Graciela Facorro

Synergistic encapsulation of the anti-HIV agent efavirenz within mixed poloxamine/poloxamer polymeric micelles

This study investigated the synergistic performance of mixed polymeric micelles made of linear and branched poly(ethylene oxide)-poly(propylene oxide) for the more effective encapsulation of the anti-HIV drug efavirenz. The co-micellization process of 10% binary systems combining different weight ratios of a highly hydrophilic poloxamer (Pluronic F127) and a more hydrophobic poloxamine counterpart (Tetronic T304 and T904) was investigated by means of dynamic light scattering, cloud point and electronic spin resonance experiments. Then, the synergistic solubilization capacity of the micelles was shown. Findings revealed a sharp solubility increase from 4 μg/ml up to more than 33 mg/ml, representing a 8430-fold increase. Moreover, the drug-loaded mixed micelles displayed increased physical stability over time in comparison with pure poloxamine ones. Overall findings confirmed the enormous versatility of the poloxamer/poloxamine systems as Trojan nanocarriers for drug encapsulation and release by the oral route and they entail a relevant enhancement of the previous art towards a more compliant pediatric HIV pharmacotherapy.

Long term production of reactive oxygen species during perinatal asphyxia in the rat central nervous system: Effects of hypothermia

The formation of oxygen-derived free radicals in hypoxic and ischemic/reperfused brains has been proposed as an important step that links brain injury to neuronal death. Previously, we have demonstrated that reactive oxygen species (ROS) production was significantly increased in rat neostriatum during acute perinatal asphyxia (PA) in pups. In this article, we have studied the time course of ROS production in the neostriatum and neocortex of adult rats subjected to PA using electron spin resonance spectrometry (ESR) in order to record ROS production. Further more, we analyzed the actions of hypothermia on ROS release in pups and adult rats. We used for this study 6-month-old rats that suffered sub-severe and severe PA when they were pups. The most significant production of ROS was detected either in the neostriatum or neocortex at 19 and 20 min of PA. Hypothermia during 20 and 100 min at 15°C prevented ROS formation either in pups and adult rats. These data further support the concept that free radicals may contribute to the brain injury alterations and that hypothermia can prevent long-term sequelae induced by PA.

Regulation of functional and regressing stages of corpus luteum development in mice. Role of reactive oxygen species

The endocrine and immune systems modulate ovarian function. The aim of the present work was to compare the status of various modulating factors in two well-defined stages of corpus luteum (CL) development (the functional stage and the regressing stage) by means of a gonadotropin-synchronised mouse model. At the regressing stage of CL development, we found that ovarian tissue showed increased prostaglandin (PG) F(2alpha) and diminished PGE levels concomitantly with enhanced protein abundance of ovarian cyclooxygenase 2, the inducible isoform of the limiting enzyme of PG synthesis. We also found both enhanced lipid peroxidation and enhanced total superoxide dismutase activity, as well as inhibited catalase activity and inhibited total hydroxyl radical scavenger capacity, when compared with ovaries at the functional stage. In addition, at the regressing stage we observed an increased percentage of CD8+ (cytotoxic/suppressor) T-cells and a decreased percentage of CD4+ (helper) T-cells from ovarian-draining lymph nodes. Also, the serum interleukin (IL)-2, IL-4 and IL-10 were diminished as compared with the functional stage. We conclude that a pro-oxidant status together with a pro-inflammatory response is responsible for the loss of luteal function.

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action.

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.

Effects of photooxidation on membrane integrity in Salix nigra seeds

BACKGROUND AND AIMS Salix nigra seeds are desiccation-tolerant, as are orthodox seeds, although in contrast to other orthodox seeds they lose viability in a few weeks at room temperature. They also differ in that the chloroplasts of the embryo tissues conserve their chlorophyll and endomembranes. The aim of this paper was to investigate the role of chlorophyll in seed deterioration. METHODS Seeds were aged at different light intensities and atmospheric conditions. Mean germination time and normal and total germination were evaluated. The formation of free radicals was assessed using electronic spin resonance spectroscopy, and changes in the fatty acid composition from phospholipids, galactolipids and triglycerides using gas-liquid chromatography. Membrane integrity was studied with electronic spin resonance spin probe techniques, electrolyte leakage and transmission electron microscopy. KEY RESULTS Light and oxygen played an important role in free-radical generation, causing a decrease in normal germination and an increase in mean germination time. Both indices were associated with a decrease in polyunsaturated fatty acids derived from membrane lipids as phospholipids and galactolipids. The detection of damage in thylakoid membranes and an increase in plasmalemma permeability were consistent with the decrease in both types of lipids. Triglycerides remained unchanged. Light-induced damage began in outermost tissues and spread inwards, decreasing normal germination. CONCLUSIONS Salix nigra seeds were very susceptible to photooxidation. The thylakoid membranes appeared to be the first target of the photooxidative process since there were large decreases in galactolipids and both these lipids and the activated chlorophyll are contiguous in the structure of that membrane. Changes in normal germination and mean germination time could be explained by the deteriorative effects of oxidation.

Nitric oxide increases in the rat retina after continuous illumination.

Continuous illumination (CI) of the retina induces an oxidative stress followed by the degeneration of photoreceptors. This phenomenon may be partially related to the excessive production of nitric oxide (NO). In order to confirm this hypothesis, the aims of this work are to determine NO levels during the illumination of the retina by electron paramagnetic resonance (EPR), and if an increase of NO is found, to characterize the NOS isoform responsible of the increment by using Western blot. Sprague-Dawley rats were continuously illuminated with white light (12,000 lux) for 2, 24, 48 h, 5 and 7 days while control rats were maintained at light/dark cycles of 12/12 h. Using EPR, an increase of NO signal was observed in the light exposed retinas peaking at 24 h of CI. Western blot analysis showed the expression of iNOS in the illuminated retinas with a peak after 24 h of CI, but did not show significant differences of nNOS among illuminated and control retinas. In summary, there is an increase of NO during CI. Further studies will reveal whether this mechanism is responsible for light induced photoreceptor degeneration.

Influence of serum protein and albumin addition on the structure and activity of an exogenous pulmonary surfactant

The comparative analysis of the deleterious action of albumin and total serum proteins (SP) might help to understand the nature of the interaction surfactant--SP. This study evaluated the effects of serum proteins and albumin on bulk shear viscosity, surface tension, surface area reduction, and the ratio between the light and heavy subtypes of surfactant suspensions. Our results showed a correlation between the bulk viscosity and aggregation degree of surfactant suspensions. The addition of albumin or SP induced the transformation from the heavy to the light subtype, reducing the viscosity. SP caused disaggregation and inactivation, whereas albumin caused only disaggregation without loss of surface activity. When SP were removed, the heavy fraction obtained recovered its surface activity. We conclude that the disaggregation may not be the primary cause for the loss of surface activity. Surfactant inactivation by a serum component, different from albumin, would be probably due to a physical interaction, a phenomenon that is reversed when SP are removed.

A spectrophotometric method for the determination of hydroperoxides in liposomes.

A modification of the Asakawa-Matsushita iodometric assay method for the determination of the content of lipid hydroperoxides was developed which permits the simultaneous processing of many samples of high lipid content. The method has the advantages of simplicity as well as good reproducibility, so it is not necessary to process standards with each determination. Our technique exceeds the sensitivity attained with other spectrophotometric determinations reported in the literature. The method requires the total elimination of water from the samples, and this was accomplished using an azeotropic mixture of ethanol:water of 96:4. The results obtained with liposomes indicate that the method is applicable to biological material limited to small volume samples, ranging 5-50 microliters. We want to emphasize that this method permits the study of the peroxidation process as function of time.

Effect of DHEA and metformin on corpus luteum in mice.

We evaluated the effect of hyperandrogenism in ovaries with functional and regressing corpora lutea (CL) and the action of metformin in preventing these possible alterations using a mouse model. To obtain a CL functional for 9+/-1 days, immature female mice of the BALB/c strain were injected i.p. with 10 IU/mouse of pregnant mares serum gonadotropin (PMSG). DHEA (60 mg/kg body weight s.c., 24 and 48 h prior to kill) decreased both serum progesterone (P) and estradiol (E(2)) levels and increased the activity of superoxide dismutase (SOD) from ovaries with functional CL (on day 5 after PMSG). It increased P and E(2) and the activities of SOD and catalase (CAT) and decreased lipoperoxidation of ovaries with regressing CL (on day 9 after PMSG). Treatment with DHEA did not affect the production of prostaglandin F(2alpha) (PGF(2alpha)) or PGE by ovaries with functional CL, whereas DHEA decreased PGF(2alpha) and increased PGE production by ovaries with regressing CL. Metformin (50 mg/kg body weight, orally) given together with DHEA restored E(2) levels from mice with ovaries with functional CL and serum P, PGF(2alpha) and PGE levels, and oxidative balance in mice with ovaries with regressing CL. Metformin alone was able to modulate serum P and E(2) levels, lipoperoxidation, SOD and CAT, and the 5,5-dimethyl-1-pyrroline N-oxide/(*)OH signal. These findings suggest that hyperandrogenism is able to induce or to rescue CL from luteolysis and metformin treatment is able to prevent these effects.

Effect of serum proteins on an exogenous pulmonary surfactant: ESR analysis of structural changes and their relation with surfactant activity

The study of the structural changes in surfactant microviscosity and bilayer organization would help to understand the mechanisms by which surfactant could be inactivated by serum components. The in vitro effects of human serum, albumin and gamma-globulin on dynamic and structural properties of surfactant suspensions and their heavy fractions were evaluated by electronic spin resonance and surface tension measurements. Our results showed that albumin and serum modified the aggregation state, transforming the active into inactive subtype, but only serum decreased the fluidity in the polar region and inactivated surfactant. In contrast, albumin and gamma-globulin generated a greater proportion of fluid-like disordered phase, without loss of surface activity. Statistical analysis showed that surface activity correlated with the fluidity in the polar area but not with that in the hydrophobic region. We concluded that one or more serum components different from albumin or gamma-globulin cause a structural change in the surfactant bilayer, increasing the rigidity in the polar area, which would be critical for proper physiological activity.