Enhanced microdialysis recovery of some tricyclic antidepressants and structurally related drugs by cyclodextrin-mediated transport

The enhanced microdialysis relative recovery (RR) of some hydrophobic tricyclic drugs (imipramine, desipramine, amitriptyline, carbamazepine and promethazine) is discussed. Enhanced RR was achieved by including a binding agent [beta-cyclodextrin (beta-CD) or 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD)] in the microdialysis perfusion fluid to form inclusion complexes with the drugs, which increases the analyte flux through the membrane material. The maximum effect of the RR increase for all the drugs studied was observed using a commercially available polycarbonate-polyether (PC) membrane. With a 4 mm PC membrane and 4.41 mmol l-1 (0.5% w/v) beta-CD included in the microdialysis perfusion fluid (0.9% saline, pH 7.4) at a flow rate of 0.5 microliter min-1, RR enhancements over controls were as follows: carbamazepine 136, imipramine 268, desipramine 298, amitriptyline 634, and promethazine 987%. Increasing beta-CD [up to 17.63 mmol l-1 (2% w/v)] or HP-beta-CD [up to 32.5 mmol l-1 (5% w/v)] concentration in the microdialysis perfusion fluid enhanced carbamazepine RR three (beta-CD) to four (HP-beta-CD) times compared to controls through PC microdialysis membranes. The PC membrane gave enhanced RR values that were twice those for cuprophan or AN-69 membranes. Enhanced RR with cyclodextrins was successfully applied to sampling from a protein solution containing desipramine in a 4% w/v bovine serum albumin solution. These results suggest that addition of cyclodextrins to microdialysis perfusion fluids may be used to increase microdialysis RR during blood sampling.     

Application of Cyclodextrins to the Extraction of Antioxidant Compounds from Olive Oil

Olive oil contains powerful antioxidant compounds which impart stability, contribute to various properties of it, and are valuable from the nutritional point of view. Their extraction with as mild conditions as possible led to its investigation using cyclodextrins as a tool. The inclusion ability of α-, β-, and γ-CD was estimated, and it has been demonstrated that the small cavity of α-CD as well as the wide one of γ-CD could enclose less effectively the antioxidant compounds of olive oil than the intermediate in shape cavity of β-CD. The highest yields of antioxidant compounds were achieved when olive oil was mixed with a 2%aqueous solution of β-CD and the resulting precipitate was treated with ethyl alcohol.__________Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 18–21, January–February, 2005.     

Determination of fluoxetine and norfluoxetine in rat brain microdialysis samples following intraperitoneal fluoxetine administration

Fluoxetine (FLX) and the N-desmethyl metabolite, norfluoxetine (NFLX) in rat brain microdialysis samples were determined by high-performance liquid chromatography (HPLC) with fluorescence detection using pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). In vitro experiment showed that the relative recovery of FLX across microdialysis membrane was enhanced by adding β-cyclodextrin (β-CD) or β-CD polymer to microdialysis perfusion fluid. The perfusion fluid containing β-CD polymer, which has polymeric glyceryl linkers attached to the hydroxyl group, gave the better recovery with satisfactory precisions. Using 1% β-CD polymer in Ringer’s solution as the perfusate, in vivo rat brain microdialysis experiment on intraperitoneal administration of FLX (10 mg/kg) to rats was carried out. The fluorescence peaks of FLX and NFLX appeared later than 30 min after the administration of FLX, and then, gradually increased with time. Two hours later, FLX reached a plateau level, but NFLX slowly increased, and at 24-48 h, NFLX levels were higher than FLX levels. These data suggest that long distributions of FLX and the potent metabolite, NFLX, in brain contributed to the long-term drug actions in vivo.     

Affinity-based microdialysis sampling using heparin for in vitro collection of human cytokines

Microdialysis sampling is a widely used method to sample from complex biological matrices. Cytokines are important signaling proteins that are typically recovered with low relative recovery values during microdialysis sampling. Heparin was included in the microdialysis perfusion fluid as an affinity agent to increase in vitro recovery of different cytokines through polyethersulfone (PES) microdialysis membranes with 100 kDa molecular weight cutoff. No change in fluid volumes collected from the microdialysis probes occurred when heparin was included in the perfusion fluid up to concentrations of 10 μM. The loss of heparin (10 μM) across the dialysis membrane was minimal (2.7 ± 0.9%, n = 3). Additionally, heparin at these concentrations did not interfere with the cytokine immunoassays. The control and heparin-enhanced relative recoveries for five human cytokines using 0.1 μM heparin in the microdialysis perfusion fluid flowing at 0.5 μL min⁻¹ were (n = 3): interleukin-4 (IL-4), 4.2 ± 0.5% and 7.2 ± 3.1%; interleukin-6 (IL-6), 1.4 ± 0.8% and 3.6 ± 1.3%; interleukin-7 (IL-7), 1.3 ± 0.8% and 4.8 ± 1.8%; monocyte chemoattractant protein-1 (MCP-1), 9.0 ± 1.6% and 19.5 ± 2.7%; and tumor necrosis factor-α (TNF-α), 7.4 ± 1.3% and 16.9 ± 1.6%, respectively. Heparin increased the microdialysis sampling relative recovery of several human cytokines in vitro.     

Enhancing the microdialysis recovery for sampling of Cu and Ni by incorporating humic acid in the perfusion liquid

A strategy to enhance the microdialysis relative recovery for sampling of Cu and Ni ions is presented. Enhanced recovery was achieved by incorporating humic acid, a binding agent, in the microdialysis perfusion liquid during sampling from a Cu and Ni standard solution mixture. All microdialysis sampling experiments were carried out at room temperature under quiescent conditions using a concentric type of microdialysis probe with an adjustable effective dialysis length. For all metal determinations electrothermal atomic absorption spectrometry was employed. Metal recoveries were shown to be dependent on the membrane molecular weight cut-off, perfusion rate, sample solution pH, perfusion liquid composition as well as perfusion liquid pH. Complete recoveries (100%) of Cu and Ni were obtained by microdialysis sampling using a 10 kDa molecular weight cut-off polysulfone membrane at a flow-rate of 2 μl/min employing a 0.05% (w/v) optimal composition of humic acid incorporated in the perfusion liquid. The optimal sampling pH of humic acid was determined to be 6 where most oxygen containing functional groups are dissociated and available for metal binding. These data have important ramifications for sampling and determination of metal ions in small sample solutions (∼10 ml) at very low concentrations in the ppb range.     

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 (pK_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 (, 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 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 mM S-β-CD–25 mM sodium phosphate, pH 3. Validation parameters are included.     

Recent advances in the application of capillary electrophoresis to neuroscience

With fast separation times (seconds to minutes), minimal sample requirements (nanoliters to femtoliters), and excellent mass detection limits (femtomole to zeptomole), capillary electrophoresis (CE) is ideally suited for in vitro and in vivo sampling of neurological samples with a high degree of spatial resolution. Advances in extracellular fluid analysis employing improved microdialysis and push–pull perfusion sampling methodologies has enabled the resolution of neurotransmitters present in limited amounts using CE. Great progress has been made to resolve complex neuropeptides, amino acids, and biogenic amines in tissue and cell cultures. Finally, owing largely to the small volume sampling abilities of CE, investigations of single nerve cells, both invertebrate and mammalian, have been accomplished. These applications of CE to the advancement of neuroscience are presented.     

Diffusion Measurements vs. Chemical Shift Titration for Determination of Association Constants on the Example of Camphor–Cyclodextrin Complexes

Measurements on camphor–cyclodextrin complexes reveal that precise association constants are more easily determined by chemical shift titration. Diffusion measurements using HR-DOSY allow easy following of the complex composition at different concentration ratios and estimation of the binding energy. Linear dependence of the diffusion coefficients on the molecular mass of free and associated cyclodextrins has been observed in D₂O. The solution structures of α- and β-cyclodextrin complexes of camphor in D₂O were deduced from intermolecular cross-relaxation data. Different preferential orientation in the 2:1 α-CD and 1:1 β-CD species have been derived in contrast to the loose 1:1 complex with γ-CD. Proton NMR chemical shift values proved to be much more sensitive to diastereomeric complex formation than diffusion coefficients.     

Physicochemical Study of Ribavirin Complexes with α-, β- and γ-Cyclodextrins

The aim of this work was to characterise interactions between ribavirin (RBV) and native cyclodextrins (CDs). The extent of complexation in solution has been evaluated by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Thermogravimetry (TG), differential scanning calorimetry (DSC) and infrared spectroscopy (FT-IR) were used to characterise the solid state of all the binary systems. Complexation of RBV with α-, β-, and γ-CDs was proved by FT-IR, HPLC and thermal analysis. The 1:1 stoichiometry for the complexes was obtained by HPLC. The stability constants for RBV with α-, β- and γ-CD were determined to be 1493, 2606, and 1179 M⁻¹, respectively. Consequently β-CD was the most suitable of the three complexing agents since it showed the highest stability constant. RBV appears not included inside the cavity of the CD because H-3 and H-5 protons were not shifted in the presence of the molecule as proved by NMR. The 2D ROESY spectra did not show any dipolar proton interaction of the RBV with the CDs. Thus the complexation does not seem to be a host–guest inclusion complex but an external intermolecular complex. FT-IR spectral changes due to the RBV carboxamide group vibrations with the CDs confirm this association.     

No delayed temporal response to sample concentration changes during enhanced microdialysis sampling using cyclodextrins and antibody-immobilized microspheres

The temporal response to concentration changes external to a microdialysis probe containing trapping agents in the perfusion fluid was studied. Native beta-cyclodextrin and a water-soluble beta-cyclodextrin polymer were used as trapping agents in the microdialysis perfusion fluid to study the temporal concentration response to carbamazepine, a hydrophobic analyte. The temporal response of microdialysis probes containing antibody-immobilized microspheres against five different cytokines (tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5) to concentration changes outside of the probe was also determined. In both cases, no delayed temporal response of enhanced microdialysis was observed for either carbamazepine or the cytokines as compared to standard microdialysis sampling procedures.     

A Use of Modified Cyclodextrins as a Transporter for a Radiolabeled Tracer NMR Investigation

Cyclodextrins (D. Duchêne (ed.): New Trends in Cyclodextrins and Derivatives (1991)) have long been shown to be capable of modifying the water solubility of a number of hydrophobic guests through the formation of inclusion complexes. Among the three natural cyclodextrins (α, β and γ-cyclodextrins containing 6, 7 and 8 d-glucopyranose units, respectively), β-cyclodextrin is by far the most commonly used although it exhibits a weaker solubility in water (and therefore a weaker solubilization power). This specific feature has encouraged the synthesis of derivatives exhibiting an increased solubility in water. Methylated cyclodextrins are amongst the simplest derivatives, and their properties regarding the solubility and the solubilization power for hydrophobic guests are well documented especially concerning Heptakis (2,6-di-Omethyl)-cyclodextrin (DIMEB) and Heptakis (2,3,6-tri-Omethyl)-cyclodextrin (TRIMEB) K. Koizumi et al.:J. Chromatogr. 368, 329–337 (1986). In order to avoid the use of human serum albumin (HSA), this property has been applied here to the solubilization of a very sparingly water-soluble fatty acid derivative (16-iodo-3-methylhexadecanoic acid), which is known to localise in viable myocardial cells, allowing the generation of functional images reflecting the viability of the cardiac tissue through the use of radiolabeled analog (Demaison et al.: J. Nucl-Med. 29, 1230–1236 (1998)). Nuclear magnetic resonance (NMR) was used throughout this study to evidence that the observed solubilization and stabilisation (under conditions required for sterilisation) induced by cyclodextrins are due to the formation of a true inclusion complex and not to non-specific interactions; This technique further allows to derive thermodynamic as well as structural informations for this complex. On one hand, the inclusion complex prevents thermal degradation during sterilisation process compared to HSA. On the other hand, NMR displacement experiments against HSA showed that the complex likely dissociates in vivo.     

Cyclodextrins Initiated Ring-Opening Polymerization of Lactide Using 4-Dimethylaminopyridine (DMAP) as Catalyst: Study of DMAP/β-CD Inclusion Complex and Access to New Structures

Cyclodextrins (CDs) are cyclic oligosaccharides used in many fields. Grafting polymers onto CDs enables new structures and applications to be obtained. Polylactide (PLA) is a biobased, biocompatible aliphatic polyester that can be grafted onto CDs by -OH-initiated ring-opening polymerization. Using 4-dimethylaminopyridine (DMAP) as an organocatalyst, a quantitative functionalization is reached on native α-, β-, γ- and 2,3-dimethyl- β-cyclodextrins. Narrow molecular weight distributions are obtained with the native CDs (dispersity < 1.1). The DMAP/β-CD combination is used as a case study, and the formation of an inclusion complex (1/1) is shown for the first time in the literature, which is fully characterized by NMR. The inclusion of DMAP into the cavity occurs via the secondary rim of the β-CD and the association constant (K_a) is estimated to be 88.2 M⁻¹. Its use as an initiator for ring-opening polymerization leads to a partial functionalization efficiency, and thus a more hydrophilic β-CD-PLA conjugate than that obtained starting from native β-CD. Polymerization results including also the use of the adamantane/β-CD inclusion complex as an initiator suggest that inclusion of the DMAP catalyst into the CD may not occur during polymerization reactions. Rac-lactide does not form an inclusion complex with β-CD.     

Peripheral endocannabinoid microdialysis: in vitro characterization and proof-of-concept in human subjects

In vivo endocannabinoid (EC) microdialysis has only seldom been performed, mostly in rodent brain tissue. Low solubility in aqueous media, adsorption to surfaces, and instability with co-present human serum albumin (HSA) are the major obstacles in EC microdialysis. The addition of hydroxypropyl-ß-cyclodextrine (HPCD) to the perfusion fluid has been previously described to facilitate lipid microdialysis, but the general biophysical properties of HPCD, especially with respect to peripheral EC microdialysis, have not been described before. We report on the characterization of EC microdialysis using an in vitro system using Ringer’s solution with 10% HPCD as the perfusion fluid and with fatty acid-free HSA as the matrix fluid. The endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2AG) were measured using LC-MS/MS. AEA was stable in the perfusion and matrix fluids, whereas 2AG was only stable in the perfusion fluid. In the matrix fluid, 2AG underwent rapid isomerization to 1-arachidonoyl glycerol. A relative recovery of 3.5% for AEA was found with 10% HPCD in the perfusion fluid and a flow rate of 1 μL/min. For 2AG, a similar relative recovery of 3.5% was estimated. Since 2AG was found unstable in the matrix fluid, a reliable calculation of the relative recovery rates was not possible. Delivery and recovery experiments revealed unequal inward and outward EC transport across the microdialysis membrane. Contrary to usual microdialysis findings, we observed increasing recovery rates for AEA with increasing flow rates. Long equilibration times of several hours were necessary to obtain constant relative recovery rates. In a proof-of-concept study in humans, we collected AEA from subcutaneous abdominal adipose tissue employing the described methodology. Our study suggests that the microdialysis technique is not suitable for the exact quantification of tissue EC concentrations, but it allows for their rough estimation.

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Figure Microdialysis of peripheral endocannabinoids, especially anandamide (AEA).

     

Complexation of Ketoconazole by Native and Modified Cyclodextrins

Complexation of ketoconazole (KET), a broad-spectrum antifungal drug, with β- and γ-cyclodextrins (CDs), heptakis (2,6-di-O-methyl)-β-CD (2,6-DM-β-CD), heptakis (2,3,6-tri-O-methyl)-β-CD (TM-β-CD), 2-hydroxypropyl-β-CD (2HP-β-CD) and carboxymethyl-β-CD (CM-β-CD) was studied. The stability constants were determined by the solubility method at pH = 6 and for 2,6-DM-β-CD and CM-β-CD at pH = 5. At pH = 6, the stability constants increased in the order: TM-β-D < γ-CD < 2HP-β-CD < β-CD < CM-β-CD < 2,6-DM-β-CD. At pH = 5, due to the increased ionization of KET, the stability constant with CM-β-CD increased and with 2,6-DM-β-CD decreased. For complexes of KET with 2HP-β-CD and 2,6-DM-β-CD, the thermodynamic parameters of complexation were determined from the temperature dependence of the corresponding stability constants. For β–γ and TM-β-CD complexes, calculations using HyperChem 6 software by the Amber force field were carried out to gain some insight into the host–guest geometry.     

In vitro and in vivo affinity microdialysis sampling of cytokines using heparin-immobilized microspheres

Heparin-immobilized microspheres were included in microdialysis sampling perfusion fluids under both in vitro and in vivo conditions to improve the recovery of different cytokines, acidic fibroblast growth factor, vascular endothelial growth factor, monocyte chemoattractant protein-1 (or CCL2), and regulation upon activation normal T cell express sequence (or CCL5). Different strategies to dissociate captured CCL2 and CCL5 from the immobilized heparin were attempted, and both cytokines could be quantitatively eluted from the beads using a phosphate buffer (pH 7.4) containing 25% (v/v) acetonitrile which did not interfere with the subsequent detection of cytokine using an ELISA assay. Using these heparin-immobilized microspheres, a two to fivefold increase of microdialysis relative recovery (RR) was achieved for the four cytokines from a quiescent solution. Enhanced microdialysis RR of CCL2 using the heparin-immobilized microspheres from microdialysis probes implanted into the peritoneal cavity of a rat was performed to test the in vivo application. This work suggests that the heparin-immobilized microspheres provide an alternative affinity agent to the previously used antibody-immobilized microspheres for enhanced microdialysis sampling of cytokines.     

A novel approach for fixation of β-cyclodextrin on cotton fabrics

Cyclodextrins are cyclic oligosaccharides, which form complexes with different organic substances such as drugs, odors, and etc. Due to the complexing abilities of cyclodextrins (CDs), they may also be used in textile industry as an auxiliary in washing, dyeing, and wastewater treatment. Fixation of CDs on textiles is possible using reactive derivatives of cyclodextrins or crosslinking agents. In this study we have investigated the use of polyaminocarboxylic acids (PACAs) as novel crosslinking agents for the fixation of β-CD on cotton fabrics. Fixation of β-CD on cotton fabric has been quantified by measuring the weight increase of the treated samples. The influence of the concentration of the catalyst (sodium hypophosphite) was studied, too. The presence of β-CD on the cotton has been investigated by the phenolphthalein test and host–guest complexation with organic volatile molecules: cyclohexene, chlorobenzene, cyclohexene-1-one and toluene.     

Droplet-based microdialysis-Concept, theory, and design considerations

The capability of continuously sampling the extracellular fluid opens up a wide range of applications of microdialysis in biological, pharmaceutical, and clinical studies. Existing microdialysis, however, faces challenges in sampling analytes with fast clearance and limited diffusivity because sampling resolution is limited by device size. Size reduction in probes and interconnected cannulae is a promising solution to improve temporal and spatial resolution. But the back pressure produced by resistance to laminar flows will be magnified in smaller channels, raising a concern as to whether it is feasible to operate continuous perfusion for miniaturized microdialysis. We demonstrate that a 10-fold smaller channel will exhibit 100-fold larger back pressure in response to the increase in the flow rate to maintain the relative recovery. In order to overcome the foreseen back pressure issue, this paper discusses a new concept using discrete droplets instead of continuous flows to operate dialysis in a miniaturized probe. This conceptual design is referred to as droplet-based digital microdialysis, in which droplets are produced, controlled and advanced within microchannels at a rate that in theory should allow for analytes to equilibrate with the extracellular fluid under no flow conditions. Expecting that a digital droplet design will entirely eliminate back pressure by introducing air between droplets, we numerically compare the equilibration kinematics of droplets to that of continuous flow. Results suggest equilibration of low molecular weight analytes between intermittently stationary droplets and the extracellular fluid in a few seconds. Considerations in design, prototyping, calibration and quantification, and the integration with other devices are suggested.     

Spectrophotometric and thermoanalytical study of cypermethrin/cyclodextrin complexes

Cypermethrin/β-CD complexes were prepared at 1:2 cypermethrin/β-CD molar ratio by different complexation methods: conventional coprecipitation, suspension and kneading methods as well as “melting in solution” technique, which was developed in our laboratory. The complexes were investigated by UV-spectrophotometry and thermal analysis. It was found that complexes made by coprecipitation, suspension and kneading methods contained cypermethrin not only in complexed but also in uncomplexed form. The guest molecule in the complex prepared by “melting in solution” technique showed to be completely complexed, so it was the most effective complexation method studied.Investigating the solubility of cypermethrin with different cyclodextrins (CDs), it was established that the increase of solubility of cypermethrin was the highest in case of methylated cyclodextrins. The equilibrium constants were calculated from solubility isotherms. On the basis of these results, the heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) complex was the more stable. By UV-irradiation measurements it was found that the photodegradation of cypermethrin was inhibited by methylated β-CDs.     

Water-soluble cyclodextrin polymers for enhanced relative recovery of hydrophobic analytes during microdialysis sampling

Microdialysis relative recovery (RR) enhancement using different water-soluble, epichlorohydrin-based cyclodextrin polymers (CD-EPS) was studied in vitro for different analytes, amitryptiline, carbamazepine, hydroquinone, ibuprofen, and 4-nitrophenol. When compared to the native CDs (alpha, beta, and gamma) on a per mole basis, the CD-EPS enhanced microdialysis RR was either statistically greater or the same. beta-CD-EPS was more highly retained than native beta-CD by a 20 000 Da molecular weight cutoff (MWCO) polycarbonate membrane, but showed no statistical difference for loss across a 100 000 Da MWCO polyethersulfone membrane (PES). When the same weight percent of beta-CD or beta-CD-EPS was included in the microdialysis perfusion fluid, the beta-CD-EPS produced a higher microdialysis RR than native beta-CD for all analytes across the PES membrane. However, enhancements for the PC membrane were statistically insignificant when beta-CD and beta-CD-EPS were compared on a per mole basis. These results suggest that CD-EPS may be used as effective enhancement agents during microdialysis sampling and for some membranes provide the additional advantage of being retained more than native CDs.     

Formation of cyclodextrin nanotube induced by POPOP molecule

The interaction between 2,2′-p-phenylenebis (5-phenyloxazol) (POPOP) and cyclodextrins (CDs) was investigated using UV-Vis absorption, steady-state fluorescence, and dynamic light scattering (DLS). The results indicated that POPOP could form the 1:2 (guest:host) inclusion complex with β-CD at lower concentrations, which could further form the extended nanotube at higher concentrations. POPOP could also induce the formation of the nanotube of γ-CD. The fluorescence emission of POPOP in aqueous solution of γ-CD showed obvious red shift accompanied by the disappearance of fine structure compared with that in aqueous solution of β-CD, which could be attributed to the formation of the excimer of POPOP in the larger cavity of γ-CD. It was found that at pH greater than 12, the hydrogen bond between the neighboring CDs was destroyed, which led to the collapse of the nanotubular structure. The results also showed that the nanotube structure was not stable at temperatures above 331 K.