Nano-mechanical characterization of asymmetric DLPC/DSPC supported lipid bilayers.

Asymmetric distribution of lipid molecules in the inner and outer leaflets of the plasma membrane is a common occurrence in the membrane formation. Such asymmetric arrangement is a crucial parameter to manipulate the properties of the cell membrane. It controls signal transduction, endocytosis, exocytosis in the cells. The artificial membrane is often used to study the lateral and transverse arrangement of the lipid molecules in place of the cell membrane. Nano-mechanical characterization of the model membrane helps to understand the mechanical stability of the lipid bilayer. The stability is sensitive to the variations in the lipid composition and their local organization. In this article, we present both topographical and nano-mechanical properties of lipid bilayer characterized by atomic force microscopy (AFM). The results show that the asymmetric lipid bilayer formation is an intrinsic character. We have selected a bi-component fluid-gel phase 1,2-dilauroyl-sn-glycero-3-phosphocholine:1,2-disteroyl-sn-glycero-3-phosphocholine (DLPC: DSPC) system for our studies. We have observed domain formation and phase separation in the bilayer by increasing the composition of the gel phase DSPC. In force spectroscopy studies, we determine the mechanical strength of the bilayer for unique mixtures of DLPC: DSPC by measuring the breakthrough force. These results also show the effect of asymmetry in the lipid bilayer. Besides AFM studies, we have implemented a coarse-grained (CG) molecular dynamics (MD) simulation using the gromacs package at room temperature and 1 bar pressure. The results from the simulation study have been compared with AFM study. It was found that the simulation studies corroborated the findings from AFM such as an increase in the bilayer thickness, change in the phase state, asymmetric and symmetric domain formation in the lipid bilayer.

Interleaflet Decoupling in a Lipid Bilayer at Excess Cholesterol Probed by Spectroscopic Ellipsometry and Simulations.

Artificial lipid membranes are often investigated as a replica of the cell membrane in the form of supported lipid bilayers (SLBs). In SLBs, the phase state of a lipid bilayer strongly depends on the presence of molecules such as cholesterol, ceramide, and physical parameters such as temperature. Cholesterol is a key molecule of biological membranes and it exerts condensing effect on lipid bilayers. In this paper, we demonstrate the influence of excess cholesterol content on a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid-phase) using spectroscopic ellipsometry (SE) and coarse-grained (CG) molecular dynamics (MD) simulations. The results show the condensation effect due to cholesterol addition up to 30% and interleaflet decoupling at excess cholesterol beyond 30%. SE results show the separation of individual leaflets of the bilayer and influence of cholesterol on the biophysical properties such as thickness and optical index. CG simulations were performed at different ratios of DOPC:cholesterol mixtures to explore cholesterol-driven bilayer properties and stability. The simulations displayed the accumulation of cholesterol molecules at the interface of the lower and upper leaflets of the bilayer, thus leading to undulations in the bilayer. This work reports the successful application of SE technique to study lipid-cholesterol interactions for the first time.

Ampicillin-mediated functionalized gold nanoparticles against ampicillin-resistant bacteria: strategy, preparation and interaction studies.

Antibiotic resistance is a highly challenging concern of infectious diseases, and it requires a rational approach to overcome. Through this work, we have synthesized ampicillin-capped gold nanoparticles (Amp-Au NPs) and studied its interaction with bacterial cells. In this process of synthesis, the primary amine group of ampicillin acts as both reducing as well as capping agent. In addition to synthesized gold nanoparticles, the ß-lactam ring remains free to interact with bacteria. This approach not only utilizes the maximum efficiency of nanoparticles and antibiotics towards ampicillin sensitive bacterial cells but also proves to be effective against ampicillin resistance bacteria. Our results illustrate that the optimized system of Amp-Au NPs was formulated by taking 1.25 mM ampicillin and 10-2 of gold ions concentration. UV-vis spectrum of gold nanoparticles and the presence of ampicillin were recorded at around 540 nm and 259 nm, respectively. Microscopic images indicate that particles are nearly spherical and are in size range between 25 and 50 nm. Moreover, formulated Amp-Au NPs show successful accumulation onto the surface of the bacterial cell as a result of which pores were formed into the bacterial membrane. The entry of nanoparticles into bacterial cells was validated through both atomic force microscopy and fluorescent microscopy. The adhesive properties of this coating material and its stability in various pH, i.e. pH 3, pH 7 and pH 10 conditions, could make them a good candidate in the prevention of biofilm formation. Amp-Au NPs show promising antimicrobial activity against ampicillin resistance Escherichia coli bacteria. Furthermore, antimicrobial studies indicate that the efficacy of Amp-Au NPs increased against both ampicillin sensitive and ampicillin resistance bacteria up to sixteen folds and four folds respectively.

Spectroscopic Ellipsometry of fluid and gel phase lipid bilayers in hydrated conditions.

The biological membranes play a crucial role in the various biological processes due to their characteristic physical properties. The parameters such as membrane composition, thickness undulations, and the influence of external stimuli play a crucial role in the phase state behavior of biological membranes. The supported lipid bilayer (SLBs) systems closely represent cell membranes and are often studied to understand their behavior. In the current study, we have implemented spectroscopic Ellipsometry (SE) technique to explore the optical properties of various single component lipid bilayers having different phase state (fluid and gel) under hydrated or native conditions. The measurement of ellipsometric angles ψ and Δ were obtained from SE and were utilized to realize an explicit phase state dependence. Further, we obtained the thickness (t) and refractive index (n) by modeling of these SE results. These results were found to be inter-related to the phase state and lateral organization. The bilayer thickness is further verified against values obtained from coarse-grained molecular dynamics simulations. The proposed method was tested on various lipid bilayers under their native phase state at room temperature and was found to have a correlation with AFM measurements.