Mechanisms underlying cytotoxicity induced by engineered nanomaterials: a review of in vitro studies

Engineered nanomaterials are emerging functional materials with technologically interesting properties and a wide range of promising applications, such as drug delivery devices, medical imaging and diagnostics, and various other industrial products. However, concerns have been expressed about the risks of such materials and whether they can cause adverse effects. Studies of the potential hazards of nanomaterials have been widely performed using cell models and a range of in vitro approaches. In the present review, we provide a comprehensive and critical literature overview on current in vitro toxicity test methods that have been applied to determine the mechanisms underlying the cytotoxic effects induced by the nanostructures. The small size, surface charge, hydrophobicity and high adsorption capacity of nanomaterial allow for specific interactions within cell membrane and subcellular organelles, which in turn could lead to cytotoxicity through a range of different mechanisms. Finally, aggregating the given information on the relationships of nanomaterial cytotoxic responses with an understanding of its structure and physicochemical properties may promote the design of biologically safe nanostructures.

Comparative sensitivity of tumor and non-tumor cell lines as reliable approach for in vitro cytotoxicity screening of lysine-based surfactants with potential pharmaceutical applications

Surfactants are used as additives in topical pharmaceuticals and drug delivery systems. The biocompatibility of amino acid-based surfactants makes them highly suitable for use in these fields, but tests are needed to evaluate their potential toxicity. Here we addressed the sensitivity of tumor (HeLa, MCF-7) and non-tumor (3T3, 3T6, HaCaT, NCTC 2544) cell lines to the toxic effects of lysine-based surfactants by means of two in vitro endpoints (MTT and NRU). This comparative assay may serve as a reliable approach for predictive toxicity screening of chemicals prior to pharmaceutical applications. After 24-h of cell exposure to surfactants, differing toxic responses were observed. NCTC 2544 and 3T6 cell lines were the most sensitive, while both tumor cells and 3T3 fibroblasts were more resistant to the cytotoxic effects of surfactants. IC50-values revealed that cytotoxicity was detected earlier by MTT assay than by NRU assay, regardless of the compound or cell line. The overall results showed that surfactants with organic counterions were less cytotoxic than those with inorganic counterions. Our findings highlight the relevance of the correct choice and combination of cell lines and bioassays in toxicity studies for a safe and reliable screen of chemicals with potential interest in pharmaceutical industry.

Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress.

Isolated hepatocytes incubated with [35S]-methionine were examined for the time-dependent accumulation of [35S]-glutathione (GSH) in cytosol and mitochondria, the latter confirmed by density gradient purification. In GSH-depleted and -repleted hepatocytes, the increase of specific activity of mitochondrial GSH lagged behind cytosol, reaching nearly the same specific activity by 1-2 h. However, in hepatocytes from ethanol-fed rats, the rate of increase of total GSH specific radioactivity in mitochondria was markedly suppressed. In in vivo steady-state experiments, the mass transport of GSH from cytosol to mitochondria and vice versa was 18 nmol/min per g liver, indicating that the half-life of mitochondrial GSH was approximately 18 min in controls. The fractional transport rate of GSH from cytosol to mitochondria, but not mitochondria to cytosol, was significantly reduced in the livers of ethanol-fed rats. Thus, ethanol-fed rats exhibit a decreased mitochondrial GSH pool size due to an impaired entry of cytosol GSH into mitochondria. Hepatocytes from ethanol-fed rats exhibited a greater susceptibility to the oxidant stress-induced cell death from tert-butylhydroperoxide. Incubation with glutathione monoethyl ester normalized the mitochondrial GSH and protected against the increased susceptibility to t-butylhydroperoxide, which was directly related to the lowered mitochondrial GSH pool size in ethanol-fed cells.

Platinum (II) and palladium (II) complexes with (N,N') and (C,N,N') ligands derived from pyrazole as anticancer and antimalarial agents: synthesis, characterization and in vitro activities

The study of the reactivity of three 1-(2-dimethylaminoethyl)-1H-pyrazole derivatives of general formula [1-(CH2)2NMe2}-3,5-R2-pzol] {where pzol represents pyrazole and Rdouble bond; length as m-dashH (1a), Me (1b) or Ph (1c)} with [MCl2(DMSO)2] (Mdouble bond; length as m-dashPt or Pd) under different experimental conditions allowed us to isolate and characterize cis-[M{κ2-N,N′-{[1-(CH2)2NMe2}-3,5-R2-pzol])}Cl2] {MMdouble bond; length as m-dashPtPt (2a-2c) or Pd (3a-3c)} and two cyclometallated complexes [M{κ3-C,N,N′-{[1-(CH2)2NMe2}-3-(C5H4)-5-Ph-pzol])}Cl] {Mdouble bond; length as m-dashPt(II) (4c) or Pd(II) (5c)}. Compounds 4c and 5c arise from the orthometallation of the 3-phenyl ring of ligand 1c. Complex 2a has been further characterized by X-ray crystallography. Ligands and complexes were evaluated for their in vitro antimalarial against Plasmodium falciparum and cytotoxic activities against lung (A549) and breast (MDA MB231 and MCF7) cancer cellular lines. Complexes 2a-2c and 5c exhibited only moderate antimalarial activities against two P. falciparum strains (3D7 and W2). Interestingly, cytotoxicity assays revealed that the platinacycle 4c exhibits a higher toxicity than cisplatin in the three human cell lines and that the complex 2a presents a remarkable cytotoxicity and selectivity in lung (IC50 = 3 μM) versus breast cancer cell lines (IC50 > 20 μM). Thus, complexes 2c and 4c appear to be promising leads, creating a novel family of anticancer agents. Electrophoretic DNA migration studies in presence of the synthesized compounds have been performed, in order to get further insights into their mechanism of action.

Establishment of an In Vitro Photoallergy Test Using NCTC2544 Cells and IL-18 Production

Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of eratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals. First, we identified suitable condition of UV-irradiation (3.5 J/cm2) by investigating the effect of UVAirradiation on intracellular IL-18 on untreated or chloropromazine (a representative phototoxic compound)- treated NCTC2544 cells. Then, the effect of UVA-irradiation over NCTC2544 cells treated with increasing concentrations of 15 compounds including photoallergens (benzophenone, 4-ter-butyl-4-methoxydibenzoylmethane, 2-ethylexyl-p-methoxycinnamate, ketoprofen, 6-methylcumarin); photoirritant and photoallergen (4-aminobenzoic acid, chlorpromazine, promethazine); photoirritants (acridine, ibuprofen, 8-methoxypsoralen, retinoic acid); and negative compounds (lactic acid, SDS and p-phenilendiamine) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay or LDH leakage, while ELISA was used to measure the production of IL-18. At the maximal concentration assayed with non-cytotoxic effects (CV80 under irradiated condition), all tested photoallergens induced a significant and a dose-dependent increase of intracellular IL-18 following UVA irratiation, whereas photoirritants failed. We suggest that this system may be useful for the in vitro evaluation of the photoallergic potential of chemicals.

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, we developed nanovesicles containing bioactive cationic lysine-based amphiphiles, and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. We found different cytotoxic responses among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalized by HeLa cells, and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behavior after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute to reducing the uncertainty surrounding their potential health hazards.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

Determinación in vitro de la digestibilidad de los nutrientes de la cebada. Aplicación práctica para calibración de un sistema NIRS, y para la utilización de enzimas en la alimentación porcina.

La intensificación de la producción porcina, sin duda es uno de los principales factores que han influido en la alta competitividad del sector. Dicha intensificación sin embargo debe enfrentarse a nuevos retos de acuerdo con la creciente preocupación de los estados europeos sobre problemas de bienestar animal y de contaminación medioambiental resultante de la producción animal intensiva. La excreción de nitrógeno en los purines (que es uno de los principales factores contaminantes), puede ser reducida substancialmente si se reduce el contenido proteico del pienso. Ello, sin embargo debe de hacerse de manera que la dieta aporte las cantidades mínimas de aminoácidos requeridas por los animales, y que por tanto los parámetros productivos no se vean afectados. Para asegurar un aporte mínimo adecuado de aminoácidos en el pienso, es esencial un conocimiento preciso del valor nutritivo de las materias primas utilizadas. En la actualidad se utilizan los valores de contenido en aminoácidos digestibles ileales presentes en tablas publicadas por diferentes fuentes de información. Dichas tablas, sin embargo, son valores medios que no contemplan la elevada variabilidad en el contenido de aminoácidos digestibles dentro de una misma materia prima. Debido a esta variabilidad, para asegurar que los requerimientos de aminoácidos queden cubiertos al menos en un 80% de los casos, se debe sobreformular a un 107-108% de los requerimientos. Se ha estimado que una rápida determinación del contenido de aminoácidos digestibles ileales en las materias primas mediante la utilización de espectroscopia del infrarojo cercano (NIRS), permitiría el conocimiento del valor nutritivo de cada partida de materia prima, y una reducción de la sobreformulación a un 103%, con el consiguiente beneficio medioambiental. El principal inconveniente, para la aplicación de dicha metodología NIRS, es que su calibración requiere un esfuerzo excepcional, que solamente puede conseguirse con el trabajo de varios años de determinaciones in vivo de digestibilidad ileal de aminoácidos de un elevadísimo número de muestras. El presente proyecto tenía como principal objetivo la puesta a punto de una técnica de digestibilidad ileal de nutrientes in vitro (validada con determinaciones in vivo), que nos permitiera un análisis más rápido de un número elevado de muestras para realizar la calibración del NIRS. Para ello se escogió la cebada como materia prima para realizar los estudios por su importancia económica en España.

Accions no canoniques dels Inhibidors d'Acetilcolinesterasa (IAChE ): estudis in vivo i in vitro de les huprines i heterodímers huprina X i tacrina

Projecte de recerca elaborat a partir d’una estada al Karolinska Institutet, Suècia, entre febrer i juliol del 2007. Els anticolinesteràsics són els fàrmacs aprovats actualment pel tractament pal•liatiu d’Alzheimer. En l’intent de trobar fàrmacs capaços de modificar l’evolució de la malaltia s’han sintentitzat nous compostos que conserven l’activitat anticolinesteràsica però que a més, són capaços d’interaccionar amb la cascada de formació de la proteïna beta-amiloid i la proteïna tau, principals agents patogènics d’aquesta malaltia. L’objectiu fonamental del projecte era el d’analitzar el contingut de proteina beta-amiloid, de receptors nicotínics, de GPAF i sinaptofisina en mostres de cervell de ratolins triple transgènics tractats previament amb Huprina X i Huperzina A.

Introducció experimental a la biotecnologia amb clavellina: cultiu in vitro

Treball de recerca realitzat per una alumna d'ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit científic del Jovent l'any 2009. Es tracta d’una recerca experimental en la que s’han assajat vuit tècniques de cultiu in vitro amb clavellina. El material vegetal s’ha esterilitzat per immersió en una solució diluïda de lleixiu i s’ha manipulat de manera estèril. En tots els casos el medi de cultiu utilitzat ha estat el MS amb una concentració de sacarosa i reguladors de creixement variable segons l’experiment. La incubació dels cultius s’han dut a terme en una cambra amb control de fotoperíode durant 4 setmanes. Els diferents reguladors de creixement han mostrat un clar efecte sobre les seccions de tija. Els explants cultivats en medi lliure d’hormones han crescut menys que els exposats a diverses concentracions de NAA i BA. Aquests tractaments hormonals han originat símptomes de creixement anòmals (engruiximents a la base i vitrificació). La presencia de 2,4-D ha afavorit la formació de cal•lus i d’arrels per organogènesi adventícia indirecta. L’obtenció de plàntules per germinació in vitro de llavors ha permès reduir notablement les pèrdues per contaminació, mentre que el subcultiu d’aquestes ha donat unes tases de micropropagació de 7.2 seccions/plàntula. Ha estat possible aclimatar aquestes vitroplantes per tal d’adaptar-les a les condicions de camp. No hem pogut obtenir organogènesis adventícia ni embriogènesi somàtica a partir d anteres ni hem pogut iniciar un cultiu de cèl•lules a partir dels cal•lus. Tot i la complexitat d’aquestes tècniques, és possible dur-les a terme en un laboratori escolar.