Preparation of amphiphilic copolymers for covalent loading of paclitaxel for drug delivery system

Article abstract of DOI:10.1002/pola.27009

A novel drug-polymer conjugate was prepared by the copper-catalyzed azide-alkyne cycloaddition reaction between an azide-functional diblock copolymer and an alkyne-functional paclitaxel (PTX). The well-defined azide-functional diblock copolymer, poly(ethylene glycol) (PEG)-b-P(OEGEEMA-co- AzPMA), was synthesized via the atom transfer radical polymerization of oligo(ethylene glycol) ethyl ether methacrylate (OEGEEMA) and 3-azidopropyl methacrylate (AzPMA), using PEG-Br as macroinitiator and CuBr/PMDETA as a catalytic system. The alkyne-functional PTX was covalently linked to the copolymer via a click reaction, and the loading content of PTX could be easily tuned by varying the feeding ratio. Transmission electron microscopy and dynamic light scattering results indicated that the drug loaded copolymers could self-assemble into micelles in aqueous solution. Moreover, the drug release behavior of PEG-b-P(OEGEEMA-co-AzPMA-PTX) was pH dependent, and the cumulative release amount of PTX were 50.0% at pH 5.5, which is about two times higher than that at pH 7.4. The in vitro cytotoxicity experimental results showed that the diblock copolymer was biocompatible, with no obvious cytotoxicity, whereas the PTX-polymer conjugate could efficiently deliver PTX into HeLa and SKOV-3 cells, leading to excellent antitumor activity. 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 366-374 The azide-functional diblock copolymer PEG-b-P(OEGEEMA-co-AzPMA) is synthesized, and alkyne-functional paclitaxel is conjugated to it, using click chemistry. The novel paclitaxel-polymer conjugate self-assembles into micellar nanoparticles in aqueous solution, and the loading content of paclitaxel is easily tuned by the feeding ratio. Copyright

Full text of DOI:10.1002/pola.27009

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Preparation of Amphiphilic Copolymers for Covalent Loading of Paclitaxel
for Drug Delivery System
Wulian Chen,1 Jin Z. Zhang,2 Jianhua Hu,1,3 Qisang Guo,4 Dong Yang1
1State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University,
Shanghai 200433, China
2Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064
3Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, Fudan University, Shanghai 201203, China
4Mdical Center for Diagnostics & Treat of Cervical Disease, Obstetrics and Gynecology Hospital, Fudan University, Shanghai
200011, China
Correspondence to: J. Hu (E-mail: hujh@fudan.edu.cn) or D. Yang (E-mail: yangdong@fudan.edu.cn)
Received 14 July 2013; accepted 31 October 2013; published online 18 November 2013
DOI: 10.1002/pola.27009
ABSTRACT: A novel drug-polymer conjugate was prepared by
the copper-catalyzed azide–alkyne cycloaddition reaction
between an azide-functional diblock copolymer and an alkyne-
functional paclitaxel (PTX). The well-defined azide-functional
diblock copolymer, poly(ethylene glycol) (PEG)-b-P(OEGEEMA-
co-AzPMA), was synthesized via the atom transfer radical poly-
merization of oligo(ethylene glycol) ethyl ether methacrylate
(OEGEEMA) and 3-azidopropyl methacrylate (AzPMA), using
PEG-Br as macroinitiator and CuBr/PMDETA as a catalytic sys-
tem. The alkyne-functional PTX was covalently linked to the
copolymer via a click reaction, and the loading content of PTX
could be easily tuned by varying the feeding ratio. Transmis-
sion electron microscopy and dynamic light scattering results
indicated that the drug loaded copolymers could self-assemble
into micelles in aqueous solution. Moreover, the drug release
behavior of PEG-b-P(OEGEEMA-co-AzPMA-PTX) was pH
dependent, and the cumulative release amount of PTX were
50.0% at pH 5.5, which is about two times higher than that at
pH 7.4. The in vitro cytotoxicity experimental results showed
that the diblock copolymer was biocompatible, with no
obvious cytotoxicity, whereas the PTX-polymer conjugate
could efficiently deliver PTX into HeLa and SKOV-3 cells, lead-
C
V
ing to excellent antitumor activity.
2013 Wiley Periodicals,
Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 366–374
KEYWORDS: amphiphilic copolymer; atom transfer radical poly-
merization; click reaction; covalent loading; diblock copoly-
mers; drug delivery system; micelles; prodrug
size of the polymeric micelle is about several dozens of
nanometers, which affords the polymeric micelles passive
targeting function to tumor tissues via the enhanced perme-
ability and retention (EPR) effect.23,24
INTRODUCTION Chemotherapy is one of the most widely
used strategies in the long history of combating cancer.1,2
However, most of the anti-cancer drugs, that is, paclitaxel
(PTX) and doxorubicin (DOX), often suffer from intrinsic lim-
itations, such as poor water-solubility, severe side effects,
and low therapeutic index.3–10 To solve these problems, vari-
ous drug delivery systems (DDSs) have been developed and
investigated in the past decades.11–19
Up to date, most of the DDSs using polymeric micelles as
drug carriers load drugs via noncovalent interactions.25–30
For example, Lee et al. have prepared an amphiphilic block
copolymer, poly(ethylene glycol)-b-poly(2-(4-(vinylbenzy-
loxy)-N,N-diethylnicotinamide)) (PEG-b-P(VBODENA)), that
could form a core-shell micelle in aqueous solution.31 PTX
was then incorporated in the polymeric micelles via physical
entrapment. However, to these DDSs, many factors, such as
high dilution, pH, temperature, ionic strength, large shear
forces during blood circulation, and the presence of numer-
ous charged blood components, can affect their stability.32,33
This could lead to burst release of drug,34–37 which is the
main reason for toxic and side effects and dramatically
Amphiphilic copolymers are frequently used as drug carriers
in DDSs. In aqueous solution, amphiphilic copolymers could
self-assemble to form micelles, which are composed of two
distinct domains, a hydrophobic core and a hydrophilic shell.
Thus, hydrophobic drugs could be entrapped into the core,20
and the hydrophilic shell, in general poly(ethylene glycol)
(PEG), could effectively protect the core against the external
biological media, inhibit nonspecific protein absorption, and
increase the plasma clearance half-life.21,22 Furthermore, the
Additional Supporting Information may be found in the online version of this article.
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2013 Wiley Periodicals, Inc.
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chromatography (GPC), Fourier transformed infrared (FT-IR),
ultraviolet (UV) spectra, and so on. The in vitro cellular cytotox-
icity experiments were performed to evaluate the biocompati-
bility of the copolymer, and the anticancer activity of the PTX-
polymer conjugate to HeLa and SKOV-3 cells.
EXPERIMENTAL
Materials
Paclitaxel (PTX) was purchased from Beijing Huafeng United
Technology Co., Ltd. Oligo(ethylene glycol) ethyl ether meth-
acrylate (OEGEEMA, Mn 5 246 g mol21, 99%) purchased
from Aldrich was passed through a neutral alumina column
to remove the inhibitor prior to use. Triethylamine (TEA,
ꢀ
99.5%, Aldrich) was dried over CaH2 and distilled at 110 C.
Toluene (Aldrich, 99%) was refluxed with sodium beads/
benzophenone complex and distilled until the solution
turned purple. Copper(I) bromide (CuBr, 99.999%, Aldrich),
N,N,N0,N00,N[tprime]-pentamethyldiethylenetriamine
(PMDETA, 98%, Aldrich), 2-bromoisobutyl bromide (BiBB,
98%, Acros), 3-chloropropanol (99.5%, J&K CHEMICA), and
sodium azide (NaN3, 98%, SCR) were used as received with-
out further purification.
SCHEME 1 Synthetic routes used for the preparation of
alkynyl-PTX, PEG-b-P(OEGEEMA-co-AzPMA) diblock copolymer
and PEG-b-P(OEGEEMA-co-AzPMA-PTX) prodrug. [Color figure
can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
decreases the therapeutic efficacy. Thus, an alternative DDS
based on polymer with covalent loading of drug, polymeric
micellar drug conjugate, has been proposed to overcome the
problems mentioned above. As compared to polymeric
micelle DDSs loading drug via physical entrapment, poly-
meric micellar drug conjugate DDSs are more stable and typ-
ically have continuous release without burst release effect.38–41
Commonly, the drug and the polymer was conjugated via rela-
tively stable linkers (e.g. amide and ester bonds). For example,
Kwon et al. have covalently linked PTX to poly(ethylene glycol)-
block-poly(aspartate-hydrazide-levulinic acid) (PEG-p(Asp-Hyd-
LEV)) through ester bonds and found that PTX could continu-
ously low dose release.42,43 Due to the only reactive functional
hydroxyl group in PTX, the existing strategies to synthesize
PTX-polymer conjugates are difficult and tedious. Therefore, it
is of strong interest to explore a facile and efficient way to con-
struct PTX-polymer conjugates.
Synthesis of Alkynyl-Paclitaxel
Alkyne-PTX was synthesized by the esterification of PTX
with propiolic acid. In a typical procedure, palitaxel (420 mg,
0.49 mmol), propiolic acid (0.3 mL, 4.9 mmol), 4-dimethyla-
miopyridine (DMAP) (10 mg), N,N’-diisopropylcarbodiimide
(DIC) (0.1 mL), and CH2Cl2 (20 mL) were added into a 100-
mL of flask. The mixture was stirred at room temperature
for 6 h. A yellowish solid (0.38 g, yield of 83.2%), alkyne-
PTX, was obtained by passing through silica column chroma-
tography (eluent: diethyl ether/hexane/ethyl acetate
(v:v) 5 1/1/1), and evaporating.
Block Copolymerization of AzPMA and OEGEEMA
PEG-Br macroinitiator was prepared by the esterification
of PEG-OH with 2-bromoisobutyl bromide according to
refs. 48–50, and 3-azidopropyl methacrylate (AzPMA)
were synthesized according to ref. 51 (See Supporting
Information). PEG-b-P(OEGEEMA-co-AzPMA) block copoly-
mer was obtained by ATRP of AzPMA and OEGEEMA using
PEG-Br as macroinitiator and CuBr/PMDETA as a catalytic
system.52–57 In a typical procedure, a dry 50 mL Schlenk
flask (flame-dried under vacuum prior to use) was
charged with PEG-Br (2.0 g, 0.39 mmol) and CuBr (57.2
mg, 0.4 mmol). Then, AzPMA (0.95 mL, 6.0 mmol),
OEGEEMA (2.0 mL, 10.0 mmol), dry toluene (10 mL), and
PMDETA (0.16 mL, 0.8 mmol) was introduced via a gas-
tight syringe. The mixture was degrassed with three
freeze-evacuate-thaw cycles followed by immersing into an
oil bath at 80 ꢀC. The polymerization lasted 6 h and was
terminated by immersing the flask into liquid N2. The mix-
ture was diluted with THF and passed through a short
neutral alumina column to remove the residual copper
catalyst. The solution was concentrated under vacuum and
precipitated into cold hexane. After repeated purification
The recent development of click chemistry provided a powerful
tool for synthetic polymer chemistry and the pharmaceutical
fields.44–47 Due to its high efficiency and selectivity, moderate
reaction condition and simple purification, the azide–alkyne
reaction has been widely used to prepare drug-polymer conju-
gates. Herein, we constructed a novel PTX-polymer conjugate
based on copolymer covalently linked to PTX via an azide–
alkyne reaction (Scheme 1). Well-defined azide-functional
diblock copolymer, PEG-b-P(OEGEEMA-co-AzPMA), was synthe-
sized via atom transfer radical polymerization (ATRP) of
oligo(ethylene glycol) ethyl ether methacrylate (OEGEEMA)
and 3-azidopropyl methacrylate (AzPMA). Then, alkyne-
functional PTX was covalently linked to the copolymer via an
azide–alkyne reaction. The chemical structures of the diblock
copolymer and PTX-polymer conjugate were characterized by
proton-nuclear magnetic resonance (1H NMR), gel permeation
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by dissolving in THF and precipitating in cold hexane, a
waxy solid, PEG-b-P(OEGEEMA-co-AzPMA), was obtained
by drying in vacuo overnight.
GPC: Mn 5 14,000 g mol21, Mw/Mn 5 1.29. 1H NMR (CDCl3):
0.77–0.95 (3H, CH3CCH2 of POEGMEA and PAzPMA), 1.22
(3H, CH3CH2O of POEGMEA), 1.85 (2H, CH2CCH3 of POEG-
MEA and PAzPMA), 1.92 (2H, CH2CH2N3), 3.37 (3H, CH3O of
PEG), 3.44 (2H, CH2CH2N3), 3.64 (4H, OCH2CH2 of PEG and
POEGMEA), 4.0–4.1 (2H, COOCH2 of POEGMEA and PAzPMA).
FT-IR: 2100 cm21 (mN ).
3
Synthesis of Drug-polymer Conjugate PEG-b-
P(OEGEEMA-co-AzPMA-PTX)
Drug-polymer conjugate, PEG-b-P(OEGEEMA-co-AzPMA-PTX),
was synthesized by copper(I)-mediated azide–alkyne click
reaction of PEG-b-P(OEGEEMA-co-AzPMA) with alkyne-PTX.
Briefly, to a 10-mL of flask, alkyne-PTX (0.09 g, 0.10 mmol
of alkyne groups), PEG-b-P(OEGEEMA-co-AzPMA) (0.12 g,
0.10 mmol of N3 groups), and PMDETA (0.16 mL, 0.8 mmol)
were added and dissolved with 2 mL of DMF. The mixture
was degrassed with three freeze-evacuate-thaw cycles fol-
lowed by the addition of CuBr (57.2 mg, 0.40 mmol). The
reaction mixture was stirred for 24 h at room temperature
under N2 atmosphere. Then the mixture was diluted with
CH2CCl2 and washed with 10 wt% aqueous EDTA and deion-
ized water. The organic phase was dried with sodium sulfate
and then precipitated into excess diethyl ether. After
repeated purification by dissolving in CH2CCl2 and precipitat-
ing in diethyl ether, a yellowish solid, PEG-b-P(OEGEEMA-co-
AzPMA-PTX), was obtained by drying in vacuo overnight.
FIGURE 1 GPC traces for (A) PEG-Br, (B) PEG-b-P(OEGEEMA-co-
AzPMA), (C) PEG-b-P(OEGEEMA-co-AzPMA-PTX)1, (D) PEG-b-
P(OEGEEMA-co-AzPMA-PTX)2, and (E) PEG-b-P(OEGEEMA-co-
AzPMA-PTX)3 with THF as the elute. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
ꢀ
tion for another 2 h at 37 C in dark. Afterward, the absorb-
ance at a wavelenth of 450 nm of each well was measured
using a microplate reader. The relative cell viability (in per-
centage) was determined by the following equation:
Isample 2 Iblank
Cell viability ð%Þ5
In Vitro Drug Release
Icontrol 2Iblank
The drug release of PTX from PTX-polymer conjugate, PEG-b-
P(OEGEEMA-co-AzPMA-PTX), was studied at 37 ꢀC in two
different medias, (a) PBS of pH 5 5.5 and (b) PBS of
pH 5 7.4. Typically, 5 mg of PTX-polymer conjugate was dis-
solved in 5 mL of deionized water. 2 mL of solution was
transferred to a dialysis bag with an molecular weight cut-
off (MWCO) of 3500 and then immersed in 250 mL of
where Isample, Icontrol, and Iblank represent the absorbance
intensity at 450 nm determined for cells teated with differ-
ent samples, for control cells (nontreated), for blank well
without cells, respectively.
The antitumor activity of PEG-b-P(OEGEEMA-co-AzPMA-PTX)
was studied in a similar way. Briefly, HeLa and SKOV-3 cells
were seeded in 96-well plates with a density of 1 3 104 cell
ꢀ
pH 5 5.5 or pH 5 7.4 PBS at 37 C with constant shake. Peri-
odically, 20 mL of external buffer solution was removed and
replaced with equal volume of fresh medium. Upon each
sampling, 20 mL of PBS was lyophilized and then dissolved
in acetonitrile and PTX concentration was quantified by
measuring the absorbance at 229 nm against a standard
curve.
ꢀ
per well and incubated at 37 C in an atmosphere containing
5% CO2 for 24 h to allow cell attachment. Then, the medium
was replaced with a fresh medium containing the indicated
concentration of PEG-b-P(OEGEEMA-co-AzPMA-PTX) for 24
and 48 h. The following procedure of the cell viability deter-
mined by CCK-8 assays was same as described above.
In Vitro Cell Assay
The cytotoxicity of PEG-b-P(OEGEEMA-co-AzPMA) was eval-
uated in HeLa, SKOV-3 and HEK-293 cells by CCK-8 kit
asssys. The cells were seeded in 96-well plates with a den-
sity of 1 3 104 cell per well and incubated at 37 ꢀC in an
atmosphere containing 5% CO2 for 24 h to allow cell attach-
ment. Then, the medium was replaced with a fresh medium
containing the indicated concentration of PEG-b-
P(OEGEEMA-co-AzPMA). After incubation for 24 and 48 h,
the medium was aspirated and replaced by 100 mL of fresh
medium containing 10 mL of CCK-8. The cells were incuba-
Characterization
1H NMR spectra were recorded on a Bruker Avance 500
spectrometer using CDCl3 as solvent, trimethylsilane was
used as internal standard. FT-IR measurements were per-
formed on a NEXUX-470 spectrometer. The molecular weight
and molecular weight distribution were determined by GPC.
ꢀ
The GPC was performed in THF at 35 C with an elution rate
of 1.0 mL min21 on Aginent 1100 equipped with a G1310A
pump, a G1362A refractive index detector, and a G1314A
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FIGURE 3 FT-IR spectra of (A) PEG-Br and (B) PEG-b-
P(OEGEEMA-co-AzPMA).
the higher molecular weight side, compared with that of
PEG-Br macroinitiator. The chemical structure of PEG-b-
P(OEGEEMA-co-AzPMA) diblock copolymer was character-
1
ized by H NMR. As shown in the 1H NMR spectra of Figure
2(B), characteristic signals of PEG, OEGEEMA, and AzPMA
were all present. The DP of P(OEGEEMA-co-AzPMA) was
determined to be 30 with the OEGEEMA molar fraction
being 0.67, as indicated in 1H NMR analysis from the signal
integration ratio of peaks g at 1.2 ppm (methyl protons at
the terminal of OEGEEMA units) and d at 3.4 ppm (methyl-
ene protons at the a position to the azide group) relative to
peak b at 3.3 ppm (methyl protons at the terminal of PEG).
The polymer was denoted PEG-b-P(OEGEEMA0.67-co-
FIGURE
2
1H NMR spectra of (A) PEG-Br, (B) PEG-b-
P(OEGEEMA-co-AzPMA), and (C) PEG-b-P(OEGEEMA-co-
AzPMA-PTX)3 in CDCl3. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
variable wavelength detector. Polystyrene standard samples
were used for the GPC calibration. Transmission electron
microscopy (TEM) images were obtained on a JEOL JEM
2100 F transmission electron microscope, and samples for
TEM measurements were prepared by casting one drop of
sample aqueous solution on carbon copper grids. The size
distribution of the micelles was measured by dynamic light
scattering (DLS) using a Malvern autosizer 4700 instrument.
UV-visible (Vis) absorbance spectra were measured with a
Perkin-Elmer Lambda 35 spectrophotometer.
AzPMA0.33 30 and had a Mn,NMR of 11.6 kDa, shortened as
)
PEG-b-P(OEGEEMA-co-AzPMA) in subsequent section. More-
over, FT-IR spectrum of PEG-b-P(OEGEEMA-co-AzPMA) in
Figure 3 also showed a peak at 2100 cm21 characteristic for
the azide vibration, and the intensity of ester bond at 1725
cm21 also greatly increased after polymerization.
The PEG-b-P(OEGEEMA-co-AzPMA-PTX) drug-polymer conju-
gate was prepared by copper(I)-mediated azide–alkyne click
reaction of PEG-b-P(OEGEEMA-co-AzPMA) with alkynyl-PTX
using PMDETA as ligand in DMF at room temperature for 24
h. The drug loading content of the resulting PEG-b-
P(OEGEEMA-co-AzPMA-PTX) drug-polymer conjugate could
be simply tuned by varying the ratio of alkynyl group and
azide group. Three different drug loading contents of PTX-
polymer conjugate were obtained by three different molar
ratios of alkynyl group and azide group (alkynyl:azide 5 1:2,
1:1, 2:1). The reaction progress was monitored by FT-IR (by
RESULTS AND DISCUSSION
The diblock copolymer, PEG-b-P(OEGEEMA-co-AzPMA) was
obtained via ATRP of OEGEEMA and AzPMA using PEG-Br as
macroinitiatorꢀ and CuBr/PMDETA as a catalytic system in
toluene at 80 C, as it was shown in Scheme 1. A typical GPC
trace of PEG-b-P(OEGEEMA-co-AzPMA) was shown in Figure
1, it appeared a monomodal elution peak and a clear shift to
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TABLE 1 Drug Loading Contents of PEG-b-P(OEGEEMA-co-
AzPMA-PTX)s Determined by Differents Methods
Drug loading content
Feed ratio
(wt %) determined by
of alkynyl
Samples
to azide
GPC
14.5
1H NMR
12.7
UV-Vis
12.9
PEG-b-P(OEGEEMA-
co-AzPMA-PTX)1
1:2
PEG-b-P(OEGEEMA-
co-AzPMA-PTX)2
1:1
2:1
23.4
30.5
24.5
33.2
20.6
28.5
PEG-b-P(OEGEEMA-
co-AzPMA-PTX)3
diblock copolymer. Furthermore, the peaks near 710 cm21
were also obviously increased, which were related to the
C–H bending vibrations of the benzene rings in PTX. The
PTX-polymer conjugate was also analyzed by GPC (Fig. 1).
With the increase in PTX content, the elution peaks showed
a clear shift to a higher molecular weight side. The chemical
structure of PEG-b-P(OEGEEMA-co-AzPMA-PTX) diblock
FIGURE 4 UV-Vis absorption spectra of (A) PEG-b-
P(OEGEEMA-co-AzPMA) at the concentration of 100 mg mL21
,
(B)
PEG-b-P(OEGEEMA-co-AzPMA-PTX)1,
(C)
PEG-b-
P(OEGEEMA-co-AzPMA-PTX)2, and (D) PEG-b-P(OEGEEMA-co-
AzPMA- PTX)3 at 40 mg mL21 with different PTX content. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
1
copolymer was characterized by H NMR. As it could be seen
from Figure 2(C), the aromatic signals at 7.0 to 8.1 ppm
from PTX appeared. The characteristic signals of protons,
including the 30-CH and 5-CH protons from PTX at 5.9 and
4.9 ppm, were also observed, indicating that alkynyl-PTX
was successfully covalently linked to diblock copolymer. The
signal integration of the 5-CH protons from PTX at 4.9 ppm
was selected to compared with that of methyl protons at the
terminal of PEG at 3.4 ppm, and thus the average number of
PTX in backbone could be determined. The UV-Vis
the decrease of the azide vibration at 2100 cm21; Supporting
Information Fig. S3). When 1:2 of alkynyl to azide ratio was
used, FT-IR analysis showed that the azide peak did not dis-
appear completely, as expected from the excess of azide
used. With the increasing ratio of alkynyl to azide, the azide
peak decreased and finally completely disappeared, which
indicated that more alkynyl-PTX was covalently linked to
FIGURE 5 TEM images of self-assembled micellss of (A) PEG-b-P(OEGEEMA-co-AzPMA) and (B) PEG-b-P(OEGEEMA-co-AzPMA-
PTX)3; DLS curves of micelle aqueous solutions of (C) PEG-b-P(OEGEEMA-co-AzPMA) and (D) PEG-b-P(OEGEEMA-co-AzPMA-
PTX)3, scale bar 5 200 nm.
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diameter of 50ꢁ200 nm are beneficial to maintain a lower
level of reticuloendothelial system uptake and efficient EPR
effect for passive tumor targeting. The size and morphology
of the self-assembled PEG-b-P(OEGEEMA-co-AzPMA-PTX)
conjugate were characterized by TEM and DLS. As it was
shown in the DLS results of Figure 5, both of the diblock
copolymer micelles and PEG-b-P(OEGEEMA-co-AzPMA-PTX)3
conjugate micelles were relatively monodisperse, exhibiting
an intensity-average hydrodynamic diameter of 43 nm and
150 nm, respectively. PEG-b-P(OEGEEMA-co-AzPMA) micelles
are not a single micelle, but micellar aggregates. After load-
ing PTX, the PEG-b-P(OEGEEMA-co-AzPMA-PTX)3 conjugates
aggregated to larger micelles, because of their higher molec-
ular weight and the introduction of hydrophobic PTX, which
increased the hydrophobicity of the polymers. TEM images
revealed that both the diblock copolymer micelles and PTX-
polymer conjugate micelles were fairly monodisperse and
spherical nanoparticles.
FIGURE 6 In vitro PTX release profiles recorded for the aque-
ous solution of diblock copolymer PEG-b-P(OEGEEMA-co-
AzPMA-PTX)3 conjugate at (᭿) pH 7.4 and ( ) pH 5.5. [Color
•
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
As mentioned earlier, the burst release of loaded drug is a
long-standing chanllenge, and it can lead to undesirable side
effects and reduce the therapeutic efficacy.58 Typically, DDSs
drug loading via noncovalent interactions often burst release
60–90% of their payloads within a few hours, because the
release behavior of drug is controlled solely by diffusion.59 In
contrast, drug covalently loaded DDSs appear to be more sta-
ble, as long as the covalent linkages remain intact. As
described above, PTX was covalently conjugated to PEG-b-
P(OEGEEMA-co-AzPMA) by a click reaction. The ester bond of
PTX-copolymer could be slowly hydrolyzed, and then PTX was
released. The in vitro drug release profiles of PEG-b-
P(OEGEEMA-co-AzPMA-PTX)3 conjugate were evaluated under
simulated physiological condition and mildly acidic pH. As
seen from Figure 6, the amount of PTX released from PEG-b-
P(OEGEEMA-co-AzPMA-PTX)3 was 28.8% under simulated
physiological condition within 60 h. In comparison, 50% of
PTX was released from PTX-polymer conjugate within 60 h at
pH 5.5. Signifant acceleration of PTX release was actuated
under mildly acidic condition, because an ester bond is sensi-
tive to acid and the hydrolysis of ester is more faster under
acidic environment. The above results implied that the PTX
release kinetics of PEG-b-P(OEGEEMA-co-AzPMA-PTX)3 conju-
gate was determined not only by diffusion but also by the
hydrolysis of the PTX-copolymer ester linker. The release
absorption was further used to verify the structure of PEG-b-
P(OEGEEMA-co-AzPMA-PTX) as shown in Figure 4. Com-
pared with the spectrum of PEG-b-P(OEGEEMA-co-AzPMA),
each spectrum of PEG-b-P(OEGEEMA-co-AzPMA-PTX)s
showed a characteristic absorption band at 229 nm, confirm-
ing the existence of PTX in the copolymer conjugate. The
intenisity of the absorption peak at 229 nm increased with
the PTX content. Thus, the drug loading content of PEG-b-
P(OEGEEMA-co-AzPMA-PTX)s could be determined by GPC,
1H NMR, and UV-Vis. The results were listed in Table 1, and
the drug loading contents determined by different methods
were consistent. In the following experiment, PEG-b-
P(OEGEEMA-co-AzPMA-PTX)3 was selected because of its
high drug loading level.
The resultant PEG-b-P(OEGEEMA-co-AzPMA-PTX) conjugate
was amphiphilic, and could self-assemble into micelles in
aqueous solution. The hydrophobic inner core of the micelles
was composed of hydrophobic PTX, whereas the outer shell
was composed of hydrophilic PEG, which provided good
hydrophilicity and biocompatibility. The size of the micelles
is an important parameter for drug delivery. Micelles with a
FIGURE 7 Cell cytotoxicity of PEG-b-P(OEGEEMA-co-AzPMA) against SKOV-3, Hela, and HEK-293 cells with different incubation
time of (A) 24 and (B) 48 h. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 8 In vitro cell viability of Hela and SKOV-3 cells against free PTX and PEG-b-P(OEGEEMA-co-AzPMA-PTX)3 at different
concentrations and incubation time. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
behavior of PTX could be controlled by varying the pH, and
with a significantly reduced burst release effect.
cells by cells endocytosis. Once entering into the cells, it was
conceivable that the acidic pH and the enzymes in the endo-
somes would hydrolyze the ester bond and release PTX.60 It
was worth noting that the free drug showed more potent cellu-
lar growth inhibition abilities at high concentration with the
culture time increasing to 48 h. However, the enhancements of
inhibition efficiency of polymeric micellar prodrug were more
obvious than that of free PTX at low concentration. This result
might be related that more free PTX entered the cells at high
concentration with the incubation time increasing to 48 h. The
results discussed above indicated that the PEG-b-P(OEGEEMA-
co-AzPMA-PTX) conjugate possessed excellent biocompatibility,
high drug loading and potent antitumor effect.
It is very important to evaluate the potential toxicity of poly-
meric materials for drug delivery application. The in vitro
cytotoxicity of PEG-b-P(OEGEEMA-co-AzPMA) against Hela,
SKOV-3 (human tumor cells) and HEK-293 (human normal
cells) with different concentrations and incubation times was
studied by the CCK-8 assays. As seen from Figure 7, PEG-b-
P(OEGEEMA-co-AzPMA) showed no obvious cytotoxic effect
on Hela and SKOV-3 cells at a concentration ranged from
0.01 to 5.0 mg mL21. Even at the concentration of PEG-b-
P(OEGEEMA-co-AzPMA) up to 5.0 mg mL21, the cell viability
was above 80% after incubation for 48 h. Furthermore, HEK-
293 cell could also tolerate the treatment with PEG-b-
P(OEGEEMA-co-AzPMA) at 5.0 mg mL21 for 24 h (Fig. 7).
Although a slight decrease in HEK-293 cell viability was
observed when the incubation time increased to 48 h, the
cell viability was still nearly 75%. These results suggested
the cytotoxicity of PEG-b-P(OEGEEMA-co-AzPMA) was pretty
low, and suitable to use as drug carrier in DDSs.
CONCLUSIONS
In summary, azide-functional diblock copolymer PEG-b-
P(OEGEEMA-co-AzPMA) was synthesized and alkyne-functional
PTX was conjugated to this copolymer using click chemistry. The
novel PTX-polymer conjugate could self-assemble into micellar
nanoparticles in aqueous solution, and the loading content of
PTX could be easily tuned by the feeding ratio. The chemical
structures of the diblock copolymer and PTX-polymer conjugate
The antitumor activity of PEG-b-P(OEGEEMA-co-AzPMA-PTX)
conjugate with different drug concentrations and incubation
times was studied in Hela and SKOV-3 cells using the CCK-8
assays. Free PTX was used as a control and the concentration
of drug was from 0.01 to 50 mg mL21. As shown in Figure 8,
after incubation for 24 h, the drug activity of polymeric micel-
lar prodrug was better than that of free PTX in both Hela and
SKOV-3 cells. This might because that PTX is a hydrophobic
drug and tends to precipitate out of cell medium, leading to be
difficult to enter into the cells.32,42 However, the polymeric
prodrug could self-assemble into micellar nanoparticles in
aqueous solution, which were more easily internalized into the
1
were determined by H NMR, GPC, FT-IR, and UV characteriza-
tions. The PEG-b-P(OEGEEMA-co-AzPMA-PTX) drug-polymer
conjugate showed cumulative release of PTX at pH 7.4, and
accelerated drug release at pH 5.5. In vitro CCK-8 assays demon-
strated that PEG-b-P(OEGEEMA-co-AzPMA) exhibited low cyto-
toxicity, and PTX-polymer conjugate could efficiently deliver PTX
into Hela and SKOV-3 cells, leading to excellent antitumor activ-
ity. The strategy of using polymers as drug carriers and PTX con-
jugated to polymers via click chemistry provides an important
alternative for developing new drug delivery systems.
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23 D. Peer, J. M. Karp, S. Hong, O. C. FaroKHzad, R. Margalit,
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The authors thank the financial support from the National Nat-
ural Science Foundation of China (51073042, 81000278,
51103026, 51373035, and 51373040), the Shanghai Natural
Science Funds (11ZR1403100), the Shanghai Scientific and Tech-
nological Innovation Project (11JC1400600 and 10431903000),
the Shanghai Rising Star Program (12QB1402900), the Special-
ized Research Fund for the Doctoral Program of Higher Educa-
tion (20110071120006), School of Pharmacy, Fudan University,
and The Open Project Program of Key Lab of Smart Drug Deliv-
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