RESUMEN
The interaction of amorphous silica nanoparticles with phospholipid monolayers and bilayers has received a great deal of interest in recent years and is of importance for assessing potential cellular toxicity of such species, whether natural or synthesized for the purpose of nanomedical drug delivery and other applications. This present communication studies the rate of silica nanoparticle adsorption on to phospholipid monolayers in order to extract a heterogeneous rate constant from the data. This rate constant relates to the initial rate of growth of an adsorbed layer of nanoparticles as SiO2 on a unit area of the monolayer surface from unit concentration in dispersion. Experiments were carried out using the system of dioleoyl phosphatidylcholine (DOPC) monolayers deposited on Pt/Hg electrodes in a flow cell. Additional studies were carried out on the interaction of soluble silica with these layers. Results show that the rate constant is effectively constant with respect to silica nanoparticle size. This is interpreted as indicating that the interaction of hydrated SiO2 molecular species with phospholipid polar groups is the molecular initiating event (MIE) defined as the initial interaction of the silica particle surface with the phospholipid layer surface promoting the adsorption of silica nanoparticles on DOPC. The conclusion is consistent with the observed significant interaction of soluble SiO2 with the DOPC layer and the established properties of the silica-water interface.
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Nanopartículas , Dióxido de Silicio , Adsorción , Fosfolípidos , Propiedades de Superficie , AguaRESUMEN
Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments.
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Técnicas Analíticas Microfluídicas , Preparaciones Farmacéuticas , Animales , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Dispositivos Laboratorio en un Chip , MicrofluídicaRESUMEN
This study compares the performance and output of an electrochemical phospholipid membrane platform against respective in vitro cell-based toxicity testing methods using three toxicants of different biological action (chlorpromazine (CPZ), colchicine (COL) and methyl methanesulphonate (MMS)). Human cell lines from seven different tissues (lung, liver, kidney, placenta, intestine, immune system) were used to validate this physicochemical testing system. For the cell-based systems, the effective concentration at 50 % cell death (EC50) values are calculated. For the membrane sensor, a limit of detection (LoD) value was extracted as a quantitative parameter describing the minimum concentration of toxicant which significantly affects the structure of the phospholipid sensor membrane layer. LoD values were found to align well with the EC50 values when acute cell viability was used as an end-point and showed a similar toxicity ranking of the tested toxicants. Using the colony forming efficiency (CFE) or DNA damage as end-point, a different order of toxicity ranking was observed. The results of this study showed that the electrochemical membrane sensor generates a parameter relating to biomembrane damage, which is the predominant factor in decreasing cell viability when in vitro models are acutely exposed to toxicants. These results lead the way to using electrochemical membrane-based sensors for rapid relevant preliminary toxicity screens.
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Hígado , Pruebas de Toxicidad , Humanos , Línea Celular , Pruebas de Toxicidad/métodos , Clorpromazina , Sustancias Peligrosas , FosfolípidosRESUMEN
HYPOTHESIS: The properties of stable gold (Au) nanoparticle dispersions can be tuned to alter their activity towards biomembrane models. EXPERIMENTS: Au nanoparticle coating techniques together with rapid electrochemical screens of a phospholipid layer on fabricated mercury (Hg) on platinum (Pt) electrode have been used to moderate the phospholipid layer activity of Au nanoparticle dispersions. Screening results for Au nanoparticle dispersions were intercalibrated with phospholipid large unilamellar vesicle (LUV) interactions using a carboxyfluorescein (CF) leakage assay. All nanoparticle dispersions were characterised for size, by dynamic light scattering (DLS) and transmission electron microscopy (TEM). FINDINGS: Commercial and high quality home synthesised Au nanoparticle dispersions are phospholipid monolayer active whereas Ag nanoparticle dispersions are not. If Au nanoparticles are coated with a thin layer of Ag then the particle/lipid interaction is suppressed. The electrochemical assays of the lipid layer activity of Au nanoparticle dispersions align with LUV leakage assays of the same. Au nanoparticles of decreasing size and increasing dispersion concentration showed a stronger phospholipid monolayer/bilayer interaction. Treating Au nanoparticles with cell culture medium and incubation of Au nanoparticle dispersions in phosphate buffered saline (PBS) solutions removes their phospholipid layer interaction.
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Nanopartículas del Metal , Electrodos , Oro , Fosfolípidos , PlataRESUMEN
A high-throughput, automated screening platform has been developed for the assessment of biological membrane damage caused by nanomaterials. Membrane damage is detected using the technique of analyzing capacitance-current peak changes obtained through rapid cyclic voltammetry measurements of a phospholipid self-assembled monolayer formed on a mercury film deposited onto a microfabricated platinum electrode after the interaction of a biomembrane-active species. To significantly improve wider usability of the screening technique, a compact, high-throughput screening platform was designed, integrating the monolayer-supporting microfabricated electrode into a microfluidic flow cell, with bespoke pumps used for precise, automated control of fluid flow. Chlorpromazine, a tricyclic antidepressant, and a citrate-coated 50 nm diameter gold nanomaterial (AuNM) were screened to successfully demonstrate the platform's viability for high-throughput screening. Chlorpromazine and the AuNM showed interactions with a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) monolayer at concentrations in excess of 1 µmol dm-3. Biological validity of the electrochemically measured interaction of chlorpromazine with DOPC monolayers was confirmed through quantitative comparisons with HepG2 and A549 cytotoxicity assays. The platform also demonstrated desirable performance for high-throughput screening, with membrane interactions detected in <6 min per assay. Automation contributed to this significantly by reducing the required operating skill level when using the technique and minimizing fluid consumption.
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Membrana Celular/metabolismo , Electroquímica/instrumentación , Nanoestructuras , Línea Celular , Membrana Celular/efectos de los fármacos , Clorpromazina/farmacología , Evaluación Preclínica de Medicamentos , Electrodos , Diseño de Equipo , Oro/química , Oro/farmacología , Humanos , Dispositivos Laboratorio en un Chip , Fosfolípidos/metabolismoRESUMEN
Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action.