Entropy-Driven Inclusion of Natural Protoberberine Alkaloids in Sulfobutylether-ß-Cyclodextrin.

The understanding of the relationship between molecular structure and the thermodynamics of host-guest binding is essential for the rational design of the applications of inclusion complexes. To obtain insight into the factors governing the driving force of complex formation in aqueous solutions, the encapsulation of five pharmaceutically important protoberberine alkaloids was studied in sulfobutylether-ß-cyclodextrin having on average 6.4 degrees of substitution (SBE6.4ßCD). Spectrophotometric, fluorescence spectroscopic, and isothermal calorimetric measurements showed 1:1 complexation in dilute solutions. From 1.92 × 104 M−1, about an eight-fold decrease of the association constant was observed in the series of berberine ≈ coptisine >> palmatine > epiberberine > dehydrocorydaline. The embedment of these alkaloids in the SBE6.4ßCD cavity was entropy-controlled with mildly negative enthalpy contributions. These findings suggest that the stabilization of the examined complexes arises primarily from the hydrophobic interaction between the constituents. The more than three orders of magnitude smaller association constants of protoberberine alkaloids with SBE6.4ßCD than with cucurbit[7]uril, a host having similar cavity size, originates from the much smaller exothermicity of the confinement in the former macrocycle.

Halochromic Behavior and Anticancer Effect of New Synthetic Anthocyanidins Complexed with ß-Cyclodextrin Derivatives.

Anthocyanidins, the aglycons of anthocyanins, are known, beyond their function in plants, also as compounds with a wide range of biological and pharmacological activities, including cytostatic effect against various cancer cells. The nature and position of the substituents in the flavylium cation is essential for such biological properties, as well as the equilibrium between the multistate of the different chemical species that are generated by the flavylium cation, including quinoidal base, hemiketal, and cis- and trans-chalcones. In this work, eight new flavylium derivatives were synthesized, characterized for confirmation of the structure by FT-IR and 2D-NMR, and investigated in vitro as possible cytostatic compounds against HCT116 and HepG2 cancer cells. The most active two compounds were explored for their halochromic properties that can influence the biological activity and subjected to molecular encapsulation in ß-cyclodextrin derivatives in order to increase their solubility in water and bioavailability. The anticancer effect was influenced by the position (6-, 7-, or 8-) of the methoxy group in the ß-ring of the methoxy-4'-hydroxy-3'-methoxyflavylium cation, while the study of the halochromic properties revealed the important role played by the chalcone species of the pH-dependent multistate in both the uncomplexed and inclusion complex forms of these anthocyanidins.

Encapsulation of Metronidazole in Biocompatible Macrocycles and Structural Characterization of Its Nano Spray-Dried Nanostructured Composite.

Due to the great potential of biocompatible cucurbit[7]uril (CB7) and 4-sulfonatocalix[4]arene (SCX4) macrocycles in drug delivery, the confinement of the pharmaceutically important metronidazole as an ionizable model drug has been systematically studied in these cavitands. Absorption and fluorescence spectroscopic measurements gave 1.9 × 105 M-1 and 1.0 × 104 M-1 as the association constants of the protonated metronidazole inclusion in CB7 and SCX4, whereas the unprotonated guests had values more than one order of magnitude lower, respectively. The preferential binding of the protonated metronidazole resulted in 1.91 pH unit pKa diminution upon encapsulation in CB7, but the complexation with SCX4 led to a pKa decrease of only 0.82 pH unit. The produced protonated metronidazole-SCX4 complex induced nanoparticle formation with protonated chitosan by supramolecular crosslinking of the polysaccharide chains. The properties of the aqueous nanoparticle solutions and the micron-sized solid composite produced therefrom by nano spray drying were unraveled. The results of the present work may find application in the rational design of tailor-made self-assembled drug carrier systems.

Simultaneous analyte indicator binding assay (SBA) for the monitoring of reversible host-guest complexation kinetics.

Very little information is available on the kinetics of the self-assembly and dissociation of optically silent building blocks despite the importance of such data in the rational design of tailor-made host-guest systems. We introduce here a novel time-resolved method that enables the simultaneous determination of complex formation and complex dissociation rate constants for inclusion-type host-guest complexes. The simultaneous analyte indicator binding assay (SBA) gives also direct access to binding affinities, thus largely simplifying the experimental procedure for a full kinetic and thermodynamic characterisation of host-guest systems.

Teaching indicators to unravel the kinetic features of host-guest inclusion complexes.

Both thermodynamic and kinetic insights are needed for a proper analysis of association and dissociation processes of host-guest interactions. However, kinetic descriptions of supramolecular systems are scarce in the literature because suitable experimental protocols are lacking. We introduce here three time-resolved methods that allow for convenient determination of kinetic rate constants of spectroscopically silent or even insoluble guests with the macrocyclic cucurbit[n]uril family and human serum albumin (HSA) protein as representative hosts.

Kinetics and Mechanism of Cation-Induced Guest Release from Cucurbit[7]uril.

The release of two organic guests from cucurbit[7]uril (CB7) was selectively monitored by the stopped-flow method in aqueous solutions of inorganic salts to reveal the mechanistic picture in detail. Two contrasting mechanisms were identified: The symmetric dicationic 2,7-dimethyldiazapyrenium shows a cation-independent complex dissociation mechanism coupled to deceleration of the ingression in the presence of alkali and alkaline earth cations (Mn+ ) due to competitive formation of CB7-Mn+ complexes. A much richer, unprecedented kinetic behaviour was observed for the ingression and egression of the monocationic and non-symmetric berberine (B+ ). The formation of ternary complex B+ -CB7-Mn+ was unambiguously revealed. A difference of more than two orders of magnitude was found in the equilibrium constants of Mn+ binding to B+ -CB7 inclusion complex. Large cations, such as K+ and Ba2+ , also promoted B+ expulsion from the ternary complex in a bimolecular process. This study reveals a previously hidden mechanistic picture and motivates systematic kinetic investigations of other host-guest systems.

Binding affinities of cucurbit[n]urils with cations.

High binding constants of 19 inorganic cations with the cucurbit[n]uril homologues (CBn, n = 5, 6, 7, 8) in water were determined and the far-reaching consequences and interferences of the high affinities (millimolar to micromolar) are discussed.

4-Sulfonatocalixarene-induced nanoparticle formation of methylimidazolium-conjugated dextrans: Utilization for drug encapsulation.

Methylimidazolium side groups were grafted via ether linkage to dextran and the self-assembly of these polymers with 4-sulfonato-calix[n]arenes (SCXn) was studied in aqueous solutions. Dynamic light scattering and zeta potential measurements revealed the mixing ratio ranges of the constituents where stable nanoparticles could be created. The macrocycle size of SCXn and the molecular mass of the polymer barely affected the nanoparticle diameter, but the lowering of the imidazolium degree of substitution substantially diminished the stability of the associates. The pH change from neutral to acidic also unfavourably influenced the self-organization owing mainly to the decrease of the SCXn charge. Cryogenic transmission electron microscopy images proved the spherical morphology of the nanoproducts in which the stoichiometry of the constituents was always close to the one corresponding to charge compensation. The flexible and positively charged dextran-chains are compacted by the polyanionic SCXn. Coralyne, a pharmacologically important alkaloid was efficiently embedded by self-assembly in the produced nanoparticles reaching 99% association efficiency.

Change of the kinetics of inclusion in cucurbit[7]uril upon hydrogenation and methylation of palmatine.

The inclusion of protonated (-)-tetrahydropalmatine (THP+) and dehydrocorydaline (DHC+), natural alkaloids, in the cavity of cucurbit[7]uril was monitored in real time by a spectrofluorimetric method in water at various temperatures. Both guests produced 1 : 1 complexes in enthalpy controlled processes without any detectable intermediates. The tight entrance of CB7 imposed substantial steric hindrance for encapsulation making the entry into the host several orders of magnitude slower than diffusion. Despite the ∼6 kJ mol-1 lower activation enthalpy, the rate constant of THP+ ingression into CB7 was about 44-fold smaller at 298 K than that of DHC+ as a consequence of the considerably negative activation entropy of the former binding. The egression rates of the two studied alkaloids differed to a much lesser extent because the lower energy barrier of THP+ release was almost compensated by the unfavourable activation entropy. In comparison with the kinetics of the reversible confinement of the palmatine parent compound, the presence of the methyl substituent on the aromatic heterocyclic ring in DHC+ barely modified the rate constant of entry into CB7 but caused about 10-fold increase in the dissociation rate at 298 K.

Substituent effect on the dynamics of the inclusion complex formation between protoberberine alkaloids and cucurbit[7]uril.

The kinetics of entry into and exit from the cavity of cucurbit[7]uril (CB7) was studied by the stopped-flow method in water at various temperatures using pharmaceutically important natural isoquinoline alkaloids as guest compounds. The rate constant of the alkaloid-CB7 complex dissociation was separately determined exploiting the very strong competitive binding of the 1-adamantylammonium cation to CB7. The enthalpy and entropy of activation for the release of berberine from CB7 were significantly lower than those found in the case of the other alkaloids, suggesting different dissociation dynamics. CB7 initially moved from the energetically most stable position encompassing the isoquinoline moiety of berberine to the smaller benzodioxole part, which could leave the macrocycle with less structural distortion. Despite the same thermodynamic parameters of berberine and palmatine inclusion, the latter compound was encapsulated in and set free from CB7 much slower due to the more substantial steric hindrance. The most rapid entry into CB7 and the most exothermic binding were found for epiberberine and coptisine, the alkaloids substituted with the less spacious dioxole ring on their isoquinoline moiety.

Substituent Effects on the Inclusion of 1-Alkyl-6-alkoxy-quinolinium in 4-Sulfonatocalix[n]arenes.

The effect of the chain length of the alkyl and alkoxy substituents on the binding characteristics of 1-alkyl-6-alkoxy-quinolinium cations was studied using 4-sulfonatocalix[4]arene (SCX4) and 4-sulfonatocalix[6]arene (SCX6) in neutral aqueous solutions at 298 K. Isothermal calorimetric titrations showed enthalpy-controlled inclusion with 1:1 stoichiometry. The equilibrium constants of complexation were always larger for the confinement in SCX4 than in its SCX6 homologue because the better matching between the host and guest sizes allowed more exothermic interaction. The binding affinity diminished with the lengthening of the aliphatic chain of the guests in the case of the association with SCX4, but insignificant change was found for SCX6 complexes. The most substantial change in the enthalpic and entropic contributions to the driving force of complex production occurred when the alkyl chain was linked to the heterocyclic nitrogen and the number of its carbon atoms varied between 1 and 4. 1H NMR spectra evidenced that in SCX6, the 1-alkyl-6-alkoxy-quinolinium cations could be included within the macrocycle cavity. In the case of SCX4, the quinolinium ring is always inside the host, but the alkyl chain is included within SCX4 only for a short chain length (n up to 4). In contrast, the alkoxy chain displays a very weak interaction with the cavity irrespective of the length. Because of the outward orientation from the host, the lengthening of the alkoxy substituent of the quinolinium moiety barely influenced the thermodynamics of inclusion in SCX4. Distinct linear enthalpy-entropy correlations were found for the encapsulation in SCX4 and SCX6.

Effect of Headgroup Variation on the Self-Assembly of Cationic Surfactants with Sulfonatocalix[6]arene.

The effect of headgroup variation on the association of supramolecular amphiphiles composed of 4-sulfonatocalix[6]arene (SCX6) and cationic surfactant possessing tetradecyl substituent was studied in aqueous solutions at pH 7. When the surfactant contained hydrophilic trimethylammonium, pyridinium, or 1-methylimidazolium headgroup, highly reversible temperature-responsive nanoparticle-supramolecular micelle transformation could be attained at appropriately chosen component mixing ratios and NaCl concentrations. In these cases, the substantial negative molar heat capacity change (ΔCp) rendered nanoparticle formation strongly endothermic at low temperature, whereas the assembly to supramolecular micelle was always accompanied by enthalpy gain. The ΔCp values became less negative when the charge density and the hydrophilic character of the surfactant headgroup diminished. The association of the more hydrophobic 6-methoxyquinolinium and quinolinium surfactants with SCX6 did not lead to supramolecular micelle formation because the self-assembly into nanoparticles was highly exothermic.

Extensive astrocyte synchronization advances neuronal coupling in slow wave activity in vivo.

Slow wave activity (SWA) is a characteristic brain oscillation in sleep and quiet wakefulness. Although the cell types contributing to SWA genesis are not yet identified, the principal role of neurons in the emergence of this essential cognitive mechanism has not been questioned. To address the possibility of astrocytic involvement in SWA, we used a transgenic rat line expressing a calcium sensitive fluorescent protein in both astrocytes and interneurons and simultaneously imaged astrocytic and neuronal activity in vivo. Here we demonstrate, for the first time, that the astrocyte network display synchronized recurrent activity in vivo coupled to UP states measured by field recording and neuronal calcium imaging. Furthermore, we present evidence that extensive synchronization of the astrocytic network precedes the spatial build-up of neuronal synchronization. The earlier extensive recruitment of astrocytes in the synchronized activity is reinforced by the observation that neurons surrounded by active astrocytes are more likely to join SWA, suggesting causality. Further supporting this notion, we demonstrate that blockade of astrocytic gap junctional communication or inhibition of astrocytic Ca2+ transients reduces the ratio of both astrocytes and neurons involved in SWA. These in vivo findings conclusively suggest a causal role of the astrocytic syncytium in SWA generation.

Kinetics of the reversible inclusion of flavopereirine in cucurbit[7]uril.

The temperature dependence of the kinetics of flavopereirine inclusion in cucurbit[7]uril (CB7) was studied by a stopped-flow method in water. The substantial blue-shift of the emission band and the ∼4-fold fluorescence quantum yield enhancement upon host-guest binding permitted the monitoring of the formation and dissociation of the flavopereirine-CB7 1 : 1 complex in real time. The competitive binding of the 1-adamantylammonium cation with extremely high affinity was exploited to selectively and very accurately determine the kinetic parameters of the exit of flavopereirine from the CB7 cavity. The rate constants of the ingression into and the egression from CB7 were found to be 9.0 × 107 M-1 s-1 and 1.6 s-1 at 298 K, respectively. Both processes had substantial activation enthalpy implying that a steric barrier had to be overcome in the course of the reversible encapsulation. The 31 ± 2 kJ mol-1 activation enthalpy of the entry into CB7 was comparable to the 37 ± 2 kJ mol-1 enthalpy change upon the dissociation of the complex.

Effect of Macrocycle Size on the Self-Assembly of Methylimidazolium Surfactant with Sulfonatocalix[n]arenes.

The effect of macrocycle size on the association of supramolecular amphiphiles composed of 4-sulfonatocalix[n]arene and 1-methyl-3-tetradecylimidazolium (C14mim+) was studied in aqueous solutions at pH 7. When the cavitand contained four sulfonatophenol units (SCX4), formation of spherical nanoparticles (NPs) was observed. By contrast, both supramolecular micelle (SM) and NP formation could be attained in the presence of NaCl when the larger, more flexible 4-sulfonatocalix[8]arene (SCX8) served as the host compound. The SCX8-promoted self-assembly into the SM was enthalpically more favorable than the NP production, but the molar heat capacity changes in the two processes barely differed. An addition of 50 mM NaCl significantly increased the enthalpy of C14mim+-SCX8 NP formation, thereby making the self-organization into the SM more favorable. The transformation of SM into NP at high temperatures was due to the substantial entropic contribution to the driving force behind the NP formation. The critical micelle concentration (cmc) and the local polarity in the headgroup domain were considerably lower for the SM compared with those of the conventional C14mim+Br- micelle.

Reversible Nanoparticle-Micelle Transformation of Ionic Liquid-Sulfonatocalix[6]arene Aggregates.

The effect of temperature and NaCl concentration variations on the self-assembly of 1-methyl-3-tetradecylimidazolium (C14mim(+)) and 4-sulfonatocalix[6]arene (SCX6) was studied by dynamic light scattering and isothermal calorimetric methods at pH 7. Inclusion complex formation promoted the self-assembly to spherical nanoparticles (NP), which transformed to supramolecular micelles (SM) in the presence of NaCl. Highly reversible, temperature-responsive behavior was observed, and the conditions of the NP-SM transition could be tuned by the alteration of C14mim(+):SCX6 mixing ratio and NaCl concentration. The association to SM was always exothermic with enthalpy independent of the amount of NaCl. In contrast, NPs were produced in endothermic process at low temperature, and the enthalpy change became less favorable upon increase in NaCl concentration. The NP formation was accompanied by negative molar heat capacity change, which further diminished when NaCl concentration was raised.

Sequential inclusion of two berberine cations in cucurbit[8]uril cavity: kinetic and thermodynamic studies.

A combination of absorption and fluorescence spectroscopic studies with isothermal calorimetric titrations and stopped-flow measurements is a powerful way to reveal the thermodynamics and kinetics of inclusion complex formation with cucurbit[8]uril (CB8). The unique photophysical characteristics of berberine (B(+)), a pharmaceutically important natural alkaloid, were exploited to distinguish the consecutive encapsulation processes, and to examine the confinement in the CB8 cavity. The highly environment sensitive fluorescence lifetime of B(+) permitted the selective detection of various cucurbituril complexes, and indicated to what extent the embedded guest was available for interaction with water. Highly stable 1 : 1 and 2 : 1 B(+) : CB8 complexes were produced due to the release of the high energy water molecules from the CB8 interior, and the second binding step proved to be almost 3 times more exothermic. The favorable entropy change contributed appreciably to the driving force of 1 : 1 encapsulation. In contrast, the embedment of the second B(+) in CB8 led to substantial entropy diminution. The kinetics of encapsulation was followed in real time by recording the fluorescence intensity change after rapid mixing of B(+) and CB8. No evidence was found for intermediates. The rate constants of (64 ± 9) × 10(6), and (5.0 ± 0.5) × 10(6) M(-1) s(-1) were found for the 1 : 1 and 2 : 1 associations, whereas 3.8 ± 0.6, and 0.6 ± 0.1 s(-1) were obtained as the rate constants of the reverse processes at 283 K, respectively.

Kinetics and thermodynamics of berberine inclusion in cucurbit[7]uril.

The kinetics and thermodynamics of berberine inclusion in cucurbit[7]uril was studied by stopped-flow method, fluorescence titrations, and isothermal calorimetry in neat water. The ~500-fold fluorescence intensity enhancement upon encapsulation was exploited to monitor the complex formation in real time at various temperatures. The increase in the fluorescence intensity could be fitted well by assuming a simple 1:1 binding equilibrium without any intermediate formation. For the rate constants of association and dissociation, (1.9 ± 0.1) × 10(7) M(-1) s(-1) and (0.81 ± 0.08) s(-1) were found at 298 K, respectively. The ingression into the cavity of CB7 had 32 ± 2 kJ mol(-1) activation enthalpy, implying a constrictive binding, whereas 69 ± 2 kJ mol(-1) was obtained for the activation enthalpy of the egression. Substantial structural change had to occur when berberine passed through the tight carbonyl-rimmed portal of the macrocycle to reach the transition state. An enthalpy-driven complexation took place with a slight entropy gain.

Effect of torsional isomerization and inclusion complex formation with cucurbit[7]uril on the fluorescence of 6-methoxy-1-methylquinolinium.

Inclusion of 6-methoxy-1-methylquinolinium (C1MQ) in the cavity of cucurbit[7]uril (CB7) was studied by absorption, fluorescence, NMR and isothermal calorimetric methods in aqueous solution at 298 K. The free C1MQ exhibited dual-exponential fluorescence decay kinetics due to the two torsional isomers differing in the orientation of the methoxy moiety relative to the heterocyclic ring. The enthalpy-driven encapsulation of the heterocycle in CB7 led to a very stable 1 : 1 complex with a binding constant of (2.0 ± 0.4) × 10(6) M(-1). The rate of C1MQ-CB7 complex dissociation was found to be comparable to the NMR timescale. Because the methoxy moiety is oriented outward from the host, its s-cis-s-trans isomerization is slightly affected by the confinement. Inclusion complex formation significantly slowed down the photoinduced electron transfer from I(-) and N3(-) to the singlet-excited C1MQ, but did not preclude the reaction because long distance electron transfer occurred through the wall of the CB7 macrocycle. Due to the large difference in the quenching rate constant for free and encapsulated forms, C1MQ is an excellent probe for the study of the inclusion of nonfluorescent compounds in CB7 in the presence of Cl(-) or Br(-).

4-Sulfonatocalix[6]arene-induced aggregation of ionic liquids.

The interaction of 4-sulfonatocalix[6]arene (SCX6) macrocycle with 1-alkyl-3-methylimidazolium type of ionic liquids possessing dodecyl, tetradecyl, or hexadecyl substituent was studied in aqueous solution at 298 K. Host-guest complexation promoted the spontaneous self-assembly into nanoparticles of 7:1 ionic liquid:SCX6 stoichiometry. Positively charged and stable nanoparticles were produced in solutions of 7-200-fold excess of ionic liquid as compared to the amount of SCX6. The negatively charged nanoparticles formed in solutions having 2-7 ionic liquid:SCX6 molar ratios evolved into larger species. The stability of the nanoparticles increased with the lengthening of aliphatic chain of the ionic liquid. Cryo-TEM experiments showed dense particles generally with spherical shape and multilayered structure, which has been confirmed by small-angle neutron scattering.