Synthesis, characterization and antitumor evaluation of CMCS-DTX conjugates as novel delivery platform for docetaxel

Article abstract of DOI:10.1016/j.ijpharm.2013.04.020

The purpose of this study is to synthesis and evaluate the antitumor efficacy of a novel carboxymethyl chitosan-docetaxel (CMCS-DTX) conjugates and the availability of CMCS as the polymer material in polymer-drug conjugates development. Docetaxel (DTX) wa

Full text of DOI:10.1016/j.ijpharm.2013.04.020

International Journal of Pharmaceutics 451 (2013) 41–49
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Synthesis, characterization and antitumor evaluation of CMCS–DTX
conjugates as novel delivery platform for docetaxel
Fengxi Liu, Lixia Feng, Li Zhang, Xu Zhang, Na Zhang∗
The School of Pharmaceutical Science, Shandong University, 44 Wenhua Xi Road, Ji’nan, Shandong Province 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The purpose of this study is to synthesis and evaluate the antitumor efficacy of a novel carboxymethyl
chitosan–docetaxel (CMCS–DTX) conjugates and the availability of CMCS as the polymer material
in polymer–drug conjugates development. Docetaxel (DTX) was attached to carboxymethyl chitosan
(CMCS) via biodegradable linker for the first time and the weight percentage of DTX in the CMCS–DTX
conjugates was up to 20%. The resulting CMCS–DTX conjugates could spontaneously self-assemble into
nanoparticles in aqueous buffer, with uniform size of 127.2 3.58 nm and zeta potential of −25.65 mV.
The stability test result showed that only 12.46% of DTX was released after incubation in plasma for
48 h, indicating good stability of CMCS–DTX conjugates in plasma. The results of in vitro cytotoxicity and
Hoechst staining indicated that CMCS–DTX conjugates exhibited significant cytotoxicity against B16 and
HepG2 cells. CMCS–DTX conjugates also displayed better antitumor effect than Duopafei® by inhibiting
tumor growth and prolonging the survival time of B16 melanoma bearing mice more effectively (the
median survival time was >30 days for CMCS–DTX conjugates versus 24 days for the Duopafei®). Besides,
CMCS–DTX conjugates demonstrated an excellent safety profile with a maximum tolerated dose (MTD)
of >250 mg/kg in mice, which was more than 4 fold higher than that of Duopafei® (50 mg/kg). CMCS–DTX
conjugates could be exploited as a promising platform for the effective delivery of DTX and CMCS was a
favorable choice as the polymer material in polymer–drug conjugates development.
Received 24 October 2012
Received in revised form 8 March 2013
Accepted 8 April 2013
Available online 20 April 2013
Keywords:
Carboxymethyl chitosan
Docetaxel
Polymer–drug conjugates
Antitumor effect
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
therapeutic effect (Moghimi et al., 2005). In order to overcome
these drawbacks, nanotechnology has been exploited to develop
Currently, chemotherapy is one of the most commonly used
methods in cancer treatment. Most of chemotherapeutic agents for
tumor treatment perform antitumor activity via cytotoxic or cyto-
static effect. Despite high potency, poor solubility, rapid clearance
rate and severe adverse effects to normal issues are common prob-
lems encountered during formulation and administration for most
of these low molecular anticancer drugs (Chadha et al., 2008). The
marketed formulations of these anticancer drugs usually contain
high concentration of nonionic surfactants as solubilizer, such as
Tween 80 and cremophor EL, both of which could cause severe side
effects e.g. hypersensitivity, nephrotoxicity, fluid retention, neuro-
toxicity and musculoskeletal toxicity in patients (Baker et al., 2009).
Besides, low molecular chemotherapeutic drugs could not dis-
criminate tumor tissue from normal tissues and show widespread
distribution all through the body by diffusion-dependent equilib-
rium, leading to adverse effects to normal tissues and compromised
novel and surfactant free delivery systems, for which anticancer
drugs are physically encapsulated into various nanodevices (such
as liposome, micelle, nanoparticle, microemulsions, and etc.) that
could selectively accumulate in tumor sites by passive targeting
(Conti et al., 2006). Indeed, these formulations eliminated toxic
effects associated with related surfactants (Chadha et al., 2008).
The albumin paclitaxel nanoparticle (Abraxane®), which has been
approved for treating metastatic breast cancer by FDA, did not show
any hypersensitivity reactions in patients according to results of
clinical studies (Gradishar et al., 2005). However, there are still
some disadvantages for the physical encapsulation method, such
as limited drug loading and less control over drug release kinetics
(Liu et al., 2012).
In recent years, polymer–drug conjugates, in which anticancer
drugs were conjugated to various hydrophilic and biocompatible
polymers via biodegradable linkers, are emerging as novel and
potential platform for the delivery of anticancer drugs (Canal et al.,
2011). Until now, several exciting and promising polymer–drug
conjugates including poly(l-glutamic acid)–paclitaxel (PGA–PTX)
(England et al., 2012), ␣,␤-poly[(N-carboxybutyl)-l-aspartamide]–
camptothecin (PGA–CPT) (Fan et al., 2010), HPMA-DOX (Sirova
et al., 2012), PEG–docetaxel (PEG–DTX) (Liu et al., 2008a) and
Abbreviations: CMCS, carboxymethyl chitosan; DTX, docetaxel; CMCS–DTX, car-
boxymethyl chitosan–docetaxel.
∗
Corresponding author. Tel.: +86 053188382015; fax: +86 053188382548.
E-mail address: zhangnancy9@sdu.edu.cn (N. Zhang).
0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.04.020

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F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
cyclodextrin–CPT (Cheng et al., 2004) have already advanced
to clinical trials. Amphiphilic polymer–drug conjugates can
self-assemble into nanoparticles in aqueous media, in which
water-insoluble drug and water-soluble polymer are served as
the hydrophobic core and hydrophilic out-layer, respectively.
Compared with nanoparticles that load drugs by traditional encap-
sulation method, a successful polymer–drug conjugate has many
advantages, including (1) higher drug loading and increased water
solubility for hydrophobic drugs after conjugating to hydrophilic
polymers (Banerjee et al., 2012); (2) prolonged half-life in the
body by controlled cleavage of the linkages between drugs and
polymers (Pasut and Veronese, 2007); (3) enhanced tumor tar-
geting of conjugates and tumor accumulation of free drugs by
enhanced permeability and retention (EPR) effect and selective
release of drug mediated by high local tumor esterases and/or
tumor macrophages (Vasey et al., 1999; Yang et al., 2012); (4) pro-
motion of drug entering into cells via efficient endocytosis and
bypassing membrane-localized efflux proteins (Li, 2002; Li and
Wallace, 2008); (5) no risk of burst release during delivery owing
to the stable covalent chemical bonds between drugs and polymers
(Eldar-Boock et al., 2011); (6) better safety by elimination of sol-
ubilizer for in vivo application (Duncan, 1992, 2009; Maeda et al.,
1992).
Docetaxel (DTX) displays confirmed therapeutic activity against
a broad spectrum of tumors (Lavelle et al., 1995). Marketed for-
mulations of DTX, such as Taxotere® and Duopafei®, contain high
concentration of nonionic surfactant—Tween 80, which could trig-
ger dose-limited toxicities and severe hypersensitivity reactions in
patients (Baker et al., 2009). Thus, development of surfactant free
and tumor targeted delivery system of DTX is one of the hot topics
in drug delivery researches. In this study, DTX was chosen as the
model drug to prepare polymer–drug conjugates with CMCS.
The purpose of this study is to prepare and evaluate the anti-
tumor efficacy of CMCS–DTX conjugates and the availability of
CMCS as polymer material in development of polymer–drug con-
jugates. DTX was conjugated to CMCS via biodegradable linker and
the morphology, particle size, zeta potential, in vitro stability and
cytotoxicity of CMCS–DTX conjugates were characterized. Hoechst
33342 staining was applied to detect cell apoptosis induced by
CMCS–DTX conjugates. The in vivo anti-tumor efficacy was stud-
ied in Kunming mice bearing B16 melanoma. Furthermore, the
maximum tolerated dose (MTD) of Duopafei® and CMCS–DTX con-
jugates were evaluated on Kunming mice.
2. Materials and methods
Selection of suitable polymer is vital in the design and devel-
opment of polymer–drug conjugates. An ideal polymer should
meet the following requirements: (1) be inherently nontoxic and
non-immunogenic; (2) be easily metabolized or excreted from the
body to prevent progressive accumulation in the body (Duncan,
1992); (3) with good water solubility to improve the solubility of
hydrophobic anticancer drugs; (4) bears enough functional groups
for efficient modification with drugs; (5) easy to be produced, low
in cost and stable in formulation that can be manufactured repro-
ducibly and safe for repeated administration (Duncan et al., 2001).
In recent years, polysaccharide-drug conjugates for cancer
therapy have received more and more attention in the field of poly-
mer therapeutics (Liu et al., 2008b). Many polysaccharides based
conjugates, such as paclitaxe–carboxymethyl dextran (AZ10992)
(Sugahara et al., 2007) and docetaxel–carboxymethylcellulose (Cel-
lax) (Ernsting et al., 2012), have been evaluated about their efficacy
in antitumor therapy. In contrast to the success of synthetic poly-
mers based conjugates, only few polysaccharides based conjugates
have entered into clinical trials. Carboxymethyl chitosan (CMCS)
is one of the most widely researched polysaccharides as new
biomedical materials due to its favorable physical, biological and
economic properties, including moisture retention capability, gel-
forming capability, ampholytic property, antibacterial activity and
low cost of production (Dong et al., 2010; Mourya and Nazma, 2010;
Upadhyaya et al., 2013). Furthermore, the presence of hydroxyl,
carboxyl and amino groups in CMCS allows conjugation with many
drugs directly or by foreign linkages via amine or ester bonds.
These amine and ester bonds were widely used in the synthe-
sis of many polymer–drug conjugates and proved to be cleavable
by some enzymes such as cathepsin B (Singer et al., 2003) and
thiol protease cathepsin B (Schmid et al., 2007). Due to these
advantages mentioned above, CMCS is particularly suitable for syn-
thesis of polymer–drug conjugates and CMCS based conjugates
have been exploited for delivery of several hydrophobic anti-
cancer drugs, such as doxorubicin (DOX) (Xie, 2006), methotrexate
(MTX) (Wang et al., 2011b) and 6-mercaptopurine (Zheng et al.,
2011), all of which confirmed the availability of CMCS as poly-
mer material for polymer–drug conjugates in terms of synthesis.
However, researches of these CMCS based conjugates were lim-
ited to synthesis and physiochemical characterization, whereas
pharmacological activities of these conjugates, which are essen-
tial factors for successful polymer–drug conjugates, were rarely
reported.
2.1. Materials
CMCS (average Mw = 50,000; degree of carboxymethyl substitu-
tion = 60%; degree of deacetylation = 85%) was purchased from Jinan
Haidebei Biological Engineering Co. (China). DTX and Duopafei®
were provided by Qilu Pharmaceutical Co., Ltd. (Jinan, China).
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC·HCl), N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS),
Hoechst 33342, succinic anhydride, 4-dimethylamiopryidine
(DMAP) and 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetra-
zolium bromide (MTT) were purchased from Sigma–Aldrich (USA).
Human hepatocellular liver carcinoma (HepG2) and murine malig-
nant melanoma (B16) cell lines were obtained from Shandong
Institute of Immunopharmacology and Immunotherapy (Jinan,
China). Super neonatal bovine serum was obtained from Hangzhou
Sijiqing Biological Engineering Materials Co., Ltd. (China). All the
other chemical and reagents involved were of analytical purity
grade or higher and obtained commercially. Female Kunming mice
weighing 18–22 g were supplied by the Medical Animal Test Center
of the New Drugs Evaluation Center, Shandong University. All ani-
mal experiments complied with the requirements of the National
Act on the Use of Experimental Animals (People’s Republic of
China).
2.2. Synthesis of CMCS–DTX conjugates
2.2.1. Synthesis of succinyl-DTX
DTX (100 mg, 0.12 mmol), succinic anhydride (22 mg,
1.8 equiv.) and DMAP (0.5 mg, 0.033 equiv.) were dissolved in
dry dichloromethane (DCM, 5 mL). After string for 24 h at room
temperature, DCM in the reaction mixture was evaporated under
reduced pressure and ethyl acetate (20 mL) was added. Then the
organic phase was collected and washed with HCl (1%, w/v) twice
and ultrapure water twice, respectively. Magnesium sulfate was
added to the organic phase and stayed overnight to remove the
remained water. Next, ethyl acetate was evaporated under reduced
pressure to get a concentrated solution of desired product (2 mL).
The final solution was subjected to SiO2 column chromatography
for purification with petroleum ether: ethyl acetate (1:2, v/v)
as the mobile phase. The fluid phase for chromatography was
removed under reduced pressure and dried under vacuum to get
dry powder of succinyl-DTX.

F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
43
2.2.2. Synthesis of sulfo-NHS-succinyl-DTX
(4.6 mm × 250 mm reverse phase stainless steel column packed
with 5 ␮m particles Inertdil®ODS-3, GL Sciences Inc., Japan) with
acetonitrile/water (55:45) as eluting solution at a flow rate of
1.0 mL/min.
25 mg of succinyl-DTX (0.027 mmol), 13 mg of EDC·HCl
(2.5 equiv.) and 14 mg of sulfo-NHS (2.5 equiv.) were dissolved in
3 mL mixture of THF: H2O (50:50) and reacted for 12 h at room
temperature to afford the sulfo-NHS ester form of succinyl-DTX.
2.6. In vitro cytotoxicity study
2.2.3. Conjugation of sulfo-NHS-succinyl-DTX to CMCS
50 mg of CMCS (0.001 mmol) was dissolved in 20 mL of mixture
of THF: H2O (50:50) and mixed with the solution of sulfo-NHS-
succinyl-DTX got in procedure 2.2.2. After stirring for 24 h at room
temperature, the reaction mixture was dialyzed with a dialysis bag
(Sigma–Aldrich, molecular weight cutoff: 8000–14000 Da) against
PBS (phosphate buffered saline, pH 7.4) for 48 h to remove organic
solvents and unreacted reagents. CMCS–DTX conjugates could self-
assemble into nanoparticle as the dialysis process went on. The
formed CMCS–DTX nanoparticles were sterile filtrated first by
0.45 ␮m and then by 0.22 ␮m Millipore PVDF filters and then stored
at 4 ◦C. For long-term storage, mannitol was added as cryopro-
tectant (5%, w/v) and lyophilized (Alpha 2–4 L SC, Marin Christ,
German). The structure of CMCS–DTX conjugates was confirmed
by using 1H NMR (AvanceTM DPX-300, Bruker BioSpin GmbH, Rhe-
instetten, Germany).
In vitro cytotoxic activity of CMCS–DTX conjugates was tested
in HepG2 and B16 cells by MTT assay (Wang et al., 2011a). Cells
were seeded in 96-well plates at a density of 4000 cells per well
in 100 ␮L of culture medium and incubated overnight to allow
cell attachment. Then cells were treated with CMCS, free DTX, or
CMCS–DTX conjugates, respectively. After 48 h exposure, 20 ␮L of
MTT (5 mg/mL) was added to each well and incubated for another
4 h. Then the cell plates were centrifuged at 3000 rpm for 10 min
and the culture medium was discarded. 200 ␮L of dimethyl sulfox-
ide (DMSO) was added to dissolve formazan crystals in each well.
The absorbance of the obtained DMSO solution was measured with
a test wavelength of 570 nm and a reference wavelength of 630 nm
by a microplate reader (Model 680, BIO-RAD, USA). Untreated cells
were taken as control with 100% viability and appropriate controls
with DMEM and MTT were run to subtract background absorbance.
The relative cell viability (%) compared to control groups was calcu-
lated by the following equation: cell viability (%) = Abs (sample)/Abs
(control) × 100%. Where, Abs (sample) stands for the absorbance
intensity of the cells treated with tested samples and Abs (control)
stands for the absorbance intensity of the cells treated with culture
medium. Since Tween 80 was proved to be cytotoxic (Arechabala
et al., 1999), instead of Tween 80/ethanol, DMSO was used as the
solvent for DTX in the cytotoxicity study.
2.3. Determination of DTX content in CMCS–DTX conjugates
Drug content in CMCS–DTX conjugates was quantified by
UV spectrophotometer (UV-2102PCS; UNICO [SHANG-HAI] Instru-
ments Co., Ltd, Shanghai, China) at an absorption wavelength
of 230 nm. Firstly, the absorption values of a series of standard
solutions at different known concentrations (5–50 ␮g/mL) of DTX
dissolved in mixed solvent of H2O: CH3OH (v/v = 1:1) were tested
to prepare the calibration curve. CMCS–DTX conjugates were dis-
solved in the form of individual molecular in the mixed solvents
composed of H2O: CH3OH (v/v = 1:1). Appropriate controls of CMCS
solution in H2O: CH3OH (v/v = 1:1) were run to subtract background
absorbance (Lee et al., 2009).
2.7. Apoptosis analysis
Cell apoptosis induced by CMCS–DTX conjugates was investi-
gated by assessment of nuclear morphology after staining the cells
with Hoechst 33342. Briefly, HepG2 cells were seeded on 24 well
cell plate (1 × 105 cells per well) and incubated for 24 h to allow
cell attachment. Then, cells were exposed to DTX or CMCS–DTX
conjugates. After incubation for 24 h, cells were washed with pre-
cold PBS twice gently and stained with Hoechst 33342 (10 ␮g/mL)
at 37 ◦C for 30 min in the dark. The stained cells were washed
with pre-cold PBS and photographed using a reverse fluorescence
microscopy (Olympus IX71, Japan).
2.4. Appearance, transmission electron microscopy (TEM) image,
particle size and zeta potential of CMCS–DTX nanoparticles
CMCS–DTX conjugates could self-assemble into nanoparticles
due to its amphiphilic property. The appearance of DTX suspen-
sion, CMCS solution and CMCS–DTX nanoparticles were compared
with each other to observe the effect of conjugation on increasing
the solubility of DTX. Transmission electron microscope (TEM) was
used to depict the morphology of CMCS–DTX nanoparticles after
negative staining with phosphotungstic acid solution (2%, w/v). Size
and zeta potential of CMCS–DTX nanoparticles were measured by
DelsaTM Nano Submicron Particle Size and Zeta Potential Particle
Analyzer photon correlation spectroscopy (PCS) (Beckman Coulter,
USA). Experimental values were calculated from the measurements
performed at least in triplicate.
2.8. In vivo antitumor efficacy
The antitumor effect of CMCS–DTX conjugates was evaluated
in Kunming mice (18–22 g) inoculated with B16 melanoma cells
by subcutaneously injection at the right axillary space. When the
tumor volume was measured up to approximately 100 mm3, the
mice were administered Duopafei® at a dose of 5 mg DTX/kg,
CMCS–DTX conjugates (5 mg DTX/kg equivalent) and CMCS (with
the equivalent dose of CMCS as that in CMCS–DTX conjugates) after
sterile filtration, respectively, by intravenous administration once
every three days for 30 days (Koo et al., 2012; Wang et al., 2012).
Mice of control group were administered with normal saline (NS).
Tumor weight was monitored and tumor size were measured using
calipers and calculated using an equation of (1/2) (L × W2), where
W is the tumor measurement at the widest point and L stands for
the tumor dimension at the longest point. Mice were monitored
until the tumor volume reaching about 4000 mm3 or the death of
the animals and mice with tumor volume beyond the upper limit
size were euthanized. A Kaplan Meier plot of survival probability
was generated from the survival data.
2.5. Stability of CMCS–DTX conjugates
The stability of CMCS–DTX conjugates was examined by deter-
mination the DTX release after incubation with PBS/Tween 80
mixed medium (pH 7.4, containing 0.5% Tween 80 (w/v) to increase
the solubility of DTX), cell culture medium and rat plasma. The
amount of free DTX released from conjugates in cell culture
medium and rat plasma was extracted by ethyl acetate and drug
release in PBS/Tween 80 mixed medium was analyzed without
further extraction procession. The amount of released DTX was
quantified by reverse-phase high-performance liquid chromatog-
raphy (RP-HPLC, SPD-10Avp Shimadzu pump, LC-10Avp Shimadzu
UV–vis detector, Shimadzu, Japan) analysis on a C18 column

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F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
Fig. 1. Synthesis scheme of CMCS–DTX conjugates.
2.9. Evaluation of the MTD
CMCS through a succinate linker which is confirmed to be cleav-
able under physiological conditions (Safavy et al., 2004), causing
release of free DTX to exert therapeutic effect (Esmaeili et al., 2009;
Lee et al., 2008). 1H NMR of CMCS–DTX conjugates in D2O (Fig. 2)
verified the successful conjugation of DTX to CMCS. Free DTX and
CMCS–DTX conjugates were identified by proton chemical shifts at
7.0–8.0 ppm for aromatic protons. The detailed 1H chemical shifts
for CMCS–DTX conjugates were as follows: the presence of the
peaks corresponding to DTX, such as, 7.94 ppm (HC-25 and HC-
29), 7.65 ppm (HC-27), 7.55 ppm (HC-26 and HC-28), 7.28–7.34 ppm
(HC-31 and HC-35, HC-32 and HC-34), 7.26 ppm (HC-33), 5.86 ppm
(HC-13), 0.89–0.95 ppm (HC-16 and HC-17), 1.22 ppm (HC-7ꢀ, HC-8ꢀ
and HC-9ꢀ), 1.59 ppm (HO-C-7) 1.70–1.76 ppm (HC-18 and HC-19)
confirmed the DTX conjugation. The resonance at 3–4.5 ppm of
CMCS–DTX conjugates was assigned to the protons of the glycol
backbone of CMCS.
Initial evaluation of tolerability of CMCS–DTX conjugates was
evaluated on healthy female KunMing mice (18–22 g). Mice were
administrated intravenously with Duopafei® (20 mg/kg, 50 mg/kg,
75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg of DTX equivalent) or
CMCS–DTX conjugates (20 mg/kg, 50 mg/kg, 100 mg/kg, 150 mg/kg,
200 mg/kg, 250 mg/kg of DTX equivalent), respectively. Changes
in body weight and survival of mice were monitored daily. The
MTD was defined as the highest dose that causes neither toxic
death nor greater than 10% of body weight loss within a week
after administration. Animals showing weight loss greater than 20%
were sacrificed, as changes of this magnitude often indicate lethal
toxicity (Xu et al., 2009).
2.10. Statistical analysis
By controlling the ratio of DTX to CMCS, the weight percentage
(wt%) of DTX in CMCS–DTX conjugates determined by UV spec-
trophotography was about ∼5–25 wt%. However, it was founded
that the particle size of CMCS–DTX nanoparticles increased signifi-
cantly when the drug loading was higher than ∼20 wt%, so ∼20 wt%
of drug loading was fixed for the synthesis of CMCS–DTX conjugates
in this study.
All studies were repeated at least of three times and measured
in triplicate. Data were reported as mean standard deviation (SD).
Statistical significance was analyzed using Student’s t-test. Differ-
ences between experimental groups were considered statistically
significant when P < 0.05.
3. Result and discussion
3.2. Appearance, TEM image, particle size and zeta potential of
CMCS–DTX nanoparticles
3.1. Synthesis and characterizations of CMCS–DTX conjugates
The chemical structure and synthesis steps of CMCS–DTX con-
jugates were shown in Fig. 1. DTX was covalently attached to
The solubility of DTX in water was measured to be approxi-
mately only 6–7 ␮g/mL. When DTX was dispersed in water at a

F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
45
Fig. 4. Characterization of CMCS–DTX conjugates: (a) TEM image and (b) particle
size of CMCS–DTX nanoparticles.
as well as interstitial transport in tumor issue due to EPR effect (Li
et al., 2012). Therefore, the particle size of CMCS–DTX conjugates
was beneficial for more drug accumulation in solid tumors. The
average zeta potential of CMCS–DTX nanoparticles was –25.65 mV
due to the presence of ionized carboxylic groups ( COO−) in CMCS.
3.3. Stability of CMCS–DTX conjugates
Fig. 2. 1H NMR spectra of CMCS in D2O, DTX in DMSO-d6 and CMCS–DTX conjugates
in D2O.
The stability of CMCS–DTX conjugates was studied by deter-
mination of DTX released from conjugates in a series of release
medium. As shown in Fig. 5, only 3.85% and 9.77% of DTX
was released from its conjugated form after 48 h incubation in
PBS/Tween 80 (0.5%) mixed medium and cell culture medium,
respectively. Besides, only 12.46% of DTX was released after 48 h
incubation in plasma, which indicated that CMCS–DTX conjugates
were stable in the blood and small amount of drug could release
during transport process. The good stability of CMCS–DTX con-
jugates in plasma could provide more time and opportunities for
CMCS–DTX conjugates accumulating in tumor sites by EPR effect,
which is beneficial for alleviation of side effects to bone, marrow,
heart and kidney, etc. (Vicent and Duncan, 2006).
concentration of 0.5 mg/mL, it could be only in a coarsely dispersed
condition with visible particles and precipitation of DTX as shown
in Fig. 3(a). Whereas, the appearance of CMCS–DTX conjugates in
water was clear or slightly bluish at the concentration of 0.5 mg/mL
of DTX equivalent as shown in Fig. 3(c). Besides, the mean particle
size for CMCS–DTX conjugates and DTX suspension was ∼130 nm
and ∼4460 nm, respectively. Compared with the turbid solution
of DTX, the improved transparency and decreased particle size
of CMCS–DTX conjugates indicated that the solubility of DTX was
increased greatly after conjugation with CMCS. The improved sol-
ubility of DTX allowed elimination of Tween 80/ethanol for in vivo
application, which could avoid the side effects associated with
Tween 80.
3.4. In vitro cytotoxicity studies
CMCS–DTX conjugates could self-assemble into nanoparticles in
aqueous condition due to its amphiphilic property. TEM was used
to visualize the morphology of the CMCS–DTX nanoparticles. As
shown in Fig. 4(a), CMCS–DTX nanoparticles were well dispersed
as individual particles with spherical shape. The average particle
size of the CMCS–DTX nanoparticles was 127.2 3.58 nm (polydis-
persivity index 0.218 0.15). Limiting the size of nanoparticles to
less than 200 nm could promote extravasation from microvessels
The cytotoxicity of CMCS, DTX and CMCS–DTX conjugates were
evaluated in HepG2 and B16 cells by MTT method. As shown in Fig. 6
CMCS did not cause obvious cytotoxicity against both HepG2 and
Fig. 5. Accumulative DTX release from CMCS–DTX nanoparticles in PBS/Tween 80,
cell culture medium and rat plasma, respectively, at 37 ◦C. Each data point represent
mean SD (n = 3).
Fig. 3. Appearance of (a) DTX dispersed in water; (b) CMCS solution; (c) CMCS–DTX
conjugates in water.

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F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
Table 1
IC50 values of free DTX and CMCS–DTX conjugates against HepG2 and B16 cell lines
after 48 h incubation.
Cell line
Free DTX (␮g/mL)
CMCS–DTX (␮g/mL)
HepG2
B16
0.33 0.03
0.61 0.01
1.02 0.10**
0.71 0.14
**P < 0.01 versus Duopafei®
.
found that when administrated at low doses (≤0.1 ␮g/mL), free
DTX exhibited higher cytotoxicity than CMCS–DTX conjugates, but
when treated with relative high concentration of drug, CMCS–DTX
conjugates showed comparable (1–10 ␮g/mL) or even higher
(50 ␮g/mL) cytotoxicity than free DTX.
It is worth noting that MTT test was carried out in a purified
biochemical system, without the interfering of many systematic
barriers that in the real physiological condition. For in vivo appli-
cation, CMCS–DTX conjugates could specifically localize in tumor
tissue by EPR effect and enter tumor cells efficiently by the promo-
tion of endocytosis (Shi et al., 2012). Besides, as shown in Fig. 6(c),
it is obvious that the cytotoxicity of Tween 80 was significant (the
concentration range of Tween 80 was parallel to that in Duopafei®
formulation). Thus, DTX dissolved in DMSO instead of Duopafei®
was chosen as the positive control in MTT test.
3.5. Apoptosis analysis
DTX can induce cell apoptosis by acting on tubulin through
stabilization of microtubules (Montero et al., 2005). In order to eval-
uate cell apoptosis induced by CMCS–DTX conjugates on HepG2
cells, Hoechst staining of nucleus was observed. As shown in
Fig. 7, the nucleuses of the control cells were homogeneously
stained, while the nucleuses of the cells treated with the DTX or
CMCS–DTX conjugates exhibited chromatin condensation and fur-
ther distributed into apoptotic bodies, which were typical features
of apoptosis induced by DTX (Liu et al., 2010). These results indi-
cated that CMCS–DTX conjugates could successfully induce cell
apoptosis like free DTX.
3.6. In vivo antitumor efficacy
Murine malignant melanoma carcinoma xenograft models were
prepared to assess the antitumor effect of the CMCS–DTX conju-
gates in vivo. The survival time and tumor growth of the mice in
each group were monitored. The survival rate of the malignant
melanoma bearing mice was shown Fig. 8(a). The mice in con-
trol group and CMCS group exhibited rapid death with 50% dying
within 15 days and 100% dying within 21 days. For the mice injected
with Duopafei® at 5 mg/kg of DTX equivalent, 50% of the mice died
within 21 days. While for mice administered with CMCS–DTX con-
jugates, only 37.5% of the mice died during 30 days of treatment.
The median survival time were 12, 24 and >30 days for control,
Duopafei® and CMCS–DTX conjugates, respectively.
CMCS–DTX conjugates showed strong anti-tumor effect not
only by prolonging the median survival time of the mice, but also
by inhibiting the tumor growth. As shown in Fig. 8(b), there was
no significant difference between the tumor volume of mice in
CMCS group and control one, indicating no therapeutic effect of
CMCS, while Duopafei® and CMCS–DTX conjugates could restrict
the tumor growth effectively. Besides, the tumor inhibition effect
of CMCS–DTX conjugates outperformed Duopafei® in a statistically
significant manner (P < 0.01), which might because that CMCS–DTX
conjugates could reach tumor site by EPR effect and maintain the
effective therapeutic concentration of DTX for a long period of time
by sustained cleavage of the conjugates.
Fig. 6. In vitro cytotoxicity study of CMCS–DTX conjugates against (a) HepG2 and (b)
B16 cell lines. Viability was determined by MTT assay after cells treated with CMCS,
DTX or CMCS–DTX conjugates for 48 h at 37 ◦C. (c) Viability of HepG2 and B16 Cells
incubated with Tween 80 (concentration was parallel to that in Duopafei® which is
with the same dose of DTX tested) for 48 h at 37 ◦C. Each data point represents the
value of mean SD (n = 5).
B16 cells at experimental concentrations range from 0.005 ␮g/mL
to 0.25 mg/mL (the doses of CMCS were equal to that in CMCS–DTX
conjugates tested), indicating the good safety and biocompatibility
of CMCS. Like free DTX, CMCS–DTX conjugates showed significant
cytotoxic effect against these two tumor cell lines, indicating that
DTX was released from CMCS–DTX conjugates successfully when
incubated with cells. The IC50 value of free DTX was lower than that
of CMCS–DTX conjugates (shown in Table 1). However, according
to the cell viability data (shown in Fig. 6), it was interesting to

F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
47
Fig. 7. Induction of apoptosis on HepG2 cells by DTX and CMCS–DTX conjugates. Fluorescence microscopy images of HepG2 cell nucleuses following 24 h incubation with
DTX or CMCS–DTX conjugates at equivalent concentration of DTX (a) 0.001 ␮g/mL; (b) 0.1 ␮g/mL; (c) 10 ␮g/mL, respectively. Cell nucleuses were stained with Hoechst 33342.
Table 2
accounting for the phenomenon that the body weight of the mice
in CMCS and NS group increased rapidly in early days of the treat-
Mean body weight on day 30 vs. day 0 (%) before and after removing of solid tumors.
Mean body weight change (%)
NS
CMCS
Duopafei®
CMCS–DTX
ment. The net body weight gain of the mice in CMCS–DTX group was
higher than that in Duopafei® group, indicating the lower system-
atic toxicity of CMCS–DTX conjugates. Besides, mice in Duopafei®
group were in a weak state in terms of voluntary movement and
spirit and one case of drug related death was observed, which might
be due to the adverse effects associated with Tween 80/ethanol and
the unspecific distribution of DTX (Wang et al., 2011a).
With tumor
Without tumor
118.8
104.9
119.1
108.2
128.4
119.9
130.0
127.9
The body weight change was monitored to evaluate the adverse
effects of different DTX formulations. As shown in Fig. 9 and Table 2,
although the total body weight of the mice in CMCS–DTX group
show low increasing rate during the early days (0–12th day) of the
treatment, the net body weight after removing solid tumors at the
end of the treatment showed the most significant increase versus
the original one, indicating low toxicity of CMCS–DTX conjugates. It
was found that the weight of solid tumors significantly contribute
to the weight gain/loss of experimental mice, which was the reason
3.7. MTD of CMCS–DTX conjugates
MTD of CMCS–DTX conjugates were determined by a dose esca-
lation study followed by daily body weight measurement and
observation of general signs of toxicity. No severe side effects or
sudden deaths were deserved through the period of this study.
Table 3
Determination of MTD after i.v. injection of Duopafei® and CMCS–DTX conjugates.
Formulations
Duopafei®
Dose (mg/kg)
Observation post injection
Lethality
Weight change
MTD (mg/kg)
50 mg/kg
20
50
75
100
125
150
Apathy, loss of appetite
Apathy, loss of appetite
Apathy, catatonia, prostration
Apathy, prostration 100% death within 5 days
Apathy, prostration 100% death within 4 days
Apathy, prostration 100% death within 3 days
0/3
0/3
1/3
3/3
3/3
3/3
−1.2%
−6.9%
−14.3%
−20%
−20%
−20%
20
50
100
150
200
250
Well tolerated
Well tolerated
Well tolerated
Well tolerated
Well tolerated
Loss of apeptite
0/3
0/3
0/3
0/3
0/3
0/3
+20.2%
+9.4%
+11.6%
+3.2%
+2.1%
−1.8%
CMCS–DTX conjugates
>250 mg/kg

48
F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
reactions and rapid 100% dying of the animals in 3–5 days. For
the mice treated with CMCS–DTX conjugates, there were no sig-
nificant observable signs of distress and lethal weight loss at the
dose up to 250 mg/kg. According to the result shown in Table 3, the
MTD of Duopafei® was approximately 50 mg/kg, while the MTD
of CMCS–DTX conjugates was >250 mg/kg, which was more 4 fold
higher than that of Duopafei®. The enhanced MTD for CMCS–DTX
conjugates was most likely attributed to the excellent biocompat-
ibility of CMCS and alleviation of the toxic solubilizer.
4. Conclusion
In this study, CMCS was successfully employed as the polymer
for synthesis of a novel of polymer–drug conjugates—CMCS–DTX
conjugates, which could self-aggregate into nanoparticles in aque-
ous buffer. CMCS–DTX conjugates increased the solubility of
DTX significantly, which could eliminate the toxicity associated
with Tween 80/ethanol formulation. In vivo antitumor efficacy
of CMCS–DTX conjugates was superior to that of Duopafei® by
inhibiting tumor growth and extending the survival time of tumor
bearing mice. CMCS–DTX conjugates displayed excellent safety
profile with the MTD 4 fold higher than that of Duopafei®. Based
on the results of this study, CMCS–DTX conjugates might be served
as a safe and efficient delivery platform for DTX. Furthermore,
the favorable properties of CMCS–DTX conjugates indicated that
CMCS based polymer–drug conjugates systems might be explored
as potential and promising delivery platforms for low molecular
anticancer drugs.
In our future work, more attention will be given to further study
of CMCS–DTX conjugates, such as acute toxicity test and antitumor
effect on other tumor models. More works focusing on develop-
ment of active targeting CMCS–DTX conjugates will also be carried
out. Furthermore, the presence of many functional groups makes
CMCS particularly suitable for preparation of polymer–drug con-
jugates, so CMCS based nanoconjugates involved different types of
antitumor drugs will also be researched in the future.
Fig. 8. Antitumor efficacy in KunMing mice bearing B16 melanoma. (a)
Kaplan–Meier survival plots for mice injected with NS, CMCS, Duopafei® and
CMCS–DTX conjugates, respectively. (b) Mean tumor growth curves. Data were
shown as means SD (n = 8); *P < 0.05, **P < 0.01.
Duopafei® was well tolerated at the dose of 20 mg/kg. However,
increasing the dosage of Duopafei® to 75 mg/kg resulted in apa-
thy, catatonia, prostration, lethal deaths of the treated mice and
causing weight loss exceeding than 10%. When the dose levels
reached 100 mg/kg or above, Duopafei® caused more severe toxic
Acknowledgments
The work was supported by National Natural Science Foun-
dation of China (No. 810725857) and Shandong Province Natural
Science Foundation (ZR2009CM011).
References
Arechabala, B., Coiffard, C., Rivalland, P., Coiffard, L.J.M., De Roeck-Holtzhauer, Y.D.Y.,
1999. Comparison of cytotoxicity of various surfactants tested on normal human
fibroblast cultures using the neutral red test, MTT assay and LDH release. J. Appl.
Toxicol. 19, 163–165.
Baker, J., Ajani, J., Scotte, F., Winther, D., Martin, M., Aapro, M.S., von Minckwitz, G.,
2009. Docetaxel-related side effects and their management. Eur. J. Oncol. Nurs.
13, 49–59.
Banerjee, S.S., Aher, N., Patil, R., Khandare, J., 2012. Poly (ethylene glycol)-prodrug
conjugates: concept, design, and applications. J. Drug Deliv. 2012, 1–17.
Canal, F., Sanchis, J., Vicent, M.J., 2011. Polymer–drug conjugates as nano-sized
medicines. Curr. Opin. Biotechnol. 22, 894–900.
Chadha, R., Kapoor, V., Thakur, D., Kaur, R., Arora, P., Jain, D., 2008. Drug carrier
systems for anticancer agents: a review. J. Sci. Ind. Res. (India) 67, 185.
Cheng, J., Khin, K.T., Davis, M.E., 2004. Antitumor activity of beta-cyclodextrin
polymer–camptothecin conjugates. Mol. Pharm. 1, 183–193.
Conti, M., Tazzari, V., Baccini, C., Pertici, G., Serino, L.P., De Giorqi, U., 2006. Anticancer
drug delivery with nanoparticles. In Vivo 20, 697–701.
Dong, W., Han, B., Feng, Y., Song, F., Chang, J., Jiang, H., Tang, Y., Liu, W., 2010.
Pharmacokinetics and biodegradation mechanisms of a versatile carboxymethyl
derivative of chitosan in rats: in vivo and in vitro evaluation. Biomacromolecules
11, 1527–1533.
Duncan, R., 1992. Drug-polymer conjugates: potential for improved chemotherapy.
Anticancer Drugs 3, 175–210.
Duncan, R., 2009. Development of HPMA copolymer-anticancer conjugates: clinical
experience and lessons learnt. Adv. Drug Deliv. Rev. 61, 1131–1148.
Fig. 9. Antitumor efficacy in KunMing mice bearing B16 melanoma. Body weight
change of the mice injected with NS, CMCS, Duopafei® and CMCS–DTX conjugates,
respectively.

F. Liu et al. / International Journal of Pharmaceutics 451 (2013) 41–49
49
Duncan, R., Gac-Breton, S., Keane, R., Musila, R., Sat, Y., Satchi, R., Searle, F., 2001.
Polymer–drug conjugates, PDEPT and PELT: basic principles for design and
transfer from the laboratory to clinic. J. Controlled Release 74, 135–146.
Eldar-Boock, A., Miller, K., Sanchis, J., Lupu, R., Vicent, M.J., Satchi-Fainaro, R., 2011.
Integrin-assisted drug delivery of nano-scaled polymer therapeutics bearing
paclitaxel. Biomaterials 32, 3862–3874.
Montero, A., Fossella, F., Hortobagyi, G., Valero, V., 2005. Docetaxel for treatment of
solid tumours: a systematic review of clinical data. Lancet Oncol. 6, 229–239.
Mourya, V.K., Nazma, N., 2010. Carboxymethyl chitosan and its applications. Adv.
Mater. Lett. 1, 11–33.
Pasut, G., Veronese, F.M., 2007. Polymer–drug conjugation. Prog. Polym. Sci. 32,
933–961.
England, R.M., Conejos-Sanchez, I., Vicent, M.J., 2012. Drug Delivery Strategies:
Polymer Therapeutics, Nanostructured Biomaterials for Overcoming Biological
Barriers. Royal Society of Chemistry, London, pp. 456–482.
Safavy, A., Georg, G.I., Vander Velde, D., Raisch, K.P., Safavy, K., Carpenter, M., Wang,
W., Bonner, J.A., Khazaeli, M.B., Buchsbaum, D.J., 2004. Site-specifically traced
drug release and biodistribution of a paclitaxel-antibody conjugate toward
improvement of the linker structure. Bioconjugate Chem. 15, 1264–1274.
Schmid, B., Chung, D.E., Warnecke, A., Fichtner, I., Kratz, F., 2007. Albumin-binding
prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that
are cleaved by cathepsin B: synthesis and antitumor efficacy. Bioconjugate
Chem. 18, 702–716.
Shi, N.Q., Gao, W., Xiang, B., Qi, X.R., 2012. Enhancing cellular uptake of activable
cell-penetrating peptide–doxorubicin conjugate by enzymatic cleavage. Int. J.
Nanomed. 7, 1613–1621.
Singer, J.W., Baker, B., Vries, P., Kumar, A., Shaffer, S., Vawter, E., Bolton, M., Garzone,
P., 2003. Poly-(L)-glutamic acid–paclitaxel (CT-2103)[XYOTAXTM], a biodegrad-
able polymeric drug conjugate: characterization, preclinical pharmacology, and
preliminary clinical data. Adv. Exp. Med. Biol. 519, 81–99.
Sirova, M., Mrkvan, T., Etrych, T., Chytil, P., Rossmann, P., Ibrahimova, M., Kovar, L.,
Ulbrich, K., Rihova, B., 2012. Preclinical evaluation of linear HPMA-doxorubicin
conjugates with pH-sensitive drug release, Efficacy, safety, and immunomodu-
lating activity in murine model. Pharm. Res. 27, 200–208.
Sugahara, S., Kajiki, M., Kuriyama, H., Kobayashi, T., 2007. Complete regression of
xenografted human carcinomas by a paclitaxel–carboxymethyl dextran conju-
gate (AZ10992). J. Controlled Release 117, 40–50.
Ernsting, M.J., Tang, W.L., MacCallum, N.W., Li, S.D., 2012. Preclinical pharma-
cokinetic, biodistribution, and anti-cancer efficacy studies of
a docetaxel-
carboxymethylcellulose nanoparticle in mouse models. Biomaterials 33,
1445–1454.
Esmaeili, F., Dinarvand, R., Ghahremani, M.H., Amini, M., Rouhani, H., Sepehri, N.,
Ostad, S.N., Atyabi, F., 2009. Docetaxel–albumin conjugates: preparation, in vitro
evaluation and biodistribution studies. J. Pharm. Sci. 98, 2718–2730.
Fan, N., Duan, K., Wang, C., Liu, S., Luo, S., Yu, J., Huang, J., Li, Y., Wang, D.,
2010. Fabrication of nanomicelle with enhanced solubility and stability of
camptothecin based on alpha, beta-poly [(N-carboxybutyl)-l-aspartamide]-
camptothecin conjugate. Colloids Surf. B: Biointerfaces 75, 543–549.
Gradishar, W.J., Tjulandin, S., Davidson, N., Shaw, H., Desai, N., Bhar, P., Hawkins, M.,
O’Shaughnessy, J., 2005. Phase III trial of nanoparticle albumin-bound paclitaxel
compared with polyethylated castor oil based paclitaxel in women with breast
cancer. J. Clin. Oncol. 23, 7794–7803.
Koo, A.N., Min, K.H., Lee, H.J., Lee, S.-U., Kim, K., Chan Kwon, I., Cho, S.H., Jeong,
S.Y., Lee, S.C., 2012. Tumor accumulation and antitumor efficacy of docetaxel-
loaded core–shell-corona micelles with shell-specific redox-responsive cross-
links. Biomaterials 33, 1489–1499.
Lavelle, F., Bissery, M., Combeau, C., Riou, J., Vrignaud, P., Andre, S., 1995. Preclinical
evaluation of docetaxel (Taxotere). Semin. Oncol. 22, 3–16.
Upadhyaya, L., Singh, J., Agarwal, V., Tewari, R.P., 2013. Biomedical applications of
carboxymethyl chitosans. Carbohydr. Polym. 91, 452–466.
Lee, E., Kim, H., Lee, I.H., Jon, S., 2009. In vivo antitumor effects of chitosan-conjugated
docetaxel after oral administration. J. Controlled Release 140, 79–85.
Lee, E., Lee, J., Lee, I.H., Yu, M., Kim, H., Chae, S.Y., Jon, S., 2008. Conjugated chitosan
as a novel platform for oral delivery of paclitaxel. J. Med. Chem. 51, 6442–6449.
Li, C., 2002. Poly(l-glutamic acid)-anticancer drug conjugates. Adv. Drug Deliv. Rev.
54, 695–713.
Vasey, P.A., Kaye, S.B., Morrison, R., Twelves, C., Wilson, P., Duncan, R., Thomson, A.H.,
Murray, L.S., Hilditch, T.E., Murray, T., 1999. Phase I clinical and pharmacokinetic
study of PK1 [N-(2-hydroxypropyl) methacrylamide copolymer doxorubicin]:
first member of a new class of chemotherapeutic agents—drug–polymer conju-
gates. Clin. Cancer Res. 5, 83–94.
Vicent, M.J., Duncan, R., 2006. Polymer conjugates: nanosized medicines for treating
cancer. Trends Biotechnol. 24, 39–47.
Li, C., Wallace, S., 2008. Polymer–drug conjugates: recent development in clinical
oncology. Adv. Drug Deliv. Rev. 60, 886–898.
Wang, L.L., Li, M., Zhang, N., 2012.
Folate-targeted docetaxel-lipid-based-
Li, Y.H., Wang, J., Wientjes, M.G., Au, J.L., 2012. Delivery of nanomedicines to extra-
cellular and intracellular compartments of a solid tumor. Adv. Drug Deliv. Rev.
64, 29–39.
Liu, D.H., Wang, L.L., Liu, Z.H., Zhang, C., Zhang, N., 2010. Preparation, characteriza-
tion, and in vitro evaluation of docetaxel-loaded poly (lactic acid)-poly (ethylene
glycol) nanoparticles for parenteral drug delivery. J. Biomed. Nanotechnol. 6,
675–682.
nanosuspensions for active-targeted cancer therapy. Int. J. Nanomed. 7,
3281–3294.
Wang, L.L., Liu, Z.H., Liu, D.H., Liu, C.X., Juan, Z., Zhang, N., 2011a. Docetaxel-loaded-
lipid-based-nanosuspensions (DTX-LNS): preparation, pharmacokinetics, tissue
distribution and antitumor activity. Int. J. Pharm. 413, 194–201.
Wang, Y., Yang, X., Yang, J., Wang, Y., Chen, R., Wu, J., Liu, Y., Zhang, N., 2011b.
Self-assembled nanoparticles of methotrexate conjugated O-carboxymethyl
chitosan: preparation, characterization and drug release behavior in vitro. Car-
bohydr. Polym. 86, 1665–1670.
Xie, Y.M., 2006. Synthesis of N, O-carboxylchitosan-doxorubicin conjugates and
research of their properties. Sichuan University, Cheng Du.
Xu, Z., Chen, L., Gu, W., Gao, Y., Lin, L., Zhang, Z., Xi, Y., Li, Y., 2009. The performance of
docetaxel-loaded solid lipid nanoparticles targeted to hepatocellular carcinoma.
Biomaterials 30, 226–232.
Yang, D., Van, S., Shu, Y., Liu, X., Ge, Y., Jiang, X., Jin, Y., Yu, L., 2012. Synthesis,
characterization, and in vivo efficacy evaluation of PGG–docetaxel conjugate
for potential cancer chemotherapy. Int. J. Nanomed. 7, 581.
Zheng, H., Rao, Y., Yin, Y., Xiong, X., Xu, P., Lu, B., 2011. Preparation, characteri-
zation, and in vitro drug release behavior of 6-mercaptopurine-carboxymethyl
chitosan. Carbohydr. Polym. 83, 1952–1958.
Liu, J.B., Zahedi, P., Zeng, F.Q., Allen, C., 2008a. Nanosized assemblies of
a
PEG–docetaxel conjugate as a formulation strategy for docetaxel. J. Pharm. Sci.
97, 3274–3290.
Liu, Z.H., Jiao, Y.P., Wang, Y.F., Zhou, C.R., Zhang, Z.Y., 2008b. Polysaccharides-based
nanoparticles as drug delivery systems. Adv. Drug Deliv. Rev. 60, 1650–1662.
Liu, Z.H., Wang, Y.T., Zhang, N., 2012. Micelle-like nanoassemblies based on
polymer–drug conjugates as an emerging platform for drug delivery. Expert
Opin. Drug Deliv., 1–18.
Maeda, H., Seymour, L.W., Miyamoto, Y., 1992. Conjugates of anticancer agents
and polymers: advantages of macromolecular therapeutics in vivo. Bioconjugate
Chem. 3, 351–362.
Moghimi, S.M., Hunter, A.C., Murray, J.C., 2005. Nanomedicine: current status and
future prospects. FASEB J. 19, 311–330.