Gaining Access to Bacteria through (Reversible) Control of Lipophilicity

Article abstract of DOI:10.1002/chem.201704562

The development of antimicrobial photodynamic therapy (aPDT) is highly dependent on the development of suitable photosensitizers (PSs); ideally, affinity of a PS towards bacterial cells should be much higher than that towards mammalian cells. A cationic c

Full text of DOI:10.1002/chem.201704562

A Journal of
Accepted Article
Title: Striking an access to the bacteria via (reversible) control of
lipophilicity
Authors: Anzhela Galstyan, Johannes Putze, and Ulrich Dobrindt
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Striking an access to the bacteria via (reversible) control of
lipophilicity
[a]
[b]
[b]
Anzhela Galstyan,* Johannes Putze, Ulrich Dobrindt
Abstract: Development of antimicrobial photodynamic therapy
PS are important. Ideally, the PS should be able to accumulate
in a high concentration in bacterial cells in a short time and be
phototoxic only to bacteria with minimal or no damage to the
healthy tissue. To fulfill these requirements conjugates with
different bacteria targeting units were studied. Promising results
(aPDT) is highly dependent on the development of suitable
photosensitizers (PS): ideally affinity of PS towards bacterial cells
should be much higher than towards mammalian cells. Cationic
charge of PS may lead to selective binding of PS to bacteria
mediated by electrostatic interaction; however, the photodynamic
outcome is highly dependent on the lipophilicity of PS. Herein we
report the aPDT effect of silicon(IV)phthalocyanine derivatives
bearing four positive charges and methyl, phenyl or naphthyl
substituents on the periphery of the macrocycle. We show that via
modulation of lipophilicity it is possible to find a therapeutic window
where bacteria but not mammalian cells are effectively killed. The
photobiological activity of these PSs dropped significantly when
host-guest complexes of PSs with cucurbit[7]uril (CB[7]) were used.
CB[7] blocks the hydrophobic part of the molecule and reduces
lipophilicity of the PS, indicating that a hydrophobic interaction with
the outer membrane of bacterial cells is essential for aPDT activity.
Efficiencies of obtained PSs were evaluated using different
uropathogenic E. coli isolates and human kidney epithelial
carcinoma cells.
have been obtained using antimicrobial
peptide-PS
[
7]
conjugates. Other studies have demonstrated that aPDT of
individual pathogens is very effective when the PS is conjugated
[
8]
to a specific antibody. Despite the vast amount of efforts
directed toward the development of targeted aPDT agents,
translation to clinical practice is rather slow due to a variety of
unique challenges. These include: (i) high production costs that
limit the wide use of these conjugates, (ii) the specificity of
obtained conjugates to a particular bacterial strain, which might
be advantageous in some cases, but non-beneficial when broad
spectrum activity is desired.
Whereas Gram-positive bacteria could be effectively killed with
neutral, cationic and anionic PSs, for inactivation of Gram-
negative species only cationic PSs or non-cationic PSs with
additional agents that permeabilize the outer membrane should
[
9]
be used. Cationic PSs were shown to replace divalent cations
of the bacterial surface that are responsible for the stabilization
of lipopolysaccharide (LPS) and the native organization of the
outer membrane. This replacement destabilizes the glycan coat
Introduction
[
10]
leading to the “self-promoted” uptake of PSs.
Introduction of
Destruction of multidrug-resistant pathogens via reactive oxygen
species (ROS) produced by photosensitizers (PS) of appropriate
structure could represent a valuable alternative therapeutic
modality addressing the problem of the increasing occurrence of
cationic charges promotes an association with the anionic
surface of bacteria in contrast to the almost neutral membrane
surface of healthy mammalian cells, giving a temporal selectivity
for bacteria over mammalian cells. Indeed, many naturally
[
1]
infectious diseases. Urinary tract infections (UTI) are the most
common of all community-acquired bacterial infections in
medical practice in industrialized countries, being the most
occurring antibacterial peptides also have pronounced
[11]
polycationic charge.
This approach was adopted by several
groups to carry out aPDT with a large range of Gram-negative
[
2]
common reason for antibiotic prescription. This unfortunate
excessive use of antibiotics has led to a considerable and
alarming increase in the rate of resistant isolates in many
[12]
bacteria.
However, the cationic character is not the sole
determinant required for the efficient photoinactivation
[13]
process.
For
instance,
tetra-
or
octa
or eight
cationic
[3]
countries. Considering that the era of antibiotics will eventually
come to an end, aPDT has been established as an effective
antimicrobial strategy that doesn’t rely on conventional antibiotic
[14]
[15]
silicon(IV)phthalocyanines bearing four
N-
methylated pyridoxy substituents in their periphery were proven
to be inefficient for the photodynamic inactivation of Gram-
negative E. coli. In our recent study, we show that boronic acid
functionality can confer activity to the tetra cationic
[
4]
mechanisms. Despite a number of advantages, there are
limited studies where PDT was considered as a treatment option
[5]
of bladder infections, whereas within recent years, PDT of
silicon(IV)phthalocyanine by substantially increasing the local
[6]
bladder cancer has found its way into widespread clinical use.
For clinical application of aPDT many characteristics of the used
[16]
concentration of PS.
Greater photobactericidal efficacy of
toluidine blue in comparison to methylene blue was also found to
be due to the stronger interaction of toluidine blue with LPS
[
17]
components.
Although aPDT can target both, external and
[
a]
b]
Dr. A. Galstyan
internal, structures of bacteria, and does not really require the
PSs to be internalized, factors that increase the amphiphilic
character of the PS, such as the introduction of lipophilic groups
or asymmetric charge distribution, can enhance the affinity of
Center for Nanotechnology, Physikalisches Institut
Westfälische Wilhelms-Universität Münster
Heisenbergstrasse 11, 48149 Münster (Germany)
E-mail: anzhela.galstyan@wwu.de
Dr. J. Putze, Prof. Dr. U. Dobrindt
Institut für Hygiene
[
18]
[
PSs for bacteria and increase their photocytotoxic effect.
These characteristics are associated with the ability of PSs to
permeabilize the cell membrane, where the oxygen
Westfälische Wilhelms-Universität Münster
Mendelstraße 7, 48149 Münster (Germany)
[
19]
concentration is higher than in the aqueous medium.
Thus,
Supporting information for this article is given via a link at the end of
the document
the design and synthesis of PSs with cooperative behavior is a
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[
26]
very successful approach: whereas positive charges enhance
the ability to “grip” onto the cell membrane of Gram-negative
bacteria, the lipophilicity of the PSs helps to penetrate the cell
membrane and “dip” into sensitive parts of the bacterial cell.
Tetrapyrrole-type photoensitizers carrying a moderate length of
hydrophobic alkyl chains (C6 – C10) and a positive charge are
molecules in aqueous media
and was widely used as a drug
carrier. Several research groups have developed antibacterial
agents that are able to assemble and disassemble with CB[7]
and its close analogs. However, whereas in some cases
bactericidal properties appeared to be activated upon formation
of supramolecular assemblies, the activity of other agents was
significantly reduced upon host-guest complexation. Generally,
the intrinsic efficacy of PSs was enhanced and a synergistic
effect was expected, however, the biological output of this
supramolecular interaction of a macrocyclic host and the PS was
not always additive.
Although several factors that are important in the photodynamic
efficiency of silicon(IV)phthalocyanines (SiPc) have already
[27]
[
20]
[28]
found to be very effective towards Gram-negative bacteria.
Larger number of hydrophobic side groups/moieties also found
[
21]
to affect activity of phenothiazinium derivatives.
However,
usually dark toxicity was observed when long alkyl chains were
implemented in the structures leading to polarization and rupture
[
22]
of bacterial cell membranes in dark.
Recently, based on
hydrophobic interaction an outer membrane-anchored aPDT
nano agent was synthesized via conjugation of protoporphyrin IX
[
16,29]
been described before,
they have not been consistently
[
23]
and cholesterol. The nano agent enables effective binding to
the outer membrane of Gram-negative E. coli, resulting in a PS-
enriched bacterial surface. Benefiting from the membrane
accumulating ability of aromatic constituents Bazan et al.
prepared a membrane-intercalating conjugated oligo electrolyte
evaluated. In an effort to clarify this, a comprehensive study was
undertaken to determine the impact of the lipophilicity and host-
guest
complexation on
the
molecular
features
of
2(3),9(10),16(17),23(24)-tetrakis-(3-pyridyloxy) phthalocyaninato
dihydroxy-silicon (IV) and its aPDT efficacy. The results of this
study provide a better understanding of the structure–property
relationships of the PSs and may provide guidelines to develop
more efficient and selective antimicrobial agents.
[
24]
with high antibacterial capability upon irradiation.
Present
evidence points out that PSs that exhibit high photobiological
outcome are presented by dyes that besides positive charge
have a well-balanced lipophilicity. Therefore, we introduced
aromatic rings to the periphery of the non-active
2
(3),9(10),16(17),23(24)-tetrakis-(3-pyridyloxy) phthalocyaninato
Results and Discussion
dihydroxy-silicon (IV) to modulate its lipophilicity, to confer a
photobiological activity and maximize the PDT outcome
Synthesis and photophysical characterization: The reactions
used to prepare a series of SiPcs are given in the Experimental
Section and in the Scheme S1 of the Supporting Information.
The phthalocyanine macrocycle was synthesized via
condensation of diiminoisoindoline according to previously
(
Scheme 1).
[
16,30]
published methods.
Quaternization of the pyridoxy
substituents on the non-peripheral positions of the macrocyclic
core using dimethylsulfide, benzyl bromide, and 2-
(
bromomethyl) naphthalene led to the formation of SiPcs
containing methyl (Pc1), phenyl (Pc2) and naphthyl (Pc3)
substituents, correspondingly. The proposed structures of the
newly synthesized Si(IV)Pcs were confirmed by 1D and 2D NMR
spectroscopy, high-resolution electrospray ionization mass
spectrometry (HRMS) and fourier transform infrared (FT-IR)
spectroscopy.
Phthalocyanine derivatives used in this study bear
hexacoordinate silicon (IV) in the central cavity, which is
coordinated to the four ring nitrogen atoms of the phthalocyanine
central cavity and two hydroxyl groups. Situated at axial
positions hydroxyl substituents can disrupt direct π-π stacking of
Scheme 1. Schematic representation of probable disposition of the cationic
silicon(IV)phthalocyanine molecules with different lipophilicity in the outer
membrane of Gram-negative bacteria.
the
macrocycles,
compiling
favorable
photophysical
characteristics to the PS. Their electronic absorption and basic
photophysical data were measured in N,N-dimethylformamide
(
DMF) as well as in water (H
Table 1.
Absorbance maxima in the UV-Vis spectra of all PSs correspond
2
O) and data are summarized in
There is an emerging recognition of the role that supramolecular
[
25]
interactions play in tuning the properties of molecular species.
To elucidate the function of aromatic rings we used
a
[16,30]
well with previously published data.
In DMF an intense
supramolecular self-assembly-based approach that utilizes host-
guest interaction between PS and CB[7]. CB[7] is a water
soluble donut-shape molecule possessing a hydrophobic cavity.
It is able to make host guest complexes with hydrophobic
sharp Q-band at around 672 nm is followed by a weaker at 604
nm. In water solution, their absorption spectra are also
dominated by sharp Q band with 5-7 nm bathochromic shifts of
the maxima.
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The fluorescence quantum yields (
F
) of the SiPcs were
calculated by the steady state comparative method using zinc
[
31]
phthalocyanine (ZnPc) as a reference
values of all SiPcs in water were found to be slightly lower
than in DMF: their values ranged from 0.31 to 0.37 in H O vs
.39 to 0.43 in DMF. This shows that the aqueous medium is not
and reported in Table 1.

F
2
0
inducing significant aggregation of the PSs and hence does not
quench their excited state properties (Table 1).
Singlet oxygen is commonly considered the main species taking
part in PDT, and thus PSs with higher singlet oxygen quantum
yields (Φ
The obtained PSs were assessed for their singlet oxygen
generation in DMF and H O. Φ s were measured by the relative
method, using 1,3-diphenylisobenzofuran (DPBF) in DMF and
,10-anthracenyl-bis(methylene)dimalonic acid (ABMDMA) in
Δ
) are usually regarded as the more promising ones.
2
Δ
[
32]
9
H
1
2
O, as a reactive trap for photogenerated O and excitation
2
with visible light filtered below 610 nm. The concentration of the
scavenger (DPBF) was monitored spectroscopically at 414 nm
in function of the irradiation time of the PS, from which the
Figure 1. UV/Vis absorption and fluorescence spectra of Pc1 (black), Pc2
values of Φ
as a reference compound (Φ
increase of Φ value was observed for Pc2 and Pc3 in
Δ
were determined. Non-substituted ZnPc was used
(red) and Pc3 (blue) in water (a and b) and N,N-dimethylformamide (c and d).
ZnPc
[33]
Δ
= 0.56 in DMF). A slight
Δ
Whereas in DMF molar absorption coefficients were rather
similar, in water media an increase of max for Pc2 and Pc3 was
observed (Figure S1, Supporting Information). These differences
may reflect the different arrangement of the fluorophores
dictated by the bulkiness of the substituents. Fluorescence
emission was recorded upon excitation at 610 nm. All PSs emit
comparison to Pc1 (Figure S3, Supporting Information). A
quantitative analysis of photooxidation reactions leading to the
loss of emission of the water-soluble anthracene 9,10-
1
dipropionic acid was used to measure O
2
production in aqueous
media. Methylene blue was used as a reference compound
MB
[34]
(
Φ
Δ
= 0.52 in H
2
O).
The Pc2 and Pc3 were found to be
2
strongly in the near-infrared in DMF and H O with λmax of around
better singlet oxygen generators, but the efficiency is still
comparable for all PSs (Figure S4, Supporting Information).
Relevant photophysical data are given in Table 1.
685 nm (Figure 1 and Table 1).
Table 1. Electronic absorption and photophysical data for Pc1, Pc2 and Pc3.
Addition of four equivalents of CB[7] to the aqueous solution of
Pc1 did not cause any noticeable change in the absorption
spectrum of the latter. For Pc2 and Pc3 addition of CB[7]
resulted only in a slight change of the absorption maxima and
hypochromicity, indicating that the PSs are in a monomeric state
and the binding of CB[7] did not contribute to the aggregation-
disaggregation process and consequently in the change of
[
F
b]
[c]

PS
λ
abs/nm
λem/nm


logPo/w
solvent
(log10ε)
± 0.03
± 0.03
PS/PS(CB[7])
4
6
73 (5.05)
Pc1
DMF
604 (4.43)
686
684
684
685
684
685
0.43
0.52
-
[a]
358 (4.74)
6
6
78 (5.04)
11 (4.43)
[a]
Pc1
[a]
photophysical
characteristics
(Figure
S2,
Supporting
0.37
0.41
0.36
0.39
0.31
0.17
0.59
0.22
0.56
0.25
-0.98 /-1.03
H
2
O
351 (4.77)
Information). On the contrary to our results, PSs that easily form
aggregates based on hydrophobic interactions were shown to
6
6
3
72 (5.06)
04 (4.44)
57 (4.76)
Pc2
DMF
[27b]
disaggregate upon addition of CB[7].
In these cases, the
formation of supramolecular PSs improves photophysical
characteristics, especially singlet oxygen quantum yields that
contribute to increase in antibacterial efficacy of PSs. Further,
we explored the impact of substituent and formation of host-
guest complexes with CB[7] on the lipophilicity of the PS. Highly
hydrophilic PS will dissolve well in aqueous media and remain in
the extracellular space. Replacing the methyl group with phenyl
or naphthyl substituents may enable the PS to partition into the
lipid bilayer of the cell’s membrane. However, it is important to
find a right balance, since too hydrophobic molecules tend to
aggregate in the aqueous phase and subsequently quench
production of singlet molecular oxygen. The octanol-water
partition coefficient (log P) is often quoted to give an indication of
the lipophilicity of a drug. The values are determined for all PSs
678 (5.10)
610 (4.46)
352 (4.83)
Pc2
H O
2
-0.71/-1.08
-0.24/-0.73
672 (5.07)
604 (4.45)
357 (4.82)
Pc3
DMF
679 (5.16)
611 (4.51)
349 (4.82)
Pc3
H O
2
[
a]
[b]
Data from ref 16. Quantum yields were calculated by the steady state
comparative method using zinc phthalocyanine as a reference (
F
= 0.28 in
DMF). Quantum yields were measured using the relative method using zinc
phthalocyanine ( = 0.56) or methylene blue ( = 0.52) as a reference.
[
b]
F

4
and for PS(CB[7]) supramolecular complexes and are given in
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Table 1. The obtained values indicate that the partitioning into
the lipid phase increases, as expected, in the following order
Pc1 < Pc2 < Pc3. Supramolecular encapsulation of the PSs by
CB[7] results in the shifts of the logP values to negative values
for Pc2 and Pc3. This clearly shows that CB[7] blocks
hydrophobic aromatic constituents resulting into more
hydrophilic PS.
In vitro photodynamic activities: A UTI usually starts as a
bladder infection (cystitis) but can develop into an acute kidney
infection (pyelonephritis). In some cases, UTI can result in
urosepsis, a disease with a mortality rate of 20-40% in the
setting of optimal resuscitation in wealthy countries. More than
Figure 2. The binding/uptake amounts of photosensitizers used in this study
by bacterial cells after 15 min incubation with 1µM solution. Mean ± SD is
presented.
[35]
85% of all UTIs are caused by Escherichia coli
– Gram-
negative bacteria which are commonly less susceptible to aPDT
[
9]
treatment.
Uropathogenic E. coli strains UTI89 (cystitis isolate), 536 and
CFT073 (pyelonephritis isolates) were used to determine the
most efficient PS of the series. Bacterial suspensions with
To investigate the influence of the light fluence on the PDT
activity different exposure times that correspond to light doses of
-2
9
, 18 and 36 Jcm were used. The photoinactivation of E. coli
9
-1
bacterial numbers of approx. 1x10 CFU mL were incubated
with 1 µM solutions of the corresponding PS for 15 min before
unbound PS was removed. The binding of the PSs to the
bacterial cells was determined by measuring the concentration
of the PS that remained in the culture supernatant after
removing the bacteria by centrifugation (Figure 2). All PSs and
their supramolecular complexes with CB[7] show comparable
binding to the bacterial cells. This is probably due to the same
amount and position of the charged groups. However,
uptake/binding of PSs does not appear to correlate with their
antimicrobial efficacy (vide infra).
using a 1µM solution of PS is shown in Figure 3. Although the
cationic charge of Pc1 promotes electrostatic interaction with the
negatively charged outer surface of the bacterial cells, it did not
cause any significant reduction in bacterial numbers. In contrast,
-1
Pc2 and Pc3 showed significant reduction of CFU mL upon
light activation, but to a different extend. Whereas Pc3 reduced
the survival of all E. coli strains by >log5 units at 1 µM
-2
concentration and 36 Jcm light exposure, the reduction of
bacterial survival caused with Pc2 was lower under the same
conditions. Once accumulated on the surface of the bacteria, an
important aspect of triggering the antibacterial properties is the
accessibility of the PS to the hydrophobic and oxygen-rich
membrane interior where the concentration of dissolved oxygen
is 2–4 times higher than in the extracellular space.
The antimicrobial activity of Pc1, Pc2 and Pc3 was evaluated by
the determination of the number of viable colony forming units
(
CFU) per milliliter. Under irradiation with polychromatic light of a
2
projector lamp (>610 nm, 10 mW/cm ) the viability of
microorganisms was not affected by light alone - light control,
nor by the direct effect of the PS - dark control (Figure S4,
Supporting Information).
Figure 3. Bacterial photoinactivation with 1 µM solution of Pc1, Pc2, Pc3 and their supramolecular complexes with CB[7]. Dose effect in light-induced toxicity
against E. coli 536, E. coli CFT073 and E. coli UTI89. Mean ± SD is presented.
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4
Figure 4. Representative fluorescence images of LIVE/DEAD assayed E. coli UTI89 after treatment with PS, CB[7] only or PS(CB[7]) complex and 3 min of
irradiation. Scale bar: 50 µM.
published studies showing that the photosensitizing activity of
cationic porphyrins towards Gram-negative bacteria is reduced
by their incorporation into liposomes, most likely due to the
After light excitation membrane components of the bacterial cells
become damaged when the PS is inserted into the membrane.
When the PS is less deeply bound to the membrane interface it
will be less active, since the concentration of the oxygen in the
extracellular interior is low and the larger fraction of the
generated singlet oxygen will be deactivated before interacting
with and oxidizing membrane components. Most likely Pc1 is too
hydrophilic and its interaction with the outer membrane of Gram-
negative bacteria is only of electrostatic nature. The photokilling
effect of Pc2 and Pc3 was strongly reduced when CB[7] was
present in the solution. Presumably, a supramolecular structure
[38]
hindered interaction of PS with cell binding sites.
Pc2 and
Pc3 can partly retain their photosensitizing activity after
formation of host-guest complexes with CB[7] indicating that PS
still could be displaced from the supramolecular complex and be
extracted into the bacterial cell membrane. The photokilling
efficacy of studied PSs was further confirmed by the Live/Dead
assay using E. coli strain UTI89. As seen from Figure 4 when
4
bacteria were incubated with Pc1, CB[7] or Pc1(CB[7]) complex
mostly viable, Syto 9 (green) stained cells were observed. When
Pc2 and Pc3 were used a high proportion of propidium iodide
stained (red) dead cells can be seen. As expected, the viability
consisting of PS(CB[7])
4
combination generated via the
intermolecular non-covalent association, “blocks” hydrophobic
interactions with the outer cell membrane of bacteria. This
confirms that the observed antibacterial effects of Pc2 and Pc3
are a direct consequence of primary damage to the highly
sensitive cellular targets in the bacterial membrane. Irrespective
4 4
of bacteria was increased when Pc2(CB[7]) and Pc3(CB[7])
were utilized, further confirming our results obtained by CFU
counting. The fact that the observed photoinactivation rates
correlate with the lipophilicity of PSs rather than photophysical
characteristics or cell binding extent indicates that the PS
localization is a dominant factor. Though positive charge-
induced electrostatic interaction contributes greatly to the
binding of a cationic PS onto the negatively charged bacterial
cell wall, enhancement of its hydrophilic character may restrain
further entry of the PS into the bacterial cell membrane. This
consideration should weigh heavily in optimizing the design of
PSs.
4
of the fact that the PS(CB[7]) accumulated to high levels on the
bacterial outer membrane, it did not exhibit higher phototoxic
efficiency, since the generation of ROS occurs on the site that is
less likely to lead to the cell death. Similar imbalance between
the binding of a PS and its photo-efficiency had already been
[
36]
reported for the benzylidene cyclopentanone based system. It
was shown that despite comparable Φ values and very similar
Δ
binding/uptake of cationic and anionic derivatives to bacterial
cells, the latter showed better aPDT activity. The authors
suppose that the synergy effects of concise chemical structure
and suitable logP value endow the good aPDT effect. In their
report Hamblin et al. tested poly-L-lysine chlorin(e6) conjugates
for inactivation of E. coli and show that despite higher uptake of
the 8-lysine conjugate in comparison to the 37-lysine conjugate,
only the latter was able to kill effectively Gram-negative bacteria
In order to rationally select the most suitable PS, it is important
to study the photocytotoxicity of all PSs when they are in contact
with mammalian cells. It is easy to assume that the lipophilicity
will also affect the uptake of PSs by mammalian cells and
[
39]
compromise their targeting specificity.
Viability assays using
Pc1, Pc2 and Pc3 were carried out using the human kidney
epithelial cell line A-498 as a model of the host cells that might
be damaged during the PDT treatment of UTI.
[
37]
at low concentrations.
The authors conclude that a certain
length of the polycationic chain is necessary to allow the PS to
enter the outer membrane of E. coli. In contrast, the large
polycationic carrier is not necessary or even disadvantageous
for the inactivation of Gram-positive bacteria such as S.
[
37]
aureus. Our results are also in accordance with the previously
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aluminum sheets pre-coated with silica gel 60 F254 (E. Merck). NMR
spectra were recorded on an ARX 300 or an AMX 400 from Bruker
Analytische Messtechnik (Karlsruhe, Germany) spectrometer at
a
1
constant temperature of 298 K. H NMR chemical shifts δ are given
relative to TMS (d = 0) and referenced to the solvent signal. Electrospray
ionization (ESI) mass spectra were recorded on a Bruker Daltonics
(
Bremen, Germany) MicroTof with loop injection.
[
40]
Synthesis: The starting 4-(3-pyridyloxy)phthalonitril
pyridyloxy)-1,3-diiminoisoindoline
known procedures. The
and 4-(3-
were synthesized and purified by
further reactions towards
(3),9(10),16(17),23(24)-tetrakis-(3-pyridyloxy) phthalocyaninato
dihydroxy-silicon (IV) followed the modify pathway of synthesis described
[
41]
2
[
30]
previously.
(3),9(10),16(17),23(24)-tetrakis-(3-pyridyloxy)
dihydroxy-silicon (IV) (50 mg, 0.053 mmol) and benzyl bromide (25 µl,
.212 mmol) were dissolved in dry dimethylformamide and the mixture
was stirred at 50°C overnight. After cooling to rt, CH Cl was added to
the solution to precipitate the product. It was dissolved in DFM and
precipitated with CH Cl again and the precipitate was dried under
Pc1 was synthesized according to the ref. 16. Pc2:
2
phthalocyaninato
0
2
2
2
2
reduced pressure. Yield: 94%. 1H NMR (600 MHz, DMSO-d6/MeOD): δ,
ppm: 9.75–9.63 (m, 8H), 9.48-9.39 (m, 4H), 9.29-9.19 (m, 4H), 8.67-
Figure 5. Photocytotoxic effect of Pc1, Pc2, and Pc3 on A498 cells incubated
with different PS concentrations (1 -100 µM) for 1h and irradiated for 1h (36
8.63(m, 4H), 8.39-8.31 (m, 8H), 7.80-7.69 (m, 8H), 7.51-7.44 (m, 12H),
.09 (s, 8H). 1H,1H GCOSY (600 MHz / 600 MHz, DMSO-d6/MeOD)
2
J/cm ). Mean ± SD is presented.
6
[
selected traces]: δ1H / δ1H: 9.73/6.02; 9.19/6.02; 8.64/6.02; 7.69/6.02;
2
7.48/6.02; 7.70/7.50; 7.80/7.49; 9.74/8.38; 9.65/8.34; 9.47/8.39;
By irradiation with 36 J/cm of NIR-light, no damage of A-498
9
C
.40/8.36; 9.19/8.33; 8.65/8.33; 9.74/920. HRMS m/z: Calcd. for
cells was induced when Pc1 was used, that had been incubated
at concentrations of 1-100 µM for 1h. More lipophilic Pc2 and
Pc3 were found to be photocytotoxic only at concentrations at
least one order of magnitude higher than those causing
significant bacterial cell death. At low concentration (1µM)
almost no photodamage of mammalian cells was observed
4+
80 58 12 6 58 12 6
H N O Si [M] : 327.60880, found: 327.60874; C80H N O SiBr
3+
[M] : 463.78458, found: 463.78476. FT-IR: 745; 1083; 1272; 1497;
1585; 3032; 3400. Pc3: 2(3),9(10),16(17),23(24)-tetrakis-(3-pyridyloxy)
phthalocyaninato dihydroxy-silicon (IV) (70 mg, 0.074 mmol) and 2-
(bromomethyl) naphthalene (65 mg, 0.295 mmol) were dissolved in dry
dimethylformamide and the mixture was stirred at 50°C overnight. After
cooling to rt, CH
It was dissolved in DFM and precipitated with CH
precipitate was dried under reduced pressure. Yield: 87%. H NMR (600
MHz, DMSO-d6/MeOD): δ, ppm: 9.78 – 9.61 (m, 8H), 9.49-9.43 (m, 4H),
2
Cl
2
was added to the solution to precipitate the product.
Cl again and the
(
Figure 5). Thus, the optimal combination of PS concentration
2
2
and drug-light interval can enable selective inactivation of
bacterial cells.
1
9
1
.16-9.11 (m, 4H), 8.72-8.61 (m, 4H), 8.38-8.25 (m, 8H), 7.91-7.54 (m,
2H), 7.47-7.40 (m, 16H), 6.11-5.85 (m, 8H). 1H,1H GCOSY (600 MHz /
Conclusions
600 MHz, DMSO-d6/MeOD) [selected traces]: δ1H / δ1H: 9.62/5.99;
9
9
7
.31/5.85; 9.13/5.99; 7.71/6.10; 7.64/ 5.99;9.70/8.33; 9.61/8.66;
.60/9.11; 9.43/8.37; 9.35/8.36; 8.71/8.35; 8.62/8.27; 8.32/8.12;
In this work, we performed a comprehensive study showing that
a positive charge of highly hydrophilic silicon(IV)phthalocyanine
does not necessarily ensure its antimicrobial efficacy. Readily
accessible, lipophilic analogs were prepared via quaternization
of pyridine moieties on the macrocyclic ring. We show that
Gram-negative uropathogenic E.coli strains are killed by
irradiation with NIR light when the used PS can translocate
through the bacterial membranes. Lipophilicity of the PSs was
modulated via host-guest complexation with CB[7]. Using an
optimal combination of PS concentration and drug-light interval a
substantial phototoxic effect could be achieved against bacteria,
whereas mammalian cells remained unaffected under the same
conditions. We believe that this analysis may help to understand
which factors should be considered when developing an
improved PDT protocol and also reinforces the need of new,
more effective PSs.
66 12 6
.91/7.64; 7.72/7.44; 7.65/7.45. HRMS m/z: Calcd. for C96H N O SiBr
3+
[M] : 530.47251, found: 530.47248. FT-IR:755; 1077; 1268; 1468;
1577; 3013; 3361.
Partition coefficient: 1-Octanol/water partition coefficients (logPo/w
)
were determined at 25 °C using equal volumes of water (1 mL) and 1-
octanol (1 mL). The final concentration of compound was approx. 20 µM.
The mixture was stirred for 1 h and centrifuged (10 min, 4400 rpm) to
enable a phase separation. An aliquot (50 µL) of aqueous and organic
phases were dissolved in 1 mL of N,N-dimethylformamide (DMF) and
final concentration determined by absorption spectroscopy. logPo/w was
calculated according to the following equation:
logPo/w = log ([PS]octanol / [PS]water
)
Photophysical measurements: Absorption spectra were measured on
a Varian Cary 500 or Agilent 8453 spectrophotometer and baseline
corrected. Steady-state emission spectra were recorded on a HORIBA
Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer equipped with a 450 W
xenon-arc lamp, double-grating excitation and emission monochromators
Experimental Section
(
2.1 nm/mm dispersion; 1200 grooves/mm). All solvents used were of the
spectrometric grade.
General: Synthetic procedures were carried out under dry argon
atmosphere unless otherwise specified. All reagents and solvents were
purchased at the highest commercial quality available and used without
further purification unless otherwise stated. Column chromatography was
carried out on silica gel Merck-60 (230–400 mesh, 60 A°), and TLC on
F
Fluorescence quantum yields: Fluorescence quantum yields (Φ ) were
determined by a comparative method using the following equation:
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Chemistry - A European Journal
10.1002/chem.201704562
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
= 
s
∙(n/n
s
)∙(F/F
s
s
)∙(A /A)
BA = 100-(ABSsample/ABSblank x 100)
Where F are areas under fluorescence emission curves of the Pc1, Pc2,
Pc3 and F of ZnPc as standard. A and A are the absorbances of the
samples and standard at the excitation wavelength and n and n are
refractive indices of solvents used for samples and standard,
Live/Dead staining: Cells were treated in the same way as for the
evaluation with CFU counting. Following irradiation, samples were
incubated with LIVE/DEAD® bacterial viability staining kit (BacLight™
Bacterial Viability Kit, Invitrogen L13152) for 15 min according to
manufacturer’s instructions. A 10 µL aliquot of the mixture was added to
glass slides and covered with a glass coverslip. Fluorescence images
were taken with a Nikon Eclipse Ci microscope, (40 X magnification).
Excitation at 480 nm and long pass filter were used.
s
s
s
[
31]
F
respectively. ZnPc in DMF was used as a standard, Φ =0.28. for the
determination of fluorescence quantum yields. The sample and the
standard were both excited at the same relevant wavelength (610 nm).
The absorbance of the solutions at the excitation wavelength ranged
between 0.05 and 0.1.
Cell line, growth condition and in vitro viability assay: The human
kidney epithelial cell line A-498 was cultured in Eagle´s Minimal Essential
Medium (EMEM) supplemented with 10 % fetal bovine serum (FBS),
2 mM L-glutamine, 1 mM sodium pyruvate and 1 x non-essential amino
Singlet oxygen quantum yields: Singlet oxygen quantum yields were
determined using the relative method. Polychromatic irradiation from a
projector lamp passing through a cut-off filter at 610 nm was used to
carry out the experiments. Freshly prepared dye solution in dark flasks
o
4
2
acids at 37 C in a humidified 5% CO incubator. About 2 × 10 A-498
were mixed with the PSs only immediately before taking the samples at
cells per well in the medium were incubated in 96-well plates and allowed
to adhere overnight. Photosensitizers were examined in the
concentration range of 0 – 100 μM. Cells were incubated for 1 h at 37 C
in the dark; subsequently, the medium was discarded and replaced with
new medium followed by an illumination of 1 h. The Alamar Blue assay
1
“
0 time.”
O
2
photogeneration rates in DMF were derived using 1,3-
o
diphenylisobenzofuran (DPBF). The initial absorbance of DPBF was
adjusted to about 1.0, then the PS was added to reach absorbance about
0
O
.2-0.3. The photooxidation of DPBF was monitored between 0 s to 25 s.
1
2
photogeneration rates in water were derived using 9,10-
was used to investigate the cell viability: After illumination, the cells were
o
anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) as a fluorescent
incubated at 37 C under 5 % CO
2
for 24 h. The medium was replaced
monitor (λexc = 370 nm) for photosensitized bleaching rates. The 
the samples was calculated according to the following equation:
Δ
for
with 200 μL of 10 % Alamar Blue (Sigma) solution in growth medium
followed by incubation for 3 h. The plate was shaken on a microplate
reader (Tecan, Switzerland) for 20 s before the fluorescence at each well
was measured (Ex 535 nm / Em 595 nm). The viability of A-498 cells was
then expressed as the relative viability (% control).
λ2

AR (  )
I
0
(  )(110
) dλ
) dλ
S

R
r
S


λ2
λ1
Φ
 Φ

rR
AS (  )
I (  )(110
0

λ1
Acknowledgements
where r is the slope of the monitor’s bleaching over time (plotted as ln
F/F )), λ – λ is the irradiation wavelength interval, I (λ) the incident
spectral photon flow, A(λ)the absorbance, and the subscripts R and S are
(
0
1
2
0
The authors are thankful for funding from the Deutsche
Forschungsgemeinschaft (DFG) projects GA 2362/1-1 to AG and SFB
1009, TP B05 to JP and UD. AG gratefully acknowledges financial
support from the Fonds der Chemischen Industrie and the Graduate
Center of WWU.
[
33]
the reference (zinc phthalocyanine 
Δ
= 0.56 in DMF and methylene
[
34]
Δ 2
blue,  = 0.52 in H O
) and sample, respectively. The incident
intensity can be approximated by a constant value, drawn out of the
integral and canceled. All recorded graphics are shown in Supporting
Information (Figure S2 and S3).
Keywords: phthalocyanine
• photodynamic therapy • antimicrobial
agents • E. coli • host-guest interaction
Bacteria culture conditions and in vitro photodynamic inactivation:
The E. coli strains were maintained on Luria-Bertani (LB) broth agar and
were stored at 4 C. A single isolated colony was picked from this plate,
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Location matters: The approach
described in this paper yields
photosensitizers that exhibit high
photocytotoxic effect against gram-
negative E.coli at low concentration
without harming eukaryotic cells. The
effectivity correlates with the
lipophilicity of the photosensitizer
that changes upon complexation with
CB[7].
Dr. A. Galstyan, Dr. J. Putze, Prof. Dr.
U. Dobrindt
Page No. – Page No.
Striking an access to the bacteria
via (reversible) control of
lipophilicity
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