Measuring Mitochondrial Membrane Potential with a Tetraphenylphosphonium-Selective Electrode.

Mitochondrial bioenergetics is based on the generation of the protonmotive force by the electron transport chain. The protonmotive force is used by mitochondria for different critical aspects of its normal function, ranging from calcium accumulation to the synthesis of ATP. The transmembrane electric potential (ΔΨ) is the major component of the protonmotive force and is also the main responsible for ATP synthesis by mitochondrial ATP synthase. Although several methods can be used to measure the ΔΨ, the use of the tetraphenylphosphonium cation (TPP(+))-selective electrode is still a method of election due to its sensitivity. The method is based on the accumulation of TPP(+) by energized mitochondria, which develop a negative charge in the matrix due to the ejection of protons. This unit describes how to build a custom-made TPP(+)-selective electrode and how to establish the necessary set-up to follow ΔΨ fluctuations in isolated mitochondrial fractions.

Surface engineering of silica nanoparticles for oral insulin delivery: characterization and cell toxicity studies.

The present work aimed at studying the interaction between insulin and SiNP surfaced with mucoadhesive polymers (chitosan, sodium alginate or polyethylene glycol) and the evaluation of their biocompatibility with HepG2 and Caco-2 cell lines, which mimic in vivo the target of insulin-loaded nanoparticles upon oral administration. Thus, a systematic physicochemical study of the surface-modified insulin-silica nanoparticles (Ins-SiNP) using mucoadhesive polymers has been described. The surfacing of nanoparticle involved the coating of silica nanoparticles (SiNP) with different mucoadhesive polymers, to achieve high contact between the systems and the gut mucosa to enhance the oral insulin bioavailability. SiNP were prepared by a modified Stöber method at room temperature via hydrolysis and condensation of tetraethyl orthosilicate (TEOS). Interaction between insulin and nanoparticles was assessed by differential scanning calorimetry (DSC), X-ray and Fourier-transform infrared (FTIR) studies. The high efficiency of nanoparticles' coating resulted in more stable system. FTIR spectra of insulin-loaded nanoparticles showed amide absorption bands which are characteristic of α-helix content. In general, all developed nanoparticles demonstrated high biocompatible, at the tested concentrations (50-500 µg/mL), revealing no or low toxicity in the two human cancer cell lines (HepG2 and Caco-2). In conclusion, the developed insulin-loaded SiNP surfaced with mucoadhesive polymers demonstrated its added value for oral administration of proteins.

Cationic solid lipid nanoparticles interfere with the activity of antioxidant enzymes in hepatocellular carcinoma cells.

Solid lipid nanoparticles (SLN) are colloidal drug and/or gene carriers developed from solid lipids and surfactants that are considered safe. Cationic SLN, usually used for formulating poorly water-soluble drugs and for gene delivery purposes, as positively charged particles may attach to cellular surfaces and be internalized more easily than negatively charged SLN, but they can also cause damage. The main aim of this work was to test a set of cationic SLN and investigate its influence on the amount of reactive oxygen species (ROS), on antioxidant enzymes activities and on possible oxidative damage to membrane lipids in HepG2 cells. The Dichlorofluorescein assay revealed great increase in ROS presence after cell exposure to SLN. While the exposure to SLN increased the activities of superoxide dismutase and glutathione peroxidase it decreased glutathione reductase activity. Although no significant increase in thiobarbituric reactive species was found, a decrease in sulfhydryl groups was detected. These results indicate that cationic SLN caused oxidative stress in HepG2 cells, but under reported exposure conditions HepG2 cells could attenuate the stress and thus the damage to cellular components was minimal.

Endopolysaccharides from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor affect differently the proliferation rate of HepG2 cells.

Fungi have been used for medicinal purposes for long time by Asian countries, being a putative source of powerful new phytopharmaceuticals such as polysaccharides. The aim of this study was to extract endopolysaccharides (IPS) from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor, grown under submerged culture, to compare crude IPS production, total carbohydrate, and protein yield, and to study the effect of these IPS on HepG2 cells proliferation rate. Total biomass produced by G. resinaceum, P. rufa, and T. versicolor was (in gram per liter) 3.32 ± 0.80, 5.42 ± 0.58, and 4.2 ± 1.29 and the IPS yield (as the biomass percent) was 9.9 ± 0.05, 29.0 ± 6.3, and 9.1 ± 3.1 %, respectively. Characterization of IPS has shown different proportion between total sugar and protein being, on average 6.04, 10.74, and 22.62, for G. resinaceum, T. versicolor, and P. rufa, respectively. The IPS effect, at 50, 100, and 200 µg mL(-1) on HepG2 cell growth and viability was negligible for G. resinaceum and P. rufa but, in the case of T. versicolor, 200 µg mL(-1) of IPS evoked 40 % reduction on cell growth. The results suggest that the intracellular polysaccharides from T. versicolor are a potential source for bioactive molecules with anti-proliferative properties.

Mitochondrial function is not affected by renal morphological changes in diabetic goto-kakizaki rat.

Renal disease is a common complication of diabetes mellitus. The pathogenesis of diabetic nephropathy is not well understood, but hyperglycemia seems to be a crucial factor. Recent evidence indicates that the overproduction of reactive oxygen species, observed in both clinical and experimental diabetes, and mitochondrial dysfunction are key factors in pathogenic process. The objective of this investigation was to test the hypothesis of whether hyperglycemia could affect kidney morphology and mitochondrial bioenergetics as well as susceptibility to oxidative stress in 12-month-old diabetic Goto-Kakizaki (GK) rats, a model of type 2 diabetes mellitus. We observed that there were no significant differences in the kidney respiratory function and phosphorylation capacity between GK and age-matched control Wistar rats. Mitochondria from kidneys of diabetic rats were equally susceptible to in vitro oxidative damage as those from normal rats, while coenzyme Q and alpha -tocopherol concentrations were similar in both types of preparations. However, the kidney of GK rats presented in most glomerulus a capillary basement membrane thickening with mesangial widening, in evolution to segmental glomerular sclerosis, and, in some interlobular arteries, excessive deposition of PAS-positive material at the tunica intima. The results show that the mild prolonged hyperglycemia and the kidney structural changes observed in GK rats are not sufficient to cause renal dysfunction and were not associated with functional and biochemical alterations in mitochondria.

Vitamin E or coenzyme Q10 administration is not fully advantageous for heart mitochondrial function in diabetic goto kakizaki rats.

The heart is one of the organs affected during the later stages of diabetes. Mitochondrial function has already been proposed to be affected during the course of diabetes. Nevertheless, little information is known concerning the impact of antioxidants in heart mitochondria of a milder model for diabetes, such as the Goto-Kakizaki (GK) rat, where mitochondrial function appears ameliorated. The objective of this work was to test if injections of Vitamin E and Coenzyme Q10, alone and in combination, were able to modify mitochondrial performance in the hearts of GK rats. Several aspects of mitochondrial function were measured, such as the respiratory control ratio and the electric potential, as well as the mitochondrial accumulation of Vitamin E and Coenzymes Q9 and Q10. We observed that only Vitamin E appeared to have a positive impact on the mitochondrial phosphorylation efficiency and on mitochondrial performance, namely on the ability to generate the electric transmembrane potential in the presence of supra-physiological calcium concentrations. Vitamin E administration also increased the mitochondrial concentration of Coenzyme Q10. None of the treatments was able to reverse the diabetic phenotype in GK rats. We conclude that in this model of mild hyperglycemia, administration of antioxidants may have a marginal positive impact on mitochondrial function.