Publications

Abstract As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H2O2 (at 0.6 V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16 nM, using a linear concentration range between 10 and 100 nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.

Abstract Dioctadecyldimethylammonium bromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In this work, we intend to study the effect of high MO content on the structure and physicochemical properties of pDNA/DODAB:MO lipoplexes to achieve some correlation with their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/-). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Forster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the beta-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/-) = 6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content.

Abstract Quorum sensing (QS) is an important regulatory mechanism in biofilm formation and differentiation. Interference with QS can affect biofilm development and antimicrobial susceptibility. This study evaluates the potential of selected phytochemical products to inhibit QS. Three isothiocyanates (allylisothiocyanate - AITC, benzylisothiocyanate - BITC and 2-phenylethylisothiocyanate - PEITC) and six phenolic products (gallic acid - GA, ferulic acid - FA, caffeic acid - CA, phloridzin - PHL, (-) epicatechin - EPI and oleuropein glucoside - OG) were tested. A disc diffusion assay based on pigment inhibition in Chromobacterium violaceum CV12472 was performed. In addition, the mechanisms of QS inhibition (QSI) based on the modulation of N-acyl homoserine lactone (AHLs) activity and synthesis by the phytochemicals were investigated. The cytotoxicity of each product was tested on a cell line of mouse lung fibroblasts. AITC, BITC and PEITC demonstrated a capacity for QSI by modulation of AHL activity and synthesis, interfering the with QS systems of C. violaceum CviI/CviR homologs of LuxI/LuxR systems. The cytotoxic assays demonstrated low effects on the metabolic viability of the fibroblast cell line only for FA, PHL and EPI.

Abstract The increasing occurrence of bacterial resistance to antibiotics has now reached a critical level. Finding antibiotic coadjuvants capable to inhibit the bacterial resistance mechanisms would be a valuable mid-term solution, until new classes of antibiotics are discovered. Selected plant alkaloids were combined with 5 antibiotics against 10 Staphylococcus aureus strains, including strains expressing distinct efflux pumps and methicillin-resistant S. aureus strains. The efficacy of each combination was assessed using the microdilution checkerboard, time-kill, Etest, and disc diffusion methods. The cytotoxicity of the alkaloids was evaluated in a mouse fibroblast cell line. Potentiation was obtained in 6% of all 190 combinations, especially with the combination of: ciprofloxacin with reserpine (RES), pyrrolidine (PYR), and quinine (QUIN); tetracycline with RES; and erythromycin with PYR. The highest cytotoxicity values were found for QUIN (half maximal inhibitory concentration [IC50] = 25 +/- 2.2 mg/L) and theophylline (IC50 = 100 +/- 4.7 mg/L).

Abstract The thermodynamics of the process of self-assembly of cationic gemini surfactants, [C12H25(CH3)(2)N(CH2)(S)N(CH3)(2)C12H25]Br-2, (the spacer S being 2, 6 or 10, assigned as C12CSC12Br2) and the system of oppositely charged mixture of surfactants formed by C12CSC12Br2 and sodium cholate (NaCA) in aqueous solution has been investigated by isothermal titration calorimetry (ITC), conductivity and turbidity measurements. The critical micelle concentration values (cmc) for the gemini surfactants C12CSC12Br2 obtained from calorimetry and conductivity were found to be consistent with values reported in the literature. The enthalpies of micellization (Delta H-mic) of C12CSC12Br2 are all exothermic, presenting a strong negative minimum at S = 6, corresponding to the maximum in the cmc values. For the mixed system of oppositely charged surfactants (C12CSC12Br2(S = 2,6,10)/NaCA), we did obtain from ITC the critical parameters for different events that take place as the concentration of gemini surfactant increases, such as the formation of NaCA-rich mixed micelles (cmc(mix), Delta Hmic-mix), the formation of a precipitate (immiscible liquid crystalline (LC) phase) (C-P, Delta H-P) and its re-dissolution (C-R, Delta H-R), and finally the formation of positive charge-rich mixed micelles (C-M, Delta H-M). It should be stressed that the values of cmc(mix) (gemini) are much smaller than those for pure gemini and pure NaCA. These results also show that there is a stronger synergistic effect between the two surfactants in the NaCA-rich region. The turbidity measurements proved valuable to the determination of the region of immiscible LC phase. The ITC results are combined with those obtained by conductivity and turbidity leading to a thorough discussion of the effect of the gemini spacer length on the aggregation behavior of the mixed systems.

Abstract A different approach to the preparation of microspheric particles of molecularly imprinted xerogels (MIX) is presented here. The technique consisted of filling up the pores of spherical, mesoporous, bare silica particles with a pregelification mixture by applying pressure. Upon gelification and drying, thin layers of MIX were deposited on the mesopores. Spherical composites of S-naproxen (S-NAP) imprints were produced by following this simple strategy. The performance of the imprints was quite satisfactory in terms of recognition ability (ascertained by selectivity against ibuprofen, alpha = 4.9, and an imprinting factor of 13) whereas an outstanding improvement on dynamic features (expressed as column efficiency), as compared to the corresponding bulk format MIX (9 vs. 1.2 theoretical plates/cm), was reached.

Abstract Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-D-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections.

Abstract Straightforward crushing and sieving bulk polymeric R-aminoglutethimide-imprinted materials were prepared by classical free radical polymerization, whereas nano thin walled grafted imprinted materials were prepared using RAFT mediated control polymerization technique. A stoichiometric non-covalent approach based on a triply hydrogen bonding functional monomer-template 1:1 complex (K = 599 mol(-1) L-1) led to chiral selectors far outperforming previously used selectors for resolving this racemate. The recognitive materials produced here (enantioselectivity factors, alpha similar to 10) also have no match within the previously reported enantioselective imprinted polymers (alpha 1.2-4.5). We here demonstrate a potentially generic solution to produce good quality grafted MIPs for templates interacting by hydrogen bonding alone, relying on solvent polarity tuning, significantly extending the range of templates compatible with this format.

Abstract In this work we report for the first time differential capacitance curves measured at imidazolium dicationic ionic liquid/electrode interfaces as a new strategy to understand the interfacial behaviour of ionic liquids (IL) near a charged electrode. The ionic liquid studied (1,5-bis(3-dimethylimidazolium)pentane di-bis(trifluoromethylsulfonyl)imide [C-5(MIM)(2)][Tf2N](2)) contains a cation with two positively charged imidazolium rings substituted with a methyl group connected by a pentane linkage alkyl chain length providing a new cationic structure variation. The comparison with the analogue monocationic ionic liquids showed that the dicationic liquid has a wider electrochemical polarization window and a more positive potential of zero charge at a Hg electrode. Differential capacity curves measured at dicationic ionic liquid/Hg interface show similar shape compared with analogous monocationic ionic liquids and revealed to be very sensitive to the electrode material.

Abstract The efficacy, cellular uptake and specific transport of dietary antioxidants to target organs, tissues and cells remains the most important setback for their application in the treatment of oxidative-stress related disorders and in particular in neurodegenerative diseases, as brain targeting remains a still unsolved challenge. Nanotechnology based delivery systems can be a solution for the above mentioned problems, specifically in the case of targeting dietary antioxidants with neuroprotective activity. Nanotechnology-based delivery systems can protect antioxidants from degradation, improve their physicochemical drug-like properties and in turn their bioavailability. The impact of nanomedicine in the improvement of the performance of dietary antioxidants, as protective agents in oxidative-stress events, specifically through the use of drug delivery systems, is highlighted in this review as well as the type of nanomaterials regularly used for drug delivery purposes. From the data one can conclude that the research combining (dietary) antioxidants and nanotechnology, namely as a therapeutic solution for neurodegenerative diseases, is still in a very early stage. So, a huge research area remains to be explored that hopefully will yield new and effective neuroprotective therapeutic agents in a foreseeable future.