Live cell cytoplasm staining and selective labeling of intracellular proteins by non-toxic cell-permeant thiophene fluorophores

Article abstract of DOI:10.1039/c3ob41982g

A structurally correlated series of cell-permeant thiophene fluorophores, characterized by intense green or red fluorescence inside live mouse embryonic fibroblasts, was developed. The fluorophores displayed rapid internalization, excellent retention inside the cells, and high optical stability in the cytosolic environment and did not alter cell viability and reproducibility. Depending on the molecular structure, they experienced distinct fate inside the cells: from bright and lasting staining of the cytoplasm to selective tagging of a small set of globular proteins. This journal is The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3ob41982g

Organic &
Biomolecular Chemistry
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Live cell cytoplasm staining and selective labeling
of intracellular proteins by non-toxic
cell-permeant thiophene fluorophores†
Cite this: Org. Biomol. Chem., 2014,
12, 1603
F. Di Maria,a,b I. E. Palamà,*c M. Baroncini,d A. Barbieri,b A. Bongini,d R. Bizzarri,e
G. Giglid,f and G. Barbarella*b,g
A structurally correlated series of cell-permeant thiophene fluorophores, characterized by intense green
or red fluorescence inside live mouse embryonic fibroblasts, was developed. The fluorophores displayed
rapid internalization, excellent retention inside the cells, and high optical stability in the cytosolic environ-
ment and did not alter cell viability and reproducibility. Depending on the molecular structure, they
experienced distinct fate inside the cells: from bright and lasting staining of the cytoplasm to selective
tagging of a small set of globular proteins.
Received 2nd October 2013,
Accepted 15th January 2014
DOI: 10.1039/c3ob41982g
www.rsc.org/obc
protein tagging inside live cells using non-genetically encoded
probes have been described so far, differing in labelling
1. Introduction
Synergistic advances in fluorescent probes1 and imaging tech- difficulties and versatility, specificity and toxicity. Nevertheless,
niques2 have made visualization of live cells one of the most despite the development of various smart procedures, it is
powerful techniques, at the interface of chemistry and biology, widely accepted that new and simpler methods are still needed
to obtain information on cell components in their native for the routine use of fluorescent probes in the cellular
context in real time. Among the various components present environment. Organic fluorophores have the advantage of
in live cells much interest is devoted to proteins and related being small-sized tags, easy to tune via organic synthesis, and
biological mechanisms at the basis of cell physiology and some of them can even be repeatedly imaged owing to high
dysfunction.3
brightness and resistance to photobleaching.1f They have been
Presently, chemical tagging methods to visualize specific largely employed for in vitro studies of proteins; however, the
proteins inside live cells are increasingly being employed as an transition to labelling inside live cells is often limited by the
alternative to fluorescent protein labelling.4 Genetically absence of membrane permeance, toxicity and the lack of
encoded fluorescent proteins can be covalently fused to the specificity for any particular intracellular protein.
protein of interest which is then targeted with a high pre-
In recent years Nilsson et al.5 have demonstrated that a
cision. However, given their significant size, the fusion may high selectivity in protein labelling may be achieved by thio-
alter the function of the targeted protein. Various methods for phene oligomers through non-covalent interactions. Their
in vitro, ex vivo and in vivo experiments have demonstrated that
molecules made of five to seven thiophene rings and with
aLaboratorio MIST.E-R, National Research Council, Via Gobetti 101, I-40129
terminal carboxy groups display a great affinity for fibrillar pro-
Bologna, Italy
teins and may act as specific probes for the pathological hall-
marks of protein misfolding diseases. Owing to the flexibility
of the aromatic backbone, these oligomers undergo subtle
conformational changes upon interaction with fibrillar pro-
teins, causing appreciable variations in absorption and emis-
sion features.
In our long-standing research on molecular engineering of
functional thiophene oligomers6 – fluorescent and electro-
active compounds, capable of an astonishing variety of non-
bonding interactions in the most diverse environments7 – we
have described thiophene fluorophores for the in vitro label-
ling of proteins and oligonucleotides6a,b and have initiated a
bInstitute for Organic Synthesis and Photoreactivity, National Research Council, Via
Gobetti 101, I-40129 Bologna, Italy
cNNL-CNR Nanoscience Institute, Università del Salento, Via Arnesano, I-73100
Lecce, Italy
dDip. Chimica G. Ciamician, Università di Bologna, Via Selmi 2, I-40126 Bologna,
Italy
eIstituto di Biofisica-CNR, via Moruzzi 1, I-56124 Pisa, Italy.
E-mail: giovanna.barbarella@isof.cnr.it, ilariaelena.palama@nano.cnr.it
fDip. Ingegneria Innova-zione, Università del Salento, Via Arnesano, I-73100 Lecce,
Italy
gMediteknology srl, National Research Council, Via Gobetti 101, I-40129 Bologna,
Italy
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob41982g
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search for cell-permeant and biocompatible thiophene fluoro- and ref. 6c for 5b) and one, compound 8, photobleaches inside
phores to study their behavior inside live cells.6c We have the live cells but not in solution in organic solvents.
found that some thiophene fluorophores are capable of
Compounds 1–2 and 7–8 were synthesized by means of the
specific non-bonding interactions with fibrillar type-I collagen Suzuki cross-coupling reaction8 in aqueous solvents. Although
inside live human and murine fibroblasts.6c,d In this paper we similar products have already been synthesized in our labo-
describe a thiophene fluorophore having the ability to dis- ratory by the Stille reaction,9,10 the Suzuki coupling is by far a
criminate among the innumerable globular proteins present better reaction to prepare ultrapure fluorophores. Enabling
inside living NIH·3T3 mouse fibroblast cells. These results techniques such as microwave11 and ultrasound12 assistance
open the door to the possibility of using specifically engin- make the preparation rapid (minutes instead of hours) and the
eered thiophene fluorophores, cell-permeant and biocompati- yields high. Moreover, owing to the reduced metal–halogen
ble, for easy and direct tagging of proteins in live cells and exchange and number of byproducts, reaction workup and
their detection by fluorescent techniques.
separation are much easier.
2.1. Optical properties and environment sensitivity
Table 1 shows maximum absorption and emission wave-
lengths, molar absorption coefficients, quantum yields and
fluorescence lifetimes of compounds 1–3 and 7–8 in toluene, a
solvent endowed with a low dielectric constant (2.40813).
Table 1S† reports a few data on ethyl acetate (EA) and dichloro-
methane (DCM) with dielectric constants 6.02 and 8.9,13
respectively. Fig. 7S† shows the absorption and emission
spectra of 1–3 and 7–8 in toluene, CDM and DMSO. Having
large dipolar moments (Table 2S†) and in agreement with
already reported data,6b,14 both absorption and emission wave-
lengths increase on increasing the dielectric constant of the
solvent. Owing to their absorption wavelength range, com-
pounds 1–3 and 7–8 are a blue-absorbing palette of fluoro-
phores, suitable for excitation by a blue-violet laser line.
However, they have very large Stokes shifts, as is generally
observed for thiophene oligomers.6
Thus, in DCM the emission wavelengths attain values in
the red region, 619 nm for 7 and 660 nm for 8. Note that red
fluorescent labels are particularly useful in biological fluo-
rescence microscopy to prevent problems due to autofluores-
cence of the cells. Table 1 shows that all fluorophores are
brightly fluorescent owing to high molar absorption coeffi-
cients and fluorescence quantum yields. Compound 7 stands
out among the others for its high ε value and quantum yield
(also in EA, as shown in Table 1S†), indicating very bright fluo-
rescence.15 Table 1S† shows that despite the small difference
in dielectric constant between EA and DCM, compounds 1–2
and 7–8 display significant emission solvatochromism. This
effect should be attributed to their marked hyperconjugated
push–pull structural features. The maximum absorption
2. Results and discussion
Chart 1 shows the molecular structure of the cell-permeant
thiophene N-succinimidyl esters whose synthesis is described
here for the first time, namely compounds 1–2 and 7–8. For
comparison, the molecular structure of all the other cell-per-
meant thiophene fluorophores identified so far, namely com-
pounds 3–6 and 9,6c are also reported. It can be easily seen
that a rationalization of the structural elements leading to cell
permeability is difficult and that the identification of the struc-
tures depicted in the chart is due to a trial and error procedure
on a large number of thiophene fluorophores. In Chart 1 the
fluorophores are organized according to their emission color
inside live mouse embryonic fibroblast NIH·3T3 cells. In agree-
ment with previous observations,6c the emission color of the
fluorophores inside live cells is similar to the fluorescence
color in dichloromethane (DCM) solution. Notably, most of
these fluorophores emit in the green region of the visible spec-
trum whereas only three display fluorescence in the red
region. Two fluorophores, 3 and 5b, are toxic (this paper for 3
Table 1 Maximum absorption (λmax, nm) and emission (λPL, nm) wave-
lengths, molar absorption coefficients (ε, cm−1 mol−1), quantum yields
(ϕ) and fluorescence lifetimes of 1–3 and 7–8a
Item
λmax
λPL
ε
ϕb
τ (ns)
1
2
3
7
8
381
430
418
414
443
465
509
515
530
581
28 400
22 300
33 000
59 800
19 200
1.00
0.92
0.12
0.92
0.80
2.1
2.7
1.3
2.3
2.7
Chart 1 Molecular structure and emission color inside live NIH·3T3
cells of all cell-permeant thiophene fluorophores identified so far (grey
structures are described in ref. 6c).
a In toluene. b With respect to perylene in ethanol, ϕref = 0.92.
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wavelengths always follow the trend 1 < 7 < 2 < 8, suggesting subsequent neutralization with stoichiometric amounts of
that the introduction of a phenyl ring on the piperidine side N(CH2CH3)3. Protonation induces sizeable upfield shifts of the
increases the energy of S0–S1 electron transition more than it protons of the phenyl group, adjacent to the protonation site,
decreases the same energy gap by increasing electronic conju- accompanied by a blue shift in fluorescence and its decrease
gation (compare 7 with 2). Conversely, replacement of a phenyl in intensity. These changes were reversible and after several
ring with an electron rich thienyl ring on the piperidine side cycles of acidification/neutralization both the chemical shifts
leads to a lower S0–S1 energy gap (compare 2 with 1 and 8 and the fluorescence came back to the starting values. Appar-
with 7). In all cases, fluorescence decays were found to be ently, 7 is a good acidity sensor and could be usefully checked
strictly monoexponential and falling in the 2–3 ns range. The in pathological cells to detect acidity variations.
lifetime trend in EA followed the number of aromatic rings in
2.2. Uptake of the fluorophores by live NIH·3T3 mouse
fibroblast cells
the compound, being higher in 7 and 8. Yet, an analogous
pattern could not be observed in DCM, suggesting that solute–
solvent interactions have a strong influence on the non-radia- Fluorophores 1–3 and 7–8 displayed excellent cell permeability
tive decay kinetics and, ultimately, on the fluorescence life- and were rapidly internalized, showing efficient retention
time. In most cases, emission red-shift was associated with a inside the cells. Fluorophores 1–2 and 7 showed bright and
reduced emission lifetime, in keeping with the typical photo- persisting staining of the cytoplasm with fluorescence trans-
physical behavior of solvatochromic compounds for which the mitted from mother to daughter cells during the replication
excited state energy stabilization favors fast internal conversion process. By contrast, the brilliant red emission of fluorophore
down to ground state.13
8 was quenched after a few seconds examination with both the
Conventional thiophene fluorophores are poorly sensitive laser scanning confocal microscope and the fluorescence
to pH changes. Fluorophores 1–2 and 7–8 were engineered for microscope. Initial experimental data and theoretical calcu-
enhanced pH sensitivity, owing to the presence of the nitrogen lations aimed at elucidating this behaviour are reported in ESI
atom in the piperidine moiety. As expected, these fluorophores (Fig. 8S, 14S†).
display a great sensitivity to acidity changes in organic sol-
The cytotoxicity of the fluorophores towards NIH·3T3 cells
vents. Protonation of the nitrogen atom caused reversible was analyzed by means of the MTT cytotoxicity test, a com-
changes in fluorescence emission. The process is illustrated in monly used method for measuring the viability of living cells
detail in Fig. 1 for fluorophore 7. The figure shows the vari- via mitochondrial dehydrogenase activity, whose key com-
ations of proton chemical shifts and fluorescence wavelengths ponent is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
upon stepwise addition of CF3COOH in toluene and bromide. The results are shown in Fig. 2.16
Fig. 1 (A) Variation of the chemical shifts in toluene of the aromatic protons of fluorophore 7 upon stepwise addition of CF3COOH and subsequent
neutralization with stoichiometric amounts of N(CH2CH3)3. (B) Corresponding variations in wavelength and intensity of fluorescence emission.
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that while 3 is cell-permeant but toxic to the cells, its regio-
isomer with the SCH3 substituent in the α-position6a,b is cell-
impermeant. Since for different reasons neither 8 nor 3 could
be examined in live cells by LSCM, their emission was checked
in fixed NIH·3T3cells. The relative images are shown in
Fig. 12S and 13S,† respectively.
Fig. 3 shows the LSCM images of live NIH·3T3 cells treated
with the green emitting fluorophores 1,2 and monitored after
1 h, 24 h and 192 h from treatment. Additional images are
reported in Fig. 9S and 10S.† Both fluorophores caused the
intense fluorescent staining of the cytoplasm of the cells –
deep green in the case of 1, green with a green-yellow shade in
the case of 2 – while the nucleus remained rigorously
unstained. Being activated esters, once crossed the cell mem-
brane, the fluorophores can react with the amine primary
groups of proteins present in the cytoplasm, i.e. in the basic
region of the cells, to form a covalent amide bond.6a–c
However, which ones of the NH2 groups of cytoplasmic pro-
teins have reacted and to what extent cannot be established.
Fig. 3 shows the presence of multiple intensely fluorescent
spots indicating the accumulation of the fluorophore in the
Fig. 2 MTT cytotoxicity tests on NIH·3T3 cells treated with fluoro-
phores 1–3 and 7–8 compared to untreated cells (NT). Representative
measurements of three distinct sets of data (Student’s t-test, P < 0.05).
No reduced cell viability was found for the cells treated with
fluorophores 1–2 and 7–8, while those treated with fluorophore
3 displayed drastically reduced viability. Incidentally, we note
Fig. 3 (A, B) LSCM images of live NIH·3T3 cells after 1 h, 24 h and 192 h from treatment with fluorophores 1 and 2, respectively. 63× oil immersion.
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perinuclear region, i.e. the region where protein synthesis numerous, large and diffuse. The treatment of the cells with
takes place. This behaviour is similar to that observed upon fluorophore 7 (as well as with fluorophores 1–3 and 8) was
treatment of the same type of cells with fluorophore 4 repeated several times and the results were highly reproducible.
(Chart 1). In that case the accumulation of green spots in the
The red fluorescent clusters formed after 192 hours from
perinuclear region was followed by formation of green fluo- treatment with 7 were isolated from NIH·3T3 cell lysate (see
rescent microfibers, mainly made of type-1 collagen embed- ESI†) and were found to be soluble in water. This ruled out the
ding the fluorophore, that were subsequently extruded in the possibility that they were aggregates of 7, since this fluoro-
extracellular matrix.6c,d By contrast, in the case of fluorophores phore is insoluble in H2O. The water solution of the red clus-
1 and 2, the accumulation in the perinuclear region is followed ters was used for a sodium dodecyl sulfate polyacrylamide gel
by elimination of the fluorophore by cell machinery. Indeed, electrophoresis (SDS-PAGE) and for spectroscopic analyses,
after 192 h from treatment, only feeble traces of green fluo- namely UV, PL and circular dichroism (CD). Despite the very
rescence are still present.
low concentration (we tentatively set the upper limit of concen-
Fig. 4 shows the LSCM images of live NIH·3T3 cells 1 h, tration to 10−6 M), the spectra were rather intense. All results
24 h, 48 h, 72 h and 192 h after treatment with fluorophore 7. are reported in Fig. 5.
Additional images are reported in Fig. 11S.† The spontaneous
The SDS-PAGE trace (Fig. 5A) was surprisingly simple and
uptake of fluorophore 7 by the cells caused rapid red fluore- revealed the presence of only four low weight proteins, two
scent staining of the cytoplasm while the nucleus remained around 60 kDa and two in the range 10–20 kDa. Given their
dark. As in the case of fluorophores 1–2, during cell solubility in water, these proteins must be globular ( fibrillar
proliferation the fluorescence was transmitted from mother to proteins are insoluble in water). The cells contain innumerable
daughter cells and accumulation of fluorescent spots in the components, including very many globular proteins. Thus,
perinuclear region was observed. However, contrary to what fluorophore 7 is capable of discriminating among all these
was found with 1 and 2, the appearance of red spots in the components and of being (and remaining) associated with
perinuclear region was followed by the progressive formation four specific globular proteins.
of bright red clusters. The bright red clusters accumulated
The spectra of the water solution of the red clusters were
with time and, after 192 from treatment, appeared also clean and simple and confirmed this conclusion, as
h
Fig. 4 LSCM images of live NIH·3T3 cells after 1 hour (A), 24 hours (B), 48 hours (C), 72 hours (D) and 192 hours (E,F) from treatment with fluoro-
phore 7.
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Fig. 5 (A) SDS-PAGE gel electrophoresis of the red fluorescent clusters formed by live NIH·3T3 live cells 192 hours after treatment with fluorophore
7 (a = broad range marker, b = sample). (B) Circular dichroism (a), UV-Vis absorption (b), excitation (c), and fluorescence (d, λexc = 375 nm) spectra of
the sample dissolved in water.
shown in Fig. 5B (a–d). The UV spectrum (a) displays only the bonding interactions such as H-bondings, electrostatic and
signals typical of proteins in the range 200–300 nm, corres- hydrophobic interactions. It could be argued that the proteins
ponding to the CD spectrum (b). Note that fluorophore 7 is an might be linked to the fluorophore by a covalent bond formed
achiral molecule which does not afford any CD signal when by reaction of the N-succinimidyl group with some NH2 groups
dissolved in DMSO–H2O. In the UV spectrum the signals per- of the proteins. However, this hypothesis seems unlikely due
taining to the thiophene fluorophore were barely visible. to the fact that the reactivity of the N-succinimidyl group in 7
However, these signals were observed in the excitation spec- should be very close to that in its shorter analogue 1, which is
trum (c) showing two broad peaks centered at 275 nm and instead eliminated by the cells. Clearly, establishing the nature
375 nm. The fluorescence signal (d, λexc = 375 nm) is broad, of the protein–fluorophore conjugate is a priority for further
intense and structured (maxima around 460 nm, 500 nm and studies. Identifying the proteins will help to guide rational
575 nm). The difference between absorption and emission is efforts to design more selective thiophene fluorophores. But
large and in the order of those generally observed for thio- no matter what the nature of the conjugate is, it is remarkable
phene fluorophores. We conclude that the ‘objects’ under that it does not cause any harm to the cells, as shown by the
examination, i.e. the red clusters, associate the properties viability tests reported in Fig. 1.
typical of proteins (in particular the CD spectrum) with the
fluorescence typical of a thiophene fluorophore.
The mutual recognition between thiophene fluorophores
and specific proteins inside the cells is driven by the cell’s
The identification of the four globular proteins interacting own metabolism without the need for any additional
with 7 inside live NIH 3T3 cells requires more extensive cofactor. An essential feature of molecular recognition is
studies, in particular the biochemical analysis of the red clus- the flexibility of the systems involved, facilitating binding
ters, which is currently under way. However, what matters at through a large number of weak interactions. In the present
this stage is that fluorophore 7 is selectively recognized by a case, the well known ability of proteins to adjust their
few specific globular proteins inside live cells. Most probably, conformation in order to make specific interactions with a
this fluorophore possesses the right size and stereoelectronic given ligand fits well with the geometric adaptability of the
requisites to be accommodated by the proteins via non- sulfur atom.6f
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1–5 nm band-pass. CD spectra were recorded with a spectropo-
larimeter JASCO J-715 under ambient conditions.
3. Conclusion
Besides their uncommon combination of properties – lack of
cytotoxicity, brightness, long-lasting fluorescence transmit-
table from mother to daughter cells and environment sensi-
tivity – the most striking result concerning the thiophene
fluorophores described here is that one of them (compound 7,
Chart 1) has the right size, steric and electronic properties to
be selectively recognized inside live NIH 3T3 cells by four low
weight globular proteins.
Laser scanning confocal microscopy (LSCM)
Confocal micrographs were obtained with a Leica confocal
scanning system mounted into a Leica TCS SP5 equipped with
oil immersion objectives and spatial resolution of approxi-
mately 200 nm in x–y and 100 nm in z.
Uptake of fluorophores by live NIH·3T3 cells
The importance of this result is stressed by the fact that
another thiophene fluorophore (compound 4, Chart 1) has
already been shown to be selectively recognized by the fibrillar
protein type-I collagen inside the same type of live cell.6c,d
Thus, some biocompatible thiophene fluorophores have the
capability to discriminate between protein types, fibrillar or
globular, at the molecular level, inside living cells, depending
on their molecular structure, shape and stereoelectronic
properties.
Mouse embryonic fibroblasts (NIH·3T3 cell line) were seeded
at a density of 100 000 cells in a tissue culture plate in 1 mL of
complete culture medium. The fluorophores were dissolved in
a minimum amount of DMSO in order to obtain a stock solu-
tion and were then administered to cells by adding an appro-
priate dilution in serum free DMEM to obtain the final
concentration of 0.05 mg mL−1 (∼10−7 M) and incubated at
37 °C in 5% CO2, 95% relative humidity for 1 h. At the end of
the incubation period the unbound dye was removed by
washing repeatedly the cell cultures with serum free DMEM.
To the best of our knowledge no other class of organic
(least of all inorganic) dyes has so far displayed similar
properties.
We believe that our results may open the door to the use of
thiophene fluorophores as a new tool for visualizing specific
proteins and their dynamics inside live cells. These small
traceable fluorescent dyes display indeed a great affinity to pro-
teins, a topic which is just starting to be developed.5
The derivatives of thiophene have been studied for decades
and are still a matter of intense research for application in
organic devices such as field-effect transistors, light-emitting
diodes, light-emitting transistors and photovoltaic cells.17–20
The results presented here indicate that they might have a bril-
liant future also in the fields of chemical biology and
biotechnology.
Acknowledgements
Thanks are due to the project “Molecular Nanotechnologies
for Human Health and Environment” (PON R&C 2007-2013,
code PON02_00563_3316357) and to Rete Alta Tecnologia
Emilia Romagna, Italy.
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