Cholesterol Regulates the Incorporation and Catalytic Activity of Tissue-Nonspecific Alkaline Phosphatase in DPPC Monolayers.

Matrix vesicles (MVs) are a special class of extracellular vesicles that drive bone and dentin mineralization by providing the essential enzymes and ions for the nucleation and propagation of mineral crystals. Tissue-nonspecific alkaline phosphatase (TNAP) is an integral protein of MV membrane and participates in biomineralization by hydrolyzing extracellular pyrophosphate (PPi), a strong mineralization inhibitor, and forming inorganic phosphate (Pi), necessary for the growth of mineral crystals inside MVs and their propagation once released in the extracellular matrix. MV membrane is enriched in cholesterol (CHOL), which influences the incorporation and activity of integral proteins in biologic membranes; however, how CHOL controls the incorporation and activity of TNAP in MV membrane has not yet been elucidated. In the present study, Langmuir monolayers were used as a MV membrane biomimetic model to assess how CHOL affects TNAP incorporation and activity. Surface pressure-area (π-A) isotherms of binary dipalmitoilphosphatidylcholine (DPPC)/CHOL monolayers showed that TNAP incorporation increases with CHOL concentration. Infrared spectroscopy showed that CHOL influences the conformation and orientation of the enzyme. Optical-fluorescence micrographs of the monolayers revealed the tendency of TNAP to incorporate into CHOL-rich microdomains. These data suggest that TNAP penetrates more efficiently and occupies a higher surface area into monolayers with a lower CHOL concentration due to the higher membrane fluidity. However, the quantity of enzyme transferred to solid supports as well as the enzymatic activity were higher using monolayers with a higher CHOL concentration due to increased rigidity that changes the enzyme orientation at the air-solid interface. These data provide new insights regarding the interfacial behavior of TNAP and CHOL in MVs and shed light on the biochemical and biophysical processes occurring in the MV membrane during biomineralization at the molecular level.

New look for an old molecule - Solid/solid phase transition in cholesterol monolayers.

Surface pressure (π) - molecular area (A) isotherms of cholesterol were precisely measured to get insight into the orientation of molecules in Langmuir monolayers, which allowed to obtain detailed information on their phase behaviour. This was possible from the detailed analysis of the interfacial compressibility modulus versus surface pressure (Cs-1- π) plots (obtained from the experimental surface pressure, π - area, A isotherms) and films thickness measurements (applying Brewster angle microscope, BAM) complemented with polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS). At first glance, the isotherm for cholesterol is characterized by the major slope change of surface pressure versus area per molecule. However, a more detailed analysis showed the presence of a discontinuity and slope change both upon the compression and expansion of the monolayer. This discontinuity is more accurately reflected in the Cs-1- π plot as a pseudo-plateau visible at π values between approximately 5 and 10 mN/m. This plateau was found to be temperature-dependent. Also, film thickness versus area plot (th-A) exhibits a pseudo-plateau in this region of surface pressures, in which the monolayer thickness increased gradually from 1.15 nm to 1.5 nm. Interestingly, although cholesterol has been intensively investigated in Langmuir monolayers, the existence of such a plateau have been overlooked previously. By linking experimental thickness values with theoretical molecular conformations, we have identified the presence of this plateau to the solid-solid (S-S') second-order transition. Using 2D analog of Clausius-Clapeyron equation, the thermodynamic functions (ΔH and ΔS) for this transition have been calculated. Based on monolayer experiments, the orientation of molecules in both solid phases was assumed to differ in the orientation of short alkyl chain attached to C17, which has additionally been confirmed with PM-IRRAS analysis.

Correlation of [RuCl3(dppb)(VPy)] cytotoxicity with its effects on the cell membranes: an investigation using Langmuir monolayers as membrane models.

One of the major challenges in drug design is to identify compounds with potential toxicity toward target cells, preferably with molecular-level understanding of their mode of action. In this study, the antitumor property of a ruthenium complex, mer-[RuCl3(dppb)(VPy)] (dppb = 1,4-bis(diphenylphosphine)butane and VPy = 4-vinylpyridine) (RuVPy), was analyzed. Results showed that this compound led to a mortality rate of 50% of HEp-2 cell with 120 ± 10 µmol L(-1), indicating its high toxicity. Then, to prove if its mode of action is associated with its interaction with cell membranes, Langmuir monolayers were used as a membrane model. RuVPy had a strong effect on the surface pressure isotherms, especially on the elastic properties of both the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the negatively charged dipalmitoylphosphatidylglycerol (DPPG) phospholipids. These data were confirmed by polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). In addition, interactions between the positive group from RuVPy and the phosphate group from the phospholipids were corroborated by density functional theory (DFT) calculations, allowing the determination of the Ru complex orientation at the air-water interface. Although possible contributions from receptors or other cell components cannot be discarded, the results reported here represent evidence for significant effects on the cell membranes which are probably associated with the high toxicity of RuVPy.

An intraocular dye solution based on lutein and zeaxanthin in a surrogate internal limiting membrane model: a Langmuir monolayer study.

Investigating the role of biomolecules and bioactive molecules associated with membranes is fundamental to comprehend at the molecular point-of-view biochemical and clinical processes that occur at biointerfaces. In this paper we exploit the interaction of an intraocular dye solution based on lutein and zeaxanthin in surrogate internal limiting membrane (ILM) models, consisting of dipalmitoyphosphatidylcholine (DPPC) Langmuir monolayers, pure or mixed with collagen, proteoglycan and laminin. The interactions between the film components occurring at the air-water interface were investigated with surface pressure-area isotherms and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). A natural dye solution based on lutein and zeaxanthin, employed to label ILM in ophthalmic surgery, was incorporated in the ILM model, and the data suggested non-rupture of the structure of the membrane, with predominance of interactions based on intermolecular forces.

Comparative study of liponucleosides in Langmuir monolayers as cell membrane models.

Liponucleosides may assist the anchoring of nucleic acid nitrogen bases into biological membranes for tailored nanobiotechnological applications. To this end precise knowledge about the biophysical and chemical details at the membrane surface is required. In this paper, we used Langmuir monolayers as simplified cell membrane models and studied the insertion of five lipidated nucleosides. These molecules varied in the type of the covalently attached lipid group, the nucleobase, and the number of hydrophobic moieties attached to the nucleoside. All five lipidated nucleosides were found to be surface-active and capable of forming stable monolayers. They could also be incorporated into dipalmitoylphosphatidylcholine (DPPC) monolayers, four of which induced expansion in the surface pressure isotherm and a decrease in the surface compression modulus of DPPC. In contrast, one nucleoside possessing three alkyl chain modifications formed very condensed monolayers and induced film condensation and an increase in the compression modulus for the DPPC monolayer, thus reflecting the importance of the ability of the nucleoside molecules to be arranged in a closely packed manner. The implications of these results lie on the possibility of tuning nucleic acid pairing by modifying structural characteristics of the liponucleosides.

Properties of lipophilic nucleoside monolayers at the air-water interface.

The capability of self-assembly and molecular recognition of biomolecules is essential for many nanotechnological applications, as in the use of alkyl-modified nucleosides and oligonucleotides to increase the cellular uptake of DNA and RNA. In this study, we show that a lipophilic nucleoside, which is an isomer mixture of 2'-palmitoyluridin und 3'-palmitoyluridin, forms Langmuir monolayers and Langmuir-Blodgett films as a typical amphiphile, though with a smaller elasticity. The nucleoside may be incorporated into dipalmitoyl phosphatidyl choline (DPPC) monolayers that serve as a simplified cell membrane model. The molecular-level interactions between the nucleoside and DPPC led to a remarkable condensation of the mixed monolayer, which affected both surface pressure and surface potential isotherms. The morphology of the mixed monolayers was dominated by the small domains of the nucleoside. The mixed monolayers could be deposited onto solid substrates as a one-layer Langmuir Blodgett film that displayed UV-vis absorption spectra typical of aggregated nucleosides owing to the interaction between the nucleoside and DPPC. The formation of solid films with DNA building blocks in the polar heads may open the way for devices and sensors be produced to exploit their molecular recognition properties.

Interaction of oligonucleotide-based amphiphilic block copolymers with cell membrane models.

Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides.