RESUMO
Dibucaine (DBC) is one of the most potent long-acting local anesthetics, but it also has significant toxic side effects and low water solubility. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been proposed as drug-delivery systems to increase the bioavailability of local anesthetics. The purpose of the present study was to characterize SLNs and NLCs composed of cetyl palmitate or myristyl myristate, a mixture of capric and caprylic acids (for NLCs only) plus Pluronic F68 prepared for the encapsulation of DBC. We intended to provide a careful structural characterization of the nanoparticles to identify the relevant architectural parameters that lead to the desirable biological response. Initially, SLNs and NLCs were assessed in terms of their size distribution, morphology, surface charge, and drug loading. Spectroscopic techniques (infrared spectroscopy and electron paramagnetic resonance, EPR) plus small-angle X-ray scattering (SAXS) provided information on the interactions between nanoparticle components and their structural organization. The sizes of nanoparticles were in the 180 nm range with low polydispersity and negative zeta values (-25 to -46 mV). The partition coefficient of DBC between nanoparticles and water at pH 8.2 was very high (>104). EPR (with doxyl-stearate spin labels) data revealed the existence of lamellar arrangements inside the lipid nanoparticles, which was also confirmed by SAXS experiments. Moreover, the addition of DBC increased the molecular packing of both SLN and NLC lipids, indicative of DBC insertion between the lipids, in the milieu assessed by spin labels. Such structural information brings insights into understanding the molecular organization of these versatile drug-delivery systems which have already demonstrated their potential for therapeutic applications in pain control.
Assuntos
Anestésicos Locais/química , Dibucaína/química , Portadores de Fármacos/química , Nanopartículas/química , Espectroscopia de Ressonância de Spin Eletrônica , Miristatos/química , Nanopartículas/ultraestrutura , Palmitatos/química , Tamanho da Partícula , Poloxâmero/química , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of dibucaine in toad skin, which may be interpreted as reflecting inhibition of the active transport of ions. This finding might be explained on the basis of the results obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, dibucaine induced structural perturbations in IUM, DMPC LUV and phospholipid multilayers. Scanning electron microscopy revealed that dibucaine induced erythrocyte stomatocytosis. According to the bilayer couple hypothesis an echinocytic type of shape change would have been expected given the preferential interaction of dibucaine with DMPC. Although it is still premature to define the molecular mechanism of action of dibucaine, the experimental results confirm the important role played by the phospholipid bilayers in the association of the anesthetic with cell membranes.
Assuntos
Dibucaína/química , Dibucaína/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Pele/efeitos dos fármacos , Sódio/metabolismo , Animais , Anuros , Transporte Biológico/efeitos dos fármacos , Dimiristoilfosfatidilcolina/química , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestrutura , Feminino , Humanos , Técnicas In Vitro , Bicamadas Lipídicas/química , Lipossomos/química , Masculino , Microscopia Eletrônica de Varredura , Modelos Biológicos , Fosfatidiletanolaminas/química , Pele/metabolismo , Difração de Raios XRESUMO
This work elucidates differences in the hemolytic pathway developed by the antipsychotic trifluoperazine (TFP), the local anesthetic dibucaine (DBC) and the antihelminthic praziquantel (PZQ). Their partition coefficients (P) were measured at pH 7.4 between n-octanol, microsomes, liposomes, erythrocyte ghosts and n-octanol/water. The effective drug:lipid molar ratios for the onset of membrane solubilization (ReSAT) and complete hemolysis (ReSOL) were calculated from the experimental P values and compared with a classical surface-active compound treatment Lichtenberg, D. Biochim. Biophys. Acta 821 (1985) 470-478[. The contribution of charged/uncharged forms of TFP and DBC for the hemolytic activity was also analyzed. In all cases the hemolytic phenomena could be related to the monomeric drug insertion into the membrane. Only for TFP at isosmotic condition lysis occurs at concentrations beyond the CMC of the drug, indicating that micellization facilitates TFP hemolytic effect, while DBC and PZQ reach a real membrane saturation at their monomeric form.
Assuntos
Dibucaína/farmacologia , Eritrócitos/efeitos dos fármacos , Hemólise , Microssomos Hepáticos/efeitos dos fármacos , Praziquantel/farmacologia , Trifluoperazina/farmacologia , Anestésicos Locais/química , Anestésicos Locais/farmacologia , Animais , Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Antipsicóticos/química , Antipsicóticos/farmacologia , Dibucaína/química , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/fisiologia , Eritrócitos/fisiologia , Concentração de Íons de Hidrogênio , Cinética , Bicamadas Lipídicas , Camundongos , Microssomos Hepáticos/fisiologia , Praziquantel/química , Trifluoperazina/químicaRESUMO
Binding of micromolar concentrations of the local anesthetic dibucaine to micelles of cationic, zwitterionic and anionic detergents was studied using the fluorescence emission of dibucaine. Difference in quantum yields for charged and neutral dibucaine allowed to obtain shifts of pKa values due to binding. Estimates for the electrostatic potential affecting the tertiary amine of dibucaine were obtained from the pKa shifts. Change of fluorescence emission upon binding allowed to obtain the binding constants of both charged and neutral dibucaine to the micelles. The binding constant for the neutral form is essentially independent of micelle charge and of specific differences in detergent structure. Consistency between the ratio of neutral to cationic dibucaine binding constants and the measured pKa shift was tested. For LPC micelles complete agreement was found. For CTAC, however, the ratio of binding constants does not explain the pKa shift. The discrepancy between the results is used to estimate the errors involved upon neglecting non-coulombic electrostatic interactions of drugs to charged membrane surfaces. Fluorescence quenching with sodium iodide and nitroxide stearic acid derivatives allowed a depth profiling of the drug in the micelles.