Unexpected effect of iodine atoms in heptamethine cyanine dyes on the photodynamic eradication of Gram-positive and Gram-negative pathogens

Article abstract of DOI:10.1016/j.dyepig.2021.109745

The introduction of heavy atoms such as iodine into organic dye molecules is known to improve the efficacy of photodynamic therapy (PDT) in general and antimicrobial photodynamic therapy (APDT) in particular. Such a phenomenon is attributed to the increas

Full text of DOI:10.1016/j.dyepig.2021.109745

Dyes and Pigments 195 (2021) 109745
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Unexpected effect of iodine atoms in heptamethine cyanine dyes on the
photodynamic eradication of Gram-positive and Gram-negative pathogens
,
,
Olga Semenovaa b, Dmytro Kobzeva b, Fares Yazbakc, Faina Nakonechnyc, Olga Kolosovab,
Anatoliy Tataretsb, Gary Gellermana, Leonid Patsenkera
,
*
a Department of Chemical Sciences, Ariel University, Ariel, Israel
b SSI “Institute for Single Crystals” of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine
c Department of Chemical Engineering, Ariel University, Ariel, Israel
A R T I C L E I N F O
A B S T R A C T
Keywords:
The introduction of heavy atoms such as iodine into organic dye molecules is known to improve the efficacy of
photodynamic therapy (PDT) in general and antimicrobial photodynamic therapy (APDT) in particular. Such a
phenomenon is attributed to the increasing probability of spin–orbit coupling resulting in the elevated rates of
reactive species generation. In this work, we synthesize a series of novel, near-IR, iodinated heptamethine
cyanine dyes containing carboxylic function and report on the unexpected effect of the increasing number of
iodine atoms (up to six) on the photodynamic eradication of Gram-positive (Staphylococcus aureus) and Gram-
negative (Escherichia coli and Pseudomonas aeruginosa) microbial pathogens. The efficacy of S. aureus photo-
eradication by non-charged, zwitterionic cyanines increases with increasing the number of iodine atoms up to
two, remains almost unchanged for the two-, three- and four-iodinated dyes, and reduces in the case of the hexa-
iodinated cyanine. However, the mono-iodinated dye exhibits the most pronounced phototoxic effect to E. coli
and P. aeruginosa. An additional positive charge provided by a triethylammonium group decreases photokilling of
S. aureus but improves inactivation of E. coli and P. aeruginosa.
Iodinated cyanines
Photosensitizers
Antimicrobial photodynamic therapy
Aggregation
1. Introduction
theranostic applications [17]. The chemical structures of cyanines can
be relatively easy modified to adjust their absorption and emission
Organic photosensitizing dyes are used in photodynamic antimi-
crobial therapy (APDT) [1], also called photodynamic antimicrobial
chemotherapy (PACT) [2], for killing pathogenic bacteria [3], as well as
in photodynamic therapy (PDT) of cancer [4,5] and for eradication of
viruses [6] and fungi [7]. The clinically approved photosensitizers are in
general porphyrins, which absorb light in the short wavelength range
and to a lesser extent in the red and near-IR spectrum region [8,9].
Among the most potent non-porphyrin based photosensitizers are
phthalocyanines [10–12] and cyanines [13,14]. One example of cyanine
based photosensitizers is indocyanine green (ICG) approved by the U.S.
Food and Drug Administration (FDA) and European Medicines Agency
for photodynamic treatment of several kinds of cancer [15,16].
maxima, solubility, hydrophobic-hydrophilic properties, and to intro-
duce reactive groups facilitating further linkage to biomolecules and
target specific carriers [18]. Some cyanines were reported to exhibit a
pronounced phototoxicity [19]. In recent years, there has been growing
interest, therefore, to cyanine dyes as potential photosensitizers [20].
The introduction of heavy atoms such as iodine into the organic dye
molecules is known to improve efficacy of PDT [21,22] and APDT
[23–25], which is attributed to the increasing probability of spin-orbit
coupling and triplet state population resulting in the elevated rates of
reactive species generation [26,27]. This effect has recently been noted
for cyanine dyes [28,29] among others [30].
Here, we synthesize a series of novel iodinated heptamethine cyanine
dyes containing carboxylic groups (Fig. 1) that can be further utilized for
binding to target-specific carriers. These dyes are investigated for
photodynamic eradication of representative Gram-positive (Staphylo-
coccus aureus) and Gram-negative (Escherichia coli and Pseudomonas
Due to the high extinction coefficients (up to 250,000 Mꢀ 1cmꢀ 1
)
within the optical therapeutic window, the long-wavelength absorbing
and emitting cyanine dyes, in particular heptamethine cyanines are
considered promising tools for bioimaging, photodynamic therapy, and
* Corresponding author. Department of Chemical Sciences, Ariel University, Ariel, 40700, Israel.
E-mail addresses: olga.semenova@isc.kh.ua (O. Semenova), kobzev@isc.kh.ua (D. Kobzev), faresy@ariel.ac.il (F. Yazbak), fainan@ariel.ac.il (F. Nakonechny),
kolosova@isc.kh.ua (O. Kolosova), tatarets@isc.kh.ua (A. Tatarets), garyg@ariel.ac.il (G. Gellerman), leonidpa@ariel.ac.il (L. Patsenker).
https://doi.org/10.1016/j.dyepig.2021.109745
Received 8 July 2021; Received in revised form 21 August 2021; Accepted 22 August 2021
Available online 26 August 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
I–]+ 551.82, found m/z 551.80.
5-Iodo-2,3,3-trimethyl-1-(3-(triethylammonio)propyl)-3H-
indol-1-ium dibromide (1d): 5-Iodo-2,3,3-trimethyl-3H-indole (1.0 g,
3.5 mmol) and 3-bromo-N,N,N-triethylpropan-1-aminium bromide
(1.23 g, 3.9 mmol) were stirred in acetonitrile (14 mL) at reflux for 2
days. Reaction mixture was diluted with benzene (50 mL). In 1 h the
solvent was decanted and the viscous solid was triturated with diethyl
ether, filtered, washed with ether, and dried to yield 1d (0.6 g, 29%). 1H
NMR (400 MHz, DMSO‑d6, ppm): δ 8.33 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H),
7.95 (d, J = 7.6 Hz, 1H), 4.56 (t, J = 7.7 Hz, 2H), 3.25 (m, 8H), 2.91 (s,
3H), 2.18 (m, 2H), 1.56 (s, 6H), 1.18 (t, J = 7.3 Hz, 9H). MS m/z (ESI+)
C
20H33Br2IN2 calculated [M ꢀ 2Br]+ 428.17, found m/z 428.20.
1-(5-Carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium bromide
(3a): 2,3,3-Trimethyl-3H-indole (1.0 g, 6.3 mmol) was mixed with 6-
bromohexanoic acid (1.84 g, 9.4 mmol) and heated at 120 ◦C for 15 h
in a sealed tube. The reaction mixture was cooled to RT, diluted with
benzene (5 mL), the solvent was decanted, and the residue was tritu-
rated with benzene (3 × 5 mL), and filtered. The obtained precipitate
was washed with acetone (1 mL) and dried to yield 3a (1.33 g, 60%). 1H
NMR (400 MHz, DMSO‑d6, ppm): δ 7.97 (d, J = 5.1 Hz, 1H), 7.84 (d, J =
3.4 Hz, 1H), 7.62 (t, J = 4.1 Hz, 2H), 4.45 (t, J = 7.8 Hz, 2H), 2.84 (s,
3H), 2.23 (t, J = 7.2 Hz, 2H), 1.85 (m, 2H), 1.56 (m, 2H), 1.54 (s, 6H),
1.43 (m, 2H). MS m/z (ESI+) C17H24BrNO2 calculated [M ꢀ Br]+ 274.18,
found m/z 274.20.
Fig. 1. Heptamethine cyanine dyes synthesized and investigated in this work.
aeruginosa) pathogens in comparison with the non-iodinated analog,
Cy7.
1-(5-Carboxypentyl)-5-iodo-2,3,3-trimethyl-3H-indol-1-ium
bromide (3b) was obtained by the same procedure as for 3a starting
from 5-iodo-2,3,3-trimethyl-3H-indole (1.0 g, 3.5 mmol) and 6-bromo-
hexanoic acid (1.37 g, 7.0 mmol) by heating at 90 ◦C for 2 days in a
sealed tube. Yield: 0.81 g (48%). 1H NMR (400 MHz, DMSO‑d6, ppm): δ
8.31 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 4.43 (t, J
= 7.7 Hz, 2H), 2.82 (s, 3H), 2.20 (t, J = 6.7 Hz, 2H), 1.82 (m, 2H), 1.53
(s, 6H), 1.42 (m, 2H), 1.34 (m, 2H). MS m/z (ESI+) C17H23BrINO2
calculated [M ꢀ Br]+ 400.08, found m/z 400.12.
2. Experimental
2.1. General
All chemicals were supplied by Alfa Aesar Israel and Sigma-Aldrich.
Solvents were purchased from Bio-Lab Israel and used as is. Chemical
reactions were monitored by TLC (Silica gel 60 F-254, Merck) and LC/
MS.
LC/MS analysis was performed using an Agilent Technologies 1260
Infinity (LC) 6120 quadrupole (MS), column Agilent Zorbax SB-C18, 1.8
mm, 2.1 × 50 mm, column temperature 50 ◦C, eluent water-acetonitrile
(ACN) + 0.1% formic acid.
1-(5-Carboxypentyl)-4,5,6-triiodo-2,3,3-trimethyl-3H-indol-1-
ium bromide (3c) was obtained by the same procedure as for 3a
starting from 4,5,6-triiodo-2,3,3-trimethyl-3H-indole (1.0 g, 1.9 mmol)
and 6-bromohexanoic acid (0.73 g, 3.7 mmol) by heating at 90 ◦C for 3
days in a sealed tube. Yield 0.4 g (29%). 1H NMR (400 MHz, DMSO‑d6,
ppm): δ 8.60 (s, 1H), 4.41 (t, J = 7.7 Hz, 2H), 2.83 (s, 3H), 2.20 (t, J =
6.7 Hz, 2H), 1.79 (m, 2H), 1.60 (s, 6H), 1.51 (m, 2H), 1.44 (m, 2H). MS
m/z (ESI+) C17H21BrI3NO2 calculated [M ꢀ Br]+ 651.87, found m/z
651.90.
HRMS was performed in ESI positive mode by using an Agilent 6550
iFunnel Q-TOF LC/MS instrument.
1H NMR and 13C NMR spectra were recorded on a Bruker Avance III
HD (1H 400 MHz and 13C 100 MHz) spectrometer and a BBO probe
equipped with a Z gradient coil. 1H NMR spectra of quaternized indo-
lenines 1a-1d and 3a-3c (all of them were recently reported) were
measured in DMSO‑d6 at 300K. 1H NMR and 13C NMR spectra of hep-
tamethine cyanine dyes Cy7, 1ICy7–6ICy7 were measured in a CDCl3
(80%) – CD3OD (20%) mixture at 275K.
2.2.1. General procedure for the synthesis of the dyes Cy7, 1ICy7–6ICy7
A solution of indolenine 1a–1d (0.25 mmol, 1.1 equiv.) and N-[5-
(phenylamino)-2,4-pentadienylidene]aniline
hydrochloride
(0.23
2.2. Synthesis
mmol, 1.0 equiv.) in acetic anhydride (2 mL) was heated at 90 ◦C for 15
min to form the corresponding intermediate product 2a–2d. Then, the
second indolenine 3a–3c (0.25 mmol, 1.1 equiv.) was added and dis-
solved in the reaction mixture at 90 ◦C. The reaction mixture was then
cooled to 50 ◦C, pyridine (1 mL) was added, heated at 90 ◦C for 5 min,
and the obtained dye was precipitated with ether, filtered, and washed
with ether. The raw product was column purified on Silica gel 60 using
5–10% methanol–chloroform as eluent.
1,2,3,3-Tetramethyl-3H-indol-1-ium iodide (1a), 5-iodo-1,2,3,3-
tetramethyl-3H-indol-1-ium iodide (1b) and 4,5,6-triiodo-1,2,3,3-
tetramethyl-3H-indol-1-ium iodide (1c) were synthesized by the
reported procedures [9,10].
1,2,3,3-Tetramethyl-3H-indol-1-ium iodide (1a): Yield: 74%. 1
H
NMR (400 MHz, DMSO‑d6, ppm): δ 7.91 (d, J = 5.2 Hz, 1H), 7.83 (d, J =
5.4 Hz, 1H), 7.62 (t, J = 3.6 Hz, 2H), 3.98 (s, 3H), 2.77 (s, 3H), 1.53 (s,
6H). MS m/z (ESI+) C12H16IN calculated [M-I–]+ 174.13, found m/z
174.10.
6-(3,3-Dimethyl-2-(7-(1,3,3-trimethylindolin-2-ylidene)hepta-
1,3,5-trien-1-yl)-3H-indol-1-ium-1-yl)hexanoate (Cy7). Dye Cy7 was
synthesized from 1,2,3,3-tetramethyl-3H-indol-1-ium iodide (1a) (75
mg, 0.25 mmol), N-[5-(phenylamino)-2,4-pentadienylidene]aniline hy-
drochloride (64 mg, 0.23 mmol), and 1-(5-carboxypentyl)-2,3,3-tri-
methyl-3H-indol-1-ium bromide (3a) (88.5 mg, 0.25 mmol). Yield: 119
mg (52%). 1H NMR (400 MHz, CDCl3–CD3OD, ppm): δ 7.71 (t, J = 13.1
Hz, 2H), 7.42 (t, J = 12.7 Hz, 1H), 7.40–7.34 (m, 2H), 7.32 (d, J = 7.5
Hz, 2H), 7.18 (t, J = 7.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 1H), 7.06 (d, J =
8.4 Hz, 1H), 6.47 (t, J = 12.5 Hz, 2H), 6.08 (d, J = 13.4 Hz, 1H), 6.05 (d,
J = 13.6 Hz, 1H), 3.92 (t, J = 7.4 Hz, 2H), 3.53 (s, 3H), 2.27 (t, J = 7.2
5-Iodo-1,2,3,3-tetramethyl-3H-indol-1-ium iodide (1b): Yield:
73%. 1H NMR (400 MHz, DMSO‑d6, ppm): δ 8.27 (s, 1H), 7.99 (d, J =
8.5 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 3.93 (s, 3H), 2.73 (s, 3H), 1.51 (s,
6H). MS m/z (ESI+) C12H15I2N calculated [M-I–]+ 300.02, found m/z
300.16.
4,5,6-Triiodo-1,2,3,3-tetramethyl-3H-indol-1-ium iodide (1c):
Yield: 43%. 1H NMR (400 MHz, DMSO‑d6, ppm): δ 8.52 (s, 1H), 3.92 (s,
3H), 2.76 (s, 3H), 1.59 (s, 6H). MS m/z (ESI+) C12H13I4N calculated [M-
2

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
Hz, 2H), 1.84–1.67 (m, 2H), 1.62 (s, 12H), 1.67–1.53 (m, 2H), 1.50–1.35
(m, 2H). 13C NMR (100 MHz, CDCl3–CD3OD, ppm): δ 175.63 (COOH),
171.14 (CN Ind), 170.56 (CN Ind) 155.75 (CH), 150.53 (2CH), 141.67
(Ar), 141.00 (Ar), 139.80 (Ar), 139.63 (Ar), 127.84 (2CH Ar), 124.76
(2CH), 124.26 (2CH Ar), 121.36 (CH Ar(, 121.23 (CH Ar), 109.50 (CH
Ar), 109.40 (CH Ar), 102.68 (CH), 102.42 (CH), 48.47 (2C(CH3)2), 43.09
(CH2), 32.98 (CH2), 30.23 (CH3), 26.83 (4CH3), 26.19 (CH2), 25.45
(CH2), 23.58 (CH2). HRMS m/z (ESI+) C34H40N2O2 calculated [M+H]+
509.3090 (509.3163), found m/z: 509.3168.
7.0 Hz, 2H), 3.95 (t, J = 6.8 Hz, 2H), 3.43 (t, J = 7.9, 2H), 3.26 (m, 6H)
2.30 (t, J = 7.2 Hz, 2H,), 2.04 (m, 2H), 1.77 (m, 2H), 1.66 (m, 2H), 1.64
(s, 6H), 1.61 (s, 6H), 1.45 (m, 2H), 1.27 (t, J = 7.1 Hz, 9H). HRMS m/z
(ESI+) C44H56F3I2N3O4 calculated [M–CF3COO]+ 888.2451 (888.2445),
found m/z: 888.2462.
6-(3,3-Dimethyl-2-(7-(4,5,6-triiodo-1,3,3-trimethylindolin-2-
ylidene)hepta-1,3,5-trien-1-yl)-3H-indol-1-ium-1-yl)hexanoate
(3ICy7). Dye 3ICy7 was synthesized from 4,5,6-triiodo-1,2,3,3-tetra-
methyl-3H-indol-1-ium iodide (1c) (170 mg, 0.25 mmol), N-[5-(phe-
nylamino)-2,4-pentadienylidene]aniline hydrochloride (65 mg, 0.23
mmol), and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium bro-
mide (3a) (88.0 mg, 0.25 mmol). Yield: 75 mg (37%). 1H NMR (400
MHz, CDCl3–CD3OD, ppm): δ 7.82 (t, J = 12.4 Hz, 1H), 7.54 (t, J = 13.0
Hz, 1H), 7.50 (s, 1H), 7.40 (d, J = 7.7 Hz, 2H), 7.40 (t, J = 12.6 Hz, 1H),
7.31 (t, J = 7.6 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 6.59 (t, J = 12.6 Hz,
1H), 6.43 (t, J = 12.5 Hz, 1H), 6.36 (d, J = 13.5 Hz, 1H), 5.77 (d, J =
13.1 Hz, 1H), 4.07 (t, J = 7.2 Hz, 2H), 3.31 (s, 3H), 2.22 (t, J = 7.2 Hz,
2H), 1.79 (m, 2H), 1.69 (s, 6H), 1.65 (s, 6H), 1.64 (m, 2H), 1.45 (m, 2H).
13C NMR (100 MHz, CDCl3–CD3OD, ppm): δ 177.20 (COOH), 174.33
(CN Ind), 166.58 (CN Ind), 155.22 (CH), 153.27 (CH), 146.81 (CH),
144.47 (Ar), 141.28 (Ar), 140.59 (Ar), 140.34 (Ar), 128.28 (CH Ar),
126.34 (CH), 126.11 (CH), 125.57 (CH Ar), 121.54 (CH Ar), 118.83
(2CH Ar), 116.77 (CI Ar), 111.04 (2CI Ar), 105.96 (CH), 101.98 (CH),
50.51 (C(CH3)2), 49.30 (C(CH3)2), 44.03 (CH2), 34.28 (CH2), 28.73
(CH3), 26.58 (CH2), 26.50 (2CH3), 25.42 (CH2), 23.98 (CH2), 23.29
(2CH3). HRMS m/z (ESI+) C34H37I3N2O2 calculated [M+H]+ 886.9989
(887.0062), found m/z: 887.0067.
6-(2-(7-(5-iodo-1,3,3-trimethylindolin-2-ylidene)hepta-1,3,5-
trien-1-yl)-3,3-dimethyl-3H-indol-1-ium-1-yl)hexanoate (1ICy7).
Dye 1ICy7 was synthesized from 5-iodo-1,2,3,3-tetramethyl-3H-indol-1-
ium iodide (1b) (106.75 mg, 0.25 mmol), N-[5-(phenylamino)-2,4-
pentadienylidene]aniline hydrochloride (64 mg, 0.23 mmol), and 1-(5-
carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium bromide (3a) (88.5 mg,
0.25 mmol). Yield: 74 mg (45%). 1H NMR (400 MHz, CDCl3–CD3OD,
ppm): δ 7.76 (t, J = 13.1 Hz, 1H), 7.61 (t, J = 13.1 Hz, 1H), 7.59 (d, J =
8.5 Hz, 1H), 7.54 (s, 1H), 7.40 (t, J = 12.7 Hz, 1H), 7.37 (t, J = 7.9 Hz,
1H), 7.36 (d, J = 7.8 Hz, 1H), 7.25 (t, J = 7.4 Hz, 1H), 7.15 (d, J = 7.9
Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.53 (t, J = 13.3 Hz, 1H), 6.44 (t, J =
13.3 Hz, 1H), 6.20 (d, J = 13.5 Hz, 1H), 5.92 (d, J = 13.3 Hz, 1H), 3.99
(t, J = 7.4 Hz, 2H), 3.42 (s, 3H), 2.24 (t, J = 7.2 Hz, 2H), 1.77 (m, 2H),
1.64 (m, 2H), 1.63 (s, 6H), 1.58 (s, 6H), 1.44 (m, 2H). 13C NMR (100
MHz, CDCl3–CD3OD, ppm): δ 176.41 (COOH), 172.48 (CN Ind), 168.14
(CN Ind), 155.64 (CH), 152.05 (2CH), 148.66 (CH), 141.83 (Ar), 141.46
(Ar), 140.63 (Ar), 140.16 (Ar), 136.45 (CH Ar), 130.15 (CH Ar), 128.06
(CH Ar), 125.46 (CH Ar), 125.20 (CH), 122.42 (CH Ar), 110.53 (CH Ar),
110.27 (CH Ar), 104.14 (CH), 101.49 (CH), 86.19 (CI Ar), 48.73 (C
(CH3)2), 47.05 (C(CH3)2), 43.54 (CH2), 33.74 (CH2), 29.95 (CH3), 26.86
(2CH3), 26.67 (2CH3), 26.35 (CH2), 25.42 (CH2), 23.80 (CH2). HRMS m/
z (ESI+) C34H39IN2O2 calculated [M+H]+ 635.2056 (635.2129), found
m/z: 635.2134.
6-(5-Iodo-3,3-dimethyl-2-(7-(4,5,6-triiodo-1,3,3-trimethylindo-
lin-2-ylidene)hepta-1,3,5-trien-1-yl)-3H-indol-1-ium-1-yl)hex-
anoate (4ICy7). Dye 4ICy7 was synthesized from 4,5,6-triiodo-1,2,3,3-
tetramethyl-3H-indol-1-ium iodide (1c) (170 mg, 0.25 mmol), N-[5-
(phenylamino)-2,4-pentadienylidene]aniline hydrochloride (65 mg,
0.23 mmol), and 1-(5-carboxypentyl)-5-iodo-2,3,3-trimethyl-3H-indol-
1-ium bromide (3b) (120 mg, 0.25 mmol). Yield: 116 mg (50%). 1H
NMR (400 MHz, CDCl3–CD3OD, ppm): δ 7.78 (t, J = 12.8 Hz, 1H), 7.68
(d, J = 8.3 Hz, 1H), 7.66 (s, 1H), 7.63 (t, J = 13.0 Hz, 1H), 7.57 (s, 1H),
7.43 (t, J = 12.7 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.58 (t, J = 12.6 Hz,
1H), 6.48 (t, J = 12.6 Hz, 1H), 5.23 (d, J = 13.4 Hz, 1H), 5.88 (d, J =
13.3 Hz, 1H), 3.98 (t, J = 7.8 Hz, 2H), 3.37 (s, 3H), 2.24 (t, J = 7.2 Hz,
2H), 1.74 (m, 2H), 1.70 (s, 6H), 1.62 (s, 6H), 1.52 (m, 2H), 1.42 (m, 2H).
13C NMR (100 MHz, CDCl3–CD3OD, ppm): δ 177.01 (COOH), 172.11
(CN Ind), 168.49 (CN Ind), 156.17 (CH), 152.62 (CH), 148.84 (CH),
144.13 (Ar), 142.34 (Ar), 141.45 (Ar), 140.42 (Ar), 137.03 (CH Ar),
130.63 (CH Ar), 126.44 (2CH), 119.32 (CH Ar), 118.06 (CI Ar), 112.19
(CH Ar), 105.56 (CI Ar), 104.78 (CH), 102.10 (CI Ar), 101.18 (CH),
89.21 (CI Ar), 50.99 (C(CH3)2), 48.72 (C(CH3)2), 43.70 (CH2), 33.48
(CH2), 28.42 (CH3), 26.60 (2CH3), 26.38 (CH2), 25.32 (CH2), 23.66
(CH2), 23.16 (2CH3). HRMS m/z (ESI+) C34H36I4N2O2 calculated
[M+H]+ 1012.8956 (1012.9028), found m/z: 1012.9034.
6-(5-Iodo-2-(7-(5-iodo-1,3,3-trimethylindolin-2-ylidene)hepta-
1,3,5-trien-1-yl)-3,3-dimethyl-3H-indol-1-ium-1-yl)hexanoate
(2ICy7). Dye 2ICy7 was synthesized from 5-iodo-1,2,3,3-tetramethyl-
3H-indol-1-ium iodide (1b) (106.5 mg, 0.25 mmol), N-[5-(phenyl-
amino)-2,4-pentadienylidene]aniline hydrochloride (64 mg, 0.23
mmol), and 1-(5-carboxypentyl)-5-iodo-2,3,3-trimethyl-3H-indol-1-ium
bromide (3b) (120 mg, 0.25 mmol). Yield: 65 mg (38%). 1H NMR (400
MHz, CDCl3–CD3OD, ppm): δ 7.71 (t, J = 13.0 Hz, 2H), 7.63 (d, J = 8.3
Hz, 2H), 7.60 (s, 2H), 7.45 (t, J = 12.7 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H),
6.85 (d, J = 8.1 Hz, 1H), 6.51 (t, J = 12.1 Hz, 2H), 6.08 (d, J = 12.7 Hz,
1H), 6.06 (d, J = 13.5 Hz, 1H), 3.90 (t, J = 7.2 Hz, 2H), 3.49 (s, 3H), 2.23
(t, J = 7.2 Hz, 2H), 1.72 (m, 2H), 1.64 (m, 2H), 1.60 (s, 12H), 1.42 (m,
2H). 13C NMR (100 MHz, CDCl3–CD3OD, ppm): δ 176.74 (COOH),
170.26 (CN Ind), 169.74 (CN Ind), 156.26 (CH), 150.81 (CH), 150.69
(CH), 142.00 (Ar), 141.75 (Ar), 141.53 (Ar), 140.86 (Ar), 136.73 (2CH
Ar), 130.45 (CH Ar), 130.31 (CH Ar), 125.65 (2CH), 111.38 (CH Ar),
111.25 (CH Ar), 103.08 (CH), 102.86 (CH), 87.63 (CI Ar), 88.52 (CI Ar),
48.2 (2C(CH3)2), 43.18 (CH2), 33.48 (CH2), 30.34 (CH3), 26.78 (4CH3),
26.12 (CH2), 25.43 (CH2), 23.73 (CH2). HRMS m/z (ESI+) C34H38I2N2O2
calculated [M+H]+ 761.1023 (761.1095), found m/z: 761.1101.
6-(5-Iodo-2-(7-(5-iodo-3,3-dimethyl-1-(3-(triethylammonio)
propyl)indolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3,3-dimethyl-3H-
indol-1-ium-1-yl)hexanoate trifluoroacetate (2ICy7þ). Dye 2ICy7þ
was synthesized from 5-iodo-2,3,3-trimethyl-1-(3-(triethylammonio)
propyl)-3H-indol-1-ium dibromide (1d) (147 mg, 0.25 mmol), N-[5-
(phenylamino)-2,4-pentadienylidene]aniline hydrochloride (65 mg,
0.23 mmol), and 1-(5-carboxypentyl)-5-iodo-2,3,3-trimethyl-3H-indol-
1-ium bromide (3b) (120 mg, 0.25 mmol). The product was purified on a
RP18 column (acetonitrile–water + 0.05% TFA). Yield: 34 mg (15%). 1H
NMR (400 MHz, CDCl3–CD3OD, ppm): δ 7.74 (t, J = 13.0 Hz, 1H), 7.70
(d, J = 8.3 Hz, 1H), 7.68 (t, J = 13.0 Hz, 1H), 7.66 (s, 1H), 7.64 (d, J =
8.1 Hz, 1H), 7.59 (s, 1H), 7.40 (t, J = 12.8 Hz, 1H), 7.01 (d, J = 8.3 Hz,
1H), 6.91 (d, J = 8.3 Hz, 1H), 6.60 (t, J = 12.7 Hz, 1H), 6.55 (t, J = 12.8
Hz, 1H), 6.28 (d, J = 13.2 Hz, 1H), 6.15 (d, J = 13.5 Hz, 1H), 4.08 (t, J =
6-(4,5,6-Triiodo-3,3-dimethyl-2-(7-(4,5,6-triiodo-1,3,3-trime-
thylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3H-indol-1-ium-1-yl)
hexanoate (6ICy7). Dye 6ICy7 was synthesized from 4,5,6-triiodo-
1,2,3,3-tetramethyl-3H-indol-1-ium iodide (1c) (170 mg, 0.25 mmol),
N-[5-(phenylamino)-2,4-pentadienylidene]aniline hydrochloride (65
mg, 0.23 mmol), and 1-(5-carboxypentyl)-4,5,6-triiodo-2,3,3-trimethyl-
3H-indol-1-ium bromide (3c) (183 mg, 0.25 mmol). Yield: 78 mg (27%).
1H NMR (400 MHz, CDCl3–CD3OD, ppm): δ 7.73 (t, J = 13.0 Hz, 2H),
7.65 (s, 1H), 7.62 (s, 1H), 7.45 (t, J = 12.6 Hz, 1H), 6.57 (m, 2H), 6.06
(d, J = 13.5 Hz, 2H), 3.86 (t, J = 7.6 Hz, 2H), 3.47 (s, 3H), 2.26 (t, J =
7.1 Hz, 2H), 1.72 (s, 12H), 1.68 (m, 2H), 1.63 (m, 2H), 1.42 (m, 2H). 13
C
NMR (100 MHz, CDCl3–CD3OD, ppm): 177.05 (COOH), 170.80 (2CN
Ind), 156.82 (CH), 151.90 (CH), 151.50 (CH), 143.68 (Ar), 142.93 (Ar),
141.94 (Ar), 141.70 (Ar), 120.03 (2CH), 119.95 (2CH Ar), 119.54 (2CI
Ar), 105.95 (CI Ar), 105.75 (CI Ar), 102.86 (2CI Ar), 102.45 (CH),
102.22 (CH), 51.72 (C(CH3)2), 51.57 (C(CH3)2), 43.08 (CH2), 33.57
(CH2), 28.72 (CH3), 25.81 (CH2), 25.22 (CH2), 23.68 (CH2), 23.01
3

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
(4CH3). HRMS m/z (ESI+) C34H34I6N2O2 calculated [M+H]+ 1264.6888
(1264.6961), found m/z: 1264.6967.
All preparatory operations with photosensitizers were carried out in
the dark to avoid their activation and photobleaching. The stock solu-
tions of the dyes in DMSO (3–8 mM, spectrophotometrical control by
known dilutions) were prepared and the desirable final concentrations
were prepared in up to three dilutions. Then, each dye solution in DMSO
2.3. Absorption and fluorescence measurements
(7 μL) was added to bacterial suspensions (1 mL) in 0.9% saline (Fal-
The absorption spectra were recorded on a Jasco V-730 UV–Vis
spectrophotometer and the fluorescence spectra were taken on an
Edinburgh FS5 spectrofluorometer. The absorption and fluorescence
con® 24-well polystyrene clear flat bottom plate was used). Thus, the
amount of DMSO added to the bacterial suspensions was always 0.7%
(7% was used in several experiments, as noted below). The bacterial
suspensions were then incubated in the dark at RT for 30 min and then
exposed to light with shaking (or kept in the dark for the control) for
certain periods of time according to the experimental conditions. The
light exposure was carried out by a 730-nm, 30 W LED equipped with a
60◦ lens from the distance of 8 cm (light power density 56 mW/cm2).
spectra were measured at 25 ◦C in standard 1-cm quartz cells at ~1
μM
dye concentrations in DMSO and 0.9% aqueous saline. The excitation
wavelength (λ*) was 680 nm.
To determine the absolute fluorescence quantum yields (FF), the
integrated relative intensities of the dyes were measured vs. the
commercially available disulfonated Cy7 (Zy7, SETA BioMedicals, htt
ps://www.setabiomedicals.com) in phosphate buffer pH 7.4 as the
reference (FF = 13%) [8]; and the quantum yields were calculated ac-
cording to Equation (1) [31].
After the light exposure, aliquots of each sample (100
μL) were
spread over BHA plates with a Drigalsky spreader, incubated at 37 ◦C for
24 h, and the colony forming units (CFU) were counted using a colony
`
counter Scan 500 (Interscience, Saint-Nom-la-Breteche, France).
ΦF = ΦFRef × (F / FRef) × (ARef / A) × (nD(media)2 / nD(Ref)2),
(1)
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 bacteria without dye: (i) in the dark and without DMSO,
(ii) in the dark in the presence of DMSO, (iii) exposed to light without
DMSO, and (iv) exposed to light in the presence of DMSO.
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-
sorbances at the excitation wavelength of the reference dye and the dye
under examination, and nD(Ref) and nD(media) are the refractive indices of
the solvents used for the reference dye and the dye under examination.
The quantum yield for each dye was independently measured three
times and the average value was taken.
All the experiments with bacteria were carried out in triplicate 4–5
times in different days and the average values were taken.
3. Results and discussion
3.1. Overview of the dye structures
2.4. Quantum yield of singlet oxygen formation
We synthesized a series of indolenine based heptamethine cyanine
dyes of asymmetric structure containing no iodine (Cy7) as well as one
(1ICy7), two (2ICy7), three (3ICy7), four (4ICy7) and six (6ICy7)
iodine atoms. These dyes possess a carboxypentyl group attached to the
nitrogen of one indolenine moiety and methyl group at the second
indolenine nitrogen. When the carboxylic group is deprotonated, these
dyes exist in zwitterionic form. Furthermore, one of the dyes (2ICy7þ)
bears an additional positive charge provided by the quaternized propyl
triethylaminium group substituting N-methyl.
The quantum yields of the singlet oxygen formation were measured
according to the known procedure [32]. A solution of 1,3-diphenyliso-
benzofuran (DPBF, c ~ 1.4 × 10ꢀ 5 M) and a dye under investigation
(c ~ 5ꢀ 7 × 10ꢀ 6 M) in methanol was prepared. The obtained solution
(3.0 mL) was light irradiated by a 747 nm 1 W LED in a standard 1 cm
quartz cell with stirring (LED was located in 1 mm distance from the cell
wall) and the absorption spectra were recorded over time. The total
irradiation time was in the range of 20–120 min. During this time the
absorbance of DPBF reduced to about 10% of its initial value. The cor-
responding plot representing the absorbance of DPBF at 411 nm versus
time was drawn and fitted by first-order exponential decay function.
Then, the singlet oxygen formation quantum yield ΦΔ was calculated
relative to 1,1′,3,3,3′,3′-hexamethylindotricarbocyanine iodide (HITC)
(ΦΔ = 0.0089 [32]) according to Equation (2).
3.2. Synthesis
All the dyes were synthesized by the same approach consisted in a
one-pot sequential reaction of N-[5-(phenylamino)-2,4-pentadienyli-
dene]aniline hydrochloride with the first and then the second quater-
nized indolenine (Scheme 1). In the first step, the starting aniline was
condensed in acetic anhydride with the first indolenine molecule 1a–1d
to form a corresponding N-phenylacetamide derivative 2a–2d, which
was further reacted with the second molecule, N-carboxypentyl indo-
lenine 3a–3c in the presence of pyridine to give the dyes Cy7, 1ICy7-
–6ICy7, and 2ICy7þ, respectively, in moderate yields (15–52%).
ΦΔ = ΦΔRef × (k / kRef) × (ARef / A),
(2)
where ΦΔRef is the quantum yield of the singlet oxygen formation for the
reference dye (HITC), kRef and k are the rates of DPBF degradation ob-
tained from the corresponding fitting curves of the reference dye and the
dye under examination, and ARef and A are the absorbances at the
excitation wavelength (747 nm) of the reference dye and the dye under
examination. Each experiment was carried out in triplicate and the
average ΦΔ was taken. The reproducibility in the determination of ΦΔ
was no worse that 5%.
3.3. Spectral properties and quantum yields of singlet oxygen formation
The absorption and emission spectra and the extinction coefficients
(ε) of the obtained heptamethine cyanines were measured at the dye
2.5. Antimicrobial studies
concentration cDye ~ 1 μM in DMSO and aqueous saline (Figs. S1 and S2)
and the corresponding characteristics are given in Table 1. These dyes
absorb in the near-IR, biologically transparent spectral region with the
Cultures of S. aureus (ATCC 25923), E. coli (ATCC 25922) and
P. aeruginosa (ATCC 25668) were 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 = 0.10 ± 0.02 at
660 nm, which corresponded to a final concentration of 108 cells/mL,
and diluted with commercially available sterile 0.9% saline solution to
the final concentration of 103–104 cells/mL.
high extinction coefficients (
ε
~ 144,000–176,000 Mꢀ 1cmꢀ 1), which is
beneficial for applications in the body. The difference in the extinction
coefficients between the dyes does not exceed ~20%. There is a ten-
dency that the iodination of the parent dye Cy7 results in a moderate
red-shift in the absorption (up to 18 nm for hexa-iodinated 6ICy7 in
DMSO) and a slight increase in the extinction coefficients.
The fluorescence quantum yield (ΦF) of the dyes measured in DMSO
4

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
Scheme 1. Synthesis of heptamethine cyanine dyes.
Table 1
Spectral characteristics of cyanine dyes measured at cDye ~1 μM.
Dye
Structure
DMSO
0.7% DMSO in saline
MeOH
λmaxAb, nm
ε
,
λmaxFl, nm
ΦF, %
λmaxAb,nm
λmaxFl, nm
ΦF, %
ΦΔ,
Mꢀ 1cmꢀ 1
143,300
%
Cy7
753
786
55
739
768
13
1.1
1ICy7
759
168,000
792
53
746
776
12
1.9
2ICy7
766
766
173,000
176,000
801
801
55
48
754
752
783
785
4
2.3
2.3
2ICy7þ
10
3ICy7
760
148,000
794
49
677a, 744
774
0.8
2.4
4ICy7
6ICy7
768
771
144,000
166,000
803
804
48
52
688a
779
779
0.1
3.7
7.8
718a, 777a
0.02
a
Aggregation band.
(~48–55%) is substantially reduced in aqueous saline (0.02–13%),
which can be attributed to the dye aggregation [33–35]. The aggrega-
tion bands are well recognized in the absorption spectra (Fig. S2). In the
saline solutions, a drastic (~650-fold) decrease of the quantum yield
with increasing the number of iodine atoms is observed. In general, such
an effect is known to be connected with the increasing probability of
5

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
spin–orbit coupling caused by heavy atoms, which results in the growing
population of the triplet state and the fluorescence quenching. However,
for the investigated cyanine dyes, the decrease of the quantum yield is
observed only in the aqueous solutions but not in DMSO. That is, the
decrease is associated with the dye aggregation rather than with the
heavy atom effect.
were not studied. In the next step, the experiments were performed with
light irradiation at different exposure time using a 730-nm 30 W LED
(power density 55 mW/cm2).
3.4.1. Photodynamic eradication of S. aureus
In our study, the effect on phototoxicity of the three following pa-
rameters was investigated: the number of iodine atoms in the dye mol-
ecules, the concentration of the dyes, and the light doses. The
methodological approach was as follows: The effect of the number of
iodine atoms in the dyes was first investigated on Gram-positive bacteria
S. aureus: (i) at the "medium" (100 J/cm2) and (ii) "low" (3 J/cm2) light
dose vs. the dye concentrations, and (iii) at the constant dye concen-
trations vs. the light dose. The "high" light dose (400 J/cm2) was not
applied in the initial research stage because it caused complete killing of
S. aureus by all the investigated dyes and, therefore, it was not possible
to compare efficacies of these dyes.
All the dyes except 2ICy7þ contain a delocalized positive charge and
a localized negative charge on the deprotonated carboxylic group.
Therefore, due to the slightly polar structure, these dyes are only a little
soluble in saline. The introduction of iodine atoms in the organic com-
pounds is known to reduce their solubility in aqueous media. Thus, the
solubility of dimethylaniline in water at 25 ◦C is 1 g/L [36] while for
4-iodo-dimethylaniline it is 34.64 mg/L [37]. The solubility of benzoic
acid in water is 3.44 g/L [38] while for 4-iodobenzoic acid it is 0.04 g/L
[39] and only 0.036 × 10ꢀ 3 g/L for 2,3,5-triiodobenzoic acid [40].
Hereby, the solubility of the investigated dyes decreases with the
increasing the number of iodine atoms and the aggregation accordingly
increases (Figs. S2 and a). At the same time, due to the presence of an
additional positive charge, dye 2ICy7þ is less aggregative compared to
2ICy7 containing the same number of iodines.
Following this approach, the impact of the number of iodine atoms
and the dye concentration (cDye = 0.01–1 μM) on the S. aureus survival
was first investigated at the constant light dose of 100 J/cm2. The most
pronounced and nearly equal phototoxicity was observed for 2ICy7,
3ICy7 and 4ICy7 that contained 2–4 iodine atoms, while Cy7, 1ICy7,
2ICy7þ, and, surprisingly, 6ICy7 were less effective (Fig. 2). These
results allow us to draw several conclusions.
The quantum yields of the singlet oxygen formation (ΦΔ) was
measured in methanol solutions by the decrease of the DPBF dye, as
described in [32], under the light exposure (747 nm 1 W LED). As
anticipated, the ΦΔ were found to increase with increasing the number
of iodine atoms (Table 1). Dyes 2ICy7 and 2ICy7þ both containing two
iodines exhibited about the same ΦΔ. In the dark, no pronounced gen-
eration of singlet oxygen was detected: the decrease in the DPBF
absorbance did not exceed 2% during 15 h. According to the absorption
spectra (Fig. S3), almost no aggregation was noted in methanol at the
investigated dye concentrations (c ~ 5ꢀ 7 × 10ꢀ 6 M).
First, the introduction of iodine atoms in the Cy7 molecule, which is
the parent structure for all the iodinated dyes, elevates phototoxicity,
which is an anticipated result: one iodine atom in 1ICy7 causes a distinct
increase of the phototoxicity but this increase is less pronounced as for
the 2–4 iodinated dyes (Fig. 2). Thus, the dyes with 2–4 iodines afford
almost total bacteria killing (survival 0–2.1%) at 0.01–1
μM, while
1ICy7 at 0.01 μM is much less phototoxic exhibiting the percentage of
survival of 60.8%. Obviously, this result is connected with increasing the
number of heavy atoms.
3.4. Toxicity and phototoxicity of the dyes
The second conclusion is that increasing the number of iodines from
2 to 4 has almost no effect on the photokilling while the hexa-iodinated
dye 6ICy7 surprisingly exhibits a reduced activity (the percentage of
All experiments with bacteria were carried out in aqueous saline
solution. Commercial 0.9% saline solution, used in this work, is known
to be acidic (pH ~ 5.5), which is mostly due to the presence of CO2 [41].
On the other side, the carboxypentyl group existing in all the investi-
gated dye molecules is more acidic (e.g. the pKa for pentanoic and
hexanoic acids are 4.84 [42] and 4.88 [43], respectively). It means that
the carboxylic group is basically deprotonated and the dyes Cy7, 1ICy7,
2ICy7, 3ICy7, 4ICy7, and 6ICy7 exist in saline solution in the
non-charged zwitterion form. In contrast, 2ICy7þ has a charge of +1
that is localized on the quaternized triethylammonium group.
survival is 35.6% at 0.01
μM) compared to the 2–4 iodinated dyes
(survival 0–2.1%).
Third, the non-delocalized positive charge produced by the triethy-
lammonium group (2ICy7þ vs. 2ICy7) noticeably decreases the
phototoxicity (Fig. 2).
We assume that the above phenomena, i.e. "saturation" and reduc-
tion of the bacteria photo-eradication with increasing the number of
iodine atoms from 2 to 6, can be attributed to the increasing dye ag-
gregation in aqueous media (Fig. S2) resulting in the decreasing dye
uptake (the aggregation reduces both the phototoxicity [17,44] and the
above mentioned fluorescence quantum yields [17,18,19]). This
conclusion contradicts, however, recently reported findings that iodin-
ation of small organic molecules such as 4-amino naphthalimides vice
versa causes the increase of the dye uptake by several mammalian cells
[45].
To investigate the effect of the dyes on bacteria, stock solutions of the
dyes were prepared in DMSO and added to the bacterial suspension in
saline in such a way that the concentration of DMSO in the sample was
0.7%. Then the bacterial suspensions were incubated with each dye in
the dark for 30 min (pre-irradiation incubation), exposed to light and
grown in the dark for 24 h at 37 ◦C followed by the calculation of the
number of bacterial colonies. The dye concentrations and the exposure
time were varied for each dye. To verify the dark toxicity of the dyes, the
same experiments were carried out in parallel without the light
exposure.
To check our assumption that increasing the number of iodine atoms
(as well as the positive charge) in the series of the investigated cyanines
reduces the dye uptake by S. aureus and thus decreases the photokilling
effect, we measured the dye uptake and correlated it with the survival
percentage. For this purpose, S. aureus suspensions in saline (103–104
As the control, the bacterial suspensions containing no dye were
utilized. These controls were kept (i) in the dark without DMSO, (ii) in
the dark in the presence of DMSO, (iii) exposed to light without DMSO,
and (iv) exposed to light in the presence of DMSO. The amount of DMSO
in these control samples was the same as for the dye-stained bacteria.
The number of bacterial colonies in each experiment was utilized to
calculate the survival percentage as compared to the control. Impor-
tantly, no detectable bacteria inhibition was registered for all the above
controls (i–iv) and the survival for these samples was taken as 100%.
Then, we found that the investigated dyes have no detectable dark
toxicity to Gram-positive (S. aureus) and Gram-negative (E. coli and
cells/mL) were incubated with the dyes (1 μM) for 30 min and the
fluorescence intensities of these suspensions were measured. Then, the
bacteria were separated from the solvent by centrifugation (10 min,
4,000 rpm), resuspended in saline and the fluorescence intensities were
again measured. In these experiments, the effect of light scattering on
the relative fluorescence intensities was minimized because the samples
contained the same number of cells before and after staining. The dye
uptakes quantified as the ratios between the fluorescence intensities for
the resuspended bacteria and for the initial suspensions were as follows:
Cy7 (0.85), 1ICy7 (0.28), 2ICy7 (0.98), 2ICy7þ (0.21), 3ICy7 (0.99),
4ICy7 (0.97), and 6ICy7 (0.73). These data indicate that 2ICy7, 3ICy7
P. aeruginosa) bacteria at least up to the 1
μM and 50 μM dye concen-
trations, respectively. The dark toxicities at higher dye concentrations
6

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
Fig. 2. Survival of S. aureus in 0.7% DMSO in saline, when exposed to 100 J/cm2 light dose (56 mW/cm2, 30 min) vs. the dye concentrations.
and 4ICy7 exhibited the most pronounced uptake while uptake for
6ICy7 and especially for 2ICy7þ was noticeably reduced. The uptake of
Cy7 is lower compared to 2ICy7 but much higher than for 1ICy7.
The obtained uptake values are in good agreement with the photo-
toxicities (Fig. 3). Thus, for the iodinated dyes, there is a clear correla-
tion between the uptake and the cell survival (r = 0.98). At the same
time, the non-iodinated dye Cy7 exhibits a substantially reduced
phototoxicity (elevated survival) compared to the value anticipated
from the correlation curve (Fig. 3). More likely, this is due to the fact
that the correlation curve takes into account also the contribution of the
heavy atom effect.
dye concentrations on the phototoxicity at this low light dose. Fig. 5
demonstrates that 0.05 μM is, actually, the minimal concentration at
which 2ICy7 and 3ICy7 almost completely eradicate S. aureus while the
dyes with fewer or more iodine atoms are less phototoxic. Thus, the non-
charged (zwitterionic) dyes with two and three iodine atoms (2ICy7 and
3ICy7) were found to be the most effective against S. aureus. They are
more effective against this pathogen compared to other dyes at low
concentrations and low light doses.
3.4.2. Photodynamic eradication of E. coli and P. aeruginosa
In the next step, we studied phototoxicity of the dyes towards Gram-
negative pathogens E. coli and P. aeruginosa. The data in Figs. 6–10 show
that these bacteria are much more resistant towards photodynamic
treatment compared to the Gram-positive S. aureus. Thus, all the
investigated dyes do not cause a sufficient phototoxic effect to E. coli
Furthermore, we studied the effect of the light dose on the dyes’
phototoxicity towards S. aureus at constant dye concentration of 0.05 μM
(Fig. 4). It can be seen that the phototoxicity increases with increasing
the light dose from 3 J/cm2 (1 min) to 400 J/cm2 (120 min). However,
the dyes with two (2ICy7) and three (3ICy7) iodines almost totally
eradicate bacteria even at 3 J/cm2 and 4ICy7 at 17 J/cm2. The data
presented in Fig. 4 confirm also that the dyes with 2–4 iodines bearing a
delocalized positive charge cause the more pronounced bacteria eradi-
cation while 1ICy7, 6ICy7, 2ICy7þ, and especially Cy7 are less
phototoxic.
even at 50
μ
M and light dose of 100 J/cm2 (Fig. 6). There is a trend,
however, that the mono-iodinated 1ICy7 is more active compared to
other dyes at 100 J/cm2 and this tendency is even more pronounced at
200–400 J/cm2 (Fig. 7). Surprisingly, the dyes containing four and six
iodines are less phototoxic than Cy7. The increase of the positive charge
(2ICy7þ vs. 2ICy7) has almost no effect on the dye phototoxicity at 100
J/cm2 (Fig. 6) but noticeably improves the toxic effect at higher light
dose of 200–400 J/cm2 (Fig. 7). Therefore, the mono-iodinated 1ICy7
and positively charged 2ICy7þ are considered more effective against
E. coli compared to other cyanines.
It can be seen from Fig. 4 that dyes 2ICy7 and 3ICy7 entirely erad-
icate S. aureus even at a low dye concentration (0.05 μM) and at a very
low light dose of 3 J/cm2. Therefore, we investigated the impact of the
Similar to S. aureus, the decrease in the phototoxicity towards Gram-
negative bacteria with increasing the number of iodine atoms between 2
and 6 is supposedly connected with the increasing dye aggregation
(Fig. S2) and decreasing dye uptake. The aggregation effect is, however,
even more pronounced for Gram-negative bacteria because the dyes are
used at much higher concentrations. To diminish the aggregation, we
increased the content of DMSO in saline to 10 times, from 0.7% (as in all
the above experiments) to 7%. The obtained data in Fig. 8 show that the
efficacy of Cy7, 1ICy7, and 2ICy7 to eradicate E. coli was noticeably
improved. Thus, the survival percentage for these three dyes at 50 μM
and 400 J/cm2 was decreased from 27.6%, 15.4% and 50.0%, respec-
tively, to zero. Nevertheless, the addition of even 7% DMSO to the saline
solutions was insufficient to overcome the aggregation and improve the
phototoxicity of 3ICy7, 4ICy7, and 6ICy7.
P. aeruginosa was found to be even more resistant towards the
investigated dyes than E. coli but the same tendency as for E. coli was
observed: The most phototoxic dyes are mono-iodinated 1ICy7 and
positively charged 2ICy7þ (Figs. 9 and 10), which is different from
S. aureus, where the most phototoxic are the diiodinated (2ICy7) and
triiodinated (3ICy7) cyanines.
Fig. 3. S. aureus survival vs. the dye uptake. The uptake was estimated as the
ratio between the fluorescence intensities of the stained bacteria after and
before washing.
7

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
Fig. 4. Survival of S. aureus at 0.05
μ
M dye concentrations in 0.7% DMSO in saline, when exposed to 3 J/cm2 (1 min), 17 J/cm2 (5 min), 50 J/cm2 (15 min), 100 J/
cm2 (30 min), 200 J/cm2 (60 min), and 400 J/cm2 (120 min) light doses.
Fig. 5. Survival of S. aureus in 0.7% DMSO in saline, when exposed to 3 J/cm2 light dose (56 mW/cm2, 1 min) vs. the dye concentrations.
Fig. 6. Survival of E. coli in 0.7% DMSO in saline, when exposed to 100 J/cm2 light dose (56 mW/cm2, 30 min) vs. the dye concentrations.
8

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
Fig. 7. Survival of E. coli at 50
μ
M dye concentrations in 0.7% DMSO in saline, when exposed to 100 J/cm2 (30 min), 200 J/cm2 (60 min), and 400 J/cm2 (120 min)
light doses.
Fig. 8. Survival of E. coli in 7% DMSO in saline, when exposed to 400 J/cm2 light dose (56 mW/cm2, 120 min) vs. the dye concentrations.
Fig. 9. Survival of P. aeruginosa in 0.7% DMSO in saline, when exposed to 100 J/cm2 light dose (56 mW/cm2, 30 min) vs. the dye concentrations.
4. Conclusions
spectral properties and ability for photodynamic eradication of S. aureus,
E. coli and P. aeruginosa pathogens were investigated.
A series of near-IR heptamethine cyanine dyes containing up to six
iodine atoms and an aliphatic carboxylic group was synthesized; their
The increasing of the number of iodine atoms has an unexpected and
ambiguous phototoxic effect on these bacteria, which is connected with
9

O. Semenova et al.
Dyes and Pigments 195 (2021) 109745
Fig. 10. Survival of P. aeruginosa at 50
μ
M dye concentrations in 0.7% DMSO in saline, when exposed to 100 J/cm2 (30 min), 200 J/cm2 (60 min), and 400 J/cm2
(120 min) light doses.
two opposite factors: (i) increasing the spin–orbit coupling and the rates
of reactive species generation and (ii) dye aggregation causing the
reduced dye uptake (such a correlation was also noted in Ref. [46]) that,
supposedly, is followed by decreased rates of reactive species genera-
tion. As a result, the increase of the number of iodine atoms up to two in
the series of zwitterionic cyanines increases the efficacy of S. aureus
eradication; then the efficacy remains almost unchanged for the two-,
three- and four-iodinated dyes and diminishes in the case of the
hexa-iodinated cyanine. At the same time, the mono-iodinated hepta-
methine cyanine causes the most pronounced phototoxic effect to E. coli
and Ps. aeruginosa. An additional positive charge contributed by a trie-
thylammonium group decreases efficacy of the dye towards S. aureus but
improves eradication of E. coli and Ps. aeruginosa.
0120U102660). Authors are grateful to Dr. Vered Marks (Ariel Univer-
sity) for the measurements of NMR spectra.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109745.
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