Ultraviolet irradiation of diacetylenic liposomes as a strategy to improve size stability and to alter protein binding without cytotoxicity enhancement.

Membrane-modification effects, induced by ultraviolet (UV) irradiation in diacetylenic liposomes, were analyzed upon contact with cells, biological membranes, and proteins. Liposomes formulated with mixtures of unsaturated 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine and saturated 1,2-dimyristoyl-sn-glycero-3-phosphocholine, in a 1:1 molar ratio, were compared with those that were UV-irradiated and analyzed in several aspects. Membrane polymerization inherence on size stability was studied as well as its impact on mitochondrial and microsomal membrane peroxidation induction, hemolytic activity, and cell viability. Moreover, in order to gain insight about the possible irradiation effect on interfacial membrane properties, interaction with bovine serum albumin (BSA), lysozyme (Lyso), and apolipoprotein (apoA-I) was studied. Improved size stability was found for polymerized liposomes after a period of 30 days at 4°C. In addition, membrane irradiation had no marked effect on cell viability, hemolysis, or induction of microsomal and mitochondrial membrane peroxidation. Interfacial membrane characteristics were found to be altered after polymerization, since a differential protein binding for polymerized or nonpolymerized membranes was observed for BSA and Lyso, but not for apoA-I. The substantial contribution of this work is the finding that even when maintaining the same lipid composition, changes induced by UV irradiation are sufficient to increase size stability and establish differences in protein binding, in particular, reducing the amount of bound Lyso and BSA, without increasing formulation cytotoxicity. This work aimed at showing that the usage of diacetylenic lipids and UV modification of membrane interfacial properties should be strategies to be taken into consideration when designing new delivery systems.

Structural characterization of photopolymerizable binary liposomes containing diacetylenic and saturated phospholipids.

The use of liposomes to encapsulate materials has received widespread attention for drug delivery, transfection, diagnostic reagent, and as immunoadjuvants. Phospholipid polymers form a new class of biomaterials with many potential applications in medicine and research. Of interest are polymeric phospholipids containing a diacetylene moiety along their acyl chain since these kinds of lipids can be polymerized by Ultra-Violet (UV) irradiation to form chains of covalently linked lipids in the bilayer. In particular the diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) can form intermolecular cross-linking through the diacetylenic group to produce a conjugated polymer within the hydrocarbon region of the bilayer. As knowledge of liposome structures is certainly fundamental for system design improvement for new and better applications, this work focuses on the structural properties of polymerized DC8,9PC:1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. Liposomes containing mixtures of DC8,9PC and DMPC, at different molar ratios, and exposed to different polymerization cycles, were studied through the analysis of the electron spin resonance (ESR) spectra of a spin label incorporated into the bilayer, and the calorimetric data obtained from differential scanning calorimetry (DSC) studies. Upon irradiation, if all lipids had been polymerized, no gel-fluid transition would be expected. However, even samples that went through 20 cycles of UV irradiation presented a DSC band, showing that around 80% of the DC8,9PC molecules were not polymerized. Both DSC and ESR indicated that the two different lipids scarcely mix at low temperatures, however few molecules of DMPC are present in DC8,9PC rich domains and vice versa. UV irradiation was found to affect the gel-fluid transition of both DMPC and DC8,9PC rich regions, indicating the presence of polymeric units of DC8,9PC in both areas. A model explaining lipids rearrangement is proposed for this partially polymerized system.

Liposome/DNA systems: correlation between hydrophobicity and DNA conformational changes.

In a previous work, we found that liposome hydrophobicity could affect deoxyribonucleic acid (DNA) association efficiency. Now, we have focused on the possible correlation between liposome hydrophobicity and DNA conformation. DNA lyophilized with cationic vesicles with high hydrophobicity changes its conformation into a more condensed form, probably the C form. With noncharged vesicles, it changes its conformation from B to a partial A form. These results contribute to a better understanding of the interaction between DNA and lipids, suggesting there is direct relationship between hydrophobicity and DNA conformation changes: The higher the hydrophobicity factor, the more pronounced the changes in DNA form, to a more condensed form.

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.

Effect of lindane and heptachlor on delta-aminolaevulinate synthase and its regulation.

The effect of lindane and heptachlor on haem metabolism was studied with the aim to elucidate the mechanism of their porphyrinogenic action. The effects of these compounds on delta-aminolaevulinate synthase (ALA-S) and ferrochelatase were evaluated and the mechanism of increase of ALA-S activity was especially studied. The results indicated the following: (1) Lindane and heptachlor produced increases in ALA-S activity; this effect was dependent on the drug dose, the time of treatment, and the development of the animal, the maximum response being obtained prior to hatching. Lindane was observed to have a greater effect on ALA-S than heptachlor. In fact, when effects of lindane and heptachlor were compared we observed that lindane produced: (a) greater increases in ALA-S activity (six fold vs four fold), both with respect to dimethyl sulphoxide (DMSO) controls (3.8+/-0.3 nmol ALA/g liver per h); (b) earlier ALA-S response (1.5 h vs 4 h); (c) responses at lower doses (0.3 mg/egg vs 1 mg/egg). (2) The increase in ALA-S activity produced by lindane or heptachlor is an induction and not an activation process since it depends on protein synthesis and the drugs per se have no effect. Thus, our results obtained from studies in ovo with actinomycin D and cycloheximide suggest that lindane is acting at the translational level while heptachlor interferes at the level of transcription. (3) The study of ALA-S subcellular distribution indicated no accumulation in the cytosol of DMSO controls and in the lindane or heptachlor treated embryos, neither of the chlorinated pesticides alter the normal subcellular distribution of this regulatory enzyme in the liver. (4) Exogenous haem was able to prevent or decrease the induction of ALA-S elicited by both pesticides, thus showing that lindane or heptachlor-induced ALA-S respond to haem regulation. (5) Lindane had no effect on ferrochelatase activity at the doses and times assayed, but heptachlor decreased this enzyme activity. The porphyrinogenic mechanism of lindane and heptachlor is discussed on the basis of the present results.

Liver ferrochelatase from normal and hexachlorobenzene porphyric rats. Studies on their properties.

1. The aim of the present work is to shed light on the way of action of hexachlorobenzene (HCB) on hepatic ferrochelatase the mitochondrial enzyme which catalyzes the last step of haem biosynthetic pathway. 2. Some properties of this enzyme from normal and HCB porphyric rat liver were studied. 3. The present findings indicate that HCB treatment would modify the configuration of the enzyme perhaps allowing the active center of the porphyric ferrochelatase to be more exposed. 4. As a consequence it would show: (a) its higher affinity for the iron; (b) the shorter time necessary to form the intermediate enzyme-substrate, reflected both by the existence of a shorter lag and consequently a shorter pre incubation time. 5. However this modification elicited by the fungicide does not alter the submitochondrial distribution of the enzyme nor the optimal conditions for its measurement.