Cyclodextrin-modified graphene quantum dots as a novel additive for the selective separation of bioactive compounds by capillary electrophoresis.

Highly reliable separation and determination of various biologically active compounds were achieved using capillary electrophoresis (CE) based on ß-cyclodextrin-functionalized graphene quantum dots (ßcd-GQDs) as the background electrolyte additive. ßcd-GQDs improve the separation efficiency between peaks of all analytes. No addition of surfactants or organic solvents was needed in the running buffer containing ßcd-GQDs. Up to eight consecutive runs were acquired with high precision for the separation of resveratrol, pyridoxine, riboflavin, catechin, ascorbic acid, quercetin, curcumin, and even of several of their structural analogs. Baseline separation was achieved within just 13 min as a result of the effective mobility of the analytes along the capillary owing to the differential interaction with the additive. The proposed analytical method displayed a good resolution of peaks for all species selecting two absorption wavelengths in the diode array detector. Detection limits lower than 0.28 µg mL-1 were found for all compounds and precision values were in the range of 2.1-4.0% in terms of the peak area of the analytes. The usefulness of the GQD-assisted selectivity-enhanced CE method was verified by the analysis of food and dietary supplements. The applicability to such complex matrices and the easy and low-cost GQD preparation open the door for routine analyses of food and natural products. The concept of using such a dual approach (macromolecules and nanotechnology) has been explored to tackle the separation of various bioactive compounds in nutritional supplements and food. Schematic illustration of the electrophoretic separation of the bioactive molecules in the capillary which is filled with the running solution without (top) and with ßcd-GQDs (bottom). The fused silica capillary with negatively ionizable silanol groups at the wall. The voltage is applied at positive polarity at the outlet. R, riboflavin; r, resveratrol; P, pyridoxine; C, catechin; c, curcumin; A, ascorbic acid; Q, quercetin.

Amphiphilic multi-charged cyclodextrins and vitamin K co-assembly as a synergistic coagulant.

Balancing and neutralizing heparin dosing after surgeries and hemodialysis treatment is of great importance in medical and clinical fields. In this study, a series of new amphiphilic multi-charged cyclodextrins (AMCD)s as anti-heparin coagulants were designed and synthesized. The AMCD assembly was capable of selective heparin binding through multivalent bonding and showed a better neutralizing effect towards both unfractionated heparin and low molecular weight heparin than protamine in plasma. Meanwhile, an AMCD and vitamin K (VK) co-assembly was prepared to realize heparin-responsive VK release and provide a novel VK deficiency treatment for hemodialysis patients. This AMCD-VK co-assembly for heparin neutralization & vitamin K supplementation synergistic coagulation represents a promising candidate as a clinical anti-heparin coagulant.

Formulation and evaluation of cyclodextrin complexes for improved anticancer activity of repurposed drug: Niclosamide.

Niclosamide, previously used as an anthelmintic drug is currently being repurposed for its anticancer activity. Niclosamide is a brick like biopharmaceutical classification system (BCS) class II drug with poor aqueous solubility and dissolution consequently leading to low bioavailability. By considering the physicochemical properties and geometry of niclosamide, inclusion complex with cyclodextrin was prepared by freeze drying method and characterized using FT-IR, DSC, PXRD, and 1HNMR. In silico molecular modeling study was performed to study the possible interactions between niclosamide and cyclodextrin. The anticancer activity of niclosamide formulation was evaluated through in vitro cell cytotoxicity study using various cancer cell lines. The potential of niclosamide complex for improvement of the bioavailability was evaluated in male BALB/c mice. In vitro cytotoxicity studies indicated significantly higher cytotoxicity at lower concentrations and the pharmacokinetic studies showed significant improvement in Cmax and Tmax of niclosamide from cyclodextrin complex in comparison to pure niclosamide alone.

Combining excitation-emission matrix fluorescence spectroscopy, parallel factor analysis, cyclodextrin-modified micellar electrokinetic chromatography and partial least squares class-modelling for green tea characterization.

In this study, an alternative analytical approach for analyzing and characterizing green tea (GT) samples is proposed, based on the combination of excitation-emission matrix (EEM) fluorescence spectroscopy and multivariate chemometric techniques. The three-dimensional spectra of 63 GT samples were recorded using a Perkin-Elmer LS55 luminescence spectrometer; emission spectra were recorded between 295 and 800 nm at excitation wavelength ranging from 200 to 290 nm, with excitation and emission slits both set at 10 nm. The excitation and emission profiles of two factors were obtained using Parallel Factor Analysis (PARAFAC) as a 3-way decomposition method. In this way, for the first time, the spectra of two main fluorophores in green teas have been found. Moreover, a cyclodextrin-modified micellar electrokinetic chromatography method was employed to quantify the most represented catechins and methylxanthines in a subset of 24 GT samples in order to obtain complementary information on the geographical origin of tea. The discrimination ability between the two types of tea has been shown by a Partial Least Squares Class-Modelling performed on the electrokinetic chromatography data, being the sensitivity and specificity of the class model built for the Japanese GT samples 98.70% and 98.68%, respectively. This comprehensive work demonstrates the capability of the combination of EEM fluorescence spectroscopy and PARAFAC model for characterizing, differentiating and analyzing GT samples.

Cyclodextrins in Food Technology and Human Nutrition: Benefits and Limitations.

Cyclodextrins are tasteless, odorless, nondigestible, noncaloric, noncariogenic saccharides, which reduce the digestion of carbohydrates and lipids. They have low glycemic index and decrease the glycemic index of the food. They are either non- or only partly digestible by the enzymes of the human gastrointestinal (GI) tract and fermented by the gut microflora. Based on these properties, cyclodextrins are dietary fibers useful for controlling the body weight and blood lipid profile. They are prebiotics, improve the intestinal microflora by selective proliferation of bifidobacteria. These antiobesity and anti-diabetic effects make them bioactive food supplements and nutraceuticals. In this review, these features are evaluated for α-, ß- and γ-cyclodextrins, which are the cyclodextrin variants approved by authorities for food applications. The mechanisms behind these effects are reviewed together with the applications as solubilizers, stabilizers of dietary lipids, such as unsaturated fatty acids, phytosterols, vitamins, flavonoids, carotenoids and other nutraceuticals. The recent applications of cyclodextrins for reducing unwanted components, such as trans-fats, allergens, mycotoxins, acrylamides, bitter compounds, as well as in smart active packaging of foods are also overviewed.

Clotrimazole-cyclodextrin based approach for the management and treatment of Candidiasis - A formulation and chemistry-based evaluation.

Clotrimazole (CT) is a poorly soluble antifungal drug that is most commonly employed as a topical treatment in the management of vaginal candidiasis. The present work focuses on a formulation approach to enhance the solubility of CT using cyclodextrin (CD) complexation. A CT-CD complex was prepared by a co-precipitation method. Various characterization techniques such as differential scanning calorimetry, infrared (IR) and X-ray spectroscopy, scanning electron microscopy and nuclear magnetic resonance (NMR) spectroscopy were performed to evaluate the complex formation and to understand the interactions between CT and CD. Computational molecular modeling was performed using the Schrödinger suite and Gaussian 09 program to understand structural conformations of the complex. The phase solubility curve followed an AL-type curve, indicating formation of a 1:1 complex. Molecular docking studies supported the data obtained through NMR and IR studies. Enthalpy changes confirmed that complexation was an exothermic and enthalpically favorable phenomenon. The CT-CD complexes were formulated in a gel and evaluated for release and antifungal activity. The in vitro release studies performed using gels demonstrated a sustained release of CT from the CT-CD complex with the complex exhibiting improved release relative to the un-complexed CT. Complexed CT-CD exhibited better fungistatic activity toward different Candida species than un-complexed CT.

Polymeric implants for the delivery of green tea polyphenols.

Polymeric implants (millirods) have been tested for local delivery of chemotherapeutic agents in cancer treatment. Modeling of drug release profiles is critical as it may provide theoretical insights on rational implant design. In this study, a biodegradable poly (ε-caprolactone) (PCL) polymeric implant delivery system was tested to deliver green tea polyphenols (GTPs), both in vitro and in vivo. Factors including polymer compositions, supplements, drug loads, and surface area of implants were investigated. Our data showed that GTPs were released from PCL implants continuously for long durations, and drug load was the main determining factor of GTPs release. Furthermore, rates of in vitro release and in vivo release in the rat model followed similar kinetics for up to 16 months. A mathematical model was deduced and discussed. GTP implants have the potential to be used systemically and locally at the tumor site as an alternative strategy.

Enhancing the cyclodextrin production by synchronous utilization of isoamylase and α-CGTase.

Cyclodextrins (CD) are cyclic α-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with α-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 °C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 °C, 40 °C and 60 °C, respectively. When the recombinant isoamylase and α-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of α-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 °C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with α-CGTase alone. This is the first report of synchronous bioconversion of CD by both α-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.

2D solid-state NMR analysis of inclusion in drug-cyclodextrin complexes.

The solubility of drug molecules can often be improved through preparation and delivery of cyclodextrin (CD) inclusion complexes. These drug-oligosaccharide complexes can be prepared in solution and converted to the solid state via methods such as lyophilization and spray-drying, or they can be prepared directly from solids by a variety of methods. The development of drug-CD complexes as solids allows for potential advantages in dosage form design, such as the preparation of layered formulations, and it also can yield improvements in chemical and physical stability. 2D solid-state NMR (SSNMR) methods provide a direct way to probe drug-CD interactions in solid complexes through dipolar interactions between nuclei within the drug and CD molecules. In this study, 2D heteronuclear and homonuclear correlation SSNMR experiments involving (1)H, (13)C, (19)F, and (31)P nuclei are used to demonstrate the inclusion of drug within the CD cavity in a variety of powder samples. To illustrate the general applicability of the SSNMR approach presented, examples are shown for the drugs diflunisal, adefovir dipivoxil, voriconazole, dexamethasone, and prednisolone in complexes with α-CD, ß-CD, and sulfobutylether-substituted ß-CD. The quantitative analysis of included and free drug fractions in a solid drug-CD complex using SSNMR is also demonstrated. On the basis of these results, general approaches to the characterization of these materials using SSNMR are proposed.

Development of sucrose stearate-based nanoemulsions and optimisation through γ-cyclodextrin.

Nanoemulsions aimed at dermal drug delivery are usually stabilised by natural lecithins. However, lecithin has a high tendency towards self-aggregation and is prone to chemical degradation. Therefore, the aim of this study was to develop nanoemulsions with improved structure and long-term stability by employing a natural sucrose ester mixture as sole surfactant. A thorough comparison between the novel sucrose stearate-based nanoemulsions and corresponding lecithin-based nanoemulsions revealed that the sucrose ester is superior in terms of emulsifying efficiency, droplet formation as well as physical and chemical stability. The novel formulations exhibited a remarkably homogeneous structure in cryo TEM investigations, as opposed to the variable structure observed for lecithin-based systems. The in vitro skin permeation rates of lipophilic drugs from sucrose stearate nanoemulsions were comparable to those obtained with their lecithin-based counterparts. Furthermore, it was observed that addition of γ-cyclodextrin led to enhanced skin permeation of the steroidal drug fludrocortisone acetate from 9.99±0.46 to 55.10±3.67 µg cm(-2) after 24 h in the case of sucrose stearate-based systems and from 9.98±0.64 to 98.62±24.89 µg cm(-2) after 24 h in the case of lecithin-based systems. This enhancement effect was significantly stronger in formulations based on lecithin (P<0.05), which indicates that synergistic mechanisms between the surfactant and the cyclodextrin are involved. Cryo TEM images suggest that the cyclodextrin is incorporated into the interfacial film, which might alter drug release rates and improve the droplet microstructure.

Skin penetration behaviour of liposomes as a function of their composition.

Deformable liposomes have been developed and evaluated as a novel topical and transdermal delivery system. Their mechanism of drug transport into and through the skin has been investigated but remains a much debated question. The present study concerns ex vivo diffusion experiments using pig ear skin in order to explain the penetration mechanism of classical and deformable liposomes. Classical and deformable vesicles containing betamethasone in the aqueous compartment through the use of cyclodextrin inclusion complexes were compared to vesicles encapsulating betamethasone in their lipid bilayer. Deformable liposomes contained sodium deoxycholate as the edge activator. Liposomes were characterised by their diameter, encapsulation efficiency, deformability, stability (in terms of change in diameter) and release of encapsulated drug. Ex vivo diffusion studies using Franz diffusion cells were performed. Confocal microscopy was performed to visualise the penetration of fluorescently labelled liposomes into the skin. This study showed that liposomes do not stay intact when they penetrate the deepest layers of the skin. Betamethasone is released from the vesicles after which free drug molecules can diffuse through the stratum corneum and partition into the viable skin tissue.

A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity.

A 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-beta-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, beta-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6'-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application.

Enrichment of endoplasmic reticulum with cholesterol inhibits sarcoplasmic-endoplasmic reticulum calcium ATPase-2b activity in parallel with increased order of membrane lipids: implications for depletion of endoplasmic reticulum calcium stores and apoptosis in cholesterol-loaded macrophages.

Macrophages in advanced atherosclerotic lesions accumulate large amounts of unesterified, or "free," cholesterol (FC). FC accumulation induces macrophage apoptosis, which likely contributes to plaque destabilization. Apoptosis is triggered by the enrichment of the endoplasmic reticulum (ER) with FC, resulting in depletion of ER calcium stores, and induction of the unfolded protein response. To explain the mechanism of ER calcium depletion, we hypothesized that FC enrichment of the normally cholesterol-poor ER membrane inhibits the macrophage ER calcium pump, sarcoplasmic-endoplasmic reticulum calcium ATPase-2b (SERCA2b). FC enrichment of ER membranes to a level similar to that occurring in vivo inhibited both the ATPase activity and calcium sequestration function of SERCA2b. Enrichment of ER with ent-cholesterol or 14:0-18:0 phosphatidylcholine, which possess the membrane-ordering properties of cholesterol, also inhibited SERCA2b. Moreover, at various levels of FC enrichment of ER membranes, there was a very close correlation between increasing membrane lipid order, as monitored by 16-doxyl-phosphatidycholine electron spin resonance, and SERCA2b inhibition. In view of these data, we speculate that SERCA2b, a conformationally active protein with 11 membrane-spanning regions, loses function due to decreased conformational freedom in FC-ordered membranes. This biophysical model may underlie the critical connection between excess cholesterol, unfolded protein response induction, macrophage death, and plaque destabilization in advanced atherosclerosis.

Evaluation of the antioxidant activity of new carotenoid-like compounds by electron paramagnetic resonance.

The antioxidant activity of a novel series of derivatives with a carotenoid-like structure was studied. These derivatives have recently been isolated chemically as a result of studies on the pigments present in a particular species of birds, namely parrots. These novel derivatives, which are also called parrodienes, have been proved to possess interesting biological properties that differ from those that carotenoids are known to have. The objective of this study was to demonstrate the ability of these novel compounds to inhibit the formation of reactive oxygen species, especially their ability to block the formation of hydroxyl radicals, which are among the most reactive products of oxygen reactions and which produce the greatest damage to cells and tissues. The technique used to assess this antioxidant capacity of parrodienes was electron paramagnetic resonance, which allows direct assessment of inhibition of hydroxyl radical formation (.OH). The results show that these derivatives, especially octatriene, are able to exert evident antioxidant activity, thus confirming that their antioxidant properties are important for their biological activity.

Synthesis and characterization of nitric oxide generative polyrotaxane.

L-Arginine was immobilized into a supramolecular-structured polyrotaxane to examine the generation of nitric oxide, with a view to improving antithrombosis and the blood compatibility of polymeric biomaterials. L-Arginine was immobilized to the hydroxyl groups of alpha-cyclodextrins in the polyrotaxane via an ester linkage, and the nitric oxide generation and L-arginine release behavior were characterized. L-Arginine-immobilized polyrotaxane was insoluble in water, but was found to generate nitric oxide when placed in Tris-HCI buffer supplemented with activators. L-Arginine-immobilized polyrotaxane exhibited sustained nitric oxide generation for a period of 250 h. L-Arginine was completely released by non-enzymatic hydrolysis from 200 h to 700 h, with a lag-time for the first 200 h. Consequently, after the generation of nitric oxide and the release of L-arginine from the L-arginine-immobilized polyrotaxane, the residual component will be a polyrotaxane with superior biocompatibility and mechanical properties. These results suggest that L-arginine-immobilized polyrotaxane can be useful in a wide range of medical applications, including use as a nitric oxide generative system for antithrombosis, coating and blending materials of hydrophobic extracorporeal circuits, and implantable catheters.

Integrated bioprocessing for plant cell cultures.

Plant cell suspension culture has become the focus of much attention as a tool for the production of secondary metabolites including paclitaxel, a well-known anticancer agent. Recently, it has also been regarded as one of the host systems for the production of recombinant proteins. In order to produce phytochemicals using plant cell cultures, efficient processes must be developed with adequate bioreactor design. Most of the plant secondary metabolites are toxic to cells at the high concentrations required during culture. Therefore, if the product could be removed in situ during culture, productivity might be enhanced due to the alleviation of this toxicity. In situ removal or extractive bioconversion of such products can be performed by in situ extraction with various kinds of organic solvents. In situ adsorption using polymeric resins is another possibility. Using the fact that secondary metabolites are generally hydrophobic, various integrated bioprocessing techniques can be designed not only to lower toxicity, but also to enhance productivity. In this article, in situ extraction, in situ adsorption, utilization of cyclodextrins, and the application of aqueous two-phase systems in plant cell cultures are reviewed.

A bis-cyclodextrin diselenide with glutathione peroxidase-like activity.

A diselenide, 2,2'-diseleno-bis-beta-cyclodextrin (2-SeCD), was synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). The GPX mimic accepts a variety of hydroperoxides as substrates. The GPX activities, reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, are 7.4, 4.5 and 10.2 U/micromol, respectively. In contrast to ebselen (PZ51), the diselenide displays high GPX-like activity. The reduction of hydroperoxide by glutathione in the presence of a radical trap shows that the mimic catalyzes the reaction via a non-radical mechanism. A ping-pong mechanism was observed in the steady-state kinetic studies of the 2-SeCD-catalyzed reaction.

Carotenoid:methyl-beta-cyclodextrin formulations: an improved method for supplementation of cultured cells.

BC. Two days after supplementation with 5 microM BC in MbetaCD, cellular BC levels reached a maximum of 140+/-11 pmol/microg DNA, leveling off to 100+/-15 pmol/microg DNA until day 8. Incubation with BC dissolved in THF/DMSO resulted in a lower BC uptake of 105+/-14 pmol/microg DNA and 64+/-20 pmol/microg DNA respectively. No cytotoxic effects of these formulations were detected. The results show that the MbetaCD formulation is an improved method for investigations of carotenoids and other lipophilic compounds in in vitro test systems compared to methods using organic solvents.

Cycloamylose (cyclodextrin) glucanotransferase degrades intact granules of potato raw starch.

Cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19, CGTase) originated from Bacillus macerans degraded intact granules of potato raw starch and converted them into cyclodextrins (CDs). The degradation required sufficient stirring of starch-CGTase suspension. The morphology of the degraded starch granules was unique; that is, the inner part of the granules was observed by scanning electron microscope to be more susceptible to CGTase than the outer part. Effects of pH, temperature, starch concentration, and enzyme amount on CD production were studied.

Artificial imitation of glutathione peroxidase with 6-selenium-bridged beta-cyclodextrin.

On the basis of cyclodextrin, 6-selenium bridged beta-cyclodextrin (6-beta-CD-Se-Se-beta-CD, known as 6-SeCD) was synthesized by the selective tosylation of beta-cyclodextrin and nucleophilic displacement by sodium hydroselenide to imitate glutathione peroxidase (GPX). The GPX activity of diselenide 6-SeCD is 4.3 times that of PZ51. The structure of the mimic 6-SeCD was characterized by means of laser mass spectroscopy, elemental analysis, IR and 1H NMR. The selenium content and its valence in 6-SeCD were determined by means of X-ray photoelectron spectra. Kinetics of the mimic showed that its enzymatic behavior was similar to that of native GPX.