Luísa Barreiros

Rapid assessment of endpoint antioxidant capacity of red wines through microchemical methods using a kinetic matching approach

Antioxidant capacity of food samples is usually assessed by different analytical methods, however the results attained even for the same method are strongly dependent on the selected reaction time and also on the standard compound used. To tackle this problem, we propose here a kinetic matching approach, associated to the conversion of results into equivalents of a common standard compound, as a universal way for expression of results. The methodology proposed was applied to methods based on different chemistries (Folin-Ciocalteu (F-C), CUPRAC, DPPH(•) and ABTS(•+) assays) and red wines (n=40) were chosen as a model of complex food sample. For implementation of the kinetic matching approach, the standard phenolic mixture (caffeic acid, (+)-catechin, hesperetin, morin and (-)-epigallocatechin gallate) was chosen for calibration in F-C, CUPRAC and DPPH(•) assays, while tannic acid was suitable for ABTS(•+) assay. Results showed that, for all methods, there was no statistical difference between results attained by the kinetic matching approach (after <10 min of reaction) and that at endpoint conditions (after 60 to 300 min). The repeatability and the reproducibility of the kinetic matching approach was <4.5%, for all antioxidant assays. The sample throughput increases from <18 (endpoint measurements) to >108 h(-1) using the proposed kinetic approach. Moreover, we have established here a way of converting results to equivalents of a common standard, providing values independent of its kinetic profile, by using the ratio between calibration sensitivities performed at endpoint conditions.

New insights into a bacterial metabolic and detoxifying association responsible for the mineralization of the thiocarbamate herbicide molinate.

A novel pathway of molinate mineralization promoted by a defined mixed culture composed of five bacteria (named ON1 to ON5) was proposed previously. Evidence was obtained of a metabolic association between Gulosibacter molinativorax ON4(T), capable of molinate breakdown, and the remaining bacteria. In the present study, the role of each isolate in that metabolic association was further explored and the possible synergistic effect of all the bacterial isolates for the stability of the mixed culture is discussed. The cleavage of the molinate thioester bond, whether occurring under aerobic or anaerobic conditions, releases ethanethiol (S-ethyl moiety) and an azepane moiety derivative, identified as azepane-1-carboxylic acid. This azepane moiety is degraded, in the presence of oxygen, by Pseudomonas strains ON1 and ON3 and G. molinativorax ON4(T). Ethanethiol, which inhibits G. molinativorax ON4(T), is consumed by Pseudomonas strain ON1 and Stenotrophomonas maltophilia ON2. Although a two-member mixed culture of G. molinativorax ON4(T) and Pseudomonas strain ON1 was able to promote the aerobic mineralization of molinate, after 20 successive transfers of the five-member mixed culture in mineral medium with molinate, none of these isolates were lost. The results obtained indicate that the whole mixed culture may have a higher fitness than the two-member culture, even when the basic degradative and cross-protection functions are assured.

Nanoparticles-in-film for the combined vaginal delivery of anti-HIV microbicide drugs.

Combining two or more antiretroviral drugs in one medical product is an interesting but challenging strategy for developing topical anti-HIV microbicides. We developed a new vaginal delivery system comprising the incorporation of nanoparticles (NPs) into a polymeric film base - NPs-in-film - and tested its ability to deliver tenofovir (TFV) and efavirenz (EFV). EFV-loaded poly(lactic-co-glycolic acid) NPs were incorporated alongside free TFV into fast dissolving films during film manufacturing. The delivery system was characterized for physicochemical properties, as well as genital distribution, local and systemic 24h pharmacokinetics (PK), and safety upon intravaginal administration to mice. NPs-in-film presented suitable technological, mechanical and cytotoxicity features for vaginal use. Retention of NPs in vivo was enhanced both in vaginal lavages and tissue when associated to film. PK data evidenced that vaginal drug levels rapidly decreased after administration but NPs-in-film were still able to enhance drug concentrations of EFV. Obtained values for area-under-the-curve for EFV were around one log10 higher than those for the free drugs in aqueous vehicle (phosphate buffered saline). Film alone also contributed to higher and more prolonged local drug levels as compared to the administration of TFV and EFV in aqueous vehicle. Systemic exposure to both drugs was low. NPs-in-film was found to be safe upon once daily vaginal administration to mice, with no significant genital histological changes or major alterations in cytokine/chemokine profiles being observed. Overall, the proposed NPs-in-film system seems to be an interesting delivery platform for developing combination vaginal anti-HIV microbicides.

Bacterial diversity and bioaugmentation in floodwater of a paddy field in the presence of the herbicide molinate

This work aimed at studying variations on the diversity and composition of the bacterial community of a rice paddy field floodwater, subjected to conventional management, namely by using the herbicide molinate. The promotion of the herbicide biodegradation either by the autochthonous microbiota or by a bioaugmentation process was also assessed. This study comprehended four sampling campaigns at key dates of the farming procedures (seeding, immediately and 6 days after application of the herbicide molinate, and after synthetic fertilization) and the subsequent physic-chemical and microbiological characterization (pH, DOC and molinate contents, total cells, cultivable bacteria and DGGE profiling) of the samples. Multivariate analysis of the DGGE profiles showed temporal variations in the bacterial community structure and the Shannon’s index values indicated that the bacterial diversity reached its minimum at the molinate application day. The highest bacterial diversity coincided with the periods with undetectable concentrations of the herbicide, although microcosm assays suggested that other factors than molinate may have been responsible for the decrease of the bacterial diversity. The ability of autochthonous microorganisms to degrade molinate and the influence of the herbicide on the bacterial community composition were assessed in microcosm assays using floodwater collected at the same dates. Given molinate was not degraded by autochthonous microorganisms, and considering it represents an environmental contaminant, bioaugmentation microcosms were assayed aiming the assessment of the feasibility of a bioremediation process to clean contaminated floodwater. A molinate-mineralizing culture, previously isolated, promoted molinate removal, induced alterations in the autochthonous bacterial community structure and diversity, and was undetected after 7 days of incubation, suggesting the feasibility of the process.

Molinate quantification in environmental water by a glutathione-S-transferase based biosensor

A glutathione-S-transferase (GST) based biosensor was developed to quantify the thiocarbamate herbicide molinate in environmental water. The biosensor construction was based on GST immobilization onto a glassy carbon electrode via aminosilane-glutaraldehyde covalent attachment. The principle supporting the use of this biosensor consists of the GST inhibition process promoted by molinate. Differential pulse voltammetry was used to obtain a calibration curve for molinate concentration, ranging from 0.19 to 7.9 mg L(-1) and presenting a detection limit of 0.064 mg L(-1). The developed biosensor is stable, and reusable during 15 days. The GST-based biosensor was successfully applied to quantify molinate in rice paddy field floodwater samples. The results achieved with the developed biosensor were in accordance with those obtained by high performance liquid chromatography. The proposed device is suitable for screening environmental water analysis and, since no sample preparation is required, it can be used in situ and in real-time measurements.

Methotrexate loaded lipid nanoparticles for topical management of skin-related diseases: Design, characterization and skin permeation potential

Methotrexate exhibits poor cutaneous bioavailability when administered topically using conventional vehicles. It is recommended for the treatment of skin-related diseases but its systemic side effects hamper application. The aim of this work was to formulate methotrexate in nanostructured lipid carriers using biocompatible lipids, and to investigate their potential for topical drug delivery. Methotrexate-loaded nanostructured lipid carriers were prepared via a hot ultrasonication method. Nanocarriers were evaluated for size, polydispersity, surface potential and entrapment efficiency. Shape and surface morphology of the produced lipid carriers confirmed their spherical shape. After 10h ca. 50% of methotrexate was released in vitro from the lipid nanocarriers following Peppas-Korsmeyer release kinetics. Moreover, methotrexate-loaded nanocarriers were stable at least for 3 months and less toxic towards fibroblasts and human keratinocytes than the free drug. Methotrexate within lipid nanocarriers was able to pass the experimental keratinocyte epidermal barrier at a flux rate of 2μgcm(-2)h(-1). Results demonstrated that lipid nanocarriers constitute a suitable approach for application of methotrexate in skin-related diseases topical therapy.

Development of PLGA nanoparticles loaded with clofazimine for oral delivery: Assessment of formulation variables and intestinal permeability

ABSTRACT The use of polymeric nanoparticles as delivery systems is a promising tool to overcome drawbacks related to low aqueous solubility of drugs, which limit their in vivo bioavailability. The aim of this study was to decrease clofazimine (CLZ) toxicity using experimental design to formulate CLZ loaded in PLGA nanoparticles (NPs‐CLZ) through a Plackett–Burman design (PBD). A screening PBD was constructed with twelve formulations involving six variables among process and formulation parameters and the selected responses were particle size, polydispersity index (PDI), association efficiency (AE) and drug loading (DL). The formulation was achieved based on the desirability tool, and the obtained NPs‐CLZ formulation was characterized regarding morphology, physicochemical properties, in vitro release and cellular studies. Particle size, PDI, AE and DL were found to be 211 ± 3 nm, 0.211 ± 0.009, 70 ± 5% and 12 ± 1%, respectively. Physicochemical studies confirmed the absence of chemical interactions between CLZ and other nanoparticles constituents and the amorphous state of CLZ, while morphological analysis revealed the spherical shape of the particles. In vitro release profile of CLZ from NPs‐PLGA showed a slow pattern of drug release. Cell viability studies towards intestinal cells revealed that NPs‐CLZ did not show CLZ toxicity on Caco‐2 and HT29‐MTX cells compared to free CLZ solutions. Moreover, CLZ could permeate Caco‐2 monolayers substantially at the end of 8 h. It can be concluded that the proposed NPs‐CLZ represent a promising platform to the oral delivery of CLZ as they were able to decrease its intrinsic toxicity, with improved absorption. Graphical abstract Figure. No Caption available.

Development and validation of a liquid chromatography-MS/MS method for simultaneous quantification of tenofovir and efavirenz in biological tissues and fluids

&NA; Millions of people worldwide live with human immunodeficiency virus (HIV) infection thus justifying the continuous search for new prevention and treatment strategies, including topical microbicide products combining antiretroviral drugs (ARVs) such as tenofovir (TFV) and efavirenz (EFV). Therefore, the aim of this work was to develop and validate a high performance liquid chromatography method coupled to triple quadrupole‐tandem mass spectrometry (HPLC–MS/MS) for the quantification of TFV and EFV in biological matrices (mouse vaginal tissue, vaginal lavage and blood plasma). Chromatographic separation was achieved using a reversed phase C18 column (3 &mgr;m, 100 × 2.1 mm) at 45 °C and elution in gradient mode using a combination of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at 0.35 mL min−1. Total run time was 9 min, with retention time of 2.8 and 4.1 min for TFV and EFV, respectively. The MS was operated in positive ionization mode (ESI+) for TFV and in negative ionization mode (ESI‐) for EFV detection. Data were acquired in selected reaction monitoring (SRM) mode and deuterated ARVs were employed as internal standards. Calibration curves were linear for ARV concentrations ranging from 4 to 500 ng mL−1 with LOD and LOQ for both analytes ≤0.4 and ≤0.7 ng mL−1 in sample extracts, respectively. The method was found to be specific, accurate (96.0–106.0% of nominal values) and precise (RSD < 2.4%) in all matrices. Both TFV and EFV were found to be stable in all matrices after standing 24 h at room temperature (20 °C) or in the autosampler, and after three freeze‐thawing cycles. Mean recovery values of ARVs spiked in mice tissues or fluids were ≥88.4%. Matrix effects were observed for EFV determination in tissue and plasma extracts but compensated by the use of deuterated internal standards. The proposed methodology was successfully applied to a pharmacokinetic study following intravaginal administration of both ARVs. Graphical abstract Figure. No caption available. HighlightsQuantification of tenofovir (TFV) and efavirenz (EFV) was attained for 4–500 ng mL−1.Validated LC–MS/MS method for vaginal tissue, lavage and blood plasma was developed.Matrix effects were observed for EFV in tissue (+17%) and plasma (−50%) extracts.LOD and LOQ values were ≤0.4 and ≤0.7 ng mL−1 in sample extracts.PK profile after intravaginal administration showed low systemic exposure.

Determination of salivary cotinine through solid phase extraction using a bead-injection lab-on-valve approach hyphenated to hydrophilic interaction liquid chromatography.

Cotinine, the first metabolite of nicotine, is often used as a biomarker in the monitoring of environmental tobacco smoke (ETS) exposure due to its long half-life. This paper reports on the development of an at-line automatic micro-solid phase extraction (μSPE) method for the determination of salivary cotinine followed by its analysis via hydrophilic interaction liquid chromatography (HILIC). The SPE methodology is based on the bead injection (BI) concept in a mesofluidic lab-on-valve (LOV) flow system to automatically perform all SPE steps. Three commercially available reversed-phase sorbents were tested, namely, Oasis HLB, Lichrolut EN and Focus, and the spherically shaped sorbents (i.e., Oasis HLB and Focus) provided better packing within the SPE column and hence higher column efficiency. An HILIC column was chosen based on its potential for achieving higher sensitivity and better retention of polar compounds such as cotinine. The method uses an isocratic program with acetonitrile:100mM ammonium acetate buffer, pH 5.8 in 95:5 v/v ratio as the mobile phase at a flow rate of 1.0 mL min(-1). Using this approach, the linear calibration range was from 10 to 1000 ng which corresponded to 5-500 μg L(-1). The corresponding μSPE-BI-LOV system was proven to be reliable in the handing and analysis of viscous biological samples such as saliva, achieving a sampling rate of 6h(-1) and a limit of detection and quantification of 1.5 and 3μgL(-1), respectively.

Topical co-delivery of methotrexate and etanercept using lipid nanoparticles: A targeted approach for psoriasis management

Methotrexate is indicated in psoriasis systemic therapy and its topical administration may be an option to overcome several side effects. A targeted delivery may be achieved through etanercept. Thus, a combination targeted therapy using methotrexate and etanercept could bring new perspectives for psoriasis patients. This work intended to develop and characterize co-delivery of methotrexate and etanercept using lipid nanoparticles, mediated by a carbopol hydrogel and to evaluate their potential for delivering the drug into the skin with reduced transdermal permeation. The nanoparticles were physico-chemically characterized. In vitro methotrexate release from solid lipid nanoparticles revealed a sustained release for 8h. The solid lipid nanoparticles were non-toxic towards human keratinocytes and fibroblasts. Permeation studies using pig ear as model revealed enhanced skin deposition of the applied methotrexate when incorporated within solid lipid nanoparticles in relation to free drug. Therapeutic amounts of methotrexate were delivered to psoriatic human skin after application of solid lipid nanoparticles, with reduced transdermal permeation.