ICONOTIDE (FORMERLY SNX-111, Neurex Pharmaceuticals, Menlo Park, Calif) is the synthetic equivalent of-conopeptide MVIIA, a 25-amino-acid polybasic peptide present in the venom of Conus magus, a marine snail. 1 Ziconotide produces potent antinociceptive effects 2 by selectively binding to N-type voltage-sensitive calcium channels 3,4 on neuronal somata, dendrites, dendritic shafts, and axon terminals, thus blocking neurotransmission from primary nociceptive afferents. Ziconotide is the first selective Ntype voltage-sensitive calcium channel blocking agent to be tested in clinical trials. There is no evidence of tolerance to ziconotide 5 or of addictive behavior in animals (Elan Pharmaceuticals Inc, unpublished data), and the drug must be administered intrathecally to maximize antinociceptive effectiveness and minimize sympatholysis. 6

IDDSs improved clinical success in pain control, reduced pain, significantly relieved common drug toxicities, and improved survival in patients with refractory cancer pain.

IDDS improved clinical success, reduced pain scores, relieved most toxicity of pain control drugs, and was associated with increased survival for the duration of this 6 month trial.

Permeabilization of the outer mitochondrial membrane is an integral step in apoptosis. The resulting release of pro-apoptotic signaling proteins leads to cell destruction through activation of the cysteine-aspartic protease (caspase) cascade. However, the mechanism of outer mitochondrial membrane (OMM) permeabilization remains unclear. It was recently shown that cytochrome c can induce pore formation in cardiolipin-containing phospholipid membranes, leading to large dextran and protein permeability. In this work, the interaction of cytochrome c with cardiolipin-containing phospholipid vesicles, serving as models of the OMM, is investigated to probe cytochrome c-induced permeability. Lipid vesicles having either a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixed-DPPC/cardiolipin membrane and containing a membrane-impermeable Raman tracer 3-nitrobenzenesulfonate (3-NBS) were optically trapped, translated into a solution containing cytochrome c, and monitored for 3-NBS leakage. Cytochrome-correlated leakage was observed only in cardiolipin-containing vesicles. Structural changes observed in the Raman spectra during permeabilization indicated acyl chain disordering along with decreased intensity of the cardiolipin cis-double-bond stretching modes. When the vesicle-associated cytochrome c Raman spectrum is compared with a spectrum in buffer, heme-resonance bands are absent, indicating loss of Met-80 coordination. To verify selective interactions of cytochrome c with cardiolipin, these experiments were repeated where the DPPC acyl chains were deuterated (D62-DPPC), allowing spectral resolution of the DPPC acyl chain response from that of cardiolipin. Interestingly, D62-DPPC acyl chains were unaffected by cytochrome c accumulation, while cardiolipin showed major changes in acyl chain structure. These results suggest that cytochrome-induced permeabilization proceeds through selective interaction of cytochrome c with cardiolipin, resulting in protein unfolding, where the unfolded form interacts with cardiolipin acyl chains within the bilayer to induce permeability.

A common approach to exploring the structure and dynamics of biological membranes is through the deposition of model lipid bilayers on planar supports by Langmuir-trough or vesicle-fusion methods. Planar-supported lipid bilayers have been shown to exhibit structure and properties similar to those of lipid-vesicle membranes and are suitable for biosensing applications. Investigations using these planar-membrane models are limited to high-sensitivity methods capable of detecting a small population of molecules at the interface between a planar support and aqueous solution. In this work, we present evidence that supported-lipid bilayers can be deposited by vesicle fusion onto the interior surfaces throughout the wide-pore network of chromatographic silica particles. The thickness of a 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) film and headgroup spacing are consistent with a single bilayer of DMPC deposited onto the pore surfaces. The high specific surface area of these materials generates phospholipid concentrations easily detected by confocal-Raman microscopy within an individual particle, which allows the structure of these supported bilayers to be investigated. Raman spectra of porous-silica-supported DMPC bilayers are equivalent to spectra of DMPC vesicle membranes, both above and below their melting phase transitions, suggesting comparable phospholipid organization and bilayer structure. These porous-silica-supported model membranes could share benefits that planar-supported lipid bilayers bring to biosensing applications, but in a material that overcomes the limited surface area of a planar support. To test this concept, the potential of these porous-silica-supported lipid bilayers as high-surface-area platforms for label-free Raman-scattering-based protein biosensing is demonstrated with detection of concanavalin A selectively binding to a lipid-immobilized mannose target.

Multidimensional least squares analysis is a well-established technique for resolving component vibrational spectra from mixed samples or systems. Component resolution of temperature-dependent vibrational spectra is challenging, however, due to the lack of a suitable model for the variation in sample composition with temperature. In this work, analysis of temperature-dependent Raman spectra of lipid membranes is accomplished by using "concentration" vectors independently derived from enthalpy changes determined by differential scanning calorimetry. Specifically, the lipid-bilayer phase transitions of DMPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) are investigated through Raman spectra acquired from individual, optically trapped vesicles in suspension as a function of temperature. Heat capacity profiles of the same vesicle suspension are measured using differential scanning calorimetry and numerically integrated to generate enthalpy change curves of each phase transition, which are in turn used to construct composition vectors. Multidimensional least squares analysis optimized for a fit to these composition vectors allows resolution of the component spectra corresponding to gel, ripple, and liquid-crystalline phases of the DMPC. The quality of fit of the calorimetry-derived results is confirmed by unstructured residual differences between the data and the model, and a composition variation predicted by the resolved spectra that matches the calorimetry results. This approach to analysis of temperature-dependent spectral data could be readily applied in other areas of materials characterization, where one is seeking to learn about structural changes that occur through temperature-dependent phase transitions.

With the development of single-longitudinal mode diode lasers, there has been an increase in using these sources for Raman spectroscopy. This is largely due to the cost-effectiveness of diode lasers, which offer savings not only in initial capital cost, but also electrical, cooling, and replacement costs over time, when compared with ion lasers. The use of diode-lasers in confocal Raman microscopy has remained a challenge, however, due to poor transverse beam quality. In this work, we present the design and implementation of a simple spatial filter capable of adapting a single-mode diode laser source to confocal Raman microscopy, yielding comparable spatial resolution as a gas-ion laser beam for profiling and optical-trapping applications. For profiling applications, spatial filtering improved x,y resolution of the beam by a factor 10, which in turn increased optical-trapping forces by ~90 times and yielded sevenfold greater Raman scattering signal intensity from an optically trapped phospholipid vesicle.

Interactions of lectins, proteins that selectively bind carbohydrates, play an important role in many biological processes including cell adhesion, immune response, and cell signaling. Given the range of lectin functions and their potential for application in disease detection, there is a need for methods to investigate lectin-carbohydrate interactions that are rapid, structurally specific, and sensitive to binding from low-concentration samples. In this work, we describe the preparation and application of supported phospholipid bilayers deposited in wide-pore chromatographic silica particles for confocal Raman-microscopy-based detection of specific binding of concanavalin-A to mannose-functionalized phospholipids. The high surface area of porous-silica supports provides an ample concentration of phospholipid and protein for rapid, label-free detection of lectin binding to be carried out in an individual lipid-bilayer-functionalized particle. The Raman spectrum provides structural information on the bound protein as well as the phospholipid bilayer. Using scattering from the supported-lipid bilayer as an internal standard, Raman scattering from accumulated protein can be interpreted quantitatively to determine its absolute surface coverage on the lipid bilayer. At low glycolipid fraction (<1 mol %) in the prepared bilayer, the surface coverage by protein increases linearly with mannose-lipid densities, where the lectin population corresponds to ∼96% occupancy of the mannose ligands. At increasing glycolipid site densities in excess of 1 mol %, the surface-associated protein population saturates at a coverage that is equivalent to a full monolayer of mannose-bound lectin proteins. The results suggest that Raman microscopy of supported phospholipid bilayers in high-surface-area support particles is a promising approach for in situ, label-free, and quantitative investigation of bilayer-localized protein-ligand interactions.