Biotin-tagged fluorescent sensor to visualize 'mobile' Zn2+ in cancer cells

Article abstract of DOI:10.1039/c8cc05425h

A cancer cell-targeting fluorescent sensor has been developed to image mobile Zn²⁺ by introducing a biotin group. It shows a highly selective response to Zn²⁺in vitro, no toxicity in cellulo and images 'mobile' Zn²⁺ specifically in cancer cells. We believe this probe has the potential to help improve our understanding of the role of Zn²⁺ in the processes of cancer initiation and development.

Full text of DOI:10.1039/c8cc05425h

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Trigiante, C. J. Kousseff, R. Crespo-Otero, M. Philpott and M. Watkinson, Chem. Commun., 2018, DOI:
10.1039/C8CC05425H.
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Biotin-tagged fluorescent sensor to visualize ‘mobile’ Zn2+ in
cancer cells
Le Fang,a Giuseppe Trigiante,b Christina J. Kousseff,a Rachel Crespo-Otero,a Michael P. Philpott,b
and Michael Watkinson*a,c
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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processes, it is still unclear whether these changes are a cause
or an effect of the cancer. Furthermore, it is not clear whether
zinc itself or zinc transporters are associated with the cancer-
related events.3 However, the difficulty of imaging in vivo
represents a significant barrier to understanding its role in
cancer. Therefore, the development of an effective way to
detect mobile zinc specifically in tumour cells would allow us
to achieve a better understanding of its role in the mechanism
of cancer initiation, progression, and potentially, its
prevention.
A cancer cell-targeting fluorescent sensor has been developed to
image mobile Zn2+ by introducing a biotin group. It shows a highly
selective response to Zn2+ in vitro, no toxicity in cellulo and images
‘mobile’ Zn2+ specifically in cancer cells. We believe this probe has
the potential to help improve our understanding of the role of
Zn2+ in the processes of cancer initiation and development.
Zinc, as the second most abundant d-block metal in the human
body, plays an important role in many biological processes.1
The aberration of zinc levels, which can cause the dysfunction
of these processes, is related to a wide range of diseases.2 One
of these diseases, cancer, causes millions of deaths every year
and is a major burden of disease around the world. There is
evidence that zinc is important in cancer development.3 This is
perhaps unsurprising as zinc is necessary for the human
superoxide dismutase enzyme system to function, and cancer
cells are highly dependent on superoxide dismutase for
protecting themselves from the damage induced by reactive
oxygen species, since the superoxide anion radical, O2-., is
actively produced in cancer cells.4 Zinc is also a growth factor
in cell proliferation,5 which is attenuated in the absence of
zinc.6 However, it is found that the alterations of mobile zinc
concentration in malignant cells are tissue specific. For
example, zinc concentration increases by about 72% in breast
cancer tissue,7 while it decreases by 75% in malignant prostate
tissue8 compared to their non-cancerous counterparts. These
difference have been explained by changes in expression of
zinc transporters, which directly influence the Zn2+ cellular
influx and efflux.9 In prostate cancer, the low Zn2+
concentration is due to the downregulation of the zinc
transporter ZIP1,8 whilst ZIP610, ZIP711 and ZIP1012 have been
proposed to be important in the elevated levels in breast
cancer. Though there is an increasing understanding of these
Small molecule fluorescent sensors, which can image Zn2+
with fluorescence as their output have become one of the
most predominant methods in use today due to their high
sensitivity, low toxicity, and good photophysical properties.13,14
We have developed
a modular double ‘click’ synthetic
methodology to produce biologically targeted Zn2+ sensors for
both extracellular and intracellular imaging of zinc.15 Biotin is a
vitamin essential to cancer cells and the sodium-dependent
multi-vitamin transporter (SMVT) is overexpressed in many
cancers, including breast, lung, ovarian, mastocytoma and
renal,16 meaning that cancer cells uptake more biotin than
normal cells. Based on this, some biotin tagged cancer drugs
have been developed and have shown good targeting.17 We
therefore hypothesized that by incorporating a biotin tag into
a zinc probe, using our previously reported methodology, we
could generate a small molecule probe that could detect zinc
specifically.
The cyclam ligand has previously been shown to be an
effective receptor unit for mobile zinc,18 and was therefore
chosen in this study. The synthetic route to the target probe,
7, is shown in Scheme 1. Biotin azide, 1, was prepared from D-
(+)-biotin by adapting literature procedures (see Electronic
Supplementary Information).19 The fluorophore and ligand
were introduced via alkynes
using the procedure we reported previously.15,18 After double
click reactions,20 the Boc-protected
was isolated in moderate
yield and was de-protected under standard conditions to give
sensor (see ESI). Unless otherwise noted, all photophysical
2 and 3, which were synthesized
6
7
experiments in solution were performed in 0.01 mM aqueous
HEPES buffer at pH 7.4 and ZnCl2 was chosen as the binding
metal ion source, in order to simulate the chloride-rich
biological environment.
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Fig. 2 a) The pH profile of 7 (50 μM) and 7 with 1 equivalent Zn2+ in 0.01 mM
HEPES buffer. b) Metal ion selectivity of 7. Average normalized fluorescence
intensities for 50 μM 7 in buffer (0.01 mM HEPES, pH 7.4) (black bars), after
addition of 5 equivalents of various metal ions (red bars), followed by
addition of 1 equivalent ZnCl2 (blue bars).
yield of
7 was found to be 0.02, while this increased to 0.05
after binding to 1 equivalent of Zn2+. Although this is rather
modest, it is in line with previous measurements18 and we
have shown such probes to be tractable in biological milieu.
The pH-dependent fluorescence response was measured, as
it is known that although cancer cells maintain their pH near to
neutrality, they are more acidic than normal cells.21 As shown
Scheme 1 The synthetic route to sensor 7.
The Job plot clearly showed the expected Zn2+:
7 binding
stoichiometry of 1:1, (see ESI, Fig. S2). This binding behaviour
has also been studied using DFT calculations. The optimized
structure of the complex of
7
with 1 equivalent Zn2+ in water
revealed the expected structure involving ligation of the
cyclam nitrogen donors with the triazole ligand acting as a
scorpion-like donor (Fig. S8, ESI), and is similar to the single
crystal X-ray structure of a closely related analogue.18
in Fig. 2a, sensor 7 shows a good switch-on response to 1
equivalent Zn2+ in the range of pH 5.5-10.5, indicating that it
should be able to detect Zn2+ in most cancer cells. The
apparent pKa of
fluorescence intensity of emission spectra against pH (see ESI,
Fig. S4). Through non-linear curve fitting (see ESI, Equation S3),
the four apparent pKas of
± 0.23, pKa3 =9.24 ± 0.31, pKa4 = 11.18 ± 0.18.
The selectivity of sensor
to Zn2+ over other competing
metal ions was also investigated. As shown in Fig. 2b, besides
Zn2+, the fluorescence is not switched on after the addition of
other metal ions, with the exception of Cd2+, which shows a
smaller response in line with our previous findings.18 However,
as its concentration in tissues is negligible this is not an issue.
Whilst Fe3+, Co2+ and Cu2+ induce significant quenching22 of the
7 was measured by integrating the
The fluorescence of
7
is switched on by adding Zn2+ and is
based on a mechanism restricting intramolecular vibrations
(see ESI, Fig. S9). As shown in Fig 1a, upon addition of Zn2+, the
fluorescence intensity increased gradually with an
approximately 5-fold maximum increase. The dissociation
constant with Zn2+ was measured through non-linear curve
fitting and was determined to be 1.88 × 10-8 M. The quantum
7 are: pKa1 = 1.64 ± 0.14, pKa2 = 4.18
7
fluorescence of 7, they almost exclusively exist in bound forms
in biology, rather than as the free cations tested here. The
+
cations Na+, K+, Ca2 , and Mg2+, which are the main metal ions
in cells, showed no effect on the fluorescence intensity of
sensor
and can potentially be applied in cellulo. The water-solubility
of is also an attractive feature for cell imaging as it does not
7
, meaning that it should have good selectivity for Zn2+
7
require the addition of co-organic solvents to solubilize the
probe.
Fig. 1 a) The fluorescence switch-on response of sensor 7 (50 µM) to
different equivalents Zn2+ in 0.01 mM HEPES buffer (λex = 346 nm, λem = 412
nm, slit width: 5 nm); b) the non-linear curve fitting of the fluorescence
intensity against different equivalents Zn2+
.
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With these promising properties confirmed in vitro, the
innate toxicity of sensor was then measured. The MCF-7
7
breast cancer and N-TERT keratinocytes cell lines were
incubated in cell medium solution containing different
concentrations of 7 for 24 hours. Then the solution was
washed away and alamarBlue was added as an indicator of cell
health.23 From these experiments (see ESI, Table S1), we can
conclude that the sensor has no toxicity to either cell type,
since the number of living cells does not decrease as the
concentration of sensor increases.
Confocal fluorescence microscopy was used to detect
mobile Zn2+ in MCF-7 and N-TERT keratinocytes cells and the
results are shown in Fig. 3 (see also ESI, Fig. S7). For MCF-7
breast cancer cells, when there was no sensor, the
fluorescence of the cells was very weak, resulting from their
background autofluorescence. After incubation with a 100 µM
solution of
7 for 2 hours, the fluorescence increased
considerably, and the cytoplasm can be visualized clearly, but
there was no response from the nucleus. On the addition of
zinc pyrithione, a membrane permeable zinc source,24 the
fluorescence response of the cells became stronger and a
fluorescence response was observed from the whole cell,
including the nucleus. We therefore assume that this sensor is
also permeable to the nucleus, but that the concentration of
nuclear mobile zinc in MCF-7 cells is very low. When TPEN, a
well-known zinc chelator,25 was added to remove Zn2+ inside
cells, the fluorescence of the cells decreased markedly as
expected. Pleasingly, as hypothesized, for the normal control
N-TERT keratinocytes cells, we observed no strong
fluorescence response even after the addition of zinc
pyrithione (Fig. S7, ESI), confirming the selective localization of
Fig. 3 Confocal microscopy images of MCF-7 cells treated with no sensor (a-
c), 100 µM sensor 7 solution (d-f), sensor 7 (100 µM) with saturated zinc
pyrithione (g-i), and sensor 7 (100 µM) after loading the cells with zinc
pyrithione, then TPEN (2 µM) was added (j-l).
sensor 7 in the cancer cells.
In conclusion, the biotin tagged fluorescent sensor was
developed using a double click reaction. It is water soluble, and
has high selectivity, low toxicity, shows good fluorescence
response and a low dissociation constant to Zn2+. Testing in
cells confirms it can localize selectively in cancer cells, can
image mobile Zn2+, and therefore has potential to help us
better understand the role of Zn2+ in cancer initiation and
progression.
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Acknowledgements
We acknowledge the EPSRC National Mass Spectrometry Service,
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QMUL Apocrita facility. We are grateful to the Chinese Scholarship
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Biotin-tagged fluorescent sensor to visualize
‘mobile’ Zn2+ in cancer cells
Le Fang,a Giuseppe Trigiante,b Christina J. Kousseff,a Rachel Crespo-Otero,a Michael P.
Philpott,b and Michael Watkinson*a,c
a. The Joseph Priestley Building, School of Biological and Chemical Sciences, Queen Mary University
of London, Mile End Road, London, E1 4NS, UK.
b. Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London
School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK.
c. The Lennard-Jones Laboratories, School of Chemical and Physical Sciences, Keele University, ST5
5BG, UK. Email: m.watkinson@keele.ac.uk
A biotin-tagged fluorescent sensor was developed to image Zn2+ in cancer cells specifically,
which showed no entry to normal cells.