GLP1R (glucagon-like-peptide-1 incretin receptor), diabetes and obesity phenotypes: An in silico approach revealed new pathogenic variants.

OBJECTIVE: Glucagon-like peptide-1 receptor belongs to the B family of G protein-coupled receptors, serving as a binding protein in membranes and is widely expressed in human tissues. Upon stimulation by its agonist, the glucagon-like peptide-1, the receptor plays a role in glucose metabolism, enhancing insulin secretion, and regulating appetite in the hypothalamus. Mutations in the glucagon-like peptide-1 receptor gene can lead to physiological changes that may explain phenotypic variations in individuals with obesity and diabetes. Therefore, this study aimed to evaluate missense variants of the glucagon-like peptide-1 receptor gene. METHODS: Data mining was performed on the single nucleotide polymorphism database, retrieving a total of 16,399 variants. Among them, 356 were missense. These 356 variants were analyzed using the PolyPhen-2 and filtered based on allele frequency, resulting in 6 pathogenic variants. RESULTS: D344E, A239T, R310Q, R227H, R421P, and R176G were analyzed using four different prediction tools. The D344E and A239T resulted in larger amino acid residues compared to their wild-type counterparts. The D344E showed a slightly destabilized structure, while A239T affected the transmembrane helices. Conversely, the R310Q, R227H, R421P, and R176G resulted in smaller amino acid residues than the wild-type, leading to a loss of positive charge and increased hydrophobicity. Particularly, the R421P, due to the presence of proline, significantly destabilized the α-helix structure and caused severe damage to the receptor. CONCLUSION: Elucidating the glucagon-like peptide-1 receptor variants and their potentially detrimental effects on receptor functionality can contribute to an understanding of metabolic diseases and the response to available pharmacological treatments.

MicroRNA-Mediated Antiproliferative Effects of M1 Macrophage-Derived Extracellular Vesicles on Melanoma Cells.

INTRODUCTION: Research in tumor treatment has shown promising results using extracellular vesicles (EVs) derived from immune cells. EVs derived from M1 macrophages (proinflammatory), known as M1-EVs, have properties that suppress tumor growth, making them a promising treatment tool for immune susceptible tumors such as melanoma. Here, small unaltered M1-EVs (M1-sEVs) were employed in a 3D mouse melanoma model (melanospheres) to evaluate such activity. METHODS: Macrophages were polarized and EVs were isolated by ultracentrifugation. The EVs obtained were characterized based on size, with measurements performed by dynamic light scattering and electron microscopy, and the expression profiles of microRNAs were analyzed by microarray and PCR. Melanospheres were used to evaluate the cytotoxicity of M1-sEVs. Pondering a possible future transposition from the animal model to the human, human melanoma cells were transfected with a specific miRNA, and the impact on cell proliferation was evaluated. RESULTS: The isolated EVs showed a size distribution between 50-400 nm in diameter, but preeminently in a range of 70-90 nm. M1-sEVs demonstrated a remarkable ability to reduce cell proliferation and viability in the melanospheres, leading to a decrease in their volume. M1-sEVs contained unique miRNAs, including miR-29a-3p, which exhibited significant antitumor activities according to bioinformatics analysis. Validation of the antitumor effects of miR-29a-3p was obtained by a functional evaluation, i.e., by inducing miRNA overexpression in human melanoma cells (SK-MEL-28). CONCLUSION: Although further research would be advisable, the study provides evidence supporting the potential of M1-sEVs and their miRNA load as a possible targeted immune therapy for melanoma.

Diagnosis and treatment of Acanthamoeba Keratitis: A scoping review demonstrating unfavorable outcomes.

Acanthamoeba spp. are pathogens that cause Acanthamoeba keratitis (AK), a serious cornea inflammation that can lead to gradual loss of vision, permanent blindness, and keratoplasty. The efficacy of AK treatment depends on the drug's ability to reach the target tissue by escaping the protective eye barrier. No single drug can eradicate the living forms of the amoeba and be non-toxic to the cornea tissue. The treatment aims to eradicate both forms of protozoan life but is hampered by the resistance of the cysts to the most available drugs, leading to prolonged infection and relapses. Drug therapy is currently performed mainly using diamidines and biguanides, as they are more effective against cysts. However, they are cytotoxic to corneal cells. Drugs are applied topically, and hourly. Over time, the frequency of administration decreases, but the treatment time varies from month to years. This study aims to obtain an up-to-date summary of the literature since 2010, allowing us to identify the trends and gaps and address future research involving new alternatives for treating AK. The results were divided into three phases, pre-treatment, empirical treatment, and the treatment after diagnosis confirmation. The drugs prescribed were stratified into antiamoebic, antibiotic, antifungal, antivirals, and steroids. It was possible to observe the transition in drug prescription during three different stages until the diagnosis was confirmed. There were more indications for antibiotic, antifungal, and antiviral drugs in the early stages of the disease. The antiamoebic drugs were only prescribed after exhausting other treatments. This can be directly involved in developing complications and no responsiveness to medical treatment.

Human body burden of bisphenol A: a case study of lactating mothers in Florianopolis, Brazil.

Exclusive breast milk is the diet recommended by the World Health Organization (WHO) until 6 months of age. However, breastfeeding has the potential of transferring certain toxic chemicals from the mother to the infant. Bisphenol A (BPA) is a synthetic chemical used as a monomer in polycarbonate plastics and epoxy resins. Information on BPA concentration in the breast milk of lactating mothers is very limited; thus, this study aimed to determine the concentration of BPA in the colostrum of 64 post-partum women at a university-affiliated tertiary hospital in South Brazil. The results showed that all the breast milk samples contained a high concentration of BPA with a median value of 34.18 ng/mL. Furthermore, the concentration of BPA in mothers was influenced by the consumption of foods packaged in plastic packaging, especially when the plastic is heated (p = 0.0182). The total daily intake of BPA in breastfed infants was 19.5 µg/kg/day and 28.5 µg/kg/day was recorded at the 95th percentile of body weight per day, which is higher than the maximum daily intake estimated by the European Authority of Food Safety. These data showed a high concentration of BPA in the breastmilk of the lactating mothers which might be through the use of plastic containers as food/drink packages. This is of public health importance as the high concentration of BPA in their breast milk can be an indicator of potentially serious health problems in these mothers and much more in the babies breastfed with BPA-contaminated breast milk.

Super aggregated amphotericin B with a thermoreversible in situ gelling ophthalmic system for amoebic keratitis treatment.

Acanthamoeba spp. are the causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK). Amphotericin B - deoxycholate (AB) is used in the treatment of infectious keratitis, however, its topical administration has side effects as blepharitis, iritis, and painful instillation. In this context, the preheating of AB can decrease its toxicity by the formation of super aggregates (hAB). hAB associated with a thermoreversible in situ gelling ophthalmic system is a promising option due to the latter biocompatibility, low toxicity, and high residence time on the ocular surface. Our objective was to develop a topical ocular formulation of hAB for the treatment of AK. After heating at 70°C for 20 min, hAB was incorporated into a thermoreversible gelling system. The amebicidal activity of AB and hAB was evaluated against trophozoites and cysts of A. castellanii (ATCC 50492) and a regional clinical isolate (IC01). The results showed that the preheating of AB did not change the pharmacological action of the drug, with the amebicidal effect of AB and hAB under trophozoites and cysts of Acanthamoeba spp. The thermoreversible system remained stable, allowing the increase of drug retention time. For assessment of cytotoxicity, HUVEC (ATCC® CRL-1730) cells were challenged with AB and hAB for 48h. Cell viability was assessed, and hAB did not show cytotoxicity for HUVEC cells. As far as we know this was the first study that showed the preheated AB associated with a thermoreversible in situ gelling ophthalmic system as a promising system for topical ocular topical administration of hAB for AK therapy.

Biological activity of 2α,3ß,23-trihydroxyolean-12-ene on glucose homeostasis.

We studied the effect and the mechanisms of action of 2α,3ß,23-trihydroxyolean-12-ene (THO), from Croton heterodoxus Baill. (Euphorbiaceae), in glucose uptake in hyperglycemic rats. The effect of in vivo pretreatment with THO in hyperglycemic rats was analyzed. The in vitro effects of THO were observed in adipocytes and in adipose tissue. THO reduced glycemia, in part by increasing serum insulin and augmenting the disposal of glucose as glycogen in hepatocytes but did not change the serum concentration of glucagon-like peptide-1. THO increased glucose uptake in adipocytes and in adipose tissue by a mechanism dependent on phosphatidylinositol 3-kinase vesicular traffic and on the process of vesicle fusion at the plasma membrane in regions containing cholesterol, indicating the involvement of glucose transporter-4 (GLUT4). This triterpene may act solely via the activation and translocation of GLUT4 (rather than via nuclear actions, such as upregulation of GLUT4 synthesis), since THO did not alter the amount of GLUT4 mRNA or the content of GLUT4. Consistent with these data, the stimulatory effect of this triterpene on the quantity of GLUT4 in the membrane fraction was dependent upon p38 phosphorylation. In this experimental model, orally administered 10 mg/kg THO did not modulate extracellular serum lactate dehydrogenase. In conclusion, THO decreases hyperglycemia by increasing serum insulin and hepatic glycogen content. The THO mechanism of action on adipose tissue for glucose uptake is suggested to be via GLUT4 translocation stimulation mediated by a p38-dependent mechanism. THO is a potential antihyperglycemic agent that acts in a target tissue for glucose homeostasis.

Bariatric surgery improves clinical outcomes and adiposity biomarkers but not inflammatory cytokines SAA and MCP-1 after a six-month follow-up.

Obesity is a global health problem and is associated with a chronic low-grade inflammatory state. Surgical obesity treatment is being increasingly common due to its efficacy. From this, we evaluate the metabolic state improvement and inflammation remission in patients with obesity undergoing bariatric surgery. Methods: The clinical data and serum levels of leptin and adiponectin were assessed in patients with obesity before and one, three and six months after bariatric surgery. Also, serum amyloid A (SAA), monocyte chemoattractant protein-1 (MCP-1) levels were measured during the follow-up surgery and compared with a lean group of individuals. Results: Weight loss decreased body mass index (BMI), comorbidities percentage, drugs use and leptin levels. Adiponectin levels increased after surgery. SAA and MCP-1 showed no difference after surgery, but a trend decrease for MCP-1 and a significant decrease was observed when the patients with obesity were compared to the lean participants. Conclusion: Bariatric surgery alters metabolic status improving obesity-related comorbidities and the adiposity biomarkers leptin and adiponectin, but not inflammatory cytokines SAA and MCP-1.

Three-dimensional multicellular cell culture for anti-melanoma drug screening: focus on tumor microenvironment.

ABSTRACT: The development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity.

Lipid nanoparticles for amphotericin delivery in the treatment of American tegumentary leishmaniasis.

Leishmaniasis occurs in the five continents and represents a serious public health challenge, but is still a neglected disease, and the current pharmacological weaponry is far from satisfactory. Triglyceride-rich nanoparticles mimicking chylomicrons (TGNP) behave metabolically like native chylomicrons when injected into the bloodstream. Previously we have shown that TGNP as vehicle to amphothericin B (AB) for treatment of fungi infection showed reduced renal toxicity and lower animal death rates compared to conventional AB. The aim of the current study was to test the tolerability and effectiveness of the TGNP-AB preparation in a murine model of Leishmania amazonensis infection. The in vitro assays determined the cytotoxicity of TGNP-AB, AB, and TGNP in macrophages and promastigote forms and the leishmanicidal activity in infected macrophages. The in vivo toxicity tests were performed in healthy mice with increasing doses of TGPN-AB and AB. Then, animals were treated with 2.5 mg/kg/day of AB, 17.5 mg/kg/day of TGNP-AB, or TGNP three times a week for 4 weeks. TGNP-AB formulation was less cytotoxic for macrophages than AB. TGNP-AB was more effective than AB against the promastigotes forms of the parasite and more effective in reducing the number of infected macrophages and the number of amastigotes forms per cell. TGNP-AB-treated animals showed lower hepatotoxicity. In addition, TGNP-AB group showed a marked reduction in lesion size on the paws and parasitic load. The TGNP-AB preparation attained excellent leishmanicidal activity with remarkable lower drug toxicity at very high doses that, due to the toxicity-buffering properties of the nanocarrier, become fully tolerable.

Genetic, reproductive and oxidative damage in mice triggered by co-exposure of nanoparticles: From a hypothetical scenario to a real concern.

Humans are potentially exposed to multiple nanoparticles kinds through nanotechnology-based consumer products. There is insufficient data on the in vivo toxicity of nanotechnology products, as well as no data on the possible toxicity, including genotoxicity and reproductive toxicity of co-exposure to different kind of nanoparticles. In this work, solid lipid nanoparticles (SLNs) and superparamagnetic iron oxide nanoparticles (SPIONs) were selected for evaluation of a hypothetical condition of in vivo co-exposure. Genotoxicity of SPIONs and SLNs was performed separately and in 1:1 mixture in mice. Bone marrow micronucleus assay, sperm morphology test, and sperm count were carried out. Also, the serum ALT and AST activities; and hematological parameters of the treated mice were analyzed. The results showed a significant increase (p < 0.05) in micronucleated polychromatic erythrocytes (MNPCE) and nuclear abnormalities (NA) in SPIONs, SLNs and their mixture treated mice. The mixture induced the highest frequency of MNPCE and NA. A similar result was observed in the sperm morphology test, with the mixture inducing the highest sperm abnormalities, followed by SLNs and the least by SPIONs. Significant alteration to RDW, MCHC, MCV, GRAN, and platelets, as well as increased activities of serum AST were observed in the mice treated with a mixture of the two kinds of nanoparticles. Calculation of interaction factor showed a possible synergistic effect between SPIONs and SLNs in MNPCE, NA and sperm morphology studied. Even as a hypothetical scenario of co-exposure to SLNs and SPIONs, this study showed, for the first time, that co-exposure to SPIONs and SLNs is more genotoxic to somatic and germ cells than their individual exposure.

Experimentally-induced maternal hypothyroidism alters enzyme activities and the sensorimotor cortex of the offspring rats.

In this study, we used an experimental model of congenital hypothyroidism to show that deficient thyroid hormones (TH) disrupt different neurochemical, morphological and functional aspects in the cerebral cortex of 15-day-old offspring. Our results showing decreased glutamine synthetase (GS) activity and Ca2+ overload in the cerebral cortex of hypothyroid pups suggest misregulated glutamate metabolism associated with developmentally induced TH deficiency. The 14C-MeAIB accumulation indicates upregulated System A activity and glutamine uptake by neurons. Energy metabolism in hypothyroid cortical slices was preserved, as demonstrated by unaltered glucose metabolism. We also found upregulated acetylcholinesterase activity, depleting acetylcholine from the synaptic cleft, pointing to disrupted cholinergic system. Increased reactive oxygen species (ROS) generation, lipid peroxidation, glutathione (GSH) depletion, which were associated with glutathione peroxidase, superoxide dismutase and gamma-glutamyltransferase downregulation suggest redox imbalance. Disrupted astrocyte cytoskeleton was evidenced by downregulated and hyperphosphorylated glial fibrillary acidic protein (GFAP). Morphological and structural characterization of the sensorimotor cerebral cortex (SCC) showed unaltered thickness of the SCC. However, decreased size of neurons on the layers II & III and IV in the right SCC and increased NeuN positive neurons in specific SCC layers, suggest that they are differently affected by the low TH levels during neurodevelopment. Hypothyroid pups presented increased number of foot-faults in the gridwalk test indicating affected motor functions. Taken together, our results show that congenital hypothyroidism disrupts glutamatergic and cholinergic neurotransmission, Ca2+ equilibrium, redox balance, cytoskeleton integrity, morphological and functional aspects in the cerebral cortex of young rats.

Millettia macrophylla (Fabaceae) phenolic fraction prevents differentiation of 3T3-L1 adipocytes and the increased risks of cardiovascular diseases in ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: A prolonged estrogen deficiency alters lipid metabolism and increases risks of cardiovascular diseases. Phytoestrogens, naturally occurring compounds with estrogenic properties are reported to have cardiovascular protective effects. Millettia macrophylla used in the Cameroonian traditional system to treat physiological disorders related to menopause, was previously reported to have estrogenic effects. AIM: We, therefore, proposed evaluating the in vitro and in vivo effects of M. macrophylla phenolic fraction on some risk factors for cardiovascular diseases. MATERIAL AND METHODS: In vitro, the ability of the M. macrophylla phenolic fraction (PF) as well as the 9 isolates to prevent the 3T3-L1 preadipocytes differentiation was assessed. Further, the preventive effects of PF on abdominal fat accumulation, body weight gain, lipid profile, nitric oxide level, superoxide dismutase (SOD) and catalase activities, reduced glutathione (GSH) and malondialdehyde (MDA) levels were assessed in a postmenopausal rat model. RESULTS: In vitro, PF and its isolate secundiferol I inhibited lipid accumulation in 3T3-L1 cells. Moreover, all the isolates except daidzein dimethylether prevented the interleukin IL-6 production in 3T3-L1 cells. In vivo, PF prevented ovariectomy-induced abdominal fat accumulation, body weight gain, dyslipidemia, glucose intolerance and decreased atherogenic index. In addition, it induced a vasorelaxant effect by preventing the low level of nitric oxide in the aorta. PF also exhibited antioxidant effects as it increased aorta GSH level, SOD, and catalase activities and decreased MDA level. CONCLUSIONS: Taken together, our data suggest that PF prevents the increased risks of cardiovascular diseases in ovariectomized rats.

Lycium barbarum Reduces Abdominal Fat and Improves Lipid Profile and Antioxidant Status in Patients with Metabolic Syndrome.

Natural antioxidants present in fruits have attracted considerable interest due to their presumed safety and potential nutritional value. Even though antioxidant activities of many fruits have been reported, the effects of phytochemicals of goji berry (GB) in patients with metabolic syndrome have not been investigated. In this study, we examined anthropometric and biochemical parameters in patients with metabolic syndrome after the consumption of GB. The patients were divided into two groups, control (C) and supplemented (S), and followed up for 45 days. Participants were individually instructed to carry out a healthy diet, but additionally, an inclusion of 14 g of the natural form of goji berry in the diet during 45 days for the S group was proposed. After 45 days of study, a significant reduction in transaminases as well as an improvement in lipid profile in the S group was observed. Likewise, a significant reduction in the waist circumference of the S group was observed when compared with that of the C group, and increased glutathione and catalase levels associated with a reduction of lipid peroxidation. These results suggest that this is an effective dietary supplement for the prevention of cardiovascular diseases in individuals with metabolic syndrome.

Complement component 3 (C3) as a biomarker for insulin resistance after bariatric surgery.

OBJECTIVES: Since complement system has been recently associated with metabolic and cardiovascular diseases, and closely related to insulin resistance, we investigated the association of plasma complement factor 3 (C3) and factor 4 (C4) with insulin sensibility and weight loss after bariatric surgery. METHODS: Serum levels of C3, C4, total cholesterol, triacylglycerol, HDL-cholesterol, LDL-cholesterol and homeostatic model assessment (HOMA-IR) measurements were assessed in morbidly obese patients before and after bariatric surgery, including a 6-month follow-up period, as well as a comparison with a lean group. RESULTS: Weight loss decreased body mass index (BMI), serum triacylglycerol, and increased serum HDL-cholesterol and insulin sensitivity, as expected. C3 and C4 were significantly higher in obese individuals when compared to lean subjects (p<0.001). In addition, C3 and C4 positively correlated with BMI and HOMA-IR, however, only C3 were significantly decreased 6months after surgery. CONCLUSION: C3 was strongly associated with insulin sensitivity after bariatric surgery.

Allylic isothiouronium salts: The discovery of a novel class of thiourea analogues with antitumor activity.

A series of 28 aryl- and alkyl-substituted isothiouronium salts were readily synthesized in high yields through the reaction of allylic bromides with thiourea, N-monosubstituted thioureas or thiosemicarbazide. The S-allylic isothiouronium salts substituted with aliphatic groups were found to be the most effective against leukemia cells. These compounds combine high antitumor activity and low toxicity toward non-tumoral cells, with selectivity index higher than 20 in some cases. Furthermore, the selected isothiouronium salts induced G2/M cell cycle arrest and cell death, possibly by apoptosis. Therefore, these compounds can be considered as a promising class of antitumor agents due to the potent cytostatic activity associated with high selectivity.

N-phenylmaleimides affect adipogenesis and present antitumor activity through reduction of FASN expression.

In light of the evidence that in contrast to most healthy tissues, several neoplasms overexpress fatty acid synthase (FASN) upon their dependence on increased lipogenesis; targeting of this protein is being considered as a valuable strategy in anticancer drug development. This can be particularly relevant for aggressive tumors such as melanoma in which FASN overexpression has been associated with increased depth of invasion and worse prognosis. We have previously shown that a sub-class of cyclic imides, the N-phenylmaleimides, presented antitumor activity against L1210 leukemia and B16F10 melanoma with evidences of interference in the energetic metabolism. Here, we aimed to investigate if some selected N-phenylmaleimides (M1 and M5) interfere with fatty acids metabolism and its relation with cancer. For that, a model of pre-adipocytes differentiation (3T3-L1 cells) and also human melanoma cells (SK-Mel-147) were used. As results, when 3T3-L1 cells were exposed to non-cytotoxic concentrations of M1 and M5 in the presence of an adipogenic cocktail, intracellular lipid content decreased by 26-36%, marking the inhibition of adipocyte differentiation. High selectivity indexes were obtained for both compounds for tumoral cells. Cell cycle phases analysis revealed a remarkable proportion of cells with DNA fragmentation after their exposure to M1 and M5. This was correlated to both apoptosis and necrosis, showed by Annexin-V/PI assay. Furthermore, M1 and M5 reduced FASN expression by 19-39%, respectively. In conclusion, M1 and M5 presented antiadipogenic and antitumoral activities. The antitumoral activity that was associated to apoptosis and necrosis is a possible consequence of the FASN reduction, which in turn, might result in a fuel decrease to cell proliferation. As it happens with antiangiogenic activity, reduction of fatty acid synthesis might be a potential target for cancer treatment in a strategy of hunger-strike, which valorizes these N-phenylmaleimides as candidates for drug development.

Isothiouronium salts reduce NRAS expression, induce apoptosis and decrease invasion of melanoma cells.

Melanoma is a very aggressive type of skin cancer. Mutation in BRAF and NRAS are often found in patients with this disease. Therefore, in recent years the search for new molecules that inhibit these proteins has been intensified. After many years with no new treatments for melanoma, the U.S. Food and Drug Administration (FDA) recently approved vemurafenib. However, many patients have already acquired resistance and have experienced severe side effects. Therefore, this work aims to evaluate a new set of compounds including allylic isothiouronium salts (1, 2 and 3), N-phenyl-substituted analog (4) and isothiosemicarbazide salts (5 and 6) for their potential antimelanoma activity. To this end, viability assay, cell cycle analysis, expression of NRAS and BRAF, as well as migration and invasion assay were performed with different melanoma cell lines. Isothiouronium salts 1-3 presented CC50 (concentration required to reduce the cell number by 50%) in a range of 7-28 µM. Furthermore, salt 1 significantly decreased the expression of NRAS. However, cells incubated with these salts did not disturb the cell cycle phases; instead, an increase in the number of apoptotic cells was observed. Regarding potential antiinvasion effects, both 1 and 2 prevented cell migration as well as cell invasion. Finally, when salts 1 and 2 were associated with vemurafenib, a marked decrease in cell viability was observed when compared to the compounds incubated alone. Briefly, the salts exhibited interesting results, especially 1, which decreased the expression of NRAS, increased apoptotic cells and, when combined with vemurafenib, resulted in a synergistic effect. Therefore, we intend to test compound 1 in pre-clinical studies.

Influence of surfactant and lipid type on the physicochemical properties and biocompatibility of solid lipid nanoparticles.

Nine types of solid lipid nanoparticle (SLN) formulations were produced using tripalmitin (TPM), glyceryl monostearate (GM) or stearic acid (SA), stabilized with lecithin S75 and polysorbate 80. Formulations were prepared presenting PI values within 0.25 to 0.30, and the physicochemical properties, stability upon storage and biocompatibility were evaluated. The average particle size ranged from 116 to 306 nm, with a negative surface charge around -11 mV. SLN presented good stability up to 60 days. The SLN manufactured using SA could not be measured by DLS due to the reflective feature of this formulation. However, TEM images revealed that SA nanoparticles presented square/rod shapes with an approximate size of 100 nm. Regarding biocompatibility aspects, SA nanoparticles showed toxicity in fibroblasts, causing cell death, and produced high hemolytic rates, indicating toxicity to red blood cells. This finding might be related to lipid type, as well as, the shape of the nanoparticles. No morphological alterations and hemolytic effects were observed in cells incubated with SLN containing TPM and GM. The SLN containing TPM and GM showed long-term stability, suggesting good shelf-life. The results indicate high toxicity of SLN prepared with SA, and strongly suggest that the components of the formulation should be analyzed in combination rather than separately to avoid misinterpretation of the results.

Solid lipid nanoparticles induced hematological changes and inflammatory response in mice.

Solid lipid nanoparticles (SLNs) are an alternative drug delivery system compared to emulsions, liposomes and polymeric nanoparticles. Due to their unique sizes and properties, SLNs offer possibility to develop new therapeutic approaches. The ability to incorporate drugs into nanocarriers offers a new prototype in drug delivery that could be used for drug targeting. However, toxicity of these new formulations has not been investigated thus far. In this study, we carried out an in vivo toxicity study. For that mice were divided into three groups and treated intraperitoneally with triestearin-based SLNs (TN), natural wax-based SLNs (VN) or vehicle for 10 days. After that, necropsies, histopathological and hematological analysis, as well as hepatic and renal functions were performed. Our results indicated that both TN and VN were absorbed post-exposure and induced an inflammatory response in adipose tissue. However, histopathological analysis demonstrated the absence of toxicity in both treated groups. In addition, the body weights were similar among the groups and low toxicity was also indicated by the unchanged serum biochemical parameters. This study provides a preliminary data for toxicological studies of two different SLNs in long-term in vivo exposure. However, further studies should be conducted in order to investigate the inflammatory response in order to establish the safety of these SLNs.

Cytotoxicity of PVPAC-treated bovine pericardium: a potential replacement for glutaraldehyde in biological heart valves.

Acellular biological tissues, including bovine pericardium (BP), have been proposed as biomaterial for tissue engineering. BP is usually modified chemically to improve mechanical and biological properties using glutaraldehyde, the standard reagent for preservation of fresh bioprosthetic materials. Glutaraldehyde-fixed BP (Glut-BP), the most widely used material in heart valve manufacture, has been associated with calcification in vivo. In an attempt to reduce this issue and maintain its biocompatibility, this study assesses the physical properties and cytotoxicity of lyophilized BP treated with poly (vinylpyrrolidone-co-acrolein) (PVPAC-BP), a novel copolymer, as a substitute for glutaraldehyde. For that, PVPAC-BP surface ultrastructure, elastic function, water uptake and tissue calcification were evaluated. For the analysis of biocompatibility, fibroblasts (3T3-L1) and endothelial cells (HUVEC) were cultured on PVPAC-BP, Untreated-BP and Glut-BP. Nitric oxide (NO) release assay, fluorescence and SEM images of endothelial cells adhered on scaffolds were also performed. As results, the data show some advantages of PVPAC-BP over the Glut-BP. The PVPAC-BP maintains partially the original ultrastructure and elastic properties, improves scaffold hydration, and presents less calcium phosphate deposits. The cells demonstrated strong attachment, high proliferation rate, and formation of a monolayer on PVPAC-BP. Attached cells were also able to release NO de-monstrating regular metabolism. In conclusion, PVPAC may be considered as a promising alternative to BP treatment improving the efficiency of cell attachment and proliferation and also avoid immunogenicity.