Ethanol perturbs lipid organization in models of stratum corneum membranes: An investigation combining differential scanning calorimetry, infrared and (2)H NMR spectroscopy.

Ethanol is used in a variety of topical products. It is known to enhance the permeability of the skin by altering the ability of the stratum corneum (SC) intercellular membranes to form an effective barrier. In addition, ethanol and other alcohols are key components of antiseptic gels currently used for hand wash. Using infrared and deuterium NMR spectroscopy as well as calorimetry, we have investigated the effect of ethanol on a model membrane composed of lipids representing the three classes of SC lipids, an equimolar mixture of N-palmitoylsphingosine (ceramide), palmitic acid and cholesterol. Ethanol is found to influence the membrane in a dose dependent manner, disrupting packing and increasing lipid motion at low concentrations and selectively extracting lipids at moderate concentrations.

Biophysical characterization of a liposomal formulation of cytarabine and daunorubicin.

The biophysical characterization of CPX-351, a liposomal formulation of cytarabine and daunorubicin encapsulated in a synergistic 5:1 molar ratio (respectively), is presented. CPX-351 is a promising drug candidate currently in two concurrent Phase 2 trials for treatment of acute myeloid leukemia. Its therapeutic activity is dependent on maintenance of the synergistic 5:1 drug:drug ratio in vivo. CPX-351 liposomes have a mean diameter of 107 nm, a single phase transition temperature of 55.3 degrees C, entrapped volume of 1.5 microL/micromol lipid and a zeta potential of -33 mV. Characterization of these physicochemical properties led to identification of an internal structure within the liposomes, later shown to be produced during the cytarabine loading procedure. Fluorescence labeling studies are presented that definitively show that the structure is composed of lipid and represents a second lamella. Extensive spectroscopic studies of the drug-excipient interactions within the liposome and in solution reveal that interactions of both cytarabine and daunorubicin with the copper(II) gluconate/triethanolamine-based buffer system play a role in maintenance of the 5:1 cytarabine:daunorubicin ratio within the formulation. These studies demonstrate the importance of extensive biophysical study of liposomal drug products to elucidate the key physicochemical properties that may impact their in vivo performance.

Phase behavior of an equimolar mixture of N-palmitoyl-D-erythro-sphingosine, cholesterol, and palmitic acid, a mixture with optimized hydrophobic matching.

The phase behavior and lipid mixing properties of an equimolar mixture of nonhydroxylated palmitoyl ceramide (Cer16), palmitic acid (PA), and cholesterol have been investigated using 2H NMR and vibrational spectroscopy. This mixture is formed by the three main classes of lipids found in the stratum corneum (SC), the top layer of the epidermis, and provides an optimized hydrophobic matching. Therefore, its behavior highlights the role played by hydrophobic matching on the phase behavior of SC lipids. We found that, below 45 degrees C, the mixture is essentially formed of coexisting crystalline domains with a small fraction of lipids (less than 20%) that forms a gel or fluid phase, likely ensuring cohesion between the solid domains. Upon heating, there is the formation of a liquid ordered phase mainly composed of PA and cholesterol, including a small fraction of Cer16. This finding is particularly highlighted by correlation vibrational microspectroscopy that indicates that domains enriched in cholesterol and PA include more disordered Cer16 than those found in the Cer16-rich domains. Solubilization of Cer16 in the fluid phase occurs progressively upon further heating, and this leads to the formation of a nonlamellar self-assembly where the motions are isotropic on the NMR time scale. It is found that the miscibility of Cer16 with cholesterol and PA is more limited than the one previously observed for ceramide III extracted from bovine brain, which is heterogeneous in chain composition and includes, in addition to Cer16, analogous ceramide with longer alkyl chains that are not hydrophobically matched with cholesterol and PA. Therefore, it is inferred that, in SC, the chain heterogeneity is a stronger criteria for lipid miscibility than chain hydrophobic matching.

Effect of dimethyl sulfoxide on the phase behavior of model stratum corneum lipid mixtures.

Dimethyl sulfoxide (DMSO), an efficient transdermal enhancer, is proposed to alter the skin barrier by, at least partially, disturbing the lipid phase of the stratum corneum (SC). We have investigated, using differential scanning calorimetry and vibrational microspectroscopy, the effect of DMSO on the phase behavior of a lipid mixture formed by N-palmitoyl-d-erythro-sphingosine, deuterated palmitic acid, and cholesterol, mimicking the SC lipid phase. Our results reveal that DMSO favors the disordering of the lipid acyl chains. Moreover, the effect of DMSO is strongly concentration dependent and this dependence is reminiscent of that describing the DMSO transdermal enhancement. DMSO-induced fluidification affects primarily the fatty acid in the mixture. Therefore, it is proposed that the molecular mechanism of the transdermal transport enhancement caused by DMSO is associated with its H-bonding properties; its presence alters the interfacial H-bond network involving the fatty acid molecules and consequently the cohesive lipid packing.

Fatty acids influence "solid" phase formation in models of stratum corneum intercellular membranes.

Stacked intercellular lipid membranes in the uppermost epidermal layer, the stratum corneum (SC), are responsible for skin's barrier function. These membranes are unique in composition, the major lipids being ceramides (Cer), cholesterol, and free fatty acids (FFA) in approximately equimolar proportions. Notably, SC lipids include chains much longer than those of most biological membranes. Previously we showed that Cer's small hydrophilic headgroup enabled SC model membranes composed of bovine brain ceramide (BBCer), cholesterol, and palmitic acid in equimolar proportion to solidify at pH 5.2. In order to determine the influence of FFA chain length on the phase behavior of such membranes, we used 2H NMR and FT-IR to study BBCer/cholesterol/FFA dispersions containing linear saturated FFA 14-22 carbons long. Independent of chain length, the solid phase dominated the FFA spectrum at physiological temperature. Upon heating, each dispersion underwent phase transitions to a liquid crystalline phase (only weakly evident for the membrane containing FFA-C22) and then to an isotropic phase. The phase behavior, the lipid mixing properties, and the transition temperatures are shown to depend strongly on FFA chain length. A distribution of FFA chain lengths is found in the SC and could be required for the coexistence of a proportion of solid lipids with some more fluid domains, which is known to be necessary for normal skin barrier function.

Raman spectroscopy as a tool for measuring mutual-diffusion coefficients in hydrogels.

Raman spectroscopy has been exploited to characterize the diffusion properties of solutes in hydrogels. Raman active vibrations were used as intrinsic probes of the solute concentration along gel cylinders. The resulting one-dimensional solute distribution, characterized as a function of both time and space, could be analyzed with a model based on Fick's diffusion law, and the mutual-diffusion coefficient (Dm) was then determined. To illustrate the potential of this approach, we measured the Dm of two polyethylene glycols (PEG) in Ca-alginate gels. In this case, the intensity of the CH stretching band was used to obtain the concentration profiles of PEGs, whereas the OH stretching band of water was used as an internal intensity standard. In addition to providing a straightforward approach to measuring diffusion coefficients, the Raman profile analysis provides information relative to the accessibility of gels to large molecules. As an example, it was found that the PEG penetration in Ca-alginate gels was restricted, a phenomenon that was dependent on PEG size. The Raman technique presented here effectively characterizes transport properties of solutes in gels, and such characterization is required for developing several technical applications of gels, such as their use as materials for controlled release of drugs.

Self- and mutual-diffusion coefficients measurements by 31P NMR 1D profiling and PFG-SE in dextran gels.

31P NMR 1D profiling was successfully introduced to measure macroscale mutual-diffusion coefficients (D(m)) of phosphate ions in dextran gels. Series of 1D profiles describing the phosphate concentration along cylindrical dextran gels were acquired at different times. These profiles that included over 600 points could be fitted using equations derived from Fick's law, with D(m) as the single fitting parameter. Release and penetration profiles were recorded providing two alternative approaches for allowing the determination of D(m). The D(m) values were compared with microscale self-diffusion coefficients (D(s)) measured by pulsed field gradient spin echo (PFG-SE) technique. D(m) values, measured between 25 and 45 degrees C, were systematically lower than D(s). The experimental diffusion time and the associated diffusion length of D(s) (60 ms, 10 microm) are short compared to those of D(m) (up to 18 h, 50 mm). These scale differences are considered to be the origin of different D(s) and D(m) and provide information relative to the network in these gels.