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- Proton-assisted Two-electron Transfer in Natural Variants of Tetraheme Cytochromes from Desulfomicrobium Sp.Publication . Correia, Ilídio Joaquim Sobreira; Paquete, Catarina; Coelho, Ana; Almeida, Claudia; Catarino, Teresa; Louro, Ricardo; Frazão, Carlos; Saraiva, Lígia M.; Carrondo, Maria; Turner, David; Xavier, AntónioThe tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 → Ser, Val-45 → Ala, and Phe-88 → Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.
- Preparation of end-capped pH-sensitive mesoporous silica nanocarriers for on-demand drug deliveryPublication . Moreira, André; Gaspar, Vítor Manuel Abreu; Costa, Elisabete C.; Diogo, Duarte Miguel de Melo; Machado, Paulo Filipe Brito; Paquete, Catarina; Correia, Ilídio Joaquim SobreiraNanocarriers with a pH responsive behavior are receiving an ever growing attention due to their potential for promoting on-demand drug release and thus increase the therapeutic effectiveness of anti-tumoral pharmaceutics. However, the majority of these systems require costly, time-consuming and complex chemical modifications of materials or drugs to synthesize nanoparticles with pH triggered release. Herein, the development of dual drug loaded pH-responsive mesoporous silica nanoparticles (MSNs) with a calcium carbonate-based coating is presented as an effective alternative. This innovative approach allowed the loading of a non-steroidal anti-inflammatory drug (Ibuprofen) and Doxorubicin, with high efficiency. The resulting dual drug loaded MSNs have spherical morphology and a mean size of 171 nm. Our results indicate that under acidic conditions the coating disassembles and the drugs are rapidly released, whereas at physiologic pH the release is slower and gradually increases with time. Furthermore, an improved cytotoxic effect was obtained for Doxorubicin–Ibuprofen MSNs coated with CaCO3 in comparison with non-coated particles. The cytotoxic effect of dual loaded carbonate coated particles, was similar to that of Doxorubicin + Ibuprofen free drug administration at 72 h, even with the delivery of a significantly lower amount of drug by MSNs-CaCO3. These results suggest that the carbonate coating of MSNs is a promising approach to create a pH-sensitive template for a delivery system with application in cancer therapy.
- Characterization of OmcA Mutants from Shewanella oneidensis MR‐1 to Investigate the Molecular Mechanisms Underpinning Electron Transfer Across the Microbe‐Electrode InterfacePublication . Neto, Sónia de Fátima Estevão; Diogo, Duarte Miguel de Melo; Correia, I.J.; Paquete, Catarina; Louro, RicardoElectricity production in microbial fuel cells (MFCs) is an emerging green alternative to the use of fossil fuels. Shewanella oneidensis MR‐1 (SOMR‐1) is a Gram‐negative bacterium, adapted to MFCs due to its ability to link its bioenergetic metabolism through the periplasm to reduce extracellular electron acceptors. OmcA is a highly abundant outer‐membrane cytochrome of SOMR‐1 cells and is involved in the extracellular electron transfer to solid acceptors and electron shuttles. To investigate electron transfer performed by OmcA towards final acceptors, site directed mutagenesis was used to disturb the axial coordination of hemes. Interactions between OmcA and redox partners such as iron and graphene oxides, and electron shuttles were characterized using nuclear magnetic resonance and stopped‐flow experiments. Results showed that solid electron acceptors do not come into close proximity to the hemes, in agreement with experimentally observed slow electron transfer. In contrast, mutation of the distal axial ligand of heme VII changes the driving force of OmcA towards electron shuttles and reduces the affinity of the FMN:OmcA complex. Overall, these results reveal a functional specificity of particular hemes of OmcA and provide guidance for the rational design of mutated SOMR‐1 strains optimized for operating in different microbial electrochemical devices.
- Nanoparticle mediated delivery of pure P53 supercoiled plasmid DNA for gene therapyPublication . Gaspar, Vítor Manuel Abreu; Correia, Ilídio; Sousa, Ângela; Silva, Filomena; Paquete, Catarina; Queiroz, João; Sousa, FaniThe translation of non-viral gene replacement therapies for cancer into clinical application is currently hindered due to known issues associated with the effectiveness of plasmid DNA (pDNA) expression vectors and the production of gene delivery vehicles. Herein we report an integrative approach established on the synthesis of nanoparticulated carriers, in association with the supercoiled (sc) isoform purification of a p53 tumor suppressor encoding plasmid, to improve both delivery and transfection. An arginine-based chromatographic matrix with specific recognition for the different topoisoforms was used to completely isolate the biologically active sc pDNA. Our findings showed that the sc topoisoform is recovered under mild conditions with high purity and structural stability. In addition, to further enhance protection and transfection efficiency, the naked sc pDNA was encapsulated within chitosan nanoparticles by ionotropic gelation. The mild conditions for particle synthesis used in the former technique allowed the attainment of a high encapsulation efficiency for sc pDNA (> 75%). Moreover, in vitro transfection experiments confirmed the reinstatement of the p53 protein expression and most importantly, the sc pDNA transfected cells exhibited the highest p53 expression levels when compared to other formulations. Overall, given the fact that sc pDNA topoisoform indeed enhances transgene expression rates this approach might have a profound impact on the development of a sustained nucleic acid-based therapy for cancer.
- Thermodynamic and kinetic characterization of trihaem cytochrome c3 from Desulfuromonas acetoxidansPublication . Correia, Ilídio Joaquim Sobreira; Paquete, Catarina; Louro, Ricardo; Catarino, Teresa; Turner, David; Xavier, AntónioTrihaem cytochrome c3 (also known as cytochrome c551.5 and cytochrome c7) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur-reducing bacterium. Thermodynamic and kinetic data for the trihaem cytochrome c3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D. acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini-sulfuretum. The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre. The kinetics of trihaem cytochrome c3 reduction by sodium dithionite were studied using the stopped-flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems. This analysis indicates that the electrons enter the cytochrome mainly via haem I. The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied. Thus, although the trihaem cytochrome c3 is readily reduced by hydrogenases from Desulfovibrio sp. and its haem core is similar to that of the homologous tetrahaem cytochromes c3, its physico-chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.
- Synthesis and characterization of micelles as carriers of non-steroidal anti-inflammatory drugs (NSAID) for application in breast cancer therapyPublication . Marques, João Filipe Gonçalves; Gaspar, Vítor Manuel Abreu; Costa, Elisabete C.; Paquete, Catarina; Correia, Ilídio Joaquim SobreiraNon-steroidal anti-inflammatory drugs (NSAIDS) are emerging as a particularly valuable class of drugs due to their recently reported anti-tumoral activity in colorectal cancer. However, despite this tremendous potential, their bioavailability at the tumor microenvironment remains rather limited. To overcome this issue, in this work we synthesized biocompatible micellar nanocarriers composed of amphiphilic chitosan to deliver ibuprofen into breast cancer cells and evaluate its anti-tumor activity, while avoiding side-effects. Our results reveal that the formulations produced herein self-assembly into spherical micelles with suitable sizes for tumor accumulation (108–252 nm). Furthermore, by using a vortex-sonication method, ibuprofen was successfully encapsulated with high efficiency. Cell uptake studies show that ibuprofen-loaded micelles are readily internalized by tumor cells and deliver their cargo in the intracellular compartment as demonstrated by confocal microscopy images. This fact led to a remarkable reduction in cancer cell viability (<13%), at a relatively low drug dosage, illustrating the anti-tumoral activity of ibuprofen when delivered to breast cancer cells. These findings demonstrate the promising potential of chitosan micelles as carriers of cost-effective NSAIDS for application in breast cancer therapy.