Stability of liposomal formulations in physiological conditions for oral drug delivery.

The stability of liposomal formulations is a key issue in drug delivery. Liposomes made of egg phosphatidylcholine (EPC), cholesterol (Chol), sphingomyelin (SM), and gangliosides (GM1 and GM type III) were incubated in different media to determine their stability. Mixtures containing GM1 or GM type III were found to be the most stable, and both showed similar stability trends in plasma at 37 degrees C. EPC/Chol was the most susceptible to lysis in plasma. In acid media (pH 2), the highest stability corresponded to EPC/Chol, whereas in bile and pancreatin, liposomes with GM1 and GM type III were more stable than those containing SM. This study suggests that among the formulations used as oral drug carriers, those containing GM1 and GM type III have higher possibilities of surviving through the gastrointestinal tract.

Interaction of sodium nitroprusside/liposome system with bovine serum albumin: a short-cut determination.

A rapid determination of protein-liposome binding was developed to predict the circulation time of the system within an animal, which is a function of the amount and type of protein bound. The binding pattern of albumin to liposomes, with and without sodium nitroprusside (SNP), was analyzed by SDS-PAGE. Liposomes were made of egg yolk lecithin, soybean lecithin and dimyristoyl lecithin, and contained SNP. They bound 58%, 26% and 100% bovine serum albumin, respectively, when compared to their corresponding controls lacking SNP. The method applied is simpler and significantly faster than ordinary chemical determinations.

Study of in vitro stability of liposomes and in vivo antibody response to antigen associated with liposomes containing GM1 after oral and subcutaneous immunization.

In order to evaluate the usefulness of liposomes as possible vaccine vehicles (oral and subcutaneous), the stability of liposomes in buffer, plasma and saliva at 25 and 37 degrees C was analyzed via fluorescence and enzymatic methodology. The tested mixtures included [EggPC/Chol] 1 : 1 (mixture I), [EggPC/Chol/SM] 1 : 1 : 1 (mixture II), [EggPC/Chol/SM/GM typeIII] 1 : 1 : 1 : 0.14 (mixture III), [EggPC/Chol/SM/GM1] 1 : 1 : 1 : 0.14 (mixture IV) and [DIAPC/DMPC] 1 : 1 non polymerized (mixture V) and polymerized (mixture VI); all mole ratio. Liposome mixtures I and II were more stable in buffer at 25 degrees C. On the other hand, mixtures III and IV were more stable in plasma at 37 degrees C; mixture VI was more stable in plasma at 37 degrees C than in buffer or saliva. Mixtures IV and V liposomes were both stable in saliva for at least one hour. Blood and feces anti-GM1 response to antigen associated liposomes after subcutaneous and oral administration was also examined. After mixture IV mice immunization, no detectable anti-ganglioside GM1 antibody response was detected. Negative stain transmission electron microscopy, shows that liposomes containing SM, GM1, GM typeIII and DIAPC : DMPC were twice the size of those made with EggPC/Chol. The hydrophobicity factor expressed as A(570/500) was obtained using the probe merocynine 540 (MC540). The order of fluidity increased from: mixture II < mixture I < mixture III < mixture IV < mixtureV < mixture VI. Although the high hydrophobicity factor for polymerizable lipids there are other factors like stability must be taken into account according to the administration via selected. Also, the hydrophilicity of the groups protruding from the membrane interphase into the solution in the case of subcutaneous inoculation is very relevant and for oral administration stability is the property to take into account, as long as they have to last through the different fluids of the gastrointestinal tract. The results obtained suggest that liposomes that showed stability in saliva and plasma at 37 degrees C containing GM1, GM typeIII or DIAPC/DMPC would serve effectively as a delivery vehicle for oral and subcutaneous non-viral vaccines.

Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ.

Interactions between merocyanine 540 (MC540) and nicotinic acetylcholine receptor (AChR) have been studied by visible absorption spectroscopy using native receptor-rich membranes from Discopyge tschudii electric tissue and liposomes obtained by aqueous dispersion of endogenous lipids extracted from the same tissue. The fact that merocyanine partitions into the membrane when this is in the liquid-crystalline state, exhibiting a characteristic peak at 567 nm, was exploited to obtain quantitative information about the physical state of the AChR-rich membrane. Spectra of MC540 revealed that this molecule was preferentially incorporated into AChR-rich membranes, with an affinity (Kdapp 30 microM) 10-fold higher than that in liposomes (Kdapp 290 microM). Changes were observed in the equilibrium dissociation constant of MC540 at different temperatures: the two-fold higher affinity at 8 degrees C than at 23 degrees C can be rationalized in terms of a higher value of the overall dimerization constant (Kdim) at the lower temperature. The local anaesthetic benzocaine competed for MC540 binding sites with higher potency in AChR-rich native membranes than in liposomes made with endogenous lipids. This competition was found to be AChR concentration-dependent, whereas in liposomes the displacement was constant at different lipid/MC540 molar ratios. Titration experiments yielded an apparent dissociation constant for benzocaine of 0.6 mM and 0.7 mM for liposomes and AChR-rich membranes, respectively. The possible location of the benzocaine binding site is deduced from the competition experiments to be at the lipid annulus surrounding the nicotinic AChR protein.

Properties of gel phase lipid-trehalose bilayers upon rehydration.

When dipalmitoylphosphatidylcholine bilayers dried under vacuum in different concentrations of trehalose are rehydrated in buffer without the sugar they show different physicochemical properties in the gel state in comparison to the normal gel state. Dry DPPC/trehalose mixtures are readily dispersed in buffer below the phase transition showing by electron microscopy a morphology similar to liposomes prepared by dispersing the lipids in buffer above the phase transition temperature. In these conditions, an increase in the peak at 570 nm of merocyanine after the dehydration-rehydration process in the presence of the sugar is observed and the water permeation increases to values comparable to those found in the fluid state as indicated by the activation energy values and the osmotic volume. The trehalose-dried liposomes rehydrated in buffer show a similar osmotic response to hypertonic gradient as DPPC liposomes without sugar near the phase transition temperature. In accordance with this behavior the trehalose-dried liposomes are lysed below the phase transition temperature by lysoderivatives. These modifications of the gel state of hydrated phospholipids by trehalose can only be achieved if a drastic dehydration is performed in the presence of the sugar. After rehydration the changes in the gel state can be detected after dyalizing the rehydrated membranes in media without trehalose during at least 24 h. These results suggest that trehalose is still intercalated between the phospholipids after restoring water to the dried liposomes either at temperatures below or above the phase transition.

Effect of dolichyl monophosphate on the permeability properties of lipid membranes.

The effect of dolichyl monophosphate on the permeability properties of dimyristoylphosphatidylcholine bilayers to alkaline cations, Ca2+ and glucose has been determined by stop-flow spectrophotometry. The results show that, in contrast to free dolichol effects, the monophosphate derivative increased the permeability following a decreasing order of the permeating particle size. Phase diagrams indicate that dolichyl monophosphate is fully incorporated into the phosphatidylcholine bilayer around 0.75% weight/weight ratio. For these ratios, the permeation of ions is higher in the gel than in the liquid crystalline state.

Synthesis, characterization, and black lipid membrane studies of [7-L-alanine] gramicidin A.

With a view to study the relevance of side-chain orientation in the transport of cations through a gramicidin transmembrane channel and to identify an analogue with favorable characteristics, [L-Ala7] gramicidin A was synthesized, purified, verified, and characterized by high-performance liquid chromatography, by carbon-13 and proton magnetic resonance spectra, and by circular dichroism spectra in methanol. Complete incorporation as the channel state was achieved when packaged in lysolecithin-containing lipid bilayers. The single-channel conductance data in diphytanoyllecithin/n-decane membranes are presented along with those of synthetic gramicidin A (GA). [L-Ala7] GA exhibits the highest most probable single-channel conductance so far reported for an analogue occurring at 28 pS as compared to 21 pS for GA under similar conditions. Also, a dramatic reduction in the dispersity of conducting states is observed with about 76% of the events falling in a narrow 1.75-pS conductance window as compared to about 31% of the events for GA under identical conditions. Thus, with the above characteristics, [L-Ala7]GA appears to be a very good candidate for a thorough study of ionic mechanism. The present results indicate that elements intrinsic to the channel proper are rate-limiting for GA and that there is no interfacial polarization or diffusion-controlled association at 1 M KCl and a 100-mV applied potential.

Shortened analog of the gramicidin A channel argues for the doubly occupied channel as the dominant conducting state.

A shortened analog of the gramicidin A transmembrane channel has been synthesized and its transport characterized in planar lipid bilayer membranes. General considerations of a shorter diffusional length and a shorter distance over which the voltage drop occurs (i.e., an increased electric field) would contribute to an increase in single-channel conductance. The finding of a decreased single-channel conductance supports the perspective that the dominant conducting state is the doubly occupied channel wherein distance-dependent repulsion due to the first ion in the channel impedes entry of the second ion in the shorter channel.

The source of the dispersity of gramicidin A single-channel conductances. The L X Leu5-gramicidin A analog.

The L X Leu5-gramicidin A analog has been designed, synthesized, and verified for the purpose of testing the perspective that different side-chain distributions are responsible for the dispersity of single-channel conductances. The structural analysis shows that the L . Leu5 analog would limit the possible number of side-chain distributions and that the dispersity of channel conductances would decrease. The finding is the expected decrease in less probable conductance states and the associated increase in the probability of the most probable conductance state.

Dispersity of des-L X Val7-D X Val8-Gramicidin A single channel conductances argues for different side chain orientations as basis.

Planar bilayer studies are reported on the channel activity of des-L X Val7-D X Val8-Gramicidin A. This analog is designed to provide more long-lived side chain distributions involving the Trp residues than occur with Gramicidin A. The carbonyls of these residues coordinate the permeant cation and the energetics of the coordination, which is proposed to depend on side chain orientation, determines the free energies of the rate limiting entrance-exit barriers and the binding sites. The finding of an increased dispersity of single channel conductance for the analog supports the perspective that dispersity derives from different side chain distributions on the same backbone conformation. Channel mechanism is not understood until dispersity is explained.

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel.

A study of the temperature dependence of gramicidin A conductance of K+ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation, vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.