Co-delivery of doxorubicin and paclitaxel by PEG-polypeptide
nanovehicle for the treatment of non-small cell lung cancer
Shixian Lva,e, Zhaohui Tanga, Mingqiang Lia,e, Jian Lina,e, Wantong Songa, Huaiyu Liuc,
Yubin Huangd, Yuanyuan Zhangb,**, Xuesi Chena,*
aKey Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China
bWake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA
cLaboratory Animal Center, Jilin University, Changchun 130012, PR China
dState Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022,
eUniversity of Chinese Academy of Sciences, Beijing 100039, PR China
a r t i c l e i n f o
Received 18 February 2014
Accepted 11 April 2014
Available online 1 May 2014
Controlled drug release
a b s t r a c t
Despite progress, combination therapy of different functional drugs to increase the efficiency of anti-
cancer treatment still remains challenges. An amphiphilic methoxy poly(ethylene glycol)-b-poly(L-glu-
tamic acid)-b-poly(L-lysine) triblock copolymer decorated with deoxycholate (mPEsG-b-PLG-b-PLL/
DOCA) was synthesized and developed as a nanovehicle for the co-delivery of anticancer drugs: doxo-
rubicin (DOX) and paclitaxel (PTX). The amphiphilic copolymer spontaneously self-assembled into
micellar-type nanoparticles in aqueous solutions and the blank nanoparticles possessed excellent sta-
bility. Three different domains of the copolymer performed distinct functions: PEG outer corona provided
prolonged circulation, middle biodegradable and hydrophilic PLG shell was designed for DOX loading
through electrostatic interactions, and hydrophobic deoxycholate modified PLL served as the container
for PTX. In vitro cytotoxicity assays against A549 human lung adenocarcinoma cell line demonstrated that
the DOX þ PTX co-delivered nanoparticles (Co-NPs) exhibited synergistic effect in inducing cancer cell
apoptosis. Ex vivo DOX fluorescence imaging revealed that Co-NPs had highly efficient targeting and
accumulation at the implanted site of A549 xenograft tumor in vivo. Co-NPs exhibited significantly higher
antitumor efficiency in reducing tumor size compared to free drug combination or single drug-loaded
nanoparticles, while no obvious side effects were observed during the treatment, indicating this co-
delivery system with different functional antitumor drugs provides the clinical potential in cancer
? 2014 Elsevier Ltd. All rights reserved.
The combination therapy of multiple drugs with different action
sites has been proved to be an effective strategy in clinical cancer
treatments. This combination can not only delay the cancer adap-
tation process and related cancer cell mutations, but also reduce
drug side effect by decreasing each of their doses and achieve
synergistic therapeutic efficacy [1e5]. However, it is difficult to
combine free drugs to obtain optimal anticancer effect due to their
different biochemical activities and pharmacokinetics among these
drugs . In addition, the combination of free drugs often brought
more serious toxic side effects to human bodies, which has become
a serious problem in clinical cancer treatments .
Over the past few decades, the nano-scaled drug carriers based
on amphiphilic copolymers have been attracting great attention for
the chemotherapeutic drug delivery in cancer treatment. With
synthetic versatility and proper structure design, the polymeric
nanomedicines generally have befitting size distribution, good
biocompatibility, higher stability, controllable drug release profiles,
altered pharmacokinetics and body biodistribution, which offer the
great advantages over traditional therapeutic agents. Because these
traditional anticancer drugs are limited in use due to fast degra-
dation, undesirable drug uptake by normal organs and serious
systemic toxicity [8,9]. In addition, the nano-scaled drug carriers
* Corresponding author. Tel./fax: þ86 431 85262112.
** Corresponding author. Tel.: þ1 336 713 1189; fax: þ1 336 713 7290.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/biomaterials
0142-9612/? 2014 Elsevier Ltd. All rights reserved.
Biomaterials 35 (2014) 6118e6129
also achieve better accumulation in solid tumors through the
enhanced permeability and retention (EPR) effect as compared
with free drugs . Accordingly, several promising nanomedicines
have been adopted in clinical trials, e.g., Doxil?(liposomal DOX),
Abraxane (protein-bound paclitaxel), Genexol-PM (paclitaxel-
encapsulated polymeric micelles) [11e13]. However, most of the
current nanomedicines only load a single drug, which rapidly
develop drug resistance in tumor cells .
Nanocarriers provide the possibility to simultaneously deliver
multiple therapeutic agents. Compared to the combination of free
drugs, the combination of two or more drugs within a single
nanocarrier can overcome toxicity and other side effects. Further-
more, such multiagent systems guarantee the simultaneous de-
livery of sufficient amount of drugs to tumor site, ensure the
synergistic effect and the improved antitumor efficacy [7,15].
However, the distinct water solubility and the diverse electric
properties of chemotherapeutic drugs make it difficult to co-deliver
multi-drugs. For instance, doxorubicin (DOX) and paclitaxel (PTX),
which are among the most common used chemotherapeutic drugs
in clinic against various solid tumors, have remarkably large dif-
ferences in their water solubility properties and anticancer mech-
anthracycline antitumor drugs which can interfere with DNA
through insertion and then induce cancer cell apoptosis. Clinical
commonly used DOX is hydrophilic in the form of protonated
doxorubicin hydrochloride (DOX$HCl). PTX, a representative anti-
microtubule agent, is highly hydrophobic with extremely poor
water solubility . Even the combination of DOX and PTX has
been used as the first-line treatment for metastatic breast cancer
with increased tumor regression rates compared the individual
drugs, but the co-delivery of DOX and PTX was rarely investigated
[17e19]. Wang et al. employed amphiphilic methoxy PEG-PLGA
copolymer nanoparticles as carriers to co-deliver DOX and PTX
through a double emulsion method and received a synergistic
oneof themost effective
antitumor effect in vitro. However, the stability of such polymer/
drug interactions between cationic DOX molecules and uncharged
PEG-PLGA copolymers is highly desired . The system above was
still devoid of data toprovethe invivofeasibilityof theirco-delivery
In order to co-deliver multiple antitumor drugs with strong
polymer/drug interactions and robust construct stability, an
amphiphilic triblock copolymer, methoxy poly(ethylene glycol)-b-
poly(L-glutamic acid)-b-poly(L-lysine) decorated with deoxycholate
(mPEG-b-PLG-b-PLL/DOCA) was designed and utilized as a favor-
able carrier for the co-delivery of DOX and PTX in this study. The
mPEG-b-PLG-b-PLL/DOCA copolymers are expected to undergo
spontaneous self-assembling to nanomicelles in the aqueous so-
lutions resulting in the PEG outer corona, PLG middle shell and
hydrophobic PLL/DOCA inner core. The PEG block provides the
prolonged blood circulation of the nanoparticles by reducing non-
specific interactions with blood components. The anionic poly
(glutamic acid) provides the strong electrostatic interaction with
cationic DOX$HCl. Such polymer/drug complexes through electro-
static interactions have been proved to be very effective in our
previous studies [20e22]. The hydrophobic modified PLL compo-
nent serves as a reservoir for lipophilic drug.
The physiochemical properties, stability, self-assembly, in vitro
drug release behavior were investigated. The synergistic antitumor
effect of the DOX and PTX co-delivered mPEG-b-PLG-b-PLL/DOCA
nanoparticles (Co-NPs) was evaluated both in vitro and in vivo.
2. Materials and methods
Poly(ethylene glycol) monomethyl ether (mPEG, Mn¼ 5000, Aldrich), doxoru-
bicin hydrochloride and paclitaxel (DOX$HCl and PTX, Beijing Huafeng United
Technology Corporation), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT, Sigma), 40,6-diamidino-2-phenylindole dihydrochloride (DAPI,
Sigma), fluorescein isothiocyanate (FITC, Aladdin), Cy5.5-NHS ester (Lumiprobe) and
25 ºC, 1 h
DMF, 35 ºC, 48 h
DMSO, 25 ºC, 48 h
DMF, 35ºC, 48 h
Scheme 1. Synthesis pathway of mPEG-b-PLG-b-PLL/DOCA.
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
Nε-benzyloxycarbonyl-L-lysine (H-Lys(Z)-OH, GL Biochem Co. Ltd.) were used as
received. N, N-dimetylformamide (DMF) and dimethylsulfoxide (DMSO) were stored
over calcium hydride (CaH2) and purified by vacuum distillation with CaH2. Amino-
terminated poly (ethylene glycol) methyl ether (mPEG-NH2) and g-Benzyl-L-gluta-
mate-N-carboxyanhydride (BLG-NCA) were prepared according to our previous
work . Nε-benzyloxycarbonyl-L-lysine-N-carboxyanhydride (Lys(Z)-NCA) was
synthesized as the literature report . Succinimido deoxycholate (DOCA-NHS)
was synthesized according to the literature .
2.2. Synthesis of mPEG-b-PLG-b-PLL triblock copolymer
Methoxy poly(ethylene glycol)-b-poly(L-glutamic acid)-b-poly(L-lysine) (mPEG-
b-PLG-b-PLL) triblock copolymer was synthesized via the one-pot two-step ring-
opening polymerization (ROP) of BLG-NCA and Lys(Z)-NCA monomers with mPEG-
NH2as the macroinitiator and a subsequent deprotection of benzyl groups. In brief,
mPEG-NH2(4.00 g, 0.8 mmol) was dissolved in dry DMF (40 mL) after azeotropic
dehydration process with toluene, then BLG-NCA (2.31 g, 8.8 mmol) dissolved in dry
DMF (23 mL) was added via a syringe under argon. The reaction was maintained at
35?C under argon.After 2 days, a traceof solutionwas removedfrom the system and
precipitated into ice diethyl ether for1H NMR and GPC measurements of methoxy
poly(ethylene glycol)-b-poly(g-benzyl-L-glutamate) (mPEG-b-PBLG). Then, Lys(Z)-
NCA (2.45 g, 8.0 mmol) was added, and the reaction was maintained for another 2
days. The methoxy poly(ethylene glycol)-b-poly(g-benzyl-L-glutamate)-b-poly(Nε-
benzyloxycarbonyl-L-lysine) (mPEG-b-PBLG-b-P (Lys(Z)) triblock copolymer was
obtained by the repeated precipitation from DMF into excess amount of ice diethyl
ether. Yield: 86%.
Subsequently, mPEG-b-PBLG-b-P(Lys(Z)) (4.0 g) was dissolved in trifluoroacetic
acid (TFA, 40 mL) at 0?C in a flask. And then 12 mL HBr/acetic acid (33 wt%) was
added slowly and the solution was stirred at 25?C to remove the protecting groups.
The mPEG-b-PLG-b-PLL copolymer was isolated by precipitation into excess amount
of ice diethyl ether. After dried under vacuum, the crude product was purified by
dialysis against distilled water (MWCO ¼ 3500 Da) and freeze-dried to give the pure
mPEG-b-PLG-b-PLL product, yielding a white solid. Yield: 90.0%.
2.3. Synthesis of mPEG-b-PLG-b-PLL/DOCA
acid)-b-poly(L-lysine) (mPEG-b-PLG-b-PLL/DOCA) was synthesized by chemical
couplingof mPEG-b-PLG-b-PLL with DOCA-NHS. In brief, mPEG-b-PLG-b-PLL (0.82 g,
0.1 mmol) and DOCA-NHS (0.44 g, 0.9 mmol) were dissolved in dry DMSO (15 mL),
then triethylamine (TEA) (0.275 mL, 1.9 mmol) was added into the solution. The
reaction mixture was maintained for 48 h at room temperature under a dry argon
atmosphere. The mixture then was dialyzed (MWCO 3500 Da) against DMSO to
remove unreacted small molecules and further dialyzed against distilled water to
remove DMSO. The mPEG-b-PLG-b-PLL/DOCA white powder was obtained after
To label mPEG-b-PLG-b-PLL/DOCA with FITC, mPEG-b-PLG-b-PLL/DOCA (50 mg)
and FITC (5 mg) were dissolved in dry DMF and the solution was stirred for 24 h at
room temperature in dark. Then the mixture was dialyzed against distilled water
and freeze-dried. The FITC-labeled mPEG-b-PLG-b-PLL/DOCA was obtained as a
2.4. Preparation of drug-loaded nanoparticles
The DOX-loaded mPEG-b-PLG-b-PLL/DOCA nanoparticles (DOX-NPs) were pre-
pared according to our previous report . Briefly, mPEG-b-PLG-b-PLL/DOCA
(90 mg) was dissolved in distilled water and adjusted to pH 7.0e7.5, then DOX$HCl
(10 mg) dissolved in distilled water was added dropwise. After stirring overnight in
the dark, the freeDOX was removed by dialysis using a dialysis bag (MWCO3500 Da)
against deionized water for 24 h, and then freeze-dried to obtain the DOX-NPs
The PTX-loaded mPEG-b-PLG-b-PLL/DOCA nanoparticles (PTX-NPs) were pre-
pared by a dialysis method. Briefly, 95 mg of mPEG-b-PLG-b-PLL/DOCA and 5 mg of
PTX were dissolved in 4.0 mL of DMSO, and the solution was allowed to stir at 25?C
for 4 h. Then, the solution was added to 16.0 mL of deionized water dropwise under
gentle stirring. After stirring for 12 h at 25?C, the solution was dialyzed against
excess deionized water with a dialysis bag (MWCO 3500) overnight, and then
filtered through a 0.45 mm pore-sized microporous membrane. The PTX-NPs
powders were obtained after lyophilization.
To prepare the DOX and PTX co-delivered mPEG-b-PLG-b-PLL/DOCA nano-
particles (Co-NPs), mPEG-b-PLG-b-PLL/DOCA (85 mg), DOX (10 mg) and PTX (5 mg)
were dissolved in 4.0 mL of DMSO. Then, the solution was added to phosphate
buffered saline (PBS) solution (pH ¼ 7.0, 16.0 mL) under gentle stirring. The subse-
quent steps were identical with the preparation of PTX-NPs. FITC-labeled Co-NPs
were prepared by the same procedure used to Co-NPs.
DOX inside the nanoparticles was determined by UV absorption at 480 nm. PTX
loaded in the nanoparticles was determined by high-performance liquid chroma-
tography (HPLC). The mobile phase consisted of a mixture of acetonitrile and water
(4:1, v/v) using a Waters 1525 Binary HPLC pumb, and the flow rate was 1.0 mL
i, l, m
Chemical Shift (ppm)
Chemical Shift (ppm)
Fig. 1. Characterization of the copolymers.1H NMR spectra of mPEG-b-PBLG (a), mPEG-b-PBLG-b-P(Lys(Z)) (b), mPEG-b-PLG-b-PLL (c) and mPEG-b-PLG-b-PLL/DOCA (d) in
Characterization of the copolymers.
aEstimated by1H NMR.
bMeasured by GPC.
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
min?1. 20 mL of sample was injected. The column effluent was detected at 227 nm
with a Waters 2489 UV/Visible detector. The column type was Waters Symmetry?
C18 (5 mm, 4.6 mm ? 250 mm). Drug loading content (DLC, wt%) and drug loading
efficiency (DLE, wt%) were calculated according to the following formulas:
DLC ¼ (amount of loaded drug/amount of drug-loaded nanoparticles) ? 100%
DLE ¼ (amount of loaded drug/amount of feeding drug) ? 100%
The1H NMR, critical micelle concentration (CMC), zeta potential, dynamic
laser scattering (DLS), transmission electron microscopy (TEM) measurements
were performed as our previous reported [20,21]. FT-IR spectra were recorded
on a Bio-Rad Win-IR instrument using the potassium bromide method. Molec-
ular weight distributions (polydispersity index, PDI ¼ Mw/Mn) of the mPEG-b-
PBLG and mPEG-b-PBLG-b-P(Lys(Z)) copolymers were determined by gel
permeation chromatography (GPC) under the same test condition as our pre-
vious work .
2.6. In vitro drug release assay
The release profiles of DOX and PTX from Co-NPs were assessed in PBS con-
taining 0.1% (w/v) Tween 80 at different pH values (7.4 and 5.5) by the dialysis
method. Briefly, the weighed Co-NPs were suspended in 5.0 mL of the PBS release
medium and transferred into a dialysis bag (MWCO 3500 Da). The release experi-
ment was started by placing the dialysis bag into 45.0 mL of release medium at 37?C
with continuous shaking at 110 rpm. At predetermined time, 4 mL of the incubated
solution was withdrawn and replaced with equal volume of fresh PBS. The amount
of DOX released was determined using UVeVis spectrometer at 480 nm. PTX con-
tents in the samples were measured by HPLC. The release experiments were con-
ducted in triplicate.
2.7. Cell cultures
Three types of human cancer lines, including lung carcinoma (A549), cervical
carcinoma (HeLa) and breast carcinoma (MCF-7) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) with high glucose containing 10% fetal bovine
serum (FBS), supplemented with 50 U mL?1penicillin and 50 U mL?1streptomycin,
and incubated at 37?C in 5% CO2atmosphere.
2.8. Confocal laser scanning microscopy (CLSM) observation
The cellular uptake behavior of FITC-labeled Co-NPs was determined by CLSM
toward A549 cells. The cells were seeded on the coverslips in 6-well plates with a
density of 1 ?105cells per well in 2 mL of DMEM and cultured for 24 h, and then
the original medium was replaced with free DOX and FITC-labeled Co-NPs (at a
DOX concentration of 5 mg mL?1in 2 mL of DMEM). After 1 or 3 h incubation,
the cells were washed and fixed with 4% formaldehyde for 20 min at room
temperature. The cell nuclei were stained by DAPI according to the standard
protocol provided by the supplier. The coverslips were placed onto the glass
microscope slides. The subcellular localization and intracellular DOX release of
Co-NPs were visualized under a laser scanning confocal microscope (Carl Zeiss
Fig. 2. Characterization of the copolymers. FT-IR spectra of mPEG-b-PLG-b-PLL (a) and
Scheme 2. Schematic illustrations of antitumor drug loading and intracellular performance of Co-NPs.
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
2.9. In vitro cytotoxicity assays
The in vitro cytotoxicities of mPEG-b-PLG-b-PLL/DOCA, free drugs, and drug-
loaded nanoparticles on three types of cancer cell lines were assessed by MTTassay.
A549, HeLa or MCF-7 cells were seeded in 96-wellplates at 7000 cells per well in
100 mL of DMEM medium and incubated at 37?C in a 5% CO2atmosphere for 24 h,
followed by removing culture medium and then adding mPEG-b-PLG-b-PLL/DOCA
blank nanoparticles (200 mL in DMEM medium) at the different concentrations. The
cells were subjected to MTT assay after being incubated for another 48 h. The ab-
sorbency of the solution was measured on a Bio-Rad 680 microplate reader at
492 nm. The relative cell viability was calculated by (Asample/Acontrol) ? 100, where
Asample and Acontrol denoted as the absorbances of the sample well and control
well, respectively. Data are presented as average ? SD (n ¼ 3).
Tumor cell proliferation inhibition behaviors of free drugs and drug-loaded
micelles were evaluated against human lung adenocarcinoma A549 cells
following the similar procedure, and 48 h incubation time was applied. The
inhibitory concentration (ICx) values were determined using Origin 8.0 (OriginLab,
Northampton, MA) according to the fitted data. The Combination Index (CI) was
measured according to the Chou and Talalay’s method . To distinguish syn-
ergistic, additive, or antagonistic cytotoxic effects, the following equation was
ðDxÞ1and ðDxÞ2represent the ICxvalue of drug 1 alone and drug 2 alone, respec-
tively. ðDÞ1and ðDÞ2represent the concentration of drug 1 and drug 2 in the com-
bination system at the ICxvalue. CI > 1 represents antagonism, CI ¼ 1 represents
additive and CI < 1 represents synergism. In this work, IC50(inhibitory concentra-
tion to produce 50% cell death) was applied.
Male Balb/C nude mice (6e8 weeks old) were purchased from the Experimental
Animal Center, Chinese Academy of Sciences (Shanghai, China). The animals were
maintained in specific pathogen free (SPF) animal lab. Animal Care and Use Com-
mittee of Jilin University approved to use the animals in this study.
2.11. Excised imaging
The mice bearing A549 tumor were injected with free DOX and Co-NPs via tail
vein (5 mg DOX/kg). After the injection, mice were sacrificed at 3, 10 and 24 h, the
tumor and major organs (heart, kidney, liver, lung and spleen) were collected and
washed with PBS. Ateach time point, three mice were used. The excised organs were
visualized using a Maestro in vivo Imaging System (Cambridge Research & Instru-
mentation, Inc., Woburn, MA, USA) at excitation and emission wavelengths of 523
and 560 nm, respectively.
2.12. In vivo antitumor efficiency
A human non-small cell lung cancer xenograft tumor model was generated by
the subcutaneous injection of A549 cells (1.5 ? 106,100 mL in PBS) in the right flank
of each mouse. When the tumor volume reached approximately 20e50 mm3, mice
were randomly divided into 7 groups (G) (n ¼ 6), the treatment was started and this
day was designated as day 0. Mice were treated with PBS (G1), free DOX$HCl
(4 mg kg?1, G2), DOX-NPs (4 mg DOX kg?1, G3), free PTX (1 mg kg?1, G4), PTX-NPs
(1 mg PTXkg?1, G5), free DOX þ PTX (4 mg DOX kg?1and 1 mg PTX kg?1, G6) and Co-
NPs (4 mg DOX kg?1and 1 mg PTX kg?1, G7) intravenously via tail vein on days 0, 4,
8, and 12. Tumor volume and body weight were measured every other day to
evaluate the antitumor activities and systematic toxicities of various formulations.
The estimated tumor volume (mm3) was calculated based on the following equa-
tion: V ¼a$b2/2, where a and b represented the longest and shortest diameter of the
Fig. 3. Solution behaviors of the nanoparticles. (A) Hydrodynamic radii (Rh) of blank mPEG-b-PLG-b-PLL/DOCA nanoparticles (a), DOX-NPs (b), PTX-NPs (c) and Co-NPs (d) in PBS
estimated by and DLS. (B) Critical micelle concentration (CMC) of mPEG-b-PLG-b-PLL/DOCA nanoparticles using pyrene as a fluorescence probe. (C) Typical TEM images of blank
mPEG-b-PLG-b-PLL/DOCA nanoparticles (a), DOX-NPs (b), PTX-NPs (c) and Co-NPs (d).
Characterization of the nanoparticles.
Rh(nm)aat 25?CZeta potentialb(mV)DLC of PTXc(wt%) DLE of PTX (%)DLC of DOXd(wt%)DLE of DOX (%)
16.7 ? 4.0
27.6 ? 7.0
22.5 ? 5.4
36.7 ? 12.5
?23.4 ? 3.4
?18.3 ? 1.3
?22.7 ? 2.2
?18.20 ? 1.99
aMeasured by DLS.
bEstimated at pH 7.4 at 25?C, a mean ? STD of 6 measurements.
cDetermined by HPLC.
dDetermined by UV absorption at 480 nm.
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
tumors. At day 18, mice were sacrificed. The tumors and major organs, including
heart, liver, spleen, lung and kidney were excised for histopathology analyses.
2.13. Histopathology and immunohistochemical analysis
At day 18, mice were anesthetized and the chests were excised open, PBS and
paraformaldehyde solution (4% in PBS) were perfused from the left atrium. Tumors
were removed and fixed in 4% buffered paraformaldehyde overnight, and then
embedded in paraffin. The paraffin-embedded tissue samples of the implanted tu-
mor were sliced at 5 mm thickness, and stained with hematoxylin and eosin (H&E)
for histopathological changes evaluated by microscope (Nikon TE2000U).
The immunohistochemical evaluation was performed with rabbit monoclonal
primaryantibody for cleaved PARP (Abcam, Cambridge, MA, USA) and PV-6000 two-
step immunohistochemistry kit (polymer detection system for immuno-histological
staining; Zhongshan Goldbridge Biotechnology, Beijing, China).
In situ terminal deoxynucleotidyl transferase-mediated UTP end labeling
(TUNEL) assay was performed using a FragEL? DNA fragment detection kit
(colorimetric-TdT Enzyme method) according to the manufacturer’s protocol (EMD
chemicals Inc, Darmstadt, Germany).
2.14. Statistical analysis
Statistical significances were analyzed using the Student’s t-test. P < 0.05 was
considered statistically significant, and P < 0.01 was considered highly significant.
3. Results and discussion
3.1. Synthesis of copolymers
For co-delivery of DOX and PTX, mPEG-b-PLG-b-PLL/DOCA
amphiphilic copolymer was synthesized (Scheme 1). The poly
Fig. 4. Drug release curves of anticancer drugs in vitro over time. Levels of DOX (A) and PTX (B) from Co-NPs in PBS containing 0.1% (w/v) Tween 80 were released at various pH
values (7.4 and 5.0) at 37?C. Each point was an average of three measurements.
Fig. 5. Confocal laser scanning microscopy images of A549 cells after incubation with free DOX (A) and FITC-labeled Co-NPs (B) for 1 h and 3 h.
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
(glutamic acid) block provides the strong electrostatic interaction
to load cationic DOX$HCl. The hydrophobic modified PLL domain
serves as a reservoir for PTX through hydrophobic interaction. The
mPEG-b-PLG-b-PLL triblock copolymers were first synthesized via
the one-pot two-step ring-opening copolymerization of BLG-NCA
and Lys(Z)-NCA using mPEG-NH2as the macroinitiator, followed
by deprotecting the protection groups in HBr/acetic acid. The1H
NMR spectra in trifluoroacetic acid-d (TFA-d) verified the successful
synthesis of the resulting copolymers. All peaks of the copolymers
were well assigned (Fig. 1). The degree of polymerization (DP) of
BLG units in mPEG-b-PBLG-b-P(Lys(Z)) triblock copolymer was
calculated to be 10 by comparing the integration of the methylene
peak of the glutamate (eCOCH2e) with that of the methylene peak
of poly(ethylene glycol) (eCH2eCH2e), which was nearly identical
with the designed DP, indicating the high conversion efficiency of
BLG-NCA monomer. The DPs of BLG and Lys(Z) units in mPEG-b-
PBLG-b-P(Lys(Z)) triblock copolymer were determined to be 10 and
9, respectively, bycomparing the integration of the methylene peak
of the glutamate (eCOCH2e) and lysine (eCH2eCH2eCH2eCH2e
NH) units with that of the methylene peak of poly(ethylene glycol)
(eCH2eCH2e), demonstrating the successful synthesis of the tri-
block copolymer. After deprotection, the resonances at d 4.87e
4.92 ppm disappeared in the mPEG-b-PLG-b-PLL (Fig.1c), revealing
the complete deprotection of the g-benzyl groups (C6H5CH2eOe).
The composition ratio of the monomeric repeating units in the
copolymers did not change after deprotection of g-benzyl (Table 1),
suggesting that the deprotection reaction did not lead to the scis-
sion of poly(amino acids) backbones. GPC analyses revealed that
both mPEG-b-PBLG and mPEG-b-PBLG-b-P(Lys(Z)) had a narrow
molecular weight distribution (PDI ¼ 1.10 and 1.08, respectively),
which might be attributed to the living feature of the ROP of the
mPEG-b-PLG-b-PLL/DOCA was further synthesized by the
chemical coupling of mPEG-b-PLG-b-PLL with DOCA-NHS. The
structure of mPEG-b-PLG-b-PLL/DOCA was characterized by
NMR and FT-IR. As shown in Fig. 1c and d, the typical resonances
of alkyl group in DOCA (d 0.60, 0.76, 0.97 and 1.14 ppm) appeared
comparing the integration of the methyl peak of the DOCA at
d 0.60 with that of the methylene peak of poly(ethylene glycol)
chain at d 3.68, the DOCA groups decorated on each copolymer
were calculated to be 8.33, which meant approximately 93% of the
amino groups of the mPEG-b-PLG-b-PLL/DOCA copolymer had
1H NMRmPEG-b-PLG-b-PLL/DOCA. By
been converted into DOCA groups. And deoxycholate (DOCA)
decoration was further confirmed by FT-IR, the FT-IR spectra of
mPEG-b-PLG-b-PLL and mPEG-b-PLG-b-PLL/DOCA were listed on
Fig. 2. The absorption at 1649 cm?1was attributed to the typical
amide of poly(amino acid) chain on the copolymer (Fig. 2a), and
the increased absorption of typical CeH stretching vibration at
about 2900 cm?1induced by DOCA groups was observed from the
FT-IR spectroscopy (Fig. 2b), indicating the successful synthesis of
3.2. Fabrication of the nanoparticles
Amphiphilic copolymers with hydrophilic and hydrophobic
segments can self-assemble into various types of nanoparticles in
aqueous solution, such as micelles and vesicles. In the study, the
mPEG-b-PLG-b-PLL/DOCA triblock copolymers could self-assemble
into micelles in the aqueous phase for loading DOX and PTX
(Scheme 2). The DOX-NPs were prepared by simply mixing DOX
and mPEG-b-PLG-b-PLL/DOCA in distilled water. The PTX-NPs and
Co-NPs were prepared bya nanoprecipitation technique which was
Fig. 6. Cell viabilities of different cell types after treated empty nanoparticles in vitro.
A549 (a), HeLa (b) and MCF-7 (c) cells were incubated with blank mPEG-b-PLG-b-PLL/
DOCA nanoparticles for 48 h before MTT assay (n ¼ 3, mean ? SD).
Fig. 7. Cell viabilities of lung cancer cells after treatment with different anticancer
strategies in vito. The A549 cancer cells were incubated with free DOX (a), free PTX (b),
free DOX&PTX (c), DOX-NPs (d), PTX-NPs (e) and Co-NPs (f) for 48 h. Data were pre-
sented as the mean ? standard deviation (n ¼ 3).
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
similar to the literature [27,28]. The DLC and DLE for the drug-
loaded nanoparticles were listed in Table 1. The Co-NPs had a
DOX loading content of 8.49 wt.% and a PTX loading content of
2.24%, indicating that DOX and PTX were successfully co-loaded
into the mPEG-b-PLG-b-PLL/DOCA nanoparticles. The size distri-
butions of the nanoparticles were determined by DLS measure-
ments. As shown in Fig. 3A, the blank mPEG-b-PLG-b-PLL/DOCA
nanoparticles had a narrow distribution with a hydrodynamic
radius (Rh) of 16.7 ? 4.0 nm, and the sizes of the drug-loaded
nanoparticles were listed in Table 2. All the drug-loaded nano-
particles had hydrodynamic radii of 20e40 nm. Loading of drug
leaded to increase of the particle size, while maintained a narrow
distribution (Fig. 3A). The morphologies of the nanoparticles were
measured by TEM measurement. TEM images revealed that all
(Fig. 3C). The sizes from TEM observations were slightly smaller
than that from DLS measurements, which might be attributed to
the dehydration of nanoparticles during the TEM sample prepara-
tion and shrinkage of the mPEG shell.
CMC is a key factor to determine the stability of nanoparticles in
the medium. In this study, CMC is measured by fluorescence
spectroscopy using pyrene as a probe, which exhibits a peak shift in
its excitation spectrum when it is captured into a hydrophobic in-
ner core. The CMC value was calculated from the inflection point of
fluorescence intensity ratio of I340/I335(Fig. 3B) as a function of the
logarithm concentration of the nanoparticles according to the
literature . The blank mPEG-b-PLG-b-PLL/DOCA nanoparticles
had an extremely low CMC of 0.00136 mg mL?1, indicating the
mPEG-b-PLG-b-PLL/DOCA nanoparticles exhibited great stability
against dilution. Such low CMC value could guarantee the nano-
particles to retain their construct during the in vivo diluted condi-
tions (e.g. blood stream), which is great benefit for the effective
delivery to tumors. The surface zeta potentials of the blank and
drug-loaded nanoparticles were measured on a Zeta Potential/BI-
In vitro cytotoxicities and combination index (CI) of drug formulations against
A549 cells for 48 h incubation time.
EntryIC50DOX/(mg mL?1) IC50PTX/(mg mL?1) CI50
DOX þ PTX
Fig. 8. Anticancer drugs distributed in solid organs and targeted at the implanted A549 lung cancer tumor in vivo at different time points. Ex vivo DOX fluorescence imaging of A549
tumor-bearing nude mice was performed at 3, 10, and 24 h post-injection of free DOX (a) and Co-NPs (b).
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
90Plus particle size analyzer and listed in Table 2. Because most
amino groups on the mPEG-b-PLG-b-PLL/DOCA copolymer had
been decorated with deoxycholate, the surface charges of the blank
and drug-loaded nanoparticles were negative (w ?20 mV). The
slightly negative surface charge will contribute to a better blood
compatibility and prolonged circulation time of the nanoparticles
for the reduced clearance by the reticuloendothelial system (RES)
3.3. Release behavior of drug-loaded nanoparticles
The DOX and PTX release behaviors of Co-NPs were assessed
using a dialysis method at 37?C in phosphate buffered saline (PBS)
containing 0.1% Tween 80 at different pH values (7.4 and 5.0). As
shown in Fig. 4A, the release of DOX from Co-NPs was greatly
affected by the environmental acidity. After a 108 h incubation
period, about 24% and 79% of DOX were released at pH 7.4 and 5.0,
respectively. The rapid DOX release at pH 5.0 might be attributed
to the significantly increased protonation degree of the carboxyl
groups in mPEG-b-PLG-b-PLL/DOCA polymer backbone at a lower
pH value, resulting in the weakening of electrostatic interactions
between the mPEG-b-PLG-b-PLL/DOCA nanoparticles and DOX
molecules. The release kinetics of PTX from Co-NPs displayed a
slow and sustained release pattern at different pH values, and
there were no obvious differences between the release rates at pH
7.4 and 5.0 (Fig. 4B). Approximately 67% and 58% of total PTX were
released at pH 7.4 and 5.0, respectively. The slow and sustained
PTX release from Co-NPs might be ascribed to the strong hydro-
phobic interaction between PTX and the inner core of the
3.4. Cellular uptake behavior of the dual-drug loaded nanoparticles
The cellular uptake behavior of Co-NPs was investigated in
A549 cells by confocal laser scanning microscopy (CLSM). The
cellular nuclei were stained with DAPI (blue), and mPEG-b-PLG-b-
PLL/DOCA copolymers were labeled by FITC (green) for subcellular
observation. As shown in Fig. 5B, the green fluorescence was
observed in the cells after 1 h incubation. When the incubation
period increased to3 h, thecell uptakeof Co-NPs was enhanced and
the green fluorescence was distributed widely in the cytoplasm,
suggesting that Co-NPs could be successfully internalized by tumor
cells via endocytosis.
The intracellular DOX release behavior of Co-NPs was also
assessed in A549 cells using CLSM. The red fluorescence was
performed to visualize the intracellular released DOX (Fig. 5). For
both free DOX and Co-NPs, the intracellular DOX fluorescence of
3 h incubation time was stronger than that of 1 h incubation time.
For free DOX treated cells, the red fluorescence was observed
mostly in the nuclei. Moreover, the DOX fluorescence was
distributed both in cytoplasm and nucleus for Co-NPs treated cells
at both 1 and 3 h, indicating that Co-NPs were initially located in
the intracellular compartments and subsequently released DOX to
the nuclei, which was consistent with the subcellular location of
Co-NPs. Additionally, cells treated with both free DOX and Co-NPs
showed similar DOX accumulation at 1 and 3 h. Considering that
free DOX could be quickly transported into cells through the cell
membrane via a passive diffusion mechanism , the results
revealed that Co-NPs exhibited a high level of cell uptake through
3.5. In vitro cytotoxicity studies
The biocompatibility of the mPEG-b-PLG-b-PLL/DOCA copol-
ymer was evaluated using MTT assay. A549 (human lung
adenocarcinoma), HeLa (human cervical cancer) and MCF-7 (hu-
man breast carcinoma) cell lines were utilized. As shown in Fig. 6,
the cell viabilities of A549, HeLa and MCF-7 cells treated with the
mPEG-b-PLG-b-PLL/DOCA nanoparticles at all the tested concen-
trations up to 1000 mg mL?1after 48 h incubation were all above
85%, indicating that the mPEG-b-PLG-b-PLL/DOCA copolymer had
excellent safety and biocompatibility.
To verify the synergistic effect of the co-delivery system, the
in vitro antitumor effects of free drugs and drug-loaded nano-
particles against A549 cells were tested using MTT assay. The cell
viability histograms were shown in Fig. 7. After 48 h incubation, all
free drugs and drug-loaded nanoparticles showed dose-dependent
cell proliferation inhibition behavior and the combination of DOX
and PTX leaded to enhanced cell proliferation inhibition. And Co-
NPs exhibited the best antitumor activity over a wide range of
drug concentrations among all the drug formulations. The IC50
values of free drugs and drug-loaded nanoparticles and combina-
tion index (CI50) values were summarized in Table 3. The IC50
values of DOX-NPs and PTX-NPs were larger than that of free DOX
Fig. 9. Tumor volume and body weight changes after anticancer treatment with
different drug formulations in nude mice bearing A549 human lung cancer xenograft.
Notes: PBS (a), DOX (b), DOX-NPs (c), PTX (d), PTX-NPs (e), DOX þ PTX (f) and Co-NPs
(g). The data are shown as mean ? SD (n ¼ 6), *p < 0.01.
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
and free PTX, respectively. The reason might be attributed to
different cell uptake pathways of free drugs and drug-loaded
nanoparticles, and the controlled release manner of drug-loaded
nanoparticles. In cell culture medium, most free drugs could
quickly display their effects after being transported into cells via
passive diffusion. However, the drug-loaded nanoparticles were
mainly taken up by cells via the endocytosis pathway and then
exerted the antitumor activity after the drug molecules were
released from the nanoparticles . The CI values lower than,
equal to, or higher than 1 indicate synergism, additivity, or
antagonism, respectively. The CI50of free PTX þ DOX was calcu-
lated to be 1.28, which demonstrated that the combination of free
DOX and PTX did not receive efficient synergistic effect at the ratio
ofPTX0.25/DOX. However,the DOXand PTXco-loaded
Fig. 10. Images of excised A549 tumors and histological changes of different drug delivery strategies in treatments of A549 human lung cancer implanted in an athymic mouse
model. For histological analysis, brown and blue stains represented cleaved PARP1 and nuclei, respectively, in immunohistochemical assay; Green and brown stains indicated
normal and apoptotic tumor cells, respectively, in TUNEL analysis; Nuclei were stained violet and extracellular matrix and cytoplasm were stained pink in H&E staining. Scale bars:
100 mm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
nanoparticles showed obvious synergism effect and the CI50value
was approximately 0.57, indicating co-delivery of DOX and PTX had
evident superiority as compared with free drug combination.
3.6. Excised imaging of free DOX and Co-NPs
To estimate the biodistribution of Co-NPs, ex vivo DOX fluo-
rescence imaging of the isolated major organs (heart, liver, spleen,
lung, kidney) and tumors at 3 h, 10 h and 24 h post-injection in
A549 tumor-bearing nude mice was observed. Free DOX was used
as the control group and the representative results were sum-
marized in Fig. 8. As shown in Fig. 8, for free DOX group, DOX
fluorescence was mainly observed in kidney at 3 h post-injection
and rapidly faded away after injection. The result suggested that
the free DOX molecules were metabolized and excreted fast from
the body by liver and kidney, which might resulted in the low
drug efficacy and the side toxic effect to the organs. Conversely,
the strongest DOX fluorescence was first distributed in liver at 3 h
post-injection and then detected mainly in kidney at 10 h post-
injection, indicating that Co-NPs exhibited longer blood circula-
tion time. However, stronger fluorescence at tumor site for Co-
NPs group was observed in comparison with that for free DOX
group at 10 and 24 h. The improved delivery of DOX to tumor for
Co-NPs might be attributed to reduced uptake by the RES, the
excellent construct stability during the blood circulation and the
EPR effect, which would contribute to the enhanced antitumor
3.7. In vivo antitumor efficiency
On the basis of the above results, the in vivo antitumor efficacy
and systemic toxicity of the dual-drug loaded nanoparticles were
further investigated on A549 human lung tumor-bearing nude
mice. Mice were treated with PBS and different drug formulations
every four days via the intravenous injection, and the tumor vol-
ume and the body weight were measured every two days. As
shown in Fig. 9A, compared with the rapid tumor growth of PBS
treatment group, all the drug formulations showed efficacy in
inhibiting the tumor growth to different degrees. For the free drugs
and the drug-loaded nanoparticles treated groups, two conclusions
could be summarized as follows: (1) The combination of DOX and
PTX was more effective than the use of single drug. (2) For the same
drugs, the loaded-drug showed better antitumor effect compared
with free drug, and the similar results were observed in the drug
combination. The best antitumor activity was observed in the Co-
NPs treated group, with the almost completely inhibition of tu-
mor growth and no obvious tumor recrudescence during the whole
treatment. The tumor volume of Co-NPs treated group was only
9.0% of control group at the end of experiment, which was 3.2-fold,
6.3-fold and 2.4-fold smaller than that treated with free DOX, free
PTX and freeDOX þ PTX,respectively. The superiorantitumoreffect
of Co-NPs might be attributed to the enhanced nanoparticle sta-
bility during the blood circulation, the sufficient and coinstanta-
neous delivery of two drugs to the tumor site, the efficient cellular
uptake in the tumor tissue and the synergistic effect of DOX and
PTX on tumor inhibition. The excised A549 tumors after the
treatment were dissected and photographed. And the result was
consistent (Fig. 10A).
Body weight change is an indicator of systemic toxicity. As
shown in Fig. 9B, the body weights of PBS, free PTX and PTX-NPs
treated mice showed a continuous and slow increase, which
might be partly ascribed to the tumor growth and low toxicity of
PTX at the dose of 1 mg kg?1. Obvious weight loss (about 10%) was
observed in mice treated with free DOX alone at 4 mg kg?1or in
combination with PTX. On the contrary, the treatment with DOX-
NPs or Co-NPs did not lead to any significant body weight loss,
demonstrating the reduced systemic toxicity of the loaded drugs.
With the high antitumor efficacy and the low drug-related toxicity,
the dual-drug loaded system is promising in cancer therapy. The
principle of drug combination is to achieve efficient antitumor ef-
fect at lower drug doses. Therefore, our next goal is to reduce drug
doses to obtain the maximal therapeutic effect and further bring
down their side effect.
3.8. Histological and immunohistochemical analyses
To further investigate the antitumor activity of Co-NPs, A549
tumor-bearing nude mice were sacrificed after the treatment (day
18) and the tumors were dissected and stained with H&E, TUNEL
and PARP for pathology analysis. The data of PBS, DOX-NPs, PTX-
NPs and Co-NPs treated groups were shown in Fig. 10B.
For H&E staining, the normal tumor cells had large nuclei with
spherical or spindle shape and more chromatin. Whereas the
necrotic cells did not have clear cell morphology, and the chromatin
became darker and pyknotic or absent outside the cellular. As
shown in Fig. 10B, the tumor cells with normal shape and more
chromatin were observed in the PBS group, revealing a vigorous
tumor growth. However, the various degrees of tissue necrosis
were observed in different drug formulation treated groups. The
Co-NPs treated group had larger necrosis area as compared with
the groups treated with DOX-NPs and PTX-NPs, indicating that
most tumor cells were necrotic in the Co-NPs treated group.
The TUNEL assay could detect DNA fragmentation in the nuclei
of tumor cells. Little apoptosis was detected in the PBS treated tu-
mor tissues. While in the DOX-NPs, PTX-NPs and Co-NPs treated
groups, obvious cell apoptosis areas were observed. The treatment
of Co-NPs obviously increased apoptosis level compared with the
signal drug-loaded nanoparticles, which was consistent with the
Poly-ADP-ribose polymerase (PARP) was one of the essential
substrates cleaved by both caspase-3 and -7. The presence of
cleaved PARP1 could further detect DNA strand breaks in many cell
types . To further confirm the tumor apoptosis, the cleaved
25 kDa fragment of PARP1 was analyzed in the tumor sections by
immunohistochemistry. The obvious cleavage products were
observed in the tumor tissues treated with various drug-loaded
nanoparticles. And compared with DOX-NPs and PTX-NPs treated
groups, more cancer cells underwent apoptosis in the grouptreated
with Co-NPs (Fig. 10B).
Together, these data clearly confirmed that the highest level of
necrosis and tumor apoptosis was observed in the tumor tissue
treated with Co-NPs, which was consistent with the in vivo anti-
In summary, we developed a polypeptide-based copolymer,
mPEG-b-PLG-b-PLL/DOCA, for the co-delivery of DOX and PTX. The
robust construct stability, efficiently delivering capacity, good
biocompatibility and favorable size distribution of mPEG-b-PLG-b-
PLL/DOCA revealed its great potential for delivering antitumor
drugs via intravenous injection in the cancer treatment. FITC-
labeled Co-NPs could be successfully internalized by A549 cells
tumor cells via endocytosis. Co-NPs had synergistic effect in sup-
pression of A549 lung tumor cell growth. Co-NPs exhibited high
tumoraccumulation, superiorantitumor efficiencyand much lower
toxicity in vivo. The present studies indicate that the co-delivery
system provides a promising platform as a combination therapy
in the treatment of lung cancer, and possible other type of cancer as
well. Further studies will be required to investigate an ultimate
S. Lv et al. / Biomaterials 35 (2014) 6118e6129
dose-dependent response, the optimal doses of both anticancer Download full-text
drugs with maximal anticancer efficacy but less side effect, and the
application of this strategy to treat different tumors.
This research was financially supported by National Natural
Science Foundation of China (Project numbers 51173184, 51373168,
51390484, 51233004 and 51321062), Ministry of Science and
Technology of the People’s Republic of China (International Coop-
eration and Communication Program 2011DFR51090), and the
Programof Scientific Development
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