Chitooligosaccharides Modified Reduction-Sensitive Liposomes: Enhanced Cytoplasmic Drug Delivery and Osteosarcomas-Tumor Inhibition in Animal Models

Article abstract of DOI:10.1007/s11095-017-2225-0

Purpose: To investigate the potential of a reduction-sensitive and fusogenic liposomes, enabled by surface-coating with chotooligosaccharides (COS) via a disulfide linker, for tumor-targeted cytoplasmic drug delivery. Methods: COS (MW2000-5000) were chemically tethered onto the liposomes through a disulfide linker (-SS-) to cholesterol (Chol). Doxorubicin (DOX) was actively loaded in the liposomes. Their reduction-sensitivities, cellular uptake, cytotoxicity, pharmacokinetics and antitumor efficacy were investigated. Results: The Chol-SS-COS/DOX liposomes (100?nm) had zeta potential of 33.9?mV and high drug loading (13% w/w). The liposomes were stable with minimal drug leakage under physiological conditions but destabilized in the presence of reducing agents, dithiothreitol (DTT) or glutathione (GSH) at 10?mM, the cytosolic level. MTT assay revealed that the cationic Chol-SS-COS/DOX liposomes had higher cytotoxicity to MG63-osteosarcoma cells than non-reduction sensitive liposome (Chol-COS/DOX). Flow cytometry and confocal microscopy revealed that Chol-SS-COS/DOX internalized more efficiently than Chol-COS/DOX with more content to cytoplasm whereas Chol-COS/DOX located around the cell membrane. Chol-SS-COS/DOX preferentially internalized into MG63 cancer cell over LO2 normal liver cells. In rats both liposomes produced a prolonged half-life of DOX by 4 - 5.5 fold (p?

Full text of DOI:10.1007/s11095-017-2225-0

Pharm Res
DOI 10.1007/s11095-017-2225-0
RESEARCH PAPER
Chitooligosaccharides Modified Reduction-Sensitive
Liposomes: Enhanced Cytoplasmic Drug Delivery
and Osteosarcomas-Tumor Inhibition in Animal Models
1
1
2
1
Xuelei Yin & Yingying Chi & Chuanyou Guo & Shuaishuai Feng
&
1
1
1,3
Jinhu Liu & Kaoxiang Sun & Zimei Wu
Received: 26 February 2017 /Accepted: 28 June 2017
Springer Science+Business Media, LLC 2017
#
ABSTRACT
cell membrane. Chol-SS-COS/DOX preferentially internal-
ized into MG63 cancer cell over LO2 normal liver cells. In
rats both liposomes produced a prolonged half-life of DOX by
4 - 5.5 fold (p < 0.001) compared with the DOX solution.
Chol-SS-COS/DOX exhibited strong inhibitory effect on tu-
mor growth in MG63 cell-bearing nude mice (n = 6), and
extended animal survival rate.
Purpose To investigate the potential of a reduction-sensitive
and fusogenic liposomes, enabled by surface-coating with
chotooligosaccharides (COS) via a disulfide linker, for
tumor-targeted cytoplasmic drug delivery.
Methods COS (MW2000-5000) were chemically tethered on-
to the liposomes through a disulfide linker (-SS-) to cholesterol
(
Chol). Doxorubicin (DOX) was actively loaded in the lipo-
Conclusions Reduction-responsive Chol-SS-COS liposomes
may be an excellent platform for cytoplasmic delivery of antican-
cer drugs. Conjugation of liposomes with COS enhanced tumor
cell uptake, antitumor effect and survival rate in animal models.
somes. Their reduction-sensitivities, cellular uptake, cytotoxici-
ty, pharmacokinetics and antitumor efficacy were investigated.
Results The Chol-SS-COS/DOX liposomes (100 nm) had
zeta potential of 33.9 mV and high drug loading (13% w/w).
The liposomes were stable with minimal drug leakage under
physiological conditions but destabilized in the presence of
reducing agents, dithiothreitol (DTT) or glutathione (GSH)
at 10 mM, the cytosolic level. MTT assay revealed that the
cationic Chol-SS-COS/DOX liposomes had higher cytotox-
icity to MG63-osteosarcoma cells than non-reduction sensitive
liposome (Chol-COS/DOX). Flow cytometry and confocal
microscopy revealed that Chol-SS-COS/DOX internalized
more efficiently than Chol-COS/DOX with more content
to cytoplasm whereas Chol-COS/DOX located around the
KEY WORDS Chotooligosaccharides (COS) . Cationic
liposomes . Reduction-sensitivity . Cytoplasmic delivery .
Tumor-suppression . Survival rate
ABBREVIATIONS
Chol
Cholesterol
COS
DCC
CLSM
DOX
Chitooligosaccharides
N,N′-dicyclohexylcarbodiimide
Confocal laser scanning microscopy
Doxorubicin
DTOP 3,3′-dithiodipropionic acid
*
*
Kaoxiang Sun
sunkaoxiang@luye.cn
DL
DLS
Drug loading
Dynamic light scattering
Zimei Wu
z.wu@auckland.ac.nz
DMAP 4-Dimethylaminopyridine
DMEM Dulbecco’s Modified Eagle Medium
DTOP 3,3′-Dithiodipropionic acid
DTT
EDC
1
School of Pharmacy, Key Laboratory of Molecular Pharmacology and
Drug Evaluation, (Yantai University), Ministry of Education, Collaborative
Innovation Center of Advanced Drug Delivery System and Biotech Drugs
in Universities of Shandong, Yantai University, Yantai 264005, People’s
Republic of China
Dithiothreitol
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride
EE
Entrapment efficiency
2
3
EPR
FBS
FCM
FTIR
Enhanced permeability and retention
Fetal bovine serum
Flow cytometry
Department of Bone Joint, Qingdao Municipal Hospital,
Qingdao, Shandong Province 266011, China
School of Pharmacy, University of Auckland, Auckland 1142, New
Zealand
Fourier transform infrared

Yin et al.
GSH
MTT
Glutathione
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide
N-hydroxysuccinimide
Polydispersity index
Polyethylene glycol
Reticuloendothelial system
Succinic anhydride
Soybean phosphatidylcholine
Thin-layer chromatography
modify nanocarriers for targeted drug delivery [21, 23, 24].
N-palmitoyl chitosan (MW 65 kDa) modified liposomxes in-
creased systemic circulation of free drug docetaxel by approx-
imately 5.5 times, although not as significant as PEGylation
(10 times) [20]. More recently, glycol-chitosan coated lipo-
somes were demonstrated to have pH-sensitivity due to the
NHS
PDI
PEG
RES
SA
protonation of -NH groups which rendered positive charge
2
to liposomes at tumor extracellular low pH (6.5). This charge
effect enhanced cellular uptake of liposomes and thus antitu-
mor efficacy compared with non-coated liposome (p < 0.05)
SPC
TLC
[25].
Chitooligosaccharides (COS) is a class of hydrolytic prod-
INTRODUCTION
ucts of chitosan. The hydrophilic backbone and low degree of
polymerization (n ≈ 2-20) renders COS high water-solubility
[26] and wider biological activities than the large molecular
chitosan at cellular and molecular levels. The free amino
groups allow interactions between COS and the cells conse-
quently increasing cell adhesion. Moreover, their widely re-
ported anti-angiogenesis and radical scavenging efficacy
[26–28] of COS, partially by induction of GSH level in the
cells [29], made them excellent candidate polymer for modi-
fication of tumor-targeted liposomes.
In this work, we aimed to prepare liposomes with COS
conjugated on the surface of liposomes via a disulfide linker
(-SS-). The liposomes were designed to be stable under normal
physiological conditions; the cell adhesive property of COS
will facilitate internalization of liposomes into tumor cells.
Upon entering cells, the cleavage of disulfide bond by the
intracellular GSH would allow destabilization of the vesicles,
facilitating rapid release of the payload in cytoplasm.
Doxorubicin (DOX) was used as a model drug as well as a
fluorescent marker. The in vitro cellular uptake and cytotoxic-
ity of liposomes were studied in human osteosarcoma cell lines
(MG63). Osteosarcoma is a heterogeneous malignant bone
tumor that has the ability to produce osteoid or immature
bone, most prevalent in children and young adults. Non-
reducible Chol-COS coated liposomes were prepared and
used as controls for the proof of concepts. In this paper, we
report the synthesis of cholesterol derivatives, Chol-SS-
COOH and Chol-COOH, that were used to conjugate
COS on liposomes.
Nano-liposomes are perceived as most biocompatible and bio-
degradable vesicular carriers for tumor-targeted drug delivery
[1–3] through the enhanced permeability and retention (EPR)
effect [4]. Recent effort has been devoted to confer liposomes
with stimuli-responsiveness [5] to exploit the special features
of tumors such as redox potential [3, 6, 7] tumor acidity [8, 9]
and special enzyme [10] as stimuli to selectively trigger drug
release at the target. In particular, disulfide bond has been
widely used in the design of redox sensitivity nanocarriers by
exploiting the glutathione (GSH) in cancer cells as a stimulus
[
3, 6, 7]. The concentration of GSH inside cancer cells is
significantly higher (up to 10 mM) than the extracellular level
2-10 μM) [3] or in the normal cells (0.1 mM) [5]. The pres-
(
ence of pendant thiol group (-SH) in GSH makes it a powerful
reducing agent and can intracellularly triggered cleavage of
disulfide bonds (-SS-).
To exert EPR effect, liposomes (and drug) need to stay in
systemic circulation for a sufficient prolonged period of time
[11]. This can is achieved by coating the carriers with a hy-
drophilic polymer which sterically hinders opsonization and
thus preventing destruction by reticuloendothelial system
(RES) [12, 13]. The gold-standard polymer to date is polyeth-
ylene glycols (PEG) [11, 14]. However, recent research
showed major drawbacks of PEGylation, including reduced
cellular uptake and slow release from endosomes [15, 16], and
induction of anti-PEG IgM antibodies which results in accel-
erated blood clearance (ABC) phenomenon following repeat-
ed administration [17]. Alternative polymers poly [N-(2-hy-
droxypropyl) methacrylamide] (HPMA), poly(vinylpyrroli-
done) (PVP) [18], and chitosan derivatives [19, 20] have been
explored to prolong blood circulation of liposomes.
Chitosan, a class of naturally occurring polysaccharides
consisting of β-(1→4)-linked d-glucosamine (deacetylated unit)
and N-acetyl-d-glucosamine (acetylated unit), have been pop-
ular polymers for drug delivery due to their excellent biocom-
patibility, biodegradability, and mucoadhesiveness [21], and
biological activities, including antitumoral, antimicrobial, and
antiviral activities [21, 22]. The amphiphilic chitosan deriva-
tives have recently been widely used to construct or surface-
MATERIALS AND METHODS
Materials
Soybean phosphatidylcholine (SPC), cholesterol (Chol), doxoru-
bicin hydrochloride (DOX∙HCl) and chemical agents, 3,3′-
dithiodipropionic acid (DTOP), succinic anhydride (SA), 1-eth-
yl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC∙HCl), N-hydroxysuccinimide (NHS), 4-
dimethylaminopyridine (DMAP), and glutathione (GSH) were

Chitooligosaccharides Modified Reduction-Sensitive Liposomes
all purchased from Aladdin (Shanghai, China).
Chitooligosaccharides (MW 2000-5000 and degree of de-
acetylation 75%) were purchased by Fengan Bio-
Pharmaceutical Co. (Zhejiang, China).
above solution to allow unreacted DCC and the by-product
1,3-dicyclohexylurea to precipitate. The precipitate was re-
moved by filtration while the filtrate containing Chol-SS-
COOH was dialyzed against deionized water using a dialysis
bag of MWCO ≈ 2000 Da and lyophilized to obtain Chol-SS-
COOH. The crude product was re-dissolved in ethyl acetate
before loaded on a silica gel column for further purification.
The column was washed with mixture of petroleum ether and
ethyl acetate (1:10, v/v). The eluted product was dried under
reduced pressure to obtain pure Chol-SS-COOH.
The Chol-COOH was synthesized successfully according
to the method reported by Yu et al. [30]. Briefly, cholesterol
and succinic anhydride (SA) were dissolved in pyridine and
kept for 24 h with constant stirring at room temperature.
Then, deionized water was added to the above solution. The
produced precipitate was obtained by filtration and dried.
The crude product was recrystallized in acetone to obtain
the white powder, Chol-COOH.
MG-63 cell line was purchased from BeNa Cell Collection
(Beijing, China), and LO2 cells were gift from Medicine and
Pharmacy Research Center, Binzhou Medical University.
The chemical agents, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyl tetrazolium bromide (MTT) and Hoechst 33,342
were obtained from Sigma–Aldrich (USA) while Dulbecco’s
Modified Eagle Medium (DMEM), Roswell Park Memorial
Institute (RPMI) 1640 Medium, and fetal bovine serum (FBS)
were purchased from Hyclone (LA, USA). All other chemicals
were of analytical reagent grade and used without further
purification.
Both Sprague-Dawley (SD) male rats, for pharmacokinetic
study, and male BALB/c nude mice, for anti-tumour efficacy,
were purchased from Beijing Vital River Laboratory Animal
Technology Co., Ltd. and treated according to the protocols
prior to use. Animal studies were approved by the
Experimental Animals Administrative Committee of Yantai
University.
The structures of the compounds, Chol-SS-COOH and
Chol-COOH, were characterized by nuclear magnetic reso-
1
nance spectrometer ( H NMR, Bruker ARX-400,
Switzerland) using deuterated DMSO-d6 as the solvent.
Synthesis of Chol-SS-COOH and Chol-COOH
Preparation of DOX-loaded liposomes conjugated
with COS
To coat COS on liposomes, Chol-SS-COOH and Chol-
COOH were first synthesized (Scheme 1) before used for li-
posome preparation.
Chol-SS-COOH was synthesized using a DCC/DMAP
coupling method. Briefly, 3,3′-dithiodipropionic acid
The liposomes, Chol-SS-COS-L and Chol-COS-L, were pre-
pared by ethanol injection method followed by conjugation
with COS. Briefly, SPC and Chol-SS-COOH or Chol-
COOH (molar ratio 10:1.4, total lipids about 30 mg) were
added in 1 ml ethanol and sonicated for 10 min. The lipid
solution was added into 10 ml PBS (pH 7.4) slowly dropwise
with stirring 1 h to obtain a liposomes colloidal suspension.
COS polymer (30 mg) was added to the above liposomes, and
tethered through a EDC/NHS coupling reaction by which an
amide bond was formed between the free -COOH groups of
(DTOP) dissolved in dimethyl sulfoxide (DMSO) was added
to a solution of N,N′-dicyclohexylcarbodiimide (DCC) and 4-
dimethylaminopyridine (DMAP) in DMSO under N atmo-
2
sphere. Upon complete dissolution, a certain amount of cho-
lesterol was added dropwise at room temperature with con-
stant stirring. The progress of reaction was monitored by thin-
layer chromatography (TLC) with a mobile phase of
methylbenzene/ethyl acetate 1:1, v/v. After 24 h reaction
was complete and excess deionized water was added into the
liposomes and free -NH of COS [25]. Briefly, the coupling
2
agents, EDC (4.6 mg) and NHS (2.7 mg) were added to the
liposomes suspension and kept under stirring at room
Scheme 1 Synthesis of a simple
and less expensive liposome system
as a potential tumor targeted drug
delivery platform with three
targeting features united: long
circulation, fusogenicity, and
glutathione (GSH)-responsiveness.

Yin et al.
temperature overnight, allowing COS to be conjugated with
liposomes.
placed into a dialysis bag (MWCO 10 KDa) and immersed
in 30 ml of PBS (10 mM, pH 7.4) containing 10 mM or 20 μM
GSH. The samples were maintained at 37 ▫C in a shaking
bed. At designed time intervals, aliquots (1 ml) of dialysis me-
dium were taken and replaced with an equivalent volume of
fresh medium. The concentrations of released DOX were
determined as described above.
Conjugation was confirmed by Fourier transform infrared
(FTIR) spectrometer (PerkinElmer Frontier, UK).
DOX was loaded into the liposomes using the widely re-
ported ammonium sulfate transmembrane gradient method.
The DOX-free liposomes was prepared as described above
with 120 mM (NH ) SO during film hydration. The
4
2
4
untrapped external (NH ) SO was removed by dialysis
against a 10% glucose solution. DOX solution (5 mg/ml)
was added to the above liposomes solution and incubated at
4
2
4
Physical stability of DOX-loaded liposomes
The physical stability of DOX-encapsulated liposomes in PBS
4
0 ▫C for 3 h while stirring and protected from light. The free
(
pH 7.4, 10 mM) containing 10% FBS stored at 4 ▫C was
DOX was removed from the DOX-loaded liposomes by
dialysis.
monitored by the measurement of particle size using
Malvern Zetasizer over 15 days.
Characterization of liposomes
In vitro cytotoxicity assay
The particle size and zeta-potential of DOX-loaded liposomes
were measured by dynamic light scattering (DLS, Malvern
Instruments Ltd., UK) after dispersed in PBS (pH 7.4). The
pH effect on zeta-potential was also tested PBS (pH 6, 6.5, and
MG63 human osteoblast-like osteosarcoma cells and human
being’s liver cell strain LO2 cells were maintained in 5% CO2
at 37 ▫C in McCoy’s 5A medium (FBS 15%, v/v) and RPMI-
1640 medium (10% FBS, v/v), respectively. Both media were
7
). The morphology of liposomes was observed by transmis-
supplemented with penicillin-streptomycin (1%, v/v). The
medium was routinely replaced with fresh medium every
day. The cells in logarithmic phase of growth were cryopre-
served for cytotoxicity using 3-(4,5-dimethythiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) cell viability assay.
Cells were seeded in 96-well plates in the abovementioned
medium. The cells were cultured for 24 h hours to allow cells
to attach before exposed to COS polymer, non-coated lipo-
somes, free DOX or drug-loaded liposomes. Each of the sam-
ples was dispersed in culture medium before adding to the
wells. After 24 h or 48 h of incubation, MTT assay was
perfosrmed to determine the cytotoxicity. The optical density
sion electron microscope (TEM, JEM-1400, Japan).
To estimate the amount of DOX encapsulated into lipo-
somes, DOX-loaded liposomes were ultra-centrifuged, and
freeze-dried before dissolved in ethanol. After centrifuge the
DOX concentration in ethanol was measured by fluorescence
spectrophotometric measurement (LS-55, Perkin Elmer,
USA), with excitation wavelength of 488 nm and an
emission wavelength of 593 nm. The linear concentration
range of DOX with the fluorescence method was 0.05-1.
2
3
μg/ml (R > 0.99). The encapsulation efficiency (EE) and
drug loading (DL) was calculated using the following equa-
tions:
(OD) of each well was measured at 490 nm using a microplate
reader (Spectra Max M2, Molecular Devices, USA). Cell vi-
ability was assessed using the following equation:
Amount of drug in liposomes
Amount of drug loaded liposomes
DL ð%Þ ¼
EE ð%Þ ¼
 100
OD treated
Amount of drug in liposomes
Total amount of feeding drug
Cell viability ð%Þ ¼
 100
 100
OD control
ODtreated and ODcontrol represented the OD of treated and
untreated (medium) wells, respectively.
Reduction-sensitivity of DOX-loaded Chol-SS-COS
liposomes
Reduction-induced destabilization of Chol-SS-COS lipo-
somes was observed by mornitoring the change in size of the
liposomes which were suspended in a PBS (0.1 M, pH 7.4)
containing 10 mM dithiothreitol (DTT), a reducing agent [8].
The mixture was placed in shaking water bath at 37 ▫C and
the change in particle size over 4 h was monitored using a
Malvern Zetasizer (DLS). Chol-COS liposomes were used as
controls.
In vitro cellular uptake
To understand the mechanism of liposome-cell interactions
and thus cytotoxicity, cellular uptake of Chol-SS-COS/
DOX by MG63 and LO2 cells was compared using flow
cytometry (FCM) and confocal laser scanning microscopy
(CLSM). For both studies, formulations (free DOX, Chol-
COS/DOX, Chol-SS-COS/DOX) were dispersed in medi-
um with a final DOX concentration of 20 μg/ml before
added into the cells.
In another experiment, to simulate the tumor cellular mi-
croenvironments, 1 ml of each liposomal formulation was

Chitooligosaccharides Modified Reduction-Sensitive Liposomes
For the FCM studies, cells were seeded in 6-well plates
10 /well) in the medium and cultured in 5% CO2 at 37
Anti-tumor efficacy in MG63 tumor bearing nude mice
6
(
▫
C, overnight. Then, the cells were treated with different for-
Male BALB/c nude mice (20 ± 2 g) were used to estab-
lish MG63 tumors by subcutaneous inoculation of
1 × 10 cells in the left hind leg of nude mice. The
mulations. At different time points, 0, 1, 2, 3, and 4 h, the cells
were washed twice with 2 ml of PBS, and detached with tryp-
sin. After centrifuged at 1500 rpm for 5 min, the cells in pellets
were re-suspended in 0.5 ml of PBS before analyzed using a
FACS Calibur flow cytometer (EPICS XL, Beckman, USA).
For CLSM analysis, cells were seeded onto clean and sterile
7
tumor sizes were closely monitored every second day.
Tumor volume was calculated using the formula:
2
0.5 × L× W , with length (L) and width (W) being the
largest and smallest diameters of tumors, respectively.
3
coverslips placed in a 12-well plates at an initial density of
5
Once the tumor size reached approximately 100 mm
5
× 10 cells/well and cultured overnight. Cells were then
(Day 0), the MG63 tumor bearing mice were randomly
divided into four treatment groups (n = 6); untreated
control (saline), free DOX, CHOL-COS/DOX, and
CHOL-SS-COS/DOX. Animals were administered with
saline or DOX formulations at 5 mg/kg via the tail veins
every 3 days for four times.
treated with free DOX, or each of the DOX-containing lipo-
somes, respectively. After incubation at 37 ▫C for 2 h or 4 h,
the cells were washed with PBS for three times, fixed with
paraformaldehyde solution (4%) for 30 min, stained nucleus
with Hoechst 33,342 solution (10 μg/ml) for 10 min before
observed under a confocal microscope (TCS SPE, Leica,
Germany).
Statistical analysis
Pharmacokinetic study
All studies were carried out in triplicate. One-way analysis of
variance (ANOVA) was used to determine the difference of
groups, and data was considered to be significant at p˂0.05.
Male SD rats (190 - 210 g) were randomly divided into three
treatment groups for pharmacokinetic studies (n = 3): free Dox
solution, CHOL-COS/DOX, and CHOL-SS-COS/DOX.
Drug was administrated intravenously at a dose of 2 mg/kg
via tail vein. At the designed time (0.5, 1, 2,4, 6, 8, 24 and
RESULTS
4
8 h), blood samples were collected from the vena
Structural characterization of Chol-SS-COOH
and Chol-COOH
ophthalmica to hepatized tubes. Plasma samples were obtain-
ed after centrifuged and drug was extracted by solvent precip-
itation with chromatographic pure methanol (plasma to meth-
anol ratio 1: 15, v/v). After votexing for 10 min, the mixture
was centrifuged at 5000 rpm for 20 min and supernatant at
fixed volume was dried using Termovap Sample
Concentrator (Wuxi Voshin Instruments Co., Ltd., Wuxi,
China). The residual was dissolved in 0.2 ml mobile phase
and centrifuged and DOX concentration was quantified using
high performance liquid chromatography (HPLC) (Agilent
Technologies 1260 series, USA). The mobile phase was com-
posed of acetonitrile, methanol and NaH PO (10 mM,
1
H NMR of Chol-SS-COOH (500 MHZ, DMSO-d6) δ
(ppm): 5.57 (d, J = 8.0 Hz, 3H), 5.27 (d, J = 5.2 Hz, 1H),
4.59 (d,J = 4.6 Hz, 1H), 0.95 (s, 3H), 0.90 (d, J = 6.5 Hz,
3H), 0.86 (d, J = 2.4 Hz, 3H), 0.85 (d, J = 2.4 Hz, 3H), 0.66
(s, 3H).
1
Chol-COOH H NMR (500 MHZ DMSO-d6) δ (ppm):
12.18 (s, 1H), 5.35 (dd, J = 5.0, 2.0 Hz, 1H), 4.47 (dtd,
J = 12.4, 8.6, 4.4 Hz, 1H), 3.32 (s, 4H), 0.98 (s, 3H), 0.90
(d,J = 6.5 Hz,3H), 0.86 (d,J = 2.4 Hz,3H), 0.85
(d,J = 2.3 Hz,3H), 0.66 (s, 3H). The results indicated the
formation of the chemical structures.
2
4
pH 4.2) at volume ratios of 28: 20: 52. The flow rate and
column temperature were set at 1 ml/min and 25°C, respec-
tively. The UV absorbance at 254 nm was determined and
injection a volume was 20 μl.
The yield of Chol-SS-COOH and Chol-COOH was
76.1% and 80.1%, respectively.
Pharmacokinetic parameters were estimated using non-
compartmental analysis based on statistical moment theory.
Elimination half-life (t1/2) was calculated ast1/2 = 0.693/λ,
where λ is the slope obtained from the regression of natural
log concentration versus time in the terminal phase over the
last three measurable data points. The linear trapezoidal rule
was used to calculate area under the curve (AUC) and the area
under the first moment curve (AUMC) without extrapolation.
The mean residence time (MRT) was further obtained as the
ratio of AUMC/AUC.
Physico-chemical characteristics of COS conjugated
liposomes
Liposomes containing Chol-SS-COOH or Chol-COOH
were successfully prepared used for ethanol injection method
followed by chemical conjugation with COS. The FTIR spec-
tra of Chol-SS-COOH containing liposomes before (Chol-
SS-COOH-L) and after coating with COS (Chol-SS-COS-
L) were shown in Fig. 1. The COS-tethered liposomes exhib-
−1
ited an intense peak was observed at 1637.53 cm (NH bend

Yin et al.
15 days with no significant change in the average size and
range (PDI data not shown).
To evaluate the redox-sensitive drug release behavior, the
liposomes were placed at 10 mM DTT in PBS (10 mM,
pH 7.4), and the size change was measured by DLS. At
10 mM DTT, Chol-SS-COS liposomes, not Chol-COS lipo-
somes, aggregated rapidly, as evidenced with the size of lipo-
somes increasing from 119 ± 1.4 nm to 125 ± 1.5 nm at 2 h
and 228.1 ± 4.9 nm after 4 h. At 24 h, the size was increased
to 254 ± 3 nm. These results confirmed that Chol-SS-COS
liposomes were dissociated in the presence of DTT, most like-
ly due to the cleavage of the -SS- bonds, resulting in detach-
ment of the COS layers. Without the steric repulsion of the
COS polymer the uncoated liposomes tend to aggregate or
fuse.
Fig. 1 FT-IR spectra of Chol-SS-COOH-liposomes (without COS) and
Chol-SS-COS liposomes, confirming the conjugation of COS via formation
of amide bond (corresponding to the new peak at 1637.53 cm ).
−1
In vitro GSH-triggered drug release
vibration), corresponding to amino bond, confirming effective
coupling of amine groups of COS with carboxylic groups
The drug release behavior of DOX-loaded liposomes,
assessed in PBS (pH 7.4) at 37 ▫C exhibited a sustained-
release profile with less than 40% of DOX released after
72 h (Fig. 4). In contrast, the drug release from Chol-SS-
COS in presence of 10 mM of GSH (pH 7.4) was fast with
>50% of DOX released within the first 10 h whearas in the
absence of or low concentration of GSH (20 μM), the accu-
mulative release of DOX was both lower than 40%. For Chol-
COS liposomes, the drug release rate was lower than 20%
with slight increase in the presence of GSH (<30%). In gen-
eral, DOX release from Chol-SS-COS-L were faster than
from Chol-COS liposomes.
(C = O stretch1732.82 cm-1) in the surface of liposomes.
DOX as the most commonly used chemotherapeutic drug
was loaded into the liposomes using the ammonium sulfate
gradient method with high EE and DL. The particle
size and zeta potential of Chol-SS-COS and Chol-
COS liposomes are shown in Table I. The average size
of nanoparticles was ~70-80 nm with a narrower size
distribution (PDI ˂ 0.2) than non-coated liposomes. The
particle size of both liposomes increased following con-
jugation with COS, but this size was suitable to exploit
the EPR effect. The ζ potential of the liposomes with-
out COS(CL)was negative, but was turned into positive
values after COS was tethered on the surface of both
liposomes, confirming the success conjugation of COS
In vitro cytotoxicity assay
The cytotoxic effects of blank liposomes on MG63 and LO2
using MTT assay were shown in Fig. 5A and B, respectively.
Compared with the medium treated cells, the cell viability
remained more than 85% at all the tested concentration up
to 100 μg/ml after 48 h incubation, suggesting that empty
liposomes were nontoxic. Similarly, COS polymer itself had
no cytotoxicity toward the two cell lines, consistent with the
wide report in the literature.
which contains –NH groups. In addition, TEM images
2
(
Fig. 2) show that both Chol-COS and Chol-SS-COS
liposomes were spherical. The particle size was smaller
than that obtained by DLS, possibly due to shrinkage of
the COS polymer layer in a dry state.
Stability and reduction-sensitivity of liposomes
To evaluate the cytotoxicity of various DOX formulations,
cells were treated with different DOX formulations at a con-
centration range of 0.001 to 10 μg/ml. After 24 h incubation,
the cell proliferation was measured with MTT assay. All
As shown in Fig. 3, the COS modified liposomes in PBS
(pH 7.4, 10 mM) containing 10% FBS was stable for at least
Tab le I The physicochemical char-
acteristics of non-coated liposomes
Liposomes
Particle size (nm) PDI
zeta potential (mV) DL (% w/w)
EE (% w/w)
(
containing Chol-SS-COOH) and
COS coated liposomes. Data are
mean ± SD, n = 3
Chol-SS-COOH-L
74.17 ± 2.22 0.279 ± 0.003 −6.52 ± 1.86
15.2 ± 0.87 96.1 ± 4.99
12.9 ± 2.34 85.4 ± 1.36
13.3 ± 2.97 91.3 ± 26.5
Chol-SS-COS/DOX 105.7 ± 0.42
Chol-COS/DOX 81.27 ± 1.13
0.197 ± 0.008
0.216 ± 0.007
33.9 ± 0.737
37.7 ± 1.33

Chitooligosaccharides Modified Reduction-Sensitive Liposomes
Fig. 2 Transmission electron
microscopy (TEM) images of
conventional liposomes with no
COS coating (a), and Chol-SS-
COS-L, similar to Chol-COS-L (b).
formulations showed DOX dose-dependent toxicity to both
cells (Figs. 5C and D). The order of cytotoxicity to MG63 of
the formulations was ranked as: free DOX ˃ Chol-SS-COS/
DOX-L ˃ Chol-COS/DOX-L, as shown by the half inhibi-
tory concentrations (IC ) (p < 0.01). Moreover, significantly
higher cytotoxicity against cancerous MG63 cells than to LO2
liver cells were observed with all formulations at all concen-
tration levels (p < 0.01).
The cellular uptake of different formulations was further
visually observed in MG63 and LO2 cells with confocal laser
scanning microscopy (Figs. 6 and 7). Similar to the flow cy-
tometry, the fluorescence intensity of DOX enhanced over
time but the signals in MG63 were much stronger than in
LO2 cells. The formulations were ranked in the same order
as per the fluorescence intensity in the cells as ranked in the
flow cytometry. Interestingly, DOX was seen mainly distrib-
uted in cellular nuclei of MG63 cells following the treatment
with free DOX, but mainly distributed in cytoplasm, and in
particular, surrounding the nuclear areas following the treat-
ment with Chol-SS-COS/DOX-L.
5
0
In vitro cellular uptake
The kinetics of cellular uptake of Chol-SS-COS/DOX-L and
Chol-COS/DOX-L compared with free DOX by MG63 and
LO2 cells was quantified using flow cytometry (Fig. 6). The
cellular uptake of all formulations by both cell types increased
in a time-dependent manner within the 4 h, and the cellular
fluorescence intensity being ranked in the same order: free
DOX > Chol-SS-COS/DOX-L > Chol-COS/DOX-L.
Moreover, the mean fluorescence intensity of DOX in
MG63 cells was significantly higher than that in LO2 cells,
particularly Chol-SS-COS/DOX-L, suggesting their selectiv-
ity for tumor cells.
Pharmacokinetics
Following i.v. injection of DOX solution, the plasma drug
concentration in rats dropped rapidly with only a minimal
concentration was observed in the chromatograms at 4 h
(< 50 ng/ml). In contrast, the drug concentrations from
both liposomes were measurable after 8 h (Fig. 8).
Compared with the free DOX, the AUC of liposomal
formulations were increased by approximately 3-fold
Fig. 3 (A) Stability of the particle
size of DOX-encapsulated
liposomes in PBS (pH 7.4, 10 mM)
containing 10% FBS stored at 4 ▫C
over 15 days (n = 3). (B) Change in
the particle size of Chol-SS-COS
liposomes in PBS (pH 7.4) after
incubation for 2 h or 4 h in 10 mM
DTTsolutions at 37▫C. Chol-COS
liposomes did not show significant
change.

Yin et al.
Anit-tumor efficacy and survival rate
In MG63 tumor-bearing nude mice, the tumor size of untreat-
ed group (injected with saline only) increased rapidly over time
(Fig. 9 A). Intravenous injection of DOX solution every 3 days
for four times (Day 0 - 12) resulted in significant inhibitory
effect on tumor growth (p < 0.01). However, unfortunately
animal death was found from Day 14 with only 50% of the
animals survived during the experiment (Fig. 9 B). In compar-
ison, the tumor growth rate was remarkably suppressed with
Chol-COS/DOX and particularly Chol-SS-COS/DOX li-
posomes. Furthermore, Chol-SS-COS/DOX liposomes
demonstrated the most distinguished effect on extending sur-
vival of MG63-bearing mice (Fig. 9B).
Fig. 4 DOX release profiles from Chol-COS-L and Chol-SS-COS-L in the
absence or presence of GSH at concentrations of 20 μM (extracellular level)
or 10 mM (intracellular level) in PBS (10 mM, pH 7.4) at 37 ▫C (n = 3).
while T1/2 increased by 4.0 and 5.6-fold for Chol-SS-
COS/DOX-L and Chol-COS/DOX-L, respectively
DISCUSSION
(
(
p < 0.01) (Table II). Maximum drug concentration
Cmax) as observed at 0.5 h were similar for the two lipo-
Targeted drug delivery with liposomes has played a major
role in improving cancer chemotherapy. Recent effort has
been made to confer liposomes long circulation property,
fusogenicity, and tumor microenvironment-responsiveness
to enhance the EPR effect, cellular uptake and cytoplas-
mic delivery efficiency, respectively. In this study, a simple
and less expensive liposome system by tethering
some groups but significantly higher than the free DOX.
Apart from a significant longer T1/2 (p < 0.05), the AUC
and MRT values of the Chol-COS/DOX-L group were
higher than those of the Chol-SS-COS/DOX-L group,
but the differences were not statistically significant
(
p > 0.05).
Fig. 5 In vitro cell viability of blank
liposomes at different
concentrations of lipids against
MG63 (A) and LO2 (B) following
4
8 h incubation. In vitro cytotoxicity
of free DOX, Chol-COS/DOX-L
and Chol-SS-COS/DOX-L against
MG63 (C) and LO2 (D) cells
following 24 h exposure, showing
for all DOX formulations had higher
cytotoxicity against cancerous
MG63 cells than to normal liver cells
LO2 (p < 0.01). The cytotoxicity
was measured by MTTassay, and
data are mean ± SD (n = 6).

Chitooligosaccharides Modified Reduction-Sensitive Liposomes
Fig. 6 Flow cytometry analyses of MG63 (A) and LO2 (B) cells incubated with different formulations, showing the fluorescence intensity of DOX in the cells
increasing over time but at different rates in the order: free DOX > Chol-SS-COS/DOX-L > Chol-COS/DOX-L for both cells. DOX dosage was 20 μg/ml for
all cases but the uptake of the two liposome formulations by MG63 was significantly different. * p ˂ 0.05 and **p ˂ 0.01. Data are mean ± SD (n = 3).
chitooligosaccharides (COS) (MW 2000-5000), a hydro-
lytic product of chitosan, on the surface via a disulfide
bond was designed to unite the three targeting features
(Scheme 1).
Fig. 7 Confocal microscopy
images of (A) MG63 cells and (B)
LO2 cells. Cells were treated with
free DOX (a), Chol-COS/DOX-L
(
b), Chol-SS-COS/DOX-L (c)
incubated for 2 h and 4 h,
respectively, for both cases. Red:
DOX; Blue: nuclear staining with
Hoechst dye.

Yin et al.
in zeta potential over pH 6 - 7.4 with the COS modified
liposomes. This can be attributed to the higher density of -
NH groups resulted from high degree of de-acetylation (75%)
2
of COS, which are positively charged within the pH range.
Cationic liposomes have shown to selectively target tumor
endothelial cells with a preferential uptake in angiogenic tu-
mor vessels [31, 32]. Furthermore, the cellular uptake of lipo-
somes is generally believed to be mediated by adsorption of
liposomes onto the cell surface, subsequently facilitating endo-
cytosis [33]. It is well documented that the surface of cancer
cells are negatively charged due to secretion of a large amount
of lactate anions across membranes, whereas the surfaces of
normal cells remain charge-neutral or slightly positive [34].
Therefore, positively charged liposomes can selectively inter-
act with cancer cells and facilitate cellular uptake via endocy-
tosis [25, 33]. This was also observed with Chol-SS-COS/
DOX-L and the non-reduction sensitive Chol-COS/DOX-
L in this study.
Fig. 8 Mean plasma DOX concentration-time profiles following intravenous
injection of the DOX liposomes or a DOX solution to rats at a dose of
2
mg/kg. Data are mean ± SD (n = 3).
To enable construction of the liposomes, a novel cholester-
ol derivative, Chol-SS-CCOH, was first synthesized and its
structure was confirmed with NMR. The successful conjuga-
tion of COS with Chol-SS-COOH pre-inserted in the lipo-
some bilayer was verified with FTIR (Fig. 1), and also evi-
denced by the increases in size and zeta potential (from nega-
tive to positive values) of both resulted liposomes (Table I).
However, due to the lack for quantitative assay the conjuga-
tion efficiency of COS on the liposomes were not determined.
The stable Chol-SS-COS/DOX liposomes with a high drug
loading of DOX had a size of 105 nm, close to Doxil®. The
liposomes were cationic with zeta potential values of 34-
Chol-SS-COS/DOX-L was stable in PBS (pH 7.4) con-
taining 10% FBS (Fig. 3) and exhibited a sustained drug re-
lease property at pH 7.4 in the absence of GSH (Fig. 4). This
suggested the good drug retention in the liposomes with min-
imal leakage during blood circulation. Drug leakage in vivo is
often a major challenge in liposomes development [35] while
crucial to achieve EPR effect [36]. Further, the in vitro study
indicated that Chol-SS-COS-L were reduction-sensitive, evi-
denced by the destabilization of the liposome structure in re-
ducing agent DTT, most likely due to disulfide bond breakage
(Fig. 3). The cleavage of COS might have resulted in fusion of
liposomes given the samples looked more ‘milky’. However, it
is unknown how much of the COS was cleaved off. Further
study showed that the drug release from Chol-SS-COS/
DOX-L could be sufficiently trigged by the presence of
GSH at the tumoral intracellular concentration (10 mM),
3
7 mV, which is higher than N-palmitoyl chitosan anchored
liposomes (20 mV) [20] or glycol-chitosan modified liposomes
25]. In contrast to glycol-chitosan coated liposomes which
[
demonstrated a pH-dependent zeta potential (+10 mV at
pH 6, −15 mV at pH 7.4) [25], there was no obvious change
Fig. 9 Tumor growth curves (A) and survival rates (B) of MG63 tumor-bearing mice following i.v. injection of different DOX formulations. The treatment started
3
on Day 0 (when the tumor volume reached 100 mm ) and was repeated on Day 4, 8 and 12 at a dose of 5 mg/kg. Data in (A) are mean ± SD (n = 6 or less as a
result of animal death). **p<0.01.

Chitooligosaccharides Modified Reduction-Sensitive Liposomes
but not the extracellular level (20 μM) (Fig. 4). Figure 4 also
shows that Chol-COS/DOX-L also gave slightly higher drug
release in the first a few hours in response to addition of
enhanced the cellar uptake of cationic liposomes but also
attracted the cationic DOX (basic pK 8.4).
a
Long circulation property of nanocarriers is critical for pas-
sive tumor targeting through the potential EPR effect [11].
Following i.v. injection to rats at a dose of 2 mg/kg, the Chol-
SS-COS/DOX-L and Chol-COS/DOX-L resulted in ap-
proximately 3-fold increase in AUC, and 4.0 and 5.5-fold
1
0 mM GSH to PBS, possibly due to the higher osmotic
pressure of the release medium.
MTT assay of COS polymer and COS modified liposomes
showed good biocompatibility with cells (Fig. 5A). Different
form Chol-COS/DOX-L, Chol-SS-COS/DOX-L was dem-
onstrated to more cytotoxic to MG63 cells, but no difference
to LO2 (Table II). A few reasons can explain this selective
cytotoxicity: 1) the charge effect on the two different cells with
MG63 surface being more negatively charged than LO2
resulting in enhanced cellular uptake of the cationic lipo-
somes, and 2) the higher concentration of GSH in cancer cells
MG63 than normal cells. The intracellular cleavage of -SS-
bond by GSH resulted in detachment of COS from lipo-
somes, and subsequent destabilization of liposomes, promot-
ing endosome escape into the cell cytoplasm [3]. In addition,
compared with free DOX, the IC50 of Chol-SS-COS/DOX-
L was higher which reflects the different cellular uptake mech-
anisms and sustained release from liposomes. Nanocarriers
entered tumor cells via endocytosis, which may be slower than
diffusion of free DOX. However, in vitro cytotoxicity tests do
not always reflect the in vivo circumstances such as the EPR
effect. In the body, free DOX will be widely distributed into
tissues with only a small amount reaching tumor cells.
Cellular uptake by MG63 and LO2 cells was quantified
using flow cytometry, and confocal microscopy, the forma-
tions were ranked as DOX > Chol-SS-COS/DOX-
L > Chol-COS/DOX-L. This is consistent with the cytotox-
icity data. The significant improvement of Chol-SS-COS/
DOX-L over Chol-COS/DOX-L demonstrated the crucial
roles of the intracellular detachment of COS in the bioavail-
ability of DOX and the cytotoxic effects. Confocal images of
Chol-COS/DOX-L treated MG63 showed some small dots,
indicating the internalized liposomes might have been
entrapped inside endosome-lysosomal vesicles. It was also not-
ed that DOX formulations, particularly Chol-SS-COS/
DOX-L, produced stronger fluorescence intensity in MG63
cells than LO2. This may be due to the more significant neg-
ative charge of cancer cells than normal cells which not only
increases in the elimination T , compared to DOX solution
½
(Fig. 8 and Table II). The relatively shorter T1/2 of Chol-SS-
COS/DOX than Chol-COS/DOX may partially be caused
by the faster DOX release (Figure 4) [36] and partially due to
the positive charge which led to non-specific interactions with
anionic proteins in the blood and subsequent clearance by the
RES [12]. Additionally, a study in tumor-bearing mice by
Gabizon et al. [37] using PEGylated liposome of DOX
(Doxil) at doses ranging 2.5-20 mg/kg demonstrated that the
liposomes clearance by the RES system is a saturated process,
with lower dose being cleared disproportional faster.
However, in this study the drug concentration was measurable
even at 48 h post injection of Doxil at all dose elvels.
Therefore, longer circulation may be expected should the
dose was increased for the COS-coated liposomes, however,
long-circulation property of COS-coating did not appear to
be as effective as PEGylation. Another note is that redox-
responsive liposomes with detachable PEG coating via disul-
fide bond were previously developed [38, 39] but pharmaco-
kinetic studies suggested that PEG layer was rapidly cleaved in
circulation [39]. To our best knowledge, this paper is the first
study using chitooligosaccharides (COS) to modify liposomal
surface, and the coating through disulfide bond was GSH-
sensitive and stable in blood circulation.
After four i.v. in the MG63 tumor-bearing nude mice at a
DOX dose of 5 mg/kg both liposomes resulted in not only sig-
nificantly enhanced inhibitory effect on tumor growth than
the free DOX, but also the survival rate of animals (Fig. 9).
Compared with Chol-COS/DOX-L, the GSH-sensitive lipo-
somes, Chol-SS-COS/DOX-L was more efficient in tumor
suppression, due to accelerated release of DOX from cyto-
plasmic by GSH-induced cleavage of -SS- bonds. These re-
sults also indicated that the inexpensive polymer COS had
good biocompatibility, and may be a promising alternative
polymer to PEG for stabilization of liposomes, evidenced from
the high survival rate of animals. The 20% death (n = 1) from
Chol-COS/DOX-L was likely due to the tumor growth
effect.
Table II Pharmacokinetic parameters of DOX following intravenous injec-
tion of DOX solution or liposomes at a dose of 2 mg/kg to rats. Data present
as mean ± SD (n = 3)
Parameter
Free DOX Chol-SS-COS/
DOX
Chol-COS/
DOX
CONCLUSIONS
C
max (μg/ml)
4.00 ± 0.93 5.99 ± 0.60
5.89 ± 0.10
14.8 ± 2.1
AUC (μg /ml∙h) 6.5 ± 1.1
1/2 (min) 16.8 ± 0.4
MRT (min)
17.8 ± 1.2
68.0 ± 9.1
Conjugation of liposomes with chitooligosaccharides (COS)
T
93.4 ± 16.9
165.4 ± 13.1
(
MW 2000-5000) extended the liposomal blood circulation
62.1 ± 13.8 148.2 ± 13.6
time and enhanced the efficiency of intracellular delivery, thus

Yin et al.
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financially supported by Taishan Scholar Project carried out
at School of Pharmacy, Yantai University, funded by
Shandong Province, China. The authors declare that they
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