Aggregation of Cyclodextrins as an Important Factor to Determine Their Complexation Behavior

Aggregation of Cyclodextrins as an Important Factor to Determine TheirComplexation Behavior

by Zsolt Bikadia), Robert Kurdib), Sandor Baloghb), Julianna Szemanc), and Eszter Hazai*a)

a) Virtua Drug, Ltd., Csalogany st. 4c, H-1015 Budapest (e-mail: [email protected])b) Delta Elektronik, Ltd., Szentendrei st. 39–53, H-1033 Budapest

c) CycloLab Cyclodextrin Research and Development Laboratory, Ltd., P. O. Box 435, H-1525 Budapest

Here, we report a study on the complexation behavior of carotenoids with cyclodextrins (CDs) usingsolubility experiments and molecular-modelling methods. Carotenoids are an important group ofnaturally occurring dyes found in vegetables and fruits. Their antioxidant property has initiatedinvestigations on their possible use as drugs. However, carotenoids are lipophilic molecules with verylittle inherent aqueous solubility. Cyclodextrin complexation has been widely used in order to increasethe potential applications of hydrophobic compounds. Thus, the aim of our investigation was to designcarotenoids with enhanced water solubility by cyclodextrin complexation. Molecular modelling ofcarotenoid–cyclodextrin complexes with a 1 :1 stoichiometry successfully explained the experimentallyobserved capability of b-cyclodextrins (b-CDs) to form complexes with carotenoids as opposed to a-cyclodextrins (a-CDs) and g-cyclodextrins (g-CDs). Furthermore, molecular-dynamics calculationsrevealed that the aggregation properties of CD derivatives significantly influence their complexationbehavior. Our docking calculations showed that RAMEB (random methylated b-CD) is the b-CDderivative that possesses the lowest tendency to aggregate. Solubility experiments yielded the sameresults, namely, RAMEB complexes possess the best water solubility. Our results showed thatcomplexation of a ligand not buried inside of the CD cavity is dependent on two factors: i) the geometryof the inclusion part of the complex; ii) the self-aggregation property of the CD itself. The lower affinitythe CDs possess for self-aggregation, the more likely are they involved in interactions with carotenoids.These results suggest that self-aggregation of CDs should be considered as an important parameterdetermining complexation in general.

Introduction. – Carotenoids are natural pigments, widely distributed in nature. Todate, over 750 natural carotenoids have been identified and characterized [1]. They areknown to be a main dietary source of vitamin A in humans. The recognition ofprotective effects of carotenoids against serious disorders such as cancer, heart disease,and degenerative eye disease has stimulated intensive research on their role asantioxidants and regulators of the immune system. The vast majority of carotenoidshave limited or undetectable solubility in aqueous solution [2] [3]. Our previous studyshowed that the limited solubility is the consequence of the formation of large card-pack and head-to-tail carotenoid aggregates [4]. To increase the utility of thesecompounds for evaluation in aqueous-phase model systems and potential clinicalapplications, various techniques have been developed to improve the solubility and/ordispersibility in various vehicles [3] [5–8]. Among solubilizers, cyclodextrin complex-ation has been widely used [9].

CHEMISTRY & BIODIVERSITY – Vol. 3 (2006)1266

C 2006 Verlag Helvetica Chimica Acta AG, ZFrich

Cyclodextrins (CDs) are cyclic oligosaccharides obtained by the enzymaticconversion of starch [10]. The parent CDs are a-, b-, and g-cyclodextrins (a-CD, b-CD, g-CD, resp.) containing six, seven, or eight glucopyranose units, respectively. CDsform a truncated cone, and, therefore, the molecule provides a hydrophobic cavity in anaqueous environment. When the solubility of a drug is the limiting factor for its oraldelivery, its aqueous solubility [11], stability [12], and the bioavailability [13] may beimproved by forming CD–drug inclusion complexes. By complexation, the hydro-phobic drug is sequestered inside the cavity of the cyclodextrin. In many cases [13–15],complexation of drugs by CDs improves their delivery characteristics withoutinterfering with their activities, because complexation is a rapidly reversible dynamicprocess. The parent CDs with appropriate cavity diameter are suitable for complexingmany pharmaceutical compounds; however, they have less than optimal solubility andsafety data [15]. Thus, numerous chemically modified CDs have been developed toimprove the solubility limits of the parent CD [13–15].

The possible factors and the various molecular forces which may play a role incyclodextrin complexation have been widely discussed [1], and several hypotheses havebeen proposed to account for cyclodextrin complex formations [14] [16] [17]. Computersimulations are widely used to rationally explain the experimental findings concerninginclusion and recognition [18]. Faucci et al. have recently developed a mathematicalmodel for predicting the stability of drug–cyclodextrin complexes with the aid ofmolecular-modelling techniques [19]. Their model was appropriate for predicting thestability of CD–small-drug complexes, which are able to totally intrude into thehydrophobic cavity of cyclodextrins. However, in spite of the efforts, the relativecontributions of the different forces involved in the process of complexation are still notknown. Evidence has been shown that interactions among cyclodextrins are alsoimportant factors in determining the cyclodextrin-complexation behavior. Recently,experimental evidence established the self-aggregation of CDs in H2O well below themaximum solubility [20]. Moreover, the self-assembly of CDs has been shown to affecttheir aggregation properties.

Previous studies on short-chain analogues of carotenoids, b-ionone retinoids[21] [22], demonstrated the formation of stable inclusion complexes with CDs. It wasshown that the terminal cyclohexene fragment of b-ionone, which is present in mostcarotenoids, has the requisite size for incorporation into the CD cavity. Thus, it seemslikely that carotenoids also form complexes. Recently, experimental evidence of a realcarotenoid–cyclodextrin inclusion-complex formation was reported [21]. However,because of the size and shape of carotenoids and CDs, carotenoids will not be totallyburied in the cyclodextrin cavity. Therefore, it is reasonable to assume that, even whenforming an inclusion complex, a part of the carotenoid molecule is still able to interacteither with another carotenoid or with a cyclodextrin molecule.

In the present work, molecular docking and molecular-dynamics (MD) calculationswere utilized as a tool for designing carotenoid–cyclodextrin complexes with enhancedcarotenoid H2O solubility. Typical carotenes and carotenoids were selected forinvestigation: open-chain carotene lycopene, b-carotene with two b-rings, andcarotenoids lutein, zeaxanthin, and a slightly H2O-soluble carotenoid derivative,crocetin (Fig. 1). Our experimental results showed that, among the parent CDs,only b-CD raises carotenoid solubility at an excess cyclodextrin concentration.

CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 1267

MD Calculations were carried out to get deeper insight into the complexationmechanism. Our calculations revealed that carotenoids are able to form complexeswith CDs with a stoichiometry greater than 1 :1. Moreover, we showed that thetendency of CDs to aggregate inversely correlates with their ability to form H2O-soluble complexes with carotenoids. Finally, our results showed that the CD of choicefor complexation with carotenoids possesses a low affinity for self-aggregation andforms large contact surface area complex.

Results and Discussion. – The complexation of various molecules with CDs is ofhigh industrial importance, as many applications exist in pharmaceutical and environ-mental research. Numerous investigations have been carried out on the mechanism ofhost–guest inclusion processes in order to obtain some information about the forceswhich lead to the association of a molecule into the cavity of CDs. In our study, theinteractions between the guest molecules and the interior of various CDs wereinvestigated by both experimental methods and molecular-modelling calculations. Theaim of our project was to find the most appropriate CD derivative which enhances thesolubility of the investigated carotenoids. Representative carotenes and carotenoidsselected for investigation were lycopene, b-carotene, lutein, zeaxanthin, and crocetin(Fig. 1).

Fig. 1. Chemical structures of the carotenoids investigated


Solubility of Carotenoids in Parent a-CD, b-CD, and g-CD Solutions. First, thecomplexation behavior of different carotenoids with parent CDs were examined(Table 1) in order to determine the appropriate cavity size. With the exception oflutein, none of the investigated carotenoids (lycopene, b-carotene, zeaxanthin,crocetin; Fig. 1) was able to form soluble complexes with any of the parent CDs.Lutein was the only carotenoid forming b-CD complex that showed a slight solubility,while it was not soluble in a-CD and g-CD. These data demonstrate that underivatizedCDs are unable to significantly enhance carotenoid solubility. It was anticipated thatmolecular modelling might help predicting which CD is the most likely to formcomplexes of greater solubility. Therefore, molecular-modelling calculations have beencarried out in order to identify the molecular mechanism underlying the complexationmechanism.

Molecular-Docking Studies of Carotenoids to Parent CDs. Molecular-modellingstudies were performed in order to determine the relationship between the dockingenergies of drug–cyclodextrin complexes and their corresponding solubility. Thedocking energies were calculated as the sum of the van der Waals forces andelectrostatic interaction energies. The only carotenoid that showed solubility in any ofthe parent CDs was lutein; therefore, this molecule was applied in our dockingcalculations. The best docking-energy geometries of lutein with a-CD, b-CD, and g-CDare shown in Fig. 2. In the case of a-CD, the size of the lutein ring does not entirelyallow the carotenoid ring to intrude into the cavity, therefore, the ring-OH group is notable to form a H-bridge with a OH group of the CD. Nonspecific hydrophobicinteractions are formed between lutein and a-CD. In contrast, in case of b-CD and g-CD, the lutein ring is buried inside the cavity and is able to form a H-bond with a OHgroup of the CD. However, the docking energy of lutein complexes with a-CD, b-CD,and g-CD do not differ substantially (Table 2), indicating that analyzing the dockingenergy itself is not sufficient in explaining which CD forms a complex with lutein. Muraand co-workers explained the solubility of inclusion complexes with three theoreticalparameters: intermolecular interaction fields, docking energy, and contact surface [19].They acquired the most accurate theoretical model for the computational prediction ofthe aqueous solubility of the inclusion complex. Therefore, the contact surface of thecomplexes, as a measure of the degree of fit of the guest molecule into the hostmolecule (Table 3), was examined. This was possible first by evaluating the solvent-accessible surface of the single molecules and then of the complexes formed.Accordingly, the decrease in the solvent-accessible surface of the complex as compared

Table 1. Aqueous Solubility [mg/ml] of Carotenoids with 5% Solution of Parent CDs. The only complexshowed higher aqueous solubility than limit of detection (LOD) is lutein–b-CD.

Carotene/CD a-CD g-CD b-CD

Lutein <LOD <LOD 0.55�0.05Lycopene <LOD <LOD <LODb-Carotenoid <LOD <LOD <LODZeaxanthin <LOD <LOD <LODCrocetin <LOD <LOD <LOD


to that of the single molecules was analyzed. Our results showed that the greatestcontact surface is observed in the case of the lutein–b-CD complex. The ring of thelutein cannot totally intrude into the cavity of a-CD. In contrast, there is an unfilledvolume in the g-CD cavity, and the lutein ring forms interactions with only a part of theCD ring. Indeed, solubility experiments showed that lutein is dissolved in aqueoussolutions of b-CD, but not in those of a-CD or g-CD (Table 1).

Fig. 2. Best energy docking results of lutein to a) a-CD b) b-CD, and c) g-CD from two viewpoints (leftand right panels)

Table 2. Docking Energies of Lutein to Parent CDs. The differences of the calculated values arenegligible.

CD–Lutein complex Docking energy [kcal/mol]

a-CD–Lutein �5.43b-CD–Lutein �5.41g-CD–Lutein �5.44


Contact-surface calculations of all investigated carotenoid–b-CD complexes werecarried out. These calculations were based on the assumption that a moleculepossessing similar or higher contact surface than lutein is able to form a H2O-solublecomplex with a b-CD derivative. The resulting contact surfaces of the calculatedminimum-energy complexes are shown in Table 4. These data show that zeaxanthin andlutein are the most promising carotenoids to form complexes with b-CDs. Otherinvestigated carotenoids without a substituent on their six-membered ring and open-chain carotenoids do not fill up the available space inside the CD cavity.

Stepwise-Docking Calculations of Carotenoids to Parent CDs and CD Derivatives.The fact that the use of derivatized CDs greatly enhances solubility has beendemonstrated by several examples. Therefore, interaction energies of carotenoids withdifferent b-CD derivates were examined. The docking of carotenoids to the different b-CD derivatives, assuming 1 :1 complexes, led to almost identical energy values andidentical contact-surface values as for the parent CDs (data not shown). This finding isreasonable if we consider that the substituted groups lie at the surface of the CDs,therefore, they are not expected to influence the interaction with the ligand. Thus, thefollowing factors should be taken into consideration when explaining the differentsolubility behaviors of CD derivatives as compared to the parent CDs: a) Onepossibility is that carotenoids form complexes with CDs with greater than 1 :1stochiometry. In this case, it is possible that not only the CD cavity, but also the polarsurface of the CD molecule interacts with the carotenoids. b) The other possibility isthat CD self-aggregation influences the complexation behavior. The CD moleculesalready in interaction with other CDs are not freely available for interaction withcarotenoids. c) Our previous study showed that carotenoids form large card-pack andhead-to-tail carotenoid aggregates [4]. Although this fact is likely to influencecarotenoid–CD complexation, in general, it does not explain differences of solubility inparent CDs and CD derivatives for a given carotenoid.

Table 4. Docked Energies and Contact-Surface Areas of Carotenoid–b-CD Complexes. Zeaxanthin andlutein were calculated to form complexes with b-CD with the highest contact-surface area.

Crocetin Lutein Lycopene b-Carotene Zeaxanthin

Docked energy [kcal/mol] �5.32 �5.71 �5.74 �5.58 �5.35Complex surface [I2] 1345.0 1626.9 1666.5 1624.5 1597.0Ligand surface [I2] 563.3 892.1 915.0 858.7 883.1b-CD surface [I2] 1058.5 1058.5 1058.5 1058.5 1058.5Contact surface [I2] 276.8 323.7 307.0 292.7 344.6


Table 3. Calculated Contact-Surface Areas of Lutein–CD Complexes. The Lutein–b-CD complexpossesses the largest contact-surface area.

Surface area [I2] Sum of CD and lutein [I2] CD–Lutein complex [I2] Contact surface [I2]

a-CD 895.0 1787.1 1518.3 268.8b-CD 1058.5 1950.6 1626.9 323.7g-CD 1177.1 2069.2 1793.4 275.8Lutein 892.1

a) The first possibility was examined as follows: another CD molecule was dockedinto the lutein–CD 1 :1 complex (data not shown). The results showed that the secondCD forms interactions with the first CD molecule rather than with the carotenoid.Subsequent dockings yielded differently arranged CDs around the previously dockedCDs rather than around the carotenoid molecule. These results indicate thatinteractions between the polyene chain of the carotenoid and the polar surface ofthe CD are of nonspecific nature. Derivatization of b-CD does not substantiallyinfluence this nonspecific interaction. These results point to the possibility of CD�CDcomplexation influencing solubility rather than more CD molecules interacting withone carotenoid molecule.

b) According to the docking calculations, the effect of CD self-aggregation shouldbe taken into account in prediction of complexation behavior of CD derivatives.Indeed, the self-aggregation of CDs in H2O has recently been postulated [20].

Molecular Dynamics Calculations (MD) of the Carotenoid–CD System. MDSimulations were carried out to simulate the dynamical behavior of CDs in the presenceof carotenoids. A simulation box including H2O and different CDs at a concentration of0.05m was constructed (Jin silico CD solutionK). The simulation was carried out in the insilico CD solution including a carotene ligand. This way, both the self-aggregationproperties of CDs and the possibility of formation of a carotene–CD complex with morethan 1 :1 stoichiometry could be simultaneously analyzed. Three parallel runs werecarried out for a simulation time of 3 ns. The organization and geometry of b-CDs andthe carotenoid was analyzed at the end of the run for i) stoichiometry of the lutein–b-CDcomplex, and for ii) b-CD aggregation. Every run differed in the stoichiometry of thelutein–b-CD complex, with 3–12 CDs being in interaction distance of lutein. Oneexample of a complex geometry after 3 ns molecular dynamic run is shown in Fig. 3.

The MD simulation revealed that, in b-CD solution, specific interactions mightform between the lutein ring and b-CD cavity. Furthermore, interactions between thepolyene chain of lutein and b-CDs can be formed; however, these interactions are non-specific in nature. The exact stoichiometry of the complex cannot be determined.Besides interacting with the carotenoid, CDs aggregated during the simulation. Thebeginning and the end of a MD simulation is shown in Fig. 4. It can clearly be seen thatthe self-aggregation of CDs influenced the number of b-CDs available for interactionwith lutein. The aggregation of CDs in H2O is also confirmed by experimental data.Namely, experimental evidence has recently been reported of b-CDs formingaggregates in aqueous solution at low concentrations well below the maximumsolubility [20]. Additionally, it has been shown that aggregation of CDs significantlyinfluences their complexation behavior. Analyzing b-CD aggregates reveals that b-CDsare held together through a large number of H-bonds. It is reasonable to assume thatsubstitution of the OH groups (as is in the case of dimethylated b-cyclodextrin(DIMEB) and random methylated b-cyclodextrin (RAMEB)) hinders the ability ofCDs to form aggregates. Thus, at the same CD concentration, a higher number ofRAMEB andDIMEBmolecules are available for interaction with lutein than in case ofb-CDs. This possibility can be further examined by calculating the interaction energy ofCDs with CDs by molecular docking.

Molecular Modelling of CD Dimerization. The interaction energy of CD derivativesin dimers was calculated using the Autodock program. The results of the docking


studies are shown in Fig. 5, and the estimated free energies of dimerization are shown inTable 5. Significant differences in interaction energies of CDs were observed. Namely,aggregation of b-CDs yielded the lowest energy, followed by 3-hydroxypropylated b-cyclodextrin (HPb-CD), DIMEB, and RAMEB.

The degree of average substitution of b-CD is as follows: HPb-CD 3, DIMEB 14,RAMEB 14 (random distribution). In the cases of DIMEB and RAMEB, substitutionof CD decreases its ability to donate H-bonds, whereas 3-hydroxypropyl substitution(HPb-CD) does not strongly influence the ability of forming intermolecular H-bonds. AsCDs form H-bonds during self-aggregation, it is reasonable to assume that the numberof H-bond donors available in CDs determine their self-aggregation properties. Indeed,the best energy of interaction was observed for b-CD, followed by HPb-CD, DIMEB,and RAMEB. The interaction energy of CDs inversely correlates with their tendency toself-aggregate which, in turn, inversely correlates with the complexation behavior.Accordingly, the investigated CDs are predicted to form H2O-soluble complexes withcarotenoids in the following order: RAMEB, followed by DIMEB, HPb-CD and b-CD.

Aqueous Solubility of Carotenoids in Complex with CD Derivatives. Lycopene andb-carotene were not soluble in aqueous solutions of any of the investigated CDs, whilecrocetin and zeaxanthin showed an improved aqueous solubility in RAMEB (Table 6).Lutein was slightly H2O-soluble in all investigated b-CD derivatives. The solubility


Fig. 3. The final geometry of a lutein–b-CD system after 3 ns molecular-dynamics simulation. The luteinmolecule is buried inside an aggregate formed by b-CDs.

rank order of lutein in the investigated b-CD derivatives was as follows: RAMEB wasthe most effective solubilizing agent for lutein, followed by DIMEB, HPb-CD, andb-CD, in accordance with the calculations.

Overall, RAMEB was by far the most efficient b-CD derivative that was capable offorming H2O-soluble complexes with the investigated carotenoids. The calculatedinteraction energy of CD dimerization inversely correlated with the results of solubilityexperiments (Fig. 6). Thus, our study shows that formation of CD complexes ofcarotenoids depends on the aggregation properties of CDs themselves. The less theCDs aggregate, the more likely are they involved in interactions with carotenoids. Thedegree of substitution of a b-CD derivative correlates with its ability to form complexeswith carotenoids.


Fig. 4. Initial structure (a) and the resulting geometry (b) of CD solvent after a 3 ns MD run. Formation ofCD aggregates can be observed, which influences their ability to form complexes with carotenoids.

Conclusions. – In this article, we reported a study on the complexation behavior ofcarotenoids with CDs using molecular-modelling and experimental methods. The aimof the investigation was to assist designing carotenoids with enhanced H2O solubility bydifferent CD derivatives. Our results indicate that the investigated carotenoids interactwith b-CD derivatives but not with a-CD or g-CDs. Molecular-docking studies withcarotenoid–CD complexes with a 1 :1 stoichiometry could explain the capability of b-CDs to form complexes with carotenoids as opposed to a-CDs and g-CDs. However,


Fig. 5. Best energy results of CD�CD dockings for a) RAMEB, b) DIMEB, c) HPb-CD, and d) b-CD.The number of H-bonds formed between CDs increases in the order of RAMEB<DIMEB<HPb-CD<

b-CD.

Table 5. Estimated Free Energy of Binding of CD Dimerization. The calculated energy correlates withthe H-bond forming ability of CDs.

Estimated free energy of binding [kcal/mol]

b-CD �2.60HPb-CD þ1.81DIMEB þ3.44RAMEB þ5.49

docking studies assuming a 1 :1 carotenoid–CD complex cannot differentiate betweenthe complexation behavior of different b-CD derivatives. MD Calculations revealedthat the aggregation properties of CD derivatives significantly influence theircomplexation behavior. The less the CDs aggregate, the more likely are they involvedin interactions with carotenoids. Docking calculations identified RAMEB as the b-CDderivative with lowest tendency to aggregate. Indeed, solubility experiments confirmedthat RAMEB complexes show by far the best H2O solubility. To summarize, our resultsshowed that complexation of a ligand not buried inside of the CD cavity is dependenton two factors: i) the geometry of the inclusion part of the complex; ii) the self-


Table 6. Aqueous Solubility [mg/ml] of Carotenoids with 5% Solution of b-CD Derivatives. RAMEBComplexes show by far the best aqueous solubility.

Carotene/CD HPb-CD DIMEB RAMEB

Lutein 0.87�0.09 10.3�0.6 29.0�1.6Lycopene <LOD <LOD <LODb-Carotenoid <LOD <LOD <LODZeaxanthin <LOD <LOD 20.0�1.2Crocetin <LOD <LOD 3.6�0.3

Fig. 6. Lutein solubility in solution of b-CD derivatives in function of calculated free energy of CDdimerization

aggregation property of the CD itself. Moreover, our results indicate that self-aggregation of CDs should be considered as an important parameter determiningcomplexation in general.

Experimental Part

General. Abbreviations. CD: Cyclodextrin; a-CD: a-cyclodextrin; b-CD: b-cyclodextrin; g-CD: g-cyclodextrin; RAMEB: random methylated b-cyclodextrin; DIMEB: dimethylated b-cyclodextrin;HPb-CD: hydroxypropylated b-cyclodextrin.

Docking Calculations. MMFF94 Force field [23] with conjugate gradient method was used forenergy minimization of the carotenoids using the SpartanK04 program. Initial coordinates of a-, b-, and g-CDs were extracted from published X-ray structures of CD–protein complexes (PDB entries: 1CXF,3CGT, 1P2G, resp.). Gasteiger partial charges were added to ligand and CD atoms with the aid ofAutoDock tools [24]. Nonpolar H-atoms were merged, and rotatable bonds and solvation parameterswere defined with AutoDock tools. Each carotenoid was docked onto each CD using the same parameterset.

Affinity (grid) maps of 100�40�40 grid points and 0.375 I spacing were generated using theAutoGrid program [24]. Preliminary studies showed that, without restrictions, AutoDock results in anartificial complex, where a CD molecule is trapped between the end groups of carotenoids. Box centreswere defined to rule out this kind of complex conformation. AutoDock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostaticterms, resp. Docking simulations were performed using the Lamarckian genetic algorithm (LGA), andthe Solis & Wets local search method [25] of AutoDock. Initial position, orientation, and torsions of theligand molecule were set randomly. All rotatable torsions were released during docking. Each dockingexperiment was derived from 100 different runs that were set to terminate after a maximum of 1,500,000energy evaluations. The population size was set to 250. During the search, a translational step of 0.2 I,and quaternion and torsion steps of 5 were applied. CD�CD Docking calculations were performed forb-CD, RAMEB, DIMEB, and HPb-CD. The free energy of binding was calculated with AutoDock [24].

Contact-Surface Calculations. The programVEGAZZ [26] was used to calculate the contact-surfacearea of carotenoid–CD complexes. A probe radius of 1.0 I and a density of 20 were applied. Surfaceareas were calculated for CDs and carotenoids independently and in the docked complex. The contactsurface area is derived from the difference between the sum of independent surfaces and the area of thecomplex surface.

Molecular Dynamics (MD) Calculations. MD Calculations were carried out using the GROMACSprogram package [27] to simulate the behavior of the lutein molecule in aqueous solutions of differentCDs. The topology files of the solvent molecules (cyclodextrin) and the solute (lutein) were generatedusing the Dundee Prodrug server [28]. Water–CD solvent boxes at 0.05m CD concentration weregenerated for a-CD, b-CD, and g-CD using the editconf and genbox commands of GROMACS. Thesolvent boxes were energy-minimized by unrestrained MD calculations. The simulation time was 1 ns.These simulation boxes were applied to solvate lutein and to calculate the dynamic behavior of lutein–CD systems. Complexes were energy-minimized using the GROMOS force field [29] implemented in theprogram package. Unrestrained MD simulations were performed for 3 ns for each lutein–CD system.

Solubility Experiments. The used CDs and CD derivatives were the products of CycloLab Ltd. (H-Budapest). The fine-chemical-grade CD derivatives are tested, identified, and characterized by anal.methods. The average degree of substitution of statistically substituted derivatives is calculated fromNMR data. For characterization of isomer distribution, various methods (TLC, MS, NMR; for somederivatives, GC, HPLC, or cap. electrophoresis) are used. The average molecular weights of parent andderivatized CDs were determined (a-CD: 972.9, b-CD: 1135.0, g-CD: 1297.2, RAMEB: 1303, DIMEB:1331.4, HPb-CD: 1309.0) The phase-solubility studies were performed in deionized H2O at 258. Excessamounts (more than those assumed to be dissolved by the cyclodextrins) of the studied carotenoids wereapplied to the CD-containing aqueous solns. The CD concentration generally varied between 1.0 and20.0 g/100 ml. The aq. suspensions were stirred with magnetic stirrer (200 rpm) for 24 h. After sufficient


equilibration, the suspensions were filtered across a 0.45-mm membrane and assayed with or withoutappropriate dilution for dissolved carotene concentration by UV/VIS spectrophotometry.

REFERENCES

[1] G. Britton, S. Liaaen-Jensen, H. Pfander, JCarotenoids: HandbookK, BirkhOuser, Basel, 2004.[2] G. Britton, S. Liaaen-Jensen, H. Pfander, JCarotenoids: Isolation and AnalysisK, BirkhOuser, Basel,

1995, Vol. Ia, p. 81–108.[3] J. S. Bertram, Nutr. Rev. 1999, 57, 182.[4] Z. Bikadi, F. Zsila, J. Deli, G. Mady, M. Simonyi, Enantiomer 2002, 7, 67.[5] W. L. Jorgensen, E. M. Duffy, Adv. Drug Delivery Rev. 2002, 54, 355.[6] S. F. Lockwood, S. OKMalley, G. L. J. Mosher, J. Pharm. Sci. 2003, 92, 922.[7] A. W. Williams, T. W. Boileau, S. K. Clinton, J. W. Erdman, J. Nutr. Cancer 2000, 36, 185.[8] B. J. Foss, H. R. Sliwka, V. Partali, C. Kopsel, B. Mayer, H. D.Martin, F. Zsila, Z. Bikadi, M. Simonyi,

Chem.–Eur. J. 2005, 11, 4103.[9] A. R. Hedges, Chem. Rev. 1998, 98, 2035.

[10] J. Szejtli, T. Osa, in JComprehensive Supramolecular Chemistry, (CDs)K, J. Szejtli, T. Osa, Eds,Vol. 3, Pergamon, Oxford, 1996, p. 5.

[11] J. Peeters, P. Neeskens, J. Adriaensen, M. Brewster, J. Inclusion Phenom. Mol. Recognit. Chem. 2002,44, 75.

[12] H. S. Lin, C. S. Chean, Y. Y. Ng, S. Y. Chan, P. C. Ho, J. Clin. Pharm. Ther. 2000, 25, 265.[13] N. Nasongkla, A. F.Wiedmann, A. Bruening,M. Beman, D. Ray,W. G. Bornmann, D. A. Boothman,

J. Gao, Pharm. Res. 2003, 20, 1626.[14] T. Loftsson, E. M. Brewster, J. Pharm. Sci. 1996, 85, 1017.[15] A. R. Rajewski, V. J. Stella, J. Pharm. Sci. 1996, 85, 1142.[16] D. Duchene, in JCDs and Their Industrial UsesK, De Sante, Paris, 1987.[17] K. A. Connors, Chem. Rev. 1997, 97, 1325.[18] K. B. Lipkowitz, Chem. Rev. 1998, 98, 1829.[19] M. T. Faucci, F. Melani, P. Mura, Chem. Phys. Lett. 2002, 358, 383.[20] M. Bonini, S. Rossi, G. Karlsson, M. Almgren, P. Lo Nostro, P. Baglioni, Langmuir 2006, 22, 1478.[21] N. E. Polyakov, T. V. Leshina, T. A. Konovalova, E. O. Hand, L. D. Kispert, Free Radical Biol. Med.

2004, 36, 872.[22] S. Munoz-Botella, M. A. Martin, B. del Castillo, D. A. Lerner, J. C. Menendez, Anal. Chim. Acta

2002, 468, 161.[23] T. A. Halgren, J. Am. Chem. Soc. 1992, 114, 7827.[24] G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew, A. J. Olson, J.

Comput. Chem. 1998, 19, 1639.[25] F. J. Solis, R. J. B. Wets, Math. Oper. Res. 1981, 6, 19.[26] A. Pedretti, L. Villa, G. Vistoli, J. Mol. Graphics 2002, 21, 47.[27] H. J. C. Berendsen, D. van der Spoel, R. van Drunen, Comput. Phys. Commun. 1995, 91, 43.[28] A. W. Schuettelkopf, D. M. F. van Aalten, Acta Crystallogr., Sect. D 2004, 60, 1355.[29] J. Hermans, H. J. C. Berendsen, W. F. Van Gunsteren, J. P. M. Postma, Biopolymers 1984, 23, 1513.

Received August 10, 2006


Aggregation of Cyclodextrins as an Important Factor to Determine Their Complexation Behavior

Documents

Transcript of Aggregation of Cyclodextrins as an Important Factor to Determine Their Complexation Behavior