Rational construction of probes rendering ratiometric response to the cancer-specific enzyme NQO1

Article abstract of DOI:10.1016/j.dyepig.2016.09.031

Pursuit of fluorescent probes for identification of cancer biomarkers benefits reliable predictions of early cancer detection. Here, a strategy employing polymeric micelle assembly for encapsulation of a new enzyme (NQO1)-responsive small molecule was est

Full text of DOI:10.1016/j.dyepig.2016.09.031

Accepted Manuscript
Rational construction of probes rendering ratiometric response to the cancer-specific
enzyme NQO1
Qiang Fei, Li Zhou, Feiyi Wang, Ben Shi, Chunbao Li, Rui Wang, Chunchang Zhao
PII:
S0143-7208(16)30721-5
10.1016/j.dyepig.2016.09.031
DYPI 5483
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 27 June 2016
Revised Date: 3 September 2016
Accepted Date: 12 September 2016
Please cite this article as: Fei Q, Zhou L, Wang F, Shi B, Li C, Wang R, Zhao C, Rational construction of
probes rendering ratiometric response to the cancer-specific enzyme NQO1, Dyes and Pigments (2016),
doi: 10.1016/j.dyepig.2016.09.031.
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Graphical abstract
Rational Construction of Probes Rendering
Ratiometric Response to the Cancer-Specific
Enzyme NQO1
Qiang Fei, Li Zhou, Feiyi Wang, Ben Shi,
Chunbao Li, Rui Wang, Chunchang Zhao

ACCEPTED MANUSCRIPT
Rational Construction of Probes Rendering Ratiometric Response to the
Cancer-Specific Enzyme NQO1
Qiang Fei,a,b Li Zhou,c Feiyi Wang,b Ben Shi,b Chunbao Li,a,* Rui Wang,c Chunchang Zhaob,*
a. Department of Chemistry, School of Science, Tianjin University, Tianjin 300072 (China), E-Mail:
lichunbao@tju.edu.cn
b. Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of
Science & Technology, Shanghai 200237 (China), E-Mail: zhaocchang@ecust.edu.cn
c. Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science
& Technology, Shanghai 200237 (China)
Keywords: cancer biomarker, polymeric micelle, nanoprobe, ratiometric, cancer-specific NQO1
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Abstract
Pursuit of fluorescent probes for identification of cancer biomarkers benefits reliable predictions of early
cancer detection. Here, a strategy employing polymeric micelle assembly for encapsulation of a new
enzyme (NQO1)-responsive small molecule was established for facile access to nanoprobes with
favorable features in bioimaging, such as good aqueous solubility, enhanced photo-stability and
biocompatibility. In the presence of the enzyme NQO1, these nanoprobes underwent ratiometric
fluorescence changes from yellow-green fluorescence signals to red fluorescence signals. By exploiting
this favorable ratiometric response to NQO1, we explored these nanoprobes for evaluation of enzyme
activity in living cells, which showed distinct differentiation between NQO1-positive cancer cells and
NQO1-negative cells.
1. Introduction
Growing evidence indicates that identification of cancer biomarkers can render great promise for
highly reliable predictions of early cancer detection [1-2]. Thereby, there currently exists extensive interest
in the pursuit of fluorescent probes for the detection of crucial species, such as enzymes, intending to
target the bio-imaging of tumor cells [3-25]. Among many potential enzyme biomarkers,
NAD(P)H:quinone oxidoreductase isozyme 1 (NQO1) is an endogenous enzyme that is overexpressed in
a number of tumors, including colon, breast, liver and lung cancers, at levels of 2- to 50 fold greater than
in surrounding normal tissue [26]. NQO1 can specifically catalyze a two-electron reduction of various
quinones directly to the hydroquinone [27], inferring that profluorophores or prodrugs containing a
quinone moiety that are activated by NQO1 should show dramatic tumor-specific activity. Indeed, it has
been realized that the catalytic property of NQO1 makes this enzyme an ideal biomarker for cancer
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diagnosis as well as a therapeutic target [28, 29].
Despite the great implication of NQO1 as a biomarker for early diagnosis, only a few
fluorescent probes, to our knowledge, were reported for evaluation of this cancer-linked enzyme
[30-33]. Furthermore, these reported probes fulfill fluorescence turn-on functions by specific
NQO1-mediated removal of the quinone cage. However, such fluorescence turn-on mode is
susceptible to experimental conditions. It is therefore highly desirable to develop more reliable
and versatile probes with ratiometric response which can greatly eliminate experimental
interferences through self-calibration effect [34-38]. Another bottleneck encountered with common
organic probes lies in the fact that they always suffer from the limitation of low aqueous solubility,
poor photo- and chemical stability, which are detrimental to the practical application in native
biological media.
With all these considerations in mind, we report the polymeric micelle assembly for facile
access to nanoprobes rendering a ratiometric response to cancer-specific NQO1, representing a
design strategy that has been unexplored. To this end, we firstly designed a small molecule probe
BOD-NQO1 by tethering an NQO1 trigger (a trimethyl-locked quinone propionic acid) to a
6-hydroxylphenol-BODIPY platform through a linker p-aminobenzyl alcohol (Scheme 1).
Interestingly, reductant-mediated conversion of the trimethyl-locked quinone to hydroquinone
triggered lactonization to liberate p-aminobenzyloxycarbonate, which then underwent
self-immolation to deliver the red fluorescent 6-hydroxylphenol-BODIPY dye. Unfortunately, the
poor solubility of BOD-NQO1 in aqueous solution (common issue encountered with organic
probes) seriously retards its direct assay of NQO1 activity. To surpass this inconvenience,
BOD-NQO1 was embedded into the hydrophobic interior of a polymeric micelle assembly based
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on
an
amphiphilic
copolymer
mPEG-DSPE
glycol)-2000))
(1,2-dimyristoyl-sn-glycero-3-phosphorethanolamine-N-(methoxy(polyethylene
[39-41], and a nanoprobe (in short Nano-NQO1) was thus created (Scheme 1). This
encapsulation endowed Nano-NQO1 excellent aqueous solubility, significantly enhanced
chemical and photo-stability as well as biocompatibility. Satisfyingly, Nano-NQO1 displayed
favorable ratiometric response to NQO1, which has been successfully exploited for discriminative
imaging of NQO1-positive cells.
2. Experimental Section
2.1.Materials and instruments
All chemical reagents and solvents for synthesis were purchased from commercial suppliers and were
used without further purification. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400
spectrometer with chemical shifts reported in ppm at room temperature. Mass spectra were measured on a HP
1100 LC-MS spectrometer.
UV-vis absorption spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence spectra
were measured with a Varian Cary Eclipse Fluorescence spectrophotometer.
2.2.Synthesis
Compound 1 was synthesized according to literature procedures [42]. HO-BODIPY was obtained
according to previous work in our group [43].
2.2.1. 4-(((tert-butyldimethylsilyl)oxy)methyl)aniline 2
To a solution of 4-aminobenzyl alcohol (2.0 g, 16.24 mmol) in dry DMF (20 mL) were added
tert-butyldimethylsilyl chloride (3.67 g, 24.35 mmol) and imidazole (2.21 g, 32.46 mmol), the resulting
mixture was further stirred for 5 h at room temperature. The reaction mixture was then diluted with
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saturated brine solution and extracted with CH2Cl2. The organic layer was separated and washed with
brine solution for several times, then dried over anhydrous Na2SO4, evaporated under vacuum, the
obtained brown residue was purified by silica gel chromatography (petroleum ether-ethyl acetate), a
brown oil was obtained (3.15 g, 82 %). 1H NMR (CDCl3, 400 MHz) δ7.12 (d, 2H, J = 8.4 Hz), 6.67 (d, 2H, J
= 8.0 Hz), 4.62 (s, 2H), 3.80 (brs, 2H), 0.91 (s, 9H), 0.08 (s, 6H).
2.2.2. N-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa
-1,4-dien-1-yl)butanamide 3
Compound 1 (512 mg, 2.05 mmol) was dissolved in dry CH2Cl2 (15 mL), to which was added
N-methylmorpholine (676 ꢀL, 6.14 mmol). The mixture was cooled to -10 °C, an d stirred for 30 min. Then
isobutyl chloroformate (311ꢀL, 2.46 mmol) was added. After Stirring for 15 min, the solution of compound
2 (874 mg, 3.68 mmol) in dry CH2Cl2 was added. The resulting reaction mixture was stirred at room
temperature for 12 h. The organic layer was washed with water, 5 % HCl, 5 % NaHCO3, and saturated
brine solution, respectively, dried over anhydrous Na2SO4 and filtered. The solvent was removed by a
rotary evaporator, and the residue was purified by silica gel column chromatography (petroleum ether -
1
ethyl acetate) to provide compound 3 as a yellow-brown solid (336.3 mg, 35 %). H NMR (CDCl3, 400
MHz) δ7.36 (d, 2H, J = 8.4 Hz), 7.28 (brs, 1H), 7.22 (d, 2H, J = 8.0 Hz), 4.67 (s, 2H), 3.00 (s, 2H), 2.15 (s,
3H), 1.95 (s, 6H), 1.48 (s, 6H), 0.92 (s, 9H), 0.07 (s, 6H); m. p. 101.2 - 102.4 °C.
2.2.3. N-(4-(hydroxymethyl)phenyl)-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)
butanemide 4
To a solution of compound 3 (235 mg, 0.5 mmol) in methanol (20 mL) was added p-toluenesulfonic
acid (PTSA) (17.2 mg, 1.0 mmol), and the reaction mixture was stirred at room temperature for 3 h. After
evaporated the solvent, the residue was dissolved in CH2Cl2, washed with brine, dried over anhydrous
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Na2SO4, and filtered. The crude mixture was purified by column chromatography on silica gel (petroleum
ether- ethyl acetate) to afford compound 4 as a yellow solid (138 mg, 78 %). 1H NMR (CDCl3, 400 MHz)
δ7.39 (d, 2H, J = 8.4 Hz), 7.27 (d, 2H, J = 8.0 Hz), 7.15 (s, 1H), 4.63 (s, 2H), 3.01(s, 2H), 2.15 (s, 3H),
1.94 (s, 6H), 1.49 (s, 6H). HRMS (ESI): calcd for C21H26NO4: 356.1862; found: 356.1864 [M+H]+; m. p.
144.1-145.7 °C.
2.2.4. 4-(3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanamido)benzyl(4-nitrophenyl)
carbonate 5
Compound 4 (250 mg, 0.7 mmol) was dissolved in dry CH2Cl2 (25 mL), and 4-nitrophenyl
chloroformate (212.7 mg, 1.06 mmol) was added. To the above solution, Et3N (106.8 mg, 1.06 mmol) was
added dropwise. The resultant mixture was kept stirring for 6 h at room temperature. After completion, the
reaction solution was washed with water (3 × 100 mL), dried over anhydrous Na2SO4. The solvent was
removed, the residue was purified by column chromatography on silica gel (petroleum ether- ethyl
acetate) to obtain compound 5 (305 mg, 83 %) as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ8.27 (d, 2H,
J = 8.0 Hz), 7.46 (d, 2H, J = 8.4 Hz), 7.38-7.35 (m, 4H), 7.16 (s, 1H), 5.23 (s, 2H), 3.04(s, 2H), 2.16 (s,
3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.50 (s, 6H). HRMS (ESI): calcd for C28H28N2NaO8: 543.1743; found:
543.1746 [M+Na]+; m. p. 141.7-142.6 °C.
2.2.5. 2-ethyl-5,5-difluoro-1,3,11-trimethyl-8-((((4-(3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-
dien-1-yl)butanamido)benzyl)oxy)carbonyl)oxy)-12-phenyl-5H-pyrrolo[1',2':3,4][1,3,2]diazaborinino[1,6-a]i
ndol-4-ium-5-uide BOD-NQO1
Compound 5 (93.7 mg, 0.18 mmol) and 4-dimethylaminopyridine (DMAP) (22.0 mg, 0.18 mmol) were
dissolved in dry CH2Cl2, then added HO-BODIPY (50 mg, 0.12 mmol), and the reaction mixture was
stirred for 10 h at room temperature. The reaction mixture was extracted with CH2Cl2 and water. The
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organic layer was separated and dried over anhydrous Na2SO4. After the solvents was removed by rotary
evaporation, the crude product was purified by silica gel column chromatography (petroleum ether-
dichloromethane- ethyl acetate) to provide compound BOD-NQO1 (21 mg, 22 %) as a red solid. 1H NMR
(CDCl3, 400 MHz) δ7.56- 7.54 (m, 4H), 7.44 (d, 2H, J = 8.8 Hz), 7.41 (d, 1H, J = 8.8 Hz), 7.36 (d, 2H, J =
8.4 Hz), 7.34-7.32 (m, 2H), 7.18 (s, 1H), 6.81 (dd, 1H, J1 = 8.8 Hz , J2 = 2.0 Hz), 5.21(s, 2H), 3.02 (s, 2H),
2.69 (s, 3H), 2.38 (q, 2H, J = 7.6 Hz), 2.16 (s, 3H), 1.96 (d, 6H, J = 2.4 Hz), 1.59 (s, 3H), 1.49 (s, 6H), 1.39
(s, 3H), 1.03 (t, 3 H, J = 7.6 Hz); 13C NMR (CDCl3, 100 MHz) δ 191.6, 187.5, 171.2, 170.3, 165.2, 153.5,
153.0, 151.5, 149.8, 148.2, 148.1, 144.3, 143.3, 142.9, 142.0, 138.3, 138.2, 138.0, 136.9, 134.9, 133.8,
130.6, 129.5, 129.4, 128.1, 121.7, 119.7, 114.9, 106.7, 69.8, 50.5, 38.4, 29.1, 17.2, 14.2, 14.1, 12.7, 12.3,
12.2, 11.1; HRMS (ESI): calcd for C46H46BF2N3NaO6: 808.3345; found: 808.3352 [M+Na]+; calcd for
C46H46BF2N3KO6: 824.3085; found: 824.3112 [M+K]+; m. p. 122.8-123.5 °C.
2.3. Preparation of Nano-NQO1 for NQO1 assay. BOD-NQO1 (0.03 mg) was poured into the micelle
solution of mPEG-DSPE (6 mg) in distilled, deionised water (9 mL) under vigorous sonication for 30 min.
Then the aqueous solution was filtered through a 0.22 µm polyvinylidene fluoride (PVDF) syringe driven
filter and dialyzed against distilled water for 24h, and the Nano-NQO1 solution was obtained. The initial
micelles solution was then diluted in PBS buffer for further studies.
3. Results and discussion
BOD-NQO1 was successfully synthesized by conjugation of HO-BODIPY and trimethyl-locked quinone
propionic acid functionalized p-aminobenzyl alcohol through a carbonate linkage (Scheme 2). The
chemical structure of BOD-NQO1 was identified by high resolution mass spectrometry (HRMS), 1H NMR
and 13C NMR spectroscopy.
With BOD-NQO1 in hand, we initially evaluated the absorption and emission spectral changes in the
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presence of a reductant under model conditions (acetonitrile/PBS buffer, 1:1, v/v, 20 mM, pH 7.4, room
temperature). To improve the aqueous solubility of BOD-NQO1, acetonitrile was here chosen as a
co-solvent. It should be mentioned that BOD-NQO1 only is stable for at least 48 h in the testing
conditions, indicative that BOD-NQO1 is suitable for monitoring analytes in the buffer solutions (Fig. S1).
As expected, the probe afforded distinct optical changes when sodium dithionite was employed as a
reducing chemical. In the absence of sodium dithionite, an intensive absorption at 509 nm was noted for
free BOD-NQO1 (quantum yield: 0.04). Notably, addition of sodium dithionite introduced a dramatic
red-shift from 509 to 542 nm (Fig. 1A) with an obvious isosbestic point at 517 nm. The changes in the
fluorescence spectra upon addition of sodium dithionite were further investigated. Time dependent
fluorescence enhancement was observed with the emission wavelength red-shift from 550 nm to 575 nm
(Fig. 1). These optical changes resulted from reductant triggered the lactonization and self-immolation to
liberate the red fluorescent HO-BODIPY dye (Scheme 1). Dithionite initiated the production of
hydroquinone, followed by a cyclic cleavage to give p-aminobenzyloxycarbonate. Self-immolation
eventually occurred to generate the red fluorescent dye. This cascade reaction sequence was also
verified by both high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry
(HRMS) experiments (Fig. S2). BOD-NQO1 had a HPLC retention time at 21.79 min and HRMS signal [M
+ Na]+ at 808.3352. Likewise, the spectra of BOD-NQO1 in the presence of dithionite showed a dominant
retention time at 8.94 min and peak of [M - H]- with 402.1830, identical to that of HO-BODIPY. The identity
of the fluorescent product to be HO-BODIPY was further revealed 1H NMR spectroscopy (Fig. S2).
It should be mentioned that BOD-NQO1 is not an ideal probe to assay the NQO1 activity, since
organic solvents must be used as co-solvents to facilitate the solubility in aqueous solution. Nevertheless,
organic solvents are detrimental to enzymes. Generally, small organic probes always suffer from the
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limitation of low aqueous solubility. Appending hydrophilic substituents to the chromophore core is
commonly employed, which requires tedious synthesis procedures. In contrast, a supramolecular
assemble strategy offers an easy-to-achieve tool to afford nanoparticles with excellent aqueous solubility.
Therefore, supramolecular micelle of amphiphilic polymers was explored to encapsulate BOD-NQO1 due
to the establishment capability of amphiphilic building blocks assembling to form hydrophilic nanoparticles
capable of entrapment hydrophobic dyes [39-41]. In this way, we constructed a nanoprobe Nano-NQO1
for interrogating the fluorescence imaging applications.
As is well known, entrapment of organic dyes with small Stokes shifts within a confined space always
introduces self-quenching of fluorescence behaviour [34]. Thus, we systematically investigated the effects
of encapsulating amount of BOD-NQO1 on the fluorescence of Nano-NQO1. In general, the amount of
the encapsulated BOD-NQO1 can be fine-tuned in a broad range. As shown in Fig. S3, the change of the
loading amount induced varied fluorescence intensity, and relatively strong emission could be obtained
when the ratio BOD-NQO1/ mPEG-DSPE was empirically optimized as 0.004−0.02 by weight. In the
optimized entrapment protocol, the ratio of BOD-NQO1/ mPEG-DSPE is about 0.005 at pH 7.4. The
morphologies and hydrodynamic size of Nano-NQO1 were then determined by transmission electron
microscopy and dynamic light scattering, respectively. The as-prepared nanoparticles are uniform with an
average hydrodynamic size of 25 nm, which was in accordance with the diameters given by TEM (Fig.
S4). Besides, the photo- and chemical stability of Nano-NQO1 was tested (Fig. S5). No obvious changes
in absorption and emission spectra were observed after storage for 48 h. Good photostability was also
revealed by continuous irradiation with an Hg/Xe lamp for 2 h, during which time only a minimal change
minimal change was observed in the optical spectra. Overall, the outstanding stability and good aqueous
solubility endow superiority of Nano-NQO1 in bioimaging.
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We next evaluated the fluorescence changes of Nano-NQO1 toward a reducing reagent in
PBS buffer. Fig. S6 represents the time-dependent fluorescence spectral changes upon addition
of 1 mM sodium dithionite. Gratifyingly, dithionite elicited a distinct ratiometric fluorescence
change identified by the observation of a gradual increase in the emission intensity at 585 nm and
a concomitant loss of emission at 550 nm. Obviously, this remarkable ratiometric fluorescence
signal change arose from reductant-promoted conversion of BOD-NQO1 to HO-BODIPY within
the hydrophobic interior of micellar assembly. After the successful demonstration of Nano-NQO1
as a suitable ratiometric fluorescent probe for sensing reducing reagent, we sought to establish
this newly designed nanoprobe is indeed promising fluorescent probe capable of NQO1 activity
assay. As shown in Fig. 2A, in the absence of NQO1, Nano-NQO1 displayed a relatively weak
emission band at 550 nm. Upon incubation with NQO1, the emission maximum at 550 nm
underwent a reduction accompanied by a remarkable fluorescence enhancement at 585 nm. An
obvious isoemission point was also noted at 557 nm. Obviously, the reaction rate between
Nano-NQO1 and NQO1 is slower than that of Nano-NQO1 with Na2S2O4. This result can be
ascribed to the fact that the steric hindrance of the biomacromolecule NQO1 limited its free
accessibility to BOD-NQO1 located in the core of the assemblies [12]. It is worth mentioning that
the fluorescence intensity ratio of the two emission bands at 550 and 585 nm (I585/I550) increased
from 0.7 to 2.5 as the incubation time proceeded (Fig. 2B), showing a 3-fold enhancement factor,
which allowed the assay of NQO1 activity by an efficient ratiometric fluorescence mode. In
contrast, minimal ratiometric fluorescence change was observed by pretreatment of NQO1 with
dicoumarol (3,3’-methylene-bis(4-hydroxycoumarin)), a commonly used inhibitor of NQO1,
confirming the key role of NQO1 in introducing the ratiometric fluorescence signal change (Fig.
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2B, Fig. S7). The kinetic values of Nano-NQO1 against NQO1 were subsequently determined by
incubating NQO1 with Nano-NQO1 in PBS buffer at 37 °C (Fig. 2C). According to Michaelis–
Menten equation, the Km and Vmax values were then calculated to be 6.77 µM and 0.043 µM min-1,
respectively (Fig.S8).
Nano-NQO1 afforded good selectivity for NQO1 over other related species when utilizing
I585/I550 as the detection signals. As shown in Fig. 3, when other species such as phosphatase,
aprotinin, collagenase , lipase, glucoamylase, 10% FBS, 10% human plasma were incubated
with Nano-NQO1 in the presence of 500 ꢀM NADH, minimal fluorescence changes were noted.
After demonstration of the promising responsive behavior of Nano-NQO1 for specific
monitoring the NQO1 activity, we then explored the ratiometric characteristic of Nano-NQO1 for
evaluation of enzyme activity in living cells (Fig. 4 and Fig. S9). Since HT-29 cells are known to
show high NQO1 activity and H596 cells are identified to have negligible NQO1 activity, here
HT-29 cells and H596 cells were explored as NQO1-positive and NQO1-negative model cell
systems, respectively. HT-29 cells incubated with NaNo-NQO1 for 30 minutes gave moderate
fluorescence signals in both green channel and red channel. The ratio of the two images
generated from red to green channel is estimated to be about 0.8 (Fig. 4A). As the incubation time
increases, the image ratio enhanced to be 2 (Fig. 4B). Such time-dependent ratio change can be
attributed to NQO1-induced reduction of Nano-NQO1. In sharp contrast, no time-dependent ratio
enhancement was observed upon incubation with NQO1-negative H596 cells. When H596 cells
were treated with Nano-NQO1 for 30 min (Fig. 4C) and 120 min (Fig. 4D), the image ratio
remained constant value at about 0.6, inferring no occurrence of reduction. These results indicate
that Nano-NQO1 can serve as an efficient tool for differentiating NQO1-positive cancer cells from
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NQO1-negative cells. An enzyme inhibition test was then performed to validate that the obtained
imaging signal indeed derived from NQO1 activity. When HT-29 cells were pretreated with various
concentrations of dicoumarol and followed by incubation with Nano-NQO1 for 120 minutes, the
ratio was greatly reduced when compared to that without dicoumarol (Fig. 4B). As shown in Fig.
4E-F, with increasing the concentration of dicoumarol from 0 to 1 mM, the fluorescence ratio from
red to green channel ultimately decreased to be 1. Collectively, Nano-NQO1 possesses the
capability of differentiating NQO1-expressing and NQO1-nonexpressing cancer cell via the
preferable ratiometric measurement of NQO1 activity.
4. Conclusion
In summary, we report the polymeric micelle assembly based nanoprobes for ratiometric monitoring
cancer-specific NQO1. To this end, we firstly designed a small molecule probe BOD-NQO1, wherein
reductant can mediate cascade reactions to deliver the red fluorescent 6-hydroxylphenol-BODIPY dye.
Unfortunately, the poor solubility of BOD-NQO1 in aqueous solution seriously retards its direct assay of
NQO1 activity. Encapsulation of BOD-NQO1 within the hydrophobic interior of a polymeric micelle
assembly was then employed to create a nanoprobe with excellent aqueous solubility, significantly
enhanced chemical and photo-stability as well as biocompatibility. Satisfyingly, such nanoprobe
possesses the capability of differentiating NQO1-positive cancer cells from NQO1-negative cells via the
preferable ratiometric measurement of NQO1 activity. We expect that the present design strategy would
benefit the establishment of versatile fluorescence probes for practical assay of cancer biomarkers.
Acknowledgements
We gratefully acknowledge the financial support by the National Science Foundation of China (grant
numbers 21172071, 21190033, 21372083), National 973 Program (2013CB733700) and the Programme
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of Introducing Talents of Discipline to Universities (B16017).
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Figure Captions
Fig. 1. Time dependent spectra changes of BOD-NQO1 (10 µM) in the presence of 5 mM Na2S2O4 under
model conditions (acetonitrile/PBS buffer, 1:1, v/v, 20 mM, pH 7.4, room temperature), (A) absorption, (B)
fluorescence; and (C) Time dependent fluorescence intensity changes at 575 nm.
Fig. 2. (A) Time dependent fluorescence spectra changes of Nano-NQO1 (1.1 mg/mL, 10 ꢀM
BOD-NQO1) in the presence of NQO1 (80 µg) and NADH (500 µM); (B) the ratiometric fluorescence
intensity changes of Nano-NQO1 + NQO1 (80 µg) + NADH (500 µM) in the absence and presence of
dicoumarol (1 mM) in PBS (pH 7.4) containing 0.007% bovine serum albumin (BSA) at 37 °C. (C) Kinetic
course of Nano-NQO1 in the presence of NQO1 (80 µg) in PBS (pH 7.4) containing 0.007% BSA at 37 °C;
Solid red line represents best fit to Michaelis–Menten equation. Here, the concentration of Nano-NQO1
(mg/mL) was converted to that of BOD-NQO1 (ꢀM) when calibrating the plot, thus the obtained plot can
be used for comparison with the reported ones. λex = 510 nm.
Fig. 3. Time-dependent ratiometric fluorescence changes of Nano-NQO1 (10 ꢀM BOD-NQO1) toward
various substrates in PBS (pH 7.4) at 37 ℃. λex = 510 nm. These analytes of interest contain
phosphatase, aprotinin, collagenase Ⅰ , lipase, glucoamylase, 10% FBS, 10% human plasma,
nitroreductase (NTR) and NQO1.
Fig. 4. Confocal microscopy images of HT-29 (NQO1-positive) (A, B), H596 (NQO1-negative) (C, D) cells,
HT-29 cells in the presence of dicoumarol 0.2 mM (E), 1 mM (F), λex = 514 nm, the emission was
collected at 520–560 nm (assigned to green channel) and 570–610 nm (red channel), ratio image
generated from red to green channel: A and C) cells were incubated with Nano-NQO1 (1.1 mg/mL) for 30
min; B, D, E, F for 120 min.
Scheme 1. Schematic representations of reductant-mediated cascade reactions in
conversion of
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BOD-NQO1 to HO-BODIPY and polymeric micelle assembly for facile access to nanoprobes with
ratiometric response to NQO1.
Scheme 2. Synthetic route for BOD-NQO1.
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Figures
Fig. 1
Fig. 2
Fig. 3
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Fig. 4
Scheme 1.
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Scheme 2.
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Rational Construction of Probes Rendering Ratiometric Response to the
Cancer-Specific Enzyme NQO1
Qiang Fei,†,‡ Li Zhou,# Feiyi Wang,‡ Ben Shi,‡ Chunbao Li,*,† Rui Wang,# He Tian,‡ Chunchang Zhao*,‡
† Department of Chemistry, School of Science, Tianjin University, Tianjin 300072 (China), E-Mail:
lichunbao@tju.edu.cn
‡ Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science &
Technology, Shanghai 200237 (China)
E-mail: zhaocchang@ecust.edu.cn
# Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science &
Technology, Shanghai 200237 (P. R. China.)
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1. Cells culture and imaging. HT-29 cells were cultured in McCoy’s 5A medium supplemented with 10 %
fetal bovine serum (FBS) at 37 °C in a humidified a tmosphere of 5/95 CO2/air incubator.
For fluorescence imaging, cells were incubated in glass bottom dishes and stabilized for 24 h. For imaging
HT-29 cells, the cells were incubated with Nano-NQO1 (1.1 mg/mL) for 30 min and 120 min, respectively,
subsequently the cells were washed three times with PBS. Then, the confocal imaging was performed using
Nikor AIR with a 60 × oil objective. The excitation wavelength was at 514 nm, the emission was collected at
520-560 nm for green fluorescence and emission at 570-610 nm for red channel.
The same procure was applied to H596 cells with the exception of using Roswell Park Memorial Institute
1640 medium (RPMI-1640) as the culture medium.
In inhibition assay, the cells were incubated with various concentrations of dicoumarol prior to treatment
with Nano-NQO1.
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2. Chemical stability of BOD-NQO1 in testing buffer solution.
Fig. S1. Chemical stability of BOD-NQO1 in PBS-CH3CN (V/V, 1:1) determined by time dependent
fluorescence spectra changes.
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3. Identification of the product by HPLC and HRMS.
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Fig. S2 HPLC profiles of (a) BOD-NQO1, (b) BOD-NQO1 + Na2S2O4, (c) HO-BODIPY. HPLC was carried
out on a ZORBAX 80A Extend-C18 column (4.6 × 250 mm, 5 ꢀm, Agilent). The conditions were as follows:
volume ratio of acetonitrile/H2O = 90:10; flow rate is 0.5 mL·min−1; detection under UV light at 509 nm and 542
nm. (d) High-resolution mass spectrometry of HO-BODIPY. (e) 1H NMR spectrometry of the fluorescent product
of BOD-NQO1 + Na2S2O4.
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4. The effects of encapsulating amount of BOD-NQO1 on the fluorescence.
Fig. S3 The effects of encapsulating amount of BOD-NQO1 in mPEG-DSPE (6 mg) on the fluorescence.
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