Amphiphilic block copolymer micelles with fluorescence as nano-carriers for doxorubicin delivery

Article abstract of DOI:10.1039/c3ra47026a

Well-defined and nontoxic core-shell polymeric micelles, containing fluorescence units, were employed for efficient drug delivery of doxorubicin (DOX). The self-assembled structures were generated from triblock copolymers of poly(ε-caprolactone)-block-poly(glycidyl methacrylate)-block- poly(poly(ethylene glycol)methyl ether methacrylate) (PCL-b-PGMA-b-P(PEGMA)) with fluorescence units. Various experiments like structural characterization, fluorescence properties, cell viability studies, encapsulation studies, measuring cytotoxicity against fibroblasts and bladder cancer cells are performed on these polymeric micelles. All of these results demonstrate that these self-assembled micelles may be promising carriers for intravesical delivery of DOX for bladder cancer therapy.

Full text of DOI:10.1039/c3ra47026a

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PAPER
Amphiphilic block copolymer micelles with
fluorescence as nano-carriers for doxorubicin
Cite this: RSC Adv., 2014, 4, 9684
delivery
a
Jiucun Chenab and Mingzhu Liu
*
Well-defined and nontoxic core–shell polymeric micelles, containing fluorescence units, were employed
for efficient drug delivery of doxorubicin (DOX). The self-assembled structures were generated from
triblock copolymers of poly(3-caprolactone)-block-poly(glycidyl methacrylate)-block-poly(poly(ethylene
glycol)methyl ether methacrylate) (PCL-b-PGMA-b-P(PEGMA)) with fluorescence units. Various
experiments like structural characterization, fluorescence properties, cell viability studies, encapsulation
studies, measuring cytotoxicity against fibroblasts and bladder cancer cells are performed on these
polymeric micelles. All of these results demonstrate that these self-assembled micelles may be
promising carriers for intravesical delivery of DOX for bladder cancer therapy.
Received 26th November 2013
Accepted 21st January 2014
DOI: 10.1039/c3ra47026a
www.rsc.org/advances
techniques, it is still a challenge to nd feasible means to
introduce functional groups onto polymer chains. These func-
1 Introduction
tional polymers, for example, uorescent groups labeled poly-
mers, biofunctionalized polymers and drug containing polymers,
can nd wide applications in chemistry, materials science and
biomedical science elds.17–19 As an efficient coupling reaction,
the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), i.e. “click
reaction”, as termed by Sharpless and coworkers,20 has gained
increasing attention because of its excellent functional group
tolerance, high specicity and nearly quantitative yields under
mild experimental conditions.21 Therefore, click chemistry is
particularly important in chemical synthesis in which high
conversion of functional groups is desired. Combining CRP with
click chemistry is an efficient way to prepare functional polymeric
materials.22–26
Bladder cancer is the second most common genitourinary
malignancy and ranks ninth in worldwide cancer incidence. It is
also the most expensive disease to treat from diagnosis to death
(US data), due to the long-term survival associated with non
muscle-invasive disease combined with life-long surveillance.27
Therefore, it represents an important public health problem.
However, few studies have reported the use of core–shell poly-
meric micelles as drug delivery vehicles for bladder cancer
therapy. In this article, novel and well-dened amphiphilic
poly(3-caprolactone)-block-poly(glycidyl methacrylate)-block-poly-
(poly(ethylene glycol)methyl ether methacrylate) (PCL-b-PGMA-
b-P(PEGMA)) with uorescence units was synthesized by
combination of ring-opening polymerization (ROP), RAFT and
click chemistry. The polymeric micelles were obtained by self-
assembly of the triblock copolymer prepared via RAFT poly-
merization. Incorporation of a block with uorescence unit as a
pendant group between the shell and core-forming blocks
displays different uorescent proles at the interface between
Currently, polymeric micelles as promising nanosized anti-
tumor drug carriers are being extensively studied.1–5 Polymeric
micelles offer many unique advantages, such as passive accu-
mulation in tumors, prolonged circulation time in blood and
the enhanced uptake by tumors.6 In particular, micelles formed
from block copolymers consisting of poly(3-caprolactone) (PCL)
and poly(ethylene glycol)methyl ether methacrylate (PEGMA)
have drawn considerable interest.7–12 The PEGMA with excellent
biocompatibility forms the hydrophilic corona in the micelles,
thereby reducing nonspecic adhesion of micelle surfaces to
blood components. PCL, as the most commonly used hydro-
phobic polyesters, has several particular properties, such as
biocompatibility and biodegradability. Thus, PCL, serving as
hydrophobic core in the micelles, can be used to load water-
insoluble drugs.
The self-assembly of amphiphilic block copolymers usually
requires that copolymers have well-dened structures and
narrow polydispersities. Remarkable advancements in
controlled radical polymerization (CRP) techniques including
reversible addition-fragmentation chain transfer (RAFT)13–15
polymerization now allow the precise construction of sophisti-
cated architectures appropriate for use as delivery vehicles for
diagnostics and therapeutic agents in the rapidly expanding
area of nanomedicine.16 Despite the versatility of CRP
aState Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous
Metal Chemistry and Resources Utilization of Gansu Province and Department of
Chemistry, Lanzhou University, Lanzhou 730000, P. R. China. E-mail: mzliu@lzu.
edu.cn; Fax: +86-931-8912582; Tel: +86-931-8912387
bInstitute for Clean Energy & Advanced Materials, Southwest University, Chongqing,
400715, P. R. China. E-mail: chenjc@swu.edu.cn
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the hydrophobic core and the hydrophilic shell. Moreover, dissolved in 9 mL of toluene, and the mixture was degassed by
loading and in vitro release of DOX, as well as anti-tumor activity purging with nitrogen for 30 min. Polymerization was carried out
of DOX-loaded polymeric micelles were investigated.
at 110 ꢁC for 24 h. The resulting PCL-CTA was precipitated in an
excess of cold ether, ltered, and dried under vacuum for 24 h.
2 Experimental section
2.1 Materials
2.4 Synthesis of PCL-b-PGMA by RAFT
The linear diblock copolymer of PCL-b-PGMA was synthesized
by the polymerization of GMA in the presence of the PCL-CTA
macro-RAFT agent. A 25 mL Schlenk ask with a magnetic stir
bar and a rubber septum was charged with PCL-CTA (3.5 g,
0.20 mmol), GMA (1.29 g, 10.0 mmol), AIBN (0.006 g,
0.04 mmol), and toluene (10 mL). The ask was deoxygenated by
three freeze–pump–thaw cycles, and then sealed followed by
immersing the ask into an oil bath preheated at 60 ꢁC to start
the polymerization. Aer reaction for 6 h, the mixture was then
quickly cooled to room temperature with cool water. The reac-
tion mixture was diluted with a little THF and precipitated in a
large amount of ether. The polymer was obtained by ltration
and dried at room temperature in vacuum to a constant weight.
Poly(ethylene glycol)methyl ether methacrylate (PEGMA, Sigma-
Aldrich, Mn ¼ 475 g molꢀ1, Mw/Mn ¼ 1.03) and glycidyl methac-
rylate (GMA, Sigma-Aldrich) were passed through a basic alumina
column and stored at ꢀ20 ꢁC prior to use. 2,20-Azobisisobutyr-
onitrile (AIBN, Sigma-Aldrich, 97%) was recrystallized with meth-
anol. Tetrahydrofuran (THF), doxorubicin hydrochloride (DOX),
4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol (CDP),
3-caprolactone (CL), 7-hydroxy coumarin, copper(I) bromide
(CuBr), stannous octanoate (Sn(Oct)2), N,N,N0,N00,N00-pentamethyl-
diethylenetriamine (PMDETA) (98%), 1,4-dioxane, Dulbecco's
modied Eagle medium (DMEM), fetal bovine serum (FBS), 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),
dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF)
were purchased from Sigma-Aldrich Chem. Co. All other regents
and solvents were purchased either from Sigma-Aldrich or Merck
Chem. Co. and were used as received. The articial urine used in
the experimental work was based on that devised by Griffith et al.28
Fibroblasts (3T3) and Human bladder cancer cells (UMUC3)
were purchased from American Type Culture Collection.
2.5 Synthesis of triblock copolymers PCL-b-PGMA-b-
P(PEGMA)
The triblock copolymers were synthesized via chain-extending
RAFT polymerization of PEGMA using the previously synthe-
sized PCL-b-PGMA copolymers as macromolecular chain
transfer agents (Scheme 1). PCL-b-PGMA (1.386 g, 0.06 mmol),
AIBN (2 mg, 0.012 mmol) and PEGMA (2.85 g, 6.0 mmol) were
dissolved in 1,4-dioxane (10 mL) in a 25 mL round-bottom ask
under stirring. This ask was capped with rubber septa. The
solution was deoxygenated by purging with nitrogen gas for 30
2.2 Characterization
The nuclear magnetic resonance (1H NMR) spectra were
obtained from a Bruker Avance 400 spectrometer (Bruker Bio-
Spin, Switzerland) (400 MHz). The molecular mass and molec-
ular weight distribution were measured by gel permeation
chromatography (GPC) on a Hitachi L-2130 pump with a Waters
2410 refractive index detector and a Waters 2487 ultraviolet
detector with the combination of Hersteller MZ-Gel SDplus
5 mm, and the THF was used as eluent at a ow rate of
1.0 mL minꢀ1. Fourier transform infrared (FTIR) spectroscopy
patterns were performed on a Nicolet 670 (U.S.A.) IR spec-
trometer. The spectra were collected at 64 scans with a spectral
resolution of 2 cmꢀ1. Transmission electron microscopy (TEM)
was performed using a Tecnai G220 TEM operated at an accel-
erating voltage of 200 kV. The samples were prepared by drop-
ping 10 mL of 0.1 mg mLꢀ1 polymersome dispersion on the
copper grid. The size of polymersomes was determined using
dynamic light scattering (DLS). Measurements were carried out
at 25 ꢁC using Zetasizer Nano ZS from Malvern Instruments
equipped with a 633 nm He–Ne laser using back-scattering
detection. The UV-visible absorption spectra were obtained
from a Shimadzu UV-3600 spectrophotometer. The uorescence
spectra were measured on a Shimadzu RF-5031 spectropho-
tometer at an excitation wavelength of 330 nm.
ꢁ
min. Polymerization was carried out at 80 C for 24 h and the
polymer was precipitated into cold hexane. The polymer was
reprecipitated three times from THF–hexane and dried under a
vacuum at room temperature for 12 h.
2.6 Reaction of PCL-b-PGMA-b-P(PEGMA) with sodium azide
PCL-b-PGMA-b-P(PEGMA) (1.2 g, 0.54 mmol of epoxide groups)
was dissolved in DMF (10 mL). Sodium azide (0.07 g, 1.08 mmol)
and ammonium chloride (0.06 g, 1.1 mmol) were added to this
ꢁ
solution, and the mixture was stirred at 50 C for 24 h. The nal
reaction solution was diluted with DMF and puried by dialysis
against water to remove any traces of salts and unreacted reactants
using a membrane with molecular weight cutoff of 14 000 Da. The
azide-containing polymers were collected by freeze-drying.
2.7 Synthesis of 7-propinyloxy coumarin (PC)
The synthesis procedure was carried out according to ref. 29. A
mixture of 7-hydroxy coumarin (1.62 g, 10 mmol) in acetone
(25 mL), K2CO3 (1.38 g, 10 mmol), KI (0.083 g, 0.5 mmol) and
propargyl bromide (1.2 mL, 15 mmol) was added to a ask, and
ꢁ
the mixture was stirred over night at 80 C. Then the reaction
2.3 Synthesis of the PCL-CTA macro-RAFT agent
mixture was cooled to room temperature and extracted with
The ROP of CL was performed at a feed ratio of [CL]0/[CDP]0/ CH2Cl2 (3 ꢂ 50 mL). The combined organic extracts were
[Sn(Oct)2]0 ¼ 300/2/1 at 110 ꢁC. CL (9.2 g, 80.70 mmol), CDP washed with water (2 ꢂ 50 mL), dried over anhydrous MgSO4
(0.210 g, 0.54 mmol), and Sn(Oct)2 (0.111 g, 0.27 mmol) were and evaporated to afford a crude product, which was puried
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Scheme 1 Synthesis of PCL-b-[PGMA-g-PC]-b-P(PEGMA) conjugates by ROP, RAFT and click chemistry.
by recrystallization from anhydrous ethanol to give a white solid Triethylamine (5 mL) was added and the solution was allowed for
(71% yield).
equilibration 12 h. Then the mixture was added dropwise using a
syringe pump to water (170 mL) under high-speed stirring. The
DOX-containing suspension was then equilibrated under stirring
at 25 ꢁC for 5 h, followed by thorough dialysis (MWCO 3500 Da)
against deionized water for 2 days to remove unloaded DOX. To
determine the drug loading level, a small portion of DOX-loaded
micelles was withdrawn and diluted with DMF to a volume ratio
of DMF–H2O ¼ 9/1. The amount of DOX encapsulated was
quantitatively determined by a UV/vis spectrophotometer. The
emission spectrum was recorded in the range 400–550 nm. The
calibration curve used for drug loading characterization was
established by the intensity of DOX with different concentrations
in DMF–H2O (9/1 (v/v)) solutions. Drug loading content (DLC)
and drug loading efficiency (DLE) were calculated according to
the following formulae:
2.8 Click graing of PC units onto azide-containing
polymeric backbones
The synthetic pathway is shown in Scheme 1. Azide-containing
polymers (1.0 g, 0.017 mmol), PMDETA (83 mL, 0.40 mmol), and
PC (0.16 g, 0.80 mmol) was purged with nitrogen to remove the
dissolved oxygen. CuBr (57 mg, 0.40 mmol) was added under
nitrogen atmosphere. Then the ampoule was ame-sealed and
stirred at 25 ꢁC in the absence of oxygen for 24 h. The reaction
mixture was exposed to air, and diluted by DMF, then passed
through a column of neutral alumina. The resultant polymer
(PCL-b-[PGMA-g-PC]-b-P(PEGMA)) was precipitated into ether
and hexane, respectively, ltered and dried under vacuum.
2.9 Self-assembly of triblock copolymer
DLC (wt%) ¼ (weight of loaded drug/weight of polymer) ꢂ 100%
DLE (%) ¼ (weight of loaded drug/weight of drug in feed) ꢂ 100%
PCL-b-[PGMA-g-PC]-b-P(PEGMA) (100 mg) was dissolved in DMF
(8 mL), which is a good solvent for the hydrophobic and the
hydrophilic block. Then, the solution was added dropwise using
a syringe pump (3 mL hꢀ1) to water (80 mL) under high-speed
stirring at room temperature. The observation of a cloudy
solution indicated the formation of polymeric micelles in the
mixture. Aer 5 h, the mixture was then dialyzed against water
for 48 h using a dialysis membrane (MWCO 3500 Da) to remove
The drug release proles were determined by the dialysis tech-
nique. The DOX-loaded micelles dispersion (15 mL) was placed
within a dialysis tube (MWCO 12 000), followed by dialysis against
articial urine (pH 6.1) at 37 ꢁC. At prescribed time intervals, 5 mL
of the external urine was withdrawn and replaced with an equiva-
lent volume of fresh medium. The concentration of DOX was
determined by the UV/vis using the pertinent calibration curve of
DOX with various concentrations in articial urine. The experi-
mental results presented herein represent an average of at least
triplicate measurements.
DMF. The targeted nal polymer concentration was 1 mg mLꢀ1
.
2.10 DOX encapsulation and release studies
The encapsulation, 200 mg of PCL-b-[PGMA-g-PC]-b-P(PEGMA)
and 20 mg of DOX, were dissolved in 8 mL of DMF separately
and the two solutions were mixed in a vial and stirred for 30 min.
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synthesize the PCL-CTA. Then, PCL-b-PGMA-b-P(PEGMA) was
synthesized by consecutive RAFT polymerization of GMA and
PEGMA using PCL-CTA as a RAFT agent. The ring-opening
reaction was applied to prepare polymers with azide groups
from the GMA copolymers. Subsequently, the obtained azido-
functionalized copolymer was involved in “click” chemistry with
7-propinyloxy coumarin to prepare uorescent polymers.
The FTIR spectrum of the PCL-CTA is shown in Fig. 1a. The
intensive absorption peak at 1735 cmꢀ1 was assigned to the
carbonyl band of PCL. Compared to the IR spectrum of PCL-
CTA, it can be clearly observed the new peak at 904 cmꢀ1 from
the epoxide ring of PGMA. 1H NMR spectrum of the PCL-CTA is
shown in Fig. 2a. The typical signals of the methylene protons of
the initiator CDP could be clearly detected at 1.27 ppm. The
major resonance peaks (a–e) were attributed to PCL. The peak of
the methylene protons was detected at 4.07 ppm when the peak
of the protons of the terminal methylene could be discovered at
3.70 ppm which indicated that PCL was terminated by hydroxyl
groups. The degrees of polymerization (DP) for the PCL could be
calculated about 135 from the integration ratio between the
methylene protons (2.32 ppm) in the repeat units and those
2.11 Cell culture
Fibroblasts (3T3) and Human bladder cancer cells (UMUC3) were
maintained in DMEM with containing 10% FBS with penicillin
(100 U mLꢀ1) and streptomycin (100 mg mLꢀ1). The cells were
grown in a 5% CO2 atmosphere in an incubator at 37 ꢁC.
2.12 Cytotoxicity assay of the polymeric micelles
The relative cytotoxicity of polymeric micelles in 3T3 cell lines
were quantitatively determined using MTT assay. All cell lines
were seeded into 96 well plates at 1 ꢂ 104 cells per well and
ꢁ
maintained in culture for 24 h at 37 C in the medium. Aer
24 h medium was removed, cells were washed with PBS and
medium containing different concentration of polymeric
micelles were added to the designated wells. The whole exper-
imental plate was incubated for 24 h or 72 h. A fresh 10 mL
sample of MTT from 5 mg mLꢀ1 stock solution was added to
each well, followed by incubation for 4 h at 37 ꢁC. Aer 4 h,
medium from the wells were removed and 100 mL of DMSO were
added to each well and incubated for 15 min, and the absor-
bance of the resulting solution was measured at 590 nm. The
cell survivals were determined by comparison of optical density
with untreated respective control cell cultures.
UMUC3 cells were seeded in a 96-well plate at a density of 5000
cells per well in DMEM (100 mL) containing 10% FBS and 1%
penicillin and incubated at 37 ꢁC for 24 h. The medium was then
replaced with 100 mL of fresh medium containing either polymeric
micelles or DOX-loaded micelles at varying micelles concentrations,
and cells were incubated for 24 h or 72 h. Thereaer, 10 mL of MTT
(5.0 mg mLꢀ1) was added into each well, followed by incubation at
37 ꢁC for 4 h. Aer 4 h, medium from the wells were removed and
100 mL of DMSO were added to each well and incubated for 15 min,
and the absorbance of the resulting solution was measured at
590 nm. The cell survivals were determined by comparison of
optical density with untreated respective control cell cultures.
1
(3.70 ppm) in the terminal unit based on H NMR spectrum.
Fig. 2B exhibits a typical 1H NMR spectrum of copolymer
PCL-b-PGMA. It was observed that besides the dominant PCL
signals, the new peaks i–k in the region of 2.6–4.4 ppm were
attributable to the epoxy protons of the glycidyl groups of
PGMA. The chemical shis at 0.9–1.4 ppm (h) were associated
with the methyl protons of the PGMA block. These results
certicated that new PGMA segments were connected to PCL.
The DP of PGMA blocks were obtained based on 1H NMR
spectrum. GPC analysis and Mn,NMR values based on the 1H
NMR spectrum were shown in Table 1.
According to GPC results, the apparent molecular weight
(Mn) and the polydispersity (Mw/Mn) of PCL-CTA are 17 500 and
1.26, respectively (Fig. 3). PCL-b-PGMA GPC trace shows that the
elution peak shis to higher molecular weight aer the RAFT
polymerization of GMA. The diblock copolymer elution peak is
relatively symmetric and no discernible tailing at the lower
2.13 Intracellular release of DOX
The cellular uptake and intracellular release behaviors of DOX-
loaded micelles were followed using UMUC3 cells. The cells
were seeded on microscope slides in a 24-well plate (5 ꢂ 104
cells per well) using DMEM supplemented with 10% FBS for
24 h. 1 mL of either polymeric micelles or DOX-loaded micelles
were added. The cells were incubated with either polymeric
micelles or DOX-loaded micelles for 4 h at 37 ꢁC in a humidied
5% CO2-containing atmosphere. The culture media were
removed and the cells were rinsed two times with PBS. The cells
were xed with 4% paraformaldehyde for 30 min. The solution
was removed and the cells were rinsed three times with PBS.
The images of cells were obtained using confocal laser scanning
microscopy (CLSM) (Zeiss LSM 510 Meta, Germany).
3 Results and discussion
3.1 Synthesis and characterization of PCL-b-[PGMA-g-PC]-b-
P(PEGMA)
Fig. 1 FTIR spectra of (a) PCL-CTA, (b) PCL-b-PGMA, (c) PCL-b-
PGMA-b-P(PEGMA), (d) PCL-b-[PGMA-N3]-b-P(PEGMA), and (e) PCL-
The overall experiment is illustrated in Scheme 1. In this study,
the hydroxyethyl-terminated CDP was used as initiator to b-[PGMA-g-PC]-b-P(PEGMA) in CDCl3.
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Fig. 2 1H NMR spectra of (a) PCL-CTA and (b) PCL-b-PGMA.
Table 1 Characterization data of copolymers synthesized by RAFT polymerization
Conv.a (%)
Mn,th (kDa)
DPNMR
Mn,NMR (kDa)
Mn,GPCd (kDa)
PDId
b
c
c
Polymer
PCL-CTA
PCL-b-PGMA
PCL-b-PGMA-b-P(PEGMA)
PCL-b-[PGMA-N3]-b-P(PEGMA)
PCL-b-[PGMA-g-PC]-b-P(PEGMA)
92
46
61
—
—
16.1
20.8
52.1
—
135 (PCL)
24 (PGMA)
72 (P(PEGMA))
—
15.8
19.2
53.4
—
17.5
23.1
58.4
59.4
64.1
1.26
1.29
1.35
1.35
1.36
—
—
—
a
b
c
Conversion of monomer obtained from gravimetry. Theoretical molecular weight (Mn,th) calculated by monomer conversion. DP of block
copolymers and Mn,NMR were calculated on the basis of 1H NMR results. Number-average molecular weight (Mn,GPC) and molecular weight
d
distribution (PDI ¼ Mw/Mn) estimated by GPC.
molecular weight side is found, conrming
consumption of macro-RAFT agent.
a
complete containing epoxide groups with sodium azide at relatively
moderate reaction conditions is one of the most convenient routes
By employing macro-CTA PCL-b-PGMA, PEGMA was poly- for preparation of polymers with azides group. Aer that, the
merized to yield PCL-b-PGMA-b-P(PEGMA) (Scheme 1). 1H NMR epoxide groups of PCL-b-PGMA can be opened with sodium azide.
analysis indicates the characteristic peaks of P(PEGMA) at 4.1, As shown in Fig. 5A, proton signals from epoxide ring disappeared
3.9–3.5, 3.4, 1.9, and 1.3–0.9 ppm attributed to CH2O ester, completely. Instead, newly formed resonances at 3.27, 3.35 ppm
CH2O ether, CH3O, CH3–C, and CH2–C backbone, respectively (t, –CH2N3) and enhanced resonance at 3.73–4.10 ppm demon-
(Fig. 4A). The DP for the P(PEGMA) could be calculated about 72 strated the successful transformation of epoxide to azide and
from 1H NMR. As shown in Fig. 3, GPC curves of triblock hydroxyl groups. On the other hand, from the IR spectra shown in
copolymer shis to higher molecular weight direction Fig. 1d, copolymer containing azide groups exhibited a typical
compared with precursor polymer with slightly increased poly- absorbance at 2103 cmꢀ1, which is the characteristic absorbance
dispersity. Synthesis of 7-propinyloxy coumarin (PC) was carried of azide groups. By GPC analysis, the synthesis of the triblock
out similar to the procedure described in previous publica- copolymer containing azide groups precursors involved no
tions.29 Resonance at 4.8 ppm was the characteristic signal of molecular weight reduction, based on the observation of narrow
the methylene protons of propargyl group, while resonance of symmetrical signals present at the same position as the starting
the alkynyl proton was observed at 2.6 ppm (Fig. 4B).
PCL-b-PGMA-b-P(PEGMA) precursors (Fig. 3).
The synthesis of polymer containing azide groups for further
The obtained azido-functionalized copolymer was subse-
modication is still challenging. Reaction of polymers quently involved in “click” chemistry with 7-propinyloxy
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Fig. 3 GPC traces of (a) PCL-CTA, (b) PCL-b-PGMA, (c) PCL-b-PGMA-
b-P(PEGMA), (d) PCL-b-[PGMA-N3]-b-P(PEGMA), and (e) PCL-b-
[PGMA-g-PC]-b-P(PEGMA).
Fig. 5 1H NMR spectra of (a) PCL-b-[PGMA-N3]-b-P(PEGMA) and (b)
PCL-b-[PGMA-g-PC]-b-P(PEGMA).
azide group almost disappeared. Further characterization with
GPC (Fig. 3) conrmed a small increase in molecular weight aer
coupling due to addition of the 200 g molꢀ1 PC.
3.2 Self-assembly and uorescence properties of the triblock
copolymers
The main objective of this work was to demonstrate the stability
and drug reservoir capabilities of the novel amphiphilic
micelles in physiological conditions and also to study their
performance as a drug delivery carrier. It is well known that PCL
homopolymer is hydrophobic, whereas P(PEGMA) is water-
soluble at room temperature. Thus, PCL-b-[PGMA-g-PC]-b-
P(PEGMA) micelles with core of PCL block and shell of
P(PEGMA) were formed by adding polymer solution in DMF into
Fig. 4 1H NMR spectra of (a) PCL-b-PGMA-b-P(PEGMA) and (b) PC.
ꢁ
10 folds of water at 25 C (Scheme 2). To conrm the micelles
nature of PCL-b-[PGMA-g-PC]-b-P(PEGMA), the following char-
coumarin to prepare uorescent polymers. The copper(I) and its acterization were carefully done. Light scattering measure-
ligands were reported to be very efficient to catalyze the ments conrmed the presence of micelles in solution and
1,3-dipolar cycloaddition of organic azides with terminal provided an average diameter of 91 nm (Fig. 6), which implies
alkynes. Herein, CuBr/PMDETA was used as the catalytic micelles were excellent carrier for hydrophobic drug. The
system, and DMF as solvent for the 1,3-dipolar cycloaddition of micelles of the amphiphilic triblock copolymers were studied by
azido-functionalized copolymer and 7-propinyloxy coumarin. transmission electron microscopy (TEM). From Fig. 7, we can
Fig.
5
shows the 1H NMR spectrum recorded for nd that the micelles from the self-assembled PCL-b-[PGMA-g-
PCL-b-[PGMA-g-PC]-b-P(PEGMA). Compared with Fig. 5A, PC]-b-P(PEGMA) in water exit excellent disperse, and the
besides the signals were assigned to the PCL and P(PEGMA), diameter of the spherical micelles vary from about 20 to 40 nm.
neonatal signals at 5.17 ppm due to the methylene proton
Fig. 8a shows the UV-vis spectra of the PCL-b-[PGMA-g-PC]-b-
neighboring the 1,2,3-triazole group, and signals at 7.80 ppm P(PEGMA), which was obtained by coupling of azide-containing
were assigned to the proton of the 1,2,3-triazole ring. The polymers with 7-propinyloxy coumarin, in water at room
characteristic signals at around 6.70–7.10 ppm, 7.40–7.50 ppm, temperature. The solution of polymeric micelles showed a
7.60–7.85 ppm and 6.25–6.30 ppm were assigned to the protons characteristic UV-vis absorption peak of coumarin moiety at
of the coumarin group. Moreover, the success of the “click” 320 nm. The uorescence spectra of PCL-b-[PGMA-g-PC]-b-
reaction can also be conrmed from FTIR spectroscopy. In Fig. 1e, P(PEGMA) in aqueous solution are displayed in Fig. 8b. It can be
the IR spectra showed that the signal at 2103 cmꢀ1 assigned to the observed that the triblock copolymer, obtained by coupling of
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Fig. 7 TEM images of PCL-b-[PGMA-g-PC]-b-P(PEGMA) micelles.
ꢁ
simulated physiological condition at 37 C. In articial urine,
the DOX release behaviors of the micellar carriers had the
typical two-phase release prole (Fig. 9). In the rst stage, about
60% DOX encapsulated in micelles released within 24 h, fol-
lowed by a sustained and slow release over a prolonged time.
Drug release was enhanced slightly in 72 h, wherein approxi-
mately 82% drug was released from the micelles.
Scheme 2 Illustration of polymer micelles based on PCL-b-[PGMA-g-
PC]-b-P(PEGMA) triblock copolymers for active loading as well as
release of DOX.
3.4 Anti-tumor activity and intracellular drug release of
DOX-loaded PCL-b-[PGMA-g-PC]-b-P(PEGMA) micelles
The cytotoxicity of PCL-b-[PGMA-g-PC]-b-P(PEGMA) was
assessed by determining the viability of 3T3 broblasts aer
incubation in a medium containing various concentrations of
pristine micelles for 24 and 72 h (Fig. 10). The MTT assay has
been described as a very suitable method for the detection of
biomaterial toxicity.30 Fig. 10 demonstrates that the drug-free
micelles are virtually nontoxic to 3T3 cells. The in vitro cyto-
toxicity of DOX-loaded micelles against UMUC3 cells in
comparison with pristine micelles determined by MTT assay is
shown in Fig. 11. Cells without any DOX treatment were used as
a negative control. Compared to the control, the viability of
UMUC3 cells was reduced to about 10% following 24 h incu-
bation with 2.0 mg mLꢀ1 DOX-loaded micelles. The high anti-
tumor activity of DOX-loaded micelles indicates that DOX has
been efficiently delivered and released into the nuclei of
UMUC3 cells. While under otherwise the same conditions,
pristine micelles were practically non-toxic (cell viability > 90%)
up to a tested concentration of 2.0 mg mLꢀ1 (Fig. 11), suggest-
ing that pristine micelles possess good biocompatibility.
Fig. 6 Plots of hydrodynamic diameter (Dh) of micelles and DOX-
loaded micelles.
azide-containing polymers with 7-propinyloxy coumarin,
exhibited a strong uorescence peak at about 390 nm.
3.3 Loading and in vitro release of DOX
Generally, DOX, one of the most potent anticancer drugs, is
It is well known that uorescent units with amphiphilic
extensively used to treat different types of solid malignant structures could self-assemble into vesicle/micelle-like aggre-
tumors through interacting with DNA by interaction and inhi- gates, which are resulted from the intermolecular interaction
bition of macromolecular biosynthesis. To investigate the within hydrophobic and hydrophilic moieties, respectively.
potential of multiblock copolymer micelles based on self- Since copolymers could be designed to have different functions
assembly, DOX was selected to evaluate the drug loading and in the block sections, amphiphilic polymers are generally
release properties. Loading of DOX into micelles was performed inclined to self-assemble into uniform structures. Therefore,
at a theoretical drug loading content (DLC) of 7.2 wt% and a uorescent amphiphilic structures are usually designed as
high DOX loading efficiency of 72%. Aer drug loading, the size gra31 or block32,33 copolymers. For instance, Chang et al.34
of micelles shows a slightly increased about 95 nm (Fig. 6). In synthesized a Rhodamine B (RhB)-anchored amphiphilic poly-
vitro release proles of the loaded DOX in micelles of PCL-b- (poly(ethylene glycol) methacrylate)-b-poly(glycidyl methacry-
[PGMA-g-PC]-b-P(PEGMA) copolymers were studied under a late) block copolymer (PPEGMA-b-PGMA/RhB), in which the
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Fig. 8 (a) UV-vis spectra and (b) fluorescence spectra of PCL-b-[PGMA-g-PC]-b-P(PEGMA) micelles solution.
Fig. 11 Cytotoxicity of different concentration of. PCL-b-[PGMA-g-
PC]-b-P(PEGMA) polymer micelles to UMUC3 after 24 h or 72 h.
Fig. 9 Plots of release behavior of DOX-loaded micelles.
the uorescent polymeric micelles at the concentration of
0.5 mg mLꢀ1 for 4 h. The uorescence images of UMUC3 cells are
shown in Fig. 12a. The loaded cells displayed the bright blue
uorescence corresponding to the coumarin dye, implying that
the uorescent polymeric micelles have been effectively inter-
nalized and accumulated in the cytoplasm. Furthermore, the
images of UMUC3 cells treated with doxorubicin-based anti-
cancer drug loaded PCL-b-[PGMA-g-PC]-b-P(PEGMA) micelles
further conrmed their cellular internalization (Fig. 12b), which
further exhibited their promising application for bladder cancer
treatment. These results demonstrated that the uorescent
polymeric micelles have the widespread application not only in
advanced bioimaging, but also in intracellular drug delivery.
Fig. 10 Cytotoxicity of PCL-b-[PGMA-g-PC]-b-P(PEGMA) polymer
micelles after 24 h or 72 h.
inter-molecular interaction of RhB resulted in the assembled
uorescent nanoparticles. The nanoassembly PPEGMA-b-PGMA/
RhB was then introduced into HeLa cells and the HeLa cells
exhibited good uorescence images. Li et al.31 also reported the
similar results by using comb-like gra copolymer poly((N-vinyl-
carbazole)-co-(4-vinylbenzyl chloride))-comb-poly(((2-dimethyla-
mino)ethylmethacrylate)-co-(acrylic acid)) to produce self-
assembled hollow vesicles with multi-walls, which even exhibited
good uorescence intensity in aqueous media. In this work, cell
imaging based on uorescent polymeric micelles was subse-
Fig. 12 CLSM images of UMUC3 cells incubated with (a) polymeric
quently investigated by CLSM. UMUC3 cells were incubated with micelles and (b) DOX-loaded micelles (0.5 mg mLꢀ1) for 4 h.
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4 Conclusions
Amphiphilic PCL-b-[PGMA-g-PC]-b-P(PEGMA) with uorescence
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Micelles obtained from the self-assembly of amphiphilic
copolymers have been used as a nano reservoir for controlled
release of DOX, a model anticancer drug. These novel micelles
are proved to be capable of loading high drug quantities with a
reasonable loading efficiency (about 70%). The release prole of
DOX from micelles in articial urine shows a slow release in 3
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Acknowledgements
The authors gratefully acknowledge the nancial support of the
National Natural Science Foundation of China (grant no.
51273086), the Fund for Fostering Talents from National
Natural Science Foundation of China (grant no. J1103307) and
Special Doctorial Program Fund from the Ministry of Education
of China (grant no. 20090211110004).
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