Small-scale structure in fluid cholesterol–lipid bilayers

Cholesterol is the single most abundant molecule in animal plasma membranes, in the range of 20–30 mol%, where it is known to modulate the lipid-bilayer component of the membrane and lead to increased mechanical stability, lower permeability, larger thickness, and a distinct lateral organization. The phase equilibria of membranes with cholesterol and the associated large- and small-scale structure have turned out to be a particularly elusive problem. With the proposal that lipid domains and so-called ‘rafts’, characterized by high local levels of cholesterol in a liquid-ordered phase, are important for a wide range of cellular functions, an understanding and a quantitative assessment of the nature of these cholesterol-induced structures and their types of ordering have become urgent. Recent progress in neutron diffraction studies of lipid–cholesterol model membranes has now revealed details of the lateral ordering, and combined with earlier molecular model studies a picture emerges of the membrane as a locally structured liquid with small ordered ‘domains’ of a highly dynamic nature.     

Fluorescent detection of cholesterol using β-cyclodextrin functionalized graphene

A fluorescence based cholesterol detection method has been developed using competitive host-guest interaction between graphene bound β-cyclodextrin (β-CD) with rhodamine 6G (R6G) and cholesterol. Fluorescence of β-CD incorporated R6G is quenched by graphene but is 'turned on' by cholesterol as it replaces R6G from the β-CD host.     

Investigation of side chain liquid crystal polymers bearing cholesterol and bile acid derivatives

Cholic acid (or 3α,7α,12α-trihydoxyl-5β-cholan-24-oic acid) and lithocholic acid (or 3α-hydroxyl-5β-cholanic-24-oic acid) are commonly occurring bile acids synthesized from cholesterol in the liver in mammals. They all possess a steroid skeleton containing four rings, three with six carbons and one with five carbons. The transformation of cholesterol to cholic acid results in two major structural changes that affect the steroid skeleton. The first is the hydrogenation of the double bond …     

Synthesis of conjugates of closo-dodecaborate dianion with cholesterol using a “click” reaction

The nucleophilic ring-opening reaction of tetrahydropyran derivative of the closo-dode-caborate dianion with sodium azide in the presence of tetrabutylammonium bromide led to the novel azido-derivatives of[B₁₂H₁₂]^2?. A Cu-catalyzed 1,3-dipolar [3+2] cycloaddition reaction of the closo-dodecaborate dianion azido-derivatives with alkynyl-cholesterol led to 1,4-disubstituted 1,2,3-triazoles with the closo-dodecaborate fragment at position 1. The resulting conjugates are potentially suitable for the development of liposomal drugs to selectively deliver boron into a tumor cell for boron neutron capture therapy of cancer.

     

Synthesis and characterization of sol–gel-derived molecular imprinted polymeric materials for cholesterol recognition

The sol–gel-derived host matrices are well known for biosensor applications where various types of organic and biological molecules can be immobilized and can act as recognition elements. The molecular imprinting technology is an attractive alternative method where expensive and labile biorecognition elements can be replaced by molecular imprinted polymers (MIPs), which are capable of recognizing a target molecule of an interest. In the present study, hybrid sol–gel MIPs were synthesized in the form of crushed powder (CP) by both non-hydrolytic and hydrolytic method for cholesterol recognition. These MIPs were characterized by scanning electron microscopy (SEM), fourier transform-infrared (FT-IR), liquid chromatography-mass spectrometry (LC–MS) and nitrogen adsorption–desorption isotherm measurements. The template molecule was extracted by means of soxhlet extraction and calcination method. The cholesterol adsorption experiments were performed by using non-imprinted (NI) and extracted crushed powder (ExCP) and the percentage of adsorption was determined by measuring the residual quantity in the analyte solution using Liebermann-Burchard (L-B) reagent. The adsorption studies with non-imprinted crushed powder (NICP) showed interference with L-B reagent as well as non-specific binding between analyte molecules and silica matrix. The percentage of adsorption or rebinding was found to be higher for phenyl triethoxysilane (PhTEOS)-derived ExCP (composition 3) which was synthesized by the aqueous sol–gel processing method at low pH as compared to PhTEOS-derived (composition 1) and 3-aminopropyltriethoxysilane (APTES)-derived ExCP (composition 2) prepared by non-hydrolytic method. The reusability of used ExCP after re-extraction was also investigated. The various factors affecting rebinding of template molecules were discussed along with interference study. The study provided information on molecular imprinting of cholesterol in sol–gel matrix and highlighted the importance of characterization of MIPs before applying it for sensing applications.     

Synthesis of molecularly imprinted polymer of βcyclodextrin for the efficient recognition of cholesterol

Polymeric receptors for cholesterol were synthesized by crosslinking β-cyclodextrin (β-CyD) with hexamethylene diisocyanate or toluene 2,4-diisocyanate in dimethyl sulfoxide (DMSO) in the presence of cholesterol as the template. Non-imprinted β-CyD polymers were much poorer in the cholesterol adsorption. When β-CyD was cross-linked by epichlorohydrin in aqueous alkaline solutions (even in the presence of cholesterol), the cholesterol adsorbing activity was nil. Use of DMSO as the cross-linking solvent is necessary for the imprinting, since β-CyD molecules form inclusion complexes with cholesterol in this solvent and thus their mutual conformation in the polymer is regulated appropriately for cholesterol binding. The adsorbed cholesterol was completely removed from the polymers by treating the adducts with ethanol, indicating a strong potential for practical applications.     

Volumetric studies of cholesterol in basic and inert solvents from 25 to 55°C

The densities of cholesterol solutions in several proton acceptor solvents (benzonitrile, N,N-dimethylacetamide, N,N-dimethylformamide, N,N,N',N'-tetramethylurea, hexamethylphosphoramide, dibutylether, dibutylamine) and in benzene,n-heptane and 1-heptanol, from 25 to 55°C have been measured. The calculated apparent molar volumes of cholesterol are independent of concentration in solution. The standard partial molar volumes of cholesterol in the solvents whose molecules contain linear hydrocarbon chains (dibutylether, dibutylamine,n-heptane and 1-heptanol) show considerably lower values than those in the remaining media. The observed volume contraction is likely to be due to interactions of van der Waals type. The measured thermal expansion coefficient of cholesterol solutions and of cholesterol in an infinitely dilute solution in all the examined systems, are higher than the thermal expansion coefficients of the pure solvents. It has been found that this difference between the limiting thermal expansion coefficients of cholesterol containing systems and that for the pure solvent is affected by the solvent polarity.     

Synthesis of β-cyclodextrin conjugated superparamagnetic iron oxide nanoparticles for selective binding and detection of cholesterol crystals

Water-soluble, β-cyclodextrin conjugated superparamagnetic nanoparticles have been constructed. These particles showed selective binding to cholesterol crystals, which opens the door for the detection of cholesterol crystal-related diseases such as atherosclerosis by magnetic resonance imaging (MRI).     

Stereoselective,solid phase-based synthesis of trans 3-alkyl-substituted β-lactams as analogues of cholesterol absorption inhibitors

A straightforward solid phase-based strategy for the rapid generation of two small libraries of trans 3-alkyl-substituted β-lactams is described. For the glycine-derived library, a controlled excess of nonactivated acid chlorides was used to prevent oxazinone formation. The second library involved the attachment of Fmoc-protected p-aminophenol to Wang resin for the preparation of structurally-closed analogues of known cholesterol absorption inhibitors. This strategy allowed us to introduce diversity in the three variable positions of the β-lactam ring.     

Effect of β-cyclodextrin and its derivatives on caveolae disruption, relationships with their cholesterol extraction capacities

Endothelial cells (HUVEC) were treated with β-cyclodextrin (β-CD) and hydroxypropylated or methylated derivatives solutions to confirm their lack of affinity with phospholipids and their specificity towards cholesterol. Further studies were performed on bovine aortic endothelial cells to assess the effect of β-CDs (mainly methylated derivatives) on membrane microdomains (lipid rafts or caveolae), by detecting the caveolae marker caveolin-1 in fractions of sucrose gradients. A displacement from the lighter to the heavier fractions, characteristic of caveolae disruption, was observed using CDs. The strongest effect was obtained with dimethyl-β-CD, for which an accumulation of caveolin-1 was observed in the bottom of the gradient. Crysmeb^® and trimethyl-β-CD seemed to have the weaker effects as a significative amount of caveolin-1 was still detected in the light fraction corresponding to caveolae. β-CD and CDs having a degree of methylation a bit lower than 2 showed intermediate effects. The results of the present study on microdomains seem in good correlation with the cell cholesterol extraction capacities of CDs previously determined.     

Solubilization of cholesterol in aqueous solution by two β-cyclodextrin dimers and a negatively charged β-cyclodextrin derivative

Two βCD dimers (linked by succinic acid, 2, or ethylenediaminetetraacetic acid, EDTA, 3, bridges) and a negatively charged monomer derivative of βCD, 1, have been synthesized and their ability to solubilize cholesterol in aqueous solution was studied. The three compounds exhibit a great capacity in solubilizing cholesterol as, for instance, concentrations up to 6 mM of cholesterol were measured in the presence of 25 mM of 3. The phase-solubility diagrams of the two dimers exhibit A _L type profiles while the monomer 1 follows an A _P isotherm. The cholesterol/dimer complexes have 1:1 stoicheiometries while monomer 1 forms two complexes with molar ratios of 1:1 and 1:2 (cholesterol/1). The equilibrium constants are K _1:1 = (5.9 ± 0.3) × 10⁴ M^?1 and K _1:1 = (8.8 ± 0.2) × 10⁴ M^?1 for 2 and 3, respectively, and K _1:1 = 73 ± 19 M^?1 and K _1:2 = 204 ± 65 M^?1 for 1. The comparison of K _1:1(3) with the product K _1:1 × K _1:2 (1) reveals that a chelate effect in binding the cholesterol by 3 exists. The structure of the cholesterol/3 complex was studied by ROESY experiments and by molecular dynamics simulations.     

Chemoenzymatic synthesis of cholesteryl-6-O-tetradecanoyl-α-d-glucopyranoside: a product of host cholesterol efflux promoted by Helicobacter pylori

In a three-step protocol involving regioselective enzymatic acylation, per-O-trimethylsilylation, and a one-pot α-glycosidation-deprotection sequence, cholesteryl-6-O-tetradecanoyl-α-d-glucopyranoside (α-CAG) of Helicobacter pylori is afforded starting from glucose in an overall yield of 45%. The production of CAG can be scaled to make purified quantities available to the biological community for the first time.     

The development of a cholesterol biosensor using a liquid crystal/aqueous interface in a SDS-included β-cyclodextrin aqueous solution

Sodium dodecyl sulphate (SDS) including β-cyclodextrin (β-CD) (β-CD_SDS) was used to detect cholesterol at the 4-cyano-4′-pentylbiphenyl (5CB)/aqueous interface in transmission electron microscopy (TEM) grid cells. The β-CD acts as a host for SDS (guest). The guest SDS enclosed within the β-CD cavity was replaced with cholesterol by injecting cholesterol solution into the TEM cell at concentrations greater than 3 μM. The replacement of SDS with cholesterol was confirmed by pH measurement and high performance liquid chromatography (HPLC). The SDS excluded from the β-CD altered the planar orientation of the 5CB confined within the TEM grid cell to a homeotropic orientation. This planar-to-homeotropic transition was observed using a polarized optical microscope under crossed polarizers. This convenient TEM grid cell provides a new method for the selective detection of cholesterol without immobilization of the detecting receptors (enzyme, antibody, or aptamer) or the use of sophisticated instruments.     

Construction of supramolecular nanofbers through electrostatic interaction between perylene and cholesterol derivatives简

The self-assembly of cationic perylene diimide(PDI) and anionic cholesterol derivatives(CHOL) was conveniently achieved by the electrostatic attraction and p–p stacking interactions, exhibiting the well-defined supramolecular nanofibers ranging from hundreds of nanometers to micron dimension.     

Electrochemiluminescent disposable cholesterol biosensor based on avidin–biotin assembling with the electroformed luminescent conducting polymer poly(luminol-biotinylated pyrrole)

An electrochemiluminescent cholesterol disposable biosensor has been prepared by the formation of assembled layers on gold screen-printed cells. The detection layer is based on the electro-formation of new luminol copolymers with different synthesized biotinylated pyrroles prepared by click-chemistry, offering a new transduction layer with new electroluminescent properties on biosensors. The electrochemiluminescence (ECL) luminol copolymers are electroformed by cyclic voltammetry (five cycles) at pH 7.0 uses a10⁻³ M biotinylated pyrrole–luminol ratio of 1:10 in PBS buffer. With respect to the recognition layer, cholesterol oxidase was biotinylated by incubation with biotin vinyl sulfone, and immobilized on the copolymer by avidin–biotin interaction. The analytical signal of the biosensor is the ECL enzymatic initial rate working in chronoamperometric mode at 0.5 V excitation potential with 10 s between pulses at pH 9.5. The disposable device offers a cholesterol linear range from 1.5 × 10⁻⁵ M to 8.0 × 10⁻⁴ M with a limit of detection of 1.47 × 10⁻⁵ M and accuracy of 7.9% for 9.0 × 10⁻⁵ M and 14.1% for 2.0 × 10⁻⁴ M, (n = 5). Satisfactory results were obtained for cholesterol determination in serum samples compared to a reference procedure.     

Novel room-temperature perylene liquid crystals: synthesis of 1,7-dibrominated cholesterol–perylene bisimides with different ester-bridging chains and their mesomorphic properties

Three novel 1,7-dibrominated cholesterol–perylene liquid crystals 6a, 6b and 6c with different ester-bridging chains were designed and synthesised in yields of 30–40%. Their structures were characterised by FT-IR, ¹H NMR and HR-MS spectra. Their mesomorphic behaviours were studied by differential scanning calorimetry (DSC), polarising optical microscopy (POM) and X-ray diffraction (XRD). Compounds 6a, 6b and 6c exhibit hexagonal columnar liquid crystalline phase at room temperature. Their mesomorphic temperature ranges are as wide as 140–162°C. Their fluorescence spectra suggested that they possess good fluorescence properties in solution. The soft ester-bridging chains are more favourable for room-temperature mesophase and high fluorescence than the rigid ester-bridging chain.     

Estimating the size of laterally phase separated cholesterol domains in model membranes with Förster resonance energy transfer: a simulation study

In this work, we use two vertically-coupled square two-dimensional lattices to simulate membrane bilayers containing a uniform size distribution of cholesterol immiscible domains of a predetermined size distribution. We substitute cholesterols and phospholipids with their fluorescent analogs and calculate the efficiency of energy transfer as a function of acceptor concentration for four membrane configurations. The simulated efficiency of energy transfer as a function of acceptor concentration data is then fit with an analytical FRET model to estimate the domain size, in the same manner in which experimental FRET data is analyzed. The fitted model parameters (domain size and donor partition coefficient) are compared to the simulation inputs to test the applicability of the FRET model to estimating the size of laterally phase separated cholesterol domains. We show that the FRET model yields good size estimates for domains that range between 1 and 25 nm. We also find that the assumed fluorophore configuration in the FRET model leads to a constant under-prediction of these values. Finally, we demonstrate that when two parameters are open to the fit, the FRET model adequately predicts the donor partition coefficient in addition to the domain size.     

Trace-level determination of eight cholesterol oxidation products in human plasma by dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry

7α-Hydroxy cholesterol (7α-OHC), 25-hydroxy cholesterol (25-OHC), 27-hydroxy cholesterol (27-OHC), 4β-hydroxy cholesterol (4β-OHC), 7α-hydroxy-4-cholesten-3-one (7α-C4), 5β-cholestane-3α, 7α, 12α-triol (5β-Triol), cholic acid (CA), and chenodeoxycholic acid (CDCA) are known biomarkers of neurodegenerative diseases. A method for their simultaneous determination in human plasma has been optimized using dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry. The limits of quantification of the target compounds were in the range of 0.3–3.3?µg/L. The precision achieved by this method was less than 13.4% for intraday and interday analyses. The proposed method was used to analyze eight cholesterol oxidation products in 30 human plasma samples. The analytical results were in a concentration range of 1.6–87.4?µg/L for 7α-OHC, 6.3–58.2?µg/L for 25-OHC, 12.1–98.5?µg/L for 27-OHC, 5.7–64.8?µg/L for 4β-OHC, 1.5–124.1?µg/L for 7α-C4, 0.5–16.5?µg/L for 5β-Triol, 13.1–245?µg/L for CA, and 19.6–487?µg/L for CDCA in the samples. The method may be used for the analysis of biomarkers of neurodegenerative diseases.     

Study of the cholesterol extraction capacity of β-cyclodextrin and its derivatives, relationships with their effects on endothelial cell viability and on membrane models

Endothelial cells (HUVEC) were treated with β-cyclodextrin and hydroxypropylated or methylated derivatives solutions in order to quantify their cholesterol extraction capacity. Non-toxic concentrations of cyclodextrins (CDs) were determined following methyl thiazol tetrazolium (MTT) assays, total protein measurements, morphological observations and trypan blue assays. The residual cholesterol content of cells was measured and the extraction power of CDs compared to results obtained by phase solubility diagrams. Cholesterol was extracted with a dose-response relationship, the lowest residual cholesterol content being obtained with β-CD at 10 mM. Low substituted derivatives (Crysmeb^® and hydroxypropyl-β-CD) maintained liposomes integrity (as shown before), were the less cytotoxic and presented the lowest affinity for cholesterol contrary to methylated derivatives with degrees of substitution around 2.