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Projeto de investigação
Research Institute for Medicines
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Synthesis of glutathione as a central aspect of PAH toxicity in liver cells
Publication . Branco, Vasco; Matos, Beatriz; Mourato, Carolina; Diniz, Mário; Carvalho, Cristina; Martins, Marta; MARE - Centro de Ciências do Mar e do Ambiente; DCEA - Departamento de Ciências e Engenharia do Ambiente; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Academic Press | Elsevier
Polycyclic Aromatic Hydrocarbons (PAH) are a class of organic pollutants normally found as mixtures with effects often hard to predict, which poses a major challenge for risk assessment. In this study, we address the effects of Phenanthrene (Phe), benzo[b]fluoranthene (B[b]F) and their mixtures (2 Phe:1 B[b]F; 1 Phe: 1 B[b]F; 1 Phe: 2 B[b]F) over glutathione (GSH) synthesis and function in HepG2 cells. We analyzed the effects on cellular viability, ROS production, glutathione (GSH) levels, protein-S-glutathionylation (PSSG), the activity of glutathione peroxidase (GPx), glutathione-S-transferases (GST) and glutathione reductase (GR). Transcript (mRNA) levels of glutathione synthesis enzymes - glutathione cysteine ligase catalytical (GCLC) and modifying (GCLM) sub-units and glutathione synthetase (GS) – and Nrf2 translocation to the nucleus were analyzed. Phe showed a higher cytotoxicity (IC50 = 130 µM after 24 h) than B[b]F related to a higher ROS production (up-to 50% for Phe). In agreement, GSH levels were significantly increased (up-to 3-fold) by B[b]F and were accompanied by an increase in the levels of PSSG, which is a mechanism that protect proteins from oxidative damage. The upregulation of GSH was the consequence of Nrf2 signaling activation and increased levels of GCLC, GCLM and GS mRNA observed after exposure to B[b]F, but not during exposure to Phe. Most interestingly, all mixtures showed higher cytotoxicity than individual compounds, but intriguingly it was the 1 Phe: 1B[b]F mixture showing the highest cytotoxicity and ROS production. GSH levels were not significantly upregulated not even in the mixture enriched in B[b]F. These results point to the role of GSH as a central modulator of PAH toxicity and demonstrate the idiosyncratic behavior of PAH mixtures even when considering only two compounds in varying ratios.
In Vitro, Ex Vivo, and In Vivo Evaluation of Silver Nanoparticles Synthesized Using Green Tomato Extract
Publication . Cunha, Daniela; Faria-Silva, Catarina; Carvalho, Filomena A.; Ascensão, Lia; Simões, Pedro; Carvalheiro, Manuela; Simões, Sandra; LAQV@REQUIMTE; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
Biogenic silver nanoparticles (AgNP) are among the fastest-growing nanomaterials due to the simplicity, efficiency, and sustainability of their biosynthesis using phytochemicals as reducing and coating agents. The agro-food industry generates large quantities of organic waste, a renewable source of biomolecules for AgNP biosynthesis. The main objective of this work was to prepare and characterize biogenic AgNP using a green tomato waste extract (TE) obtained by subcritical water extraction. To the best of our knowledge, this is the first report on the use of such an extract in the synthesis of AgNP. The effects of the TE and AgNO3 concentrations, reaction time, pH, and temperature on AgNP physico-chemical characteristics and on in vitro cytotoxicity against HaCaT and THP-1 cells were assessed. Antimicrobial activity was determined in vitro and ex vivo. The wound-healing capability of AgNP was evaluated in vivo in an incisional wound mouse model. The developed AgNP have a Surface Plasmon Resonance (SPR) band between 402 and 406 nm and a size of ±60 nm, and they are negatively charged (−42 mV) and spherical. In vitro and ex vivo studies prove that AgNP do not compromise skin cells and can decrease cutaneous irritation. The AgNP formulated in a gel revealed similar wound-healing properties to a commercial silver-containing topical ointment. Overall, the biogenic synthesis of AgNP employing an extract of agricultural waste obtained by an eco-friendly method is simple and cost-effective and presents the potential for application in skin disease management.
Studies on the Interaction between Model Proteins and Fluorinated Ionic Liquids
Publication . Alves, Márcia M. S.; Melo, Manuel N.; Mertens, Haydyn D. T.; Pereiro, Ana B.; Archer, Margarida; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); DQ - Departamento de Química; MDPI AG
Proteins are inherently unstable, which limits their use as therapeutic agents. However, the use of biocompatible cosolvents or surfactants can help to circumvent this problem through the stabilization of intramolecular and solvent-mediated interactions. Ionic liquids (ILs) have been known to act as cosolvents or surface-active compounds. In the presence of proteins, ILs can have a beneficial effect on their refolding, shelf life, stability, and enzymatic activities. In the work described herein, we used small-angle X-ray scattering (SAXS) to monitor the aggregation of different concentrations of ILs with protein models, lysozyme (Lys) and bovine serum albumin (BSA), and fluorescence microscopy to assess micelle formation of fluorinated ILs (FILs) with Lys. Furthermore, coarse-grained molecular dynamics (CG-MD) simulations provided a better understanding of Lys–FIL interactions. The results showed that the proteins maintain their globular structures in the presence of FILs, with signs of partial unfolding for Lys and compaction for BSA with increased flexibility at higher FIL concentrations. Lys was encapsulated by FIL, thus reinforcing the potential of ILs to be used in the formulation of protein-based pharmaceuticals.
Topical eye drug delivery hybrid nanoparticles with improved bioactivity and mucoadhesive properties
Publication . Duarte, César Daniel Marques; Gonçalves, Lídia
The eye topical drug delivery continues to be challenging due to the eye's biological mechanisms preventing the achievement of optimal drug concentration with therapeutic effect. Various strategies can be made to enhance the bioavailability, retention time, and penetration of the drug into the eye. In this work, it was developed the use of naturally available polymers like chitosan and hyaluronic acid (HA) with mucoadhesive and targeting properties in a hybrid nanoparticulated system with lipids (hyLNPs), taking advantage to deliver both lipophilic and hydrophilic drugs.
The hyLNPs were optimized by surface modulation properties and lipophilic drug (Curcumin-Curc) incorporation using the high shear homogenization technique. The HA and ceftazidime (CZ) were adsorbed to the LNPs’ surface. The characterization was done in terms of size, polydispersity index (PDI) and ζ-potential, mucoadhesion and permeation properties, in vitro cell uptake, cell viability, the efficiency of drug encapsulation and loading (%EE, %DL), and drugs’ bio-efficacy.
The optimized formulations comprising solid and liquid lipids, chitosan, and HA presents an average particle size of 256.2 ± 6.0 nm, PDI of 0.386 ± 0.013, and ζ-potential -33.3 ± 0.5 mV and mucoadhesive properties. The %EE was 86.5 ± 0.9 % and 48.4 ± 7.9 %, with a %DL of 0.135 ± 0.009 % and 2.0 ± 0.3 % for Curc and CZ, respectively. Both drugs were released from the nanoparticles and successfully permeated through membranes within 2 h after instillation. The empty and drug-loaded hyLNPs didn’t present cytotoxicity in human retinal pigment epithelia cell line (ARPE-19). The drug-loaded hyLNPs reduced oxidative stress and were effective in inhibiting P. aeruginosa growth. CZ-Curc-hyLNPs are cell internalized being biocompatible which potentiates its application for the treatment of ocular pathologies.
Diarylethene-Based Ionic Liquids: Synthesis and Photo-Driven Solution Properties
Publication . Soromenho, Mário R. C.; Afonso, Carlos A. M.; Esperança, José M. S. S.; LAQV@REQUIMTE; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
In this work, the design and synthesis of a series of photochromic gemini diarylethene-based ionic liquids (GDILs) with different cationic motifs is reported. Several synthetic pathways were optimized for the formation of cationic GDILs with chloride as the counterion. The different cationic motifs were achieved through the N-alkylation of the photochromic organic core unit with different tertiary amines, including different aromatic amines such as imidazole derivatives and pyridinium, and other non-aromatic amines. These novel salts present surprising water solubility with unexplored photochromic features that broaden their known applications. The covalent attachment of the different side groups dictates their water solubility and differences upon photocyclization. The physicochemical properties of GDILs in aqueous and in imidazolium-based ionic liquid (IL) solutions were investigated. Upon irradiation with ultraviolet (UV) light, we have observed changes in the physico-chemical properties of distinct solutions containing these GDILs, at very low concentrations. More specifically, in aqueous solution, the overall conductivity increased with the time of UV photoirradiation. In contrast, in IL solution, these photoinducible changes are dependent on the type of ionic liquid used. These compounds can improve non-ionic and ionic liquids’ solutions since we can change their properties, such as conductivity, viscosity or ionicity, only by UV photoirradiation. The electronic and conformational changes associated with these innovative stimuli GDILs may open new opportunities for their use as photoswitchable materials.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UIDP/04138/2020