Cholesteryl Hemisuccinate Research Articles

Effect of Formulation and Preparation Methods on Naproxen Liposome Performance

Purpose: The aim of this study was to evaluate the effects of formulation composition and methods of preparation on the physico-chemical properties and drug release of naproxen liposome. Methods: The ratio of lipid to drug, lipid to lipid, organic to aqueous phase and methods of preparation were varied. Results: Formulation composed of 0.9 g lecithin:3.6 g cholesterol: 0.9 g cholesteryl hemisuccinate and drug : lipid ratio of 1:3 gave the highest entrapment efficiency (83.6%). Formulations prepared by using different ratios of organic to aqueous phase showed different drug entrapment efficiency depending on the ratio of the volume of the two phases. Also, the method used for preparation of the liposome had an effect on drug entrapment efficiency. The drug release data indicated that liposome prepared by the heating method and composed of 9 g lecithin:3.6 g cholesterol:0.9 g cholesteryl hemisuccinate using a ratio of 3 lipid:1 drug showed the best control release profile. The percent drug released was 14.9% and 71.5% at 15 minutes and 360 minutes of testing dissolution. Original Research Article Lugo and Ghaly; JSRR, 4(4): 292-305, 2015; Article no.JSRR.2015.033 293 Conclusion: The physico-chemical properties, drug entrapment and drug release from naproxen liposome were influenced by formulation composition, ratio of aqueous to organic phase and methods of preparation.

Cathepsin B-sensitive cholesteryl hemisuccinate–gemcitabine prodrug nanoparticles: enhanced cellular uptake and intracellular drug controlled release

Cholesteryl hemisuccinate–gemcitabine prodrug: enhanced cellular uptake and intracellular drug controlled release.

Ursolic acid incorporation does not prevent the formation of a non-lamellar phase in pH-sensitive and long-circulating liposomes.

Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low water solubility, which limits its biological applications. In this context, our research group has proposed the incorporation of UA in long-circulating and pH-sensitive liposomes (SpHL-UA).These liposomes, composed of dioleylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethylene glycol2000 (DSPE-PEG2000), were shown to be very promising carriers for UA. Considering that the release of UA from SpHL-UA and its antitumor activity depend upon the occurrence of the lamellar to non-lamellar phase transition of DOPE, in the present work, the interactions of UA with the components of the liposomes were evaluated, aiming to clarify their role in the structural organization of DOPE. The study was carried out by differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS) under low hydration conditions. DSC studies revealed that DOPE phase transition temperatures did not shift significantly upon UA addition. On the other hand, in SAXS studies, a different pattern of DOPE phase organization was observed in the presence of UA, with the occurrence of the cubic phase Im3m at 20 °C and the cubic phase Pn3m at 60 °C. These findings suggest that UA interacts with the lipids and changes their self-assembly. However, these interactions between the lipids and UA were unable to eliminate the lamellar to non-lamellar phase transition, which is essential for the cytoplasmic delivery of UA molecules from SpHL-UA.

Experimental determination and computational interpretation of biophysical properties of lipid bilayers enriched by cholesteryl hemisuccinate

Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water–membrane interface. The implications of these findings for considerations of protein–cholesterol interactions are briefly discussed.

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Expression, purification and reconstitution of the 4-hydroxybenzoate transporter PcaK from Acinetobacter sp. ADP1

The aromatic acid:H+ symporter family of integral membrane proteins play an important role in the microbial metabolism of aromatic compounds. Here, we show that the 4-hydroxybenzoate transporter from Acinetobacter sp. ADP1, PcaK, can be successfully overexpressed in Escherichia coli and purified by affinity chromatography. Affinity-purified PcaK is a stable, monodisperse homotrimer in the detergent n-dodecyl-β-d-maltopyranoside supplemented with cholesteryl hemisuccinate. The purified protein has α-helical secondary structure and can be reconstituted to a functional state in synthetic proteoliposomes. Asymmetric substrate transport was observed when proteoliposomes were energized by applying an electrochemical proton gradient (Δμ‾H+) or a membrane potential (ΔΨ) but not by ΔpH alone. PcaK was selective in transporting 4-hydroxybenzoate and 3,4-dihydroxybenzoate over closely related compounds, confirming previous reports on substrate specificity. However, PcaK also showed an unexpected preference for transporting 2-hydroxybenzoates. These results provide the basis for further detailed studies of the structure and function of this family of transporters.

How well does cholesteryl hemisuccinate mimic cholesterol in saturated phospholipid bilayers?

Cholesteryl hemisuccinate is a detergent that is often used to replace cholesterol in crystallization of membrane proteins. Here we employ atomistic molecular dynamics simulations to characterize how well the properties of cholesteryl hemisuccinate actually match those of cholesterol in saturated protein-free lipid membranes. We show that the protonated form of cholesteryl hemisuccinate mimics many of the membrane properties of cholesterol quite well, while the deprotonated form of cholesteryl hemisuccinate is less convincing in this respect. Based on the results, we suggest that cholesteryl hemisuccinate in its protonated form is a quite faithful mimic of cholesterol for membrane protein crystallization, if specific cholesterol-protein interactions (not investigated here) are not playing a crucial role.

Ammonium glycyrrhizinate-loaded niosomes as a potential nanotherapeutic system for anti-inflammatory activity in murine models.

BackgroundLiquorice extracts demonstrate therapeutic efficacy in treating dermatitis, eczema, and psoriasis when compared with corticosteroids. In this work, nonionic surfactant vesicles (niosomes, NSVs) containing polysorbate 20 (Tween 20), cholesterol, and cholesteryl hemisuccinate at different molar concentrations were used to prepare monoammonium glycyrrhizinate (AG)-loaded NSVs. The anti-inflammatory properties of AG-loaded NSVs were investigated in murine models.MethodsThe physicochemical properties of the NSVs were characterized using dynamic light scattering. The fluidity of the lipid bilayer was evaluated by measuring the fluorescence intensity of diphenylhexatriene. The drug entrapment efficiency of AG was assessed using high-performance liquid chromatography. The physicochemical stability of the NSVs was evaluated as a function of time using dynamic light scattering combined with Turbiscan Lab® Expert analysis. Serum stability was determined by incubating the NSVs with 10% v/v fetal bovine serum. The cytotoxic effects of the NSVs were investigated in human dermal fibroblasts using the Trypan blue dye exclusion assay (for cell mortality) and an MTT assay (for cell viability). Release profiles for the AG-loaded NSVs were studied in vitro using cellulose membranes. NSVs showing the most desirable physicochemical properties were selected to test for in vivo anti-inflammatory activity in murine models. The anti-inflammatory activity of the NSVs was investigated by measuring edema and nociception in mice stimulated with chemical agents.ResultsNSVs showed favorable physicochemical properties for in vitro and in vivo administration. In addition, they demonstrated long-term stability based on Turbiscan Lab Expert analysis. The membrane fluidity of the NSVs was not affected by self-assembling of the surfactants into colloidal structures. Fluorescence anisotropy was found to be independent of the molar ratios of cholesteryl hemisuccinate and/or cholesterol during preparation of the NSVs. The anti-inflammatory AG drug showed no effect on the stability of the NSVs. In vivo experiments demonstrated that AG-loaded NSVs decreased edema and nociceptive responses when compared with AG alone and empty NSVs. In vitro and in vivo results demonstrated that pH sensitive and neutral NSVs show no statistical significant difference.ConclusionNSVs were nontoxic and showed features favorable for potential administration in vivo. In addition, neutral NSVs showed signs of increased anti-inflammatory and antinociceptive responses when compared with AG.

Plasma stable, pH-sensitive non-ionic surfactant vesicles simultaneously enhance antiproliferative effect and selectivity of Sirolimus

Objective: The purpose of the present investigation was to prepare a plasma stable, pH-sensitive niosomal formulation to enhance Sirolimus efficacy and selectivity.Materials and methods: pH-sensitive niosomal formulations bearing PEG-Poly (monomethyl itaconate)-CholC6 (PEG-PMMI-CholC6) copolymers and cholesteryl hemisuccinate (CHEMS) were prepared by a modified ethanol injection method and characterized with regard to pH-responsiveness and stability in human serum. The ability of pH-sensitive niosomes to enhance the Sirolimus cytotoxicity was evaluated in vitro using human erythromyeloblastoid leukemia cell line (K562) and compared with cytotoxicity effect on human umbilical vein endothelial cells (HUVEC).Results and discussion: This study showed that both formulations can be rendered pH-sensitive property and were found to rapidly release their contents under mildly acidic conditions. However, the CHEMS-based niosomes lost their pH-sensitivity after incubation in plasma, whereas, PEG-PMMI-CholC6 niosomes preserved their ability to respond to pH change. Sirolimus encapsulated in pH-sensitive niosomes exhibited a higher cytotoxicity than the control conventional formulation on K562 cell line. On the other hand, both pH-sensitive niosomes showed lower antiproliferative effect on HUVEC cells.Conclusion: Plasma stable, pH-sensitive PEG-PMMI-CholC6-based niosomes can improve the in vitro efficiency and also reduce the side effects of Sirolimus.

Multifunctional Tumor-Targeting Nanocarriers Based on Hyaluronic Acid-Mediated and pH-Sensitive Properties for Efficient Delivery of Docetaxel

The objective of this work was to develop a multifunctional tumor-targeting nanocarrier based on the mechanism of CD44-mediated endocytosis and pH-induced drug release to improve the therapeutic efficacy of docetaxel (DTX). Hyaluronic acid-coated docetaxel-loaded cholesteryl hemisuccinate vesicles (HA-CHEMS vesicles) were prepared. The physiochemical properties and pH-dependent drug release of HA-CHEMS vesicles were evaluated. The HA-CHEMS vesicles were investigated for CD44-mediated internalization and in vitro cell viability using MCF-7,A549 and L929 cells.In addition,tissue distribution as well as antitumor efficacy was also evaluated in MCF-7 tumor-bearing mouse model. The particle size and zeta potential of HA-CHEMS vesicles were 131.4 ± 6.2nm and -13.3 ± 0.04mV,respectively. The in vitro drug release results demonstrated a pH-responsive drug release under different pH conditions. In vitro cell viability tests suggested that the encapsulation of DTX in HA-CHEMS vesicles led to more than 51.6-fold and 46.3-fold improved growth inhibition in MCF-7 and A549 cell lines,respectively compared to Taxotere®. From the cell uptake studies,the coumarin 6-loaded HA-CHEMS vesicles enhanced intracellular fluorescent intensity in the CD44-overexpressing cell line (MCF-7). Biodistribution studies revealed selective accumulation of HA-CHEMS vesicles in the MCF-7 bearing BalB/c nude mice as a result of passive accumulation and active targeting (CD44-mediated endocytosis). Compared to Taxotere®,HA-CHEMS vesicles exhibited higher antitumor activity by reducing tumor volume (P < 0.05) and drug toxicity,demonstrating the success of the multifunctional targeting delivery. This work corresponds to the preparation of a multifunctional tumor-targeted delivery system. Our investigation shows that hyaluronan-bearing docetaxel-loaded cholesteryl hemisuccinate vesicles (HA-CHEMS vesicles) is a highly promising therapeutic system,leading to tumor regression after intravenous administration without visible toxicity.

Saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.

Computational Prediction and in Vitro Analysis of Potential Physiological Ligands of the Bile Acid Binding Site in Cytochrome c Oxidase

A conserved bile acid site has been crystallographically defined in the membrane domain of mammalian and Rhodobacter sphaeroides cytochrome c oxidase (RsCcO). Diverse amphipathic ligands were shown previously to bind to this site and affect the electron transfer equilibrium between heme a and a3 cofactors by blocking the K proton uptake path. Current studies identify physiologically relevant ligands for the bile acid site using a novel three-pronged computational approach: ROCS comparison of ligand shape and electrostatics, SimSite3D comparison of ligand binding site features, and SLIDE screening of potential ligands by docking. Identified candidate ligands include steroids, nicotinamides, flavins, nucleotides, retinoic acid, and thyroid hormones, which are predicted to make key protein contacts with the residues involved in bile acid binding. In vitro oxygen consumption and ligand competition assays on RsCcO wildtype and its Glu101Ala mutant support regulatory activity and specificity of some of these ligands. An ATP analog and GDP inhibit RsCcO under low substrate conditions, while fusidic acid, cholesteryl hemisuccinate, retinoic acid, and T3 thyroid hormone are more potent inhibitors under both high and low substrate conditions. The sigmoidal kinetics of RsCcO inhibition in the presence of certain nucleotides is reminiscent of previously reported ATP inhibition of mammalian CcO, suggesting regulation involving the conserved core subunits of both mammalian and bacterial oxidases. Ligand binding to the bile acid site is noncompetitive with respect to cytochrome c and appears to arrest CcO in a semioxidized state with some resemblance to the "resting" state of the enzyme.

β2‐Adrenergic Receptor Activation by Agonists Studied with 19F NMR Spectroscopy

β₂‐Adrenergic Receptor Activation by Agonists Studied with ¹⁹F NMR Spectroscopy

Contextual inhibition of fatty acid synthesis by metformin involves glucose-derived acetyl-CoA and cholesterol in pancreatic tumor cells

Metformin, a generic glucose lowering drug, inhibits cancer growth expressly in models that employ high fat/cholesterol intake and/or low glucose availability. Here we use a targeted tracer fate association study (TTFAS) to investigate how cholesterol and metformin administration regulates glucose-derived intermediary metabolism and macromolecule synthesis in pancreatic cancer cells. Wild type K-ras BxPC-3 and HOM: GGT(Gly) → TGT(Cys) K12 transformed MIA PaCa-2 adenocarcinoma cells were cultured in the presence of [1,2-13C2]-d-glucose as the single tracer for 24 h and treated with either 100 μM metformin (MET), 1 mM cholesteryl hemisuccinate (CHS), or the dose matching combination of MET and CHS (CHS–MET). Wild type K-ras cells used 11.43 % (SD = ±0.32) of new acetyl-CoA for palmitate synthesis that was derived from glucose, while K-ras mutated MIA PaCa-2 cells shuttled less than half as much, 5.47 % [SD = ±0.28 (P < 0.01)] of this precursor towards FAS. Cholesterol treatment almost doubled glucose-derived acetyl-CoA enrichment to 9.54 % (SD = ±0.24) and elevated the fraction of new palmitate synthesis by over 2.5-fold in MIA PaCa-2 cells; whereby 100 μM MET treatment resulted in a 28 % inhibitory effect on FAS. Therefore, acetyl-CoA shuttling towards its carboxylase, from thiolase, produces contextual synthetic inhibition by metformin of new palmitate production. Thereby, metformin, mutated K-ras and high cholesterol each contributes to limit new fatty acid and potentially cell membrane synthesis, demonstrating a previously unknown mechanism for inhibiting cancer growth during the metabolic syndrome.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-013-0555-4) contains supplementary material, which is available to authorized users.

Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Metformin inhibits the TCA cycle and fatty acid synthesis in MIAPaCa-2 pancreatic cancer cells

M.J. Cantoria , L.G. Boros , E.J. Meuillet . Dept. of Nutritional Sciences, The University of Arizona, Tucson, AZ, USA 2 SiDMAP, LLC, Los Angeles, CA, USA Harbor-UCLA Research and Education Institute, UCLA School of Medicine, Torrance, CA, USA Arizona Cancer Center, Tucson, AZ, USA Pancreatic adenocarcinoma is an aggressive malignancy that is very difficult to diagnose and treat. Systemic treatment of pancreatic cancer, either with a single agent or a combination of agents, has shown only modest benefits. Epidemiological studies have reported a lower incidence of pancreatic cancer in diabetics treated with the biguanide metformin but the mechanism bywhich it exerts its putative anti-cancer effects has yet to be elucidated. Using 1,2-13C glucose labeling we show for the first time that metformin (100mM) exerts metabolic control on the pancreatic cancer cell lines BxPC-3 (WT KRAS) and MIAPaCa-2 (G12C-KRAS). Compared to control MIAPaCa-2 cells, metformin inhibits tricarboxylic acid (TCA) cycle turnover in cells grown in media supplemented with 1mM cholesteryl hemisuccinate (CHS), as indicated by decreased CO2 production in the MIAPaCa-2-CHS-metformin group (8.70+0.82 vs 5.31+0.82, p1⁄40.01). Compared to their cell line-specific controls, metformin increases extracellular lactate labeling in CHS-BxPC-3 (8.79+0.78 vs 11.11+0.45, p1⁄40.02) and CHS-MIAPaCa-2 (10.75+0.65 vs 17.79+1.31 vs, p1⁄40.004) cells, providing evidence that metformin prevents pyruvate from entering the TCA cycle. Metformin also inhibits succinate formation and the activities of pyruvate dehydrogenase and pyruvate carboxylase as measured by glutamate labeling. Metformin decreases new fatty acid synthesis in CHStreated BxPC-3 (3.86+0.42 vs 8.17+0.78, p1⁄40.002) and MIAPaCa-2 (11.30+0.81 vs 15.30+0.96, p1⁄40.006) compared to the groups that were not metformin-treated. Understanding the mechanism behind the phenotypic differences in metformin's control of metabolic fluxes in these two cell lines will help support the application of metformin as a potential anticancer drug and potentially discover novel drug targets for pancreatic cancer.

A Conserved Amphipathic Ligand Binding Region Influences K-Path-Dependent Activity of Cytochrome c Oxidase

A conserved, crystallographically defined bile acid binding site was originally identified in the membrane domain of mammalian and bacterial cytochrome c oxidase (CcO). Current studies show other amphipathic molecules including detergents, fatty acids, steroids, and porphyrins bind to this site and affect the already 50% inhibited activity of the E101A mutant of Rhodobacter sphaeroides CcO as well as altering the activity of wild-type and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine, and CHOBIMALT detergents further inhibit RsCcO E101A, with lesser inhibition observed in wild-type. The detergent inhibition is overcome in the presence of micromolar concentrations of steroids and porphyrin analogues including deoxycholate, cholesteryl hemisuccinate, bilirubin, and protoporphyrin IX. In addition to alleviating detergent inhibition, amphipathic carboxylates including arachidonic, docosahexanoic, and phytanic acids stimulate the activity of E101A to wild-type levels by providing the missing carboxyl group. Computational modeling of dodecyl maltoside, bilirubin, and protoporphyrin IX into the conserved steroid site shows energetically favorable binding modes for these ligands and suggests that a groove at the interface of subunit I and II, including the entrance to the K-path and helix VIII of subunit I, mediates the observed competitive ligand interactions involving two overlapping sites. Spectral analysis indicates that ligand binding to this region affects CcO activity by altering the K-path-dependent electron transfer equilibrium between heme a and heme a(3). The high affinity and specificity of a number of compounds for this region, and its conservation and impact on CcO activity, support its physiological significance.

Supramolecular dendrimers based on the self-assembly of carbazole-derived dendrons and triazine rings: liquid crystal, photophysical and electrochemical properties

Two novel families of dendrimers have been synthesized by hydrogen bonding between a triazine moiety (M), as an electron-transporting central core, and three peripheral dendrons of generations 1 and 2 derived from bis(hydroxymethyl)propionic acid (bis-MPA). The dendrons contain both carbazole-based groups as hole-transporting moieties and promesogenic units derived from 5-[4-(4-butoxybenzoyloxy)phenyloxy]pentanoic acid (family A) or cholesteryl hemisuccinate (family B) (in a 1 : 3 ratio). The formation of the complexes was confirmed by IR and NMR data. All synthesized complexes displayed mesogenic properties and their liquid crystalline properties have been investigated by means of differential scanning calorimetry (DSC), polarized optical microscopy (POM) and X-ray diffractometry (XRD). Complexes derived from CzAn dendrons (family A) exhibit nematic mesomorphism whereas complexes derived from CzBn dendrons (family B) exhibit a smectic A phase. The electrochemical behavior of the dendritic complexes was investigated by cyclic voltammetry (CV) and the dendrimers show irreversible or quasi-reversible electrochemical behavior in the range 0.0–2.0 V at a scan rate of 100 mV s−1. The UV-vis absorption and emission properties of the compounds and the photoconductive properties of the dendrons and dendrimers have also been investigated.

Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors

pH-sensitive liposomes represent an effective gene vector in cancer therapy. However, their use is greatly hampered by their relatively low transfection efficiency. To improve the transfection efficiency of pH-sensitive liposomes, we prepared complexes containing 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidyl ethanolamine (DOPE) liposomes and pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and DOPE, and evaluated the influence of various factors on plasmid DNA (pDNA) transfection efficiency. All DC-Chol/DOPE liposome/pDNA and pH-sensitive liposome complexes showed similarly potent pH sensitivity. In the presence of serum-containing medium, two optimized complexes of DC-Chol/DOPE liposomes/pDNA and pH-sensitive PEGylated liposomes showed high transfection efficiency of 22.94% and 20.07%, respectively. Notably, DC-Chol/DOPE (2:3) liposomes/pH-sensitive PEGylated (1%) liposome complexes with a charge ratio of 1:1 (m/m [+/−]) showed enhanced accumulation in tumors in vivo. Our results show the influence of various factors on pDNA transfection efficiency in complexes of DC-Chol/DOPE liposomes and pH-sensitive PEGylated liposomes. Understanding of such mechanisms will lead to better design of complexes of DC-Chol/DOPE liposomes and pH-sensitive liposomes for gene therapy.

Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor

The steroid cholesterol is an essential component of eukaryotic membranes, and it functionally modulates membrane proteins, including G protein-coupled receptors. To reveal insight into how cholesterol modulates G protein-coupled receptors, we have used dynamic single-molecule force spectroscopy to quantify the mechanical strength and flexibility, conformational variability, and kinetic and energetic stability of structural segments stabilizing the human β(2)-adrenergic receptor (β(2)AR) in the absence and presence of the cholesterol analog cholesteryl hemisuccinate (CHS). CHS considerably increased the kinetic, energetic, and mechanical stability of almost every structural segment at sufficient magnitude to alter the structure and functional relationship of β(2)AR. One exception was the structural core segment of β(2)AR, which establishes multiple ligand binding sites, and its properties were not significantly influenced by CHS.