Methyl Β-cyclodextrin Research Articles

Preparation of trimethoprim/methyl β-cyclodextrin complexes, in vitro and in vivo pharmacokinetic study, and evaluation of antibacterial activity in combination with the complex of sulfamethoxazole/methyl β-cyclodextrin

Through cyclodextrin selection from phase solubility study, complexation method screening, and complexation condition optimization by using single and orthogonal strategy, the complexes of trimethoprim with methyl β-cyclodextrin, HP β-cyclodextrin and HP-γ-cyclodextrin with the best solubility increasing so far were prepared and confirmed by FTIR, DSC and proton NMR spectra. The in vitro pharmacokinetic study showed that trimethoprim was 80 %–90 % released in three complexes in 20 min., and in their dog's in vivo pharmacokinetic study, the Cmax was increased from 0.22 µg/mL to 0.85 µg/mL, 0.7 µg/mL and 0.72 µg/mL, the AUC0∼40 was increased from 2.35 µg*h/mL to 3.94 µg*h/mL, 5.06 µg*h/mL and 6.73 µg*h/mL, the LC was shorten from 2.17 L/kg/h to 0.89 L/kg/h, 0.56 L/kg/h and 1.07 L/kg/h for the complexes with methyl β-cyclodextrin, HP β-cyclodextrin and HP-γ-cyclodextrin respectively. After combination with sulfamethoxazole cyclodextrin complex, the antibacterial activity of the complex of trimethoprim with methyl β-cyclodextrin was increased significantly compared with the pure drug alone, indicated that this complex can be used as new potent antibacterial agent.

Design and development of a chitosan-based nasal powder of dimethyl fumarate-cyclodextrin binary systems aimed at nose-to-brain administration. A stability study

The nasal administration route has been studied for the delivery of active molecules directed to the Central Nervous System, thanks to the anatomical connection between the nasal cavity and the brain.Dimethyl fumarate is used to treat relapsing-remitting multiple sclerosis, with a role as an immunomodulator towards T- T-cells and a cytoprotector towards neurons and glial cells. Its use in therapy is hindered by its low aqueous solubility, and low stability, due to hydrolysis and sublimation at room temperature. To overcome this limitation, in this study we evaluated the feasibility of using two amorphous β-cyclodextrin derivatives, namely hydroxypropyl β-cyclodextrin and methyl β-cyclodextrin, to obtain a nasally administrable powder with a view to nose-to-brain administration.Initially, the interaction product was studied using different analytical methods (differential scanning calorimetry, Fourier transform infrared spectroscopy and powder X-ray diffraction) to detect the occurrence of binary product formation, while phase solubility analysis was used to probe the complexation in solution.The dimethyl fumarate-cyclodextrin binary product showing best solubility and stability properties was subsequently used in the development of a chitosan-based mucoadhesive nasally administrable powder comparing different preparative methods. The best performance in terms of both hydrolytic stability and DMF recovery was achieved by the powder obtained via freeze-drying.

Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and β-cyclodextrin

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (μ = 0.036 day−1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM β-CD + 100 μM MeJA (9.57 mg/g DW) and 5.0 mM β-CD + 100 μM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined β-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and β-CD on withaferin A production.

Cordyceps fungus-mediated enantioselective synthesis of chiral alcohol and enhanced biocatalytic efficiency in cyclodextrin/surfactant-buffer system

Asymmetric synthesis of chiral alcohols by biocatalysts holds promising prospect. In this study, a medicinal Cordyceps fungus, namely Tolypocladium inflatum, was adopted as a novel biocatalyst for the synthesis of chiral alcohols. Among the assessed prochiral ketone substrates, T. inflatum exhibited the best catalytic activity towards 1-(4-bromophenyl)−2,2,2-trifluoroethanone (BPFO), yielding a valuable pharmaceutical intermediate (S)−1-(4-bromophenyl)−2,2,2-trifluoroethanol ((S)-BPFL) with >99.9 % enantiomeric excesses (ee). For more efficient biosynthesis of (S)-BPFL, cyclodextrins or surfactants were introduced to the reaction system as additives to strengthen this bioprocess. Among which methyl β-cyclodextrin (MCD) improved cell membrane permeability and alleviated product inhibition, while Triton X-100 enhanced BPFO solubility. Furthermore, synergistic promotion for BPFO bioreduction by MCD and Triton X-100 was observed. Under the optimal reaction conditions, 50 mM BPFO was converted into (S)-BPFL with 89.1 % yield and >99.9 % ee in 12 h, showing 16-fold increase of space-time yield in the developed MCD/Triton X-100-buffer system compared to neat buffer solution. This study illustrated the potential of the enantioselective preparation of chiral alcohols mediated by Cordyceps fungus. Additionally, the biocatalytic efficiency was significantly improved in the constructed synergistic reaction system, and the underlying mechanism was also explored.

Incorporation of Loratadine-Cyclodextrin Complexes in Oral Thin Films for Rapid Drug Delivery

Rapidly dissolving polymer thin films, or oral thin films (OTFs), have recently emerged as an improved oral drug delivery vehicle with its ability to bypass liver first pass metabolism, longer shelf-life, and simpler transport and distribution requirements, compared to traditional tablets and liquid formulations. Loratadine (LOR), an antihistamine commonly used to treat allergic rhinitis, undergoes liver first pass metabolism and is a prime candidate for incorporation within an OTF. However, loratadine is a BCS II drug with low aqueous solubility. Herein, the solubility of loratadine was improved by complexation with methyl β-cyclodextrin (MBCD) by co-evaporation of 2:1, 1:1, and 1:2 LOR:MBCD ratios and incorporation into a pullulan-based OTF at 4 wt% by solvent casting at 50 °C for 30 – 35 min. A therapeutically relevant 10 mg LOR dose could be prepared in a 3 cm by 3 cm OTF. The feasibility of complexation was observed with a Bs-type phase solubility diagram, and complexation itself was confirmed via differential scanning calorimetry (DSC) by disappearance of the LOR melting peak, Fourier-transform infrared spectroscopy (FTIR) by shifting of the C=O peak, via 1H NMR spectroscopy by downfield shifting and change in peak multiplicity of the LOR aromatic protons, and via diffusion-ordered spectroscopy by a decrease in the diffusion coefficient of LOR:MBCD complex. LOR:MBCD could be incorporated homogeneously throughout an OTF, and LOR:MBCD OTFs exhibited reasonable mechanical strength and endured 12 ± 3 folds before breaking. LOR:MBCD OTFs disintegrated within 38 ± 10 s. The cumulative in vitro release of LOR:MBCD OTFs peaked at 80 % within 3–4 min of dissolution, and LOR in LOR:MBCD OTFs exhibited permeability across a 0.22 μm nitrocellulose membrane, demonstrating its applicability as a rapid drug delivery vehicle.

Endothelial cells differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes.

Endothelial cells (ECs) experience two different blood flow patterns: laminar and disturbed flow. Their responses to laminar flow contribute to vascular homeostasis, whereas their responses to disturbed flow result in EC dysfunction and vascular diseases. However, it remains unclear how ECs differentially sense laminar and disturbed flow and trigger signaling that elicits different responses. Here, we showed that ECs differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes in opposite directions. This results in distinct changes in mitochondrial function, namely, increased adenosine triphosphate (ATP) production for laminar flow and increased hydrogen peroxide (H2O2) release for disturbed flow, leading to ATP- and H2O2-mediated signaling, respectively. When cultured human aortic ECs were subjected to laminar or disturbed flow in flow-loading devices, the lipid order of their plasma membranes immediately decreased in response to laminar flow and increased in response to disturbed flow. Laminar flow also decreased the lipid order of mitochondrial membranes and increased mitochondrial ATP production. In contrast, disturbed flow increased the lipid order of mitochondrial membranes and increased the release of H2O2 from the mitochondria. The addition of cholesterol to the cells increased the lipid order of both membranes and abrogated laminar flow-induced ATP production, while treatment of the cells with a cholesterol-depleting reagent, methyl-β cyclodextrin, decreased the lipid order of both membranes and abolished disturbed flow-induced H2O2 release, indicating that changes in the membrane lipid order and/or cholesterol content are closely linked to flow-induced changes in mitochondrial functions.NEW & NOTEWORTHY How vascular endothelial cells (ECs) differentially sense laminar and disturbed flows and trigger intracellular signaling remains unclear. Here, we show that EC plasma membranes act as mechanosensors to discriminate between laminar and disturbed flows by undergoing opposite changes in their lipid order. Similar lipid order changes occur simultaneously in the mitochondrial membranes, which are linked to changes in mitochondrial function, that is, increased ATP production for laminar flow and increased H2O2 release for disturbed flow.

Enhanced photostability and biocompatibility of sunscreen formulation of 2-phenylbenzimidazole-5-sulfonic acid with methyl-beta-cyclodextrin

Ensuring the photostability of sunscreen lotions is crucial for their effectiveness and safety when exposed to sunlight. Photostability refers to the ability of a substance to remain stable and undergo minimal chemical degradation or photochemical reactions upon exposure to ultraviolet (UV) radiation, especially from sunlight. In this study, we focused on 2-phenylbenzimidazole-5-sulfonic acid (PBS), one of the leading sunscreen agents, and aimed to form inclusion complexes (IC) with methyl-β-cyclodextrin (MCD). Through phase solubility measurements, we determined the stoichiometry of the IC to be 1:1, with a binding constant of 155.4 M−1. Proton Nuclear Magnetic Resonance (1H NMR) and ROESY analysis provided evidence that the benzimidazole part of PBS is successfully included in the cavity of MCD. Quantum mechanical calculations using the B3LYP/6-31G(D, P) method in the Gaussian 16 package further supported the formation of the IC by providing energy data. Notably, the photostability of the IC is maintained for up to four hours of UV exposure, showing no significant structural changes in PBS. Moreover, we conducted biocompatibility tests, and the results are encouraging for the superior activity, and suggest the potential of the IC for promoting its cosmetic application as a UVB sunscreen.

Altered Caveolin-1 Dynamics Result in Divergent Mineralization Responses in Bone and Vascular Calcification.

Though vascular smooth muscle cells adopt an osteogenic phenotype during pathological vascular calcification, clinical studies note an inverse correlation between bone mineral density and arterial mineral-also known as the calcification paradox. Both processes are mediated by extracellular vesicles (EVs) that sequester calcium and phosphate. Calcifying EV formation in the vasculature requires caveolin-1 (CAV1), a membrane scaffolding protein that resides in membrane invaginations (caveolae). Of note, caveolin-1-deficient mice, however, have increased bone mineral density. We hypothesized that caveolin-1 may play divergent roles in calcifying EV formation from vascular smooth muscle cells (VSMCs) and osteoblasts (HOBs). Primary human coronary artery VSMCs and osteoblasts were cultured for up to 28days in an osteogenic media. CAV1 expression was knocked down using siRNA. Methyl β-cyclodextrin (MβCD) and a calpain inhibitor were used, respectively, to disrupt and stabilize the caveolar domains in VSMCs and HOBs. CAV1 genetic variation demonstrates significant inverse relationships between bone-mineral density (BMD) and coronary artery calcification (CAC) across two independent epidemiological cohorts. Culture in osteogenic (OS) media increased calcification in HOBs and VSMCs. siRNA knockdown of CAV1 abrogated VSMC calcification with no effect on osteoblast mineralization. MβCD-mediated caveolae disruption led to a 3-fold increase of calcification in VSMCs treated with osteogenic media (p < 0.05) but hindered osteoblast mineralization (p < 0.01). Conversely, stabilizing caveolae by calpain inhibition prevented VSMC calcification (p < 0.05) without affecting osteoblast mineralization. There was no significant difference in CAV1 content between lipid domains from HOBs cultured in OS and control media. Our data indicate fundamental cellular-level differences in physiological and pathophysiological mineralization mediated by CAV1 dynamics. This is the first study to suggest that divergent mechanisms in calcifying EV formation may play a role in the calcification paradox. The online version contains supplementary material available at 10.1007/s12195-023-00779-7.

A Comparison between the Molecularly Imprinted and Non-Molecularly Imprinted Cyclodextrin-Based Nanosponges for the Transdermal Delivery of Melatonin.

Melatonin is a neurohormone that ameliorates many health conditions when it is administered as a drug, but its drawbacks are its oral and intravenous fast release. To overcome the limitations associated with melatonin release, cyclodextrin-based nanosponges (CD-based NSs) can be used. Under their attractive properties, CD-based NSs are well-known to provide the sustained release of the drug. Green cyclodextrin (CD)-based molecularly imprinted nanosponges (MIP-NSs) are successfully synthesized by reacting β-Cyclodextrin (β-CD) or Methyl-β Cyclodextrin (M-βCD) with citric acid as a cross-linking agent at a 1:8 molar ratio, and melatonin is introduced as a template molecule. In addition, CD-based non-molecularly imprinted nanosponges (NIP-NSs) are synthesized following the same procedure as MIP-NSs without the presence of melatonin. The resulting polymers are characterized by CHNS-O Elemental, Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric (TGA), Differential Scanning Calorimetry (DSC), Zeta Potential, and High-Performance Liquid Chromatography (HPLC-UV) analyses, etc. The encapsulation efficiencies are 60-90% for MIP-NSs and 20-40% for NIP-NSs, whereas melatonin loading capacities are 1-1.5% for MIP-NSs and 4-7% for NIP-NSs. A better-controlled drug release performance (pH = 7.4) for 24 h is displayed by the in vitro release study of MIP-NSs (30-50% released melatonin) than NIP-NSs (50-70% released melatonin) due to the different associations within the polymeric structure. Furthermore, a computational study, through the static simulations in the gas phase at a Geometry Frequency Non-covalent interactions (GFN2 level), is performed to support the inclusion complex between βCD and melatonin with the automatic energy exploration performed by Conformer-Rotamer Ensemble Sampling Tool (CREST). A total of 58% of the CD/melatonin interactions are dominated by weak forces. CD-based MIP-NSs and CD-based NIP-NSs are mixed with cream formulations for enhancing and sustaining the melatonin delivery into the skin. The efficiency of cream formulations is determined by stability, spreadability, viscosity, and pH. This development of a new skin formulation, based on an imprinting approach, will be of the utmost importance in future research at improving skin permeation through transdermal delivery, associated with narrow therapeutic windows or low bioavailability of drugs with various health benefits.

Cortisol Rapidly Facilitates Glucocorticoid Receptor Translocation to the Plasma Membrane in Primary Trout Hepatocytes

Glucocorticoids (GCs) stimulate rapid cell signalling by activating the membrane-anchored intracellular glucocorticoid receptor (GR). However, the recruitment of the GR to the plasma membrane to facilitate nongenomic signalling is far from clear. As cytosolic free calcium ([Ca2+]i) is involved in intracellular protein dynamics, we tested the hypothesis that acute elevation in cortisol levels rapidly stimulates GR translocation to the plasma membrane via a calcium-dependent process in rainbow trout (Oncorhynchus mykiss) hepatocytes. To test this, we monitored temporal changes in intracellular GR distribution in response to cortisol exposure. Immunofluorescence labelling showed that the GR was present in cytosolic and nuclear compartments in trout hepatocytes. However, upon cortisol exposure, the GR rapidly (within 5 min) formed punctate and colocalized with caveolin-1, suggesting plasma membrane localization of the receptor. This redistribution of the GR to the plasma membrane was transient and lasted for 30 min and was evident even upon exposure to cortisol-BSA, a membrane-impermeable analogue of the steroid. The rapid cortisol-mediated GR translocation to the plasma membrane involved F-actin polymerization and was completely abolished in the presence of either EGTA or Cpd5J-4, a calcium release-activated calcium (CRAC) channel blocker. Additionally, the modulation of the biophysical properties of the plasma membrane by cholesterol or methyl β-cyclodextrin, which led to changes in ([Ca2+]i) levels, modified GR translocation to the plasma membrane. Altogether, acute cortisol-mediated rise in ([Ca2+]i) levels rapidly stimulated the translocation of intracellular GR to the plasma membrane, and we propose this as a mechanism promoting the nongenomic action of the GR for hepatocyte stress resistance.

Effect of Lipid Raft Disruptors on Cell Membrane Fluidity Studied by Fluorescence Spectroscopy.

Lipid rafts are specialized microdomains in cell membranes, rich in cholesterol and sphingolipids, and play an integrative role in several physiological and pathophysiological processes. The integrity of rafts can be disrupted via their cholesterol content-with methyl-β-cyclodextrin (MCD) or with our own carboxamido-steroid compound (C1)-or via their sphingolipid content-with sphingomyelinase (SMase) or with myriocin (Myr). We previously proved by the fluorescent spectroscopy method with LAURDAN that treatment with lipid raft disruptors led to a change in cell membrane polarity. In this study, we focused on the alteration of parameters describing membrane fluidity, such as generalized polarization (GP), characteristic time of the GP values change-Center of Gravity (τCoG)-and rotational mobility (τrot) of LAURDAN molecules. Myr caused a blue shift of the LAURDAN spectrum (higher GP value), while other agents lowered GP values (red shift). MCD decreased the CoG values, while other compounds increased it, so MCD lowered membrane stiffness. In the case of τrot, only Myr lowered the rotation of LAURDAN, while the other compounds increased the speed of τrot, which indicated a more disordered membrane structure. Overall, MCD appeared to increase the fluidity of the membranes, while treatment with the other compounds resulted in decreased fluidity and increased stiffness of the membranes.

Free Cholesterol Affects the Function and Localization of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1).

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are associated with obesity. They are accompanied by increased levels of free cholesterol in the liver. Most free cholesterol resides within the plasma membrane. We assessed the impact of adding or removing free cholesterol on the function and localization of two hepatocellular uptake transporters: the Na+/taurocholate cotransporting polypeptide (NTCP) and the organic cation transporter 1 (OCT1). We used a cholesterol–MCD complex (cholesterol) to add cholesterol and methyl-β-cyclodextrin (MCD) to remove cholesterol. Our results demonstrate that adding cholesterol decreases NTCP capacity from 132 ± 20 to 69 ± 37 µL/mg/min and OCT1 capacity from 209 ± 66 to 125 ± 26 µL/mg/min. Removing cholesterol increased NTCP and OCT1 capacity to 224 ± 65 and 279 ± 20 µL/mg/min, respectively. In addition, adding cholesterol increased the localization of NTCP within lipid rafts, while adding or removing cholesterol increased OCT1 localization in lipid rafts. These results demonstrate that increased cholesterol levels can impair NTCP and OCT1 function, suggesting that the free cholesterol content of the liver can alter bile acid and drug uptake into the liver. This could explain the increased plasma bile acid levels in NAFLD and NASH patients and potentially lead to altered drug disposition.

Artificial Palmitoylation of Proteins Controls the Lipid Domain-Selective Anchoring on Biomembranes and the Raft-Dependent Cellular Internalization.

Protein palmitoylation, a post-translational modification, is universally observed in eukaryotic cells. The localization of palmitoylated proteins to highly dynamic, sphingolipid- and cholesterol-rich microdomains (called lipid rafts) on the plasma membrane has been shown to play an important role in signal transduction in cells. However, this complex biological system is not yet completely understood. Here, we used a combined approach where an artificial lipidated protein was applied to biomimetic model membranes and plasma membranes in cells to illuminate chemical and physiological properties of the rafts. Using cell-sized giant unilamellar vesicles, we demonstrated the selective partitioning of enhanced green fluorescent protein modified with a C-terminal palmitoyl moiety (EGFP-Pal) into the liquid-ordered phase consisting of saturated phospholipids and cholesterol. Using Jurkat T cells treated with an immunostimulant (concanavalin A), we observed the vesicular transport of EGFP-Pal. Further cellular studies with the treatment of methyl β-cyclodextrin revealed the cholesterol-dependent internalization of EGFP-Pal, which can be explained by a raft-dependent, caveolae-mediated endocytic pathway. The present synthetic approach using artificial and natural membrane systems can be further extended to explore the potential utility of artificially lipidated proteins at biological and artificial interfaces.

Synergistic promotion for microbial asymmetric preparation of (R)-2-chloro-1-(2,4-dichlorophenyl)ethanol by NADES and cyclodextrin

Microbial whole-cell catalysis has been a popular method for chiral alcohols preparation. In this study, Cyberlindnera saturnus ZJPH1807 was successfully employed as a biocatalyst to reduce 2-chloro-1-(2,4-dichlorophenyl)ethanone (CPE) to (R)-2-chloro-1-(2,4-dichlorophenyl)ethanol ((R)-CPEO), a key chiral intermediate of antifungal drug miconazole. In order to further improve the bioprocess yield, natural deep eutectic solvent and cyclodextrin were introduced into the reaction system as additives. It was found that carnitine/trehalose (C/Tre, 1:1 mol/mol) could increase the permeability of cell membrane appropriately and improve the mass transfer rate. And the addition of methyl-β-cyclodextrin (MCD) could improve the solubility of CPE in buffer solution, simultaneously promoting cofactor recycling. Furthermore, MCD and C/Tre exerted a synergistic effect on the acceleration of CPE bioreduction, producing 91.0% yield in 1.5 h at 35.8 mM CPE in a C/Tre-MCD-containing system, with a 12.4-fold increase in productivity compared to neat buffer solution only. When the substrate loading was increased to 67.1 mM, the yield could also achieve 84.2%. The established bioreduction process was also feasible at 100 mL preparation scale with a 88.5% (R)-CPEO yield. The integration of C/Tre and MCD can significantly improve catalytic efficiency for hydrophobic substrates and be applied in biocatalysis.

Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents.

A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F’-[K(DipR)5]) alone and F’-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)5] alone or F’-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

Enhanced-solubilization and dissolution of multicomponent DNAPL from homogeneous porous media

The presence of multicomponent dense nonaqueous phase liquid (DNAPL) mixtures in porous media can significantly limit the effectiveness of groundwater remediation. A series of column transport and flushing experiments were conducted to quantify the impact of various enhanced flushing agents on dissolution and removal of a multicomponent DNAPL source within a macroscopically homogeneous porous medium. The columns were established with NAPL saturations consisting of an equal mole mixture of cis-1,2-dichloroethene, trichloroethene, and tetrachloroethene. The solubilization agents used included two complexing sugars - hydroxypropyl-β-cyclodextrin (HPCD and methyl-β-cyclodextrin (MCD); a surfactant - sodium dodecyl sulfate (SDS); and a cosolvent - ethanol (EtOH). The chemical flushing agents greatly reduced the time needed to remove each DNAPL component, compared to flushing with water alone. Initial DNAPL-component elution concentrations were successfully predicted using Raoult's Law for MCD, HPCD, and water flushing, indicating that ideal dissolution was initiated by the lower-power enhanced-solubilization agents. EtOH was most efficient at removing the contaminants in terms of normalized mass recovery but least efficient based on a mass-ratio and mole-ratio of contaminant to reagent analysis. SDS was most efficient for contaminant removal when analyzed based on mass-contaminant to mass-reagent recovered and MCD was most effective based on a moles-contaminant to moles-reagent recovered efficiency evaluation. However, in terms of mass flux reduction analysis (i.e. removal metric), SDS was least efficient for contaminant removal compared to all other enhanced-flushing agents tested, especially during the initial stages of DNAPL removal. Results from this study indicate that ideal dissolution was initiated during enhanced-solubilization and that several criteria should be used to evaluate the removal effectiveness of flushing agents for multi-component NAPL systems.

Lipid rafts integrity is essential for prolactin-induced mitogenesis in mouse embryonic stem cells.

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constraints and regulations applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using two-dimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergent-free preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency, we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs act in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

Methods for Studying Cholesterol-Dependent Regulation of P2X7 Receptors.

Cholesterol dynamically regulates P2X7 receptor function in both physiological and pathological conditions. Studies suggest that cholesterol suppresses P2X7 receptor activity through direct binding or through indirect effects on the biophysical properties of the membrane. Notably, the palmitoylated C-terminus seems to counteract the action of cholesterol to make it less inhibitory. However, the mechanism underlying cholesterol-dependent regulation of P2X7 receptor remains unclear. Here we describe detailed methods that facilitate the quantification of P2X7 channel activity while controlling the amount of cholesterol in the system. We will first describe the use of methyl-β-cyclodextrin (MCD), a cyclic oligosaccharide consisting of seven glucose monomers, to decrease or increase plasma membrane cholesterol levels. We will then describe protocols for the reconstitution of purified P2X7 in proteoliposomes of defined lipid composition. These methods can be combined with commonly used techniques such as dye-uptake assays or electrophysiology. We also describe a fluorescence assay to measure cholesterol-binding to P2X7. These approaches are complementary to cryo-EM or molecular dynamics simulations, which are also powerful tools for investigating cholesterol-P2X7 interactions. An improved understanding of the mechanisms of action of cholesterol on P2X7 may contribute to elucidate the roles of this receptor in ageing, inflammation, and cancer, whose progression correlates with the level of cholesterol.

A Critical Role for the IKKβ/Lipid Rafts Axis in Regulating Platelet Exocytosis, Via the Spatial Regulation of SNARE Proteins

Lipid rafts, which are membrane microdomains that are rich in cholesterol and glycosphingolipids, have been implicated in the regulation of intracellular protein trafficking. Furthermore, we have previously shown that SNAREs mediate granule-plasma membrane fusion during exocytosis in platelets, and that IKKβ regulates SNAREs and granule exocytosis. Based on these considerations, we sought to investigate the regulatory relationship between IKKβ and lipid rafts, by employing the PF4-Cre/IKKβflox/flox platelets. We demonstrate that lipid rafts play a critical role in mediating IKKβ-/--dependent regulation of SNARE proteins and SNARE complexes. Specifically, we found that methyl β-cyclodextrin (MβCD, which disrupts lipid rafts) is able to inhibit platelet aggregation, dense and alpha granule secretion, PS exposure and αIIbβ3 integrin activation. We also observed that MβCD is able to inhibit platelet spreading, clot retraction, as well as 7S complex formation. In terms of co-localization, the SNARE protein SNAP-23 was found to associate with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in control platelets, but not in the IKKβ-/- platelets. Furthermore, in resting platelets, plasma membrane SNARE proteins such as syntaxin-11 and SNAP-23 or the granule-associated SNARE vesicle-associated membrane protein VAMP-8 were neither present in the microdomains of control platelets, nor in those from IKKβ-/- mice. Interestingly however, agonist/thrombin stimulation led to the relocation of syntaxin-11 and VAMP-8 into the lipid rafts, and as part of raft-associated phospho-SNAP-23/syntaxin-11/VAMP-8 complexes in control platelets. This seems to be IKKβ dependent, as these associations did not take place in the IKKβ-/- platelets. Taken together, these data reveal a critical role for the IKKβ/lipid rafts axis in regulating platelet exocytosis, via the spatial regulation of SNARE proteins. DisclosuresNo relevant conflicts of interest to declare.

Antinociceptive Effects of Lipid Raft Disruptors, a Novel Carboxamido-Steroid and Methyl β-Cyclodextrin, in Mice by Inhibiting Transient Receptor Potential Vanilloid 1 and Ankyrin 1 Channel Activation

Transient Receptor Potential Vanilloid 1 and Ankyrin 1 (TRPV1, TRPA1) cation channels are expressed in nociceptive primary sensory neurons, and play an integrative role in pain processing and inflammatory functions. Lipid rafts are liquid-ordered plasma membrane microdomains rich in cholesterol, sphingomyelin, and gangliosides. We earlier proved that lipid raft disintegration by cholesterol depletion using a novel carboxamido-steroid compound (C1) and methyl β-cyclodextrin (MCD) significantly and concentration-dependently inhibit TRPV1 and TRPA1 activation in primary sensory neurons and receptor-expressing cell lines. Here we investigated the effects of C1 compared to MCD in mouse pain models of different mechanisms. Both C1 and MCD significantly decreased the number of the TRPV1 activation (capsaicin)-induced nocifensive eye-wiping movements in the first hour by 45% and 32%, respectively, and C1 also in the second hour by 26%. Furthermore, C1 significantly decreased the TRPV1 stimulation (resiniferatoxin)-evoked mechanical hyperalgesia involving central sensitization processes, while its inhibitory effect on thermal allodynia was not statistically significant. In contrast, MCD did not affect these resiniferatoxin-evoked nocifensive responses. Both C1 and MCD had inhibitory action on TRPA1 activation (formalin)-induced acute nocifensive reactions (paw liftings, lickings, holdings, and shakings) in the second, neurogenic inflammatory phase by 36% and 51%, respectively. These are the first in vivo data showing that our novel lipid raft disruptor carboxamido-steroid compound exerts antinociceptive and antihyperalgesic effects by inhibiting TRPV1 and TRPA1 ion channel activation similarly to MCD, but in 150-fold lower concentrations. It is concluded that C1 is a useful experimental tool to investigate the effects of cholesterol depletion in animal models, and it also might open novel analgesic drug developmental perspectives.