New cationic heptamethinecyanine-graphene hybrid materials

Full text of DOI:10.1016/j.dyepig.2019.108047

Journal Pre-proof
New cationic heptamethinecyanine-graphene hybrid materials
Kyriakos C. Prousis, Ruben Canton-Vitoria, Georgia Pagona, Maria Goulielmaki,
Vassilis Zoumpourlis, Nikos Tagmatarchis, Theodora Calogeropoulou
PII:
S0143-7208(19)32318-6
DOI:
https://doi.org/10.1016/j.dyepig.2019.108047
DYPI 108047
Reference:
To appear in: Dyes and Pigments
Received Date: 30 September 2019
Revised Date: 6 November 2019
Accepted Date: 12 November 2019
Please cite this article as: Prousis KC, Canton-Vitoria R, Pagona G, Goulielmaki M, Zoumpourlis V,
Tagmatarchis N, Calogeropoulou T, New cationic heptamethinecyanine-graphene hybrid materials,
Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.108047.
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New cationic heptamethinecyanine-graphene hybrid materials
#
#
Kyriakos C. Prousis,a, Ruben Canton-Vitoria,b, Georgia Pagona,b Maria Goulielmaki,a Vassilis
Zoumpourlis,a Nikos Tagmatarchisb* and Theodora Calogeropoulou a*
a Institute of Chemical Biology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, Athens
11635, Greece
b Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou
Avenue, Athens 11635, Greece
# Equally contributed.
*Corresponding authors
1. Introduction
Near-infrared (NIR) fluorophores that absorb and emit within the 700–900 nm region have attracted
considerable attention by the scientific community due to their diverse applications in biomedical
materials and related fields. These dyes have been applied widely as active ingredients in
semiconducting and laser materials, optical recording media, paints, and bio probes[[1]]. More
specifically, NIR fluorescent dyes have emerged as promising modalities for monitoring the levels of
various biologically relevant species in cells and organisms[2,3]. The advantages of NIR probes include
minimal interfering absorption and fluorescence from biological samples, inexpensive laser diode
excitation, reduced scattering and enhanced tissue penetration depth [[3]]. However, there are only
relatively few classes of NIR dyes that are readily available. These include phthalocyanines, cyanines,
squaraines and more recently sulfone-rhodamines [5]. In particular, cyanine dyes encompass a large and
diverse class of molecules with two aromatic nitrogen-containing heterocycles connected by a
polymethine linker. They exhibit large extinction coefficient and high quantum yield values and possess
highly tunable structures. The monomethine and trimethinecyanines (Cy3) generally show absorption in
the visible region while, heptamethinecyanines (Cy7) may show absorption beyond 1000 nm. In
particular, the NIR dye Indocyanine Green (ICG) has been approved by the FDA and has been
1

implemented in clinical use [6]. Currently, there is an increasing demand for photostable NIR dyes
presenting large Stokes shifts, due to the emerging interest in optical in vivo imaging as well as for their
applications in the modification of nanomaterials [7].
Graphene is a single graphite plane in which the sp2 hybridized carbon atoms form a hexagonal lattice.
Graphene can be considered as a `building block' for graphite, nanotubes and other carbon materials.
Recent progress has shown that graphene-based materials can have a profound impact on electronic
and optoelectronic devices, chemical sensors, nanocomposites and energy storage [8-10]. Furthermore,
during the past few years, graphene has attracted considerable attention for potential applications in
biosensing, biomedical diagnosis and therapy and biotechnological applications due to its unique
physical properties and high surface-to-volume ratio allowing the introduction of numerous species
upon functionalization [11,12]. Thus, graphene has been functionalized with avidin–biotin, peptides,
NAs, proteins, aptamers, small molecules, bacteria, and cells through physical adsorption or chemical
conjugation. The functionalized graphene biosystems have been used to generate biological platforms,
biosensors, and biodevices [13,14]. Photothermal therapy (PTT) has been pursued during recent years as
an alternative cancer therapeutic strategy which relies on the capability of NIR-photoabsorbers to
produce heat upon NIR laser irradiation, which in turn results in the thermal ablation of malignant cells.
PTT advantages include specificity, minimal invasiveness and spatial-temporal selectivity [15]. The
ability of graphene to absorb in the NIR region has prompted its application in the photothermal therapy
of cancer. Thus, a variety of functional compounds have been used to decorate graphene aiming to
increase the efficiency of the nanocomposites towards cancer therapy and/or imaging modalities [16-
20].
In the current study we have synthesized four new near-infrared fluorescent indolenine-based cationic
heptamethinecyanine dyes, which were further covalently conjugated to exfoliated graphene. The
rationale for the synthesis of the graphene-based hybrid materials is their potential application as an
2

optical imaging modality characterised by high cell viability with respect to the cyanine dyes
counterparts. Additionally, future applications could involve their application as a delivery platform for
high loading efficiency of additional components for theranostic applications.
Specifically, the conjugation of two of the dyes was accomplished via a condensation reaction on pre-
modified graphene, while the other two heptamethinecyanines were directly anchored onto exfoliated
graphene sheets via the methodology of in-situ generated aryl diazonium salt addition. The optical
properties of the dyes and the respective graphene-based hybrids were studied using absorbance and
fluorescence spectroscopy. In order to get an insight on the potential for biomedical applications two
cell lines with distinct characteristics regarding their physiologic state were employed. Thus, a
representative of a cancer cell line (RKO), and an undifferentiated, very sensitive non-cancerous
progenitor cell line (WJ-MSC human umbilical cord mesenchymal stem cells) were used to evaluate the
in vitro cytotoxicity and the cell penetrating capacity of the new hybrids and corresponding NIR dyes.
The current work goes beyond the state-of-the-art as previous efforts were focused only on the
assessment of charge-transfer interactions developed within modified graphene with anionic
tricyanofuran-based heptamethinecyanine dyes, formed either through covalent derivatisation [21] or
supramolecular functionalisation [22].
2. Experimental section
2.1. Materials and instrumentation
All reactions were carried out under scrupulously dry conditions. THF was distilled over sodium in the
presence of benzophenone, DCM was distilled over calcium hydride. 1H NMR spectra were recorded at
13
600 or 300 MHz, C NMR spectra at 150.9 or 75.5 MHz and were internally referenced to residual
solvent signals. Data for 1H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet,
br s = broad singlet, d = doublet, dt = doublet of triplets, dd = doublet of doublet, t = triplet, m =
13
multiplet), coupling constant and integration. Data for C NMR are reported in terms of chemical shift
3

(δ ppm). HR-mass spectra were recorded on a UHPLC LC-MSn Orbitrap Velos-Thermo instrument. Thin
Layer Chromatography (TLC) was carried out on precoated silica gel (0.2 mm, 60 F254) glass plates.
Chromatographic purification was performed using silica gel (200–400 mesh). Microwave reactions were
run on a CEM instrument. Steady-state UV-Vis electronic absorption spectra were recorded on a Perkin
Elmer (Lambda 19) UV-Vis-NIR spectrophotometer. Steady-state emission spectra were recorded on a
Fluorolog-3 JobinYvon-Spex spectrofluorometer (model GL3-21). Pico-second time-resolved
fluorescence spectra were measured by the time-correlated-single-photon-counting (TCSPC) method on
a Nano-Log spectrofluorometer (Horiba JobinYvon), by using a laser diode as an excitation source
(NanoLED, 654 and 784 nm) and a UV-Vis detector TBX-PMT series (250-850 nm) by Horiba JobinYvon.
Lifetimes were evaluated with the DAS6 Fluorescence-Decay Analysis Software. Mid-infrared spectra in
the region 500−4500 cm-1 were obtained on a Fourier transform IR spectrometer (Equinox 55 from
Bruker Optics) equipped with a single reflection diamond ATR accessory (DuraSamp1IR II by SensIR
Technologies). Typically, 100 scans were acquired at 2 cm−1 resolution. Micro-Raman scattering
measurements were performed at room temperature in the backscattering geometry using a RENISHAW
inVia Raman microscope equipped with a CCD camera and a Leica microscope. A 2400 lines mm-1 grating
was used for all measurements, providing a spectral resolution of ± 1 cm-1. As an excitation source the
Ar+ laser (633 nm with less than 2.65 mW laser power) was used. Measurements were taken with 45
seconds of exposure times at varying numbers of accumulations. The laser spot was focused on the
sample surface using a long working distance 50x objective. Raman spectra were collected on numerous
spots on the sample and recorded with Peltier cooled CCD camera. The intensity ratio ID/IG was obtained
by taking the peak intensities following any baseline corrections. The data were collected and analyzed
with Renishaw Wire and Origin software. Thermogravimetric analysis was performed using a TGA Q500
V20.2 Build 27 instrument by TA in a nitrogen (purity >99.999%) inert atmosphere. Scanning electron
4

microscope (SEM) imaging and energy dispersive X-ray spectroscopy (EDS) were performed using a FE-
SEM (model JSM-7610F) equipped with an EDAX (X-ACT, Oxford instrument).
2.2 Synthetic procedures
2.2.1 Synthesis of (3E)-2-chloro-2-(hydroxymethylene)cyclohex-1-enecarbaldehyde (2).
o
To a solution of dimethylformamide (20 mL, 273 mmol) in 20 mL CH2Cl2 at 0 C, a solution of POCl3
(17.5 mL, 115 mmol) in 20 mL CH2Cl2 was added dropwise. After 30 min, cyclohexanone was added (5 g,
50 mmol), the resulting mixture was refluxed with vigorous stirring for 3 h at 80 °C, was poured into ice-
cold water, and was let it stand overnight to obtain 2 as a yellow solid (7.6 g, 85%) after filtration and
1
drying. H NMR (300 MHz, DMSO-d6) δ: 1.54 – 1.52 (m, 2H), 2.35 (m, 4H), 7.52 (s, 1H), 10.08 (s, 1H),
10.82 (s, 1H). ESI m/z [M+H]+ 173.1.
2.2.2. Synthesis of 1,2,3,3-tetramethyl-3H-indolium iodide (3).
A mixture of 2,3,3-trimethyl-3H-indole (2.0 g, 12.6 mmol) and methyl iodide (4.71 mL, 75.3 mmol) in
acetonitrile (7.8 mL) was heated at reflux, using microwave irradiation for 15 minutes (130W) (open
vessel). The reaction mixture was cooled to room temperature and the solvent evaporated under
reduced pressure. The residue obtained was triturated with diethyl ether (20 mL) and the solid was
recrystallized from methanol-diethyl ether. The pale yellow crystalline product was filtered, washed
1
with diethyl ether and dried under vacuo over P2O5. Yield 3.42 g, 91%. H NMR (300 MHz, DMSO-d6) δ:
1.53 (s, 6H), 2.77 (s, 3H), 3.97 (s, 3H), 7.64 – 7.62 (m, 2H), 7.82 – 7.84 (m, 1H), 7.90 – 7.92 (m, 1H).
2.2.3. Synthesis of 2-(2-[2-chloro-3-([1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene]ethylidene)-1-
cyclohexen-1-yl]ethenyl)-1,3,3-trimethylindolium iodide (4)
1,2,3,3-Tetramethyl-3H-indolium iodide (3) (2.5 g, 8.31 mmol), dialdehyde 2 (0.478 g, 2.77 mmol) and
CH3COONa (1.36 g, 16.62 mmol) were added to a mixture of acetic acid and acetic anhydride (1:1, 108
mL) and the mixture was heated for 4 h at 120 °C. Subsequently, the solvent was evaporated and the
residue was partitioned between CH2Cl2 (50.0 mL) and water (30 mL) and the organic layer was dried
5

over sodium sulphate and the solvent was evaporated in vacuo. The crude product was purified by flash
column chromatography to yield compound 4 as a green solid (2.84 g, 74%), [eluting solvent system:
CH2Cl2/CH3OH, (98:2 to 95:5)]. 1H NMR (300 MHz, DMSO-d6) δ: 1.67 (s, 12H), 1.92 – 1.79 (dt, J = 11.8, 6.3
Hz, 2H), 2.71 (t, J = 6.0 Hz, 4H), 3.68 (s, 6H), 6.30 (d, J = 14.2 Hz, 2H), 7.25 – 7.33 (m, 2H), 7.44 (d, J = 4.1
13
Hz, 4H), 7.62 (d, J = 7.3 Hz, 2H), 8.25 (d, J = 14.2 Hz, 2H). C NMR (600 MHz, DMSO-d6) δ: 20.4, 25.9,
27.3, 31.6, 48.8, 101.8, 111.4, 122.3, 125.1, 126.0, 128.5, 140.9, 142.6, 142.8, 147.6, 172.6.
2.2.4. Synthesis of 6-aminohexanoic acid (5)
6-Bromohexanoic acid (1.0 g, 51.3 mmol) was dissolved in 25% aqueous NH4OH (40 mL) and the
solution was stirred for 24 hours at ambient temperature. The reaction mixture was then evaporated
1
under reduced pressure to give the desired product 5 as a white solid (7.26 g, 99% yield). H NMR (300
MHz, CD3OD) δ: 1.32 – 1.45 (m, 2H), 1.54 – 1.70 (m, 4H), 2.20 (t, J = 6.7 Hz, 2H), 2.87 (t, J = 7.0 Hz, 2H),
3.28 (s, 2H).
2.2.5. Synthesis of NIR-dye 7
Compound 4 (25.0 mg, 0.05 mmol), 6-aminohexanoic acid (5) (38.5 mg, 0.33 mmol) and triethylamine
o
(109 μL, 0.82 mmol) were dissolved in CH3OH (2.0 mL) and refluxed at 70 C for 4 h. The solvent was
evaporated under reduced pressure and the residue was purified by flash column chromatography
1
(95:4:1 CH2Cl2/CH3OH/CH3COOH) to yield NIR-dye 7 as a blue solid (30 mg, 52%); H NMR (300 MHz,
CD3OD) δ: 1.44 – 1.51 (m, 2H), 1.65 (s, 12H), 1.58 – 1.69 (m, 2H), 1.77 – 1.89 (m, 4H), 2.25 (t, J = 7.2 Hz,
2H), 2.56 (t, J = 6.3 Hz, 4H), 3.44 (s, 6H), 3.78 (t, J = 6.8 Hz, 2H), 5.78 (d, J = 13.1 Hz, 2H), 7.05 – 7.11 (m,
13
4H), 7.29 – 7.38 (m, 4H), 7.77 (d, J = 12.9 Hz, 2H). C NMR (150 MHz, CD3OD) δ: 171.1, 168.9, 145.0,
141.2, 140.2, 129.4, 123.9, 123.0, 121.7, 109.8, 95.6, 51.5, 35.0, 32.2, 29.1, 27.5, 26.0, 25.8, 22.9. ESI-
MS: m/z 578.31 [M]+; ESI-MS: m/z 578.39 [M]+ HR-MS (ESI+): [M]+ C38H48N3O2 calculated for 578.3741,
.
found 578.3737.
6

2.2.6. Synthesis of NIR-dye 8
Compound 4 (50 mg, 0.082 mmol) and thioglycolic acid (9 μL, 0.1 mmol) were dissolved in
acetonitrile (10 mL), and one drop of triethylamine was added. The reaction mixture was stirred at room
temperature for 15 min, and then the solvent was evaporated. The crude product was purified by flash
column chromatography to give NIR-dye 8 (18.7 mg, 70%) as green solid. [eluting solvent system:
CH2Cl2/CH3OH, (98:2 to 80:20)]. 1H NMR (300 MHz, CD3OD) δ: 1.74 (s, 12H), 1.86 – 1.90 (m, 2H), 2.56 (t, J
= 6.1 Hz, 4H), 3.58 (s, 2H), 3.62 (s, 6H), 5.97 (d, J = 14.1 Hz, 2H), 7.05 (d, J = 8.1 Hz, 2H), 7.20 (t, J = 7.4 Hz,
13
4H), 7.33 – 7.36 (m, 4H), 9.12 (d, , J = 14.1 Hz, 2H). C NMR (150 MHz, CD3OD) δ: 175.1, 174.5, 159.1,
147.5, 144.4, 142.8, 142.5, 134.4, 129.7, 126.0, 123.3, 111.6, 102.1, 50.4, 43.6, 31.6, 28.3, 27.1, 22.7;
ESI-MS: m/z 539.29 [M]+. HR-MS (ESI+): [M]+ C34H39N2O2S calculated for 539.2727, found 539.2724.
2.2.7. Synthesis of 4-(2-aminoethyl)benzenamine (10)
To a solution of 2-(4-nitrophenyl)ethanamine hydrochloride (9) (1 g, 4.94 mmol) in methanol (25 mL)
was added ammonium formate (1.56 g, 24.7 mmol) followed by 10% palladium on carbon (130 mg), and
the reaction mixture was refluxed for 24 h. The reaction mixture was filtered through Celite and washed
with methanol. The filtrate and washings were combined and the solvent was then evaporated. The
crude product was purified by flash column chromatography (CH2Cl2/CH3OH: 80:20) to give 4-(2-
aminoethyl)benzenamine hydrochloride salt as a brown solid. The hydrochloride salt was then treated
with aqueous NaOH 5N (15 mL), extracted with dichloromethane, dried over Na2SO4 and concentrated
under reduced pressure to give 4-(2-aminoethyl)benzenamine 10 as brown sticky foam. Yield 0.51 g,
80%. 1H NMR (300 MHz, CDCl3) δ: 2.84 (t, J = 6.8 Hz, 2H), 2.90 (t, J = 6.9 Hz, 2H), 3.57 (br s, 2H), 6.64 (d, J
= 8.4Hz, 2H), 6.99 (d, J = 8.4Hz, 2H).
2.2.8. Synthesis of NIR-dye 11
Compound 4 (50 mg, 0.082 mmol) and 4-(2-aminoethyl)benzenamine (10) (21.8 mg, 0.164 mmol)
were dissolved in acetonitrile (5 mL). The reaction mixture was heated at reflux for 2h and then stirred
7

at room temperature for 12h (consumption of starting material was monitored by ESI-MS).
Subsequently the solvent was evaporated and the residue was purified by column chromatography
1
(CH2Cl2/CH3OH: 98:2 to 95:5) to give NIR-dye 11 (35 mg, 61%) as green solid. H NMR (600 MHz, CDCl3)
δ: 1.57 – 1.68 (m, 14H), 2.39 (t, J = 6.2 Hz, 4H), 3.17 (t, J = 6.7 Hz, 2H), 3.34/3.57 (br 2s, 6H), 4.10 (dd, J =
5.9, 12.0 Hz, 2H), 5.48 (d, J = 9.5 Hz, 2H), 6.58 (d, J = 8.1 Hz, 2H), 7.02 – 7.04 (m, 4H), 7.23 – 7.26 (m, 4H),
7.61 (br s, 2H). 13C NMR (300 MHz, CD3OD) δ: 21.3, 25.2, 29.0, 29.7, 36.2, 50.7, 93.9, 108.0, 115.4, 122.1,
122.6, 128.1, 129.8, 137.3, 140.8, 169.7, 172.6; ESI-MS: m/z 583.40 [M]+. HR-MS (ESI+): [M]+ calculated
for C40H47N4 583.3795, found 583.3792.
2.2.9. Synthesis of tert-butyl 4-(2-hydroxyethyl)phenylcarbamate (13)
To a solution of 2-(4-aminophenyl) ethanol (12) (1 g, 7.28 mmol) in ethyl acetate (10 mL) was added
di-tert-butyl dicarbonate (1.75 g, 8.02 mmol), and the reaction mixture was stirred for 24 hours at room
temperature. Subsequently, water and ethyl acetate were added to the reaction mixture. The organic
layer was dried over anhydrous magnesium sulphate and the solvent was removed under reduced
pressure. The residue was purified by crystallization with ethyl acetate and petroleum ether to give tert-
1
butyl 4-(2-hydroxyethyl)phenylcarbamate (13) (1.64 mg, 95%) as white solid. H NMR (300 MHz, CDCl3)
δ: 1.52 (s, 9H), 2.81 (t, J = 6.6 Hz, 2H), 3.82 (br s, 2H), 6.45 (br s, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.29 (d, J =
8.4 Hz, 2H).
2.2.10. Synthesis of dye 16
Diethyl azodicarboxylate (0.63 mL, 4 mmol) was added to an efficiently stirred solution of triphenyl
phosphine (1.50 g, 4 mmol) in 10 mL of freshly distilled tetrahydrofuran at 0 °C. The mixture was stirred
at 0 °C for 30 min. A solution of tert-butyl 4-(2-hydroxyethyl)phenylcarbamate (13) (0.475 g, 2 mmol)
and thioacetic acid (0.28 mL, 4 mmol) in 5 mL tetrahydrofuran was added dropwise over 10 min, and the
mixture was stirred for 1 h at 0 °C and 1 h at ambient temperature and a clear yellowish solution was
8

formed. The reaction mixture was concentrated and the crude product was purified by flash column
chromatography (petroleum ether /ethyl acetate: 90:10) to afford thioacetate 14 (0.53, 90% yield).
To a suspension of lithium aluminum hydride (0.37 g, 9.75 mmol) in 2.5 mL of anhydrous
tetrahydrofuran was added dropwise a solution of thioacetate 14 (0.53 g, 1.8 mmol) in 4 mL of
anhydrous tetrahydrofuran and the reaction mixture was stirred at room temperature for 30 min. The
reaction was quenched by the dropwise addition of a solution 95:5 tetrahydrofuran / water (5 mL), then
diluted with ethyl acetate (20 mL), dried over sodium sulphate and the solid was filtered through a short
celite pad and washed with ethyl acetate (80 mL). The filtrate was evaporated in vacuo to afford tert-
butyl 4-(2-mercaptoethyl)phenylcarbamate (15) as an oil, which was used directly without any further
purification.
Compound 4 (100 mg, 0.164 mmol) and tert-butyl 4-(2-mercaptoethyl)phenylcarbamate (101 mg, 0.4
mmol) were dissolved in acetonitrile (100 mL), and triethylamine (0.5 mL) was added. The reaction
mixture was stirred at room temperature for 12 h (consumption of starting material was monitored by
ESI-MS), and the solvent was evaporated. The crude product was purified by flash column
chromatography to give dye 16 (85 mg, 64 %) as green solid. [eluting solvent system: CH2Cl2/CH3OH,
1
(98:2 to 95:5)]. H NMR (600 MHz, CD3OD) δ: 1.50 (s, 9H), 1.66 (s, 12H), 1.89 – 1.93 (m, 2H), 2.66 (t, J =
5.8 Hz, 4H), 2.92 (t, J = 6.9 Hz, 2H), 3.09 (t, J = 6.9 Hz, 2H), 3.63 (s, 6H), 6.24 (d, J = 14.2 Hz, 2H), 7.10 (d, J
= 8.4 Hz, 2H), 7.25 (t, J = 7.4 Hz, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 7.41 (t, J = 7.3 Hz,
13
2H), 7.50 (d, J = 7.3 Hz, 2H), 8.82 (d, J = 14.2 Hz, 2H). C NMR (300 MHz, CD3OD) δ: 22.3, 27.1, 28.2,
28.7, 31.6, 36.4, 40.4, 50.3, 80.8, 102.0, 111.7, 120.1, 123.4, 126.1, 129.8, 130.0, 134.5, 135.0, 139.3,
142.4, 144.4, 147.2, 155.3, 158.5, 174.5. ESI-MS: m/z 700.30 [M]+. HR-MS (ESI+): [M]+ calculated for
C45H54N3O2S 700.3931, found 700.3931.
9

2.2.11. Synthesis of NIR-dye 17
Compound 16 (42 mg, 0.073 mmol) was dissolved in a solution of trifluoroacetic acid and
dichloromethane (9:1, 2 mL) and stirred for 1 h at room temperature. The solvents were evaporated and
the residue was recrystallized with methanol/diethyl ether to give NIR-dye 17 (45 mg, 75%) as green
solid. 1H NMR (300 MHz, CD3OD) δ: 1.65 (s, 12H), 1.80 – 1.85 (m, 2H), 2.65 (br s, 4H), 2.72 (br s, 2H), 2.96
(br s, 2H), 3.65 (s, 6H), 6.27 (d, J = 14.1 Hz, 2H), 6.51 (br s, 2H), 6.84 (br s, 2H), 7.26 (t, J = 6.6 Hz, 2H),
7.37 – 7.44 (m, 4H), 7.59 (d, J = 7.0 Hz, 2H), 8.71 (d, J = 14.1 Hz, 2H). 13C NMR (300 MHz, CD3OD) δ: 22.3,
27.2, 28.2, 31.6, 36.9, 40.1, 50.3, 102.1, 111.7, 120.8, 123.3, 126.2, 129.8, 130.8, 134.6, 136.7, 138.3,
142.3, 144.4, 147.1, 158.4, 174.5. 19F NMR (300 MHz, CD3OD) δ: -76.9. The trifluoroacetic salt of 17 was
transformed to the free amino compound 17 by treatment with triethylamine . ESI-MS: m/z 600.39 [M]+.
HR-MS (ESI+): [M]+ C40H46N3S calculated for 600.3407, found 600.3407.
2.3 Preparation of graphene-based hybrid materials
2.3.1 Synthesis of hybrid materials 21 and 22.
In a typical reaction, 15 mg of derivatized exfoliated graphene 20 [24,25] were dispersed by
sonication in 10 mL of dry dichloromethane under nitrogen atmosphere. Then, the cyanine dye (15 mg,
0.021 mmol for 7 and 10 mg, 0.015 mmol for 8) was added, followed by the quick addition of 4-
dimethylaminopyridine (8.2 mg, 0.067 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (5.5
mg, 0.035 mmol), and stirred at reflux for 96 hours. After that period, the reaction mixture was cooled
down to room temperature, filtered over a PTFE membrane filter (pore size 0.1 μm) and washed with a
large amount of dichloromethane to remove completely any organic residual matter. The newly
prepared graphene-based hybrid materials 21 and 22 were obtained as solids and stored under dark.
2.3.2. Synthesis of hybrid materials 23 and 24.
In a typical reaction, 10 mg of exfoliated graphene were dispersed by sonication in 10 mL of 1,2-
dichlorobenzene, under a nitrogen atmosphere. Then, a solution of the NIR dye (30 mg, 0.042 mmol for
10

aniline derivative cyanine dye 11 and 35 mg, 0.049 mmol for aniline derivative cyanine dye 17) in 5 mL
of acetonitrile was added, followed by the quick addition of isoamyl nitrite (10 mg, 0.085 mmol) and the
o
reaction mixture was stirred at 60 C, under N2, for 96 hours. After cooling down to room temperature,
the reaction mixture was diluted with 30 mL of dimethylformamide, filtered over a PTFE membrane
filter (pore size 0.1 μm) and washed with a large amount of dimethylformamide and CH2Cl2 to remove
completely any organic residuals. The newly prepared graphene-based hybrid materials 23 and 24 were
obtained as solids and stored under dark.
2.4. In vitro cytotoxicity
2.4.1 Cell lines
RKO human colon adenocarcinoma cell line was obtained from American Type Culture Collection
(ATCC). Cells were grown in 75-cm2 flasks (Corning) in D-MEM medium supplemented with 10% foetal
bovine serum (FBS), antibiotics and amino acids (all from Invitrogen). Wharton’s Jelly Mesenchymal
Stem Cells (WJ-MSC) were isolated from donated Umbilical Cord tissue samples from normal, full-term
deliveries following approval by the institutional bioethics review board. Isolation and culture protocols
followed for WJ-MSC are fully described in Christodoulou et al., 2013 [23]. Briefly, cells were cultured in
75-cm2 flasks (Corning), in DMEM/F12 (with 3.5ꢀg/L glucose, ultraglutamine I, and Na pyruvate; Lonza)
supplemented with 10% batch-tested FBS, 15ꢀmM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic
acid (HEPES), 1x nonessential amino acids, and 1% penicillin/streptomycin (all from Invitrogen). All cells
were maintained in a humidified chamber with 5% CO2 in air at 37 °C with media changes every 3-4 days
until 70%–80% confluence.
2.4.2. Cell viability assay
For growth studies the crystal violet (Sigma Aldrich) assay was used. Firstly, cells were seeded into 96-
well micro titre plates (Corning) in a density of 4000 cells/well and were allowed to attach overnight
(cancer cells) or for 48h (stem cells). The cells were then treated with different concentrations of the
11

dyes diluted in DMSO and the correspondent graphene-hybrid material solutions (dH2O + 0.4% DMSO)
for 24 h. Thereafter, cells were fixed with methanol and stained with 0.2% (w/v) crystal violet and the
dye was released in 33% (v/v) acetic acid. The absorbance was measured at 595 nm (Tecan, Austria). The
cell number in treated versus control wells was estimated and the percentage of viable cells was plotted
each time. SD was used for error bar generation. Statistical analysis was performed with one-way
ANOVA, post-hoc Bonferroni test in Graphpad Prism 6.0.
2.5. Two-dimensional culture for immunocytochemistry (ICC)
For the ICC 2D culture experiments, cells (10000 cells/well) were grown on coverslips in 24 well plates
(Corning) in the appropriate culture medium, at 37 °C for 24 h and then treated with the selected dyes
diluted in DMSO and the correspondent graphene-hybrid material solutions (dH2O + 0.4% DMSO) for 4h.
Cells were fixed with 4% paraformaldehyde and washed with PBS (Invitrogen) to remove dead cells.
Photographs of the 2D cultures were taken under confocal microscope (Leica 626 TCS SPE confocal laser
scanning microscope) after the appropriate staining. LAS AF software was used for image acquisition
(Leica Lasertechnik, Heidelberg, Germany). Cell nuclei were stained with Hoechst No. 33342 (Sigma
Aldrich, B2261).
3. Results and discussion
3.1. Chemistry
Our initial efforts were focused on the design and synthesis of new NIR dyes, exploiting the
tricarbocyanine scaffold as template. Previous studies have shown that compounds of this general
structure possess high photostabilities and high polar extinction coefficients [7]. Taking advantage of the
facile displacement of the chloro substituent, at the central vinyl carbon of the tricarbocyanine scaffold,
by various nucleophiles, appropriate linkers were introduced, which could in turn be covalently ligated
to the exfoliated graphene surface either through amide bond formation or via in-situ generated aryl
diazonium salt addition [24]. Variation of the heteroatom linkage to the carbocyanine core (Nitrogen or
12

Sulfur) enabled us to tune the photophysical properties of the synthesised NIR-fluorescent dyes. The
synthesis of the new dyes is described in Schemes 2-4 and in Scheme 5 of the corresponding hybrids.
The symmetrical tricarbocyanine core 4, which was employed as the key common starting material for
all the NIR dyes of the present study was prepared as described in Scheme 1. Thus, all (3E)-2-chloro-2-
(hydroxymethylene)cyclohex-1-enecarbaldehyde (2) was prepared via a Vilsmeier–Haack reaction from
cyclohexanone (1), using phosphorus oxychloride and dimethylformamide in dichloromethane under
reflux, followed by condensation with 1,2,3,3- tetramethyl-3H-indolium iodide (3) to afford compound
4.
Scheme 1. Synthesis of chloro tricarbocyanine core scaffold 4.
NIR-fluorescent dyes 7 and 8 bearing a terminal carboxyl group suitable for an amide linkage to the
amino-functionalised exfoliated graphene, were prepared from the chloro-tricarbocyanine derivative 4
and 6-aminohexanoic acid or thioacetic acid, respectively (Scheme 2).
13

Scheme 2. Synthesis of NIR fluorescent dyes 7 and 8.
The synthesis of NIR-dyes 11 and 17, suitable for covalent attachment to exfoliated graphene through
the in-situ generated aryl diazonium salt, was effected as described in Schemes 3 and 4, respectively.
Thus, reaction of 4-(2-aminoethyl)aniline (10), prepared by the catalytic transfer hydrogenation of p-
nitrophenethylamine hydrochloride (9) using palladium on carbon and ammonium formate, with the
chloro-tricarbocyanine derivative 4 afforded the desired NIR fluorescent dye 11 (Scheme 3).
Scheme 3. Synthesis of NIR fluorescent dye 11.
14

The thio congener of 11 dye 17, was prepared as shown in Scheme 4. Protection of the amino group in
2-(4-aminophenyl)ethanol (12) using tert-butyloxycarbonyl (BOC) anhydride afforded BOC-protected
amino alcohol 13, which was transformed to the thioacetate ester 14 via Mitsunobu reaction which in
turn, was reduced using lithium aluminium hydride to afford thiol 15. Reaction of thiol 15 with the
chloro-tricarbocyanine derivative 4 gave the thio NIR-fluorescent dye 16. Removal of the BOC protecting
group using trifluoroacetic acid in dichloromethane afforded the desired dye 17 bearing a free aniline
moiety (Scheme 4).
Scheme 4. Synthesis of NIR fluorescent dye 17.
3.2. Optical properties of NIR dyes 7, 8, 11 and 17
Following the synthesis of the new dyes we studied their optical properties. The fluorescence
spectrum (λexc. 620 nm) of carboxy bearing NIR-dye 7 shows an emission band centred at 750 nm, while
in the excitation spectrum a band at 620 nm is evident (Supplementary Information, Fig. S1). Conversely,
the presence of a thio group in NIR dye 8 results in a red-shift of both the excitation and the emission
wavelength compared to the amino–substituted congener, NIR-dye 7. As shown in Supplementary
Information, Fig. S2, in the excitation spectrum of dye 8 a band at 775 nm with a shoulder at 700 nm is
evolved, while the corresponding emission band (λexc. 775 nm) is observed at 800 nm. For amino-
substituted aniline NIR-dye 11 the excitation and emission (λexc. 650 nm) bands are observed at 657 and
15

761 nm (Supplementary Information, Fig. S3), respectively. Similar to the case of NIR dye 8, the
excitation and emission (λexc. 720 nm) bands of dye 17 are further red-shifted at 787 and 808 nm
(Supplementary Information, Fig. S4), respectively.
3.3. Synthesis of exfoliated graphene-based hybrids 21-24
The preparation of graphene-based hybrid materials 21-24 covalently modified with cyanine dyes 7,
8, 11 and 17, respectively, is shown in Scheme 5. The main routes to obtain graphene involve either
physical processes (mechanical cleavage, epitaxial growth etc) or chemical means (intercalation,
oxidation-reduction, surfactants-polymers, functionalization etc), with each method having its own
scope and limitations [25].
For the synthesis of hybrids 21 and 22 exfoliated graphene, obtained upon treatment of graphene with
chlorosulfonic acid according to a previously reported procedure [26], was functionalized through a
diazotization reaction [27]. employing N-BOC protected aniline derivative 18 and isoamyl nitrite to in-
situ form the aryl diazonium salt capable to covalently add to the graphene skeleton. Cleavage of the
BOC protecting group by trifluoroacetic acid resulted to a plethora of amino groups onto the graphene
sheets that were further reacted with the carboxylic acid group of cyanine dyes 7 and 8, following
activation using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), yielding hybrids
21 and 22, respectively.
In turn, hybrids 23 and 24 were prepared from exfoliated graphene [26], and the aryl diazonium salts of
cyanine dyes 11 and 17 formed in situ upon treatment with isoamyl nitrite.
16

Scheme 5. Preparation of graphene-based hybrid materials 21-24.
3.4. Characterisation of exfoliated graphene-based hybrids 21-24
The characterization of hybrid materials 21-24 was accomplished by complementary spectroscopic,
thermal and microscopy imaging means.
Initially, Raman spectroscopy was employed to corroborate the successful covalent modification of
exfoliated graphene with the in-situ formed aryl diazonium salt, resulting in material 19 which was
further utilised for the preparation of hybrids 21 and 22. In the Raman spectrum of 19, three bands
located at 1330, 1580 and 2670 cm-1 are discernible as shown in Figure 1. The G-band at 1580 cm-1 is
attributed to vibrations due to sp2 hybridized carbon atoms, while the D-band at 1330 cm-1 is ascribed to
vibrations due to defect sites and sp3 hybridized carbon atoms. The shape and frequency of the 2D band
17

located at 2670 cm-1 is sensitive to the number of graphene sheets [28,29]. Evidently, an increased D/G
ratio is found for 19 as compared with that owed to exfoliated graphene (ID/IG = 0.42 for 19; ID/IG = 0.20
for exfoliated graphene), highlighting the success of the covalent functionalization reaction on graphene
sheets. Furthermore, the D/G ratio for materials 21 and 22 was found unaltered (ID/IG = 0.42) as
compared to that of 19, in accordance with the fact that the cleavage of the BOC unit followed by
condensation with cyanine dyes 7 and 8, respectively, does not disrupt further the graphene skeleton. In
addition, the G-band for hybrids 21 and 22 was marginally shifted by ca. 3 cm-1, as compared to the
value registered for exfoliated graphene, implying the development of charge-transfer phenomena
between the NIR dyes and the graphene sheets. The 2D band for hybrids 21 and 22 is deconvoluted and
fitted with four and one Lorentzians, respectively (Supplementary Information, Fig. S5). The full-width-
half-maximum value for the 2D band of 21 and 22 was calculated to be 82 and 79 cm-1, respectively,
thereby, accordingly indicating the presence of monolayered and bilayered graphene sheets [28,30,31]..
Notably, the first indication for the successful preparation of 21 and 22 arose by employing the Kaiser
test (c.f. Supplementary Information). The latter enabled us to quantify the amount of free amine
groups in 21 and 22, ca. 15 μmol g−1, which was found significantly lower as compared with the one
found in the precursor modified graphene 20, ca. 140 μmol g−1, hence proving the success of the
condensation reaction between 20 and cyanines 7 and 8. Furthermore, the work up procedure followed
for the isolation of all hybrids including 21 and 22 ensures the absence of any physisorbed dye 7 and 8
onto graphene. More specifically, the work up procedure involves filtration over PTFE membrane filter
with pore size 100 nm, to remove organic materials and impurities, followed by extensive washing with
dichloromethane, a solvent that dissolves the dyes 7 and 8, until examination of the filtrate shows no
absorbance due to dyes.
18

Figure 1. Raman spectra for exfoliated graphene (black) and graphene-based materials 19 (grey), 21
(green), and 22 (red), normalized at the G-band and obtained upon 633 nm excitation.
The covalent bonding of cyanines dyes 11 and 17 on exfoliated graphene yielding hybrid materials 23
and 24, respectively, is corroborated based on Raman spectroscopic studies. Μore specifically, in the
Raman spectra of 23 and 24, the evolvement of the characteristic D-band, which is due to the presence
of sp3 hybridized carbon atoms, in both hybrids was evidenced. In turn, this band is absent in the Raman
spectrum of exfoliated graphene (Supplementary Information, Fig. S6).
Subsequently, attenuated-total-reflectance infra-red (ATR-IR) studies further confirmed the effective
coupling of dyes 7 and 8 onto exfoliated graphene. In graphene-based material 19, characteristic strong
carbonyl vibrations at 1698 and 1630 cm-1 attributed to the presence of benzamide and BOC units,
respectively, were evident. Markedly, in 20 the latter was absent, reflecting the effective cleavage of
BOC group that gives rise to the presence of free –NH2 groups. In graphene-based hybrid material 21,
the carbonyl amide vibration was found enhanced as a result of the formation of new amide band at
1640 cm1, while the vibration at 1720 cm-1 owed to the carboxylic acid moiety of the free cyanine dye 7
had accordingly disappeared (Figure 2). Similar observations can be made for the ATR-IR spectrum of
19

hybrid 22 (Supplementary Information, Fig. S7). In the ATR-IR spectra of dyads 23 and 24 only the C-H
stretching vibration modes were discernible at 2800-2900 cm-1 (Supplementary Information, Fig. S8).
Carboxylic
Figure 2. ATR-IR spectra for graphene-based materials 19 (black), 20 (grey), 21 (green) and cyanine dye 7
(blue).
The degree of graphene functionalization for 21 and 22 was evaluated by thermogravimetric analysis
(TGA). Graphene is thermally stable at high temperatures under inert atmosphere and this is shown in
the corresponding TGA graph (Figure 3, black line). Covalently functionalized graphene-based materials
possess characteristic TGA graphs since organic addends thermally decompose in a temperature range
of 200-550 ºC, while beyond that temperature the thermal decomposition of the graphene skeleton at
sites with sp3 hybridized carbon atoms takes place. This explains the continuous mass loss observed in
the TGA graphs for functionalized graphene-based materials 21-24. For modified graphene-based
material 20, a mass loss of 12% was observed in the temperature range 200-560 °C, while for hybrids 21
20

and 22 the corresponding mass loss was 22 % and 17 %, respectively (Figure 3). On this basis, the
loading of cyanine dyes 7 and 8 onto graphene was calculated as 1 per every 270 and 360 carbon atoms
for 21 and 22, respectively. Analogously, from the thermal decomposition of 23 and 24 and the mass
loss observed, 2 % and 8 %, respectively, the loading of 11 and 17 was calculated to be 1 unit per every
1560 and 900 carbon atoms for 23 and 24, respectively (Supplementary Information, Fig. S9). Although
this represents a rather small amount of dyes incorporated onto the graphene sheets, it is consistent
with the small perturbation of graphene lattice in 23 and 24 upon the covalent incorporation of 11 and
17, as revealed with the small enhancement of the D-band in the corresponding Raman spectra (cf.
Supplementary information, Fig. S6).
Figure 3. TGA graphs for exfoliated graphene (black), graphene-based material 20 (grey), and hybrids 21
(green) and 22 (red), obtained under nitrogen atmosphere.
The morphology of graphene-based hybrid materials 21-24 was examined by FE-SEM imaging. Briefly,
after dispersing the material by mild sonication in hexane, a drop was deposited onto the sample holder
and examined. A representative image for 21, shown in Figure 4, reveals the presence of overlapping
oligolayered graphene sheets, of various sizes, with typical folding and ripple patterns. Although the
exact number of layers cannot be accurately estimated, the absence of large graphitic particles is
21

obvious, as it is also proven by Raman spectroscopy (cf. Figure 1). Similar were the morphological
findings for the hybrids 22, 23 and 24 (Supplementary Information, Fig. S10). The SEM image of
exfoliated graphene (Supplementary Information, Fig. S11) is very similar to the images obtained for
hybrids 21-24, which further supports that 21-24 are based on graphene sheets.
Figure 4. Representative FE-SEM image for graphene-based hybrid 21.
The dispersibility of hybrids 21-24 was enhanced in polar solvents such as DMF, allowing to perform
spectroscopic studies in solution. Hence, examining possible ground-state interactions between cyanine
dyes 7 and 8 with graphene within hybrid materials 21 and 22, respectively, UV-Vis-NIR studies were
performed. The electron absorption spectrum for 21 obtained in DMF, showed the characteristic
continuous absorption in the NIR region owed to graphene, followed by a broadened band centred at
660 nm (Figure 5a), corresponding to free cyanine dye 7 possibly possessing different conformational
structures on graphene. This finding not only guarantees the successful integration of 7 onto graphene,
by covalently forming hybrid 21, but also indicates appreciable electronic interactions between the two
species at the ground state. In the case of hybrid 22, the UV-Vis-NIR spectrum is a simple superposition
of the absorption spectra owed to graphene and cyanine dye 8 (Figure 5b), suggesting the absence of
appreciable interactions in the ground state. The low loading of 11 and 17 within hybrids 23 and 24 (cf.
22

Supplementary Information, Fig. S9), respectively, handicapped the electronic absorption
measurements. In the latter, the characteristic absorption bands due to 11 and 17, centred at 657 and
787 nm, respectively, were masked under the continuous and strong absorbance of graphene
(Supplementary Information, Fig. S12).
Figure 5. UV-Vis electronic absorption spectra for (a) free cyanine dye 7 (red) and graphene-based
hybrid material 21 (black), and (b) free cyanine dye 8 (red) and graphene-based hybrid material 22
(black), obtained in DMF.
Next, focusing on excited state interactions, photoluminescence assays for hybrids 21 and 22 in
comparison with the corresponding free cyanine dyes 7 and 8, for samples displaying equal absorbance
at the excitation wavelength, were performed. Upon 660 nm excitation, 7 revealed a broad emission
band centred at 757 nm (Figure 6a). Markedly, in hybrid 21, the latter emission was found significantly
depressed, indicating strong electronic interactions between graphene and 7, within 21 at the excited
state, following electron and/or energy transfer as the decay mechanism of the singlet excited state of
17*. Then, on the basis of the time-correlated-single-photon-counting method, the photoluminescence
lifetime profiles for 7 were acquired. Analysis of the time-profile of the decay at 757 nm upon excitation
1
at 654 nm for the 7* was monoexponentially fitted with a lifetime of 0.84 ns. On the other hand, the
decay component in hybrid 21 was found to be 500 ps. Analogously, the photoluminescence of hybrid
22, at 810 nm was found quenched as compared to the one attributed to the free cyanine dye 8 (Figure
6b), while the corresponding lifetimes for 8 and 22 were evaluated as 0.81 ns and 250 ps, respectively.
23

The photoluminescence spectra for hybrids 23 and 24, as compared with those of the corresponding
free cyanine dyes 11 and 17 are presented in the Supplementary Information, Fig. S13. In addition, the
emission time profiles for graphene-based hybrids 23 and 24 were characterized by quicker decays, as
compared with those registered for the free cyanine dyes 11 and 17, respectively, and in analogy with
those belonging to hybrids 21 and 22. Collectively, those findings are related with intra-hybrid
transduction of electron and/or energy from the electron donors 7, 8, 11 and 17 to graphene in hybrids
21-24, respectively. The emission rate constant (kS) and quantum yield (ΦS) for free cyanine dyes 7, 8, 11
and 17 and graphene-based hybrid materials 21-24 were calculated based on equations (1) and (2),
respectively, and are presented in Table 1.
Equation 1: kS = (1/τf)hybrid - (1/τf)dye , where τf is the fluorescence lifetime
Equation 2: ΦS = kS/(1/τf)hybrid
Figure 6. Photoluminescence spectra (excitation at 660 nm) for (a) free cyanine dye 7 (red) as compared
with hybrid 21 (black), and (b) free cyanine dye 8 (red) as compared with hybrid 22 (black), obtained in
DMF.
Next, shedding light on the redox properties of the hybrid materials, cyclic voltammetry (CV)
measurements performed on acetonitrile employing Bu4NPF6 as electrolyte in a standard three-
electrode cell with glassy carbon as working electrode and platinum wires as counter and
pseudoreference electrodes were conducted. Hybrid materials 21 and 22 exhibit a reversible oxidation
24

at 0.80 and 0.11 V versus Fc/Fc+, cathodically shifted by approximately 10 and 4 mV, respectively, as
compared with the oxidation processes registered for the corresponding free cyanine dyes 7 and 8.
These shifts are attributed to intra-hybrid electronic interactions between the dyes and graphene
sheets, rendering easier the oxidation of the latter within the graphene-based hybrid materials.
Furthermore, a peak at -0.45 V associated with the reduction of graphene similarly with other reports
[9,32], as well as additional reversible reduction processes centred at -1.24 and -1.06 V, were identified
in hybrids 21 and 22. Disappointingly, due to the limited solubility of 23 and 24 we were not able to
register suitable and reliable redox data. Collectively, the redox data are presented in Table 1.
Table 1. Photophysical and electrochemical data for cyanine dyes 7, 8, 11 and 17 and graphene-based
hybrids 21-24.
Material
Cyanine dye 7
Hybrid 21
τf (ps)
840
kS (s-1)
ΦS
EOx. (V) ERed.1 (V) ERed.2 (V)
0.90
0.80
0.15
0.11
0.35
N/A a
0.20
N/A a
-1.19
-1.24
-1.16
-1.06
-1.34
500
8.10 x 108
2.77 x 109
1.26 x 109
1.81 x 109
0.40
0.69
0.48
0.67
-0.45
-0.45
N/A a
N/A a
Cyanine dye 8
Hybrid 22
810
250
Cyanine dye 11
Hybrid 23
1183
475
Cyanine dye 17
Hybrid 24
1074
366
-1.35
a not registered due to limited solubility
3.5. Cytotoxicity and in vitro imaging studies.
25

3.5.1 Cell viability study
The biocompatibility of dyes 7, 8, 11, 17 and the corresponding graphene-based hybrid materials 21-
24 was examined using the RKO human colon adenocarcinoma cell line and WJ-MSC human umbilical
cord mesenchymal stem cells. The crystal violet cell viability assay was used to assess cytotoxicity. The
free cyanine dyes 7, 8, 11 and 17 were examined in a range of concentrations (1, 2, 5, 10, and 20 μΜ).
The corresponding hybrids for comparison were tested at the same g/mL concentrations as the
respective dyes. The small differences between the percentages, either above or below 100%, are
attributed to statistical errors (Figures 7A and 7B). In general, the graphene-hybrid materials are well-
tolerated by both cancer and stem cells, since a reduction in cell viability not higher than 20% was
observed only for hybrid 23 for the examined concentration range. Regarding the NIR dyes, the results
are diverse for the tested cell lines. NIR dyes 7 and 8 bearing a terminal carboxylic acid functionality are
well tolerated by cancer cells showing some toxicity at the high concentration (20 μΜ). However, there
is a difference with respect to stem cells with dye 7 substituted by an aminohexanoic acid moiety, being
non toxic, versus dye 8 which possesses a thioacetic acid functionality exhibiting some toxicity at
concentrations higher than 2μΜ. Conversely, dye 17 bearing a para-thioethylaniline moiety exhibits
toxicity against RKO and WJ-MSC cells at concentrations higher than 2 μΜ which is more pronounced
against the latter cell line. Finally, dye 11 substituted by a para-aminoethylaniline group is less toxic
against stem cells versus cancer cells. Apparently, the presence of the sulfur versus the nitrogen
heteroatom increases toxicity in this compound class. In addition, cell morphology was also not affected
by the non-toxic concentration treatment, as checked under light microscope (data not shown). These
results indicate that the incorporation of the above dyes on graphene renders the resulting hybrid
materials more biocompatible and well-tolerated by cultured human cells.
26

RKO
A
140
120
100
80
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
***
60
40
20
0
7
21
ctr
dH20-DMSO
0.7μg/ml
1.4μg/ml
3.5μg/ml
7.1μg/ml
14.1μg/ml
140
120
100
80
ns
ns
ns
ns
ns
ns
ns
ns
**
*
*** ***
***
60
40
20
0
8
22
ctr
dH20-DMSO
0.66μg/ml
1.33μg/ml
3.33μg/ml
6.65μg/ml
13.3μg/ml
120
100
80
60
40
20
0
ns
ns
**
**
**
**
**
*
***
** ***
*
**
*
**
*
11
23
7.1μg/ml
ctr
dH20-DMSO
0.7μg/ml
1.4μg/ml
3.6μg/ml
14.2μg/ml
120
100
80
60
40
20
0
ns
ns
ns
ns
ns
ns
ns
**
**
***
***
***
17
0,9μg/ml
24
8,3μg/ml
ctr
dH20-DMSO
1,7μg/ml
4,2μg/ml
16,6μg/ml
27

WJ-MSC
B
140
120
100
80
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
60
40
20
0
7
21
ctr
dH20-DMSO
0.7μg/ml
1.4μg/ml
3.5μg/ml
7.1μg/ml
14.1μg/ml
140
ns
ns
120
100
80
60
40
20
0
ns
ns
ns
ns
ns
**
*
***
***
***
8
22
ctr
dH20-DMSO
0.66μg/ml
1.33μg/ml
3.33μg/ml
6.65μg/ml
13.3μg/ml
160
**
140
120
100
80
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
60
***
40
20
0
11
23
ctr
dH20-DMSO
0.7μg/ml
1.4μg/ml
3.6μg/ml
7.1μg/ml
14.2μg/ml
160
ns
ns
140
120
100
80
ns
ns
ns
ns
ns
ns
ns
60
***
***
***
40
20
0
17
24
ctr
dH20-DMSO
0.9μg/ml
1.7μg/ml
4.2μg/ml
8.3μg/ml
16.6μg/ml
28

Figure 7. Cell viability in A. RKO colon cancer cell line and B. Wharton’s Jelly Mesenchymal Stem Cells
(WJ-MSC) after 24h treatment with different concentrations of the four graphene-hybrid materials 21,
22, 23, 24 (right side of each plot) and the respective free cyanine dyes 7, 8, 11, 17 (left side of each
plot). The concentrations of the dyes can also be expressed as micromolar (1, 2, 5, 10, 20 μΜ). As a
negative control, cell viability after treatment with the correspondent solvent (DMSO for the dyes,
dH2O+ 0.4% DMSO for the graphene-based hybrids) was measured. Data are representative of two
independent experiments, each one performed in duplicates. SD was used for error bar generation.
*p<0.05, **p<0.01, ***p<0.001, ns: p>0.05. Statistical significance shown represents the comparison of
each sample with the control.
3.5.2. Intracellular imaging
In order to explore their potential application for intracellular imaging and drug delivery protocols,
the hybrid materials and the correspondent dyes were observed under confocal fluorescence
microscope, after their incubation with RKO or WJ-MS cells for 4 hours. Cell nuclei became visible after
DNA staining with Hoechst in order to check whether the above materials are able to enter the cell
cytoplasm and in that case to further visualize their dispersion within the cells. All of the examined dyes
were able to enter the cells with a high efficacy resulting in intense fluorescence signals which were
markedly reduced in the case of the respective graphene-based materials as observed in Figures 8A and
8B. However, stronger signals could be achieved after increasing the tested concentrations, since higher
concentrations of the hybrids were not cytotoxic, as discussed above. Pink color stains can be observed
in the images of dyes 7, 8 and 11 in RKO cells, which result from the overlapping of the blue (Hoechst)
and red (dyes) staining, and thus could indicate an interaction of the dyes with the nuclear DNA.
However, the inability of the other materials to overlap with the nuclear DNA could be attributed to a
higher quantity needed in order to achieve this effect. This was not tested, since we opted to employ
concentrations that didn’t induce cytotoxic effects (c.f. Figures 7A and 7B).
29