Formação de complexos supramoleculares entre estruturas carreadoras e moléculas bioativas.

Bioactive compounds have the property of modulating several metabolic processes, but their bioactivity is dependent on their stability and bioavailability. Thus, the formation of supramolecular complexes between bioactive molecules and natural macromolecules, such as proteins and cyclodextrins, is an interesting strategy for the incorporation of bioactive substances in different matrices to maintain their bioactivity. For this, it is essential to characterize the interactions that promote and sustain the formation ofthese complexes, as well as to know their formation and dissociation kinetics. This work was divided into three scientific articles and aimed to characterize the intermolecular interactions of the following systems: micelle of casein-curcumin (MC-CUR), P-casein-quercetin (B-cas-Qet), P-cyclodextrinresveratrol/structural analog (B-CD-RES and B-CD-RESAnl).In the first article, the formation of the MC-CURcomplex, the carrying capacity of MC and its thermal protection for CUR at a pH of 6.6 was studied. It has been shown that an MC can carry around 18,000 molecules of CUR. The thermodynamic parameters indicated an enthalpically driven complex formation process (AHº = —64.63 kJ-mol! and TASº ranging from —42.45 to —44.46 kJ-mol"!). Theincrease in temperature reduced the rate of formation of the MC-CUR complex and increased its rate of dissociation. The activation energy for the formation of activated MC-CUR complexes was negative for the association of free MC and CUR molecules (-62.8 kJ-mol!) and positive for the dissociation of thermodynamically stable complexes (1.80 kJ:mol). MC protected CUR against its thermal degradation. In the second article, it was demonstrated that B-cas and Qct form thermodynamically stable supramolecular complexes in a process driven by an increase in the entropy ofthe system (AHº= 25.86 and TASº= 53.49 kJ-mol! at 25 ºC),via formation of an activated complex driven by an entropic reduction (TAS*a= -15.31 kJ-mol! and TAS*a= - 68.80 kJ-mol! at 25 ºC) and an enthalpic increase (AH*(a = 30.87 and AH*(q = 5.0 kJ-mol! at 25 “C). The addition of both salts, KCl or KSCN,increased the stability of the complex by decreasing the values of the kinetic and thermodynamic enthalpies. In the third article, the interaction kinetics between modified B-CD (B-CD-NH;,) and RES, as well as its structural analog (RESAnl) was determined. The inclusion of RESAnl, as well as its dissociation from B-CD-NH,, were faster than the same processes that occurred with RES as a guest molecule (kBTCD=NH2/RESANI340,101 Mis, KBTCD=NHaIRES4 97410) Mig; kbP“CD-NH2/RESANI039 gl, kBCD=NHa/RES-0,30 sl, at 25 ºC). The structural differences between the polyphenols (RES and RESAnl) affected the energetic parameters of formation of the activated complex more via the association of free molecules than via dissociation from the E$TCD-NHa/RES =1481 k-mol'! EBTCD- 2/RESAn1 thermodynamically stable complexes (Eqc+a acta 15.01 to 82.35 kJmol!), (EÉZO-NH/RES 5.19 kJmol! and EÉSONHRESA 6,29 kJ-mol!). These differences are mainly due to the desolvation process of the interacting molecules. It is concluded that the thermodynamic and kinetic parameters obtained for each of the studied systems, as well as the results of the complementary analyzes, are important foundations for optimizing the application of these bioactive molecular complexes in different food or pharmaceutical systems. Keywords: Bioactive compounds. Supramolecular complexes. Caseins. B-cyclodextrin. Curcumin. Quercetin. Resveratrol. Thermodynamics. Kinetics.Os compostos bioativos apresentam a propriedade de modular diversos processos metabólicos, mas sua bioatividade é dependente de suas estabilidade e biodisponibilidade. Dessa forma, a formação de complexos supramoleculares entre moléculas bioativas e macromoléculas naturais, como proteínas e ciclodextrinas, é uma estratégia interessante para a incorporação dos bioativos em diferentes matrizes de forma a manter sua bioatividade. Para isso, é fundamental que se caracterize as interações que promovem e sustentam a formação desses complexos, assim como se conheça sua cinética de formação e dissociação. Este trabalho foi dividido em três artigos científicos e objetivou caracterizar as interações intermoleculares dos seguintes sistemas: micela de caseína-curcumina (MC-CUR), -caseíina-quercetina (P-cas-Qet), Pciclodextrina-resveratrol/análogo estrutural (B-CD-RES e B-CD-RESAnl). No primeiro artigo, estudou-se a formação do complexo MC-CUR, a capacidade carreadora da MC e sua proteção térmica para CUR em um pH de 6,6. Mostrou-se que uma MC pode transportar cerca de 18.000 moléculas de CUR. Os parâmetros termodinâmicos indicaram um processo de formação de complexo entalpicamente dirigido (AHº = —64,63 kJ/mole TASº variando de —42,45 a —44,46 kJ/mol) e o aumento da temperatura reduziu a taxa de formação do complexo MC-CUR e aumentou sua taxa de dissociação. A energia de ativação para a formação dos complexos ativados MC-CURfoi negativa para a etapa de associação de moléculas livres de MC e CUR (62,8 kJ/mol) e positiva para a dissociação dos complexos termodinamicamente estáveis (1,80 kJ/mol). A MC protegeu a CUR contra sua degradação térmica. No segundo artigo, foi demonstrado que B-cas e Qct formam complexos supramoleculares termodinamicamente estáveis em um processo dirigido por um aumento na entropia do sistema (AHº = 25,86 e TASº = 53,49 kJ/mol a 25 ºC), via formação de um complexo ativado dirigido por uma redução entrópica (TAS*a= -15,31 kJ/mol e TAS*g= -68,80 kJ/mol a 25 ºC) e um ganho entálpico (AH*(a = 30,87 e AH*(a) = 5,0 kJ/mol a 25 ºC). A adição de ambos os sais, KCl ou KSCN, aumentou a estabilidade do complexo por diminuição dos valores das entalpias cinética e termodinâmica. No terceiro artigo, a cinética de interação entre B-CD modificada (B-CD-NH,) e RES, bem como seu análogo estrutural (RESAnl) foi determinada. A inclusão de RESAnl, bem como sua dissociação da B-CD-NH,, foram mais rápidos que os mesmos processos que ocorreram tendo RES como molécula hóspede (kÉT 2/RES 310x10! Mis, KÉTCD-NH2/RES487 0)MiIsl, KÉO 285 g3gsi kiOP 2803051225 ºC). As diferenças estruturais entre os polifenóis (RES e RESAnl) afetaram mais os parâmetros energéticos de formação do complexo ativado entre estes e a B-CD-NH, via associação das moléculas livres que via dissociação dos complexos termodinamicamente estáveis (EÊRCD= act,a 2/RES 1481 KkJmol, EBC act,a 2/RESAN 1501 a 8235 kJmol, Ela 2/RES —5,19 kJ/mol e Elia 2/RES 6,29 kJ/mol). Essas diferenças são devidas, principalmente, ao processo de dessolvatação das moléculas que interagiram. Concluise que os parâmetros termodinâmicose cinéticos obtidos para cada um dos sistemas estudados, bem como os resultados das análises complementares, são importantes fundamentos para otimização da aplicação desses complexos moleculares bioativos em diferentes sistemas alimentícios ou farmacêuticos. Palavras-chave: Compostos bioativos. complexo supramoleculares. Caseínas. pciclodextrina. Curcumina. Quercetina. Resveratrol. Termodinâmica. Cinética.CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológic

Bovine serum albumin as nanocarrier of curcumin

A curcumina exibe diversas atividades biológicas, mas suas características de baixa solubilidade e pouca estabilidade termodinâmica limitam sua biodisponibilidade. Portanto, é indispensável estudar sua interação com a albumina do soro bovino (BSA), que é conhecida por aumentar a solubilidade e estabilidade de compostos hidrofóbicos em diversas formulações. Neste trabalho estudou-se a interação entre a curcumina e a BSA em suas conformações nativa e desnaturada, em pH 7,0, por meio de diferentes técnicas analíticas, e avaliou- se a cinética de fotodegradação da curcumina na presença da BSA. Pela análise de espectroscopia de fluorescência (EF) e de calorimetria de titulação isotérmica (ITC) os valores da constante de ligação (K a ) entre a proteína e o composto bioativo obtidos foram na ordem de 10 5 L.mol -1 , enquanto que por ressonância plasmônica de superfície (RPS) esse valor foi 3,88x10 3 L.mol -1 . Essas diferenças são explicadas pelas limitações apresentadas por cada técnica. O experimento de competição realizado indicou que a curcumina compete com os marcadores utilizados pelos sítios I, II e III da BSA. Os valores obtidos para ΔH° por EF (-8,67 kJ.mol -1 ) e por ITC (-29,11 kJ.mol -1 ) indicaram que o processo de formação dos complexos foi exotérmico. Nos estudos com a BSA desnaturada houve um ligeiro aumento nos valores de K a (4,53x10 5 L.mol -1 e 2,00x10 6 L.mol -1 , obtidos por EF e ITC, respectivamente, a 25 °C), comparados aos valores encontrados com a BSA nativa, o que indicou que há outros locais na BSA para ligação da curcumina além dos mostrados no experimento de competição. O perfil termodinâmico foi semelhante ao obtido para a conformação nativa, mas ΔH° foi mais negativo (- 16,12 kJ.mol -1 e -42,63 kJ.mol -1 , por EF e ITC, respectivamente), indicando que a mudança de conformação da BSA proporcionou uma interação mais exotérmica. No experimento de fotodegradação, à medida que a concentração de BSA aumentou, os valores da constante de degradação (K d ) da curcumina diminuíram exponencialmente e seu tempo de meia-vida (t 1/2 ) aumentou linearmente, o que indicou que a BSA protege a curcumina da fotodegradação. Esse trabalho contribui para um conhecimento mais aprofundado da interação intermolecular entre curcumina e BSA por trazer resultados obtidos por técnicas com diferentes fundamentos e parâmetros cinéticos da degradação da curcumina na presença de diferentes concentrações de BSA, o que ainda era ausente na literatura, fornecendo informações importantes para o sucesso da aplicação tecnológica da curcumina em diferentes formulações.Curcumin exhibits various biological activities, but its low solubility and stability characteristics limit its bioavailability. Therefore, it is indispensable to study its interaction with bovine serum albumin (BSA), which can increase the solubility and stability of several hydrophobic compounds in various formulations. Here, we studied the interaction between curcumin and BSA in its native and denatured conformations, at pH 7.0, was studied through different analytical techniques, and the kinetics of curcumin photodegradation was evaluated in the presence of BSA. By the analysis of fluorescence spectroscopy (FS) and isothermal titration calorimetry (ITC) the values of the binding constant (K a ) between the protein and the bioactive compound obtained were in the order of 10 5 L.mol -1 , whereas by surface plasmonic resonance (SPR), this value was 3.88x10 3 L.mol -1 . These differences are explained by the limitations presented by each technique. The competition experiment performed indicated that curcumin competes with the markers used by sites I, II and III of the BSA. The values obtained for ΔH ° by FS (-8.67 kJ.mol -1 ) and by ITC (-29.11 kJ.mol -1 ) indicated that the process of forming the complexes was exothermic. In the studies with denatured BSA there was a slight increase in K a values (4.53x10 5 L.mol -1 and 2.00x10 6 L.mol -1 , obtained by FS and ITC, respectively, at 298 K), compared to the values found with the native BSA, which indicated that there are other BSA sites for curcumin binding in addition to those shown in the competition experiment. The thermodynamic profile was like that obtained for the native conformation, but ΔH ° was more negative (- 16.12 kJ.mol -1 and -42.63 kJ.mol -1 , by FS and ITC, respectively), indicating that the change in conformation of the BSA provided a more exothermic interaction. In the photodegradation experiment, as the BSA concentration increased, the values of the curcumin degradative constant (K d ) decreased exponentially and its half-life (t 1/2 ) increased linearly, which indicated that BSA protected the curcumin from photodegradation. This work contributes to a more deepened knowledge of the intermolecular interaction between curcumin and BSA by bringing results obtained by techniques with different fundamentals and kinetic parameters of curcumin degradation in the presence of different BSA concentrations, which was still absent in the literature, providing important information to the success of the technological application of curcumin in different formulations.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Lactoferrin denaturation induced by anionic surfactants : the role of the ferric ion in the protein stabilization.

Here, investigation was made of the interaction between lactoferrin (Lf) and the anionic surfactants sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), and sodium decyl sulfate (DSS), using isothermal titration calorimetry, Nano differential scanning calorimetry (NanoDSC), and fluorescence spectroscopy. The Lf-surfactant interaction was enthalpically favorable (the integral enthalpy change ranged from ?5.99?kJ?mol?1, for SDS at pH?3.0, to ?0.61?kJ?mol?1, for DSS at pH?12.0) and promoted denaturation of the protein. The Lf denaturation efficiency followed the order DSS?<?SDS?<?SDBS. The adsorption capacity of the protein with respect to surfactant strongly depended on pH and the surfactant structure, reaching a maximum value of 505 SDBS molecules per gram of Lf at pH?3.0. The different efficiencies of the surfactants in denaturing Lf were attributed to the balance of hydrophobic and electrostatic interactions, which also depended on pH and the surfactant structure, highlighting the SDBS-tryptophan residue specific interaction, where SDBS acted as a quencher of fluorescence. Interestingly, the NanoDSC and fluorescence measurements showed that the ferric ion bound to Lf increased its stability against denaturation induced by the surfactants. The results have important implications for understanding the influence of surfactants on structural changes in metalloproteins

Solvophobic effect of 1-alkyl-3-methylimidazolium chloride on the thermodynamic of complexation between ?-cyclodextrin and dodecylpyridinium cation.

Preferential solvation participate in various supramolecular self-assembly processes, whose thermodynamic properties can be modulated by the addition of ionic liquids (ILs). However, the effects of these liquids on the thermodynamics of the host-guest complexation process remain unexplored. In this study, the thermodynamic properties of the complexation between 1-dodecylpyridinium cations (C12Py+) and ?-cyclodextrin (?CD) species in aqueous solutions with different concentrations of 1-alkyl-3-methylimidazolium halides (CnmimX) were investigated by isothermal titration calorimetry. In water, C12Py+ and ?CD form a 1:1 inclusion complex, which is enthalpically ( ?9.2 ? 0.1?kJ mol?1) and entropically ( 16.1 ? 0.2?kJ mol?1) favorable. However, in IL aqueous solutions, all the ?CD?C12Py+ thermodynamic parameters of the complexation change and this IL effect is dependent on the carbon chain length of Cnmim+ cations. ILs with shorter alkyl chains (Cnmim+, n ? 4) decreases the system entropy, while ILs with longer alkyl chains (Cnmim+, n ? 6) reduce the enthalpy values. These effects are attributed to i) preferential solvation of surfactant tails by ILs; ii) ability of the ILs to modify the 3D water structure and iii) inclusion of IL molecules into the inner cavities of ?CD

Aggregation of sodium dodecylbenzene sulfonate : weak molecular interactions modulated by imidazolium cation of short alkyl chain length.

Ionic liquids (ILs) can modify cooperative process in aqueous solutions to a large extent, including anionic surfactant aggregation. Here, the micellization of sodium dodecylbenzene sulfonate (SDBS) was evaluated in low concentrations of 1-alkyl-3-methylimidazolium chloride (CnmimCl, n = 0, 2, and 4) aqueous solutions through fluorescence spectroscopy, isothermal titration calorimetry, dynamic light scattering, and conductometry. The thermodynamic stability of SDBS aggregates strongly depended on the IL structure and concentration, following the order C4mim+ > C0mim+ ? C2mim+. At 1.0 mmol L?1 of the ILs, the increase of the hydrophobicity of the imidazolium cation decreased the enthalpic favorableness, changing from ?3.75 ? 0.07 kJ mol?1, for C0mim+, to ?2.69 ? 0.01 kJ mol?1, for C4mim+. On the other hand, the entropic feasibility showed an opposite trend, i.e., the higher hydrophobicity of C4mim+ overcame the kosmotropic effect of IL cations in the bulks. We suggested that the imidazolium cations interact with the SDBS monomers on the micellar surface, mainly through hydrophobic, ?-?, and electrostatic interactions for C4mim+ and C2mim+, and through electrostatic interactions and hydrogen bonds for C0mim+

Thermodynamic and kinetic analyses of curcumin and bovine serum albumin binding

Bovine serum albumin (BSA)/curcumin binding and dye photodegradation stability were evaluated. BSA/curcumin complex showed 1:1 stoichiometry, but the thermodynamic binding parameters depended on the technique used and BSA conformation. The binding constant was of the order of 105 L·mol−1 by fluorescence and microcalorimetric, and 103 and 104 L·mol−1 by surface plasmon resonance (steady-state equilibrium and kinetic experiments, respectively). For native BSA/curcumin, fluorescence indicated an enthalpic and entropic driven process based on the standard enthalpy change (ΔH○F = −8.67 kJ·mol−1), while microcalorimetry showed an entropic driven binding process (ΔH○cal = 29.11 kJ·mol−1). For the unfolded BSA/curcumin complex, it was found thatp ΔH○F = −16.12 kJ·mol−1 and ΔH○cal = −42.63 kJ·mol−1. BSA (mainly native) increased the curcumin photodegradation stability. This work proved the importance of using different techniques to characterize the protein-ligand binding

β-Carotene and milk protein complexation: a thermodynamic approach and a photo stabilization study

The demand for bioactive molecules, such as β-carotene (β-car), has increased, but some characteristics such as low water solubility and low photo stability limit its application in many formulations. The bioactive entrapment into milk proteins may overcome this barrier. Thus, the aim of this work was to study the interaction between β-car and bovine serum albumin (BSA) or β-casein and the photo stability of this bioactive in the presence of the proteins. Fluorescence spectroscopy showed that at pH 7.0, increasing concentrations of β-carotene reduced the fluorescence intensity of both proteins, and the fluorescence-quenching mechanism is mainly static. The stoichiometry of the β-car/protein complex varied between proteins, being 1:1 to native BSA, 1:3 to denatured BSA (d-BSA), and 1:2 for β-casein. The standard Gibbs-free energy (ΔG°) of complex formation was negative for all systems studied and followed the order ΔG°BSA < ΔG°β-casein < ΔG°d-BSA. The formation of β-car/protein complex was driven by entropy increasing in all studied conditions. Both proteins improved β-car photo stability, but β-casein micelle was more efficient, reducing and increasing four times, respectively, the bioactive degradation constant and the half-time of β-car. The overall results pointed to the strategic use of milk proteins, especially β-casein micelles as nanovehicle for β-car in food and other systems

Binding thermodynamics of synthetic dye Allura Red with bovine serum albumin

The interaction between Allura Red and bovine serum albumin (BSA) was studied in vitro at pH 7.4. The fluorescence quenching was classified as static quenching due to the formation of AR–BSA complex, with binding constant (K) ranging from 3.26 ± 0.09 to 8.08 ± 0.06 104 L.mol−1, at the warfarin binding site of BSA. This complex formation was driven by increasing entropy. Isothermal titration calorimetric measurements also showed an enthalpic contribution. The Allura Red diffusion coefficient determined by the Taylor-Aris technique corroborated these results because it reduced with increasing BSA concentration. Interfacial tension measurements showed that the AR–BSA complex presented surface activity, since interfacial tension of the water-air interface decreased as the colorant concentration increased. This technique also provided a complexation stoichiometry similar to those obtained by fluorimetric experiments. This work contributes to the knowledge of interactions between BSA and azo colorants under physiological conditions