Desenvolvimento de sistemas poliméricos nanoparticulados de PS-b-PAA com superfície modificada com derivados de quitosana para a encapsulação de minoxidil

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas, Programa de Pós-Graduação em Química, 2015.O presente trabalho descreve a síntese e caracterização de três derivados de quitosana que foram utilizados para decorar vesículas produzidas a partir da auto-associação do copolímero em bloco PS404-b-PAA63 visando a encapsulação do minoxidil. As vesículas foram obtidas usando o método de co-solvente orgânico (dioxano) em água e caracterizadas quanto ao seu tamanho por Espalhamento de Luz Dinâmico (DLS), morfologia por Microscopia Eletrônica de Transmissão (TEM) e potencial zeta. Essas vesículas apresentam morfologia bem definida com diâmetros inferiores a 200 nm e, devido à dissociação das porções de ácido carboxílico do bloco hidrofílico, estas vesículas exibem carga de superfície negativa, o que possibilita o revestimento da sua superfície com polissacarídeos de carga oposta, tais como quitosana e derivados de quitosana. Após a decoração, estas vesículas são claramente diferentes. Com a adição e o aumento da concentração de quitosana ou derivados de quitosana, o valor de potencial zeta diminui, passando por uma região de transição, próximo do potencial zero, onde são observadas a formação de agregados. Depois de alcançar o máximo de adsorção, os parâmetros de potencial zeta se estabilizam, as vesículas se re-dispersam e o diâmetro hidrodinâmico fica ligeiramente maior quando comparado com as vesículas não decoradas. A eficiência de encapsulação (EE) do minoxidil, quantificada por eletroforese capilar, resulta numa EE máxima de 50,7 %, o que está em boa concordância com a capacidade de carga de tais vesículas.Abstract : The present work describes the synthesis and characterization of three chitosan derivatives which were used to decorate vesicles made from the self-assembly of the block copolymer PS404-b-PAA63 aiming encapsulation of minoxidil. The vesicles were obtained by using the organic (dioxane) co-solvent method in water and characterized according to their size by Dynamic Light Scattering (DLS), morphology by Transmission Electron Microscopy (TEM) and zeta potential. These vesicles have well-defined morphology with diameters lower than 200 nm and, due to the dissociation of the carboxylic acid moieties of the hydrophilic block, these vesicles have a negative surface charge, which allows the coating of its surface with oppositely charged polysaccharides, such as chitosan and chitosan derivatives. After the decoration, these vesicles were clearly different. With the addition and the concentration increases of chitosan or chitosan derivatives, the zeta potential value decreases, passing through a transition region, near zero potential, where?s observed the formation of aggregates. After reaching maximum adsorption, zeta potential parameters are stabilized, the vesicles are re-dispersed and the hydrodynamic diameter is mildly larger when compared to the vesicles without decoration. The encapsulation efficiency (EE) of minoxidil, quantified by capillary electrophoresis, results in an EE maximum of 50,7 %, which is in good agreement with the cargo ability of these vesicles

Voltage-gated Na⁺ channel activity increases colon cancertranscriptional activity and invasion via persistent MAPK signaling

© 2015 Macmillan Publishers Limited. All rights reserved. Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes

Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated

Re-Evaluation of the Action Potential Upstroke Velocity as a Measure of the Na+ Current in Cardiac Myocytes at Physiological Conditions

Background: The SCN5A encoded sodium current (INa) generates the action potential (AP) upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of INa with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of INa, which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC) technique might provide an alternative to traditional voltage clamp (VC) technique for the determination of INa properties under physiological conditions. Principal Findings: We studied INa under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak INa during a depolarizing VC step or maximal upstroke velocity, dV/dtmax, during VC/CC served as an indicator of available INa. In HEK cells, biophysical properties of INa, including current density, voltage dependent (in)activation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied INa in left ventricular myocytes isolated from control or failing rabbit hearts

A Critical Review of Properties and Analytical Methods for the Determination of Docetaxel in Biological and Pharmaceutical Matrices

Docetaxel (DTX) is an antineoplastic agent of the second generation of the taxoid family. It is a semi-synthetic drug prepared from a precursor extracted of the plant Taxus baccata. The commercial formulation of DTX, Taxotere®, employs the surfactant polysorbate 80, due to the low water solubility of the drug, causing several side effects. Therefore, there is a need to develop delivery systems to reduce the side effects of DTX. In addition, this drug has been qualitative and quantitatively analyzed in pharmaceutical formulations and biological samples. Thus, several techniques and analytical methods have been reported with the aim of optimizing the analytical signal, increasing sensitivity, selectivity and reducing the effects of interference. Herein, we highlight immunoassay, capillary electrophoresis and chromatographic methods. This review presents a summary of physicochemical and pharmacokinetics properties, mechanisms of action, drug delivery systems and analytical methods used in quantification of DTX in diverse matrices such as blood, plasma, oral fluid, urine, carcinoma cells, pharmaceutical formulations and delivery systems.São Paulo State University (UNESP) School of Pharmaceutical SciencesSão Paulo State University (UNESP) School of Pharmaceutical Science