Assessment of penetration of quantum dots through in vitro and in vivo human skin using the human skin equivalent model and the tape stripping method

Quantum dots (QDs) are rapidly emerging as an important class of nanoparticles (NPs) with potential applications in medicine. However, little is known about penetration of QDs through human skin. This study investigated skin penetration of QDs in both in vivo and in vitro human skin. Using the tape stripping method, this study demonstrates for the first time that QDs can actually penetrate through the stratum corneum (SC) of human skin. Transmission electron microscope (TEM) and energy diverse X-ray (EDX) analysis showed accumulation of QDs in the SC of a human skin equivalent model (HSEM) after dermal exposure to QDs. These findings suggest possible transdermal absorption of QDs after dermal exposure over a relatively long period of time.     

Comparison of molecular interactions of Ag2Te and CdTe quantum dots with human serum albumin by spectroscopic approaches

Ag₂Te quantum dots (QDs) have attracted great attention in biological applications due to their superior photoluminescence qualities and good biocompatibility, but their potential biotoxicity at a molecular biology level has been rarely discussed. In order to better understand the basic behavior of Ag₂Te QDs in biological systems and compare their biotoxicity to cadmium‐containing QDs, a series of spectroscopic measurements was applied to reveal the molecular interactions of Ag₂Te QDs and CdTe QDs with human serum albumin (HSA). Ag₂Te QDs and CdTe QDs statically quenched the intrinsic fluorescence of HSA by electrostatic interactions, but Ag₂Te QDs exhibited weaker quenching ability and weaker binding ability compared with CdTe QDs. Electrostatic interactions were the main binding forces and Sudlow's site I was the primary binding site during these binding interactions. Furthermore, micro‐environmental and conformational variations of HSA were induced by their binding interactions with two QDs. Ag₂Te QDs caused less secondary structural and conformational change in HSA, illustrating the lower potential biotoxicity risk of Ag₂Te QDs. Our results systematically indicated the molecular binding mechanism of Ag₂Te QDs with HSA, which provided important information for possible toxicity risk of these cadmium‐free QDs to human health.     

Interaction and energy transfer studies between bovine serum albumin and CdTe quantum dots conjugates: CdTe QDs as energy acceptor probes

In this paper, a systematic investigation of the interaction of bovine serum albumin (BSA) with water‐soluble CdTe quantum dots (QDs) of two different sizes capped with carboxylic thiols is presented based on steady‐state and time‐resolved fluorescence measurements. Efficient Förster resonance energy transfer (FRET) was observed to occur from BSA donor to CdTe acceptor as noted from reduction in the fluorescence of BSA and enhanced fluorescence from CdTe QDs. FRET parameters such as Förster distance, spectral overlap integral, FRET rate constant and efficiency were determined. The quenching of BSA fluorescence in aqueous solution observed in the presence of CdTe QDs infers that fluorescence resonance energy transfer is primarily responsible for the quenching phenomenon. Bimolecular quenching constant (k_q) determined at different temperatures and the time‐resolved fluorescence data provide additional evidence for this. The binding stoichiometry and various thermodynamic parameters are evaluated by using the van ‘t Hoff equation. The analysis of the results suggests that the interaction between BSA and CdTe QDs is entropy driven and hydrophobic forces play a key role in the interaction. Binding of QDs significantly shortened the fluorescence lifetime of BSA which is one of the hallmarks of FRET. The effect of size of the QDs on the FRET parameters are discussed in the light of FRET parameters obtained.     

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K_a were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as ?3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA–STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three‐dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α‐helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study. Copyright © 2013 John Wiley & Sons, Ltd.     

Interaction of cromolyn sodium with human serum albumin: a fluorescence quenching study

The interaction between cromolyn sodium (CS) and human serum albumin (HSA) was investigated using tryptophan fluorescence quenching. In the discussion of the mechanism, it was proved that the fluorescence quenching of HSA by CS is a result of the formation of a CS–HSA complex. Quenching constants were determined using the Sterns–Volmer equation to provide a measure of the binding affinity between CS and HSA. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated. The distance r between donor (Trp²¹⁴) and acceptor (CS) was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, synchronous fluorescence spectroscopy data and UV–vis absorbance spectra have suggested that the association between CS and HSA changed the molecular conformation of HSA and the electrostatic interactions play a major role in CS–HSA association.     

Comparative studies on the interactions of baicalein and Al(III)–baicalein complex with human serum albumin

A new potential drug aluminum(III)–baicalein complex (ALBC) was synthesized and characterized. The binding mechanisms of baicalein (BC) and ALBC to human serum albumin (HSA) under simulative physiological conditions were investigated, in order to understand the pharmacokinetics of BC and ALBC. Fluorescence spectroscopy results suggested that the binding level of BC is higher than that of ALBC. Results of UV–vis, synchronous fluorescence, 3D fluorescence, circular dichroism and Fourier transform infrared spectroscopic analyses consistently demonstrated that the conformation of HSA was altered when bound to BC or ALBC. The distance between HSA as a donor and BC (or ALBC) as an acceptor was determined via fluorescence resonance energy transfer. The results of competitive experiments and molecular docking studies indicated that BC was located in site I (subdomain IIA) on HSA and that ALBC was bound to HSA mainly within site II (subdomain IIIA). Copyright © 2015 John Wiley & Sons, Ltd.     

Binding mechanism of trans-N-caffeoyltyramine and human serum albumin: Investigation by multi-spectroscopy and docking simulation

trans-N-Caffeoyltyramine (TNC), which was isolated from the Cortex Lycii in our laboratory, is a phenolic amide compound with multiple pharmacological activities. The interaction between TNC and human serum albumin (HSA) was studied by Nuclear magnetic resonance (NMR) relaxation experiment, fluorescence spectroscopy, and docking simulation. NMR methodology is based on the analysis of selective and non-selective spin-lattice relaxation rate enhancements of TNC protons in the presence of the HSA. Result indicated that the interaction occurred between HSA and TNC, and changed the proton magnetic environment of TNC. Fluorescence spectroscopy confirmed that TNC displayed a strong capability to quench the fluorescence of HSA, and the acting forces for binding were hydrogen bonds and van der Waals forces. Furthermore, the circular dichroism, synchronous, and three-dimensional fluorescence spectra, which were employed to determine the conformation of protein, revealed that binding of TNC with HSA could induce conformational changes in HSA. In addition, the molecular modeling results exhibited that TNC mainly bonded to site I in sub-domain IIA of HSA.     

The effect of non-enzymatic glycation on the unfolding of human serum albumin

We monitored the unfolding of human serum albumin (HSA) and glycated human serum albumin (gHSA) subjected to guanidine hydrochloride (GndHCl) by using fluorescence and circular dichroism (CD) spectroscopy. A two-state model with sloping baselines best described the Trp-214 fluorescence unfolding measurements, while a three-state model best described the far-UV CD unfolding data. Glycation of HSA increased the [D](50%) point by approximately 0.20M. This corresponded to an increase in the free energy of unfolding of gHSA relative to HSA of 2.6kJ/mol. The intrinsic fluorescence of Trp-214 in gHSA is 0.72 of that of HSA and the far-UV CD spectrum of gHSA is nearly identical to that of HSA. These results showed that glycation altered the local structure around Trp-214 while not significantly impacting the secondary structure, and this alteration translated into an overall change in the stability of gHSA compared to HSA.     

New fluorescence of nonenzymatically glucosylated human serum albumin

Glucosylated human serum albumin (G-HSA) obtained under incubation with glucose at 37 degrees C for 8 days showed a new fluorescence with a maximum at 430 nm, resulting in quenching of the fluorescence of only one tryptophan residue on HSA. The quantum yield of new fluorescence is 0.024 at 25 degrees C. The analysis of the excitation spectra allowed us to conclude the absence of energy transfer. In G-HSA, non-disulfide cross-linking hexamer was confirmed by SDS-PAGE.     

New mathematical approach for the evaluation of drug binding to human serum albumin by high-performance liquid affinity chromatography

A novel mathematical approach for investigation of drug–human serum albumin (HSA) interactions by means of high-performance liquid affinity chromatography is developed. The model is based on the assumption that two types of competitive binding sites exist on the HSA molecule. The widely used single-site binding equation is extended and a proper mathematical analysis is proposed allowing the determination of the major parameters characterizing the multisite binding (cobinding) process. The utility of the new approach is proved by competitive studies on HSA binding of two model drugs, diazepam and diclofenac.     

Direct interactions in the recognition between the environmental estrogen bisphenol AF and human serum albumin

Bisphenol AF (BPAF) was used as a model compound to investigate the binding mechanism between the endocrine disrupting compound and human serum albumin (HSA) using multispectroscopic techniques and molecular modeling method at the protein level. The results indicated that BPAF was indeed bound to HSA and located in the hydrophobic pocket of HSA on subdomain IIA through hydrogen bond and van der Waals interactions. The fluorescence quenching data showed that the binding of BPAF and HSA quenched the intrinsic fluorescence of HSA, and the static quenching constants were acquired. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on the interaction between Oxaprozin-E and bovine serum albumin by spectroscopic methods

The interaction between Oxaprozin-E and bovine serum albumin (BSA) was studied by spectroscopic methods including fluorescence and UV–vis absorption spectroscopy. The quenching mechanism of fluorescence of BSA by Oxaprozin-E was discussed to be a dynamic quenching procedure. The number of binding sites n and apparent binding constant K was measured by fluorescence quenching method. The thermodynamics parameter ΔH, ΔG, ΔS were calculated. The results indicate the binding reaction was mainly entropy-driven and hydrophobic forces played major role in the binding reaction. The distance r between donor (BSA) and acceptor (Oxaprozin-E) was obtained according to Förster theory of non-radioactive energy transfer.     

Diosmin binding to human serum albumin and its preventive action against degradation due to oxidative injuries

Diosmin is a glycosylated polyphenolic compound, commonly found in fruits and vegetables, which is utilized for the pharmacological formulation of some drugs. The interactions of diosmin to human serum albumin have been investigated by fluorescence, UV–visible, FTIR spectroscopy, native electrophoresis and protein–ligand docking studies. The fluorescence studies indicate that the binding site of the additive involves modifications of environment around Trp214 at the level of subdomain IIA. Combining the curve-fitting results of infrared Amide I′ band, the modifications of protein secondary structure have been estimated, indicating a decrease in α-helix structure following flavonoid binding. Data obtained by fluorescence and UV–visible spectroscopy, FTIR experiments and molecular modeling afforded a clear picture of the association mode of diosmin to HSA, suggesting that the primary binding site of diosmin is located in Sudlow's site I. Computational mapping confirms this observation suggesting that the possible binding site of diosmin is located in the hydrophobic cavity of subdomain IIA, whose microenvironment is able to help and stabilize the binding of the ligand in non-planar conformation. Moreover the binding of diosmin to HSA significantly contributes to protect the protein against degradation due to HCLO and Fenton reaction.     

Study the interactions between human serum albumin and two antifungal drugs: Fluconazole and its analogue DTP

Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern–Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10⁴ L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug–protein. The effect of metal ions on the binding constants of fluconazole–HSA complex suggested that the presence of Mg²⁺ and Zn²⁺ ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.     

Binding of all-trans retinoic acid to human serum albumin: fluorescence, FT-IR and circular dichroism studies

All-trans retinoic acid derived from vitamin A is an essential component for the modulation of angiogenesis, the process of blood vessel formation. We have investigated the binding of all-trans retinoic acid to the carrier protein, human serum albumin (HSA) under physiological conditions. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy were used for the biophysical studies. The binding parameters were determined by a Scatchard plot and the results found to be consistent with those obtained from a modified Stern–Volmer equation. From the thermodynamic parameters calculated according to the van’t Hoff equation, the enthalpy change ΔH⁰ and entropy change ΔS⁰ are found to be 106.17 and 106.14 J/mol K, respectively. These values suggest that apart from hydrophobic interactions electrostatic interactions are present. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. Docking studies performed substantiated our experimental findings and it was observed that all-trans retinoic acid hydrogen bonded with Trp 214 and Asp 451 residues of subdomain IIA and IIIA of HSA, respectively.     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

Spectroscopic and molecular docking study on the structure–affinity relationship and mechanism in the interaction of genistein and its derivatives with bovine serum albumin

In this paper, the interaction of genistein (GEN) and its four derivatives (GEN1–4) with bovine serum albumin (BSA) were investigated by ultraviolet–visible absorption spectra, fluorescence, synchronous fluorescence, three‐dimensional fluorescence spectroscopy, circular dichroism and molecular docking techniques. The experimental results showed that the intrinsic fluorescence of BSA was quenched by genisteins and was due to the formation of a genisteins–BSA complex. The quenching constant, binding constants, binding sites, intermolecular distances and thermodynamic properties were calculated at 298 K, 306 K and 310 K. Site marker competitive experiments indicated that the binding site of genisteins to BSA was mainly located in subdomain IIA. The conformational investigation showed that the presence of 0020 genisteins led to changes in the secondary structure of BSA and induced the slight unfolding of protein polypeptides, which confirmed some micro‐environmental and conformational changes of BSA molecules. Furthermore, the binding affinity decreased in the order GEN1 > GEN > GEN4 > GEN3 > GEN2, which revealed that different type and position of substituents of genistein significantly influenced the affinity of compounds to BSA. The number of hydroxyl groups on the ring A was the most important factor because increasing the hydroxyl groups on ring A clearly enhanced the binding affinity. However, trifluoromethylation did not much affect the affinity, alkylation, esterification and difluoromethylation slightly enhanced the binding affinity. The results obtained herein will provide valuable information about the pharmacokinetics at a molecular level and be a useful guideline for the further design of much more suitable genistein derivatives.     

Investigations of the effects of ethanol on warfarin binding to human serum albumin

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.     

Spectroscopic analysis of the binding interaction between tinidazole and bovine serum albumin (BSA)

The interaction between bovine serum albumin (BSA) and tinidazole (Tindamax; 1) in aqueous solution was investigated in detail by means of UV/VIS and fluorescence spectroscopy, as well as through resonance light-scattering (RLS) spectroscopy. The apparent binding constant and number of binding sites were determined at three different temperatures, as well as the average binding distances between 1 and the nearest amino acid residue(s) of BSA, as analyzed by means of F?rster's theory of non-radiation energy transfer. Compound 1 was found to quench the inner fluorescence of BSA by forming a tight 1:1 aggregate, based on both static quenching and non-radiation energy transfer. The entropy change upon complexation was positive, and the enthalpy change was negative, indicating that the observed spontaneous binding is mainly driven by electrostatic interactions.