Diffusion and partitioning of fluorescent lipid probes in phospholipid monolayers.

The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Pi < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems.

Single-molecule transport in three-terminal devices.

Transport through single molecules has been studied using different test beds. In this paper we focus on three-terminal devices in which a molecule bridges the gap between two gold electrodes and a third electrode-the gate-is able to modulate the conduction properties of the junction. Depending on the electronic coupling, Γ, between the molecule and the gold electrodes, different transport regimes can be distinguished. We show measurements on junctions incorporating different single-molecule systems which demonstrate the distinction between these regimes, as well as the experimental limitations in controlling the exact value of Γ.

Next generation adoptive immunotherapy--human T cells as carriers of therapeutic nanoparticles.

An important step in adoptive immunotherapy in general and specifically with respect to cancer treatment is the initiation of an inflammatory T cell response at the tumor site. Here we suggest a new concept for a controlled inflammatory response in which the intrinsic cytotoxic properties of T cells are upgraded with the properties of nanoparticles transfected into the T cells during the ex vivo expansion process. We report in vitro upgrading of human T cells using PEGylated boron carbide nanoparticles functionalised with a translocation peptide aimed at Boron Neutron Capture Therapy (BNCT). A key finding is that the metabolism of such upgraded human T cells were not affected by a payload of 0.13 pg boron per cell and that the nanoparticles were retained in the cell population after several cell divisions. This is vital for transporting nanoparticles by T cells to the tumor site.

Functionalization and cellular uptake of boron carbide nanoparticles. The first step toward T cell-guided boron neutron capture therapy.

In this paper we present surface modification strategies of boron carbide nanoparticles, which allow for bioconjugation of the transacting transcriptional activator (TAT) peptide and fluorescent dyes. Coated nanoparticles can be translocated into murine EL4 thymoma cells and B16 F10 malignant melanoma cells in amounts as high as 0.3 wt. % and 1 wt. %, respectively. Neutron irradiation of a test system consisting of untreated B16 cells mixed with B16 cells loaded with boron carbide nanoparticles were found to inhibit the proliferative capacity of untreated cells, showing that cells loaded with boron-containing nanoparticles can hinder the growth of neighboring cells upon neutron irradiation. This could provide the first step toward a T cell-guided boron neutron capture therapy.

Preparation and characterization of Boron carbide nanoparticles for use as a novel agent in T cell-guided boron neutron capture therapy.

Boron carbide nanoparticles are proposed as a system for T cell-guided boron neutron capture therapy. Nanoparticles were produced by ball milling in various atmospheres of commercially available boron carbide. The physical and chemical properties of the particles were investigated using transmission electron microscopy, photon correlation spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, vibrational spectroscopy, gel electrophoresis and chemical assays and reveal profound changes in surface chemistry and structural characteristics. In vitro thermal neutron irradiation of B16 melanoma cells incubated with sub-100 nm nanoparticles (381.5 microg/g (10)B) induces complete cell death. The nanoparticles alone induce no toxicity.

Novel methods for studying lipids and lipases and their mutual interaction at interfaces. Part I. Atomic force microscopy.

Mono-layers of lipids and their interaction with surface active enzymes (lipases) have been studied for more than a century. During the past decade new insight into this area has been obtained due to the development of scanning probe microscopy. This novel method provides direct microscopic information about the system in question and allows in situ investigations under near physiological conditions. In the present review the theory, experimental set-up and sample requirements of atomic force microscopy (AFM) are described. An overview of recent results is also presented with special emphasis on lipase hydrolysis and kinetics investigated in situ using AFM.

Novel methods for studying lipids and lipases and their mutual interaction at interfaces. Part II. Surface sensitive synchrotron X-ray scattering.

Monolayers of lipids have been studied for more than a century. During the past decade new insight into the field has resulted from the development of surface sensitive X-ray scattering methods utilizing synchrotron radiation: grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). These novel methods provide direct microscopic information about the systems in question and allow in situ investigations under near physiological conditions. GIXD gives information about the in-plane molecular structure, e.g., lattice symmetry and structural parameters; XR provides the electron density profile across the interface. The present review describes the theory, experimental procedures and sample requirements for surface sensitive X-ray scattering. An overview of recent results is presented as well, with special emphasis on biologically important systems, e.g., investigations by GIXD and/or XR of lipid and protein structures at interfaces and of lipid/protein interactions.

Langmuir and Langmuir-Blodgett films of amphiphilic hexa-peri-hexabenzocoronene: new phase transitions and electronic properties controlled by pressure.

We present the synthesis as well as the structural and electronic properties of an amphiphilic derivative of hexaalkylhexa-peri-hexabenzocoronene (HBC), which contains one alkyl substituent that is terminated with a carboxylic acid group. The molecules form well-defined Langmuir films when spread from a solution at the air-water interface. Grazing-incidence X-ray diffraction (GIXD) and X-ray reflectivity studies of the Langmuir monolayer reveal two crystallographic phases at room temperature which depend on the surface pressure applied to the film. Scattering from very well-ordered (zeta = 200-400 A) pi-stacked lamellae of HBC molecules tilted approximately 45 degrees relative to the surface normal is observed in the low-pressure phase. In this phase, the HBC molecules pack in a rectangular two-dimensional unit cell with a = 22.95 A and b = 4.94 A. In the high-pressure phase, coherence from the pi stack is lost. This is a consequence of stress induced by the crystallization of the substituent alkyl chains into a hexagonal lattice, which has a trimerized superstructure in one direction: a = 3 x b = 15.78 A, b = 5.26 A, gamma = 120 degrees, A = 71.9 A2 = 3 x 23.9 A2. Thin monolayer films can be transferred to solid supports by the Langmuir-Blodgett (LB) technique. Atomic force microscopy (AFM) with atomic resolution reveals the crystalline packing of alkyl chains in the high-pressure phase. Kelvin force microscopy (KFM) shows a clear potential difference between the high- and low-pressure phases. This is discussed in terms of orbital delocalization (band formation) in the highly coherent low-pressure phase, which is in contrast to the localized molecular orbitals present in the high-pressure phase. The highly coherent pi stack is expected to sustain a very high charge-carrier mobility.

Lag-burst kinetics in phospholipase A(2) hydrolysis of DPPC bilayers visualized by atomic force microscopy.

The lag-burst phenomenon in the phospholipase A(2) mediated hydrolysis of phospholipid bilayers is for the first time demonstrated in an atomic force microscopy (AFM) study. Simultaneous AFM measurements of the degree of bilayer degradation and the physical-chemical state of the membrane reveals growing nanoscale indentations in the membrane during the lag phase. It is argued that these indentations are domains of hydrolysis products (lysoPC/PC) which eventually trigger the burst. The rate of the rapid hydrolysis following the burst is found to be proportional to the length of the edge between membrane adsorbed and desorbed to the mica base. The observed maximal rate of membrane degradation is approx. 0.2 mmol lipid/min/mol lipase in solution.

Structure and dynamics of lipid monolayers: implications for enzyme catalysed lipolysis.

We have investigated the role of the substrate on the interfacial activation of lipases by an interdisciplinary study of the structure and dynamics of 1,2-sn dipalmitoylglycerol monolayers at distinct surface pressures. The diglyceride Langmuir film undergoes two phase transitions occurring at 38.3 and 39.8 A2 per molecule. The first transition is unique for diglyceride molecules and is driven by a reorganization of the headgroups causing a change in the hydrophobicity of the oil-water interface. X-ray diffraction studies of different mesophases shows that in the two highest pressure phases, the alkyl chains pack in an hexagonal structure relaxing to a distorted-hexagonal lattice in the lowest pressure phase with the alkyl chains tilted by approximately 14 degrees in a direction close to a nearest neighbour direction.

Langmuir-blodgett films of a functionalized molecule with cross-sectional mismatch between head and tail.

A functionalized surfactant has been investigated as floating monolayers by synchrotron x-ray diffraction and as bilayers transferred to solid supports by the Langmuir-Blodgett technique through atomic force microscopy. The transfer process is accompanied by an increase of the unit cell area (about 17 percent) and by an increase of the average domain diameter of nanometer-scale domains (about three times). The unit cell area of the floating monolayer corresponds to close packing of the head groups and a noncharacteristic packing of the tifted alkyl chains. The larger unit cell area of the bilayer film is consistent with a particular ordered packing of the alkyl chains, leaving free space for the head groups.