Y-shaped block copolymer (methoxy-poly(ethylene glycol))2-b-poly(l-glutamic acid): Preparation, self-assembly, and use as drug carriers

Article abstract of DOI:10.1039/c4ra07890j

Y-shaped amphiphilic block copolymers, (methoxy-poly(ethylene glycol))₂-block-poly(l-glutamic acid) ((mPEG)₂-PGA) and its precursor (methoxy-poly(ethylene glycol)₂-block-poly(γ-benzyl-l-glutamate) ((mPEG)₂-PBG), were prepared in three steps: (1) macroinitiator (methoxy-poly(ethylene glycol))₂-NH₂((mPEG)₂-NH₂) was synthesized by coupling two methoxy-poly(ethylene glycol)s with serinol and diisocyanate. (2) (mPEG)₂-PBG was synthesized by ring opening polymerization of γ-benzyl-l-glutamate-N-carboxyanhydride initiated with the macroinitiator ((mPEG)₂-NH₂); (3) the protective benzyl groups in (mPEG)₂-PBG were removed to obtain (mPEG)₂-PGA. The properties of both (mPEG)₂-PBG and (mPEG)₂-PGA were characterized by ¹H NMR, FT-IR, GPC, and DLS. In aqueous solution (mPEG)₂-PBG tends to form more stable micelles compared to linear mPEG-PBG copolymer. The size of (mPEG)₂-PBG decreases with increasing length of hydrophobic PBG in (mPEG)₂-PBG. Paclitaxel and cisplatin were grafted onto (mPEG)₂-PGA to form (mPEG)₂-PGA-PTX (MPTX) with a grafting ratio of near 90% and (mPEG)₂-PGA-Pt (MPt) conjugates with a loading efficacy of 15% (w/w). MPTX can greatly improve the solubility of PTX. Both conjugates can self-assemble into micelles with a mean diameter of about 50 nm and show enhanced anti-cancer activity against MCF-7, HeLa, and SMMC cell lines. The in vivo anticancer evaluation in mice shows MPt showed a desirable antitumor activity and allowed us to deduce the system toxicity. Therefore, both MPTX and MPt have a great potential as a polymer drug in cancer chemotherapy. This journal is

Full text of DOI:10.1039/c4ra07890j

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PAPER
Y-shaped block copolymer (methoxy-
poly(ethylene glycol)) -b-poly(L-glutamic acid):
2
Cite this: RSC Adv., 2014, 4, 41588
preparation, self-assembly, and use as drug carriers
a
a
ab
ab
ab
a
Lixin Yang, Xiuli Hu, Weiqi Wang, Shi Liu, Tingting Sun, Yubin Huang,
a
a
Xiabin Jing and Zhigang Xie*
Y-shaped amphiphilic block copolymers, (methoxy-poly(ethylene glycol))
2
-block-poly(L-glutamic acid)
(
(
(
(mPEG)
(mPEG)
(mPEG)
2
2
2
-PGA) and its precursor (methoxy-poly(ethylene glycol)
-PBG), were prepared in three steps: (1) macroinitiator (methoxy-poly(ethylene glycol))
-NH ) was synthesized by coupling two methoxy-poly(ethylene glycol)s with serinol and
-PBG was synthesized by ring opening polymerization of g-benzyl-L-
glutamate-N-carboxyanhydride initiated with the macroinitiator ((mPEG) -NH ); (3) the protective benzyl
-PGA. The properties of both (mPEG) -PBG
2
-block-poly(g-benzyl-L-glutamate)
2
-NH
2
2
diisocyanate. (2) (mPEG)
2
2
2
groups in (mPEG)
2
-PBG were removed to obtain (mPEG)
2
2
1
and (mPEG)
2
-PGA were characterized by H NMR, FT-IR, GPC, and DLS. In aqueous solution (mPEG)
2
-
PBG tends to form more stable micelles compared to linear mPEG-PBG copolymer. The size of
mPEG) -PBG decreases with increasing length of hydrophobic PBG in (mPEG) -PBG. Paclitaxel and
cisplatin were grafted onto (mPEG) -PGA to form (mPEG) -PGA-PTX (MPTX) with a grafting ratio of near
0% and (mPEG) -PGA-Pt (MPt) conjugates with a loading efficacy of 15% (w/w). MPTX can greatly
(
2
2
2
2
9
2
improve the solubility of PTX. Both conjugates can self-assemble into micelles with a mean diameter of
about 50 nm and show enhanced anti-cancer activity against MCF-7, HeLa, and SMMC cell lines. The in
vivo anticancer evaluation in mice shows MPt showed a desirable antitumor activity and allowed us to
deduce the system toxicity. Therefore, both MPTX and MPt have a great potential as a polymer drug in
cancer chemotherapy.
Received 31st July 2014
Accepted 27th August 2014
DOI: 10.1039/c4ra07890j
www.rsc.org/advances
addition fragmentation chain transfer (RAFT) and “click
chemistry” reactions, and so on, miktoarm amphiphilic block
Introduction
Poly(ethylene glycol)-containing amphiphilic block copolyesters copolymers have been designed and synthesized such as mPEG-
7
–9
(
PEG-PE), such as copolycarbonate (PEG-PC) and copoly(amino (PLA)
n
(n ¼ 2, 4), mPEG
-PLA , mPEG -PLA and mPEG-PCL .
2 2 3 2
acid) (PEG-PAA) have been synthesized and employed as drug Compared to linear counterparts, miktoarm amphiphilic block
1–3
carriers. With the use of these polymeric drug carriers, it is compolymers always have unique self-assembly abilities,
possible to effectively prolong the circulation time of clinical resulting in interesting pharmacologic behaviors because of
anti-cancer drugs, to shield the recognition by immune system, their unique topological structure. Cinziacarofalo et al.
to escape the capture of reticuloendothelial system (RES), and to prepared three shapes of copolymers of general formula mPEG-
accumulate at tumor site via the enhanced permeability and (PLA)
retention (EPR) effect. Among these polymer carriers, linear isotactic) to evaluate the architecture and chemical composition
diblock or triblock copolymers have been extensively investi- effect on the micelles formation and stability. Compared to
n
(n ¼ 1, 2 or 4; mPEG: mPEG2k or mPEG5k; PLA: atactic or
4,5
gated and some of them have entered clinical trials and shown other copolymers, mPEG2k-(PD,LLA)
encouraging clinical results.
was able to form mono-
dispersive and stable micelles. Sun et al. investigated the effect
Recently, thanks to the development of synthetic methods, of solvent and the relative length of the PBG on the self-
such as atom transfer radical polymerization (ATRP), reversible assembly of Y-shaped copolymer, poly(L-lactide) -b-poly-
g-benzyl-L-glutamate). At a xed composition of copolymer, the
2
6
10
2
(
copolymer formed a transparent gel in toluene, while became
a
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of homogeneous dispersion containing nano-scale brous aggre-
Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People's
11
gates in benzyl alcohol.
Republic of China. E-mail: xiez@ciac.ac.cn; Fax: +86 431-85262779; Tel: +86 431-
In the past few decades, linear amphiphilic PEG-PAA copoly-
mers, mostly PEG-b-poly(aspartic acid), PEG-b-poly(glutamic
acid) (PEG-PGA), and PEG-b-poly(lysine) (PEG-PLL), have been
8
5262779
b
Graduate School of Chinese Academy of Sciences, Beijing 100049, People's Republic of
China
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successfully used to physically encapsulate or chemically bond (PDI) of the polymers were determined by size exclusion chro-
anti-cancer drugs, such as cisplatin, oxaliplatin, paclitaxel and matography (SEC), which was performed with a Waters 1525
1
2–14
doxorubicin, etc.
Compared with other drug carriers, PEG- tted with two columns (Styragel HT3 and HT4, 7.8 ꢁ 300 mm),
ꢀ1
PAA copolymers have their specic features: (1) many amino with THF as mobile phase at a ow rate of 1.0 ml min and an
acids can be chosen and available easily; (2) several amino acids operating temperature of 35 C. PS standards were used for
ꢂ
contain functional groups and special functionalization is not calibration. Fourier transform infrared (FTIR) spectra were
needed; (3) some amino acids can exist in different charge recorded on a Bio-Rad Win-IR instrument using potassium
states such as neutral in protected form and positive or negative bromide (KBr) method. The size of micelles was measured by
in deprotected form; (4) amino acids have different isoelectric DLS measurements carried out with a DAMN EOS instrument
ꢂ
potentials and their solubility is pH dependent; (5) chiral equipped with a He–Ne laser at the scattering angle xed at 90 .
ꢀ1
molecular structure and ordered secondary structures of amino The micelle solution of about 0.5 mg ml in water was ltered
acids are oen associated with special aggregation behaviors of through a 0.45 mm of lter membrane before measurement. The
them. In short, PEG-PAAs are more versatile than other drug morphologies of the nanoparticles were conrmed by using
carriers. For this reason, many PAAs and PEG-PAAs have been transmission electron microscopy (TEM) measurement on a
developed to demonstrate their special aggregates and possible JEOL JEM-1011 transmission electron microscope with an
application in drug delivery. Nano PAAs or PEG-PAAs prodrugs accelerating voltage of 100 kV. A drop of the nanoparticle
ꢀ
1
such as poly(L-glutamic acid)-paclitaxel, (PGA-PTX, XYOTAX™, solution (1 mg ml ) was deposited onto a 230-mesh copper
CT2103), PEG-polyaspartate-DOX (NK911) and PEG-poly- grid coated with carbon, and was air-dried at room temperature.
aspartate-PTX (NK105), etc., have been developed and evaluated
15,16
in preclinical or clinical trials.
From the above discussion Preparation of 2-(tert-butyloxycarbamino)-1,3-propanediol
about the effect of the topological structure of miktoarm (N-Boc-serinol, NBS)
amphiphilic polymers on the self-assemble behaviors and drug
2-(tert-Butyloxycarbamino)-1,3-propanediol (N-Boc-Serinol) was
delivery, it can be concluded that the variability of amphiphilic
PAA with different topologic structures has an ability of
extending the scope of amphiphilic PAA prodrugs. Unfortu-
nately, no reports focus on the preparation of Y-shaped
amphiphilic PAA prodrugs having two mPEG arms till now,
which is expected to have excellent water solubility, unique self-
assembly and pharmaceutical behaviors.
synthesized according to the ref. 18. Briey, serinol (2-amino-
1,3-propanediol) (9.1 g, 0.1 mol) was dissolved in EtOH (90 ml),
and then di-tert-butyl dicarbonate (23.9 g, 0.11 mol) was drop-
ped. The solution was stirred for 1 hour at room temperature,
and the solvent was removed under reduced pressure. The
yellow solid was recrystallized from hexane/EtOAc to give NBS as
1
1
7.1 g of white solid (yield: 83%). H NMR (400 MHz, DMSO): d
Herein, Y-shaped block copolymers of (mPEG)
variety of PBG block lengths, their deprotection products
mPEG) -b-PGA, and their conjugates with paclitaxel (PTX) and
cisplatin, i.e., (mPEG) -b-PGA-PTX and (mPEG) -b-PGA-Pt were
synthesized. The self-assembly behaviors of (mPEG) -b-PBG and
2
-b-PBG with a
¼
5.25 (1H, NH), 3.70–3.79 (5H, m, 2 ꢁ CH OH and CHN), 1.44
2
(
9H, s, 3 ꢁ CH ).
3
(
2
2
2
Synthesis of macroinitiator mPEG5k-NH
(mPEG) -NH
2
-mPEG5k
2
(
2
2
)
(
mPEG) -b-PGA-PTX were investigated. The in vitro and in vivo
2
Dried mPEG5k (15.0 g, 3 mmol) and HDI (0.53 g, 3.15 mmol)
anti-cancer efficacy of and (mPEG) -b-PGA-cisplatin conjugates
2
were added into a dried ask, 20 ml 1,4-dioxacyclohexane and
was further elucidated.
ꢂ
0.1 g Sn(Oct)
2
were added. The solution was stirred at 70 C
under nitrogen for 1 hour, and then NBS (0.29 g, 1.5 mmol) was
added. The solution was stirred for 2 h and then precipitated in
cold ether. The precipitate was collected and dried to obtain
Experimental section
Materials
1
4.5 g white product (mPEG ) -NHBoc (yield: 91.7%). To a 20
5k 2
ꢀ1
Methoxy-poly(ethylene glycol) (mPEG) (Mn ¼ 5 kg mol ) (from
ml solution of (mPEG ) -NHBoc (10.0 g) in dichloromethane,
5
k 2
Sigma) was dried by azeotropic distillation with toluene before
2
0 ml triuoroacetic acid was added. The solution was stirred in
ice bath for 1 h and poured into cold ether. The precipitate was
ltered out and dried. The product was dissolved in water and
2
use. Stannous octoate (Sn(Oct) ) (form Sigma Chemical Co.), di-
tert-butyl dicarbonate (from J&K Scientic Ltd.), 2-amino-1,3-
propanediol (serinol, from J&K Scientic Ltd.) and hexamethy-
lenediisocyanate (HDI, from Aladdin Industrial Co.) were used
as received. 1,4-Dioxacyclohexane was dried over CaH2 and
distilled over sodium just before use. g-Benzyl-L-glutamate-N-
carboxyanhydride (BLG-NCA) were synthesized according to the
ref. 17.

neutralized by saturated aqueous sodium bicarbonate. The pH
value of the solution was adjusted to 7–8. The solution was
dialyzed against water using a dialysis bag (MWCO 3500 g
ꢀ1
mol ) for 2 days and frozen-dried to obtain 8.5 g of white
product (mPEG ) -NH (yield: 85%).
5
k 2
2
Synthesis of Y-shaped copolymer mPEG -PBG-mPEG
5k
5
k
Characterizations
2
((mPEG5k) -PBG) and linear copolymer mPEG5k-PBG
1
H NMR spectra were measured by an AV-400 instrument Three kinds of (mPEG ) -PBG with different PBG lengths, i.e.,
5
k 2
(
Bruker BioSpin GmbH, Rheinstetten, Germany). The number- (mPEG5k
)
2
-PBG14, (mPEG5k
)
2
-PBG20 and (mPEG5k
)
2
-PBG35, were
-NH . A
average molecular weights (Mn) and polydispersity indices prepared by the ROP of BLG-NCA initiated by (mPEG5k
)
2
2
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2 2
typical synthetic procedure for (mPEG5k) -PBG20 is as follows: and its amount was equal to that used in (mPEG5k) -PGA35-PTX.
dried (mPEG ) -NH (3.0 g) as a macroinitiator and BLG-NCA All of the drugs with PTX were modulated to a series of nal
5
k 2
2
(
1.5 g) were dissolved in 5 ml dried DMF. The solution was equivalent PTX concentrations ranging from 0.005 to 50 mg
ꢂ
ꢀ1
stirred at 25 C for 72 h and then precipitated in cold ether. The ml . The incubation of each drug formulation was continued
precipitate was collected and dried to obtain white product for 48 h or 72 h. Then, 20 ml of MTT solution of 5 mg ml in PBS
ꢀ
1
(
mPEG5k
)
2
-PBG20. Linear copolymers mPEG5k-PBGs with DP of was added and the plates were incubated for another 4 h at
ꢂ
PBG of 14, 20, and 35, respectively, were prepared by the same 37 C, followed by removal of the culture medium containing
way, but with mPEG5k-NH
2
as the macroinitiator.
MTT and addition of 150 ml of DMSO to each well to dissolve the
formazan crystals formed. Finally, the plates were shaken for
10 minutes, and the absorbance of formazan formed was
measured at 492 nm by a microplate reader. The cell viability
against HeLa and SMMC lines was determined by the
similar way.
Synthesis of methoxy-poly(ethylene glycols)
2
-b-poly-
(
L-glutamic acid) ((mPEG ) -PGA)
5k 2
2
(mPEG5k) -PBG (1.5 g) was dissolved in a mixed solution of 6 ml
of CF COOH and 2 ml of 33 wt% HBr/acetic acid. The solution
3
was stirred at room temperature for 2.5 h and poured into
excessive ether. The precipitate was ltered out and dried to In vivo evaluation
2
obtain white product (mPEG5k) -PGA.
Animal model preparation. Chinese Kunming (KM) female
mice were obtained from Jilin University, China (56–84 days old,
2
Synthesis of (mPEG) -PGA-PTX prodrug (MPTX)
20–25 g in weight) and maintained under required conditions.
To a 20 ml solution of (mPEG ) -PGA (200 mg, 0.012 mmol) All animal studies were conducted in accordance with the
5
k 2
35
and PTX (70 mg, 0.08 mmol), DCC (105 mg, 0.51 mmol) and principles and procedures outlined in “Regulations for the
DMAP (30 mg, 0.25 mmol) were added. The solution was stirred Administration of Affairs Concerning Laboratory Animals”,
at room temperature for 8 h, and then precipitated in a mixture approved by the National Council of China on October 31, 1988,
of cold ether–ethanol. The product was collected and dried. PTX and “The National Regulation of China for Care and Use of
1
content in the prodrug was determined by H NMR. The PTX Laboratory Animals”, promulgated by the National Science and
content and its gra efficacy were calculated according to the Technology Commission of China, on November 14, 1988. To
following formula: PTX (wt%) ¼ (weight of PTX/weight of pro- develop the tumor xenogras, H22 cells were injected into the
6
drug) ꢁ 100% and PTX gra efficiency (wt%) ¼ (weight of PTX lateral aspect of the right anterior limb of the mice (5 ꢁ 10 cells
3
in prodrug/weight of PTX in feed) ꢁ 100%, respectively.
in 0.1 ml PBS). Aer the tumor volume reached 100–200 mm ,
the hair of the mice was removed with a sodium sulde solution
ꢀ1
(80 g L in 30 vol% aqueous alcohol).
Synthsis of (mPEG)
Cisplatin (150 mg, 0.5 mmol) was dissolved into 20 ml water,
and then AgNO (170 mg, 1.0 mmol) was added, the solution
2
-PGA-cisplatin prodrug (MPt)
Anti-tumor efficacy. Thirty two KM mice bearing H22 tumor
nodules were randomly divided into four groups for (a) normal
saline (control); (b) cisplatin, 3 mg Pt per kg; (c) MPt, 3 mg Pt per
kg and (d) MPt, 6 mg Pt per kg. Before injection, all the mice
were marked and weighed, and the length and width of the
tumors were measured as the initial size on day 1. The day of the
3
was stirred under dark for 24 h. To the clear solution obtained
by removal of the white precipitate formed via ltration, was
added (mPEG5k
2
) -PGA35 (300 mg, 0.018 mmol). The solution
was stirred under dark for 8 h, and then dialyzed against water

rst injection was designated as day 1. For groups (b) to (d),
ꢀ
1
using a dialysis bag (MWCO 3500 g mol ) for 1 day and frozen-
dried to obtain 450 mg of white product MPt.
designed doses of Pt were intravenously injected via tail vein on
day 1, day 3, and day 5, separately. For group (a), the mice were
injected with equivalent volume of normal saline. The tumor
Cell culture
size was measured every other day and the tumor volume (V)
2
MCF-7, HeLa, and SMMC cell lines were purchased from the was calculated by the formula of V ¼ ab /2, where a and b were
Institute of Biochemistry and Cell Biology, Chinese Academy of the length and width of tumor, respectively.
Sciences, Shanghai, China and were grown in Dulbecco's
modied Eagle's medium (DMEM, GIBCO) supplemented with deviation (SD). The Student's t-test was used to determine the
0% heat-inactivated fetal bovine serum (FBS, GIBCO), 100 U statistical difference between various experimental and control
Statistics. The data were expressed as the mean ꢃ standard
1
ꢀ1
ꢀ1
ml penicillin, and 100 mg ml streptomycin (Sigma), and the groups. Differences were considered statistically signicant at a
culture medium was replaced once very day.
level of p < 0.05.
In vitro MTT assay
Results and discussion
Synthesis of Y shaped amphiphilic copolymers
MCF-7 cells harvested in a logarithmic growth phase were
seeded in 96-well plates at a density of 10 cells per well and
5
incubated in RPMI 1640 for 24 h. The medium was then In order to prepare Y shaped amphphilic block polymer, that is,
replaced by various drug formulations of PTX, (mPEG ) -PGA methoxy-poly(ethylene glycol) -block-poly(g-benzyl-L-glutamate)
5
k 2
35
2
and (mPEG5k
)
2
-PGA35-PTX. To test the cytotoxicity of the free ((mPEG5k
2
) -PBG), two main synthetic strategies were adopted.
drug carrier, just (mPEG5k
)
2
-PGA35 was used to treat the cells One is that the coupling reaction between isocyanate group and
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hydroxyl group is always adopted to prepare copolymers con-
taining accurate structure, such as hydrogels and drug
19,20
carriers.
The other is the ring-opening polymerization (ROP)
of corresponding amino acid NCA monomers initiated by
primary amines. Various poly(ethylene glycol)-containing
copolypeptides with well controlled degrees of polymerization
2
1,22
(
DP) have been synthesized by this way,
preparation of the accurate structure of drug carriers.
2
In this work, three Y-shaped (mPEG5k -PBG and (mPEG5k) -
which helps to the
)
2
PGA copolymers with different PBG or PGA lengths were
prepared in three steps as shown in Scheme 1: (1) synthesis of
macroinitiator (mPEG ) -NH . Briey, mPEG was reacted
5
k 2
2
5k
with HDI to form mPEG5k-NCO, then two mPEG5k-NCOs were
coupled with NBS to obtain (mPEG5k -NHBoc, and nally,
complete removal of Boc group from (mPEG5k -NHBoc by HBr/
HAc/CF COOH to afford (mPEG5k -NH . (2) (mPEG5k -PBG
)
2
)
2
1
Fig. 1 H NMR spectra of (mPEG5k
B) in CDCl
2 2 2
) -NHBoc (A) and (mPEG5k) -NH
3
)
2
2
)
2
(
3
.
was synthesized by the ROP of N-carboxyanhydride of g-benzyl-
L-glutamate (BLG-NCA) initiated by (mPEG ) -NH . (3) The
5
k 2
2
benzyl groups on the PBG block were removed by HBr/HAc/
CF COOH to afford (mPEG ) -PGA.
ꢀ1
in (mPEG5k
)
2
-NHBoc, the peak at 3352 cm appeared in Fig. 3C
3
5k 2
2 5k 2 2
and was assigned to free NH group of (mPEG ) -NH .
The structures of (mPEG5k
)
2
-NHBoc and (mPEG5k
)
2
-NH
were conrmed by H NMR and FT-IR. The H NMR spectra of
mPEG5k -NHBoc and (mPEG5k -NH are shown in Fig. 1. As
shown in Fig. 1A, peaks a at 3.38 ppm and b at 3.62 ppm are
assigned to the protons of CH O– and –OCH CH O– units of
mPEG of (mPEG ) -NHBoc. The peaks marked with c (3.14
2
2
The structure of (mPEG5k) -NHBoc can also be proved by
GPC. As shown in Fig. 3 and Table 1, mPEG5k and mPEG10k gave
GPC peaks at 17.1 and 15.7 min, respectively, while that of
1
1
(
)
2
)
2
2
(
mPEG5k
This is understandable, because (mPEG5k
(mPEG )s coupled with an HDI-seritol-HDI linkage. Of course,
)
2
-NHBoc was at 15.7 min, close to that of mPEG10k
.
3
2
2
)
2
-NHBoc is two
5
k 2
5
k
ppm), d (1.47 ppm) and e (1.32 ppm) are assigned to the protons
of methylene units of HDI. Peak h at 1.42 ppm is assigned to
the molecular weight values determined by GPC were not
exactly 5k or 10k, but their two-fold relationship did exist (8.4 vs.
–
O–C(CH
group (Boc) in (mPEG5k
appeared completely in Fig. 1B, indicating successful synthesis
of macroinitiator (mPEG5k -NH
FT-IR spectra of (mPEG5k 2-NHBoc and (mPEG5k
shown in Fig. 2B and C. Compared to that of mPEG5k in Fig. 2A,
3
)
3
unit in NBS. Aer the removal of the protecting
ꢀ1
1
6.4 kg mol , Table 1). Moreover, the unimodal peak shape of
)
2
-NHBoc, peak h at 1.42 ppm dis-
(
mPEG ) -NHBoc and the narrow distribution (PDI ¼ 1.02) in
5
k 2
Fig. 3C also provided evidence for absence of mPEG residues
5
k
)
2
2
.
and successful synthesis of (mPEG5k
comparing integral area of peak b (Fig. 1) at 3.62 ppm assigned
to the protons of –CH CH O– units in mPEG with that of peak c
at 3.14 ppm assigned to the protons of –NH–CH CH CH CH
CH CH –NH– in HDI, no change was found before and aer the
2
) -NHBoc. Additionally, by
)
2 2
) -NH are
2
2
ꢀ
1
ꢀ1
the peaks at 1721 cm (C]O stretching) and 1533 cm (amide
II mode) in (mPEG ) -NHBoc appeared and the peak at 3475
cm
2
2
2
2
-
5
k 2
2
2
ꢀ
1
(OH stretching) disappeared, indicating successful
-NHBoc. Aer the removal of Boc group
deprotection (data not shown), indicating that CF
3
COOH did
synthesis of (mPEG5k
)
2
not cause chain cleavage of (mPEG ) -NHBoc during its
5
k 2
deprotection. In a word, well dened (mPEG ) -NH was
5
k 2
2
2 2 2 2
Scheme 1 Synthetic routes of (mPEG5k) -NH , (mPEG5k) -PBG and (mPEG5k) -PGA.
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1
Fig.
4
2 3
H NMR spectra of (mPEG5k) -PBG35in CDCl (A) and
Fig. 2 FT-IR spectra of mPEG5k (A), (mPEG5k
2
) -NHBoc (B), (mPEG5k
2
) -
(mPEG5k)2-PGA35 (B) in CDCl /CF COOD (v/v, 1 : 1).
3
3
2 2 2
NH (C), (mPEG5k) -PBG35 (D), and (mPEG5k) -PGA35 (E).
marked with c, d and e are assigned to the protons of the
hexamethylene group in HDI. The peaks i at 8.34 ppm, m at 7.25
ppm, l at 5.03 ppm, k at 2.62 ppm and j at 2.28 ppm are char-
acteristic of the protons on the PBG main chain of (mPEG ) -
5
k 2
PBG . The peaks a and b are assigned to the protons of mPEG
3
5
block of (mPEG5k
ppm) are assigned to the protons of –(OCH
(OCH –CH–NH– units in serinol residue. For the preparation
of (mPEG5k -PGA35, a solution of CF COOH and HBr 33 wt% in
)
2
-PBG35. The peaks f (4.18 ppm) and g (4.05
2 2
)
–CH–NH–,
–
2 2
)
)
2
3
acetic acid was used as a deprotection reagent to remove the
benzyl groups in (mPEG ) -PBG . Aer deprotection, the
5
k 2
35
peaks at 5.03 and 7.25 ppm assigned to the benzyl group in PBG
disappeared almost completely in Fig. 4B, indicating complete
removal of the protective groups.
The degree of polymerization (DP) of BLG in (mPEG5k
can be calculated by comparing the relative intensities of peak l
and peak b in Fig. 4A. The DP of PGA in (mPEG5k -PGA is
2
) -PBG
Fig. 3 The GPC traces of mPEG5k (A), PEG10k (B), (mPEG5k
C) and (mPEG5k -PBG20 (D).
2
) -NHBoc
)
2
(
)
2
calculated by a similar way. The DP data of PBG and PGA are
listed in Table 1. It is clear that the DP of PBG of (mPEG ) -PBG
5
k 2
is well in accordance with feed composition, indicating almost
complete conversion of BLG-NCA during the ROP of BLG-NCA.
Table 1 GPC results of mPEG5k, PEG10k, (mPEG5k
)
2
-NHBoc, mPEG5k
-
PBGs and (mPEG5k
)
2
-PBGs
The little DP reduction of (mPEG5k
demonstrates that CF COOH/HBr/acetic acid has no harmful
effect on the degradation of the main chain of PBG segment of
mPEG5k -PBG. Relatively, the molecular weight obtained by
GPC is larger than that determined by H NMR (Table 1), but Y-
2
) -PBG aer deprotection
ꢀ
1
a
ꢀ1
b
Samples
Mn (kg mol
)
PDI
Mn (kg mol
)
DP
3
mPEG5k
mPEG10k
8.44
16.2
-NHBoc 16.4
1.02
5.0
(
)
2
1.02 10.0
1.02 10.0
1.03 11.7
1.08 12.5
1.05 14.1
1
(mPEG5k
(mPEG5k
(mPEG5k
(mPEG5k
)
)
)
)
2
2
2
2
-PBG14
-PBG20
-PBG35
14.8
12.7
15.5
11.2
12.6
17.5
13/12 shaped copolymers shows low correction factor, and linear
19/19
32/31
13/–
17/–
32/–
copolymer gives higher results. This result indicated the
difference of various topologies.
The FT-IR spectra of (mPEG5k) -PBG35 and (mPEG5k) -PGA35
2 2
are shown in Fig. 2D and E. In comparison with that of
mPEG5k-PBG14
mPEG5k-PBG20
mPEG5k-PBG35
1.05
1.06
1.07
6.7
7.2
9.1
ꢀ
1
ꢀ1
(
2 2
mPEG5k) -NH in Fig. 2C, the peaks at 3297 cm , 1651 cm
a
1
b
The molecular weight determined by H NMR. The degree of
polymerization (DP) of PBG and PGA was calculated by H NMR.
ꢀ
1
1
and 1549 cm appeared and they are characteristic of NH
stretching and amide I and amide II modes of PBG, respectively,
indicating the formation of the polypeptide block. Moreover,
ꢀ1
ꢀ1
prepared and it was competent for acting as a macroinitiator for the absorption peaks at 750 cm and 700 cm are attributed
23
ROP of NCA.
to the phenyl groups in the PBG block The disappearance of
these two peaks from Fig. 4E implies successful removal of the
1
The H NMR spectra of (mPEG5k
)
2
-PBG35 and (mPEG5k
)
2
-
PGA35 are collected in Fig. 4A and B, respectively. The peaks benzyl groups, and the red shi of the peak (C]O stretching)
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from 1734 cm to 1727 cm and the blue shi of the peak
RSC Advances
ꢀ
1
ꢀ1
ꢀ
1
ꢀ1
(amide I mode) from 1651 cm to 1655 cm in Fig. 2E with
respect to those in Fig. 2D can be ascribed to the enhanced
hydrogen bonding between repeat units in PGA of (mPEG ) -
5
k 2
2
4
PGA35
terized by GPC. As shown in Fig. 3 and Table 1, the GPC of
mPEG5k -PBG20 (Curve D) showed even less retention time
compared to that of (mPEG5k -NHBoc (Curve C) and the Mw of
mPEG5k -PBGs thus obtained were in the range of 12.7–15.5 kg
.
2
The molecular weight of (mPEG5k) -PBG was charac-
(
)
2
)
2
(
)
2
ꢀ
1
mol , indicating different hydrodynamic behaviors of them
from that of linear PEG counterparts. Nevertheless, the PDIs of
(
mPEG ) -PBGs calculated from GPC proles were less than
5k 2
1.08 (Table 1), indicating absence of side reactions and
approximately living polymerization of BLG-NCA under the
polymerization conditions employed. In conclusion, Y-shaped
Fig. 5 TEM images of (mPEG5k
mPEG5k-PBG20 (C), and mPEG5k-PBG35 (D).
2 2
) -PBG20 (A), (mPEG5k) -PBG35 (B),
block copolymers (mPEG5k
successfully synthesized.
2 2
) -PBG and (mPEG5k) -PGA were
Self-assembly of copolymers in aqueous solution
and 88 nm), respectively. The possible reason is that at the same
length of PBG, the molar ratio of mPEG/PBG in (mPEG ) -PBG
5
k 2
A steady-state uorescent spectroscopic method using pyrene as
a probe was used to study self-assembly of linear and Y-shaped
copolymers in aqueous solution. It is well known that the CMC
values reect the stability of micelles, i.e., a low CMC value
is two times that in mPEG -PBG, resulting in a larger size of
5
k
micelles. Comparatively, the size of mPEG -PBG and
5
k
(
mPEG5k
determined by DLS because of the micelle shrinkage during
TEM sample preparation. The size of (mPEG5k -PBG ranges
2
) -PBG determined by TEM is smaller than those
25
means a high stability of micelles. It was reported that linear
)
2
mPEG20k-PBGs with the different molar ratio of PBG (14.9 to
from 75 nm to 82 nm, and that of mPEG5k-PBG is in the range
from 60 to 62 nm. This is in agreement with DLS
measurements.
4
.8%) self-assembled into micelles in water, their CMC values
ꢀ
1
26
ranged from 7.8 to 19.9 mg L
. As listed in Table 2, the CMC
values of (mPEG5k
)
2
-PBGs are in the range from 4.1 to 4.9 mg
ꢀ
1
L
, close to those of mPEG -PBGs, indicating that (mPEG ) -
5
k
5k 2
PBGs can be a competent drug carrier.
mPEG5k -PBG consists of hydrophilic segments of mPEG5k
Synthesis and characterization of (mPEG5k
mPEG5k -PGA–cisplain conjugates
2
) -PGA-PTX and
(
)
2
(
)
2
and hydrophobic segment of PBG, and thus can self-assemble
into micelles in aqueous solution, mPEG forms the outer shell
while PBG forms the inner core. TEM and DLS were used to
characterize the morphology and size of the micelles. As shown
in Fig. 5, the micelles of both Y-shaped and linear mPEG-PBG
show a spherical shape with a PDI value of 0.15–0.18, which
shows a narrow distribution. As shown in Table 2, the sizes of
Polymer prodrugs based on mPEG-PGA has been widely inves-
tigated and some of these polymer prodrugs have entered
preclinical studies. These polymer prodrugs can enhance the
solubility of hydrophobic drugs, such as paclitaxel, campto-
thecin, prolong the circulation time of loaded drugs, reduce
system toxicity caused by anti-cancer small molecule drugs, and
27,28
improve the quality of patients' lives.
further exploring the potential of (mPEG5k
In this work, for
-PGA on the eld of
mPEG5k-PBG and (mPEG5k
length of hydrophobic PBG segment. The sizes of (mPEG5k
PBG14, (mPEG5k -PBG20, and (mPEG5k -PBG35 determined by
2
) -PBG both decrease with increasing
)
2
2
) -
polymer prodrugs, Paclitaxel (PTX) and Cisplatin (Pt) were
selected as model drugs and loaded into (mPEG5k -PGA to
prepare (mPEG5k -PGA-PTX and (mPEG5k -PGA-Pt conjugates,
)
2
)
2
)
2
DLS are 123, 117, and 99 nm, respectively, higher than that of
mPEG5k-PBG14, mPEG5k-PBG20 and mPEG5k-PBG35 (108, 87,
)
2
)
2
the in vitro and in vivo anti-cancer activities of them were further
investigated.
Paclitaxel (PTX) is widely used in treatment of advanced and
refractory ovarian, breast, lung, and head and neck cancers.
Because of its low water solubility, it is usually administrated in
the dose form of Taxol, i.e., a suspension in Cremophor EL (a
polyoxyethylene castor oil)/anhydrous ethanol (1 : 1) solution.
Cremophor EL leads to severe side effects and lowers the anti-
Table 2 Parameters of linear and Y-shaped micelles
Size (nm)
by DLS
PDI
ꢀ1
Copolymers
by TEM
by DLS
CMC (mg L )
(
(
(
mPEG5k
mPEG5k
mPEG5k
)
)
)
2
2
2
-PBG14
-PBG20
-PBG35
123
117
99
108
87
80
82
75
75
60
62
0.15
0.16
0.18
0.17
0.18
0.16
4.89
4.55
4.67
3.56
4.61
3.68
29
tumor efficacy of PTX. In order to overcome these side effects,
polymer–drug conjugates have been developed. For example,
mPEG5k-PBG14
mPEG5k-PBG20
mPEG5k-PBG35
poly(L-glutamic
acid)-paclitaxel
conjugate
(PGA-PTX,
XYOTAX™, CT2103), now under phase III clinical trials,
presents improved water solubility, self-assembles into ꢄ80 nm
88
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nanoparticles in aqueous solution, and shows enhanced anti- relative integral areas of the protons of benzyl groups of PTX
cancer activity and targeted accumulation at tumor site with the between 7.34 and 8.03 ppm with respect to that of the protons of
help of both cathepsin B and the enhanced permeability and –CH CH O– units of mPEG at 3.53 ppm. Similarly, (mPEG) -
2
2
2
retention (EPR) effect, which improve the efficacy of chemo- PGA-Pt conjugate was prepared by virtue the coordination of the
30
therapy and reduce the side effects as compared to free Taxol.
carboxyl groups in PGA segment and the central Pt atom of
Considering that PEG has merits of good water solubility, non- cisplatin. The Pt content in the conjugate was determined by
immunogenicity, chain mobility and long blood circulation ICP-MS to be 15% (w/w).
time through prevention of renal elimination and avoidance of
2 2
(mPEG5k) -PGA35-PTX and (mPEG5k) -PGA35-Pt were allowed
receptor-mediated protein uptake by cells of the reticuloendo- to self-assemble into nano-micelles (MPTX and MPt) in aqueous
31,32
thelial system (RES),
PTX was conjugated to (mPEG )-PGA
35
solution. TEM micrographs in Fig. 7A and B revealed the
5
k
for the preparation of (mPEG) -PGA-PTX conjugates. The spherical shape of the micelles formed. The average particle size
2
structure, morphology, and in vitro anti-cancer efficacy of was about 50 nm for MPTX and about 45 nm for MPt. It implies
mPEG5k-PGA-PTX were further investigated.
By virtue of the reactivity of hydroxyl groups in the 2 position PGA35-PTX or PGA35-Pt as a whole is indeed hydrophobic.
of PTX, it was conjugated onto (mPEG5k -PGA35 with the help of Therefore, the mPEG segments forms the outer shell and the
DCC and DMAP to form an ester linkage. The structure of hydrophobic PGA-PTX or PGA-Pt segment forms the core of
that although PGA is hydrophilic to some extent, the conjugate
0
)
2
33
1
(mPEG5k
2
) -PGA35-PTX was characterized by H NMR. As shown micelles. To conrm this structure, the frozen-dried MPTX
1
1
in Fig. 6, the H NMR spectrum (6A) of (mPEG ) -PGA -PTX micelles were re-dissolved in D O for H NMR measurement. As
5
k 2
35
2
contains all feature peaks of PTX (6B) and (mPEG ) -PGA (6C) shown in Fig. 7C, the peak at 3.64 ppm assigned to the protons
5
k 2
35
0
except that of the proton (4.80 ppm) at the 2 position of PTX, of –CH CH O– units remained, while the characteristic signals
2
2
indicating successful synthesis of (mPEG)
content of PTX in (mPEG5k -PGA35-PTX is about 20%, and the
coupling efficiency of PTX was calculated to be 90% by using the SMMC cell lines was evaluated by MTT assay in comparison
with that of (mPEG) -PGA35, PTX, and Pt. As shown in Fig. 8, the
cell viability of (mPEG5k -PGA35 treated cells was higher than
0% no matter at 48 or 72 h, indicating very low toxicity of
mPEG) -PGA as a drug carrier material. As shown in Fig. 9, the
2
-PGA-PTX. The associated with the PGA-PTX segment were all absent.
)
2
The cytotoxicity of MPTX, and MPt against MCF-7, HeLa and
2
)
2
9
(
2
1
Fig. 6 H NMR spectra of (mPEG5k
COOD (v/v, 1 : 1), PTX in CDCl (B) and (mPEG5k
CF COOD (v/v, 1 : 1).
)
2
-PGA35-PTX (A) in DMSO/CF
3
-
3
2
) -PGA35 (C) in CDCl
3
/
2
Fig. 8 In vitro cytotoxicity of (mPEG5k) -PGA35 for 48 h and 72 h
against MCF-7 cell line.
3
1
Fig. 7 TEM images of (mPEG5k
PTX micelles in D O.
2
) -PGA35-PTX (A) and (mPEG5k
)
2
-PGA35-Pt (B) particles. (C) H NMR spectrum of frozen-dried (mPEG5k
)
2
-PGA35
-
2
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Fig. 9 MTT assay results at 48 h: (A) MPTX and PTX for MCF7; (B)MPTX and PTX for HeLa; (C) PTX and PTX for SMMC; (D) Pt and cisplatin for
MCF7; (E) Pt and cisplatin for HeLa; (F) MPt and Pt for SMMC.
cell viability showed obvious concentration dependence and intervals at the same dose of 3 mg and 6 mg Pt per kg for
cell line dependence for PTX, MPTX, Pt, and MPt. Based on the cisplatin and MPt. The relative tumor volume (RTV) vs. time and
concentration dependence, IC50 was calculated and collected in the relative body weight vs. time are shown in Fig. 10. As shown
Table 3. Firstly, given a formulation, IC50 of MPTX varied from in Fig. 10A, the RTV increment of four groups took an order of
ꢀ
1
ꢀ1
ꢀ1
less than 0.005 mg ml for MCF-7 to 1.24 mg ml for HeLa cells, Saline [ MPt (3 mg Pt per kg ) > Pt (3 mg Pt per kg) > MPt
th
approximately corresponding to that of pure PTX; while IC50 of (6 mg Pt per kg). For example, on the 14 day, the RTV values
ꢀ
1
ꢀ1
MPt varied from 9.11 mg ml for MCF-7 to 1.76 mg ml for were 343.9 ꢃ 25.2, 81.5 ꢃ 10.7, 34.7 ꢃ 8.0, and 16.9 ꢃ 3.8,
SMMC cells, approximately in parallel to that of pure Pt. This is respectively. Notably, increasing the dose of MPt from 3 mg Pt
understandable, because every anti-cancer drug is not effective
to all cancers. Secondly, for each cell line tested, MPTX and MPt
displayed comparable or a little bit less cytotoxicity than pure
PTX and Pt, respectively. Because MPTX and MPt as nano-
micelles have other advantages over PTX and Pt, e.g., reduced
systemic toxicity, this comparable cytotoxicity implies possi-
bility for them to be used as effective drug delivery systems.
In vivo antitumor activity of MPt
Cisplatin (abbr. as Pt) is a rst line anti-cancer drug for treat-
ment of many malignancies, such as ovarian, bladder, non-
small-cell lung and liver cancers. H22 is a well-known cell line
obtained from human liver carcinoma. In this work, H22
bearing Kunming mice were treated i.v. three times at 2 day
Table 3 IC50 values of MPTX, PTX, MPt, and Pt against MCF7, HeLa,
and SMMC cell lines
ꢀ1
IC50 (mg ml
)
Drugs
MCF7
HeLa
SMMC
MPTX
PTX
MPt
Pt
<0.005
<0.005
9.11
1.24
0.34
4.58
1.42
0.01
<0.005
1.76
Fig. 10 Relative tumor volume (A) and relative body weight (B) of the
H22 cancer bearing mice as a function of time. (a) saline; (b) cisplatin
5.28
1.34
(3 mg Pt per kg); (c) MPt (3 mg Pt per kg); (d) MPt (6 mg Pt per kg).
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per kg to 6 mg Pt per kg led to enhanced anti-cancer efficacy
9 G. Lapienis, Prog. Polym. Sci., 2009, 34, 852–892.
while similar increase in cisplatin dose caused death of all test 10 C. Garofalo, G. Capuano, R. Sottile, R. Tallerico, R. Adami,
th
mice on the 4 day post rst injection, indicating the lower
E. Reverchon, E. Carbone, L. Izzo and D. Pappalardo,
systemic toxicity of MPt than cisplatin. This conclusion was
Biomacromolecules, 2014, 15, 403–415.
supported by the body weight measurement. As shown in 11 J. Sun, X. S. Chen, J. S. Guo, Q. Shi, Z. G. Xie and X. B. Jing,
Fig. 10B, the relative body weight took an order of Saline [ Polymer, 2009, 50, 455–461.
MPt (3 mg Pt per kg) > MPt (6 mg Pt per kg) > Pt (3 mg Pt per kg) 12 H. Cabral, M. Murakami, H. Hojo, Y. Terada, M. R. Kano,
on day 14.
U. I. Chung, N. Nishiyama and K. Kataoka, Proc. Natl.
Acad. Sci. U. S. A., 2013, 110, 11397–11402.
1
3 M. Yokoyama, T. Okano, Y. Sakurai, S. Suwa and K. Kataoka,
J. Controlled Release, 1996, 39, 351–356.
Conclusions
A novel strategy for preparing Y-shaped amphiphilic (mPEG)
PBG copolymer containing two hydrophilic arms and one
2
-
14 H. Cabral, N. Nishiyama and K. Kataoka, J. Controlled
Release, 2007, 121, 146–155.
hydrophobic arm was successfully developed, that is, to prepare 15 J. Schulz, H. Burris, C. Redfern, M. Warner and C. Paradise,
mPEG) -PBG by using a macroinitiator with a middle primary
Eur. J. Cancer, 2002, 38, S132.
to initiate the ROP of g-benzyl-L- 16 Y. Matsumura and K. Kataoka, Cancer Sci., 2009, 100, 572–
glutamate-N-carboxyanhydride. Due to its amphiphilic nature,
579.
mPEG) -PBG can self-assemble into micelles and can encap- 17 J. X. Ding, C. L. He, C. S. Xiao, J. Chen, X. L. Zhuang and
sulate hydrophobic drugs. Aer removal of the benzyl groups on
X. S. Chen, Macromol. Res., 2012, 20, 292–301.
the PBG segment, the copolymer became (mPEG) -PGA con- 18 C. Neri and J. M. J. Williams, Adv. Synth. Catal., 2003, 345,
taining free carboxyl groups. By virtue of these carboxyl groups,
835–848.
mPEG) -PGA could conjugate with hydrophobic drugs like PTX 19 L. X. Yang, H. H. Xiao, L. S. Yan, R. Wang, Y. B. Huang,
(
2
2 2
amino group (mPEG) -NH
(
2
2
(
2
and Cisplatin. The conjugates formed were also amphiphilic
and were allowed to self-assemble into nanoscale drug formu- 20 L. X. Yang, J. Z. Wei, L. S. Yan, Y. B. Huang and X. B. Jing,
lations, which showed good water solubility, satisfactory cyto-
Biomacromolecules, 2011, 12, 2032–2038.
toxicity to cancer cells and excellent antitumor efficacy to H22 21 L. S. Yan, L. X. Yang, H. Y. He, X. L. Hu, Z. G. Xie, Y. B. Huang
Z. G. Xie and X. B. Jing, J. Mater. Chem. B, 2014, 2, 2097.
cancers. Therefore, polymer nano-prodrug based on (mPEG)
2
-
and X. B. Jing, Polym. Chem., 2012, 3, 1300–1307.
PGA has a potential in the treatment of solid-tumors.
22 L. S. Yan, W. B. Wu, W. Zhao, R. G. Qi, D. M. Cui, Z. G. Xie,
Y. B. Huang, T. Tong and X. Jing, Polym. Chem., 2012, 3,
2
403–2413.
Acknowledgements
2
2
2
2
2
2
2
3 J. Yue, R. Wang, S. Liu, S. H. Wu, Z. G. Xie, Y. B. Huang and
X. B. Jing, So Matter, 2012, 8, 7426–7435.
4 C. Deng, G. Z. Rong, H. Y. Tian, Z. H. Tang, X. S. Chen and
X. B. Jing, Polymer, 2005, 46, 653–659.
5 C. Agatemor and M. P. Shaver, Biomacromolecules, 2013, 14,
699–708.
6 Y. I. Jeong, J. W. Nah, H. C. Lee, S. H. Kim and C. S. Cho, Int.
J. Pharm., 1999, 188, 49–58.
7 H. H. Xiao, R. G. Qi, S. Liu, X. L. Hu, T. C. Duan, Y. H. Zheng,
Y. B. Huang and X. B. Jing, Biomaterials, 2011, 32, 7732–7739.
8 X. L. Hu, S. Liu, Y. B. Huang, X. S. Chen and X. B. Jing,
Biomacromolecules, 2010, 11, 2094–2102.
9 L. Sheihet, O. B. Garbuzenko, J. Bushman, M. K. Gounder,
T. Minko and J. Kohn, Eur. J. Pharm. Sci., 2012, 45, 320–329.
0 J. Gligorov and J. P. Lotz, Oncologist, 2004, 9(suppl. 2), 3–8.
1 J. P. Nam, J. K. Park, D. H. Son, T. H. Kim, S. J. Park,
S. C. Park, C. Choi, M. K. Jang and J. W. Nah, Colloids
Surf., B, 2014, 120, 168–175.
Financial support was provided by the National Science Foun-
dation of China (Project no. 51003101, 91227118, and
51373167).
Notes and references
1
R. Gref, M. L u¨ ck, P. Quellec, M. Marchand, E. Dellacherie,
S. Harnisch, T. Blunk and R. H. M u¨ ller, Colloids Surf., B,
2000, 18, 301–313.
2
3
4
5
6
7
W. Chen, F. H. Meng, F. Li, S.-J. Ji and Z. Y. Zhong,
Biomacromolecules, 2009, 10, 1727–1735.
Y. Bae and K. Kataoka, Adv. Drug Delivery Rev., 2009, 61, 768–
784.
K. Cho, X. Wang, S. Nie, Z. G. Chen and D. M. Shin, Clin.
Cancer Res., 2008, 14, 1310–1316.
H. Maeda, J. Wu, T. Sawa, Y. Matsumura and K. Hori,
J. Controlled Release, 2000, 65, 271–284.
3
3
J. Khandare and T. Minko, Prog. Polym. Sci., 2006, 31, 359–
3
2 H. Otsuka, Y. Nagasaki and K. Kataoka, Adv. Drug Delivery
Rev., 2003, 55, 403–419.
3 C. Li, D. F. Yu, R. A. Newman, F. Cabral, L. C. Stephens,
N. Hunter, L. Milas and S. Wallace, Cancer Res., 1998, 58,
397.
J. X. Ding, C. S. Xiao, L. Zhao, Y. L. Cheng, L. L. Ma,
Z. H. Tang, X. L. Zhuang and X. S. Chen, J. Polym. Sci., Part
A: Polym. Chem., 2011, 49, 2665–2676.
3
2404–2409.
8
L. Y. Qiu and Y. H. Bae, Pharm. Res., 2006, 23, 1–30.
41596 | RSC Adv., 2014, 4, 41588–41596
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