Journal of Photochemistry & Photobiology, B: Biology 229 (2022) 112414

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Journal of Photochemistry & Photobiology, B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

In vitro evaluation of the cis-[Ru(phen)2(pPDIp)]2+** complex for

antimicrobial photodynamic therapy against Sporothrix brasiliensis and
Candida albicans
M.A. Tiburcio a, *, A.R. Rocha b, c, R.A. Romano b, N.M. Inada b, V.S. Bagnato b, d, R.M. Carlos a, H.
H. Buzzá b, e, *
a
Chemistry Department, Federal University of São Carlos, Brazil
b
São Carlos Institute of Physics, University of Sao Paulo, Brazil
c
PPG Biotec, Federal University of São Carlos, Brazil
d
Hagler Fellow, Texas A&M University, College Station, TX, USA
e
Institute of Physics, Pontificia Universidad Católica de Chile, Santiago, Chile

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Photodynamic therapy (PDT) activates a photosensitizer by visible light to generate cytotoxic ox­
Antimicrobial photodynamic therapy ygen species that lead to cell death. With proper illumination, PDT is often used in applications on superficial and
Photodynamic inactivation sub-surface lesions. Sporotrichosis infection occurs by Sporothrix fungi which causes a skin wound, worsened by
Photodynamic therapy
Candida albicans infections. This study investigated the photosensitizing efficiency of the Ru(phen)2(pPDIp)
Ru(II) complex
S. brasiliensis
(PF6)2 complex, RupPDIp, against S. brasiliensis and C. albicans.
C. albicans Material and methods: RupPDIp efficiency against these fungi was tested using 450 nm (blue light and 36 J/cm2)
Abbreviations and 525 nm (green light, 25.2 J/cm2) at 0.05–20 μM concentrations. To ensure PDT effectiveness, control groups
[**] (phen) were tested in the absence and in the presence of RupPDIp under light irradiation and in the dark.
1,10-phenanthroline Results: RupPDIp eliminated both fungi at ≤5.0 μM. Green light showed the best results, eliminating S. brasiliensis
(pPDIp) and C. albicans colonies at RupPDIp 0.5 μM and 0.05 μM, respectively.
bis-phenanthroline-perylenediimide derivative Conclusion: RupPDIp is a promising photosensitizer in aPDT, eliminating 106 CFU/mL of both fungi at 450 nm
and 525 nm, with lower light doses and concentrations when treated with the green light compared to the blue
light.

1. Introduction
Sporotrichosis is a subacute or chronic fungal infection that has
increased exponentially in several parts of the world. In addition to
contact with contaminated plants or organic matter, one of the most
common forms of contamination is through inoculation of the fungus by
contact with infected animals, especially cats, horizontally (cat-cat) or
zoonotic (animal-human). The increase of sporotrichosis cases shows the
endemic character of this disease [1,2].
The thermal dimorphism of Sporothrix clade is an important viru­
lence factor, it appears in mycelial form in environments rich in organic
matter and yeast form when inoculated in a host at 37 ◦ C, making
tropical countries susceptible to the spread of this disease. In Brazil,
sporotrichosis is spread nearly throughout the entire national territory
and the most recurrent strains in the diagnosis of this disease are
S. schenckii, S. globosa, S. iuriei and, in particular, S. brasiliensis, which is
almost exclusive of this country [3,4].
Candida species also cause topical infections and are the fungi that
affect humans the most, present in the mucous microbiota [5]. In Brazil,
infections caused by Candida species are considered a public health
problem, because C. albicans is still the main cause of chronic infections,
accounting for almost half of the cases [6]. Especially in immunocom­
promised people, these species cause mucocutaneous infections such as
candidiasis in the gastrointestinal and respiratory tracts and onycho­
mycosis (nail infection) [5,7].
For Sporothrix and Candida infections, the most common treatments
involve the administration of antifungal drugs such as fluconazole,
itraconazole, ketoconazole in veterinary treatments, amphotericin B and

* Corresponding authors at: Rod. Washington Luiz, s/n, São Carlos, SP 13565-905, Brazil.
E-mail addresses: tiburcioamarco@gmail.com (M.A. Tiburcio), hilde.buzza@uc.cl (H.H. Buzzá).

https://doi.org/10.1016/j.jphotobiol.2022.112414
Received 26 May 2021; Received in revised form 10 February 2022; Accepted 21 February 2022
Available online 24 February 2022
1011-1344/© 2022 Elsevier B.V. All rights reserved.
M.A. Tiburcio et al.
caspofungin for more severe cases [8,9,10]. However, adverse effects
can occur during clinical therapy and lead to the development of strains
resistant to commercial antifungal medication, because most of them
have a fungistatic and non-fungicidal effect, resulting in long treatments
that can last weeks and even months [8,11]. Another consequence of the
long treatment with these antifungal drugs is the adverse effects, such as
hepatotoxicity, which limit their use in some patients [12].
The increasing incidence of these topic infections shows that more
effective therapeutic agents with lower toxicity and methods for
routinely applied diagnosis are needed to improve patient well-being
and adherence to treatment. Photodynamic inactivation (PDI) or Anti­
microbial Photodynamic Therapy (aPDT) uses light at a specific wave­
length and a photosensitizing compound to generate highly cytotoxic
species to result in cell death [13]. This technique is an effective and
promising option to treat several types of cancer, with reliable and safe
results for non-melanoma skin cancer [14], and has been proved helpful
in bacterial and fungal infections, in particular for topical applications
[13].
Some fundamental issues that should be considered in planning a
photosensitizer for PDT application are their spectroscopic properties,
solubility and susceptibility to photobleaching [15]. In this regard,
ruthenium complexes have good biological and medicinal properties:
different oxidation states, excellent light absorption, easily absorbed and
excreted by the organism, soluble in an aqueous medium and have a
binding affinity with biomolecules [16].
In aPDT, Ru(II) photosensitizers are also quite new, the complex Ru
(phen)3Cl2 proved to be effective in combating C. albicans in the absence
and presence of light [17]. Although, the spectroscopic properties of the
Ru(II) complexes are easily altered with modifications on ligand field,
for example, the addition of a perylenediimide component to one of the
coordinated phenanthrolines to [Ru(phen)3]2+ results in the dyad [Ru
(phen)2(pPDIp)]PF6)2, (RupPDIp, phen = phenantroline; pPDIp = bis-
phenanthroline perylenediimide derivative), and which benefits from
the spectroscopic and biological properties of both parts. This complex
has high absorption in the visible region, emission in the near IR region
and under irradiation promotes electronic transitions of MLCT (Ru, dπ
→ pPDIp, π*) and ILCT (pPDIp, π → pPDIp, π*) that decays to reach the
long-lifetime triplet excited state (1.8 μs) of the pPDIp ligand [18]. In an
aqueous medium, the triplet excited state can be quenched by molecular
oxygen to generate singlet oxygen with quantum yield Φ = 0.57. When
applied in PDT against B16F10 murine melanoma cancer cells, showed
an IC50 = 1.2 μM with irradiation at 518 nm (0.41 J/cm2) [19].
Based on that, we hypothesize that the RupPDIp complex can be used
as photosensitizer in photodynamic therapy tests in vitro against
S. brasiliensis and C. albicans.
2. Materials and Methods
2.1. [Ru(phen)2(pPDIp)](PF6)2 Complex - Synthesis and
Characterization
Perylene-3,4,9,10-tetracarboxylic dianhydride, RuCl3•3H2O, 1,10-
phenanthroline and tetrabutylammonium hexafluorophosphate were
purchased from Sigma-Aldrich.
Optical spectra were recorded in an Agilent 8453A spectrophotom­
eter, and emission spectra were obtained using a Shimadzu RF-5301PC
spectrofluorometer, exciting the sample at 450 and 500 nm, read be­
tween 470 and 900 nm.
The precursor complex Ru(phen)2Cl2 was synthesized starting from
RuCl3•3H2O and 1,10-phenanthroline (1:2,1), with LiCl (5.44 mM) as
catalyst in deaerated dimethylformamide (DMF) solution [18]. The
pPDIp ligand was obtained by combining perylene-3,4,9,10-
tetracarboxylic dianhydride and two parts of 5-amino-phenanthroline
in distilled quinoline [18], after 24 h under reflux at 230 ◦ C the ligand
was filtered and washed with NaH2CO3 5%, distilled water and finally
methanol.
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Journal of Photochemistry & Photobiology, B: Biology 229 (2022) 112414
RupPDIp final complex was synthesized by reacting the Ru
(phen)2Cl2 and pPDIp (1:2) in deaerated DMF, after 24 h under reflux
the complex was precipitated using tetrabutylammonium hexa­
fluorophosphate (0.134 mM), the final product was solubilized in
acetone to separate from insoluble solids (pPDIp) and recrystallized in
ether [18]. The purity and structure of the complex were guaranteed by
elemental and 1H NMR analysis, as well as UV–vis spectroscopic and
steady-state fluorescence.
The spectroscopic analysis of the RupPDIp complex was performed in
a phosphate buffer solution (PBS, pH 7.4) at a concentration of 20 μM
under nitrogen atmosphere.
2.2. Culture of the Microorganisms
Sporothrix brasiliensis: 50 μL of a pre-inoculum solution of
S. brasiliensis (ATCC MYA4823 - supplied by the College of Pharma­
ceutical Sciences - USP) was incubated in 15 conical centrifuge tubes
containing 10 mL of YPD liquid medium at pH 7.8 and stored at 37 ◦ C for
monitoring the growth of the yeast phase. For the growth of mycelial
form, 50 μL of the same pre-inoculum solution (ATCC MYA4823) was
incubated in 15 conical centrifuge tubes containing 10 mL of YPD liquid
medium at pH 4.5 and left at 25 ◦ C. The optical density (OD) of the
suspension was measured daily, and this inoculum was plated on Sab­
ouraud dextrose agar to relate the day of growth to the daily OD of the
suspension and the number of microorganisms in colonies forming units
per milliliter (CFU/mL).
After monitoring the fungal growth, the best day for cultivation was
determined when the culture reached a range of 106–107 CFU/mL.
C. albicans: The growth of C. albicans (ATCC 90028) colonies was
conducted as described in the literature [20]. 50 μL of a pre-inoculum
were plated on a new Sabouraud dextrose agar and left overnight in
an incubator at 37 ◦ C. The OD was measured until it reached 0.38,
corresponding to a range of 106–107 CFU/mL.
2.3. Preparation of the Fungal Suspension
The colonies of S. brasiliensis and C. albicans were collected from the
plates and suspended in 10 mL distilled water. The suspension was
centrifuged at 4000 RPM for 10 min and the supernatant was discarded.
This procedure was repeated three times to remove any interference
from the culture medium. After resuspending in 10 mL of distilled water,
1 mL of the washed suspension was placed in a cuvette containing 1 mL
of 4% formaldehyde and taken to the spectrophotometer (Varian Cary®
50 UV–Vis Spectrophotometer-Agilent, Santa Clara, California, USA).
The OD was measured at 580 nm and adjusted until reaching the value
of 0.38, corresponding to 106 CFU/mL of S. brasiliensis and C. albicans.
2.4. Analysis of Incubation Time Using Fluorescence Confocal
Microscopy (FCM)
For analysis of the best drug-light interval, 500 μL of a suspension
with previously adjusted OD of the S. brasiliensis fungus in its mycelial
form was incubated in a multi-well plate with 10 μM of RupPDIp. The
incubation times were 0, 10, 20, 30, 40, 50 and 60 min.
One drop from each well with different incubation times was placed
on a slide and taken to the fluorescence confocal microscope (Zeiss,
Germany). Acquisition of the images through FCM was performed using
an excitation wavelength of 450 nm and the spectrum was collected in
the 500–700 nm range.
2.5. Photodynamic Inactivation (PDI)
Suspensions for S. brasiliensis in yeast and mycelial forms, as well as
for C. albicans, were prepared as described in the previous sections. The
PDI tests were performed in multi-well plates with 500 μL of the fungal
suspension and the following groups were formed – Control groups -
M.A. Tiburcio et al.
Only fungi: microorganism alone in PBS; Only Light: microorganism
under irradiation; Only DMSO: microorganism with 2% of DMSO
(maximum percentage from the complex stock solution); Only photo­
sensitizer (PS): microorganism and photosensitizer in the dark; PDI
groups – PS + Light: microorganism and photosensitizer at different
concentrations under light irradiation. In all experiments conducted
with the photosensitizer, RupPDIp was added varying the concentration
at 0.05, 0.5, 1, 2, 3, 4, 5, 10 and 20 μM, always with a final volume of 1
mL per well.
After 20 min of incubation, the plates were irradiated in equipment
developed by the Technological Support Laboratory (LAT – IFSC/USP).
The device consists of an array of 24 LEDs (light-emitting diode) with a
fixed distance from the multi-well plate and lenses that correct the
divergence of emitting light, ensuring that each well receives irradiance
homogeneously [21,22]. Current controls and heat dissipation modu­
lators also ensure controlled emission [21,22]. For different wave­
lengths, the parameters obtained were different: at 450 nm, with 40
mW/cm2 irradiance, for 15 min to deliver a total dose of 36 J/cm2. Using
the same device, the plates were irradiated at 525 nm, with 7 mW/cm2
irradiance, for 15 and 60 min to deliver total doses of 6.3 and 25.2 J/
cm2.
After incubation and/or irradiation, according to each group, 100 μL
of each well were diluted in series (10− 1 to 10− 4) and 20 μL of 10− 4,
10− 3 and 10− 2 dilutions were plated on Sabouraud agar. The colonies
were counted after three days and all tests were performed in triplicate.
Data were analyzed using ANOVA, a one-way analysis of variance and
denoted significance (*) p < 0.05.
3. Results
3.1. Characterization of [Ru(phen)2(pPDIp)]2+ Complex
The RupPDIp complex, Fig. 1A, was synthesized through the reaction
of cis-[Ru(phen)2Cl2] and the pPDIp ligand (1:2) in deaerated DMF and
precipitated as a hexafluorophosphate salt, with 62% yield [18]. The
purity and structure of RupPDIp complex were analyzed by elemental
analysis (C72H38F12N10O4P2Ru – 1498.14 g.mol− 1), calc: C 57.72, H
2.55, N 9.35; Found: C 56,97, H 2.84, N 9.14. And H1 NMR (Fig. S1),
with perylene 1H signals appearing in the chemical shift range 8.7 < δ <
9.0 and the signals from 1H of phenanthrolines appearing between 8.7 <
δ < 7.5 [18].
The spectrum of RupPDIp in buffer solution (pH 7.4) on Fig. 1A
shows a wide absorption in the visible region with characteristic MLCT
and ILCT bands already reported in these conditions [19]. Also, RupP­
DIp has an emission with maximum at 600 nm from the 3*MLCT
Fig. 1A. Illustrative chemical structure of the [Ru(phen)2(pPDIp)](PF6)2 complex. 1B. Absorption (black) and emission (red, λex: 500 nm.) spectra of a deaerated
solution of 20 μM of [Ru(phen)2(pPDIp)]2+ in PBS solution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
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Journal of Photochemistry & Photobiology, B: Biology 229 (2022) 112414
deactivation, as shown in fig. 1B.
The addition of pPDIp to [Ru(phen)3]2+ promotes a red-shift in ab­
sorption spectra, sum of individual spectra of each part. Studies by our
group have shown that the RupPDIp complex acts only by FRET sensi­
tizing singlet oxygen, without undergoing photobleaching. This fact is
very relevant when dealing with PDT, where there is an incessant search
for photosensitizers that act within the therapeutic window. This
motivated us to investigate the efficiency of the RupPDIp complex,
which has one of the phenanthrolines linked to a perylene ligand, with
two light sources, at 450 nm and 525 nm.
3.2. Growth Curve of Sporothrix Brasiliensis
The growth of the fungus was analyzed to determine the best day for
colony cultivation and collection. Over 14 days of culture, the
S. brasiliensis forms presented distinct growths. The yeast form grown on
Sabouraud dextrose agar at pH 7.8 and stored at 37 ◦ C showed growth
stabilization around day 9, with 106 CFU/mL related to OD = 0.38.
For the mycelial form stored at 25 ◦ C at pH 4.5, growth stabilization
occurred around day 7 (106 CFU/mL and OD = 0.38). These growth
curves were like those found in the literature [23].
3.3. Fluorescence Confocal Microscopy (FCM)
The light-drug interval is defined as the minimal time for drug
accumulation in the target cells. This interaction between the photo­
sensitizer and the target is important to ensure that most of the complex
is adhered to or inserted into the microorganism because the singlet
oxygen lifetime is short and causes cytotoxic damage only close to where
it is generated [24]. When excited at 450 nm, RupPDIp showed an
intense emission band with a maximum around 600 nm, which allows its
application in biologic medium without the interference of absorption
and emission of biomolecules [25].
Fig. 2 shows the FCM images of S. brasiliensis in its mycelial form
alone as a Control group (Fig. 2A) and the presence of RupPDIp during
the incubation periods of 10, 20 and 60 min (Fig. 2B, c and D,
respectively).
The confocal images (Fig. 2) show the elongated and fine shapes
characteristic of the S. brasiliensis mycelial form. These structures are
hyphae and serve as a support for fungal replication [26]. In the pres­
ence of RupPDIp, interaction with the microorganism is well defined by
a strong red emission, indicating that the photosensitizing agent has
adhered to or penetrated the fungal membrane.
The best incubation time was defined as 20 min, when the fluores­
cence of RupPDIp remains adhered to the surface of the microorganism.
M.A. Tiburcio et al. Journal of Photochemistry & Photobiology, B: Biology 229 (2022) 112414

Fig. 2. Fluorescent confocal microscopy images of the S. brasiliensis fungus in its mycelial (filamentous) form in the absence (A) and presence of RupPDIp with
incubation times of (B) 10; (C) 20 and (D) 60 min. λEX: 450 nm.

At shorter times, there are some regions of the fungal without any also the best period found for other analysis of C. albicans inactivation
fluorescence, suggesting that there is no compound in these regions. In through PDT [27].
contrast, the fluorescence can be observed around the microorganism at
longer times (Fig. 2d). One of the reasons for that may be the diffusion of
the complex, which begins to be slowly released into the solution after
adhering to the surface of the microorganism. The 20-min interval was

Fig. 3. Sporothrix brasiliensis photodynamic inactivation in mycelial (red) and yeast (blue) forms irradiated at 450 nm, with light dose of 36 J/cm2. *The growth
reduction was statistically significant (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

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M.A. Tiburcio et al. Journal of Photochemistry & Photobiology, B: Biology 229 (2022) 112414

3.4. Photodynamic Inactivation (PDI) – In Vitro Test for Sporothrix 0.05 μM and a total reduction at the concentrations of 0.5 and 5.0 μM
Brasiliensis were observed. When a dose of 6.3 J/cm2 is delivered, the Sporothrix
colonies in both forms are reduced to zero at a concentration of 5.0 μM.
PDI experiments were performed against S. brasiliensis in its mycelial All of these results are shown in Fig. 4.
and yeast forms. By comparison, the efficiency of RupPDIp was tested on
450 nm (blue) and 525 nm (green) light systems.
3.5. Photodynamic Inactivation (PDI) – In Vitro Test for Candida
Control groups were the reference for the analysis of antifungal effect
Albicans
counting the CFU/mL. Fig. 3 showed that the Only Light group at 450
nm, with a total dose of 36 J/cm2, showed no effect compared with the
Photodynamic Inactivation (PDI) experiments were performed
Only Fungi group, demonstrating that the light dose used did not cause
against C. albicans in its yeast form also under irradiation of 450 nm
any damage to the microorganism in its both forms. In addition, no
(blue light) and 525 nm (green light) and showed no effect on C. albicans
cytotoxic effect was observed when testing microorganisms with 2%
growth using either of two wavelengths for irradiation, as shown in
DMSO - the solvent condition of maximum concentration of RupPDIp.
Fig. 5 (0 μM group).
In the dark (Only PS groups), the S. brasiliensis mycelial form at pH
For C. albicans, in the absence of irradiation (Dark groups), RupPDIp
4.5 presented reductions of 1 and 2 log10 with 5.0 and 10.0 μM,
showed no toxicity at lower concentrations and presented a reduction of
respectively, whereas for the yeast form both concentrations showed a
about 2 log10 for 5.0 μM, a similar behavior to that observed for
reduction of 2 log10. These results show that the complex alone presents
S. brasiliensis.
a little fungicidal activity depending on the concentration used, since
The effects of PDI were enhanced against C. albicans compared with
concentrations <5.0 μM did not show any effect.
S. brasiliensis since a total reduction of colonies was achieved at lower
When irradiated at 450 nm, in PDI groups, the mycelial form showed
concentrations. As shown in Fig. 5 when irradiated at 450 nm, with a
a reduction of 2 log10 with 5.0 μM in relation to the control. At the
light dose of 36 J/cm2, concentrations from 0.5 μM presented total
concentration of 10.0 μM, it presented total elimination, which means a
elimination of the microorganism (6 log10 in relation to the Control
reduction of 6 log10 in relation to the Control group and 4 log10 in
groups). With the green light at 525 nm, when a dose of 25.2 J/cm2 was
relation to the Control PS groups.
delivered, a total reduction was achieved from a concentration of 0.05
The yeast form presented a reduction at lower concentrations
compared with the mycelial form. With 0.5 μM in the PDI group, there
μM of RupPDIp (Fig. 5).
Just like in Sporothrix, when a dose of 6.3 J/cm2 was delivered,
was a reduction of 2 log10 compared with the Control groups. With 5.0
higher concentrations were needed to reduce 6 log10 of fungal colonies,
and 10.0 μM, there was a reduction of the order of 6 log10 compared with
and this concentration started from 0.5 μM for C. albicans (Fig. 5).
the Controls, presenting total elimination of fungal colonies. The results
for 450 nm for both mycelial and yeast forms are summarized in Fig. 3.
When irradiated at 525 nm (Fig. 4), with a low light dose, the con­ 4. Discussion
centrations needed for aPDT effect are lower than those used for 450 nm
light, showing a greater efficiency in this wavelength. For the yeast As in sporotrichosis, treatments for candidiasis in humans are long
form, despite showing a reduction of 1 log10 in the dark (Only PS and expensive, leading to premature discontinuation of antifungal
Group), the concentration of 5.0 μM also eliminated the fungal colonies agents, promoting fungal resistance [28]. These factors are aggravating
in the PDI group (PS + Light). With 0.5 μM, no cytotoxic effect was when it comes to infections by microorganisms and, in 2020 in Brazil, a
observed in the absence of light (Only PS Group), but the S. brasiliensis super fungus of the Candida spp., Corynebacterium auris was reported for
colonies were eradicated when irradiated (representing 6 log10 in rela­ the first time [29]. The mass contamination of both fungi can be con­
tion to the Control groups). tained with effective, fast and safe treatments, avoiding abandonment
For the mycelial form, the same behaviors were observed: compared and the development of resistant strains [14,30]. aPDT is an emerging
with the Control groups, a reduction of 1 log10 in the PDI group with and promising technique that can be applied to infections at early stages
without the need for a microbial diagnosis, preventing disease

Fig. 4. Sporothrix brasiliensis photodynamic inactivation in yeast (blue) and mycelium (red) form with 25.2 J/cm2 light dose at 525 nm and yeast (green) and
mycelium (yellow) form with 6.3 J/cm2 light dose at 525 nm. *The growth reduction was statistically significant (p < 0.05). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

5
M.A. Tiburcio et al.
Fig. 5. Candida albicans photodynamic inactivation in absence of light (grey) and using two protocols: 450 nm with light dose of 36 J/cm2 (blue) and 525 nm with
light dose of 6.3 J/cm2 (red) and 25.2 J/cm2 (green). *The growth reduction was statistically significant (p < 0.05). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
progression, decreasing treatment time and adverse effects caused by
systemic and oral treatments with antifungal drugs, in addition to
reducing the cost of treatment, when the long conventional treatments
are compared with the aPDT procedure [30,31].
Fungi of the Schenckii complex have the production of melanin as
their virulence agent, which provides them with resistance to antifungal
drugs and presents great absorption in the nearby UV region, affecting
aPDT rates as it also absorbs in the visible range of the spectrum
(300–650 nm) [32], thus reducing the photoinactivation of the micro­
organism. C. albicans can also produce melanin, but in smaller quantities
compared with fungi of the Schenckii complex [33], which can make
them more susceptible to the effects of aPDT and justify the lower
concentrations needed for their elimination. Aiming to increase the ef­
ficiency and, for clinical application, increase the biological tissue
penetration, both forms of S. brasiliensis and C. albicans were irradiated
with a light source of 450 nm in the absorption of Ru(II) and 525 nm in
the absorption region of the pPDIp ligand.
Souza et al. [34] compared photosensitizers such as methylene blue,
toluidine blue, and malachite green against C. albicans. Radiating at 660
nm and delivering a dose of 39 J/cm2, a 3 log10 reduction of the Candida
colonies was observed with 0.1 mg/mL of photosensitizers [34].
Studies on the application of aPDT in sporotrichosis are scarce and
the most used photosensitizer is methylene blue (MB). Mario (2014)
showed a reduction of 4 log10 during the photoinactivation of Sporothrix
spp., including S. brasiliensis, at a dose of 28 J/cm2 (685 nm) and 2 mg/
mL of MB [35]. Li (2019) also demonstrated 4 log10 reductions with 2
mg/mL and irradiation at 640 nm with a dose of 40 J/cm2 against
S. globosa in vitro, while the same dose was used for in vivo tests to treat
infected mice paws, presenting visual improvement and wound reduc­
tion after a few sections [36]. A study using 5-ALA shows a reduction of
6 log10 against S. globosa using a dose of 162 J/cm2 at 633 nm [37].
Some clinical studies for sporotrichosis were performed using 1%
methylene blue. With solar irradiation (400–700 nm), Galicia-Malinis
(2018) demonstrated an improvement in the wounds found in a 41-
year-old man, while Gilaberte (2014) showed clinical improvement in
the wounds of a 65-year-old woman with irradiation at 640 nm and 37
J/cm2 [38,39].
MB has been used in aPDT against Sporothrix sp.; however, when
given to cats it can cause depression, difficulty breathing, bluish color­
ation of urine, feces and tissues such as kidneys and livers [40,41].
Another aggravating factor in the administration of MB in cats is the
deposition of this species and its reduced form (leuko-methylene blue) in
neuronal tissues, causing severe damages [42]. It is remarkable the need
for new photosensitizing drugs for use in various applications of aPDT,
especially against sporotrichosis, which has the cat as the main vector.
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Journal of Photochemistry & Photobiology, B: Biology 229 (2022) 112414
As shown in Fig. 4 the RupPDIp complex presents a 6 log10 reduction
of S. brasiliensis colonies with 0.5 μM (0.75 μg/mL) and 25.2 J/cm2,
while MB had shown a reduction of 4 log10 using 2 mg/mL and light dose
28 J/cm2 [35]. Although methylene blue has the advantage of illumi­
nation at wavelengths >600 nm, the RupPDIp complex has shown
promising results against S. brasiliensis with 525 and 450 nm irradiation,
which are wavelengths that have been used in PDI.
In fungi such as F. solarium and A. fumigatus using 0.1% rose bengal
as a photosensitizer, green light irradiation (518 nm - 5.4 J/cm2) showed
a reduction of 78.2 and 79.8%, respectively [43]. Applications of PDI on
T. rubrum with rose Bengal (280 μM) and two green light sources, a laser
at 532 nm with 228 J/cm2 of fluence and a LED array with 24 J/cm2 at
530 nm, reached 85% and 100% reduction on fungal colonies, respec­
tively [44]. T. rubrum with blue light treatment uses a combination of
curcumin and curcuminoids with irradiation at 417 nm (10 J/cm2) to
completely inhibit the growth [45].
For C. albicans the use of green light shows promising results. For
example, with 0.1% rose bengal, there was a 96% inhibition of
C. albicans growth using a dose of 5.4 J/cm2 at 518 nm [43]. And studies
involving biofilms also demonstrate significant reduction with rose
bengal (12.5 μM) and irradiation at 532 nm (90 mW – 16.2 J) [46].
Using 3.12 μM of erythrosine, Costa (2011) showed a total reduction of
the fungus with a light dose of 42.63 J/cm2 at 532 nm [47] and similar
parameters of the present study.
The natural photosensitizer curcumin has been shown to completely
inactivate C. albicans with 7.4 mg/L and a light dose of 37.5 J/cm2 (455
nm) [34]. However, after delivering a dose of 0.4 J/cm2, almost 70% of
the curcumin molecules underwent photobleaching, losing their pho­
tosensitizing properties [48]. Clinical tests with 1.5% curcumin gel and a
mixture of curcumins and curcuminoids used light doses around 120 J/
cm2 at 450 nm and 470, respectively, showing improvements in patients
with nail onychomycosis without reports of pain [49,50].
Therefore, there is a wide array of literature using parameters similar
to those used in the present study. Our in vitro tests showed that the
RupPDIp complex is a good photosensitizer against S. brasiliensis, elim­
inating the fungal colonies at low concentrations of 5.0 μM (450 nm) and
0.5 μM (525 nm) and using similar or lower light doses (36 J/ cm2–450
nm and 25.2 J/cm2–525 nm) than those found for in vitro tests. This
greater efficiency at a green light is probably due to the absorption of the
complex in this region, leading to the formation of reactive oxygen
species (ROS) by activating both the perylene ring and the metal-to-
ligand charge transfer (MLCT).
The RupPDIp complex has been applied to B16F10 non-melanoma
cancer cells [19], but there is no literature involving ruthenium com­
plexes in the treatment of sporotrichosis through PDT. In other hand, for
M.A. Tiburcio et al.
photodynamic inactivation of C. albicans, the Ru(phen)3Cl2 complex,
despite showing a strong fungicidal character in the dark (MIC = 0.05
mg/mL), has a photodynamic effect with MFC = 0.025 mg/mL (white
light 250 W, 60 min) [17]. This complex has a maximum absorption at
~450 nm. The RupPDIp complex is a combination between the [Ru
(phen)3]2+ moiety and the pPDIp ligand and the addition of the perylene
ligand shifts the absorption to a maximum at 535 nm [18], improving
the absorption range and the photodynamic effect, according to Fig. 5
RupPDIp eliminates 6 log10 of C. albicans colonies with 0.05 μM (0.075
μg/mL), while the irradiation upon [Ru(phen)3]Cl2 complex has a
minimal fungicidal concentration of 0.025 mg/mL [17].
Despite the light doses may be different for each application, many
studies have shown that higher light doses are common for treating
fungal infections. This fact occurs because fungi are larger than non-
melanoma cancer cells, in addition to having structural and biochem­
ical differences that can interfere with the efficiency of photodynamic
therapy [51]. Fungi have a rigid membrane formed by soluble and
insoluble polymers and glycoproteins, ensuring that the membrane
permeability is regulated by the lipophilicity/hydrophilicity of the
molecule, hindering the passage of the photosensitizer [50]. Basically,
the biochemical process involved in photodynamic therapy is enzyme
inactivation and lipid peroxidation, culminating in cell and mitochon­
drial lysis. Fungi may have greater control over the production of anti­
oxidants and DNA repair mechanisms [51,52,53]. Studies report that
this difference is also seen between fungi and bacteria, as yeasts need
higher concentrations of PS and higher doses of light to be eliminated
than Gram-positive bacteria [53].
Although Ru(II) complexes are not as affordable in cost, their use at
low concentrations with efficiency, as presented in this study, and
because of their no photobleaching properties enables their application
in aPDT [18]. Moreover, the photodynamic effects after the addition of
pPDIp ligand motivate us to find new ligands or modifications that can
improve the spectroscopy properties of Ruthenium complexes.
5. Conclusion
The RupPDIp complex has a mild fungicidal character when used at
high concentrations (≥5 μM). However, the combination with the light
sources showed that the complex can eliminate colonies (6 log10 ~ 106
CFU/mL) of both fungi using two wavelengths as illumination. We
conclude from this study that the RupPDIp complex performs better
under green light irradiation since required lower light doses than those
used for the blue one (450 nm – 36 J/cm2 and 525 nm – 25.2 J/cm2) and
lower concentrations of RupPDIp complex. For S. brasiliensis and
C. albicans yeasts, the total elimination occurs at ≥5.0 μM and ≥ 0.5 μM,
respectively, at 450 nm irradiation. With the 525 nm treatment, the total
elimination occurs at lower concentrations, ≥0.5 μM for S. brasiliensis
and ≥ 0.05 μM for C. albicans.
Funding
This study was supported by São Paulo Research Foundation –
FAPESP (Proc. n◦ 2018/03424-0, Proc. n◦ 2016/14033-6), National
Institute of Optics and Photonics, by INCT program - National Council
for Research and Development (CNPq - grant 465360/2014-9 and
FAPESP - grant 2014/50857-8, 2019/21143-0), Center for Research in
Optics and Photonics – CePOF (FAPESP grant 2013/07276-1) and
CAPES (Cód. 001).
CRediT authorship contribution statement
M.A. Tiburcio: Methodology, Validation, Investigation, Formal
analysis, Writing – original draft, Writing – review & editing, Visuali­
zation. A.R. Rocha: Methodology, Validation, Investigation, Formal
analysis. R.A. Romano: Investigation, Formal analysis, Writing – review
& editing. N.M. Inada: Conceptualization, Methodology, Resources,
7
Journal of Photochemistry & Photobiology, B: Biology 229 (2022) 112414
Writing – review & editing. V.S. Bagnato: Conceptualization, Re­
sources, Writing – review & editing, Project administration, Visualiza­
tion, Funding acquisition. R.M. Carlos: Conceptualization,
Methodology, Validation, Resources, Writing – review & editing, Project
administration, Funding acquisition. H.H. Buzzá: Conceptualization,
Methodology, Validation, Formal analysis, Resources, Writing – original
draft, Writing – review & editing, Supervision, Project administration,
Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
We are grateful for all the professionals who were involved in this
study, especially Dr. Rafael Cavalieri Marchi and Dr. Tiago Venâncio for
the discussion in the NMR experiments and to the funding agencies
(CAPES, CNPq and FAPESP).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jphotobiol.2022.112414.
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