Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for ß-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support.

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.

Material properties of lipid microdomains: force-volume imaging study of the effect of cholesterol on lipid microdomain rigidity.

The effect of cholesterol (CHOL) on the material properties of supported lipid bilayers composed of lipid mixtures that mimic the composition of lipid microdomains was studied by force-volume (FV) imaging under near-physiological conditions. These studies were carried out with lipid mixtures of dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and sphingomyelin. FV imaging enabled simultaneous topology and force measurements of sphingomyelin-rich domains (higher domain (HD)) and phospholipid-rich domains (lower domain (LD)), which allowed quantitative measurement of the force needed to puncture the lipid bilayer with or without CHOL. The force required to penetrate the various domains of the bilayer was probed using high- and low-ionic-strength buffers as a function of increasing amounts of CHOL in the bilayer. The progressive addition of CHOL also led to a decreasing height difference between HD and LD. FV imaging further demonstrated a lack of adhesion between the atomic force microscope tip and the HD or LD at loads below the breakthrough force. These results can lead to a better understanding of the role that CHOL plays in the mechanical properties of cellular membranes in modulating membrane rigidity, which has important implications for cellular mechanotransduction.

AFM study of the interaction of cytochrome P450 2C9 with phospholipid bilayers.

Cytochromes P450 (CYP) are key enzymes involved in the metabolism of drugs and other lipophilic xenobiotics and endogenous compounds. In this study, atomic force microscopy was applied to characterise the association of CYP2C9 to dimyristoylphosphatidylcholine (DMPC) supported phospholipid bilayers. CYP2C9 was found to exclusively localise in the gel domains of partially melted DMPC bilayers. Despite lacking the N-terminus transmembrane spanning domain, the CYP2C9 protein appeared to partially embed into the membrane bilayer, as evidenced by an increase in melting temperature of surrounding phospholipids. Reversible binding of CYP2C9 via an engineered His tag to a phospholipid bilayer was facilitated using nickel-chelating lipids, presenting potential applications for biosensor technologies.

Nanomechanical characterization of phospholipid bilayer islands on flat and porous substrates: a force spectroscopy study.

In this study, we compare for the first time the nanomechanical properties of lipid bilayer islands on flat and porous surfaces. 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers were deposited on flat (silicon and mica) and porous silicon (pSi) substrate surfaces and examined using atomic force spectroscopy and force volume imaging. Force spectroscopy measurements revealed the effects of the underlying substrate and of the lipid phase on the nanomechanical properties of bilayers islands. For mica and silicon, significant differences in breakthrough force between the center and the edges of bilayer islands were observed for both phospolipids. These differences were more pronounced for DMPC than for DPPC, presumably due to melting effects at the edges of DMPC bilayers. In contrast, bilayer islands deposited on pSi yielded similar breakthrough forces in the central region and along the perimeter of the islands, and those values in turn were similar to those measured along the perimeter of bilayer islands deposited on the flat substrates. The study also demonstrates that pSi is suitable solid support for the formation of pore-spanning phospholipid bilayers with potential applications in transmembrane protein studies, drug delivery, and biosensing.

Kinetics membrane disruption due to drug interactions of chlorpromazine hydrochloride.

Drug-membrane interactions assume considerable importance in pharmacokinetics and drug metabolism. Here, we present the interaction of chlorpromazine hydrochloride (CPZ) with supported phospholipid bilayers. It was demonstrated that CPZ binds rapidly to phospholipid bilayers, disturbing the molecular ordering of the phospholipids. These interactions were observed to follow first order kinetics, with an activation energy of approximately 420 kJ mol(-1). Time-dependent membrane disruption was also observed for the interaction with CPZ, such that holes appeared in the phospholipid bilayer after the interaction of CPZ. For this process of membrane disruption, "lag-burst" kinetics was demonstrated.

Thermosensitive copolymer coatings with enhanced wettability switching.

Expanded cross-linked copolymers of poly(N-isopropylacrylamide) (PNiPAAm) and poly(acrylic acid) (PAAc) of varying monomer ratios were grafted from a crystalline silicon surface. Surface-tethered polymerization was performed at a slightly basic pH, where electrostatic repulsion among acrylic acid monomer units forces the network into an expanded polymer conformation. The influence of this expanded conformation on switchability between a hydrophilic and a hydrophobic state was investigated. Characterization of the copolymer coating was carried out by means of X-ray photoelectron spectroscopy (XPS) ellipsometry, and diffuse reflectance IR. Lower critical solution temperatures (LCSTs) of the copolymer grafts on the silicon surfaces were determined by spectrophotometry. Temperature-induced wettability changes were studied using sessile drop contact angle measurements. The surface topography was investigated by atomic force microscopy (AFM) in Milli-Q water at 25 and 40 degrees C. The reversible attachment of a fluorescently labeled model protein was studied as a function of temperature using a fluorescence microscope and a fluorescence spectrometer. Maximum switching in terms of the contact angle change around the LCST was observed at a ratio of 36:1 PNiPAAm to PAAc. The enhanced control of biointerfaces achieved by these coatings may find applications in biomaterials, biochips, drug delivery, and microfluidics.

Lateral heterogeneities in supported bilayers from pure and mixed phosphatidylethanolamine demonstrating hydrogen bonding capacity.

The phase behavior and lateral organization of saturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) bilayers were investigated using atomic force microscopy (AFM) and force-volume (FV) imaging for both pure and two component mixed layers. The results demonstrated the existence of unexpected segregated domains in pure PE membranes at temperatures well below the transition temperature (T(m)) of the component phospholipid. These domains were of low mechanical stability and lacked the capacity for hydrogen bonding between lipid headgroups. Temperature dependent studies for different PC/PE ratios using AFM also demonstrated the mixing of these phospholipid bilayers to exhibit only a single gel to liquid transition temperature. Further work performed using FV imaging and chemically modified probes established that no lipid segregation exists at the PC/PE ratios investigated.

Characterisation of the binding of cationic amphiphilic drugs to phospholipid bilayers using surface plasmon resonance.

The interactions of three cationic amphiphilic drugs (CPZ, AMI, PROP) with phospholipid vesicles comprising DOPC, DMPC, or DSPC were investigated using surface plasmon resonance (SPR). Responses for CAD concentrations in the range 15.625 to 1500 microM were measured. The greatest uptake by each phospholipid bilayer occurred with CPZ. Inclusion of CAD concentrations between 750 and 1500 microM provided evidence for a second nonsaturable binding process, which may arise from intercalation of the drugs within the lipid bilayer. CAD binding was additionally shown to be dependent on membrane fluidity. Responses were initially fitted over a concentration range of 15.625 to 500 microM using a model which incorporated terms for a saturable binding site. This yielded very poor values of K(D) and nonsensible values of saturation responses. Subsequently, responses were fit to the expression for a model which incorporated terms for both a saturable binding site and second nonsaturable site. Measurable binding affinities (K(D) values ranged from 170 to 814 microM) were obtained for DOPC and DMPC bilayers which are similar to values reported previously. This work demonstrates that SPR studies with synthetic phospholipid bilayers provide a potentially useful approach for characterising drug-membrane binding interactions and for providing insight into the processes that contribute to drug-membrane binding.