Affinity For Human Serum Albumin Research Articles

Structure-relaxivity relationships of serum albumin targeted MRI probes based on a single amino acid Gd complex.

The Gd(III) complex of DO3A-N-α-aminopropionate, Gd(DOTAla), was used to generate a small library of putative MRI probes targeted to human serum albumin (HSA). Ten compounds were synthesized via multistep organic synthesis, and the corresponding Gd complexes were investigated for their affinity to HSA, lipophilicity, and relaxivity in the absence and presence of HSA. Negative charge and moderate lipophilicity correlate with increased HSA affinity and relaxivity.

Methylation of genistein and kaempferol improves their affinities for proteins

Methylation of flavonoids appears to be a simple and effective way to improve metabolic resistance and transport of flavonoids. Serum albumins are major soluble proteins serving as transport proteins for many exogenous compounds. This work in here mainly concerns about the effect of methylation of flavonoids on the affinity for human serum albumin (HSA) and ovalbumin. One isoflavone (genistein) and one flavonol (kaempferol) and their monomethylated derivatives at position 4′ (biochanin A and kaempferide) were studied for their affinities for ovalbumin and HSA. The methylation of flavonoids significantly affects the binding process. In general, the methylation of flavonoids improved the affinities for proteins by 2–16 times. This result supports that the methylation of genistein and kaempferol enhanced the transporting ability, which leads to facilitated absorption and greatly increased bioavailability. The methylation increases the hydrophobicity of genistein and kaempferol, and the hydrophobic interaction plays an important role in binding flavonoids to HSA and ovoalbumin.

High performance affinity chromatography (HPAC) as a high-throughput screening tool in drug discovery to study drug–plasma protein interactions

Drug–plasma protein binding is an important parameter that, together with other physicochemical properties such as lipophilicity and pKa, greatly influences drug absorption, distribution, metabolism, and excretion (ADME). Therefore, it is important for pharmaceutical companies to develop a rapid screening assay to examine plasma protein binding during the early stages of the drug discovery process. Human serum albumin (HSA) and α1-acid glycoprotein (AGP) are the most important plasma proteins that are capable of binding drugs. In this work, an automated and high-throughput (<3min/compound) strategy was developed using high performance affinity chromatography (HPAC) with commercial HSA and AGP columns to evaluate drug–plasma protein interactions for drug screening. A generic gradient was used throughout the study to separate drugs that were weakly and tightly bound to HSA and AGP. To accelerate the analysis time, the system was calibrated in a single run by pooling reference compounds without overloading the column. For both HSA and AGP studies, the developed methods were successfully transferred from HPAC–UV to HPAC–MS with single quadrupole MS detection and ammonium acetate, pH 7.0 as a volatile mobile phase. The MS detection enhanced the sensitivity, selectivity, and throughput of the method by pooling unknown compounds. For HSA analyses, the binding percentages obtained using HPAC were well correlated with the binding percentages from the literature. This method was also able to rank compounds based on their affinity for HSA. Concerning the AGP analyses, the quality of the correlation between the binding percentages obtained in HPAC and those from the literature was weaker. However, the method was able to classify compounds into weak, medium, and strong binders and rank compounds based on their affinity for AGP.

Interactive Associations of Drug–Drug and Drug–Drug–Drug with IIA Subdomain of Human Serum Albumin

Owing to advantageous biochemical and pharmacological properties of human serum albumin (HSA), HSA-based drug carrier is playing an increasing role in the clinical setting. Since the IIA subdomain of HSA is a big hydrophobic cavity, we proposed that HSA delivers multiple drugs simultaneously docking on the IIA subdomain and that drugs may influence each other's binding affinity to albumin when cobinding the HSA IIA subdomain. Therefore, we studied the interactive association of drugs with the IIA subdomain of HSA by fluorescence spectroscopy and X-ray crystallography, in order to elucidate the molecular mechanism and behavior of drugs cobinding the IIA subdomain of HSA, and develop a universal structure-based model for HSA carrier design. We solved HSA-fatty acid-cinnamic acid, HSA-fatty acid-cinnamic acid-indometacin and HSA-fatty acid-cinnamic acid-indometacin-lamivudine complex structures, respectively. HSA complex structures and fluorescence quenching of HSA revealed that different drugs can regulate binding sites, binding mode and binding affinity of each other. For example, indometacin renders cinnamic acid to make reposition, and decreases binding affinity of HSA for cinnamic acid, but increases binding affinity of itself to HSA. Lamivudine makes cinnamic acid and indometacin to bind new subsites. Cinnamic acid-indometacin enhances binding affinity of lamivudine, and cinnamic acid-lamivudine increases binding affinity of indometacin, but indometacin-lamivudine decreases binding affinity of cinnamic acid to HSA. The study provided a biochemical basis for structure-guided development of HSA delivery system.

In Vitro Study of Ketoprofen and Indapamide Used in Multidrug Therapy: 1H-NMR Analysis

ABSTRACT Interaction of ketoprofen (KP, a nonsteroidal anti-inflammatory drug) and indapamide (IDP, a thiazide-related diuretic) with human serum albumin (HSA) in low-affinity binding sites (LAS) and competition between these medicines used in multidrug therapy have been studied by proton nuclear magnetic resonance (1H-NMR) spectroscopy. The analysis of chemical shifts, Δσ (ppm), of drug proton resonances and the values of the association constant, Ka [M−1], were used to estimate the effect of IDP and KP on the KP–HSA and IDP–HSA complexes, respectively. The results showed the changes in the affinity of human serum albumin toward the drug with different mechanisms of action (KP and IDP) and the competition in the binding to HSA.

Effects of green-, yellow- and red-emitting CdTe QDs on the interaction between 7-hydroxyflavone/quercetin with human serum albumin in vitro

In this study, 7-hydroxyflavone and quercetin were studied for their affinities for human serum albumin (HSA) in the presence and absence of quantum dots (QDs). Three typical CdTe QDs with maximum emissions of 535 nm (green-emitting, G-QDs), 598 nm (yellow-emitting, Y-QDs) and 654 nm (red-emitting, R-QDs) were tested. The fluorescence intensities of HSA decreased remarkably with addition of QDs. Quercetin resulted in obvious blue-shifts of the maximum λ em of HSA from 340.8 to 332.6 nm and 7-hydroxyflavone hardly changed the maximum λ em of HSA in the absence of QDs. The extents of shifts of the maximum λ em of HSA induced by 7-hydroxyflavone or quercetin in the presence of QDs were larger than that in the absence of QDs. When 8.00 µmol/L of 7-hydroxyflavone was added, the fluorescence decreased by 68.1%, 65.7% and 71.8% in the presence of G-QDs, R-QDs and Y-GDs, respectively, from the original G-QDs, R-QDs and Y-GDs that resulted in 42%, 43% and 55%, respectively. When 8.00 µmol/L of quercetin was tested, the fluorescence decreased by 72.4%, 69.5% and 75.6% in the presence of G-QDs, R-QDs and Y-GDs, respectively. G- and R-QDs hardly affected the affinities of 7-hydroxyflavone and quercetin for HSA. However, Y-QDs decreased the affinities of 7-hydroxyflavone and quercetin for HSA by about 13.61% and 6.8%, respectively.

Effect of Nonenzymatic Glycosylation on the Magnetic Resonance Imaging (MRI) Contrast Agent Binding to Human Serum Albumin

Enhanced nonenzymatic glycosylation (NEG) of human serum albumin (HSA) is observed in diabetic patients. This modifies some of the physiological functions of HSA, as the binding of ligands. Some gadolinium complexes, commonly used as MRI contrast agents, have a high affinity for HSA, which enhances their efficacy. The aim of this study is to evaluate the possible influence of the NEG of HSA on its affinity for some gadolinium chelates.

Analysis of cause of failure of new targeting peptide in PEGylated liposome: Molecular modeling as rational design tool for nanomedicine

Drug nanocarriers are often derivatized with targeting moieties to achieve site specific delivery, however, the results from this approach have, as yet, not reached expectations. We have tested a new phage display based targeting moiety, the activated endothelium targeting peptide (AETP), for its vascular endothelium directed targeting efficiency, when anchored to a PEGylated liposome via maleimide chemistry. Our results have, however, not shown any evidence of improved targeting. We have hypothesized that the failure of the AETP moiety is due to its availability to target receptors being restricted, as a result of steric hindrance due to the PEG polymer, and possibly affinity for bloodstream proteins, particularly human serum albumin (HSA). In this context, molecular modeling was used to contrast the properties of the AETP moiety to those of the RGD targeting peptide, already found to be effective in previous trials. Our molecular dynamics simulation results indicate the AETP moiety is located within the PEG layer, and its hydrophobic nature causes it to be obscured by PEG to a greater extent than the more hydrophilic RGD targeting peptide. Protein–ligand docking results indicated similar affinities for HSA of both the AETP moiety and a PEG fragment, and a significantly lower affinity for the RGD peptide. We know of no means to investigate this experimentally with atomic level resolution, thus our use of computational methods to investigate this can be seen as a new tool for rational design in nanomedicine.

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 . The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

Results of molecular docking as descriptors to predict human serum albumin binding affinity

Pharmacokinetic properties of a compound are important in drug discovery and development. These properties are most often estimated from the structural properties of a compound with a structural–activity relationship (QSAR) approach. Rapid advances in molecular pharmacology have characterized a number of important proteins that shape the pharmacokinetic profile of a compound. Previous studies have shown that molecular docking, which is capable of analyzing compound–protein interactions, could be applied to make a categorical estimation of a pharmacokinetic property. The present study focused on the binding affinity of human serum albumin (HSA) as an example to show that docking descriptors might also be useful to estimate the exact value of a pharmacokinetic property. A previously reported dataset containing 94 compounds with log K HSA values was analyzed. A support vector regression model based on the docking descriptors was able to approximate the observed log K HSA in the training and validation dataset with an R 2 = 0.79. This accuracy was comparable to known QSAR models based on compound descriptors. In this case study, it was shown that an account of protein flexibility is essential to calculate informative docking descriptors for use in the quantitative estimation of log K HSA.

High‐throughput analysis of drug dissociation from serum proteins using affinity silica monoliths

A noncompetitive peak decay method was used with 1 mm×4.6 mm id silica monoliths to measure the dissociation rate constants (kd) for various drugs with human serum albumin (HSA) and α1-acid glycoprotein (AGP). Flow rates up to 9 mL/min were used in these experiments, resulting in analysis times of only 20-30 s. Using a silica monolith containing immobilized HSA, dissociation rate constants were measured for amitriptyline, carboplatin, cisplatin, chloramphenicol, nortriptyline, quinidine, and verapamil, giving values that ranged from 0.37 to 0.78 s(-1). Similar work with an immobilized AGP silica monolith gave kd values for amitriptyline, nortriptyline, and lidocaine of 0.39-0.73 s(-1). These kd values showed good agreement with values determined for drugs with similar structures and/or affinities for HSA or AGP. It was found that a kd of up to roughly 0.80 s(-1) could be measured by this approach. This information made it possible to obtain a better understanding of the advantages and possible limitations of the noncompetitive peak decay method and in the use of affinity silica monoliths for the high-throughput analysis of drug-protein dissociation.

Effect of CdTe QDs on the protein-drug interactions

Recently, investigations of biological toxicity of cadmium QDs and their toxic interaction with plasma proteins have attracted great interest. In this work, flavonoids were studied for the affinities for human serum albumin (HSA) in the presence and absence of CdTe G-QDs by fluorescence quenching method. CdTe G-QDs obviously enhanced the binding affinities of kaempferol, genistein and biochanin A by 3.78 to 154.88 times depending on the QDs concentration. However, the affinity of kaempferide for HSA was slightly weakened in the presence of G-QDs. The non-methylated flavonoids were more sensitive to G-QDs than their methylated forms. The affinities of kaempferide and kaempferol for HSA at first were slightly improved and then obviously decreased with increasing G-QDs concentration. For genistein, the affinities for HSA decreased with increasing G-QDs concentration. However, the G-QDs concentration showed no obvious effect on the affinity of biochanin A. The binding affinities of flavonoids for HSA improved with increasing QDs size.

Surface plasmon resonance biosensor for microalbumin detection

Human serum albumin (HSA) is an important biomarker for diagnosing nephropathy. Traditional microalbumin detection is easy affected by the intake of certain medicines, such as aspirin, corticosteroids and antibiotics. In this research, the surface plasmon resonance (SPR) biosensor technique was used to detect HSA. The anthraquinone dye Cibacron Blue F3G-A (CB) is used as a receptor which has a specific affinity for HSA. The purpose of this study is establishing the optimum conditions for SPR to detect HSA. The results exhibited that the sensor chip has high affinity as well as high capacity at pH 4 with 0.05 M sodium chloride. In addition, linear response appeared in the range of 0.01–0.1 mg/ml HSA and the detecting limit was 4 μg/ml. This methodology may prove to be useful in clinical diagnosis and may serve as an assay for HSA binding ability.

Steric and Allosteric Effects of Fatty Acids on the Binding of Warfarin to Human Serum Albumin Revealed by Molecular Dynamics and Free Energy Calculations

Human serum albumin (HSA) binds with drugs and fatty acids (FAs). This study was initiated to elucidate the relationship between the warfarin binding affinity of HSA and the positions of bound FA molecules. Molecular dynamics simulations of 11 HSA-warfarin-myristate complexes were performed. HSA-warfarin binding free energy was then calculated for each of the complexes by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. The results indicated that the magnitude of the binding free energy was smaller in HSA-warfarin complexes that had 4 or more myristate molecules than in complexes with no myristate molecules. The unfavorable effect on the HSA-warfarin binding affinity was caused sterically by the binding of a myristate molecule to the FA binding site closest to the warfarin binding site. On the other hand, the magnitude of HSA-warfarin binding free energy was largest when 3 myristate molecules were bound to the high-affinity sites. The strongest HSA-warfarin binding was attributable to favorable entropic contribution related to larger atomic fluctuations of the amino acid residues at the warfarin binding site. In the binding of 2 myristate molecules to the sites with the highest and second-highest affinities, allosteric modulation that enhanced electrostatic interactions between warfarin and some of the amino acid residues around the warfarin binding site was observed. This study clarified the structural and energetic properties of steric/allosteric effects of FAs on the HSA-warfarin binding affinity and illustrated the approach to analyze protein-ligand interactions in situations such that multiple ligands bind to the other sites of the protein.

Interactions between Antimalarial Indolone-N-oxide Derivatives and Human Serum Albumin

The binding affinity of human serum albumin (HSA) to three antimalarial indolone-N-oxide derivatives, INODs, was investigated under simulated physiological conditions using fluorescence spectroscopy in combination with UV-vis absorption and circular dichroism (CD) spectroscopy. Analysis of fluorescence quenching data of HSA by these compounds at different temperatures using Stern-Volmer and Lineweaver-Burk methods revealed the formation of a ground state indolone-HSA complex with binding affinities of the order 10(4) M(-1). The thermodynamic parameters ΔG, ΔH, and ΔS, calculated at different temperatures, indicated that the binding reaction was endothermic and hydrophobic interactions play a major role in this association. The conformational changes of HSA were investigated qualitatively using synchronous fluorescence and quantitatively using CD. Site marker competitive experiments showed that the binding process took place primarily at site I (subdomain IIA) of HSA. The number of binding sites and the apparent binding constants were also studied in the presence of different ions.

Use of peak decay analysis and affinity microcolumns containing silica monoliths for rapid determination of drug–protein dissociation rates

This report examined the use of silica monoliths in affinity microcolumns containing human serum albumin (HSA) to measure the dissociation rates for various drugs from this protein. Immobilized HSA and control monolith columns with dimensions of 1 mm × 4.6 mm i.d. were prepared for this work and used with a noncompetitive peak decay method. Several drugs known to bind HSA were examined, such as warfarin, diazepam, imipramine, acetohexamide, and tolbutamide. Items that were studied and optimized in this method included the sample volume, sample concentration, and elution flow rate. It was found that flow rates up to 10 mL/min could be used in this approach. Work with HSA silica monoliths at these high flow rates made it possible to provide dissociation rate constants for drugs such as warfarin in less than 40 s. The dissociation rate constants that were measured gave good agreement with values reported in the literature or that had been obtained with other solutes that had similar binding affinities for HSA. This approach is a general one that should be useful in examining the dissociation of other drugs from HSA and in providing a high-throughput method for screening drug–protein interactions.

Binding Citrus flavanones to human serum albumin: effect of structure on affinity

Much of the bioactivities of Citrus flavanones significantly appear to impact blood and microvascular endothelial cells. It is essential to investigate the interaction between Citrus flavanones and serum albumin to verify the effect of flavanone structures on the distribution and transportation in blood. The interactions between flavonoids and proteins have attracted great interest among researchers. The work in here mainly concerns about the binding interaction between Citrus flavanones and human serum albumin (HSA) in vitro. The methoxylation of tangeretin improved the affinity for HSA by 100 times. The 2,3-double bond in conjugation with a 4-oxo group plays an important role for the affinity for HSA. The affinity of apigenin for HSA is about 10,000-times higher than that of naringenin. It was found that the hydroxylation on position 3' of flavonol significantly improves the binding affinity for HSA. The affinity of quercetin (3', 4') for HSA is about 100-times higher than that of kaempferol (4'). The hydroxylation on position 3' of flavone slightly improves the binding affinity for HSA. The affinity of luteolin for HSA is about 1.38-times higher than that of apigenin. The values of log10(Ka) are proportional to the number of binding sites (n), which confirms the method used here is suitable to study the interaction between Citrus flavanones and HSA.

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme-albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA-heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0°C. The affinity of isoniazid and rifampicin for HSA [K (0)=(3.9±0.4)×10(-4) and (1.3±0.1)×10(-5)M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K (h)=(4.3±0.4)×10(-3) and (1.2±0.1)×10(-4)M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H (0)=(1.9±0.2)×10(-8)M] decreases by about 1order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H (d)=(2.1±0.2)×10(-7)M]. In the absence and presence of CO(2), the values of the second-order rate constant (l (on)) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1×10(5) and 4.3×10(5)M(-1)s(-1), respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO(2). Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free L: -tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow's site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow's site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.

CdTe quantum dots (QDs) improve the affinities of baicalein and genistein for human serum albumin in vitro

Baicalein and genistein were studied for the affinities for human serum albumin (HSA) in the presence and absence of three CdTe quantum dots (QDs) with different sizes. Three typical CdTe QDs with maximum emissions of 535 nm (green-emitting, G-QDs), 598 nm (yellow-emitting, Y-QDs), and 654 nm (red-emitting, R-QDs) were tested. The fluorescence intensities of HSA decreased remarkably with increasing concentration of QDs. Baicalein resulted in an obvious blue-shift of the λ em of HSA from 340 to 334 nm. However, the extents of blue-shifts induced by baicalein and genistein in the presence of QDs were much bigger than that in the absence of QDs. The quenching process of baicalein for HSA was easily affected by the QDs size than that of genistein. QDs increased the quenching constant from 136.97% to 162.24% for baicalein. However, QDs only increased the quenching constants from 20.56% to 32.23% for genistein. G-QDs, Y-QDs, and R-QDs increased the affinities of baicalein for HSA about 3.02%, 6.38% and 9.40%. G-QDs, Y-QDs, and R-QDs increased the affinities of genistein for HSA about 2.56%, 13.46% and 19.44%. The binding affinities of baicalein and genistein for HSA increased with increasing QDs size.