Customized cationic nanoemulsions loading triamcinolone acetonide for corneal neovascularization secondary to inflammatory processes.

Customized cationic oil-in-water nanoemulsions (NEs) have been produced to improve the bioavailability of poorly water-soluble drugs, such as triamcinolone acetonide (TA). TA is a synthetic glucocorticoid with anti-inflammatory and antiangiogenic therapeutic properties and it is widely used as an effective treatment in ocular disorders. In this work, TA-NEs were characterized using two different custom-made cationic surfactants, showing a high positive surface charge favouring corneal penetration and a particle size below 300 nm. Both TA-NE formulations demonstrated to be stable at 4 °C during the first months of storage. Furthermore, TA-NEs were able to produce antiangiogenic effects in chicken membranes. The TA-NEs safety profile was evaluated using in vitro and in vivo ocular tolerance tests. Out of the two formulations, the one showing no irritant effects was screened in vivo demonstrating capacity to ameliorate ocular inflammation in New Zealand rabbits significantly, specially to reduce the risk of ocular inflammation processes, with antiangiogenic activity, and can therefore be exploited as a suitable formulation to avoid inflammatory reactions upon ocular surgical procedures, such as cataracts.

Permeability, anti-inflammatory and anti-VEGF profiles of steroidal-loaded cationic nanoemulsions in retinal pigment epithelial cells under oxidative stress.

Age-related macular degeneration (AMD) is defined as a degenerative, progressive and multifactorial disorder that affects the macula with a complex etiology. The retinal pigment epithelium is a monolayer of cells that has the function to separate the surface of the choroid from the neural retina that is involved in the signal transduction leading to vision. The blood-aqueous barrier and the blood retinal barrier limit the permeation of drugs into the retina and thereby reducing their efficacy. Triamcinolone acetonide (TA) is widely used as anti-inflammatory and immunomodulatory drug that promotes the inhibition of the inflammatory processes. The factors that stimulate or inhibit angiogenesis in AMD create a local balance that is responsible for the growth of sub-retinal neovascularization. In AMD, the main angiogenic stimulus is the vascular endothelial growth factor (VEGF). In this work, nanoemulsions with cationic surfactants (mono- and dicationic DABCO and quinuclidine) were produced to deliver TA, and were found to reduce the production of tumor necrosis factor alpha (TNF-α), which stimulates the choroidal neovascularization development by upregulating the VEGF production, and consequently decreased the VEGF levels. Our results support the potential use of mono- and dicationic DABCO and quinuclidine-based cationic nanoemulsions for the delivery of TA in the treatment of AMD.

Physicochemical and biopharmaceutical aspects influencing skin permeation and role of SLN and NLC for skin drug delivery.

The skin is a complex and multifunctional organ, in which the static versus dynamic balance is responsible for its constant adaptation to variations in the external environment that is continuously exposed. One of the most important functions of the skin is its ability to act as a protective barrier, against the entry of foreign substances and against the excessive loss of endogenous material. Human skin imposes physical, chemical and biological limitations on all types of permeating agents that can cross the epithelial barrier. For a molecule to be passively permeated through the skin, it must have properties, such as dimensions, molecular weight, pKa and hydrophilic-lipophilic gradient, appropriate to the anatomy and physiology of the skin. These requirements have limited the number of commercially available products for dermal and transdermal administration of drugs. To understand the mechanisms involved in the drug permeation process through the skin, the approach should be multidisciplinary in order to overcome biological and pharmacotechnical barriers. The study of the mechanisms involved in the permeation process, and the ways to control it, can make this route of drug administration cease to be a constant promise and become a reality. In this work, we address the physicochemical and biopharmaceutical aspects encountered in the pathway of drugs through the skin, and the potential added value of using solid lipid nanoparticles (SLN) and nanostructured lipid vectors (NLC) to drug permeation/penetration through this route. The technology and architecture for obtaining lipid nanoparticles are described in detail, namely the composition, production methods and the ability to release pharmacologically active substances, as well as the application of these systems in the vectorization of various pharmacologically active substances for dermal and transdermal applications. The characteristics of these systems in terms of dermal application are addressed, such as biocompatibility, occlusion, hydration, emollience and the penetration of pharmacologically active substances. The advantages of using these systems over conventional formulations are described and explored from a pharmaceutical point of view.

Development and Characterization of Nanoemulsions for Ophthalmic Applications: Role of Cationic Surfactants.

The eye is a very complex organ comprising several physiological and physical barriers that compromise drug absorption into deeper layers. Nanoemulsions are promising delivery systems to be used in ocular drug delivery due to their innumerous advantages, such as high retention time onto the site of application and the modified release profile of loaded drugs, thereby contributing to increasing the bioavailability of drugs for the treatment of eye diseases, in particular those affecting the posterior segment. In this review, we address the main factors that govern the development of a suitable nanoemulsion formulation for eye administration to increase the patient's compliance to the treatment. Appropriate lipid composition and type of surfactants (with a special emphasis on cationic compounds) are discussed, together with manufacturing techniques and characterization methods that are instrumental for the development of appropriate ophthalmic nanoemulsions.

DABCO-Customized Nanoemulsions: Characterization, Cell Viability and Genotoxicity in Retinal Pigmented Epithelium and Microglia Cells.

Quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and of quinuclidine surfactants were used to develop oil-in-water nanoemulsions with the purpose of selecting the best long-term stable nanoemulsion for the ocular administration of triamcinolone acetonide (TA). The combination of the best physicochemical properties (i.e., mean droplet size, polydispersity index, zeta potential, osmolality, viscoelastic properties, surface tension) was considered, together with the cell viability assays in ARPE-19 and HMC3 cell lines. Surfactants with cationic properties have been used to tailor the nanoemulsions' surface for site-specific delivery of drugs to the ocular structure for the delivery of TA. They are tailored for the eye because they have cationic properties that interact with the anionic surface of the eye.

Mono- and Dicationic DABCO/Quinuclidine Composed Nanomaterials for the Loading of Steroidal Drug: 32 Factorial Design and Physicochemical Characterization.

Oil-in-water nanoemulsions (NEs) are considered a suitable nanotechnological approach to improve the eye-related bioavailability of lipophilic drugs. The potential of cationic NEs is prominent due to the electrostatic interaction that occurs between the positively charged droplets with the negatively charged mucins present in the tear film. This interaction offers prolonged NEs residence at the ocular surface, increasing the drug absorption. Triamcinolone acetonide (TA) is one of the first pharmacologic strategies applied as an intravitreal injection in the treatment of age-related macular degeneration (AMD). Newly synthesized quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and quinuclidine surfactants have been screened with the purpose to select the best compound to formulate long-term stable NEs that combine the best physicochemical properties for the loading of TA intended for ocular administration.

Evaluation of Structurally Distorted Split GFP Fluorescent Sensors for Cell-Based Detection of Viral Proteolytic Activity.

Cell-based assays are essential for virus functional characterization in fundamental and applied research. Overcoming the limitations of virus-labelling strategies while allowing functional assessment of critical viral enzymes, virus-induced cell-based biosensors constitute a powerful approach. Herein, we designed and characterized different cell-based switch-on split GFP sensors reporting viral proteolytic activity and virus infection. Crucial to these sensors is the effective-yet reversible-fluorescence off-state, through protein distortion. For that, single (protein embedment or intein-mediated cyclization) or dual (coiled-coils) distortion schemes prevent split GFP self-assembly, until virus-promoted proteolysis of a cleavable sequence. All strategies showed their applicability in detecting viral proteolysis, although with different efficiencies depending on the protease. While for tobacco etch virus protease the best performing sensor was based on coiled-coils (signal-to-noise ratio, SNR, 97), for adenovirus and lentivirus proteases it was based on GFP11 cyclization (SNR 3.5) or GFP11 embedment distortion (SNR 6.0), respectively. When stably expressed, the sensors allowed live cell biosensing of adenovirus infection, with sensor fluorescence activation 24 h post-infection. The structural distortions herein studied are highly valuable in the development of cellular biosensing platforms. Additionally highlighted, selection of the best performing strategy is highly dependent on the unique properties of each viral protease.

Cationic Surfactants: Self-Assembly, Structure-Activity Correlation and Their Biological Applications.

The development of biotechnological protocols based on cationic surfactants is a modern trend focusing on the fabrication of antimicrobial and bioimaging agents, supramolecular catalysts, stabilizers of nanoparticles, and especially drug and gene nanocarriers. The main emphasis given to the design of novel ecologically friendly and biocompatible cationic surfactants makes it possible to avoid the drawbacks of nanoformulations preventing their entry to clinical trials. To solve the problem of toxicity various ways are proposed, including the use of mixed composition with nontoxic nonionic surfactants and/or hydrotropic agents, design of amphiphilic compounds bearing natural or cleavable fragments. Essential advantages of cationic surfactants are the structural diversity of their head groups allowing of chemical modification and introduction of desirable moiety to answer the green chemistry criteria. The latter can be exemplified by the design of novel families of ecological friendly cleavable surfactants, with improved biodegradability, amphiphiles with natural fragments, and geminis with low aggregation threshold. Importantly, the development of amphiphilic nanocarriers for drug delivery allows understanding the correlation between the chemical structure of surfactants, their aggregation behavior, and their functional activity. This review focuses on several aspects related to the synthesis of innovative cationic surfactants and their broad biological applications including antimicrobial activity, solubilization of hydrophobic drugs, complexation with DNA, and catalytic effect toward important biochemical reaction.

Optimization of nimesulide-loaded solid lipid nanoparticles (SLN) by factorial design, release profile and cytotoxicity in human Colon adenocarcinoma cell line.

The aim of this work is development of a nontoxic, long-term stable solid lipid nanoparticles (SLN) formulation for the loading of Nimesulide (NiM) by a 22 factorial design. The optimized formulation was composed of 10 wt% of glyceryl behenate and 2.5 wt% of poloxamer 188. Immediately after production, Z-Ave of NiM-SLN was 166.1 ± 0.114 nm, with a polydispersity index (PI) of 0.171 ± 0051 and zeta potential nearly neutral (-3.10 ± 0.166 mV). A slight increase of Z-Ave was recorded for NiM-SLN stored at 25 °C for a period of 15 days, whereas at 4 °C particles kept size within similar range. Long-term stability was monitored using TurbiscanLab®, showing a high stability of the nanoparticles with variations in the backscattering profiles below 10%. The release profile of NiM-SLN followed a sustained pattern with ca. 30% of drug released up to 24 h. Empty-SLN and NiM-SLN were nontoxic after exposing Caco-2 cells to the highest concentration (100 µg/mL) up to 48 hours (cell viability higher than 80%). NiM-SLN were lyophilized using different cryoprotectants, producing particles of 463.1 ± 36.63 nm (PI 0.491 ± 0.027) with 5% trehalose. Solid character of NiM-SLN was confirmed by DSC, recording a recrystallization index of 83% for NiM-SLN and of 74% for lyophilized SLN.

Phase Behavior of Polymorphic Fats in Drug Delivery Systems - A Review of the State of Art.

Fats are essential nutrients that have a significant role in the human diet and are essential to provide energy. Fatty acids are present in several types of lipids, such as triglycerides and phospholipids. Fatty acids differ among them, depending on the number of double bonds and on the length of the hydrocarbon chains. If there are no double bonds, the fatty acids are considered saturated and show a linear structure. Compounds with double bonds are unsaturated and have bent structure. The saturated fatty acids are usually solid at room temperature and the unsaturated fatty acids are liquid at that very same temperature. These compounds are of recognized value as raw materials for drug delivery systems, such as lipid nanoparticles. The behaviour of the macroscopic aspects of fat polymorphisms is directly influenced by the melting point, the crystallization and their polymorphic transformations. In this work, we revise the most critical factors contributing for the long-term stability of lipid nanoparticles, as well as the influence of the polymorphism on the loading capacity for drug molecules.

Neoplastic Multifocal Skin Lesions: Biology, Etiology, and Targeted Therapies for Nonmelanoma Skin Cancers.

Neoplastic skin lesions are multifocal, diffuse skin infiltrations of particular relevance in the differential diagnosis of ulcerative, nodular, or crusting skin lesions. Nonmelanoma skin cancers (NMSCs), namely, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and also actinic keratosis (AK), are the most common malignant tumors in humans. BCCs do not proliferate rapidly and most of the times do not metastasize, while SCCs are more infiltrative, metastatic, and destructive. AKs are precursor lesions of cutaneous SCCs. The classical therapy of NMSCs makes use of photodynamic therapy associated with chemotherapeutics. With improved understanding of the pathological mechanisms of tumor initiation, progression, and differentiation, a case is made towards the use of targeted chemotherapy with the intent to reduce the cytotoxicity of classical treatments. The present review aims to describe the current state of the art on the knowledge of NMSC, including its risks factors, oncogenes, and skin carcinogenesis, discussing the classical therapy against new therapeutic options.

Outcomes of acute kidney injury in a nephrology ward.

Acute kidney injury (AKI) is a global problem which predicts immediate and long-term adverse outcomes. We evaluated the AKI progression to end-stage renal disease, as well as the mortality associated with AKI and the in-hospital readmission rate because of a cardiovascular event in AKI patients admitted in a nephrology ward. A 5-year retrospective study was set in a nephrology department, with a follow-up period of up to 8 years. In a total of 191 patients, mean age was 73.83 ± 12.49 years, and 137 (71.7%) patients had history of chronic kidney disease. One hundred and twenty-four (65%) patients needed RRT and two (1%) needed surgery. Upon discharge, 107 (56%) patients had recovered the renal function, 41 (21.6%) patients had partial recovery, 25 (13%) patients were RRT dependent, 16 (8.4%) died, and two (1%) patients had outcomes unknown to us, because they were transferred to other hospitals. The median survival time free of RRT was 74 months. The median survival time of the followed patients was 34 months (95% CI 23.3-44.7). The mortality rate in the follow-up period in this sample was 18 deaths/100 patients-years, and the incidence of a composite cardiovascular endpoint of heart failure, acute coronary syndrome, and stroke was 6 events/100 patients-years. The mortality rate in the follow-up period was higher than usually described for patients outside intensive care unit, probably because our patients were old and had many comorbidities.

Improved regeneration and de novo bone formation in a diabetic zebrafish model treated with paricalcitol and cinacalcet.

Bone changes related to diabetes have been well stablished, but few strategies have been developed to prevent this growing health problem. In our work, we propose to investigate the effects of calcitriol as well as of a vitamin D analog (paricalcitol) and a calcimimetic (cinacalcet), in fin regeneration and de novo mineralization in a zebrafish model of diabetes. Following exposure of diabetic transgenic Tg(ins:nfsb-mCherry) zebrafish to calcitriol, paricalcitol and cinacalcet, caudal fins were amputated to assess their effects on tissue regeneration. Caudal fin mineralized and regenerated areas were quantified by in vivo alizarin red staining. Quantitative real-time PCR was performed using RNA from the vertebral column. Diabetic fish treated with cinacalcet and paricalcitol presented increased regenerated and mineralized areas when compared with non-treated diabetic group, while no significant increase was observed in non-diabetic fish treated with both drugs. Gene expression analysis showed an up-regulation for runt-related transcription factor 2b (runx2b), bone gamma-carboxyglutamic acid-containing protein (bglap), insulin a (insa) and insulin b (insb) and a trend of increase for sp7 transcription factor (sp7) in diabetic groups treated with cinacalcet and paricalcitol. Expression of insra and vdra was up-regulated in both diabetic and nondiabetic fish treated with cinacalcet. In nondiabetic fish treated with paricalcitol and cinacalcet a similar increase in gene expression could be observed but not so pronounced. The increased mineralization and regeneration in diabetic zebrafish treated with cinacalcet and paricalcitol can be explained by increased osteoblastic differentiation and increased insulin expression indicating pro-osteogenic potential of both drugs.

Prognostic significance of AKT/mTOR signaling in advanced neuroendocrine tumors treated with somatostatin analogs.

INTRODUCTION: Somatostatin analogs (SSAs) are used as part of standard treatment for advanced neuroendocrine tumors (NETs). The mechanisms behind the antiproliferative action of SSAs remain largely unknown, but a connection with the mammalian target of rapamycin (mTOR) signaling pathway has been suggested. Our purpose was to evaluate the activation status of the AKT/mTOR pathway in advanced metastatic NETs and identify biomarkers of response to SSA therapy. PATIENTS AND METHODS: Expression of phosphatase and tensin homolog (PTEN), phosphorylated (p)-AKT(Ser473), and p-S6(Ser240/244) was evaluated using immunohistochemistry in archival paraffin samples from 23 patients. Expression levels were correlated with clinicopathological parameters and progression-free survival under treatment with SSAs. RESULTS: A positive association between p-AKT and p-S6 expression was identified (P = 0.01) and higher expression of both markers was observed in pancreatic NETs. AKT/mTOR activation was observed without the loss of PTEN expression. Tumors showing AKT/mTOR signaling activation progressed faster when treated with SSAs: higher expression of p-AKT or p-S6 predicted a median progression-free survival of 1 month vs 26.5 months for lower expression (P = 0.02). CONCLUSION: Constitutive activation of the AKT/mTOR pathway was associated with shorter time-to-progression in patients undergoing treatment with SSAs. Larger case series are needed to validate whether p-AKT(Ser473) and p-S6(Ser240/244) can be used as prognostic markers of response to therapy with SSAs.

Metodologias laboratoriais para o diagnóstico de hemoglobinas variantes / The laboratory methodology for diagnosis of hemoglobin variants

Among the electrophoretical methodologies for hemoglobin examinations under different experimental conditions, isoelectrofocusing and high performance liquid chromatography are the most frequently used for the detection of hemoglobin variants. Moreover, molecular techniques are also developed in many laboratories. The correct laboratorial diagnosis of hemoglobin alterations is especially important for the interactive forms principally in Brazil, because of the many associations of hemoglobin alterations and variants with co-migration. The association of classical and molecular techniques is essential for accurate identification of hemoglobin variants.

Contribuição do estudo molecular de hemoglobinas S-like para o conhecimento da diversidade genética da população brasileira: [carta ao editor] / The contribution of molecular studies of S-like hemoglobins to knowledge of the genetic diversity of the Brazilian population: [letter to editor]

Over 900 hemoglobin variants have already been described, withmore than 100 having electrophoretic migration similar to Hb S inalkaline pHs. In the analysis of 98 samples that presented with thismigration pattern, Hb D-Los Angeles in heterozygosis was prevalentand associations with Hb S and Hb Lepore were identified, apreviously unpublished fact and with beta-thalassemia, which isalso rare. In addition, carriers of Hb Korle-Bu and Hb Hasharonwere found amongst other hemoglobinopathies. This heterogeneityreflects the special colonization conditions and the high racialadmixture in Brazil. The correct laboratorial diagnosis is veryimportant for hemoglobinopathy carriers because of the manyassociations of hemoglobin alterations and the variants with comigration.An accurate identification of hemoglobin variants isessential for genetic counseling and appropriate therapy.

Avaliação eletroforética, cromatográfica e molecular da Hb D Los Angeles no Brasil / Electrophoretical, chromatographic and molecular valuations of Hb D Los Angeles in Brazil

A variante de hemoglobina (Hb) D mais comum, Hb D Los Angeles ou D Punjab, é originada de uma transversão GAA->CAA no códon 121 da globina beta; essa mutação resulta na substituição do ácido glutâmico por glutamina na proteína. É a terceira variante de hemoglobina mais freqüente da população brasileira. Como as hemoglobinas D apresentam migração similar à hemoglobina S em pH alcalino, e com a hemoglobina A em pH ácido, são necessários vários testes para o correto diagnóstico. No presente estudo objetivou-se relacionar os diferentes procedimentos laboratoriais de rotina diagnóstica, além da análise molecular, para estabelecer o perfil de Hb D Los Angeles no Brasil. Foram analisados 47 indivíduos da população brasileira com provável Hb D Los Angeles, por vários procedimentos eletroforéticos em diferentes condições de pH, além da cromatografia líquida de alta pressão, e testes moleculares para confirmação da mutação. Foram encontrados quatro tipos de combinações de hemoglobinas: 42 indivíduos portadores de hemoglobina AD Los Angeles, dois indivíduos com doença de Hb S/D Los Angeles, dois indivíduos com Hb D Los Angeles e talassemia beta e um indivíduo com Hb D Los Angeles e Hb Lepore. Os indivíduos heterozigotos para D Los Angeles são assintomáticos, entretanto, em associação com outras variantes e talassemias podem apresentar graus variáveis de manifestações clínicas. Os resultados apresentados enfatizaram a necessidade da associação de várias metodologias para a identificação da Hb D Los Angeles, além de auxiliar na elucidação de combinações raras

The most common Hb D variant, the Hb D-Los Angeles,also know as Hb D-Punjab, originates through aGAA¨CAA change at the 121 codon of the ƒÀ globingene; this mutation results in the replacement ofglutamic acid for glutamine in the protein. It is thethird most common hemoglobin variant in theBrazilian population. This variant has electrophoreticmigration in alkaline pHs similar to Hb S and identicalmigration to hemoglobin A in acidic pHs. Thus, severaltechniques are necessary for its correct diagnosis. Thepurpose of this work was to relate the differentlaboratorial techniques and molecular analyses to determinethe profile of Hb D Los Angeles in Brazil. Fortysevenindividuals from the Brazilian population withHb D Los Angeles were studied. Multiple electrophoresisin several experimental conditions were carried out,in addition to high performance liquid chromatography (HPLC) and molecular analysis to confirm thismutation. Four compound heterozygotes were observed:42 individuals heterozygous Hb AD Los Angeles, twowith Hb S/D Los Angeles disease, two individuals withHb D Los Angeles and beta-thalassemia and one withHb D Los Angeles and Hb Lepore. The heterozygoushemoglobin D Los Angeles is asymptomatic, even thoughits association with other variants and thalassemias maypresent varying degrees of clinical manifestations. Theresults presented emphasize the significance of theassociation of different laboratorial techniques for DLos Angeles diagnosis, and help to elucidate rarecombinations.