Calcein release from polymeric vesicles in blood plasma and PVA hydrogel.

PURPOSE: The main goal of this study was to show the long-term stability of vesicles from poly(2-methyl oxazoline-block-polydimethylsiloxane-block poly(2-methyl oxazoline) (PMOXA-PDMS-PMOXA) in PBS, blood plasma and the feasibility to use these vesicles for drug release from PVA hydrogels. METHODS: The vesicle formation properties and loading efficiency was evaluated using fluorescent dyes. The stability of the vesicles was evaluated in buffer at pH 7 at room temperature and in 50% blood plasma at 37 degrees C. The calcein loaded vesicles were dispersed in a UV crosslinked PVA hydrogel. The stability of the vesicles in the hydrogel was observed over one week, before the vesicles were ruptured with Triton X-100. RESULTS: The vesicles are very stable in buffer, blood plasma, and the PVA hydrogel. In plasma 50% of the calcein is released in 48 h in the presence of sodium azide. The vesicles can be evenly dispersed in PVA and are stable. The release can be triggered and the calcein diffuses afterwards quickly throughout the gel. CONCLUSION: Polymeric vesicles can be used as diffusion barrier in hydrogels for the controlled release of water soluble drugs.

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.

Screening for novel drug effects with a microphysiometer: a potent effect of clofilium unrelated to potassium channel blockade.

Changes in cellular metabolism in response to pharmacological compounds can be detected using a biosensor known as a microphysiometer, which measures the rate at which cells release acidic metabolites. We have applied this technique to screen for effects of cation channel blockers on the metabolism of a variety of human and murine cell lines. At concentrations sufficient for cation channel blockade, most of these drugs have little or no effect on cellular metabolism as measured by acid release. In contrast, the potassium channel blocker clofilium triggers sustained increases in acid release at low (> or = 3 microM) concentration. Acid release persists in media containing high (150 mM) extracellular potassium. This release is not triggered by chemically similar potassium channel blockers. Thus these metabolic effects reflect a potent and specific function of clofilium which is unrelated to potassium channel blockade. Attempts to identify physiological correlates to this response revealed that low concentrations of clofilium but not other potassium channel blockers cause lymphoma apoptosis. These findings demonstrate that effects of clofilium found in other studies may not be due to changes in plasma membrane potassium conductance.