Vertical ordering sensitivity of solid supported DPPC membrane to alamethicin and the related loss of cell viability.

BACKGROUND: Experimental studies of antimicrobial peptides interacting with lipid membranes recently attracted growing interest due to their numerous biomedical applications. However, the influence of such peptides on the structural organisation of lipid membranes in connection with the actual cell response still remains an elusive issue. METHODS: X-ray diffraction was employed on detecting the sensitivity of the periodical spacing of dipalmitoyl-phosphatidyl-choline stacked as solid-supported bilayers to the presence of varying amounts of the peptide alamethicin in a wide range of peptide-to-lipid molar ratios. These results were then correlated with the effects of alamethicin on biological membranes in vitro as observed by optical microscopy and microculture tetrazolium assay on the tumour cells HeLa to provide a comprehensive and quantitative analysis of these effects, based on a dose-response relationship. RESULTS: The experiments allowed correlating the periodical spacing and the peptide-to-lipid molar ratio on alamethicin-dipalmitoyl-phosphatidyl-choline samples. Two different trends of periodical spacing vs. peptide-to-lipid molar ratio clearly appeared at low and high hydration levels, showing intriguing non-linear profiles. Unexpected correspondences were observed between the peptide-to-lipid molar ratio range where the changes in dipalmitoyl-phosphatidyl-choline structure occur and the alamethicin doses which alter the viability and the plasma membrane morphology of HeLa. CONCLUSIONS: Alamethicin might induce either mechanical or phase changes on dipalmitoyl-phosphatidyl-choline bilayers. Such easily accessible ordering information was well-calibrated to predict the alamethicin doses necessary to trigger cell death through plasma membrane alterations. GENERAL SIGNIFICANCE: This benchmark combined study may be valuable to predict bioeffects of several antimicrobial peptides of biomedical relevance.

Temperature-dependent structural changes on DDAB surfactant assemblies evidenced by energy dispersive X-ray diffraction and dynamic light scattering.

Cationic amphiphile DDAB (dimethyl-dioctadecyl-ammonium-bromide) can spontaneously form water-dispersed and solid supported mimicking biomembrane structures as well as valuable DNA delivery vehicles whose shape, stability and transfection efficiency can be easily optimized on varying temperature, water content and chemical composition. In this framework, disclosing the thermotropic behavior of DDAB assemblies can be considered as an essential step in conceiving and developing new non-viral vector systems. Our work has been focused primarily on understanding the mesophase structure of silicon supported DDAB thin film on varying temperature at constant relative humidity by energy dispersive X-ray diffraction (EDXD). Diffraction results have then been employed in providing a more comprehensive dynamic light scattering (DLS) analysis of corresponding thermotropic water dispersed vesicles made up of DDAB alone and in combination with helper lecithin DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) liposomes. We found that above 55 °C silicon-supported DDAB films undergo a significant thinning effect, whilst DDAB-water vesicles exhibit a reduction in size polydispersity. Upon cooling to 25 °C a distinct silicon supported DDAB mesophase, exhibiting a relative humidity-dependent spacing, has been pointed out, and modeled in terms of a lyotropic metastable gel-crystalline phase.DDAB/DOPC-water vesicles show a temperature-dependent switching in size distribution, leading to promising biomedical applications.

Silicon supported lipid-DNA thin film structures at varying temperature studied by energy dispersive X-ray diffraction and neutron reflectivity.

Non-viral gene transfection by means of lipid-based nanosystems, such as solid supported lipid assemblies, is often limited due to their lack of stability and the consequent loss of efficiency. Therefore not only a detailed thermo-lyotropic study of these DNA-lipid complexes is necessary to understand their interaction mechanisms, but it can also be considered as a first step in conceiving and developing new transfection biosystems. The aim of our study is a structural characterization of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-dimethyl-dioctadecyl-ammonium bromide (DDAB)-DNA complex at varying temperature using the energy dispersive X-ray diffraction (EDXD) and neutron reflectivity (NR) techniques. We have shown the formation of a novel thermo-lyotropic structure of DOPC/DDAB thin film self-organized in multi-lamellar planes on (100)-oriented silicon support by spin coating, thus enlightening its ability to include DNA strands. Our NR measurements indicate that the DOPC/DDAB/DNA complex forms temperature-dependent structures. At 65°C and relative humidity of 100% DNA fragments are buried between single lamellar leaflets constituting the hydrocarbon core of the lipid bilayers. This finding supports the consistency of the hydrophobic interaction model, which implies that the coupling between lipid tails and hypo-hydrated DNA single strands could be the driving force of DNA-lipid complexation. Upon cooling to 25°C, EDXD analysis points out that full-hydrated DOPC-DDAB-DNA can switch in a different metastable complex supposed to be driven by lipid heads-DNA electrostatic interaction. Thermotropic response analysis also clarifies that DOPC has a pivotal role in promoting the formation of our observed thermophylic silicon supported lipids-DNA assembly.

Structural changes induced in proteins by therapeutic ultrasounds.

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV-VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and alpha-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (alpha-helix or beta-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.