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This paper discusses the formation of eLiposomes, defined as a liposome with internal emulsion droplets. Liposomes have been investigated as passively targeted drug carriers due to their ability to deliver drugs to a cancerous tumor via the enhanced permeability and retention (EPR) effect. The enclosed emulsion droplets in an eLiposome add the ability to further control the location and time of release from the liposome with ultrasound. Emulsion droplets were formed from perfluorohexane (PFC6) by sonication at 20 kHz and stabilized with dipalmitoyl phosphatidyl choline (DPPC). The size of the resulting droplets was reduced to approximately 100 nm or 50 nm by extrusion through polycarbonate filters of the same size at 50°C. Small unilamellar vesicles (SUVs) were prepared from DPPC by thin film hydration and extrusion through a 50 nm filter. Interdigitated DPPC sheets were prepared from the SUVs by the addition of ethanol to a concentration of 3M. Excess ethanol was removed by centrifugation washing. The sheets were mixed with emulsion and the solution was heated to 50°C, resulting in the refolding of the DPPC sheets into closed vesicles. Emulsion droplets were encapsulated inside of the newly formed eLiposomes. The size of the eLiposomes was reduced by extrusion. Cryogenic transmission electron microscopy (cryoTEM) and negative-staining TEM were used to image the emulsion droplets and the eLiposomes. Encapsulation of emulsion droplets was verified by rotating the microscope stage of cryoTEM samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The behavior of Listeria monocytogenes in pasteurized milk during fermentation with starter and nonstarter lactic acid bacteria was investigated. Pasteurized milk was co-inoculated with approximately 10(4) CFU/ml of L. monocytogenes and 10(6) CFU/ml of Lactococcus lactis, Lactococcus cremoris, Lactobacillus plantarum, Lactobacillus bulgaricus, or Streptococcus thermophilus. Inoculated milks were incubated at 30 degrees C or 37 degrees C for 24 to 72 h. Listeria monocytogenes survived and also grew to some extent during incubation in the presence of all starter cultures; however, inhibition ranged from 83 to 100% based on maximum cell populations. During incubation with L. bulgaricus and L. plantarum, L. monocytogenes was completely inactivated after 20 h and 64 h of incubation at 37 degrees C and 30 degrees C, respectively. The pH of the fermenting milks declined steadily throughout the fermentation periods and was approximately 4.2 at the conclusion of the experimental period regardless both of the starter culture and pathogen combination or the temperature of incubation.

The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls., (Copyright 1999 Academic Press.)

The objective of this research was to elucidate the factors effecting the permeability of cell membranes of gram-negative bacteria toward hydrophobic compounds. Ultrasound treatment, cell age, and the phase state of phospholipid membranes were considered. Spin-labeling EPR method was used to quantify the penetration and distribution of a lipophilic spin probe, 16-doxylstearic acid (16-DS), in Pseudomonas aeruginosa cell membranes. This bacterium was chosen because of its reported resistance to the action of hydrophobic antibiotics caused by the low permeability of the outer cell membrane for hydrophobic compounds. EPR spectra were collected from cell pellets and cell lysates. The overall spin probe uptake was measured in 10% SDS-cell lysates. Lysis with 0.6% SDS revealed the fraction of the probe located in membrane sites readily accessible to the surfactant. The results indicated a structural heterogeneity of P. aeruginosa membranes, with the presence of structurally "stronger" and "weaker" sites characterized by different susceptibility to the SDS treatment. The intracellular concentration of 16-DS was higher in insonated cells and increased linearly with the sonication power. EPR spectra indicated that ultrasound enhanced the penetration of the probe into the structurally stronger sites of the inner and outer cell membranes. The effect of ultrasound on the cell membranes was transient in that the initial membrane permeability was restored upon termination of the ultrasound treatment. These results suggest that the resistance of gram-negative bacteria to the action of hydrophobic antibiotics was caused by a low permeability of the outer cell membranes. This resistance may be reduced by the simultaneous application of antibiotic and ultrasound. This hypothesis was confirmed in our experiments with P. aeruginosa exposed to erythromycin.

A high-pressure anion-exchange chromatographic system has been modified to provide measurement of large numbers of molecular constituents in serum and for direct comparison to similar measurement in urine. Operating parameters have been adopted which greatly extend the range of elution for strongly retained anionic constituents and limit resolution of early-eluting basic and neutral compounds which were of less interest in this study. Dual monitoring by UV absorption and fluorescence produced by cerate oxidation provides both sensitive and wide-ranging detection capability. Comparative serum and urine chromatograms for a clinically normal subject, a subject after ingesting the drug 4-hydroxyacetanilide, and an infant suffering from extreme acidosis, illustrate the potential usefulness of this analysis in studying the origin, transport, in vivo reactions, and disposition of metabolites.