M. A. Busquets

Interaction of E2 and NS3 synthetic peptides of GB virus C/ hepatitis G virus with model lipid membranes

The membrane-interacting properties of two potential epitopes of the GB virus C/Hepatitis G virus, located respectively at the regions (99-118) of the E2 structural protein and (440-460) of the NS3 non-structural protein were studied. Changes in the intrinsic fluorescence of Trp and Tyr residues after the addition of DPPC-LUV revealed that the peptide-membrane interaction was optimal above the gel-liquid crystalline transition temperature of the lipid. Differential scanning calorimetry studies showed that the E2 peptide incorporated into lipid bilayers perturbs the packing of lipids and affects their thermotropic properties. Moreover, the 20-mer structural peptide induced a slow leakage of vesicular contents at 55 degrees C.

Peptides and Liposomes: From Biophysical to Immunogenic Studies

Synthetic peptide sequences constitute a useful tool to understand protein related diseases. A preliminary study consists of the analysis of peptide interaction with model membranes. The simplest one is based on monomolecular films of lipids spread at the air-water interface that imitate the interfacial environment in which some proteins function. Monolayer methodology provides a reliable screen of the extent to which hydrophobic interactions, charges, dipole potentials and subphase composition drive protein-lipid interaction. One step forward is based on the use of liposomes (lipid-based vesicles) that were originally introduced in 1965 as models of lipid bilayer membranes. Later, they have been widely studied as drug delivery systems mainly due to their safety, structural versatility, composition, fluidity and also because of their ability to incorporate almost any molecule regardless of its structure. In this sense, liposomes have been used as carriers of proteins and peptide antigens. Antigenic materials can be attached to the outer surface, encapsulated within the internal aqueous spaces or reconstituted within the lipid bilayers of the liposomes. In the present review we describe the steps going from the selection of peptides related to viral hepatitis proteins to its diagnostic and therapeutic application, with special emphasis on the use of model membranes to predict peptide mode of interaction with the target cell.

Spectroscopic analysis of the interaction of a peptide sequence of Hepatitis G virus with bilayers.

Merocyanine 540 (MC540) has been used as external probe to determine the interaction of the peptide sequence 125-139 corresponding to the E2 protein of Hepatitis G virus, with lipid bilayers. The probe was incorporated into large unilamellar vesicles (LUVs) or small unilamellar vesicles (SUVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). When incorporated into bilayers, MC540 shows two absorption maxima corresponding to the monomer (570 nm) and dimer (530 nm) form of the probe. Changes in the probe microenvironment are reflected by a modification in the position and/or intensity of these maxima. Addition of increasing amounts of peptide resulted in a slight decrease of the ratio A570/A530 thus indicating a change in MC540 partition into the membrane, going from a hydrophobic to a more hydrophilic environment. This effect was concomitant with an increase in dimer formation as stated from the values of the apparent dimerization constant (K(app)) obtained. Fluorescence spectra as well as steady state anisotropy measurements were in agreement with the above results indicating that the peptide was able to relocate the probe and displacing MC540 from its initial location into the bilayer. Results with SUVs or LUVs were similar for what curvature does not seem to play any role on peptide activity. These results reflect the ability of peptide to interact with biomimetic membranes in the lipid head group region.

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus†

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

Gentamicin Encapsulation in Liposomes: Factors Affecting the Efficiency

AbstractGentamicin was encapsulated in small unilamellar vesicles (SUV), multilamellar vesicles (ML V), and dehydration-rehydration vesicles (DRV). The encapsulation efficiency of the three liposomal preparations was compared. The values corresponding to DRV are ten to fifteen times greater than those for MLV or SUV. For dehydration-rehydration vesicles, the influence of lipid constituents, mixtures of PC, PS, SA, Chol, in encapsulation efficiency was also determined. The stability of the DRV preparations yielding the maximum encapsulation value, as a function of osmotic gradients and incubated in the presence of blood, was determined.

Fluorescence analysis of the interaction of two peptide sequences of hepatitis GB virus C with liposomes

The physicochemical characterization of the peptide sequences E2 (39-53) and E2 (32-59) corresponding to the structural protein E2 of the GB virus C was done by studying their interaction with model membranes. The peptides showed surface activity concentration dependent when injected beneath a buffered solution. This tendency to accumulate into the air/water interface suggested a potential ability of these peptides to interact with bilayers. For that reason, Small Unilamellar Liposomes (SUVs) of 1,2-dimyiristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-dimyiristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG) were chosen as a mimetic membranes. A series of fluorescence experiments based on tryptophan peptide fluorescence or with fluorescence labeled SUVs, were done to cover different aspects of peptide interaction with bilayers. Steady state fluorescence anisotropy studies with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) dioleoylphosphatidylethanolamine (NBD-PE) or 1-[4-(trimethylammonium) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) labeled SUVs indicated that only the long peptide was able to change the lipid microenvironment of DMPG vesicles by slightly increasing the rigidity of the bilayer both above and under the lipid main transition temperature. These results were concordant with the slight blue shift of the maximum tryptophan wavelength emission after E2 (32-53) peptide incubation with DMPG vesicles. Our data provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with a potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host.