Breaching the wall: morphological control of efficacy of phthalocyanine-based photoantimicrobials

Article abstract of DOI:10.1039/c8tb01357h

An efficient treatment of infections using antimicrobial photodynamic therapy (aPDT) anticipates that uptake of photosensitizer (PS) by bacterial cells is very fast and effective. In this work, the design, synthesis, characterization, and photodynamic activity of amphiphilic, water-soluble zinc(ii)phthalocyanine (Zn(ii)Pc) molecules bearing none, three or six thiophenyl moieties are described. We show that PSs that contain no or flexible substituents on non-peripheral positions can photoinactivate microbes at very low loading concentrations and low light doses. In contrast, a PS derivative that contains non-flexible substituents is rendered less effective, despite an increased generation of cytotoxic singlet oxygen, higher lipophilicity and a lower tendency to aggregate. Our unexpected finding emphasizes the role of the morphology of PSs in bacterial cell-molecule interactions and suggests another relevant and hitherto disregarded characteristic to improve PS design.

Full text of DOI:10.1039/c8tb01357h

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Regulating the stemness of mesenchymal stem cells by tuning
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Breaching the wall: morphological control of efficacy of
phthalocyanine-based photoantimicrobials
Anzhela Galstyan*a and Ulrich Dobrindt b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Efficient treatment of infections using antimicrobial photodynamic therapy (aPDT) anticipates that uptake of
photosensitizer (PS) by bacterial cells is very fast and effective. In this work design, synthesis, characterization, and
photodynamic activity of amphiphilic, water-soluble zinc(II) phthalocyanines (Zn(II)Pc) bearing none, three or six
thiophenyl moieties are described. We show that PSs that contain no or flexible substituents on non-peripheral positions
can photoinactivate microbes at very low loading concentrations and low light doses. Contrary, a PS derivative that
contains non-flexible substituents is rendered less effective, despite an increased generation of cytotoxic singlet oxygen,
higher lipophilicity and lower tendency to aggregate. Our unexpected finding emphasizes the role of the morphology of PS
in bacterial cell-molecule interaction and suggests another relevant and hitherto disregarded characteristic to improve PS
design.
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complex: it contains two membranes, with
a
thin
Introduction
peptidoglycan layer spanning the periplasm and
lipopolysaccharide (LPS) chains embedded on the surface layer
of the outer membrane (Figure 1b). In order to be sensitive to
the photosensitized destruction, PS should efficiently bind to
or pass through one or more of these barriers.8 Despite the
evidence that internalization of PS is not required per se,
literature review indicates that PS, which can plunge into the
oxygen-rich lipid bilayer, is able to inactivate bacteria at very
low concentrations and low light doses.9,10 While Gram-
negative bacteria frequently produce outer membrane
vesicles, endocytosis-like membrane-trafficking is hindered by
its asymmetric lipid architecture and the peptidoglycan layer.
Protein transport machineries such as porin channels can
mediate PS uptake,11 however, they have threshold sizes of
Antimicrobial photodynamic therapy (aPDT) is a gradually
expanding approach that enables fast and very effective
treatment of bacterial infections.1,2 Efficient and reliable PS for
aPDT have emerged over the last few decades to meet this
demand.3-5 A good PS is expected to fulfill many essential
characteristics, such as ability to absorb light energy in the
spectral rage of visible and near infrared light (600 – 800 nm)
characterized by a high penetration depth into the tissues and
transfer it to substrate (e.g. oxygen), high extinction
coefficients, tunable photophysical properties, low dark
toxicity, selectivity towards bacterial cells, etc. Localization and
distribution of PSs are significant in determining antimicrobial
activity and it is assumed that these depend on two PS
features: affinity for the cellular components and amphiphilic
character.6 An important challenge remains beyond the simple
correlation of PS lipophilicity and photodynamic efficacy,
namely establishing the role of the molecule’s shape and
flexibility of the substituents on photodynamic efficacy.
transportable molecules12 (generally
< 600 Da). Though,
positive charges of PS promote interactions with anionic
groups at the external surfaces of the microbial target it was
considered that further translocation via self-promoted uptake
pathway is determined by the hydrophobicity of the PS. In the
present study, we clearly show for the first time, that the
morphology of the chromophore plays an important role in PS
translocation across bacterial cell membranes and
consequently has a big impact on its photodynamic efficacy.
The amphiphilic nature of PSs is one of the most desired
features and is frequently used to promote hydrophobic
interactions with the outer membrane of bacteria.13 Most
commonly the chromophore unit carries a positive charge
whereas hydrophobic side groups are introduced to direct the
PS to the interior of the bacterial cell.14-16 However, an
enormous increase of activity was observed when charged
groups were a part of an auxochromic side chain.9,17 Most
likely direct and efficient interaction of the chromophore with
Numerous studies have shown that photoinactivation of Gram-
negative bacteria is particularly challenging.7 Although
bacterial cells are far less advanced than mammalian cells, the
composition of the cell wall of Gram-negative bacteria is very
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the PDT-sensitive membrane is contributing to the increased core results in a large red shift of the Q-band.19 Additionally,
antibacterial effect.
such a ring modification also contributes to the efficient
photosensitized generation of singlet oxygen due to the
increase in intersystem crossing efficacy and fast formation of
lowest triplet state due to the “heavy atom effect”.20
In this paper photophysical, theoretical and biological studies
are combined, highlighting importance of different
characteristics for design of new and more effective PSs.
Results and discussion
A series of amphiphilic low-symmetry Zn(II)Pcs with A3B type
construction that contain no (ZnPc-0S), three (ZnPc-3S) or six
(ZnPc-6S) thioaryl substituents on the unit A and one 2,4,6-tris
(dimethyl aminomethyl)phenyl substituent on the unit B were
prepared. The starting 3-[2,4,6-tris(N,N-dimethylaminomethyl)
phenoxy] phthalonitrile,21 3-phenylthio phthalonitrile,22 3,6-
bis(tosyloxy)
phthalonitrile
and
3,6-bis(phenylthio)
phthalonitrile19a were synthesized and purified by known
procedures. The classical synthetic approach was used towards
unsymmetrically substituted phthalocyanines, which implies
the cross-condensation of unit A and unit B in a 4 : 1 molar
ratio following so-called lithium method. Zn(II)Pcs were
obtained through metal insertion reaction of the free base
phthalocyanine with zinc acetate salt and the complexes ZnPc-
0S, ZnPc-3S and ZnPc-6S were isolated by silica gel column
chromatography. To achieve solubility in a wide pH range
dimethylamino groups were further quaternized with methyl
iodide leading to the formation of ZnPc-0SQ, ZnPc-3SQ, and
ZnPc-6SQ, correspondingly (Scheme 1).
Fig. 1. (a) Optimized structures of ZnPc-0S, ZnPc-3S, ZnPc-6S at the B3LYP/6-31G(d,p)
level of theory, (b) schematic representation of cell wall composition of Gram-negative
bacteria and its interaction with ZnPcs.
Phthalocyanines (Pc) are one of the most studied PSs with an
intense
π→
π* absorption in the visible spectrum.18 By
changing number, position, and nature of the substituents it is
possible to shift the Q band in Uv-vis spectra to the longer
wavelength that corresponds to the tissue transparency
window and is highly desirable for PDT applications. Kobayashi
et al. demonstrated that the incorporation of group 16 (S, Se
and Te) elements at the non-peripheral positions of the Pc
Scheme 1. Synthetic pathway towards Zn(II)Pcs used in this study.
2 | J. Name., 2012, 00, 1-3
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introduction of sterically demanding substituents can
substantially suppress chromophore stacking.23
To gain insights into the stability of PSs, we compared
photobleaching (photodegradation) of ZnPc-0S, ZnPc-3S and
ZnPc-6S upon continuous illumination with λ > 610 nm light
with 30 mW/cm2 flow rate and recorded the decrease in the
Q-band of the Uv-vis spectra. Photodegradation was found to
be more pronounced in DMF for all Zn(II)Pc derivatives since
DMF is known to be less efficient in stabilizing the ring against
oxidative attack. Notably, comparing photobleaching of PSs in
H2O, the largest photobleaching was observed for ZnPc-0S (Fig.
S5, ESI†).
A varying number of the thiophenyl substituents (0-3-6)
impacted the absorption and emission maxima of Zn(II)Pcs.
Compared to Q band absorption of ZnPc-0S at 678 nm in DMF,
the introduction of the electron-donating sulfur atoms in α-
positions of the macrocycle induced a red-shift of the Q band
absorption; ZnPc-3S at 702 nm and ZnPc-6S at 754 nm in DMF.
Fig. 2. a. Optical absorbance of ZnPc-0S (black), ZnPc-3S (red) and ZnPc-6S (blue)
in DMF normalized to the same absorptivity for the lowest energy Q-band; b.
fluorescence emission spectra of ZnPc-0S (black), ZnPc-3S (red) and ZnPc-6S
The quaternized derivatives exhibited their
Q
band
(blue) in DMF (normalized for absorbance values
~ 0.08), c. normalized
absorptions at very similar positions to that of uncharged
derivatives (Table 1). A similar trend in the red-shift was
observed in the fluorescence spectra of Zn(II)Pcs (Fig. 2b). The
absorption spectra of ZnPc-0S (black), ZnPc-3S (red) and ZnPc-6S (blue) and d.
ZnPc-0SQ (black), ZnPc-3SQ (red) and ZnPc-6SQ (blue) at the concentration 1 x
10-5 M in water.
highest fluorescence quantum yield (
ZnPc-0S. Dyes containing sulfur bridges showed an obvious
decrease in F, while at the same time singlet oxygen
quantum yields ( ꢀ) were increased, which points to a very
ΦF) was observed for
Φ
As expected, introduction of thiophenyl substituents into α–
positions of the Pc macrocycle had a substantial influence on
the aggregating behaviour and photophysical characteristics of
Φ
rapid intersystem crossing from the singlet to the triplet
excited state. Quaternization of dimethylamino groups
Zn(II)Pcs. Whereas these compounds were present in
a
affected the photophysical properties such as ΦF and
ZnPc-0SQ and ZnPc-3SQ the values of ΦF were decreased,
while the increase of values of ꢀ was observed. For ZnPc-6SQ
Φꢀ. For
monomeric form in DMF, as indicated by a sharp absorption Q-
band, they were substantially aggregated in aqueous media
(Fig. 2). A broad shoulder at lower wavelengths, which is
typical for H-type aggregate formation, was more pronounced
for ZnPc-0S, ZnPc-0SQ and ZnPc-3S. In contrast, the intensity
ratio of the first two vibronic peaks in the absorption spectra
of ZnPc-3SQ, ZnPc-6S and ZnPc-6SQ increased (Fig. 2c and 2d).
Thus the quaternization of the dimethyl amino groups and/or
Φ
differences to unquaternized derivative were not significant. In
respect to the number of thiophenyl substituents, both values
followed the rule of the heavy atom effect as in case of an-
quaternized derivatives (Table 1).
Table 1. Photophysical data for zinc(II)phthalocyanines used in this study
PS
λabs/nm (log10ε)
λem/nm[a]
Φ
F ± 0.02 [b]
Φꢀ ± 0.03
[c]
LogPo/w
ZnPc-0S
678 (5.01), 611 (4.36), 336 (4.58)
675 (5.08), 609 (4.48), 342 (4.67)
702 (5.02), 631 (4.32), 332 (4.39)
701 (5.00), 630 (4.33), 333 (4.42)
754 (4.90), 678 (4.39), 346 (4.52)
753 (4.93), 677 (4.65), 349 (4.80)
683
682
708
705
758
758
0.25
0.20
0.09
0.06
0.02
0.02
0.43
0.05
-0.53
0.14
-0.44
0.23
-0.19
[c]
0.66 /0.61
[c]
[d]
[d]
[d]
ZnPc-0SQ
ZnPc-3S
0.61
[c]
0.72 /0.76
[c]
ZnPc-3SQ
ZnPc-6S
0.93
[c]
0.89 /0.81
ZnPc-6SQ
[a]Excited at 610 nm (ZnPc-0S), 630 nm (ZnPc-3S) and 660 nm (ZnPc-6S). [b]Quantum yields were calculated by the steady state comparative method using
zinc(II)phthalocyanine as a reference ( F = 0.56) as
F = 0.28 in DMF). [c]Quantum yields were measured in DMF using the relative method and zinc(II)phthalocyanine (
a reference. [d] Quantum yields were measured in H2O using the relative method and methylene blue (
F = 0.52) as a reference.
Φ
Φ
Φ
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