A self-referenced nanodosimeter for reaction based ratiometric imaging of hypochlorous acid in living cells

Article abstract of DOI:10.1039/c2sc21485g

Hypochlorous acid (HOCl) is biosynthesized from hydrogen peroxide via catalysis of myeloperoxidase in lysosomes of immunological cells. Despite being harnessed by immune systems against invading pathogens, biogenic HOCl can also damage host tissues and has been associated with a number of diseases. In this edge article, Foerster resonance energy transfer based ratiometric imaging of lysosomal HOCl was achieved with silica nanoparticles comprising FITC (donor dye) and a nonfluorescent chemodosimeter which turned into rhodamine (acceptor dye) upon HOCl triggered tandem oxidation and β-elimination of the doped chemodosimeter. The nanodosimeter exhibited distinct biochemical properties relative to small molecule-based free chemodosimeters, e.g. lysosome-homing specificity and compatibility with aqueous media, enabling facile monitoring of lysosomal HOCl by conventional flow cytometry. The nanoprobe would be of broad utility for studies on in vivo generation and the impact of lysosomal HOCl in living cells or even in animals.

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ARTICLE TYPE
A self-referenced nanodosimeter for reaction based ratiometric imaging
of hypochlorous acid in living cells
Xuanjun Wu,a Zhu Li,a Liu Yang,a Jiahuai Hanb and Shoufa Hana,*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Hypochlorous acid (HOCl) is biosynthesized from hydrogen peroxide via catalysis of myeloperoxidase in
lysosomes of immunological cells. Despite being harnessed by immune systems against invading
pathogens, biogenic HOCl also could damages host tissues and has been associated with a number of
diseases. In this report, Förster resonance energy transfer based ratiometric imaging of lysosomal HOCl
10 was achieved with silica nanoparticles comprising FITC (donor dye) and a nonfluorescent
chemodosimeter which turned into rhodamine (acceptor dye) upon HOCl triggered tandem oxidation and
β-elimination of the doped chemodosimeter. The nanodosimeter exhibited distinct biochemical properties
relative to small molecule-based free chemosodimeter, e. g. lysosome-homing specificity and
compatibility with aqueous media, enabling facile monitoring of lysosomal HOCl by conventional flow
15 cytometry. The nanoprobe would be of broad utility for studies on in vivo generation and impacts of
lysosomal HOCl in living cells or even in animals.
cells. Here we report Förster resonance energy transfer (FRET)
based ratiometric imaging of lysosomal HOCl with mesoporous
silica nanoparticles (MSN) functionalized with FITC, which
50 serves as the donor dye, and a highly selective rhodamine-derived
chemodosimeter which turns into highly fluorescent rhodamine
acceptor upon analyte triggered oxidative opening of the
intramolecular thioether (Fig. 1).
Introduction
Hypochlorous acid (HOCl) is a natural oxidant that can be
synthesized from hydrogen peroxide and chloride with the aide of
20 myeloperoxidase in a number of immunological cells[1]. Despite
being harnessed by immune systems against microorganism
infections, in vivo generated HOCl could also be detrimental and
has been implicated in multiple diseases such as rheumatoid
arthritis and neuron degeneration.[2] Chemosensors that allow
25 imaging or quantitation of HOCl will be valuable for
investigations on HOCl related biological processes. In line with
this, a number of imaging agents that become emissive upon
analyte triggered oxidation have been developed by integration of
fluorophores with HOCl-responsive functionalities.[3] Single-
30 intensity based probes are often hindered in quantitative assays
due to many uncertainties, e.g. uneven distribution of imaging
agents in biological specimens. Ratiometric probes that could
normalize these interferences has been largely unexplored for
HOCl.[4]
488 nm
525 nm
488 nm
590 nm
HO
O
O
HO
O
O
COOH
COOH
N
O
N
N
O
N
O
HOCl
O
S
N
H
N
H
RB-FITC-MSN
ThioRB-FITC-MSN
55
Fig. 1 FRET based ratiometric imaging of HOCl with ThioRB-
FITC-MSN via analyte triggered oxidative opening of the
intramolecular spirothioether to give acceptor fluorophore.
35
Lysosomes, hallmarked by acidic intracompartmental pH
(6.0-4.5), are the major degradation machinery for internalized
microorganisms. Given the roles of HOCl in inactivation of
microorganism in host defense and the fact that myeloperoxidase
is a lysosomal enzyme dedicating to the biogenesis of HOCl,
60 Experimental
Material and methods
40 probes that can selectively report levels of lysosomal HOCl will
be of tremendous interest for real time studies of in vivo
generation and impacts of HOCl. With pKa of 7.46, HOCl readily
dissociates into hypochlorite (OCl-) in cytosol. Small molecule
based probes are cell membrane permeable and diffuse into cells.
45 Due to the lack of the capability to target lysosomes, existing
chemosensors mostly responded to cytosolic hypochlorite inside
LysoTracker Blue DND 22 was purchased from Invitrogen.
Methoxy-PEG-succinimidyl propionate was obtained from
Jiaxing Biomatrix Inc. The aqueous solution of sodium
65 hypochlorite (NaOCl) and other chemicals were obtained from
Alfa Aesar. Column chromatography was performed on silica gel
(100-200 mesh). NMR spectra (1H at 400 MHz and 13C at 100
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dichloromethane/ triethylamine (10/1) as the eluent to afford RB-
MHz) were recorded on a Bruker instrument using tetramethyl
silane as the internal reference. Mass analysis was performed in
Bruker En Apex ultra 7.0T FT-MS. Fluorescence spectra and
1
CM in 85% yield. HNMR (400 MHz, CD3OD): δ 8.34 (dd, 1H,
J1= 1.04 Hz, J2 = 1.16 Hz), 7.81 (m, 3H), 7.56 (m, 3H), 7.06 (m,
UV-vis absorption spectra were recorded on a spectrofluorometer 60 4H), 5.51 (s, 1H), 3.68 (q, 8H, J = 7.12 Hz), 3.21 (q, 3H, J = 7.32
5
(SpectraMax M5, Molecular Device). L929 cells and HeLa cells
were obtained from American Type Culture Collection (ATCC).
Confocal fluorescence microscopy images were obtained on
Leica SP5 using the following filters: λex@543 nm and
Hz), 1.31 (t, 12H, J = 4.16 Hz) ; 13C-NMR (100 MHz, CD3OD):
165.56, 158.90, 157.90, 155.71, 133.81, 132.77, 132.46, 130.90,
130.85, 130.26, 130.13, 129.99, 127.61, 125.78, 114.04, 113.34,
+
95.86, 51.56, 46.30, 45.44, 11.43, 7.80; HRMS (C31H35N2O3 )
λem@565-625 nm for rhodamine B signal; λex@488 nm and 65 calculated (M+): 483.2642, found: 483.2646.
10 λem@500-530 nm for FITC signal. The fluorescence of
LysoTracker blue and that of fluorescein inside cells were
merged by using Photoshop CS 5.0. Flow cytometric data were
obtained on Beckman Coulter. The fluorescence emission
intensity of FITC was recorded with filter FL1 (510-535 nm)
15 while that of ThioRB was recorded by filter FL2 (565-625 nm).
10000 cells were analyzed and the data were processed by Origin
8.5.
Synthesis of ThioRB-ester
Sodium hydrosulfide (0.2 g) was added to a flask containing
DMF (10 ml) and RB-CM (1 g). The mixture was stirred at rt for
10 min. The solution was poured into water (200 ml) and then
70 extracted by dicholomethane/water (200 ml). The organic layer
was collected, dried over sodium sulfate and then evaporated in
vacuo. The residue was purified by silica gel chromatography
using hexanes/triethylamine (10/1) as the eluent to afford
Synthesis of compound 1
1
ThioRB-ester as the desired product in 55% yield. H-NMR (400
To the solution of rhodamine
B (10 g) in anhydrous 75 MHz, DMSO): δ 7.49 (d, 1H, J1 = 7.68 Hz), 7.32 (t, 1H, J = 7.68
20 tetrahydrofuran (50 ml) was added lithium aluminium hydride (4
g) in portions. The mixture was stirred at rt for 48 hrs and then
quenched with dropwise addition of ethyl acetate (50 ml). The
mixture were poured into water (200 ml) and then extracted with
Hz), 7.22 (t, 1H, J = 7.52 Hz), 6.71 (m, 3H), 6.36 (m, 2H), 6.23
(m, 2H), 5.22 (dd, 1H, J1 = 3.36 Hz, J2 = 3.44 Hz), 3.64 (s, 1H),
3.35 (s, 3H), 3.30 (q, 8H, J = 2.28 Hz), 1.08 (t, 12H, J = 3.80 Hz)
;
13C-NMR (100 MHz, DMSO): 171.94, 151.32, 150.79, 149.88,
ethyl acetate (200 ml). The organic layer was collected, dried 80 147.82, 147.73, 141.85, 131.89, 131.15, 128.80, 127.81, 127.05,
25 over sodium sulfate, and then concentrated by rotary evaporation.
The residue was purified by silica gel chromatography using
ethyl acetate/hexanes/triethylamine (10/2/1) as the eluent to
afford the desired product (6.5 g, 65%). 1H-NMR (400 MHz,
CDCl3): δ 7.45 (m, 1H), 7.29 (m, 3H), 6.71 (dd, 2H, J1 = 0.64
30 Hz, J2 = 0.64 Hz), 6.42 (m, 2H), 6.30 (m, 2H), 4.59 (s, 2H), 3.35
(q, 8H, J = 7.08 Hz), 1.18 (t, 12H, J = 7.04 Hz); 13C-NMR (100
MHz, CDCl3): 151.67, 147.77, 138.66, 131.38, 129.96, 129.61,
129.47, 127.96, 126.89, 124.24, 120.45, 111.43, 107.85, 107.42,
98.77, 97.78, 62.94, 46.18, 44.39, 12.64, 11.46.
124.54, 114.95, 114.11, 108.54, 96.97, 62.20, 52.04, 48.60,
44.15, 44.05, 12.92; HRMS (C31H36N2O3S): calculated (M+H+):
517.2519, found: 517.2521.
Preparation of ThioRB-FITC-MSN
85 Synthesis of ThioRB-APTS: ThioRB-ester (0.1 g) was added in
(3-aminopropyl)triethoxysilane (APTS) (2 ml) in a capped vial.
The mixture was stirred at rt in dark for 7 days. The resultant
product, ThioRB-APTS, was directly used for the preparation of
mesoporous silica nanoparticles.
90
Synthesis of FITC-APTS: FITC (50 mg) was added in DMF
(500 ꢀl) containing APTS (300 ꢀl) in a capped vial. The mixture
was stirred at rt in dark for 2 hrs. The resultant product, FITC-
APTS, was directly used for the preparation of mesoporous silica
nanoparticles.
35 Synthesis of compound 2
To the solution of compound 1 (5 g) in dichloromethane (30 ml)
was slowly added PCC (5 g). The mixture was stirred at rt for 20
min, poured into water (150 ml) and then extracted with
dichloromethane (200 ml). The organic layer was collected, dried
40 over sodium sulfate, and then concentrated by rotary evaporation.
The residue was purified by silica gel chromatography using
ethyl acetate/hexanes/triethylamine (10/2/1) as the eluent to
95
Preparation of ThioRB-FITC-MSN: For MSNs assayed in
Na2HPO4-citrate buffer (100 mM, pH 5), the as-prepared
solutions of ThioRB-APTS (100 ꢀl) and FITC-APTS (100 ꢀl)
and tetraethyl orthosilicate (TEOS, 2.3 ml) were added to a clean
1
afford compound 2 as the desired product (4 g, 80%). H-NMR
flask
containing
deionized
water
(240
ml),
(400 MHz, CDCl3): δ 7.55 (d, 1H), 7.40 (m, 2H), 6.94 (dd, 2H, J1 100 cetyltrimethylammonium bromide (CTAB) (0.5 g) and 1.75 ml
o
45 = 6.0 Hz, J2 = 8.4 Hz), 6.65 (m, 2H), 6.41 (m, 4H), 3.37 (q, 8H, J
= 6.92 Hz), 1.19 (t, 12H, J = 7.02 Hz); 13C-NMR(100 MHz,
CDCl3): 152.66, 152.33, 148.66, 130.50, 130.06, 129.27, 128.18,
of aqueous NaOH (2 M) at 80 C. The mixture was stirred at 80
oC for 1 hr and then centrifuged at 10,000 rpm for 10 min to
collect the silica MSNs. The MSNs were further treated with
methanol containing HCl (5 %) under reflux for 3 hrs. The
123.95, 122.31, 111.68, 108.00, 107.90, 99.34, 97.66, 97.50,
+
44.50, 12.71; HRMS (C28H31N2O2 ) calculated (M+): 427.2380, 105 sample was centrifuged and the pellet was further subjected to
50 found: 427.2359.
repeated resuspension in methanol by ultrasonication,
centrifugation and decantation to remove the unreacted chemicals
and CTAB.
Synthesis of RB-CM
(Methoxycarbonylmethylene)-triphenylphosphorane (4 g) was
added to a flask containing methanol (20 ml) and compound 2 (4
g). The mixture was stirred at rt for 12 hrs. The solution was
55 concentrated by rotary evaporation to remove the solvent, and the
residue was purified by silica gel chromatography using
For MSNs assayed in PBS (pH 7.4), the nanoparticles were
110 prepared using the same procedure as described above with the
exception that a lower volume of FITC-APTS (30 ꢀl) was utilized
in co-condensation with ThioRB-APTS (100 ꢀl) and tetraethyl
orthosilicate (2.3 ml).
2
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Surface modification of MSNs with poly(ethylene glycol)
55 addition of NaOCl.
The as-prepared MSNs (200 mg) were added to
dimethylformamide (20 ml) containing methoxy-PEG-
succinimidyl propionate (MW 5000, 0.145 g) and triethylamine
(200 ꢀl). The mixture was sonicated for 90 min followed by
addition of saturated NaHCO3 solution (30 ml). The mixture was
further sonicated for 90 min, and then centrifuged. The
nanoparticles were collected, extensively washed with deionized
water to remove any residual chemicals, and then stored in water
10 (50 mg ml-1) for subsequent cell assays. The nanoparticles before
and after pegylation were analyzed by transmission electron
microscopy (TEM), scanning electron microscopy (SEM) and
dynamic light scattering.
Characterization of ThioRB-ester for HOCl with High
Resolution Mass Spectrometry
To aqueous acetonitrile (50%, v/v) containing ThioRB-ester (100
mg) was added NaOCl solution (100 ꢀl). The mixture was
60 directly analyzed by high resolution mass spectrometry. The
solution was evaporated and the residue was purified by silica gel
chromatography using dichloromethane/triethylamine (10/1).
1HNMR (400 MHz, CD3OD): δ 8.10 (d, 1H, J = 7.60 Hz), 7.67
(m, 4H), 7.18 (m, 2H), 7.05 (m, 4H), 6.60 (m, 1H), 5.50 (s, 1H),
65 3.71 (q, 8H, J = 7.16 Hz), 3.21 (m, 3H), 1.31 (t, 12H, J = 2.8 Hz);
5
+
HRMS (C31H35N2O3 ) calculated (M+): 483.2642, found:
483.2650.
Kinetic profiles on the reaction of ThioRB-FITC-MSN with
15 HOCl
Ratiometric titration of ThioRB-FITC-MSNs for HOCl
Aliquots of ThioRB-FITC-MSN stock solution (50 mg ml-1) in
70 water were added to Na2HPO4-citrate buffer (100 mM, pH 5) or
PBS (pH 7.4) supplemented with various amounts of NaOCl to a
final concentration of 1 mg ml-1. The fluorescence emission
spectra were recorded as a function of NaOCl concentrations
using λex@490 nm or λex@560 nm.
Aqueous NaOCl solution was respectively added into PBS (pH
7.4) containing ThioRB-FITC-MSN (1 mg ml-1) to make a serial
of assay solutions containing NaOCl (0-100 ꢀM). The rates of
fluorescence development in the reaction solutions were recorded
20 on SpectraMax M5 by λex@560 nm and λem@586.
pH titration of ThioRB-FITC-MSN
75 Selectivity of ThioRB-FITC-MSN for HOCl over selected
chemical species
An aliquot of ThioRB-FITC-MSN stock solution in water was
added to Na2HPO4-H3PO4 buffer (200 mM) of various pH values
(8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, or 4.5) to a final concentration of
25 1 mg ml-1. The fluorescence emission at 586 nm was recorded as
a function of pH using λex@560 nm for activated ThioRB doped
in MSN.
The reactive nitrogen species and reactive oxygen species were
-
prepared following published procedures.[3c] Briefly, O2 • was
generated by adding KO2 to the assay solutions. •OH radical was
80 genrated in-situ by addition of Fe(ClO4)2 and H2O2 into assay
solutions. In case of ROO•, ammonium persulfate was added
directly into the assay solution. Nitric oxide was formed from
sodium nitroprusside added to the assay solution.
Effects of pH on HOCl dependent activation of ThioRB-
FITC-MSN
For assays performed in Na2HPO4-citrate buffer (100 mM, pH 5):
85 To a serial of solutions of ThioRB-FITC-MSN (1 mg ml-1) were
respectively added each of the following compounds: KCl (1
mM), NaCl (1 mM), CuSO4 (1 mM), MnCl2 (1 mM), MgCl2 (1
mM), CaCl2 (1 mM), ZnCl2 (1 mM), Fe(ClO4)3 (1 mM),
Fe(ClO4)2 (1 mM), CoCl2 (1 mM), NiCl2 (1 mM), Pb(NO3)2 (1
90 mM) or NaOCl (0.08 mM); H2O2 (1 mM), •OH radical generated
from H2O2 (1 mM) with Fe(ClO4)2 (1 mM), ammonium
persulfate (1 mM), sodium nitroprusside (1 mM), KO2 (1 mM) or
NaOCl (0.08 mM). The samples were mixed and incubated at rt
for 30 min. The fluorescence emission spectra of the samples
95 were recorded using λex@490 nm.
30 An aliquot of NaOCl solution were added to a serial of Na2HPO4-
H3PO4 buffers of different pH (8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, or
4.5) containing ThioRB-FITC-MSN (1 mg ml-1) to a final
concentration of 100 ꢀM. The fluorescence emission intensity at
586 nm was recorded as a function of pH using λex@560 nm for
35 ThioRB doped in MSN.
Effects of biomacromolecules on activation of ThioRB-FITC-
MSN by NaOCl
NaOCl solution was added to DMEM culture medium spiked
with or without ThioRB-FITC-MSN (1 mg ml-1) to a final
40 concentration of 500 ꢀM. The samples were mixed and then
aqueous HCl (1M, 10 ꢀl) was then added to both samples. Visual
images of the samples were recorded by a digital camera. In
parallel, NaOCl solution was added to PBS spiked with or
without ThioRB-FITC-MSN (1 mg ml-1) to a final concentration
45 of 500 ꢀM. The samples were mixted and then recorded by a
digital camera for visual images.
For assays performed in PBS (pH 7.4): the assays were
performed using the same procedure as described above with the
exception that 0.5 mg ml-1 ThioRB-FITC-MSN and 0.1 mM
NaOCl were used in the assays.
100 Endocytosis of ThioRB-FITC-MSN into lysosomes in living
cells
Effects of media on activation of ThioRB-ester by NaOCl
L929 cells were grown at 37 °C under 5% CO2 in DMEM
supplemented with 10% fetal bovine serum. Cells were seeded on
35 mm glass-bottom dishes (NEST) and incubated for 24 hrs. The
105 cells were further incubated in DMEM medium supplemented
with ThioRB-FITC-MSN (25 ꢀg ml-1) for 2 hrs. The cells were
washed with PBS and then respectively incubated in PBS spiked
with or without NaOCl (200 ꢀM) for 10 min. The cells were
further incubated in DMEM containing LysoTracker Blue DND-
An aliquot of ThioRB-ester stock solution in dimethylsulfoxide
was respectively added to PBS, acetonitrile, or PBS buffered
50 acetonitrile (25%, 50%, and 75% v/v) to a final concentration of
10 ꢀM. NaOCl was spiked to the samples to a final concentration
of 100 ꢀM. The samples were mixed and then recorded by a
digital camera for visual images of the samples. Control
experiments were carried out under identical conditions without
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22 (1 ꢀM) for 10 min. Cells were then analyzed by confocal
fluorescence microscopy.
N
O
N
N
O
N
N
O
N
PCC oxidation
O
Wittig reaction
O
OH
Imaging of HOCl with ThioRB-ester in L929 cells
OCH3
2
1
RB-CM
L929 cells were seeded on 35 mm glass-bottom dishes (NEST)
and incubated for 24 hrs, and then further incubated in DMEM
containing ThioRB-ester (10 ꢀM) for 1 hr. The cells were then
incubated in PBS spiked with or without NaOCl (200 ꢀM) for 15
min. The cells were then cultured in DMEM containing Lyso
Tracker Blue DND-22 for 10 min. Cells were washed with fresh
5
Michael addition
NaHS
N
O
N
N
O
N
O
Cyclization
O
S
SH
OCH3
OCH3
10 DMEM medium and then analyzed by confocal fluorescence
microscopy.
ThioRB-ester
50
Scheme 1. Synthesis of HOCl activatable rhodamine-
spirothioether based chemodosimeter for incorporation into silica
nanoparticles.
Ratiometric assay of lysosomal HOCl with ThioRB-FITC-
MSN by flow cytometry
Preparation and characterization of the dual-colored
55 nanodosimeter containing HOCl responsive motif
L929 cells and HeLa cells were respectively cultured in DMEM
15 containing ThioRB-FITC-MSN (25 ꢀg ml-1) for 2 hrs, and then
incubated in PBS supplemented with various amounts of NaOCl
(0, 0.1, 0.5, 1 mM) for 10 min. Cells harvested by trypsin
digestion were washed with PBS and then analyzed by flow
cytometry.
Aminolysis of ThioRB-ester in (3-aminopropyl)triethoxy-
silane (APTS) afforded ThioRB-APTS (ESI†, Scheme S1). Co-
condensation of ThioRB-APTS with FITC-APTS and tetraethyl
orthosilicate (TEOS) in the presence of cetyltrimethylammonium
60 bromide (CTAB) readily afforded silica nanoparticles doped with
FITC and ThioRB, which were further processed following a
published procedure to remove CTAB.[5] Transmission electron
microscopy (TEM) and scanning electron microscopy (SEM)
images showed that the as-prepared porous nanoparticles are
65 uniform in size (Fig. 2, A and B). To minimize nonspecific
interactions in subsequent cell studies and increase the collodial
stability, the dual colored nanoparticles was further pegylated to
give the desired nanodosimeter (referred to as ThioRB-FITC-
MSN). Dynamic light scattering analysis revealed that the
70 average diameter of pegylated MSNs increased to 140 nm as
compared to unmodified MSN (91.3 nm) (Fig. 2, D). Consistently
zeta potential of the pegylated MSN decreased from 28.8 mv to -
33.9 mv after modification (Fig 2, C), indicating efficient
pegylation of the nanoparticles.
20 Cytotoxicity of ThioRB-FITC-MSN
L929 cells were cultured with DMEM medium containing
ThioRB-FITC-MSNs (0, 10, 25, 50, 100 ꢀg ml-1) for various
periods of time (0-24 h) at 37 oC with 5% CO2. Cell viability was
determined by trypan blue exclusion test.
25
Results and discussion
Design and synthesis of
chemodosimeter for HOCl
a small molecule fluorogenic
To develop a selective chemodosimeter for HOCl that can be
30 immobilized into silica nanoparticles, ThioRB-ester was designed
and synthesized as described in Scheme 1. Historically, the
rhodamine derivative containing intramolecular spirothioether
moiety has been reported for fluorogenic sensing of OCl- ion via
analyte triggered oxidative opening of the spirothioether.[3b]
75
To probe the reactivity of ThioRB attached in MSN, various
amounts of NaOCl were spiked into phosphate buffered saline
(PBS) containing ThioRB-FITC-MSN. Formation of fluorescent
species in solutions was monitored by fluorometry. Time course
studies showed that the reactions completed immediately upon
35 ThioRB-ester is
a bifunctional molecule comprised of a
nonfluorescent rhodamin-spirothioether functionality which is
HOCl responsive and an ester group that can be exploited for
subsequent immobilization in MSN. Reduction of rhodamine B
with lithium aluminium hydride in diethyl ether gave 1 in 65%
40 yield. Oxidation of 1 with pyridinium chlorochromate (PCC) in
dichloromethane afforded 2 in 80% yield. Treatment of 2 with
(methoxycarbonylmethylene)-triphenylphosphorane in methanol
at room temperature gave rhodmaine-cinnamate, methyl ester
(RB-CM) as a deep red colored solid in 85% yield. Treatment of
45 RB-CM with hydrosulfide (HS-) in dimethylformamide readily
afforded nonfluorescent and colorless ThioRB-ester via analyte
triggered tandem Michael addition and intramolecular cyclization
(Scheme 1).
80 addition of various levels of NaOCl (5-100 ꢀM) (ESI†, Fig. S1).
The intense fluorescence centered at 586 nm suggested opening
of the spirothioether of ThioRB doped in MSN. In contrast, albeit
responsive to HOCl in acetonitrile and PBS buffered acetonitrile
(ESI†, Fig. S5), ThioRB ester, the free small molecule
85 chemodosimeter, failed to detect NaOCl in PBS (Fig. 3, A and D).
The findings revealed the unexpected and yet essential role of the
silica platforms of the reactive nanoprobe in sensing HOCl in
aqueous media.
90
95
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(1 mg ml-1) and ThioRB-ester (10 ꢀM) in PBS or acetonitrile
30 supplemented with or without NaOCl (100 µM).
B
A
To probe the assay mechanism, the fluorescent species
generated from ThioRB-ester and HOCl in aqueous acetonitrile
was characterized by high resolution mass spectrometry. A major
35 peak located at 483.2650 was revealed which was consistent with
the theoretical molecular weight of RB-CM (ESI†, Fig. S2).
Formation of RB-CM was further confirmed by 1H-NMR
analysis of the colored species generated in the assay solution,
indicating β-elimination of in-situ generated rhodamine-sulfonyl
40 chloride intermediate (Scheme 2B). Compared with reported
hydrolysis of rhodamine-sulfonyl chloride intermediate generated
in HOCl sensing with a structurally analogous chemodosimeter
(Scheme 2A),[3b] the occurring of β-elimination in ThioRB-ester
based assay was presumably due to the increased acidity of the
45 proton next to the carbonyl group (Scheme 2B).
200 nm
50 nm
C
7
6
5
4
3
2
1
0
-100
0
100
200
Zeta potential (mV)
20
D
15
10
5
A
0
N
O
N
N
O
N
N
O
N
1
10
100
1000
10000
HOCl
hydrolysis
Size (d. nm)
S
O
SO3H
S
Fig. 2 Physical properties of ThioRB-FITC-MSN. SEM (A) and
TEM (B) images of the dual colored nanodosimeter before
pegylation; (C) zeta potential of the nanodosimeter (1 mg ml-1) in
water before (in red) and after pegylation (ThioRB-FITC-MSN,
in green) as compared to ThioRB-FITC-MSN (1 mg ml-1) in
water supplemented with NaOCl (100 µM) (in blue), and (D)
10 diameter size of unpegylated nanodosimeter (1 mg ml-1) (in green)
and ThioRB-FITC-MSN (in red) in water as measured by
dynamic light scattering.
Cl
O
5
B
N
O
N
N
O
N
N
O
N
O
β-elimination
HOCl
OCH3
Cl
S
S
O
O
O
H
O
OCH3
OCH3
RB-CM
Scheme 2. Sensing mechanism of ThioRB-ester (B) to HOCl as
comapred to that of a reported chemodosimeter (A).[3b]
50
A
B
3000
2500
2000
1500
1000
500
Ratiometric responses and selectivity of ThioRB-FITC-MSN
to HOCl
100µ M , CH3CN
5000
4000
3000
2000
1000
0
100µ M , PBS
300
200
100
0
100µ M , PBS
To access the ratiometric responses of ThioRB-FITC-MSN
towards HOCl, various amounts of NaOCl were supplemented to
0µ M , PBS
0
100 200 300 400 500 600
Time (senconds)
0µ M , CH3CN
55 PBS (pH 7.4) or Na2HPO4-citrate buffer (pH 5.0) in the presence
of ThioRB-FITC-MSN. Fig. 4 showed that fluorescence emission
of activated ThioRB which was centered at 586 nm intensified as
a function of NaOCl concentrations whereas a concomitant
decrease in fluorescein emission was observed in Na2HPO4-
60 citrate buffer of lysosomal pH, proving efficient FRET between
fluorescein donor and the newly formed rhodamine acceptor by
single wavelength excitation of fluorescein donor or dual
wavelength exitation (ESI†, Fig. S3). The ratios of fluorescence
0µ M , PBS
0
0
100 200 300 400 500 600
Time (senconds)
0
100 200 300 400 500 600
Time (seconds)
3500
3000
2500
2000
1500
1000
500
C
D
+ HOCl
- HOCl
0
4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
pH
intensities (I586nm/I526nm
)
increased significantly, enabling
15 Fig. 3 Chemical properties of ThioRB-FITC-MSN. (A) HOCl
mediated turn-on fluorescence of ThioRB-ester (10 ꢀM) in PBS
(dark line) or acetonitrile (blue line) spiked with or without
NaOCl (100 µM); the insert showed the zoom of kinetic profiles
of ThioRB-ester in PBS; (B) kinetic profiles of HOCl triggered
20 activation of the spirothioether of ThioRB-FITC-MSN (1 mg ml-1)
in PBS spiked with or without NaOCl (100 µM); (C) Titration of
ThioRB-FITC-MSN (1 mg ml-1) in Na2HPO4-H3PO4 buffer (200
mM) of various pH values supplemented with or without NaOCl
(100 µM); pH profile of ThioRB-FITC-MSN was shown in dark
25 while pH correlated titration curve on activation of ThioRB-
FITC-MSN by HOCl was shown in red; the fluorescence
intensity at 586 nm was monitored using an excitation
wavelength of 560 nm; (D) visual images of ThioRB-FITC-MSN
65 estimation of HOCl concentrations by ratios of the fluorescence
intensities of the dual-colored nanoprobe. In parallel experiments,
ratiometric responses of ThioRB-FITC-MSN towards HOCl were
also shown to be efficient in PBS of cytosolic pH (pH 7.4) (ESI†,
Fig. S4). As low as 5 ꢀM HOCl can be detected in buffers of pH
70 5.0 or 7.4.
Lysosomes are intracellular vesicles with acidic luminal pH
(6.0-4.5). Rhodamine (deoxy)lactams, the structural analogs of
ThioRB-ester, have been employed for fluorescent staining of
lysosomes via proton mediated opening of the intramolecular
75 lactams.[6] To image lysosomal HOCl, it is essential that ThioRB
moiety displayed on the nanodosimeter is inert to lysosomal
acidity. pH titration of ThioRB-FITC-MSN in buffers of different
This journal is © The Royal Society of Chemistry [year]
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pH showed that ThioRB group remained non-emissive in both
cytosolic pH and the pH window of lysosomes (Fig. 3C). Being
capable of forming gaseous chlorine or OCl- ion in aqueous
Ca2+, Zn2+, Fe3+, Fe2+, Co2+, Ni2+, Pb2+, or HOCl (0.08 mM, in
red); (B) fluorescence spectra of ThioRB-FITC-MSN (1 mg ml-1)
in Na2HPO4-citrate buffer with no addition, or with each of the
-
solution, HOCl is the predominant species in acidic media of pH 50 following species (1 mM): H2O2, •OH, ROO•, NO, O2 • or HOCl
5
4-6.[7] The pH dependent activation of the nanodosimeter with
HOCl revealed that HOCl is an effective species to activate
ThioRB moiety in the nanoprobe (Fig. 3C), which is consistent
with the documentation that HOCl is a superior oxidizer relative
to OCl- ion in certain organic transformations.[8] Collectively,
(0.08 mM, in red) (λex@490 nm).
Site-specific internalization of ThioRB-FITC-MSN into
lysosomes in living cells
With the advantageous features demonstrated, we next
55 investigated the feasibility of imaging lysosomal HOCl with
ThioRB-FITC-MSN. MSNs, readily internalized into cells by
endocytosis, are often accumulated in lysosomes,[9] which
underlies our proposed regio-selective detection of lysosomal
HOCl with ThioRB-FITC-MSN in living cells. To determine the
60 intracellular locations of internalized ThioRB-FITC-MSN, L929
cells were co-stained with ThioRB-FITC-MSN and LysoTracker
Blue DND-22 (referred to as LysoTracker blue) which is an
established lysosome marker, and then incubated in PBS
supplemented with NaOCl for 10 min. The cells were rinsed and
65 then visualized by confocal fluorescence microscopy. Rhodamine
and FITC signals, both clearly present in cells, overlayed with the
fluorescence of LysoTracker blue, demonstrating that ThioRB-
FITC-MSN was site-specifically internalized in lysosomes in
living cells (Fig. 6).
10 these results suggested the applicability of the nanodosimeter in
ratiometric and real-time imaging of HOCl under both cytosolic
and lysosomal pH.
ThioRB-FITC-MSN was further evaluated for its selectivity
towards potential interferents that could be present in biological
15 specimens, e.g. reactive oxygen species and reactive nitrogen
species. Compared with the dramatic fluorescence response of
ThioRB-FITC-MSN towards HOCl, negligible emission of
ThioRB-FITC-MSN was observed in buffers containing H2O2,
-
nitric oxide (NO), •OH, ROO• or O2 • at concentrations up to 1
20 mM in buffers of pH 5.0 or pH 7.4 (Fig. 5B, ESI†, Fig. S7).
Additionally, no influence on ThioRB-FITC-MSN was observed
in buffers (pH 5 or 7.4) supplemented with various metal ions
such as Na+, K+, Ca2+, Zn2+, Pb2+, Fe3+ and Fe2+ (Fig. 5A, ESI†,
Fig. S8). These findings demonstrated the stringent sensitivity of
25 ThioRB-FITC-MSN to HOCl over a variety of interfering species
that could be present in cells.
70
ThioRB-MSN
FITC-MSN
LysoTracker blue
Merged
Bright field
A
B
1.5
1.0
0.5
5000
4000
3000
2000
1000
0
0 μM
80 μM
80 μM
0 μM
Fig. 6 Intracellular distribution of ThioRB-FITC-MSN in L929
cells treated with NaOCl. Cells pre-loaded with LysoTracker blue
(1 ꢀM) and ThioRB-FITC-MSN (25 ꢀg ml-1) were incubated in
75 PBS containing NaOCl (200 ꢀM) for 10 min, and then probed by
0
20
Concentration of NaClO (10 M)
40
60
80
-6
500 520 540 560 580 600 620 640
Wavelength (nm)
confocal fluorescence microscopy. Activated
ThioRB
Fig 4. Titration of ThioRB-FITC-MSN with HOCl in Na2HPO4-
30 citrate buffer (100 mM, pH 5.0) by fluorometry. A) Fluorescence
emission spectra of ThioRB-FITC-MSN (1 mg ml-1) in Na2HPO4-
citrate buffer spiked with NaOCl was recorded using an
excitation wavelength of 490 nm. Analyte concentrations used: 0,
5, 10, 20, 30, 40, 50, 60, and 80 ꢀM; B) the titration curve was
35 plotted by fluorescence emission intensity at 586 nm over that at
526 nm as a function of HOCl concentrations.
fluorescence was shown in red, FITC signal was shown in green
and that of LysoTracker blue was shown in blue. Merging of
three signals was shown in white. Bars, 5 ꢀm.
80
Ratiometric imaging of lysosomal HOCl with ThioRB-FITC-
MSN
To probe the effectiveness of HOCl imaging, L929 cells pre-
loaded with ThioRB-FITC-MSN or ThioRB-ester were
5000
A
B
5000
4000
3000
2000
1000
0
85 respectively incubated in PBS supplemented with or without
NaOCl for 10 min. No rhodamine signal was observed in control
cells that were incubated in PBS in the absence of NaOCl
whereas intense rhodamine fluorescence was recorded in virtually
all cells treated with ThioRB-FITC-MSN and NaOCl, suggesting
90 efficient incorporation of the nanoparobe into the targeted cells
populations (Fig. 7, A: broad view; B: single cell image) and
HOCl mediated opening of the spiorthioether of ThioRB-FITC-
MSN in lysosomes. In contrast, no obvious rhodmaine
fluorescence was observed in cells stained with the small
95 molecule chemodosimeter of ThioRB-ester in the absence or
presence of HOCl (Fig. 8), highlighting the superior efficacy of
ThioRB-FITC-MSN in sensing lysosomal HOCl over ThioRB-
ester.
4000
3000
2000
1000
0
500 520 540 560 580 600 620 640
Wavelength (nm)
500 520 540 560 580 600 620 640
Wavelength (nm)
Fig. 5 Selectivity of ThioRB-FITC-MSN for HOCl over selected
40 cations or reactive oxygen species in Na2HPO4-citrate buffer (100
mM, pH 5.0). The fluorescence emission of the nanodosimeter in
buffers spiked with various analytes which was due to opening of
the thioRB moiety was recorded using an excitation wavelength
of 490 nm. (A) Fluorescence spectra of ThioRB-FITC-MSN (1
45 mg ml-1) in Na2HPO4-citrate buffer with no addition or with each
of the following species (1 mM): K+, Na+, Cu2+, Mn2+, Mg2+,
6
| Journal Name, [year], [vol], 00–00
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levels of NaOCl (Fig. 9A, ESI†, Fig. S9). Since the slopes of
cells populations represent FL2/FL1 values (Fig. 9A), monitoring
lysosomal HOCl levels in different cell samples could be readily
achieved by comparison of the relative slopes of the cell
40 populations in flow cytometric images. It is noteworthy that the
assay is compatible with conventional flow cytometers as single
wavelength excitation of ThioRB-FITC-MSN (fluorescein donor)
can be achieved by the 488 nm blue laser which is equipped with
all flow cytometers.
A
ThioRB-MSN
FITC-MSN
Merged
Bright field
45
HOCl has been reported to oxidize a wide variety of
biomolecules, including proteins, cholesterol, and NADH, etc.[10]
ThioRB-FITC-MSN, albeit effectively detected HOCl in aqueous
buffers, failed to detect NaOCl in Dulbecco’s Modified Eagle
Medium (DMEM) which is a cell culture medium containing a
B
ThioRB-MSN
FITC-MSN
Merged
Bright field
50 myriad of biomolecules (ESI†, Fig. S6), suggesting the depletion
of NaOCl by ingredients of DMEM. Hence, the relaxed assay
efficiency for exogenous HOCl in the cell-based flow cytometric
assays as compared to that in PBS is partially due to consumption
of the spiked NaOCl by cellular components. It is anticipatable
55 that endogenous HOCl produced in lysosomes could be more
efficiently imaged by the lysosome-residing ThioRB-FITC-MSM.
5
Fig. 7 Imaging of lysosomal HOCl in L929 cells with ThioRB-
FITC-MSN. Cells loaded with ThioRB-FITC-MSN (25 ꢀg ml-1)
were incubated in PBS spiked with or without NaOCl (200 ꢀM)
for 10 min. The cells were analyzed by confocal fluorescence
microscopy. Merging of the fluorescence of activated ThioRB
0.1 mM
1000
800
0 mM
0 mM
A
B
0.1 mM
1 mM
1 mM
600
10 (shown in red) and FITC (in green) was shown in yellow. Bars:
400
200
0
20 ꢀm for A, and 10 ꢀm for B.
ThioRB-ester LysoTracker blue
Merged
Bright field
0
200 400 600 800 1000
FL1-H (FITC signal)
0
200
FL2-H (ThioRB signal)
400
600
800 1000
Fig. 9 Flow cytometric analysis of lysosomal HOCl in L929 cells
60 with ThioRB-FITC-MSN under single wavelength excitation
(λex@488 nm) (A). (B) Dose-dependent formation of ThioRB
fluorescence in L929 cells treated with different levels of NaOCl
as indicated. The fluorescence of FITC (FL1) was collected
@510-535 nm while that of ThioRB signal was collected @565-
65 625 nm (FL2).
Fig. 8 Detection of HOCl with ThioRB-ester. L929 cells were
15 incubated with ThioRB-ester (10 ꢀM). The cells were washed
and then treated with or without NaOCl in PBS for 10 min,
following by supplemented with LysoTracker blue (1 ꢀM) in
DMEM medium for 10 min. The cells were analyzed by confocal
fluorescence microscopy. ThioRB-ester fluorescence was shown
20 in red and that of LysoTracker blue was shown in blue. Bar: 20
ꢀm.
Cytotoxicity of ThioRB-FITC-MSN
The cytotoxicity of ThioRB-FITC-MSN was evaluated in L929
cells by trypan blue exclusion test. No toxic effects were
70 observed on cell viability after incubation with the nanoprobe for
24 hrs at doses up to 100 ꢀg ml-1 (Fig. 10). Taken together, the
studies demonstrated the utility of the reactive nanoprobes in
ratiometric reporting of lysosomal HOCl in living cells.
To explore the efficacy of ratiometric reporting of lysosomal
HOCl with ThioRB-FITC-MSN, L929 and HeLa cells were
25 respectively loaded with ThioRB-FITC-MSN and then cultured
in PBS containing different concentrations of NaOCl. The cells
were then analyzed by flow cytometry. As can be seen in Fig. 9B,
rhodamine fluorescence (FL2 value) increased as a function of
NaOCl concentrations, suggesting dose dependent turn-on
30 fluorescence of ThioRB moiety. FL2/FL1 values, ratios of
fluorescein signal over that of rhodamine, are the indicator of
FRET efficiency of the nanoprobe inside cells. Analysis revealed
that the average FL2/FL1 value of L929 and HeLa cell
populations treated with higher levels of NaOCl was significantly
35 elevated relative to cells with no treatment or treated with lower
0 hr
100
50
0
4 hrs
8 hrs
24 hrs
0
10
25
50
100
Dose of ThioRB-FITC-MSN (µg ml-1
)
75 Fig. 10 Cytotoxicity of ThioRB-FITC-MSN. L929 cells were
incubated with various amounts of ThioRB-FITC-MSN (0, 10, 25,
50, 100 ꢀg ml-1) for 0-24 hrs. Cell viability was determined by
the trypan blue exclusion assay.
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Chemical Science
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H. J. Cho, J. Lee, I. Shin, J. Tae, Org. Lett., 2009, 11, 859; (l) X.
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Conclusions
Self-referenced silica nanoparticles featuring HOCl activatable
chemodosimeter were fabricated for FRET based ratiometric
detection of HOCl in aqueous media of different pH values.
Particularly, the nanodosimeter is suitable for ratiometric
reporting of HOCl levels in lysosomes in living cells. The assay
is compatible with conventional flow cytometry via single-
wavelength excitation of the fluorescein-rhodamine dye pair in-
situ generated upon HOCl triggered oxidation, allowing facile
70
75
80
4
5
6
5
10 monitoring of alterations of intralysosomal HOCl. Disruption on
homeostasis of HOCl generated in lysosomes of immune cells
was linked in a number of chronic diseases. The distinguished
biochemical features of the nanodosimeter, e. g. compatibility
with aqueous media and stringent chemo-/regio-selectivity for
15 lysosomal HOCl, suggested its broad utility for biomedical
studies on biogenesis or impacts of HOCl in living cells or in
animals.
7
8
9
(a) J. Lu, Liong, M., Li, Z., Zink, J. I., and Tamanoi, F., Small, 2010,
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a Department of Chemical Biology, College of Chemistry and Chemical
20 Engineering, and the Key Laboratory for Chemical Biology of Fujian
Province, Xiamen University, Xiamen, China;
bState Key Laboratory of Cellular Stress and School of Life Sciences,
Xiamen University, Xiamen, China;
c Central Laboratory of Medical College, Xiamen University, Xiamen,
25 China
Tel: 86-0592-2181728; E-mail: shoufa@xmu.edu.cn
Acknowledgments: Dr. S. Han was supported by grants from NSF China
21272196 and 21072162, Natural Science Foundation of Fujian Province
2011J06004 and the Fundamental Research Funds for the Central
30 Universities 2011121020; Dr. J. Han was supported by grants from NSF
China 30830092, 30921005, 91029304, 81061160512 and 973 program
2009CB522200.
† Electronic Supplementary Information (ESI) available on
characterization of HOCl triggered activation of ThioRB-ester in different
35 media, selectivity of ThioRB-FITC-MSN in cytosolic pH,
characterization of the assay mechanism, and cytotoxicity of HOCl on
L929 cells. See DOI: 10.1039/b000000x/
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