A series of water-soluble photosensitizers based on 3-cinnamoylcoumarin for: In vitro antimicrobial photodynamic inactivation

Article abstract of DOI:10.1039/c8ra02557f

A series of novel water-soluble photosensitizers (PSs; M1-M5) based on 3-cinnamoylcoumarin derivatives, incorporating carboxylic acid salt (M1, M2), pyridine salt (M3, M4) and quaternary ammonium salt (M5) groups, were designed and synthesized. Their phot

Full text of DOI:10.1039/c8ra02557f

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A series of water-soluble photosensitizers based on
3-cinnamoylcoumarin for in vitro antimicrobial
photodynamic inactivation†
Cite this: RSC Adv., 2018, 8, 17073
a
Zhiyuan Sun,‡ac Shaona Zhou,‡b Haixia Qiu,b Ying Gub and Yuxia Zhao
*
A series of novel water-soluble photosensitizers (PSs; M1–M5) based on 3-cinnamoylcoumarin derivatives,
incorporating carboxylic acid salt (M1, M2), pyridine salt (M3, M4) and quaternary ammonium salt (M5)
groups, were designed and synthesized. Their photophysical and photochemical properties and in vitro
antimicrobial photodynamic inactivation (PDI) were investigated. M2, modified with two carboxylic acid
salts, was unstable in phosphate-buffered saline (PBS). The four other PSs all showed higher binding/
uptake to methicillin-resistant Staphylococcus aureus (MRSA), A. baumannii and C. albicans compared
with the clinical drug methylene blue (MB), except for M1 to A. baumannii. Furthermore, the three
cationic PSs (M3–M5) exhibited equivalent antibacterial PDI efficacies against MRSA and A. baumannii
compared with MB. The antifungal efficacies of M4 and M5 to C. albicans were both significantly higher
than that of MB, especially for M5, indicating that the quaternary ammonium-salt-modified coumarin
derivative has substantial potential for antifungal PDI.
Received 24th March 2018
Accepted 19th April 2018
DOI: 10.1039/c8ra02557f
rsc.li/rsc-advances
more negatively charged outer layer, which serves as a barrier to
prevent the entry of outer species,17 meaning that only cationic PSs
can easily be taken up. However, fungi are different from bacteria.
Although fungi have an even thicker negatively charged outer wall
than Gram-negative bacteria, anionic PSs still can inactivate them
effectively.18 The available evidence shows that antifungal photo-
dynamic therapy (PDT) involves more complex mechanisms, such
as the presence of drug efflux pumps, the induction of enzymes
and the formation of biolms.19,20
In our previous work, a series of PSs modied by poly(-
ethylene glycol) (PEG), pyridinium or carboxylic acid salt based
on benzylcyclopentanone were synthesized and investigated.21
The results showed that pyridinium- and carboxylic-acid-salt-
modied PSs both had better antimicrobial PDI efficacies.22
Additionally, quaternary ammonium-salt-modied PSs have
been used as antibacterial agents for a long time, and most
could effectively inactivate bacteria.23–25 Coumarin is a natural
substance found in many plants.26 Many clinical drugs contain
coumarin or its derivatives.27,28 In cancer chemotherapy,
coumarin and 7-hydroxycoumarin act as cytostatic agents
against renal cell carcinoma.29,30 Furthermore, coumarins
exhibit potent antitumor activities at different stages of cancer
formation through various mechanisms, such as inducing cell
apoptosis or inhibiting DNA-associated enzymes.31 In antitumor
PDT, it was also proved that coumarin derivatives have good
curative effects.32,33
Introduction
Since their discovery in the early 20th century, antibiotics have
been widely used to treat a variety of infectious diseases in
clinical practice all over the world.1–3 However, with the exten-
sive use of antibiotics, many drug-resistant or multidrug-
resistant bacteria, even superbacteria, have emerged.4,5
Consensus has now been reached on the importance of
reducing the abuse of antibiotics and nding new methods of
treating infection diseases.6–9 Antimicrobial photodynamic
inactivation (PDI) uses light to excite a disease-localized
photosensitizer (PS) to generate reactive oxygen species (ROS),
such as singlet oxygen (1O2), which can inactivate bacteria,
viruses and fungi in situ.10–12 Unlike antibiotics, ROS has strong
oxidizing properties with respect to all organic substrates, thus
avoiding the problem of drug resistance.13–15
Because of the wide variety of microorganisms, there is no clear
guide for the selection of PSs for antimicrobial PDI at present.
Generally, the outer walls of Gram-positive bacteria are relatively
porous, so can readily be penetrated by cationic, anionic, neutral
or nonionic PSs.16 Gram-negative bacteria have an additional and
aTechnical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing
100190, P. R. China. E-mail: yuxia.zhao@mail.ipc.ac.cn; Tel: +86 10 82543569
bDepartment of Laser Medicine, Chinese PLA General Hospital, Beijing, 100853, P. R.
China
In this article, we designed and synthesized ve PSs based on
3-cinnamoylcoumarin derivatives through introduction of
carboxylic acid salt (M1 and M2), cationic pyridinium salt (M3
and M4) and quaternary ammonium salt (M5) groups. Their
cUniversity of Chinese Academy of Sciences, Beijing 100049, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra02557f
‡ These authors contributed equally to this work.
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photophysical and photochemical properties and in vitro anti- dimethylformamide
(DMF).34
1,3-Diphenylisobenzofuran
microbial PDI toward Gram-positive bacteria methicillin- (DPBF) was used as the scavenger.35 Mixed solutions of PSs (M1–
resistant Staphylococcus aureus (MRSA), negative bacteria M5 or RB) and DPBF were prepared. The concentrations of all
A. baumannii and fungus C. albicans were investigated.
solutions were adjusted to the same absorbance (0.1) at 473 nm,
and the initial concentration of DPBF was consistent. To ensure
the saturation of air, all solutions were stirred vigorously during
the experimental procedure. The bleaching of the absorption
band of DPBF in each solution at 415 nm was monitored under
Experimental
Synthesis and characterization
The synthetic routes towards M1–M5 are shown in Fig. 1. 3-Acetyl- the irradiation of a 473 nm diode laser. The DPBF solution
7-(dimethylamino)-2H-chromen-2-one (1) and 3-(ethyl(4- alone was also irradiated to diminish the errors origin from the
formylphenyl)amino) propanoic acid (2) were synthesized accord- photo-activation. The FD of each PS was calculated from the
ing to our previous work.33 4-Aminobenzaldehyde (3), 2-(ethyl(- slope of the decay curve.
phenyl)amino)ethanol (6), 2,20-(phenylazanediyl) diethanol (9) and
4-(dimethylamino)benzaldehyde (12) were purchased from Energy
Chemical. All targeted compounds were obtained by an initial
high-yield aldol reaction between 3-acetyl-7-(dimethylamino)-2H-
chromen-2-one and the corresponding p-aminobenzaldehyde,
followed by the modication of different anions or cations.
Synthetic details and characterization data are provided in ESI.†
The uptakes of PSs by microorganism cells
The binding of M1, M3, M4 and M5 to the three strains was
measured as previously described.36,37 Briey, the concentra-
tions of bacteria or fungus were maintained at McF ¼ 0.5, 108
cells per mL for a bacteria suspension and 106 cells per mL for
a fungus suspension with 20 mM PSs. Aer 1 h of incubation in
the dark at room temperature, the suspension was centrifuged
at 5000 rpm for 10 min to harvest the bacteria. The obtained
bacteria were washed with PBS three times and then lysed with
lysozyme (100 mg mLꢀ1) for 1 h, followed by sonication for 2 h at
37 ꢁC. The lysed bacterial solution was centrifuged again to
obtain the supernatant. The uorescence intensities of the
samples were measured for the different strains. Then the
uptakes of various PSs were calculated according to the stan-
dard curve.
Instruments
1H nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker AV400 (400 MHz) spectrometer with deuterated
reagents, CDCl3 or D2O, using tetramethylsilane as an internal
standard. Mass spectra were measured using Bruker APEX 7.0E.
Ultraviolet-visible (UV-Vis) spectra were measured using a Hita-
chi U-3900 spectrophotometer. Steady-state uorescence was
carried out on a Hitachi F-4500 spectrometer. Phosphate-
buffered saline (PBS) buffer solution (0.01 M sodium phos-
phate, pH ¼ 7.2–7.4) was purchased from Solarbio. Other
chemical reagents were from Energy Chemical or J&K chemical
Ltd. MRSA, A. baumannii and C. albicans were provided by
Chinese PLA General Hospital.
In vitro antimicrobial PDI
In the PDI group (PS + laser), MRSA and A. baumannii (ꢂ108
colony-forming unit [CFU] mLꢀ1) and C. albicans cells (ꢂ106
CFU mLꢀ1) were incubated with PSs at different concentrations
(2.5, 5, 7.5, 10 and 25 mM) for 1 h in the dark and then irradiated
under a laser with an exposure dose of 24 J cmꢀ2 (irradiated for
10 min by a laser with 40 mW cmꢀ2 power density, 457 nm for
M1, M3–M5 and 630 nm for methylene blue (MB) as the refer-
ence). A blank group and a laser group were also included.
Incubation and illumination were carried out in 96-well plates
(Costar, USA) with 40 mL of mixtures in each well. The viability of
the microbial cells was determined by the dilution plating
method, as described previously.38 In brief, 10 mL aliquots of
each sample were serially diluted and spread over the surfaces
of Sabouraud's dextrose agar plates. All plates were aerobically
incubated in an incubator for 24 h. log10 reductions in micro-
bial cells were compared with the untreated control. All exper-
iments were performed three times.
Singlet oxygen quantum yields (FD)
The singlet oxygen quantum yield (FD) was determined using
Rose Bengal (RB) as the reference with a yield of 0.47 in
Results and discussion
Solubility and octanol–water partition coefficient
The UV-Vis absorption spectra and uorescence emission
spectra of PSs M1–M5 in PBS are shown in Fig. 2, and their
corresponding data are listed in Table 1. All of these PSs have
a strong charge transfer (CT) absorption peak at around 470–
abs
Fig. 1 Synthetic routes to targeted PSs M1–M5.
17074 | RSC Adv., 2018, 8, 17073–17078
490 nm. For each PS, both the absorbance maximum l
and
max
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Fig. 2 Absorption (A) and fluorescence (B) spectra of PSs M1–M5 in PBS (5.0 ꢃ 10ꢀ5 mol Lꢀ1).
the uorescence emission maximum (lmax) in PBS present red-
The binding/uptake of PSs by bacteria and fungus cells
shis compared with the data in DMF, which can be ascribed
to the solvent effects of the reduction of excited state energy in
polar proton solvents. The octanol–water partition coefficient
(log P) are 1.49, ꢀ1.20, 0.05, ꢀ0.48 and 0.11 for M1–M5,
respectively. These results indicate that the water solubility of
these PSs is enhanced with the increase in the number of
pyridinium or carboxylic acid salts. Additionally, the FD of PSs
M1–M5 were determined as 0.22, 0.10, 0.06, 0.15, and 0.16,
respectively, in DMF, which are acceptable for PDT. However,
the data in PBS could not be obtained due to the fast decay of
singlet oxygen in water or PBS.35
The binding/uptake amounts of these PSs by bacteria (MRSA
and A. baumannii) and fungus (C. albicans) were tested. As
shown in Fig. 4, M1 and M3–M5 can be uptaken more efficiently
by the three strains compared with MB, which has almost no
clear uptake of uorescence signal. The reason may be ascribed
to the good water solubility of MB (log P ¼ ꢀ0.96 (ref. 39 and
40)), which affects its penetration in lipid cell membranes.
Moreover, the intakes of M1, M3–M5 by C. albicans (665–3320
pmol/106 cells) are somewhat larger than the corresponding
amounts taken by bacteria (2–118 pmol/106 cells), which is
probably because of the larger size of the Candida species, about
25–50 times of the bacteria size.41 For the negatively charged cell
walls, the cellular intakes of cationic PSs M3 and M5 by both
bacteria and fungus are much higher than the corresponding
amounts of anionic PS M1, such as 118 pmol/106 cells of M5
versus 38 pmol/106 cells of M1 in the MRSA group and 2260
pmol/106 cells of M3 versus 665 pmol/106 cells of M1 in the C.
albicans group. The cellular uptake of M1 and M4 by MRSA and
C. albicans are similar, while the cellular uptake of M1 by A.
baumannii is obviously lower than that of M4. These results all
Photostability
The photostability of these PSs was studied under irradiation of
a 473 nm laser. As shown in Fig. 3, compared with the four other
PSs, M2 was bleached 15% aer 120 s. Additionally, even when
kept in the dark, the PBS solution of M2 was still unstable aer
8 h, and was therefore excluded in the follow-up in vitro
experiments.
Table 1 Photophysical and photochemical parameters of PSs M1–M5 in different solventsa
abs
Com
M1
M2
M3
M4
M5
Solvent
log P
1.49
l
(nm)
3max (104 Mꢀ1 cmꢀ1
)
lmax (nm)
Dnss (10ꢀ4 nmꢀ1
)
Ff
FD
max
DMF
PBS
DMF
PBS
DMF
PBS
DMF
PBS
469
490
437
486
473
485
470
481
464
470
1.63
1.21
1.98
1.48
3.46
2.60
5.15
4.50
3.94
3.86
551
641
566
643
602
618
529
609
559
615
3.17
4.81
5.22
5.02
4.53
4.44
2.37
4.37
3.66
5.01
0.018
0.22
—
0.10
—
0.06
—
0.15
—
0.0023
0.0087
0.0031
0.0039
0.0036
0.011
0.0039
0.0098
0.0022
ꢀ1.20
0.05
ꢀ0.48
0.11
DMF
PBS
0.16
—
a
abs
max
log P is the octanol–water partition coefficient; l
is the absorption band maximum; 3max is molar absorption coefficient at lambasx; lm ax is
uorescence emission maximum; Dnss is the Stokes shi; Ff is uorescence quantum yield; FD is singlet oxygen quantum yield measured by
photochemical trap method.
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In vitro antimicrobial PDI
The toxicities of PSs toward the three strains with or without
laser irradiation were tested. For the dark toxicity, aer incu-
bating the microorganisms with 100 mM PSs M1, M3–M5 or MB
in the dark for 24 h, the bacteria or the fungus viabilities had no
signicant decrease in all groups. As shown in Fig. 5, except for
the anionic PS M1, the cationic PSs M3–M5 all have equivalent
antibacterial PDI efficacies against MRSA and A. baumannii
compared with the clinical drug MB, with an over 3 log CFU
mLꢀ1 decrease of the bacterial viability when the concentration
of M3–M5 is up to 5 mM. The results correlate well with the
cellular uptake and the FD of the PSs. For C. albicans, cationic
PSs M4 and M5 both exhibited much better antifungal PDI
efficacies compared with MB, especially for M5. There was an
over 3 log CFU mLꢀ1 decrease of the fungal viability when the
concentration of M5 reached 2.5 mM. However, the antimicro-
bial PDI efficacy of the anionic PS M1 was poor, effective only for
MRSA, with an over 3 log CFU mLꢀ1 decrease of the bacterial
viability when its concentration reached 25 mM. For C. albicans,
the difference may be caused by different mechanisms for
various PSs. As mentioned in the introduction, the antifungal
PDT process involves more complex mechanisms compared
with antibacterial PDT.42 Unfortunately, until now the specic
reasons are still not clear. Additionally, based on the
Fig. 3 Photostability of M1–M5 with irradiation time in DMF solution.
indicate that the cationic PSs M3–M5 have good potential in
antimicrobial PDI. Compared with M3 and M5, the binding/
uptake of M4 is lower, which indicates that the charge is not
the only determinant. According to our previous report,22 the
cellular uptake also correlated with the log P value of PSs. Based
on the lower log P data of ꢀ0.48 for M4 versus 1.49 for M3 and
0.11 for M5, we speculate that the lower binding/uptake of M4 is
due to its enhanced water solubility by introducing two cationic
groups.
Fig. 4 The cellular uptakes of PSs M1, M3–M5 and reference MB by MRSA, A. baumannii (A) and C. albicans (B).
Fig. 5 Relative cell viability of three strains MRSA (A), A. baumannii (B) and C. albicans (C) after being irradiated with laser (40 mW cmꢀ2) for 10 min
and then incubated with PSs M1, M3–M5 and MB for another 24 h. The error bars denote the standard deviation of three replicates.
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comparable intake of M1 and M4 (Fig. 4), the antimicrobial PDI
effect of M1 is much lower than that of M4. Except for the
difference between cations and anions, we speculate that the
antimicrobial PDI effect of a PS may also correlate with its
binding site or subcellular localization, which will be more
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Conclusions
In conclusion, a series of novel water-soluble PSs (M1–M5)
based on 3-cinnamoylcoumarin derivatives were synthesized,
and their properties were investigated. The results show that
with the introduction of two carboxylic acid salts, the photo-
stability of the PSs declined. The cationic PSs M3–M5 exhibited
higher binding/uptake to MRSA, A. baumannii and C. albicans
compared with the clinical drug MB. Additionally, the in vitro
experiments showed that three cationic PSs (M3–M5) had
equivalent antibacterial PDI efficacies against MRSA and A.
baumannii compared with MB. The antifungal PDI efficacies of
M4 and M5 to C. albicans were both much higher than that of
MB, especially for M5. However, anionic PS M1 was only effec-
tive for MRSA. Although the cationic PSs M4 and M5 showed an
excellent antifungal PDI performance, the specic reasons are
still not clear. Further study needs to be carried out to elucidate
the mechanism. This work indicates the potential of coumarin
derivatives in antimicrobial PDI.
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Conflicts of interest
There are no conicts of interest to declare.
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Acknowledgements
The work was supported by the National Natural Science
Foundation of China (No. 61575208).
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