Metastable Amorphous Dispersions of Hydrophobic Naphthalene Compounds Can Be Formed in Water without Stabilizing Agents via the "Ouzo Effect".

Hydrophobic molecules dissolved in water-miscible organic solvents are used in vitro for biological membrane studies and for testing of potential pharmaceuticals in high-throughput screenings. When these solutions are introduced into an aqueous environment, it is possible that metastable "ouzo-like" dispersions form from liquid-liquid phase separation. It is therefore hypothesized that when solutions of naphthalene compounds in water-miscible solvents are added to water, metastable dispersions will form. Millimolar solutions of naphthalene, N-phenyl-1-naphthylamine (NPN), 1-aminonaphthalene, 1-iodonaphthalene (INAP), 1,4-dimethoxynaphthalene, and 1-naphthol were prepared in either dimethyl sulfoxide, ethanol, or acetone at concentrations similar to those used in biological membrane studies. Each solution was diluted 10-fold in water. Particle formation was characterized by qualitative observations, dynamic light-scattering, nephelometry, and optical microscopy. It was discovered that two of the compounds tested made metastable dispersions: INAP and NPN. The initial particle sizes were ∼400 nm (radius), with turbidity ranging from 1,000 to 20,000 NTU, depending on the initial concentrations used. Fluorescence microscopy imaging showed spherical particles that do not aggregate while under observation. Slow-nucleating crystallization occurs over days, presumably from a heterogeneous nucleation process. The formation of these dispersions has implications for in vitro delivery of hydrophobic molecules to biological membranes.

Orthogonal inactivation of influenza and the creation of detergent resistant viral aggregates: towards a novel vaccine strategy.

BACKGROUND: It has been previously shown that enveloped viruses can be inactivated using aryl azides, such as 1-iodo-5-azidonaphthalene (INA), plus UVA irradiation with preservation of surface epitopes in the inactivated virus preparations. Prolonged UVA irradiation in the presence of INA results in ROS-species formation, which in turn results in detergent resistant viral protein fractions. RESULTS: Herein, we characterize the applicability of this technique to inactivate influenza. It is shown that influenza virus + INA (100 micromolar) + UVA irradiation for 30 minutes results in a significant (p < 0.05) increase in pelletablehemagglutinin after Triton X-100 treatment followed by ultracentrifugation. Additionally, characterization of the virus suspension by immunogold labeling in cryo-EM, and viral pellet characterization via immunoprecipitation with a neutralizing antibody, shows preservation of neutralization epitopes after this treatment. CONCLUSION: These orthogonally inactivated viral preparations with detergent resistant fractions are being explored as a novel route for safe, effective inactivated vaccines generated from a variety of enveloped viruses.

Effects of UVA irradiation, aryl azides, and reactive oxygen species on the orthogonal inactivation of the human immunodeficiency virus (HIV-1).

Previously we reported that hydrophobic aryl azides partition into hydrophobic regions of the viral membrane of enveloped viruses and inactivate the virus upon UVA irradiation for 2 min. Prolonged irradiation (15 min) resulted in viral protein aggregation as visualized via Western blot analysis, due to reactive oxygen species (ROS) formation, with preservation of the surface antigenic epitopes. Herein, we demonstrate that these aggregates show detergent resistance and that this property may be useful towards the creation of a novel orthogonal virus inactivation strategy for use in preparing experimental vaccines. When ROS-modified HIV virus preparations were treated with 1% Triton X-100, there was an increase in the percent of viral proteins (gp41, p24) in the viral pellet after ultracentrifugation through sucrose. Transmission electron microscopy (TEM) of these detergent-resistant pellets shows some recognizable virus fragments, and immunoprecipitation studies of the gp41 aggregates suggest the aggregation is covalent in nature, involving short-range interactions.

Quantitative analysis of phospholipids using nanostructured laser desorption ionization targets.

Since its introduction as an ionization technique in mass spectrometry, matrix-assisted laser desorption ionization (MALDI) has been applied to a wide range of applications. Quantitative small molecule analysis by MALDI, however, is limited due to the presence of intense signals from the matrix coupled with non-homogeneous surfaces. The surface used in nano-structured laser desorption ionization (NALDI) eliminates the need for a matrix and the resulting interferences, and allows for quantitative analysis of small molecules. This study was designed to analyze and quantitate phospholipid components of liposomes. Here we have developed an assay to quantitate the DPPC and DC(8,9)PC in liposomes by NALDI following various treatments. To test our method we chose to analyze a liposome system composed of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DC(8,9)PC (1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine), as DC(8,9)PC is known to undergo cross-linking upon treatment with UV (254 nm) and this reaction converts the monomer into a polymer. First, calibration curves for pure lipids (DPPC and DC(8,9)PC) were created using DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) as an internal standard. The calibration curve for both DPPC and DC(8,9)PC showed an R(2) of 0.992, obtained using the intensity ratio of analyte and internal standard. Next, DPPC:DC(8,9)PC liposomes were treated with UV radiation (254 nm). Following this treatment, lipids were extracted from the liposomes and analyzed. The analysis of the lipids before and after UV exposure confirmed a decrease in the signal of DC(8,9)PC of about 90%. In contrast, there was no reduction in DPPC signal.

Characterization of the effects of aryl-azido compounds and UVA irradiation on the viral proteins and infectivity of human immunodeficiency virus type 1.

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4'-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non-azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.

Ion complexation: a route to enhanced block copolymer alignment with electric fields.

We investigate the enhanced alignment of lamellar microdomains under an electric field by addition of lithium chloride (LiCl) into polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) copolymers. A significant increase of dielectric contrast resulting from the formation of lithium-PMMA complexes markedly reduces the critical electric field strength required to overcome the preferential interactions of one block with the substrate, providing a route to achieve the complete alignment of microdomains in block copolymer thin films.