Publications

Abstract Iron is essential for the formation, maturation and dispersal of bacterial biofilms, playing a crucial role in the physiological and metabolic functions of bacteria as well as in the regulation of virulence. Limited availability of iron can impair the formation of robust biofilms by altering cellular motility, hydrophobicity and protein composition of the bacterial surface. In this study, the antibiofilm activity of two natural iron chelating agents, kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) and maltol (3-hydroxy-2-methyl-4-pyrone), were investigated against Staphylococcus aureus and Pseudomonas aeruginosa. In addition, the ability of these 2-hydroxy-4-pyrone derivatives in preventing and eradicating S. aureus and P. aeruginosa biofilms through the enhancement of the efficacy of two antibiotics (tobramycin and ciprofloxacin) was explored. The iron binding capacity of the kojic acid and maltol was confirmed by their affinity for iron (III) which was found to be about 90 %, comparable to the regular chelating agent ethylenediaminetetraacetic acid (EDTA, 89 %). The antibiofilm efficacy of 2-hydroxy-4-pyrone derivatives, alone and in combination with antibiotics, was evaluated by measuring the total biomass, metabolic activity, and culturability of biofilm cells. Furthermore, their impact on the membrane integrity of S. aureus biofilm cells was investigated using flow cytometry and epifluorescence microscopy with propidium iodide staining. It was also examined the ability of 2-hydroxy-4-pyrone derivatives and 2-hydroxy-4-pyrone derivate-antibiotic dual-combinations in inhibiting the production of virulence factors (total proteases, lipases, gelatinases and siderophores) by S. aureus. Regarding biofilm formation, the results showed that 2-hydroxy-4-pyrone derivatives alone reduced the metabolic activity of S. aureus biofilm cells by over 40 %. When combined with tobramycin, a 2-log (CFU cm-2) reduction in S. aureus biofilm cells was observed. Moreover, the combination of maltol and kojic acid with ciprofloxacin prevented P. aeruginosa biomass production by 60 %, compared to 36 % with ciprofloxacin alone. In pre-established S. aureus and P. aeruginosa biofilms, selected compounds reduced the metabolic activity by over 75 %, and a 3-log (CFU cm-2) reduction in the culturability of biofilm cells was noted when kojic acid and maltol were combined with antibiotics. Moreover, 2-hydroxy-4-pyrone derivatives alone and in combination with tobramycin, damaged the cell membranes of pre-established biofilms and completely inhibited total proteases production. Despite the increasing of reactive oxygen species production caused by the cellular treatment of maltol, both 2-hydroxy-4-pyrone derivatives showed good safe profile when tested in human hepatocarcinoma (HepG2) cells. The pre-treatment of HepG2 cells with both compounds was crucial to prevent the cellular damage caused by iron (III). This study demonstrates for the first time that the selected 2-hydroxy-4-pyrone derivatives significantly enhance the antibiofilm activity of tested antibiotics against S. aureus and P. aeruginosa, highlighting their potential as antibiotic adjuvants in preventing and eradicating biofilm-related infections.

Abstract New psychoactive substances (NPS) are designed to evade legal regulation while mimicking the effects of classic illicit drugs such as 3,4-methylenedioxymethamphetamine (MDMA). This category includes phenethylamine derivatives, such as the psychedelic 2C and NBOMe drugs. Given the lack of data regarding the toxicological profile of these substances, the goal of this study was to evaluate the neurotoxicity of 2C-I and 25I-NBOMe and explore their neurotoxic pathways. Lower EC50 values, in both NR uptake and MTT reduction assays in differentiated SH-SY5Y cells and primary rat cortical cultures, revealed that 25I-NBOMe is significantly more cytotoxic than 2C-I, likely due to its higher lipophilicity. Both drugs triggered severe mitochondrial dysfunction, characterized by decreased intracellular ATP levels and mitochondrial membrane depolarization, although no significant changes in intracellular ROS/RNS levels were observed. Additionally, 25I-NBOMe increased the intracellular Ca2* levels. Apoptosis was an observed mechanism of cell death for both drugs, as demonstrated by a significant increase in the number of cells undergoing early apoptosis (AnV+/PI-) and late apoptosis/necrosis (AnV+/PI+). However, only 2C-I induced autophagy and strongly triggered caspase-3 activation. This suggests that 2C-I induces caspase-3-dependent apoptosis, whereas 25I-NBOMe may also induce apoptosis through a caspase-3-independent pathway, possibly involving increased intracellular Ca2* levels and direct mitochondrial damage. These findings underscore the complex interplay between mitochondrial dysfunction, calcium dysregulation, and cell death pathways, highlighting the central role of mitochondria in the cytotoxicity of 2C-I and 25INBOMe.

Abstract Edaravone is one of the treatment options for Amyotrophic Lateral Sclerosis, but its therapeutic efficacy is limited due to the incapacity to cross the blood-brain barrier, as well as its short life span and poor stability, which is ultimately caused by its tautomerism in physiological condions. This work presents an overview about the use of several nanoformulations based on polymeric, protein, lipidic, or hybrid structure as suitable and stable drug delivery systems for encapsulating edaravone. We also evaluated the functionalization of nanoparticles with pegylated chains using the polyethylene glycol or tocopherol polyethylene glycol succinate and the possibility of preparing polymeric nanoparticles at different pH (7.4, 9, and 11). Edaravone was sucessfully encapsulated in polymeric, lipid-polymer hybrid, and lipidic nanoparticles. The use of higher pH values in the synthesis of polymeric nanoparticles has led to a decrease in nanoparticle size and an increase in the percentage of encapsulation efficiency. However, the resulting nanoformulations are not stable. Only polymeric and hybrid nanoparticles showed good stability over 80 days of storage, mainly at 4 degrees C. Overall, the nanoformulations tested did not show cytotoxicity in the SH-SY5Y cell line except the nanostructured lipid carrier formulations that showed some cytotoxicity possibly due to lipidic peroxidation. In conclusion, this work shows that edaravone can be encapsulated in different nanocarriers that could act as an interesting alternative for the treatment of Amyotrophic Lateral Sclerosis.

Abstract Antibacterial resistance poses a critical public health threat, challenging the prevention and treatment of bacterial infections. The search for innovative antibacterial agents has spurred significant interest in quaternary heteronium salts (QHSs), such as quaternary ammonium and phosphonium compounds as potential candidates. In this study, a library of 49 structurally related QHSs was synthesized, varying the cation type and alkyl chain length. Their antibacterial activities against Staphylococcus aureus, including antibiotic-resistant strains, were evaluated by determining minimum inhibitory/bactericidal concentrations (MIC/MBC) <= 64 mu g/mL. Structure-activity relationship analyses highlighted alkyl-triphenylphosphonium and alkyl-methylimidazolium salts as the most effective against S. aureus CECT 976. The length of the alkyl side chain significantly influenced the antibacterial activity, with optimal chain lengths observed between C-10 and C-14. Dose-response relationships were assessed for selected QHSs, showing dose-dependent antibacterial activity following a non-linear pattern. Survival curves indicated effective eradication of S. aureus CECT 976 by QHSs at low concentrations, particularly compounds 1e, 3e, and 5e. Moreover, in vitro human cellular data indicated that compounds 2e, 4e, and 5e showed favourable safety profiles at concentrations <= 2 mu g/mL. These findings highlight the potential of these QHSs as effective agents against susceptible and resistant bacterial strains, providing valuable insights for the rational design of bioactive QHSs.

Abstract Tolcapone is an orally active catechol-O-methyltransferase (COMT) inhibitor used as adjuvant therapy in Parkinson's disease. However, it has a highly hepatotoxic profile, as recognized by the U.S. Food and Drug Administration. As a possible solution, nanoscience brought us several tools in the development of new functional nanomaterials with tunable physicochemical properties, which can be part of a solution to solve several drawbacks, including drug's short half-life and toxicity. This work aims to use PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a stable carrier with lower hydrodynamic size and polydispersity to encapsulate tolcapone in order to overcome its therapeutic drawbacks. Using the nanoprecipitation method, tolcapone-loaded nanoparticles with a DLC% of 5.7% were obtained (EE% of 47.0%) and subjected to a lyophilization optimization process to obtain a final shelf-stable formulation. Six different cryoprotectants in concentrations up to 10% (w/v) were tested. A formulation of PLGA nanoparticles with 3% hydroxypropyl-beta-cyclodextrin (HP beta CD) as a cryoprotectant (PLGA-HP@Tolc), presenting sub-200 nm sizes and low polydispersity (PdI < 0.200) was selected. Cytotoxicity assays, namely, MTT and SRB, were used to study the metabolic activity and cell density of tolcapone and PLGA-HP@Tolc-treated cells. In both assays, a hepatocarcinoma cell line (HepG2) growing in glucose or glucose-free media (galactose-supplemented medium) was used. The results demonstrated that the treatment with the PLGA-HP@Tolc formulation led to a decrease in cytotoxicity in comparison to free tolcapone-treated cells in both media tested. Moreover, the elected formulation also counteracted ATP-depletion and excessive ROS production induced by tolcapone. The results suggest that HP beta CD might have a dual function in the formulation: cryoprotectant and anticytotoxic agent, protecting cells from tolcapone-induced damage. Using an in vitro COMT inhibition assay, the PLGA-HP@Tolc formulation demonstrated to inhibit COMT as efficiently as free tolcapone. Overall, the results suggest that tolcapone-loaded PLGA NPs could be an interesting alternative to free tolcapone, demonstrating the same in vitro efficacy in inhibiting COMT but with a safer cytotoxic profile.

Abstract Nitrocatechols are the standard pharmacophore to develop potent tight-binding inhibitors of catechol O-methyltransferase (COMT), which can be used as coadjuvant drugs to manage Parkinson's disease. Tolcapone is the most potent drug of this class, but it has raised safety concerns due to its potential to induce liver damage. Tolcapone-induced hepatotoxicity has been attributed to the nitrocatechol moiety; however, other nitrocatechol-based COMT inhibitors, such as entacapone, are safe and do not damage the liver. There is a knowledge gap concerning which mechanisms and chemical properties govern the toxicity of nitrocatechol-based COMT inhibitors. Using a vast array of cell-based assays, we found that tolcapone-induced toxicity is caused by direct interference with mitochondria that does not depend on bioactivation by P450. Our findings also suggest that (a) lipophilicity is a key property in the toxic potential of nitrocatechols; (b) the presence of a carbonyl group directly attached to the nitrocatechol ring seems to increase the reactivity of the molecule, and (c) the presence of cyano moiety in double bond stabilizes the reactivity decreasing the cytotoxicity. Altogether, the fine balance between lipophilicity and the chemical nature of the C1 substituents of the nitrocatechol ring may explain the difference in the toxicological behavior observed between tolcapone and entacapone.

Abstract Catechol-O-methyltransferase inhibitors (iCOMT), such as entacapone, have been successfully employed to treat tremor-related symptoms of Parkinson's disease. However, iCOMT has been associated with a short half-life and poor oral bioavailability. Nanobased drug delivery systems have often been used to overcome this type of setbacks. Therefore, entacapone was encapsulated in PEGylated poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) via a nanoprecipitation process, as well as in PEGylated nanostructured lipid carriers (NLCs) using a solvent emulsification/evaporation method. Both nanoformulations presented sub-200 nm populations, with zeta-potential (ZP) values close to -30 mV, and showed stability at different pHs, while maintaining their physicochemical properties mostly intact, presenting only a change in their superficial charge (ZP values), indicating their interaction. Both nanoformulations presented interaction with mucins, which anticipates good permeation and bioavailability for oral and topical administration. No cytotoxic effects were observed for lyophilized PLGA NPs encapsulating entacapone, in which 2-hydroxypropyl-ss-cyclodextrin (HP beta CD) was used as a cryoprotectant at 3% concentration (HP-PLGA@Ent), in human hepatocellular carcinoma (HepG2), human neuroblastoma (SH-SY5Y), or human epithelial colorectal adenocarcinoma (Caco-2) cell lines. Conversely, NLCs encapsulating entacapone (W-NLCs@Ent) presented cytotoxic effects on the HepG2 cell line, likely due to intracellular lipid accumulation or storage. Both nanoformulations maintained a COMT inhibition effect in HepG2 cells, using 3-BTD as the COMT probe. An increase of entacapone permeability in both monolayer and coculture models (Caco-2 and Caco-2/HT29-MTX, respectively) was observed for the developed nanoformulations. Overall, this work shows that encapsulated entacapone in different nanocarriers could be a stimulating alternative to solve entacapone setbacks, since they improve its physicochemical properties and permeability while still maintaining the COMT inhibitory activity.