In situ fabrication of paclitaxel-loaded core-crosslinked micelles via thiol-ene "click" chemistry for reduction-responsive drug release

Article abstract of DOI:10.1002/pola.27778

In this study, a facile method to fabricate reduction-responsive core-crosslinked micelles via in situ thiol-ene "click" reaction was reported. A series of biodegradable poly(ether-ester)s with multiple pendent mercapto groups were first synthesized by melt polycondensation of diol poly(ethylene glycol), 1,4-butanediol, and mercaptosuccinic acid using scandium trifluoromethanesulfonate [Sc(OTf)₃] as the catalyst. Then paclitaxel (PTX)-loaded core-crosslinked (CCL) micelles were successfully prepared by in situ crosslinking hydrophobic polyester blocks in aqueous media via thiol-ene "click" chemistry using 2,2′-dithiodiethanol diacrylate as the crosslinker. These PTX-loaded CCL micelles with disulfide bonds exhibited reduction-responsive behaviors in the presence of dithiothreitol (DTT). The drug release profile of the PTX-loaded CCL micelles revealed that only a small amount of loaded PTX was released slowly in phosphate buffer solution (PBS) without DTT, while quick release was observed in the presence of 10.0 mM DTT. Cell count kit (CCK-8) assays revealed that the reduction-sensitive PTX-loaded CCL micelles showed high antitumor activity toward HeLa cells, which was significantly higher than that of reduction-insensitive counterparts and free PTX. This kind of biodegradable and biocompatible CCL micelles could serve as a bioreducible nanocarrier for the controlled antitumor drug release.

Full text of DOI:10.1002/pola.27778

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In Situ Fabrication of Paclitaxel-Loaded Core-Crosslinked Micelles via
Thiol-Ene “Click” Chemistry for Reduction-Responsive Drug Release
Ying Huang,1 Rui Sun,2 Qiaojie Luo,3 Ying Wang,2 Kai Zhang,3,4 Xuliang Deng,1 Weipu Zhu,2
Xiaodong Li,3 Zhiquan Shen2
1Department of Geriatric Dentistry, School and Hospital of Stomatology, Peking University, Beijing 100081,
People’s Republic of China
2Department of Polymer Science and Engineering, MOE Key Laboratory of Macromolecular Synthesis and Functionalization,
Zhejiang University, Hangzhou 310027, People’s Republic of China
3Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital, College of Medicine, Zhejiang University,
Hangzhou, 310006, People’s Republic of China
4Zhoushan Stomatology Hospital, Zhoushan 316000, People’s Republic of China
Correspondence to: W. P. Zhu (E-mail: zhuwp@zju.edu.cn) or X. D. Li (E-mail: cisarli@zju.edu.cn)
Received 29 April 2015; accepted 20 July 2015; published online 11 August 2015
DOI: 10.1002/pola.27778
ABSTRACT: In this study, a facile method to fabricate reduction-
responsive core-crosslinked micelles via in situ thiol-ene
“click” reaction was reported. A series of biodegradable poly
(ether-ester)s with multiple pendent mercapto groups were first
synthesized by melt polycondensation of diol poly(ethylene
glycol), 1,4-butanediol, and mercaptosuccinic acid using
scandium trifluoromethanesulfonate [Sc(OTf)3] as the catalyst.
Then paclitaxel (PTX)-loaded core-crosslinked (CCL) micelles
were successfully prepared by in situ crosslinking hydrophobic
polyester blocks in aqueous media via thiol-ene “click” chemis-
try using 2,20-dithiodiethanol diacrylate as the crosslinker.
These PTX-loaded CCL micelles with disulfide bonds exhibited
reduction-responsive behaviors in the presence of dithiothreitol
(DTT). The drug release profile of the PTX-loaded CCL micelles
revealed that only a small amount of loaded PTX was released
slowly in phosphate buffer solution (PBS) without DTT, while
quick release was observed in the presence of 10.0 mM DTT.
Cell count kit (CCK-8) assays revealed that the reduction-
sensitive PTX-loaded CCL micelles showed high antitumor
activity toward HeLa cells, which was significantly higher than
that of reduction-insensitive counterparts and free PTX. This
kind of biodegradable and biocompatible CCL micelles could
serve as a bioreducible nanocarrier for the controlled antitumor
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drug release. 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part
A: Polym. Chem. 2016, 54, 99–107
KEYWORDS: antitumor; biodegradability; drug delivery; polycon-
densation; reduction response; thiol-ene “click” chemistry
Stimuli-responsive micelles can release the loaded drugs at
target sites in response to some specific stimuli, such as pH,
temperature, and reductive environment.11–14 Disulfide
bonds are reductively degradable linkages, known to be
destroyed rapidly due to the intracellular reductive environ-
ment.15–17 Hence, the reduction-sensitive disulfide bonds
have been applied to nanoparticles for controlled release of
loaded drugs.18–23
INTRODUCTION In the past few decades, great interests have
been attracted by micelles self-assembled from amphiphilic
block copolymers as nanosized drug carriers.1–5 However,
under extreme dilution conditions, such micelles will disas-
sociate and release the encapsulated drugs at undesired
sites. Therefore, one crucial and important question is how
to stabilize the polymeric micelles. Nowadays, crosslinking
the core or the shell of polymeric micelles after assembly
has emerged as a viable strategy.6–9 By providing covalent
linkages between the self-assembled polymer chains, the sta-
bility of polymeric micelles has been greatly improved.10
As a FDA approved water soluble polymer, PEG has been
widely investigated in both academia and industry for chemi-
cal and biological applications.24–30 The PEG-based drug car-
riers have a few advantages: a prolonged residence in body,
a decreased degradation by metabolic enzymes and a reduc-
tion or elimination of protein immunogenicity, etc.31 Hence,
For further applications in the clinic, overly stable micelles
are not ideal for drug release at desired sites. Many kinds of
stimuli-responsive micelles have been developed recently.
Ying Huang and Rui Sun contributed equally to this work.
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2016 Wiley Periodicals, Inc.
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SCHEME 1 In situ fabrication of reduction-responsive CCL micelles.
micelles self-assembled from PEG-based aliphatic amphi-
philic multifunctional poly(ether-ester)s are attractive candi-
dates for smart drug carriers due to their excellent
biodegradability, biocompatibility, and crosslinkability.32–34
PEG-based multifunctional poly(ether-ester)s are usually syn-
thesized by the ring-opening polymerization of cyclic esters
with functional groups in the presence of PEG as a macroini-
tiator, which always involves burdensome protections and
deprotections.35–37
paclitaxel (PTX)-loaded PEG–PBMS micelles in aqueous
media via Michael addition type thiol-ene “click” reaction
under physiological conditions (Scheme 1). These PTX-
loaded CCL micelles with disulfide bonds exhibited
reduction-responsive behaviors in the presence of dithiothre-
itol (DTT). This study provides a universal strategy to pre-
pare drug-loaded CCL micelles, which is suitable for almost
all hydrophobic drugs.
EXPERIMENTAL
Our research group has developed a “one-pot” strategy to
synthesize multifunctional biodegradable PEG derivatives by
direct polycondensation of PEG diol and functional diacid
employing scandium trifluoromethanesulfonate [Sc(OTf)3] as
Materials
Poly(ethylene glycol) (Mn 5 1000, Sinopharm Chemical Rea-
gent Co., China) was dried by azeotropic distillation in the
presence of dry toluene. Scandium trifluoromethanesulfonate
[Sc(OTf)3] was synthesized according to our previous
work.50 1,4-dithiothreitol (DTT, 99%; Aladdin), mercaptosuc-
cinic acid (98%; Aladdin, China), paclitaxel (PTX, 99.5%;
Haoxuan Bio-technique Co., China), 1,4-butanediol (BD,
99.5%; Wulian Chemical Engineering Co., China), 2,20-dithio-
diethanol (90%; Alfa Aesar), acryloyl chloride (98%; Energy
Chemical, China), and nile red (99%; Acros Organics) were
used as received. All other reagents were purchased from
Sinopharm Chemical Reagent Co. and used as received.
a
chemoselective catalyst under mild conditions.38–41
Recently, we have published a communication, in which 1,4-
butanediol was used as hydrophobic monomer to copolymer-
ize with PEG diol and mercaptosuccinic acid, resulting in
amphiphilic poly(ethylene glycol)–poly(butylene mercapto-
succinate) (PEG–PBMS) multiblock copolymer containing
multiple mercapto groups.42 This amphiphilic polymer could
self-assemble into sphere micelles in aqueous solution for
encapsulation of hydrophobic antitumor drugs. The hydro-
phobic PBMS block with multiple mercapto groups in the
micellar core could be crosslinked in the presence of H2O2,
to give disulfide bonds, which led to reduction-responsive
drug release from the micelles. However, this strategy is not
suitable for loading oxidation-sensitive drugs into the
micelles due to the violent oxidability of H2O2. Therefore, it
is meaningful to explore new crosslinking agents in order to
introduce disulfide crosslinkages into the micelles. The rise
of thiol-ene “click” chemistry as a toolbox gathering only
simple, high yielding, easily workable transformations, and
mild reaction conditions has facilitated an extraordinary
increase in the fabrication of biomaterials.42–49 In this work,
2,20-dithiodiethanol diacrylate was used to in situ crosslink
Synthesis of Multiblock Copolymers
A series of multiblock copolymers with different PEG and BD
molar ratios were synthesized by an improved method
according to our previous report.43 In a 100 mL Schlenk
flask equipped with mechanical stirrer, a typical feeding
molar ratio of monomers was as follows: PEG diol (6.0 g, 6.0
mmol), BD (2.72 g, 30.0 mmol), mercaptosuccinic acid (5.41
g, 36.0 mmol), and Sc(OTf)3 (0.36 g, 0.72 mmol) were added.
The flask was purged with argon and then put into an oil
bath thermostated at 80 8C. Vigorous stirring was carried
out to ensure the uniformity after PEG was melted. After
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esterification for 10 h at 80 8C, polycondensation at 90 8C
was carried out under lower pressure for 4 h, and then the
mixture continuous reacted at high vacuum (< 100 Pa) for
another 6 h. Thereafter, the reaction was stopped and the
products were dissolved in a small amount of THF and then
precipitated in cold diethyl ether. Yield: 8.5 g (66%).
linker (2,20-dithiodiethanol diacrylate or 1,6-hexamethylene
diacrylate, the molar ratio of mercapto groups and double
bonds is 1:1) were dissolved in 5 mL of THF, and 5 mL of
phosphate buffer solution (PBS, pH 5 7.4) was added drop-
wise into the solution under vigorous stirring to induce NR
and crosslinker incorporated into the hydrophobic micellar
core. After stirring for 2 h, the solution was evaporated to
remove most of the THF. Thereafter, the solution was stirred
for another 2 h to make the “click” reaction completely. The
solution was dialyzed against distilled water for 24 h using a
tubular dialysis membrane (MWCO 5 3500 Da) and filtrated
to remove residual THF, DMF (all of the added NR was
loaded into the CCL micelles).
Synthesis of 2,20-Dithiodiethanol Diacrylate and 1,6-
Hexamethylene diacrylate
The 2,20-dithiodiethanol (3 g, 17.5 mmol) was dissolved in
30 mL of THF in a 100 mL Schlenk flask. Then excess trie-
thylamine (9.76 mL, 70 mmol) was added to the flask, and
the mixture was stirred for 20 min at 0 8C. Thereafter, acryl-
oyl chloride (2 equal, 5.72 mL, 70 mmol) was introduced by
dripping slowly and the reaction mixture was stirred at
room temperature for 12 h. THF was evaporated and then
30mL of dichloromethane was added. The solution was
poured into a separatory funnel and washed with saturated
NaHCO3 and NaCl solutions for three times separately. The
organic layer was dried by anhydrous MgSO4, evaporated to
give yellow liquids, which was dried under reduced pressure
for 12 h. Yield: 2.6 g (59%). 1,6-hexamethylene diacrylate
was synthesised by the same method. Yield: 1.8 g (53%).
Determination of Crosslinking Degrees of the CCL
Micelles
The crosslinking degree of CCL micelles was determined
using Ellman’s reagent as described in literature.51 Briefly, 1
mg/mL solution of polymer or CCL micelles was prepared in
PBS (pH 7.4). Then Ellman’s reagent (0.5 mL, 10 mM) was
added into the solutions and the reactions were allowed to
proceed for 2 h at room temperature. Afterward, the absorb-
ance was measured at a wavelength of 412 nm. The amount
of thiols was calculated from the corresponding standard
curve elaborated between 0.25 and 2 mM of L-cysteine solu-
tion in PBS (pH 7.4). The crosslinking degrees were calcu-
lated at last.
Preparation of PTX- or NR-Loaded CCL Micelles
The PTX-loaded CCL micelles were in situ prepared by the
solvent evaporation method. CCL micelles with disulfide
bond (crosslinked by 2,20-dithiodiethanol diacrylate) was
named as CCL micelles-SS, CCL micelles crosslinked by 1,6-
hexamethylene diacrylate was named as CCL micelles-CC.
Briefly, 5 mg of PTX, 50 mg of PEG–PBMS, and crosslinker
(2,20-dithiodiethanol diacrylate or 1,6-hexamethylene diacry-
late, the molar ratio of mercapto groups and double bonds is
1:1) were dissolved in 30 mL of THF, and 30 mL of phos-
phate buffer solution (PBS, pH 5 7.4) was added dropwise
into the solution under vigorous stirring to induce the
hydrophobic PTX and crosslinker incorporated into the
hydrophobic micellar core. After stirring for 2 h, the solution
was evaporated to remove most of the THF. Thereafter, the
solution was stirred for another 2 h to make the “click” reac-
tion complete. The solution was dialyzed against distilled
water for 24 h using a tubular dialysis membrane (MWCO 5
3500 Da) and filtrated to remove residual THF and unloaded
PTX. The final concentration of PTX-loaded CCL micelles
aqueous solution was adjusted to 1.0 mg/mL. The solid state
of PTX-loaded CCL micelles was recovered by lyophilization.
The drug-loading capacity of the CCL micelles was investi-
gated by RP–HPLC analysis. The drug loading content (DLC)
and the drug loading efficiency (DLE) were calculated as
below:
In Vitro Drug Release
In vitro release profiles of PTX from the CCL micelles were
investigated by dialysis method. Concretely, 4 mL of PTX-
loaded CCL micelles solution was introduced into a dialysis
bag (MWCO 5 14 kDa) against 20 mL PBS buffer solution
(pH 7.4, 10 mM) containing 10 mM DTT (mimicking intracel-
lular reductive condition). Another group was conducted
without DTT (nonreductive condition). Tween 80 (0.1 wt %)
was introduced into the PBS buffer solution to solubilize
released PTX. At desired time intervals, 5 mL of release
mediums were taken out and replenished with an equal vol-
ume of corresponding fresh media. The amounts of released
PTX were determined by RP–HPLC analysis.
Cell Uptake and Internalization Analysis
NR-loaded CCL micelles-SS and NR-loaded CCL micelles-CC
were prepared separately. The internalization and intracellu-
lar distribution of NR-loaded CCL micelles in HeLa cells were
monitored using confocal laser scanning microscopy
(BX61W1-FV1000; OLYMPUS, Japan). Autoclave sterilized
coverslips were placed in 6-well plate. The HeLa cells were
seeded in 6-well plates with a concentration of 1 3 105 cells
per well in 1.8 mL of complete a-MEM and cultured for 24
h. Thereafter, 200 lL NR-loaded CCL micelles-SS, NR-loaded
CCL micelles-CC, or free NR suspension were added and cul-
tured for another 1 h and 4 h. Then the culture media was
removed, followed by washing the cells thrice with PBS and
fixed with4 % paraformaldehyde for 15 min. Cell nucleus
were stained by Hochest 33324 (Sigma-Aldrich, blue fluores-
cence). After being mounted with neutral balsam, samples
DLC ð%Þ 5 ðweight of loaded drug = weight of polymerÞ3 100
DLE ð%Þ 5 ðweight of loaded drug = weight of drug in feedÞ3 100
NR was loaded into the micelles using the same strategy in
the dark. NR-loaded CCL micelles-SS and NR-loaded CCL
micelles-CC were prepared separately. Briefly, 0.2 mL of NR’s
DMF solution (1.0 mg/mL), 10 mg of PEG–PBMS, and cross-
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were observed with 603 confocal laser scanning microscopy
(CLSM).
Microplate reader was used for quantitative determination of
cellular uptake of NR-loaded CCL micelles. 180 lL suspen-
sion of the HeLa cells were seeded into each well of 96-well
plate (5 3 103 cells) and incubated for 24 h. Then 20 lL
PBS containing NR-loaded CCL micelles-SS, NR-loaded CCL
micelles-CC, or free NR solutions were added. After incu-
bated for 1 h and 4 h, cells were washed with PBS twice,
and finally 100 lL PBS was added. Cells incubated with
equivalent amount PBS was used as negative control. Fluo-
rescence intensity was measured with a microplate reader
(SpectraMax, molecular devices, AmericaX) with excitation
wavelength 550 nm and emission wavelength of 605 nm.
Each group was tested in quintuplicate.
FIGURE 1 1H NMR spectra (400MHz, CDCl3) of PEG–PBMS mul-
tiblock copolymers. The feed molar ratio of PEG and BD is 1:3
(A), 1:4 (B), and 1:5 (C).
Cell Viability Assay
The in vitro relative cytotoxicities of PEG–PBMS against HeLa
cells were evaluated by the Cell Counting Kit-8 (CCK-8) assay
(Dojindo Laboratories, Japan). Appropriate amounts of PEG–
PBMS solution were dissolved in PBS to obtain their extracts
with concentration of 100, 10, 1, and 0.1 mg/L. HeLa cells
were seeded into 96-well plates at a density of 10,000 cells
per well in 180 lL of culture medium. Twenty-four hours
later, 20 lL extracts of various concentrations were added to
the well. Cells were exposed to the gel precursor for 48 h.
Before 100 lL fresh medium and 10 lL CCK-8 reagent were
added, each well was washed three times with PBS. After a
further incubation of 1 h, the optical density (OD) of the
wells was measured at 450 nm with a Microplate Spectro-
photometer (SpectraMax M5). Nontreated cells were used as
a negative control. Relative cell viability was calculated as
following equation:
Tetrahydrofuran (THF) was used as the mobile phase at a
flow rate of 1.0 mL/min at 40 8C. The hydrodynamic diame-
ter and size distribution of micelles were determined by
dynamic light scattering (DLS) at 173 8 angle to the incident
beam and at 37 8C on a Malvern 2000 zetasizer analyzer. All
micelles solutions had a final polymer concentration of 1.0
mg/mL and were filtered through a 0.45 lm filter. Transmis-
sion electron microscopy (TEM) images were obtained using
JEM-1230 operating at an acceleration voltage of 60 kV. A
drop of 1.0 mg/mL micelles solution was dropped onto the
surface of Formvar-carbon film-coated copper grids. Excess
solution was quickly wicked away with a filter paper. All
grids were finally stained by 2 wt % phosphotungstic acid.
The amount of released PTX was investigated by reverse-
phase high-performance liquid chromatography (RP–HPLC)
analysis. A mobile phase of methanol/water (70/30, v/v)
was used, and the flow rate was set at 1 mL/min (35 8C).
ꢀ
ꢁ
ðOD450sample 2OD450blank
ðOD450control 2OD450blank
Þ
Þ
Relative cell viability ð%Þ 5
3 100
The cytotoxicities of PTX-loaded CCL micelles-SS and PTX-
loaded CCL micelles-CC were also evaluated by CCK-8 assay.
Micelles were diluted to final PTX concentrations of 5, 1, 0.1,
0.05, and 0.005 mg/L. Similarly, after HeLa cells incubated
for 24 h in 96-well plates, the cells were incubated with vari-
ous concentrations of PTX-loaded CCL micelles for another
48 h. As a comparison, another HeLa cell line was incubated
with PTX stock solutions (free PTX) under the same condi-
tions. PTX stock solutions were prepared according to a ref-
erence method.52
RESULTS AND DISCUSSION
Synthesis and Characterization of PEG–PBMS Multiblock
Copolymers
PEG–PBMS multiblock copolymers with different PEG and
BD molar ratios were synthesized by one-pot melt polycon-
densation reaction in the presence of Sc(OTf)3. The chemical
composition of multiblock copolymers was characterized by
proton nuclear magnetic resonance (1H NMR). As shown in
Figure 1, the characteristic proton signals of PEG and PBMS
segments are presented and marked. All peaks can be well
assigned with their chemical structures. Notably, the charac-
teristic signal of mercapto proton (Ha) was clearly detected
at 2.38 ppm, demonstrating the stability of mercapto groups
during the polycondensation process. The molar ratios of
butyl units to PEG segments could be calculated by the inte-
gral of corresponding proton (Hb and Hf), which is very close
to the initial feed ratio (Table 1), indicating the similar activ-
ity of butanediol and diol PEG in polycondensation with mer-
captosuccinic acid. Moreover, the Mn of PEG–PBMS measured
by GPC analysis was also summarized in Table 1.
Characterization
Proton nuclear magnetic resonance spectroscopy (1H NMR)
spectrum was recorded on a Bruker Avance DMX500 spec-
trometer in CDCl3 with tetramethylsilane as internal stand-
ard. The molecular weight and molecular weight distribution
were determined by gel permeation chromatography (GPC).
The GPC system consisted of a Waters degasser, a Waters
1525 HPLC pump with 717 plus autosampler, Waters 2410
RI detector and columns: Styragel, HT 3, HT 4. The calibra-
tion was performed with commercial polystyrene standards.
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TABLE 1 Multiblock Copolymers With Various PEG and BD Molar Ratio
PEG:BD
Copolymers
In Feeda
In Polymerb
Yield (%)
Mn (g/mol)c
MWDc
DLEd (%)
DLCd (%)
Diametere (nm)
PDIe
1
2
3
1:3
1:4
1:5
1:3.1
1:3.9
1:4.9
77
72
66
6100
6300
6900
1.31
1.34
1.41
73.0
74.1
75.5
6.8
6.9
7.0
127.2
129.7
119.5
0.425
0.316
0.185
a
d
Feeding molar ratio.
Measured by HPLC.
Measured by DLS.
b
c
e
Measured by 1H NMR.
From GPC using polystyrene standards.
Synthesis and Characterization of the Crosslinkers
The 2,20-dithiodiethanol diacrylate and 1,6-hexamethylene
diacrylate were synthesized through the acylation reaction.
even after 30 h with 10 mM DTT [Fig. 3(B)], which could be
attributed to stability of carbon–carbon bonds in reductive
environment. The sizes and morphologies of PTX-loaded CCL
micelles were further characterized by TEM measurements
as shown in Figure 3(C,D). The self-assembled micelles are
well dispersed as individual nanoparticles with regularly
1
The H NMR spectra of 2,20-dithiodiethanol diacrylate and
1,6-hexamethylene diacrylate are shown in Figure 2, in
which the characteristic proton signals are presented and
marked.
Preparation and Characterization of PTX-Loaded CCL
Micelles
The PTX-loaded CCL micelles were prepared by the solvent
evaporation method and dialysis. The PTX-loaded micelles
were in situ crosslinked via thiol-ene “click” reaction under
physiological conditions. The drug-loading capacity of the
PTX-loaded CCL micelles was investigated by HPLC. The DLC
and DLE were calculated as summarized in Table 1. The DLC
and DLE of the PTX-loaded CCL micelles did not changed
much upon the varying of PEG/BD molar ratio. The diame-
ters of the PTX-loaded CCL micelles were measured by DLS,
which also did not changed much upon the varying of PEG/
BD molar ratio. However, the PTX-loaded CCL micelles with
lowest PDI were obtained using copolymer 3 with longer
average PBMS chain length. More stable and uniform PTX-
loaded CCL micelles could be prepared from PEG–PBMS mul-
tiblock copolymer with high hydrophobic content. PTX-
loaded CCL micelles based on copolymer 3 were chosen to
perform the follow-up experiments.
The crosslinking degrees of CCL micelles were measured by
using Ellman’s reagent, which were 97.3% for CCL micelles-
SS and 94.8% for CCL micelles-CC. The high crosslinking
degrees demonstrate high efficiency of thiol-ene “click” reac-
tion in micellar cores and the successful syntheses of CCL
micelles. To investigate whether the disulfide bond could be
degraded under reductive conditions, the changes of CCL
micelles sizes in response to 10 mM DTT was monitored by
DLS measurement at time intervals. As shown in Figure
3(A), the size of CCL micelles-SS increased slightly with 10
mM DTT during the experimental period. This phenomenon
should be attributed to the gradual decrosslinking of the
CCL micelles-SS caused by the cleavage of disulfide bonds,
resulting in larger micelles with looser structure. Besides,
the size of CCL micelles-SS in PBS did not change during the
time intervals, which indicated the stability of micelles. How-
ever, the size of CCL micelles-CC did not increase sharply
1
FIGURE 2 H NMR spectra (400MHz, CDCl3) of 2,20-dithiodiethanol
diacrylate (A) and 1,6-hexamethylene diacrylate (B).
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FIGURE 3 Size distribution and TEM images of PTX-loaded CCL micelles. Change in the size distribution of PTX-loaded CCL
micelles in response to 10 mM DTT, determined by DLS at 37 8C, CCL micelles-SS (A) and CCL micelles-CC (B). TEM images of
CCL micelles-SS (C) and CCL micelles-CC (D).
spherical shapes. The diameters of PTX-loaded CCL micelles
observed from TEM images are smaller than those by DLS,
which is due to the dehydration of micelles during the prep-
aration process of TEM samples.53,54
In Vitro PTX Release From CCL Micelles
The PTX release profiles from CCL micelles in phosphate
buffer solution (PBS, pH 5 7.4) with or without 10 mM DTT
were obtained using dialysis method. Tween 80 (0.1 wt %)
was introduced into the PBS buffer solution to solubilize
released PTX. As shown in Figure 4, the release of PTX from
PTX-loaded CCL micelles-SS in PBS with 10 mM DTT was
rapid and about 80% of loaded PTX was released within 10
h. In contrast, PTX release from the CCL micelles-SS in PBS
without DTT was much slower and only about 20% of loaded
PTX was released even after 60 h. Furthermore, the PTX
release from CCL micelles-CC was also carried out either in
pure PBS or in PBS with 10 mM DTT. The release profiles are
FIGURE 4 PTX release kinetics from PEG–PBMS CCL micelles
very similar to that using PTX-loaded CCL micelles-SS in pure
in 10 mM PBS (pH 5 7.4, containing 0.1% Tween 80) with and
PBS, indicating that PTX-loaded CCL micelles-CC have no
reductive-responsibility. It should be noted that the concentra-
tion of glutathione (GSH) in tumor cells (8–10 mM) is about
2-fold higher than that in normal cells (2–8 mM) and much
without 10 mM DTT at 37 8C. In the presence of a reducing
agent (DTT, 10 mM), the hydrophobic drug molecules can be
easily released from the core due to the cleavage of the disul-
fide bonds.
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in cell nuclei [Fig. 5(B)]. As a comparison, the fluorescence of
NR was much weaker when HeLa cells were incubated with
NR-loaded CCL micelles-CC than those incubated with CCL
micelles-SS for the same time [Fig. 5(C,D)]. Moreover, no fluo-
rescence was detected when incubating free NR with HeLa
cells even after 4 h [Fig. 5(F)], which indicates that free NR
could not be taken in cells. All of the results by CLSM imply
that PEG–PBMS CCL micelles-SS could be used as drug car-
riers to delivery hydrophobic drugs into cells, and the loaded
drug could be rapidly released in response to the high GSH
concentration. Microplate reader was used for quantitative
determination of cellular uptake of NR-loaded CCL micelles.
As shown in Figure 6, NR-loaded CCL micelles-SS showed
much higher fluorescence intensity than NR-loaded CCL
micelles-CC and free NR, which further confirms that PEG–
PBMS CCL micelles-SS is an efficient nanocarrier to deliver
hydrophobic molecules into tumor cells, and quickly release
them upon the intracellular reductive environment.
Relative Cell Viabilities of PEG–PBMS and PTX-Loaded
CCL Micelles
To evaluate the cytotoxicities of PEG–PBMS, a HeLa cell line
was exposed to PEG–PBMS solutions at a series of concen-
trations ranging from 0.1 to 100 mg/L for 48 h by CCK-8
assay. As shown in Figure 7(A), compared with the control,
the presence of all the tested samples did not significantly
inhibit cell growth, which suggested that PEG–PBMS was
almost nontoxic against HeLa cells. Even when the concen-
tration was as high as 100 mg/L, the relative cell viability
still remained over 90% after 48 h incubation. The nontoxic-
ity of PEG–PBMS may arise from the biocompatibility of
PEG.56
FIGURE 5 CLSM images of HeLa cells after 1 or 4 h incubation
with free NR or NR-loaded CCL micelles. For each panel, the
images from left to right showed cell nuclei stained by Hoechst
33342 (blue), NR fluorescence in cells (red), overlays of two left
images and bright field. The scale bars correspond to 10 lm in
all the images. (A) NR-loaded CCL micelles-SS, 1 h. (B) NR-
loaded CCL micelles-SS, 4 h. (C) NR-loaded CCL micelles-CC, 1
h. (D) NR-loaded CCL micelles-CC, 4 h. (E) Free NR, 1 h. (F)
Free NR, 4 h. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
Furthermore, the antitumor activity of the PTX-loaded CCL
micelles against HeLa cells upon their reduction-responsive
was also investigated CCK-8 assay, and free PTX was used as
a control. As shown in Figure 7(B), the cytotoxicities of free
higher than that in blood plasma (1–2 lM)55. The PTX-loaded
CCL micelles-SS could be stable enough in simulative human
blood plasma environment, passively targeted to the tumor
tissue through the EPR effect, and disassociated in tumor cells
upon the reductive environment to release PTX.
Cell Uptake and Internalization Analysis
In order to investigate the cell uptake and internalization of
CCL micelles into HeLa cells, NR was used as a fluorescence
probe to replace PTX for preparation of CCL micelles. NR-
loaded CCL micelles were incubated in HeLa cells for 1 h and
4 h, which were monitored using CLSM. As shown in Figure
5(A), after incubating NR-loaded CCL micelles-SS in HeLa cells
for 1 h, weak fluorescence of NR was accumulated around the
nuclei of HeLa cells. Then, after 4 h incubation, strong red flu-
orescence was observed even in the cell nuclei, indicating that
NR has been efficiently delivered into HeLa cells and released
FIGURE 6 Quantitative determination of cellular uptake of NR-
loaded CCL micelles and free NR by microplate reader.
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PTX-loaded CCL micelles-SS could be prepared easily by in
situ crosslinking hydrophobic PBMS segments with 2,20-
dithiodiethanol diacrylate via thiol-ene “click” reaction under
physiological conditions. PTX-loaded CCL micelles-SS could be
stable during blood circulation, efficiently be taken up by
tumor cells through EPR effect, quickly release antitumor
drugs in response to high GSH concentration in tumor cells,
and finally kill the tumor cells, which made it a promising
candidate as a biodegradable and biocompatible nanocarrier
for antitumor chemotherapy.
ACKNOWLEDGMENTS
The authors thank Mr. Dingcheng Zhu, Prof. Jianbin Tang, and
Prof. Youqing Shen for HPLC measurements. The financial sup-
ports from the National Natural Science Foundation of China
(21274121 and 51173163), the Major State Basic Research
Project (2011CB606001), the Fundamental Research Funds
for the Central Universities (2015QNA4036), and the Science
and Technology Project of Zhoushan (2011C12054) are grate-
fully appreciated.
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