Green synthesis and physical characterization of Au nanoparticles and their interaction with bovine serum albumin

Accepted Manuscript Title: Green Synthesis and Physical Characterization of Au Nanoparticles and Their Interaction with Bovine Serum Albumin Author: Hua-Li Yue Yan-Jun Hu Jun Chen Ai-Min Bai Yu Ouyang PII: DOI: Reference:

S0927-7765(14)00348-8 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.06.055 COLSUB 6501

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

22-2-2014 2-6-2014 24-6-2014

Please cite this article as: H.-L. Yue, Y.-J.H. [email protected], J. Chen, A.-M. Bai, Y. Ouyang, Green Synthesis and Physical Characterization of Au Nanoparticles and Their Interaction with Bovine Serum Albumin, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.06.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Green Synthesis and Physical Characterization of Au

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Nanoparticles and Their Interaction with Bovine Serum Albumin

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Hua-Li Yue, Yan-Jun Hu*, Jun Chen, Ai-Min Bai, Yu Ouyang

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Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Department of Chemistry, Hubei Normal University,

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Huangshi 435002, PR China

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Corresponding Author:

E–mail: [email protected], [email protected] (Y.J. Hu);

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Tel.: +86–714–6515602; Fax: +86–714–6573832.

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Abstract In this study, we used morin as a reducing agent for the synthesis of stable and nearly spherical Au nanoparticles (M–AuNPs), which were characterized by UV–vis, transmission

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electron microscopy (TEM) and X–ray diffraction (XRD). The binding characteristics and molecular mechanism of the interaction between the M–AuNPs and bovine serum albumin

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(BSA) were explored by UV–Vis absorbance, fluorescence spectroscopy, and circular

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dichroism spectra (CD). The results showed that the quenching mechanisms were based on static quenching. The thermodynamic parameters ΔG, ΔH and ΔS, suggested that the reaction

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was spontaneous, and mainly driven by electrostatic interactions. Site marker competitive displacement experiments indicated that M–AuNPs bound with high affinity to site I

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(subdomain IIA) of BSA. Synchronous fluorescence and CD spectra demonstrated that BSA conformation was slightly altered in the presence of M–AuNPs. In addition, the effect of pH,

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temperature, morin quantity, and reaction time were investigated.

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Keywords: Morin; Au nanoparticles; BSA; Spectroscopic method; Molecular mechanism

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1. Introduction With the development of nanotechnology, metal nanoparticles have drawn remarkable interest due to their unusual chemical and physical properties showing great promises for

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potential applications in biology, medicine, therapeutic and sensors [1–4]. For instance, in biological and medical applications, nanoparticles have been considered as drug carriers, as

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well as key ingredients in curing diseases and as diagnostic tools for in vivo imaging [5–6].

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Targeted drug delivery, a controlled release of a drug at the desired site, not only improves the therapeutic potential of a drug, but also lowers the overall dose and undesired side effects.

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Advances in this direction will have an invaluable impact on disease management and improve the quality of life for patients [7]. In recent years, many researchers have tried to

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investigate nanomaterials as platforms for drug delivery [8–11].

Owing to intriguing optoelectronic properties and biocompatibility, Au nanoparticles

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(AuNPs) have widely been exploited for diagnostics and therapeutic applications [12–14].

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AuNPs add onto the group of delivery systems that involve attachment of drugs or ligands to carriers. The interest for Au lies in its inert and nontoxic nature, controllable size and ease of

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functionalization with desired ligands [15]. AuNPs have been developed as drug delivery vehicles to improve bioavailability, efficiency and specificity of pharmaceutical drugs, in particular anticancer agents [16]. Metal Au complexed with breynia rhamnoides and stoechospermum marginatum have been used as an antibacterial and an antifungal drugs. It has also been against drug–resistant cardiovascular diseases [17, 18]. In spite of these broad uses of Au, the activity of Au–morin complex is poorly understood. Morin (3,5,7,2’,4’–pentahydroxyflavone; molecular structure: Figure 1) is a flavonoid that has been identified in fruits, vegetables, tea, wine, and many Chinese herbs [19]. It is reported that morin has antioxidant, antinociceptive, and anti–tumor activity [20–22]. These novel properties of AuNPs and morin led us to design morin–conjugated Au nanoparticles for

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potential cancer therapeutic applications. Therefore, we used the nontoxic, ecofriendly, antioxidant, and anti–tumorigenic aqueous drug morin as a reducing agent for the synthesis of stable and nearly spherical M–AuNPs.

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It is known that AuNPs have been used as a model for understanding nanoparticle– protein interactions because of their promise for diverse biomedical applications, including

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their use as probes in many biodiagnostic systems, as well as photothermal and targeted drug delivery treatments of cancer [23, 24]. Albumin is the most abundant protein in plasma and

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the major soluble protein constituent present in the blood stream of vertebrates. It serves as a

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transport carrier of drugs due to its great ability to bind reversibly with a large variety of endogenous and exogenous ligands present in blood. This property has very important

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applications in cell culture, clinical diagnosis, electrophoresis chromatography and immune biochemistry [25, 26]. BSA is used as the model protein for drug delivery because of its

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medical importance, abundance, low cost, ease of purification, unusual ligand–binding

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properties and the fact that it shares almost identical structure, function, pH–dependent transitions with its human counterpart. It is also widely accepted in the pharmaceutical

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industry [27]. Therefore, the binding interaction of M–AuNPs with BSA will be an excellent model to understand the therapeutic applications of these bioconjugated nanomaterials in vivo.

For the first time, we report an environmentally friendly and rapid method, using

aqueous morin as reducing agent to synthesize M–AuNPs, which have been verified using XRD, UV–vis and TEM techniques. We also investigate the effects of reaction conditions such as morin volume, reaction temperature, time and pH on the rate of synthesis and stabilization of M–AuNPs. In view of the fact that nanoparticles synthesized with morin have a high surface area, enhanced binding interaction with BSA can be expected, leading to a rapid transfer of drugs to the tissues. This confirms the fundamental importance of studying in

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vitro binding interactions between BSA and morin synthesized Au nanoparticles. In this article, we present a spectroscopic approach to investigate the binding of M–AuNPs to BSA. For this, we studied the interaction information regarding quenching mechanisms, binding

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parameters, thermodynamic parameters, binding modes, binding site, effect of pH, and

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conformation.

2. Materials and Methods

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2.1. Materials

Chloroauric acid (HAuCl4·4H2O) was obtained from Sinopharm Chemical Reagent Co.,

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Ltd (Shanghai, China); morin (CAS Num: 654055–01–3) was obtained from Acros Organics (New Jersey, USA); BSA and warfarin were obtained from Sigma–Aldrich (St. Louis, MO,

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USA) and a solution was prepared in 0.1 mol·L–1 phosphate buffered solution (PBS, pH = 7.4); ibuprofen was obtained from Hubei biocause pharmaceutical Co., Ltd. (Hubei, China;

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the purity no less than 99.7%); All other reagents used in these experiments were of analytical

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grade; deionized water was used throughout the experiments.

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2.2. Synthesis of morin–conjugated Au Nanoparticles Morin (2.5×10–4 mol·L–1) was prepared using deionized water as a solvent. M–AuNPs

were synthesized in a according to the following procedure. Five different amounts of aqueous morin (1.0, 1.5, 2.0, 2.5, and 3.0 mL) were separately and added to five different 5 mL solutions of 5×10–4 mol·L–1 aqueous HAuCl4 at 75 °C under reflux conditions while stirring vigorously Multiple color changes of the solution were observed within 5 min. Then the solution was allowed to cool to room temperature under stirring. The resulting aqueous M–AuNPs were stored 4 °C for further characterization. 2.3. Sample Preparation for M–AuNPs Conjugated BSA BSA protein solution (concentration 5×10–6 mol·L–1) used in the experiments, consisted of 0.1 mol·L–1 of sodium phosphate at pH 7.4. The concentrations of the nanoparticles varied 5

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from 0 to 1.5×10–10 mol·L–1. M–AuNPs–BSA conjugates with different molar ratios of BSA/M–AuNPs were prepared by mixing the BSA solution with different concentration of M–AuNPs, and incubated at 4 °C for at least two hours before the spectra were obtained.

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2.4. Equipment and spectral measurements XRD (D8 Advanced, Germany) used Cu Kα radiation in a θ–2θ configuration; UV–vis

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spectra was recorded at room temperature on a U–3010 spectrophotometer (Hitachi, Japan) equipped with 1.0 cm quartz cells; TEM images were taken on a JEM–2100F transmission

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electron microscope operating at an acceleration voltage of 200 keV; fluorescence spectra

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were recorded on F–4500 Spectrofluorimeter (Hitachi, Japan) equipped with 1.0 cm quartz cells and a thermostat bath. The widths of both the excitation slit and the emission slit were

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set to 5.0 nm, and the excitation wavelength was 295 nm; CD spectra were measured with a Jasco J–810 Spectropolarimeter (Jasco, Tokyo, Japan) at room temperature Quartz cells

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3. Results and Discussion

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having path lengths of 1.0 cm were used at a scanning speed of 200 nm/min.

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3.1. Characterization of M–AuNPs 3.1.1. XRD analysis

X–ray diffraction was used to confirm the crystalline nature of the nanoparticles. The

X–ray diffraction pattern for the M–AuNPs films deposited on a glass substrate (Supporting Information, Figure S1). According to the XRD pattern of M–AuNPs, it is clear that five diffraction peaks are observed. They can be indexed as 38.20° (1 1 1), 44.44° (2 0 0), 64.82° (2 2 0), 77.74° (3 1 1), 81.75° (2 2 2) reflections of face–centered–cubic (fcc) structure. These agreed well with the reported standards in JCPDS file no.04–0784. This study reveals that the synthesized M–AuNPs are of pure crystalline Au. 3.1.2. TEM analysis

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Transmission electron microscopy (TEM) has been used to identify the morphology and size of nanoparticles. The TEM images of M–AuNPs presented in Figure 2 show that the M–AuNPs that were formed were monodispesed, well defined and almost spherical (Figure

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2A–D). This indicated the stabilization of the nanoparticles by the reducing agent morin. The NPs size distribution was analyzed using the ImageJ software [17] (See Figure 2A), and the

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resulting data were plotted in histograms, which were displayed in Figure 2E. From the histograms, we found that M–AuNPs form in the narrow size range of 30–40 nm. However,

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the mean particle size measured for the M–AuNPs was observed to be 35 nm.

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3.1.3. UV-vis analysis

3.1.3.1. Effect of morin quantity about M–AuNPs synthesis

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UV–vis spectroscopy is an important technique to obtain information on the formation and stabilization of aqueous metal nanoparticles. One of the most important features in optical

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absorbance spectra of metal nanoparticles is surface plasmon resonance (SPR), which is due

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to collective oscillation of free conduction electrons around the surface mode of the

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nanoparticles. According to Mie theory [28], metallic nanoparticles with a radius much smaller than the incident wavelength of light strongly absorb strongly certain wavelengths because of the resonant excitation of the surface plasmons resonance. Furthermore, the position and intensity of the absorption bands (SPR bands) is strongly influenced by particle size and shape, the surrounding medium, and the boundary conditions imposed by adjacent metallic particles [29].

Figure 3 shows the SPR bands of M–AuNPs in the UV–vis spectra at room temperature when systematically increasing the volume of aqueous morin in the solution of HAuCl4 (5×10–4 mol·L–1, 5 mL). It was apparent that the addition of aqueous morin resulted in the SPR peak for M–AuNPs underwent a red–shift after a blue–shift, indicating a decrease–increase phenomenon in the particle size [17]. Thus morin plays a crucial role in the 7

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size distribution of synthesized M–AuNPs. The broad SPR peak (Figure 3A) obtained with 1 mL of aqueous morin was due to to the formation of large anisotropic particles resulting from the absence of sufficient morin for capping and efficient stabilization [30]. According to Mie

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theory, the sharp and symmetrical SPR band for M–AuNPs shown in Figures 3B–E, suggested the formation of spherical nanoparticles which were dispersed into the aqueous

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solution with no sign of aggregation. For a volume of morin of 2 mL (Figure 3C), the SPR of the Au nanoparticles formed corresponded to 530 nm, which indicated that the nanoparticles

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size reached a minimum of about 35 nm. For volumes of morin of 2.5 mL and 3 mL, the SPR

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bands of the M–AuNPs increased from 532 nm to 536 nm and the corresponding SPR intensities were found to increase, which suggested the formation of large nanoparticles and

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the increase in the yield of nanoparticles [31]. For larger Au nanoparticles the extinction cross section is dependent on higher–order multipole modes within the full Mie equation and the

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extinction spectrum is then also dominated by quadrupole and octopole absorption as well as

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scattering. These higher oscillation modes explicitly depend on the particle size and increasing size causes the plasmon absorption maximum to shift to longer wavelength and the

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bandwidth to increase [32].

3.1.3.2. Effect of reaction temperature and time about M–AuNPs synthesis Figure 4 shows M–AuNPs synthesized in aqueous morin (2 mL) at 25 °C, 50 °C, 75 °C,

100 °C within 60 minutes. It was clear that the reductions were very slow, SPR intensity were very poor at 25 °C and 50 °C as time increases, while at 100 °C the reaction was quite faster, but stability was very poor. Phenomenon of successive decline–rise–decline in absorbance value, was observed, suggesting the formation of Poly–Au nanoparticles in the aqueous solutions as reaction time increased. Therefore, longer reaction time is disadvantageous for biosynthetic procedures [17]. However, the absorbance value was maximum at 5 min, which indicated that Au3+ ions were fully reduced to Au0 at 75 °C, in addition the M–AuNPs was

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stable for at least two months. Thus, temperature and time are found to play a critical role in the size, dispersity and morphology of M–AuNPs. 3.1.3.3. Effect of pH about M–AuNPs synthesis

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The experiment was also carried out by varying the pH of the chloroauric acid solution and keeping the amount of morin (2 mL) constant at 75 °C under reflux conditions. The

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UV–vis absorption spectra of M–AuNPs at different pHs from 3 to 10 are provided (Supporting Information, Figure S2). The formation of M–AuNPs is considered to be difficult

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at high pH (Supporting Information, Figure S2 A). Decrease in the pH of the system favored

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the formation of M–AuNPs. At pH = 5, formation of the M–AuNPs is remarkable. However, peak intensity increased with decreasing pH, suggesting an increase in the yield of the

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reaction formation of nanoparticles [31]. We found a change of wavelength from 526 nm to 536 nm when varying pH from 3 to 10 (Supporting Information, Figure S2 B), but absorbance

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the best synthesis condition.

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intensity was larger at pH = 5 as well as the SPR peak centered at 536 nm, which suggesting

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3.2. Characteristics of BSA with M–AuNPs 3.2.1. Fluorescence quenching mechanism Fluorescence spectroscopy is a useful tool to obtain information on the conformational

changes of protein. The different mechanisms of fluorescence quenching can be divided into static quenching and dynamic quenching. Static and dynamic quenchings can be distinguished by their different dependence on temperature and viscosity or preferably by lifetime measurements [33]. Higher temperatures result in faster diffusion and hence larger amounts of dynamic quenching; higher temperatures will typically result in the dissociation of weakly bound complexes and, hence, smaller amounts of static quenching. In other words, increasing temperature increases dynamic quenching while it decreases static quenching. In this work, the concentration of BSA solutions were stabilized at 5.0×10–6 mol·L–1, and 9

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the concentrations of M–AuNPs varied from 0 to 1.5×10–10 mol·L–1 with increments of 0.15×10–10 mol·L–1. The effect of M–AuNPs on BSA fluorescence intensity at 298 K is shown in Figure 5. Figure 5 shows that a progressive decrease in the fluorescence intensity is

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caused by quenching. In order to understand the quenching mechanism, we studied the interaction between M–AuNPs and BSA at different temperatures. The fluorescence

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quenching data were analyzed to obtain the quenching constant by using the well–known Stern–Volmer equation [19]:

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F0 = 1 + K SV [Q] F

(1)

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Where F0 and F denote the steady–state fluorescence intensities in the absence and presence

[Q] is the concentration of M–AuNPs.

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of the quencher (M–AuNPs), respectively, Ksv is the Stern–Volmer quenching constant, and

From the Stern–Volmer equation, we calculated the Stern–Volmer quenching constant

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Ksv at four different temperatures and the data were compiled in Table 1. The result showed

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that the Stern–Volmer quenching constant Ksv was inversely correlated with temperature,

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which indicated that the probable quenching mechanism was the formation of M–AuNPs–BSA complex (static quenching) rather than by dynamic quenching.

3.2.2. UV–vis absorption spectra

Dynamic quenching can also be distinguished from static quenching through the careful

examination of the fluorophore examination spectra. Collisional quenching only affects the excited states of the fluorophores and thus no changes in the absorption spectra are expected. In contrast, ground–state complex formation will frequently result in perturbation of the absorption spectrum of the fluorophore [34]. Therefore, we used the difference absorption spectroscopy to validate the quenching mechanism. The absorption spectra of BSA in absence and presence of different concentration M–AuNPs at room temperature (298 K) are shown in Supporting Information, Figure S3 A. The UV–vis absorption spectra of M–AuNPs (Figure 10

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S3 B, curve b) and the difference absorption spectra (Figure S3 B, curve d) between M–AuNP–BSA complex (Figure S3 B, curve c) and BSA (Figure S3 B, curve a) at the same concentration could not be superposed. This result confirmed that the probable quenching

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mechanism of fluorescence of BSA by M–AuNPs was a static quenching procedure.

3.2.3. Binding parameters

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Since the quenching is a static mechanism, the data could be analyzed according to the modified Stern–Volmer equation [34, 35].

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F0 1 1 1 = + ΔF f a K a [Q] fa

(2)

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Where ΔF is the difference in fluorescence in the absence and presence of the quencher at concentration [Q], Ka is the effective quenching constant for the accessible fluorophores,

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which is analogous to associative binding constants for the quencher (M–AuNPs)–acceptor (BSA) system, and fa is the fraction of accessible fluorescence. Therefore, the modified

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Stern–Volmer equation was applied to determine Ka by a linear regression of F0/ΔF versus

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1/[Q], and the results were listed in Table 1. The data shows that the decreasing trend of Ka

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with increasing temperature was in accord with Ksv’s dependence on temperature, which further confirmed that the fluorescence quenching of BSA was static quenching. When a small molecule binds independently to a set of equivalent sites on a

macromolecule, the apparent binding constant (Kb) and the number of binding sites (n) can be obtained according to the following equation [36]:

log

F0 − F = log K b + n log[Q] F

(3)

where Kb is the binding constant and n is the number of binding sites per BSA. According to Equation 3, the binding constant Kb and the number of binding sites n can be obtained (Table 2). We found that the binding constant Kb decreased with increasing temperature, indicating a reduction of the binding capacity of M–AuNPs to BSA. Increasing temperature resulted in an 11

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increase of the diffusion coefficient and a reduction of the stability of the M–AuNPs–BSA complex. The values of n are approximately equal to 1 suggesting that there is a single class of binding sites on BSA for M–AuNPs. The high linear correlation coefficient at different

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temperatures, indicated that the mathematical model (Eq. 3) used to characterize the experiment was suitable to study the interaction between M–AuNPs and BSA. We also have

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analyzed the interaction free morin drug with BSA [19], by comparing the binding constants Ka and Kb, we find that the Ka and Kb of M–AuNPs and BSA are higher than those of free

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morin drug and BSA. Hence, we can infer that M–AuNPs enhance the binding force with

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BSA. 3.2.4. Thermodynamic parameters and binding mode

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The interaction forces between drugs and biomolecules may include electrostatic interactions, multiple hydrogen bonds, van der Waals interactions, hydrophobic and steric

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contacts within the antibody–binding site, and so on [37]. Ross and Subramanian have

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characterized the signs and magnitudes of the thermodynamic parameters (ΔH and ΔS) associated with various individual kinds of interactions that may take place in protein

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association processes. That is, if ΔH > 0 and ΔS > 0, the main force is hydrophobic interaction. If ΔH < 0 and ΔS < 0, van der Waals and hydrogen–bonding interactions play major roles in the reaction. Electrostatic forces are more important when ΔH < 0 and ΔS > 0 [38]. If the enthalpy change (ΔH) does not vary significantly over the studied temperature

range, then its value and that of entropy change (ΔS) can be evaluated from the van’t Hoff equation [39, 40]: ln K = −

ΔH ΔS + RT R

(4)

where K is analogous to the binding constant at the corresponding temperature and R is the gas constant. In order to elucidate the interaction between M–AuNPs and BSA (Supporting Information, Figure S4), the thermodynamic parameters were calculated from the van’t Hoff 12

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plots. The enthalpy change (ΔH) was calculated from the slope of the van’t Hoff relationship. The free energy change (ΔG) was then estimated from the following relationship:

ΔG = ΔH − TΔS = − RT ln K

(5)

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The thermodynamic parameters for the interaction of M–AuNPs with BSA were shown in Table 2. A negative value for ΔH indicates the exothermicity of the binding reaction while a

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negative free energy change (ΔG) represents a thermodynamically favorable process. A positive entropy change (ΔS) and negative enthalpy change (ΔH) generally indicate that

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electrostatic forces play crucial role in the reaction process between M–AuNPs and BSA.

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3.2.5. Identification of the binding site of M–AuNPs on BSA

To ascertain M–AuNPs binding location on the BSA, competitive binding experiments

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have been carried out using drugs (warfarin and ibuprofen) that specifically bind to a known site or domain. It is reported that the warfarin site marker binds to the subdomain IIA

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(Sudlow’s site I) and the ibuprofen binds to the subdomain IIIA (Sudlow’s site II) [41, 42].

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Therefore, information about the M–AuNPs binding site can be gained by monitoring the changes in the fluorescence of M–AuNPs bound BSA.

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In the site competitive replacement experiments, M–AuNPs was gradually titrated with a

1:1 solution of BSA and site marker. The addition of the site marker (warfarin or ibuprofen) drew the fluorescence intensity below what it was without the marker (Supporting Information, Figure S5). To compare the effects of warfarin and ibuprofen on the binding of M–AuNPs to BSA, the binding constants in the presence of site markers and other parameters were calculated using the modified Stern–Volmer equation and the Double–Logarithm equation (Table 3). The results showed that the binding constant in the presence of warfarin was much smaller than in its absence, whereas the binding constant in the presence of ibuprofen was almost the same as without it. The results suggest that the binding site of M–AuNPs on BSA is the same as warfarin and therefore, M–AuNPs is mainly located in

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region of subdomain IIA (Sudlow site I). 3.2.6. Synchronous fluorescence spectra

The synchronous fluorescence spectra give information about the molecular environment

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in the vicinity of the chromospheres molecules. When the D–value (Δλ) between excitation and emission wavelength is selected at 15 nm or 60 nm, the synchronous fluorescence gives

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the characteristic information of tyrosine or tryptophan residues [33]. The red shift indicates that the residues move to a more hydrophilic environment, while the blue shift suggests that a

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more hydrophobic environment appear around the residues [43]. The effect of M–AuNPs on

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BSA synchronous fluorescence spectroscopy is shown in Figure 6.

It was apparent that the maximum emission wavelength had a blue shifts (from 289.8 nm

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to 287 nm) at the investigated concentrations range when Δλ = 15 nm (Figure 6A), and the maximum emission wavelength had a red shifts (from 282.6 nm to 285 nm) when Δλ = 60 nm

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(Figure 6B). The blue shift indicated that the tyrosine residues in both BSA and M–AuNPs

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moved to a more hydrophobic environment. The red shift suggested that the tryptophan residues in both M–AuNPs and BSA moved to a more polar environment. These observations

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demonstrate that M–AuNPs interact with both the tyrosine and the tryptophan residue of BSA. This indicates a change in the molecular conformation of BSA as well as a modification of the microenvironment of the amino acid residues. 3.2.7. Far–UV CD spectra

Circular dichroism spectrum is a sensitive technique to monitor the conformation of

peptides and proteins because the structural characterization of proteins depends greatly on the remarkable sensitivity of CD in far–UV region [44]. The CD spectrum of BSA has two negative bands at 208 nm and 220 nm, which characterize the transition of π–π* and n–π* of the α–helical structure [45]. To ascertain the possible influence of M–AuNPs binding on the secondary structure of BSA, we measured the far–UV CD spectra in the range of 200–260 nm.

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The CD results were expressed in terms of mean residue ellipticity (MRE) in deg·cm2·dmol–1 according to the following equation [19]: observed CD (mdeg ) c p nl × 10

(6)

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MRE =

where cp is the molar concentration of the protein (BSA), n is the number of amino acid

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residues (582) and l is the path length (1 cm).

It could be observed that BSA alone showed very neat peaks at 208 and 222 nm in

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Figure 7, whereas the BSA with M–AuNPs showed a decrease in ellipticity (curve A–B), which indicated that the molecular structure of BSA was changed by M–AuNPs.

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The α–helix contents of free and combined BSA were calculated from MRE values at 208 nm using the equation [43]: − MRE 208 − 4000 × 100 33,000 − 4000

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α − Helix (%) =

(7)

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where MRE208 is the observed MRE value at 208 nm, 4000 is the MRE of the β–form and

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208 nm.

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random coil conformation cross at 208 nm, and 33000 is the MRE value of a pure α–helix at

The quantitative analysis of the α–helix content was based on Equations (6) and (7). A

major reduction of the α–helix from 56.6% (free BSA) to 53.5% (M–AuNPs–BSA complex) was observed, meaning that the peptide strand unfolded even more, while the hydrophobicity became increased. Furthermore, the CD spectra of bovine serum albumin in presence of M–AuNPs indicate that the structure of BSA is predominantly helical and that the secondary structure of bovine serum albumin have been changed. 3.2.8. Effect of pH on the binding of M–AuNPs and BSA

Solution pH plays a critically important role in a variety of chemical and biological processes. In this study, we investigated the pH (phosphate buffer solution) effect on the conformational changes of BSA in the bioconjugates since albumin was known to undergo 15

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reversible conformational isomerization as a consequence of pH changes [46]. The pH–dependent forms of albumin are classified as N, for normal or native form, which is predominant at pH range 4.5–7.5; F, for the fast migrating form produced abruptly at pH

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values less than 4.5; B, for the basic form occurring above pH 8 and E, for the expanded form at pH less than 3.5. The effect of pH on the binding of M–AuNPs and BSA (Supporting

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Information, Figure S6) are shown in Table 4. We found that both the quenching and binding constant were the highest at pH 7.4, revealing maximum adsorption of albumin molecules on

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the surface of M–AuNPs. In addition, the quenching constants and binding constants in strong

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acid or strong alkali solutions were much lower than they were a neutral pH, indicating that this type of solutions of extreme pH are unfavorable to the binding of M–AuNPs with BSA.

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These results show that the pH of the medium greatly influences the conformational changes of albumin, and they suggest different intrinsic conformational state of the protein at different

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4. Conclusions

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pH values.

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In summary, we have introduced a simple method to synthesize stable and nearly spherical Au nanoparticles (M–AuNPs) by using aqueous solutions of the the nontoxic, ecofriendly, and anti–tumorigenic drug morin as a reducing agent. The interaction between M–AuNPs and BSA was evaluated by fluorescence, UV–vis, and CD spectroscopy. The mechanism study proved that the fluorescence quenching of BSA by M–AuNPs is a result of the formation of M–AuNPs–BSA complex. It was found that the interaction between M–AuNPs and BSA was spontaneous and that electrostatic interactions played key role in the reaction process. The analysis of synchronous fluorescence, and CD spectra indicated that the secondary structure of BSA molecules changed dramatically in the presence of M–AuNPs. The effect of pH indicated that acid solutions and alkali solutions were unfavorable to the binding of M–AuNPs with BSA, and revealed that the maximum adsorption of albumin 16

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molecules on the surface of M–AuNPs at pH 7.4. The results of the bioconjugation of water–dispersed M–AuNPs (D 35 nm) with protein provide indispensable proof of their applications in bioanalysis. This work can be of importance in the field of clinical research as

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it provides information about nanoparticles and the theoretical basis for new drug designing.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (No.

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21273065), and the Research Foundation of Education Bureau of Hubei Province, China (Nos.

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Q20122205, B20132502, T201311).

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[7] M.R. Papasani, G. Wang, R.A. Hill, Nanomedicine 8 (2012) 804–814. [8] C. Schulze, U.F. Schaefer, C.A. Ruge, W. Wohlleben, C.M. Lehr, Eur. J. Pharm. Biopharm. 77 (2011) 376–383. [9] A. Bhogale, N. Patel, P. Sarpotdar, J. Mariam, P.M. Dongre, A. Miotello, D.C. Kothari, Colloids Surf. B 102 (2013) 257–264. [10] F. Tang, L. Li, D. Chen, Adv. Mater. 24 (2012) 1504–1534. 17

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[22] Z. Marković, D. Milenković, J. Ðorović, J.M.D. Marković, V. Stepanić, B. Lučić, D. Amić, Food Chem. 134 (2012) 1754–1760. [23] T. Sen, S. Mandal, S. Haldar, K. Chattopadhyay, A. Patra, J. Phys. Chem. C 115 (2011) 24037–24044. [24] S. Naveenraj, S. Anandan, A. Kathiravan, R. Renganathan, M. Ashokkumar, J. Pharm.

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e17230. [27] E. Ahmad, P. Sen, R.H. Khan, Cell Biochem. Biophys. 61 (2011) 313–325.

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[36] G.W. Zhang, L. Wang, J.H. Pan, J. Agric. Food Chem. 60 (2012) 2721–2729. [37] X.L. Li, Y.J. Hu, H. Wang, B.Q. Yu, H.L. Yue, Biomacromolecules 13 (2012) 873−880. [38] X.G. Pan, P.F. Qin, R.T. Liu, J. Wang, J. Agric. Food Chem. 59 (2011) 6650–6656. [39] Y. Zhang, X.R. Liu, Z.D. Qi, F.L Jiang, Y. Liu, J. Solution Chem. 41 (2012) 351–366. [40] Y. Zhang, J.H. Li, X.R. Liu, F.L. Jiang, Y. Liu, J. Fluoresc. 21 (2011) 475–485. [41] G. Sudlow, D.J. Birkett, D.N. Wade, Mol. Pharmacol. 11 (1975) 824–832. [42] G. Sudlow, D.J. Birkett, D.N. Wade, Mol. Pharmacol. 12 (1976) 1052–1061. [43] D. Huang, F. Geng, Y.H. Liu, X.Q. Wang, J.J. Jiao, L. Yu, Colloids Surf. A 392 (2011) 191–197. [44] Y.J. Hu, Y. Liu, X.S. Shen, X.Y. Fang, S.S. Qu, J. Mol. Struct. 738 (2005) 143–147.

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Ac ce p

te

d

M

an

us

cr

ip t

[46] L. Shang, Y.Z. Wang, J.G. Jiang, S.J. Dong, Langmuir 23 (2007) 2714–2721.

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Figure Captions: Figure 1. Chemical structure of morin. Figure 2. TEM images (A–D) and Histogram distribution (E) for M–AuNPs.

cr

shows variation of SPR band wavelength with quantity of morin.

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Figure 3. UV–vis absorption spectra of M–AuNPs prepared using morin (1–3 mL), the inset

Figure 4. Absorption spectra of M–AuNPs at different temperature (25 °C to 100 °C) within

us

60 min.

an

Figure 5. Fluorescence emission spectra of BSA in the presence of different M–AuNPs at 298

K. Inset: Relative fluorescence intensity for BSA of the M–AuNPs for the different

M

concentrations. c (BSA) = 5×10–6 mol·L–1; c (M–AuNPs)/(10–10 mol·L–1), A–K: from 0.0 to 1.5 at increments of 0.15.

d

Figure 6. Synchronous fluorescence spectrum of M–AuNPs–BSA system, (A) Δλ = 15 nm;

Ac ce p

1.5 at increments of 0.15.

te

(B) Δλ = 60 nm. c (BSA) = 5×10–6 mol·L–1; c (M–AuNPs)/ (10–10 mol·L–1), A–K: from 0.0 to

Figure 7. CD spectra of (A) BSA and (B) M–AuNPs–BSA.

21

Page 21 of 34

Table 1. Binding constants for the interaction of M–AuNPs with BSA at various

temperatures.

S.D. b

10–10Ka (L·mol–1)

Ra

S.D. b

292

1.81

0.998 4

0.0411

1.21

0.999 8

0.0247

298

1.73

0.997 0

0.0541

1.14

0.999 0

0.0574

304

1.66

0.996 1

0.0595

1.06

0.999 1

0.0535

310

1.60

0.995 5

0.0617

0.998 1

0.0815

us

Ra

an

0.99

te

d

M

R is the correlation coefficient. b S.D. is standard deviation.

Ac ce p

a

10–10Ksv (L·mol–1)

ip t

T (K)

Modified Stern–Volmer Method

cr

Stern–Volmer Method

22

Page 22 of 34

Table 2. Binding constants, binding sites and relative thermodynamic parameters of

M–AuNPs–BSA system at pH = 7.4. 10–10 Kb –1

n

Ra

ΔH (kJ·mol–1) ΔG (kJ·mol–1)

(L·mol )

292

1.43

1.22

0.996 9

–56.36

298

1.37

1.31

0.997 6

–57.37 –8.53

1.41

0.998 2

310

1.24

1.43

0.994 4

–58.34

us

1.30

163.8

–59.31

te

d

M

an

R is the correlation coefficient.

Ac ce p

a

304

(J·mol–1·K–1)

cr

(K)

ΔS

ip t

T

23

Page 23 of 34

Table 3. Binding constants of competitive experiments for M–AuNPs–BSA system at 298 K.

Modified Stern–Volmer Method R

(L·mol–1)

b

S.D.

10–10 Kb

Ra

(L·mol–1)

1.14

0.998 9

0.0574

1.37

Ibuprofen

1.06

0.999 6

0.0360

1.42

Warfarin

0.58

0.999 8

0.0336

1.21

0.997 5

0.0238

0.997 2

0.0255

0.998 2

0.0226

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/ (Blank)

S.D.b

te

d

M

an

R is the correlation coefficient. b S.D. is standard deviation.

Ac ce p

a

a

ip t

10–10 Ka

cr

Site marker

Double–Logarithm Method

24

Page 24 of 34

Table 4. Effect of pH on the binding of M–AuNPs and BSA.

Stern–Volmer Method

Double–Logarithm Method 10–10Kb (L·mol–1)

n

Ra

3.4

1.29

0.987 7

0.95

1.4 ± 0.04

0.994 0

5.4

1.31

0.993 2

0.96

1.3 ± 0.01

0.998 8

7.4

1.73

0.994 8

1.37

0.997 6

9.4

1.35

0.996 5

1.16

11.4

1.10

0.985 1

0.86

ip t

Ra

an

us

1.3 ± 0.02

0.996 7

1.4 ± 0.03

0.994 5

te

d

M

R is the correlation coefficient.

1.4 ± 0.02

Ac ce p

a

10–10Ksv (L·mol–1)

cr

pH

25

Page 25 of 34

HO O

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OH

OH

OH O

cr

OH

Ac ce p

te

d

M

an

us

Figure 1

26

Page 26 of 34

ip t

B

an

us

cr

A

M

C 70

E

d

60 50 40

te

Counts

D

30

Ac ce p

20 10

0

15

20

25

30

35

40

Particle diameter (nm)

45

50

55

Figure 2

27

Page 27 of 34

1.25

Abs

1.00

538 536 534 532 530

0.75

1.0

1.5

2.0

Volume (mL)

A 500

600

Wavelength (nm)

700

800

an

400

us

E 0.25

3.0

cr

0.50

2.5

ip t

Wavelength (nm)

540

Ac ce p

te

d

M

Figure 3

28

Page 28 of 34

1.1 1.0

ip t

Abs

0.9 0.8 0.7

10

20

30

Time (min)

40

50

60

an

0

50℃ 100℃

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25℃ 75℃

0.5 0.4

cr

0.6

Ac ce p

te

d

M

Figure 4

29

Page 29 of 34

0.8

K 0.6

Y = 0.9832-0.4998 * X R = -0.9991

0.8

0.6

0.4

0.2

0.0

0.3

0 .6

0.9

1010 [Q] (m ol/L)

1.2

cr

0.4

1.5

0.2 0.0 340

360

380

400

Wa velength (nm)

420

440

an

320

us

Fluo rescence intensity (a.u.)

A

1.0

ip t

Fluore sce nc e inte nsity (a .u. )

1.0

Ac ce p

te

d

M

Figure 5

30

Page 30 of 34

(A)

A

0.15

cr

0.10

0.05

0.00 270

280

290

M

K

Ac ce p 0.0

(B)

d

0.6

0.2

310

te

0.8

A

0.4

300

an

Wavelength (nm)

1.0

Fluorescence intensity (a.u.)

ip t

K

us

Fluorescence intensity (a.u.)

0.20

250

260

270

280

290

Wavelength (nm)

300

310

Figure 6

31

Page 31 of 34

20

-40

cr

A BSA B M-AuNPs-BSA

B -60

A

200

210

ip t

-20

220

230

us

[θ](degcm2dmol-1)

0

240

250

260

an

Wavelength (nm)

Ac ce p

te

d

M

Figure7

32

Page 32 of 34

Ac ce p

te

d

M

an

us

cr

ip t

Graphical Abstract

33

Page 33 of 34

Highlights

Ac ce p

te

d

M

an

us

cr

ip t

Morin–directed green synthesis of Au nanoparticles (AuNPs). Morin retains its biological activity after formation of Au nanoparticles (M–AuNPs). The interactions of M–AuNPs with BSA, including determining a series of binding parameters, have been explored.

34

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