In vitro photodynamic activity of lipid vesicles with zinc phthalocyanine derivative against Enterococcus faecalis

Article abstract of DOI:10.1016/j.jphotobiol.2018.04.025

Zinc(II) phthalocyanine bearing eight non-peripheral 2-propoxy substituents was subjected to physicochemical study and, after incorporation in lipid vesicles, assessed as a potential photosensitizer for antibacterial photodynamic therapy. The phthalocyani

Full text of DOI:10.1016/j.jphotobiol.2018.04.025

Accepted Manuscript
In vitro photodynamic activity of lipid vesicles with zinc
phthalocyanine derivative against Enterococcus faecalis
Lukasz Sobotta, Jolanta Dlugaszewska, Piotr Kasprzycki,
Sebastian Lijewski, Anna Teubert, Jadwiga Mielcarek, Maria
Gdaniec, Tomasz Goslinski, Piotr Fita, Ewa Tykarska
PII:
DOI:
Reference:
S1011-1344(18)30235-5
doi:10.1016/j.jphotobiol.2018.04.025
JPB 11208
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date:
Revised date:
Accepted date:
28 February 2018
10 April 2018
14 April 2018
Please cite this article as: Lukasz Sobotta, Jolanta Dlugaszewska, Piotr Kasprzycki,
Sebastian Lijewski, Anna Teubert, Jadwiga Mielcarek, Maria Gdaniec, Tomasz Goslinski,
Piotr Fita, Ewa Tykarska , In vitro photodynamic activity of lipid vesicles with zinc
phthalocyanine derivative against Enterococcus faecalis. The address for the
corresponding author was captured as affiliation for all authors. Please check if
appropriate. Jpb(2018), doi:10.1016/j.jphotobiol.2018.04.025
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ACCEPTED MANUSCRIPT
In vitro photodynamic activity of lipid vesicles with zinc phthalocyanine
derivative against Enterococcus faecalis
Lukasz Sobottaa,*, Jolanta Dlugaszewskab, Piotr Kasprzyckic, Sebastian Lijewskid, Anna
Teuberte, Jadwiga Mielcareka, Maria Gdaniecf, Tomasz Goslinskid, Piotr Fitac, Ewa Tykarskad
aDepartment of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences,
Grunwaldzka 6, 60-780 Poznan, Poland
bDepartment of Genetics and Pharmaceutical Microbiology, Poznan University of Medical
Sciences, Swiecickiego 4, 60-780 Poznan, Poland
cInstitute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5,
02-093 Warsaw, Poland
dDepartment of Chemical Technology of Drugs, Poznan University of Medical Sciences,
Grunwaldzka 6, 60-780 Poznan, Poland
eInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14,
Poznan, Poland
fFaculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan,
Poland

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ABSTRACT
Zinc(II) phthalocyanine bearing eight non-peripheral 2-propoxy substituents was
subjected to physicochemical study and, after incorporation in lipid vesicles, assessed as a
potential photosensitizer for antibacterial photodynamic therapy. The phthalocyanine
derivative obtained in the macrocyclization reaction was characterized by MS and NMR
techniques. Moreover, its chemical purity was confirmed by HPLC analysis. X-ray structural
analysis revealed that overcrowding of the phthalocyanine derivative leads to a strong out-of-
plane distortion of the π-system of the macrocycle core. In the UV-Vis absorption spectra of
zinc(II) phthalocyanine two characteristic bands were found: the Soret (300–450 nm) and the
Q band (600–800 nm). Photophysical properties of mono- and diprotonated forms of
phthalocyanine derivative were studied with time-resolved fluorescence spectroscopy. Its tri-
and tetraprotonated forms could not be obtained, because compound decomposing in higher
acid concentrations. The presented zinc(II) phthalocyanine showed values of singlet oxygen
generation ΦΔ = 0.18 and 0.16, the quantum yield of the photodecomposition ΦP = 3.06∙10-4
and 1.23∙10-5 and the quantum yield of fluorescence ΦFL = 0.005 and 0.004, designated in
DMF and DMSO, respectively. For biological studies, phthalocyanine has been incorporated
into modified liposome vesicles containing ethanol. In vitro bacteria photoinactivation study
revealed no activity against Escherichia coli and 5.7 log reduction of the Enterococcus
faecalis growth.
KEYWORDS: phthalocyanines, singlet oxygen, X-ray, PACT, Enterococcus faecalis
*Corresponding author
E-mail address: lsobotta@ump.edu.pl (L. Sobotta).

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1. Introduction
Phthalocyanines (Pcs) belong to a group of aromatic macroheterocycles and consist of
four isoindole units linked together with azamethine bridges. Due to their complex structure
Pcs exhibit unique spectro-, photo- and electrochemical properties. Therefore, they can be
applied in many fields of technology and industry, i.e. as dyes, in photovoltaics, in medicine,
etc. [1–6]. Pcs excited with visible light in a photodynamic process can generate singlet
oxygen and other equally reactive oxygen species. Especially, highly energetic singlet oxygen
can react in living organisms with biologically active substrates disturbing its functions and
finally leading to apoptotic and/or necrotic cell death [7]. This phenomenon enables the
application of Pcs in medicine as photosensitizers (PS) for photodynamic therapy (PDT) [8,9].
PDT is a treatment method of cancer lesions, psoriasis, actinic keratosis, in dermatology for
photorejuvenation and many others. PDT possesses some advantages in comparison to
conventional treatment methods, especially chemotherapy and radiotherapy. Moreover,
antimicrobial PDT reveals potential applications in vitro and in vivo study against viral and
bacterial infections [10–13]. The challenge would be to develop novel antibiotics and
antimicrobial therapies. The amount of reports on the bacterial resistance to antibiotics is
rising. For this reason, current scientific research concerns methods of treatment in the
upcoming post-antibiotic era [11]. Photodynamic Antimicrobial Chemotherapy (PACT) is a
medical approach worth developing, as it can provide interesting alternatives and solutions to
diminish growing bacterial resistance. The potential clinical use of this procedure has been so
far limited mainly to the treatment of local skin or mucosa infections, but the amount of other
clinical studies has been rapidly growing, among others for the treatment of malaria and
trypanosomiasis [14–17]. Recently, photosensitizers synthesized and researched for modern
PACT in many in vitro and in vivo studies revealed the potential of a synergistic effect in
combination with antibiotics [18].
Photosensitizers often possess a highly hydrophobic nature, which complicates drug
administration. This limitation can be overcome by modifying macrocycle molecules or
applying pharmaceutical formulations, for instance, liposomes or cyclodextrins [19,20]. Our
group has developed many porphyrazine and phthalocyanine photosensitizers and evaluated
their biological activity in vitro against various cancer cells [21–23], bacteria [24] and viruses
[22]. Lately, for the delivery of photosensitizers, various vesicular systems embodying
ethanol, have been proposed, including modified liposome vesicles known as invasomes and
ethosomes. Invasomes besides common phospholipid ingredients and ethanol contain

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terpenes. The optimal concentration of ethanol makes the lipid vesicle membrane more
flexible and increases the delivery of biologically active ingredients into the skin or enhances
its penetration through the biological membranes [25]. The flexibility of invasomes is a
desirable property. These carriers containing photosensitizer mTHPC were proposed for the
inactivation of Enterococcus faecalis, which is the leading pathogen causing periapical
changes in teeth as it penetrates into dentinal tubules. Dentinal tubules diameters are in the
range of 0.9 up to 2.5 µm, which enable bacteria to go inside the tubules and persist the
treatment. It has been proved that invasomes can be considered as a useful carrier for the
delivery of photosensitizer [26]. In this study, we have focused on photodynamic inactivation
of bacteria with novel zinc(II) phthalocyanine derivative. Therefore, we have synthesized a
zinc phthalocyanine derivative and assessed its photophysical and photochemical properties.
After that, the in vitro antibacterial PACT potential of this photosensitizer incorporated in
lipid vesicular systems embodying the optimal concentration of ethanol was evaluated.
2. Experimental
2.1. General
All reactions were carried out in oven-dried glassware under inert gas atmosphere.
Reaction temperature refers to external bath temperature. Solvents were distilled before use.
Other solvents and all reagents were obtained from commercial suppliers and used without
further purification. Flash column chromatography was carried out on Merck silica gel 60,
particle size 40-63 µm. Reversed-phase column chromatography was performed on Fluka
Silica gel C18. Thin layer chromatography (TLC) was performed on silica gel Merck Kieselgel
60 F254 plates and visualized with UV (λmax 254 or 365 nm). UV-Vis spectra were recorded on
1
a Hitachi UV-Vis U-1900 spectrophotometer: λmax [nm] (log ε). H NMR, 13C NMR spectra
were recorded using a Bruker 400 spectrometer. Chemical shifts (δ) are quoted in parts per
million (ppm) and are referred to a residual solvent peak. Coupling constants (J) are quoted in
Hertz (Hz) to the nearest 0.5 Hz. The abbreviations s, d and m refer to singlet, doublet, and
multiplet, respectively. MALDI TOF mass spectrometry was carried out by Advanced
Chemical Equipment and Instrumentation Facility at the Faculty of Chemistry, Adam
Mickiewicz University in Poznan. Analytical HPLC was performed on Agilent 1200 Series
instrument coupled with DAD detector.
2.2. Chemical synthesis and characterization

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1,4,8,11,15,18,22,25-Octakis[(2-propoxy)phthalocyanine] zinc(II) (Pc-1)
Phthalonitrile derivative 1 was obtained according to the literature procedure [27].
Phthalonitrile 1 (1.952 g, 8 mmol), zinc acetate (826 mg, 4.5 mmol), DBU (1.34 mL, 9 mmol)
were stirred in n-pentanol (5 mL) at 140 °C for 20 h. After that time, the solvent was
evaporated and dry residue was chromatographed. Phthalocyanine Pc-1 was isolated using
silica gel (dichloromethane : methanol; 100:1) and repurified using reverse phase column
chromatography (methanol : dichloromethane; 7:2) to give a solid green (260 mg, 12.5%
yield). TLC Rf = 0.47 (dichloromethane : methanol, 10:1); UV–Vis, λ(ε); DMF: 325 nm
(4.13), 408 (4.00), 746 nm (4.64); DMSO: 325 (4.05), 405 (3.88), 750 nm (4.52); MS
1
(MALDI) m/z 1040 [M+H]+; H NMR (400 MHz, Pyr) δ (ppm) 7.77 (s, 8H, ArH), 5.33 (m,
3
8H, OCH), 1.73 (d, 48H, J= 6.0 Hz, OCH(CH3)2; 13C NMR (100 MHz, Pyr) δ (ppm) 153.34,
151.74, 130.53, 123.15, 75.78, 23.54; Chemical purity assessed by HPLC method: 97.35–
99.04% (Supplementary data). Crystal Data for Pc-1: C61H69N9O8Zn (M =1121.62 g/mol):
monoclinic, space group P21/c, a = 11.2922(2) Å, b = 17.0375(3) Å, c = 29.5576(5) Å,
β = 98.909(2)°, V = 5618.01(17) Å [28], Z = 4, T = 100.15 K, μ(MoKα) = 0.501 mm-1,
Dcalc = 1.326 g/cm3, 38236 reflections measured (5.642° ≤ 2Θ ≤ 51.364°), 10616 unique
(Rint = 0.0349, Rsigma = 0.0488) which were used in all calculations. The final R1 was 0.0365
(I > 2σ(I)) and wR2 was 0.0847 (all data).
2.3. X-ray studies
Green single crystals of Pc-1 (CCDC 1815755) were grown by slow evaporation of
pyridine solution at room temperature. The diffraction intensities were collected at 100 K with
an Oxford Diffraction Xcalibur diffractometer using fine focus Mo Enhance X-ray source.
The data were processed using the CrysAlisPro software [29]. The structure was solved by
direct methods and refined by the full-matrix least-squares on F2 using SIR2014 [30] and
SHELXL-2016 [28] programs, respectively. Non-hydrogen atoms were refined
anisotropically. All H-atoms were placed at idealized positions and included in the refinement
as riding on their pivot atoms with Uiso(H) = 1.2eq(C) or 1.5eq (CH3). Summary of the Pc-1
structure determination is given in Tab. S1. The asymmetric unit with atom labeling scheme is
shown in Fig. S1.
2.4. Optical study
2.4.1. Singlet oxygen generation measurements

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Singlet oxygen generation assay was performed according to the procedure described in
details previously [31–33]. Irradiation was performed at a wavelength corresponding to the Q-
band maximum of studied Pc-1. Experiments were carried out at ambient temperature under
air conditions in DMF and DMSO solutions.
2.4.2. Photostability determination
Evaluation of photodegradation processes was performed following the method
described before [31,32,34]. Experiments were carried out at ambient temperature under air
conditions in DMF and DMSO.
2.4.3. Emission study
Steady-state fluorescence study was performed according to the method described
before [33-35]. Experiments were carried out at ambient temperature under air conditions in
DMF and DMSO. Time-resolved fluorescence studies were carried out in anhydrous DMF at
the same conditions using a home-built experimental setup based on a femtosecond laser
source (Spectra Physics MaiTai) tunable in the range of 690–1020 nm and a Time-Correlated
Single Photon Counting (TCSPC) module HydraHarp400 from PicoQuant. Details of the
experimental setup are presented in the Supplementary data.
2.4.4. Lipid vesicles preparation
Lipid vesicles were prepared according to the method described by Dragicevic-Curic
and co-workers [35]. These systems consist of phospholipids supplied by Avanti Polar Lipids
Inc. (Alabaster, AL, USA): POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and
DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, 25 mg/mL).
POPC and DOTAP dissolved in chloroform were mixed in molar ratio 8:2. Next, chloroform
solution of Pc-1 was added to lipids mixture in chloroform. The obtained mixture was taken
under reduced pressure at 30 °C and chloroform was evaporated. In the next step, 3.3% w/v of
ethanol was added to the dried residue and the mixture was vortexed for 5 min. Then, sterile
phosphate-buffered saline (PBS) was added to the mixture and subjected to additional 5 min
of vortexing. The final stage was the extrusion of formed lipid vesicles through polycarbonate
membranes of 200 nm pore size with syringe extruder (Avanti Polar Lipids) (21 times).
Formed unified vesicles were stored in a refrigerator at 2–8 °C and protected from light.
Vesicles size and their distribution were measured with Zetasizer Nano ZS (Malvern).
2.4.5. In vitro photodynamic activity against bacteria
2.4.5.1. Bacterial cultures

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Enterococcus faecalis and Escherichia coli strains were purchased from American
Type Culture Collection (ATCC; E. faecalis ATCC 29212; E. coli ATCC 25922). Tested
strains were cultured aerobically in BHI broth at 36°C ± 1°C for 18–20 h. Then,
microorganisms were harvested by centrifugation (3000 rpm for 15 min), re-suspended in 10
mM phosphate buffered saline (PBS, pH = 7.0) and diluted in PBS to a final concentration of
ca. 107 colony forming units (CFU)/mL.
2.4.5.2. Dark activity
Aliquots of a standardized microbial suspension were placed in the wells of a
microtitration plate, then solutions containing different concentrations of the studied
compound incorporated in the lipid vesicles formulation were added to each well except
controls and bacteria, and incubated for 20 min. Bacterial suspension from each well, after
dilution, was grown on tryptic soy agar (TSA) plates. After the incubation period (20 h at
36°C ± 1°C), the viability of bacteria was calculated by counting the number of colony
forming units (CFUs). The log reduction of living bacteria in each sample was determined.
Experiments were performed in triplicates.
2.4.5.3. Light-dependent activity
Light activity evaluation was performed similarly to the dark one, with irradiation of
the culture after incubation time of 20 min. Bacteria were irradiated at room temperature with
high power LED MultiChip Emitters (60 high-efficiency AlGaAs diode chips, Roithner
LaserTechnik GmbH, Vienna, Austria) with maximum wavelength 735 nm at a fixed total
light dose 30 J/cm2.
2.5. Microtox® analysis
Experiments have been performed according to 81.9% Microtox® test protocol with
Microtox Model 500 analyzer and MicrotoxOmni software (Modern Water, Inc.) [36,37].
3. Results and discussion
Phthalocyanine derivative Pc-1 (Fig. 1) was synthesized according to a two-step
procedure. Firstly, 3,6-dihydroxyphthalonitrile was treated with 2-bromopropane in the
presence of potassium carbonate as a base to give phthalonitrile derivative 1 following the
literature method [27]. Compound 1 was subjected to the macrocyclization reaction with zinc
acetate and 1,8-diazabicyclo(5.4.0)undec-7-ene as a base in n-pentanol to give phthalocyanine

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derivative Pc-1 (13% yield). The identity of the obtained compound was confirmed using
UV-Vis spectrophotometry, MS MALDI spectrometry, and NMR spectroscopy.
O
O
O
O
O
O
O
O
N
N
N
N
N
N
Zn
N
N
N
N
O
O
1
Pc-1
Fig. 1. Synthesis of Pc-1: (i) Zn(OAc)2, DBU, n-pentanol, reflux
X-ray structural analysis revealed that zinc(II) phthalocyanine Pc-1 crystallizes in the
space group P21/c with one molecule per asymmetric unit. The central Zn(II) cation is situated
0.3800(8) Å from the N4-plane formed by four aza-nitrogens and exhibits distorted square-
pyramidal coordination geometry being coordinated by the isoindole nitrogen atoms and the
N atom of the apical pyridine ligand. Zn—N distances range from 2.017(1) to 2.031(2) Å
(Tab. S2). Phthalocyanine Pc-1 is highly overcrowded due to eight non-peripheral 2-propoxy
substituents at the isoindole groups resulting in a strong non-planarity of the -system of the
Pc-1 core (Fig. 2). Diverse orientations of 2-propoxy groups relative to the heterocycle reduce
the highest possible C4 symmetry of the molecule to C1. A steric hindrance between the
neighboring 2-propoxy groups is released mainly by the twist along the C-N bonds connecting
the isoindole units and the distortion of planar isoindole system (combination of a propeller-
like distortion and a bending of the fused rings). The Pc-1 core adopts a saddle-like shape
with the isoindole units A/C and the units B/D tilted in the opposite directions of the aza-N4-
plane. The dihedral angles between the planes of the pyrrole rings A and C, and B and D, are
21.4(1) and 11.9(1), respectively. Analysis of Pc-1 supramolecular architecture reveals that
in the absence of functional groups able to form strong intermolecular interactions only weak
forces stabilize the 3D crystal structure. Zig-zag chains are formed by - stacking
interactions between virtually coplanar six-membered isoindole A and the axial pyridine rings
of the two fold screw axis related molecules. The distance between ring centroids is 3.52 Å.
Parallel chains are arranged into layers perpendicular to the cell c axis (Fig. S2). Weak intra-
and interlayer C-H…O interactions between 2-propoxy substituents stabilize the 3D crystal
network.

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Fig. 2 Out-of-plane distortion of Pc-1 core. 2-Propoxy substituents are drawn in
wireframe style. Hydrogen atoms are omitted for clarity
UV-Vis absorption spectra of the studied phthalocyanine in various solvents reveal two
characteristic bands, first in the blue region and the second in the red one (Fig. 3c). Shape and
bands intensities are typical for phthalocyanine derivatives previously described [31,38,39].

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Fig. 3. UV-Vis absorption and emission spectra recorded for Pc-1: (a) photostability
measurements in DMF; (b) singlet oxygen generation study with DPBF as a chemical
quencher in DMSO; (c) fluorescence measurements in DMSO.
Steady-state emission properties of the studied phthalocyanine were investigated in DMF
and DMSO. The fluorescence spectrum profile is a mirror image of that one for absorption
(Fig. 3c). It suggests that geometry of an excited molecule does not differ from a molecule in
the ground state. Pc-1 shows very low quantum yield of fluorescence, reaching the value of
ΦFL = 0.005 and 0.004 in DMF and DMSO, respectively. Low values of fluorescence
quantum yield of Pc-1 are reflected in its sub-nanosecond fluorescence lifetimes, e.g. the
fluorescence decay recorded in DMF (Fig. 4a) can be quite well fitted with a

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monoexponential function with a decay time of 0.67  0.02 ns. However, the residuals
obtained for the mono-exponential fit are apparently structured (Fig. 4b). A biexponential fit
results in completely flat residuals (Fig. 4c) and decay times equal to 0.75  0.03 ns
(normalized pre-exponential factor 0.62) and 0.44  0.03 ns (normalized pre-exponential
factor 0.38).
Nevertheless, the very small difference in the quality of the mono- and biexponential
fits suggests that the structure of residuals of the monoexponential fit results not from the
presence of two distinct fluorescence decay components of different fluorophores, but rather
from a distribution of fluorescence lifetimes of a single chemical species. The distribution can
be due to different rotamers with respect to the orientation of 2-propoxy substituents.
Presence of aggregates in the sample, which could result in the appearance of the shorter
decay component, was excluded by fluorescence lifetime measurements in DMF with various
concentrations of pyridine. It is therefore justified to identify the fluorescence lifetime of Pc-1
in DMF with the decay time obtained from the monoexponential fit. A more precise approach
is the calculation of the fluorescence lifetime as a weighted average of two decay times of the
biexponential fit function (with the fractional intensities of the decay components as weights),
[37], equal to 0.63  0.03 ns (Tab. 2).
Fig. 4. (a) Fluorescence decay of Pc-1 in DMF solution recorded with excitation at
740 nm and detection at 760 nm; Residuals of fits of the fluorescence decay of Pc-1: (b)
monoexponential; (c) biexponential.

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Previous studies on the non-peripherally alkoxy-substituted phthalocyanines have
revealed that the macrocycle core can be easily protonated [40–46]. The above-mentioned
observation applies to Pc-1 as well. Titration of Pc-1 in DMF solution with sulfuric acid leads
to a gradual conversion of the non-protonated molecule into its monoprotonated form and
subsequently into the trans-diprotonated form, seen as the origin of new broadened and red-
shifted absorption bands (Fig. 5). Two pronounced isosbestic points, at 773 and 845 nm, can
be distinguished in the spectral evolution (Supplementary data, Fig. S3 a, b), which indicates
that the protonation of the molecule within the acid concentrations in the range of
41 μM – 56 mM is not accompanied by other processes. Further addition of the acid resulted
in decomposition of the phthalocyanine. Therefore its tri- and tetraprotonated forms could not
be obtained.
Fig. 5. Absorption spectra of Pc-1 in DMF solution recorded during titration with
sulfuric acid. Spectra that can be attributed to mono- and diprotonated forms recorded at acid
concentrations shown are printed with thick red and blue lines, respectively. All spectra are
normalized to the maximum of the absorption of the non-protonated form (green).
Analysis of the absorption spectra obtained as a result of the titration according to the
Hill formalism [42,47], following the scheme described in detail in reference [43], allows for
estimation of the formation constants of the mono- and diprotonated forms, log K1 = 4.8, log
K2 = 2.8, respectively (corresponding Hill plots are shown in Fig. S3 c, d). Value of log K1 is
significantly larger than obtained earlier for analogues of Pc-1 with longer alkoxy chains [43]
indicating stronger tendency for protonation. This effect can be explained by a more electron-
donating character of 2-propoxy groups than that of 1,4,7-trioxanonyl and n-butoxy
substituents, resulting in higher reactivity of the Pc-1 macrocycle than of the other molecules.
This trend is not maintained for log K2 which for Pc-1 is slightly smaller than the value
obtained for its n-butoxy analogue but is significantly larger for the trioxanonyl analogue.

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This could be as a result of a disturbance of the macrocycle by monoprotonation, whose effect
prevails over small differences in the electron-donating character of the 2-propoxy and n-
butoxy substituents. Nevertheless, the effect of the monoprotonation does not override the
much larger difference between the electron-donating ability of trioxanonyl and n-propoxy
substituents.
Protonation of Pc-1 significantly shortens the fluorescence lifetime due to the increase
in the non-radiative deactivation rate, according to the energy gap law [48] in protonated
molecules (Fig. 6). Fluorescence decays, measured in different conditions, including acid
concentration, excitation/detection wavelengths, in which the monoprotonated form should
bring the main contribution, are dominated by the fast component (0.36 ns) with only a small
(normalized pre-exponential factor 0.03) contribution of the longer one (0.78 ns). The former
decay time should be identified with the fluorescence lifetime of the monoprotonated form,
whereas the latter most probably results from the presence of a small population of non-
protonated molecules or reflects a small distribution of the fluorescence lifetimes, as in the
case of non-protonated molecules. Fluorescence decay recorded for the diprotonated form is
clearly monoexponential with a decay time shortened down to 0.14 ns. Summary of the
fluorescence lifetimes of the protonated forms of Pc-1 is shown in Tab. 2 and plots of the
residuals are shown in supplementary data – Fig. S4.
Fig. 6. Fluorescence decays of mono- [HPc-1]+ and diprotonated [H2Pc-1]2+ forms of Pc-1,
compared with the fluorescence decay of the parent molecule, recorded at conditions
indicated in Tab. 1.

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Tab. 1. Fluorescence lifetimes of Pc-1 and its protonated forms measured in DMF solution at
indicated excitation and detection wavelengths
Reduced 2
Acid
conc.
Excitation
wavelength wavelength
Detection
Decay
times (ns)
(nm)
(nm)
0
740
760
Monoexp.
Pc-1
0.67±0.02
1.77
1.34
Biexp.
0.75±0.03
(0.62)
0.44±0.03
(0.38)
Average
0.63±0.03
0.36±0.01
(0.97)
0.78±0.02
(0.03)
[HPc-1]+
790
890
830
910
1.43
1.14
41 M
[H2Pc-1]2+
56 mM
0.14±0.01
Singlet oxygen generation study showed the moderate potential of Pc-1 in singlet
oxygen production. The changes recorded in the spectra of Pc-1 and DPBF mixture are
presented in Fig. 3b. For the photodynamic therapy, the formation of singlet oxygen is crucial
[22]. Quantum yields of singlet oxygen generation calculated for Pc-1 were equal to 0.18 and
0.16 in DMF and DMSO, respectively (Tab. 2).
Tab. 2. Photodynamic parameters, antibacterial activity and size of lipid vesicles for studied
zinc phthalocyanine Pc-1
reduction in
Size of lipid
photochemical properties
bacterial growth
[log]
vesicles
30
Mean
Enterococ
cus
faecalis
3.06 ·
10
0.1
8
0.00
5
J/c 5. diamet PDI
DMF
DMF
DMF
m2
7
er ± SD index
[nm]
-4
Φ
Φ
Φ
F
735
nm
100
µM
Δ
P
L
1.23 ·
DMS 0.1
DMS
O
DMS 0.00 Escherich
a coli
0.
5
-5
O
6
O
4
10
174±4
0.23

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Also, the "heavy atom effect" was observed comparing the singlet oxygen quantum
yield for Pc-1 with the results of our group's latest published work on magnesium
phthalocyanine containing 2-propoxy substituents at non-peripheral positions. The
magnesium derivative showed singlet oxygen quantum yield ΦΔ = 0.03 and 0.04 in DMSO
and DMF, respectively [49]. An introduction eight of 2-propoxy groups to the structure of
phthalocyanine (Pc-1) results in lowering the quantum yield values in comparison to
unsubstituted zinc(II) phthalocyanine (ΦΔ values equal 0.56 in DMF and 0.67 in DMSO). The
above phenomenon can be related to an increasing non-radiative deactivation due to the
presence of alkyl and alkoxy substituents in the structure [50].
During irradiation of Pc-1 dissolved in DMF or DMSO with visible light, the intensity
of their absorption spectra decreased (Fig. 3a). Moreover, a formation of an additional band
was not noticed. It suggests that photodecomposition phenomenon reveals photobleaching
pathway. For Pc-1, it was assessed that the quantum yields of photodecomposition (ΦP)
processes equal 3.1·10-4 and 1.2·10-5 in DMF and DMSO, respectively. The photostability of
the studied phthalocyanine Pc1 is comparable with the stability of peripherally substituted
phthalocyanines with oxy moieties presented by Chauke and co-workers (I, II; Fig. 7) [33].
Earlier our group studied trioxanonyl zinc phthalocyanine (III; Fig. 7) [22], which in
comparison with Pc-1 revealed 2.7-fold better photostability in DMF. Interestingly, Pc-1
dissolved in DMSO showed the same photostability level as [1,4,8,11,15,18,22,25-
octakis(1,4,7-trioxanonyl)phthalocyanine] zinc(II) (III) previously studied in the same solvent
[43].
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
Cl
Cl
O
O
O
O
O
O
O
O
Ga
Ga
Mg
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
I
II
III
Fig. 7. Chemical structures of phthalocyanines studied by Chauke and co-workers – I,
II [33]; Sobotta and co-workers – III [22]
For photodynamic inactivation of bacteria, novel phthalocyanine Pc-1 was
incorporated into lipid vesicles containing ethanol in relatively low concentration. According

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to the literature, many types of lipid vesicles embodying ethanol have been considered so far
[25]. The amount of ethanol in lipid formulation is known to influence many features of
vesicles, including diameter and skin penetration potential. Therefore in many literature
studies, ethanol has been used in lipid mixtures in various concentrations [51]. On the one
hand, vesicular systems, known as ethosomes, containing ethanol in relatively high
concentrations (20-50%) revealed enhanced skin permeation to many active pharmaceutical
ingredients. On the other hand, invasomes containing permeability enhancers, such as
terpenes and ethanol at lower concentrations (typically from 0% to 3.3%) were also efficient
in delivering of pharmaceutically active ingredients [25]. Invasomes in comparison to
liposomes are more flexible and enable the delivery of drug molecules more effectively. Some
initial studies have been conducted showing the effectiveness of invasomes for PACT [26].
As a model bacteria to in vitro photodynamic inactivation tests, Enterococcus faecalis as a
Gram-positive bacteria and Escherichia coli as a Gram-negative were chosen. E. faecalis is
known as the main problem in dental pulp infections. It is a facultative anaerobe, and
therefore it does not suffer from a lack of oxygen [52,53]. Usually in dental practice sodium
chlorate(I) (NaClO) is applied as a bactericidal drug [54]. It was reported that the dental
tubules can be penetrated by E. faecalis up to 300 – 1000 µm, whereas NaClO reaches only
inside to 150 µm. Unfortunately, this phenomenon enables the bacteria to survive the
treatment procedure [55–57]. It was found that the treatment of dentine tubules with
invasomes can be considered as an alternative approach for the conventional endodontic
treatment procedure [35,57]. Because of rising bacterial resistance in conventional treatment,
the development of novel techniques for bacteria eradication is becoming increasingly
important. It should be pointed out that some Enterococcus faecalis strains reveal resistance
to β-lactams (cephalosporins), clindamycin, macrolides, sulfonamides and may reveal
resistance to other antibiotics, e.g. glycopeptides (vancomycin), tetracyclines [58–60]. In our
study, researched Pc-1 in lipid vesicles containing the optimal concentration of ethanol
inactivated E. faecalis at the concentration of 100 µM, when excited with 735 nm light at light
dose 30 J/cm2. For mentioned dosimetry, the number of viable cells were reduced to the level
of 5.7 log.
Escherichia coli was studied as an example of Gram-negative bacteria, which is
present in the gastrointestinal tract in humans. Commonly E. coli exhibit a positive influence
on the human digestive process. Besides, there are known E. coli strains with specific
virulence, which can cause diseases including enteric/diarrhoeal problems, urinary system
infections or even sepsis[61]. As it was expected, studied Pc-1 revealed no significant

ACCEPTED MANUSCRIPT
photoinactivation ability against E. coli. It can be connected with a lack of positive charges on
the periphery of the molecule, which is more important for photodynamic inactivation of
Gram-negative than positive bacteria [62].
As an extension of our study, 81.9% screening test with Microtox® system was
performed, which allowed us to determine the dark toxicity of the studied compound.
Microtox® system is based on bioluminescence measurement of Vibrio fischeri, which under
exposition to a toxic agent dies and loses its bioluminescence ability [36,37,63]. Studied
phthalocyanine Pc-1 has revealed very low level of dark toxicity with luminescence inhibition
at 7.9% after 5 min exposition and 5.3% after 15 min.
4. Conclusions
In this paper, we presented the synthesis of novel zinc(II) phthalocyanine bearing
eight 2-propoxy substituents (Pc-1). Phthalocyanine macrocycle Pc-1 (Fig. 1) was
synthesized according to a two-step procedure, starting from 3,6-di(2-propoxy)phthalonitrile.
The obtained phthalocyanine was characterized by UV-Vis spectrophotometry,
spectrofluorimetry, MS MALDI spectrometry and NMR spectroscopy. As shown by X-ray
crystallography, a steric hindrance between eight non-peripheral 2-propoxy substituents leads
to distortion of the planar π-electron system of the macrocycle core. The UV-Vis absorption
spectra in various solvents of the studied phthalocyanine reveal two characteristic bands, first
in the blue region and second in the red one. The bands shapes and intensities in the UV-Vis
are typical of phthalocyanine derivatives. Low values of fluorescence quantum yield of Pc-1
are reflected in its sub-nanosecond fluorescence lifetimes.
Titration of the studied phthalocyanine with sulfuric acid in DMF solution led to a
gradual conversion of the non-protonated molecule into its monoprotonated form and
subsequently into the trans-diprotonated form. Further addition of the acid resulted in
decomposition of the phthalocyanine. Therefore its tri- and tetraprotonated forms could not be
obtained. Moreover, the usability of PACT against bacteria was assessed. The presented
phthalocyanine studied, has revealed moderate singlet oxygen generation ability equal to 0.18
and 0.16 in DMF and DMSO, respectively. Moderate photostability has been noticed with
quantum yield of photodecomposition equal 3.1·10-4 and 1.2·10-5 in DMF and DMSO,
respectively. Also, the phthalocyanine studied revealed the low potential for fluorescence
emission with yields 0.005 and 0.004 in DMF and DMSO, respectively. In vitro PACT
biological study indicates moderate inactivation of Enterococcus faecalis equal to 5.7 log

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reduction in bacterial growth and no activity against Escherichia coli at 100 μM for lipid
vesicles embodying zinc(II) phthalocyanine bearing eight 2-propoxy substituents.
Acknowledgments
This work was supported by the National Science Centre, Poland under Grant No.
2016/21/B/NZ9/00783.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
The crystallographic data in CIF format have been deposited at the Cambridge
Crystallographic Data Centre, CCDC 1815755. They can be obtained free of charge via
https://www.ccdc.cam.ac.uk/structures/

ACCEPTED MANUSCRIPT
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Graphical abstract
Zinc(II) phthalocyanine with eight non-peripheral 2-propoxy substituents was synthetized. X-ray
analysis indicated that overcrowding of the compound leads to a strong out-of-plane distortion of the
π-system of the macrocycle core. Although compound excited with red light formed singlet oxygen
with a moderate yield, it reduced Enterococcus faecalis growth by 5.7 log.

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Highlights
 Zinc(II) phthalocyanine bearing eight non-peripheral 2-propoxy substituents was
synthetized
 The presented zinc(II) phthalocyanine showed ability for singlet oxygen generation
 X-ray analysis revealed that overcrowding of phthalocyanine leads to strong out-of-
plane distortion of macrocycle π-system
 In vitro bacteria photoinactivation study revealed the high potential of researched
macrocycle against Enterococcus faecalis