Functionalization of cationic porphyrins with peripheral platinum(II) complexes to optimize photodynamic therapy against Candida-associated infections: a focus on denture stomatitis and burn wounds

ABSTRACT

Candida spp. are opportunistic pathogens associated with mucosal and cutaneous infections. Its increased resistance to antifungals has instigated the development of adjunct treatments, such as antimicrobial photodynamic therapy (aPDT). This study evaluated the antifungal effects of aPDT mediated by two tetra-cationic porphyrins with peripheral platinum(II) complexes (3-Pt and 4-Pt). A thorough investigation was performed using in vitro and in vivo assays to determine their antifungal activity on Candida albicans and toxicity in human cells. Next, specific models were employed to search for understanding of the action of aPDT on Candida-associated infections. As a result, a MIC value of 16 μmol/L was found for both porphyrins, with low toxicity to keratinocytes even in higher concentrations. Planktonic cultures of C. albicans treated by aPDT with 3-Pt achieved complete inhibition in 40 s, while 4-Pt reduced 1.3 Log10 (CFU) within 80 s. These effects were also extended to C. albicans biofilms, in which 3-Pt and 4-Pt reduced 4 and 0.8 Log₁₀ (CFU), respectively. The mechanisms of action of 3-Pt were related to hyphae inhibition, increased ROS production, and cell wall damage. Finally, 3-Pt showed efficacy against denture stomatitis biofilms in a microcosm model and burn wounds in Galleria mellonella, indicating its potential for treating Candida-associated infections.

GRAPHICAL ABSTRACT

1. Introduction

The manifestations caused by Candida albicans can vary from superficial lesions to potentially fatal systemic infections (Costa-de-Oliveira and Rodrigues 2020). The wide range of C. albicans infections is associated with its high adaptability to host tissues and expression of several virulence factors. Amongst them, the formation of hyphae, the secretion of proteolytic enzymes, and the ability to form biofilms are considered the most important factors for the infection process. The transition from planktonic cells to biofilm stage occurs due to a complex remodelling of Candida phenotypic behaviour, supported by changes in the expression of virulence genes, which makes biofilms more resistant to antifungal treatments (Pereira et al. 2021; Ponde et al. 2021).

Superficial Candida infections often occur in mucous membranes as the oral cavity or cutaneous surfaces such as burn wounds. In relation to oral fungal infections, denture stomatitis is one of the most common diseases associated with Candida biofilms. Denture stomatitis is a chronic inflammatory process of the oral mucosa that results from contamination of the denture by a mature biofilm composed of Candida species and bacteria (McReynolds et al. 2023; Ribeiro et al. 2024). In these biofilms, the different species establish a complex interaction, which makes treatment more difficult and leads to frequent relapses (McReynolds et al. 2023). Burn wounds are also frequently associated with Candida and bacterial infections since they facilitate microbial colonisation and induce immune dysfunction (Van Bang et al. 2020; Ji et al. 2024). In the course of time, the wound fungal colonisation results in biofilm formation, providing a scaffold for bacterial attachment and an increased resistance to antifungal drugs (Kalan and Grice 2018). A recent study showed that 66.7% of the patients with burn injuries had infections by Candida species (Van Bang et al. 2020).

The available drugs for the treatment of biofilm-associated Candida infections are restricted and challenging. This limitation has been exacerbated by the rapid emergence of antifungal resistant strains (Vila et al. 2020). Therefore, antimicrobial photodynamic therapy (aPDT) has gained significant attention as an adjuvant treatment for superficial infections caused by Candida spp. (Wiench et al. 2021). aPDT is a photochemical and photophysical reaction that comprises three components: a chemical compound named photosensitiser, a light source at specific wavelength, and molecular oxygen. During this process, the photosensitiser absorbs light photons and can interact with oxygen in the tissue through two distinct processes: Type I reaction, involving electron transfer leading to the generation of radical species; and Type II reaction, in which energy transference results in the singlet oxygen formation (Garcia et al. 2021b; Terra-Garcia et al. 2021). Therefore, both reactions generate reactive oxygen species (ROS) that initiate oxidative stress and react with various cellular components, resulting in microbial cell death. Unlike conventional antibiotic and antifungal drugs, which typically act on specific targets of action (key-lock mechanism), aPDT exerts its effects through multiple targets simultaneously. aPDT can act on proteins, lipids, and nucleic acids of fungal cells, and even in the extracellular matrix of biofilms. Consequently, the development of resistance by microbial cells is unlikely (Cieplik et al. 2018; Hu et al. 2018).

The photosensitiser plays a fundamental role in aPDT reaction as it absorbs the light energy and transfers it to molecular oxygen (da Silveira et al. 2020). Among different photosensitisers investigated, porphyrins stand out as macrocyclic tetrapyrrolic compounds capable of absorbing energy in the visible spectrum (400–800 nm) and generate a significant amount of ROS (Basso et al. 2019; Guterres et al. 2020). The macrocyclic tetrapyrrolic structure of porphyrins allows specific structural modifications through substitution of the peripheral groups, resulting in neutral, anionic, or cationic porphyrins. Cationic porphyrins may have superior efficacy in aPDT due to their enhanced solubility in water and improved ability to penetrate into fungal cells (Ries et al. 2020), as a result of electrostatic interactions with the negatively charged membrane of microbial cells (Jornada et al. 2021; Liu et al. 2024).

In this context, the purpose of our study was to evaluate the antifungal activity of aPDT mediated by tetra-cationic porphyrins containing peripheral Pt(II) complexes. We hypothesised that these porphyrins would enhance the antifungal activity of aPDT. Derivatives containing platinum(II) have well-founded photobiological properties, such as high solubility, low aggregation tendency, and effective generation of reactive oxygen species in solution mainly singlet oxygen. Also, these Pt(II) derivatives exhibit lower toxicity to animal cells when compared to tetra-methylated porphyrins (Oliveira et al. 2024). More specifically, we investigated the effects of aPDT with tetra-cationic porphyrins containing peripheral Pt(II) complexes on planktonic cultures, filamentation, and biofilms of C. albicans, seeking to understand its mechanism of action on fungal cells and cytotoxicity in human keratinocytes. After that, the efficacy of aPDT with the functionalised porphyrins was tested on microcosm biofilms of denture stomatitis and burn wounds in the G. mellonella model.

2. Material and methods

2.1. Candida albicans strain

The reference strain of C. albicans SC5314, stored at −80 °C in the Laboratory of Oral Microbiology and Immunology at UNESP, was employed for both in vitro and in vivo assays. For activation, the strain was cultured in Sabouraud Dextrose Broth (Difco, Detroit, USA) for 48 h at 37 °C.

2.2. Photosensitizer and light source

Tetra-cationic porphyrins containing peripheral bipyridyl Pt(II) complexes, named 3-Pt and 4-Pt (Figure 1), were synthesised following the methodology previously described by Naue et al. (2009). Then, these photosensitisers were diluted in dimethyl sulphoxide (DMSO-Sigma) and stored protected from light at 4 °C. Prior to use, the porphyrins were diluted in distilled water to obtain various concentrations. The light source used was a blue LED (Emitter G, Schuster), emitting light at a wavelength of 420 nm with a power output of 1,250 mW, a power density of 0.16 W/cm² and an energy density ranging from 0.79–12.78 J/cm².

Figure 1.

Representative chemical structure. Tetra-cationic porphyrins containing peripheral Pt(II) complexes, named 3-Pt and 4-Pt.

2.3. Study of aPDT mediated by 3-Pt and 4-Pt on planktonic cultures of C. albicans

2.3.1. Determination of minimum inhibitory concentration

The minimum inhibitory concentrations (MIC) of porphyrins were determined using the broth microdilution technique, as described by Pinheiros et al. with modifications (Rosa Pinheiro et al. 2023). Briefly, 100 µL of RPMI broth were added to each well of a 96-well microplate. Subsequently, 100 µL of the tested treatment were added. Serial dilutions of porphyrins (3-Pt or 4-Pt) were performed using RPMI 1640 (Sigma-Aldrich) supplemented with 2% glucose in 96-well microplates until reaching final concentrations ranging from 64 µmol/L to 0.062 µmol/L. Negative control (200 µL of RPMI broth), and positive control (100 µL of RPMI broth plus 100 µL of fungal inoculum) were included. The inoculum was prepared in sterile phosphate buffered saline (PBS) and standardised at 10³ cells/mL. After preparation, an inoculum of 100 µL was added to the wells, except for the negative control. Then, the plates were irradiated with blue light for 20 s or kept in the dark for the same period (group without irradiation), with subsequent incubation in the dark for 24 h/37 °C. The MIC value was determined as the lowest concentration capable of visually inhibiting the fungal growth. Candida krusei ATCC 6258 was used as a quality control. This test was conducted in duplicate (technical replicate) and was carried out 3 different times (biological replicate).

2.3.2. Kill curve assay of C. albicans at different light-irradiation times

The MIC values previously determined were used to assess the kill curve of C. albicans at different light-irradiation times. Initially, C. albicans was cultured in Yeast Extract Peptone Dextrose broth (YPD; Himedia) at 37 °C for 24 h. Following centrifugation (2,000 r/min for 10 min), the supernatant was discarded, and the pellet was resuspended in 6 mL PBS. This process was repeated twice to ensure thorough washing. The cell density of the C. albicans suspension was then adjusted to 10⁷ cells/mL using a haemocytometer and trypan blue stain, based on the number of viable cells (Garcia et al. 2024). Then, 100 µL of the standardised suspension and 100 µL of 3-Pt, 4-Pt, or PBS were added to a 96-well plate, followed by blue LED irradiation for 5, 10, 20, 40, and 80 s, or kept in the dark, according to the experimental group. Finally, serial dilutions and plating on Sabouraud Dextrose agar (SDADifco, Detroit, USA) were performed to enumerate colony forming units (CFU/mL). Kill curve assay was conducted with 10 technical replicates at 3 different times (biological replicate).

2.3.3. Candida albicans filamentation assay

For the filamentation assay, a suspension containing 10⁷ cells/mL of C. albicans was prepared as described above. In a 24-well plate, 1 mL of distilled water supplemented with 10% foetal bovine serum (FBS) (Invitrogen, New York, USA) was added, followed by 100 μL of the standardised C. albicans suspension. Next, an inoculum of 100 μL of 3-Pt, 4-Pt, or PBS was placed in each well, and the light groups were irradiated for 20 s. The plates were then incubated at 37 °C. After 6 h, 10 μL of the inoculum from each well were dispersed over a Neubauer chamber. The material was covered with a coverslip and observed under an optical microscope at 400× magnification. To quantify the hyphae, 64 fields of the chamber were analysed and photographed, and the presence of hyphae or yeast was counted. This experiment was performed with 6 technical replicates at 3 different times (biological replicate).

2.4. Analysis of the toxicity of porphyrins (3-Pt and 4-Pt) to keratinocytes

The cytotoxicity of the 3-Pt or 4-Pt porphyrins was carried out using human keratinocytes (HaCaT) cell line obtained from the Rio de Janeiro Cell Bank (APABCAM, Rio de Janeiro, Brazil), following the methodology of Meccatti et al. (2023). For this, Keratinocytes were cultivated at 96-well microplates at a concentration of 2 × 10⁵/well and cultured in 200 µL of Dulbecco’s modified Eagle’s medium (DMEM-LGC Biotechnology, Cotia, Brazil), supplemented with a high concentration of glucose and 10% foetal bovine serum (FBS) (Invitrogen, New York, USA). The plates were incubated at 37 °C with 5% CO₂ for 24 h to allow cell adhesion. Then, the cells were exposed to different concentrations of the photosensitisers (3-Pt or 4-Pt) or DMEM for 5 min or 24 h. DMEM was used as the negative control group.

Metabolic activity was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric test, using a suspension of 0.5 mg of powdered MTT (Sigma-Aldrich) in 1 mL of DMEM supplemented with 10% FBS. The MTT solution was transferred to 96-well microplates at a volume of 100 µL/well, followed by incubation in the dark at 37 °C with 5% CO₂ for 4 h. Then, the solution was discarded, and 100 µL/well of dimethyl sulphoxide (DMSO, Sigma) was added. After 10 min of incubation in a shaker, the absorbance of the wells was measured using a microplate reader at a wavelength of 570 nm. The optical densities (OD) obtained were then converted into percentage of cell viability. This assay was conducted in duplicate (technical replicate) at 2 different times (biological replicate). Percentages of cell viability above 80% were considered as non-cytotoxicity; within 80%–60% weak; 60%–40% moderate; and below 40% strong cytotoxicity (López-García et al. 2014).

2.5. Study of aPDT mediated by 3-Pt and 4-Pt on C. albicans biofilms

Candida albicans biofilms were formed on the bottom of 96-well plates according to methodology of Lapena et al. (2022). Initially, 200 μL of C. albicans suspension was added to each well, and the plates were incubated for 1 h and 30 min at 37 °C at 75 r/min (Quimis, São Paulo, Brazil) to promote initial adhesion. Following this period, the cells were washed twice with PBS, and 200 μL of Yeast Nitrogen Base (YNB, Difco) broth with 100 mmol/L glucose was added to each well. The plates were incubated at 37 °C for 24 h. Next, the broth was removed and replaced with fresh broth, and the biofilms were incubated for an additional 24 h, resulting in a 48 h biofilm.

After biofilm formation, the broth was aspirated, and the biofilms were washed twice with 200 μL of PBS. Then, the biofilms were treated with 3-Pt (2× MIC), 4-Pt (2× MIC), or PBS solution according to the experimental groups. Subsequently, the biofilms of the light groups were irradiated with blue LED light source for 40 s. Then, the biofilms were detached from the bottom of the wells using an ultrasonic homogeniser (Sonopuls HD 2200, Bandelin Electronic, Berlin, Germany) operating at 7.0 W for 30 s. Serial dilutions were then prepared and plated in Petri dishes containing SDA. The plates were stored for 24 h at 37 °C to enumerate the CFU. This test had 10 technical replicates and was carried out 3 times (biological replicate).

2.6. Effects of aPDT with 3-Pt on fungal cells: determination of ROS production

After the assays in planktonic cultures of C. albicans, toxicity in keratinocytes and biofilms, 3-Pt porphyrin was selected for further investigation related to its mechanism of action. The effects of aPDT associated with 3-Pt on ROS production by C. albicans cells were determined by a fluorimetric assay using the 2,7-dichlorofluorescin diacetate probe (DCFH-DA; 10 µmol/L, Invitrogen, Life Technologies, Carlsbad, USA) (Carmo et al. 2022). To this end, previously standardised suspension of C. albicans (10⁷ cells/mL) was placed in 96-well microdilution plates containing RPMI 1640 medium along with 3-Pt and/or light, according to the experimental groups. The probe was added 30 min before each time point designated for ROS measurement (2-, 6- and 24 h post-treatment). Fluorescence intensity was measured using a fluorimeter (Varioskan Lux multimode microplate reader, Thermo Scientific; Waltham, MA, EUA) with excitation and emission wavelengths of 485 and 530 nm, respectively. This assay was conducted with 8 technical replicates at 3 different times (biological replicate).

2.7. Effects of aPDT mediated by 3-Pt on fungal cells: analysis by Transmission Electron Microscopy (TEM)

Standardized suspensions of C. albicans (10⁸ cells/mL) in RPMI medium were treated with LED alone (P-L+), 3-Pt alone (3-Pt+L-) or aPDT (3-Pt+L+) at their MIC values. A non-treated group was included as negative control (P-L-). Then, C. albicans cells were fixed for at least 2 h at room temperature in paraformaldehyde 4.0%, glutaraldehyde 2.5% in cacodylate buffer 0.1 mol/L, pH 7.2. After fixation, cells were washed in PBS and postfixed in osmium tetroxide (OsO₄) 1.0% cacodylate buffer 0.1 mol/L, pH 7.2 with potassium ferricyanide 1.0%, and calcium chloride (CaCl₂) 5.0 mmol/L at room temperature, in the dark. Cells were then washed in PBS, dehydrated in acetone, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed in a Tecnai G2 Spirit BioTWIN 120 kV (FEI) Transmission Electron Microscope. This test was performed in duplicate (technical replicate) at 2 different times (biological replicate).

2.8. Study of aPDT mediated by 3-Pt on denture stomatitis biofilms using a microcosm model

2.8.1. Collection of clinical samples

For this part of the study, clinical samples were obtained from two patients diagnosed with denture stomatitis, who received care at a Basic Health Unit located in the city of São José dos Campos, SP, Brazil. The experimental protocol was approved by the Ethics Committee of the Institute of Science and Technology (CEPh of ICT/Unesp) under reference number 5.827755. After collection using a cytobrush, the samples were cultured on Brain Heart Infusion (BHI, Sigma) for 24 h. The presence of yeast and hyphae, suggestive of Candida cells, were verified by microscopic analysis using Gram stain. Finally, these samples were stored in a freezer at −80 °C.

2.8.2. Microcosm biofilm formation

Microcosm biofilms were formed according to the methodology described by Garcia et al. (2021b) with modifications. Samples of denture stomatitis stored in a freezer were thawed and homogenised. Then, 400 µL were transferred to 20 mL of BHI broth supplemented with 5% sucrose and incubated at 37 °C in 5% CO₂ for 48 h. After incubation, the suspension was centrifuged and washed twice with PBS. The pellet was then resuspended in 20 mL of BHI broth supplemented with 5.0% sucrose. In a 24-well microplate containing acrylic resin discs, 2 mL of this microbial suspension was added to cover all the acrylic resin. The plates were incubated at 37 °C for 120 h for biofilm formation, under 5% CO₂ pressure. Every 24 h, the wells were washed with PBS, and 2 mL of fresh broth were added.

2.8.3. Application of aPDT on microcosm biofilms

After biofilm formation, the specimens were transferred to new wells and submerged in 200 µL of 3-Pt photosensitiser or PBS. Then, the plates were irradiated with blue LED light at a wavelength of 420 nm for 40 s. The non-irradiated groups were kept in the dark for the same period.

2.8.4. Analysis of biofilms by counting the viable cells

The specimens containing the treated biofilms (five per group) were transferred to Falcon tubes containing 4 mL of PBS. The adhered biofilms were detached from the bottom of the wells using homogenising ultrasound (Sonopuls HD2200, Bandelin Electronic, Berlin, Germany) at 7.0 W for 30 s. Biofilm suspensions were serially diluted and plated on BHI agar and on selective culture media, including Mitis Salivarius agar for streptococci, Mannitol salt agar for staphylococci, Rogosa agar for lactobacilli and SDA with chloramphenicol for yeast. The plates were incubated at 37 °C for 48 h at 5% CO₂ for enumeration of CFU/mL.

2.8.5. Analysis of biofilms by Scanning Electron Microscopy (SEM)

For this analysis, the specimens containing the treated biofilms (two per group) were submerged in 1 mL of 2.5% glutaraldehyde for 1 h to fix the cells. Next, they were dehydrated using a series of increasing ethanol concentrations (10%, 25%, 50%, 75%, and 90%), with each concentration maintained for 20 min. Finally, the biofilms were submerged in absolute ethanol for 1 h. The dehydrated biofilms were incubated at 37 °C for 24 h, and then mounted in aluminium stubs and gold-coated for 160 s at 40 mA (Denton Vacuum Desk II, Denton Vacuum, Moorestown, USA). After metallization, the biofilms were analysed using a scanning electron microscope (JEOL JSM-7900, Peabody, USA).

2.9. Action of aPDT mediated by 3-Pt on burn wounds infected by C. albicans using G. mellonella model

2.9.1. Galleria mellonella larvae

Galleria mellonella larvae in their final larval stage served as the host model. These larvae were obtained from the Invertebrate Laboratory of ICT/Unesp. Each experimental group consisted of 10 larvae, with a body weight ranging between 200 and 250 mg. Throughout the experiment, the larvae were not fed. Additionally, for each trial, a control group consisting of larvae without any intervention was included to ensure the quality of larval rearing (Garcia et al. 2024).

2.9.2. Induction of burn injury and C. albicans infection in G. mellonella

Burn injury induction was performed following the methodology introduced by Figueiredo-Godoi et al. (2022). The larvae were placed individually in Petri dishes (35 cm × 10 cm) and maintained at 37 °C. Four hours prior to the experiment, the larvae were refrigerated at 4 °C until use. Antisepsis of the larval cuticle was carried out using 70% ethanol. Subsequently, a steel instrument was heated until it reached a red-hot stage and applied to the dorsal portion of the larvae for 4 s. For infection, immediately after the induced burn injury, 10 μL of a standardised suspension of C. albicans was locally applied to the lesion site. The larvae were kept immobile for 30 s before being returned to the Petri dish, then they were incubated at 37 °C.

2.9.3. Application of aPDT on the infected wound of G. mellonella larvae

Thirty minutes after inducing burn injury and C. albicans infection, the larvae were submitted to aPDT, that involved the application of 10 μL of 3-Pt porphyrins on the burn lesion and LED irradiation (in predetermined parameters). The following groups were performed: healthy larvae; larvae with burn alone; larvae with burn and infection with no treatment; larvae with burn and infection treated with 3-Pt; and larvae with burn and infection subjected to 3-Pt and LED irradiation (aPDT). Each group consisted of 15 larvae, and assays were repeated at 2 different times.

2.9.4. Survival analysis of G. mellonella larvae

Following the treatments, the larvae were reintroduced on the plates and incubated at 37 °C in the dark. Survival analysis was carried out daily for a duration of 168 h (7 d). The mortality rate of larvae was assessed daily to create the survival curve. Larvae exhibiting no movement upon tactile stimulation with metal tweezers were considered dead.

2.9.5. Assessment of G. mellonella larvae health index

Larvae were monitored based on a pathological scoring system proposed by Loh et al. (2013), based on the following parameters: movement activity, extent of silk production (cocoon formation), melanisation and survival. A score was attributed to each parameter as follow: a) Movement activity: 0 (no movement); 1 (minimal movement upon stimulation); 2 (movement upon stimulation); 3 (movement without stimulation); b) Cocoon formation: 0 (absence); 0.5 (incomplete); 1 (complete); c) Melanisation: 0 (complete); 1 (dark spots in brown larvae); 2 (more than three spots in beige larvae); 3 (less than three spots in beige larvae); 4 (no melanisation); d) Survival: 0 (dead larvae); 2 (alive). These scores were combined to generate a comprehensive larval health index. The average scores obtained for each attribute were normalised to percentages (100%) and represented graphically.

2.9.6. Recovery of C. albicans from G. mellonella haemolymph

Following 2 h after aPDT application, larvae were euthanised through a ventral incision along the cephalocaudal axis using a scalpel. Haemolymph was then extracted by gently squeezing the larvae’s body through the incision. Subsequently, the haemolymph of the larvae was serially diluted and plated onto SDA supplemented with chloramphenicol (0.1 mg/mL). After 48 h of incubation, CFU/mL were enumerated for each experimental group (Garcia et al. 2024).

2.10. Statistical analysis

One-way ANOVA followed by Tukey’s multiple comparison test were used to determine significant differences between groups. Kaplan-Meier survival analysis was employed to generate survival curves in G. mellonella assays. These analyses were conducted using GraphPad Prism 9.0, considering a significant level of 5%.

3. Results

3.1. 3-Pt and 4-Pt porphyrins had strong antifungal activity in aPDT against planktonic cells of C. albicans

For the study in planktonic cultures of C. albicans, initially the MIC values for 3-Pt or 4-Pt porphyrins were determined in the presence of LED irradiation (aPDT) and in dark conditions. No MIC values were observed for 3-Pt or 4-Pt (>64 µmol/L) in the dark, indicating that these photosensitisers alone had no antifungal activity. However, when irradiated with LED source for 20 s, a MIC value of 16 µmol/L was found for both 3-Pt and 4-Pt porphyrins.

Then, the concentration of 16 µmol/L for 3-Pt and 4-Pt porphyrins was adopted to determine the kill curve of C. albicans at different irradiation times. aPDT mediated by 3-Pt porphyrin caused statistically significant reduction in C. albicans cell viability within just 5 s of irradiation, that corresponded to an inhibition of 1.48 Log₁₀ (CFU). This inhibition was gradually intensified, reaching a complete inhibition of fungal cells after 40 s of irradiation. In relation to aPDT mediated by 4-Pt porphyrin, the first statistically significant inhibition was observed at 20 s of irradiation, with only 0.5 Log₁₀ of reduction. The greatest inhibition (1.5 Log₁₀ CFU) was reached within 80 s of irradiation (Figure 2).

Figure 2.

Time-kill curve of Candida albicans. CFU/mL (Log₁₀) count after different irradiation times using blue LED source (0.79–12.78 J/cm²). The following groups were evaluated: non-treated group without porphyrin and not irradiated by LED (P-L-); group irradiated by LED in the absence of porphyrin (P-L+); group treated with 3-Pt porphyrin in dark conditions (3-Pt+L-); group treated with 4-Pt porphyrin in dark conditions (4-Pt+L-); group treated with aPDT using 4-Pt porphyrin and LED irradiation (4-Pt+L+); and group treated with aPDT using 3-Pt porphyrin and LED irradiation (3-Pt+L+).

In the study of planktonic cultures of C. albicans, the hyphae formation after a PDT was also quantified by optical microscopy. The non-treated group (P-L-) and group treated only with LED irradiation (P-L+) exhibited yeasts and hyphae in a proportion of approximately 52% and 49%, respectively. On the other hand, the groups treated with 3-Pt porphyrin in the presence (3-Pt+L+) or absence of irradiation (3-Pt+L-) did not exhibit hyphae formation. Only yeasts were observed (100% of the C. albicans cells). Similar results were observed for the assays with 4-Pt porphyrin, in which only 1% of hyphae formation was found in the 4-Pt+L- and 4-Pt+L+ groups (Figure 3). These results suggested that the 3-Pt and 4-Pt porphyrins have influence in the dimorphic transition of C. albicans cells independently of exposure to light.

Figure 3.

Filamentation assay. Percentage of hyphae and yeasts in planktonic cells of Candida albicans after aPDT treatments or other experimental conditions. The following groups were included: group without porphyrin and not irradiated by LED (P-L-); group irradiated by LED in the absence of porphyrin (P-L+); group treated with 3-Pt porphyrin in dark conditions (3-Pt+L-); group treated with 4-Pt porphyrin in dark conditions (4-Pt+L-); group treated with aPDT using 4-Pt porphyrin and LED irradiation (4-Pt+L+); and group treated with aPDT using 3-Pt porphyrin and LED irradiation (3-Pt+L+).

3.2. 3-Pt porphyrin was non-cytotoxic to keratinocytes, while 4-Pt porphyrin showed weak cytotoxicity

Human keratinocytes were exposed to different concentrations of 3-Pt and 4-Pt porphyrins (48 to 0.06 µmol/L) for a period of 5 min or 24 h. Promisingly, all concentrations of 3-Pt porphyrins maintained the cell viability above 80%, indicating non-cytotoxicity. More specifically, treatment with 3-Pt at 48 μmol/L maintained the cell viability in 84% and 83% after 5 min and 24 h, respectively. For 4-Pt porphyrin (48 μmol/L), a weak cytotoxicity was observed, with 71% and 72% of keratinocytes viable after 5 min and 24 h, respectively (Figure 4).

Figure 4.

Toxicity for human keratinocytes. Cell viability percentage relative to the treatments with different concentrations of porphyrins. (a) Treatment with 3-Pt porphyrin by 5 min. (b) Treatment with 3-Pt porphyrin by 24 h. (c) Treatment with 4-Pt porphyrin by 5 min. (d) Treatment with 4-Pt porphyrin by 24 min.

3.3. aPDT mediated by 3-Pt porphyrin had greater antifungal activity than 4-Pt porphyrin on C. albicans biofilms

In this study, within 48 h of incubation, C. albicans formed a mature biofilm composed of approximately 8 Log₁₀ (CFU). The specific values of C. albicans counts for the biofilms not treated with a PDT were 8.33 Log₁₀ (CFU) for P-L- group, 8.33 Log₁₀ (CFU) for P-L+ group, 8.50 Log₁₀ (CFU) for 3-P+L- group, and 8.49 Log₁₀ (CFU) for 4-P+L- group. Promisingly, the biofilms treated with aPDT exhibited a count of 4.38 Log₁₀ (CFU) for 3-Pt+L+ group and 7.47 Log₁₀ (CFU) for 4-Pt+L+ group. Compared to P-L- group, a PDT mediated by 3-Pt and 4-Pt porphyrins lead to a reduction of approximately 4 Log₁₀ (CFU) and 0.8 Log₁₀ (CFU), respectively. Therefore, a PDT with 3-Pt porphyrin had higher antibiofilm activity compared to 4-Pt porphyrin (Figure 5).

Figure 5.

Candida albicans biofilms. Mean and standard deviation values of the C. albicans count (CFU). The following groups were included: group without porphyrin and not irradiated by LED (P-L-); group irradiated by LED in the absence of porphyrin (P-L+); group treated with 3-Pt porphyrin in dark conditions (3-Pt+L-); group treated with aPDT using 3-Pt porphyrin and LED irradiation (3-Pt+L+); group treated with 4-Pt porphyrin in dark conditions (4-Pt+L-); and group treated with aPDT using 4-Pt porphyrin and LED irradiation (4-Pt+L+). Different letters represent statistically significant differences (p ≤ 0.05).

3.4. Antifungal activity of aPDT with 3-Pt porphyrin was accompanied by increased ROS production

The high antifungal activity of aPDT with 3-Pt porphyrin, in both planktonic and biofilm stages of C. albicans, instigated a further investigation about its mechanisms of action on fungal cells. For this, the intracellular levels of ROS were measured at 2, 6, and 24 h after treatments. Across all time points analysed, the P-L- and P-L+ groups showed similar results of ROS production. However, treatments with 3-Pt porphyrin in dark condition (3-Pt+L- group) and mainly in the irradiation condition (3-Pt+L+ group) led to an increase in ROS production. Notably, the 3-Pt+L+ group showed elevated ROS levels in the three observation times, which were superior to all other groups, with statistically significant difference among them (Figure 6).

Figure 6.

ROS production by Candida albicans cells. Mean and standard deviation values of ROS levels measured by fluorescence analysis (arbitrary units: AU) at different observation times (2, 6, and 24 h after treatments). The following groups were analysed: group without porphyrin and not irradiated by LED (P-L-); group irradiated by LED in the absence of porphyrin (P-L+); group treated with 3-Pt porphyrin in dark conditions (3-Pt+L-); and group treated with aPDT using 3-Pt porphyrin and LED irradiation (3-Pt+L+). Different letters represent statistically significant differences (p ≤ 0.05).

3.5. aPDT using 3-Pt porphyrin caused ultrastructural damages in C. albicans cells

To complete the investigation of mechanisms of action of aPDT on Candida cells, TEM images were obtained for the P-L-, 3-Pt+L-, and 3-Pt+L+ groups. Cells of C. albicans from the untreated group (P-L-) and the group treated solely with the photosensitiser (3-Pt+L-) exhibited well-preserved morphological features, characterised by typical and distinct cell membrane, cell wall, and nucleus structures. Conversely, cells subjected to aPDT (3-Pt+L+) displayed intracellular alterations with a loss of their oval form and modifications on the cell wall. These cells also exhibited cytoplasmic retraction, cell wall rupture, and nuclear fragmentation (Figure 7). These findings suggest that the increased ROS production by aPDT was able to affect multiple cell targets.

Figure 7.

Transmission Electron Microscopy (TEM) analysis. (a) Group without porphyrin and not irradiated by LED (P-L-). (b) Group treated with 3-Pt porphyrin in dark conditions (3-Pt+L-). (c) Group treated with aPDT using 3-Pt porphyrin and LED irradiation (3-Pt+L+). Candida albicans cells of the 3-Pt+L+ group showed cytoplasmic retraction, cell wall rupture, and nuclear fragmentation.

3.6. Denture stomatitis biofilms subjected to aPDT with 3-Pt porphyrin had significantly reduction of yeasts, lactobacilli, streptococci, and staphylococci

With well-successful results of aPDT mediated by 3-Pt porphyrin on monotypic biofilms of C. albicans, this research advanced to more complex biofilms associated with Candida spp. For this, microcosm biofilms were formed in vitro using samples collected from two different patients (sample 1 and sample 2). Both samples 1 and 2 generate complex biofilms formed by yeasts and bacteria, including lactobacilli and streptococci. Biofilm from sample 1 was also composed of staphylococci.

Promisingly, these biofilms were significantly reduced with aPDT (3-Pt+L+) application compared to the groups without aPDT (P-L-, P-L+, and 3-Pt+L-). For the biofilm obtained from sample 1, it was found a reduction of approximately 0.8 Log₁₀ (CFU) for lactobacilli, 1.8 Log₁₀ (CFU) for staphylococci, 4.3 Log₁₀ (CFU) for streptococci, and 4.4 Log₁₀ (CFU) for yeasts (Figure 8a). In relation to biofilm obtained from sample 2, aPDT caused inhibitions of approximately 1.5 Log₁₀ (CFU) for lactobacilli, 3.0 Log₁₀ (CFU) for streptococci, and 2.0 Log₁₀ (CFU) for yeasts (Figure 8b). Therefore, aPDT with 3-Pt porphyrin had antifungal activity against yeast even in mixed biofilms with bacteria. The antimicrobial effects were also extended to different groups of bacteria, embracing lactobacilli, streptococci, and staphylococci. Amongst these microorganisms, yeasts and streptococci showed higher susceptibility to aPDT in relation to lactobacilli.

Figure 8.

Viability of microcosm biofilms from patients with denture stomatitis. Mean and standard deviation values of CFU count of microorganisms in non-selective medium BHI, lactobacilli in Rogosa agar, staphylococci in Mannitol agar, streptococci in MS agar and yeasts in Sabouraud agar. (a) Microcosm biofilm formed from sample of patient 1. (b) Microcosm biofilm formed from sample of patient 2. The following groups were compared: group without porphyrin and not irradiated by LED (P-L-); group irradiated by LED in the absence of porphyrin (P-L+); group treated with 3-Pt porphyrin in dark conditions (3-Pt+L-); and group treated with aPDT using 3-Pt porphyrin and LED irradiation (3-Pt+L+). Different letters represent statistically significant differences (p ≤ 0.05).

Besides the viable cell count, the formation of a mature denture stomatitis biofilm was confirmed by SEM analysis. The SEM images of the untreated group (P-L-) displayed a dense and compact biofilm characterised by a complex three-dimensional architecture. These biofilms were composed of numerous microbial aggregates embedded in an extracellular matrix, covering the entire acrylic resin surface. Various microbial morphologies were found, such as yeasts, fungal hyphae, cocci, coccobacilli, and filamentous bacilli, reflecting the microbial diversity of denture stomatitis biofilms. The 3-Pt+L- group presented a biofilm density comparable to that of the P-L- group, although slightly reduced. However, when biofilms were subjected to aPDT (3-Pt+L+ group), a substantial reduction in adhered microorganisms was evident, leading to a disaggregation of the biofilms, and exposing the acrylic resin surface. In these images, only a few cellular aggregates and isolated cells were observed (Figure 9).

Figure 9.

SEM analysis of microcosm biofilms from patients with denture stomatitis. (a–c) Biofilms obtained from sample of patient 1. (d–f) Biofilms obtained from sample of patient 2. The following groups were analysed: group without porphyrin and not irradiated by LED (P-L-); group treated with 3-Pt porphyrin in dark conditions (3-Pt+L-); and group treated with aPDT using 3-Pt porphyrin and LED irradiation (3-Pt+L+). Images at 5 K× magnification, indicating the presence of biofilms composed of yeasts, hyphae, and bacteria on acrylic resin surface.

3.7. 3-Pt porphyrin-mediated aPDT showed efficacy in treating burn wounds infected by C. albicans in G. mellonella

In this study, aPDT with 3-Pt porphyrin was also investigated as a possible treatment for burn wounds associated with C. albicans, using an experimental infection model in G. mellonella. In the results, larvae with infected burn wounds showed 100% mortality when treated only with PBS (P-L- group) or 3-Pt porphyrin in dark condition (3-Pt+L- group) in 120 and 168 h post-treatment, respectively. When the larvae were treated with aPDT (3-Pt+L+ group), the mortality rate was only 25% at the end of the experiment (Figure 10a).

Figure 10.

In vivo study in Galleria mellonella model. Different experimental conditions were evaluated as follows: healthy larvae (Control group); larvae subjected to burn injury without infection (Burn group); larvae subjected to burn injury and infection by C. albicans (Infected burn group); larvae with burn wound infected and treated with 3-Pt in dark conditions (Infected burn 3-Pt+L- group); and larvae with burn wound infected and treated with 3-Pt in the presence of LED irradiation (Infected burn 3-Pt+L+ group). (a) Survival rate monitoring of G. mellonella larvae over 168 h, with statistically significant difference between Infected burn group and Infected burn 3-Pt+L+ group (p = 0.0001). (b) Measurement of the health index of G. mellonella larvae over 168 h, with a statistically significant difference between the Infected burn group and the 3-Pt+L+ group (p = 0.0001).

In addition to tracking the survival curve of G. mellonella, the health status of the larvae was also monitored, assessing attributes such as movement activity, cocoon formation, and melanisation. Larvae with wound infections treated only with PBS (P-L- group) or with 3-Pt in dark condition (3-Pt+L- group) showed a deterioration in health status, characterised by increased melanisation and impaired wound healing. These larvae displayed a gradual decline in the score, reaching zero as all larvae died. On the other hand, larvae treated by aPDT (3-Pt+L+ group) remained healthy, exhibiting lighter colour pigmentation and effective wound healing. Their attributed scores ranged between 80% and 90% until the final day of assessment (Figures 10b and 11).

Figure 11.

Stereomicroscope images obtained from Galleria mellonella larvae. Images were captured at 6, 24, and 48 h after the burn injury. The following conditions were analysed: burn non-treated; burn and infection without treatment; burn and infection treated with 3-Pt porphyrin in dark condition (3-Pt+L-); burn and infection treated with 3-Pt porphyrin in irradiation condition (3-Pt+L+).

Next, G. mellonella larvae with wound infection were treated, and after 2 h their haemolymph was collected to recover C. albicans cells. Groups treated with PBS alone (P-L-) or with 3-Pt in dark condition (3-Pt+L-) had a recovery of fungal cells of 5.4 and 5.6 Log₁₀ (CFU/mL), respectively. However, the group treated with aPDT (3-Pt+L+) displayed a recovery of only 2.8 Log₁₀ (CFU/mL) of C. albicans, demonstrating the efficacy of aPDT in inhibiting Candida invasion and aggressive infection (Figure 12).

Figure 12.

Recovery of Candida albicans cells from haemolymph of Galleria mellonella larvae. Mean and standard deviation values of the C. albicans count (CFU). The following groups were analysed: larvae subjected to burn and infection without treatment (Infected burn group); larvae subjected to burn and infection treated with 3-Pt porphyrin in dark condition (Infected burn 3-Pt+L- group); larvae subjected to burn and infection treated with 3-Pt porphyrin in irradiation condition (Infected burn 3-Pt+L+ group). Different letters represent statistically significant differences (p ≤ 0.05).

Taken together, the localised application of aPDT on infected wounds decreased the number of C. albicans in haemolymph and protected them from a systemic infection, consequently increasing the survival rate by 75%.

4. Discussion

Currently, available therapies for Candida infections face challenges related to bioavailability, side effects, and microbial resistance. In localised infections, such as denture stomatitis and skin wounds, aPDT has been studied as a promising adjuvant therapy (Mardani and Kamrani 2021). To identify new photosensitisers for aPDT, the present study investigated the antifungal effects of two isomers of platinum tetra-cationic porphyrin (3-Pt and 4-Pt) against C. albicans and associated infections.

Despite the extensive exploration of aPDT, no single photosensitiser has been deemed ideal for clinical use (Chibebe Junior et al. 2013; Sasaki et al. 2017; Rossi et al. 2021; Lapena et al. 2022). Porphyrins, heterocyclic organic compounds with a tetrapyrrolic structure comprising four pyrrolic rings linked by methine groups, can play a crucial role in various biological processes (Donnelly et al. 2008; Rosa Pinheiro et al. 2023). Notably, porphyrins can absorb light at wavelengths of 400–800 nm, thereby initiating the photodynamic therapy process. The absorption profile at 420 nm (blue region) makes the porphyrins particularly suitable for clinical application as LED equipment in this specific wavelength is easily available in healthcare settings (Kanpittaya et al. 2021). In addition, porphyrins do not require a pre-irradiation time, as porphyrins bind to the microbial cell wall only upon irradiation, unlike other photosensitisers such as methylene blue (Lapena et al. 2022), toluidine blue (Souza et al. 2010), and chlorin (Figueiredo-Godoi et al. 2019), which require pre-irradiation time for penetration before irradiation.

The antifungal action of isomers of platinum tetra-cationic porphyrins has been little explored, so the initial test of this study was the MIC determination. In this analysis, it was found a MIC value of 16 µmol/L for both 3-Pt and 4-Pt porphyrins against C. albicans. Employing the same Pt porphyrins, Rosa Pinheiro et al. (2023) tested the effects of aPDT with a white light source against C. albicans, finding a MIC value of 0.45 μmol/L though the irradiation time of 120 min. In another study (Cormick et al. 2011), the action of 5-(4-trifluorophenyl)-10,15,20-tris(4-N,N,N-trimethylammoniumphenyl)porphyrin at a concentration of 5 μmol/L was evaluated on planktonic culture of C. albicans. As a result, a 5 Log₁₀ (CFU) inhibition was observed after 30 min of irradiation with white light. In the present study, 3-Pt porphyrin at 16 μmol/L caused complete inhibition of fungal cells with just 40 seconds of blue light irradiation. Although less impactful, 4-Pt porphyrin also inhibited C. albicans in these irradiation conditions, leading to 1 Log₁₀ (CFU) reduction. These results highlight the critical influence of aPDT parameters on treatment efficacy and the need for optimisation in clinical applications.

Since 3-Pt and 4-Pt porphyrins inhibited the growth of C. albicans, we studied the effects of aPDT mediated by these photosensitisers against virulence factors of C. albicans, such as filamentation. The capacity of C. albicans in forming hyphae is considered an important virulence factor, as it is directly related to biofilm formation and pathogenicity (Honorato et al. 2022; Ganser et al. 2023). In this study, when evaluating the action of aPDT associated with 3-Pt and 4-Pt porphyrins in reducing filamentation, we observed an inhibition of 100% and 99% for the 3-Pt+L+ and 4-Pt+L+ groups, respectively. An interesting fact about this test is that the groups without irradiation (3-Pt+L- and 4-Pt+L-) also showed action against the filamentation of this fungus. Despite the need for further studies to understand the mechanisms of action, these results suggest that Pt porphyrins are capable of interfering with C. albicans filamentation, even without irradiation.

Biofilm formation is another important virulence factor of C. albicans since it can occur on various solid structures, such as medical devices and host tissues (Rodrigues et al. 2023). In biofilms, Candida cells are embedded in an extracellular matrix, which increases the fungus’s resistance to antimicrobials (Xu et al. 2019). Given this, the present study evaluated the impact of Pt porphyrins on the reduction of C. albicans biofilm. Mature biofilms treated with aPDT using 3-Pt porphyrin or 4-Pt porphyrin 32 µmol/L (2× MIC) had a decrease of 4 Log₁₀ (CFU) in the 3-Pt+L+ group and 1.2 Log₁₀ (CFU) in the 4-Pt+L+ group. Although aPDT with Pt porphyrins did not achieve complete inhibition in biofilms as in planktonic cultures, these results are quite significant since fungal biofilms can have high tolerance to aPDT. Using different photosensitisers and irradiation parameters, previous studies have found reductions of C. albicans biofilms ranging from 0.5 to 5 Log₁₀ (CFU) (Costa et al. 2012; Dovigo et al. 2013; Garcia et al. 2021a). Amongst them, methylene blue stands out as the most studied photosensitiser, but its dye characteristics can limit the medical and dental applications. The use of porphyrins can be more advantageous than methylene blue, as they do not stain human tissues.

Cytotoxicity assays are commonly required by researchers to explore the antimicrobial properties of new compounds (Filipić et al. 2024). Additionally, an ideal photosensitiser should exhibit low cytotoxicity in the absence of light (Yan et al. 2023). In this study, the dark toxicity of 3-Pt porphyrin and 4-Pt porphyrin at different concentrations in human keratinocytes (HaCat) was consistently below 30%. These results are in accordance with the ISO 10993-5 (2009) protocol, which defines cellular damage as occurring when viability falls below 70%. These findings are similar to those reported by Wang et al. (2021), who also evaluated the cytotoxicity of the photosensitiser aloe-emodin on HaCat cells. However, Bila et al. (2021) found a cytotoxicity greater than 50% in keratinocytes for a possible photosensitiser in the study (2-chalcone).

Since Candida cells survive in relatively low levels of ROS, these molecules can act as second messengers, promoting fungal growth and facilitating antifungal resistance. However, at high levels, ROS exerts cytotoxic effects, disrupting homoeostasis and causing severe damage to fungal cells (Komalapriya et al. 2015). The production of ROS is a fundamental mechanism of aPDT (Kanpittaya et al. 2021). Tetra-cationic porphyrins have been considered effective compounds in generating ROS when activated by appropriate light irradiation (Pourhajibagher et al. 2020). According to the literature, these derivatives can produce various species, such as singlet oxygen (¹O₂) and free radicals, including hydroxyl radicals (•OH), superoxide (O₂^•–), and hydroperoxyl (•OOH) radicals (Mascio et al. 2019; Pinto et al. 2021). In the present study, analysis of Candida cells, conducted 2 h after aPDT, showed a 242% increase in intracellular ROS production compared to the control group, with further increases observed in the subsequent hours. These data confirm the high capacity of ROS production by the porphyrins studied.

In line with other findings, the present study observed through TEM analysis that the cell wall of C. albicans cells treated with aPDT exhibited ruptures. Similar damages were found in Candida cells treated with 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (Quiroga et al. 2020), Nano-TiO₂ (Damrongrungruang et al. 2024), and 5-aminolevulinic acid (Shi et al. 2021). These findings indicated that aPDT with 3-Pt porphyrin is capable of increasing cell membrane permeability and impairing cell wall function.

In the oral cavity, Candida species coexist with other microorganisms such as Streptococcus and Lactobacillus, resulting in the formation of multispecies biofilms that can be more resistant to antimicrobial therapies. In this context, microcosm biofilms serve as a more accurate in vitro representation of the microbiological complexity present in the oral cavity (Araujo et al. 2022). This study evaluated the efficacy of aPDT associated with 3-Pt porphyrin on microcosm biofilms obtained from two patients with denture stomatitis. To our knowledge, this is the first study assessing aPDT’s action on denture stomatitis microcosm biofilms.

Promisingly, our results showed that aPDT mediated by 3-Pt porphyrin was able to act on microcosm biofilms, decreasing the number of yeast and even bacteria. Fungal cells, the focus of this investigation, showed inhibitions of 4.3 and 2 Log₁₀ (CFU) for samples of patients 1 and 2, respectively. These findings surpass the results reported by Garcia et al. (2021b), who evaluated aPDT with Fotoenticine (chlorin e-6) on microcosm biofilms of dental caries, verifying a maximum reduction of 3.2 Log₁₀ (CFU) for yeasts. In relation to bacteria observed in our study, lactobacilli showed the lowest susceptibility to aPDT (reduction of 0.8 and 1.5 Log₁₀ CFU), staphylococci were present only in the sample from patient 1 (reduction of 1.8 Log₁₀ CFU), and streptococci had higher inhibitions (reduction of 4.3 and 3 Log₁₀ CFU). These results were different from those found by Méndez et al. (2018), who applied aPDT with methylene blue on dental caries microcosm biofilms and found a greater reduction in the count of lactobacilli than streptococci.

SEM images confirmed the yeasts and bacteria reductions of microcosm biofilms after aPDT. In both samples of patients 1 and 2, it was possible to observe a dense biofilm in the 3-Pt-L- and 3-Pt+L- groups, with a large variety of well-aggregated microorganisms and extracellular matrix production. On the other hand, the treatment with aPDT (3-Pt+L+ group) showed a different structure, containing free areas without microbial colonisation, and areas with adhered cells not covered by the extracellular matrix. These findings demonstrated that aPDT with Pt-porphyrin was able to disaggregate the microcosm biofilms from patients 1 and 2.

Expanding the scope of aPDT beyond the oral application, this study evaluated the effect of aPDT with 3-Pt porphyrin against C. albicans cutaneous infection in G. mellonella. This is the first study that analyzes cutaneous infection by C. albicans in an invertebrate model. In a previous study conducted by our research group (Figueiredo-Godoi et al. 2022), aPDT was investigated for Acinetobacter baumannii cutaneous infections using methylene blue, which resulted in 80% of larval survival. Similarly, in the present study, a survival rate of 75% was achieved.

To broaden the investigations into G. mellonella survival, additional health parameters were analysed, such as locomotor activity, melanisation, and cocoon formation. Taken together, these parameters can provide a comprehensive assessment of microbial virulence and infection progression (Loh et al. 2013). aPDT showed notable efficacy in maintaining the larval health, with treated larvae scoring between 80% and 90% on the health index. In contrast, the untreated group exhibited a steady decline in health score, reaching their lowest levels by the last day of survival. As expected, and consistent with previous findings, the recovery of fungal cells from the haemolymph in untreated groups was significantly higher compared to the treated group, indicating reduced capacity for fungal penetration and systemic larval infection post-irradiation.

In summary, we concluded that aPDT mediated by tetra-platinated porphyrins 3-Pt and 4-Pt had activity against planktonic cells, biofilms, and filamentation of C. albicans. Particularly noteworthy was the efficacy of 3-Pt porphyrin, which reached complete inhibition of planktonic cell growth and high anti-biofilm action. The effects of aPDT with 3-Pt porphyrin against Candida cells were associated with increased ROS production and damage to the fungal cell wall. Furthermore, 3-Pt porphyrin-mediated aPDT led to significant reductions in denture stomatitis microcosm biofilm, presenting a broad spectrum against oral microorganisms including yeasts, streptococci, staphylococci, and lactobacilli. Their inhibition effects against C. albicans were further confirmed in an in vivo study, in which aPDT mediated by 3-Pt porphyrin was effective to treat burn infections in G. mellonella larvae. Therefore, 3-Pt porphyrin can be a promising effective photosensitiser for aPDT targeted to treat C. albicans related infections.

Funding Statement

This research was funded by UNESP [PROPe 13/2022], National Council for Scientific and Technological Development [CNPq 407032/2023-1], and São Paulo Research Foundation [FAPESP 2020/09101-8].

Disclosure statement

No potential conflict of interest was reported by the author(s).