE-introduced liposomes showed content

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release responding to pH decrease and were taken up by tumor cells at a rate 2.5 times higher
than that of liposomes without CHexPG-PE.
Fig 6. was inserted here.

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There is obvious difference of reduced glutathione between plasma (2.8µM) and
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cytoplasm (1-10µM) [66]. Chi et al. [67] aimed to develop redox-sensitive and CD44-targeted
liposomes to improve chemotherapy of osteosarcoma. Cationic liposomes were prepared and
stabilized with a novel detachable polyethylene glycol (PEG2000) conjugated with cholesterol
through a bio-reducible disulfide linker (Chol-SS-mPEG). Hyaluronic acid (HA, MW 20-40
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kDa), a ligand to CD44, was non-covalently coated on the cationic liposomes. Doxorubicin
(DOX) was actively loaded in the liposomes as a model drug. In vitro release of DOX was well-
controlled at physiological conditions, but a burst release of 60% was observed in the presence of
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10 mM glutathione (GSH), in contrast to non-redox sensitive liposomes (Chol-mPEG/ HA-L and

Chol-mPEG-L). And in a MG63 xenograft mouse model, Chol-SS-mPEG/HA-L showed the
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most effective tumor suppression with minimal uptake by the liver compared with other
liposomes.
The expression of phospholipase A2, cathepsin or matrix metalloproteinase (MMP) in
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tumor tissues was much higher than that in normal tissues [68-70]. In MMPs, MMP-2 and
MMP-9 are closely related to the growth of human tumors. Ji et al.[71] synthesized MMP-2
sensitive liposomes modified with β-cyclodextrins, which were divided into two functional
groups by MMP-2 enzyme after entering human tumor tissue. One of them could release β-
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cyclodextrin for anti fibrosis drugs, the other could release RGD modified liposomes. Liposomes
triggered by this enzyme release drugs in stellate pancreatic cells can significantly enhance the
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ability of drugs to enter the cell, then improve the anti-tumor efficiency.
Hyperthermia has been extensively used for antitumor treatment, and it is generally
combined with chemotherapy [72]. Hyperthermia can be applied via several approaches, such as
radiofrequency [73], microwaves [74], focused ultrasound [75], or intracavitary perfusion (i.e.
with heated chemotherapy-containing solutions) [76], but is limited to locally well-defined, solid
tumors. Hyperthermia generally leads to an increase in tumor blood flow and to an enhanced
vascular permeability, thus promoting drug and oxygen supply to tumors [77, 78]. In non-
ablative settings, the applied temperatures typically range between 39 and 42 °C. While
increasing the temperature, the extravasation of liposomes was enhanced significantly [79], and
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for thermosensitive liposomes, they could release drug immediately, making drug accumulated
in tumor tissues. Zeng et al. [80] constructed Oxaliplatin (OXP) thermosensitive liposomes
(TSL). The results showed that more than 90% of OXP was released within 10 min at 42℃ and
less than 15% was released within 60 min at temperatures below 39℃. OXP-TSL were stable at
37℃for 96 h and at 4℃ for 6 months. The anti-tumor activity of OXP-TSL at the dose of 2.5
mg/kg was certified to be equal to those of OXP injection and non-thermosensitive liposomes

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(NTSL) at the dose of 5 mg/kg.
Photodynamic therapy (PDT) refers to the treatment of tissues, typically tumors, with a
photosensitizing agent, followed by activation via locally applied laser light [81]. In an

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exemplary preclinical study, a monoclonal antibody-photosensitizer (i.e. panitumumab fused
with the photosensitizer IR700; directed against the human epidermal growth factor receptor
(EGFR; HER1)) was combined with laser light and with liposomal daunorubicin, to treat mixed

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tumors [82]. Lajunen et al. [83] have developed light triggered liposomes with clinically
approved indocyanine green (ICG) as the light sensitizing compound. This study focused on the
interactions of lipid bilayer, light sensitizer (ICG) and PEG coating on the liposome stability.
The results showed that localization of the light triggering agent significantly alters the structure

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of the liposomes and it is important to consider these aspects in triggered drug delivery system
design.
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3.4. Multifunctional liposomes
Although the monofunctional liposomes exhibit good transport efficiency, the changes in
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the body's internal environment are complex and changeable, and they could not completely cope
with the obstacles in the transmission process. Therefore, more and more researchers have
rationally stacked different functional methods to enable liposomes to respond more intelligently
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to complex organism environment, thus achieving better therapeutic effect. Based on the
different chemical properties of therapeutic agents, drugs, genes, molecular probes or proteins
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can be loaded in different parts of liposomes by mixing, adsorption and covalent linking. This
characteristic makes liposomes very easy to prepare into a co-carrier system, through the synergy
of different therapeutic agents to obtain better therapeutic effect or achieve "diagnosis and
treatment integration". In view of the differences in the optimal route of administration, duration
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of action and toxicity of different liposomes in vivo, more and more researchers have focused on
the design of a co-carrier system of liposomes capable of simultaneously loading different
therapeutic agents [84]. Ying et al. constructed dual-targeting daunorubicin liposomes by
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modifying with p-aminophenyl-α-D-mannopyranoside and transferrin [85]. The transport ratio

across the BBB model was significantly increased because of the abundant glucose transporters
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and transferrin receptors on the BBB [86, 87]. Li et al. [88] constructed cell penetrating peptide
dNP2 and tumor microenvironment-cleavable folic acid (FA) dual modified, paclitaxel (PTX)
loaded liposome for the targeted delivery of glioma. The acid-cleavable dual modified strategy
retained the BBB penetrating and tumor targeting ability, meanwhile, the cleavage of FA further
maximized the cell permeability of dNP2, exhibiting enhanced tumor targeting effect. The multi-
targeting strategy provides a promising approach towards targeted chemotherapy for glioma.
In the course of tumor chemotherapy, the existence of multidrug resistance (MDR) will
hinder the therapeutic effect of drugs. MDR can be divided into acquired MDR and intrinsic
MDR according to the mechanism. Acquired MDR is mainly due to overexpression of drug
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efflux pump in cells, while intrinsic MDR is related to mitochondria. Gao et al. developed a kind
of dual-functional liposomes by co-encapsulating paclitaxel with chloroquine to treat paclitaxel-
resistant carcinoma. They confirmed that the dual functional paclitaxel plus chloroquine could
block the efflux of paclitaxel by ABC transporters [89].
Gene therapy for tumor does not produce multidrug resistance and other problems, but the
slow onset and low transfection efficiency seriously limit the its application. Using gene-drug co-

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carrier system can make up for each other's deficiencies by using their different therapeutic
mechanisms and achieve the goal of synergistic treatment [90]. Studies have shown that the
sequence and timing of the release of different therapeutic agents from the co-carrier system is a

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major factor affecting the therapeutic effect, for example, the early release of chemotherapeutic
drugs to kill cells will seriously hinder the gene transfection [91]. Yang et al. [92] combines
features of biological (cell-permeable peptides, CPPs) and physical (magnetic) siRNA targeting

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for use in magnetic hyperthermia-triggered release. A siRNA-CPPs conjugate (siRNA-CPPs)
was loaded into thermal and magnetic dual-responsive liposomes (TML) (siRNA-CPPs/TML),
which exhibited good physicochemical properties, effective cellular uptake, endosomal escape
and a significant gene silencing efficiency in MCF-7 cells in vitro. Additionally, in the in

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vivo study, siRNA-CPPs/TML under an alternating current (AC) magnetic field displayed a
superior in vivo targeted delivery efficacy, antitumor efficacy and gene silencing efficiency in a
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MCF-7 xenograft murine model.

4. Biomedical applications of liposomes

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4.1. Liposomes as drug carriers

Liposomes usually form vesicles in water with a diameter of 25-500 nm and a thickness of
about 5 nm. Hydrophilic drugs are encapsulated in the aqueous phase of the liposomes, and
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hydrophobic drugs are inserted into the bilayer composed of lipid molecules. The morphology of
vesicles can be changed by adjusting the ratio of hydrophilic to hydrophobic parts of lipid
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molecules, thus increasing the loading of different drugs [93]. Liposomes as drug carriers have
the following advantages: (1) Increasing the solubility of hydrophobic drugs; (2) Increasing the
stability of drugs in vivo; (3) Prolonging the release time of therapeutic agents; (4) Reducing the
uptake of drugs by normal tissues, and reducing the side effects of therapeutic agents to a certain
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extent; (5) Through functional modification of liposomes to increase drug sensitivity to disease
location, fixed-point drug release and cell phagocytosis [94].
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4.1.1. Delivery of small molecule drugs by liposomes

Generally, the small molecular drugs loaded by liposomes can be divided into two
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categories: anti-infective drugs and anti-tumor drugs. Hydrophilic drugs are contained in the
aqueous phase of liposomes, while lipophilic drugs are loaded in lipid bilayers. The first
liposome which has been approved for cancer treatment was Doxil®/Caelyx®, injectable
PEGylated liposomes (∼100 nm) encapsulating doxorubicin (DOX) [95]. And so far, many
liposome products have been approved for listing (Table 1).
Table 1 was inserted here.

4.1.2. Delivery of bioactive macromolecule by liposomes

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Initially, the research object of gene therapy was DNA, and the commonly used gene
carriers were traditional cationic liposomes. Lipofectamine 2000 is a typical commercialized
cationic liposome for gene transfection. In view of the cytotoxicity of traditional liposomes and
their easy removal in vivo, various bio-inspired or viroid liposomes have been designed. Jiang et
al. [106] used a new liposome with a large dendrimer rich in arginine as its head for intratumoral
injection and found that in vivo transfection results were still higher than the control cationic
liposome group. Just as drug delivery, cationic carriers are blocked by various physiological

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barriers during transfection. In order to mimic the response of viruses, long-circulating liposomes,
environmentally sensitive liposomes, bio-targeted liposomes and viroid multifunctional
liposomes were designed, and they optimized the gene transfection efficiency in varying degrees.

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DNA as a therapeutic agent has a large molecular weight, not easy to load, must be
transcribed in the human nucleus and other adverse factors, so RNA interference (RNAi) therapy

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as an alternative to gene therapy has gradually attracted attention. RNAi is a method of treating
diseases by using double-stranded RNA including small interfering RNA (siRNA), micro RNA
(miRNA), mRNA and short hairpin RNA (shRNA) to down-regulate or silence sequence-
specific target genes in the body, rapidly blocking gene activity. Different from DNA, siRNA,

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which usually consists of 19-23 base pairs, only needs to enter the cytoplasm to silence disease-
related genes for therapeutic purposes [107]. However, the direct application of siRNA can
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induce immune stimuli and has a low therapeutic effect, so it is very important to find a highly
efficient and low toxicity carrier. In 2003, it was first reported that siRNA was encapsulated in
DOTAP liposomes and injected intraperitoneally and intravenously into rats. The results showed
that siRNA could be successfully transported into cells and mediated the production of TNF-α
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and IL-6 [108]. Since then, there have been more than 20,000 reports of liposomes for siRNA
delivery, suggesting that liposomes have become an important carrier for siRNA delivery.
MicroRNAs are typically composed of 18 to 25 nucleotides, which can accelerate the
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degradation of mRNA in the body, thereby impeding transcription. The biggest difference
between miRNA and siRNA is that some miRNA can regulate more than one mRNA. Lotfabadi
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et al. [109] prepared a new formulation of miRNA-loaded cationic liposomes (CL/miR-101) for
bone marrow cells. The results have shown that the size and charge of the prepared CLs with
new formulation were 84.5nm and 20.1mV and for CL/miR-101 were 126.6nm and 4.31mV.
CL/miR-101 were proved to be cytotoxic for cancer cells and could be considered as a novel
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gene therapy system.

After decades of research, bioactive ingredients derived from proteins or peptides have
also been used as drugs to treat diseases, such as enzymes, peptide hormones and cytokines. And
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significant progress has been made in the delivery of these protein drugs by liposomes. Lin et al.
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[110] designed a dumbbell-shaped probe, including magnetic beads (MBs), double-

stranded DNA (dsDNA), and enzyme-encapsulated liposome. In the presence of OTA, the
aptamer preferred to combine with OTA to form G-quadruplex, resulting in the release of the
detection probe and the enzyme-encapsulated liposome. Each liposome contained a large amount
of HRP. Thus, when the liposome was lysed by adding the mixed solution of 3,3′,5,5′-
tetramethylbenzidine (TMB) and H2O2, a large number of HRP were released. Besides protein,
many peptides could also be efficiently delivered by liposomes. Hwang et al. [111] designed
PEGylated nanoliposomes encapsulating angiogenic peptides to improve perfusion defects. And
the data demonstrated that PEGylated nanoliposomes loaded with angiogenic peptides improved
myocardial perfusion defects and increased vascular density.
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4.2. Application of liposomes in medical imaging

Magnetic resonance imaging (MRI) and Near-infrared fluorescence (NIRF) are widely used
accurate diagnostic imaging method. For some diseases, the use of simple imaging molecular
probes will be limited by physiological barriers. For example, the presence of a blood-brain
barrier prevents molecular probes from entering the brain, limiting the diagnosis of gliomas.
Then we loaded molecular probes in classical liposomes or functionalized liposomes, which

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efficiently and directionally delivered them to liver, kidney, spleen, cardiovascular, tumor and
inflammation sites [112]. Imaging molecular probes are loaded with liposomes in three ways: 1)
loading the probe into the aqueous or organic phase of the liposomes by the principle of "similar

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solubility"; 2) adsorbing the probe onto the surface of the liposomes by the principle of
"electrostatic adsorption"; 3) covalently attaching the probe to the liposomes [113]. Chen et al.
[114] synthesized a novel dual-modality MRI and NIRF probe and verify its feasibility in nude

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mouse models with liver cancer. The probes are comprised of superparamagnetic iron oxide
(SPIO) nanoparticles coated with liposomes to which a tumor-targeted agent, Arg-Gly-Asp
peptides (RGD), and a NIRF dye (indocyanine green, ICG) have been conjugated. The study
demonstrated that both MRI and fluorescent images showed clear tumor delineation after probe

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injection (SPIO@Liposome-ICG-RGD) (as shown in Fig 7).
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Fig 7. was inserted here.

4.3. Liposomes as vaccine carriers

Since antigens or vaccine adjuvants can be adsorbed or covalently attached to liposomes,
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liposomes have gradually become an important vaccine carrier. In general, the basic process of
immune response by intramuscular or subcutaneous injection of the antigens encapsulated in
liposomes is as follows: First, use liposome to penetrate tissue and reach the lymphatic system,
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then the liposome slowly releases antigen as well as maintains the integrity of their conformation.
The released antigen combines with B cell receptor, thus activating the immune response of the
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body. In addition to these processes, it is possible to cross-present antigens to CD8+ cells by the
major histocompatibility type I complex [115]. Molecules used as antigens are diverse, including
peptides, proteins, DNA, RNA, and glycoproteins.
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Immune adjuvants are components that enhance the immune response of vaccines. They are
usually injected with antigens together or in advance to prolong the release time of antigens and
increase the chance of contact between antigens and antigen presenting cells. Liposome is an
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immune adjuvant itself. In the last 20 years, liposome vaccine technology has matured and now
several vaccines containing liposome-based adjuvants have been approved for human use or
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have reached late stages of clinical evaluation (as shown in Table 2) [116]. Virosomes
demonstrate the characteristics of an adjuvant system and are biodegradable, non-toxic and do
not induce antibodies against themselves [117]. The virosomes are reconstituted influenza virus
envelopes devoid of inner core and genetic information. During the production process, the
influenza virus surface antigens neuraminidase and hemagglutinin are integrated into
phosphatidylcholine bilayer liposomes, yielding unilamellar virosomes with an average diameter
of 150 nm (as shown in Fig 8) [118]. The particulate structure and the function of the surface
hemagglutinin protein of binding to the cell receptor, mediating pH-dependent fusion with
endosomes and stimulating the immune system are responsible for the adjuvant function [119-
123]. The composition of Inflexal®V consists of a mixture of three monovalent virosome pools,
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each formed with one influenza strain’s specific hemagglutinin and neuraminidase glycoproteins
[124, 125].
Table 2 was inserted here.
Fig 8. was inserted here.

4.4. Application of liposomes in cosmetics

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In addition to these areas of application, liposomes as cosmetics have the following
advantages: (1) increasing skin moisture; (2) improving cell membrane fluidity, and make the

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skin more vibrant; (3) Liposomes encapsulating oil-soluble and water-soluble active ingredients
penetrate deep through the skin and deposit in the dermis to form a reservoir, which can sustain
and prolong the action time of active ingredients [126, 127]. As early as 1987, the famous French

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Christian Dior company launched the first liposome cosmetics "Capture". Soon after, another
French company, L'Oreal, launched "Nactosome", and Applied Genetics, which also uses
liposomes, launched a sunscreen called "Photosome". Lee et al. [128] synthesized spherical silica
hybrid liposome which contained citrus unshiu extract as an antioxidant cosmetic ingredient. The

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silicified liposomes particles effectively entrapped as much as 314.2 mg/L solution of the citrus
unshiu peel extract, and the controlled release profile showed that the maximum release was
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41.4%.

5. Conclusion and perspectives

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After more than 60 years of research, liposomes have developed from a concept to the
mainstream nano drug delivery system. Liposome-based strategies are the first nano delivery
systems already successfully translated into the clinical use, and many are in different stages of
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clinical evaluation. In addition to being biocompatible, biodegradable and non-immunogenic,

liposomes have well-established metabolism, pharmacokinetic and biodistribution profiles via
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different routes of administration. Liposomes have developed into long-circulating liposomes,

environmentally sensitive liposomes and actively targeting liposomes from the original classical
composition. Through organic combination, multifunctional liposomes achieve better clinical
application potential. Very recently, the FDA approved a liposome encapsulating a combination
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of daunorubicin and cytarabine (VYXEOS®, Jazz Pharmaceuticals, Inc.) for the treatment of
acute myeloid leukemia [129]. This is the first nanomedicine entrapping two drugs approved for
biomedical applications, opening new avenues for the clinical development and regulatory
approval of advanced combinatorial approaches using a single carrier. In considering the
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potential clinical applications, further development of liposomes may involve the following three
aspects: (i) a simple manufacturing procedure for industrial production; (ii) a safe, stable and
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controllable final product in view of quality control and drug specification; and (iii) a further
demonstrated efficacy in clinical trials. With the development of science and technology, more
and more efficient liposomes will be widely used in clinic and play an important role in the
treatment of various diseases.

Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content
and writing of this paper.
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Acknowledgements
Authors are thankful to the support of Tianjin Natural Science General Project Fund
(2018KJ124).

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Reference
[1] A.D. Bangham, M.M. Standish, J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids, J.

RI
Mol. Biol. 13 (1965) 238-252.

[2] K. Greish, Enhanced permeability and retention effect for selective targeting of anti-cancer nanomedicine: are we there yet?
Drug. Discov. Today. Technol. 9 (2012) e161-e166.

SC
[3] WHO | Cancer, WHO (n.d.) http://www.who.int/mediacentre/factsheets/fs297/en/

(Accessed 07/06/2018).

U
[4] M.J.A. Jonge, J. Verweij, Renal toxicities of chemotherapy, Semin. Oncol. 33 (2006) 68-73.
AN
[5] L. Harris, G. Batist, R. Belt, D. Rovira, R. Navari, N. Azarnia, L. Welles, E. Winer, TLC D-99 study group, liposome-
encapsulated doxorubicin compared with conventional doxorubicin in a randomized multicenter trial as first-line therapy of
metastatic breast carcinoma, Cancer. 94 (2002) 25-36.

[6] K.S. Golombek, M. Jan-Niklas, B. Theek, L. Appold, Na. Drude, F. Kiesslinga, T. Lammers. Tumor targeting via EPR:
M

Strategies to enhance patient responses, Adv. Drug. Deliv. Rev. 130 (2018) 17-38.

[7] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of
tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer. Res. 46 (1986) 6387-6392.
D

[8] V. Torchilin, Tumor delivery of macromolecular drugs based on the EPR effect, Adv. Drug. Deliv. Rev. 63 (2011) 131-135.
TE

[9] U. Prabhakar, H. Maeda, R.K. Jain, E.M. Sevick-Muraca, W. Zamboni, O.C. Farokhzad, S.T. Barry, A. Gabizon, P.
Grodzinski, D.C. Blakey, Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine
drug delivery in oncology, Cancer. Res. 73 (2013) 2412-2417.

[10] H. Maeda, H. Nakamura, J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor
EP

uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo, Adv. Drug. Deliv. Rev. 65 (2013) 71-79.

[11] H. Hashizume, P. Baluk, S. Morikawa, J.W. McLean, G. Thurston, S. Roberge, R.K. Jain, D.M. McDonald, Openings
between defective endothelial cells explain tumor vessel leakiness, Am. J. Pathol. 156 (2000) 1363-1380.
C

[12] S.D. Steichen, M. Caldorera-Moore, N.A. Peppas, A review of current nanoparticle and targeting moieties for the delivery of
cancer therapeutics, Eur. J. Parm. Sci. 48 (2013) 416-247.
AC

[13] J.R. Morgan, L.A. Williams, C.B Howard, Technetium-labelled liposome imaging for deep-seated infection, Brit. J. Radiol.
58 (1985) 35-39.

[14] P.L. Felgner, T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M. Wenz, J.P. Northrop, G.M. Ringold, M. Danielsen,
Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 7413-7417.

[15] B.K Kim, Y.U.Bae, K.O. Doh, G.B Hwang, SH Lee, H. Kang, YB. Seu. The synthesis of cholesterol-based cationic lipids
with trimethylamine head and the effect of spacer structures on transfection efficiency, Bioorg. Med. Chem. Lett. 21 (2011) 3734-
3737.

[16] M. Mintzer, E.E. Simanek, Nonviral vectors for gene delivery, Chem. Rev. 109 (2009) 259-302.
 ACCEPTED MANUSCRIPT

[17] D. Niculescu-Duvaz, J. Heyes, C.J. Springer, Structure-activity relationship in cationic lipid mediated gene transfection,
Curr. Med. Chem. 10 (2003) 1233-1261.

[18] J.A. Wolff, R.W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, P.L. Felgner, Direct gene transfer into mouse muscle in
vivo, Science. 247 (1990) 1465-1468.

[19] Q. Jiang, D. Yue, Y. Nie, X. Xu, Y. He, S. Zhang, E. Wagner, Z. Gu, Specially-Made Lipid-Based Assemblies for Improving
Transmembrane Gene Delivery: Comparison of Basic Amino Acid Residue Rich Periphery, Mol. Pharm. 13 (2016) 1809-1821.

PT
[20] F. Tang, J.A. Hughes, Synthesis of a single-tailed cationic lipid and investigation of its transfection, J. Control. Release. 62
(1999) 345-358.

[21] S.H. Alavizadeh, F. Gheybi, A.R. Nikpoor, A. Badiee, S. Golmohammadzadeh, M.R. Jaafari, Therapeutic Efficacy of
Cisplatin Thermosensitive Liposomes upon Mild Hyperthermia in C26 Tumor Bearing BALB/c Mice, Mol. Pharm. 14 (2017)

RI
712-721.

[22] R. Koynova, B. Tenchov, L. Wang, R.C. Macdonald, Hydrophobic moiety of cationic lipids strongly modulates their
transfection activity, Mol. Pharm. 6 (2009) 951-958.

SC
[23] L. Li, S. Hongmei, L. Kui, H. Bin, N. Yu, Y. Yang, W. Yao, G. Zhongwei, Gene transfer efficacies of serum-resistant amino
acids-based cationic lipids: Dependence on headgroup, lipoplex stability and cellular uptake, Int. J. Pharm. 408 (2011) 183-190.

[24] S. Kaddah, N. Khreich, F. Kaddah, L. Khrouz, C. Charcosset, H. Greige-Gerges, Corticoids modulate liposome membrane

U
fluidity and permeability depending on membrane composition and experimental protocol design, Biochimie. 153 (2018) 33-35.
AN
[25] J.S. Choi, E.J. Lee, H.S. Jang, J.S. Park, New cationic liposomes for gene transfer into mammalian cells with high efficiency
and low toxicity, Bioconjug. Chem. 12 (2001) 108-113.

[26] S.J. Freedland, R.W. Malone, H.M. Borchers, Z. Zadourian, J.G. Malone, J.B. Michael, H.N. Michael, J.H. Li, H.G. Paul,
L.E. Kent, Toxicity of Cationic Lipid–Ribozyme Complexes in Human Prostate Tumor Cells Can Mimic Ribozyme Activity,
M

Mol. Genet. Metab. 59 (1996) 144-153.

[27] L. Rania, R.S. John, Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic
amphiphiles, BBA-Biomembranes. 1023 (1990) 124-132.
D

[28] T. Fuxing, J.A. Hughes, Introduction of a Disulfide Bond into a Cationic Lipid Enhances Transgene Expression of Plasmid
DNA, Biochem. Bioph. Res. CO. 242 (1998) 141-145.
TE

[29] S. Wang, S. Zhang, J. Liu, Z. Liu, L. Su, H. Wang, J. Chang, pH- and reduction-responsive polymeric lipid vesicles for
enhanced tumor cellular internalization and triggered drug release, ACS. Appl. Mater. Interfaces. 6 (2014) 10706-10713.

[30] K. Oumzil, S. Benizri, G. Tonelli, C. Staedel, A. Appavoo , M. Chaffanet, L. Navailles, P. Barthélémy, pH-Cleavable
EP

Nucleoside Lipids: A New Paradigm for Controlling the Stability of Lipid-Based Delivery Systems, ChemMedChem. 10 (2015)
1797-1801.

[31] D. Liu, J. Hu, W. Qiao, Z. Li, S. Zhan, L. Cheng, Synthesis and characterization of a series of carbamate-linked cationic
lipids for gene delivery, Lipids. 40 (2005) 839-848.
C

[32] A. Aissaoui, B. Martin, E. Kan, N. Oudrhiri, M. Hauchecorne, J.P. Vigneron, J.M. Lehn, P. Lehn, Novel cationic lipids
incorporating an acid-sensitive acylhydrazone linker: synthesis and transfection properties, J. Med. Chem. 47 (2004) 5210-5223.
AC

[33] A. Wicki, D. Witzigmann, V. Balasubramanian, J. Huwyler, Nanomedicine in cancer therapy: challenges, opportunities, and
clinical applications, J. Control. Release. 200 (2015) 138-157.

[34] Y.J. Kao, R.L. Juliano, Interactions of liposomes with the reticuloendothelial system effects of reticuloendothelial blockade
on the clearance of large unilamellar vesicles, BBA-Gen. Subjects. 677 (1981) 453-461.

[35] M.C. Woodle, Sterically stabilized liposome therapeutics, Adv. Drug. Deliv. Rev. 16 (1995) 249-265.

[36] G. Blume, G. Cevc, Liposomes for the sustained drug release in vivo, BBA-Biomembranes. 1029 (1990) 91-97.

[37] L.L. Klibanov, K. Maruyama, P.V. Torchilin, L. Huang, Amphipathic polyethyleneglycols effectively prolong the circulation
time of liposomes, FEBS. Lett. 268 (1990) 235-237.
 ACCEPTED MANUSCRIPT

[38] M.C. Woodle, D.D. Lasic, Sterically stabilized liposomes, Biochim. Biophys. Acta. 1113 (1992) 171–199.

[39] X.Z. Tang, J. Sun, T. Ge, K.Q. Zhang, Q.Y. Gui, S.T. Zhang, W.D. Chen, PEGylated liposomes as delivery systems for
Gambogenic acid: Characterization and in vitro/in vivo evaluation, Colloid. Surface. B. 172 (2018) 26–36.

[40] D.C. Drummond, O. Meyer, K. Hong, D.B. Kirpotin, D. Papahadjopoulos, Optimizing liposomes for delivery of
chemotherapeutic agents to solid tumors, Pharmacol. Rev. 51 (1999) 691-743.

[41] E.T. Dams, P. Laverman, W.J. Oyen, G. Storm, G.L. Scherphof, J.W. Der Meer, F.H. Corstens, O.C. Boerman, Accelerated

PT
blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes, J. Pharmacol. Exp. Ther. 292
(2000) 1071-1079.

[42] L. Chaoyang, L. Cong, L. Mengyang, Q. Qiujun, L. Xiang, L. Xinrong, H. Ling, D. Yihui, S. Yanzhi, Effect of Kupffer cells
depletion on ABC phenomenon induced by Kupffer cells-targeted liposomes, Asian. J. of Pharm. Sci. 2018, DOI:

RI
https://doi.org/10.1016/j.ajps.2018.07.004

[43] Z. Zhang, J. Yao, Preparation of irinotecan-loaded folate-targeted liposome for tumor targeting delivery and its antitumor
activity, AAPS. Pharm. Sci. Tech. 13 (2012) 802-810.

SC
[44] K.M. Laginha, E.H. Moase, N. Yu, A. Huang, T.M. Allen, Bioavailability and therapeutic efficacy of HER2 scFv-targeted
liposomal Doxorubicin in a murine model of HER2-overexpressing breast cancer, J. Drug. Target. 16 (2008) 605-610.

[45] X. Li, L. Ding, Y. Xu, Y. Wang, Q. Ping, Targeted delivery of DOXorubicin using stealth liposomes modified with

U
transferrin, Int. J. Pharm. 373 (2009) 116-123.
AN
[46] G.W. Sledge, K.D. Miller, Exploiting the hallmarks of cancer: the future conquest of breast cancer. Eur. J. Cancer. 39 (2003)
1668-1675.

[47] Y. Bi , F. Hao, G. Yan, L. Teng, R.J. Lee, J. Xie, Actively targeted nanoparticles for drug delivery to tumor, Curr. Drug.
Metab. 17 (2016) 763-782.
M

[48] G. Weissmann, D. Bloomgarden, R. Kaplan, C. Cohen, S. Hoffstein, T. Collins, A. Gotlieb, D. Nagle, A general method for
the introduction of enzymes, by means of immunoglobulin-coated liposomes, into lysosomes of deficient cells, Proc. Natl. Acad.
Sci. U. S. A. 72 (1975) 88-92.
D

[49] A. Rodallec, B. Jean-Miche, S. Giacometti, H. Maccario, F. Correard, E. Mas, C. Orneto, A. Savina, F. Bouquet, B.
Lacarelle, J. Ciccolini, R. Fanciullino, Docetaxel-trastuzumab stealth immunoliposome: development and in vitro proof of
TE

concept studies in breast cancer. Int. J. Nanomed. 13 (2018) 3451-3465.

[50] X.G. Frank, R. Karnik, Z. W. Andrew, F. Alexis, C.F. Omid , Targeted nanoparticles for cancer therapy, Nano. Today. 2
(2007) 14-21.
EP

[51] E. Moghimipour, M. Rezaeic, Z. Ramezani, M. Kouchak, M. Amini, K. A. Angali, F. A. Dorkoosh, S. Handali, Folic acid-
modified liposomal drug delivery strategy for tumor targeting of 5-fluorouracil, Eur. J. Pharm. Sci. 114 (2018) 166-174.

[52] F. Ravar, E. Saadat, M. Gholami, P. Dehghankelishadi, A.D. Farid, Hyaluronic acid-coated liposomes for targeted delivery of
paclitaxel, in-vitro characterization and in-vivo evaluation, J. Control. Release. 229 (2016) 87-95.
C

[53] H.S. Yoo, T.G. Park, Folate receptor targeted biodegradable polymeric doxorubicin micelles, J. Control. Release. 96 (2004)
273-283.
AC

[54] A. Jhaveri, P. Deshpande, B. Pattni, V. Torchilin, Transferrin-targeted, resveratrol-loaded liposomes for the treatment of
glioblastoma, J. Control. Release. 277 (2018) 89-101.

[55] S.D. Steichen, M. Caldorera-Moore, N.A. Peppas, A review of current nanoparticle and targeting moieties for the delivery of
cancer therapeutics, Eur. J. Parm. Sci. 48 (2013) 416-247.

[56] C.B. Peter, M. P. M. Anthony, M. Rosenfeld, R.A. Reisfeld, A.D. Cheresh, Integrin αvβ3 antagonists promote tumor
regression by inducing apoptosis of angiogenic blood vessels, Cell. 79 (1994) 1157-1164.

[57] K. Jiang, M.J. shen, W.H. Xu. Arginine, glycine, aspartic acid peptide-modified paclitaxel and curcumin co-loaded liposome
for the treatment of lung cancer: in vitro/vivo evaluation, INT. J. Nanomed. 13 (2018) 2561-2569.
 ACCEPTED MANUSCRIPT

[58] S.S.K. Dasa, G. Diakova, R. Suzuki, A.M. Mills, M.F. Gutknecht, A.L. Klibanov, J.K. Slack-Davis, K.A. Kelly, Plectin-
targeted liposomes enhance the therapeutic efficacy of a PARP inhibitor in the treatment of ovarian cancer, Theranostics. 8 (2018)
2782-2798.

[59] F.X Gu, R. Karnik, A.Z. Wang, F. Alexis, E. Levy-Nissenbaum, S. Hong, R.S. Langer, O.C. Farokhzad, Targeted
nanoparticles for cancer therapy, Nano. Today. 2 (2007) 14-21.

[60] L.Y. Peddada, O.B. Garbuzenko, D.I. Devore, T. Minko, C.M. Roth, Delivery of antisense oligonucleotides using
poly(alkylene oxide)-poly(propylacrylic acid) graft copolymers in conjunction with cationic liposomes, J. Control. Release. 194

PT
(2014) 103-112.

[61] K. Engin, D.B. Leeper , J.R. Cater, A.J. Thistlethwaite, L. Tupchong, J.D. McFarlane, Extracellular pH distribution in human
tumours, Int. J. Hyperthermia. 11 (1995) 211-216.

RI
[62] I. Koltover, T. Salditt, J.O. Rädler, C.R. Safinya, An inverted hexagonal phase of cationic liposome-DNA complexes related
to DNA release and delivery, Science. 281 (1998) 78-81.

[63] K.M.G. Taylor, M.R. Morris, Thermal analysis of phase transition behaviour in liposomes, Thermochimica. Acta. 248 (1995)

SC
289-301.

[64] L. Jiang, B. He, D. Pan, K. Luo, Q. Yi, Z. Gu, Anti-Cancer Efficacy of Paclitaxel Loaded in pH Triggered Liposomes, J.
Biomed. Nanotechnol. 12 (2016) 79-90.

U
[65] E. Yuba, T. Osaki, M. Ono, S. Park, A. Harada, M. Yamashita, K. Azuma, T. Tsuka, N. Ito, T. Imagawa, Y. Okamoto,
Bleomycin-Loaded pH-Sensitive Polymer–Lipid-Incorporated Liposomes for Cancer Chemotherapy, Polymers. 10 ( 2018) 1-15.
AN
[66] S.D. Manickam, J. Li, D.A. Putt, Z. Qing-Hui, D. Oupický, Effect of innate glutathione levels on activity of redox-
responsive gene delivery vectors, J. Control. Release. 141 (2010) 77-84.

[67] C. Yingying, Y. Xuelei, S. Kaoxiang, F. Shuaishuai, L. Jinhu, C. Daquan, C. Chuanyou, W. Zimei, Redox-sensitive and
M

hyaluronic acid functionalized liposomes for cytoplasmic drug delivery to osteosarcoma in animal models, J. Control. Release.
261 (2017) 113-125.

[68] N. Li, N. Li, Y. Qiangying, K. Luo, G. Zhongwei, Amphiphilic peptide dendritic copolymer-doxorubicin nanoscale
D

conjugate self-assembled to enzyme-responsive anti-cancer agent, Biomaterials. 35 (2014) 9529-9545.

[69] C.M. Overall, C. López-Otín, Strategies for MMP inhibition in cancer: innovations for the post-trial era, Nat. Rev. Cancer. 2
TE

(2002) 657-672.

[70] G. Kong, M.W. Dewhirst, Hyperthermia and liposomes, Int. J. Hyperthermia. 15 (1999) 211-216.

[71] T. Ji, S. Li, Y. Zhang, J. Lang, Y. Ding, X. Zhao, R. Zhao, Y. Li, J. Shi, J. Hao, Y. Zhao, G. Nie, An MMP-2 Responsive
EP

Liposome Integrating Antifibrosis and Chemotherapeutic Drugs for Enhanced Drug Perfusion and Efficacy in Pancreatic Cancer,
ACS. Appl. Mater. Interfaces. 8 (2016) 3438-3445.

[72] H.S. Koops, M. Vaglini, S. Suciu, B.B. Kroon, J.F. Thompson, J. Göhl, A.M. Eggermont, F.D. Filippo, E.T. Krementz, D.
Ruiter, F.J. Lejeune, Prophylactic isolated limb perfusion for localized, high-risk limb melanoma: results of a multicenter
C

randomized phase III trial. European Organization for Research and Treatment of Cancer Malignant Melanoma Cooperative
Group Protocol 18832, the World Health Organization Melanoma Program Trial 15, and the North American Perfusion Group
Southwest Oncology Group-8593, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 16 (1998) 2906-2912.
AC

[73] P. Wust, M. Seebass, J. Nadobny, P. Deuflhard, G. Mönich, R. Felix, Simulation studies promote technological development
of radiofrequency phased array hyperthermia, Int. J. Hyperth. 12 (1996) 477-494

[74] L. Dubois, J.P. Sozanski, V. Tessier, J.C. Camart, J.J. Fabre, J. Pribetich, M. Chive, Temperature control and thermal
dosimetry by microwave radiometry in hyperthermia, IEEE. Trans. Microw. Theory. Tech. 44 (1996) 1755-1761

[75] C.J. Diederich, K. Hynynen, Ultrasound technology for hyperthermia, Ultrasound. Med. Biol. 25 (1999) 871-887

[76] A.J. Witkamp, E. Bree, R.V.Goethem, F.A. Zoetmulder, Rationale and techniques of intra-operative hyperthermic
intraperitoneal chemotherapy, Cancer. Treat. Rev. 27 (2001) 365-374
 ACCEPTED MANUSCRIPT

[77] W.C. Dewey, D.E. Thrall, E.L. Gillette, Hyperthermia and radiation–a selective thermal effect on chronically hypoxic tumor
cells in vivo, Int. J. Radiat. Oncol. Biol. Phys. 2 (1977) 99-103.

[78] R. Burd, T.S. Dziedzic, Y. Xu, M.A. Caligiuri, J.R. Subjeck, E.A. Repasky, Tumor cell apoptosis, lymphocyte recruitment
and tumor vascular changes are induced by low temperature, long duration (fever-like) whole body hyperthermia, J. Cell.
Physiol. 177 (1998) 137-147.

[79] G. Kong, R.D. Braun, M.W. Dewhirst, Characterization of the effect of hyperthermia on nanoparticle extravasation from
tumor vasculature, Cancer. Res. 61 (2001) 3027-3032.

PT
[80] Z. Chunying, Y. Fanglin, Y. Yang, C. Xiaohui, L. Yan, Z. Hui, Z. Shiqing, Y. Zhenbo, L. Mingyuan, L. Zhiping, M. Xingguo,
Preparation and Evaluation of Oxaliplatin Thermosensitive Liposomes with Rapid Release and High Stability, PLoS. ONE. 11
(2016) e0158517.

RI
[81] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, S.M. Hahn, M.R. Hamblin, A. Juzeniene, D.
Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B.C. Wilson, J. Golab, Photodynamic therapy of cancer: an update,
CA. Cancer. J. Clin. 61 (2011) 250-281.

SC
[82] K. Sano, T. Nakajima, P.L. Choyke, H. Kobayashi, The effect of photoimmunotherapy followed by liposomal daunorubicin
in a mixed tumor model: a demonstration of the super-enhanced permeability and retention effect after photoimmunotherapy,
Mol. Cancer. Ther. 13 (2014) 426–432.

[83] T. Lajunen, R. Nurmi, D. Wilbie, T. Ruoslahti, N.G. Johansson, O. Korhonen, T. Rog, A. Bunker, M. Ruponen, A. Urtti, The

U
effect of light sensitizer localization on the stability of indocyanine green liposomes, J. Control. Release. 284 (2018) 213–223.
AN
[84] N.S. Gandhi, R.K. Tekade, M.B. Chougule, Nanocarrier mediated delivery of siRNA/miRNA in combination with
chemotherapeutic agents for cancer therapy: Current progress and advances, J. Control. Release. 194 (2014) 238-256.

[85] X. Ying, H. Wen, W.L. Lu, J. Du, J. Guo, W. Tian, Y. Men, Y. Zhang, R.J. Li, T.Y. Yang, D.W. Shang, J.N. Lou, L.R. Zhang,
Q. Zhang, Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals, J. Control. Release
M

141 (2010) 183–192.

[86] D.C.D. Vivo, R.R. Trifiletti, R.I. Jacobson, G.M. Ronen, R.A. Behmand, S.I. Harik, Defective glucose transport across the
blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay, N. Engl. J. Med. 325 (1991)
D

703–709.

[87] W.A. Jefferies, M.R. Brandon, S.V. Hunt, A.F. Williams, K.C. Gatter, D.Y. Mason, Transferrin receptor on endothelium of
TE

brain capillaries, Nature. 312 (1984) 162–163.

[88] L. Man, S. Kairong, T. Xian, W. Jiaojie, C. Xingli, C. Xiaoxiao, Y. Qianwen, Z. Zhirong, H. Qi, pH-sensitive folic acid and
dNP2 peptide dual-modified liposome for enhanced targeted chemotherapy of glioma, Eur. J. Pharm. Sci. 124 (2018) 240-248.
EP

[89] M. Gao, Y. Xu, L. Qiu, Enhanced combination therapy effect on paclitaxel-resistant carcinoma by chloroquine co-delivery
via liposomes, Int. J. Nanomed. 10 (2015) 6615–6632.

[90] Z. Yang, D. Gao, Z. Cao, C. Zhang, D.Cheng, J. Liu, X.Shuai, Drug and gene co-delivery systems for cancer treatment,
Biomater. Sci. 3 (2015) 1035-1049.
C

[91] M.J. Lee, A.S. Ye, A.K. Gardino, A.M. Heijink, P.K. Sorger, G. MacBeath, M.B. Yaffe, Sequential Application of Anticancer
Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks, Cell. 149 (2012) 780-794.
AC

[92] Y. Yanfang, X. Xiangyang, X. Xueqing, X. Xuejun, W. Hongliang, L. Lin, D. Wujun, M. Panpan, Y. Yang, L. Yuling, M.
Xingguo. Thermal and magnetic dual-responsive liposomes with acell-penetrating peptide-siRNA conjugate for enhanced and
targetedcancer therapy, Colloid. Surface. B. 146 (2016) 607-615.

[93] A. Bunker, A. Magarkar, T. Viitala, Rational design of liposomal drug delivery systems, a review: Combined experimental
and computational studies of lipid membranes, liposomes and their PEGylation, BBA-Biomembranes. 1858 (2016) 2334-2352.

[94]Yingchoncharoen, Kalinowski, Richardson, Lipid-Based Drug Delivery Systems in Cancer Therapy: What Is Available and
What Is Yet to Come, Pharmacol. Rev. 68 (2005) 701-787.

[95] P.D. Brown, P.R. Patel, Nanomedicine: a pharma perspective, Wires. Nanomed. Nanobiotechnol. 7 (2015) 125–130.
 ACCEPTED MANUSCRIPT

[96] T.J. Walsh, J.W. Hiemenz, N.L. Seibel, J.R. Perfect, G. Horwith, L. Lee, J.L. Silber, M.J. DiNubile, A. Reboli, E. Bow, J.
Lister, E.J. Anaissie, Amphotericin B lipid complex for invasive fungal infections: analysis of safety and efficacy in 556 cases,
Clin. Infect. Dis. 26 (1998) 1383.

[97] R. Bowden, P. Chandrasekar, M.H. White, X. Li, L. Pietrelli, M. Gurwith, J.A. van Burik, M. Laverdiere, S. Safrin, J.R.
Wingard, A double-blind, randomized, controlled trial of amphotericin B colloidal dispersion versus amphotericin B for
treatment of invasive aspergillosis in immunocompromised patients, Clin. Infect. Dis. 35 (2002) 359-366.

[98] I.M. Hann, H.G. Prentice, Lipid-based amphotericin B: a review of the last 10 years of use, Int. J. Antimicrob. Ag. 17 (2001)

PT
161-169.

[99] N.D. James, R.J. Coker, D. Tomlinson, J.R.W. Harris, M. Gompels, A.J. Pinching, J.S.W. Stewart, Liposomal doxorubicin
(Doxil): An effective new treatment for Kaposi's sarcoma in AIDS, Clin Oncol-UK. 6 (1994) 294-296.

RI
[100] C.E. Petre, D.P. Dittmer, Liposomal daunorubicin as treatment for Kaposi's sarcoma, Int. J. Nanomed. 2 (2007) 277-288.

[101] G. Batist, G. Ramakrishnan, C.S. Rao, A. Chandrasekharan, J. Gutheil, T. Guthrie, P. Shah, A. Khojasteh, M.K. Nair, K.
Hoelzer, K. Tkaczuk, Y.C. Park, L.W. Lee, Reduced cardiotoxicity and preserved antitumor efficacy of liposome-encapsulated

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doxorubicin and cyclophosphamide compared with conventional doxorubicin and cyclophosphamide in a randomized,
multicenter trial of metastatic breast cancer, J. Clin. Oncol. 19 (2001). 1444-1454.

[102] M.J. Glantz, S. LaFollette, K.A. Jaeckle, W. Shapiro, L. Swinnen, J.R. Rozental, S. Phuphanich, L.R. Rogers, J.C. Gutheil,
T. Batchelor, D. Lyter, M. Chamberlain, B.L. Maria, C. Schiffer, R. Bashir, D. Thomas, W. Cowens, S.B. Howell, Randomized

U
trial of a slow-release versus a standard formulation of cytarabine for the intrathecal treatment of lymphomatous meningitis, J.
Clin. Oncol. 17 (1999) 3110-3116.
AN
[103] Shah NN, Cole DE, Lester-McCully CM, Wayne AS, Warren KE, Widemann BC. Plasma and cerebrospinal fluid
pharmacokinetics of vincristine and vincristine sulfate liposomes injection (VSLI, marqibo®) after intravenous administration in
Non-human primates.Invest New Drugs. 2016; 34(1): 61-65.
M

[104] DiGiulio, Sarah. FDA Approves Onivyde Combo Regimen for Advanced Pancreatic Cancer. FDA Action & Updates,
2015-10-25 [2017-11-05]. http: //journal.lww.com/oncology-times/blog/fdaactionsandupdates/pages/post.aspx?PostID=124.

[105] D. Gambling, T. Hughes, G. Martin, W. Horton, G. Manvelian, A comparison of Depodur, a novel, single-dose extended-
D

release epidural morphine, with standard epidural morphine for pain relief after lower abdominal surgery, Anesth. Analg. 100
(2005) 1065-1074.
TE

[106] Q. Jiang, D. Yue, Y. Nie, X. Xu, Y. He, S. Zhang, E. Wagner, Z. Gu, Specially-Made Lipid-Based Assemblies for Improving
Transmembrane Gene Delivery: Comparison of Basic Amino Acid Residue Rich Periphery, Mol. Pharm. 13 (2016) 1809-1821.

[107] N.S. Gandhi, R.K. Tekade, M.B. Chougule, Nanocarrier mediated delivery of siRNA/miRNA in combination with
chemotherapeutic agents for cancer therapy: Current progress and advances, J. Control. Release. 194 (2014) 238-256.
EP

[108] M.Sioud, D.R. Sørensen, Cationic liposome-mediated delivery of siRNAs in adult mice, Biochem. Bioph. Res. 312 (2003)
1220-1225.

[109] N.N. Lotfabadi, H.M. Kouchesfehani, M.H. Sheikhha, S.M. Kalantar, Development of a novel cationic liposome:
C

Evaluation of liposome mediated transfection and anti-proliferative effects of miR-101 in acute myeloid leukemia, J. Drug. Deliv.
Sci. Tec. 45 (2018) 196-202.
AC

[110] L. Cuiying, Z. Huixia, S. Mi, G. Yajuan, L. Fang, G. Longhua, Q. Bin, L. Zhenyu, C. Guonan, Highly sensitive colorimetric
aptasensor for ochratoxin A detection based on enzyme-encapsulated liposome, Anal. Chim. Acta. 1002 (2018) 90-96.

[111] H. Hwang, J. Hwan-Seok, O. Phil-Sun, K. Minjoo, L. Tai-Kyoung, J. Kwon, K. Hyeon-Soo, L. Seok Tae, S. Myung-Hee, J.
Hwan-Jeong, PEGylated nanoliposomes encapsulating angiogenic peptides improve perfusion defects: Radionuclide imaging-
based study, Nucl. Med. Biol. 43 (2016) 552-558.

[112] V.P. Torchilin, Liposomes as delivery agents for medical imaging, Mol. Med. Today. 2 (1996) 242-249.

[113] V. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat. Rev. Drug. Discov. 4 (2005) 145-160.

[114] C. Qingshan, S. Wenting, Z. Chaoting, W. Kun, L. Xiaoyuan, C. Chongwei, L. Xiao, Y. Jian, F. Chihua, T. Jie, Theranostic
imaging of liver cancer using targeted optical/MRI dual-modal probes, Oncotarget. 8 (2017) 32741-32751.
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[115] D.M. Smith, J.K. Simon, J.R.Baker, Applications of nanotechnology for immunology, Nat. Rev. Immunol. 13 (2013) 592-
605.

[116] D.S. Watson, A.N. Endsley, L. Huang, Design considerations for liposomal vaccines: Influence of formulation parameters
on antibody and cell-mediated immune responses to liposome associated antigens, Vaccine. 30 (2012) 2256-2272.

[117] S.J. Cryz, J.U. Que, R. Gluck. A virosome vaccine antigen delivery system does not stimulate an antiphospholipid antibody
response in humans, Vaccine. 14(1996) 1381-3.

PT
[118] C. Herzog, K. Hartmann, V. Künzi, O. Kürsteiner, R. Mischler, H. Lazar, R. Glück, Eleven years of Inflexal®V—a
virosomal adjuvanted influenza vaccine, Vaccine. 27 (2009) 4381-4387.

[119] C. Moser, M. Amacker, A.R. Kammer, S. Rasi, N. Westerfeld, R. Zurbriggen, Influenza virosomes as a combined vaccine
carrier and adjuvant system for prophylactic and therapeutic immunizations, Exp. Rev. Vaccines. 6 (2007) 711-21.

RI
[120] Arkema A, Huckriede A, Schoen P, Wilschut J, Daemen T. Induction of cytotoxic T lymphocyte activity by fusion-active
peptide-containing virosomes. Vaccine 2000;18:1327-33.

SC
[121] M.G. Cus, Applications of influenza virosomes as a delivery system, Hum. Vaccin. 2 (2006) 1-7.

[122] L. Bungener, K. Serre, L. Bijl, L. Leserman, J. Wilschut, T. Daemen, P. Machy, Virosome-mediated delivery of protein
antigens to dendritic cells, Vaccine. 20(2002) 2287-95.

U
[123] A. Huckriede, L. Bungener, T. Stegmann, T. Daemen, J. Medema, A.M. Palache, J. Wilschut, The virosome concept for
influenza vaccines, Vaccine. 23(2005) S26-38.
AN
[124] J.S.J. Cryz, R. Gluck, Immunopotentiating reconstituted influenza virosomes as a novel antigen delivery system, Dev. Bio.
Stand. 92(1998) 219-23.

[125] R. Mischler, I.C. Metcalfe, Inflexal®V a trivalent virosome subunit influenza vaccine: production, Vaccine. 20 (2002) B17-
M

23.

[126] G.M.E. Maghraby, B.W. Barry, A.C. Williams, Liposomes and skin: from drug delivery to model membranes, Eur. J.
Pharmaceut. Sci. 34 (2008) 203-222.
D

[127] J.M. Chan, L. Zhang, K.P. Yuet, G. Liao, J.W. Rhee, R. Langer, O.C. Farokhzad, PLGA-lecithin-PEG core-shell
nanoparticles for controlled drug delivery, Biomaterials. 30 (2009) 1627-1634.
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[128] L. Hyesun, J.H. Chang, Spherical silica hybrid liposome particles with controlled release of citrus unshiu peel extracts.
Mater. Chem. Phys. 208 (2018) 183-188.

[129] C.S. Gondi, J.S. Rao, Cathepsin B as a cancer target, Expert Opin. Ther. Targets. 17 (2013) 281-291.
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Fig 1. Conventional low-molecular-weight (MW) chemotherapy versus EPR-based
liposome.
A: Conventional small molecule chemotherapeutic drugs show high levels of off-
target accumulation in healthy tissues during the distribution and elimination phase
(upper parts of the panels on the left) and low levels of tumor accumulation (lower
parts of the panels on the left). Conversely, nanodrugs (liposomes) prevent
chemotherapy accumulation in healthy tissues (upper parts of the panels on the right),
and promote accumulation at pathological sites (lower parts of the panels on the

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right). B: Typical pharmacokinetic profiles of small molecule drugs (left) and
nanodrugs (right) in blood and tumors, exemplifying prolonged circulation properties
and enhanced tumor accumulation over time. [6]

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Fig 2. The shape of cationic lipids.

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Fig 3. Cationic lipids with steroid hydrophobic domain

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Fig 4. Chemical structure of amino acid-based cationic lipids [23]

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Fig 5. In vitro cytotoxicity analysis of free PTX/CUR, PTX/CUR LPs and RGD-
PTX/CUR LPs on A549 cell lines. Notes: (A) 24 hours, or (B) 48 hours. cell viability
assay was performed by MTT assay. *p<0.05 RGD-PTX/CUR LPs vs PTX/CUR
LPs. [57]

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Fig 6. Intracellular drug delivery. (i) Accumulation of liposomes to tumor tissue via
EPR effect. (ii) BLM release responding to weakly acidic pH a tumor tissue. (iii)
Some liposomes are taken up by tumor cells and release the drug in response to
endosomal. [65]

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Fig 7. In vivo comparison of the biodistribution of SPIO@Liposome-ICG-RGD and
SPIO@Liposome-ICG. A, B. In vivo continuous observations (120 h) of liver cancer
xenografts administrated with SPIO@Liposome-ICG-RGD (A) or SPIO@ Liposome-
ICG (B) using FMI. Black circles indicate the regions of interest for calculating the
tumor to background ratio (TBR) in each time point. The quantification of
fluorescence intensity at the tumor sites reveals a higher accumulation of
SPIO@Liposome-ICG-RGD at all observation points C. Comparison of TBR profiles
of the two probes. The peak and maximum difference both occurred at 72 h post-
injection, suggesting the optimal surgical window time D. Experiments were run in

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triplicate.[114]

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Fig 8. Virosomes are reconstituted influenza virus envelopes devoid of inner core and
genetic information. By mimicking native influenza viruses, the virosomes maintain
the cell entry and membrane-fusion properties that allow presentation to the MHC
class I and class II pathway. [118]

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Caption of graphical abstract
Liposomes are nano drug carriers, their aqueous phase could contain hydrophilic drugs
and their phospholipid bilayer should localize the lipophilic drugs. In order to make the
common liposome more fit the human and animal body’s complex environment, the
structural variation strategy in the head, tail and bond of lipid molecules have been
employed to develop the different functionalized liposomes, including PEGylated long-
circulation liposomes, active targeting liposomes, environmental sensitive liposomes
and multifunctional liposomes.

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Table 2 Selected liposome and lipid-based vaccines approved for human use or in
clinical trials

Name Company Disease Description Status

Virosomes-reconstituted influenza viral membranes

Inflexal®V Crucell influenza (phospholipids, hemagglutinin, and neuraminidase) Marketed
supplemented with PC

Formalin-inactivated Hepatitis A virus adsorbed to

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Epaxal® Crucell Hepatitis A Marketed
virosomes

Non-small cell BLP25 (palmitoylated MUC1), MPL, DPPC,

Stimuvax Merck KGaA, Oncothyreon Phase 3
lung cancer DMPG, Chol

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Recombinant fusion of P. falciparum
RTS,S/AS01a GlaxoSmithKline Malaria circumsporozoite protein and Hepatitis B surface Phase 3

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antigen, PC, Chol, MPL, QS21

Vaxisome NasVax influenza Inactivated influenza vaccine, CCS, Chol Phase 2

Inactivated influenza vaccine, DOTIM, Chol,

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JVRS-100 Juvaris BioTherapeutics Influenza Phase 2
non-coding plasmid DNA
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Plasmid DNA-encoded influenza proteins,
Vaxfectin Vical Influenza Phase 1
GAP-DMORIE, DPyPE

CAF01 Statens Serum Institut Tuberculosis Subunit protein antigen Ag85B-ESAT, DDA, TDB Phase 1
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Table 1 Marketed liposome loaded with small molecular drugs

Product Name Drug Approved Indication Year Approved Ref.

Abelcet Amphotericin B Sever fungal infections 1995 [96]

Amphotec Amphotericin B Sever fungal infections 1996 [97]

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Ambisome Amphotericin B Sever fungal infections 1997 [98]
Doxil Doxorubicin Kaposi’s sarcoma, ovarian 1995 [99]
cancer, breast cancer

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DaunoXome Doxorubicin Kaposi’s sarcoma 1996 [100]

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Myocet Doxorubicin Breast cancer 2000 [101]
Depocyt Cytarabine Neoplastic meningitis, 1999 [102]
lymphomatous meningitis
Marqibo Vincristine
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Acute lymphoblastic 2012 [103]
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leukemia
Onivyde Irinotecan Pancreatic cancer 2015 [104]
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DepoDur Morphine Pain following surgery 2004 [105]

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Highlights
Medicinal chemistry in liposome design
Types of liposomes now available
Medical application of Liposomes

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