Cationic benzylidene cyclopentanone photosensitizers for selective photodynamic inactivation of bacteria over mammalian cells

Article abstract of DOI:10.1039/c5ra06143a

To inactivate both standard strains as well as antibiotic-resistant strains of bacteria with minimum damage to host cells, three new pyridyl cationic-modified benzylidene cyclopentanone photosensitizers (PSs), P1 (with one cationic group), P2 (with two ca

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Cationic benzylidene cyclopentanone photosensitizers for
selective photodynamic inactivation of bacteria over mammalian
cells
Received 00th January 20xx,
Accepted 00th January 20xx
Yanyan Fang,a, b Tianlong Liu,a Qianli Zou,a Yuxia Zhao*a and Feipeng Wu*a
DOI: 10.1039/x0xx00000x
To inactivate both standard strains as well as antibiotic-resistant strains with minimum damage to host cells, three new
pyridyl cationic-modified benzylidene cyclopentanone photosensitizers (PSs), P1 (with one cationic group), P2 (with two
cationic groups bilaterally), and P3 (with two cationic groups unilaterally) were synthesized and characterized. Their
selective uptakes by bacteria over HepG2 cells and their photodynamic inactivation efficiencies against Staphylococcus
aureus (ATCC 6538), Escherichia coli (ATCC 25922), and the drug-resistant Escherichia coli (CA-31) were studied using
methylene blue (MB) and hematoporphyrin monomethyl ether (HMME) as references. The results showed that the uptake
amounts of P1, P2, and P3 by all strains were at least 2, 20, and 18 times more than those by HepG2 cells, respectively. All
PSs exhibited good antimicrobial photodynamic therapy (aPDT) effects towards three strains with low concentrations of ≤
8.0 μM, while MB was invalid towards three strains and HMME was invalid toward Escherichia coli (CA-31) with the
concentration up to 32.0 μM. Especially, the minimum inhibitory concentrations (MICs) of P3 against these strains were all
≤ 2.0 μM, under which about 94% HepG2 cells were still alive, indicating P3 had high aPDT selectivity for bacterial cells
over mammalian cells. The relationships between structure and antimicrobial properties of these cationic PSs were
www.rsc.org/
discussed
to
reveal
their
high
photodynamic
inactivation
selectivity
of
bacterial
cells.
Instead, Gram-negative (Gram-(−)) bacteria have an addiꢀonal,
more negatively charged outer layer that serves as
1. Introduction
a
permeability barrier to prevent the entry of noxious
compounds.15 For this reason, neutral and anionic PSs are
often failed to inactivate Gram-(−) bacteria effecꢀvely while
cationic PSs can still bind to their outer membrane strongly
and damage their integrity.16-19 Therefore, cationic PSs are
considered to be broad-spectrum antimicrobial agents having
more potential for the development of aPDT.
The discovery of antibiotics has greatly enhanced the abilities
of humans to resist microbial infections. However, the
worldwide rapidly increasing antibiotic resistance among
pathogenic bacteria is becoming a serious problem.1 In this
3
regard, photodynamic therapy (PDT),2, a powerful clinical
protocol for the treatment of tumors, has been proposed as a
promising alternative method to inactivate bacteria, viruses,
fungi, and protozoa.4, 5 Unlike antibiotics, in antimicrobial PDT
(aPDT) process, photosensitizer (PS) is irradiated to generate
reactive oxygen species (ROS), such as superoxide (O2·-),
For an ideal aPDT process, the bacterial cells should be
selectively inactivated with low or even no PDT damage to
host tissues.16 One strategy to achieve this goal is to combine
photoactive compounds with bacteria-targeted agents or
carriers. In previous studies, Friedberg et al.20 reported that
binding PSs to a monoclonal antibody (Mab) could specifically
kill P. aeruginosa. Gross et al.21 obtained a highly selective
aPDT against Staphylococcus aureus by linking a PS with a non-
specific IgG. However, the broad-spectrum antimicrobial
potencies of these PSs showed decreases after such
modifications. Another strategy to promote selectivity is to
take advantage of the differences in membrane composition
and transmembrane potential between bacterial cells and
mammalian cells. As some studies revealed, the negative
transmembrane potential in bacterial cells was much higher
than that in mammalian cells, and more importantly, the
negative charges were located on the outer wall of bacteria
but in the inner leaflets of mammalian cells.19 Recently, Zhu et
hydroxyl radical (HO·) and/or singlet oxygen (1O2) to kill
7
microbial cells.6,
The multi-target nature and non-
involvement of the genetic material property of aPDT make it
less chance to induce the bacterial resistance or trigger the
bacterial repair mechanisms.8-11 In recent years, aPDT has
proved its potential in disinfection of contaminated blood12
and environmental waters.13
Generally, Gram-positive (Gram-(+)) bacteria are more
sensitive to aPDT because their outer walls are relatively
porous and all types of PSs can diffuse through them readily.14
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al.22 reported that a cationic conjugated polymer with a study subjects. Both S. aureus and E. coli were revived with
relatively high quaternary ammonium ratio could selectively Luria Bertani (LB) broth and nutrient agar at 37°C for 24 hours.
combine with bacterial cells but did not bind to Jurkat T cells, The OD600 value, the optical density at 600 nm, was
indicating that the membrane charge difference between monitored to determine the density of bacterial cells.
bacteria and mammalian cells was large enough to be clearly
2.5. The uptakes of PSs by bacterial cells. For the uptake test,
a bacterial suspension (~ 108 CFU per mL) of each strain with
distinguished by cationic PSs.
In our previous studies, we reported two series of water- 10 μM PSs was prepared. After 1 h of incubation in dark at
soluble benzylidene cyclopentanone PSs modified by room temperature, the suspension was centrifuged at 5000
polyethylene glycol (PEG) and carboxylate anionic groups, and rpm for 10 min to harvest the bacteria. The obtained bacteria
proved that several of them were good candidates for were washed with PBS for three times and then lysed with
anticancer PDT.23, 24 Moreover, we found that the anticancer lysozyme (100 μg/mL) for 1h followed by sonication for 2h at
PDT effects of these PSs sharply decreased with their 37°C. The lysed bacterial solution was centrifuged again to
increasing hydrophilicity, and proved that this was related to obtain the supernatant. The fluorescence intensity of the
their decreased uptake by mammalian cells. Here, we design samples was measured, excited at 460 nm and monitored from
three
novel
pyridyl
cationic-modified
benzylidene 475 nm (for methylene blue, excited at 580 nm and monitored
cyclopentanone PSs (P1
–P3, shown in Figure 1). By introducing from 595 nm). The fluorescence intensity of PSs at the
different numbers of pyridyl cationic groups at different concentration of 10 μM without bacteria was taken as the
positions, we expect that the uptake capabilities of these PSs baseline for the calculation of the uptake amount.
by bacteria will be significantly affected, and the detailed
2.6. Agarose diffusion assay. The agarose diffusion assay was
structure-antimicrobial property relationships of these cationic
used to analyse the antibacterial PDT activity of PSs, S. aureus,
PSs are analysed and discussed. The significant photodynamic
E. coli and the drug resistant strain E. coli (CA-31) were used as
inactivation efficiencies of these PSs against the Gram-(+)
the test organisms. According to the method reported by
Lehrer et al.34, 3 mL cell suspensions (~ 105 CFU per mL) were
Staphylococcus aureus (S. aureus, ATCC 6538), Gram-(–)
Escherichia coli (E. coli, ATCC 25922), and drug resistant strain
E. coli (CA-31) are studied. Methylene blue (MB),25-28 one of
added into 100 mL of warmed (50°C) agarose, then poured
into a sterile Petri dish (Φ = 10 cm) and allowed to harden. A
the most widely used cationic PS in clinical practice right now,
sterile puncher (diameter ~ 4.5 mm) was used to bore holes,
and hematoporphyrin monomethyl ether (HMME),29-31 an
and then 20 μL samples at six different concentrations of 4.0,
effective second-generation anionic PS, which has been used
8.0, 16.0, 32.0, 50.0 and 64.0 ꢀM were added to each well.
in the treatment of various clinical diseases for years, are used
as reference compounds. What’s more, the high selective
photodynamic inactivation of these PSs towards bacteria over
mammalian cells is studied and analysed.
After incubating the plates upright at 37°C for 1 h, the control
group (without light) was put into the incubator for continuous
incubation. For the experimental group, a 532 nm laser (30 J
cm-2, 10 min) was used to irradiate each hole for 10 min. The
inhibition zones of both the control group and the
experimental group were evaluated after another 24 h of
incubation at 37°C in the dark.
2. Experimental
2.1. Synthesis and characterization. The synthesis routes of
2.7. Minimum inhibitory concentration (MIC) test. In MIC test,
a modified resazurin method reported by Elavarasan et al. was
utilized.35 As shown in the schematic diagram (Figure S2), the
concentrations of four neighboring wells in a sterile 96-well
plate, for example C1, C2, D1, and D2, are same. 100 μL of the
photosensitizers at different concentrations were added into
the corresponding wells on 96-well plates. The GM group (100
μL 1% gentamicin in each well) and the B+B group (100 μL
broth with bacteria) were used as the positive-control and
negative-control groups, respectively. All the wells described
above contained 10 μL of bacterial suspension (~ 5×106 CFU
per mL) to achieve a final concentration of 5×105 CFU per mL
except of the Broth group, which contained 10 μL of nutrient
broth as the blank-control group. These 96-well plates were
irradiated with a 532 nm laser for 10 min (the total light dose
was approximately 30 J cm-2) and incubated at 37°C for
another 18 h. For methylene blue, a 635 nm laser (30 J cm-2)
was used. A 0.0675% resazurin solution was prepared. 10 μL of
resazurin indicator solution was added into each well and then
plates were incubated at 37°C. After 4 h of chromogenic
reaction, the lowest concentration showing no pink colour
photosensitizers (P1–P3) are shown in Scheme 1. All final
compounds were characterized by 1H NMR spectra, HR-MS,
element analysis, and HPLC analysis confirming a purity of
>95%. DEA was synthesized and reported in our previous
work.23
2.2. Fluorescence quantum yield. The fluorescence quantum
yields of P1–P3 in PBS were measured using the fluorescein
disodium salt as reference (Φf = 0.9).32
2.3. Singlet oxygen production. The singlet oxygen quantum
yields (ΦΔ) of P1–P3 were measured using 9,10-
anthracenediyl-bis(methylene)dimalonic acid (AMDA) and
Rose Bengal as 1O2 scavenger and the reference,
respectively.33 The experimental details are given in supporting
information.
2.4. Bacterial growth. In this study, the Gram-(+) S. aureus
(ATCC 6538), Gram-(–) E. coli (ATCC 25922) and a drug
resistant strain, β-lactamase producing E. coli (CA-31), which
was isolated from swine and provided by the College of
veterinary, China Agricultural University, were selected as
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change was considered as the complete growth inhibition
concentration of a PS. Each test was carried out for three times
at different days. The relative death rate was calculated by
equation (1) after monitoring the optical density (OD) value of
each well at 630 nm.
3. Results and discussion
3.1. Synthesis and characterization. Scheme 1 shows the
synthetic route of P1 P2, and P3. Compounds C1 and C2
,
containing one or two chlorinated ethyl groups were
synthesized via a Vilsmeier-Haack reaction of the hydroxyl
groups in B1 and B2 with phosphorus chloride and DMF
according to the report of Massin et al.37 Intermediate
ꢃꢄꢅꢆꢃꢄꢇ
ꢀ
ꢁ
Relative death rate % = ꢂ1 −
ꢉ × 100% (1)
ꢃꢄꢈꢆꢃꢄꢇ
products D1
base-catalysed condensation reported in our previous
work.23 The final target cationic PSs were synthesized by the
alkylation of pyridine with D1 D3. Purifications of these
–D3 were obtained in excellent yields (90–95%) via
Where OD0, OD1, and OD2 are the average OD values of Broth
group, B+B group, and experimental group, respectively. The
data of GM group was used as reference to normalize the
results.
a
–
cationic PSs were achieved by re-crystallization from toluene,
and the characterization data, including 1H NMR, HR-MS,
elemental analysis and HPLC spectra of these PSs, are provided
in supporting information.
2.8. Cell culture. HepG2 cells and mouse melanoma B16 cells
were cultured with the growth media (DMEM supplemented
with 10% FBS, 100 unit/mL penicillin, 100 μg/mL streptomycin
at 37°C in a humidified atmosphere containing 5% CO2). All the
PSs were dissolved in PBS (pH=7.4), as 1.0 mM stock solutions,
and then diluted in the growth media to the final working
concentrations.
3.2. Solubility and octanol-water partition coefficient. As data
shown in Table 1, after introducing one or two pyridyl groups,
the water solubility of all PSs P1–
P3 (> 4 mg mL-1) is
significantly improved compared with the prototype
compound 2,5-bis(4-(diethylamino)benzylidene cyclopean-
tanone (BDEA).23 Additionally, the water solubility enhances
with the increasing number of pyridyl groups. The Log P values
are 1.8 for P1, −1.0 for P2, and −0.1 for P3, respectively. It is
interesting that the Log P values of these PSs are affected not
only by the number of the pyridyl groups, but also by the
position of the modification. P2, bilaterally modified by two
pyridyl groups is less lipophilic than P3 modified unilaterally.
For P3 still has one lipophilic end, it may make it easier to
enter the lipid phase. In this study, the Log P values of MB and
HMME were also measured as 0.1 and 0.8, respectively.
2.9. The uptakes of PSs by HepG2 cells. The HepG2 cells were
incubated with 10 μM PSs in 12-well plates for certain hours,
and then, after washing the cells with PBS for three times, 200
mL lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-
100) was used to lyse the cells.36 The intracellular
concentration of PSs at different incubation times was
calculated based on the measurement of fluorescence
intensity.
2.10. Subcellular localization. To detect the subcellular
localization, a confocal laser scanning microscope (Nikon AIR
MP) was used. HepG2 cells were incubated in a cover glass
chamber (diameter 35 mm) for 24 h to reach a density of
1×105 cells per well. Media containing 10 μM PSs was used to
replace the original culture solution. After 8 h incubation, the
chambers were washed with PBS for three times. Then the
culture media with 100 nM Mito-tracker green was used to
incubate the cells for 20 min and the chambers were washed
with PBS again. A light of 488 nm was used to excite Mito-
tracker green, while the PSs were excited by a 561 nm light.
3.3. Photophysics and singlet oxygen production. The
absorption and fluorescence emission properties of all PSs
were carried out in PBS (phosphate buffered saline) (Table 1).
As shown in Figure 2, the absorption and emission bands both
blue-shift from P1 to P2 and P3, which indicates that the
electron-donating capability of the terminal dialkyl-amino
group decreases after modification by the pyridyl cationic
group. Compound P2 exhibits the largest absorption band gap
because its two terminal amino groups are affected.
2.11. Cytotoxicity of PSs. For the dark cytotoxicity, HepG2 cells
and B16 cells were dispersed and cultured in 96-well plates for
24 h. Then, 100 μL culture media containing different
concentrations of PSs were used to replace the original culture
solution. After another 24 h of incubation, the cell viability was
determined by CCK-8 assay. For photo-cytotoxicity, a 532 nm
laser (30 J cm-2, 10 min) was used to irradiate the cells after
incubating them with PSs for 8 h. Then, after 10 min
irradiation, the cells were incubated in the dark for another 24
h before testing. Cells without PSs were also treated with the
same laser and were used as the 100% cell survival base line.
As results shown in Table 1 and Figure S3, the ΦΔ values of P1
and P3 are 0.036 and 0.027, respectively, and no significant
changes are found when increasing the number of unilaterally
modified groups. Compound P2, which is bilaterally modified
by two pyridyl groups, has an obviously reduced ΦΔ value
(0.007), likely as a result of its enhanced radiative transition of
fluorescence emission (Table 1).
3.4. The uptakes of PSs by bacterial cells. Bacteria cellular
uptake was examined by incubating suspensions of Gram-(+) S.
aureus, Gram-(−) E. coli, and the drug resistant strain of E. coli
(CA-31) with PSs, respectively, at the concentration of 10 μM
in dark for 1 h. After centrifugation to remove the supernatant,
the bacterial cells were lysed and the fluorescence intensity of
these PSs in the lysed solution was detected. As shown in
2.12. Statistical analysis. Independent assays and replicates of
the same sample were done to obtain the representative
results. Data were statistically analyzed by one-way analysis of
variance (ANOVA) test using SPSS 16.0 (SPSS Inc., Chicago, IL).
The p value of <0.05 was considered to be statistically
significant.
Figure 3, P1, P2, and P3 are taken up by the three strains more
readily than MB and HMME, and the difference is significant (P
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< 0.05). Moreover, the order of uptake amount is the same as For S. aureus and E. coli, the photodynamic inactivation
P3 P2 P1 MB HMME in all these strains. For example, abilities of P1 P3 are close to or equivalent to HMME. The MIC
the uptake amounts of P1 P2 P3 MB, and HMME by S. values of P1 P2 P3, and HMME against S. aureus are 2.0, 2.0,
>
>
>
≈
–
,
,
,
,
,
aureus are 1003, 1308, 1457, 236, and 189 pmol/106 cells, 1.0, and 1.0 μM, respectively, while against E. coli, they are
respectively, and by E. coli (CA-31), they are 434, 765, 1200, 4.0,
46, and 15 pmol/106 cells, respectively. As expected, the 4.0, 2.0, and 2.0 μM, respectively. It is worth to mention that
positive charges in cationic PSs enhance their electrostatic although HMME is an anionic PS, its activity to inactivate
interaction with the negatively charged outer membranes of Gram-(−) E. coli is still comparable to the cationic PSs, P1
bacteria, so it is reasonable to find that the uptake amounts of which is likely as a result of its advantage of relatively high ΦΔ
cationic PSs P1
P3 are more than those of the anionic PS values (~ 0.13 in PBS43). However, for the drug resistant strain
–P3,
–
HMME, and the uptake of P2 and P3, with two cationic groups, E. coli (CA-31), the photodynamic inactivation ability of HMME
are higher than that of P1 with one cationic group. However, is significantly decreased (MIC > 32 μM), and this MIC is
the large difference of uptake between P1 and MB indicates significantly different with those toward S. aureus and E. coli (P
that the charge is not the only determinant. It seems that the < 0.05). The more strict control of the outer membrane
bacteria cellular uptake also correlated with the Log P value of permeability against the entrance of noxious compounds and
these PSs, so P1 with the higher Log P (1.8) is taken up more the enhanced ability of the efflux pump are likely
than MB (Log P = 0.1) by bacteria, and a similar trend is also explanations.38
found when comparing P3 with P2. Additionally, owing to the
Most interestingly, the cationic PSs P1
antimicrobial properties against not only standard strains but
the antibiotic resistant strain E. coli (CA-31), especially for P3
–P3 show obvious
enhanced efflux pump function and the more strict control of
outer membrane permeability in drug resistant strains,38 the
uptake amounts of PSs by E. coli (CA-31) are the lowest when
compared with the corresponding data obtained for the other
two strains. P3, the PS taken up the best by all three strains, is
promising as a potential anti-bacteria agent.
,
which is the best among all three newly synthesized cationic
PSs. The reasons for the effective antimicrobial PDT effects of
these cationic PSs include both the destructive effect of 1O2,
and the destabilization and interruption of native organized
3.5. Antimicrobial PDT. Inhibition zones tests were carried out bacterial cell walls caused by cationic charges.44
at six different concentrations. The results are listed in Table
3.6. The uptakes of PSs by HepG2 cells and their subcellular
S1 and partly shown in Figure S5. A typical result with PSs as
localization. To evaluate the safety of these cationic PSs
4.0 μM is shown in Figure 4. Under the light conditions (532
towards mammalian cells, the test of cellular uptake and
nm, 30 J cm-2, 10 min), it is clear that no obvious bacterial cell
subcellular localization toward HepG2 cells was carried out.
death is found in the blank groups. However, for the
HepG2 cells were incubated with PSs for 1, 2, 4, 6, 8, 12 and 24
experimental groups, P1-P3 showed obvious antibacterial
h, respectively. The results are shown in Figure 6. The
concentrations of the three PSs in cells reach a plateau after
8h incubation. Because of its relatively high Log P value, the
mono-cationic PS P1 accumulates the most in cells. However,
the uptake process is hindered when there is more than one
cationic group in the PS. The amounts of P2 and P3 taken up
by the cells are much lower compared with P1, which is also
demonstrated by the images of subcellular localization
abilities even without light, and these results were consistent
with the natural antibacterial property of cationic PSs.39, 40 P3
has the best aPDT effect because of its higher bacterial uptake
efficiency, although its ΦΔ value is a little lower than that of
P1. Additionally, despite its more effective uptake results, P2
has inferior aPDT effects compared with P1 because of its
obviously decreased ΦΔ value.
For quantitative evaluation, the MIC assay was carried out obtained by confocal laser scanning microscope (CLSM).
using a modified resazurin method. The relative death rate of
Although the numbers of modified groups in P2 and P3 are the
the bacterial cells was calculated by monitoring the optical
same, P3, modified unilaterally, possesses a higher rate of
density of the wells at 630 nm (Figure 5). The wells in which
uptake compared with that of P2 (modified bilaterally). This
the blue color did not turn to pink were considered to
subtle change may be attributed to the high lipophilicity of the
correspond to the MIC, and the relative death rate of GM
unilateral bis-ethyl agents in P3 favoring to the cellular uptake
group (> 97%) was used as references. A 532 nm laser (30 J cm-
process. The uptake amounts of P1, P2, and P3 are 190, 38,
and 67 pmol/106 cells, respectively, after incubating HepG2
cells with 10 μM PSs for 1 h. It is worth to mention that the
2) was used to irradiate P1
–
P3 and HMME, and a 635 nm laser
(30 J cm-2) was used for MB. The results are shown in Table 2
and Figure S6, P1 P3 (MIC 8 μM) exhibits more excellent
–
≤
uptake amounts of P1, P2, and P3 by the three strains are at
aPDT effects than those of MB (MIC > 32 μM) against S.
aureus, E. coli, and even the drug resistant strain of E. coli (CA-
31). Though MB has much higher ΦΔ value (~ 0.5 in PBS41) than
least about 2, 20, and 18 times more than that by HepG2 cells
under parallel conditions (10 μM PSs in dark for 1 h). The high
selectivity of P2 and P3 for bacterial cells over HepG2 cells
indicates the possibility for such PSs to selectively inactivate
bacterial cells with low PDT damage to host tissues.
those of P1–P3, its lower uptake amounts will limit its effect.
Moreover, as reported previously, MB could readily form
dimmers on the bacterial cell surface, which would sharply
decrease its ability to photo-inactivate microbes.42
Additionally, as shown in Figure 6b, P1 accumulates mainly in
mitochondria, the cellular energy manufacturing factory,
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indicating its high photo-cytotoxicity toward HepG2 cells. P2 2.0 μM, at which about 94% mammalian cells were still viable,
and P3 accumulate mainly in cell membranes. Compared with indicating its potential as aPDT agent to be used in clinic.
the size of mammalian cells (~ 20 μm), the lifetime and the
action radius of 1O2 is short in biologic systems (<0.04 ms and <
0.02 μm),45 so the damages of PSs P2 and P3 toward important
Acknowledgements
organelles inside cells are limit.
This work was supported by the National Natural Science
Foundation of China (Grant No. 60978057). The authors are
grateful to Dr. Meiling Zheng for her expertise assistance with
the confocal fluorescence microscopy.
3.7. Cytotoxicity of PS. To further understand cytotoxicities of
these cationic PSs, the dark- and photo-cytotoxicity of them
toward HepG2 cells were studied. As shown in Table 2 and
Figure 7, both P2 and P3 show negligible dark-cytotoxicity
towards HepG2 cells at the concentrations up to 40 μM.
However, the cytotoxicity of P1 in the dark (IC50(dark)=
33.5μM) is relatively significant (P < 0.05, compared with the
results of P2 and P3), likely as a result of its high cellular
uptake. From the photo-cytotoxicity study, the IC50(light) value
of P1 (4.4 μM) was relatively low compared with those of P2
(9.6 μM) and P3 (7.7 μM). Obviously, P1 shows less chance to
be considered as an effective aPDT agent because its MIC
value to photo-inactivate E. coli (CA-31) is as high as 8.0 μM
and only 21% of HepG2 cells are detected to survive at this
concentration. Although P3 seems more toxic than P2 owing
to its ΦΔ value and cellular uptake efficiency, it is the most
likely candidate for aPDT after taking its antimicrobial results
as well as dark- and photo-cytotoxicity towards HepG2 cells
into consideration. At 8.0 μM (the MIC for P2 to photo-
inactivate E. coli (CA-31)), the cell viability of P2 towards
HepG2 cells was about 68% after irradiation. By comparison,
about 94% of HepG2 cells still survive at the MIC value of P3
Notes and references
‡
Electronic Supplementary Information (ESI) available:
[Characterization methods, 1H NMR spectra of PSs, HPLC analysis
of PSs, the measurement of singlet oxygen quantum yield in PBS,
results of inhibition zones tests, MIC tests, cytotoxicity of MB
and HMME, and cytotoxicity of new synthesized PSs toward B16
cells]. See DOI: 10.1039/x0xx00000x.
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(all
≤ 2.0 μM), which indicates that bacterial cells can be
selectively photo-inactivated while minimizing the damage to
mammalian cells. This selectivity is further confirmed by B16
cells, a cell line from skin. As shown in Figure S7, the IC50(light)
8.
values of P1, P2, and P3 are 4.2, 8.1, and 10.9 μM,
respectively. While, the IC50(light) values of MB and HMME are
either comparable with their MICs or even much lower, so
they have no such a merit (Figure S8). Therefore, P3 is more
competitive to be an effective aPDT agent to selectively
inactivate bacterial cells under conditions in which host tissues
can be spared.
9.
10.
11.
12.
Conclusions
Three new cationic benzylidene cyclopentanone derivatives,
P1, P2, and P3, were synthesized to study the selective
photodynamic inactivation of bacteria over mammalian cells.
The structure-antimicrobial property relationships of these PSs
were discussed based on the influences of the modified charge
numbers and positions of pyridyl cationic groups. It was found
13.
14.
that P1-P3 all presented much better aPDT effects compared
to MB (cationic PS used in clinical practice) towards three
strains and HMME (a second-generation porphyrin derivative
used in clinic) towards E. coli (CA-31). Furthermore, compound
P3, modified by two pyridyl groups unilaterally, was found
perform best to inactivate not only standard strains but also
the antibiotic-resistant E. coli (CA-31) with the MIC as low as
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Table 2. The results of minimum inhibitory concentration (MIC) test for
aPDT and dark- and photo-cytotoxicity of PSs toward HepG2 cells
MIC (μM)
IC50
IC50
PS
(light) (dark)
E.coli
S. aureus
E. coli
(CA-31)
P1
P2
2.0
2.0
4.0
4.0
8.0
8.0
4.4
9.6
7.7
7.8
2.3
33.5
>40.0
>40.0
40.0
P3
1.0
2.0
2.0
MB
>32.0
1.0
>32.0
2.0
>32.0
>32.0
HMME
>40.0
IC50 is the concentration of the photosensitizer that is
necessary to achieve 50% growth inhibition of HepG2 cells in
the dark or after irradiation by 532 nm light.
Scheme 1. Synthetic Routes of PSs P1 – P3. Conditions: (a) PCl5, DMF, 45°C,
12 h; (b) MeOH, LiOH·H2O, 35 °C, 18 h; (c) Pyridine, 100°C, 24 h.
Table 1. Solubility, octanol-water partition coefficient, photophysics, and
singlet oxygen production of PSsa
Figure 1. Chemical structures of P1–P3, MB and HMME.
εmax
Solubility
PS (mg mL-
ab
fl
log
P
λmax
(nm) M−1
cm−1)
4.1
(104
λmax
Φf
ΦΔ
(nm)
1
)
P1 4.3
P2 >10
P3 >10
1.8
-1.0 489
501
643
623
630
0.015 0.036
0.024 0.007
0.015 0.027
5.9
-0.1 498
4.5
aThe solvent was phosphate buffered saline (PBS) unless
otherwise noted; Log
P is the octanol-water partition
coefficient. λmaxab is the absorption maximum.
εmax is the molar
Figure 2. The UV-Vis absorption (a) and normalized fluorescence emission
spectra (b) of P1-P3 in PBS.
absorption coefficient at λmaxab. λmax is the fluorescence
emission maximum. Φf is the fluorescence quantum yield. ΦΔ
is the singlet oxygen quantum yield measured by
photochemical trap method in PBS, Rose Bengal (RB) is used as
the reference (ΦΔ = 0.75 in PBS).
fl
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Figure 4. Diameters of the inhibition zone of PSs (4 μM) against S. aureus, E.
coli, and E. coli (CA-31) after incubation for 24 h.
Figure 3. PS uptake by S. aureus, E. coli, and a drug resistant strain of E. coli
(CA-31) after incubation with 10 μM PSs for 1h. (** P < 0.05 compared with
the results of P1, P2, and P3).
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Figure 5. Relative death rate of three strains after being irradiated with laser (30 J cm-2) for 10 min and incubated for another 24 h. (** P < 0.05 compared
with the corresponding data of S. aureus and E. coli).
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Figure 7. Viability of HepG2 cells incubated with different
concentrations of PSs. (a) In dark. (b) Irradiated with a 532 nm laser
(30 J cm-2) for 10 min. (** P < 0.05 compared with the corresponding
data of P2 and P3).
Figure 6. (a) PS uptake by HepG2 cells with 10 μM PSs for different
times. The error bars denote the standard deviation of three replicates.
(b) Confocal fluorescence images obtained by incubating HepG2 cells
with 10 μM PSs for 8 h and 100 nM Mito-tracker green for 20 min.
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