Bile Salt Aggregates Research Articles

Femtosecond Solvation Dynamics in Different Regions of a Bile Salt Aggregate: Excitation Wavelength Dependence

Solvation dynamics of coumarin 480 (C480) in the secondary aggregate of a bile salt (sodium deoxycholate, NaDC) is studied using femtosecond up-conversion. The secondary aggregate resembles a long (approximately 40 A) hollow cylinder with a central water-filled tunnel. Different regions of the aggregate are probed by variation of the excitation wavelength (lambdaex) from 375 to 435 nm. The emission maximum of C480 displays an 8 nm red shift as the lambdaex increases from 345 to 435 nm. The 8 nm red edge excitation shift (REES) suggests that the probe (C480) is distributed over regions of varied polarity. Excitation at a short wavelength (375 nm) preferentially selects the probe molecule in the buried locations and exhibits slow dynamics with a major (84%) slow component (3500 ps) and a small (16%) contribution of the ultrafast component (2.5 ps). Excitation at lambdaex=435 nm (red end) corresponds to the exposed sites where solvation dynamics is very fast with a major (73%) ultrafast component (<or=2.5 ps) and relatively minor (27%) slow (2000 ps) component. In sharp contrast to solvation dynamics, the anisotropy decay becomes slower as lambdaex increases from 375 to 435 nm. It is proposed that the buried locations (lambdaex=375 nm) offer lower friction because of the rigid sheetlike structure of the bile salt.

Ultrafast fluorescence resonance energy transfer in a bile salt aggregate: Excitation wavelength dependence

Fluorescence resonance energy transfer (FRET) from Coumarin 153 (C153) to Rhodamine 6G (R6G) in a secondary aggregate of a bile salt (sodium deoxycholate, NaDC) is studied by femtosecond up-conversion. The emission spectrum of C153 in NaDC is analysed in terms of two spectra-one with emission maximum at 480 nm which corresponds to a non-polar and hydrophobic site and another with maximum at ∼530 nm which arises from a polar hydrophilic site. The time constants of FRET were obtained from the rise time of the emission of the acceptor (R6G). In the NaDC aggregate, FRET occurs in multiple time scales — 4 ps and 3700 ps. The 4 ps component is assigned to FRET from a donor (D) to an acceptor (A) held at a close distance (R DA ∼ 17 Å) inside the bile salt aggregate. The 3700 ps component corresponds to a donor-acceptor distance ∼48 Å. The long (3700 ps) component may involve diffusion of the donor. With increase in the excitation wavelength (λ ex) from 375 to 435 nm, the relative contribution of the ultrafast component of FRET (∼4 ps) increases from 3 to 40% with a concomitant decrease in the contribution of the ultraslow component (∼3700 ps) from 97 to 60%. The λ ex dependence is attributed to the presence of donors at different locations. At a long λ ex (435 nm) donors in the highly polar peripheral region are excited. A short λ ex (375 nm) ‘selects’ donor at a hydrophobic location.

Effect of Solvent Polarity and Viscosity on the Guest Binding Dynamics with Bile Salt Aggregates†

Bile salts form supramolecular aggregates with two binding sites with different properties. The guest binding dynamics to the aggregates and guest protection from species in the aqueous phase were investigated using fluorescence and laser flash photolysis experiments. Sodium cholate, deoxycholate and taurodeoxycholate were used as bile salts and acetonitrile or ethylene glycol were added as co-solvents to water in order to alter the binding properties of 1-ethylnaphthalene and 1-naphthyl-1-ethanol with the aggregates. The binding dynamics are faster and protection efficiencies are lower for guests bound to cholate and in the presence of either co-solvent.

Berberine Alkaloid as a Sensitive Fluorescent Probe for Bile Salt Aggregates

Effect of sodium cholate (NaC) bile salt on the absorption and fluorescence properties of berberine cation was studied in aqueous solution and water-cosolvent mixtures. The alteration of the fluorescent behavior with increasing NaC concentration showed an entirely different trend from that found previously in the presence of sodium dodecylsulfate. Binding to bile salt agglomerates led to significant fluorescence intensity enhancement, and the fluorescence lifetime of berberine proved to be highly sensitive to the structure and size of the aggregates. The dual exponential decay kinetics above 10 mM NaC concentration showed that the probe resided in two totally different binding sites. At 2-10 mM NaC concentrations, only primary aggregates were detected. The aggregate disrupting power of cosolvents decreased in the series of dimethylformamide, acetonitrile, formamide, and methanol. These compounds enhanced the water accessibility of berberine bound to aggregates and diminished the number of secondary aggregates.

Spectroscopic probing of bile salt–albumin interaction

Interaction of the bile salts, sodium cholate and sodium deoxy cholate with albumin has been probed by fluorescence and circular dichroism studies. Both covalently and non-covalently labeled protein have been used to follow the aggregation of bile salts in presence of protein and to study bile salt–protein interactions in general. Time resolved studies, in agreement with steady-state fluorescence and circular dichroism studies, indicate alteration of protein secondary structure due to positive co-operative effects in bile salt binding to protein. These studies also indicate that covalent labeling may not always be good for studying proteins as it causes alteration of protein secondary structure.

Supramolecular Dynamics Studied Using Photophysics

Dynamics is an essential feature of supramolecular systems, and it's understanding will be central in achieving new chemical function. The methodology to obtain association and dissociation rate constants for fast binding of guests to host systems in real time is described. Examples are provided for binding of guests to cyclodextrins or bile salt aggregates with an emphasis on the type of information and mechanistic insight that can only be uncovered from kinetic studies and is not apparent in thermodynamic investigations.

Influence of Planarity and Size on Guest Binding with Sodium Cholate Aggregates

Bile salt aggregates are supramolecular structures with two types of binding sites, called primary and secondary sites. The objective of this work was to explore how the nonplanarity and size of guests (biphenyl [BP], 1-1'-binaphthyl [BNP] and dibenz[b,f]oxepin [DBX]) affected their binding affinity and dynamics to sodium cholate (NaC) aggregates. Fluorescence and laser-flash photolysis experiments were performed to obtain information on the binding environment for the guests, the accessibility of quenchers to guests in the aggregate and the dissociation rate constants of the guests from the aggregates. All guests were bound to the more hydrophobic primary aggregate, showing that this site can accommodate nonplanar molecules. However, the structure of the guest affects the structure of the primary aggregates, leading to changes in the accessibility of anions to aggregate-bound guests and to changes for the guest dissociation rate constants from the aggregates.

Molecular dynamics simulations of spontaneous bile salt aggregation

Bile salts are surfactants in bile that facilitate digestion, adsorption and excretion of various compounds. They have planar hydrophobic and hydrophilic faces and therefore exhibit some unusual properties; including the shape and size of the micelles that they form. Molecular dynamics simulations of the spontaneous aggregation of six bile salts (cholate (CHD), glycocholate (GCH), taurocholate (TCH), glycochenodeoxycholate (GCD), glycodeoxycholate (GDX) and glycolithocholate (GLC)) were performed in an aqueous phase to gain insight into their micellar structure. The aggregates that formed spontaneously from a random distribution of molecules ranged in size from 8 to 17 molecules. The structures are highly dynamic in nature and are on average oblate, but can vary from oblate, to spherical or prolate. Intermolecular hydrogen bonding within the micelles was found to be an important factor in determining the micelle size, structure and dynamics. The molecular arrangement within the micelles maximises the hydration of the hydrophilic chains and some favourable orientations for adjacent molecules were acquired. The dynamics of the micelles were investigated using the hydrogen-bond lifetime autocorrelation function correlation time, which exhibited a relationship with the degree of hydroxylation. Comparison of the proposed model to the three literature models showed some features of the disk shaped models of Cary and Small [M.C. Cary, D.M. Small, Arch. Intern. Med. 130 (1972) 506–527] and Kawamura et al. [H. Kawamura, Y. Murata, T. Yamaguchi, H. Igimi, M. Tanaka, G. Sugihara, J.P. Kratohvil, J. Phys. Chem. 93 (1989) 3321–3326], whereas the third, inverted helix model of Giglio et al. [E. Giglio, S. Loreti, N.V. Pavel, J. Phys. Chem. 92 (1988) 2858–2862] can be discounted. The proposed model is better than the existing models, which assumed a rigid and structured molecular arrangement.

Micellization of conjugated chenodeoxy- and ursodeoxycholates and solubilization of cholesterol into their micelles: comparison with other four conjugated bile salts species

Micelle formations of sodium glyco- and taurochenodeoxycholate (NaGCDC and NaTCDC) and sodium glyco- and tauroursodeoxycholates (NaGUDC and NaTUDC) was studied at 308.2 K for their critical micelle concentrations at various NaCl concentrations by pyrene fluorescence probe, and the degree of counterion binding to micelle was determined using the Corrin–Harkins plots. The degree of counterion binding was found to be 0.37–0.38 for chenodeoxycholate conjugates, while the determination of the degree was quite difficult for ursodeoxycholate conjugates. The change of I 1/ I 3 values on the fluorescence spectrum with the conjugate bile salt concentration suggested two steps for their bile salt aggregation. The first step is a commencement of smaller aggregates, the first cmc, and the second one is a starting of stable aggregates, the second cmc. The aggregation number was determined at 308.2 K and 0.15 M NaCl concentration by static light scattering: 16.3 and 11.9 for sodium NaGCDC and NaTCDC, and 7.9 and 7.1 for NaGUDC and NaTUDC, respectively. The solubilization of cholesterol into the bile salt micelles in the presence of coexisting cholesterol phase and the maximum additive concentration (MAC) of cholesterol was determined against the bile salt concentration. The standard Gibbs energy change for the solubilization was evaluated, where the micelles were regarded as a chemical species. The solubilization was stabilized in the order of NaGUDC ≅ NaTUDC < NaTC < NaGC < NaTCDC < NaGCDC < NaTDC < NaGDC, where the preceding results were taken into the order.

Photo-induced intermolecular electron transfer from electron donating solvents to Coumarin dyes in bile salt aggregates: Role of diffusion in electron transfer reaction

The photo-induced electron transfer between Coumarin dyes and aromatic amines has been investigated using steady state and time-resolved fluorescence quenching studies. We have observed a Marcus type inversion in the electron transfer rate in correlation of quenching constant to the free energy change occurred during reaction. To justify the “inverted region” obtained in the correlation of quenching constant versus free energy curve, we have performed anisotropy measurement and estimated the several diffusional parameters. The translational diffusion coefficients exhibit a similar picture like electron transfer rate constant when it is plotted against free energy. Thus we argued that the diffusion has played an important role in the electron transfer kinetics.

Modulation with Acetonitrile of the Dynamics of Guest Binding to the Two Distinct Binding Sites of Cholate Aggregates

Bile salt aggregates are supramolecular systems containing two different binding sites. The effect of the addition of acetonitrile on the specificity and dynamics of guest binding to the two binding sites of cholate aggregates was studied. The protection of guests included in the aggregate from interaction with ions in the aqueous phase was evaluated from quenching of the singlet and triplet excited states of guest molecules bound to the cholate aggregates. The dynamics of guest binding to the primary and secondary binding sites of the cholate aggregates were determined at increasing acetonitrile mole fractions. The structure of the aggregates was not significantly altered provided the cholate concentrations were higher than 20 mM and the acetonitrile mole fraction did not exceed 0.033 (9.1% v/v). These results show that acetonitrile can be used to modulate the solubility of guests in the aggregates and to manipulate the residence time of guests in the primary and secondary binding sites.

Micellar Aggregation and Membrane Partitioning of Bile Salts, Fatty Acids, Sodium Dodecyl Sulfate, and Sugar-Conjugated Fatty Acids: Correlation with Hemolytic Potency and Implications for Drug Delivery

The co-administration of a drug with a penetration enhancer (PE) is one method by which the membrane permeability of a drug can be improved. To facilitate PE design, it is important that the molecular basis of PE toxicity and efficacy be examined, so we investigated the membrane affinity and micellar aggregation of a series of synthetic liposaccharide PEs and correlated these properties with hemolytic potency. The influence of liposaccharide alkyl chain length (nc) on the system was studied, and comparisons were made with conventional PEs such as bile salts, fatty acids, and surfactants. The liposaccharides were each synthesized in eight steps in good overall yield. Their critical micelle concentrations (CMCs) in phosphate-buffered saline ranged from 0.207 to 20.2 mM, and it was found that increasing nc by 2 afforded a 1 order of magnitude decrease in the CMC. Immobilized artificial membrane (IAM) chromatography was used to determine each PE's affinity for biological membranes, and an increase in nc caused a significant increase in the extent of membrane binding. A study of hemolytic activity revealed that liposaccharides with an nc of < or = 12 are the most likely to be biocompatible. The CMC values for all PEs showed a negative correlation with hemolytic potency; however, it was PE monomers, not micelles, that were responsible for the onset of hemolysis. The affinity of all enhancers for the IAM displayed a positive correlation with hemolytic potency, and therefore, IAM chromatography can be used to predict PE hemolytic activity. It was concluded that the biocompatibility of liposaccharides can be modulated by minor alterations in nc.

Characteristics of conjugate bile salt–phosphatidylcholine–cholesterol–water systems

Solubilization of l-α-phosphatidylcholine (PC) to micelles of conjugate bile salts, sodium glycocholate (NaGC), sodium taurocholates (NaTC), and sodium glycodeoxycholate (NaGDC), was studied at different bile salt concentrations by enzymatic method and by transmittance of the suspension system at 308.2 K. The solubility of PC increased in the order of NaTC<NaGC<NaGDC. An average molar ratio of PC/NaTC decreased with increasing bile salt concentration at different temperatures. Hydrodynamic diameters ( d h) of PC–saturated conjugate bile salt aggregates were measured by dynamic light scattering (DLS or QELS) as a function of the bile salt concentration at 308.2 K. Change of the d h values with increasing bile salt concentration indicated shape transition from vesicle ( d h: 150 nm) to micelle ( d h: 10 nm). The maximum additive concentrations of cholesterol into the aqueous micellar solutions of PC–conjugate bile salt mixtures were spectrophotometrically determined using enzymatic analysis. The cholesterol solubility increased in the order of NaTC<NaGC at the same concentration, and in addition, cholesterol can be more solubilized at higher molar ratio of PC/bile salt. An addition of cholesterol to the PC–bile salt systems was found to become a trigger that gave rise to generation of polydisperse distribution of larger aggregates or vesicle formation for PC–bile salt systems. The critical cholesterol concentration of vesicle formation was 1.2 mM for NaTC (47.5 mM)–PC (36.5 mM) system and 0.7 mM for NaTC (47.5 mM)–PC (23.8 mM) system.

Probing the binding dynamics to sodium cholate aggregates using naphthalene derivatives as guests.

The binding dynamics with bile salt aggregates for a series of naphthalene derivatives of different polarities was studied using fluorescence and laser flash photolysis. Fluorescence was employed to determine the nature of the binding site for each guest and the accessibility of the bound guest to quenchers. Laser flash photolysis was employed to study the mobility of the triplet states of the naphthalenes between the sodium cholate aggregates and the aqueous phase. Primary aggregates, which provide an environment protected from quenchers in the aqueous phase, bind 1- and 2-ethylnaphthalene as guests. The complexation dynamics with this type of aggregate is slow. 1- and 2-Naphthyl-1-ethanol, and 1- and 2-acetonaphthone bind to the secondary aggregates, which provide moderate protection from quenching and faster binding dynamics. The addition of salts lowered the cholate concentration at which primary aggregates were formed, but did not influence the formation of secondary aggregates.

Micelle formation of sodium glyco- and taurocholates and sodium glyco- and taurodeoxycholates and solubilization of cholesterol into their micelles

Micelle formation of sodium glyco- and taurocholates and sodium glyco- and taurodeoxycholates was studied at 308.2 K for the critical micelle concentration at various NaCl concentrations by pyrene fluorescence and scattered light intensity, and the degree of counterion binding to micelle was calculated using the Corrin–Harkins plots. The change of I 1/ I 3 values of the fluorescence spectrum with the conjugate bile salt concentration suggested two steps for the bile salt aggregation, which was supported by the scattered light intensity. The first step is a commencement of smaller aggregates, the first CMC, and the second one is a beginning of stable aggregates, the second CMC. Between the first and the second CMCs, the aggregates grow in size with the conjugate concentration. The aggregation number above the second CMC was determined at 308.2 K and 0.15 mol dm −3 NaCl concentration by the static light scattering: 8.7 and 6.0 for sodium glyco- and taurocholate, respectively, and 15.7 and 15.9 for sodium glyco- and taurodeoxycholate, respectively. The solubilization of cholesterol into the bile salt micelles in the presence of coexisting cholesterol phase or the maximum additive concentration (MAC) of cholesterol was determined against the bile salt concentration, and then, the standard Gibbs energy change for the solubilization was evaluated, where the micelles were regarded as a chemical species. The solubilization was stabilized in the order of sodium taurocholate<sodium glyco-cholate<sodiun taurodeoxycholate<sodium glycodeoxycholate.

Diffusivity Study of Dihydroxy−Trihydroxy Bile Salt Systems

Previously, structural units inferred from X-ray diffraction analysis of fibers, crystals, and concentrated aqueous solutions were identified as models of dihydroxy and trihydroxy bile salt micellar aggregates. Dimers andtheir multiples (mainly octamers and hexadecamers) or structures derived from 7/1 helices (formed by trimers) were proposed for trihydroxy salts sodium glycocholate (NaGC) and taurocholate (NaTC) or for dihydroxy salts sodium glycodeoxycholate (NaGDC) and taurodeoxycholate (NaTDC), respectively, on the basis of strong experimental evidences. However, the radii of the cylindrical structures derived from 7/1 helices were unknown. This paper deals with small-angle X-ray scattering (SAXS) measurements that have permitted to estimate the cylindrical aggregate radius of NaGDC (about 22 A), whereas that of NaTDC is very probably about 3 A longer. SAXS and quasi-elastic light-scattering (QELS) data have shown a linear increase of the apparent mean hydrodynamic radius (within the approximate range 40-70 A) with the height of NaGDC and NaTDC cylindrical aggregates, suggesting that the main interparticle correlation effects, ascribed to charge, van der Waals, and volume excluded interactions, are small or compensate each other. Moreover, aqueous solutions containing one dihydroxy and one trihydroxy bile salt (NaGDC and NaGC, NaTDC and NaTC, NaGDC and NaTC, NaTDC and NaGC) and 0.8 M NaCl have been studied. QELS measurements give information on the growing processes of NaGDC and NaTDC aggregates as a function of NaGC and NaTC concentration. Experimental data and calculations performed using the above-mentioned models support that a small amount of the bile salt forming smaller size aggregates (trihydroxy salt) inhibits the growth of the bile salt forming bigger size aggregates (dihydroxy salt), giving rise to smaller size aggregates. Reasonably, this phenomenon is due to the very different and not interchangeable structures of dihydroxy and trihydroxy bile salt aggregates. It seems also that the aggregate growth is a little more inhibited when the trihydroxy molecule polar head is different from that of the aggregate molecules. Trihydroxy salt monomers and oligomers compete with dihydroxy salt trimers in the growth process and interact with their cylindrical aggregates especially by means of polar forces.

Microenvironmental properties and chiral discrimination abilities of bile salt micelles by fluorescence probe technique.

The microenvironmental properties as well as the chiral discrimination abilities of four kinds of bile salt micelles were investigated by the fluorescence probe technique. A new fluorescence probe, 1,1'-bi-2-naphthol, was exploited to study the aggregation of bile salts, size, micropolarity, and microrigidity of the micelles with or without the presence of inorganic salts. Based on these results, the chiral discrimination abilities of bile salt micelles were further investigated by using (R)- and (S)-1,1'-bi-2-naphthol as chiral fluorescence probes. Different chiral discrimination ability was revealed by fluorescence spectra, fluorescence increase rate, and fluorescence quenching constants. The chiral discrimination mechanism of bile salt micelles was discussed.

Solvation Dynamics in Bile Salt Aggregates

The solvation dynamics inside an aggregate of a bile salt, sodium deoxycholate (NaDC), in aqueous solution has been studied using picosecond time-resolved emission spectroscopy and two fluorescent probes, 4-(dicyanomethylene)-2-methyl-(p-dimethylaminostyryl)-4H-pyran (DCM) and 2,6-p-toluidinonaphthalenesulfonate (TNS). Addition of NaDC to an aqueous solution causes a nearly 110-fold increase in the emission quantum yield (φf) of TNS. From the variation of φf of TNS, two critical micellar concentrations of NaDC have been detected at around 7 and 60 mM, respectively. The solvation dynamics of TNS in 100 mM NaDC solution is described by three components, 95 ps (17%), 380 ps (16%), and 1.9 ns (67%). The solvation dynamics of DCM bound to 100 mM NaDC is also found to be triexponential with components of 110 ps (19%), 700 ps (17%), and 2.75 ns (64%). The substantially slow solvation dynamics of water in the vicinity of NaDC indicates restricted solvation dynamics of water inside the bile salt aggregate.

Thermodynamic Characterization of Bile Salt Aggregation as a Function of Temperature and Ionic Strength Using Isothermal Titration Calorimetry

The critical micellar concentration (cmc) and the demicellization enthalpy ΔHdemic of the primary aggregates of sodium cholate (NaC) and sodium deoxycholate (NaDC) in water and 0.1 M NaCl at pH 7.5 were determined by isothermal titration calorimetry (ITC). The cmc of NaC and NaDC in water and 0.1 M NaCl at pH 7.5 shows a minimum between 295 and 300 K. With increasing ionic strength, the cmc of the bile salts decreases. ΔHdemic is strongly temperature-dependent but shows almost no dependence on the ionic strength. For comparison with other systems, the thermodynamic parameters ΔGdemic and ΔSdemic associated with the demicellization process were calculated using the pseudo-phase-separation model. From the temperature dependence of ΔHdemic, the change in heat capacity ΔCpdemic for the demicellization process was determined. The data obtained for ΔCpdemic are positive and at 298 K have values of 250 J·mol-1·K-1 for NaC and 350 J·mol-1·K-1 for NaDC. These values correspond to changes in the exposed hydrophobic...

On the role of hydronium ions in the protonated micellar aggregates of bile salts

In previous work, structural units observed in bile salt crystals and fibres have been successfully used to represent bile salt micellar aggregates in aqueous solutions and electromotive force measurements have shown that protonated micellar species are present below some critical values of pH. This paper deals with the crystal structures of 3α,12α-dihydroxy-5β-cholanoylglycine (HGDC), 3α,12α-dihydroxy-5β-cholanoyltaurine (HTDC) and 3α,7β-dihydroxy-5β-cholanoyltaurine (HTUDC), which were solved to obtain models of protonated micellar aggregates. The models are compared with those found in crystals and fibres of sodium and rubidium salts of HGDC and HTDC (NaGDC, NaTDC, RbGDC, RbTDC) in order to verify whether the acid structures match with the salt structures. The HGDC packing resembles that of a NaTDC crystal and is stabilized mainly by hydrogen bonds as well as by dipoledipole interactions between acetone molecules and carboxylic groups. Three different 31 helices are identified. One of these can be easily transformed into the 7/1 helix which satisfactorily describes the NaGDC, NaTDC, RbGDC and RbTDC micellar aggregates. The HTDC and HTUDC crystal structures are practically the same. Strong hydrogen bonds between H3O+ (hydronium ion) and three oxygen atoms of the anions show O· · ·O distances within the range 2.42.6 Å, owing to additional ionion and iondipole interactions. Very probably, H3O+ replaces Na+ in the micellar aggregates without remarkably changing their structure because the H3O+· · ·O and Na+· · ·O distances are very close. Inspection of previous electromotive force data indicates that the glycodeoxycholate and taurodeoxycholate micellar aggregates' proton affinities increase as their sizes increase and that those of the bigger aggregates seem to converge, even though the proton affinity of COO− is greater than that of SO3−. These findings strongly suggest that micellization induces H3O+ binding. HTDC and HTUDC form micellar aggregates which increase their apparent hydrodynamic radius by adding HCl.