Calorimetric and spectroscopic studies on the interaction of anticancer drug mitoxantrone with human serum albumin

Binding of the anticancer drug mitoxantrone with the protein human serum albumin (HSA) has been studied by using isothermal titration calorimetry (ITC), in combination with fluorescence, UV–visible, and circular dichroism spectroscopy. The thermodynamic parameters of binding have been evaluated from ITC and spectroscopic results and compared. The ITC results demonstrate that the binding of mitoxantrone with HSA occurs according to two sets of binding sites on the protein as opposed to the fluorescence and UV–visible spectroscopic results. Blockage of one binding site on HSA for mitoxantrone in the presence of NaCl indicates strong involvement of electrostatic interactions in the binding of the drug with the protein. An insignificant temperature dependence of the association constant observed in fluorescence measurements suggests a very low enthalpy of binding which is in close agreement with the results obtained from ITC measurements. Fluorescence life time measurements suggest formation of a static complex between mitoxantrone and HSA. The discrepancies in the ITC and fluorescence results suggest that one of the binding sites on the protein for mitoxantrone does not contain tryptophan residue in its immediate vicinity. The calorimetric and spectroscopic results have provided quantitative information on the binding of mitoxantrone with HSA and suggest that the binding is dominated by electrostatic interactions.     

Biophysical analysis of partially folded state of α-lactalbumin in the presence of cationic and anionic surfactants

The role of different types of interactions and their contribution in the stabilization of bovine α-lactalbumin (α-LA) molten globule in presence of cationic surfactant, hexadecyl trimethyl ammonium bromide (HTAB) and anionic surfactant, sodium dodecyl sulphate (SDS) have been examined using a combination of spectroscopic, light scattering and calorimetric techniques. The results correlated well with each other and were used to characterize the partially folded states of the protein both qualitatively and quantitatively. At lower concentration of the surfactants, the thermodynamic parameters obtained from UV-visible spectroscopy suggested an increased exposure of non-polar groups in HTAB while a possible restructuring of non-polar groups were indicated in SDS. The fluorescence and circular dichroism spectroscopy showed the formation of an intermediate state at various concentrations of HTAB and SDS while the lifetime measurements supported the assumption of protein-surfactant complex stability in HTAB as compared to SDS. The hydrodynamic diameter and the ζ-potential were analyzed by dynamic light scattering (DLS) which also implicated the combined influence of electrostatic and hydrophobic interactions in protein unfolding in HTAB and only hydrophobic interactions in SDS. The binding parameters for ANS obtained from isothermal titration calorimetric (ITC) measurements suggested a high stability of α-LA molten globule and the role of enthalpic and entropic contribution in the binding of ANS in HTAB. It also indicated the fragility of α-LA molten globule in SDS. The possible binding sites as well as the interactions of ANS with the partially folded protein were also studied from the thermodynamic parameters obtained from the ITC.     

Energetics of binding and protein unfolding upon amphiphilic drug complexation with a globular protein in different aqueous media

The interactions and complexation process of the structurally related amphiphilic phenothiazines promazine and triflupromazine hydrochlorides with horse myoglobin in aqueous buffered solutions of pH 2.5, 5.5 and 9.0 have been examined by zeta-potential, isothermal titration calorimetry (ITC), UV-vis spectroscopy and dynamic light-scattering techniques with the aim of analyzing the effect of hydrophobic and electrostatic forces, the alteration of protein conformation and the effect of substituents in the drug molecular structure on the binding mechanism and structure of the resulting complexes. The energetics and stoichiometry of the binding process was derived from ITC. The enthalpies of binding obtained are small and exothermic, and the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Binding isotherms were obtained from microcalorimetric data by using a theoretical model based on the Langmuir isotherm. zeta-Potential data showed a reversal in the sign of the protein charge at pH 9.0 as a consequence of drug binding. Gibbs energies of drug binding per mole of drug were also derived from zeta-potential data. On the other hand, binding of the phenothiazines causes a conformational transition on protein structure which was followed as a function of drug concentration by using UV-vis spectroscopy. These data were analyzed to obtain the Gibbs energy of the transition in water (DeltaG(w)(degrees)) and in a hydrophobic environment (DeltaG(hc)(degrees)). Finally, the population distribution of the different species in solution and their size was analyzed through dynamic light scattering. The existence of an aggregation process of drug/protein complexes, mainly at pH 2.5, was observed. We think this is a consequence of the already expanded structure of the protein at this pH and the subsequent binding of drug molecules to the protein.     

Binding of naproxen and amitriptyline to bovine serum albumin: biophysical aspects

Binding of the drugs naproxen (which is an anti-inflammatory) and amitriptyline (which is an anti-depressant) to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC), in combination with fluorescence and circular dichroism spectroscopies. Naproxen is observed to bind more strongly to BSA than amitriptyline. The temperature-dependent ITC results indicate the interaction of one molecule of naproxen with more than one protein molecule. On the other hand, amitriptyline binds to BSA with a reaction stoichiometry that varies from 1:1.2 to 1:2.9. The van't Hoff enthalpy, which is calculated from the temperature dependence of the binding constant, agrees well with the calorimetric enthalpy in the case of naproxen binding to BSA, indicating adherence to a two-state binding process. However, their disagreement in the case of amitriptyline indicates conformational changes in the protein upon ligand binding, as well as with the rise in temperature. The spectroscopic results did not suggest appreciable conformational changes as a result of binding; hence, the discrepancy could be attributed to the temperature-induced conformational changes. With increases in the ionic strength, a reduction in the binding affinity of naproxen to BSA is observed. This suggests the prevailing electrostatic interactions in the complexation process. The preponderance of the hydrophobic interactions in the binding of amitriptyline to BSA is indicated by the absence of any dependence of the ionic strength. A predominance of electrostatic interactions in the case of naproxen binding to BSA and that of hydrophobic interactions in the case of amitriptyline binding to BSA is further strengthened by the results of the binding experiments performed in the presence of ionic and nonionic surfactants. The binding parameters indicate that Triton X-100 blocks the hydrophobic binding sites on BSA, thereby altering the binding affinity of amitriptyline toward BSA. A partial overlap of the binding sites for these drugs is indicated by the binding parameters obtained in the titration of naproxen to the amitriptyline-BSA complex and vice versa. Thus, the results provide a quantitative understanding of the binding of naproxen and amitriptyline to BSA, which is important in understanding their effect as therapeutic agents individually and in combination therapy.     

Studies on the Antagonistic Behavior Between Cyclophosphamide Hydrochloride and Aspirin with Human Serum Albumin: Time-Resolved Fluorescence Spectroscopy and Isothermal Titration Calorimetry

The interactions between cyclophosphamide hydrochloride (CYC) and aspirin (ASA) with human serum albumin (HSA) were investigated by measuring fluorescence anisotropy, poly-dispersity index, and time-resolved fluorescence. Also, isothermal titration calorimetry (ITC) was performed. The fluorescence spectra of the drugs exhibited an appreciable hypsochromic shift along with an enhancement in the fluorescence intensity. The gradual addition of HSA led to a marked increase in fluorescence anisotropy (r), and from this value it is argued that the drugs were located in a restricted environment of the protein. The binding constants for the ASA–HSA and CYC–HSA complexes were found to be 1.27 × 10⁸ and 4.23 × 10⁸ mol·L^?1, respectively, as calculated from the relevant fluorescence data. The polydispersity index and size distribution of the protein–drug complex were measured at several concentrations of the drugs by the zeta potential technique, which confirmed the already obtained experimental results. From the analysis of the steady-state and time-resolved fluorescence quenching of the drugs in aqueous solutions in the presence of HSA, it was found that the quenching is static in nature. ITC experiments revealed that, in the absence of drugs, the dominant forces are electrostatic, whereas hydrophobic and weak electrostatic forces became significant in the presence of the drug. The primary binding pattern between the drugs and HSA was interpreted as a combined effect of hydrophobic association and electrostatic interactions.     

Temperature-dependent simultaneous ligand binding in human serum albumin

Human serum albumin (HSA) is a soluble protein in our circulatory system, which is known to bind a variety of drugs and ligands. Since Sudlow's pioneering works on the ligand-binding sites, a major effort of the biophysical/biochemical research has been directed to characterize the structural, functional, and dynamical properties of this protein. Structural studies on HSA have revealed distinct temperature-induced folded states. Despite knowing about the ligand-binding properties and residues important for the binding, less is understood about the temperature-dependent molecular recognition of the protein. Here, we have prepared thermally induced unfolded states of the protein and characterized those by circular dichroism (CD) and differential thermal analysis (DTA) techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering (DLS) measurements. We have used two fluorescent ligands (4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran) (DCM; hydrophobic; neutral) and Nile blue (NB; cationic) of different natures to characterize the ligand-binding properties of the protein in the native and thermally unfolded states. The possible binding sites of the ligands have been characterized by competitive binding with other drug molecules having definite binding sites in HSA. Picosecond-resolved F?rster resonance energy transfer (FRET) studies along with steady-state and polarization-gated spectroscopies on the ligands in the protein reveal the dynamics of the binding sites at various temperatures. From the FRET studies, an attempt has been made to characterize the simultaneous binding of the two ligands in various temperature-dependent folded states of HSA.     

Thermodynamic studies on the interaction of folic acid with bovine serum albumin

Binding of the vitamin folic acid with bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, in the presence of nonionic surfactants triton X-100, tetrabutylammonium bromide, and sucrose. The values of the van’t Hoff enthalpy calculated from the temperature dependence of the binding constant agree with the calorimetric enthalpies indicating that the binding of folic acid to the BSA is a two state process without involving intermediates. These observations are supported by the intrinsic fluorescence and circular dichroism spectroscopic measurements. With increase in the ionic strength, reduction in the binding affinity of folic acid to BSA is observed suggesting predominance of electrostatic interactions in the binding. The contribution of hydrophobic interactions in the binding is also demonstrated by decrease in the binding affinity in the presence of tetrabutylammonium bromide (TBAB). The value of binding affinity in the presence of sucrose indicates that hydrogen bonding also plays a significant contribution in the complexation process. The calorimetric and spectroscopic results provide quantitative information on the binding of folic acid to BSA and suggest that the binding is dominated by electrostatic interactions with contribution from hydrogen bonding.     

Physicochemical characteristics of protein-NP bioconjugates: the role of particle curvature and solution conditions on human serum albumin conformation and fibrillogenesis inhibition

Gold nanoparticles (Au NPs) from 5 to 100 nm in size synthesized with HAuCl(4) and sodium citrate were complexed with the plasma protein human serum albumin (HSA). Size, surface charge, and surface plasmon bands of the Au NPs are largely modified by the formation of a protein corona via electrostatic interactions and hydrogen bonding as revealed by thermodynamic data. Negative values of the entropy of binding suggested a restriction in the biomolecule mobility upon adsorption. The structure of the adsorbed protein molecules is slightly affected by the interaction with the metal surface, but this effect is enhanced as the NP curvature decreases. Also, it is observed that the protein molecules adsorbed onto the NP surface are more resistant to complete thermal denaturation than free protein ones as deduced from the increases in the melting temperature of the adsorbed protein. Differences in the conformations of the adsorbed protein molecules onto small (<40 nm) and large NPs were observed on the basis of ζ-potential data and FTIR spectroscopy, also suggesting a better resistance of adsorbed protein molecules to thermal denaturing conditions. We think this enhanced protein stability is responsible for a reduced formation of HSA amyloid-like fibrils in the presence of small Au NPs under HSA fibrillation conditions.     

人血白蛋白结合位点II的分子结合模式研究

人血白蛋白是血浆中含量最高的蛋白,是一种重要的载体蛋白,能与多种内源性和外源性物质结合。人血白蛋白主要有两个药物结合位点,位点I和位点II,其中位点II的柔性较大,对药物分子的亲和性较高。本文采用分子模拟方法,基于12种结合于位点II的小分子-人血白蛋白晶体结构,分析了相互作用能,发现12种分子的结合以疏水作用为主,静电作用为辅。进一步采用丙氨酸扫描和结合能评价,分析结合部位的关键氨基酸残基,探究结合模式的规律性,发现从位点入口到空腔内部存在静电、疏水和杂合的三层相互作用分布,共同形成了稳定的分子结合。最后采用分子对接和分子动力学模拟,预测了L-色氨酸的结合模式。研究结果有助于深入了解人血白蛋白药物位点II的小分子结合模式,为基于位点II的药物和分离配基的优化设计提供指导。     

Interaction of Globular Plasma Proteins with Water‐Soluble CdSe Quantum Dots

The interactions between water‐soluble semiconductor quantum dots [hydrophilic 3‐mercaptopropionic acid (MPA)‐coated CdSe] and three globular plasma proteins, namely, bovine serum albumin (BSA), β‐lactoglobulin (β‐Lg) and human serum albumin (HSA), are investigated. Acidic residues of protein molecules form electrostatic interactions with these quantum dots (QDs). To determine the stoichiometry of proteins bound to QDs, we used dynamic light scattering (DLS) and zeta potential techniques. Fluorescence resonance energy transfer (FRET) experiments revealed energy transfer from tryptophan residues in the proteins to the QD particles. Quenching of the intrinsic fluorescence of protein molecules was noticed during this binding process (hierarchy HSA<β‐Lg <BSA, lower binding affinity for hydrophobic protein molecules). Upon binding with QD particles, the protein molecules underwent substantial conformational changes at the secondary‐structure level (50 % helicity lost), due to loss in hydration.     

Binding of chitosan to phospholipid vesicles studied with isothermal titration calorimetry

We thermodynamically characterize the interaction of chitosan with small liposomes and the binding and organization of the polysaccharide on the membrane of the vesicles. By means of isothermal titration calorimetry (ITC), we obtain the enthalpy variations arising from binding of the positively ionized chitosan to neutral and negatively charged liposomes. The strong electrostatic interaction of the polysaccharide with the negative charges at the membrane gives rise to highly exothermic signal until charge compensation is reached. The equilibrium constant, the interaction stoichiometry, and the molar enthalpy of binding chitosan monomers to phospholipids from the external leaflet of the vesicle membrane are obtained from the isotherm curve fitting assuming independent binding sites. The strong exothermic signal indicates that the electrostatically driven binding of chitosan to the membrane is energetically favored, leading to further stabilization of the vesicle suspension. The higher the net negative charge of the vesicles, the more pronounced the adsorption of chitosan is, leading to weaker chain organization of the adsorbed chitosan at the membrane. At the point of charge saturation, vesicle aggregation takes place and we show that this behavior does not always lead to charge reversal at the membrane. Models for the binding behavior and structural organization of chitosan are proposed based on the experimental results from ITC, ζ-potential, and dynamic light scattering.     

Detergent binding as a sensor of hydrophobicity and polar interactions in the binding cavities of proteins

To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein-ligand binding, we studied the interaction of human serum albumin (HSA) and beta-lactoglobulin with various aliphatic (C10-C14) cationic and zwitterionic detergents. We find that cationic detergents, at levels that do not cause unfolding, interact with a single site on beta-lactoglobulin and with two primary and five to six secondary sites on HSA with an affinity that is approximately the same as that with which zwitterionic (dimethylamineoxide) detergents interact, suggesting the absence of significant electrostatic interactions in the high-affinity binding of these compounds. The binding affinity for all of the groups of compounds was dependent upon hydrocarbon chain length, suggesting the predominant role of hydrophobic forces, supported by polar interactions at the protein surface. A distinct correlation between the binding energy and the propensity for micelle formation within the group of cationic or noncharged (nonionic and zwitterionic) detergents indicated that the critical micellar concentration (CMC) for each of these detergent groups, rather than the absolute length of the hydrocarbon chain, can be used to compare their hydrophobicities during their interaction with protein. Intrinsic fluorescence data suggest that the two primary binding sites on serum albumin for the zwitterionic and cationic compounds are located in the C-terminal part of the albumin molecule, possibly in the Sudlow II binding region. Comparisons with previous binding data on anionic amphiphiles emphasize the important contribution of ion bond formation and other polar interactions in the binding of fatty acids and dodecyl sulfate (SDS) by HSA but not by beta-lactoglobulin. Electrostatic interactions by cationic detergents played a significant role in destabilizing the protein structure at high binding levels, with beta-lactoglobulin being more susceptible to unfolding than HSA. Zwitterionic detergents, in contrast to the cationic detergents, had no tendency to unfold the proteins at high concentrations.     

Self-assembled micelles based on hydrophobically modified quaternized cellulose for drug delivery

Novel amphiphilic cationic cellulose (HMQC) derivatives carrying long chain alkyl groups as hydrophobic moieties and quaternary ammonium groups as hydrophilic moieties were synthesized. Structure and properties of the amphiphilic cellulose derivatives were characterized by elemental analysis, FT-IR, (1)H NMR, ζ-potential measurement, dynamic light scattering (DLS), fluorescence spectroscopy and transmission electron microscopy (TEM). The results revealed that HMQCs can be self-assembled into cationic micelles in distilled water with the average hydrodynamic radius of 320-430 nm. The cytotoxicity study showed that the HMQC exhibited low cytotoxicity. Prednisone acetate, a water insoluble anti-inflammation drug, was chosen as a model drug to investigate the utilization of self-assembled HMQC micelles as a delivery carrier for poorly water-soluble drugs. The study indicated that the prednisone acetate could be incorporated effectively in the self-assembled HMQC micelles and be controlled released.     

Insulin complexes with PEGylated basic oligopeptides

Biodegradable oligolysine and oligoarginine-type homopeptides functionalized with PEG of two different molecular weights interact with insulin, at physiological pH, affording complexes studied by dynamic light scattering, ζ-potential, circular dichroism, FTIR spectroscopy, and isothermal titration calorimetry (ITC). High levels of insulin complexation efficiencies (>99.5%) were determined for all derivatives. FTIR spectra suggest that the positively charged homo-oligopeptide derivatives interact with B chain C-terminus of insulin leading to the formation of nanoparticles than can be traced even at low oligopeptide/insulin molar ratios. The ITC profiles are complex, displaying significant endothermic and exothermic contributions. Oligoarginine-type derivatives exhibit the strongest interactions, while PEGylation of either oligopeptide with the high molecular weight chains significantly affects the ITC profiles and leads to larger enthalpy changes. This may be attributed to PEG-induced aggregation of insulin due to the depletion attraction effect leading to the formation of stable nanocomplexes. Stabilization of complexed insulin against enzymatic degradation by trypsin and α-chymotrypsin is observed especially for the high molecular weight PEGylated arginine-based derivative. Insulin release rates in simulated intestinal fluid are controlled by the length of PEG chains and the presence of arginine end-groups. Released insulin retains its secondary structure as established by circular dichroism spectroscopy.     

Studies on the interaction of caffeic acid with human serum albumin in membrane mimetic environments

In this work, fluorescence quenching technique, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) technique were used to gain the binding information of caffeic acid and human serum albumin (HSA) in AOT/isooctane/water microemulsions. The interaction of HSA with caffeic acid at 296, 303, and 310 K in omega(0) 20 microemulsions was characterized by one binding site with the affinity constant K at (3.23+/-0.01) x 10(4), (3.06+/-0.03) x 10(4) and (2.82+/-0.05) x 10(4)M(-1), respectively. The affinities in microemulsions are much higher than that in buffer solution. The CD spectra and FT-IR spectra with qualitative and quantitative results proved that the protein secondary structure changed in the microemulsions in the absence and presence of caffeic acid compared with the free form of HSA in buffer. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses. These data indicated that hydrophobic interaction played a major role in the binding of caffeic acid to HSA in microemulsions and electrostatic interaction can not be excluded. The displacement experiments confirmed that caffeic acid could bind to the site I of HSA, which was in agreement with the result of the molecular modeling study. Furthermore, the DLS data suggested that HSA may locate at the interface of the microemulsion and caffeic acid could interact with them.     

Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water

In the present study we examine the enthalpy of binding of 2-methoxy-3-isobutylpyrazine (IBMP) to the mouse major urinary protein (MUP), using a combination of isothermal titration calorimetry (ITC), NMR, X-ray crystallography, all-atom molecular dynamics simulations, and site-directed mutagenesis. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than a classical entropy-driven signature that might be expected given that the binding pocket of MUP-1 is very hydrophobic. The only ligand-protein hydrogen bond is formed between the side-chain hydroxyl of Tyr120 and the ring nitrogen of the ligand in the wild-type protein. ITC measurements on the binding of IBMP to the Y120F mutant demonstrate a reduced enthalpy of binding, but nonetheless binding is still enthalpy dominated. A combination of solvent isotopic substitution ITC measurements and all-atom molecular dynamics simulations with explicit inclusion of solvent water suggests that solvation is not a major contributor to the overall binding enthalpy. Moreover, hydrogen/deuterium exchange measurements suggest that there is no significant contribution to the enthalpy of binding derived from "tightening" of the protein structure. Data are consistent with binding thermodynamics dominated by favorable dispersion interactions, arising from the inequality of solvent-solute dispersion interactions before complexation versus solute-solute dispersion interactions after complexation, by virtue of poor solvation of the binding pocket.     

Supramolecular complex induced by the binding of sodium dodecyl sulfate to PAMAM dendrimers

Isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) were employed to study the spontaneous supramolecular complexation of amine terminated PAMAM dendrimer (G3[EDA] PAMAM-NH2) induced by the binding of an anionic surfactant, sodium dodecyl sulfate (SDS). At pHor=10, the electrostatic binding ceased because the deprotonated PAMAM dendrimer was uncharged, and hence the surfactant-induced supramolecular assembly could not be formed.     

Monovalent salt enhances colloidal stability during the formation of chitosan/tripolyphosphate microgels

Chitosan micro- and nanoparticles are routinely prepared through ionotropic gelation, where sodium tripolyphosphate (TPP) is added as a cross-linker to dilute chitosan solutions. Despite the wide use of these gel-like particles, their preparation currently relies on trial and error. To address this, we used isothermal titration calorimetry (ITC), dynamic light scattering (DLS), transmission electron microscopy (TEM), and ζ-potential measurements to investigate how the formation, structure, and colloidal stability of chitosan microgels are linked to the molecular interactions that underlie their self-assembly. The strength of the chitosan/TPP interactions was systematically varied through the addition of monovalent salt (NaCl). Remarkably, and contrary to other colloidal systems, this revealed that moderate amounts of NaCl (e.g., 150 mM) enhance the colloidal stability of chitosan/TPP microgels during their formation. This stems from the weakened chitosan/TPP binding, which apparently inhibits the bridging of the newly formed microgels by TPP. The enhanced colloidal stability during the ionic cross-linking process yields microgels with dramatically narrower size distributions, which hitherto have typically required the deacetylation and fractionation of the parent chitosan. Conversely, at high ionic strengths (ca. 500 mM) the chitosan/TPP binding is weakened to the point that the microgels cease to form, thus suggesting the existence of an optimal ionic strength for ionotropic microgel preparation.     

Analysis of the interactions between human serum albumin/amphiphilic penicillin in different aqueous media: an isothermal titration calorimetry and dynamic light scattering study

The complexation process of the amphiphilic penicillins sodium cloxacillin and sodium dicloxacillin with the protein human serum albumin (HSA) in aqueous buffered solutions of pH 4.5 and 7.4 at 25 °C was investigated through isothermal titration calorimetry (ITC) and dynamic light scattering. ITC experiments were carried out in the very dilute regime and showed that although hydrophobic interactions are the leading forces for complexation, electrostatic interactions also play an important role. The possibility of the formation of hydrogen bonds is also deduced from experimental data. The thermodynamic quantities of the binding mechanism, i.e, the enthalpy, , entropy, , Gibbs energy, , binding constant, and the number of binding sites, n_i, were obtained. The binding was saturable and is characterised by Langmuir adsorption isotherms. From ITC data and following a theoretical model, the number of bound and free penicillin molecules was calculated. From Scatchard plots, and n_i were obtained and compared with those from ITC data. The interaction potential between the HSA–penicillin complexes and their stability were determined at pH 7.4 from the dependence of the diffusion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drug–protein complexes with increase in the concentration of added drug.     

Hematite nanoparticle monolayers on mica preparation by controlled self-assembly

In this research, a simple, green and effective strategy was developed to produce long-term stable oil in water emulsion from soy protein and soy polysaccharide. Soy protein and soy polysaccharide formed dispersible complexes at pH around 3.25 aqueous solution through electrostatic and hydrophobic interactions. A high pressure homogenization produced the protein/polysaccharide complex emulsion having a droplet size about 250 nm. A heat treatment of the emulsion resulted in the protein denaturation, forming irreversible oil-water interfacial films composed of soy protein/soy polysaccharide complexes. The droplets of the emulsion were characterized by dynamic light scattering, ζ-potential, transmission electron microscopy, polysaccharide digestion via pectinase, and confocal laser scanning microscopy observation via dual fluorescence probes. As a result of the polysaccharide being fixed on the droplet surface, the emulsions exhibited long-term stability in the media containing pH values of 2-8 and 0.2 mol/L NaCl. The stable soy protein/soy polysaccharide complex emulsion is a suitable food-grade delivery system in which lipophilic bioactive compounds can be encapsulated.