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Livne L.,Technion - Israel Institute of Technology | Epand R.F.,McMaster University | Papahadjopoulos-Sternberg B.,NanoAnalytical Laboratory | Epand R.M.,McMaster University | Mor A.,Technion - Israel Institute of Technology
FASEB Journal | Year: 2010

Antibiotic resistance has become a worldwide medical problem. To find new ways of overcoming this phenomenon, we investigated the role of the membraneactive oligo-acyl-lysyl (OAK) sequence C12K- 7α8, in combination with essentially ineffective antibiotics. Determination of minimal inhibitory concentration (MIC) against gram-negative multidrug-resistant strains of Escherichia coli revealed combinations with sub-MIC OAK levels that acted synergistically with several antibiotics, thus lowering their MICs by several orders of magnitude. To shed light into the molecular basis for this synergism, we used both mutant strains and biochemical assays. Our results suggest that bacterial sensitization to antibiotics was derived mainly from the OAK's capacity to overcome the efflux-enhanced resistance mechanism, by promoting backdoor entry of otherwise excluded antibiotics. To facilitate simultaneous delivery of the pooled drugs to an infection site, we developed a novel OAK-based cochleate system with demonstrable stability in whole blood. To assess the potential therapeutic use of such cochleates, we performed preliminary experiments that imitate systemic treatment of neutropenic mice infected with lethal inoculums of multidrug resistance E. coli. Single-dose administration of erythromycin coencapsulated in OAK-based cochleates has decreased drug toxicity and increased therapeutic efficacy in a dose-dependent manner. Collectively, our findings suggest a potentially useful approach for fighting efflux-enhanced resistance mechanisms. © FASEB.

Vinod Kaimal,University of Cincinnati | Chu Z.,University of Cincinnati | Mahller Y.Y.,University of Cincinnati | Papahadjopoulos-Sternberg B.,NanoAnalytical Laboratory | And 3 more authors.
Molecular Imaging and Biology | Year: 2011

Purpose: Nanovesicles composed of the phospholipid dioleylphosphatidylserine (DOPS) and a fusogenic protein, saposin C (SapC), selectively target and induce apoptotic cell death in a variety of human cancer cells in vitro and in vivo. We tested whether such tumor-homing nanovesicles are capable of delivering fluorescent probes and magnetic resonance (MR) contrast agents to cancerous tissue to aid in earlier detection and improve visualization. Procedures: SapC-DOPS nanovesicles labeled with either a far-red fluorescent probe (CellVue® Maroon, CVM) or conjugated with a dextran coated MR contrast agent, ultrasmall superparamagnetic iron oxide (USPIO), were systemically administrated into xenografts for tumor detection using optical and MR imaging systems. Results: SapC-DOPS nanovesicles were effectively detected in vivo in tumor-bearing animals using both optical and MR imaging techniques, thereby demonstrating the cancer-selective properties of these nanovesicles. Conclusions: SapC-DOPS nanovesicles offer promise as a new and robust theranostic agent for broad cancer-selective detection, visualization, and potential therapy © Academy of Molecular Imaging and Society for Molecular Imaging, 2010.

Epand R.F.,McMaster University | Sarig H.,Technion - Israel Institute of Technology | Ohana D.,Technion - Israel Institute of Technology | Papahadjopoulos-Sternberg B.,NanoAnalytical Laboratory | And 2 more authors.
Journal of Physical Chemistry B | Year: 2011

Several cationic antimicrobial oligo-acyl-lysyl (OAK) peptide mimetics can form cochleate structures, that is, elongated multilayered cylindrical structures, with lipid mixtures mimicking the composition of bacterial cytoplasmic membranes. These cochleate structures do not require divalent cations for their assembly. In the present work, we use light microscopy to screen for cochleate formation in several OAK-lipid systems and freeze-fracture electron microscopy to assess their morphological features and size. We identify several factors that facilitate a structural change in these assemblies. Dehydration of the membrane interface and a high melting temperature are features of the lipids that enhance cochleate formation in OAK-based lipid systems. In addition, we observed that there is a specific length of the hydrocarbon linker in the OAK of 8-9 carbon atoms that provides optimal formation of these structures. The biophysical properties established in this study will allow for a better understanding of their role and suitability for biological studies. © 2011 American Chemical Society.

Papahadjopoulos-Sternberg B.,NanoAnalytical Laboratory
Methods in molecular biology (Clifton, N.J.) | Year: 2010

Freeze-fracture electron microscopy (FFEM) as a cryo-fixation, replica, and transmission electron microscopy technique is unique in membrane bilayer and lipid monolayer research because it enables us, to excess and visualize pattern such as domains in the hydrophobic center of lipid bilayer as well as the lipid/gas interface of the lipid monolayer. Since one of the preparatory steps of this technique includes fracturing the frozen sample and, since during this fracturing process the fracture plane follows the area of weakest forces, these areas are exposed allowing us to explore the pattern built up by lipids and/or intrinsic proteins and which are also initiated by peptides, drugs, and toxins reaching into these normally hard to access areas. Furthermore, FFEM as a replica technique is applicable to objects of a large size range and combines detailed imaging of fine structures down to nano-resolution scale within images of larger biological or artificial objects up to several ten's of micrometers in size.Biological membranes consist of a multitude of components which can self-organize into rafts or domains within the fluid bilayer characterized by lateral inhomogeneities in chemical composition and/or physical properties. These domains seem to play important roles in signal transduction and membrane traffic. Furthermore, lipid domains are important in health and disease and make an interesting target for pharmacological approaches in cure and prevention of diseases such as Alzheimer, Parkinson, cardiovascular and prion diseases, systemic lupus erythematosus and HIV. As a cryofixation technique FFEM is a very powerful tool to capture such domains in a probe-free mode and explore their dynamics on a nano-resolution scale.

Epand R.M.,McMaster University | Epand R.F.,McMaster University | Arnusch C.J.,Weizmann Institute of Science | Papahadjopoulos-Sternberg B.,NanoAnalytical Laboratory | And 2 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2010

Three Arg-rich nonapeptides, containing the same amino acid composition but different sequences, PFWRIRIRR-amide (PR-9), RRPFWIIRR-amide (RR-9) and PRFRWRIRI-amide (PI-9), are able to induce segregation of anionic lipids from zwitterionic lipids, as shown by changes in the phase transition properties of lipid mixtures detected by differential scanning calorimetry and freeze fracture electron microscopy. The relative Minimal Inhibitory Concentration (MIC) of these three peptides against several strains of Gram positive bacteria correlated well with the extent to which the lipid composition of the bacterial membrane facilitated peptide-induced clustering of anionic lipids. The lower activity of these three peptides against Gram negative bacteria could be explained by the retention of these peptides in the LPS layer. The membrane morphologies produced by PR-9 as well as by a cathelicidin fragment, KR-12 that had previously been shown to induce anionic lipid clustering, was directly visualized using freeze fracture electron microscopy. This work shows the insensitivity of phase segregation to the specific arrangement of the cationic charges in the peptide sequence as well as to their tendency to form different secondary structures. It also establishes the role of anionic lipid clustering in the presence of zwitterionic lipids in determining antimicrobial selectivity. © 2010 Elsevier B.V.

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