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Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA-virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA-virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA-virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA-virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn2+ system exhibits superior drug retention over the nigericin/K+ system, and compares favorably with vesicles loaded by the standard DeltapH or amine methods. The unique features of this methodology and possible benefits are discussed.

A new procedure for loading doxorubicin into large unilamellar vesicles (LUVs) is characterized. It is shown that doxorubicin can be loaded into LUVs composed of sphingomyelin/cholesterol (55:45 mole/mole) in response to a transmembrane MnSO4 gradient in the absence of a transmembrane pH gradient. Complex formation between doxorubicin and Mn2+ is found to be a driving force for doxorubicin uptake. Uptake levels approaching 100% can be achieved up to a drug-to-lipid molar ratio of 0.5 utilizing an encapsulated MnSO4 concentration of 0.30 M. In vitro leakage assays show excellent retention properties over a 24 h period. The possible advantages of a liposomal formulation of doxorubicin loaded in response to entrapped MnSO4 are discussed.

The interactions between beta-lactoglobulin and 1-monostearoyl-glycerol were studied in order to gain insight into protein-gel-phase monoglyceride interactions. Using a monomolecular layer at the air-water interface, we determined the insertion of beta-lactoglobulin into the monoglycerides under different conditions of protein and surface charge by varying the pH and/or incorporating charged amphiphiles into the monolayer, respectively, and using subphases with either a low or high ionic strength. The interactions were quantified by determining the binding of 14C-labeled beta-lactoglobulin to the monolayer. Our results show the importance of electrostatics for binding of beta-lactoglobulin to condensed monoglycerides. Moreover, electrostatic interactions were found to be important for specific insertion of beta-lactoglobulin into the monolayer. A negatively charged surface in particular allowed positively charged beta-lactoglobulin to insert in a surface charge density-dependent manner, even at surface pressures as high as 36 mN/m, whereas under other conditions, the limiting insertion pressure was 32 mN/m. The rheological properties of the monolayer were not affected by the interactions with beta-lactoglobulin.

The orientation of a mitochondrial-presequence peptide, associated with anionic lipid-containing model membranes, was investigated. The peptide inserts with its N-terminal alpha-helical part into cardiolipin (CL) monolayers so that the N-terminal 14 residues are protected from proteinase K. In phosphatidylglycerol (PG) monolayers the inserted peptide was fully accessible to the protease. A consequence of the different orientations of the peptide was that membrane potential-dependent protection from trypsin was much faster for the peptide bound to PG-containing vesicles compared to CL-containing membranes, suggesting that in the mitochondrial protein import process other components of the import apparatus are involved in the efficient potential-driven translocation of presequences across the inner mitochondrial membranes.

In this study the secondary structure and topology of the peptide, corresponding to the presequence of cytochrome oxidase subunit IV (p25) in a negatively charged membrane-mimetic environment, were assessed by circular dichroism and two-dimensional nuclear magnetic resonance. The micelles used consisted of dodecylphosphoglycol (DPG), a mild anionic detergent with a headgroup resembling that of phosphatidylglycerol. The secondary structure was analyzed by interresidue nuclear Overhauser enhancement measurements and chemical shifts of backbone protons. The data revealed alpha-helix formation of the peptide upon interaction with the micelles, both in the N- and in the C-terminal halves, which are separated from each other by the proline residue at position 13. The topology of the peptide was studied by determining the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances. This method revealed the insertion of hydrophobic residues of both the N- and the C-terminal halves of p25 into the hydrophobic environment of the micelles, demonstrating the orientation of the amphiphilic helix.

The secondary structure of the presequence of cytochrome oxidase subunit IV (p25) was studied by circular dichroism and 2D nuclear magnetic resonance in micelles of dodecylphosphocholine (DPC) and mixed micelles of DPC and mitochondrial cardiolipin (CL). In both systems, alpha-helix formation was observed. The alpha-helix stretches from the N- to the C-terminus with a break at the proline residue at position 13. Upon introduction of CL in the DPC micellar system, an increased stability of the helix was observed around proline13 and in the C-terminal half. This observation, together with reported results on specific interactions between CL and p25, led to the proposal of a two-state equilibrium of the alpha-helical conformation of p25, modulated by CL.

The effect of anionic lipids on the membrane insertion of a carboxyl group on a specially designed palmitoylated peptide was studied, using tryptophan fluorescence. It is demonstrated that the negatively charged membrane surface of mixed phosphatidylcholine/phosphatidylglycerol small unilamellar vesicles enhances the protonation of the C-terminal carboxyl group, and the subsequent insertion of that part of the peptide.

The present study demonstrates that the permanently positively charged, lipid-conjugated rhodamine, R18, can be transported from the outer to the inner leaflet of lipid bilayers in response of a transmembrane potential (negative inside). This conclusion was based on the following observations. (i) A fast decrease of the R18 fluorescence, when present at self-quenching concentrations in DOPC large unilamellar vesicles, was revealed upon induction of a valinomycin-induced K(+)-diffusion potential. (ii) Iodide quenching experiments demonstrated that R18 was no longer accessible to externally added aqueous quencher after application of a transmembrane potential. (iii) 2H-NMR measurements, using DOPC, specifically deuterated at the alpha-position of the phosphocholine head group, revealed a massive transbilayer movement of R18 upon induction of a membrane potential. The extent of the fluorescence changes were found to be dependent on the magnitude of the applied transmembrane potential, which opens possibilities for novel applications of R18 as an internal lipid-conjugated membrane potential probe.

The peptide specificity of both presequence-monolayer interactions and the ability of presequences to induce interbilayer contacts between large unilamellar vesicles was investigated. A range of different synthetic peptides that are documented for their mitochondrial protein import abilities were used for this purpose. Both monolayer insertion and vesicle aggregation were found to be strongly dependent on the primary structure of the studied presequence peptides. The combination of monolayer data and results of vesicle aggregation experiments leads to the overall suggestion that monolayer insertion and interbilayer contact formation are mechanistically related. For maximal effects the full length of a presequence peptide is required. The cardiolipin specificity of presequence-induced interbilayer contact formation previously reported was found to be a more general property among presequence peptides. The peptide's ability to induce vesicle-vesicle contacts seems to parallel the efficiency of its import ability into mitochondria. These results lead to an extended hypothesis on the role of presequence-induced contact site formation during the mitochondrial protein import process.

The ability of a synthetic peptide corresponding to the presequence of cytochrome c oxidase subunit IV from yeast to cause intermembrane contacts was investigated using monolayer techniques. The presequence inserted efficiently into the monolayer with a specificity for the mitochondrial cardiolipin. In the inserted form, the peptide strongly promoted the formation of close contacts with large unilamellar lipid vesicles present in the subphase, a property which was also specific for cardiolipin. The contacts formed were stable and tight and resulted in the flow of lipids from the vesicles to the monolayer. These results led to new suggestions on the involvement of intermembrane contact formation in mitochondrial protein import and membrane biogenesis.

In this study the effect of a transmembrane electrical potential on the phospholipid headgroup conformation was investigated using the 2H-NMR technique. Large unilamellar vesicles were prepared of dioleoylphosphatidylcholine, specifically 2H-labeled at the alpha- or beta-position of the choline group. No conformational change of the phosphocholine headgroup could be detected after induction of a valinomycin-induced K(+)-diffusion potential across the bilayer. However, this method could be used to measure the redistribution of tetraphenylphosphonium across the bilayer in response to delta psi, which reorients the phosphocholine headgroups in the opposite bilayer-water interfaces.

A new property of the presequence of the mitochondrial precursor protein cytochrome oxidase subunit IV is presented. This mitochondrial presequence induces interbilayer contacts between large unilamellar vesicles consisting of phosphatidylcholine and cardiolipin. The presequence-vesicle aggregates can be dissociated by applying a membrane potential across the bilayers (negative inside). These effects require the presence of cardiolipin and are not observed for other negatively charged phospholipids. We propose a role for the presequence in the formation and dissociation of mitochondrial contact sites.

A method was developed for the well-defined coupling of phosphoethanolamine group (PEA)- and carboxylic acid group-containing polysaccharides and oligosaccharides to proteins without the need for extensive modification of the carbohydrate antigens. The carboxylic acid group of the terminal 2-keto-3-deoxyoctulosonic acid moiety was utilized to introduce a thiol function in meningococcal immunotype L2 and L3,7,9 lipopolysaccharide-derived oligosaccharides. The thiol group-containing oligosaccharides were subsequently coupled to bromoacetylated proteins. Immunotype L2 and L3,7,9 PEA group-containing oligosaccharide-tetanus toxoid conjugates were prepared, and their immunogenicities were studied in rabbits. Both the immunotype L2 and immunotype L3,7,9 conjugates evoked high immunoglobulin G (IgG) antibody titers after the first booster injection. These conjugates also displayed an ability to induce long-lasting IgG antibody levels which could be detected until 9 months after one booster injection at week 3. The adjuvant Quil A enhanced the immune response to all the conjugates to a minor extent, which is in contrast with reported adjuvant effects of Quil A on these types of antigens in mice. A conjugate prepared from the dephosphorylated L3,7,9 oligosaccharides evoked a significantly lower IgG response than a similar PEA-containing conjugate, and enzyme-linked immunosorbent assay inhibition studies indicated a different epitope specificity. Furthermore, antisera elicited with the complete bacteria contained antibodies directed against PEA-containing epitopes, which stresses the importance of the presence of unmodified PEA groups in meningococcal lipopolysaccharide-derived oligosaccharide-protein conjugates. The procedure developed offers an elegant solution for the specific coupling of meningococcal PEA-containing oligosaccharides to proteins and may therefore be a very useful tool in the development of a vaccine against group B meningococci.