Éva Fenyvesi

Evaluation of the cytotoxicity of β-cyclodextrin derivatives: evidence for the role of cholesterol extraction.

Several beta-cyclodextrin (beta-CD) derivatives have been synthesized recently to improve the physicochemical properties and inclusion capacities of the parent molecule, however, there is limited information available about their cytotoxic effects. In this study we investigated the cytotoxic and hemolytic properties of various beta-CDs in correlation with their cholesterol-solubilizing capacities to expose the mechanism of toxicity. MTT cell viability test, performed on Caco-2 cells showed significant differences between the cytotoxicity of beta-CD derivatives. Cell toxicity of methylated-beta-CDs was the highest, while ionic derivatives proved to be less toxic than methylated ones. Most of the second generation beta-CD derivatives, having both ionic and methyl substituents showed less cytotoxicity than the parent compounds both on Caco-2 cells and human erythrocytes. Inclusion of cholesterol into the ring of randomly methylated-beta-CD and heptakis(2,6-di-O-methyl)-beta-CD abolished the cell toxicity indicating the role of cholesterol extraction in cytotoxicity. These data demonstrate the correlation between the cytotoxic effect, hemolytic activity and the cholesterol complexation attributes of beta-CD derivatives and we propose that cholesterol-solubilizing properties can be a predictive factor for beta-CD cell toxicity.

Release of alpha-tocopherol from antioxidative low-density polyethylene film into fatty food simulant: influence of complexation in beta-cyclodextrin.

The release of alpha-tocopherol from two formulations (with and without complexation with beta-cyclodextrin) of low-density polyethylene (LDPE) film was examined. Specific migration studies were performed at 7.0 ± 0.5°C using plastic bags filled with 95% ethanol as a fatty food simulant. The amount of complexed and free (non-complexed) alpha-tocopherol migrating into the food simulant was followed by high-performance liquid chromatography (HPLC). It was concluded that complexation with beta-cyclodextrin had a significant effect on the release rate of the antioxidant. Using a mathematical model for the description of the migration, a decrease in diffusion coefficient (D) of one order of magnitude was calculated in the case of complexed alpha-tocopherol compared with the free form. Total migration of alpha-tocopherol from both films was observed, meaning that the partition coefficient of tocopherol was not influenced by incorporation with cyclodextrin. Thus, complexation might be the key to a long-lasting antioxidative effect of such kind of active packaging.

P-glycoprotein inhibition by membrane cholesterol modulation

P-glycoprotein (Pgp) is a transmembrane protein that actively exports lipophilic chemotherapeutics from the cells causing multidrug resistance. Pgp molecules are partially localized in TX-100-resistant rafts, and the activity of the transporter is highly sensitive to the presence of cholesterol. To better understand these relationships, the influence of membrane cholesterol content on Pgp function, as measured via calcein accumulation, was studied in correlation with changes elicited in membrane structure. Membrane cholesterol was modulated by heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB) and the cholesterol inclusion complex of DIMEB (Chol-DIMEB). Changes in membrane cholesterol level were reflected by alterations in the overall lipid packing as measured by Merocyanine 540 (MC540) staining and were also accompanied by changes in the raft association of the pump. DIMEB and Chol-DIMEB treatments have also lead to increased permeability of the cell membrane in both directions, raising the possibility that the effects on pumping efficiency reflect leakage of ATP also from the non-permeabilized cells. However, the treatments did not influence the intracellular ATP levels of the non-permeabilized cells. Our data suggest that Pgp inhibition by cyclodextrin treatments arises through modulation of its membrane microenvironment, rather than as a result of concomitant cytotoxicity.

Cyclodextrins, Blood–Brain Barrier, and Treatment of Neurological Diseases

Biological barriers are the main defense systems of the homeostasis of the organism and protected organs. The blood-brain barrier (BBB), formed by the endothelial cells of brain capillaries, not only provides nutrients and protection to the central nervous system but also restricts the entry of drugs, emphasizing its importance in the treatment of neurological diseases. Cyclodextrins are increasingly used in human pharmacotherapy. Due to their favorable profile to form hydrophilic inclusion complexes with poorly soluble active pharmaceutical ingredients, they are present as excipients in many marketed drugs. Application of cyclodextrins is widespread in formulations for oral, parenteral, nasal, pulmonary, and skin delivery of drugs. Experimental and clinical data suggest that cyclodextrins can be used not only as excipients for centrally acting marketed drugs like antiepileptics, but also as active pharmaceutical ingredients to treat neurological diseases. Hydroxypropyl-β-cyclodextrin received orphan drug designation for the treatment of Niemann-Pick type C disease. In addition to this rare lysosomal storage disease with neurological symptoms, experimental research revealed the potential therapeutic use of cyclodextrins and cyclodextrin nanoparticles in neurodegenerative diseases, stroke, neuroinfections and brain tumors. In this context, the biological effects of cyclodextrins, their interaction with plasma membranes and extraction of different lipids are highly relevant at the level of the BBB.

Fluorescently Labeled Methyl-Beta-Cyclodextrin Enters Intestinal Epithelial Caco-2 Cells by Fluid-Phase Endocytosis

Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB) on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10−8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

Uptake of a fluorescent methyl-β-cyclodextrin via clathrin-dependent endocytosis.

Cyclodextrins (CDs) are widely used both in pharmaceutical applications to improve drug bioavailability and in cell biology as cholesterol-depleting and -delivering agents. Recently, it was shown that β-CD covalently coupled to fluorescent dextran polymers accumulates in cholesterol-enriched lysosomal storage organelles of human fibroblasts (Rosenbaum et al., 2010). By employing a methyl-βCD tagged with fluorescein (FMβCD), we have characterized the cellular trafficking of the CD in mammalian cell lines and its distribution into the endocytic compartments within the first minutes following addition to cells. FMβCD enters mammalian cells via endocytosis. The colocalization of FMβCD with transferrin-containing endosomes and the inhibition of FMβCD internalization by chlorpromazine or by an antisense RNA against clathrin heavy chain indicate that FMβCD is taken up via receptor-mediated, clathrin-dependent endocytosis. These results not only highlight the possibility of using CDs to target drugs intracellularly, but also warn about potential unwanted effects on cell physiology other than cholesterol extraction/loading at high concentrations, high temperatures and prolonged incubation times.

A host-guest supramolecular complex with photoregulated delivery of nitric oxide and fluorescence imaging capacity in cancer cells.

Herein we report the design, preparation, and properties of a supramolecular system based on a tailored nitric oxide (NO) photodonor and a rhodamine-labeled β-cyclodextrin conjugate. The combination of spectroscopic and photochemical experiments shows the absence of significant interchromophoric interactions between the host and the guest in the excited states. As a result, the complex is able to release NO under the exclusive control of visible light, as unambiguously demonstrated by direct detection of this transient species through an amperometric technique, and exhibits the typical red fluorescence of the rhodamine appendage. The supramolecular complex effectively internalizes in HeLa cancer cells as proven by fluorescence microscopy, shows a satisfactory biocompatibility in the dark, and induces about 50% of cell mortality upon irradiation with visible light. The convergence of all these properties in one single complex makes the present host-guest ensemble an appealing candidate for further delevopment of photoactivatable nanoscaled systems addressed to photostimulated NO-based therapy.

Endocytosis of fluorescent cyclodextrins by intestinal Caco-2 cells and its role in paclitaxel drug delivery

Cyclodextrins are widely used excipients in pharmaceutical formulations. They are mainly utilized as solubilizers and absorption enhancers, but recent results revealed their effects on cell membranes and pharmacological barriers. In addition to the growing knowledge on their interaction with plasma membranes, it was confirmed that cyclodextrins are able to enter cells by endocytosis. The number of the tested cyclodextrins was limited, and the role of this mechanism in drug absorption and delivery is not known. Our aim was to examine the endocytosis of fluorescently labeled hydroxypropyl-β-cyclodextrin, random methyl-β-cyclodextrin and soluble β-cyclodextrin polymer, and the cellular uptake of the fluorescent paclitaxel derivative-random methyl-β-cyclodextrin complex. The studied cyclodextrin derivatives were able to enter Caco-2 intestinal cells and localized in vesicles in the cytoplasm, while their permeability was very limited through Caco-2 monolayers. We demonstrated for the first time that the fluorescent paclitaxel derivative and rhodamine-labeled random methyl-β-cyclodextrin were detected in the same intracellular vesicles after treating cells with their inclusion complex. These results indicate that the endocytosis of cyclodextrin complexes can contribute to drug absorption processes.

Characterization of cyclodextrin containing nanofilters for removal of pharmaceutical residues

Due to the increasing amount of persistent organic pollutants (POPs) in general and pharmaceutical residues in particular in municipal wastewater, the efficiency of water treatment technologies should be improved. Following the biological treatment of wastewater nanofiltration offers a possible way for the removal of POPs. In this study β-cyclodextrin containing nanofilters having different chemical composition and thickness (1.5-3.5mm) were investigated. For their characterization, their adsorption capacity was determined applying ibuprofen containing model solution and total organic carbon (TOC) analyzer. It could be established that the regeneration of nanofilters with ethanol and the application of inorganic additives (NaCl, NaHCO3, NH4HCO3) increased the adsorption capacity of nanofilters. The best results were achieved with chemical composition of 30m/m% β-cyclodextrin polymer beads and 70m/m% ultra-high molecular weight polyethylene in the presence of 12mmol ammonium hydrogen carbonate/nanofilter.

Nanoencapsulation of flavors and aromas by cyclodextrins

Abstract Cyclodextrins are natural carbohydrates useful for molecular encapsulation of organic guest molecules such as flavors and aromas resulting in enhanced physical stability (via reducing volatility) and chemical stability (via reducing sensitivity against light, heat, oxidation, and hydrolysis). They are approved for use in food (listed as GRAS by FDA). Further advantages of cyclodextrin formulations are the enhanced solubility and improved sensorial properties. The technological advantages, such as stable, standardized compositions, improved wettability, simple dosing, and handling of dry powders, reduced packaging and storage costs, more economical manufacturing processes, and reduced labor costs justify their application in the food industry. The possible methods for formulation of flavors and aromas are overviewed and the analytical methods for their characterization are described. The role of cyclodextrins in active packaging of food (eg, aroma-tight packaging) is discussed, too. Several examples on the application in foods and beverages are given.