Anionic Cyclodextrin Research Articles

Chitosan/cyclodextrin nanoparticles can efficiently transfect the airway epithelium in vitro

The main goal of the present study was to investigate the potential of a new generation of hybrid polysaccharide nanocarriers, composed of chitosan (CS) and anionic cyclodextrins (CDs), for gene delivery to the airway epithelium. More specifically, these nanocarriers were investigated with regard to their ability to enter epithelial cells and promote gene expression in the Calu-3 cell culture model. In the search for the most suitable nanocarrier composition for gene delivery, the effect of CS molecular weight (Mw) on the nanocarriers characteristics and their ability to transfect cells was investigated. Thus, hybrid CS/CD nanoparticles were prepared with two different CS Mw, medium (110 kDa) and low (10 kDa), and loaded with pSEAP (plasmid DNA model that encodes the expression of secreted alkaline phosphatase). The resulting nanoparticles presented an adequate size range (100–200 nm, depending on CS Mw), a positive surface charge (+22 to +35 mV) and very high DNA association efficiency values (>90%). Cellular uptake studies showed that the nanoparticles were effectively internalized by the cells, providing a good indication of their potential as gene carriers. The transfection efficiency of the different formulations, measured by the concentration of secreted gene product (SEAP), indicated that all the nanoparticles were able to elicit a significantly higher response than the naked DNA (control), the transfection efficiency being more important for low MwCS nanoparticles than for those composed of medium MwCS. Overall, this report is the first evidence of the potential of a new generation of safe polysaccharide nanocarriers for gene delivery to the airway epithelium.

Cyclodextrin‐based nonaqueous electrokinetic chromatography with UV and mass spectrometric detection: Application to the impurity profiling of amiodarone

The potential of nonaqueous electrokinetic chromatography (NAEKC) using cyclodextrins (CD) for the analysis of basic drugs and related compounds was evaluated. Both UV absorbance and mass spectrometric (MS) detection were employed. Addition of neutral CD to the NA background electrolyte did not significantly enhance the separation of a test mixture of basic drugs, and no change in selectivity was observed. In contrast, anionic single-isomer-sulfated CD strongly added to the selectivity of the NAEKC system inducing an improved resolution among the test compounds and increasing the migration time window. The applicability of the NAEKC system using anionic CD is demonstrated by the profiling of a sample of the drug amiodarone that had been stored for 1 year at room temperature. Amiodarone is poorly soluble in water. NAEKC-UV analysis indicated the presence of at least seven impurities in the amiodarone sample. In order to identify these compounds, the NAEKC system was coupled directly to electrospray ionization (ESI) ion-trap MS. The total of detected impurities increased to 12 due to the added sensitivity and selectivity of MS detection. Based on the acquired MS/MS data, three sample constituents could be identified as 'known' impurities (British Pharmacopoeia), whereas for three unknown impurities molecular structures could be proposed. Estimated limits of detection for amiodarone using the NAEKC method were 1 microg/mL with UV detection and 15 ng/mL with ESI-MS detection (full-scan). Based on relative responses, the impurity content of the stored drug substance was estimated to be 0.33 and 0.47% using NAEKC-UV and NAEKC-ESI-MS, respectively.

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation ( R s > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.

Development of a chitosan‐based nanoparticle formulation for delivery of a hydrophilic hexapeptide, dalargin

Nanoparticle based delivery systems can offer opportunities for targeting, controlled release, and enhanced stability of their drug, protein, or gene therapy payload. This study investigated the use of chitosan in combination with the ionic additives sulfobutyl-ether-7-beta-cyclodextrin (SB-CD) or SB-CD/dextran sulfate (SB-CD/DS) mixture in comparison with chitosan: DS in the formulation of nanoparticles incorporating the hexapeptide dalargin. The physical characteristics (particle size, zeta potential), entrapment and loading efficiency, and release of dalargin were quantified. It was demonstrated that anionic cyclodextrin, SB-CD, can be used in complex coacervation with chitosan, with and without the presence of DS, to form nanoparticles. The presence of SB-CD or DS in the nanoparticle formulation and the weight ratio of chitosan to anionic additive(s) influenced the physical properties of the nanoparticles and their ability to carry dalargin. In addition, the particle size of nanoparticles was also affected by the molecular weight of chitosan and DS. The use of either DS or SB-CD/DS mixture produced chitosan nanoparticles with small particle size, high dalargin entrapment efficiency, enhanced peptide stability, and sustained release characteristics.

New Insights in Cyclodextrin: Surfactant Mixed Systems from the Use of Neutral and Anionic Cyclodextrin Derivatives

The kinetics of the hydrolysis of 4-methoxybenzenesulfonyl chloride (MBSC) have been studied in mixed systems made up of surfactant, sodium dodecyl sulfate (SDS) or tetradecyltrimethylammonium bromide (TTABr), and cyclodextrin, beta-CD or SBE-beta-CD(Captisol). The use of SBE-beta-CD instead of beta-CD allowed us to indicate certain characteristics of the mixed cyclodextrin-surfactant system: (a) The percentage of uncomplexed cyclodextrin is higher for SBE-beta-CD than for beta-CD when we use SDS, but the opposite effect was observed when we use TTABr. This behavior can be explained by taking into account the increase in salinity when we add SBE-beta-CD, and the electrostatic forces between the SBE-beta-CD and the surfactant that have influence on the complexation. (b) The presence or even the charge of cyclodextrin has no effect on the properties of surfactant micelles once they have been formed; in particular, it does not alter K(s)(m) or k(m), parameters very sensitive to the micellar system structure. Therefore, we can conclude that for surfactants concentrations lower than the micellization point, the charge of cyclodextrin modifies the cyclodextrin-surfactant interactions but once the micelles have been formed there is no interaction between them and the cyclodextrins.

Study on the supramolecular system of tetrakis(2-hydroxy-5-sulfonatophenyl)porphyrin with cyclodextrins by spectroscopy

The supramolecular systems of meso-tetrakis(2-hydroxy-5-sulfonatophenyl)porphyrin (THSPP) with six cyclodextrins (CDs) have been examined by means of absorption, fluorescence and 1H NMR spectroscopy in phosphate buffer (pH 7.15). The formation of inclusion complexes has been confirmed on the base of change of spectroscopy properties. Meso-tetrakis(2-hydroxy-5-sulfonatophenyl)porphyrin can form 1 : 1 inclusion complexes with the six CDs. The inclusion constants have been estimated from the absorbance and fluorescence intensity changes in neutral phosphate buffer solutions. The results show that the inclusion ability of anionic CD SBE-β-CD is weaker and the abilities of hydroxypropyl modified CDs are stronger compared to native CDs. It suggests that the interaction of hydrogen bond and charge attraction between CD and porphyrin play important roles in the inclusion procedure except for hydrophobic effect. The mechanism of inclusion interaction was carried out by 1H NMR technique. Furthermore, the inclusion constants of hydroxypropyl-γ-CD with THSPP were measured at 15–30°C. The values decrease with increasing temperature and the thermodynamic parameters have been calculated.

Nonaqueous electrokinetic chromatography–electrospray ionization mass spectrometry using anionic cyclodextrins

The set-up of an on-line method for coupling nonaqueous electrokinetic chromatography (NAEKC) and electrospray ionization mass spectrometry (ESI-MS) is presented. It allows the use of the single-isomer derivative anionic cyclodextrins heptakis(2,3-di- O-methyl-6- O-sulfo)-β-cyclodextrin (HDMS-β-CD) and heptakis(2,3-di- O-acetyl-6- O-sulfo)-β-cyclodextrin (HDAS-β-CD) for chiral and achiral separations of positively charged analytes. The effect of the cyclodextrins (CDs) on the MS signal intensities of model compounds was studied. When a voltage is applied over the CE capillary, the overall mobility of the CDs is towards the inlet vial preventing CDs from entering the ion source. However, the sodium counter ions of the CDs still enter the ion source and appeared to cause ionization suppression. Nevertheless, significant analyte signals could still be detected with detection limits in the sub-μg/ml. System parameters such as sheath liquid composition and flow rate, nebulizing gas pressure, capillary position in the sprayer and the drying gas flow and temperature were studied and optimized. The selection of a relatively low nebulizing gas pressure appeared to be important to achieve optimum sensitivity. The chiral selectivity of the NAEKC–ESI-MS system could be improved by addition of camphorsulfonate to the background electrolyte. Using mixtures of drugs and drug-related compounds, the NAEKC–ESI-MS system is shown to offer potential for (chiral) drug profiling.

Analysis of diterpenoic compounds in natural resins applied as binders in museum objects by capillary electrophoresis

The exudates of conifers consist mainly of diterpenoic acids of the abietane and pimarane type (abietic, neoabietic, dehydroabietic, palustric, pimaric, isopimaric, levopimaric and sandaracopimaric acid) and larixol acetate. These natural resins were used as adhesives, coatings, varnishes or plasticizers in artistic and historic works since ancient times. For the purpose of conservation and restoration and for art historic examination of such museum objects the identification of the binding media used is undoubtedly of paramount importance. In the present paper, the characterization of these resins based on the pattern of their diterpenoid constituents is carried out by capillary electrophoresis. For separation a background electrolyte which has been initially introduced for the analysis of chlorinated and natural resin acids in waste water was modified and the experimental conditions were adjusted in terms of resolution and analysis time. Separation was carried out in borate buffer at pH 9.25 (ionic strength 20 mmol L −1) with methyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin as additives to increase selectivity and enhance the solubility of the analytes. With this electrophoretic system the resin acids of interest and larixol acetate – all as anionic cyclodextrin complexes – were separated within 5 min and detected at 200, 250 and 270 nm with a diode array detector. The electrophoretic patterns served for the characterisation of the relevant diterpenoic resins, balsams and copals. Sample pre-treatment was limited to sonication in methanol at 55 °C for 30 min. This enables the identification of the resins in mixtures with other binders like plant gums, animal glues or drying oils, even when these media are present in excess. Colophony was identified as resinous constituent of a modelling mass for gilded frames originating from the 19th century.

Enantioseparation of cis and trans nucleosides, aromatic analogues of stavudine, by capillary electrophoresis and high-performance liquid chromatography

Compounds 1–4 are diastereoisomeric thymine derivatives of isochroman aromatic analogues of stavudine, an approved drug. Both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) techniques were used to separate these species with high resolution and thus permit the determination of enantiomeric excess. Chiral selectivity was developed using anionic (highly sulfated) cyclodextrins as chiral selectors in CE and amylose, cellulose and cyclodextrin chiral stationary phases by HPLC. The HPLC method was found to be more efficient than the CE method and was applied, after validation (repeatability, limit of detection, limit of quantification) to follow and quantify the kinetics of a stereoselective esterification.

Capillary Electrophoretic Resolution of Enantiomers of Aromatic Amino-Acids with Highly Sulfated α-, β- and γ-Cyclodextrins

Using cyclodextrin capillary electrokinetic chromatography (CD-EKC), baseline separation of aromatic amino acids was achieved. The variations in amino acids side chain, in the nature of the aromatic moiety and its substitution, and their derivatives at the amino or carboxylic terminal provide an ideal model for the systematic study of the structural interactions with the chiral selectors:anionic cyclodextrins (highly sulfated-CD or highly S-CD). The use of negatively charged cyclodextrins provides a driving force in the opposite direction of the positively charged or neutral molecules in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3 (w/v) of highly S-CD at 25 °C with a applied field of 0.30 kV cm−1 with capillaries dynamically coated with polyethylene oxide (PEO). A comparison was possible to investigate the structural arrangement responsible for regiospecific interactions with the enantiomers of the different amino acids.

Enantiomeric analysis of baclofen analogs by capillary zone electrophoresis, using highly sulfated cyclodextrins: Inclusion ionization constant pKa determination

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen phaclofen, saclofen, and hydroxy-saclofen, potent gamma-aminobutyric acid(B) (GABA(B)) agonist or antagonists was achieved. A method for the enantioresolution of those analogs of GABA was developed using anionic cyclodextrins (highly sulfated CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). With charged CDs we observed good resolutions due to the large electrophoretic mobility of these chiral selectors opposite to the mobility of the solutes. The highly S-alpha-CD and S-beta-CD were found to be complementary and the most effective complexing agent, allowing good enantiomeric resolution in short runtimes. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3% w/v of highly S-alpha-CD or S-beta-CD at 25 degrees C with an applied field of 0.30 kV/cm. The apparent binding constants of the inclusion complexes were evaluated and the migration order was determined. A comparison was possible to investigate the importance of the anionic group of the molecules in the separations. The pK(a) values were determined for all four compounds in order to explain relative electrophoretic migration of the solutes.

Enantiomeric separation of organophosphorus pesticides by capillary electrophoresis: Application to the determination of malathion in water samples after preconcentration by off-line solid-phase extraction

The separation of the enantiomers of a group of organophosphorus pesticides (OPs) has been investigated by electrokinetic chromatography (EKC) using different anionic cyclodextrins as chiral selectors. The use of a 25 mM Tris buffer (pH 7.0), 20 mM in CM-β-CD together with an applied voltage of 24 kV and a temperature of 25 °C enabled the individual enantiomeric separation of malathion and phenthoate each one into its two enantiomers, the partial separation of the enantiomers of phenamiphos and the separation of three of the four enantiomers of isomalathion. Since naled is very reactive in aqueous solutions, its enantiomeric separation achieved in about 8 min in 25 mM borate buffer at pH 9 with 10 mM CM-β-CD resulted in the observation of a broad peak due to degradation products. A preconcentration step by disk solid-phase extraction was studied and used together with the chiral EKC method developed for the enantiomeric separation of malathion in order to determine this pesticide in tap water samples spiked at the μg mL −1 level. Finally, the precision and recovery of the method was established.

Nonaqueous Capillary Electrophoretic Chiral Separations of Anticholinergic Drugs with Heptakis(2,3-di-O-Methyl-6-O-Sulfato)-β-Cyclodextrin

This paper reports the use of an anionic cyclodextrin, heptakis(2,3-di-O-methyl-6-O-sulfato)-β-cyclodextrin (HDMS-β-CD), for chiral separations of pharmaceutical enantiomers by nonaqueous capillary electrophoresis (NACE). Enantiomer resolution was affected mainly by HDMS-β-CD concentration and the acidity of the background electrolyte (BGE). The effects of capillary length and applied voltage on enantiomer resolution were also investigated. Results showed that in a methanol solution of 20 mM phosphoric acid, 10 mM sodium hydroxide, and 10 mM HDMS-β-CD, seven anticholinergic drugs were separated to baseline but no chiral separation was obtained for three other similar drugs. NACE is suitable for routine, rapid separation of the enantiomers of pharmaceutical compounds.

Chiral separation of the four stereoisomers of a novel antianginal agent using a dual cyclodextrin system in capillary electrophoresis

Reported here is an analytical method enabling the stereochemical resolution of a new antianginal compound possessing two stereogenic centers, leading to four stereoisomers. Only one of these isomers is currently under development as a novel antianginal agent and consequently, the other three isomers are considered as unwanted chiral impurities. Therefore, an enantioselective method is required in order to check its enantiomeric purity. This paper presents a method exploiting the high efficiency of capillary electrophoresis and the complexing properties of cyclodextrins to achieve the separation of the four stereoisomers of this weakly basic compound (pKa = 7.4). For this purpose, the combination of a neutral cyclodextrin, hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD), and an anionic cyclodextrin, carboxymethyl-beta-cyclodextrin (CM-beta-CD), was added to the separation buffer running in an uncoated silica capillary. After selection of the suitable cyclodextrin system, satisfactorily separation conditions were as follows: 30 mM phosphate buffer (pH 6.4) containing 10 mM of HP-gamma-CD and 10 mM of CM-beta-CD, running voltage +30 kV. The resulting run time and resolutions were respectively about 17 min and between 1.95 and 2.84. Linearity curves (0.993 < r2 < 0.998) are also shown.

Simultaneous Chiral Analysis of Methamphetamine and Related Compounds by Capillary Electrophoresis/Mass Spectrometry Using Anionic Cyclodextrin

A capillary electrophoresis/mass spectrometry method for the simultaneous chiral analysis of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA), ephedrine (EP), norephedrine (NE) and methylephedrine (ME) in urine has been developed. The background electrolyte was 1 M formic acid (pH 1.7). Using 0.85 mM heptakis(2,6-diacethyl-6-sulfato)-beta-cyclodextrin as the chiral selector, the 12 enantiomers were completely separated within 25 min. The detection limits were 0.01 microg mL(-1) for the enantiomers of MA, AP, DMA, EP and ME, and 0.02 microg mL(-1) for the enantiomers of NE using selected ion monitoring. The reproducibilities of within-run (n = 4) for the migration times and peak areas of the standard mixture were under 0.58% and 7.83%, respectively. The calibration curves of the peak areas of the 12 enantiomers were linear in the range of 0.05 - 10 microg mL(-1). This method was applicable to the analysis of urine samples.

Chiral cyclodextrin-modified microemulsion electrokinetic chromatography.

Cyclodextrin (CD)-modified microemulsion electrokinetic chromatography (MEEKC) or CD-MEEKC has not previously been applied to the area of chiral separations. Herein, the results of investigations of various microemulsions with CD additives are presented. Two different microemulsions are explored: an ethyl acetate sodium dodecyl sulfate microemulsion, and a chiral dodecoxycarbonylvaline (DDCV) microemulsion. Each microemulsion is paired separately with a neutral CD (hydroxypropyl-beta-CD) and an anionic CD (sulfated-beta-CD). In addition, the chiral DDCV microemulsion is investigated in both the R- and S- form. By varying simple parameters such as buffer system, applied voltage, surfactant enantiomer, and type of cyclodextrin, dramatic improvements in the chiral separations were noted. Resolution was found to be highly dependent on buffer identity and concentration, and somewhat dependent on whether the CDs used were randomly or highly sulfated. Under optimized conditions, the resolution ranged from 0.8 to 4.8, with plate counts ranging from 4000 to 26 000. Additionally, S- and R-levetiracetam, which had never before been enantioseparated via capillary electrophoresis (CE) methodologies, were separated in less than 8 min, with a resolution of 1.1.

Enantioseparation of four cis and trans diastereomers of 2′,3′-didehydro-2′,3′-dideoxythymidine analogs, by high-performance liquid chromatography and capillary electrophoresis

Compounds 1– 4 are the four stereoisomers of a synthetic new potential antiviral agent (d4T analog) containing two chiral centers and a base (uracil). Both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) techniques were used to separate and quantify enantiomers with high resolution. The determination of enantiomeric purity of the compounds was developed using both amylose chiral stationary phase by HPLC and anionic cyclodextrins (highly S-CD) as chiral selectors in CE. The HPLC method was found to be superior in sensitivity to the CE method.

Binding constants for aromatic amino acids and their derivatives with sulfobutyl ether β-cyclodextrin determined using capillary electrophoresis

The binding interaction of aromatic amino acids, their glycine dipeptides and some tryptophan derivatives with sulfobutyl ether fi-cyclodextrin (SBE7-fi-CD), an anionic cyclodextrin, in phosphate buffer (pH3, 5, 7 and 9) was studied using capillary electrophoresis (CE) chromatography. L-Trp, L-Tyr and L-Phe were the amino acids evaluated. Gly-L-Trp, L-Trp-Gly, Gly-L-Phe, L-Phe-Gly, Gly-L-Tyr and L-Tyr-Gly were studied as glycine dipeptides. N-acetyl-L-Trp, N-acetyl-L-Trp ethylester and L-Trp ethylester were used as L-Trp derivatives. For all the amino acids and the dipeptides, binding constants were higher at pH3 presumably because of the interaction between largely cationic amino acids/peptides at this pH and the anionic SBE7-fi-CD. The order of binding with the Trp series was Trp ethylester>N-acetyl-Trp ethylester>Trp>N-acetyl-Trp. These results support the conclusion that the interaction between amino acids/peptides and SBE7-fi-CD depends on charge and the neighboring functional groups. This study shows that SBE7-fi-CD can interact with amino acids and their derivatives and that a positive charge on the molecule facilitates the interaction.

Enantioseparation of basic pharmaceutical compounds by capillary electrophoresis using sulfated cyclodextrins: Application to E-6006, a novel antidepressant

In this study, a chiral capillary electrophoresis method was optimized and validated for E-6006, a thienylpyrazolylethanamine derivative (p K a 8.9). Enantioselectivity of neutral and anionic cyclodextrins (CDs) was evaluated at acid pH (3), obtaining cathodic and anodic migration, respectively. Hydroxypropyl-β-CD, carboxymethyl-β-CD and sulfobutyl ether-β-CD led to similar and partial selectivity, whereas sulfate (S)-β-CD produced baseline separation of the enantiomers. Four types of sulfated CDs were compared considering: cavity size (α, β, γ) and random substitution versus unique derivative (S-β-CD, 6-heptakis-S-β-CD). Complete peak separation was obtained in all cases, but with different affinity and binding strength. Some factors that play a role in the complex formation include: position/region/degree of substitution, size of CD cavity and proportion of derivatives in mixtures. Enantioaffinity and enantioselectivity increased with the average of sulfate groups/mol. β Cavity size complexed better, although α and γ cavities did not compromise separation. 6-Heptakis-S-β-CD had less affinity and separation efficiency, attributed to its lower degree and unique position of substitution. The method was optimized with S-β-CD (Aldrich, randomly substituted, 7–11 groups/mol). With this selector, the effect of pH value (3–9) was evaluated. Around pH 7 the cross-over point with change in the direction and order of migration was observed, associated with great enantioselectivity and long migration times. Fine tuning was done by adjusting the CD concentration and the buffer counterion. Definitive conditions were: uncoated silica capillary, 10 m M S-β-CD–25 m M sodium phosphate, pH 3. Validation parameters are included.

Synergistic effects of ion-pairing in the enantiomeric separation of basic compounds with cyclodextrin derivatives in nonaqueous capillary electrophoresis.

The enantiomeric separation of various kinds of basic pharmaceuticals has been investigated in nonaqueous capillary electrophoresis (NACE) systems using an ion-pairing reagent in combination with cyclodextrins (CDs). The simultaneous addition to the methanolic background electrolyte (BGE) of (+)-S-camphorsulfonate or alkanesulfonates and an anionic beta-cyclodextrin derivative, heptakis(2,3-dimethyl-6-sulfato)-beta-cyclodextrin (HDMS-beta-CD), led to partial or complete enantioresolution in most cases. In the absence of ion-pairing reagent, the enantiomeric resolution obtained with this CD derivative was most often completely lost or strongly reduced, indicating the important role of ion-pairing in the chiral recognition mechanism in these NACE systems. The influence of the nature and concentration of the counterion and the anionic CD derivative on the enantioseparation of basic compounds was studied. Synergistic effects between these two kinds of charged additives were clearly observed.