Spatiotemporal control of photothermal heating using pH sensitive near-infrared croconaine-based dyes

Article abstract of DOI:10.1016/j.jphotochem.2019.111936

Near-infrared (NIR) absorbing dyes sensitive to pH are promising small molecules that can be used in photothermal therapy for intracellular cancer treatment. In this study, a croconaine dye molecule protected within a macrocycle has been prepared to be te

Full text of DOI:10.1016/j.jphotochem.2019.111936

Accepted Manuscript
Title: Spatiotemporal control of photothermal heating using
pH sensitive near-infrared croconaine-based dyes
Authors: Teresa Alejo, Clara Font, Miriam Abad, Vanesa
Andreu, Victor Sebastian, Gracia Mendoza, Manuel Arruebo
PII:
S1010-6030(19)30017-6
DOI:
Article Number:
https://doi.org/10.1016/j.jphotochem.2019.111936
111936
Reference:
JPC 111936
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Journal of Photochemistry and Photobiology A: Chemistry
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Revised date:
Accepted date:
3 January 2019
10 June 2019
11 June 2019
Please cite this article as: Alejo T, Font C, Abad M, Andreu V, Sebastian V, Mendoza G,
Arruebo M, Spatiotemporal control of photothermal heating using pH sensitive near-
infrared croconaine-based dyes, Journal of Photochemistry and amp; Photobiology, A:
Chemistry (2019), https://doi.org/10.1016/j.jphotochem.2019.111936
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Spatiotemporal control of photothermal heating using pH
sensitive near-infrared croconaine-based dyes
Teresa Alejoa,b*, Clara Fontb, Miriam Abadc, Vanesa Andreub, Victor Sebastianb,d,
Gracia Mendozab, Manuel Arruebob,d
aFoundation Institute of Nanoscience of Aragón (FINA), 50018- Zaragoza, Spain.
bDepartment of Chemical and Environmental Engineering. Aragon Institute of
Nanoscience (INA), University of Zaragoza, Campus Río Ebro - Edificio I+D, C/ Poeta
Mariano Esquillor S/N, 50018-Zaragoza, Spain; Aragon Health Research Institute (IIS
Aragón), 50009 Zaragoza, Spain.
cDepartment of Organic Chemistry, Faculty of Science, Aragon Institute of Material
Science (ICMA), University of Zaragoza-CSIC, 50009 Zaragoza, Spain
dNetworking Research Center on Bioengineering, Biomaterials and Nanomedicine,
CIBER-BBN, 28029-Madrid, Spain.
* Corresponding author: Teresa Alejo: teresaal@unizar.es
GRAPHICAL ABSTRACT
HIGHLIGHTS

Synthesis and characterization of a novel pH sensitive NIR absorbing croconaine-based
dye for its use as photothermal agent against tumoral cells.
1



Croconaine dye internalized within the acidic endosomal-lysosomal interior of tumoral
cells was activated and efficiently absorbed the NIR light resulting in cell death
exclusively produced on irradiated cells, without any damage observed in neighboring
cells.
The cytotoxic effect was exclusively attributed to photothermia as ROS generation
observed for the croconaine dye was negligible.
ABSTRACT
Near-infrared (NIR) absorbing dyes sensitive to pH are promising small molecules that
can be used in photothermal therapy for intracellular cancer treatment. In this study, a
croconaine dye molecule protected within a macrocycle has been prepared to be tested
as photothermic agent. This pH sensitive croconaine dye allowed a selective NIR-light
absorption only when the molecule was located in an acidic environment like the one
found in intracellular vesicles; meanwhile it remained inactivated under physiological
conditions. Results showed that photothermic heating produced upon 808 nm laser
irradiation on mouse melanoma cells treated with this croconaine-based dye resulted in
cell death which may be attributed to a dye uptake into acidic intracellular vesicles (i.e.,
endosomal-lysosomal pathway). Cell damage was exclusively produced on irradiated
cells, without any damage observed in neighboring cells. Negligible reactive oxygen
species release was detected, thus the effect was attributed to a pure photothermic
process. Croconaine dye presented a subcytotoxic concentration of 100 M on
macrophages and tumoral cells. From the results obtained it can be concluded than these
types of near-infrared dyes have shown great promise as photothermal agents, providing
a great opportunity for achieving a spatiotemporal control on photothermal cancer
therapy.
Keywords: Croconaine dye, photothermal therapy, cancer treatment, NIR-light heating,
cytotoxicity.
2

1. Introduction
Currently there are many lines of research concerning advanced therapies applied to
cancer treatment, and some of them are under investigation in a clinical level. Some
examples include tissue, cell and gene or RNA interference (RNAi) therapies, other
treatments use light to eliminate solid tumors including photodynamic (PDT) or
photothermal (PTT) therapies, among others [1-6]. Regarding this last one,
photothermal therapy basis consists on an agent (usually a nanoparticle or a dye)
capable of absorbing light at a specific wavelength and release vibrational energy
producing a local heat increase. When this heat rises up above 43ᵒC, the denaturation of
proteins is induced and therefore the apoptosis of tumoral cells occurs [7, 8]. Along all
the thermal therapies, PTT is known as a minimal invasive technique with a high
specificity and efficacy and low toxicity to healthy tissues, that it can be combined with
existing cancer therapies [9, 10]. These advantages are achieved by using laser
irradiation at near-infrared (NIR) wavelengths (700 - 1100 nm). Compared to ultraviolet
(UV) or visible (VIS) light, NIR light is able to penetrate deeply into the tissues without
harming healthy cells as neither water, blood cells nor biological tissues can strongly
absorb NIR wavelengths [7, 10, 11]. Regarding to the NIR light absorbing agents used
in PTT, they should be non-toxic and biocompatible at the doses used and include
inorganic nanoparticles (gold nanostructures, carbon materials, copper sulfide
nanoparticles, etc.) or organic dyes (namely photosensitizers including indocyanine
green (ICG), IR780, IR-808, etc.) [8-11]. Small molecular organic dyes show relevant
advantages, including a good reproducibility in their effects, a very well defined
molecular structure, strong absorption at NIR wavelengths and enhanced
biodegradability [9, 10]. However, even if ICG has been approved by the Food and
Drug Administration (FDA) and it is widely used in PTT, it shows poor photostability,
high fluorescence quenching, reduced tumor accumulation and its chemical
modification is not straightforward [12, 13]. Hence, recent studies have developed new
synthetic chromophores with absorption in the NIR region to address these issues. In
this regard, croconaine dyes are croconaine acid based-molecules with the ability to
alter their structure depending on the local chemical environment [14, 15]. They present
an intense NIR absorption and high photothermal conversion efficiency [16].
Croconaine dyes actually have a dual behavior depending on the environmental pH,
switching their structure and absorption profile from an alkaline form (absorption peak
3

in the visible region under slightly alkaline conditions, i.e., pH 7.4) to an acidic form
(high absorption peak in the NIR region) [14]. Moreover, croconaine dyes have a strong
resistance to photobleaching, good chemical and thermal stability and show a short-
excited state lifetime which underlies highly efficient relaxation to the ground state [17].
They also have very little fluorescence emission and reduced singlet oxygen generation;
thus, they constitute ideal dyes to decouple the photodynamic from the photothermal
effects [18]. This structural shift determined by the pH of the medium can be very
useful for achieving a controlled, effective and safe therapy. The extracellular pH, under
physiological conditions, is about 7.3-7.4 [19], keeping the croconaine molecule on its
alkaline structure. On the other hand, since the uptake of molecules occurs mainly via
endocytosis, more specifically following the non-specific pinocytic pathway (for
molecules smaller than 0.15 µm) [20], the pH inside the endosome-lysosome would
reach 5.5, meaning that under those conditions an acidic structure of the croconaine
molecules would be acquired and their consequent high absorption around 800 nm
when located inside intracellular vesicles [21]. This fact not only reinforces the
hypothesis that this therapy will not cause harm to neighboring healthy cells having a
reduced dye uptake, but it also suggests that cell-death would be exclusively triggered
upon light illumination only when the dye was located inside the target cell [21, 22],
proving to be promising candidates for PTT. It is important to point out that several NIR
dyes have demonstrated preferential uptake by cultured, circulating and disseminated
tumor cell lines over normal cells [23-27].
ICG has been used as photothermal agent and also as photosensitzer to produce
cytotoxic reactive oxygen species (ROS) upon irradiation with NIR laser on
hepatocellular carcinoma cells [28]. However, the combination of PTT and PDT
presents some drawbacks, as the ROS produced leads to bleaching of the dye reducing
its effect, and also chemotherapy drugs used in synergistic therapies can be decomposed
by the ROS generated [18]. Therefore, it is interesting to produce exclusively PTT
without simultaneous ROS generation that could damage adjacent cells or inactivate
drugs. Croconaine-based molecules have been previously used as photothermal agents
in a mixed population of adherent CHO-K1 cells and non-adherent Jurkat cells, where
only CHO-K1 cells where incubated with the croconaine dye. Results showed a high
selectivity in cell destruction after irradiation only on dye-labeled cells without any
damage to adjacent cells [21]. Those authors tested three different croconaine dye
molecules, all of them based on croconic acid, but with different flanking units to adjust
4

the degree of lipophilicity. They showed that the highly lipophilic croconaine dyes
permeated into the cells and accumulated into organelle membranes and liphophilic
intracellular vesicles, whereas the hydrophilic ones were impermeable to cells and thus
had no effect on cell viability [21]. Consequently, a high lipophilicity of the dye is
desired for achieving cell penetration to be able to effectively inactivate cells
intracellularly. Smith and coworkers [14, 29] reported a modification of croconaine
dyes trapping the molecule within a macrocycle to form a croconaine rotaxane, Croc-
cycle, in order to avoid self-aggregation and electronic coupling of the dye. The dye
protected with the macrocycle was incorporated into the membrane of liposomes to
carry out ratiometric photoacoustic imaging and photothermal heating in the acidic
peritoneal cavity [14]. Nevertheless, this Croc-cycle dye has not been previously used
as photothermic agent against cancer cells. Herein, we have prepared and characterized
the free lipophilic croconaine dye protected with a macrocycle to be used as
photothermal agent. ROS release produced by this Croc-cycle dye was analyzed and its
cytotoxicity evaluated in murine melanoma cells (B16F1 cell line) and macrophages.
The in vitro PTT effect of Croc-cycle was tested for both cell lines. The pH-dependent
absorption behavior of the dye was used to selectively induce cell death only when
irradiating with the NIR laser. This molecule could be internalized within the cells and
in their interior it was activated and efficiency absorbed the NIR light to generate heat
producing selective and local cell death.
2. Experimental Section
2.1. Materials and methods
2,3,3-Trimethylindolenine (98%), sodium nitrate (NaNO3, 98%), sulfuric acid
(H2SO4, 98%), sodium hydroxide (NaOH, ≥98%), ethyl acetate (EtOAc), magnesium
sulfate (MgSO4, ≥99.99%), croconic acid (98%), 1-butanol (anhydrous, 99.8%), toluene
(anhydrous, 99.8%), ether (≥99.7%), anthracene, aqueous hydrobromic acid (HBr,
47%), glacial acetic acid (100%), 1,3,5-trioxane (≥99%), tetradecyltrimethylammonium
bromide (TTA), ethanol (95%), hexamethylenetetramine (≥99%), chloroform (CHCl3,
99.9%), chloroform (CHCl3, anhydrous, ≥99%) hydrochloric acid (HCl, 37%), sodium
carbonate (Na2CO3, ≥99%), 5-tert-butylisophthalic acid (98%), benzene (99.8%),
dimethylformamide (DMF), thionyl chloride (SOCl2, ≥99%), acetone (C3H6O, ≥99%),
5

deuterated chloroform (CDCl3, 99.96%), dihydrorhodamine 123 (DHR123, ≥95%) and
phosphotungstic acid (PTA) were purchased from Sigma-Aldrich. All of these
chemicals were used as received without further purification. Human monocytes (THP-
1) (ATCC® TIB-202™) were obtained from the American Type Culture Collection
(ATCC), while B16F1 mouse melanoma cells were kindly donated by Dr Pilar Martin-
Duque. Dulbecco’s modified Eagle’s medium high glucose (DMEM w/stable
Glutamine, Biowest), RPMI-1640 medium (w/stable Glutamine, Biowest), fetal bovine
serum (FBS) (Gibco), penicillin-streptomycin-amphotericin B (Biowest), non-essential
aminoacids (Biowest), sodium pyruvate (Biowest), HEPES (Biowest), 2-
mercaptoethanol (Gibco), CellQuanti-Blue reagent (Abnova), phorbol 12-myristate 13-
acetate (PMA) and dimethyl sulfoxide (DMSO, ≥99.7%) (Sigma-Aldrich) were used as
reagents for cell culture experiments.
Nuclear magnetic resonance (NMR) spectroscopy was carried out on a 400 MHz
Bruker AV-400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) using
CDCl3 as solvent.
UV-vis spectra were recorded using a Jasco V670 spectrophotometer (JASCO
Corporation, Tokyo, Japan) in a quartz cuvette with 1 cm path length, to evaluate the
characteristic absorption bands of the molecules in the electromagnetic spectrum.
Fluorescence spectra were recorded using a Perkin Elmer Luminescence
Spectrometer Model LS-55 (Massachusetts, USA) with a slit width of 10 nm and
excitation and emission wavelengths set at 480 nm and 530 nm, respectively.
Croc-cycle dye irradiation was performed using an 808 nm-laser diode (model
MDL-III-808-2W, Changchun New Industries Optoelectronics Technology Co., Ltd.,
Changchun, P.R. China). Samples of 100 μM Croc-cycle concentration in DMEM and
RPMI media were irradiated using a laser irradiance of 5.2 W/cm2 during 10 minutes.
The ROS generation study was performed at 16 μM Croc-cycle concentration in
ethanol using an irradiance of 5.2 W/cm2.
2.2. Synthetic procedures
2.2.1. Synthesis of croconaine dye Croc
Croc was synthesized following the procedure reported by Guha et al. (Scheme
S1) [14]. An ice-salt water bath at -5ᵒC was prepared for the 5 min stirring of the 2,3,3-
Trimethylindolenine (2.0 mL, 12.5 mmol). Then, 1.09 g (13 mol) of NaNO3 were added
6

at once and 31 mL (591 mmol) of H2SO4 were added drop wise for 10 min. The mixture
was left stirring for 1h in the same cold bath (-5ᵒC). It was then neutralized with the
slow addition of NaOH checking the pH of the mixture. The crude formed was cooled
and dissolved in 50 mL of EtOAc. The biphasic solution was separated, and the organic
layer generated was washed three times with 40 mL of distilled water. The final product
was dried with MgSO4, filtered and concentrated under reduced pressure.
This final product (205.3 mg, 1 mmol) and croconic acid (69.5 mg, 0.49 mmol) were
mixed and dissolved in 8 mL of 1-Butanol and 8 mL of toluene. The solution was
heated at 95ᵒC under reflux for 18 h. The mixture was then cooled at -23ᵒC for 2h. The
precipitated solid was filtered and washed with ether three times and finally dried under
reduced pressure until the solvents were removed. Croc was obtained as a dark green-
grey solid (150 mg, yield 58%). 1H NMR (400 MHz, CDCl3): ppm 8.19-8.3 (m, 2H),
8.08-8.17 (m, 2H), 7.3-7.42 (m, 2H), 6.0-6.27 (m, 2H), 1.53- 1.55 (m, 12H).
2.2.2. Synthesis of croconaine-macrocycle Croc-Cycle
Synthesis of 5-(tert-Butyl)isophthaloyl dichloride 1
5-tert-butylisophthalic acid (10 mg, 56 mmol) was dissolved in 40 mL of benzene and a
single drop of DMF. 30 mL of SOCl2 were carefully added in the mixture placed in an
ice bath (0 ᵒC). The mixture was then refluxed for 5 h in an oil bath at 70 ᵒC (Scheme
S1). The SOCl2 was removed by distillation, and the remaining benzene was
evaporated, obtaining a white solid 1 [30] (11 g, yield 94 %). 1H NMR (400 MHz,
CDCl3): ppm 8.63 (s, 1H), 8.34 (s, 2H), 1.34 (s, 9H); 13C NMR (100 MHz, CDCl3):
ppm167, 154, 134.2, 134, 131.5, 35.5, 31.
9,10-bis(aminomethyl)anthracene 3 synthesis
Anthracene (5 g, 28 mmol) was dissolved in 25 mL of glacial acetic acid and 100 mL of
hydrobromic acid. 1,3,5-trioxane (5 g, 56 mmol) and TTA (0.2 g) were added and
stirred at 75 ᵒC under reflux with a condenser sealed with an argon balloon for 24h. The
mixture was then cooled, filtered and washed with water. The crude was dried and
dissolved with toluene in a 70 ᵒC bath, and then recrystallized in an ice bath. Three
more washes with hot ethanol at 60 ᵒC were needed to remove the remaining
anthracene. A green-yellow solid of 9,10-bis(bromomethyl)anthracene 2 was obtained
7

[31] (1.3 g, yield 60 %). 1H NMR (400 MHz, CDCl3): ppm 8.4 (dd, J = 6.9 Hz, J = 3.2
Hz, 4H), 7.7 (dd, J = 6.9 Hz, J = 3.2 Hz, 4H), 5.53 (s, 4H); 13C NMR (100 MHz,
CDCl3): ppm 130.2, 129.6, 126.7, 124.4, 26.6.
Hexamethylenetetramine (0.43 g, 3.04 mmol) was dissolved in 100 mL of chloroform.
Then 2 (0.5 g, 1.37 mmol) was added and heated at 50 ᵒC to reflux overnight while
sealed with an argon balloon. The mixture was allowed to cool down to precipitate, then
it was filtered and washed with chloroform. The dried solid was dissolved with a
mixture of ethanol and HCl (80 mL ethanol and 10 mL of pure HCl) and heated at 70 ᵒC
to vigorously reflux for 48 h, sealed again with an argon balloon. The mixture was
cooled in an ice bath at 0 ᵒC and the precipitate formed was filtered, washed with cold
ethanol and dried in the open air. The crude was dispersed and stirred into 50 mL of a
10% Na2CO3 solution. 20 mL of chloroform were added into the solution and all of it
separated by liquid-liquid extraction, washing three times the aqueous layer with 30 mL
of chloroform to extract the product. The final chloroform solution was dried with
MgSO4, filtered and dried under high vacuum, obtaining a yellow solid of bisamine 3
[32] (117 mg, yield 50%). 1H NMR (400 MHz, CDCl3): ppm 8.34 (dd, J = 6.9 Hz, J =
3.2 Hz, 4H), 7.49 (dd, J = 6.9 Hz, J = 3.2 Hz, 4H), 4.76 (s, 4H).
Croconaine-macrocycle Croc-cycle synthesis
Briefly, Croc (24.35 mg, 0.16 mmol) was dissolved in 20 mL of chloroform and mixed
with 1 (128.35 mg, 0.64 mmol) dissolved in 40 mL of chloroform and 3 (101.95 mg,
0.64 mmol), dissolved in 40 mL of chloroform and 1 mL of triethylamine injected using
a mechanical syringe pump in 4 hours (Scheme S1). The mixture was then left stirring
overnight and concentrated under reduced pressure. The final crude product obtained
was then purified via silica gel column chromatography. In order to find the adequate
proportion of eluents (acetone and chloroform) and find out the polarity of the
precursors to separate them, thin-layer chromatography was used. Two columns were
needed, a first one using a solution of chloroform and acetone (98% - 2%), and a second
one using just chloroform (100%). A dark green-black solid Croc-cycle was obtained
(18 mg, yield 10%). 1H NMR (400 MHz, CDCl3): ppm 9.32-9.51 (m, 2H), 8.44-8.55
(m, 4H), 8.34-8.39 (m, 2H), 8.15-8.2 (d, 2H), 7.63-8.13 (m, 8H), 7.38-7.62 (m, 4H),
7.08-7.13 (m, 2H), 6.52-7.06 (m, 8H), 5.1-5.39 (m, 8H), 4.55-4.99 (m, 2H), 1.44-1.73
(m, 18H), 0.73-0.83 (m, 12H).
8

2.3. Cell culture and biological studies
The cytocompatibility of the Croc-cycle dye was evaluated in B16F1 mouse
melanoma cells and in THP1 human macrophages.
B16F1 cells were grown using DMEM supplemented with 10% FBS and 1%
antibiotic-antimycotic in a humidified atmosphere containing 5% CO2 at 37ºC. THP-1
cell line (human acute monocytic leukemia suspension cell) was cultured in RPMI 1640
medium supplemented with 10% FBS, 1% non-essential aminoacids (v/v), 1% sodium
pyruvate, 1% HEPES, 0.1% 2-mercaptoethanol and 1% and antibiotic-antimycotic at 37
°C in a 5% CO2 humidified incubator. Monocytic cells were differentiated into the
adherent macrophage-like state in supplemented RPMI 1640 medium containing 1 µM
phorbol 12-myristate 13-acetate (PMA) for 72 h.
2.3.1. Cell viability assay
In vitro cytotoxicity of Croc-cycle dye was evaluated using the Blue Cell Viability
assay according to the manufacturer’s instructions. B16F1 cells and macrophages
(density of 5000 and 70000 cells/well, respectively) were seeded in a 96-well plate prior
to incubation with the corresponding Croc-cycle dye doses.
Croc-cycle dye was added to the cells in complete growth medium at different
concentrations (75 - 400 μM) and, then, cells were maintained in standard culture
conditions for 24 h. After incubation, cells were washed with Dulbecco's Phosphate
Buffered Saline (DPBS, Biowest) and incubated with complete growth medium
containing 10% (v/v) Blue Cell Viability assay reagent for 4 h (37 ºC, 5% CO2). The
fluorescence intensity of the medium was measured in a multi-mode Synergy HT
Microplate Reader at excitation and emission wavelengths of 530 nm and 590 nm,
respectively (Biotek). Cell viability was calculated by interpolation of the emission data
displayed by the treated samples and the control ones (untreated samples = 100%
viability). The percentages obtained are depicted as the average of five values.
2.3.2. Photothermic in vitro effects
In order to evaluate the photothermally induced cell death mediated by the Croc-
cycle dye upon NIR-light irradiation, B16F1 and human macrophages were seeded on
24-well plates at cell densities of 40000 and 500000 cells/well, respectively. Then, cells
were treated with Croc-cycle dye solutions in DMSO mixed with growth medium to
reach the desired concentrations (100 μM final concentration, subcytotoxic
concentration obtained from the Blue Cell Viability assay) and no precipitates were
9

observed in any case. After incubation for 30 min, cells were irradiated with a NIR laser
(808 nm; 10 min at 5.2 W/cm2). Changes in cell well temperature were also monitored.
Then, cell damage was evaluated by fluorescence microscopy just after irradiation by
using the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo-Fisher Scientific, USA)
following the manufacturer instructions. Briefly, a solution containing 2 μM calcein
AM and 4 μM ethidium homodimer-1 was added. After incubation at room temperature
(30 min), cells were visualized under an inverted fluorescence microscope Olympus
IX81 through the composition of 20 fields on each well acquired at 4X magnification to
obtain a mosaic of each well. Control samples (treated and not irradiated, not treated
and irradiated, not treated and not irradiated) were also assayed to evaluate the basal
status of cells. Quantitative analysis of cell dead area was done from microscopy images
using Image J software.
2.3.3 ROS generation using a red-ox sensitive probe.
To analyze the ability of Croc-cycle dye to release ROS, and to ensure that cell
damage was exclusively produced by a photothermic effect we evaluated the Croc-
cycle ROS generation using a fluorescence probe. Dihydrorhodamine 123 (DHR123)
was used as indicator of ROS presence as it reacts with ROS producing its oxidized and
cationic form (i.e., rhodamine 123) [33-35]. DHR123 is nonfluorescent but its oxidized
derivative (R123) emits strong green fluorescence at 530 nm (Imax 530 nm) [36]. A
solution containing 6.6 µM DHR123 and 16 µM Croc-cycle dye in an acidic medium
(ethanol-HCl) was tested. The final dispersion was irradiated (808 nm, 5.2 W/cm2) to
evaluate the amount of ROS generated. The generation of R123 was monitored by using
fluorescence spectroscopy using excitation and emission wavelengths of 485 nm and
530 nm, respectively. As control, the influence of the light and heating caused by the
irradiation were also evaluated in order to discriminate the ROS generation attributed
exclusively to the Croc-cycle dye. Sample fluorescence was quantified before and after
5 and 10 minutes of irradiation or heating.
3. Results and discussion
3.1. Characterization of Croc dye and Croc-cycle dye.
Croc dye (Fig. 1a) was synthesized following a procedure described by Guha et al.
(Scheme S1) [14]. The chemical structure of the final compound was confirmed by
proton nuclear magnetic resonance spectroscopy (1H-NMR), as shown Fig. 1a. The
10

macrocycle used for the inclusion of croconaine inside was composed of two synthetic
molecules (Scheme S1): 5-(tert-Butyl)isophthaloyl dichloride 1 was obtained from the
transformation of a 5-tert-butylisophthalic acid into an acyl chloride as described by
Bugarin et al. [30] and on the other side, 9,10-bis(aminomethyl)anthracene 3, which
was synthetized in two steps. First, a 9,10-bis(bromomethyl)anthracene 2 was obtained
from a procedure reported by Altava et al. [31] improving the purification process. In
order to remove the remaining anthracene, the solid was filtered and washed with warm
ethanol (60ᵒC) three times, acquiring a 90% yield. Finally, the crude 2 was reacted with
hexamethylenetetramine retrieving 117 mg (50% yield) of 3 as reported by Gassensmith
et al. [32]. The chemical structure of each compound obtained was confirmed by
magnetic resonance spectroscopy. The croconaine dye trapped within the macrocycle to
form a croconaine rotaxane, Croc-cycle (Fig. 1b), was synthetized according to the
protocol described by Guha et al. (Scheme S1) [14], using a modification in the
purification process. Two chromatographic columns were needed to purify the final
product, a first one using a solution of chloroform and acetone (98% - 2%) as eluent,
and a second one using just chloroform (100%). The adequate portion of solvents
(acetone and chloroform), which separates the precursors by their polarity, was
previously established using thin-layer chromatography (TLC). To ensure the product
obtained was the croconaine trapped into the macrocycle, proton nuclear magnetic
resonance spectroscopy (1H NMR) (Fig. 1b) and two-dimensional nuclear magnetic
resonance spectroscopy (COSY) (Fig. S1) were performed. The product desired was
obtained as a result of a 10% yield.
2
(a)
(b)
11
7
11
7
12
1
3
1
2
1
2
1
5
1
6
1
6
4
4
3
3
4
9
4
9
8
8
7
8
8
8
5
10
5
10
3
5
5
5
5
5
5 5
8
8
8
7
4
4
12
2
11
11
Fig. 1. 1H NMR spectrum of Croc (a) and Croc-cycle (b) in CDCl3. (*) stands for the solvent CDCl3 (7.2
ppm). (**) stands for the remaining solvents used including toluene, 1-butanol and ethanol.
11

Croc and Croc-cycle were characterized by UV/vis spectroscopy to evaluate
their absorption spectra profile and the influence of pH in their absorption properties.
Therefore, acidic and alkaline Croc dye and Croc-cycle solutions were prepared for
UV/vis analysis. Croc and Croc-cycle were tested in ethanol-HCl (pH 5) and ethanol-
NaOH (pH 8) solutions, having a concentration of 16 µM of the dye molecule.
Absorbance was measured in the range from 200 nm to 1000 nm. As shown in Fig. 2,
both molecules have a specific high absorption in the NIR region around 800 nm in the
presence of acidic pH, and a lower one around 600 nm in the presence of alkaline pH.
Moreover, the Croc-cycle (Imax (ethanol-HCl) = 782 nm and Imax (ethanol-NaOH) = 640
nm), presents about 10 nm red-shift of both Croc absorption maxima (Imax (ethanol-
HCl) = 778 nm and Imax (ethanol-NaOH) = 630 nm), Fig. 2b. A similar red-shift effect
was previously observed by Guha et al. due to croconaine encapsulation inside the
macrocycle [14].
0.3
0.2
0.1
0.0
1.0
0.8
0.6
0.4
0.2
0.0
(a)
(b)
778 nm
Alkaline (pH 8)
Acidic (pH 5)
Alkaline (pH 8)
Acidic (pH 5)
782 nm
630 nm
640 nm
400
600
800
400
600
Wavelength (nm)
800
Wavelength (nm)
Fig. 2. UV-Vis spectra of acidic and alkaline Croc (a) and Croc-cycle (b) in ethanol solutions at 16 µM
concentration.
3.2. Reactive oxygen species (ROS) study
A reactive oxygen species (ROS) study was also performed to verify if Croc-
cycle dye generates ROS, and if consequently the potential therapy based on those
organic dyes would be exclusively based on thermal ablation. Croc-cycle ROS release
was evaluated following a modification of the procedure reported by Brawek et al. [33].
Dihydrorhodamine 123 was used as the indicator given that in the presence of ROS it
suffers an oxidation turning into its cationic form (rhodamine 123), a fluorescent dye,
therefore, a fluorescence emission reading can be carried out showing an emission
maximum at 530 nm [34, 35]. A solution containing 6.6 µM DHR123 and 16 µM Croc-
cycle in an acidic medium (ethanol-HCl) was tested. The samples’ fluorescence was
12

quantified in a fluorescence spectrometer set at 480 nm for excitation, after 5 min and
10 min of laser irradiation (800 nm, 5.2 W/cm2). Controls were performed using a
solution of DHR123 (6.6 µM) after 5 min stirring and after 5 min of irradiation, also
using a solution of Croc-cycle (16 µM) after 5 min stirring and after 5 min of
irradiation, using a solution of DHR123 (6.6 µM) and Croc-cycle (16 µM) after 5 min
and 10 min stirring, and after 5 min warmed up in a water bath at 46ᵒC. As shown in
Fig. 3a, there is a correlation between the irradiation time and the increase in
fluorescence emission at 530 nm. After 5 minutes of irradiation, the mixture
fluorescence increased about 60 units, and after 5 more minutes under irradiation, the
fluorescence increased 110 more units. Additionally, several controls were performed in
order to assure if the ROS released were just related to the irradiation. First of all, it was
measured the fluorescence emitted by the Croc-cycle itself after 5 min of irradiation,
and the acidic ethanol solution by itself again after 5 min irradiation. In both cases, no
fluorescence emission was measured (Fig. S2a). Secondly, the fluorescence emitted by
the DHR123 was tested under different scenarios: after 5 min stirring, after 5 min
irradiation and after 5 min under a 46ᵒC water bath, which is the same temperature
reached on the irradiated Croc-cycle solution (Fig. S2b). As observed in Fig. S2b,
DHR123 suffers a spontaneous oxidation when heated at 46ᵒC (about 80 units increase),
which leaded to a final control to quantify this DHR123 influence on the results
previously obtained. Fig. 3b shows that there was a ROS generation increase only when
a temperature (46ᵒC) was applied on the sample, Croc-cycle with DHR123 for 5 min
(20 units increase), and even some increase when only stirring it for 5 min (10 units
increase). Hence, the real irradiation influence on the ROS release was measured in
about 30 units (Fig. 3b). Spence el al. [17] asserted a low oxygen photosensitization
ability of a different croconaine dye with two appended thiophene units, using the
singlet oxygen trap 1,3-Diphenylisobenzofuran (DPBF). Herein, our results by means of
the ROS sensor DHR123 pointed out that our Croc-cycle dye produces a reduced
singlet oxygen generation. Therefore, it has been demonstrated that the presence of ROS
when the sample containing Croc-cycle was irradiated seems to be almost negligible
when compared it with the ROS generated by the controls.
13

Fig. 3. Fluorescence emission spectra for Croc-cycle and DHR123 solution (acidic ethanol solution), (a)
after 5 min irradiation (t1) and 10 min irradiation (t2) at 808 nm and 5.2 W/cm2 (b) after 5 min stirring
(t1), 5 min stirring in a water bath at 46ᵒC and 5 min irradiation at 808 nm and 5.2 W/cm2.
3.3. Cell viability assay
The cytocompatibility of the Croc-cycle dye was studied in the cell lines used for the
subsequent laser-induced photothermic assays, being B16F1 melanoma cells and human
macrophages, which are the cell models for the proposed biomedical application. The
treatment of the cell lines incubated with different Croc-cycle concentrations (75-400
µM) involved detrimental effects on viability on a dose dependent manner at the higher
concentrations assayed as it was determined by the Blue Cell Viability assay (Fig. 4a).
The increase in Croc-cycle concentration (Fig. 4a) beyond 100 µM implied a decrease
in viability higher than 50% for macrophages, and higher than 70% in the case of
B16F1 cells. At the highest concentration assayed (400 µM), the viability percentages
obtained were 24% and 66% on macrophages and B16F1 cells, respectively. According
to these results and the recommendations of the ISO 10993-5 [37], 100 µM was
considered the subcytotoxic concentration for further studies as this concentration
displayed viability percentages higher than 70% in all the cell lines assayed. Compared
to the existing cytotoxicity tests concerning other croconaine dyes, reported by
Harmatys et al. [21] or by Tang et al. [12], which rendered cytotoxicity up to 25-30 µM,
herein it has been corroborated that even at higher concentrations (100 µM), this
molecule is not cytotoxic. Therefore, we used the subcytotoxic concentration previously
obtained, 100 µM, for the following experiments to test the photothermic behavior of
the Croc-cycle dye on cells.
14

(a)
(b)
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
B16F1
Macrophages
DMEM_ pH 5.5_Croc100M
RPMI_pH 5.5_Croc100M
0
2
4
6
8
10
75
100
200
400
Time / min
Concentration (µM)
Fig. 4. (a) Cell viability of Croc-cycle dye in the two cell lines assayed after 24 h. Percentages are
displayed as mean ± SD (N = 5). (b) Heating curve of 100 μM Croc-cycle dye in DMEM and RPMI
media at pH 5.5 upon irradiation at 808 nm and 5.2 W/cm2.
3.4. Laser-Induced Heat Generation Studies
Photothermal properties of the Croc-cycle dye alone were initially studied.
Croc-cycle dye was dissolved in DMSO and added to DMEM and RPMI media at pH
5.5 to obtain final solutions of 100 µM concentration. 1 mL of the solution was
irradiated using an 808 nm-laser diode with the beam passing through it in a plate well,
in the same conditions than in the ones used in the subsequent cell assays. The laser
irradiance was set at 5.2 W/cm2. The temperature was monitored by a thermocouple
immersed into the solution, recording the temperature every 0.017 s. When the Croc-
cycle dye was irradiated with the laser (808 nm, 5.2 W/cm2), a 20 ᵒC temperature
increase was achieved using 100 µM Croc-cycle solution (Fig. 4b). Laser irradiation
assays were carried out in order to monitor the temperature increase when Croc-cycle
was induced to its acidic structure, reaching a final temperature of ~50 ᵒC in 10 min
(Fig. 4b), which can be used for photothermic in vitro tests with cells. Controls were
performed for the irradiation of DMEM and RPMI media at pH 5.5 in the same
experimental conditions, but without Croc-cycle dye, presenting the absence of heating
(Fig. S3).
3.5. Evaluation of laser-induced cell damage
Photothermal effects of Croc-cycle dye on melanoma cells (B16F1) and human
macrophages were measured using fluorescence microscopy (Fig. 5) after staining live
cells with calcein and dead cells with ethidium bromide. B16F1 and human
15

macrophages were incubated with Croc-cycle dye (100 µM) during 30 min at 37ºC.
After incubation, cells were subsequent irradiated for 10 minutes with the NIR-laser
operating at 808 nm (5.2 W/cm2). The rise of temperature in the well after irradiation
was measured using a near-infrared sensor, showing a final temperature below 44ºC in
all cases. For both cell lines treated with Croc-cycle dye, cell damage was focalized on
the irradiated area, Fig. 5, showing a central area of dead cells (red) corresponding with
the laser spot surrounded by live cells (green). Furthermore, control samples treated
with Croc-cycle dye and no irradiated, and cells not treated with Croc-cycle and
irradiated, did not exert cell damage (Fig. S4). The increase of temperature in the
controls cell wells was always < 3 ºC, indicating the absence of a thermal effect. With
the same laser spot projected area cell death seemed to be more accentuated in
melanoma cells (22.3 % cell dead area) than in macrophages (7.7 % cell dead area),
which can be attributed to the lower pH of intracellular vesicles of cancer cells
compared to the one of normal cells as previously reported [38-40]. These results
highlight the selective cellular ablation effect of our Croc-cycle dye, showing high
viability in neighboring cells not irradiated though displaying a local photothermal cell
damage after NIR irradiation when the dye is placed in the acidic intracellular milieu,
necessary to activate its absorption in the NIR region of the spectrum. Overall, Croc-
cycle dye has demonstrated to be a potential agent for PTT, producing no cell death
without irradiation and a selective cell destruction when the dye is located inside the
cells.
(a)
(b)
2 mm
2 mm
Fig. 5. Photothermal effects on murine melanoma cells (B16F1 cell line) and macrophages treated with
Croc-cycle dye using the same laser spot projected area on the culture wells. Composition of 20 pictures
(4X magnification) obtained by fluorescence microscopy to show the whole culture well when cells were
treated for 30 min with Croc-cycle dye (at a subcytotoxic concentration, 100 μM). (a) Macrophages and
(b) B16F1 cells laser irradiated (808 nm, 5.2 W/cm2) for 10 min and images acquired immediately after
irradiation. Live cells are stained in green while dead cells in red. Scale bar 2 mm.
16

4. Conclusions
In summary, we have synthesized and characterized a croconaine-based dye
trapped within a macrocycle as photothermal therapeutical agent. The Croc-cycle dye
was capable of heating up a medium in a pH-dependent manner, therefore in the case of
acidic pH Croc-cycle dye absorbs the irradiation of a laser in the NIR region, efficiently
increasing the medium temperature at very low concentrations. Croc-cycle dye was
internalized within the acidic interior of tumoral cells and macrophages, and only in the
intracellular milieu it was activated and efficiently absorbed the NIR light resulting in
cell death. Consequently, cell damage was exclusively produced intracellularly on
irradiated cells, without damage observed in neighboring cells, which remained alive.
The presence of the molecule in the extracellular media did not produce cytotoxic effect
at the concentrations tested. ROS generation observed for the Croc-cycle dye was
negligible so the reported cytotoxic effect was exclusively driven by photothermia.
Results demonstrated that Croc-cycle dye could have great potential in photothermal
therapy, and could be not only potentially used for the selective damage of cancer cells
labeled, but also for a wide range of materials science applications in which its pH-
responsiveness could be used.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors thank financial support from the ERC Consolidator Grant program (ERC-
2013-CoG-614715, NANOHEDONISM). CIBER-BBN is an initiative funded by the VI
National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the
assistance of the European Regional Development Fund. T. Alejo thanks the Fundation
Institute of Nanoscience for her postdoctoral fellowship. We also acknowledge Dr. Pilar
Martin-Duque for donating B16F1 mouse melanoma cells.
17

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