Synthesis, characterization and applications of selenocysteine-responsive nanoprobe based on dinitrobenzene sulfonyl-modified poly(carbonate) micelles

Article abstract of DOI:10.1039/c5ra12314c

In trace amounts, selenium (Se) participates in different physiological functions of the human body. Its biological significance is manifested in its presence as selenocysteine (Sec) in genetically encoded selenoproteins that are important in redox regulation, anti-inflammation, and cancer treatment. Recently, stimuli-responsive micelles were developed as biosensors, but there are no nanomicelle probes for the selective imaging of Sec under biological conditions (pH = 7.4). Herein, we report the design and preparation of a 2,4-dinitrobenzenesulfonyl-decorated block poly(carbonate) copolymer, viz. PMPC-Dns, for Sec imaging. We found that PMPC-Dns trapped with the fluorescent drug doxorubicin (DOX) selectively responds to Sec, while getting little interference from biological thiols, amines or alcohols. We applied the PMPC-Dns probe successfully to image endogenous Sec in cervical cancer tissues as well as in HeLa cells. In the course of these studies, we observed simultaneous release of the trapped DOX. Hence, besides Sec imaging, the probe can be used for controlled delivery of hydrophobic molecules for biomedical applications.

Full text of DOI:10.1039/c5ra12314c

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Synthesis, Characterization and Applications of
Selenocysteine Responsive Nanoprobe Based on
Dinitrobenzene Sulfonyl-modified Poly(carbonate)s
Micelles
Cite this: DOI: 10.1039/x0xx00000x
a,†
a,†
a,*
a
a,*
Yanxia Nan , Wenjie Zhao , Xinhua Xu , Chak-Tong Au , Renhua Qiu
In trace amount, selenium (Se) participates in different physiological functions of human body.
Its biological significance is manifested in its presence as selenocysteine (Sec) in genetically
encoded selenoproteins that are important in redox regulation, anti-inflammation, and cancer
treatment. Recently stimuli-responsive micelles were developed as biosensors but there are no
nano micelle probes for the selective imaging of Sec under biological condition (pH = 7.4).
Herein we report the design and preparation of 2,4-dinitrobenzenesulfonyl-decorated block
poly(carbonate) copolymer, viz. PMPC-Dns, for Sec imaging. We found that the PMPC-Dns
trapped with the fluorescence doxorubicin (DOX) drug selectively responds to Sec, while
getting little interference from biological thiols, amines or alcohols. We applied the PMPC-
Dns probe successfully to image endogenous Sec in cervical cancer tissues as well as in Hela
cells. In the course of studies, we observed simultaneous release of the trapped DOX. Hence,
besides Sec imaging, the probe can be used for controlled delivery of hydrophobic molecules
for biomedical applications.
Received 00th June 2015,
Accepted 00th June 2015
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
redox signalling and anti-inflammation as well as in the production
of active thyroid hormones. It is hence essential to find out the Sec is
Selenium (Se) is considered as an essential micronutrient and its a pathological roles of Sec because the majority of in vivo functions
1
participation in physiological functions is well recognized. Various of the Se-containing entities are performed by it. For this kind of
kinds of Se metabolites such as hydrogen selenide, selenocysteine studies, a biocompatible tool is needed to monitor the metabolites in
(
Sec), selenite, selenophosphate, selenodiglutathione, and charged living systems.
2
,3
Sec-tRNA are biosynthesized in animals. The anticancer ability of In terms of stability, simplicity, sensitivity, portability, cost-
Se was discovered in 1969. Nonetheless, it is noted that a number of effectiveness, and storage convenience, stimuli-responsive micelles
diseases are related to Se intakes of abnormal levels. Selenium have advantages, and were studied for the development of biosensors.
compounds of low molecular weight such as Sec and The commonly used stimuli are pH, temperature, light, redox
methylselenol are key metabolites in cancer prevention. Sec is a potential, ultrasound, charge, gases, biomolecules, and enzymes.
2
2
,4
5
,6
7,8
10–18
9
Se-containing amino acid encoded by a UGA stop codon that To the best of our knowledge, the use of nanomicelle probes for
locates in the active sites of selenoproteins (SePs). It functions in
selective recognition of Sec under biological condition (pH = 7.4)
has never been reported. Up to now, only a few fluorescence probes
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of of small molecules were studied for the detection and imaging of
1
9, 20
21
22
Chemistry & Chemical Engineering, Hunan University, Changsha 410082, Sec
China.
or SePs. In 2006, Maeda et al. reported the use of a
fluorescence probe to make discrimination between Sec and cysteine
(Cys) based on the fact that the selenol group of Sec is inclined to
ionize rather than to stay as the thiol group of Cys at pH 5.8. In 2014,
*
E-mail: renhuaqiu@hnu.edu.cn (R. Qiu)
xhx1581@hnu.edu.cn (X. Xu)
†
1
9
The authors contributed equally to this work.
Zhang et al. reported the design, synthesis, and biological
See evaluation of a series of potential Sec probes based on the same
Electronic
Supplementary
Information
(ESI)
available:
2
0
DOI: 10.1039/b000000x/
mechanism under physiological conditions. Recently, Kong et al.
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developed a fluorescence method to investigate the inhibitory
mechanism of Se in tumor cells. But due to enhanced permeability
2
3
and retention (EPR) effect, the reported methods cannot be widely
applied in biomedical researches as a result of poor biocompatibility
2
4
and low photobleaching resistance.
In this paper, we report the use of amphiphilic polymer micelles for
the detection of Sec. The 2,4-dinitrobenzenesulfonyl-decorated block
poly(carbonate) copolymer, viz. PMPC-Dns, was synthesized. Then
by means of ring-opening polymerization (ROP) of cyclic carbonates,
we generated amphiphilic block copolymers with poly(carbonate)s
hydrophobic chains. Finally by means of copper-catalysed azide-
alkyne Huisgen 1,3-dipolar cycloaddition (a coupling reaction), we
produced the Sec-responsive micelles in which the fluorescence
doxorubicin (DOX) drug was trapped. The copolymer that self-
assembles into micelles consists of a hydrophobic core surrounded
by a hydrophilic shell. It should be noted that the hydrophobic ends
are the sites for Sec recognition. In the presence of Sec, 2,4-
dinitrobenzenesulfonyl is activated and there is the generation of
hydrophilic ends in the destabilized micelles. The consequence is the
release of DOX for fluorescence imaging. In this study, we
investigated the synthesis and cytotoxicity of the micelle probe, as
well as the release of DOX as induced by Sec.
Figure 1.Working principle and response mechanism of the PMPC-
Dns probe for Sec detection.
2 2
that at δ = 4.28 (–C(O)OCH CCH O–, the methylene protons of the
carbonate units), which is close to the theoretical value of the
protons of carbonate units as shown in Table S1. The results of GPC
investigation reveal that the Sec-responsive copolymer has a narrow
polydispersity index (PDI) of 1.04 and number-average molecular
-
1
weight(M ) of 7263 g moL , which are in good agreement with the
n
1
results of H NMR end group analysis. Secondly, we modified the
PEG-b-poly(MPC) to prepare the PMPC-Dns copolymer via click
reaction (Scheme 1c), and purified the product by dialysis. We
Results and discussion
1
compared the H NMR spectra of Dns-N , PMPC-Dns, and PEG-b-
3
Synthesis and self-assembling of amphiphilic copolymers
poly(MPC) (Figure S1). The signal at δ = 2.54 in Figure
S1cassignable to the protons of the alkynyl groups of PEG-b-
The monomers of azide-functional dinitrobenzenesulfonate(Dns-N3) poly(MPC) disappears after click chemistry modification (Figure
and 5-Methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one(MPC) were S1b), indicating that there is complete reaction of the propargyl
synthesized according to the procedure shown in Scheme 1. Sec- groups of PEG-b-poly(MPC) with Dns-N . With reference to Figure
3
responsive copolymer PMPC-Dns was prepared through a two-step S1a and c, Figure S1b reveals that over the PMPC-Dns copolymer,
method. Firstly, PEG-b-poly(MPC) copolymer was synthesized via the Dns-N signals are at δ 2.21, 3.47, 4.24-4.43, 8.49, 8.74 and 9.07,
3
ROP of MPC with poly(ethylene glycol) (PEG) being the while signals owing to PEG-b-poly(MPC) are at δ 1.22, 3.37, 3.65
macroinitiator (Scheme 1b). The copolymer was characterized using and 4.26. Moreover, a weak signal at δ 7.72 that is attributable to the
1
the H NMR and Gel permeation chromatography (GPC) techniques. proton of triazole ring provides evidence for the attachment of Dns-
1
The H NMR spectrum of PEG-b-poly(MPC) in CDCl is shown in N3 to the polyester backbone. The GPC results reveal that the
3
Figure S1c. The degree of polymerization (DP) of the polycarbonate PMPC-Dns copolymer has a narrow PDI of 1.07 and M of 11353 g
n
-1
1
backbone was determined to be 10 by comparing the integrals of mol , close to that calculated by H NMR end group analysis (Table
peaks at δ =3.36 (CH O–, methyl protons of PEG end group) with
S1). The GPC trace of the graft copolymer shows a slight shift to
lower retention time while maintaining narrow distribution with
dispersity similar to that of the unmodified copolymer PEG-b-
poly(MPC) (Figure S2). It is apparent that the PMPC-Dns graft
copolymer can be readily prepared from propargyl-functionalized
polycarbonate via the click reaction.
The amphiphilic copolymers PEG-b-poly(MPC) and PMPC-Dns
self-assemble into micelles of hydrophobic core that are stabilized
with hydrophilic PEG coronae (Figure 1), and their self-assembly
behaviours were investigated in detail by fluorescence spectroscopy,
3
N
3
aN3
DCM, Et
3
N
O
S
N
a
HO
Br
HO
N
3
O
2
N
O
)
)
O
S
O
O
O
2N
Cl
O
1
NO
2
NO
2
Dns-N3
O
OH OH
O
OH OH
O
O
O
O
H
O
O
O
KOH, DMF
Br
O
TEA, THF
O
ROP
113
10
b
O
O
O
O
O
H
OH
O
1
13
Cl
O
PEG
2
MPC
PEG-b-poly(MPC)
O
O
H
O
O
O
O
N
3
10 as well as by dynamic light scattering (DLS) and scanning electron
microscopy (SEM) techniques. Using hydrophobic Nile Red (NR) as
fluorescence dye, the use of fluorescence spectroscopy can
conveniently monitor the self-assembly of micelles and determine
O
H
O
S
1
13
O
O
O
O
10
c
113
Click reaction
O
)
O
O
2
N
O
O
O
O
S
NO
2
O
O
N
O
2
N
N
N
NO
2
PMPC-Dns
30
the critical micelle concentration (CMC) of amphiphiles. As shown
Scheme 1.Synthesis of Dns-N
amphiphilic copolymer PMPC-Dns.
3
, MPC, PEG-b-poly(MPC) and in Figure S3, the emission fluorescence intensity gradually increases
with increasing amphiphile concentration, suggesting the
spontaneous self-assembly of micelles. PMPC-Dns shows a CMC
2
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-1
value similar to that of PEG-b-poly(MPC) (0.01245 mg mL vs. react with Sec and there is the cleavage of 2,4-
-1
0
.01159 mg mL ), suggesting that the self-assembled micelles are dinitrobenzenesulfonate. In the process, the hydrophobic ends
thermodynamically stable. These values are consistent with literature change into hydroxyl ends that are hydrophilic, leading to the
3
0-32
data reported for graft copolymers.
Then SEM (Figure 2a and b) breaking up of micelles and unwrapping of DOX. Since the intensity
and DLS (Figure S4a and b) were applied to measure the size and of fluorescence reflects the amount of released DOX, the
morphology of the self-assembled micelles. In the SEM images, concentration of Sec can be measured as a degree of fluorescence
spherical morphology was observed with an average size of 60 and intensity enhancement.
9
0 nm for PEG-b-poly(MPC) and PMPC-Dns micelles, respectively. To validate the feasibility of the copolymer micelles for Sec
Despite being common in morphology, the PMPC-Dns micelles are detection, the Sec-responsive PMPC-Dns micelles were prepared
larger than the PEG-b-poly(MPC) micelles in DLS-determined with DOX trapped inside. The drug loading efficiency (DLE) and
diameter, which can be attributed to the expanded hydrophobic drug loading content (DLC) of the DOX-loaded PMPC-Dns micelles
nucleus of the former. In the presence of Sec, the PMPC-Dns are found to be 6.5% and 7.63%, respectively. The fluorescence
micelles with the recognition site of 2,4-dinitrobenzenesulfonyl is response of PMPC-Dns micelles towards Sec was examined (Figure
activated, generating intertwining hydrophilic ends in the S6a). Compared to that of Sec absence, there is a 2.78-fold
destabilized micelles. One can see that there are micellesthat are not enhancement of fluorescence intensity at 590 nm when Sec (1 mM)
-1
activated in Figure 2d, and upon destabilization of micelles there is is introduced to the solution of micelles (0.5 mg mL ). To further
an obvious change of DLS average size from 118 (Figure S4b) to 13 evaluate the feasibility, time-dependent fluorescence changes of the
nm (Figure S4d). After Sec activation, the product dialyzes in micelles at 590 nm were monitored in the absence and presence of
1
aqueous solution and lyophilizes. In H NMR analysis, one can see Sec (Figure S6b). Without Sec, no fluorescence changes at 590 nm
that the signal at δ 8.49, 8.74, and 9.07 disappears and a weak signal are observed within 10 min, which indicates that there is no release
at δ 7.72 appears (Figure S5), meaning the reaction goes along with of DOX and the micelles are stable. On the contrary, in the presence
the release of 2,4-dinitrobenzenesulfonyl. However the PEG-b- of Sec, there is obvious enhancement of fluorescence intensity that
poly(MPC) micelles with no recognition sites show unchanged of reaches a plateau after 1.6 h, indicating the release of DOX. The
morphology (Figure 2c) and DLS average size (Figure S4c) results clearly prove that the PMPC-Dns micelles respond to Sec.
compared to the Figure 2a and Figure S4a, respectively.
Sensitive and specific detection of Sec
a)
b)
To demonstrate the applicability of the approach for qualitative
identification of Sec, we measured the fluorescence spectra of
PMPC-Dns micelles in phosphate-buffered saline (PBS) solution
treated with different concentrations of Sec. As shown in Figure 3,
with increasing concentration of Sec, there is increase of
fluorescence intensity, displaying a concentration-dependent trend.
A linear range is obtained between 0 and 25 µM. With further
increase in Sec concentration, the fluorescence intensity reaches a
plateau. Based on the 3σ rule, the detection limit is calculated to be
2
50 nm
250 nm
c)
d)
0
.05 µM. The results reflect the cleavage of 2,4-
dinitrobenzenesulfonateand destabilization of micelles in the
presence of Sec. With higher concentration of Sec, more DOX is
released and hence the higher fluorescence intensity.
2
50 nm
250 nm
Due to the high selectivity of 2,4-dinitrobenzenesulfonate interaction
1
9
with Sec as described by Zhang et el., the detection of Sec using
the PMPC-Dns micelles is highly selective. In the presence of high
concentration of various reducing reagents including thiols, beta-
mercaptoethanol (b-ME), thioredoxinreductase (TrxR), dithiothreitol
Figure2. SEM photographs of the micelles: (a) PEG-b-poly(MPC),
b) PMPC-Dns, (c) PEG-b-poly(MPC) treated by Sec, and (d)
PMPC-Dns treated by Sec.
(
Working principle and feasibility of PMPC-Dns micelles for Sec
(
(
DTT), N-acetyl-L-cysteine (NAC), Vitamin C, hydrogen sulfide
H S), Na SeO , and a mixture of Na SeO , Cys, and GSH, there is
Detection
2
2
3
2
3
no detectable fluorescence changes as shown in Figure 4. The
The working principle of the Sec-responsive PMPC-Dns copolymer
is schematically shown in Figure 1. The Sec recognition site 2,4-
dinitrobenzenesulfonate is copolymerized with the polycarbonate
backbone at the hydrophobic end. When the PMPC-Dns copolymer
and the DOX drug are put in the aqueous solution, the micelles
assemble and the hydrophobic DOX is wrapped inside. In the
absence of Sec, the Sec recognition sites are stable under
physiological conditions. In the presence of Sec, however, the sites
1
selectivity was further verified by H NMR analysis as shown in
1
Figure S7. There is no obvious change in H NMR features of
PMPC-Dns after the PMPC-Dns micelles was treated with the thiols
and sulfur compounds. The results confirm that the PMPC-Dns
probe shows no response toward the reducing reagents. It is apparent
that H S and the reducing reagents with a sulfhydryl group do not
2
response to the probe. It is noted that vitamin C also shows negative
response while DTT (50 μM) only exhibits slight response. One
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probable reason for TrxR not responding is that due to steric effect,
it is hard for the Sec in SePs to get close to the recognition sites.
a)
b)
c)
d)
e)
6
4
2
0
e+5
e+5
e+5
e+0
3.0
2.5
2.0
1.5
1.0
0
5
10 15 20 25
[
Sec]µM
5
00
550
600
650
700
Figure5. The Hela cells were treated with Sec (5 µM) for 2 to 12 h,
-1
followed by incubation with PMPC-Dns micelles (0.5µg mL ), and
Wavelength/nm
there was the appearance of a bright red fluorescence signal (Figure
-1
Figure3. Fluorescence spectra of PMPC-Dns micelles (0.5µg mL )
in PBS (pH 7.4, 20 mM) solution treated with increasing amounts of
Sec. The arrow indicates the signal changes with increase of Sec
concentration (0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 10.0, 12.0, 13.0,
4.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0 and 24.0µM).
Inset: The F/F ratio of micelles as a function of Sec concentration,
5
b, 5c), whereas in the case of control cells there was no obviously
fluorescence enhancement (Figure 5a). For the cells stimulated with
sodium selenite for 12 h, there was notable appearance of
fluorescence (Figure 5e). A short time treatment (2 h) resulted in
weak but obviously enhanced signal (Figure 5d). The images of the
last row were acquired by Image Pro Plus software. The colour strip
shows the pseudo colour change with Sec.
1
0
where F
5
0
and F are fluorescence intensity of PMPC-Dns micelles at
95 nm in the absence and presence of Sec, respectively.
6
e+5
a)
Sec
4
2
0
e+5
e+5
e+0
GSH, Cys, NAC,
DTT, H S,
Na SeO , TrxR,
2
b)
c)
2
3
2
β-ME, Na Se,
VC
5
00
550
600
650
700
Wavelength/nm
Figure6. The cervical tumor cells were treated with Sec (5 μM) for
-1
Figure4. The fluorescence of PMPC-Dns micelles (0.5µg mL ) in
PBS (pH 7.4, 20 mM) solution treated with thiols and other sulfur
compounds for 90 min (1µM).
-
1
)
2 h, followed by incubation with PMPC-Dns micelles (0.5 mg mL
, and there was the appearance of a bright red fluorescence signal
1
(Figure 6c), whereas in the case of control cells there was no obvious
fluorescence enhancement (Figure 6a). When the cells were
stimulated with sodium selenite for 12 h, there was notable
appearance of fluorescence (Figure 6b). The images of the last row
were acquired by Image Pro Plus software. The colour strip shows
the pseudo colour change with Sec.
PMPC-Dns micelles detection of Sec in cells and tissues
For suitability of biological applications, the probe has to be
nontoxic. The long-term cellular toxicity of PEG-b-poly(MPC) and
PMPC-Dns micelles towards the Hela cell lines was determined by
means of a standard MTT (methyl thiazolyl tetrazolium) assay. As
shown in Figure S8a, when the micelles concentration is 1 mg mL ,
the cell viability still remains at ca. 93%, demonstrating its low
cytotoxicity. The result can be attributed to the excellent
biocompatibility of PEG and poly(carbonate)s. Moreover, the
stability of PMPC-Dns micelles under condition of high
concentration of Fetal Bovine Serum (FBS) (Figure S8b) is also of
importance, especially in the detection of Se in tissues. We recorded
the fluorescence spectra of the probe after different periods of
storage time. The results show that the PMPC-Dns micelles are
stable under biological condition for a long time. Hence, the PMPC-
Dns micelles can serve as a selective and sensitive probe for
qualitative analysis of Sec. In addition, the Sec can be used as a
target agent for drug delivery in cancer therapy.
The imaging of Sec in live cells was performed using Hela cells as a
model. Since the physiological concentration of Sec is low in cells,
we first determined whether the PMPC-Dns micelles could respond
-1
4
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to exogenous Sec. After culturing the Hela cells with Sec for 2 to 12 Monomethoxypoly (ethyleneglycol) (PEG5k
,
n
M =5000), ethyl
h, we detect a bright red fluorescence signal when the probe is chloroformate, 2,4-dinitrobenzenesulfonyl chloride, 3-bromo-1-
applied (Figure 5b, 5c), while there is no obvious enhancement of propanol and 2,2-bis(hydroxyl methyl)propionic acid were
fluorescence intensity in the case of control cells (Figure 5a). We purchased from Alfa Aesar and used as received. We dried 1,8-
further determined the endogenous Sec in the cells. Sodium selenite diazabicyclo[5.4.0]undec-7-ene (DBU), dichloromethane (DCM),
(
Na SeO ) is a precursor of Sec biosynthesis, and the supplement of triethylamine, dimethylformamide (DMF) over calcium hydride for
2 3
cells with sodium selenite could significantly increase the Sec level 24 h at room temperature (RT) and distilled them under reduced
3
3
in the cells. After the cells were stimulated with sodium selenite for pressure. The thiourea catalyst (TU) was synthesized as reported
2
5
1
2 h, there is notable appearance of fluorescence (Figure 5e). A short previously and recrystallized from dry methylene chloride. We
time treatment of 2 h gives weak enhancement of fluorescence signal synthesized (Sec) according to the procedure reported by Braga et
2
2
6
(
Figure 5d). The results demonstrate that the probe is suitable for al. The Sec was generated from (Sec) in the presence of DTT (100
2
imaging Sec in live cells.
µM, from 50 µM (Sec) and 50 µM DTT). Tetrahydrofuran (THF)
2
To further investigate the response of PMPC-Dns micelles under was dried by means of refluxing over a benzophenone-sodium
biological condition, we selected cervical neoplasm for imaging. mixture until a deep blue appearance, and then subject to distillation.
Figure 6 shows the micelles staining of frozen slices of the cervical
tumor tissues. Strong fluorescence signals (Figure 6c) was observed 2 Characterization
over the cervical neoplasm slices that were treated with Sec for 12 h.
The tissue slice (Figure 6b) treated with Na SeO for the same time UV-Vis absorption spectra were recorded over Hitachi U-4100
2
3
period also gives fluorescence signal but slightly weaker in intensity. UV/Vis spectrophotometer using quartz cuvettes of 1 cm path length.
As for the neoplasm slice not treated with Sec or Na SeO , there is The steady-state fluorescence emission spectra were recorded over
2
3
no obvious fluorescence enhancement (Figure 6a).The results are in an equipment of Photon Technology International. Fluorescence
good agreement with those of Hela cells imaging. The confocal emission spectra were collected using a bandwidth of 5 nm and
2
1
13
images clearly demonstrate that the PMPC-Dns micelles can be used 0.2×1 cm quartz cuvettes containing 500 µL of solution. H and
for qualitative analysis of the Sec level of cervical cancer cells under NMR spectra were recorded on an Invoa-400 (Invoa 400)
C
biological condition.
spectrometer and referenced to solvent signals. The number-average
molecular weight (M ) and molecular weight distribution (PDI =
n
Conclusions
M /M
w
n
) of polymers were determined at RT using a Waters GPC
liquid chromatograph equipped with four TSK HXL series of
We designed and prepared a novel 2,4-dinitrobenzenesulfonyl- polystyrene divinylbenzene gel columns (300×7.8 mm). Calibration
decorated block poly(carbonate) copolymer, viz. PMPC-Dns, via a was established with polystyrene standards from Polymer
-1
convenient
click
conjugation
of
Sec-responsive
2,4- Laboratories. THF having a flow rate of 1 mL min was used as
dinitrobenzenesulfonyl molecules to propargyl-functionalized solvent. SEM images were obtained on an S-4800 scanning electron
poly(carbonate)s. DLS and SEM measurements revealed that the microscope (Hitachi) with a working voltage of 5 kV. A drop of the
-1
polymer self-assembles in aqueous solution into spherical micelles micelle aqueous solution (0.05 mg mL ) was deposited onto a
with an average diameter of 100 nm. The CMC of the micelles was silicon slice and allowed to dry at RT before measurement. The
-1
determined to be 0.01245 mg mL by fluorescence spectroscopy mean size of the micelles was determined by DLS using a Malvern
using NR as fluorescence dye. The follow-up studies demonstrated Nano S instrument (Malvern, UK).
that PMPC-Dns with the fluorescence drug DOX trapped inside
3
Synthesis of 3-bromopropyl 3-azido-propanol (1)
could selectively respond to Sec and other selenols under biological
conditions (pH = 7.4). It was found that there is little interference
from the biological thiols, amines, or alcohols. The PMPC-Dns
probe was successfully applied to image the endogenous Sec in Hela
cells as well as that in live cervical neoplasm. It is noted that during
Sec imaging, there is simultaneous release of the entrapped
hydrophobic DOX molecules under physiological conditions. To the
best of our knowledge, PMPC-Dns is the first Sec-induced probe
selective for Sec imaging. The work opens up a way to study the role
of Sec in biological, pathological as well as tumor xenograft model
systems. Furthermore, the approach provides a methodology for
controlled delivery of hydrophobic molecules in biomedical
applications.
To a solution of 3-bromo-propan-1-ol (1.02 g, 7.3 mmol) in water
10 mL) was added sodium azide (0.95 g, 14.6 mmol) and the
(
o
solution was heated at 80 C for 18 h. The aqueous solution was
extracted with EtOAc (4×20 mL). The organic layers were washed
with brine (30 mL), dried over MgSO , subject to filtration, and
4
concentrated under reduced pressure to give pure 3-azido-propanol
as colourless oil (0.57 g, yield = 76.5%).
1
3 2
H NMR (400 MHz, CDCl ): δ=3.73 (t, 2H, OHCH ), 3.44 (t, 2H,
CH CH N ), 2.51 (s, 1H, OH), 1.80-1.86 (m, 2H, CH CH CH );
2
2
3
2
2
2
1
3
3
C NMR (100 MHz, CDCl ): δ =59.65, 48.41, 31.43.
4
Synthesis of 3-azidopropyl 2,4-dinitrobenzenesulfonate
(
Dns-N3)
Experimental Section
A solution of 3-azido-propanol (0.57 g, 5.6 mmol) and Et
3
N
1
Materials
(
1.56 mL, 11.2 mmol) in dry CH Cl (10 mL) was cooled to
2
2
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Journal Name
o
0
C. With the addition of 2, 4-dinitrobenzene-1-sulfonyl The ROP of MPC was carried out under an inert atmosphere of
chloride (1.7 g, 6.7 mmol) in dry dichloromethane (10 mL), the nitrogen using the standard Schlenk-line technique. In a typical
solution was stirred at RT for 18 h. After quenching of the experiment, MPC (0.594 g, 3 mmol), PEG (0.601 g, 0.12 mmol),
5
k
reaction with water (40 mL), the organic layer was separated, TU (0.055 g, 0.15 mmol), DBU (0.005 g, 0.03 mmol) and dried
dried over MgSO , subject to filtration, and concentrated under DCM (10 mL) were placed in a dried Schlenk tube fitted with a
4
reduced pressure. The as-obtained crude oil was purified by rubber septum. The solution was further degassed through three
column chromatography (eluent: ethyl acetate/pet ether = 1/5, freeze–pump–thaw cycles. The resulting mixture was stirred at RT
v/v) to yield 3-bromopropyl 2,4-dinitrobenzenesulfonate as for 7 h, followed by precipitation in ice-cold diethyl ether and
colourless oil (1.33 g, yield = 71.1%).
centrifugation. The resulting product was collected by filtration and
): δ= 9.00 (s, 1H, ArH) , 8.67 (d, 1H, dried under vacuum to yield a white powder. Yield: 1.1 g (92.0%).
3
1
H NMR (400 MHz, CDCl
ArH), 8.45 (d, 1H, ArH), 4.03-4.25(m, 2H, CH CH CH ), 3.32
1
2
2
2
H NMR (400 MHz, CDCl ): δ = 4.73 (d, OCH CCH), 4.28-4.32 (m,
3
2
(
t, 2H, OCH CH ), 1.86(t, 2H, CH CH N );
C NMR (100 MHz, CDCl ): δ =152.19, 145.22, 142.19, CH CCH), 2.19 (s, OH), 1.29 (s, CH ).
3 2 3
23.88, 123.47, 115.71, 62.00, 42.49, 24.34.
OC(O)OCH ), 3.65 (s, OCH CH O), 3.38 (s, CH O), 2.55 (s,
2 2 2 3
2
2
2
2
3
1
3
-1
1
GPC (THF, RI): M (PDI) =7263 g mol (1.04).
n
5
Synthesis of 2,2-Bis (hydroxyl methyl) propionate (2)
8 Synthesis of PMPC-Dns via ‘‘click’’ chemistry
In a 250 mL round-bottom flask, 2,2-bis(hydroxyl methyl)-propionic
In a Schlenk tube, PEG-b-poly(MPC) (200mg, propargyl group,
.023 mmol), Dns-N (76 mg, 0.23 mmol), sodium ascorbate (4.5
acid (2.24 g, 16.72 mmol), KOH (1.01 g, 18.04 mmol), and DMF
0
3
o
(
100 mL) were added. The mixture was stirred at 100 C for 2 h, and
mg, 0.023 mmol), and DMF (4 mL) were introduced. The tube was
fitted with a rubber septum. The solution was further degassed
through three freeze-pump-thaw cycles. A DMF solution of copper
sulfate (2.8 mg, 0.012 mmol) was then added to the Schlenk tube.
The solution was stirred at RT for 24 h. The crude material was
purified by dialysis (dialysis tubing 3500 MWCO) against deionised
then propargyl bromide (2.13 g, 18.04 mmol) was added dropwise
over a 30-min period. After 72 h of reaction, the reaction mixture
was subject to filtration, and the solvent was evaporated under
reduced pressure. The residues were dissolved in 50 mL of DCM
and extracted three times with saturated salt water (20 mL×3). The
organic phase was concentrated to yield the crude product, which
was purified by column chromatography (eluent: ethyl
(DI) water that was renewed regularly. After 3 days, the final
product, PMPC-Dns, was obtained by lyophilization. Yield: 190.9
acetate/petroleum ether = 1/5, v/v). Yield: 1.3 g (45.1%).
mg (81.3%).
1
1
H NMR (400 MHz, CDCl
3 2
): δ = 4.76 (d, 2H, CHCCH CO), 3.93 (d,
3
H NMR (400 MHz, CDCl ): δ = 9.07 (s, ArH), 8.75 (d, ArH), 8.49
2
H, CH OH), 3.73 (d, 2H, CH OH), 2.87 (s, 2OH), 2.51 (t, 1H,
2
2
(s, ArH), 7.72 (s, N CHC), 4.73 (s, C(O)OCH ), 4.31 (s, C(O)OCH ),
3 2 2
CHCCH CO), 1.11 (s, 3H, CH CC);
2
3
4.28-4.24 (m, OCH CH CH N), 3.64 (s, OCH CH O), 3.47 (t，
2 2 2 2 2
1
3
C NMR (100 MHz, CDCl
3
): δ = 175.01, 75.20, 67.82, 60.37, 52.43,
CH CH O), 3.38 (s, CH O), 2.17 (s, NCH CH ), 1.29 (s, CH ).
2
2
3
2
1
2
3
-
4
9.29, 16.95.
GPC (THF, RI): M (PDI) =11353 g mol (1.07).
n
6
Synthesis of 5-Methyl-5-propargylxycarbonyl-1,3-dioxane-2-
9
Preparation of micelles
one (MPC)
Micelles of PEG-b-poly(MPC) and PMPC-Dns were prepared by a
dialysis method. First 25.0 mg copolymer was dissolved in DMF (1
mL), and then DI water (10 mL) was slowly added with vigorous
stirring. After vigorous stirring for another 2 h at RT, the micelles
were obtained and subject to further dialysis against DI water for 24
h to remove DMF (MWCO 1000 Da). The final polymer
Compound
2 (1.18 g, 6.88 mmol) was mixed with ethyl
chloroformate (1.48 g, 13.76 mmol) and THF (20 mL) in a sealed
vessel that was purged with nitrogen and cooled in an ice bath. After
stirring for an hour, triethylamine (1.39 g, 13.76 mmol) was added
dropwise over a 30-min period under nitrogen atmosphere. The
reaction was conducted at 0 C with stirring for 3 h, then at 25 C
under stirring overnight. The solution was then subject to filtration,
evaporated to dryness, and the as-obtained product was precipitated
in a mixture of ethyl acetate and diethyl ether (1:1) as white crystals.
o
o
-1
concentration was adjusted by adding DI water to 0.5 mg mL .
1
0 Measurement of the critical micelle concentration (CMC)
Yield: 1.24 g (91.1%).
The CMC of PEG-b-poly(MPC) and PMPC-Dns amphiphiles were
1
3 2
H NMR (400 MHz, CDCl ): δ = 4.82 (d, 2H, CHCCH CO), 4.75
determined by a dye solubilization method using NR as probe
-1
(
d, 2H, CH OCO), 4.27 (d, 2H, CH OCO), 2.57 (t, 1H,
2
2
molecule. NR in THF (0.1 mg mL , 30 µL) was added to a glass vial
using a microsyringe. After the evaporation of THF, a portion of the
micelle solution (2 mL) was added. The concentration of micelle
CHCCH CO), 1.39 (s, 3H, CH CC);
2
3
1
3
C NMR (100 MHz, CDCl
2.70, 53.47, 40.17, 17.39.
3
): δ = 170.31, 147.21, 76.35, 75.94,
-4
-1
7
solution was varied from 0.1 to 5×10 mg mL . Then, the solution
was stirred for 24 h. Fluorescence measurements were taken at an
excitation wavelength of 550 nm and the emission was monitored
from 570 to 750 nm.
7
Synthesis of PEG-b-poly(MPC)
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1
1Preparation of DOX-Loaded PMPC-Dns micelles and washing the cells three times with PBS solution (pH =7.4), fresh
calculation of drug loading content (DLC)
medium was added. All the fluorescence images were acquired using
an Olympus FV1000 laser confocal microscope and analyzed by
Image Pro Plus software.
In brief, PMPC-Dns (15.0 mg) and DOX (1.0 mg) were
simultaneously added into dimethyl sulfoxide (DMSO, 5 mL) under
vigorous stirring until complete dissolution. Then, the mixture was 14Preparation and staining of Hela cancer tissue slices
transferred into a dialysis bag (MWCO 3500 Da) and subject to
Tissue slices were prepared from Hela cancer neoplasm. A total of
dialysis in aqueous solution for 3 days for the formation of the
DOX-loaded PMPC-Dns micelles. During the 3-day period, the DI
water was renewed regularly. To determine the DLC and DLE, the
DOX-loaded PMPC-Dns micelles were incubated in Sec solution for
6
2
×10 Hela cancer diluted in 100 mL of serum-free DMEM was
injected subcutaneously into the right flank of 6 to 8-week-old
BALB/c nude mice for tumor inoculation. After 15-20 days, the
mice were sacrificed, and the tumors were transferred and embedded
with O.C.T (Sakura Finetek, USA, Torrance, CA) for frozen sections.
The tissues were cut into 50 µm thick slices using a vibrating-blade
microtome. For detection of exogenous produced Sec, the slices
were co-incubated with Sec (20 µM) for 12 h. After washing three
times with PBS solution (pH = 7.4) to remove the remaining Sec, the
2
4 h, then lyophilized, and dissolved in DMSO again. The drug
concentration was determined by measuring the fluorescence
intensity of DOX (excited at 490 nm).The DLC and DLE were
calculated according to the following equations:
DLC (%) = Wloaded/(Wpolymer + Wloaded) × 100%
DLE (%) = Wloaded/Wtotal × 100%
-1
slices were further incubated with PMPC-Dns micelles (0.2 µg mL )
Where Wtotal, Wloadedand Wpolymer are the weight of total DOX used,
theloaded DOX, and PMPC-Dnsmicelles, respectively.
o
for another 12 h at 4 C. Afterward, the incubation slices were
washed with 3×3 mL of PBS solution (pH = 7.4) before imaging. To
induce the endogenous Sec, the tissues were exposed to sodium
selenite (20 µM) for 12 h, and then the slices were further incubated
with the PMPC-Dns micelles (0.2 µg mL ) for 12 h at 4 C. After
washing three times with PBS solution (pH = 7.4), the slices were
mounted with 10% glycerol and sealed with nail varnish on a glass
substrate. The fluorescence images of Hela cancer tissue were
carried out on an Olympus FV1000 laser confocal microscope and
analysed by Image Pro Plus software.
1
2Cytotoxicity assay
-1
o
Using Hela cells as model, the cellular cytotoxicity of the probe was
evaluated by standard cell viability MTT assay. Hela cells were
seeded into a 96-well plate at a concentration of 5×10 cells well in
1
in a 5% CO 95% air incubator for 24 h. Then, the medium was
removed and replaced with 200 µL polymer micelles. The aggregate
2
7
3
-1
o
00 µL of MEM with 10% FBS. The plates were maintained at 37 C
2
concentrations of each formulation were prepared by serial dilution
with DMEM. The cells incubated with the culture medium only were Acknowledgement
used as controls. The cells were washed with PBS solution for three
-1
times, and then 100 µL MTT solution (0.5 mg mL in PBS solution) This work was supported by the National Natural Science
-1
was added to each well. After the addition of DMSO (150 µL well ), Foundation of China (Nos. 21273068 and 21373003). C.T. Au
the assay plate was shaken at RT for 10 min. Experiments were done thanks the HNU for an adjunct professorship.
in triplicate. The cell viability was calculated based on measurement
of UV-Vis absorption at 570 nm using the following equation, where Notes and references
2
8, 29
OD570 represents the optical density.
Cell viability = [OD570(sample) − OD570(blank)] / [OD570(control)
OD570(blank)
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