Iodinated xanthene-cyanine NIR dyes as potential photosensitizers for antimicrobial photodynamic therapy

Article abstract of DOI:10.1016/j.dyepig.2020.108854

Photosensitizers (PSs) are chemical entities that upon light exposure are able to produce cytotoxic species such as singlet oxygen superoxide and free radicals. PSs are used in photodynamic therapy applications (PDT) to eradicate cancer cells, pathogenic bacteria, fungi, and viruses. Development of the NIR-activatable photosensitizes with high phototoxicity and low toxicity in dark conditions is challenging. Here we first report on the synthesis and antimicrobial testing of the new NIR photosensitizers based on the iodinated xanthene-cyanine dyes. These new photosensitizers exhibit high antimicrobial efficacy on Staphylococcus aureus pathogens at low dye concentration (~0.5 μM) and low NIR light dose (24.3 J/cm²).

Full text of DOI:10.1016/j.dyepig.2020.108854

Dyes and Pigments 184 (2021) 108854
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Iodinated xanthene-cyanine NIR dyes as potential photosensitizers for
antimicrobial photodynamic therapy
,
*
T.M. Ebastona, Faina Nakonechnyb, Efrosiniia Talalaia, Gary Gellermana, Leonid Patsenkera
a Department of Chemical Sciences, The Faculty of Natural Sciences, Ariel University, Ariel, 40700, Israel
b Department of Chemical Engineering, The Faculty of Engineering, Ariel University, Ariel, 40700, Israel
A R T I C L E I N F O
A B S T R A C T
Keywords:
Photosensitizers (PSs) are chemical entities that upon light exposure are able to produce cytotoxic species such as
singlet oxygen superoxide and free radicals. PSs are used in photodynamic therapy applications (PDT) to
eradicate cancer cells, pathogenic bacteria, fungi, and viruses. Development of the NIR-activatable photosensi-
tizes with high phototoxicity and low toxicity in dark conditions is challenging. Here we first report on the
synthesis and antimicrobial testing of the new NIR photosensitizers based on the iodinated xanthene-cyanine
dyes. These new photosensitizers exhibit high antimicrobial efficacy on Staphylococcus aureus pathogens at
Xanthene-cyanine dye
Iodinated dye
Photosensitizer
Antimicrobial photodynamic therapy
APDT
Staphylococcus aureus
low dye concentration (~0.5
μ
M) and low NIR light dose (24.3 J/cm2).
1. Introduction
photodynamic therapy (PDT) applications [19–21].
The main goal of this work is to evaluate phototoxicity of the parent
xanthene-cyanine dye XCy (Fig. 1,B) and explore possibility of devel-
oping highly phototoxic PSs based on this structure. One of the known
methods to increase photosensitizing efficacy and phototoxicity of
organic dyes is the introduction of heavy atoms such as iodine [22]. This
approach has recently been demonstrated in the example of cyanine
[23] and squaraines [24] dyes.
Recently, the synthesis and spectral properties of several xanthene-
cyanine dyes with a core structure shown in Fig. 1, A were reported
[1]. These dyes absorb (679 nm) and emit light (705 nm) within the
near-IR (NIR) spectral region, which is beneficial for probing in bio-
logical systems, in particular in vivo. Due to the high brightness (high
extinction coefficients and fluorescence quantum yields), these dyes
were proposed for biological sensing and imaging applications [2,3].
Several fluorogenic dyes with activatable emission have been developed
based on the xanthene-cyanine core and evaluated as fluorescent re-
porters for targeted anticancer drug delivery monitoring [1] and other
numerous imaging applications such as determination of hepatoxicity
[4], activity of alkaline phosphatase [5], azoreductase [6] and leucine
aminopeptidase [7], detection of cysteine [8] and keloid [9], imaging of
biological nitroxyl [10] and hydrogen polysulphides [11], glutathione
(GSH) activated cancer imaging [12], monitoring of thiol flux [13],
photoacoustic visualization of peroxynitrite [14], probing of cancer
specific enzyme hNQO1 [15], hypochlorous acid recognition [16], and
pH sensing imaging in vivo [17]. However, phototoxicity of
xanthene-cyanines on biological specimens has never been investigated,
although this parameter seriously limits suitability of the dyes as the
fluorescent reporters in vitro and in vivo [18]. Nevertheless, phototox-
Herein, we report on the synthesis, spectroscopic characterization
and phototoxicity of the new NIR-excitable mono- (I-XCy) and di-
iodinated (I2-XCy) xanthene-cyanine dyes (Fig. 1,B) versus the parent
non-halogenated xanthene-cyanine XCy and FDA approved [25–27]
heptamethine cyanine photosensitizer ICG [28], which is also activat-
able in the NIR range.
Phototoxicities of XCy, I-XCy, I2-XCy, and ICG were tested on gram-
positive Staphylococcus aureus (S. aureus) bacteria, which is a major
human pathogen that causes a wide range of clinical infections. S. aureus
can be commonly found on the skin and nose in most humans and can
lead to death when entering bloodstream, joints, bones, lungs or heart
[29]. Treatment usually involves antibiotics and drainage of the infected
area. However, some staphylococcal infections no longer respond to
common antibiotics [30] and antibacterial photodynamic therapy
(APDT) is, therefore, an alternative beneficial method for eradication of
this pathogen [31].
icity is
a mandatory feature for photosensitizers (PSs) used in
* Corresponding author. author.
E-mail addresses: ebaston@ariel.ac.il (T.M. Ebaston), fainan@ariel.ac.il (F. Nakonechny), ftalalay@gmail.com (E. Talalai), garyg@ariel.ac.il (G. Gellerman),
leonidpa@ariel.ac.il (L. Patsenker).
https://doi.org/10.1016/j.dyepig.2020.108854
Received 9 May 2020; Received in revised form 26 July 2020; Accepted 11 September 2020
Available online 14 September 2020
0143-7208/© 2020 Elsevier Ltd. All rights reserved.

T.M. Ebaston et al.
Dyes and Pigments 184 (2021) 108854
2. Experimental
indolium (5a): Catalytic amount of sodium acetate was added to a
mixture of 2-chloro-3-hydroxymethylene-cyclohex-1-enecarbaldehyde
(1 eq., 500 mg, 2.9 mmol) and 1,2,3,3-tetramethyl-3H-indolium 3a
(2.2 eq.,1.1 g, 6.38 mmol) in ethanol and stirred for 4 h at 50 ◦C at N2
atmosphere. The resulting green solution was evaporated and purified
by flash column chromatography using EtOAc/CH3OH as eluent to
afford compound 5a as a dark green solid. Yield 900 mg (65%). MS m/z
(ESI+) C32H36ClN2+ calculated 483.25, found m/z: 483.0.
2.1. General
All chemicals were supplied by Alfa Aesar Israel and Sigma-Aldrich.
2,3,3-Trimethylindolenine (2a) was purchased from Aldrich and used as
is. Solvents were purchased from Bio-Lab Israel and used as is. Chemical
reactions were monitored by thin layer chromatography (TLC) (Silica
gel 60 F-254, Merck) and by LC/MS.
2-(2-{2-Chloro-3-[2-(5-iodo-1,3,3-trimethyl-1,3-dihydro-indol-
2-ylidene)-ethylidene]-cyclohex-1-enyl}-vinyl)-5-iodo-1,3,3-tri-
methyl-3H-indolium (5b): Dye 5b was synthesized from 3b (2.2 eq.,
43 mg, 0.14 mmol) according to the same procedure as 5a affording a
dark green solid. Yield 50 mg (47%). MS m/z (ESI+) C32H34ClI2N2+
calculated 735.05, found m/z: 735.1.
LC/MS analysis was performed using an Agilent Technologies 1260
Infinity (LC) 6120 quadruple (MS), column Agilent SB-C18, 1.8 mm, 2.1
× 50 mm, column temperature 50 ◦C, eluent water-acetonitrile (ACN) +
0.1% formic acid.
HRMS was performed in ESI positive mode by using an Agilent 6550
iFunnel Q-TOF LC/MS instrument.
2-(2-{2-Chloro-3-[2-(4,6-diiodo-1,3,3-trimethyl-1,3-dihydro-
indol-2-ylidene)-ethylidene]-cyclohex-1-enyl}-vinyl)-4,6-diiodo-
1,3,3-trimethyl-3H-indolium (5c): Dye 5c was synthesized from 3c
(2.2 eq., 108 mg, 0.25 mmol) according to the same procedure as 5a.
Dark green solid, yield 40 mg (35%). MS m/z (ESI+) C32H32ClI4N2+
calculated 986.84, found m/z: 986.8.
1H NMR and 13C NMR spectra were measured in CD3OD at 300 K on a
Bruker AvanceIII HD (1H 400 MHz and 13C 100 MHz) spectrometer and
a BBO probe equipped with a Z gradient coil.
2.2. Synthesis
Dyes 5a–5c were used for further synthesis without purification.
2-[2-(6-Hydroxy-2,3-dihydro-1H-xanthen-4-yl)-vinyl]-1,3,3-tri-
methyl-3H-indolium (XCy): Potassium carbonate (276 mg, 2 mmol)
and resorcinol (220 mg, 2 mmol) were dissolved in acetonitrile (20 mL)
and stirred for 15 min under N2 atmosphere. The above mixture was
added to a solution of 5a (683 mg, 1 mmol) in acetonitrile (15 mL) and
stirred for 8 h at 50 ◦C. The reaction was monitored by TLC. After the
reaction was complete, the solvent was evaporated under reduced
pressure and the crude product was column purified (silica gel 70–230
mesh, DCM—methanol, 90:10, v.v.). XCy was obtained as a blue solid.
Yield 295 mg (61%). Purity 98% (LC/MS, 254 nm). 1H NMR (400 MHz,
CD3OD): δ 8.64 (d, J = 16 Hz, 1H), 7.62 (d, J = 7.62 Hz, 1H), 7.51 (m,
2H), 7.40 (s, 1H), 7.32 (m, 2H), 6.74 (m, 2H), 6.33 (d, J = 16 Hz, 1H),
3.71 (s, 3H), 2.69 (t, J = 12 Hz, 2H), 2.62 (t, J = 12 Hz, 2H), 1.84 (m,
2H), 1.71 (s, 6H). 13C NMR (100 MHz, CD3OD): δ 164.64, 163.94,
146.11, 144.32, 137.29, 132.72, 130.88, 130.41, 128.03, 123.89,
117.31, 116.64, 116.05, 113.42, 103.74, 103.44, 100.62, 51.78, 32.71,
31.09, 30.26, 28.68, 25.47 MS m/z (ESI+) C26H26NO2+ calculated
384.1964, found m/z: 384.1968.
4-Iodophenylhydrazine 1b: Solution containing 4-iodoaniline (20
g, 91.3 mmol) and hydrochloric acid (5.5 M, 15 mL) was cooled to
ꢀ 10 ◦C and NaNO2 (12.6 g, 182.6 mmol) in 45 mL of water was added
dropwise with continuous stirring. The suspension was stirred for 30
min and then an ice-cold solution of SnCl2⋅2H2O (67.99 g, 301.3 mmol)
in 40 mL of concentrated HCl was added dropwise keeping the tem-
perature at ꢀ 10 ◦C. The reaction mixture was stirred at that temperature
for 1.5 h and then 18 h at 5 ◦C. The light brown precipitate was filtered
and washed three times with water and extracted with ether. After
drying over anhydrous MgSO4, the ether layer was evaporated to dry-
ness to afford 1b as brown powder. Yield 17.94 g (84%). MS m/z (ESI+)
C6H7IN2 calculated 233.96, found m/z: 233.90.
3,5-Diiodophenylhydrazine (1c): Phenyl hydrazine 1c was ob-
tained by the same procedure as for 1a starting from 3, 5-diiodoaniline
(5 g, 14.5 mmol) [32]. The product 1c was isolated as fine brown nee-
dles. Yield 3.7 g, (72%). MS m/z (ESI+) C6H6I2N2 calculated 359.86,
found m/z: 359.9.
5-Iodo-2,3,3-trimethyl-3H-indole (2b) and 4,6-diiodo-2,3,3-tri-
methyl-3H-indole (2c) were prepared according to the literature pro-
cedure [33].
2-[2-(6-Hydroxy-2,3-dihydro-1H-xanthen-4-yl)-vinyl]-5-iodo-
1,3,3-trimethyl-3H-indolium (I-XCy): Dye I-XCy was synthesized
similar to XCy, starting from 5b (50 mg, 1 mmol). The product was
isolated as a blue solid. Yield 15 mg (44%). Purity 80% (LC/MS 254 nm).
1H NMR (400 MHz, CD3OD): δ 8.59 (d, J = 16 Hz, 1H), 7.86 (s, 1H), 7.72
(dd, J = 8 Hz 1H), 7.45 (s, 1H), 7.36 (d, J = 8 Hz 1H), 7.13 (d, J = 8 Hz
1H), 6.76 (m, 2H), 6.23 (d, J = 16 Hz 1H), 3.62 (s, 3H), 2.70 (t, J = 12
Hz, 2H), 2.63 (t, J = 12 Hz, 2H), 1.93 (m, 2H), 1.69 (s, 6H). 13C NMR
(100 MHz, CD3OD): δ 164.49, 157.07, 145.58, 145.17, 144.11, 139.73,
139.21, 138.32, 132.95, 131.01, 130.89, 126.89, 117.64, 116.73,
114.77, 103.24, 102.90, 90.77, 51.24, 36.70, 33.22, 32.34, 28.42,
23.89 MS m/z (ESI+) C26H25INO2+ calculated 510.0930, found m/z:
510.0935.
General procedure for the synthesis of 1,2,3,3-tetramethyl-3H-
indolium (3a), 5-iodo-1,2,3,3-tetramethyl-3H-indolium (3b) and
4,6-diiodo-1,2,3,3-tetramethyl-3H-indolium (3c): 3a, 3b, 3c were
synthesized according to the procedure [23]. Corresponding indolenine
2a–2c (1 g, 500 mg and 500 mg, respectively, 6.3, 1.8 and 1.2 mmol)
was dissolved in toluene (1 mL) and a large excess of methyl iodide (3
eq.) was added. The reaction mixture was heated in a pressure tube at
80 ◦C for 2 h, cooled to RT, and the precipitate was filtered and dried in a
vacuum desiccator. Yields 760 mg (70%) (3a); 289 mg (55%) (3b); 222
mg (43%) (3c).
2-Chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde
(4) was synthesized according to the procedure [34].
2-[2-(6-Hydroxy-2,3-dihydro-1H-xanthen-4-yl)-vinyl]-4,6-
diiodo-1,3,3-trimethyl-3H-indolium I2-XCy: Dye I2-XCy was synthe-
sized similar to XCy, starting from 5c (40 mg, 1 mmol). The product was
2-(2-{2-Chloro-3-[2-(1,3,3-trimethyl-1,3-dihydro-indol-2-yli-
dene)-ethylidene]-cyclohex-1-enyl}-vinyl)-1,3,3-trimethyl-3H-
Fig. 1. Previously reported XCy-based dyes (A) and the dyes investigated in this work (B).
2

T.M. Ebaston et al.
Dyes and Pigments 184 (2021) 108854
isolated as a blue solid. Yield 8 mg (32%). Purity 81% (LCMS, 254 nm).
1H NMR (400 MHz, CD3OD): δ 8.52 (d, J = 16 Hz, 1H), 7.94 (s, 1H), 7.60
(s, 1H), 7.52 (m, 1H), 7.41 (d, J = 8 Hz, 1H), 6.77 (m, 2H), 6.10 (d, J =
12 Hz 1H) 3.52 (s, 3H), 2.72 (t, J = 12 Hz, 2H), 2.64 (t, J = 12 Hz, 2H),
1.85 (m, 2H), 1.81 (s, 6H). 13C NMR (100 MHz, CD3OD): δ 157.4, 157.1,
155.3, 147.3, 146.2, 139.9, 137.3, 135.8, 131.0, 128.1, 119.6, 117.1,
116.4, 115.5, 108.7, 104.6, 96.2, 97.6, 51.0, 35.7, 33.2, 31.8, 29.9, 27.5,
25.1 MS m/z (ESI+) C26H24I2NO2+ calculated 635.9897, found m/z:
635.9810.
the dark to avoid their activation and photobleaching. The stock solu-
tions of 0.1 mM of XCy, I-XCy, I2-XCy and ICG in DMSO were prepared
and the aliquots were added to 1 mL of S. aureus in BH to the final dye
concentrations 0.1 μM, 0.5 μM and 1.0 μM. The amount of the DMSO dye
solutions added to the bacteria suspensions did not exceed 0.5%. The
cells were incubated in the dark for 15 min and then exposed to light for
5 min, 15 min and 30 min by a 660-nm, 12 W LED (light intensity 27
mW/cm2; light dose 8.1 J/cm2, 24.3 J/cm2 and 48.6 J/cm2,
respectively).
After the light exposure, 100 μL aliquots of each sample at various
decimal dilutions were spread over BHA plates with a Drigalsky
spreader, incubated at 37 ◦C for 24 h, and the colony forming units
(CFU) were counted by the ImageJ software [35]. To verify the dark
toxicity of the dyes, the same experiments were carried out in parallel
without light exposure. As the control we utilized the samples of
S. aureus without dye (i) in the dark and without DMSO, (ii) in the dark
in the presence of 1% DMSO, (iii) light irradiated without DMSO, and
(iv) light irradiated in the presence of 1% DMSO.
2.3. Absorption and fluorescence measurements
Absorption spectra were recorded on a Jasco V-730 UV–Vis spec-
trophotometer and the fluorescence spectra were taken on an Edinburgh
FS5 spectrofluorometer. The absorption and fluorescence spectra were
measured at 25 ◦C in standard 1-cm quartz cells at ~0.5
μM dye con-
centrations in 10 mM phosphate buffer pH 7.4 (PB) containing 5%
methanol to facilitate solubility of the investigated compounds. Excita-
tion wavelength (λex) was 650 nm.
All the experiments were carried out in triplicate and the average
value was taken.
To determine the absolute fluorescence quantum yield (FF), the in-
tegrated relative intensities were measured in PB vs. non-iodinated XCy
dye in PB as the reference (FF = 37%) [8], and the quantum yield was
calculated according to Equation (1).
2.5. Fluorescence imaging
The fluorescence images were acquired by Photometrics CoolSNAP
HQ2 camera mounted on an Olympus iX81 fluorescent microscope. The
microscope was equipped with a 120 W metal halide discharge lamp.
For the fluorescence images, a cube comprising an ET620/60x bandpass
excitation filter, ET700/75 m bandpass emission filter and T66lpxr
dichroic filter was used. All images were recorded with the same in-
strument settings. The images were taken with, exposure time 900 ms,
gain 20 dB and × 20 magnification.
ΦF = ΦFRef × (F / FRef) × (ARef / A),
(1)
where ΦFRef is the quantum yield of the reference, FRef and F are the
∫
areas (integral intensities) of the emission spectra (F = I(λ)dλ) of the
reference dye and the dye under examination, ARef and A, are the ab-
sorbancies at the excitation wavelength of the reference and the dye
under examination, respectively.
The quantum yield for each dye was independently measured three
times and the average value was taken.
3. Results and discussion
2.4. Antimicrobial studies
3.1. Synthesis
Culture of S. aureus (ATCC 25923) was grown on Brain Heart agar
plates (BHA, Acumedia, Lansing, MI, USA) for 24 h, transferred into
Brain Heart broth (BH, Acumedia, Lansing, MI, USA), grown at 37 ± 1 ◦C
with shaking at 170 rpm until reaching the absorbance A = 0.1 at 660
nm, which corresponded to a final concentration of 108 cells/mL, and
diluted then to the final concentration of 104–105 cells/mL.
We synthesized the known, non-iodinated xanthene-cyanine dye
XCy [2] and its two new derivatives, mono-iodinated I-XCy and
di-iodinated I2-XCy (Scheme 1). The synthetic pathway was via the
hydrazines 1a–1c obtained as described in Ref. [32]. The hydrazines
1a–1c were subjected to cyclocondensation with methyl-isopropyl ke-
tone by the Fisher method to corresponding indolenines 2a–2c. The last
All preparatory operations with photosensitizers were carried out in
Scheme 1. Synthesis of XCy, I-XCy and I2-XCy dyes.
3

T.M. Ebaston et al.
Dyes and Pigments 184 (2021) 108854
ones were quaternized to indolenines 3a–3c which were then reacted
example of the dye XCy, therefore, we investigated the pH dependent
absorption and emission spectra. It can be seen that in the neutral (pH
7.4) and alkali (pH 11.0) media the dye exists preferably in the fluo-
rescent deprotonated form with λmaxAb ~ 679 nm and λmaxFl ~ 705 nm
(Fig. 3). In the acidic environment (pH 4.0), the absorption maximum is
blue-shifted and the dye shows no detectable fluorescence. Importantly,
in our further experiments the phototoxicities of the dyes are tested on
the S. aureus bacteria, which have the intracellular pH in the range of
8.4–8.7 [37,] where the dyes exist mostly in the fluorescent deproto-
nated forms.
with cyclohexene carbaldehyde
4
to yield the intermediate
chloro-cyanine dyes 5a–5c, as described in Ref. [36]. The reported
procedure was modified by using ethanol (solvent) instead of a mixture
of dioxane and toluene (1:1), which allowed us to improve the synthetic
yield and purity of the products. Cyanines 5a–5c were then subjected to
straightforward nucleophilic substitution of the chlorine atom with
resorcinol to eliminate the indolenine moiety by a retro-Knovenagel
reaction followed by cyclization and dehydration, as suggested in
Ref. [2], to give the aimed xanthene-cyanines XCy, I-XCy and I2-XCy in
satisfactory yields (~40–60%).
3.3. Antimicrobial activity
3.2. Spectral properties
Our APDT experiments included the following three major steps
(Scheme 2): (i) incubation of bacteria with PS in the dark (pre-irradia-
tion incubation); (ii) irradiation of bacteria with various light doses (a)
while, in parallel, bacteria were kept in the dark for the same time to be
used as the reference (b); and (iii) growth of bacteria in the dark at 37 ◦C
followed by the calculation of bacteria population.
The spectral properties of XCy, I-XCy and I2-XCy (cDye = 0.5 μM)
were measured in phosphate buffer pH 7.4 (PB) containing 5% methanol
to facilitate the dye solubility. All these dyes exhibit strong aggregation,
which is observed as the broadened absorption bands and the violation
of mirror symmetry between the absorption and fluorescence bands
(Fig. 2). The aggregation predictably increases with increasing the
number of iodine atoms. While the absorption (~680 nm) and emission
(~705 nm) maxima for all these dyes are about the same, the extinction
coefficients (ε) and the fluorescence quantum yields (ΦF) noticeably
decrease in the order: XCy > I-XCy > I2-XCy (see Table 1).
Dyes XCy, I-XCy and I2-XCy contain a conjugated hydroxyl group,
which is subjected for protonation-deprotonation in respect to the sol-
vent acidity. Obviously, the protonated and deprotonated forms possess
different spectral properties and photosensitizing activity. In the
Fig. 3. Absorption and emission spectra of XCy (cDye = 0.5 μM) at pH 4.0 (solid
line), 7.4 (dashed line) and 11.0 (dotted line). λex = 650 nm. T = 25 ◦C. The
fluorescence intensity at pH 11.0 is normalized to the absorption band at the
same pH. The fluorescence intensities of the bands measured at pH 4.0 and pH
7.4 are shown relative to the fluorescence intensity of the band at pH 11.0.
Fig. 2. Normalized absorption and emission spectra of XCy (solid line), I-XCy
(dashed line) and I2-XCy (dotted line) dyes (cDye = 0.5 μM) measured in 0.1 M
PB pH 7.4 containing 5% methanol. λex = 650 nm. T = 25 ◦C.
Table 1
Spectral properties of XCy, I-XCy and I2-XCy (cDye = 0.5 μM) in phosphate buffer
pH 7.4 (PB) containing 5% methanol: the absorption (λmaxAb) and emission
(λmaxFl) maxima, extinction coefficient at the maximum (
and the fluorescence quantum yield (ΦF).
ε) and at 660 nm (ε660),
Dye
λmaxAb, nm
ε
(
ε660), Mꢀ 1cmꢀ 1
λmaxFl, nm
ΦF, %
XCy
679
684
684
789
88,000 (71,000)
27,500 (23,300)
21,000 (18,700)
194,000 (18,400)
705
705
705
810
37
27
16
5
I-XCy
I2-XCy
ICG
Scheme 2. APDT experiment.
4

T.M. Ebaston et al.
Dyes and Pigments 184 (2021) 108854
First, we investigated the effect of the pre-irradiation incubation time
for the ICG photosensitizer recently reported for its photodynamic
eradication of S. aureus in vitro and in vivo [38–40]. It can be seen from
the Table, that at the activation wavelength 660 nm, ICG absorbs about
the same number of photons (ε660 ~ 18,400 Mꢀ 1cmꢀ 1) as I2-XCy but this
excitation is a bit less pronounced as compared to I-XCy (ε660 ~ 23,300
Mꢀ 1cmꢀ 1) and about four times smaller than that for XCy (ε660 ~ 71,
000 Mꢀ 1cmꢀ 1). Nevertheless, in all our experiments ICG did not show a
detectable eradication of S. aureus at least at a concentration of up to 1.0
(in the dark) on the PS uptake. For this, S. aureus bacteria were incu-
bated with 1 μM I2-XCy for 5, 15, 30 and 60 min, irradiated with 24.3 J/
cm2 light dose and cell survival was estimated (Fig. 4,A). It was found
that even 5 min incubation is sufficient for the PS uptake. Nevertheless,
in our further experiments we applied 15 min dark incubation to ensure
the uptake of the dyes by cells. Because all the dyes are fluorescent, we
were able to verify their uptake by using the fluorescence microscopy
images acquired in 15 min after incubation. The corresponding images
showing accumulation of the dyes in cells are presented in Fig. 5.
In the next step, the photodynamic eradication of S. aureus was
μ
M and light dose of up to 48.6 J/cm2, while XCy, I-XCy and I2-XCy
exhibit noticeable phototoxicity.
A comparative study of the investigated dyes demonstrates that their
phototoxicity towards S. aureus noticeably increases with increasing the
number of the iodine atoms: XCy < I-XCy < I2-XCy. Thus, I2-XCy
exhibited the most significant eradication of S. aureus with only 1.1%
investigated at three different PS concentrations (0.1, 0.5, 1.0 μM) and
at three different light doses of 8.1 J/cm2, 24.3 J/cm2 and 48.6 J/cm2 (5,
15, 30 min, respectively) using a 660-nm 12 W LED that produced 27
mW/cm2 for each sample. The obtained results were compared to those
cell survival at 1
μ
M and 24.3 J/cm2 light dose; and almost no survival
Fig. 4. APDT experiment. A: Effect of pre-irradiation incubation time on the S. aureus survival (step “i" on Scheme 2): I2-XCy (1 μM) was incubated in the dark
followed by a 24.3 J/cm2 light exposure. B–D (step “ii” on Scheme 2): S. aureus survival after 15 min of dark incubation followed by a 8.1 J/cm2 (5 min, B), 24.3 J/
cm2 (15 min, C), and 48.6 J/cm2 (30 min, D) light exposure. ICG (0.1–10
μM) does not eradicate S. aureus at the investigated light doses. Irradiation was performed
by a 660-nm 12 W LED (27 mW/cm2).
Fig. 5. Fluorescence microscopy images of S. aureus taken in 15 min dark incubation with 1 μM of XCy (a), I-XCy (b) and I2-XCy (c).
5

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Dyes and Pigments 184 (2021) 108854
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cells were found at 0.5
μ
M and 48.6 J/cm2 (Fig. 4,B–C). Remarkably, the
reference dye ICG does not show any detectable eradication of S. aureus
even at 10
μM concentration. Recently, ICG was reported to cause a
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pronounced eradication of S. aureus at 32
μ
M (25
μ
g/mL) and 100 J/cm2
NIR light dose [38,39], which is a much more severe condition
compared to those in our experiments. Importantly, all the investigated
dyes were found to exhibit no dark toxicity to the bacteria at the con-
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4. Conclusions
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The two iodinated xanthene-cyanine dyes I-XCy and I2-XCy were
synthesized, their spectral properties were investigated and the ability
for the photoinduced eradication of S. aureus pathogens was studied in
comparison with the non-iodinated xanthene-cyanine XCy and well
established photosensitizer ICG. An increase in the number of iodine
atoms was shown to enhance phototoxicity of the dyes. Due to the
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cm2), low dark toxicity even at 10
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CRediT authorship contribution statement
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T.M. Ebaston: Experimental work: synthesis, spectroscopic charac-
terization, photodynamic treatment, and fluorescence imaging. Faina
Nakonechny: Supervision, and experimental work on the biological
part of the research: Planning of biological experiments, cell culture
preparation and cell analysis after the treatment. Efrosiniia Talalai:
Experimental work: assistance in cell culture preparation and analysis of
photodynamic treatment results. Gary Gellerman: Writing - original
draft, Formulation of the problem, selection of the cells for the photo-
dynamic experiments, and writing the article. Leonid Patsenker: Su-
pervision, Writing - original draft, of the work in general, development
of photosensitizers, planning synthetic, spectroscopic and photody-
namic experiments, formulating conclusions, and writing the article.
[13] Yang Y, Zhou T, Jin M, Zhou K, Liu D, Li X. Thiol–chromene “click” reaction
triggered self-Immolative for NIR visualization of thiol flux in physiology and
pathology of living cells and mice. J Am Chem Soc 2020;142:1614–20. https://doi.
org/10.1021/jacs.9b12629.
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fluorescence and photoacoustic imaging of peroxynitrite. Anal Chem 2018;90.
https://doi.org/10.1021/acs.analchem.8b01879. 9301–7.
[15] Zhang C, Zhai BB, Peng T, Zhong Z, Xu L, Zhang QZ. Design and synthesis of near-
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Dyes Pigments 2017;143:245–51. https://doi.org/10.1016/j.dyepig.2017.04.043.
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recognition mechanism based on chlorine–oxygen bond (Clꢀ O) formation. Chem
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Anal Chem 2015;87:2495–503. https://doi.org/10.1021/ac5045498.
[18] Kiepas A, Voorand E, Mubaid F, Siegel PM, Brown CM. Optimizing live-cell
fluorescence imaging conditions to minimize phototoxicity. J Cell Sci 2020;133:
jcs242834. https://doi.org/10.1242/jcs.242834.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[19] Jenni S, Sour A. Molecular theranostic agents for photodynamic therapy (PDT) and
magnetic resonance imaging (MRI). INORGA 2019;7:10. https://doi.org/10.3390/
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Acknowledgements
[20] Ghorbani J, Rahban D, Aghamiri S, Teymouri A, Bahador A. Photosensitizers in
antibacterial photodynamic therapy: an overview. Laser Ther 2018;27:293–302.
https://doi.org/10.5978/islsm.27_18-RA-01.
This work was supported by the Israel Scientific Foundation (ISF),
project 810/18 and the Ariel University, Isreal (Joint Research Project
between Ariel University and Florida Atlantic University), FAU project
RA2000000056. Leonid Patsenker is thankful to the Center for Ab-
sorption in Science of the Ministry of Immigrant Absorption of Israel for
the financial support under the KAMEA program. Gary Gellerman is
thankful to the Gale Foundation for the generous donation to support
this work.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2020.108854.
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