Influence of porphyrin cationic charges on photoinactivation of Candida albicans morphotypes

Influence of porphyrin cationic charges on photoinactivation of Candida albicans morphotypes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of porphyrin cationic charges on photoinactivation of Candida albicans morphotypes María G. Alvarez, Paula V. Cordero, Jesica M. González, María E. Pérez, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7992830/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Dec, 2025 Read the published version in Photochemical & Photobiological Sciences → Version 1 posted 11 You are reading this latest preprint version Abstract The increasing resistance of Candida albicans to conventional antifungal therapies highlights the need for alternative treatment strategies. In this study, photodynamic inactivation (PDI) was evaluated using four cationic porphyrins (AB 3 + , A 2 B 2 2+ , A 3 B 3+ , and A 4 4+ ), differing in symmetry and number of positive charges. These compounds were tested against C. albicans in its planktonic, pseudohyphal, and biofilm forms. Upon incubation with 1.0 µM porphyrin, rapid cellular uptake was observed, with A 3 B 3+ and A 4 4+ , showing the highest accumulation (0.65 and 0.50 nmol/10⁶ cells, respectively). The amount of porphyrin bound to cells remained stable over time, with no significant changes beyond 5 min of incubation. PDI was performed using different porphyrin concentrations (0.5–5.0 µM) and light exposure times (5–30 min). A 3 B 3+ and A 4 4+ exhibited potent photoinactivation, reducing cell viability by over 5 log (> 99.999%) after 5 min of irradiation using 2.5 µM porphyrin. Reactive oxygen species quenching experiments indicated that singlet molecular oxygen was the primary cytotoxic agent. Additionally, A 3 B 3+ and A 4 4+ effectively eradicated C. albicans cells on agar surfaces. These porphyrins also inactivated pseudohyphal suspensions of C. albicans , achieving a reduction greater than 5 log, when incubated with 5 µM porphyrin and 5 min of irradiation. Using this concentration, C. albicans biofilms were completely photoinactivated after 60 min of light exposure. These findings demonstrate that highly charged cationic porphyrins are promising photosensitizers for the targeted elimination of C. albicans across its major morphological states. photodynamic inactivation cationic porphyrin Candida albicans pseudohyphae biofilms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Candidiasis is an opportunistic fungal infection and is considered the most prevalent mycosis in humans [1]. In recent years, the incidence of infections caused by Candida spp. has increased significantly on a global scale. This rise is attributed to multiple factors, including the widespread and often inappropriate use of antifungal drugs, which has contributed to the development of resistance, as well as the expanded use of medical devices such as vascular grafts, prosthetic heart valves, catheters, and dental implants [2]. These surfaces provide ideal conditions for microbial colonization and biofilm formation. This is primarily caused by species of the Candida genus, with Candida albicans being the most frequently implicated pathogen. During the course of infection, C. albicans exhibits remarkable morphological plasticity, transitioning from a unicellular yeast form to filamentous structures, such as pseudohyphae and hyphae [3]. This morphological switching plays a critical role in the pathogenesis of candidiasis by facilitating adhesion, tissue penetration, and the establishment of persistent biofilms on both biotic and abiotic surfaces. Candida biofilms are typically composed of dense agglomerates of yeast and hyphal cells embedded within an extracellular polymeric matrix, which confers enhanced protection against environmental stressors, antifungal agents, and host immune defences [4]. The architecture and composition of the biofilm matrix significantly impair drug penetration and promote cell survival under hostile conditions. Due to these characteristics, Candida biofilms exhibit markedly increased resistance to conventional antifungal therapies compared to their planktonic (free-floating) counterparts [5]. Moreover, the biofilm acts as a physical and biochemical shield, not only limiting the efficacy of antifungal drugs but also preventing recognition and clearance by the immune system of the host [6,7]. The persistence of such infections, particularly on medical devices, increases the risk of chronic and systemic disease. In this context, photodynamic inactivation (PDI) of pathogens has gained increasing attention as a non-conventional antifungal approach with promising therapeutic potential [8]. PDI involves the use of a photosensitizer (PS) that selectively associates with microbial cells. Upon activation by visible light in the presence of molecular oxygen, the PS generates reactive oxygen species (ROS) [9]. These reactive intermediates interact with biological components, damaging multiple vital structures within the microorganism, which ultimately leads to cell death. Therefore, this therapy represents a promising alternative for the treatment of multi-resistant pathogens [10]. In this study, the photoinactivation of C. albicans in its yeast, pseudohyphal, and biofilm forms was systematically investigated using a series of cationic porphyrins as PSs. The compounds evaluated, AB 3 + , A 2 B 2 2+ , A 3 B 3+ , and A 4 4+ (Fig. 1 ), differ in the number and spatial distribution of their positive charges. The cationic groups are located at the ends of the flexible spacers and they are combined with lipophilic trifluoromethyl substituents to enhance amphiphilicity. These structural variations were designed to modulate cellular uptake and photodynamic efficacy. First, photokilling activity was tested in C. albicans planktonic cell suspensions, in cells localized on agar surfaces, and in colonies immobilized on solid media. Further mechanistic insights were obtained through the use of ROS scavengers to elucidate the predominant photochemical pathways involved in fungal inactivation. Considering that reversible cell morphogenesis is an important virulence factor, these compounds were also tested for their ability to eliminate C. albicans pseudohyphae. Finally, the photoinactivating efficacy of these porphyrins was assessed in C. albicans biofilms, to explore their potential in the prevention and control of these clinical pathogens. 2. Materials and Methods Materials and instrumentation are provided in the Supplementary Materials. 2.1. Cationic porphyrins The cationic porphyrins, 5-[4-(3- N , N , N -trimethylammoniopropoxy)phenyl]-10,15,20-tris(4-trifluoromethylphenyl)porphyrin (AB 3 ⁺), 5,15-di[4-(3- N , N , N -trimethylammoniopropoxy)phenyl]-10,20-di(4-trifluoromethylphenyl)porphyrin (A 2 B 2 ²⁺), 5,10,15-tris[4-(3- N , N , N -trimethylammoniopropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin (A 3 B 3 ⁺), and 5,10,15,20-tetrakis[4-(3- N , N , N -trimethylammoniopropoxy)phenyl]porphyrin (A 4 ⁴⁺) were synthesized according to previously reported procedures [11]. Stock solutions of each porphyrin (0.5 mM) were prepared in N,N -dimethylformamide (DMF). The concentrations were determined spectrophotometrically using the molar extinction coefficients corresponding to their Soret bands in DMF (1.71 x 10 5 M − 1 cm − 1 for AB 3 + , 1.67 x 10 5 M − 1 cm − 1 for A 2 B 2 2+ , 1.69 x 10 5 M − 1 cm − 1 for A 3 B 3+ , and 1.64 x 10 5 M − 1 cm − 1 for A 4 4+ ) [11]. The final concentration of DMF in all experimental conditions did not exceed 1% v/v, a level confirmed to be non-toxic to C. albicans cells. 2.2. C. albicans cultures The C. albicans strain PC31 used in this study was previously isolated and characterized [12]. For cultivation, yeast cells were grown aerobically in 4 mL of Sabouraud broth (SB) at 37°C for 18–24 h until reaching the stationary growth phase. After incubation, cells were collected by centrifugation at 1200 × g for 15 min and subsequently washed and resuspended in 4 mL of phosphate-buffered saline (PBS; 10 mM, pH 7.2). This procedure yielded a suspension containing approximately 10 7 colony-forming units (CFU)/mL. To obtain the working inoculum, the suspension was diluted tenfold in PBS to reach a final concentration of ~ 10 6 CFU/mL. Cell density was confirmed by serial dilution and plating on Sabouraud agar (SA), followed by incubation at 37°C for 48 h to determine viable CFUs using the spread plate technique [13]. 2.3. Binding of porphyrins to yeast cells C. albicans cell suspensions (2 mL, ~10 6 CFU/mL) in PBS were incubated with 1 µM of the porphyrin in the dark at 37°C for varying durations (2, 5, 15, and 30 minutes). After incubation, the cells were collected by centrifugation at 1200 × g for 5 min and resuspended in 2 mL of 2% (w/v) aqueous sodium dodecyl sulfate (SDS) solution. These suspensions were maintained at 4°C overnight and then subjected to sonication for 30 min to release cell-associated porphyrins. The concentration of porphyrin in the resulting supernatants was determined via spectrofluorimetric analysis (λ exc = 419 nm, λ em = 655 nm), using calibration curves prepared from standard porphyrin solutions in 2% SDS (0.05–0.20 µM). The fluorescence values obtained from each sample were referenced to the total number of cells contained in the suspension [14]. Microscopic fluorescence images of C. albicans cells were acquired using a green filter (EX BP480-550, DM570, BA590). A brightfield image was captured for each region to verify the presence of yeast cells. All images were obtained using a 100× magnification objective and recorded with a CMOS camera [13]. 2.4. Photoinactivation of C. albicans yeast cells Suspensions of C. albicans planktonic cells (2 mL, ~10 6 CFU/mL) in PBS were incubated with varying concentrations of porphyrin (0.5, 1.0, 2.5, and 5.0 µM) for 15 min in the dark at 37°C. Subsequently, 200 µL of each suspension was transferred to the wells of a 96-well microtiter plate and irradiated with white light (90 mW/ cm 2 ) for different periods (5, 15, and 30 min). When required, sodium azide (50 mM), diazabicyclo[2.2.2]octane (DABCO, 50 mM), D-mannitol (50 mM) and L-cysteine (50 mM) were added to cell suspensions from stock solutions 1 M in water [15]. After that, cells were incubated for 30 min at 37 ºC in the dark previous to the treatment with the porphyrins. Studies in deuterated water (D 2 O) were performed using 2 mL of cell suspensions (∼10 6 CFU/mL) in PBS, which were centrifuged (3000 rpm for 15 min) and re-suspended in 2 mL of D 2 O. Then, the cell suspensions in D 2 O were incubated with 1.0 µM PS for 15 min in the dark at 37 ºC. The number of viable C. albicans cells was determined as described above [16]. 2.5 Photoinactivation of C. albicans plated on agar surfaces Yeast cell suspensions (~ 10 6 CFU/mL, 2 mL) were incubated with 5.0 µM PS in the dark for 30 min at 37°C. After treatment, the suspensions were diluted 1:1000 in PBS, and 100 µL of each dilution was uniformly spread onto SA surfaces. The plates were incubated for 15 min at 37°C in the dark and then exposed to white light for different times (15, 30, and 60 min). After that, the plates were incubated at 37°C for 48 h, after which the number of colonies was counted to evaluate cell viability [17]. 2.6. Photoinactivation of C. albicans by agar surface-bound PS Aliquots with different amounts of PSs (0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 nmol) were spread on SA plates (5 cm in diameter) to cover an area of 0.6 cm 2 . Then, plates were spread with 100 µL of a C. albicans cell suspension (~ 10 6 CFU/mL) in PBS. The plates were incubated at 37°C for 30 min in the dark. After that, the cultures were irradiated with white light for 30 min, the plates were incubated at 37°C in the dark. Yeast growth was observed 48 h post-treatment [17]. 2.7. Studies in C. albicans pseudohyphae C. albicans pseudohyphae were obtained as previously described [18]. Yeast cell suspension (~ 10 6 CFU/mL) was incubated with human serum (HS) during 4 h at 37°C to induce the formation of pseudohyphae. After incubation, the germ tube formation was verified trough optical microscopy. Pseudohyphae were harvested and washed three times to eliminate all HS by centrifugation (1200 × g for 15 min) and suspended in PBS until appropriated cell density to obtain ~ 10 6 CFU/mL. Then, 2 mL of the suspension was placed in Pyrex brand culture tubes (13 x 100 mm) and incubated with 0.5, 1.0, 2.5 and 5.0 µM porphyrin for 30 min in the dark at 37°C. After that, 200 µL of culture were placed in wells of a 96-wells microtiter plate and exposed to visible light for different time (2, 5, 15 and 30 min). Subsequently, pseudohyphal viability was assessed by CFU/mL enumeration following incubation at 37°C for 48 h, as previously reported [16]. 2.8. Experiments in C. albicans biofilms A C. albicans cell suspension (~ 10 7 CFU/mL) was prepared in PBS supplemented with 7% v/v human serum (HS). Then, 900 µL of this culture was placed in wells of a 48-wells microtiter plate containing one polyvinylchloride (PVC) disc (5 mm Ø x 1.5 mm) in each well. The plate was incubated for 90 min at 37°C with gentle shaking (~ 75 rpm) to allow cell adhesion to the discs. After incubation, the discs were removed and washed by successive immersion in PBS to eliminate non-adherent cells. For the proliferation step, the PVC discs were transferred to fresh wells containing 5.0 µM porphyrin in SB supplemented with 7% v/v HS and incubated for 18 h at 37°C. After that, the discs were carefully washed twice with PBS and placed into fresh wells containing 900 µL of PBS. Biofilms were then irradiated with white light for 60 min. Following irradiation, each disc along with the corresponding well content was transferred to a test tube, sonicated for 1 min, and vigorously vortexed for 2 min to detach the biofilm cells from the disc [18]. Viable C. albicans cells were determined by CFU counting, as described above. 2.9. Controls and statistical analysis Control groups included C. albicans cultures incubated with or without the PS under dark conditions, as well as cell samples exposed to light in the absence of PS. All experiments were performed independently in triplicate. Error bars represent the standard deviation of the mean. Statistical significance was determined using one-way analysis of variance (ANOVA) at a 95% confidence level (p < 0.05). 3. Results and discussion 3.1. Binding of porphyrin to C. albicans planktonic cells The binding ability of cationic porphyrins to C. albicans cells was evaluated using cell suspensions (~ 10 6 CFU/mL) in PBS. C. albicans cultures were incubated with 1.0 µM PS at 37°C in the dark for different periods. The amount of porphyrin recovered from the cells after each incubation time is presented in Fig. 2 . Notably, the highest level of cell-bound PS was observed after a short incubation time of 5 min. Furthermore, the amount of cell-bound porphyrin did not significantly change with longer incubation times, such as 15 and 30 min. The recovered porphyrin levels were 0.66 and 0.50 nmol/10 6 cells for A 3 B 3+ and A 4 4+ , respectively. In contrast, lower values were observed for AB 3 + and A 2 B 2 2+ with 0.10 and 0.16 nmol/10 6 cells, respectively. In previous investigation, the binding of 5,10,15,20-tetra(4- N -methylpyridyl)porphyrin (TMPyP 4+ ) to the same C. albicans strain used in the present study was evaluated, yielding 1.7 nmol/10 6 cells after incubation with 5 µM PS [17]. Comparable binding kinetics were also reported for 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin (TFAP 3+ ) and 5,10,15,20-tetra(4- N , N , N -trimethylammoniumphenyl)porphyrin (TMAP 4+ ) [12]. Under these conditions, both cationic porphyrins showed similar bounding efficiencies. In the present study, the uptake value obtained for A 4 4+ was consistent with that previously informed [14]. Also, the uptake of A 3 B 3+ was slight higher than that found for A 4 4+ , possibly influenced by the amphiphilic character of the tricationic porphyrin derivate, which may favour interactions with both hydrophobic and polar domains of the fungal cell surface. Moreover, the intracellular distribution of the porphyrins in yeast cells was examined by fluorescence microscopy. The micrographs revealed that C. albicans cells incubated with 1.0 µM PS for 15 min in the dark exhibited the characteristic red fluorescence emission of porphyrin derivatives (Fig. 3 ). The images indicated that the porphyrins were predominantly localized in the cell envelope. A similar peripheral fluorescence pattern was previously reported for a tricationic porphyrin in C. albicans cells [19]. Consistently, fluorescence microscopy studies demonstrated the accumulation of TMPyP 4+ in the cell wall and plasma membrane, but not in the cytoplasm [20]. Likewise, C. albicans cells treated with TMAP 4+ displayed the typical red fluorescence of porphyrin derivatives, whereas incubation with an anionic porphyrin produced no detectable intracellular signal [21]. Furthermore, the fluorescence images of A 4 4+ in yeast cells were compared with those produced by its non-cationic analogue [13]. All porphyrins evaluated in this study exhibited comparable fluorescence quantum yields of approximately 0.1 in DMF [22]. Accordingly, the observed fluorescence intensity was proportional to the extent of porphyrin uptake by the cells. In particular, a weak emission was observed for the monocationic porphyrin AB 3 + , whereas no significant differences in red fluorescence intensity were detected between yeast cells treated with A 3 B 3+ and A 4 4+ . Thus, the incorporation of three or four cationic ammonium substituents into the porphyrin macrocycle markedly enhanced its binding affinity toward C. albicans cells. 3.2. Photosensitized inactivation of C. albicans planktonic cells The cytotoxic effect photosensitized by porphyrins was fist evaluated in C. albicans cell suspensions in PBS. The cultures of yeast (~ 10 6 UFC/mL) were incubated with 5.0 µM PS for 15 min in the dark at 37°C and irradiated with white light for 5, 15 and 30 min. At this concentration of porphyrin, the viability of the C. albicans was not affected by incubation in the dark (Figure S1 ). Furthermore, cell survival was not modified by irradiation of the culture without porphyrin (Fig. 4 ). Therefore, these control experiments confirm that the photoinactivation of C. albicans was caused by porphyrin-induced photodynamic activity. As shown in Fig. 4 , photoinactivation of C. albicans was dependent on the porphyrin derivative and the irradiation times. When cultures were incubated with 5.0 µM PS, the photokilling effect induced by AB 3 + and A 2 B 2 2+ resulted in a reduction of about 1.0 log after 5 min of irradiation, while increased to 2.0 log after 30 min of exposure to white light. Under the same conditions, the most effective porphyrins were A 3 B 3+ and A 4 4+ , achieving over 6 log reductions in cell viability after 5 min of irradiation. Based on these results, the photodynamic activity of A 3 B 3+ and A 4 4+ was further investigated by varying both the treatment concentration and irradiation time. Both porphyrins exhibited similar photoinactivation capacities to eliminate C. albicans cells (Fig. 5 ). After 15 min of irradiation, a decrease in cell viability greater than 1 log and 3 log was achieved in cultures treated with 0.5 µM and 1.0 µM PS, respectively. The reduction in cell survival increased to 5 log for yeasts incubated with 2.5 mM PS and irradiated for only 5 min exposure, while no viable cells were detected after 15 min of irradiation. Previous PDI studies were determined using tetracationic porphyrins in C. albicans strain under comparable experimental conditions. When yeast cultures were incubated with 5 µM TMPyP 4+ , a reduction of approximately 1.7 log in cell survival was observed upon an irradiation of 5 min [17]. A markedly higher photoinactivation for C. albicans was found for cells treated with 5 µM TMAP 4+ , achieving about a 3.8 log decrease after 5 min of white light irradiation [12]. Under these conditions, A 4 4+ exhibited strong photodynamic activity against C. albicans in suspension, producing a 3.6 log reduction in cell viability upon irradiation in culture tubes [14]. Therefore, both A 3 B 3+ and A 4 4+ proved to be effective photosensitizers for the inactivation of C. albicans planktonic cells, even at low concentrations and under relatively low light fluence. 3.3. Effect of scavengers of ROS on the photoinactivation of C. albicans planktonic cells With the purpose of obtaining information about the photodynamic mechanism of action sensitized by A 3 B 3+ and A 4 4+ in C. albicans cell suspensions, PDI studies were carried out in presence of ROS scavengers (sodium azide, DABCO, D-mannitol, and L-cysteine) and D 2 O. Cultures were first treated with the additives for 30 min at 37 ºC in the dark and then with 1.0 µM porphyrin for 30 min at 37 ºC in the dark. Cell viability was not affected in yeast cultures incubated with 50 mM of these compounds in the dark and exposed for 15 min to white light in absence of PSs (Figure S2). Likewise, no toxicity was observed in irradiated cell suspensions prepared in D 2 O (Figure S2, line 4). In addition, cultures incubated with the scavenger and the porphyrin, or suspended in D 2 O with PS, showed no reduction in viability after incubation in the dark for 15 min (Figures S3). In these experiments, a PS concentration of 1.0 µM and 15 min of irradiation were chosen to produce a photoinactivation of C. albicans of approximately 3 log and thus, to be able to visualize the effect produced by the additives or the D 2 O medium. The PDI results after different treatments are shown in Fig. 6 . Sodium azide and DABCO were used as quenchers of O 2 ( 1 Δ g ) [16,]. With both additives, a reduction in the photoinactivation was found in PDI treatments of C. albicans . The azide ions produced a reduction of about 2.0 log in the inactivation of yeast cells treated with A 3 B 3+ (Fig. 6 A, line 3), while this effect was slightly higher with A 4 4+ , reaching 2.5 log of protection (Fig. 6 B, line 3). Similar photoprotective results were found for cultures incubated with DABCO and porphyrin (Fig. 6 B, line 4), although with a decrease in inactivation somewhat less than that produced by sodium azide. Therefore, both scavengers produced a significant decrease in porphyrin-sensitized photodynamic action by quenching O 2 ( 1 Δ g ). To confirm the involvement of a type II mechanism, D 2 O was used instead of water in order to increase the O 2 ( 1 Δ g ) lifetime [23]. PDI treatments of C. albicans cell suspensions in D 2 O with porphyrin produced a significant increase in the yeast photoinactivation relative to cells in PBS (Fig. 6 , line 5). The greatest effect was observed for the A 4 4+ , producing about 2 log increase in cell inactivation. These results also suggest the participation of O 2 ( 1 Δ g ) in the photodynamic pathway that produces cell death. On the other hand, D-mannitol and L-cysteine can act as radical scavengers and thus these compounds can be used as inhibitors of type I photoprocess [24,25]. For both porphyrins, the addition of D-mannitol produced about 0.5 log in C. albicans cell protection (Fig. 6 , line 6). Comparable behaviour was found in yeast cultures when L-cysteine was used as free radical scavenger (Fig. 6 , line 7). Therefore, the presence of D-mannitol and L-cysteine in C. albicans cell suspensions caused only slight changes in yeast photokilling, suggesting that a type I photoprocess makes only a minor contribution. It was previously reported that the photoinactivation of C. albicans sensitized by tri- and tetra-cationic porphyrins occurs predominantly through a type II mechanism, with only a minor contribution from type I pathways [12]. A similar pattern in ROS involvement was also observed for the inactivation of C. albicans sensitized by A 4 4+ and [16]. Moreover, photoinactivation was negligible when the oxygen atmosphere was replaced by argon, indicating an insignificant contribution of oxygen-independent pathways to the photokilling of yeast cells. These results confirm that the photoinactivation of C. albicans sensitized by A 3 B 3+ and A 4 4+ proceeds mainly through of O 2 ( 1 Δ g )-mediated mechanism. The minor protective effects of D-mannitol and L-cysteine indicate a limited participation of type I pathways. 3.4. Photoinactivation of C. albicans on agar surfaces In this assay, C. albicans cell suspensions (~ 10 6 CFU/mL, 2 mL) were incubated with 5.0 µM A 3 B 3+ or A 4 4+ in the dark for 30 min at 37°C to allow porphyrin binding to yeast cells. After treatment, 100 µL aliquots containing ~ 100 cells were uniformly spread onto SA plates. Following 15 min of incubation in the dark, the plates were exposed to white light for different periods (15, 30, and 60 min). Control experiments confirmed the absence of cytotoxic effects in cells irradiated without porphyrin (Fig. 7 ) or treated with PS and maintained in the dark (Figure S4). After 15 min of irradiation, yeast viability was reduced to 53% with A 3 B 3+ and to 75% with A 4 4+ . With 30 min of irradiation, survival decreased to 35% for A 4 4+ , while no colony formation was detected for A 3 B 3+ . A comparable outcome was observed after 60 min of light exposure for both PSs, confirming the potent photoinactivating activity of these porphyrins under these conditions. A comparable photokilling effect was also observed for C. albicans cells treated with 5 µM TMPyP 4+ in cell suspension and irradiated on SA [17]. The results show that complete loss of C. albicans viability was achieved with A 3 B 3+ after 30 min of irradiation, whereas A 4 4+ required longer exposure to produce a comparable effect. These findings highlight the high photodynamic efficiency of the surface-bound porphyrins and their potential for effective antifungal applications. 3.5. Photoinactivation of C. albicans by surface-bound PSs These experiments were used to evaluate the ability of A 3 B 3+ and A 4 4+ deposited on the SA surfaces to photoinhibit the growth of C. albicans colonies. Therefore, different amounts of PSs (0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 nmol) were uniformly applied onto the surface of SA plates, as illustrated in Fig. 8 . A cell suspension of C. albicans (~ 10 6 CFU/mL, 100 µL) in PBS was spread on the SA surfaces. The cultures were maintained in the dark for 30 min at 37°C to allow interaction between the PS and the cells. Subsequently, the plates were irradiated with white light for 30 min and then incubated at 37°C for 72 h. As shown in Fig. 8 , these amounts of PSs were not toxic to C. albicans cells maintained in the dark, since confluent growth was observed in the areas containing the porphyrin. In contrast, no cell growth of C. albicans was found in the areas treated with A 3 B 3+ and A 4 4+ and irradiated for 30 min. The area of the inhibition zone increased with the amount of PS deposited. Furthermore, colony growth was not detected even 3 days after PDI treatment. To confirm the results, samples of these areas treated with PSs and irradiated were aseptically transferred to fresh SB medium and then to a new SB agar plate. After additional 48 h incubation at 37°C, no formation of colonies of C. albicans cells was detected, indicating a complete inactivation of yeast cells. Likewise, areas spread with different amounts of TMPyP⁴⁺ effectively photoinactivated C. albicans cells immobilized on SA surfaces [17]. These results demonstrate the potential of surface-bound porphyrins as effective photoactive antimicrobial coatings. Their strong photoinactivation capacity against C. albicans supports possible applications in medical devices and hospital surfaces to prevent microbial contamination. This approach offers a sustainable strategy for light-activated disinfection. 3.6. Photoinactivation of C. albicans pseudohyphae C. albicans is capable of transitioning from a yeast-like morphology to germ tubes, progressing through a series of elongated forms known as pseudohyphae, and ultimately developing into hyphae []. At the microbiological level, this dimorphic capacity is regarded as a key virulence factor, as it enables the yeast to colonize and invade mucosal tissues [26]. In light of this, the photodynamic activity sensitized by A 3 B 3+ and A 4 4+ was specifically evaluated against C. albicans pseudohyphae suspended in PBS. To induce the dimorphic state, yeast cultures were incubated in HS for 4 h at 37°C, after which the formation and morphology of pseudohyphae were confirmed by optical microscopy [18]. Suspensions of C. albicans pseudohyphae (~ 10 6 CFU/mL) in PBS were treated with 0.5, 1.0, 2.5, and 5.0 µM porphyrin for 30 min in the dark at 37°C. The survival of pseudohyphae after irradiation with white light for 2, 5, 15, and 30 min is presented in Fig. 9 . Control experiments showed that irradiation alone did not affect pseudohyphal viability, and no toxicity was observed in cells incubated with 5.0 µM porphyrin in the dark for 30 min (Figure S5). These results indicate that the observed photokilling of pseudohyphae upon light exposure was specifically attributable to the photosensitizing action of A 3 B 3+ and A 4 4+ . As shown in Fig. 9 , the photoinactivation of C. albicans pseudohyphae was dependent on both the porphyrin concentration and the light irradiation. Both A 3 B 3+ and A 4 4+ exhibited similar photodynamic activity. Treatment with 1.0 µM porphyrin resulted in a reduction in viability of more than 3 log after 30 min of irradiation, while a comparable effect was observed for cultures treated with 2.5 µM porphyrin and exposed to light for 15 min. Under these conditions, no colony formation was detected when the irradiation time was extended to 30 min. Using 5.0 µM porphyrin, a reduction of approximately 5 log was achieved after 5 min of irradiation. Moreover, no cell survival was observed after 15 min of irradiation, corresponding to a decrease of about 6 log for pseudohyphae treated with the tricationic porphyrin. Under comparable conditions, this cationic porphyrin was also more effective at photoinactivating C. albicans pseudohyphae than glycoporphyrins containing two tertiary amino groups as precursors of cationic centers [27]. The results demonstrate that A 3 B 3+ and A 4 4+ effectively photoinactivate C. albicans pseudohyphae in a concentration- and irradiation-dependent manner. These findings highlight the potent photodynamic activity of both porphyrins against the virulent pseudohyphal form of the yeast. 3.7. Photokilling of C. albicans biofilms C. albicans biofilms pose serious disadvantages for clinical management due to their strong antifungal resistance, capacity for immune evasion, and persistence on medical devices [28]. These biofilms readily colonize implant surfaces, such as catheters, prosthetic valves, and dental materials, leading to device failure, systemic infections, and high mortality rates [29]. Biofilm-associated cells display multifactorial drug resistance mediated by the extracellular matrix, efflux pump upregulation, and the formation of persistent cells, which collectively render biofilm infections difficult to eradicate with conventional antifungal agents [5,30]. Therefore, C. albicans biofilms represent a therapeutic challenge, causing persistent infections, treatment failures, and increased healthcare burdens. The photoinactivation of C. albicans by AB 3 + , A 2 B 2 2+ , A 3 B 3+ , and A 4 4+ was evaluated in biofilms grown on PVC discs, which is one of the materials commonly used in the manufacture of several medical devices [31]. After the adhesion step, the cultures were treated with 5.0 µM porphyrin in SB supplemented with 7% HS for 18 h at 37 ºC in the dark during biofilm proliferation phase. The cell survival after PDI treatments are shown in Fig. 10 . Biofilms not treated with PSs and irradiated with white light for 60 min showed no reduction in cell viability. Likewise, controls incubated with PSs in the dark did not exhibit any cytotoxic effects. The photodynamic effect mediated by AB 3 + and A 2 B 2 2+ resulted in a lower photokilling capacity, reaching 1.0 log (90%) and 2.5 log (99.7%) reduction, respectively, in the viability of C. albicans . In contrast, A 3 B 3+ and A 4 4+ produced the eradication of C. albicans cells in the biofilms upon 60 min of irradiation. In particular for A 3 B 3+ , this denotes a greater than 6 log decrease in yeast survival, which represents 99.9997% photoinactivation. These results show that the efficiency of porphyrins increased in the following order: A 3 B 3+ ∼ A 4 4+ >A 2 B 2 2+ >AB 3 + . These tendency in the photokilling activity was similar to those previously demonstrated in Escherichia coli [11]. To confirm the efficacy of the photodynamic action sensitized by these porphyrins, after the PDI treatments, the PVC discs were deposited on a SA plate and incubated for 48 h at 37 ºC in the dark. After that, significant proliferation of viable C. albicans cells was observed in cultures treated with AB 3 + and A 2 B 2 2+ . In contrast, no cell growth was found on the PVC discs treated with A 3 B 3+ and A 4 4+ after an irradiation time of 60 min. Therefore, the photodynamic activity inhibited biofilm formation, and the PDI treatment was able to decrease the survival of yeast cells in the biofilm. The photodynamic treatment with cationic porphyrins has also proven effective in inhibiting biofilm formation by clinical C. albicans strains [32]. Furthermore, a porphyrinic formulation composed of five cationic porphyrins (FORM) combined with KI provided a highly efficient photodynamic strategy for eradicating C. albicans biofilms [33]. While FORM alone exhibited limited efficacy, its combination with KI markedly enhanced fungal inactivation, leading to complete biofilm destruction. PDI of C. albicans biofilms was also achieved using the tetracationic metalloporphyrin ZnTnHex-2-PyP⁴⁺ [34]. Upon blue light irradiation, this Zn(II) porphyrin induced up to an 89% reduction in biofilm viability and extensive structural disruption, characterized by decreased hyphal density and biofilm disorganization. These results highlight the importance of porphyrin lipophilicity and cationic charge in promoting biofilm penetration and oxidative damage generation. Furthermore, tetracationic porphyrin derivatives significantly interfered with biofilm formation by reducing adhesion forces and altering the nanomechanical properties of the cell wall [35]. Among the porphyrins tested, A 3 B 3+ and A 4 4+ exhibited a high photokilling efficiency, completely eradicating C. albicans biofilms on PVC surfaces. 4. Conclusions The present study demonstrates that the tricationic and tetracationic porphyrins A 3 B 3+ and A 4 4+ are highly effective photosensitizers for the PDI of C. albicans under various experimental conditions and morphological forms. Both porphyrins exhibited rapid cellular uptake and strong association with the yeast cell envelope, promoting efficient ROS-mediated photodamage. In planktonic cultures, A 3 B 3+ and A 4 4+ achieved complete eradication of viable cells after low light fluence at micromolar concentrations. ROS quenching experiments confirmed that O 2 ( 1 Δ g ) was the primary cytotoxic species involved in cell photokilling. These porphyrins also proved highly effective against C. albicans grown on agar surfaces or immobilized within solid media, where surface-bound PSs induced full inhibition of fungal proliferation. Moreover, both A 3 B 3+ and A 4 4+ efficiently photoinactivated the pseudohyphal morphotype, achieving up to six orders of magnitude reduction in viability, highlighting their ability to target the virulent filamentous form. Remarkably, these porphyrins completely eradicated mature biofilms grown on PVC, a material commonly used in medical devices, underscoring their potential for antifungal coatings and device disinfection. The superior photoinactivating performance of A 3 B 3+ and A 4 4+ can be ascribed to the optimized arrangement of cationic charges at the termini of flexible aliphatic chains, which confers greater mobility to the cationic groups enhancing electrostatic interactions with the yeast cell envelope and penetration into the biofilm matrix. Overall, these findings support A 3 B 3+ and A 4 4+ as potent, broad-spectrum porphyrinic photosensitizers capable of eliminating C. albicans across its major morphotypes through O 2 ( 1 Δ g )-driven photodynamic mechanisms. Declarations Supplementary Information The online version contains supplementary material available at https://doi.org/ ... Author Contribution M.G.A. conceptualization, methodology, data analysis, validation, investigation, data curation, original draft preparation. P.V.C., J.M.G. and M.E.P.: conceptualization, methodology, data analysis, validation, investigation, data curation. E.N.D. conceptualization, methodology, validation, resources, supervision, writing-reviewing and editing, project administration, and funding acquisition. All authors reviewed the manuscript. Acknowledgements This work was supported by SECYT-UNRC (PPI C612) and CONICET of Argentina (PIP 11220200101208CO). M.E.P. thanks CONICET for the postdoctoral fellowship. P.C.G., M.G.A. and E.N.D. are Scientific Members of CONICET. References . Schroeder, J. A., Wilson, C. M., Pappas, P. G. (2025). Invasive candidiasis. Infectious Disease Clinics of North America , 29, 93–119. . Xu, Z., Wang, K., Min, D., Soteyome, T, Lan, H., Hong, W., Peng, F., Fu, X., Peng, G., Huang, T, Liu, J. Kjellerup, B. V. (2022). Regulatory network controls microbial biofilm development, with Candida albicans as a representative: from adhesion to dispersal. Bioengineered , 13, 253–267. . Mba, I. E., Nweze, E. I., Eze, E. A., Anyaegbunam, Z. K. G. (2022). Genome plasticity in Candida albicans : a cutting-edge strategy for evolution, adaptation, and survival. Infection, Genetics and Evolution , 99, 105256. . Kaur, J., Nobile, C. J. (2023). Antifungal drug-resistance mechanisms in Candida biofilms. Current Opinion in Microbiology , 71, 102237. . Roy, S., Gow, N. A. R. (2023). The role of the Candida biofilm matrix in drug and immune protection. The Cell Surface , 10, 100111. . Atriwal, T., Azeem, K., Husain, F. M., Hussain, A., Khan, M. N., Alajmi, M. F., Abid, M. (2021). Mechanistic understanding of Candida albicans biofilm formation and approaches for its inhibition. Frontiers in Microbiology , 12, 638609. . Tits, J., Cammue, B. P. A., Thevissen, K. (2020). Combination therapy to treat fungal biofilm based infections. International Journal of Molecular Sciences , 21, 22. . Rodríguez-Cerdeira, C., Martínez-Herrera, E., Fabbrocini, G., Sanchez-Blanco, B., López-Barcenas, A., Samahy, M., Juárez-Durán, E. R., González-Cespón J. L. (2021). New applications of photodynamic therapy in the management of candidiasis. Journal of Fungi , 7, 1025. . Aroso, R. T., Schaberle, F. A., Arnaut, L. G., Pereira, M. M. (2021). Photodynamic disinfection and its role in controlling infectious diseases. Photochemical and Photobiological Sciences , 20, 1497–1545. . Pourhajibagher, M., Bahrami, R., Moghaddam, E. K., Bahador, A. (2025). Boosting the antibacterial potency of antimicrobial photodynamic therapy against oral pathogens through supplement agents: a narrative review. Journal of Dental Sciences , 20, 2058–2065. . Caminos, D. A., Durantini, E. N. (2005). Synthesis of asymmetrically meso-substituted porphyrins bearing amino groups as potential cationic photodynamic agents. Journal of Porphyrins and Phthalocyanines , 9, 334–342. . Cormick, M. P., Alvarez, M. G., Rovera, M., Durantini, E. N. (2009). Photodynamic inactivation of Candida albicans sensitized by tri- and tetra-cationic porphyrin derivatives. European Journal of Medicinal Chemistry , 44, 1592–1599. . Quiroga, E. D., Cordero, P., Mora, S. J., Alvarez, M. G., Durantini, E. N. (2020). Mechanistic aspects in the photodynamic inactivation of Candida albicans sensitized by a dimethylaminopropoxy porphyrin and its equivalent with cationic intrinsic charges. Photodiagnosis and Photodynamic Therapy , 31, 101877. . Quiroga, E. D., Mora, S. J., Alvarez, M. G., Durantini, E. N. (2016). Photodynamic inactivation of Candida albicans by a tetracationic tentacle porphyrin and its analogue without intrinsic charges in presence of fluconazole. Photodiagnosis and Photodynamic Therapy , 13, 334–340. . Agazzi, M. L., Durantini, J. E., Quiroga, E. D., Alvarez, M. G., Durantini, E. N. (2021). A novel tricationic fullerene C 60 as broad‑spectrum antimicrobial photosensitizer: mechanisms of action and potentiation with potassium iodide. Photochemical and Photobiological Science , 20, 327–341. . Cordero, P. V., Alvarez, M. G., Gonzalez Lopez, E. J., Heredia, D. A., Durantini, E. N. (2023). Photoinactivation of planktonic cells, pseudohyphae, and biofilms of Candida albicans sensitized by a free-base chlorin and its metal complexes with Zn(II) and Pd(II). Antibiotics , 12, 105. . Quiroga, E. D., Alvarez, M. G., Durantini, E. N. (2010). Susceptibility of Candida albicans to photodynamic action of 5,10,15,20-tetra(4- N -methylpyridyl)porphyrin in different media. FEMS Immunology and Medical Microbiology , 60, 123–131. . Cordero, P.V., Ferreyra, D. D., Pérez, M. E., Alvarez, M. G., Durantini, E. N. (2021) Photodynamic effect of 5,10,15,20-tetrakis[4-(3- N , N -dimethylaminopropoxy) phenyl]chlorin towards the human pathogen Candida albicans under different culture conditions. Photochem , 1, 505–522. . Lambrechts, S. A. G., Aalders, M. C. G., Van Marle, J. (2005). Mechanistic study of the photodynamic inactivation of Candida albicans by a cationic porphyrin. Antimicrobial Agents and Chemotherapy , 49 2026–2034. . Voit, T. Cieplik, F., Regensburger, J., Hiller, K. A., Gollmer, A., Buchalla, W., Maisch, T. (2021). Spatial distribution of a porphyrin-based photosensitizer reveals mechanism of photodynamic inactivation of Candida albicans . Frontiers in Medicine , 8, 641244. . Novaira, M., Cormick, M. P., Durantini, E. N. (2012). Spectroscopic and time-resolved fluorescence emission properties of a cationic and an anionic porphyrin in biomimetic media and Candida albicans ce lls . Journal of Photochemistry and Photobiology A: Chemistry , 246, 67–74. . Caminos, D. A., Spesia, M. B., Durantini, E. N. (2006). Photodynamic inactivation of Escherichia coli by novel meso -substituted porphyrins by 4-(3- N , N , N -trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups. Photochemical and Photobiological Science , 5 56–65. . Di Palma, M. A., Alvarez, M. G., Durantini, E. N. (2015). Photodynamic action mechanism mediated by zinc(II) 2,9,16,23-tetrakis [4-( N -methylpyridyloxy)]phthalocyanine in Candida albicans cells. Photochemical and Photobiology , 91, 1203–1209. . Gsponer, N. S., Agazzi, M. L., Spesia, M. B., Durantini, E. N. (2016). Approaches to unravel pathways of reactive oxygen species in the photoinactivation of bacteria induced by a dicationic fulleropyrrolidinium derivative. Methods , 109, 167–174. . Costa, L., Faustino, M. A. F., Neves, M. G. P. M. S., Cunha, Â., Almeida, A. (2012). Photodynamic inactivation of mammalian viruses and bacteriophages. Viruses , 4, 1034–1074. . Noble, S. M., Gianetti, B. A., Witchley, J. N. (2017). Candida albicans cell-type switching and functional plasticity in the mammalian host. Nature Reviews Microbiology , 15, 96–108. . Palacios, Y. B., Simonetti, S. O., Hernández Chavez, C., Álvarez, M. G., Cordero, P., Cuello, V. E. A., González López, E. J., Larghi, E. L., Agazzi, M. L., Durantini, E. N., Heredia, D. A. (2025). “Illuminated glycoporphyrins”: a photodynamic approach for Candida albicans inactivation. Journal of Photochemistry and Photobiology B: Biology , 264, 113105. . Fan, F., Liu, Y., Liu, Y., Lv, R., Sun, W., Ding, W., Cai, Y, Li, W, Liu, X., Qu, W. (2022). Candida albicans biofilms: antifungal resistance, immune evasion, and emerging therapeutic strategies. International Journal of Antimicrobial Agents , 60, 106673. . Le, P. H., Linklater, D. P., Medina, A. A., MacLaughlin, S., Crawford, R. J., Ivanova, E. P. (2024). Impact of multiscale surface topography characteristics on Candida albicans biofilm formation: from cell repellence to fungicidal activity. Acta Biomaterialia , 177, 20–36. . Kaur, J., Nobile, C. J. (2023). Antifungal drug-resistance mechanisms in Candida biofilms. Current Opinion in Microbiology , 71, 102237. . Duarte-Peña, L., López-Saucedo, F., Concheiro, A., Alvarez-Lorenzo, C., Bucio, E. (2022). Modification of indwelling PVC catheters by ionizing radiation with temperature and pH-responsive polymers for antibiotic delivery. Radiation Physics and Chemistry , 193, 110005. . Orlandi, V. T., Martegani, E., Bolognese, F., Trivellin, N., Mat’átková, M., Paldrychová, O., Andreina Baj, Caruso, E. (2020). Photodynamic therapy by diaryl-porphyrins to control the growth of Candida albicans . Cosmetics , 7, 31. . Vieira, C., Bartolomeu, M., Santos, A. R., Mesquita, M. Q., Gomes, A. T. P. C., Neves, M. G. P. M. S, Faustino, M. A. F., Almeida, A. (2022). Photoinactivation of bacterial and fungal planktonic/biofilm forms using the combination of a porphyrinic formulation with potassium iodide. Medical Sciences Forum , 12, 13. . Souza, S. O., Raposo, B. L., Sarmento-Neto, J. F., Rebouças, J. S., Macêdo, D. P. C., Figueiredo, R. C. B. Q., Santos, B. S., Freitas, A. Z., Cabral Filho, P. E., Ribeiro, M. S., Fontes, A. (2022). Photoinactivation of yeast and biofilm communities of Candida albicans mediated by ZnTnHex-2-PyP 4+ porphyrin. Journal of Fungi , 8, 556. . Amorim, C. F., Iglesias, B. A., Pinheiro, T. R ., Lacerda, L. E., Sokolonski, A. R., Pedreira, B. O., Moreira, K. S.,. Burgo, T. A. L., Meyer, R., Azevedo, V., Portela, R. W. (2023). Photodynamic inactivation of different Candida species and inhibition of biofilm formation induced by water-soluble porphyrins. Photodiagnosis and Photodynamic Therapy , 42 103343. Additional Declarations No competing interests reported. Supplementary Files manuscriptAlvarezSI.docx Cite Share Download PDF Status: Published Journal Publication published 22 Dec, 2025 Read the published version in Photochemical & Photobiological Sciences → Version 1 posted Editorial decision: Revision requested 20 Nov, 2025 Reviews received at journal 19 Nov, 2025 Reviews received at journal 18 Nov, 2025 Reviews received at journal 15 Nov, 2025 Reviewers agreed at journal 06 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers agreed at journal 04 Nov, 2025 Reviewers invited by journal 03 Nov, 2025 Editor assigned by journal 03 Nov, 2025 Submission checks completed at journal 31 Oct, 2025 First submitted to journal 30 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7992830","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":541162921,"identity":"8c7b3f6e-66e6-481a-bba7-8113d8d3b70a","order_by":0,"name":"María G. Alvarez","email":"","orcid":"","institution":"Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"G.","lastName":"Alvarez","suffix":""},{"id":541162922,"identity":"19b9c0eb-3dbc-40bf-9fe6-66aa4e9563e4","order_by":1,"name":"Paula V. Cordero","email":"","orcid":"","institution":"Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"V.","lastName":"Cordero","suffix":""},{"id":541162923,"identity":"ad638e59-39c9-4e4c-8cc1-843793553ad0","order_by":2,"name":"Jesica M. González","email":"","orcid":"","institution":"Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Jesica","middleName":"M.","lastName":"González","suffix":""},{"id":541162929,"identity":"2570a5ba-19d8-4f68-9f50-6942aa4773ad","order_by":3,"name":"María E. Pérez","email":"","orcid":"","institution":"Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"E.","lastName":"Pérez","suffix":""},{"id":541162930,"identity":"0ca12538-db3d-484e-9d2f-7616442f5893","order_by":4,"name":"Edgardo N. Durantini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYFCCBDDJw8DMfABJlI2gFgOgFrYE0rSALDIgTgs/e47Zh587/siYs/N8fMxTw5C4XfrwA4YPZYcZ+GcfwKpFsueN8czeMwY8ls28mw1nHGNI3NmXZsA449xhBolzCVi1GNzIMWbgbTPgMTjMu03iAxtD4oYzQH/xth1mYDiD3WH2QC2Mf8FaeJ7/SPgH1fIXqEUehxYDiRxjZogtPGwMH9ugWhiBWgxwaJE486yYWbbNGKiFzVhyZp+E8YYzbAYHe86l8xji0MLfnryZ8W2bnL3B+cMPP/N8s5HdcIb54YMfZdZycji0YNgKJg8wgNLDKBgFo2AUjAKyAQAF1VYYkXx+lQAAAABJRU5ErkJggg==","orcid":"","institution":"Instituto para el Desarrollo Agroindustrial y de la Salud (IDAS), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)","correspondingAuthor":true,"prefix":"","firstName":"Edgardo","middleName":"N.","lastName":"Durantini","suffix":""}],"badges":[],"createdAt":"2025-10-30 22:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7992830/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7992830/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43630-025-00835-3","type":"published","date":"2025-12-22T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96239880,"identity":"47e7917e-bbce-4fe6-9b68-e9c4f4ad9edd","added_by":"auto","created_at":"2025-11-19 07:07:52","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1530913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscriptAlvarez.docx","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/30665e49778903411c1ae51f.docx"},{"id":95832523,"identity":"8ab554e1-c7d4-400a-b877-f3e389df1598","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6805,"visible":true,"origin":"","legend":"","description":"","filename":"168b1b42ded94bb0a4fb89ecb2e24361.json","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/2c1258a80a55f79523a55752.json"},{"id":95832533,"identity":"4e20a9ba-67c8-4ed7-8ddc-81e6c778a907","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscriptAlvarezSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/f6098b666270efaa34eac4ce.docx"},{"id":95832529,"identity":"af7061a2-217d-4573-906b-2019e4dc5778","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81544,"visible":true,"origin":"","legend":"","description":"","filename":"168b1b42ded94bb0a4fb89ecb2e243611enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/99334093cde27199afdb0376.xml"},{"id":95832531,"identity":"c353f9db-8971-49ab-a545-132450b1b29b","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"emf","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125784,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.emf","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/8f6f0856ede67cc4a79bd337.emf"},{"id":96239513,"identity":"5b82868c-d7b1-4c9d-9de9-faeba8755310","added_by":"auto","created_at":"2025-11-19 07:06:51","extension":"emf","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50072,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.emf","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/20a1460d49e074dbe7b9ae19.emf"},{"id":96239706,"identity":"79fe0397-6877-4345-bc2e-58fab8aecc23","added_by":"auto","created_at":"2025-11-19 07:07:24","extension":"emf","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":43928,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.emf","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/6f0d76aa1b6e4b3eddaf1ba3.emf"},{"id":96239610,"identity":"f5471cd3-c761-48f9-b7e1-713e0c2ea365","added_by":"auto","created_at":"2025-11-19 07:07:09","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":218520,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/033a1739178a12a3607de442.png"},{"id":96239627,"identity":"daf9c0d9-3730-4704-9cff-481fe473231c","added_by":"auto","created_at":"2025-11-19 07:07:13","extension":"emf","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55024,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.emf","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/761ffcac7597586551de3f36.emf"},{"id":95832551,"identity":"6c6bbf9b-2b27-4288-a85a-c86449e3224b","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":256383,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/b12f7b1137e0aa975ee75e64.jpeg"},{"id":95832539,"identity":"15c21dfd-8774-4d9a-8627-0a18c2123e3f","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":196164,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/aa06bcb157b1f41775fb4a35.jpeg"},{"id":95832548,"identity":"e1150831-dc18-4272-9131-2ede67cbc9f9","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"emf","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42824,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.emf","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/ecb31b18483c93a4d8fdb089.emf"},{"id":95832555,"identity":"9d6ed173-3a91-44ec-b21b-06d84d347498","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1012679,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/913a2763e911518549383ffb.png"},{"id":96239762,"identity":"9afa5ccf-4643-4ca6-9d0d-8fd675065cb5","added_by":"auto","created_at":"2025-11-19 07:07:34","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":292024,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/6de531f12a4b8ac3dd1aa846.jpeg"},{"id":95832534,"identity":"c3c47f96-4d81-4622-ad5e-835902d77dfd","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9036,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/d875e27e046c886c989217b4.png"},{"id":95832535,"identity":"56fc0930-bb44-43f6-881a-e4980e70c4d2","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2755,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/4cee8e9573032f97a8bdd23e.png"},{"id":96239593,"identity":"2a2014fa-2090-4e44-a1ce-50bba24a4884","added_by":"auto","created_at":"2025-11-19 07:07:04","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2867,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/a120a61c50f48e0f1186f1b9.png"},{"id":96241042,"identity":"3bb46829-2e57-4b09-bd1e-2a758f1b848c","added_by":"auto","created_at":"2025-11-19 07:09:54","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20516,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/e198f4c3d80ccb6729234d1a.png"},{"id":96239731,"identity":"870092dc-adb3-4237-9f0f-c299d84044bf","added_by":"auto","created_at":"2025-11-19 07:07:26","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3134,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/4c036a073f79ddc57320e948.png"},{"id":95832546,"identity":"dac0b01a-b4f3-4e11-9038-5e6d590a0105","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55772,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/1bb9c538429bde2068a46f01.png"},{"id":96239928,"identity":"97b030fc-43fc-41f2-b25d-b0fe2c41095c","added_by":"auto","created_at":"2025-11-19 07:08:01","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40308,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/d890e888d69db447946d853d.png"},{"id":95832550,"identity":"ac07cf26-b040-4e48-aba8-7184934acaea","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3093,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/6e0cca48554bfd551aef69bb.png"},{"id":95832556,"identity":"9245be00-479b-4f6f-a883-db2561f84a9c","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106473,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/fb847aca8c56360bbe54d824.png"},{"id":96239688,"identity":"7516f8b1-b3b0-4922-aae1-4f8ee7481eb3","added_by":"auto","created_at":"2025-11-19 07:07:21","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65112,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/f6e9ebf0b62e41757f2c0415.png"},{"id":95832553,"identity":"129567db-531f-4f6d-b8c9-261c5abed075","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82737,"visible":true,"origin":"","legend":"","description":"","filename":"168b1b42ded94bb0a4fb89ecb2e243611structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/88f3e22c8e122058f64b11ea.xml"},{"id":95832557,"identity":"63f76bf9-b8b2-4062-b9f2-60555c8b9af0","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91267,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/b869b0870294f677beabb87d.html"},{"id":96240686,"identity":"a39b90b6-bf68-4150-ba8e-50496879b777","added_by":"auto","created_at":"2025-11-19 07:09:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67284,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of cationic porphyrins.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/07338340fbbe47ef8afce48b.png"},{"id":95832520,"identity":"f5e6127d-8982-422c-8ef3-5edad778e310","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43894,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of porphyrins recovered from \u003cem\u003eC. albicans\u003c/em\u003e planktonic cells incubated with 1.0 µM AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (squares), A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e (circles), A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e (upward triangles), and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e (downward triangles) for different periods at 37 °C in the dark.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/33c37da4254247eb7d445dfa.png"},{"id":95832525,"identity":"f9b86747-8ffa-4876-8667-036a0c9a8f89","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":178698,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopic observation of \u003cem\u003eC. albicans\u003c/em\u003e treated with 1.0 μM porphyrin for 15 min at 37 °C in the dark. Inset: Cells under a bright field (100× microscope objective).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/6374e5d6672f6918a06c4708.png"},{"id":96239932,"identity":"d5fa5541-2501-4657-85f2-4bcd0cde1602","added_by":"auto","created_at":"2025-11-19 07:08:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45474,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eC. albicans\u003c/em\u003e cell suspensions (~10\u003csup\u003e6 \u003c/sup\u003eCFU/mL) incubated with 5.0 µM AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (squares), A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e (circles), A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e (upward triangles), and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e (downward triangles) for 15 min at 37 °C in the dark and irradiated with white light (90 mW/cm\u003csup\u003e2\u003c/sup\u003e) for different periods. Control of \u003cem\u003eC. albicans\u003c/em\u003e untreated with PS and irradiated (open circles); *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to the control.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/275798ef64b7937f2276d2ba.png"},{"id":96239708,"identity":"7b102e48-7021-480e-974d-d4ef22da3ebb","added_by":"auto","created_at":"2025-11-19 07:07:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":113456,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eC. albicans\u003c/em\u003e cell suspensions (~10\u003csup\u003e6 \u003c/sup\u003eCFU/mL) incubated with 0.5 (squares), 1.0 (upward triangles) and 2.5 µM (downward triangles) of (A) A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and (B) A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e for 15 min at 37 °C in the dark and irradiated with white light (90 mW/cm\u003csup\u003e2\u003c/sup\u003e) for different periods. Control of \u003cem\u003eC. albicans\u003c/em\u003e untreated with PS and irradiated (open circles); *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to the control.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/d4e96d6b019ed7240c3f52b2.png"},{"id":96240956,"identity":"4b174154-91df-40f0-9e33-a00a303d1c2f","added_by":"auto","created_at":"2025-11-19 07:09:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":96589,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eC. albicans\u003c/em\u003e planktonic cells (∼10\u003csup\u003e6\u003c/sup\u003e CFU/mL) treated with 1.0 μM (A) A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and (B) A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e for 30 min at 37 °C in the dark and irradiated with white light for 15 min; (1) cells; (2) cells treated with PS; (3) cells treated with 50 mM sodium azide and PSs; (4) cells treated with 50 mM DABCO and PSs; (5) cells suspended in D\u003csub\u003e2\u003c/sub\u003eO and treated with PSs; (6) cells treated with 50 mM D‑mannitol and PSs; (7) cells treated with 50 mM L-cysteine and PSs.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/e64c5a3c7a480a77f56b4e93.png"},{"id":96240542,"identity":"ca8a0d59-f78b-4682-b11f-ba5a6e8e6ed7","added_by":"auto","created_at":"2025-11-19 07:09:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":19123,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eC. albicans\u003c/em\u003e cell suspension (~10\u003csup\u003e6 \u003c/sup\u003eCFU/mL) treated with 5.0 mM A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e (upward triangles) and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e (downward triangles) for 30 min at 37 °C in the dark, plated on SA surfaces and irradiated with white light (90 mW/cm\u003csup\u003e2\u003c/sup\u003e) for different periods. Control of \u003cem\u003eC. albicans\u003c/em\u003e untreated with PS and irradiated (open circles); *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to the control.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/d66ef9078256f8762b2d3e68.png"},{"id":96239517,"identity":"b27cf7d9-82fe-41dc-b994-c45d43820d01","added_by":"auto","created_at":"2025-11-19 07:06:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":640392,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eC. albicans\u003c/em\u003e cells on SA plates containing different amounts of (A) A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and (B) A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e embedded in the agar surface and irradiated with white light (90 mW/cm\u003csup\u003e2\u003c/sup\u003e) for 30 min. The dashed circles indicate the area where different amounts of PS were applied.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/f88f44912d3847bb8736c80d.png"},{"id":96239744,"identity":"0d9f06f9-460b-4abd-9798-9b10d382265b","added_by":"auto","created_at":"2025-11-19 07:07:32","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":123921,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae (~10\u003csup\u003e6 \u003c/sup\u003eCFU/mL) incubated with 0.5 (squares), 1.0 (upward triangles), 2.5 µM (downward triangles), and 5.0 µM (circles) of (A) A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and (B) A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e for 15 min at 37 °C in the dark and irradiated with white light (90 mW/cm\u003csup\u003e2\u003c/sup\u003e) for different periods. Control of \u003cem\u003eC. albicans\u003c/em\u003e untreated with PS and irradiated (open circles); *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to the control.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/d6921edf8c678662c0844d7c.png"},{"id":95832536,"identity":"9bc848a3-c746-4183-895e-a32e5500e0fb","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":49952,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of \u003cem\u003eC. albicans\u003c/em\u003e biofilm incubated with 5.0 mM PS for 18 h at 37 ºC in the dark and irradiated for 60 min with white light (90 mW/cm\u003csup\u003e2\u003c/sup\u003e); 1) cells in the dark; 2) irradiated cells; 3) cells treated with AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in the dark; 4) irradiated cells treated with AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e; 5) cells treated with A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e in the dark; 6) irradiated cells treated with A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e; 7) cells treated with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e in the dark; 8) irradiated cells treated with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e; 9) cells treated with A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e in the dark; 10) irradiated cells treated with A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to the control.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/ed3be2007dcf19f907607f83.png"},{"id":99172444,"identity":"57eb0b85-a6d5-4b35-8a01-7496cc1235c7","added_by":"auto","created_at":"2025-12-29 16:09:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2507712,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/1517eec7-d771-48b0-a24b-1fb38f2ad9d1.pdf"},{"id":95832521,"identity":"38b6eb29-3c90-4ef8-96f2-bdb6f9f95e07","added_by":"auto","created_at":"2025-11-13 12:41:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":135469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscriptAlvarezSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7992830/v1/7b9ff93a4b33d285ed38f131.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of porphyrin cationic charges on photoinactivation of Candida albicans morphotypes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCandidiasis is an opportunistic fungal infection and is considered the most prevalent mycosis in humans [1]. In recent years, the incidence of infections caused by \u003cem\u003eCandida\u003c/em\u003e spp. has increased significantly on a global scale. This rise is attributed to multiple factors, including the widespread and often inappropriate use of antifungal drugs, which has contributed to the development of resistance, as well as the expanded use of medical devices such as vascular grafts, prosthetic heart valves, catheters, and dental implants [2]. These surfaces provide ideal conditions for microbial colonization and biofilm formation. This is primarily caused by species of the \u003cem\u003eCandida\u003c/em\u003e genus, with \u003cem\u003eCandida albicans\u003c/em\u003e being the most frequently implicated pathogen.\u003c/p\u003e\u003cp\u003eDuring the course of infection, \u003cem\u003eC. albicans\u003c/em\u003e exhibits remarkable morphological plasticity, transitioning from a unicellular yeast form to filamentous structures, such as pseudohyphae and hyphae [3]. This morphological switching plays a critical role in the pathogenesis of candidiasis by facilitating adhesion, tissue penetration, and the establishment of persistent biofilms on both biotic and abiotic surfaces. \u003cem\u003eCandida\u003c/em\u003e biofilms are typically composed of dense agglomerates of yeast and hyphal cells embedded within an extracellular polymeric matrix, which confers enhanced protection against environmental stressors, antifungal agents, and host immune defences [4]. The architecture and composition of the biofilm matrix significantly impair drug penetration and promote cell survival under hostile conditions. Due to these characteristics, \u003cem\u003eCandida\u003c/em\u003e biofilms exhibit markedly increased resistance to conventional antifungal therapies compared to their planktonic (free-floating) counterparts [5]. Moreover, the biofilm acts as a physical and biochemical shield, not only limiting the efficacy of antifungal drugs but also preventing recognition and clearance by the immune system of the host [6,7]. The persistence of such infections, particularly on medical devices, increases the risk of chronic and systemic disease.\u003c/p\u003e\u003cp\u003eIn this context, photodynamic inactivation (PDI) of pathogens has gained increasing attention as a non-conventional antifungal approach with promising therapeutic potential [8]. PDI involves the use of a photosensitizer (PS) that selectively associates with microbial cells. Upon activation by visible light in the presence of molecular oxygen, the PS generates reactive oxygen species (ROS) [9]. These reactive intermediates interact with biological components, damaging multiple vital structures within the microorganism, which ultimately leads to cell death. Therefore, this therapy represents a promising alternative for the treatment of multi-resistant pathogens [10].\u003c/p\u003e\u003cp\u003eIn this study, the photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e in its yeast, pseudohyphal, and biofilm forms was systematically investigated using a series of cationic porphyrins as PSs. The compounds evaluated, AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e, A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e, and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), differ in the number and spatial distribution of their positive charges. The cationic groups are located at the ends of the flexible spacers and they are combined with lipophilic trifluoromethyl substituents to enhance amphiphilicity. These structural variations were designed to modulate cellular uptake and photodynamic efficacy. First, photokilling activity was tested in \u003cem\u003eC. albicans\u003c/em\u003e planktonic cell suspensions, in cells localized on agar surfaces, and in colonies immobilized on solid media. Further mechanistic insights were obtained through the use of ROS scavengers to elucidate the predominant photochemical pathways involved in fungal inactivation. Considering that reversible cell morphogenesis is an important virulence factor, these compounds were also tested for their ability to eliminate \u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae. Finally, the photoinactivating efficacy of these porphyrins was assessed in \u003cem\u003eC. albicans\u003c/em\u003e biofilms, to explore their potential in the prevention and control of these clinical pathogens.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eMaterials and instrumentation are provided in the Supplementary Materials.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cationic porphyrins\u003c/h2\u003e\u003cp\u003eThe cationic porphyrins, 5-[4-(3-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-trimethylammoniopropoxy)phenyl]-10,15,20-tris(4-trifluoromethylphenyl)porphyrin (AB\u003csub\u003e3\u003c/sub\u003e⁺), 5,15-di[4-(3-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-trimethylammoniopropoxy)phenyl]-10,20-di(4-trifluoromethylphenyl)porphyrin (A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u0026sup2;⁺), 5,10,15-tris[4-(3-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-trimethylammoniopropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin (A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3\u003c/sup\u003e⁺), and 5,10,15,20-tetrakis[4-(3-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-trimethylammoniopropoxy)phenyl]porphyrin (A\u003csub\u003e4\u003c/sub\u003e⁴⁺) were synthesized according to previously reported procedures [11]. Stock solutions of each porphyrin (0.5 mM) were prepared in \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide (DMF). The concentrations were determined spectrophotometrically using the molar extinction coefficients corresponding to their Soret bands in DMF (1.71 x 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, 1.67 x 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e, 1.69 x 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e, and 1.64 x 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ecm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e) [11]. The final concentration of DMF in all experimental conditions did not exceed 1% v/v, a level confirmed to be non-toxic to \u003cem\u003eC. albicans\u003c/em\u003e cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. C. albicans cultures\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eC. albicans\u003c/em\u003e strain PC31 used in this study was previously isolated and characterized [12]. For cultivation, yeast cells were grown aerobically in 4 mL of Sabouraud broth (SB) at 37\u0026deg;C for 18\u0026ndash;24 h until reaching the stationary growth phase. After incubation, cells were collected by centrifugation at 1200 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min and subsequently washed and resuspended in 4 mL of phosphate-buffered saline (PBS; 10 mM, pH 7.2). This procedure yielded a suspension containing approximately 10\u003csup\u003e7\u003c/sup\u003e colony-forming units (CFU)/mL. To obtain the working inoculum, the suspension was diluted tenfold in PBS to reach a final concentration of ~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL. Cell density was confirmed by serial dilution and plating on Sabouraud agar (SA), followed by incubation at 37\u0026deg;C for 48 h to determine viable CFUs using the spread plate technique [13].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Binding of porphyrins to yeast cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e cell suspensions (2 mL, ~10\u003csup\u003e6\u003c/sup\u003e CFU/mL) in PBS were incubated with 1 \u0026micro;M of the porphyrin in the dark at 37\u0026deg;C for varying durations (2, 5, 15, and 30 minutes). After incubation, the cells were collected by centrifugation at 1200 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min and resuspended in 2 mL of 2% (w/v) aqueous sodium dodecyl sulfate (SDS) solution. These suspensions were maintained at 4\u0026deg;C overnight and then subjected to sonication for 30 min to release cell-associated porphyrins. The concentration of porphyrin in the resulting supernatants was determined via spectrofluorimetric analysis (λ\u003csub\u003eexc\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;419 nm, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;655 nm), using calibration curves prepared from standard porphyrin solutions in 2% SDS (0.05\u0026ndash;0.20 \u0026micro;M). The fluorescence values obtained from each sample were referenced to the total number of cells contained in the suspension [14]. Microscopic fluorescence images of \u003cem\u003eC. albicans\u003c/em\u003e cells were acquired using a green filter (EX BP480-550, DM570, BA590). A brightfield image was captured for each region to verify the presence of yeast cells. All images were obtained using a 100\u0026times; magnification objective and recorded with a CMOS camera [13].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Photoinactivation of C. albicans yeast cells\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSuspensions of \u003cem\u003eC. albicans\u003c/em\u003e planktonic cells (2 mL, ~10\u003csup\u003e6\u003c/sup\u003e CFU/mL) in PBS were incubated with varying concentrations of porphyrin (0.5, 1.0, 2.5, and 5.0 \u0026micro;M) for 15 min in the dark at 37\u0026deg;C. Subsequently, 200 \u0026micro;L of each suspension was transferred to the wells of a 96-well microtiter plate and irradiated with white light (90 mW/ cm\u003csup\u003e2\u003c/sup\u003e) for different periods (5, 15, and 30 min). When required, sodium azide (50 mM), diazabicyclo[2.2.2]octane (DABCO, 50 mM), D-mannitol (50 mM) and L-cysteine (50 mM) were added to cell suspensions from stock solutions 1 M in water [15]. After that, cells were incubated for 30 min at 37 \u0026ordm;C in the dark previous to the treatment with the porphyrins. Studies in deuterated water (D\u003csub\u003e2\u003c/sub\u003eO) were performed using 2 mL of cell suspensions (\u0026sim;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) in PBS, which were centrifuged (3000 rpm for 15 min) and re-suspended in 2 mL of D\u003csub\u003e2\u003c/sub\u003eO. Then, the cell suspensions in D\u003csub\u003e2\u003c/sub\u003eO were incubated with 1.0 \u0026micro;M PS for 15 min in the dark at 37 \u0026ordm;C. The number of viable \u003cem\u003eC. albicans\u003c/em\u003e cells was determined as described above [16].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Photoinactivation of C. albicans plated on agar surfaces\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eYeast cell suspensions (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 2 mL) were incubated with 5.0 \u0026micro;M PS in the dark for 30 min at 37\u0026deg;C. After treatment, the suspensions were diluted 1:1000 in PBS, and 100 \u0026micro;L of each dilution was uniformly spread onto SA surfaces. The plates were incubated for 15 min at 37\u0026deg;C in the dark and then exposed to white light for different times (15, 30, and 60 min). After that, the plates were incubated at 37\u0026deg;C for 48 h, after which the number of colonies was counted to evaluate cell viability [17].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Photoinactivation of C. albicans by agar surface-bound PS\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAliquots with different amounts of PSs (0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 nmol) were spread on SA plates (5 cm in diameter) to cover an area of 0.6 cm\u003csup\u003e2\u003c/sup\u003e. Then, plates were spread with 100 \u0026micro;L of a \u003cem\u003eC. albicans\u003c/em\u003e cell suspension (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) in PBS. The plates were incubated at 37\u0026deg;C for 30 min in the dark. After that, the cultures were irradiated with white light for 30 min, the plates were incubated at 37\u0026deg;C in the dark. Yeast growth was observed 48 h post-treatment [17].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Studies in C. albicans pseudohyphae\u003c/h2\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae were obtained as previously described [18]. Yeast cell suspension (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) was incubated with human serum (HS) during 4 h at 37\u0026deg;C to induce the formation of pseudohyphae. After incubation, the germ tube formation was verified trough optical microscopy. Pseudohyphae were harvested and washed three times to eliminate all HS by centrifugation (1200 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min) and suspended in PBS until appropriated cell density to obtain\u0026thinsp;~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL. Then, 2 mL of the suspension was placed in Pyrex brand culture tubes (13 x 100 mm) and incubated with 0.5, 1.0, 2.5 and 5.0 \u0026micro;M porphyrin for 30 min in the dark at 37\u0026deg;C. After that, 200 \u0026micro;L of culture were placed in wells of a 96-wells microtiter plate and exposed to visible light for different time (2, 5, 15 and 30 min). Subsequently, pseudohyphal viability was assessed by CFU/mL enumeration following incubation at 37\u0026deg;C for 48 h, as previously reported [16].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Experiments in C. albicans biofilms\u003c/h2\u003e\u003cp\u003eA \u003cem\u003eC. albicans\u003c/em\u003e cell suspension (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e CFU/mL) was prepared in PBS supplemented with 7% v/v human serum (HS). Then, 900 \u0026micro;L of this culture was placed in wells of a 48-wells microtiter plate containing one polyvinylchloride (PVC) disc (5 mm \u0026Oslash; x 1.5 mm) in each well. The plate was incubated for 90 min at 37\u0026deg;C with gentle shaking (~\u0026thinsp;75 rpm) to allow cell adhesion to the discs. After incubation, the discs were removed and washed by successive immersion in PBS to eliminate non-adherent cells. For the proliferation step, the PVC discs were transferred to fresh wells containing 5.0 \u0026micro;M porphyrin in SB supplemented with 7% v/v HS and incubated for 18 h at 37\u0026deg;C. After that, the discs were carefully washed twice with PBS and placed into fresh wells containing 900 \u0026micro;L of PBS. Biofilms were then irradiated with white light for 60 min. Following irradiation, each disc along with the corresponding well content was transferred to a test tube, sonicated for 1 min, and vigorously vortexed for 2 min to detach the biofilm cells from the disc [18]. Viable \u003cem\u003eC. albicans\u003c/em\u003e cells were determined by CFU counting, as described above.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Controls and statistical analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eControl groups included \u003cem\u003eC. albicans\u003c/em\u003e cultures incubated with or without the PS under dark conditions, as well as cell samples exposed to light in the absence of PS. All experiments were performed independently in triplicate. Error bars represent the standard deviation of the mean. Statistical significance was determined using one-way analysis of variance (ANOVA) at a 95% confidence level (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Binding of porphyrin to C. albicans planktonic cells\u003c/h2\u003e\u003cp\u003eThe binding ability of cationic porphyrins to \u003cem\u003eC. albicans\u003c/em\u003e cells was evaluated using cell suspensions (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) in PBS. \u003cem\u003eC. albicans\u003c/em\u003e cultures were incubated with 1.0 \u0026micro;M PS at 37\u0026deg;C in the dark for different periods. The amount of porphyrin recovered from the cells after each incubation time is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Notably, the highest level of cell-bound PS was observed after a short incubation time of 5 min. Furthermore, the amount of cell-bound porphyrin did not significantly change with longer incubation times, such as 15 and 30 min. The recovered porphyrin levels were 0.66 and 0.50 nmol/10\u003csup\u003e6\u003c/sup\u003e cells for A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e, respectively. In contrast, lower values were observed for AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e with 0.10 and 0.16 nmol/10\u003csup\u003e6\u003c/sup\u003e cells, respectively.\u003c/p\u003e\u003cp\u003eIn previous investigation, the binding of 5,10,15,20-tetra(4-\u003cem\u003eN\u003c/em\u003e-methylpyridyl)porphyrin (TMPyP\u003csup\u003e4+\u003c/sup\u003e) to the same \u003cem\u003eC. albicans\u003c/em\u003e strain used in the present study was evaluated, yielding 1.7 nmol/10\u003csup\u003e6\u003c/sup\u003e cells after incubation with 5 \u0026micro;M PS [17]. Comparable binding kinetics were also reported for 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin (TFAP\u003csup\u003e3+\u003c/sup\u003e) and 5,10,15,20-tetra(4-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-trimethylammoniumphenyl)porphyrin (TMAP\u003csup\u003e4+\u003c/sup\u003e) [12]. Under these conditions, both cationic porphyrins showed similar bounding efficiencies. In the present study, the uptake value obtained for A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e was consistent with that previously informed [14]. Also, the uptake of A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e was slight higher than that found for A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e, possibly influenced by the amphiphilic character of the tricationic porphyrin derivate, which may favour interactions with both hydrophobic and polar domains of the fungal cell surface.\u003c/p\u003e\u003cp\u003eMoreover, the intracellular distribution of the porphyrins in yeast cells was examined by fluorescence microscopy. The micrographs revealed that \u003cem\u003eC. albicans\u003c/em\u003e cells incubated with 1.0 \u0026micro;M PS for 15 min in the dark exhibited the characteristic red fluorescence emission of porphyrin derivatives (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The images indicated that the porphyrins were predominantly localized in the cell envelope. A similar peripheral fluorescence pattern was previously reported for a tricationic porphyrin in \u003cem\u003eC. albicans\u003c/em\u003e cells [19]. Consistently, fluorescence microscopy studies demonstrated the accumulation of TMPyP\u003csup\u003e4+\u003c/sup\u003e in the cell wall and plasma membrane, but not in the cytoplasm [20]. Likewise, \u003cem\u003eC. albicans\u003c/em\u003e cells treated with TMAP\u003csup\u003e4+\u003c/sup\u003e displayed the typical red fluorescence of porphyrin derivatives, whereas incubation with an anionic porphyrin produced no detectable intracellular signal [21]. Furthermore, the fluorescence images of A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e in yeast cells were compared with those produced by its non-cationic analogue [13]. All porphyrins evaluated in this study exhibited comparable fluorescence quantum yields of approximately 0.1 in DMF [22]. Accordingly, the observed fluorescence intensity was proportional to the extent of porphyrin uptake by the cells. In particular, a weak emission was observed for the monocationic porphyrin AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, whereas no significant differences in red fluorescence intensity were detected between yeast cells treated with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e. Thus, the incorporation of three or four cationic ammonium substituents into the porphyrin macrocycle markedly enhanced its binding affinity toward \u003cem\u003eC. albicans\u003c/em\u003e cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Photosensitized inactivation of C. albicans planktonic cells\u003c/h2\u003e\u003cp\u003eThe cytotoxic effect photosensitized by porphyrins was fist evaluated in \u003cem\u003eC. albicans\u003c/em\u003e cell suspensions in PBS. The cultures of yeast (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e UFC/mL) were incubated with 5.0 \u0026micro;M PS for 15 min in the dark at 37\u0026deg;C and irradiated with white light for 5, 15 and 30 min. At this concentration of porphyrin, the viability of the \u003cem\u003eC. albicans\u003c/em\u003e was not affected by incubation in the dark (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, cell survival was not modified by irradiation of the culture without porphyrin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, these control experiments confirm that the photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e was caused by porphyrin-induced photodynamic activity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e was dependent on the porphyrin derivative and the irradiation times. When cultures were incubated with 5.0 \u0026micro;M PS, the photokilling effect induced by AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e resulted in a reduction of about 1.0 log after 5 min of irradiation, while increased to 2.0 log after 30 min of exposure to white light. Under the same conditions, the most effective porphyrins were A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e, achieving over 6 log reductions in cell viability after 5 min of irradiation.\u003c/p\u003e\u003cp\u003eBased on these results, the photodynamic activity of A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e was further investigated by varying both the treatment concentration and irradiation time. Both porphyrins exhibited similar photoinactivation capacities to eliminate \u003cem\u003eC. albicans\u003c/em\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). After 15 min of irradiation, a decrease in cell viability greater than 1 log and 3 log was achieved in cultures treated with 0.5 \u0026micro;M and 1.0 \u0026micro;M PS, respectively. The reduction in cell survival increased to 5 log for yeasts incubated with 2.5 mM PS and irradiated for only 5 min exposure, while no viable cells were detected after 15 min of irradiation.\u003c/p\u003e\u003cp\u003ePrevious PDI studies were determined using tetracationic porphyrins in \u003cem\u003eC. albicans\u003c/em\u003e strain under comparable experimental conditions. When yeast cultures were incubated with 5 \u0026micro;M TMPyP\u003csup\u003e4+\u003c/sup\u003e, a reduction of approximately 1.7 log in cell survival was observed upon an irradiation of 5 min [17]. A markedly higher photoinactivation for \u003cem\u003eC. albicans\u003c/em\u003e was found for cells treated with 5 \u0026micro;M TMAP\u003csup\u003e4+\u003c/sup\u003e, achieving about a 3.8 log decrease after 5 min of white light irradiation [12]. Under these conditions, A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e exhibited strong photodynamic activity against \u003cem\u003eC. albicans\u003c/em\u003e in suspension, producing a 3.6 log reduction in cell viability upon irradiation in culture tubes [14]. Therefore, both A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e proved to be effective photosensitizers for the inactivation of \u003cem\u003eC. albicans\u003c/em\u003e planktonic cells, even at low concentrations and under relatively low light fluence.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effect of scavengers of ROS on the photoinactivation of C. albicans planktonic cells\u003c/h2\u003e\u003cp\u003eWith the purpose of obtaining information about the photodynamic mechanism of action sensitized by A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e in \u003cem\u003eC. albicans\u003c/em\u003e cell suspensions, PDI studies were carried out in presence of ROS scavengers (sodium azide, DABCO, D-mannitol, and L-cysteine) and D\u003csub\u003e2\u003c/sub\u003eO. Cultures were first treated with the additives for 30 min at 37 \u0026ordm;C in the dark and then with 1.0 \u0026micro;M porphyrin for 30 min at 37 \u0026ordm;C in the dark. Cell viability was not affected in yeast cultures incubated with 50 mM of these compounds in the dark and exposed for 15 min to white light in absence of PSs (Figure S2). Likewise, no toxicity was observed in irradiated cell suspensions prepared in D\u003csub\u003e2\u003c/sub\u003eO (Figure S2, line 4). In addition, cultures incubated with the scavenger and the porphyrin, or suspended in D\u003csub\u003e2\u003c/sub\u003eO with PS, showed no reduction in viability after incubation in the dark for 15 min (Figures S3). In these experiments, a PS concentration of 1.0 \u0026micro;M and 15 min of irradiation were chosen to produce a photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e of approximately 3 log and thus, to be able to visualize the effect produced by the additives or the D\u003csub\u003e2\u003c/sub\u003eO medium.\u003c/p\u003e\u003cp\u003eThe PDI results after different treatments are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Sodium azide and DABCO were used as quenchers of O\u003csub\u003e2\u003c/sub\u003e(\u003csup\u003e1\u003c/sup\u003eΔ\u003csub\u003eg\u003c/sub\u003e) [16,]. With both additives, a reduction in the photoinactivation was found in PDI treatments of \u003cem\u003eC. albicans\u003c/em\u003e. The azide ions produced a reduction of about 2.0 log in the inactivation of yeast cells treated with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, line 3), while this effect was slightly higher with A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e, reaching 2.5 log of protection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, line 3). Similar photoprotective results were found for cultures incubated with DABCO and porphyrin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, line 4), although with a decrease in inactivation somewhat less than that produced by sodium azide. Therefore, both scavengers produced a significant decrease in porphyrin-sensitized photodynamic action by quenching O\u003csub\u003e2\u003c/sub\u003e(\u003csup\u003e1\u003c/sup\u003eΔ\u003csub\u003eg\u003c/sub\u003e). To confirm the involvement of a type II mechanism, D\u003csub\u003e2\u003c/sub\u003eO was used instead of water in order to increase the O\u003csub\u003e2\u003c/sub\u003e(\u003csup\u003e1\u003c/sup\u003eΔ\u003csub\u003eg\u003c/sub\u003e) lifetime [23]. PDI treatments of \u003cem\u003eC. albicans\u003c/em\u003e cell suspensions in D\u003csub\u003e2\u003c/sub\u003eO with porphyrin produced a significant increase in the yeast photoinactivation relative to cells in PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, line 5). The greatest effect was observed for the A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e, producing about 2 log increase in cell inactivation. These results also suggest the participation of O\u003csub\u003e2\u003c/sub\u003e(\u003csup\u003e1\u003c/sup\u003eΔ\u003csub\u003eg\u003c/sub\u003e) in the photodynamic pathway that produces cell death.\u003c/p\u003e\u003cp\u003eOn the other hand, D-mannitol and L-cysteine can act as radical scavengers and thus these compounds can be used as inhibitors of type I photoprocess [24,25]. For both porphyrins, the addition of D-mannitol produced about 0.5 log in \u003cem\u003eC. albicans\u003c/em\u003e cell protection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, line 6). Comparable behaviour was found in yeast cultures when L-cysteine was used as free radical scavenger (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, line 7). Therefore, the presence of D-mannitol and L-cysteine in \u003cem\u003eC. albicans\u003c/em\u003e cell suspensions caused only slight changes in yeast photokilling, suggesting that a type I photoprocess makes only a minor contribution.\u003c/p\u003e\u003cp\u003eIt was previously reported that the photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e sensitized by tri- and tetra-cationic porphyrins occurs predominantly through a type II mechanism, with only a minor contribution from type I pathways [12]. A similar pattern in ROS involvement was also observed for the inactivation of \u003cem\u003eC. albicans\u003c/em\u003e sensitized by A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e and [16]. Moreover, photoinactivation was negligible when the oxygen atmosphere was replaced by argon, indicating an insignificant contribution of oxygen-independent pathways to the photokilling of yeast cells. These results confirm that the photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e sensitized by A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e proceeds mainly through of O\u003csub\u003e2\u003c/sub\u003e(\u003csup\u003e1\u003c/sup\u003eΔ\u003csub\u003eg\u003c/sub\u003e)-mediated mechanism. The minor protective effects of D-mannitol and L-cysteine indicate a limited participation of type I pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Photoinactivation of C. albicans on agar surfaces\u003c/h2\u003e\u003cp\u003eIn this assay, \u003cem\u003eC. albicans\u003c/em\u003e cell suspensions (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 2 mL) were incubated with 5.0 \u0026micro;M A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e or A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e in the dark for 30 min at 37\u0026deg;C to allow porphyrin binding to yeast cells. After treatment, 100 \u0026micro;L aliquots containing\u0026thinsp;~\u0026thinsp;100 cells were uniformly spread onto SA plates. Following 15 min of incubation in the dark, the plates were exposed to white light for different periods (15, 30, and 60 min). Control experiments confirmed the absence of cytotoxic effects in cells irradiated without porphyrin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) or treated with PS and maintained in the dark (Figure S4). After 15 min of irradiation, yeast viability was reduced to 53% with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and to 75% with A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e. With 30 min of irradiation, survival decreased to 35% for A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e, while no colony formation was detected for A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e. A comparable outcome was observed after 60 min of light exposure for both PSs, confirming the potent photoinactivating activity of these porphyrins under these conditions. A comparable photokilling effect was also observed for \u003cem\u003eC. albicans\u003c/em\u003e cells treated with 5 \u0026micro;M TMPyP\u003csup\u003e4+\u003c/sup\u003e in cell suspension and irradiated on SA [17]. The results show that complete loss of \u003cem\u003eC. albicans\u003c/em\u003e viability was achieved with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e after 30 min of irradiation, whereas A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e required longer exposure to produce a comparable effect. These findings highlight the high photodynamic efficiency of the surface-bound porphyrins and their potential for effective antifungal applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Photoinactivation of C. albicans by surface-bound PSs\u003c/h2\u003e\u003cp\u003eThese experiments were used to evaluate the ability of A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e deposited on the SA surfaces to photoinhibit the growth of \u003cem\u003eC. albicans\u003c/em\u003e colonies. Therefore, different amounts of PSs (0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 nmol) were uniformly applied onto the surface of SA plates, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. A cell suspension of \u003cem\u003eC. albicans\u003c/em\u003e (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 100 \u0026micro;L) in PBS was spread on the SA surfaces. The cultures were maintained in the dark for 30 min at 37\u0026deg;C to allow interaction between the PS and the cells. Subsequently, the plates were irradiated with white light for 30 min and then incubated at 37\u0026deg;C for 72 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, these amounts of PSs were not toxic to \u003cem\u003eC. albicans\u003c/em\u003e cells maintained in the dark, since confluent growth was observed in the areas containing the porphyrin. In contrast, no cell growth of \u003cem\u003eC. albicans\u003c/em\u003e was found in the areas treated with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e and irradiated for 30 min. The area of the inhibition zone increased with the amount of PS deposited. Furthermore, colony growth was not detected even 3 days after PDI treatment. To confirm the results, samples of these areas treated with PSs and irradiated were aseptically transferred to fresh SB medium and then to a new SB agar plate. After additional 48 h incubation at 37\u0026deg;C, no formation of colonies of \u003cem\u003eC. albicans\u003c/em\u003e cells was detected, indicating a complete inactivation of yeast cells. Likewise, areas spread with different amounts of TMPyP⁴⁺ effectively photoinactivated \u003cem\u003eC. albicans\u003c/em\u003e cells immobilized on SA surfaces [17]. These results demonstrate the potential of surface-bound porphyrins as effective photoactive antimicrobial coatings. Their strong photoinactivation capacity against \u003cem\u003eC. albicans\u003c/em\u003e supports possible applications in medical devices and hospital surfaces to prevent microbial contamination. This approach offers a sustainable strategy for light-activated disinfection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Photoinactivation of C. albicans pseudohyphae\u003c/h2\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e is capable of transitioning from a yeast-like morphology to germ tubes, progressing through a series of elongated forms known as pseudohyphae, and ultimately developing into hyphae []. At the microbiological level, this dimorphic capacity is regarded as a key virulence factor, as it enables the yeast to colonize and invade mucosal tissues [26]. In light of this, the photodynamic activity sensitized by A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e was specifically evaluated against \u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae suspended in PBS. To induce the dimorphic state, yeast cultures were incubated in HS for 4 h at 37\u0026deg;C, after which the formation and morphology of pseudohyphae were confirmed by optical microscopy [18].\u003c/p\u003e\u003cp\u003eSuspensions of \u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae (~\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) in PBS were treated with 0.5, 1.0, 2.5, and 5.0 \u0026micro;M porphyrin for 30 min in the dark at 37\u0026deg;C. The survival of pseudohyphae after irradiation with white light for 2, 5, 15, and 30 min is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Control experiments showed that irradiation alone did not affect pseudohyphal viability, and no toxicity was observed in cells incubated with 5.0 \u0026micro;M porphyrin in the dark for 30 min (Figure S5). These results indicate that the observed photokilling of pseudohyphae upon light exposure was specifically attributable to the photosensitizing action of A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae was dependent on both the porphyrin concentration and the light irradiation. Both A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e exhibited similar photodynamic activity. Treatment with 1.0 \u0026micro;M porphyrin resulted in a reduction in viability of more than 3 log after 30 min of irradiation, while a comparable effect was observed for cultures treated with 2.5 \u0026micro;M porphyrin and exposed to light for 15 min. Under these conditions, no colony formation was detected when the irradiation time was extended to 30 min. Using 5.0 \u0026micro;M porphyrin, a reduction of approximately 5 log was achieved after 5 min of irradiation. Moreover, no cell survival was observed after 15 min of irradiation, corresponding to a decrease of about 6 log for pseudohyphae treated with the tricationic porphyrin. Under comparable conditions, this cationic porphyrin was also more effective at photoinactivating \u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae than glycoporphyrins containing two tertiary amino groups as precursors of cationic centers [27]. The results demonstrate that A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e effectively photoinactivate \u003cem\u003eC. albicans\u003c/em\u003e pseudohyphae in a concentration- and irradiation-dependent manner. These findings highlight the potent photodynamic activity of both porphyrins against the virulent pseudohyphal form of the yeast.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Photokilling of C. albicans biofilms\u003c/h2\u003e\u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e biofilms pose serious disadvantages for clinical management due to their strong antifungal resistance, capacity for immune evasion, and persistence on medical devices [28]. These biofilms readily colonize implant surfaces, such as catheters, prosthetic valves, and dental materials, leading to device failure, systemic infections, and high mortality rates [29]. Biofilm-associated cells display multifactorial drug resistance mediated by the extracellular matrix, efflux pump upregulation, and the formation of persistent cells, which collectively render biofilm infections difficult to eradicate with conventional antifungal agents [5,30]. Therefore, \u003cem\u003eC. albicans\u003c/em\u003e biofilms represent a therapeutic challenge, causing persistent infections, treatment failures, and increased healthcare burdens.\u003c/p\u003e\u003cp\u003eThe photoinactivation of \u003cem\u003eC. albicans\u003c/em\u003e by AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e, A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e, and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e was evaluated in biofilms grown on PVC discs, which is one of the materials commonly used in the manufacture of several medical devices [31]. After the adhesion step, the cultures were treated with 5.0 \u0026micro;M porphyrin in SB supplemented with 7% HS for 18 h at 37 \u0026ordm;C in the dark during biofilm proliferation phase. The cell survival after PDI treatments are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Biofilms not treated with PSs and irradiated with white light for 60 min showed no reduction in cell viability. Likewise, controls incubated with PSs in the dark did not exhibit any cytotoxic effects.\u003c/p\u003e\u003cp\u003eThe photodynamic effect mediated by AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e resulted in a lower photokilling capacity, reaching 1.0 log (90%) and 2.5 log (99.7%) reduction, respectively, in the viability of \u003cem\u003eC. albicans\u003c/em\u003e. In contrast, A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e produced the eradication of \u003cem\u003eC. albicans\u003c/em\u003e cells in the biofilms upon 60 min of irradiation. In particular for A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e, this denotes a greater than 6 log decrease in yeast survival, which represents 99.9997% photoinactivation.\u003c/p\u003e\u003cp\u003eThese results show that the efficiency of porphyrins increased in the following order: A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e \u0026sim; A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e \u0026gt;A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e \u0026gt;AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. These tendency in the photokilling activity was similar to those previously demonstrated in \u003cem\u003eEscherichia coli\u003c/em\u003e [11]. To confirm the efficacy of the photodynamic action sensitized by these porphyrins, after the PDI treatments, the PVC discs were deposited on a SA plate and incubated for 48 h at 37 \u0026ordm;C in the dark. After that, significant proliferation of viable \u003cem\u003eC. albicans\u003c/em\u003e cells was observed in cultures treated with AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e. In contrast, no cell growth was found on the PVC discs treated with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e after an irradiation time of 60 min. Therefore, the photodynamic activity inhibited biofilm formation, and the PDI treatment was able to decrease the survival of yeast cells in the biofilm.\u003c/p\u003e\u003cp\u003eThe photodynamic treatment with cationic porphyrins has also proven effective in inhibiting biofilm formation by clinical \u003cem\u003eC. albicans\u003c/em\u003e strains [32]. Furthermore, a porphyrinic formulation composed of five cationic porphyrins (FORM) combined with KI provided a highly efficient photodynamic strategy for eradicating \u003cem\u003eC. albicans\u003c/em\u003e biofilms [33]. While FORM alone exhibited limited efficacy, its combination with KI markedly enhanced fungal inactivation, leading to complete biofilm destruction. PDI of \u003cem\u003eC. albicans\u003c/em\u003e biofilms was also achieved using the tetracationic metalloporphyrin ZnTnHex-2-PyP⁴⁺ [34]. Upon blue light irradiation, this Zn(II) porphyrin induced up to an 89% reduction in biofilm viability and extensive structural disruption, characterized by decreased hyphal density and biofilm disorganization. These results highlight the importance of porphyrin lipophilicity and cationic charge in promoting biofilm penetration and oxidative damage generation. Furthermore, tetracationic porphyrin derivatives significantly interfered with biofilm formation by reducing adhesion forces and altering the nanomechanical properties of the cell wall [35]. Among the porphyrins tested, A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e exhibited a high photokilling efficiency, completely eradicating \u003cem\u003eC. albicans\u003c/em\u003e biofilms on PVC surfaces.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe present study demonstrates that the tricationic and tetracationic porphyrins A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e are highly effective photosensitizers for the PDI of \u003cem\u003eC. albicans\u003c/em\u003e under various experimental conditions and morphological forms. Both porphyrins exhibited rapid cellular uptake and strong association with the yeast cell envelope, promoting efficient ROS-mediated photodamage. In planktonic cultures, A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e achieved complete eradication of viable cells after low light fluence at micromolar concentrations. ROS quenching experiments confirmed that O\u003csub\u003e2\u003c/sub\u003e(\u003csup\u003e1\u003c/sup\u003eΔ\u003csub\u003eg\u003c/sub\u003e) was the primary cytotoxic species involved in cell photokilling. These porphyrins also proved highly effective against \u003cem\u003eC. albicans\u003c/em\u003e grown on agar surfaces or immobilized within solid media, where surface-bound PSs induced full inhibition of fungal proliferation. Moreover, both A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e efficiently photoinactivated the pseudohyphal morphotype, achieving up to six orders of magnitude reduction in viability, highlighting their ability to target the virulent filamentous form. Remarkably, these porphyrins completely eradicated mature biofilms grown on PVC, a material commonly used in medical devices, underscoring their potential for antifungal coatings and device disinfection. The superior photoinactivating performance of A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e can be ascribed to the optimized arrangement of cationic charges at the termini of flexible aliphatic chains, which confers greater mobility to the cationic groups enhancing electrostatic interactions with the yeast cell envelope and penetration into the biofilm matrix. Overall, these findings support A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e as potent, broad-spectrum porphyrinic photosensitizers capable of eliminating \u003cem\u003eC. albicans\u003c/em\u003e across its major morphotypes through O\u003csub\u003e2\u003c/sub\u003e(\u003csup\u003e1\u003c/sup\u003eΔ\u003csub\u003eg\u003c/sub\u003e)-driven photodynamic mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eSupplementary Information\u003c/h2\u003e\u003cp\u003eThe online version contains supplementary material available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/\u003c/span\u003e\u003cspan address=\"https://doi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e...\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.G.A. conceptualization, methodology, data analysis, validation, investigation, data curation, original draft preparation. P.V.C., J.M.G. and M.E.P.: conceptualization, methodology, data analysis, validation, investigation, data curation. E.N.D. conceptualization, methodology, validation, resources, supervision, writing-reviewing and editing, project administration, and funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by SECYT-UNRC (PPI C612) and CONICET of Argentina (PIP 11220200101208CO). M.E.P. thanks CONICET for the postdoctoral fellowship. P.C.G., M.G.A. and E.N.D. are Scientific Members of CONICET.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e. Schroeder, J. A., Wilson, C. M., Pappas, P. G. (2025). Invasive candidiasis. \u003cem\u003eInfectious Disease Clinics of North America\u003c/em\u003e, 29, 93\u0026ndash;119.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Xu, Z., Wang, K., Min, D., Soteyome, T, Lan, H., Hong, W., Peng, F., Fu, X., Peng, G., Huang, T, Liu, J. Kjellerup, B. V. (2022). Regulatory network controls microbial biofilm development, with \u003cem\u003eCandida albicans\u003c/em\u003e as a representative: from adhesion to dispersal. \u003cem\u003eBioengineered\u003c/em\u003e, 13, 253\u0026ndash;267.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Mba, I. E., Nweze, E. I., Eze, E. A., Anyaegbunam, Z. K. G. (2022). Genome plasticity in \u003cem\u003eCandida albicans\u003c/em\u003e: a cutting-edge strategy for evolution, adaptation, and survival. \u003cem\u003eInfection, Genetics and Evolution\u003c/em\u003e, 99, 105256.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Kaur, J., Nobile, C. J. (2023). Antifungal drug-resistance mechanisms in \u003cem\u003eCandida\u003c/em\u003e biofilms. \u003cem\u003eCurrent Opinion in Microbiology\u003c/em\u003e, 71, 102237.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Roy, S., Gow, N. A. R. (2023). The role of the \u003cem\u003eCandida\u003c/em\u003e biofilm matrix in drug and immune protection. \u003cem\u003eThe Cell Surface\u003c/em\u003e, 10, 100111.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Atriwal, T., Azeem, K., Husain, F. M., Hussain, A., Khan, M. N., Alajmi, M. F., Abid, M. (2021). Mechanistic understanding of Candida albicans biofilm formation and approaches for its inhibition. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, 12, 638609.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Tits, J., Cammue, B. P. A., Thevissen, K. (2020). Combination therapy to treat fungal biofilm based infections. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e, 21, 22.\u003c/span\u003e \u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Rodr\u0026iacute;guez-Cerdeira, C., Mart\u0026iacute;nez-Herrera, E., Fabbrocini, G., Sanchez-Blanco, B., L\u0026oacute;pez-Barcenas, A., Samahy, M., Ju\u0026aacute;rez-Dur\u0026aacute;n, E. R., Gonz\u0026aacute;lez-Cesp\u0026oacute;n J. L. (2021). New applications of photodynamic therapy in the management of candidiasis. \u003cem\u003eJournal of Fungi\u003c/em\u003e, 7, 1025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Aroso, R. T., Schaberle, F. A., Arnaut, L. G., Pereira, M. M. (2021). Photodynamic disinfection and its role in controlling infectious diseases. \u003cem\u003ePhotochemical and Photobiological Sciences\u003c/em\u003e, 20, 1497\u0026ndash;1545.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Pourhajibagher, M., Bahrami, R., Moghaddam, E. K., Bahador, A. (2025). Boosting the antibacterial potency of antimicrobial photodynamic therapy against oral pathogens through supplement agents: a narrative review. \u003cem\u003eJournal of Dental Sciences\u003c/em\u003e, 20, 2058\u0026ndash;2065.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Caminos, D. A., Durantini, E. N. (2005). Synthesis of asymmetrically meso-substituted porphyrins bearing amino groups as potential cationic photodynamic agents. \u003cem\u003eJournal of Porphyrins and Phthalocyanines\u003c/em\u003e, 9, 334\u0026ndash;342.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Cormick, M. P., Alvarez, M. G., Rovera, M., Durantini, E. N. (2009). Photodynamic inactivation of \u003cem\u003eCandida albicans\u003c/em\u003e sensitized by tri- and tetra-cationic porphyrin derivatives. \u003cem\u003eEuropean Journal of Medicinal Chemistry\u003c/em\u003e, 44, 1592\u0026ndash;1599.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Quiroga, E. D., Cordero, P., Mora, S. J., Alvarez, M. G., Durantini, E. N. (2020). Mechanistic aspects in the photodynamic inactivation of \u003cem\u003eCandida albicans\u003c/em\u003e sensitized by a dimethylaminopropoxy porphyrin and its equivalent with cationic intrinsic charges. \u003cem\u003ePhotodiagnosis and Photodynamic Therapy\u003c/em\u003e, 31, 101877.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Quiroga, E. D., Mora, S. J., Alvarez, M. G., Durantini, E. N. (2016). Photodynamic inactivation of \u003cem\u003eCandida albicans\u003c/em\u003e by a tetracationic tentacle porphyrin and its analogue without intrinsic charges in presence of fluconazole. \u003cem\u003ePhotodiagnosis and Photodynamic Therapy\u003c/em\u003e, 13, 334\u0026ndash;340.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Agazzi, M. L., Durantini, J. E., Quiroga, E. D., Alvarez, M. G., Durantini, E. N. (2021). A novel tricationic fullerene C\u003csub\u003e60\u003c/sub\u003e as broad‑spectrum antimicrobial photosensitizer: mechanisms of action and potentiation with potassium iodide. \u003cem\u003ePhotochemical and Photobiological Science\u003c/em\u003e, 20, 327\u0026ndash;341.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Cordero, P. V., Alvarez, M. G., Gonzalez Lopez, E. J., Heredia, D. A., Durantini, E. N. (2023). Photoinactivation of planktonic cells, pseudohyphae, and biofilms of \u003cem\u003eCandida albicans\u003c/em\u003e sensitized by a free-base chlorin and its metal complexes with Zn(II) and Pd(II). \u003cem\u003eAntibiotics\u003c/em\u003e, 12, 105.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Quiroga, E. D., Alvarez, M. G., Durantini, E. N. (2010). Susceptibility of \u003cem\u003eCandida albicans\u003c/em\u003e to photodynamic action of 5,10,15,20-tetra(4-\u003cem\u003eN\u003c/em\u003e-methylpyridyl)porphyrin in different media. \u003cem\u003eFEMS Immunology and Medical Microbiology\u003c/em\u003e, 60, 123\u0026ndash;131.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Cordero, P.V., Ferreyra, D. D., P\u0026eacute;rez, M. E., Alvarez, M. G., Durantini, E. N. (2021) Photodynamic effect of 5,10,15,20-tetrakis[4-(3-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylaminopropoxy) phenyl]chlorin towards the human pathogen \u003cem\u003eCandida albicans\u003c/em\u003e under different culture conditions. \u003cem\u003ePhotochem\u003c/em\u003e, 1, 505\u0026ndash;522.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Lambrechts, S. A. G., Aalders, M. C. G., Van Marle, J. (2005). Mechanistic study of the photodynamic inactivation of \u003cem\u003eCandida albicans\u003c/em\u003e by a cationic porphyrin. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e, 49 2026\u0026ndash;2034.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Voit, T. Cieplik, F., Regensburger, J., Hiller, K. A., Gollmer, A., Buchalla, W., Maisch, T. (2021). Spatial distribution of a porphyrin-based photosensitizer reveals mechanism of photodynamic inactivation of \u003cem\u003eCandida albicans\u003c/em\u003e. \u003cem\u003eFrontiers in Medicine\u003c/em\u003e, 8, 641244.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Novaira, M., Cormick, M. P., Durantini, E. N. (2012). Spectroscopic and time-resolved fluorescence emission properties of a cationic and an anionic porphyrin in biomimetic media and \u003cem\u003eCandida albicans\u003c/em\u003e ce\u003cem\u003ells\u003c/em\u003e. \u003cem\u003eJournal of Photochemistry and Photobiology A: Chemistry\u003c/em\u003e, \u003cem\u003e246, 67\u0026ndash;74.\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Caminos, D. A., Spesia, M. B., Durantini, E. N. (2006). Photodynamic inactivation of \u003cem\u003eEscherichia coli\u003c/em\u003e by novel \u003cem\u003emeso\u003c/em\u003e-substituted porphyrins by 4-(3-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups. \u003cem\u003ePhotochemical and Photobiological Science\u003c/em\u003e, 5 56\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Di Palma, M. A., Alvarez, M. G., Durantini, E. N. (2015). Photodynamic action mechanism mediated by zinc(II) 2,9,16,23-tetrakis [4-(\u003cem\u003eN\u003c/em\u003e-methylpyridyloxy)]phthalocyanine in \u003cem\u003eCandida albicans\u003c/em\u003e cells. \u003cem\u003ePhotochemical and Photobiology\u003c/em\u003e, 91, 1203\u0026ndash;1209.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Gsponer, N. S., Agazzi, M. L., Spesia, M. B., Durantini, E. N. (2016). Approaches to unravel pathways of reactive oxygen species in the photoinactivation of bacteria induced by a dicationic fulleropyrrolidinium derivative. \u003cem\u003eMethods\u003c/em\u003e, 109, 167\u0026ndash;174.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Costa, L., Faustino, M. A. F., Neves, M. G. P. M. S., Cunha, \u0026Acirc;., Almeida, A. (2012). Photodynamic inactivation of mammalian viruses and bacteriophages. \u003cem\u003eViruses\u003c/em\u003e, 4, 1034\u0026ndash;1074.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Noble, S. M., Gianetti, B. A., Witchley, J. N. (2017). \u003cem\u003eCandida albicans\u003c/em\u003e cell-type switching and functional plasticity in the mammalian host. \u003cem\u003eNature Reviews Microbiology\u003c/em\u003e, 15, 96\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Palacios, Y. B., Simonetti, S. O., Hern\u0026aacute;ndez Chavez, C., \u0026Aacute;lvarez, M. G., Cordero, P., Cuello, V. E. A., Gonz\u0026aacute;lez L\u0026oacute;pez, E. J., Larghi, E. L., Agazzi, M. L., Durantini, E. N., Heredia, D. A. (2025). \u0026ldquo;Illuminated glycoporphyrins\u0026rdquo;: a photodynamic approach for \u003cem\u003eCandida albicans\u003c/em\u003e inactivation. \u003cem\u003eJournal of Photochemistry and Photobiology B: Biology\u003c/em\u003e, 264, 113105.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Fan, F., Liu, Y., Liu, Y., Lv, R., Sun, W., Ding, W., Cai, Y, Li, W, Liu, X., Qu, W. (2022). \u003cem\u003eCandida albicans\u003c/em\u003e biofilms: antifungal resistance, immune evasion, and emerging therapeutic strategies. \u003cem\u003eInternational Journal of Antimicrobial Agents\u003c/em\u003e, 60, 106673.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Le, P. H., Linklater, D. P., Medina, A. A., MacLaughlin, S., Crawford, R. J., Ivanova, E. P. (2024). Impact of multiscale surface topography characteristics on \u003cem\u003eCandida albicans\u003c/em\u003e biofilm formation: from cell repellence to fungicidal activity. \u003cem\u003eActa Biomaterialia\u003c/em\u003e, 177, 20\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Kaur, J., Nobile, C. J. (2023). Antifungal drug-resistance mechanisms in \u003cem\u003eCandida\u003c/em\u003e biofilms. \u003cem\u003eCurrent Opinion in Microbiology\u003c/em\u003e, 71, 102237.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Duarte-Pe\u0026ntilde;a, L., L\u0026oacute;pez-Saucedo, F., Concheiro, A., Alvarez-Lorenzo, C., Bucio, E. (2022). Modification of indwelling PVC catheters by ionizing radiation with temperature and pH-responsive polymers for antibiotic delivery. \u003cem\u003eRadiation Physics and Chemistry\u003c/em\u003e, 193, 110005.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Orlandi, V. T., Martegani, E., Bolognese, F., Trivellin, N., Mat\u0026rsquo;\u0026aacute;tkov\u0026aacute;, M., Paldrychov\u0026aacute;, O., Andreina Baj, Caruso, E. (2020). Photodynamic therapy by diaryl-porphyrins to control the growth of \u003cem\u003eCandida albicans\u003c/em\u003e. \u003cem\u003eCosmetics\u003c/em\u003e, 7, 31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Vieira, C., Bartolomeu, M., Santos, A. R., Mesquita, M. Q., Gomes, A. T. P. C., Neves, M. G. P. M. S, Faustino, M. A. F., Almeida, A. (2022). Photoinactivation of bacterial and fungal planktonic/biofilm forms using the combination of a porphyrinic formulation with potassium iodide. \u003cem\u003eMedical Sciences Forum\u003c/em\u003e, 12, 13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Souza, S. O., Raposo, B. L., Sarmento-Neto, J. F., Rebou\u0026ccedil;as, J. S., Mac\u0026ecirc;do, D. P. C., Figueiredo, R. C. B. Q., Santos, B. S., Freitas, A. Z., Cabral Filho, P. E., Ribeiro, M. S., Fontes, A. (2022). Photoinactivation of yeast and biofilm communities of \u003cem\u003eCandida albicans\u003c/em\u003e mediated by ZnTnHex-2-PyP\u003csup\u003e4+\u003c/sup\u003e porphyrin. \u003cem\u003eJournal of Fungi\u003c/em\u003e, 8, 556.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e. Amorim, C. F., Iglesias, B. A., Pinheiro, T. R ., Lacerda, L. E., Sokolonski, A. R., Pedreira, B. O., Moreira, K. S.,. Burgo, T. A. L., Meyer, R., Azevedo, V., Portela, R. W. (2023). Photodynamic inactivation of different Candida species and inhibition of biofilm formation induced by water-soluble porphyrins. \u003cem\u003ePhotodiagnosis and Photodynamic Therapy\u003c/em\u003e, 42 103343.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"photodynamic inactivation, cationic porphyrin, Candida albicans, pseudohyphae, biofilms","lastPublishedDoi":"10.21203/rs.3.rs-7992830/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7992830/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe increasing resistance of \u003cem\u003eCandida albicans\u003c/em\u003e to conventional antifungal therapies highlights the need for alternative treatment strategies. In this study, photodynamic inactivation (PDI) was evaluated using four cationic porphyrins (AB\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, A\u003csub\u003e2\u003c/sub\u003eB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e, A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e, and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e), differing in symmetry and number of positive charges. These compounds were tested against \u003cem\u003eC. albicans\u003c/em\u003e in its planktonic, pseudohyphal, and biofilm forms. Upon incubation with 1.0 \u0026micro;M porphyrin, rapid cellular uptake was observed, with A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e, showing the highest accumulation (0.65 and 0.50 nmol/10⁶ cells, respectively). The amount of porphyrin bound to cells remained stable over time, with no significant changes beyond 5 min of incubation. PDI was performed using different porphyrin concentrations (0.5\u0026ndash;5.0 \u0026micro;M) and light exposure times (5\u0026ndash;30 min). A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e exhibited potent photoinactivation, reducing cell viability by over 5 log (\u0026gt;\u0026thinsp;99.999%) after 5 min of irradiation using 2.5 \u0026micro;M porphyrin. Reactive oxygen species quenching experiments indicated that singlet molecular oxygen was the primary cytotoxic agent. Additionally, A\u003csub\u003e3\u003c/sub\u003eB\u003csup\u003e3+\u003c/sup\u003e and A\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e4+\u003c/sup\u003e effectively eradicated \u003cem\u003eC. albicans\u003c/em\u003e cells on agar surfaces. These porphyrins also inactivated pseudohyphal suspensions of \u003cem\u003eC. albicans\u003c/em\u003e, achieving a reduction greater than 5 log, when incubated with 5 \u0026micro;M porphyrin and 5 min of irradiation. Using this concentration, \u003cem\u003eC. albicans\u003c/em\u003e biofilms were completely photoinactivated after 60 min of light exposure. These findings demonstrate that highly charged cationic porphyrins are promising photosensitizers for the targeted elimination of \u003cem\u003eC. albicans\u003c/em\u003e across its major morphological states.\u003c/p\u003e","manuscriptTitle":"Influence of porphyrin cationic charges on photoinactivation of Candida albicans morphotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 12:41:13","doi":"10.21203/rs.3.rs-7992830/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T10:02:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-19T23:45:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T20:28:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-15T22:33:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7430693336716930348360096499055954430","date":"2025-11-06T18:19:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32843489773356802007095150669020016071","date":"2025-11-05T15:57:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3298157370857399750487384254495175014","date":"2025-11-04T09:22:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-03T15:54:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-03T11:17:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-31T08:07:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photochemical \u0026 Photobiological Sciences","date":"2025-10-30T21:55:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photochemical-and-photobiological-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ppss","sideBox":"Learn more about [Photochemical \u0026 Photobiological Sciences](https://link.springer.com/journal/43630)","snPcode":"43630","submissionUrl":"https://www.editorialmanager.com/ppss/","title":"Photochemical \u0026 Photobiological Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a8389cc2-9a71-4e3f-b13a-e56ac43ea84e","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:04:36+00:00","versionOfRecord":{"articleIdentity":"rs-7992830","link":"https://doi.org/10.1007/s43630-025-00835-3","journal":{"identity":"photochemical-and-photobiological-sciences","isVorOnly":false,"title":"Photochemical \u0026 Photobiological Sciences"},"publishedOn":"2025-12-22 15:57:46","publishedOnDateReadable":"December 22nd, 2025"},"versionCreatedAt":"2025-11-13 12:41:13","video":"","vorDoi":"10.1007/s43630-025-00835-3","vorDoiUrl":"https://doi.org/10.1007/s43630-025-00835-3","workflowStages":[]},"version":"v1","identity":"rs-7992830","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7992830","identity":"rs-7992830","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}