Besifloxacin liposomes with positively charged additives for an improved topical ocular delivery.

Topical ophthalmic antibiotics show low efficacy due to the well-known physiological defense mechanisms of the eye, which prevents the penetration of exogenous substances. Here, we aimed to incorporate besifloxacin into liposomes containing amines as positively charged additives and to evaluate the influence of this charge on drug delivery in two situations: (i) iontophoretic and (ii) passive treatments. Hypothesis are (i) charge might enhance the electromigration component upon current application improving penetration efficiency for a burst drug delivery, and (ii) positive charge might prolong formulation residence time, hence drug penetration. Liposomes elaborated with phosphatidylcholine (LP PC) or phosphatidylcholine and spermine (LP PC: SPM) were stable under storage at 6 ºC for 30 days, showed mucoadhesive characteristics, and were non-irritant, according to HET-CAM tests. Electron paramagnetic resonance spectroscopy measurements showed that neither the drug nor spermine incorporations produced evident alterations in the fluidity of the liposome's membranes, which retained their structural stability even under iontophoretic conditions. Mean diameter and zeta potential were 177.2 ± 2.7 nm and - 5.7 ± 0.3 mV, respectively, for LP PC; and 175.4 ± 1.9 nm and + 19.5 ± 1.0 mV, respectively, for LP PC:SPM. The minimal inhibitory concentration (MIC) and the minimal bactericide concentration (MBC) of the liposomes for P. aeruginosa showed values lower than the commercial formulation (Besivance). Nevertheless, both formulations presented a similar increase in permeability upon the electric current application. Hence, liposome charge incorporation did not prove to be additionally advantageous for iontophoretic therapy. Passive drug penetration was evaluated through a novel in vitro ocular model that simulates the lacrimal flow and challenges the formulation resistance in the passive delivery situation. As expected, LP PC: SPM showed higher permeation than the control (Besivance). In conclusion, besifloxacin incorporation into positively charged liposomes improved passive topical delivery and can be a good strategy to improve topical ophthalmic treatments.

On the interaction of bovine serum albumin with ionic surfactants: temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility.

The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations, HPS presented a smaller effect at concentrations above 1.5mM. At 10mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant-BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue.

Molecular dynamics and partitioning of di-tert-butyl nitroxide in stratum corneum membranes: effect of terpenes.

In this work, we have used electron paramagnetic resonance (EPR) spectroscopy of the small spin label di-tert-butyl nitroxide (DTBN), which partitions the aqueous and hydrocarbon phases, to study the interaction of the terpenes alpha-terpineol, 1,8-cineole, L(-)-carvone and (+)-limonene with the uppermost skin layer, the stratum corneum, and the membrane models of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The EPR spectra indicated that the terpenes increase both the partition coefficient and the rotational correlation time of the spin labels in the stratum corneum membranes, whereas similar effects were observed in the DMPC and DPPC bilayers only at temperatures below the liquid-crystalline phase. The EPR parameter associated to probe polarity inside the membranes showed thermotropically induced changes, suggesting relocations of spin probe, which were dependent on the membrane phases. While the DMPC and DPPC bilayers showed abrupt changes in the partitioning and rotational correlation time parameters in the phase transitions, the SC membranes were characterized by slight changes in the total range of measured temperatures, presenting the greatest changes or membranes reorganizations in the temperature range of approximately 50 to approximately 74 degrees C. The results suggest that terpenes act as spacers, weakening the hydrogen-bonded network at the polar interface and thus fluidizing the stratum corneum lipids.

Terpenes increase the partitioning and molecular dynamics of an amphipathic spin label in stratum corneum membranes.

In this work, the interaction of the skin penetration enhancers dl-menthol, alpha-terpineol, 1,8-cineole and (+)-limonene with the uppermost skin layer, the stratum corneum and with multilamellar vesicles from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is investigated by electron paramagnetic resonance (EPR) spectroscopy of the small spin label 2,2,6,6,-tetramethylpiperedine-1-oxyl (TEMPO), which partitions the aqueous and hydrocarbon phases. The EPR spectrum allows for the determination of the actual partition coefficient and the rotational diffusion rates of the spin probe in the two environments. The enthalpy changes, DeltaH degrees , to transfer the spin probe from the aqueous to the hydrocarbon phase, as well as the activation energies associated to its rotational motion, were considerably smaller for stratum corneum, indicating less pronounced thermal reorganization. For DPPC, the terpenes increased both the partition coefficient and the rotational diffusion rate of the spin label in the membrane, except in the liquid-crystalline phase, while these increases in stratum corneum were observed in the entire temperature range measured with the exception of the rotational motion parameter for dl-menthol and alpha-terpineol at temperatures below their melting point (32-41 degrees C). It is suggested that the terpenes effectively acting as spacers in the membrane fluidize the lipids and cause ruptures in the hydrogen-bonded network of the polar interface.

Effects of 1,8-cineole on the dynamics of lipids and proteins of stratum corneum.

The interaction of a potent percutaneous penetration enhancer, 1,8-cineole, with the stratum corneum (SC) and DPPC membranes was investigated by electron paramagnetic resonance spectroscopy (EPR) of spin-labeled analogs of stearic acid (5-DSA) and androstanol (ASL). The EPR spectra of lipid derivatives spin probes structured in stratum corneum tissue of neonatal rat containing of 0.1-10% (v/v) 1,8-cineole in the solvent indicate an abrupt increase in membrane fluidity at around 1% 1,8-cineole. These spectra of stratum corneum membranes are characterized by the presence of two spectral components differing in mobility. Component 1 was attributed to the spin labels H-bonded to the headgroups, while component 2 possibly arose from spin labels H-bonded to water molecules or temporally non-hydrogen-bonded. With the addition of 1,8-cineole, the spin probes were transferred from the motionally more restricted component 1 to the more mobile component 2, suggesting that 1,8-cineole causes ruptures in the hydrogen-bonded network of the membrane-water interface, with consequent displacements of spin probes towards the hydrophobic core. 1,8-Cineole increased the rotational diffusion rates of component 2, whereas no significant mobility changes were observed in component 1. The EPR spectra of maleimide derivative spin label (6-MSL) covalently attached to stratum corneum proteins indicate that 1,8-cineole does not alter the dynamics of protein backbones. Instead, this terpene only increases the solvent's ability to 'dissolve' and mobilize the nitroxide side chain, which is in agreement with its low irritation response.

Effects of ethanol/l-menthol on the dynamics and partitioning of spin-labeled lipids in the stratum corneum.

The interaction of ethanol as well as ethanol/L-menthol mixtures with the uppermost layer of epidermis, the stratum corneum, was investigated by electron paramagnetic resonance (EPR) spectroscopy utilizing spin-labeled analogs of androstanol (ASL), stearic acid (5-DSA) and methyl stearate (5-DMS). The EPR spectra of these spin probes structured in stratum corneum tissue of neonatal rat are characterized by the coexistence of two spectral components indicating the presence of two classes of spin labels with very different states of mobility. Probably, one class of spin labels is H-bonded to the polar surface of the membrane and another class corresponds to spin labels more deeply inserted in the hydrophobic core. EPR results showed that in the ethanol range 0-70% neither fluidity in stratum corneum membranes nor the relative fractions of these two components changes were observed. Instead, ethanol only caused a selective extraction of spin labels. The removal of the steroid ASL began at 30% ethanol, reaching extraction levels over 50% at ethanol concentrations of 60-70%, whereas the more hydrophobic 5-DMS was partially removed only with 70% ethanol. Addition of 5% L-menthol to the solvent containing 20% ethanol increases both the mobility and the fraction of those spin labels situated in the hydrophobic core (more mobile spectral component). Altogether, these findings suggest that the L-menthol stabilizes mainly in the central region of stratum corneum membranes attracting the membrane lipids and causing hydrogen bond ruptures in the polar membrane interface.