α-amyrin-loaded nanocapsules produce selective cytotoxic activity in leukemic cells.

INTRODUCTION: Amyrins are triterpenes that have attractive pharmacological potential; however, their low water solubility and erratic stomach absorption hinders their use as a drug. The aim of this paper was to develop a novel α-amyrin-loaded nanocapsule for intestinal delivery and evaluate, preliminarily, its cytotoxic ability against leukemic cells. MATERIAL AND METHODS: Five nanocapsule formulations were designed by the solvent displacement-evaporation method. Poly-ε-caprolactone, Eudragit® E100, and Kollicoat® Mae 100 P were used as film-former materials. Particle size, polydispersity index (PdI), zeta potential, and the pH of all formulations were measured. The cytotoxic potential of the nanocapsules was evaluated in vitro using different leukemic lineages RESULTS: Nanocapsules coated with Kollicoat® Mae 100 P presented the smallest particle size (130 nm), the lowest zeta-potential (-38 mV), and the narrowest size distribution (PdI = 0.100). The entrapment efficiency was 65.47%, while the loading capacity was 2.40%. Nanocapsules release 100% of α-amyrin in 40 min (pH 7.4), by using a possible mechanism of swelling-diffusion. The formulation showed excellent on-shelf physicochemical stability during one year. Additionally, nanocapsules produced a selective cytotoxic effect on a human leukemia lineage Kasumi-1, an acute myeloid leukemia cell line, and produced cell death by apoptosis CONCLUSION: α-amyrin-loaded nanocapsules appear to be a promising nanoformulation that could be used against leukemia.

Covalently Binding of Bovine Serum Albumin to Unsaturated Poly(Globalide-Co-ε-Caprolactone) Nanoparticles by Thiol-Ene Reactions.

When nanoparticles (NPs) are introduced to a biological fluid, different proteins (and other biomolecules) rapidly get adsorbed onto their surface, forming a protein corona capable of giving to the NPs a new "identity" and determine their biological fate. Protein-nanoparticle conjugation can be used in order to promote specific interactions between living systems and nanocarriers. Non-covalent conjugates are less stable and more susceptible to desorption in biological media, which makes the development of engineered nanoparticle surfaces by covalent attachment an interesting topic. In this work, the surface of poly(globalide-co-ε-caprolactone) (PGlCL) nanoparticles containing double bonds in the main polymer chain is covalently functionalized with bovine serum albumin (BSA) by thiol-ene chemistry, producing conjugates which are resistant to dissociation. The successful formation of the covalent conjugates is confirmed by flow cytometry (FC) and fluorescence correlation spectroscopy (FCS). Transmission electron microscopy (TEM) allows the visualization of the conjugate formation, and the presence of a protein layer surrounding the NPs can be observed. After conjugation with BSA, NPs present reduced cell uptake by HeLa and macrophage RAW264.7 cells, in comparison to uncoated NP. These results demonstrate that it is possible to produce stable conjugates by covalently binding BSA to PGlCL NP through thiol-ene reaction.

Polymeric nanocapsules as a technological alternative to reduce the toxicity caused by meloxicam in mice.

This study determined whether meloxicam in nanocapsules modifies stomach and liver damage caused by free meloxicam in mice. Male Swiss mice were treated with blank nanocapsules or meloxicam in nanocapsules or free meloxicam (10 mg/kg, intragastrically, daily for five days). On the seventh day, blood was collected to determine biochemical markers (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, total bilirubin, unconjugated bilirubin, albumin and alkaline phosphatase). Stomachs and livers were removed for histological analysis. There was no significant difference in the biochemical markers in the plasma of mice. Meloxicam in nanocapsules did not have an ulcerogenic potential in the stomach or cause lipid peroxidation in the stomach and liver. Free meloxicam increased the ulcerogenic potential in the stomach and lipid peroxidation in the stomach and liver. Meloxicam in nanocapsules caused less histological changes than free meloxicam. In conclusion, polymeric nanocapsules can represent a technological alternative to reduce the toxicity caused by meloxicam.

A CO2-Free Synthetic Host-Odor Mixture That Attracts and Captures Triatomines: Effect of Emitted Odorant Ratios.

Triatomines, vectors of Chagas Disease, are hematophagous insects. Efforts have been made to develop synthetic attractants based on vertebrate odor-to lure them into traps. However, because those lures are not practical or have low capture efficiency, they are not in use in control programs. Therefore, more work is needed to reach a practical and efficient odor lure. Recently, a three-component, CO2-free, synthetic blend of vertebrate odor (consisting of ammonia, l-(+)-lactic acid, and hexanoic acid), known as Sweetscent (Biogents AG, Regensburg, Germany), was shown to attract and capture triatomines in the laboratory. In this study, using a trap olfactometer and an odor blend with constituents similar to those of Sweetscent (delivered from low-density polyethylene sachets) we found that the odorant ratios of the mixtures have a strong effect in the capture of triatomines. The blend with the most efficient combination of odorant ratios evoked ca. 81% capture in two relevant triatomine species. In the case of the most effective odor mixtures, we measured the odor mass emission for the three components of the mixture and therefore were able to estimate the odorant ratios emitted that were responsible for such a high capture performance. Thus, in those mixtures, pentanoic acid was the main component (ca. 65 %) followed by ammonia (ca. 28%) and, l(+)-lactic acid (ca. 7 %). Our results are encouraging as efficient, practical, and cheap odor baits to trap triatomines in the field would be within reach. The odor-delivery system used should be improved to increase stability of odor emission.

Evaluation of a CO2 -free commercial mosquito attractant to capture triatomines in the laboratory.

Efforts have been made to develop vertebrate odor-based attractants to lure hematophagous triatomines into traps. However, more work is needed to reach a practical, cheap, and efficient odor lure. We carried out attraction and capture tests in a dual-choice olfactometer and a pitfall trap. Here we report that a three-component, CO2 -free, synthetic blend of vertebrate odor (consisting of ammonia, L(+) lactic acid and hexanoic acid, and known as Sweetscent®) significantly induces 3(rd) -instar Rhodnius prolixus and Triatoma infestans nymphs to fall into the test capture-tube of the olfactometer. Blend constituents presented singly or in two-component blends did not evoke a response and, therefore, we propose that the insects respond specifically to the three-component blend in a synergistic way. When tested in a pitfall trap in an experimental arena, this blend induced capture in 37.5% of the lured traps, whereas 9% of the nymphs tested were captured in a single night. No insects were captured in control traps. Our work represents a proof-of-concept regarding capture of triatomines using host odor-based, CO2 -free synthetic mixtures as lures for pitfall traps. CO2 -free lures are more practical for field work than natural or CO2 -containing synthetic blends.

Short chain fatty acids (propionic and hexanoic) decrease Staphylococcus aureus internalization into bovine mammary epithelial cells and modulate antimicrobial peptide expression.

Short chain fatty acids (SCFAs) are critical nutrients for ruminants and are mainly obtained from bacterial fermentation of carbohydrates. In addition to their nutrimental function, SCFAs have antimicrobial and anti-inflammatory properties, as well as immunomodulatory roles. It has been reported that sodium butyrate reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMEC) and modulates antimicrobial peptide mRNA expression. Nevertheless, it has not been evaluated if sodium propionate (NaP) and sodium hexanoate (NaH) have similar actions. Since they are present in milk, the aim of this study was to determinate the effect of both SCFAs on S. aureus internalization into bMEC and to evaluate their effects on modulation of innate immunity elements. Our data showed that both SCFAs (0.25-5mM) did not affect S. aureus growth and bMEC viability. By gentamicin protection assay (MOI 30:1) we showed that NaP and NaH reduced bacterial internalization into bMEC, which ranged 27-55% and 39-65%, respectively, in relation to non treated controls. Also, both SCFAs up-regulate tracheal antimicrobial peptide (TAP) mRNA expression; however, bovine neutrophil ß-defensin 5 (BNBD5) mRNA expression was not modified or was down-regulated. In addition, TAP and BNBD5 expression was up-regulated by S. aureus. Finally, the decrease in bacterial internalization under SCFA treatments is not related to nitric oxide production. In conclusion, NaP and NaH decrease S. aureus internalization into bMEC and modulate TAP gene expression, which may be related to the reduction in bacterial internalization.

Antimicrobial activities of components of the glandular secretions of leaf cutting ants of the genus Atta.

The secretions of the mandibular and metapleural glands of leaf cutting ants contain antimicrobial substances that protect the mutualistic fungal colony within the nest from attack by parasitic micro-organisms. The major constituents of these secretions (citral, 4-methyl-3-heptanol, 2-heptanone, 3-octanone, 4-methyl-2-heptanone, beta-citronellol, geraniol, phenylacetic, indolacetic, hexanoic and octanoic acids were tested against resistant strains of the human pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans. Assays were carried out using filter paper discs impregnated with either hexane or water solutions of the analytes in the concentration range 250-6,000 ng/microl. Although most of the tested compounds presented strong antibacterial and antifungal activities, citral, geraniol, 4-methyl-3-heptanol, hexanoic and octanoic acids were the most effective, particularly against C. albicans. The results suggest that these compounds may be of potential value as antibiotics in the treatment of human candidiasis.

Poly (epsilon-caprolactone)/propolis extract: microencapsulation and antibacterial activity evaluation.

Spherical and homogenous microparticles of poly(epsilon-caprolactone) (PCL), containing propolis were prepared by the emulsification-solvent evaporation technique. Using this method of preparation, a solid formulation of propolis, free of ethanol and suitable for manipulation and storage, was obtained from an ethanolic extract of propolis. The incorporation efficiency of propolis in the microparticles was almost 30% and around 60% of the substance was released in 48 h. In vitro propolis microparticles exhibited similar halo zones in the Petri plate test against Streptococcus mutans (GS5) with a 10-fold lower concentration than the free propolis extract showing that the encapsulated propolis in microparticles is more efficient as antibiotic.

Effects of organic acids on lipid synthesis and ecdysis in Triatoma infestans eggs.

Medium chain fatty acids are known inhibitors of the activity of lipogenic enzymes at the transcriptional level. Mature Triatoma infestans eggs incorporate [14C]-acetate into phospholipid (PL), triacylglycerol (TG), and free fatty acid (FFA) fractions. In the presence of sodium octanoate (SO) or sodium phenylethylpropionate (PHEP), lipid synthesis is inhibited in a dose-dependent manner. When eggs are incubated in vivo with [14C]-acetate, the usual fatty acid products are largely palmitic and oleic acids. However, in the presence of SO (5-10 mM), the elongation of [14C]-acetate units is interrupted at [14C] eight total carbons. Eggs incubated in vivo with [14C]-SO, accumulate most of the label in the FFA fraction. SO (> 0.1 mM) but not sodium hexanoate (SH), inhibits the activity of microsomal and cytosolic fatty acid synthetases (FAS's), measured as [14C]-malonyl-CoA incorporation. PHEP (1 mM) and SO (10 mM) also produce major alterations in egg hatching and survival of the emerged insects, after a 1 h immersion bioassay.

An in vitro model suitable for studying insulin secretion in amphibians.

The aim of this work was to develop an in vitro model suitable for studying insulin secretion in amphibians and for identifying agents capable of either blocking or stimulating such a process in this group. For this purpose, pancreases from the toad Bufo arenarum were incubated for 60 min at 25 degrees with several insulin secretagogues and blockers, and the immunoreactive insulin released into the medium was measured by radioimmunoassay. Under these experimental conditions, metabolic (glucose, ketoisocaproic acid, and arginine) and nonmetabolic (K+ and tolbutamide) agents as well as glucagon and acetylcholine significantly stimulated the release of immunoreactive insulin. Conversely, somatostatin and nifedipine blocked its secretion. All these agents exerted similar effects on the mammalian pancreas. These results prove that our model is a useful tool with which to study in vitro insulin secretion in amphibians and to identify agents which affect hormone release in this group.

Effect of different insulin secretagogues and blocking agents on islet cell Ca2+-ATPase activity.

Plasma membrane Ca2+-ATPase activity was measured in rat islet homogenates. The enzyme was inhibited, in a dose-dependent manner, when the islets were preincubated for 5 min with different concentrations of glucose (2 to 16 mM). This inhibition disappeared almost entirely after 15 min incubation, regardless of the glucose concentration in the medium. Simultaneous measurement of insulin in the medium revealed a stimulatory effect of glucose upon insulin secretion. The Ca2+-ATPase activity was also inhibited when the islets were preincubated for 3 min with other stimulators of insulin secretion such as gliclazide (76 microM), tolbutamide (1.5 mM), glucagon (1.4 microM) + theophylline (10 mM) and ketoisocaproic acid (15 mM). Conversely, the activity of the enzyme was significantly enhanced when the islets were preincubated briefly with the insulin secretion blocker, somatostatin (1.4 microM). Neither glucose nor any of the other substances tested when added directly to the enzyme assay medium modified significantly the Ca2+-ATPase activity measured in the islet homogenates. These results would suggest that the activity of the islet plasma membrane is modulated by one or more of the intracellular metabolites produced when the islets are challenged by the insulin stimulator or blocking agents.