Enhanced photo-ablation effect of positively charged phthalocyanines-detonation nanodiamonds nanoplatforms for the suppression of Staphylococcus aureus and Escherichia coli planktonic cells and biofilms

Article abstract of DOI:10.1016/j.jphotochem.2021.113200

Photodynamic antimicrobial therapy (PACT) is a powerful technic recommended to eliminate life-threatening pathogens that cause localized and superficial infections as pathogens cannot develop resistance to it. For this reason, new positively charged chalc

Full text of DOI:10.1016/j.jphotochem.2021.113200

Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Enhanced photo-ablation effect of positively charged
phthalocyanines-detonation nanodiamonds nanoplatforms for the
suppression of Staphylococcus aureus and Escherichia coli planktonic cells
and biofilms
Yolande Ikala Openda, Tebello Nyokong*
Institute for Nanotechnology Innovation, Department of Chemistry, Rhodes University, Makhanda 6140, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords:
Photodynamic antimicrobial therapy (PACT) is a powerful technic recommended to eliminate life-threatening
pathogens that cause localized and superficial infections as pathogens cannot develop resistance to it. For this
reason, new positively charged chalcone substituted zinc (3a) and indium (4a) metalated phthalocyanines (Pcs)
Detonation nanodiamonds
Chalcone phthalocyanines
Biofilms
were synthesized and were
water soluble nanoplatfoms 3a@DNDs and 4a@DNDs. The conjugates generated high singlet oxygen quantum
yields (Φ ) in water (1% DMSO, used for PACT studies) with values of 0.46 and 0.47 for 3a@DNDs and
a@DNDs, respectively. Hence, they were tested for PACT against biofilms of S. aureus and E. coli, as well as their
π
-π interacted with detonation nanodiamonds (DNDs) nanoparticles to form new
Planktonic
Photodynamic antimicrobial therapy
Δ
4
planktonic cells. The quaternized Pcs alone 3a and 4a as well as their nanoconjugates 3a@DNDs and 4a@DNDs
were effective PACT agents with log10 CFU > 9 for E. coli and S. aureus. The quaternized derivatives were found
to have higher ability to completely suppress both planktonic and biofilms of S. aureus and E. coli in vitro.
Therefore, they could be used as appropriate photosensitive agents.
1
. Introduction
Up to 60 % of severe infection cases leading to deaths globally are
efficacy of PACT [8,10]. PACT does not target a single site in bacteria,
unlike conventional antibiotics. ROS target various bacterial cell struc-
tures and different metabolic pathways [11]. Thus, bacteria do not
readily develop resistance to PACT [12].
attributed to bacterial biofilms. Free pathogenic bacteria cells that
adhere to a living or inert surface tend to form a bacterial biofilm
structure [1,2]. It is much more difficult to eradicate pathogenic bacteria
living within a biofilm as they easily develop resistance to the available
antibiotic drugs and treatments [3–5]. Additionally, bacteria in biofilms
can tolerate 10–1000 times higher dosage of antibiotics than their
planktonic forms (free bacteria cells) [6]. Therefore, the major challenge
remain to develop new antibiofilm approaches to fight against bacteria
biofilms, either by biofilm structure disruption or suppression of biofilm
Several studies have also indicated that the photoinactivation of
Gram-negative bacteria (i.e. E. coli) is quite challenging because of the
complexity in the composition of their cell wall as compared to Gram-
positive bacteria (i.e. S. aureus) [13]. Therefore, it is agreed that a
good PS should also combine characteristics such as hydrophilicity and
positive charge(s) for attachment towards negatively charged bacterial
cell walls, but also to prevent aggregation in aqueous medium and
improve cell uptake [14–17]. Metallophthalocyanines (MPcs) are
known PACT photosensitizers for planktonic cells and biofilms [18–20].
Increasing the number of positive charges on the substituents enhances
the extent of phthalocyanine binding to gram-negative bacteria such as
E. coli [21]. Thus, the development of new polycationic phthalocyanines
is needed [22] as they show successful PACT activities and high affinity
to the bacteria cells.
[
7]. Amongst many techniques based on the generation of singlet oxy-
gen, there is photodynamic antimicrobial chemotherapy (PACT).
The photodynamic effect consist of a non-toxic photosensitizer (PS)
that is activated by visible light of appropriate wavelength in the pres-
ence of oxygen leading to the production of reactive oxygen species
(
ROS) including singlet oxygen that can cause cell death [8,9]. Con-
centration, nature and spectral properties of a PS are crucial in the
Hence in this work, we synthesize new pyridine chalcone
*
Corresponding author.
E-mail address: t.nyokong@ru.ac.za (T. Nyokong).
https://doi.org/10.1016/j.jphotochem.2021.113200
Received 14 September 2020; Received in revised form 1 February 2021; Accepted 6 February 2021
Available online 12 February 2021
1
010-6030/© 2021 Elsevier B.V. All rights reserved.

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
tetrasubstituted zinc and indium Pcs and their quaternized (positively
charged) Pc derivatives. Chalcone substituted MPcs have been reported
for photodynamic therapy (PDT) of cancer, which is similar to PACT
2. Experimental
Materials and equipment items used in this work can be found in the
Supporting Information (SI).
[
23,24] and other applications [25,26]. The use of chalcone substituted
MPcs for PACT is reported in this work for the first time. In addition, the
pyridine chalcone substituent on MPcs allows for quaternization, mak-
ing the complexes water soluble. Chalcone derivatives are known for
their antibacterial activities [27], hence they can enhance PACT activ-
ities of the MPcs, by synergetic effect. In addition, chalcones are known
for their vascular disrupting effect in PDT, which destroy the tumor’s
neovasculature, leading to tumor starvation and subsequently to tumor
death by necrosis [24].
2.1. Synthesis
2.1.1. (E)-1-(4-hydroxyphenyl)-3-(pyridin-4-yl)prop-2-en-1-one (1),
Scheme 1
A solution of 4-hydroxyacetophenone (1 g, 7.3 mmol) and 4-pyridi-
necarbaxaldehyde (0.7867 g, 7.3 mmol) in ethanol (20 mL) was added
◦
drop-wise to a stirred solution of 30 % KOH cooled at 0 C in an ice bath
To further extend the range of nanotechnology applications in
biomedical fields, we used detonation nanodiamonds (DNDs) to make
nanoconjugates with MPcs. DNDs have been used recently in fighting
infectious diseases and biofilm formation and have proven to be a
possible alternative to common antimicrobials [5]. Nanodiamonds have
been widely used in many biomedical fields such as bactericides, wound
dressing agents and as components in different drug delivery matrices,
under argon atmosphere. The reaction mixture was kept at room tem-
perature for 24 h and the completion of the reaction was monitored by
thin layer chromatography (TLC) using hexane:ethylacetate (2:1, v/v).
At the completion, the reaction mixture was poured into iced water and
adjusted to neutral pH with 1 M HCl then the precipitate was filtered
out. The yellow powder product was obtained by recrystallization from
ethanol yielding 65.23 % of the desired product.
max/cm 1: 3140 (OH), 3050 (Ar C
ꢀ
etc. [28]. The presence of
π
electrons on DNDs allows for
π
-
π
interactions
IR (UATR-TWO™)
ν
–
H and
with other
this work.
π
containing molecules such as MPcs, and this is applied in
intermolecular H bonds), 2917
–
–
–
(Alph C
–
–
H), 1658 (C
C). H NMR (600 MHz, DMSO- d
8.65 (d, J =6 Hz, 2 H), 8.12 (d, J =16 Hz, 1 H), 8.09 (d, J =9 Hz, 2 H),
O), 1586 (C
–
C), 1570 (C
N), 1509ꢀ 1419
1
Thus, the objectives of the current work is to prepare a nanoplatform
composed of water soluble and positively charged Pcs and DNDs that can
display inhibition activity against planktonic cells and biofilms of both
gram-positive bacteria (e.g. S. aureus) and gram-negative bacteria (e.g.
E. coli) using photodynamic antimicrobial therapy method.
(C
6
–
OH),
1
3
7.81 (d, J =6 Hz, 2 H), 7.61 (d, J =16 Hz, 1 H), 6.93 (d, J =6 Hz, 2 H).
NMR (600 MHz, DMSO- d
C
6
): δ (ppm): 187.3, 162.9, 150.6, 142.3, 140.1,
131.7, 128.9, 126.8, 122.7 and 115.8. MALDI TOF MS m/z: Calcd:
+
2
25.24 Found: [M+H] = 226.39.
◦
Scheme 1. The synthesis route of (1), the dinitrile compound (2) and novel phthalocyanines (3, 3a, 4 and 4a). Reaction conditions: (i): KOH, EtOH, at 25 C, Ar; (ii):
◦
anhydrous K
2
CO
3
, anhydrous dry DMF, 4-nitrophthalonitrile, 60 C, Ar; (iii): dry DMAE (for 3 and quinoline for 4), DBU and anhydrous zinc acetate or indium
◦
chloride at 160 C, Ar; (iv): dry DMF, acetone and excess CH
3
I at reflux.
2

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
2
.1.2. Synthesis of (E)-4-(4-(3-(pyridin-4-yl)acryloyl)phenoxy)
and dried under reduced pressure.
ZnPc (3a): Yield: 0.048 g (92 %). IR (UATR-TWO™)
max/cm 1:
–
–
O), 1583ꢀ 1490
ꢀ
phthalonitrile (2)
ν
4
-Nitrophthalonitrile (0.369 g, 2.1 mmol) and compound 1 (0.400 g,
–
3055 (Ar-H), 2928ꢀ 2855 (Aliph C H), 1721 (C
–
–
–
– –
1
.8 mmol) were dissolved in 15 mL of N,N- dimethylformamide (DMF)
(C
1086 (Sym., Ar
8.97ꢀ 8.95 (m, 8H, ArH-pyridine ring); 8.90 (d, 8H, ArH); 8.25 (d, 2H,
–
C, C
–
N), 1411ꢀ 1362 (C C), 1228ꢀ 1164 (Asym., Ar O
Ar),
1
in a round-bottom flask and stirred under argon atmosphere. K
2
CO
3
O Ar), 832. H NMR (600 MHz, DMSO- d
– –
6
): δ (ppm):
(
0.368 g, 2.7 mmol) was added then the stirring was continued for 24 h
◦
–
–
at 60 C. The reaction mixture was afterwards poured into 50 mL iced
water. The obtained orange solid was filtered out and recrystallized in
ArH-Pc ring); 8.20 (d, 2H, ArH-Pc ring); 8.03 (d, 4H, CH
CH);
CH); 7.46ꢀ 7.44 (dd, 8H, ArH-
pyridine ring), 7.39 (d, 4H, ArH-Pc ring); 7.28 (d, 8H, ArH) and 2.90 (s,
12H, CH ). MALDI TOF MS m/z: Calcd: 1530.95; Found:
[M+H] = 1531.99. Anal. Calc. for [C92 Zn] (%): C, 72.18; H,
4.21; N, 10.98; Found (%): C, 72.26; H, 4.19; N, 10.94.
InPc (4a): Yield: 0.05 g (98 %). IR (UATR-TWO™) max/cm : 3056
–
–
7.96ꢀ 7.92 (m, 8H, ArH-Pc ring and CH
ethanol to obtain 2. Yield: 0.5175 g (83 % yield). IR (UATR-TWO™)
max/cm : 3068 (Ar C
ν
ꢀ
1
–
H and intermolecular H bonds), 2937 (Aliph
3
–
–
–
–
– –
–
–
+
C
–
H), 1667 (C
486ꢀ 1360 (C
Ar Ar), 802. H NMR (600 MHz, DMSO-d
–
O), 1583 (C
–
C), 1538 (C
–
N), 2231 (C
Ar), 1098 (Sym.,
): δ (ppm): 8.93 (d, J
N),
64 12 8
H N O
1
–
C), 1287ꢀ 1166 (Asym., Ar O
1
ꢀ 1
–
O
–
ν
6
–
H), 1722 (C
–
–
O), 1583ꢀ 1491 (C
–
–
C,
Ar), 1087
): δ (ppm):
8.97ꢀ 8.95 (m, 8H, ArH-pyridine ring); 8.90 (d, 8H, ArH); 8.25 (d, 2H,
=
=
2
8 Hz, 2 H), 8.88 (d, J =9 Hz, 2 H), 8.34 (d, J =8 Hz, 1 H), 8.03 (d, J
(Ar-H), 2925ꢀ 2856 (Aliph C
–
–
1
– –
16 Hz, 1 H), 7.95 (s, 1 H), 7.92 (d, J =16 Hz, 1 H), 7.44 (d, J =8 Hz,
C
–
N), 1413ꢀ 1362 (C C), 1228ꢀ 1164 (Asym., Ar O
1
3
H), 7.37 (d, J =8 Hz, 1 H) and 7.26 (d, J =9 Hz, 2 H). C NMR
(Sym., Ar O Ar), 832. H NMR (600 MHz, DMSO- d
– –
6
(
600 MHz, DMSO- d
6
): δ (ppm): 186.7, 162.8, 154.2, 151.8, 143.6,
–
–
1
1
31.6, 130.8, 130.7, 130.5, 129.2, 124.5, 1223, 116.3, 115.5, 115.4,
ArH-Pc ring); 8.20 (d, 2H, ArH-Pc ring); 8.03 (d, 4H, CH
CH);
CH); 7.46ꢀ 7.44 (m, 8H, ArH-
pyridine ring), 7.39 (d, 4H, ArH-Pc ring); 7.28 (d, 8H, ArH) and 2.90 (s,
2H, CH
). MALDI TOF MS m/z: Calcd: 1615.84; Found: [M+H]
64 ClInN12 ] (%): C, 68.38; H, 3.39;
–
–
11.8 and 111.1. MALDI TOF MS m/z: Calcd: 351.36; Found:
7.96ꢀ 7.92 (m, 8H, ArH-Pc ring and CH
+
[
M+H] = 352.14.
1
3
2
.1.3. Phthalocyanines (3, 4), Scheme 1
+ = 1616.39. Anal. Calc. for [C92
H
O
8
Under argon atmosphere, the dinitrile compound 2 (0.250 g,
.72 mmol) was dissolved in dry dimethylaminoethanol (DMAE, 3 mL)
N, 10.40; Found (%): C, 68.28; H, 3.30; N, 10.46.
0
and anhydrous zinc acetate (0.065 g, 0.354 mmol) was added for
phthalocyanine 3. For phthalocyanine 4, compound (0.250 g,
.712 mmol) was dissolved in dry quinoline (3 mL) and anhydrous in-
2.1.5. Conjugation of zinc phthalocyanines to detonation nanodiamonds
(DNDs), Scheme 2
2
0
The nanoconjugation of the Pc complexes as well as their quater-
nized derivatives to DNDs nanoparticles to form Pcs@DNDs nano-
dium chloride (0.472 g, 2.13 mmol) was added. Following this, a few
drops of DBU were added in each reaction mixture and these were
brought to reflux for 24 h. After completion, the mixtures were cooled to
room temperature, then ethanol was added and the reaction mixtures
were further stirred for 1 h. The obtained green crude products were
collected under centrifugation and were purified on silica gel using
conjugates was done through
π
-
π
stacking interactions accordingly to a
(0.010 g,
previously reported procedure [30] as follows: Pc
3
0.0068 mmol) or Pc 4 (0.010 g, 0.0064 mmol) or Pc 3a (0.010 g,
0.0065 mmol) or Pc 4a (0.010 g, 0.0062 mmol) were each dissolved in
dry DMSO (3 mL), then DNDs (5 mg) were added to each solution. The
mixtures were sonicated for 4 h, followed by stirring for 96 h at room
temperature. The resulting products were precipitated out by centrifu-
gation and washed repetitively with ethanol for the non-quaternized Pcs
and with acetone and THF for the quaternized Pcs, to remove the
unreacted Pcs and DNDs, then the products were air dried. The formed
nanoconjugates are represented as 3@DNDs, 3a@DNDs, 4@DNDs and
4a@DNDs.
chloroform: ethanol (9:1) as an eluting solvent mixture.
ꢀ
ZnPc (3): Yield: 0.187 g (36 %). IR (UATR-TWO™)
ν
max/cm 1
:
–
H), 2923ꢀ 2855 (Aliph C
–
H), 1721 (C
–
O), 1583ꢀ 1490
3
055 (Ar
–
–
–
– –
(
C
–
C, C
–
N), 1413ꢀ 1362 (C C), 1228ꢀ 1164 (Asym., Ar O
Ar),
): δ (ppm):
1
1
8
086 (Sym., Ar O Ar), 832. H NMR (600 MHz, DMSO- d
– –
6
.97ꢀ 8.95 (m, 8H, ArH-pyridine ring); 8.90 (d, 8H, ArH); 8.25 (d, 2H,
–
–
ArH-Pc ring); 8.20 (d, 2H, ArH-Pc ring); 8.03 (d, 4H, CH
CH);
–
7
.96ꢀ 7.92 (m, 8H, ArH-Pc ring and CH
–
CH); 7.46ꢀ 7.44 (dd, 8H, ArH-
pyridine ring), 7.39 (d, 4H, ArH-Pc ring) and 7.28 (d, 8H, ArH). MALDI
2.2. Photophysicochemical properties
+
TOF MS m/z: Calcd: 1470.81; Found: [M+H] = 1471.88. Anal. Calc. for
[
C
88
H
52
N
12
O
8
Zn] (%): C, 71.86; H, 3.56; N, 11.43; Found (%): C, 71.81;
Fluorescence (Φ ) quantum yields for both the Pcs and their nano-
F
H, 3.59; N, 11.49.
conjugates were determined by comparative methods as defined in
ꢀ
InPc (4): Yield: 0.300 g (27 %). IR (UATR-TWO™)
ν
max/cm 1: 3056
literature with equations shown in the supporting information. ZnPc
–
H), 1722 (C
–
O), 1583ꢀ 1491 (C
–
–
C,
dissolved in DMSO with reported values of Φ = 0.2 [31] was used as
(
Ar-H), 2923ꢀ 2856 (Aliph C
F
–
N), 1413ꢀ 1362 (C C), 1228ꢀ 1164 (Asym., Ar O
– –
–
C
–
Ar), 1087
): δ (ppm):
.97ꢀ 8.95 (m, 8H, ArH-pyridine ring); 8.90 (d, 8H, ArH); 8.26 (d, 2H,
standard.
1
(
Sym., Ar
–
O
–
Ar), 832. H NMR (600 MHz, DMSO- d
6
Singlet oxygen quantum yields (Φ , were determined using ZnPc (in
Δ
8
DMSO, Φ = 0.67 [32]) or AlPcSmix (in 1% DMSO aqueous media,
Δ
–
–
ArH-Pc ring); 8.20 (d, 2H, ArH-Pc ring); 8.03 (d, 4H, CH
CH);
Φ = 0.42 [32]) as references for non-quaternized and quaternized
Δ
–
–
7
.96ꢀ 7.92 (m, 8H, ArH-Pc ring and CH
CH); 7.46ꢀ 7.44 (dd, 8H, ArH-
products,
respectively.
Diphenylisobenzofuran
(DPBF)
and
pyridine ring), 7.39 (d, 4H, ArH-Pc ring) and 7.28 (d, 8H, ArH). MALDI
anthreacene-910-bis-methylmalonate (ADMA) were used as singlet ox-
ygen chemical quenchers in DMSO and aqueous media respectively.
DPBF and ADMA photobleaching effects were respectively monitored at
417 nm and 378 nm, respectively.
+
TOF MS m/z: Calcd: 1555.70; Found: [M] = 1555.26. Anal. Calc. for
88 8
[C H52ClInN12O ] (%): C, 67.94; H, 3.37; N, 10.80; Found (%): C,
6
7.96; H, 3.39; N, 10.84.
2
.1.4. Quaternized phthalocyanines (3a and 4a), Scheme 1
2.3. PACT studies
The quaternization of the Pcs was done following a procedure
described in literature with slight modifications [29] as follows: 3
0.050 g, 0.034 mmol) or 4 (0.050 g, 0.032 mmol) were each dissolved
in dry DMF (3 mL)/acetone (5 mL) solvent mixture. Excess iodomethane
4 mL) was added to the solution. The reaction mixtures were heated at
2.3.1. Antibacterial assays on planktonic cells
(
The gram positive Staphylococcus aureus and gram negative Escher-
ischia coli bacterial planktonic cells were prepared for PACT studies as
previously reported in literature [29,33,34] as follows: the inoculation
of an aliquot of each bacterial species was aseptically done in 5 mL of
freshly prepared nutrient broth, followed by an overnight cultivation in
(
reflux temperature for 72 h. The green products were precipitated out
with hot acetone and collected by centrifugation. Afterwards the prod-
ucts were washed with acetone, tetrahydrofuran (THF) and diethyl ether
◦
an incubator with shaker at 37 C. The individual bacterial inocula in the
3

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
Scheme 2. MPcs@DNDs nanoconjugates synthesis.
well of the plate, aqueous crystal violet solution (200 L of 1%) was
mid logarithmic phase of growth was stopped once they reached an
optical density (OD) value between 0.6ꢀ 0.8 recorded at 620 nm. Af-
terward, the bacteria cultures were harvested by centrifugation (4000
RPM for 15 min) and then washed with phosphate buffer solution (PBS).
The bacteria cultures were later diluted to 1/1000 in PBS to make
working stock solutions of approximately 109 colony forming units
μ
added and this was left to stain the biofilm for 30 min, then the optical
density was determined at 570 nm. Subsequently, PBS was used to rinse
the wells and remove the excess dye before air drying the plate [36,37].
In this experiment, the 96-well plates containing biofilms were
inoculated with Pcs alone or nanoconjugates (100
μ
L) at different con-
(
CFU) per mL for both type of bacteria. Photodynamic antimicrobial
centrations of 50 and 100 g/mL in triplicates. And the wells where Pcs
μ
chemotherapy studies on S. aureus and E. coli were done following
were not added, were considered as the control samples. After 30 min of
◦
literature with slight changes [33,35] where, for comparison purposes,
incubation time in the dark at 37 C, the biofilms were irradiated at
1
% DMSO in PBS solution was used as the solvent.
670 nm with the Modulight laser lamp (irradiance: 524 mV/cm and
dose: 943 J/cm) for 2 h with 30 min time intervals. After irradiation, the
The concentration optimization of the Pcs was performed by using
Pcs at different concentrations of 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and
suspensions were subjected to serial dilution using PBS, and 100 L of
μ
4
0
μ
g/mL. In all cases, firstly, solutions containing the photosensitizer
each sample was aseptically inoculated on agar plates then incubated for
◦
◦
and bacteria were incubated for 30 min in the dark at 37 C. Afterward,
18 h at 37 C to determine the number of colony-forming units per
2
.5 mL of the samples containing the photosensitizer and bacteria was
millilitre.
irradiated for 30 min at the 670 nm in 24 well plate using a Modulight
laser lamp (irradiance: 524 mV/cm and dose: 943 J/cm) while the other
2.3.3. Statistical analysis
2
.5 mL was kept in the dark for 30 min to determine dark toxicity ac-
Three independent (n = 3) experiments were conducted in triplicates
and the obtained data were compared using a 3-way factorial ANOVA.
The data are presented as means ± standard deviation (SD). A p-value of
0.05 was considered statistically significant.
tivity of the photosensitizers. For both the irradiated and non-irradiated
samples, 100
μ
L of each sample was aseptically inoculated on nutrient
◦
agar plates in triplicates, followed by incubation for 18 h at 37 C, at the
end of which the colony counting was done. The control samples con-
tained only bacteria and 1% DMSO in PBS without the drug [29].
For the time studies optimization, bacterial suspensions containing
the optimized concentration of Pcs were prepared and the antimicrobial
assays were done similarly as described above. In this case 2.5 mL of the
suspension was kept in the dark and 2.5 mL was irradiated for 120 min at
3
. Results and discussion
3
.1. Synthesis and characterization
The synthetic routes of compounds (1, 2, 3, 4, 3a and 4a) are shown
3
0 min irradiation intervals starting from 0 min. Afterwards, the dark
in Scheme 1. In the first step, compound 1 was synthesized following
Claisen-Schmidt condensation reaction of 4-hydroyacetophenone and 4-
pyridinecarbaxaldehyde [38] and in the second step, the synthesised
phthalonitrile 2 was obtained via base-catalyzed aromatic substitution
and irradiated treated suspensions were respectively inoculated on agar
and the number of the formed colonies on each petri dish was counted
◦
after a period of 18 h incubation at 37 C. A control experiment was also
performed as stated above for both the dark and irradiation studies. The
acquired CFU/mL data were then converted to the log reduction values
and percentage cell survival.
reaction of 1 under argon atmosphere using K
2
CO
3
as the base in dry
◦
DMF at 60 C for 24 h [39]. Phthalocyanines 3 and 4 were prepared by
cyclotetramerization reaction [40] of compound 2, in DMAE (for 3) or
quinoline (for 4) with few drops of DBU in the presence of anhydrous
zinc acetate and indium chloride salts, respectively, under argon at-
mosphere. For the quaternized derivatives 3a and 4a, MPcs 3 and 4 were
2
.3.2. Biofilm formation
The single-species biofilms of S. aureus and E. coli were prepared as
following: Freshly prepared suspensions of each bacterial species at 106
CFU/mL were individually suspended in tryptic soy broth to obtain a
3
respectively reacted with excess CH I in DMF and acetone at room
temperature to obtain positively charged and water soluble Pcs. The
7
1
concentration of 10 CFU/mL. From each newly prepared suspension,
structures of the synthesized compounds were analysed by FT-IR,
H
NMR, 13C NMR, MS and UV–vis techniques (see Figs. S1–S10, in the
Supporting Information, SI for NMR and mass spectra).
2
00
μ
L was removed and seeded in 96 well plates then incubated stati-
◦
cally and anaerobically at 37 C for 5 days to allow the cell adhesion to
the surface. Unbound planktonic cells were gently washed out using PBS
Compound 1 was obtained as yellow powder, with a protonated
+
and 200 L of fresh tryptic soy broth was added daily to stimulate bio-
μ
molecular ion peak [M+H] at m/z = 226.39 in the MS spectrum cor-
1
film formation whose growth was monitored by microscope.
responding to a molecular formula C14
H11NO
2
(Fig. S1 in SI). H NMR
At the incubation completion, the biofilm-coated wells of the 96
(
Fig. S5) exhibited two doublet peaks at 8.12 and 7.61 corresponding to
trans protons and a broad singlet peak at 10.58 corresponding to the OH
proton while in 13C NMR (Fig. S6) δ
142.3 and 126.8 corresponded to
well-plates were carefully washed twice with 200
μL of PBS and left to
air dry for 30 min. To determine the formation of the biofilm, to each
C
4

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
–
O). The H and 13C
1
the alpha, beta-unsaturated carbons and 187.3 (C
–
phthalocyanines in DMSO. The Q bands of all the Pcs exhibited mono-
meric behavior in DMSO. The spectra show intense absorption at
684 nm (for 3), 686 nm (4), 683 nm (for 3a) and 684 nm (for 4a)
(Table 1). InPc (4) showed a slight red spectral shift as compared to ZnPc
(3), this is supported by the non-planar effect of the indium (III) ion,
which as a relatively bigger atomic radius than the zinc (II) [41],
resulting in the red shift in the former. There were insignificant shifts in
the Q band maxima following quaternization, Table 1. Following
conjugation of Pcs to DNDs, slight red shifts are observed (Fig. 1C),
except for 4a.
NMR data (Figs. S7 and S8) of compound 2 were in agreement with the
proposed structure, exhibiting the disappearance of characteristic peaks
–
such as OH proton peak and the appearance of -CN carbon peaks at
15.5 in the 13C NMR spectrum as shown in Fig. S8 in SI with m/
(Fig. S2 in SI).
1
13 3 2
z = 352.14 corresponding to C22H N O
The 1H NMR spectra for MPcs 3, 4, 3a and 4a exhibited aromatic
proton peaks ranging from 8.97 to 7.28 ppm (Figs. S9 and S10, using
MPcs 4 and 4a as examples). And the methyl protons were observed as
singlets at around 2.90 ppm for the quaternized derivatives. In the ac-
quired mass spectra of the Pcs, the expected molecular ion peaks were
In 1% DMSO solution, aggregation was observed in the UV–vis ab-
sorption spectra of the quaternized derivatives as they exhibited two
non-vibrational peaks in the Q band region [42], Fig. 1(B), Table 1. The
red-shifted bands present at 688 (for 3a); 688 (for 3a@DNDs) nm and
691 nm (for 4a); 690 nm (for 4a@DNDs), respectively, are due to the
monomeric species, whereas the blue-shifted bands at 650 (for 3a); 651
(for 3a@DNDs) nm and 651 (for 4a); 651 (for 4a@DNDs) nm are due to
the aggregation.
+
+
obtained at m/z = 1471.88 [M+H] for 3, m/z = 1555.26 [M] for 4, m/
+
+
z = 1531.99 [M+H] for 3a and m/z = 1616.39 [M+H] for 4a. These
results were in good agreement with the suggested structures (Figs. S3
and S4 using MPcs 3 and 4 as examples). The nanoconjugates of the
synthesized Pcs and detonation nanodiamonds were acquired by
interactions between the two (Scheme 2).
π-π
3
.1.1. UV–vis spectra
Fig. 1A illustrates the UV–vis spectra of the synthesized
Following
π
-
π
interactions formed between the Pcs and DNDs
π
sys-
tems (Scheme 2), an enhancement in absorption below 600 nm, Fig. 1
(
C), due to absorption by DNDs.
Loading of Pcs unto DNDs was done spectroscopically via UV–vis
spectra as previously reported [43,44], where the quaternized
Pcs@DNDs had a greater mass loading of 737 and 536
DNDs) for 3a@DNDs and 4a@DNDs respectively, as compared to the
non-quaternized counterparts with mass loading of 383 and 146
Pc)/mg (DNDs) for 3@DNDs and 4@DNDs respectively, Table 1. This
could be justified by the electrostatic interactions between the positive
charges on the Pcs and the negative charges created by electrons on the
μg (Pc)/mg
(
μg
(
π
DNDs sheets for 3a@DNDs and 4a@DNDs, hence larger loading. And
the smaller Pc mass loadings observed in the indium derivatives is due to
the presence of chlorine axial ligand on the InPc that may limit the
number of Pcs loaded due to the bulkiness [45].
3
.1.2. FT-IR spectra
The data from FT-IR also confirmed the suggested structures. The
–
ꢀ 1
C
–
N vibration for 2 at 2231 cm , disappeared following cyclo-
tetramerization reaction to form the MPcs, Fig. 2. This is indicating the
successful formation of targeted molecules.
ꢀ 1
ꢀ 1
Peaks appearing at 3056ꢀ 3055 cm
and 2923ꢀ 2855 cm
are
attributed to stretching vibrations of aromatic C
–
H and aliphatic C
–
H
ꢀ 1
–
bonds, respectively. The peaks at 1583ꢀ 1491 cm are due to C
–
C and
–
–
N vibrations, while peaks appearing around 1722ꢀ 1721 cm are
ꢀ 1
C
–
O groups for the Pcs alone. In the FT-IR spectra of
associated to the C
the Pcs@DNDs nanoconjugates, the C
as compared to the Pcs counterparts, this can be explained by the larger
–
–
–
O peaks showed high intensities
–
number of C O groups in the conjugates which are from both the Pcs
–
and the DNDs. Broader vibration peaks with higher intensities at 3056
cm- in the spectra of the nanoconjugates spectra are associated to the
1
2
OH and NH stretching from DNDs, confirming the successful conjuga-
tion to MPcs. Shifts in FT-IR spectra are an indication that molecular
interactions have taken place.
3
.1.3. Raman spectra
In the current study, Raman spectroscopy was conducted to deter-
mine the quality of the DNDs and that of the prepared
π
-
π
stacked
2
nanoconjugates. The G-band (sp ) tangential mode was observed at
ꢀ
1
3
1
593 cm and the D-disorder band (breathing mode, sp ) was observed
ꢀ 1
at 1371 cm (Fig. 3) [46,47] for DNDs alone. In the spectra of the
conjugates, the G bands shifted to higher frequencies of 1597 and
ꢀ 1
1
595 cm for 3@DNDs and 4@DNDS respectively, while the G bands
ꢀ 1
shifted to lower frequencies of 1568 and 1574 cm for 3a@DNDs and
4
a@DNDs, respectively. On the other hand, the D bands shifted to
ꢀ 1
higher frequencies of 1379 and 1389 cm
for 3a@DNDs and
Fig. 1. Electronic absorption spectra of: (A) DNDs and phthalocyanines (3 and
4
a@DNDs, respectively. There were no shifts in the D-bands of the
4
3
) in DMSO; (B) 3 in DMSO, and 3a in 100 % DMSO and 1 % DMSO, and (C) 3,
@DNDs and 3a@DNDs in DMSO.
neutral Pcs@DNDs (Fig. 3 using 4@DNDNs as an example). The larger
5

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
Table 1
Photophysicochemical parameters of the DNDs, Pcs alone and different nanoconjugates in DMSO.
a
b
Sample
Abs.
Em.
Loading (
μ
g Pc/mg DNDs)
Raman I
D
/I
G
Φ
–
F
τ
F
(ƞs)
Φ
–
Δ
DNDs (2.4 nm)
–
684
–
0.26
–
–
3
695
693
693
689
694
693
691
689
–
0.072
0.067
0.043
0.020
0.045
0.031
0.036
0.020
2.97
2.84
2.91
2.26
2.81
2.22
2.49
2.02
0.39
b
b
b
b
3
4
4
3
3
4
4
a
683 (688, 650)
686
–
–
0.43 (0.21)
0.50
–
–
a
684 (691, 651)
687
–
–
0.53 (0.27)
0.51
@DNDs (8 nm)
a@DNDs (13 nm)
@DNDs (18 nm)
a@DNDs (22 nm)
383
737
146
536
0.34
0.59
0.34
0.59
690 (688, 651)
689
0.61 (0.46)
0.68
684 (690, 651)
0.69 (0.47)
a
Values in brackets are TEM sizes.
b
Values in brackets are in water which is used for cell studies.
Fig. 2. FT-IR spectra of 2, 4, 4a, DNDs, 4@DNDs and 4a@DNDs.
shifts and increase in the intensities of D-bands in the positively charged
nanoconjugates may be due to more interaction from both and
electrostatic interactions. As literature states, shifts in the Raman fre-
quencies are always an indication of strong electron interactions be-
tween the Pcs and carbon nanomaterial such as DNDs [48].
The ratio of the intensities of the D and G bands (I /I ) can provide
information on the quality of extent of functionalization in the DNDs.
The I /I obtained value was 0.26 for DNDs alone, while I /I values
were about 0.34 for neutral Pcs@DNDs (3, and 4) and 0.59 for the
positively Pcs@DNDs (3a and 4a), Table 1. The increase in the I /I
Fig. 3. Raman spectra of DNDs alone, 4@DNDs and 4a@DNDs (as example s).
π–π
π
3@DNDs and 3a@DNDs, 4@DNDs and 4a@DNDs respectively, Table 1.
The increase in size is due to aggregation following conjugation of Pcs to
nanoparticles.
D
G
D
G
D
G
3
3
.2. Photophysicochemical properties
D
G
3
F
.2.1. Emission, Fluorescence quantum yield (Φ ) and Fluorescence lifetime
value observed implies that there is presence of sp defects from Pcs on
2
τ
F
( )
the sp lattice of the DNDs which enhances the D-band [49].
The emission spectra were recorded in DMSO and were mirror im-
ages of the excitation spectra which are found to be similar to the ab-
sorption spectra proving that the molecules that are emitting light are
the same as those that are absorbing light (Fig. 5). The measurements of
the emission wavelengths are reported in Table 1. Slight the shifts be-
tween excitation and absorption spectra in Fig. 5, may be due to
different equipment used.
3
.1.4. Transmission Electron Microscopy (TEM)
To determine the morphology and size of the prepared DNDs and
Pcs@DNDs nanoconjugates, we made use of the TEM technique (Figs. 4
and S11 in SI). The obtained images confirmed that the DNDs were
spherical and monodispersed. Fig. 4 gave the DNDs size of 2.4 nm.
However, on conjugation, an increase on the sizes of Pcs@DNDs con-
jugates is observed as the sizes increased to ~ 8, 13, 18 and 22 nm for
F
The obtained Φ values are low (equations in Supporting Informa-
tion) at 0.072, 0.067, 0.043, 0.020, 0.045, 0.031, 0.036 and 0,020 for 3,
6

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
Fig. 5. Example of normalized absorption, excitation and emission spectra of 4
in DMSO, excitation 614 nm.
Table 1, could be due to the fact that electron donating groups are
known to increase intersystem crossing in porphyrin-type complexes
[
52], reducing fluorescence. However the decrease is not observed for
a@DNDs compared to 4a, Table 1, both with Φ value of 0.02.
Fluorescence lifetime ( ) is the average time a molecule remains in
its excited state before returning to its ground state by emitting light
53]. The of the newly synthesized Pcs and nanoconjugates were
4
F
τ
F
[
τ
F
obtained using the time correlation single photon counting (TCSPC)
method (Fig. 6 using 4@DNDs as an example). Mono-exponential decay
curves were observed in all cases. The
τ
F
values were 2.97 ns (3), 2.84 ns
(
3a), 2.91 ns (4), 2.26 ns (4a), 2.81 ns (3@DNDs), 2.22 ns (3a@DNDs),
2
τ
F
.49 ns (4@DNDs), and 2.02 ns (4a@DNDs). The decreases in in the
presence of DNDs corresponds to the decrease in Φ
change in unison.
F
values, the two
3
Δ
.2.2. Singlet oxygen quantum yield (Φ )
Similar to PDT, PACT process also relies on singlet oxygen ( O
1
2
)
generated at the end of the energy transfer between the triplet state of a
photosensitizer and ground state molecular oxygen [54]. Singlet oxygen
quantum yield (Φ
ficiency of a photosensitizer to produce
determination of Φ values was done by the means of a chemical method
using 1,3-diphenylisobenzofuran (DPBF) and 9,10-antracenediyl-bis
methylene)dimalonoic acid (ADMA) as singlet oxygen quenchers in
Δ
) is a significant parameter used to quantify the ef-
1
2
O . In the current study, the
Δ
(
DMSO and aqueous media, respectively. We monitored the decrease in
absorbance of DPBF at 417 nm and ADMA at 380 nm using UV–vis
spectrophotometer (Fig. 7A and B, 4a was used as an example). Fig. 7A
Fig. 4. TEM images of DNDs, 3@DNDs and 3a@DNDs nano-assemblies (as
examples) showing the morphology and size increase upon conjugation.
3
a, 4, 4a, 3@DNs, 3a@DNDs, 4@DNDs and 4a@DNDs in DMSO,
respectively. These low values (lower than the standard ZnPc at 0.20 in
DMSO [31]) could be to the quenching effect the substituent. The type of
substituent on the Pc macrocycle is known to affect Φ
F
F
values [50]. The
Φ
values of InPcs are lower than those of ZnPc counterparts since In (III)
is a heavier metal than Zn (II). Heavy central metals are known to
enhance the intersystem-crossing to the triplet state, thus reducing
fluorescence [51].
The Φ
were not calculated due to their non-fluorescence behavior in this
values of the quaternized Pcs were lower when compared
F
values for the quaternized Pcs measured in PBS (1% DMSO)
media. The Φ
F
to the non-quaternized entities. This may be explained by the fact that
chalcone compounds are fluorescent, and their quaternization can
F
inhibit fluorescence. The decrease in Φ values in the presence of DNDs,
Fig. 6. TCSPC fluorescence decay curve of 4@DNDs (as an example).
7

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
cell-wall, this will facilitate its penetration and ability to act within the
cell since the generation of singlet oxygen is done within the cell [58].
Hence, in the current work, we developed positively charged photo-
sensitizers that have efficient inactivation of gram-positive but espe-
cially gram-negative bacteria for strong photo-antibacterial
effectiveness.
The optimal concentrations, which are the lowest concentrations at
which compounds can still exhibit antimicrobial potency by inhibiting
more than 50 % of the bacteria, were found to be at 10
.25 g/mL for the non-quaternized and quaternized Pcs (and their
conjugates), respectively on planktonic cells.
For the E. coli and S. aureus biofilms studies, a concentration of
00 g/mL of each of the synthesized compounds were used to deter-
mine the photo-antibiofilm activity after 30 min of irradiation at
μg/mL and
1
μ
1
μ
6
70 nm.
3
.3.1. In vitro antibacterial activity on planktonic cells
Each of the non-quaternized compounds (10
μ
g/mL) or quaternized
compounds (1.25 g/mL) and their conjugates were dissolved in 1%
μ
DMSO in PBS solutions. The control solutions were made up of 1%
DMSO in PBS without the photosensitizers. The controls showed no
antibacterial effect on the studied bacteria, Fig. 8A, B, C and 9A, B, C.
The samples were also subjected to dark toxicity studies which
confirmed that the photosensitizers possessed no dark toxicity, as the
results displayed no significant change in log (CFU, colony forming
units) in Figs. 8A and 9 A for studies conducted on S. aureus and E. coli,
respectively.
For the photoinhibition studies, samples containing the synthesized
compounds and bacteria were irradiated at 670 nm using a Modulight
laser. As displayed in Figs. 8B, 9 B and Table 2, quaternized Pcs and their
Pcs@DNDs counterparts were able to effectively kill both different
bacteria strains at lower concentrations and in a short period of time.
The efficient killing of bacteria strains by the positively charged com-
pounds is expected as the compounds are not only water soluble but also
present stronger affinity to the cell-wall thus resulting in complete cell
membrane destruction and enhanced drug-cell uptake for efficient
photo-antibacterial abilities since they are producing the singlet oxygen
in a close proximity of the cell.
Fig. 7. A typical spectrum for the determination of singlet oxygen quantum
yield of (A) 4a in DMSO using DPBF and (B) 4a in 1%DMSO using ADMA.
and B indicate no significant change in the absorption intensities of the
Q-band of the phthalocyanines, this suggests that the Pcs are stable
during irradiation. Similar trends were observed for the nanoconjugates
as well. The Φ
phthalocyanines, and 1% DMSO (in PBS) (Table 1). The Φ
given in Table 1. The Φ values in Table 1 range between 0.39 and 0.69
in DMSO with the indium derivatives showing the highest values. The
higher Φ values of 0.51, 0.61, 0.68 and 0.69 obtained for 3@DNDs,
a@DNDs, 4@DNDs and 4a@DNDs nanoconjugates, respectively, in
DMSO compared to MPcs alone is due to the increased intersystem
values are higher
Δ
values were determined in DMSO for the studied
Δ
data are
Δ
Δ
For S. aureus, a log reduction of 9.60 ± 0.002 for 3a and 4a after
3
3
0 min and the same value of 9.60 ± 0.001 for 3a@DNDs and 4a@DNDs
but at a higher irradiation time of 60 min were obtained for S. aureus
showing no advantage of DNDs, Table 2. The log reductions for non-
quaternized 3, 4 and 3@DNDs were lower compared to corresponding
quaternized derivatives even at a high irradiation time of 120 min, thus
showing the importance of the positive charges as a result of quaterni-
zation, Table 2. 4@DNDs (even though containing an unquaternized Pc)
had a high log reduction value (9.27 ± 0.003) most likely due to the high
singlet oxygen quantum yield which is almost the same as for 4a@DNDs,
Table 1. Comparing 3 and 4 and their conjugates shows improvement in
terms of log reductions in the presence of DNDs, even though that was
not the case for quaternized derivatives.
crossing in the presence of DNDs discussed above. Φ
Δ
for the InPc derivatives (4 and 4a and their conjugates) compared to
corresponding ZnPc derivatives (3 and 3a and their conjugates) due to
the heavy atom effect of In. The Φ
Δ
values obtained for the quaternized
derivatives were higher compared to the non quaternized counterparts
in DMSO. This may be due to the absence of photoinduced electron
transfer (PET) process between phthalocyanine macrocycle and the
substituents since the lone pair electrons of nitrogen atoms are bonded
to methyl groups [54].
Previous reports state that the presence of nitrogen atoms in
graphitic materials generates charged sites that augment the adsorption
For E. coli, 3a and 4a gave log reductions values of 9.64 ± 0.002 after
of oxygen [55]. This may also result in the observed enhanced Φ
Δ
in the
3
0 min irradiation. These values increased in the presence of DNDs (for
presence of DNDs. Values in water (1 % DMSO), Table 1, are low due to
quenching of singlet oxygen by water [31]. The new synthesized chal-
cone substituted Pcs and conjugates have sufficient singlet oxygen
capability as photosensitizers for PACT applications.
3
a@DNDs and 4a@DNDs, respectively) corresponding to the increase in
singlet oxygen quantum yield, Table 2.
The non quaternized Pcs alone had less activities against both strains,
with compound 3 exhibiting 0.52 ± 0.001 log10 CFU reduction (28.98 %
cell survival) and 0.15 ± 0.003 log10 CFU reduction (57.16 % cell sur-
vival) when treating S. aureus and E. coli, respectively. On the other
hand, 4 presented a 1.85 ± 0.002 log10 CFU reduction (1.54 % cell
survival) and 0.48 ± 0.001 log10 CFU reduction (27.2 % cell survival)
when treating S. aureus and E. coli respectively, whereas, the DNDs alone
showed no major activity in both cases.
3
.3. PACT studies
PACT activities against S. aureus and E. coli were evaluated using the
newly prepared photosensitisers. The principle of PACT is based on
phototoxic and chemical reactions, which cause death of bacterial free
cells or biofilms following irradiation of a photosensitizer by a light in
the presence of molecular oxygen [56,57]. For the efficiency of PACT
process, the photosensitizer should be able to efficiently bind to the
The lower activities observed for the non quaternized compounds
against E. coli as compared to S. aureus is expected as literature reports
the double-layered cell membrane of gram-negative bacteria being a
8

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
Fig. 9. (A) Dark toxicity studies, (B) photoinhibition and (C) cell survival for
E. coli planktonic cells in the presence of Pcs alone and the Pcs@DNDs conju-
Fig. 8. Log CFU plots for (A) Dark toxicity studies and, (B) photoinhibition, and
gates (irradiation at 670 nm). Concentration of the Pcs and conjugates 10
μ
g/
(
C) cell survival for S. aureus planktonic cells in the presence of Pcs alone and
the Pcs@DNDs conjugates (irradiation at 670 nm). Concentration of the Pcs and
conjugates =10 g/mL for non quaternized and 1.25 g/mL for the quaternized.
Data represent the mean ± SD.
mL for non quaternized and 1.25
μ
g/mL for the quaternized. Data represent
the mean ± SD.
μ
μ
therefore this favors the failure of many known treatments [59,60].
The quantification of biofilms formation by crystal violet assays
showed that S. aureus and E. coli strains are strong biofilm producers
under the used working conditions. In Figs. 10A and 11 A, it is illustrated
barrier to neutral photosensitizers access inside the cell. Since PACT
process relies on singlet oxygen production by the photosensitizer, the
resulting data of this work are in perfect agreement with singlet oxygen
quantum yields values obtained and also this confirms the importance of
conjugation of photosensitizers to carbon nanomaterials that have also
been reported to possess intrinsic antibacterial properties. The synthe-
sized nanoconjugates showed better PACT activities corresponding to
high singlet oxygen quantum yields.
that all the compounds did not show antibiofilm activity at 50
μ
g/mL but
some activity was observed while using 100 g/mL of each compound.
μ
For this reason, the time studies to determine the therapeutic efficacy of
the new photosensitizers were carried on using the concentration of
1
00
μ
g/mL in all cases.
For S. aureus biofilms, 3a@DNDs and 4a@DNDs showed highest
value of 9.68 log10 CFU reduction with total photoinhibition of biofilm
cells, while 3a and 4a respectively gave values of 1.40 log10 CFU
reduction (cell survival of only 5.85 %) and 2.13 log10 CFU reduction
3
.3.2. In vitro biofilms eradication
Different concentrations 50 and 100 g/mL of photosensitizers were
μ
used for biofilms photoinhibition studies. The treatment doses were
increased in this case due to the non-sensitivity of biofilms toward
antimicrobial treatment compared to the planktonic cells. This is mainly
caused by the composition of the extracellular polymeric matrixes of
biofilms that prevents in most cases the penetration of the drugs and
(
cell survival of only 0.36 %), all after 120 min of irradiation (Fig. 10B,
Table 3). While 3@DNDs and 4@DNDs presented log CFU reductions of
0
.3 and 1.63 respectively.
As displayed in Fig. 11B, only the indium derivatives 4a and
9

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
Table 2
Log reduction and Cell survival (%) values of 10
μ
g/mL for non quaternized and 1.25
μ
g/mL for quaternized samples in 1% DMSO/PBS after irradiation.
Samples
S. aureus
E. coli
Log reduction
Cell Survival (%)
Time of irradiation (min)
Log reduction
Cell Survival (%)
Time of irradiation (min)
3
0.52±0.001
9.60±0.002
28.98
0
120
30
0.15±0.003
9.64±0.002
57.16
0
120
30
3
a
3
@DNDs
1.32±0.004
9.60±0.001
1.85±0.002
9.60±0.001
9.27±0.003
9.60±0.001
4.81
0.15
1.54
0
120
60
0.74±0.003
9.64±0.003
0.48±0.001
9.64±0.002
0.58±0.003
9.64±0.002
25.92
0
120
30
3
a@DNDs
4
120
30
27.2
0
120
30
4
a
4
@DNDs
0.17
0
120
60
26.23
0
120
30
4
a@DNDs
Fig. 10. Survival graph of photodynamic antimicrobial chemotherapy on S. aureus biofilm (A) after 30 min of irradiation at concentration of 50 and 100
B) variation of cell survival for all the synthesized at 100
g/mL with time up to 120 min irradiation. Data represent the mean ± SD.
μ
g/mL and
(
μ
4
a@DNDs were able to totally kill E. coli cells in biofilm at 100
μ
g/mL
Non quaternized Pcs alone 3 and 4 showed no major reductions in
biofilms in comparison to the respective untreated control groups for all
the strains. The great decrease in biofilms cell viability and the complete
eradication of the two singled biofilm species under light conditions
observed in the quaternized nanoconjugates could be due to the
upon 120 min of irradiation, with 9.80 log10 CFU reduction with no cell
survival for both compounds. And statistical significant reductions in
biofilm with 1.45, 1.43, 1.38 and 2.24 log10 CFU were obtained in viable
count for 3a, 3@DNDs, 3a@DNDs and 4@DNDs, respectively.
1
0

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
Fig. 11. Survival graph of photodynamic antimicrobial chemotherapy on E. coli biofilm (A) after 30 min of irradiation at concentration of 50 and 100 g/mL and (B)
μ
variation of cell survival for all the synthesized at 100
μ
g/mL with time up to 120 min irradiation. Data represent the mean ± SD.
with small light doses.
Table 3
Log reduction values for biofilms at 120 min.
4
. Conclusion
MPcs/conjugate
S. Aureus
E. Coli
3
0.07
1.40
0.3
0.33
1.45
1.43
1.38
0.58
9.80
2.24
9.80
Novel quaternized and non-quaternized zinc and indium chalcone
substituted phthalocyanines and their corresponding interactions to
3
3
3
4
4
4
4
a
π-π
@DNDs
a@DNDs
DNDs nanoconjugates are reported for the first time. The prepared
compounds showed the ability to produce singlet oxygen and have high
potential in the eradication of not only the bacterial planktonic cells of
S. aureus and E. coli, but they also possessed great activities against their
difficultly treated bacterial biofilms. This study also reports on the
characterization and photophysicochemical parameters of all the syn-
thesized compounds. The indium derivatives and the Pcs@DNDs
showed in most cases, high singlet oxygen quantum yields, mainly with
the quaternized compounds. The photo-antimicrobial activities of all the
complexes/conjugates using PACT with irradiation at 670 nm were
determined for all the compounds. It was revealed that the quaternized
nanoconjugates had the highest activity against planktonic cells of
S. Aureus and E. coli, with the highest log reductions causing total deaths
of the planktonic cells and cells in the biofilms. The in vitro results
indicate that at lower concentration the synthesized photosensitisers
9.68
0.15
2.13
1.63
9.68
a
@DNDs
a@DNDs
synergistic effect brought by the DNDs and the Pc alone, the positive
charges and the high singlet oxygen quantum yields.
The basic mode of action of the new compounds studied might
include inhibition of cell metabolism and growth, damage to the cyto-
plasmic membrane and increase in cell permeability due the charges
found on the moieties [61,62]. All of the obtained data further confirm
that the newly prepared nano photosensitisers could be used as potential
photoantibacterial agents against Staphylococcus aureus and Escherichia
coli planktonic cells and biofilms at low concentrations of the complexes
1
1

Y.I. Openda and T. Nyokong
Journal of Photochemistry & Photobiology, A: Chemistry 411 (2021) 113200
have a great prospective in biofilms ablation.
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Dyes Pigm. 166 (2019) 149–158.
CRediT authorship contribution statement
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Yolande Ikala Openda: Conceptualization, Investigation, Writing -
original draft. Tebello Nyokong: Supervision, Resources, Writing - re-
view & editing, Funding acquisition.
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Declaration of Competing Interest
The authors report no declarations of interest.
[19] Y. Gao, B. Mai, A. Wang, M. Li, X. Wang, K. Zhang, Q. Liu, S. Wei, P. Wang,
Antimicrobial properties of a new type of photosensitizer derived from
phthalocyanine against planktonic and biofilm forms of Staphylococcus aureus,
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This work was supported by the Department of Science and Inno-
vation (DSTI) and National Research Foundation (NRF), South Africa
through DSI/NRF South African Research Chairs Initiative for Professor
of Medicinal Chemistry and Nanotechnology (UID 62620), Rhodes
University, the Organization for Women in Science for the Developing
World (OWSD) and Swedish International Development Cooperation
Agency (Sida).
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