Safety assessment of different unloaded polymeric nanocapsules in Caenorhabditis elegans.

Nano-sized drug delivery systems have been the subject of intense research in recent years because polymeric materials allow the absorption and release of active substances in a controlled manner. Despite the benefits, the safety of nanoparticulate systems is an aspect to be understood, particularly in vivo systems. Caenorhabditis elegans is a very useful alternative model for nanotoxicology and has been recently applied in this field. The aim of this study was to evaluate toxicological endpoints in C. elegans exposed to nanocapsules (NC) prepared with different coatings: polysorbate 80 (NCP80); polyethylene glycol (NCPEG), Eudragit® RS 100 (NCEUD) and chitosan (NCCS). Nanocapsules were prepared by nanoprecipitation method and showed acceptable physico-chemical characterization. Polyethylene glycol nanocapsules and chitosan nanocapsules increased worms lethality in a dose-dependent manner in acute exposure; polysorbate 80 nanocapsules, polyethylene glycol nanocpsules and chitonan nanocapsules also increased lethality following chronic exposure. Chitosan nanocapsules were the most toxic in all exposures, demonstrating toxicity even at low concentrations. Reproduction and body length were not affected by any of the nanocapsules exposures. The expression of superoxide dismutase showed that polysorbate 80 nanocapsules at the highest concentration slightly increased SOD-3::GFP expression. On the other hand, chitosan nanocapsules exposure blunted SOD-3 expression. This work demonstrates the toxicological differences between nanocapsule produced with different coatings and indicates higher safety for the use of eugragit nanocapsule in new formulations for future drug delivery and targeting systems.

Effect of tween 40 and ethanol on the secretion, structure and antioxidant activities of exopolysaccharides from Inonotus rickii.

In this study, the effects of Tween 40 and ethanol supplementation on the secretion, structure and antioxidant activities of exopolysaccharide (EPS) from Inonotus rickii were investigated. It was observed that Tween 40 and ethanol displayed a stimulatory effect on EPS secretion. The EPSs obtained by the addition of Tween 40 (EPS-T), ethanol (EPS-E) and control (EPS-C) were purified by Sepharose CL-6B gel chromatography and molecular weights of EPS-T, EPS-E and EPS-C were estimated to be 22.1, 30.0, and 40.5 kDa, respectively. Monosaccharide composition analysis indicated that EPS-T, EPS-E and EPS-C were mainly composed of mannose and glucose. Furthermore, EPS-E exhibited better OH• and DPPH scavenging activities than those of EPS-C and EPS-T, which might be associated with its molecular characterization.

Production, characterization and toxicology assay of creatine pegylated nanoliposome with polysorbate 80 for brain delivery.

Creatine acts intracellularly as energy buffer and storage, demonstrating protective effects in animal models of neurodegenerative diseases. However, its permeability throught blood-brain barrier (BBB) is reduced. The aim of the present study was developing a carrier to facilitate the delivery of creatine to the central nervous system. Creatine nanoliposomes were produced, characterized and assayed in models of toxicity in vitro and in vivo. Particles showed negative zeta potential (-12,5 mV), polydispersity index 0.237 and medium-size of 105 nm, which was confirmed by transmission electron microscopy (TEM) images. Toxicity assay in vitro was evaluated with blank liposomes (no drug) or creatine nanoliposomes at concentrations of 0.02 and 0.2 mg/mL, that did not influence the viability of Vero cells. The result. of the comet assay that the nanoliposomes are not genotoxic, togeher with cell viability demonstrated that the nanoliposomes are not toxic. Besides, in vivo assays not demonstrate toxicity in hematological and biochemical markers of young rats. Nevertheless, increase content of creatine in the cerebral cortex tissue after subchronic treatment was observed. Altogether, results indicate increase permeability of creatine to the BBB that could be used as assay for in vivo studies to confirm improved effect than free creatine.

Co-nanoencapsulation of antimalarial drugs increases their in vitro efficacy against Plasmodium falciparum and decreases their toxicity to Caenorhabditis elegans.

Drugs used for the treatment and prevention of malaria have resistance-related problems, making them ineffective for monotherapy. If properly associated, many of these antimalarial drugs may find their way back to the treatment regimen. Among the therapeutic arsenal, quinine (QN) is a second-line treatment for uncomplicated malaria but has side effects that limit its use. Curcumin (CR) is a natural compound with anti-plasmodial activities and low bioavailability. In this context, the aim of this work was to develop and characterize co-encapsulated QN + CR-loaded polysorbate-coated polymeric nanocapsules (NC-QC) to evaluate their activity on Plasmodium falciparum and the safety of the nanoformulations for Caenorhabditis elegans. NC-QC displayed a diameter of approximately 200 nm, a negative zeta potential and a slightly basic pH. The drugs are homogeneously distributed in the NCs in the amorphous form. Co-encapsulated NCs exhibited a significant reduction in P. falciparum parasitemia, better than QN/CR. The worms exposed to NC-QC showed higher survival and longevity and no decrease in their reproductive capacity compared to free and associated drugs. It was possible to prove that the NCs were absorbed orally by the worms using fluorescence microscopy. Co-encapsulation of QN and CR was effective against P. falciparum, minimizing the toxic effects caused by chronic exposure of the free drugs in C. elegans.

In vitro genotoxicity and cytotoxicity in murine fibroblasts exposed to EDTA, NaOCl, MTAD and citric acid

The aim of the present study was to evaluate the capacity of some root canal irrigants to induce genetic damage and/or cellular death in vitro. Murine fibroblast cells were exposed to ethylenediaminetetraacetic acid (EDTA), sodium hypochlorite (NaOCl), MTAD™ and citric acid in increasing concentrations for 3 h at 37ºC. The negative control group was treated with vehicle control (phosphate buffer solution - PBS) for 3 h at 37°C, and the positive control group was treated with methylmetanesulfonate, 1 μM. for 3 h at 37°C. Cytotoxicity was assessed by the trypan blue test and genotoxicity was evaluated by the single cell gel (comet) assay. The results showed that exposure to 2.5% and 5% NaOCl and 8.5% citric acid resulted in a significant cytotoxic effect. NaOCl, EDTA and citric acid did not produce genotoxic effects with respect to the comet assay data for all evaluated concentrations. Although MTAD was not a cytotoxic agent, it showed significant genotoxic effects at all tested concentrations (ANOVA and Tukey's test; p<0.05). NaOCl, EDTA and citric acid were found to be cytotoxic in a dose-dependent manner, but they were not genotoxic. MTAD did not cause cell death, but presented genotoxic effects.

O objetivo do presente estudo foi avaliar a capacidade de alguns irrigantes endodônticos em induzir danos genéticos e/ou morte celular in vitro. Células de fibroblastos murinos foram expostas ao ácido etilenodiaminotetracético (EDTA), hipoclorito de sódio (NaOCl), MTAD™ e ácido cítrico em concentrações crescentes durante 3 h a 37°C. O grupo controle negativo foi tratado com solução tampão fosfato - PBS por 3 h a 37° C e o grupo controle positivo foi tratado com metilmetanesulfonato a 1 μM por 3 h a 37° C. A citotoxicidade foi testada pelo azul de tripan e a genotoxicidade foi avaliada pelo teste do cometa. Os resultados apontaram que a exposição ao NaOCl a 2,5% e 5%, e ácido cítrico a 21% resultou em efeitos citotóxicos significativos. O NaOCl, EDTA e o ácido cítrico não produziram efeitos genotóxicos no que diz respeito aos dados obtidos pelo ensaio do Cometa em todas as concentrações testadas. Embora o MTAD não tenha sido um agente citotóxico, mostrou efeitos genotóxicos significativos em todas as concentrações testadas (ANOVA e teste de Tuckey; p<0,05). O NaOCl, o EDTA e o ácido cítrico mostraram-se citotóxicos de maneira dose-dependente, mas não genotóxicos. Por outro lado, apesar do MTAD não ter causado a morte celular, foi genotóxico em todas as concentrações testadas.

Haloperidol-loaded polysorbate-coated polymeric nanocapsules decrease its adverse motor side effects and oxidative stress markers in rats.

Haloperidol is the most widely used antipsychotic drug in the treatment of psychiatric disorders. Despite its satisfactory therapeutic effect, its chronic use is related to severe motor side effects. Here, we investigate the incidence of motor side effects of haloperidol-loaded nanocapsules when compared to free haloperidol and the relation with oxidative stress (OS) development. Both vehicle (B-NcFO) and haloperidol loaded polysorbate-coated nanocapsules suspension (H-NcFO) prepared with fish oil as core showed uniform and rounded particles, nanometric size, negative zeta potential, low polydispersity indices and high encapsulation efficiency. Wistar rats received a single dose of free haloperidol (FH), B-NcFO or H-NcFO (0.2 mg/kg ip) and were submitted to acute motor side effects evaluation 1 h after the injection. Lower catalepsy time and oral dyskinesia were observed in H-NcFO-treated group than in FH group; however, both formulations decreased animals' locomotor activity. In a experiment performed subchronically, rats injected daily with H-NcFO (0.2 mg/kg-ip) for 28 days showed decreased oral dyskinesia frequency and catalepsy time and no impairment on locomotor activity as compared to FH group (0.2 mg/kg-ip). FH group showed higher OS, as observed by increased lipid peroxidation and reduced glutathione levels and catalase activity in extrapyramidal region. Our findings showed that nanocapsules may be an efficient form to prevent or minimize haloperidol motor side effects, which are related to OS development, ameliorating psychiatric patients' quality of life.

Hemocompatibility of poly(ɛ-caprolactone) lipid-core nanocapsules stabilized with polysorbate 80-lecithin and uncoated or coated with chitosan.

The hemocompatibility of nanoparticles is of critical importance for their systemic administration as drug delivery systems. Formulations of lipid-core nanocapsules, stabilized with polysorbate 80-lecithin and uncoated or coated with chitosan (LNC and LNC-CS), were prepared and characterized by laser diffraction (D[4,3]: 129 and 134 nm), dynamic light scattering (119 nm and 133 nm), nanoparticle tracking (D50: 124 and 139 nm) and particle mobility (zeta potential: -15.1 mV and +9.3 mV) analysis. In vitro hemocompatibility studies were carried out with mixtures of nanocapsule suspensions in human blood at 2% and 10% (v/v). The prothrombin time showed no significant change independently of the nanocapsule surface potential or its concentration in plasma. Regarding the activated partial thromboplastin time, both suspensions at 2% (v/v) in plasma did not influence the clotting time. Even though suspensions at 10% (v/v) in plasma decreased the clotting times (p<0.05), the values were within the normal range. The ability of plasma to activate the coagulation system was maintained after the addition of the formulations. Suspensions at 2% (v/v) in blood showed no significant hemolysis or platelet aggregation. In conclusion, the lipid-core nanocapsules uncoated or coated with chitosan are hemocompatible representing a potential innovative nanotechnological formulation for intravenous administration.

In vitro genotoxicity and cytotoxicity in murine fibroblasts exposed to EDTA, NaOCl, MTAD and citric acid.

The aim of the present study was to evaluate the capacity of some root canal irrigants to induce genetic damage and/or cellular death in vitro. Murine fibroblast cells were exposed to ethylenediaminetetraacetic acid (EDTA), sodium hypochlorite (NaOCl), MTAD™ and citric acid in increasing concentrations for 3 h at 37ºC. The negative control group was treated with vehicle control (phosphate buffer solution - PBS) for 3 h at 37°C, and the positive control group was treated with methylmetanesulfonate, 1 µM. for 3 h at 37°C. Cytotoxicity was assessed by the trypan blue test and genotoxicity was evaluated by the single cell gel (comet) assay. The results showed that exposure to 2.5% and 5% NaOCl and 8.5% citric acid resulted in a significant cytotoxic effect. NaOCl, EDTA and citric acid did not produce genotoxic effects with respect to the comet assay data for all evaluated concentrations. Although MTAD was not a cytotoxic agent, it showed significant genotoxic effects at all tested concentrations (ANOVA and Tukey's test; p<0.05). NaOCl, EDTA and citric acid were found to be cytotoxic in a dose-dependent manner, but they were not genotoxic. MTAD did not cause cell death, but presented genotoxic effects.

Cytotoxicity of solubilization vehicles for Trichomonas gallinae and Tritrichomonas foetus measured by the resazurin microtiter assay.

The parasites Tritrichomonas foetus and Trichomonas gallinae present veterinary and economic importance since they cause bovine and avian trichomonosis, respectively. The absence of a specific treatment and the necessity of effective and safe drugs against these etiologic agents have stimulated the search for new antiprotozoal drugs with high activity, low toxicity to the animal, and low cost. Screening of potential antiprotozoal molecules is currently a common practice and different kinds of organic solvents and surfactant vehicles are used, since many bioactive compounds have low water solubility. Thus, it is important to determine the toxicity and to provide the minimal inhibitory concentration (MIC) values of the most common solubilization vehicles used in biological activity in vitro evaluation: ethanol, methanol, isopropanol, acetone, DMSO, tween 20 and tween 80. The assays were conducted employing the resazurin microtiter assay, which demonstrated a rapid, safe, and quantitative method for the in vitro determination of T. foetus and T. gallinae trophozoites viability. In summary, all solvents and surfactants, except ethanol, could be used in cytotoxicity assays against T. foetus, and acetone, tween 20 and tween 80 are the preferential vehicles for in vitro analysis of potential bioactive compounds against T. gallinae, though these must be used with caution.