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.

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 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.

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 dodecylphosphoeholine (DPC) and mixed micelles of DPC and mitocbondrial cardiolipin (CL). In both systems, a-helix formation ~as observed. The a-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 mieellar system, an increased stability of the helix was observed around proline ~3 and in the (i-terminal hall This observation, together with reported results on specific interactions between CL and p25, led to the proposal of a two-state equilibrium of the a-helical conformation of p25, modulated by CL.