Bioimaging of peroxynitrite in MCF-7 Cells by a new fluorescent probe rhodamine b phenyl hydrazide

Article abstract of DOI:10.1007/s10895-013-1205-y

Peroxynitrite is a potent oxidizing and nitrating agent which has detrimental effects on cells by altering the structure and function of biomolecules present within. A fluorescent probe rhodamine B phenyl hydrazide (RBPH) has been proposed for peroxynitrite (ONOO^-) imaging in MCF-7 cells based on its oxidation property, which converts RBPH to pink colored and highly fluorescent rhodamine B. The fluorescence emission intensity of the rhodamine B produced in the above process is linearly related to the concentration of peroxynitrite. The method obeys Beer's law in the concentration range 2-20 nM and the detection limit has been found to be 1.4 nM. The possible reaction mechanism of peroxynitrite with RBPH to produce rhodamine B has been discussed with spectroscopic evidence. The Probe is selective to the peroxynitrite in the pH range 6-8 which is near physiological pH. Fluorescence microscopic studies suggest that the probe is cell permeable and hence peroxynitrite was imaged in MCF-7 cells.

Full text of DOI:10.1007/s10895-013-1205-y

J Fluoresc
DOI 10.1007/s10895-013-1205-y
ORIGINAL PAPER
Bioimaging of Peroxynitrite in MCF-7 Cells by a New
Fluorescent Probe Rhodamine B Phenyl Hydrazide
Gopi Ambikapathi & Suresh Kumar Kempahanumakkagari &
Babu Ramappa Lamani & Devaraju Kuramkote Shivanna &
Harish Bodagur Maregowda & Anushree Gupta & Pandurangappa Malingappa
Received: 2 February 2013 /Accepted: 24 February 2013
#
Springer Science+Business Media New York 2013
Abstract Peroxynitrite is a potent oxidizing and nitrating
agent which has detrimental effects on cells by altering the
structure and function of biomolecules present within. A
fluorescent probe rhodamine B phenyl hydrazide (RBPH)
has been proposed for peroxynitrite (ONOO−) imaging in
MCF-7 cells based on its oxidation property, which
converts RBPH to pink colored and highly fluorescent
rhodamine B. The fluorescence emission intensity of the
rhodamine B produced in the above process is linearly
related to the concentration of peroxynitrite. The method
obeys Beer’s law in the concentration range 2–20 nM and
the detection limit has been found to be 1.4 nM. The
possible reaction mechanism of peroxynitrite with RBPH
to produce rhodamine B has been discussed with spectro-
scopic evidence. The Probe is selective to the peroxynitrite
in the pH range 6–8 which is near physiological pH.
Fluorescence microscopic studies suggest that the probe is
cell permeable and hence peroxynitrite was imaged in
MCF-7 cells.
Introduction
Reactive nitrogen species such as nitric oxide (NO) and
peroxynitrite (ONOO−), oxygen species such as superoxide
(O2·−) and hydrogen peroxide (H2O2) are usually involved in
several physiological and pathological processes [1].
Peroxynitrite is a potent nitrating species formed in macro-
phages, neurons, endothelial cells and platelets by the fast
reaction (~1×1010 M−1 s−1) of nitric oxide (NO) and superox-
ide anion [2]. Peroxynitrite is a short lived species with a
biological half-life lesser than 0.1 s [3]. It can interact with
wide range of molecules in cells, including DNA, proteins and
modify them [4, 5]. Peroxynitrite possesses high affinity for
tyrosine residues in proteins and 3-nitrotyrosine serves as an
indicator of peroxynitrite mediated protein modification [6].
Protein nitration has been observed in a series of diseases like
Parkinsons, Alzheimers, Huntingtons, Atherosclerosis and
Hypertension [7–11]. At lower concentration they behave as
signaling molecules but at higher concentration they induce
cell damage [12]. Detection and quantification of peroxynitrite
localized in a cell is a challenging task which is needed to be
addressed. As peroxynitrite is produced only in trace amounts
there has to be a sensitive technique to detect the same.
Fluorescence bioimaging plays a pivotal role in such crippling
conditions which is found to be highly sensitive and safe
compared to other techniques. In recent years, various methods
have been tried to detect peroxynitrite such as fluorescence
spectrometry, UV–Vis spectrometry, chemiluminescence, elec-
trochemical methods, enzymatic methods and immunohisto-
chemistry [13–20]. But fluorescent methods find significant
among all other methods for in vivo and in vitro monitoring of
various oxidants, especially the xanthene derivatives due to
their attractive spectroscopic properties like longer emission
wavelengths, high quantum yields and their water solubility
which are the desirable properties that can be utilized in the
.
.
Keywords Peroxynitrite Rhodamine B phenyl hydrazide
.
.
Fluorimetry Bioimaging MCF-7 cells
:
:
G. Ambikapathi B. Ramappa Lamani
:
:
D. Kuramkote Shivanna (*) H. Bodagur Maregowda A. Gupta
Department of Microbiology & Biotechnology,
Bangalore University, Jnanabharathi Campus,
Bangalore 560 056, India
e-mail: ksdevaraju@bub.ernet.in
:
S. K. Kempahanumakkagari P. Malingappa
Department of Studies in Chemistry, Bangalore University,
Central College Campus,
Bangalore 560 001, India

J Fluoresc
biological systems study [21–24]. These xanthene derivatives
are colorless and non fluorescent in their spiro closed forms but
highly fluorescent and colored (either green or pink) in their
corresponding open forms [25, 26]. This property has been
utilized extensively for sensing and monitoring both biological
analytes as well as metal ions and anions by converting the
spiro closed forms (non fluorescent) into spiro open
forms (fluorescent) by the analytes involving various
strategies like specific bond cleavage followed by hydro-
lysis, oxidizing the reduced forms, by coordination lead-
ing to ring opening [27–29]. Some of the fluorescein
derivatives have been recently reported for bioimaging
of peroxynitrite and these probes are found to posses
short excitation wavelengths compared to that of rhoda-
mine derivatives [14, 30–32]. The probes possess longer
excitation wavelength found to be advantageous for
bioimaging due to low background fluorescence of them
and also longer wavelength UV excitation leads to less toxic-
ity. So the efforts are made to synthesize a new fluorescent
probe which has longer excitation and emission wavelengths
to image peroxynitrite in biological cells.
Apparatus
Absorbance measurements were made using a Shimadzu
Scanning Spectrophotometer (model UV-3101PC) with
1 cm quartz cuvettes. All the fluorescence measurements were
measured using Ocean optics (USA) spectrofluorimeter with
1 cm quartz cuvettes. All pH measurements were carried out
using a Control Dynamics digital pH meter (model APX 175).
NMR spectra were recorded using a Bruker-400 MHz
Spectrometer with chemical shifts reported as parts per million
(ppm in CDCl3, TMS as internal standard). Mass spectral data
was obtained using a Thermo Finnigin DecaQXP Mass
Spectrometer. Fluorescence imaging of MCF-7 cells were
performed with an Olympus FluoView FV1000 laser scan-
ning microscope with 20 times magnification.
Synthesis of RBPH
In a 250 mL round bottom flask, 2.4 g rhodamine B (4.8 mM)
was dissolved in 100 mL of absolute ethanol. Then 20 mL
(200 mM) of phenyl hydrazine was added and the reaction
mixture was refluxed for 4 h. The solvent was evaporated
under reduced pressure. The obtained solid was dissolved in
50 ml of dichloromethane and washed several times with
saturated NaHCO3 solution. The organic phase was dried over
anhydrous NaHSO4. Then the solution was filtered and the
filtrate obtained was evaporated to dryness. The obtained solid
was washed with acetonitrile and dried under vacuum. RBPH
was obtained as yellow powder with 84 % yield. The com-
pound was characterized by the ESI-MS and 1H NMR studies.
A new rhodamine derivative called rhodamine B phenyl
hydrazide (RBPH) bearing an active spirolactam group to
sense peroxynitrite in vivo as well as in vitro systems has
been proposed. This probe is very sensitive due to the
presence of active phenyl hydarazone group which is sus-
ceptible to oxidation by free radicals compared to hydrazine
groups and reduced forms of the dyes as in case of other
reported xanthene derivatives [13–15]. The proposed probe
is selective to the peroxynitrite in the near physiological pH
and can be utilized for sensing peroxynitrite. No other
oxidants like OCl−, H2O2 and ClO4 as well as metal ions
Cell Culture and Imaging
−
like Cu2+ and Hg2+ induce the spectroscopic changes in the
probe in this pH range studied.
Breast cancer cell line (MCF-7) was cultured on a cover slip
with RPMI 1640 with 5 % fetal bovine serum at 37º C in a
Materials and Methods
Materials
Peroxynitrite was synthesized as per the reported literature
[33]. Rhodamine B hydrazide was purchased from Sigma-
Aldrich. Phenyl hydrazide and Acetonitrile were purchased
from the SD fine Chem. Ltd., Mumbai. Stock solution of
RBPH (1.0 mM) was prepared by dissolving 0.532 g in
acetonitrile—water mixture (%v/v). 0.1 M of KH2PO4 and
Na2HPO4 solutions were used for the buffer solution prep-
aration of pH 8. Robinson buffer solutions of pH3–12 were
prepared using 0.04 M H3BO3, 0.04 H3PO4, 0.04 M
CH3COOH and 0.2 M NaOH. All the reagents were of
analar grade and double distilled water was used throughout.
MCF-7 cell lines were procured from National Centre for
Cell Sciences, Pune, India.
Fig. 1 Fluorescence emission spectra of 1 mL of RBPH (1 mM ) in
presence of different volumes of peroxynitrite (0–10 nM) (Inset showing
the calibration plot of peroxynitrite concentration from 0 to 10 nM)

J Fluoresc
(2–20 nM) were added into a series of 10 mL volumetric
flasks and diluted to the mark with distilled water and kept
at room temperature for 10 min. The solutions were excited
at 560 nm and the emitted intensities were measured at
580 nm.
Results and Discussion
The absorption and fluorescence emission spectral charac-
teristics of RBPH were studied in 10 % acetonitrile-water
mixture in the pH range 3–12. The RBPH is colorless and
non fluorescent similar to that of other xanthene derivatives
due to its spiro closed structure. The addition of
peroxynitrite to the above solution in presence of phosphate
buffer of pH 8 turns the solution into pink colored and
highly fluorescent due to the generation of rhodamine B
from RBPH by peroxynitrite through spirolactam ring
opening process (Figs. 1 and 2). The transformation of
RBPH to rhodamine B by peroxynitrite was observed in
physiological pH conditions. Hence studies have been
carried out to determine the presence of peroxynitrite in
biological systems.
Fig. 2 Absorption spectra of RBPH 1 mL (10 mM) A. In absence of
peroxynitrite (10 mM) B. In presence of peroxynitrite (10 nM)
humidified atmosphere with 5 % CO2. Then 48 h grown
culture was incubated with 10 μM RBPH for 2 h and washed
five times with phosphate buffer saline (PBS) in order to
remove the unabsorbed probe to overcome the background
fluorescence and then incubated with 0.5 μM peroxynitrite at
room temperature for 1 h and finally washed five times with
PBS to avoid fluorescence noise. These cells were subjected
for fluorescence imaging. Excitation of cells was carried out at
560 nm and the emission was measured at 570–670 nm.
Evidence for the Reaction Between RBPH and Peroxynitrite
The peroxynitrite quantitatively reacts with RBPH in the pH
range 7–8 to generate rhodamine B by cleaving the
spirolactam ring. It has been proved by characterizing the
generated molecule through various spectroscopic tools in-
cluding UV-Vis spectroscopy, fluorescence spectroscopy,
Recommended Procedure
One mL each of phosphate buffer solution (pH8) and RBPH
(1.0 mM) and different concentrations of peroxynitrite
1
Fig. 3 H nmr spectrum of RBPH

J Fluoresc
Fig. 4 1H nmr spectrum of
reaction product obtained in the
reaction RBPH and
peroxynitrite
proton NMR and mass spectrometry. The UV-Vis absorption
spectra of the RBPH in acetonitrile-water (1:10 v/v) with
phosphate buffer (pH-8) showed very little absorption
whereas upon addition of peroxynitrite to the above solution
showed a new band at 560 nm which corresponds to the
rhodamine B generated in the reaction.
Fig. 5 ESI MS spectra of RBPH

J Fluoresc
Fig. 6 ESI-MS spectrum of reaction product obtained in the reaction between RBPH and peroxynitrite
Similarly the fluorescence spectrum comparison of
the excitation and emission fluorescence spectra of the
reaction system with that of authentic rhodamine B
showed identical spectra, both having excitation maxi-
mum at 560 nm and emission maximum at 580 nm (not
shown), indicating that the fluorescent product generated
in the reaction mixture is rhodamine B. In order to
carry out spectral analysis, the reaction between RBPH
and peroxynitrite was carried out in bulk quantities at
pH8. The pink colored product obtained was extracted
using dichloromethane and the residue was isolated by
evaporation method. The solid substance was
recrystallised using methanol and the red powder
obtained was used for NMR and mass spectral study.
The 1H NMR spectra of RBPH contains the NHC6H5
signal at 5.9 ppm where as the product obtained in the reaction
lacks this peak instead its proton NMR spectra well matched
1
with that of H NMR spectra of authentic rhodamine B
(Figs. 3 and 4).
The ESI-MS spectra of RBPH has [M+H]+ around
533.21 where as the product obtained in the reaction
showed [M+H]+ 443.47 well matched with the [M+H]+
Scheme 1 Schematic representation of reaction between Rhodamine B Phenyl Hydrazide and Peroxynitrite

J Fluoresc
Fig. 9 Effect of Reaction time
Effect of Concentration of RBPH
Fig. 7 Effect of pH
The optimium concentration of RBPH required for the reac-
tion was studied. Into a series of 10 mL volumetric flasks
containing 1 mL of phosphate buffer solution ( pH8) 1 mL of
10 nM of peroxynitrite were added and different volumes of
1 mM RBPH were added and diluted up to the mark. It is clear
from the figure (Fig. 8) that the fluorescence emission inten-
sity increases with increasing concentration of RBPH and
remains constant when the overall concentration of RBPH
beyond 1 mM. Hence in all further studies 1 mL of 1 mM
RBPH was used as an optimum concentration.
443.47 of authentic rhodamine B (Figs. 5 and 6). Based
on these spectroscopic experimental evidences, we can
conclude that the reaction between RBPH and
peroxynitrite undergoes quantitatively to generate rhoda-
mine B compound. The plausible mechanism of the
reaction between the reactants has been proposed
(Scheme 1).
Optimization Study
Effect of Reaction Time
Effect of pH
The optimum time required for the reaction between RBPH
and peroxynitrite was studied by keeping all other parameters
constant. The solutions were prepared as above in 10 mL
volumetric flasks and allowed for different time intervals.
Then the fluorescence intensity of the solutions were recorded
as a function of reaction time (λex/em=560/580 nm). It was
observed that the fluorescence intensity of the solutions in-
creased up to 3 min and remained constant even after 1 h, but
the fluorescence background of the solution in the absence of
The effect of pH on the reaction between RBPH and
peroxynitrite was studied by keeping all other parameters
constant. In a set of 10 mL volumetric flasks, 1 mL of 10 nM
of peroxynitrite, 1 mL of 1 mM RBPH and 1 mL of phophate
buffer solutions of pH in the range of 3–12 were added into
10 mL volumetric flask and diluted upto the mark and allowed
for 10 min. at room temperature. The fluorescence intensity of
the solutions were measured (λex/em=560/580 nm). It is clear
that fluorescence intensity of the solution has increased with
pH up to 8 and decreased beyond 9 (Fig. 7). Hence pH 8 of
phosphate buffer was chosen as an optimum pH for the quan-
titative reaction between RBPH and peroxynitrite. Therefore
the proposed probe can be applied for bioimaging of
peroxynitrite from cells in the physiological pH condition.
Fig. 10 Fluorescence emission spectra of 1 ml of pH-8 phosphate
buffer with a 1 ml of 1 mM RBPH and 10 nM Peroxynitrite b 1 ml
of 1 mM RBPH and c 1 ml of 1 mM RBPH with other oxidizing
species
Fig. 8 Effect of RBPH concentration

J Fluoresc
Table 1 Effect of foreign ions
studies were carried out in MCF-7 breast cancer cells. In view
of its good water solubility, favorable spectroscopic properties
and the instantaneous interaction with peroxynitrite, RBPH
should be well-suited for fluorescence imaging in living cells.
The cells were incubated with RBPH (10 μM) for 2 h at
37 ° C, then followed by the addition of peroxynitrite
(0.5 μM) and incubation for another 1 h. The cells were
washed with PBS solution and their fluorescence images were
recorded before and after addition of peroxynitrite (Fig. 11).
In absence of peroxynitrite, free RBPH showed no de-
tectable fluorescence signal in living cells. After incubation
with peroxynitrite, a bright fluorescence was observed in
cells. These results suggest that the probe RBPH can pene-
trate the cell membrane and can be applied for in vitro
imaging of peroxynitrite in living cells and could be used
potentially in vivo too.
The proposed fluorescent probe has unique features in
comparison with the existing probes which suites for imag-
ing studies also in live cells viz. (1) Its specificity towards
the particular analyte in the physiological pH conditions in
presence of other potent oxidants. (2) Its long emission
wavelengths (probes with short emission wavelengths are
cytotoxic because cell have to irradiate with higher energies
which may result in cell damage) and (3) water solubility of
the probe which is essential during imaging studies.
Interference
Tolerance
limit (μM)
Glucose, Urea, Methionine, Threonine
800
Ascorbic acid, Glutamic acid, Cystein, H2O2
500
−
SO42−, NO2−, NO3
2,500
1,000
Na+, K+, Fe3+, Mg2+, Mn2+, Fe2+, Cu2+
,
Hg2+, Ca2+, Zn2+
peroxynitrite remained unchanged (Fig. 9). Hence 10 min.
reaction time was allowed in all further experimental studies.
Interference Study
In order to check the selectivity of RBPH towards the
peroxynitrite signaling, the effect of various interfering species
normally oxidising agents and other biological species present in
the cells along with analyte has been studied. Under the
optimised conditions only the analyte facilitates the transforma-
tion of RBPH to rhodamine B compound. The spectral behavior
of RBPH in presence of various interfering species at pH 8 of
phosphate buffer is shown in Fig. 10. The metal ions like Cu2+
and Hg2+ ions did not induce any fluorescence under the phys-
iological pH condition used in the present investigation. Various
other metal ions, anions and oxidizing species generally present
in the biological systems like H2O2 does not trigger any fluores-
cence changes of the probe in the pH range 7–9. The tolerance
limits of the various species studied are given in the Table 1.
Conclusion
We have proposed a novel fluorogenic probe for
peroxynitrite recognition in biological cells. The probe ex-
hibits excellent selectivity, sensitivity and it is very simple
for the determination of peroxynitrite. The fluorogenic
probe is also suitable for bioimaging of peroxynitrite be-
cause the probe specifically reacts with peroxynitrite in the
physiological pH range. Hence peroxynitrite in biological
samples can be easily detected at physiological conditions.
The response time of the proposed probe for peroxynitrite is
instantaneous with a detection limit of 1.4 nM. Other bio-
logical oxidants which are commonly present in the human
physiology do not induce the fluorescence with the
Analytical Merits
The proposed method obeyed Beer’s law in the concentration
range 2–20 nM with a regression coefficient r 2=0.9991. The
limit of detection (LOD) and relative standard deviations were
found to be 1.4×10−9 and 4.1 % respectively.
Fluorescence Imaging of Living Cells
The biological application of the probe RBPH has been dem-
onstrated through fluorescence imaging experiments. These
Fig. 11 Fluorescence images of Peroxynitrite in MCF-7 cells with
RBPH. a Bright-field transmission image of MCF-7 cells incubated with
RBPH (10 μM) for 2 h. b Fluorescence image of MCF-7 cells incubated
with RBPH (10 μM) for 2 h. c Fluorescence image of MCF-7 cells
incubated with RBPH (10 μM) for 2 h, washed five times with PBS,
and then further incubated with 0.5 μM Peroxynitrite for 30 min

J Fluoresc
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radiant energy is directly proportional to the amount of
peroxynitrite present in a particular cell or tissue.
Acknowledgments The authors acknowledge the financial support
and award of the fellowship to Gopi A by University grants Commis-
sion (UGC), New Delhi, India.
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