Multifunctional Probe Based on Cationic Conjugated Polymers for Nitroreductase-Related Analysis: Sensing, Hypoxia Diagnosis, and Imaging

Article abstract of DOI:10.1021/acs.analchem.7b00477

Nitroreductase (NTR) is overexpressed in hypoxic tumors. Moreover, hypoxia is usually considered as the most important feature of various diseases. Thus, it is important to build a sensitive and selective method for NTR detection and hypoxia diagnosis. He

Full text of DOI:10.1021/acs.analchem.7b00477

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Article
Multifunctional Probe Based on Cationic Conjugated Polymers for
Nitroreductase- Related Analysis: Sensing, Hypoxia Diagnosis and Imaging
Xiaoqian Zhang, Qi Zhao, Yanru Li, Xinrui Duan, and Yanli Tang
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Apr 2017
Downloaded from http://pubs.acs.org on April 19, 2017
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Multifunctional Probe Based on Cationic
Conjugated Polymers for Nitroreductase-
Related Analysis: Sensing, Hypoxia
Diagnosis and Imaging
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Xiaoqian Zhang, Qi Zhao, Yanru Li, Xinrui Duan, Yanli Tang*
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key
Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School
of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062,
P. R. China
[
*]Eꢀmail: yltang@snnu.edu.cn. Fax: (086) 29ꢀ81530727 (Prof. Y. L. Tang)
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ABSTRACT: Nitroreductase (NTR) is overexpressed in hypoxic tumors. Moreover,
hypoxia is usually considered as a most important feature of various diseases. Thus it
is important to build a sensitive and selective method for NTR detection and hypoxia
diagnosis. Herein, a new cationic conjugated polymer (PBFBTꢀNP) with
pꢀnitrophenyl group in the side chain was designed and synthesized as a fluorescent
probe for the detection of NTR. In the absence of NTR, the fluorescence of
PBFBTꢀNP was quenched due to photoꢀinduced electron transfer (PET). On the
contrary, in the presence of NTR, NTR can specifically react with pꢀnitrophenyl group
to form pꢀaminophenyl group, which leads to the PET being inhibited and the
polymer’s fluorescence thus significantly increasing (>110ꢀfold). The sensitive and
selective NTR sensing method in vitro is thus constructed with a low detection limit
of 2.9 ng/mL. Moreover, the hypoxic status of tumor cells can be visualized by
fluorescence bioimaging with very low cytotoxicity. Interestingly, the probe was
successfully used for imaging NTRꢀexpressed microorganism, such as E. coli, and
showed excellent antibacterial activity against E. coli under white light irradiation. In
brief, this multifunctional probe is promising for widespread use in NTRꢀrelated
biological analysis.
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KEYWORDS: Cationic conjugated polymers; Nitroreductase detection; Hypoxia
diagnosis; Photoꢀinduced electron transfer; Bioimaging
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INTRODUCTION
Hypoxia refers to a state in which oxygen supply is insufficient, which is
usually considered as a most important feature of various diseases, including
acute cerebrovascular disease, cardiac ischemia, stroke, inflammatory diseases
and the solid tumors. Clinical researches indicated that the hypoxic status of
tumors is closely related with tumor progression toward a more malignant
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phenotype with increased metastatic potential. Moreover, hypoxia cells can
reduce the efficiency of antiꢀcancer drugs to some degree compared with
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normoxic cells. Therefore, the estimation of tumor hypoxia degree is very
important for predicting anticancer efficacies. Because of this, it remains
urgently vital to develop a novel approach to detect hypoxia status selectively
and sensitively. It is known that the reductive enzymes, including
nitroreductase (NTR), azoreductase and quinone reductase, are overexpressed
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in hypoxia status cells.
Among them, NTR is considered as an indicative
biomarker because NTR level corresponds directly with the degree of
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hypoxia.
Therefore, the hypoxia status in a tumor can be directly assessed
by detecting the level of NTR.
Nitroreductase, a cytosolic enzyme, can make use of NADH or NADPH as
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an electron source to reduce the nitro groups to the corresponding amines.
Recently, several new techniques based on fluorescent probes have been
2,7,12,18,19ꢀ28
designed for NTR detection.
Among them, fluorescent probes based
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on nitroaromatic compounds are obtained widely attention because of their high
sensitivity, selectivity and in situ detection. However, most of them require long
response time, tedious procedures, expensive instruments or materials, and poor
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water solubility, which limit their practical applications.
In recent years, cationic conjugated polymers (CCPs), comprised of a large
number of conjugated repeat units, have gained more and more attention due to
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their unique photoꢀ and electroꢀcharacteristics.
Upon irradiation by
ultraviolet (UV) or visible light, exciton can be efficiently transferred to a lower
electron or energy acceptor sites along the whole backbone of the conjugated
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polymer, which gives rise to fluorescent signal amplification.
Modification
with different functional groups on sideꢀchains of CCPs can allow CPPs to
specifically detect a series of targets, such as, enzymes, oligonucleotides, toxic
metal ions, and so on. Therefore, these favorable advantages make CCPs
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applicable in biosensors and cell imaging, etc.
Inspired by the characteristics of CCPs and biological effects of NTR, a new
“turnꢀon” fluorescent CCPs probe PBFBTꢀNP was synthesized to detect NTR.
PBFBTꢀNP probe has good waterꢀsolubility and contain nitro group which
specifically reacts with NTR. It was reported that aromatic nitro group, as
strong electronꢀwithdrawing group, could modulate the fluorescence properties
of fluorophore via a photoinduced electron transfer (PET) process from the
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excited fluorophore to aromatic nitro group.
When the nitro group was
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introduced to PBFBTꢀNP on the side chains, it is reasonably to assume that the
fluorescence of PBFBTꢀNP is quenched through PET from the backbones to
the side chains (Scheme 1). However, when NTR is present, which reduces the
nitro to amine group, PET could be efficiently blocked followed by
PBFBTꢀNPemitting strong fluorescence. Thus this strategy provides us a
sensitive and simple method to detect NTR and NTRꢀrelated hypoxia diagnosis.
Moreover, it is known that NTR is usually expressed in microorganism, such as
Escherichia coli. The new probe also can be used for bacteria imaging without
the use of expensive commercial stain and used for antibacterial agent.
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EXPERIMENTAL SECTION
Materials and Measurements. All chemicals were purchased from J&K Scientific
Ltd. (Beijing, China), Aladdin Industrial Corporation (Shanghai, China) or Sinopharm
chemical reagent Co. Ltd. (Beijing, China), and were used without any further
purification. Nitroreductase from E. coli was purchased from SigmaꢀAldrich Co. Ltd.
(
St. Louis, MO, USA). βꢀNicotinamide adenine dinucleotide disodium salt hydrate
NADH) was purchased from TCI Co. Ltd. (Shanghai, China). The anaerobic culture
(
bags were purchased from Mitsubishi gas chemical Co. Ltd. (Tokyo, Japan).
Ultrapure water was collected from a MilliꢀQ reference system. All pH measurements
were carried out with a Sartorius basic pHꢀMeter. UVꢀvis absorption spectra were
taken on a Perkin Elmer Lambda 35 spectrophotometer. The fluorescence spectra
were recorded on a Hitachi Fꢀ7000 spectrophotometer equipped with a xenon lamp
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excitation source. Fluorescence imaging experiments of E. coli were performed on an
Olympus IX73; Confocal laser scanning microscopy images of A549 cells were
recorded on Olympus Fluoꢀview 1200. Copolymer molecular weight was measured
by GPCꢀMALLS. The antibacterial activities were recorded using an Accuri C6 flow
cytometry. TEM images were obtained using Tecnai G2 F20 Field Transmission
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Electron Microscopy (FEI). H NMR and C NMR spectra were recorded on Bruker
Avance 300 MHz and Ascend 600 MHz spectrometers.
Synthesis of Monomer 3. Under nitrogen, pꢀnitrophenol (0.696 g, 5 mmol) and
NaH (0.12 g, 5 mmol) were dissolved in DMF (30 mL). After stirring at room
temperature for 15 min, compound 2 (0.389 g, 0.782 mmol) was added to the mixed
solution and then stirred at room temperature for 24 h. The final mixture was
extracted with CH
2
Cl
2
and the collected organic phase was washed with HCl solution
and concentrated under
(1 M), brine and distilled water, and then dried over MgSO
4
vacuum. The residue was subjected to silica gel chromatography, eluted with
petroleum ether/ ethyl acetate (v/v=16:1), affording monomer 3 (0.121 g, 28%) as
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yellow solid. H NMR (300 MHz, CDCl
3
), δ 8.22 (d, J = 9.0 Hz, 2 H), 7.22 (s, 1 H),
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.17 (s, 1 H), 7.01 (d, J = 9.0 Hz, 2 H), 4.35 (t, J = 6.0 Hz, 2 H), 4.15 (t, J = 6.0 Hz, 2
1
3
H), 3.82 (s, 3 H), 2.33(m, 2 H). C NMR (75 MHz, CDCl
3
) δ 163.9, 153.7, 152.4,
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41.6, 125.9, 123.2, 121.4, 114.5, 85.5, 65.23, 57.2, 29.0. HRMS (ESI): m/z: calcd.
+
+
+
15 2 5
for C16H I NO Na 577.8938[M+Na] , found 577.8938 [M+Na] .
Synthesis of Br-PBFBT-NP. Monomer 1 (71.68 mg, 0.110 mmol), monomer 2
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(
117.22 mg, 0.158 mmol), monomer
3
(8.74 mg, 0.0158 mmol),
,7ꢀdibromoꢀ2,1,3ꢀbenzothiadiazole (9.26 mg, 0.0315 mmol), THF (20 mL) and
CO solution (2 M, 10 mL) were mixed and degassed with nitrogen gas for 30 min.
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PdCl (dppf) (36.60 mg, 0.05 mmol) was then added to the solution at room
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temperature under nitrogen. The mixed solution was heated at 85 C for 48 h. The
cooled mixture was extracted with CHCl
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, washed with distilled water, dried over
Na SO and concentrated under vacuum. The residue was dissolved in CHCl
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and
precipitated in methanol 3 times to give BrꢀPBFBTꢀNP as black solid (80 mg, 57%).
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H NMR (300 MHz, CDCl ), δ 8.01ꢀ7.38 (m), 7.11 (m), 6.85 (m), 4.21 (br), 4.06 (br),
3
3
.87 (br), 3.30 (br), 3.08 (br), 2.15 (br), 1.69 (br), 1.25 (br), 0.88 (br). GPCꢀMALLS:
Mw=24,215; Mn=14,679; PDI=1.65 (The solvent is THF).
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Synthesis of PBFBT-NP. Br-PBFBT-NP (30 mg) was dissolved in CHCl (45 mL)
and the reaction mixture was stirred vigorously to form a homogeneous solution.
Then, trimethylamine ethanol solution (2 mL) was added and the mixture was stirred
at room temperature for 72 h. The solid was collected by filtration, washed with
CHCl
3
3 times and dried under vacuum to give PBFBT-NP as black solid (29 mg,
1
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9% (molar yield)). H NMR (600 MHz, MeOD), δ 8.09ꢀ7.38 (m), 4.29 (t), 4.08 (m),
3
.65 (s), 3.28 (m), 3.06 (br), 2.19 (br), 1.60 (br), 1.29 (br), 0.90 (br).
General Procedure for NTR Detection. In 2.0 mL TrisꢀHCl (10 mM, pH 7.4)
solution, 0.1 µM (final concentration) PBFBTꢀNP and 100 µM (final concentration)
o
NADH were added. Then, NTR was added and incubated at 37 C. Fluorescence
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spectra were recorded with the excitation wavelength of 430 nm. Under the same
conditions, the blank solution (without nitroreductase) was prepared and measured.
Cell Culture. The A549 cells were grown in dulbecco’s modified eagle’s medium
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(DMEM) supplemented with 10% fetal bovine serum (FBS) and cultured at 37 °C
under 5% CO for normoxic condition (20% O ), otherwise A549 cells were cultured
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for 6 h at 37 °C under anaerobic conditions.
Strain and Growth Condition. Gramꢀnegative bacteria E. coli as model bacteria
was used in this study. Bacterial samples were transferred from the frozen state onto
o
agar slants (1.2% agar + Lysogeny Broth (LB) for E. coli) and incubated at 37 C
o
overnight and then held at 4 C for up to 2 weeks. Every time, a colony of bacteria
o
was put on LB (20 mL) and cultured at 37 C overnight. After growth, the bacterial
culture was centrifuged at 6000 rpm/min three times. Then the pellet was suspended
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in 0.9% NaCl solution. The final concentration of bacteria was around 2 × 10 mL .
RESULTS AND DISCUSSION
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Scheme 1 The schematic presentation of the nitro group reduced into the amino group by
NTR and cell hypoxia diagnosis.
Design and Synthesis of PBFBT-NP. The important features of suitable
fluorescent probes for NTR detection should have enhanced fluorescence, fast
and sensitive response, good selectivity. Taking all of these characteristics into
account, we designed and synthesized the copolymer PBFBTꢀNP probe by a
Suzuki coupling reaction between monomer 1, 2, 3 and 4,7ꢀdibromoꢀ2,1,3
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ꢀ
benzothiadiazole and the following quaternization reaction, which meets the
requirement that a fluorescent probe should consist of a detecting part and a
fluorescence reporting part. Herein, the monomer 1/2 with quaternary
ammonium groups linked to the side chains makes PBFBTꢀNP possessing
excellent waterꢀsolubility, monomer 3 makes PBFBTꢀNP detecting NTR
selectively, and 4,7ꢀdibromoꢀ2,1,3ꢀbenzothiadiazole makes PBFBTꢀNP having
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a absorption wavelength in visible light range.
Structurally, pꢀnitrophenol
was covalently linked to the side chain of PBFBTꢀNP, which is not only served
as an electron acceptor (a fluorescence quencher) but also as a NTR trapper. In
fact, due to pꢀnitrophenyl side groups playing key roles in target recognition
and fluorescence quenching, the proportion of pꢀnitrophenyl side groups has
important influence on the NTR sensing. If the proportion of groups is too
small, the fluorescence from main chains could not be quenched effectively,
which will increase the background signal. Otherwise, if the proportion of
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groups is too large, the fluorescence can be quenched effectively, but to
enhance the fluorescence it requires more NTR to react with the groups. Taking
these points in account, we chose the proportion as 0.1 to prepare the probe that
presented a very low background signal and an excellent response to NTR. The
main chains of conjugated polymers are fluorescence reporting part.
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The synthesis of PBFBTꢀNP was illustrated in Scheme 2. Compound 1,
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monomer 1 and 2
were synthesized according to the literatures. Reaction of
compound 2 with pꢀnitrophenol affords monomer 3. The BrꢀPBFBTꢀNP is
prepared by a Suzuki coupling reaction between monomer 1, 2, 3 and
4
,7ꢀdibromoꢀ2,1,3ꢀbenzothiadiazole in the presence of PdCl
CO solution. Then cationic PBFBTꢀNP is obtained by quaternarization
reaction of the precursor BrꢀPBFBTꢀNP (Figure S10) with trimethylamine in
CHCl . As shown in Figure S10 and Figure S11, the NMR peak attribution was
presented for every spectrum in details. In Figure S10, the peak at 4.06 and 3.30
belong to ꢀCH (Cꢀ127) in Monomer 3 side chain and ꢀCH Br in Monomer 1/2
2
(dppf) in THF and
K
2
3
3
2
2
side chain, respectively. According to the integral ratio, one can estimate
Monomer 1 and 2 to Monomer 3 ratio is around 7:1. As shown in Figure S11,
3 3 2
after quaternization, the peak at 3.06 and 2.19 belong to ꢀN(CH ) Br and ꢀCH
in Monomer 1/2 side chain, respectively. Furthermore, the ratio between them
is around 3, which means the quaternization efficiency is about 66%. Due to the
overlay of hydrogen chemical shift on aromatic rings, it is difficult to determine
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the actual ratio of 4,7ꢀdibromoꢀ2,1,3ꢀbenzothiadiazole. Therefore, we still used
the theoretical ratio as the proportion of monomer in the copolymer. Because of
copolymers with high molecular weight, it is not easy to obtain a higher
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concentration in D O to meet the requirement for NMR measurement. Because
PBFBTꢀNP copolymers are amphiphilic macromolecules, when they are
soluble in water, the copolymers may form very loose aggregates that could be
affected easily by microenvironment. The size distribution measurement was
conducted by the dynamic light scattering (DLS). As shown in Figure S1, the
size is around 161
± 18 nm. TEM image showed copolymers formed
nanoparticles, which is coincident with the DLS data.
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Scheme 2 The synthesis of PBFBTꢀNP.
As shown in Figure S1, the UVꢀvis absorption spectra and the fluorescence
spectra of PBFBTꢀNP were carried out in water without the requiring of any
organic solvent such as ethanol or DMSO as coꢀsolvent, which is advantageous
3
8ꢀ40
to application in bioanalysis.
The absorption spectra showed that
PBFBTꢀNP has two absorption peaks at 350 nm and 430 nm, respectively. In
this experiment, the excitation wavelength is set at 430 nm to avoid 350 nm UV
light damaging cells and bacteria in following hypoxia diagnosis and
antibacterial tests, and the emission wavelength is obtained around 558 nm in
the absence of NTR. To do control experiments in following researches, we
also synthesized the copolymer PBFBT devoid of pꢀnitrophenyl side groups.
The synthesis procedure was shown in Scheme S1 and the characterizations
were presented in Supporting Information. Figure S1b showed PBFBT have the
similar absorption peaks at 349 and 440 nm, respectively, and emission peak at
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58 nm, which indicated that the pꢀnitrophenyl side groups did not have much
influence on the absorption and emission spectra of copolymers.
Optical Response of PBFBT-NP to NTR. As shown in Figure 1, before
addition of NTR, PBFBTꢀNP (0.1 µM) in the presence of 100 µM NADH
emitted an ultraꢀweak fluorescence because of PET process from backbone to
pꢀnitrophenol in the side chain (Scheme 1). Therefore, the ultraꢀlow
fluorescence background signal enables PBFBTꢀNP beneficial to the highly
sensitive detection of NTR. Upon addition of an aliquot of NTR, the
fluorescence intensity of PBFBTꢀNP increased. When the concentration of
NTR was added to 14.0 µg/mL, a significant enhancement of about 113ꢀfold in
fluorescence intensity was observed for PBFBTꢀNP (The fluorescence ratio
were shown in Figure S2 in Supporting Information), which is benefit from the
NTR reducing nitro into amino substance. The reason for the enhancement of
fluorescence is to inhibit the PET process. Due to copolymers forming very
loose aggregates in aqueous solution, it is possible that part of the nitrophenyl
groups may be trapped slightly inside of the aggregates at the first. However,
when NTR is added, the nitrophenyl groups still can be accessible to NTR that
could change the microenvironment of copolymers, resulting in aggregates
disrupting. According to the previously reported NTRꢀcatalyzed reactions, NTR
performs high reactivity to aromatic substrates due to strong interaction
between them, including hydrogen bonding, π−π stacking interaction,
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hydrophobic effect, and so on. The nitro groups in substrates can be reduced
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ultimately to an amino group,
which contributes to the distinct fluorescence
enhancement of PBFBTꢀNP. In Figure 1b, the fluorescence intensity in the
presence and absence of NTR was recorded at 530 nm. The fluorescence
increased along with the increment of NTR concentration. Moreover, we can
find that this method had a wide linear range from 0 to 14.0 µg/mL. The
detection limit for NTR was calculated as low as 2.9 ng/mL (LOD calculation
equation was shown in Supporting Information).
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Figure 1 (a) Fluorescence spectra and (b) fluorescence intensity of PBFBTꢀNP (0.1 µM)
after addition to different concentrations of NTR in the presence of NADH in TrisꢀHCl (10
mM, 7.4) (inset: linear relationship between intensity to and low NTR concentration). The
error bars represent the standard deviations of three parallel measurements. λex = 430 nm,
λ
em =530 nm.
In addition, we carried out the control experiments of NTR test without
NADH and PBFBT response to NTR. As shown in Figure S3a, no obvious
fluorescence enhancement of PBFBTꢀNP was observed in the absence of
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NADH, which presented that NADH as an electron donor plays a key role in
the reduction reaction. Figure S3b showed that the fluorescence of PBFBT only
increase a little in the presence of NTR and NADH because of PBFBT devoid
of pꢀnitrophenyl side groups. These results well proved that the fluorescence
enhancement of PBFBTꢀNP in the presence of NTR and NADH benefited from
the nitro reduction into amino group by NTR. Additionally, in the presence of
NTR, the emission maximum of both PBFBTꢀNP and PBFBT blue shifts may
result from NTR disrupting the aggregates of copolymers, which leads to the
conjugation change. To support the speculation, we measured the emission
spectra of PBFBTꢀNP and PBFBT in the presence of bovine serum albumin
(BSA). As shown in Figure S3, once addition of BSA in the solution of
PBFBTꢀNP, the maximum emission of PBFBTꢀNP blue shifts around 10 nm,
whereas that of PBFBT blue shifts about 2 nm. The tendency of maximum
emission blueꢀshift is consistent with that induced by NTR. Therefore, the
addition of enzyme may change the microenvironment of copolymers, resulting
in the aggregates changing and maximum emission blueꢀshift.
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Figure 2. The fluorescence intensity ratio of PBFBTꢀNP (0.1 µM) with respect to the reaction
time in the presence of varied concentrations of NTR. The error bars represent the standard
deviations of three parallel measurements. λex = 430 nm.
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To investigate the response time, we measured the fluorescence spectra of
PBFBTꢀNP/NADH solution, and fluorescence ratio at 530 nm was set as a
function of the reaction time. First, the concentration of NTR is fixed at 14.0
µg/mL, because the activity of NTR gives the maximum response at this
concentration, which is above mentioned. As shown in Figure 2, the ratio
increases from around 1 to 88 quickly for the PBFBTꢀNP/NADH incubating
with NTR for 3 min. After NTR reaction for 6 min, the ratio increases to the
plateau, which demonstrates the enzymatic activity of NTR is desirable.
Obviously, the same results were obtained at other concentrations of NTR, such
as 1.0, 6.0 µg/mL. It should be noted that the enzyme assay is complete in a
short time (6 min) without any timeꢀconsuming process. Other method reported
by Ma needs 30 min to react with NTR and that reported by Li needs 10 min to
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reach the plateau. Thus, our method presents a more rapid and simple assay
with high sensitivity for NTR.
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Figure 3 (a) Fluorescence ratio of PBFBTꢀNP after addition different kinds of potential
interfering species and 14.0 µg/mL NTR in the presence of NADH in TrisꢀHCl; (b)
Fluorescence ratio of PBFBTꢀNP after addition various redox species and 6.0 µg/mL NTR
in the presence of NADH in TrisꢀHCl. FDH: formate dehydrogenase (10 µg/mL); GR:
glutathione reductase (10 µg/mL); Cyt c: cytochrome c (10 µg/mL). The error bars
represent the standard deviations of three parallel measurements. λ = 430 nm.
ex
To further study the reaction selectivity of PBFBTꢀNP, various potential
interfering species were added respectively to PBFBTꢀNP (0.1 µM) solution in
+
+
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the presence of 100 µM NADH, such as salts (K , Na , Ca ), amino acids
(glycine, arginine, histidine), glucose, reactive oxygen species (H ),
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lysozyme, glutathione, and BSA. As shown in Figure 3a, no apparent
fluorescence enhancement was observed no matter any of the interfering
species was present. However, when 14.0 µg/mL NTR was added in the same
condition, the significant fluorescence signal enhancement was observed as
before. Furthermore, other kinds of potential interfering species, such as
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oxidoreductases (formate dehydrogenase, glutathione reductase) and electron
transporters (cytochrome c), were also investigated. As depicted in Figure 3b,
these electron transporters and enzymes exhibited no response to PBFBTꢀNP
and don’t interfere with the fluorescence enhancement of PBFBTꢀNP in the
presence of NTR. Therefore, PBFBTꢀNP exhibits high selectivity for NTR over
other species.
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In addition, an inhibition test about the activity of NTR was also
investigated. Dicoumarin is used usually as the inhibitor of NTR. As shown in
Figure S4, when PBFBTꢀNP was preꢀtreated with 0.05 mM dicoumarin and
then mixed with NTR, the fluorescence intensity is much lower than that in the
absence of dicoumarin. Moreover, when the concentration of dicoumarin
increased to 0.5 mM, the fluorescence intensity of PBFBTꢀNP was very low.
The results demonstrate that increasing the concentration of dicoumarin can
cause a greater decrease in fluorescence intensity. Also, the test confirmed that
the fluorescence signal response of PBFBTꢀNP to NTR can be effectively
inhibited by the inhibitor dicoumarin, and the enzymeꢀcatalyzed reduction
reaction brings the fluorescence signal enhancement response of PBFBTꢀNP to
NTR.
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Figure 4 Confocal fluorescence microscopy images of A549 incubated with 3 µM
PBFBTꢀNP under normoxic or anaerobic conditions. The fluorescence imaging of Hoechst
(fluorescent stains for labeling DNA), Dil (cell membrane dye) and PBFBTꢀNP were
collected at 425 ꢀ 475 nm (λex = 405 nm), 575 ꢀ 675 nm (λex = 559 nm), and 500 ꢀ 545 nm
(λex = 488nm), respectively. The scale bar is 20 µm.
Fluorescence Imaging of Hypoxia in A549 Cells. It is well known that the
level of NTR directly corresponds with the hypoxic status, which means the
2
decrease of O level can lead to the increase of NTR concentration in tumors.
As mentioned above, the nitro group in side chain of PBFBTꢀNP can readily be
transferred into the amino group via a reduction pathway by NTR, which brings
about the great enhancement of PBFBTꢀNP fluorescence. Thus PBFBTꢀNP is
expected to be capable of monitoring the hypoxic degree by reacting with the
endogenous NTR. To carry out the test of hypoxic tumor cells fluorescence
imaging by PBFBTꢀNP, A549 cells was used here because they were known to
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express NTR. The cells were grown under anaerobic conditions for 6 h, and
then were treated with 3 µM PBFBTꢀNP for 1.5 h. As we can see in Figure 4,
A549 cells incubated under normoxic conditions showed very weak green
fluorescence background signal in nucleus. Interestingly, the cells incubated
under anaerobic conditions showed very strong green fluorescence in nucleus.
As mentioned before that the very loose aggregates of PBFBTꢀNP can be
disrupted easily once the microenvironment changes. After PBFBTꢀNP enter
inside the complex cellular environment by endocytosis, the aggregates could
be disrupted then the cationic PBFBTꢀNP may go through nuclear pores inside
cell nuclei by the electrostatic and hydrophobic interactions. The cell nuclei
thus are labeled with PBFBTꢀNP. In addition, the control experiments of cell
imaging of PBFBT were carried out according to the same procedure as
PBFBTꢀNP. As shown in Figure S5, after incubating with PBFBT for 1.5 h no
matter under normoxic conditions or under anaerobic conditions, all A549 cells
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fluorescence emission without the requirement of NTR reduction.
In
general, the PBFBTꢀNP probe can enter inside the cells by endocytosis and can
be used to in cellulo imaging the hypoxic status via its reaction with the
endogenous NTR.
Basically, low cell cytotoxicity at appropriate concentration is necessary for a
potential fluorescent probe. Herein, the cell cytotoxicity of the PBFBTꢀNP was
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tested using MTT assay against A549 cells. The cell viability is expressed by
the ratio of the absorbance of the cells incubated with PBFBTꢀNP to the cells
incubated with culture medium only. As shown in Figure S6, the cell viability
remained at ~100% even if the concentration of PBFBTꢀNP was 10.0 µM after
incubation for 48 h. These results showed that PBFBTꢀNP with such low cell
cytotoxicity has greatly potential application as fluorescent probe for cell
imaging and future biological analysis application.
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Figure 5 Fluorescence imaging of E. coli incubated without PBFBTꢀNP (a) and with
PBFBTꢀNP (b) for 30 min. Scale bar = 50 µm. (c) E. coli viability against PBFBTꢀNP at
various concentrations for 60 min under white light. (d) PBFBTꢀNP sensitized ROS upon
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white light irradiation with an excitation of 488 nm. The error bars represent the standard
deviations of three parallel measurements.
Bacteria Imaging and Antibacterial Performance of PBFBT-NP. Taking
advantage of PBFBTꢀNP probe sensing NTR with sensitivity, rapidity and low
cytotoxicity, other NTRꢀrelated analysis, such as microbial imaging and
antibacterial activity were also investigated based on this new probe. It is
reported that the NTR is expressed commonly in microorganisms, such as
Escherichia coli. As mentioned above, the PBFBTꢀNP probe can clearly detect
the endogenous NTR and image the hypoxic tumor cells. Correspondingly, the
PBFBTꢀNP is anticipated to apply for microbial imaging that usually need
expensive commercial dyes. The E. coli cells imaging tests were carried out as
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followed: PBFBTꢀNP (5 µM ) was added to bacterial suspension (2×10 mL )
o
and then the mixture was incubated for half an hour at 37 C in the shaker. E.
coli bacteria then were observed under inverted fluorescence microscope
without any commercial dye staining. As shown in Figure 5a and 5b, the
bacteria are nonꢀblooming without PBFBTꢀNP probe, however, in the presence
of PBFBTꢀNP, the bacteria emit bright green fluorescence. These data indicated
that the nitro group of PBFBTꢀNP was reduced to the amine group by NTR in
E. coli and the recovered fluorescence of PBFBTꢀNP can be used to image
bacteria. In addition, the control experiment of E. coli cells imaging after
incubating with PBFBTꢀNP and dicoumarin were carried out. As shown in
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Figure S7, in the presence of dicoumarin, almost no bacteria emit green
fluorescence due to the dicoumarin inhibiting the NTR activity inside the
bacteria cells. Therefore, the nitro group of PBFBTꢀNP can’t be reduced to the
amine group by NTR, leading to E. coli no blooming. The results proved that
the probe should enter the bacteria first and then be reduced by NTR.
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Based on the microbial imaging, antiꢀbacteria test was further carried out by
flow cytometry. Recent researches indicated cationic conjugated polymers
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CCPs) possess high antibacterial activities and can be used as photosensitizer
30, 52ꢀ55
for photodynamic therapy.
The PBFBTꢀNP is one of typical CCPs, and
also can be considered as a PDT agent for antibacterial. As shown in Figure 5c
Complementary flow cytometry data were shown in Figure S8), the red line
(
7
ꢀ1
represents the mixture solution contained PBFBTꢀNP and E.coli (2×10 mL )
was exposed to white light for 60 min. The black line shows that the mixture
solution contained PBFBTꢀNP and E.coli was incubated in dark for 60 min.
Under white light irradiation, the antibacterial activities of PBFBTꢀNP showed
the concentrationꢀdependent response. When the concentration of PBFBTꢀNP
was 0.4 µM, over 40% E. coli bacteria were dead. Interestingly, when the
concentration of probe is 0.8 µM, the disinfection efficiency can reach up to
near 100% under white light. The corresponding fluorescence images were
obtained in the presence of PBFBTꢀNP and propidium iodide (PI, red
fluorescence for dead bacteria). As shown in Figure S9, green indicates live
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bacteria, red indicates dead bacteria, and orange represents the dying bacteria.
The orange and red bacteria cells were observed more and more along with the
increasing of PBFBTꢀNP concentration, which means more and more E. coli
bacteria were killed by PBFBTꢀNP under white light irradiation. The results are
highly consistent with that obtained by flow cytometry.
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To prove that the antibacterial effect should result from reactive oxygen
species produced by PBFBTꢀNP with white irradiation, we utilized
2,7ꢀdichlorofluorescein diacetate (DCFHꢀDA) as a cellꢀpermeable fluorogenic
ROSꢀsensitive probe in our experiment. As shown in Figure 5d, an apparent
emission at 525 nm was detected upon irradiating DCFH with white light in the
presence of PBFBTꢀNP, while the control without PBFBTꢀNP emits weak
fluorescence, which confirm the efficient generation of ROS by PBFBTꢀNP.
These results indicate that the PBFBTꢀNP also can be used as a good
antibacterial photosensitizer.
CONCLUSIONS
In summary, we have designed a new cationic conjugated polymer PBFBTꢀNP
as fluorescent probe that not only can be applied to conveniently detect the
NTR level and hypoxia status of tumor cells, but also can be applied to bacteria
imaging and highly efficient antiꢀbacterial. The nitro group as NTR capturing
group was introduced into the probe, which afforded a very low fluorescence
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background due to the PET process from the backbones of probe to the side
chains. The study in this paper also verified that PBFBTꢀNP probe exhibited
rapidity, simplicity, high selectivity and sensitivity to NTR. More importantly,
the in cellulo hypoxia status of tumor cells can be visualized by the greatly
enhanced fluorescence of PBFBTꢀNP through endogenous NTR reduction
reaction. In addition, the probe can be used for microbial imaging instead of
commercial stain dyes which are usually expensive. Interestingly, the probe
presented about 100% antibacterial activity against E. coli at low concentration
with white light irradiation. All the above features demonstrate that the new
multifunctional probe is promising for widespread use in NTRꢀrelated
biological analysis.
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ASSOCIATED CONTENT
Supporting Information. Detailed synthesis procedure of PBFBT, other
experimental procedures and figures as noted in text. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*
Phone: (086) 29ꢀ81530844. Fax: (086) 29ꢀ81530727. Eꢀmail: yltang@snnu.edu.cn.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
We are grateful for the financial support from the National Natural Science
Foundation of China (Grants 21675106, 21222509, 21335005), the 111 Project
(B14041), Program for Changjiang Scholars and Innovative Research Team in
University (IRT 14R33), and the Program for Innovative Research Team in
Shaanxi Province (No. 2014KCTꢀ28).
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