Accepted Manuscript Title: Study on the interaction between bovine serum albumin and starch nanoparticles prepared by isoamylolysis and recrystallization Author: Na Ji Chao Qiu Xiaojing Li Liu Xiong Qingjie Sun PII: DOI: Reference:
S0927-7765(15)00150-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.03.016 COLSUB 6956
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-10-2014 11-2-2015 5-3-2015
Please cite this article as: N. Ji, C. Qiu, X. Li, L. Xiong, Q. Sun, Study on the interaction between bovine serum albumin and starch nanoparticles prepared by isoamylolysis and recrystallization, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.03.016 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.
Study on the interaction between bovine serum albumin and starch nanoparticles prepared
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by isoamylolysis and recrystallization
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Na Ji Chao Qiu Xiaojing Li Liu Xiong Qingjie Sun*
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School of Food Science and Engineering, Qingdao Agricultural University
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(Qingdao, Shandong Province, 266109, China)
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*Correspondence
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[email protected]), School of Food Science and Engineering, Qingdao Agricultural University,
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266109, 700 Changcheng Road, Chengyang District, Qingdao, China.
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Abstract: The current study primarily investigated the interaction of bovine serum albumin (BSA)
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with starch nanoparticles (SNPs) prepared by isoamylolysis and recrystallization using UV-visible,
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fluorescence, transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and
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circular dichroism (CD). The enhanced absorbance observed by UV-visible spectroscopy and
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decreased intensity of fluorescence spectroscopy suggested that BSA could bind to SNPs and form
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a BSA-SNP complex. The synchronous fluorescence spectra revealed that the emission maximum
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Fax:
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e-mail:
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of Tyr residue (at Δλ = 15 nm) was red-shifted at the investigated concentrations range, indicating that the conformation of BSA was changed. Quenching parameters showed that the quenching effect of SNPs was static quenching. TEM images showed that the SNPs were surrounded by protein coronae, indicating that nanoparticle-protein complexes had formed. The FTIR and CD
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characterization indicated that the SNPs induced structural changes in the secondary structure of
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BSA.
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Keywords: starch nanoparticles, bovine serum albumin, interaction, spectroscopy
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1. Introduction 1
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Nanomaterials are at the forefront of the swiftly-developing field of commercial exploration of nanotechnology [1]. The advent of nanotechnology has accelerated the evolution of materials
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with many exceptional size-dependent properties for use in numerous biological and medical
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applications [2-3]. When a nanomaterial enters a physiological environment, its surface is
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immediately covered by a layer of proteins, forming what is known as the protein “corona” [4].
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The adsorbed proteins on nanoparticle surfaces and the formation of the protein corona, have been
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identified as determining their fate in vivo [5]. Therefore, it is essential to understand the
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interactions of nanoparticles and proteins.
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As a water-soluble protein, serum albumin is the most abundant and basic protein of blood plasma. It is primarily involved in the transport of substances in the blood that are poorly soluble
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in water, including drugs, and also in the disposition of endogenous and exogenous compounds
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present in blood [6]. Albumin is also reported to associate with nanoscale particles to promote
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their transportation through cells [7-8]. Bovine serum albumin (BSA) is one of the most
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extensively-studied proteins and has structural homology to human serum albumin as a substitute
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for research [9]. As a model protein, BSA has two tryptophans, Trp-134 and Trp-212 [10] that possess intrinsic fluorescence and have commonly been utilized to study the interactions between BSA and nanoparticles [11-13].
The conformational behavior of albumin in conjugation with nanoparticles is of great
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importance in numerous biomedical applications, particularly drug delivery and receptor targeting.
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However, the structural changes of protein after conjugation with nanoparticles are not very clear
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and require some basic understanding before they can be employed for biomedical applications.
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At present, many researchers have reported their studies on the interaction of BSA with metal NPs 2
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such as Ag [14], and Au [15-16]. On the other hand, a certain amount of literature is available on
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the interaction of metal oxide nanoparticles, such as ZnO [17-18] and TiO2 [19] with BSA. Starch
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nanoparticles (SNPs), a novel product derived from starch, are widely used as a carrier in drug
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delivery systems [20-22]. Recently, there has been growing interest in SNPs for their abundant
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availability of starch, comparatively easy processability, biocompatibility, biodegradability, and
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non-toxicity [23]. However, the interaction between SNPs and BSA has not been investigated. In
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this study, we investigate on this interaction by UV-vis, fluorescence, transmission electron
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microscopy (TEM), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic
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techniques.
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The objective of this study is to investigate interaction between BSA and SNPs prepared by
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isoamylolysis and recrystallization; it is expected to provide novel insight into SNPs interaction
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with BSA, and to provide theoretical guidance for their application in the field of biomedical
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materials and nano-sized drug carriers.
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2. Materials and Methods
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2.1. Materials
Waxy rice starch (approximately 1% amylose and 99% amylopectin) was obtained from
Zhucheng Xingmao Corn Development Co., Ltd. (Shangdong, China). Pullulanase (E.C.3.2.1.41, 6000 ASPU/g, 1.15 g/mL, where ASPU is defined as the amount of enzyme that liberates 1.0 mg
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glucose from starch in 1 min at pH 4.4 and 60 °C) was supplied by Novozymes (China)
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Investment Co. Ltd. (Bagsvaerd, Denmark). BSA pH 7.0 (MW 66 kDa and > 95% purity) was
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procured from Sigma-Aldrich (St. Louis, MO). All other chemicals used were of analytical grade.
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2.2. Preparation of SNPs 3
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The SNPs were prepared using the method described by Sun et al. [24], with some
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modifications. Typically, waxy rice starch (15 g, db) was dispersed in 100 mL of disodium
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hydrogen phosphate and citric acid buffer solution (pH 5.0). The starch slurry was cooked in
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boiling water with vigorous stirring for 30 min to fully gelatinize the starch. The temperature of
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the cooked waxy rice starch was adjusted to 58 °C and pullulanase (30 ASPU/g of dry starch) was
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added. After an 8 h incubation period at 58 °C, isoamylolysis was stopped by heating at 100 °C for
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30 min to inactivate the pullulanase and the slurry was centrifuged, followed by cooling to room
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temperature. Then, the solutions were stored at 4 °C for 8 h and retrogradated to form
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nanoparticles. The suspensions were washed several times with distilled water until neutrality was
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achieved and then freeze-dried to obtain SNPs.
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2.3. Interaction of BSA with SNPs
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Various concentrations of SNPs (0, 0.2, 0.4, 0.6, 0.8, and 1.0×10-9 mol/L) interacted with a
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constant BSA (3.0×10-6 mol/L) concentration for 30 minutes on a rotary shaker (300 rpm) at room
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temperature [25]. After the interaction period, characterization studies were performed with the
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help of UV-visible, fluorescence, TEM and CD spectroscopy. Molar concentration of SNPs is hypothesized from the molecular numbers of SNPs
multiplied by Avogadro's constant. The molecular numbers of SNPs were calculated by dividing the sample weight by the weight of one SNP. The weight of one SNP was calculated by the following equation:
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m = ρv
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Where ρ is the density of starch, v is the volume of SNPs, and the average particle size of SNPs is 50 nm. 4
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2.4. UV-visible spectroscopy After the interaction period, 3 mL of each sample was used to record UV-visible spectra
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using a UV-visible spectrophotometer (Shimadzu-2600, Kyoto, Japan) from 260 to 340 nm. The
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spectra obtained for the BSA-SNPs system were analyzed to evaluate the changes occurring in the
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absorption peak of BSA at 278 nm, a characteristic peak of BSA [26].
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2.5. Fluorescence spectra analysis
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Fluorescence of BSA after interaction with SNPs was measured using a F2500
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Spectrofluorimeter (Hitachi, Tokyo, Japan). An aliquot of 3.0×10-6 mol/L of BSA interacted with
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various concentrations of SNPs (0, 0.2, 0.4, 0.6, 0.8, and 1.0×10-9 mol/L) and 3 mL of the sample
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was subjected to analysis at the excitation wavelength of 278 nm and emission spectra in the range
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of 300-500 nm. A quartz cuvette (4 cm × 1 cm × 1 cm) with path length of 1 cm was used for
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measurements.
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2.6. Synchronous fluorescence measurement
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The synchronous fluorescence spectra of the mixture solutions of SNPs and BSA were
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recorded on a F2500 Spectrofluorimeter (Hitachi, Tokyo, Japan) at room temperature. For this measurement, the difference between excitation and emission wavelength (Δλ = λemission – λexcitation) was set at 15 nm for tyrosine residue or at 60 nm for tryptophan residue. Excitation and emission slit width was set at 2.5 nm. 2.7 Resonance light scattering (RLS) In the case of RLS scans, both excitation and emission wavelengths were scanned
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simultaneously in the wavelength range of 220–800 nm with Δλ = 0.
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2.8. Transmission electron microscopy (TEM) 5
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Transmission electron micrographs of SNPs and BSA-SNPs were taken with a 7650
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transmission electron microscope (Hitachi, Tokyo, Japan) with an acceleration voltage of 80 kV. A
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drop of BSA-conjugated SNPs dispersed in double-distilled water was placed on a copper grid and
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lyophilized.
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2.9 Fourier transform infrared (FTIR)
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The FTIR spectra of BSA and the mixture of BSA and SNPs were recorded on an FTIR
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spectrophotometer (NEXUS-870; Thermo Nicolet Corporation, Madison, WI, USA) within the
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wavenumber range of 4000–600 cm-1. The background obtained from the scan of KBr was
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automatically subtracted from the sample spectra.
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2.10. Circular dichroism (CD) spectroscopy
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Circular dichroism spectroscopy was performed to analyze the changes that occurred in the
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secondary structure of BSA. For CD analysis, an aliquot of 3.0×10-6 mol/L of BSA interacted with
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various concentrations of SNPs (0, 0.2, 0.4, 0.6, 0.8, and 1.0×10-9 mol/L). After 30 min incubation,
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CD spectra were analyzed using JASCOJ-715 CD spectroscopy (Welltech Enterprises, Maryland,
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United States). Change in secondary structure of protein was confirmed by comparing the CD spectrum data of the control sample to the test sample. 3. Results and discussion 3.1 UV-visible spectra
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The UV-visible absorption spectra of BSA in the absence and in presence of SNPs of
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different concentration are shown in Fig. 1. The maximum spectrum of BSA at 278 nm is caused
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by the π→π* transition of the aromatic amino acid residues [26]. With the increase in the
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concentration of SNPs, the maximum peak at 278 nm was increased, clearly indicating the 6
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formation of a ground state complex of BSA-SNPs. Our results were in agreement with Ravindran
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et al. [26], who reported that the ground state complex of Al2O3-BSA was formed by the
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adsorption of some BSA molecules on the Al2O3 nanoparticles. Wu et al. [28] reported that the
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interaction between BSA and ZnS quantum dots (QDs) led to the formation of QDs-BSA complex.
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3.2 Steady state fluorescence spectra
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The fluorescence spectra of the SNPs-BSA system are shown in Fig. 2. Due to the presence
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of two aromatic amino acids (tryptophan (Trp) and tyrosine (Tyr)), proteins are considered to have
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intrinsic fluorescence [29]. The fluorescence spectrum of BSA presented strong emission at 345
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nm when excited at 278 nm. The fluorescence intensity of BSA showed a significant gradual
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decrease accompanied by an increase in the concentration of SNPs and there was no shift of the
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emission wavelength. This was due to the adsorption of some BSA molecules on the SNPs’
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surface and the formation of the ground state complex of BSA-SNPs. This result showed that
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SNPs could quench the intrinsic fluorescence of protein strongly, and that there was an interaction
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between SNPs and BSA.
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3.3 Synchronous fluorescence spectra The synchronous fluorescence spectra were measured when the difference between excitation
wavelength and emission wavelength were stabilized at 15 or 60 nm, as shown in Fig. 3. Generally, the shift of the maximum emission wavelength reveals the alteration of polarity microenvironment
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around Tyr and Trp residues [30]. The synchronous fluorescence characteristic of BSA protein was
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studied at different scanning intervals. The fluorescence intensities of Tyr and Trp residues in BSA
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decreased regularly with increased concentrations of SNPs. At the same time, the emission
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maximum of Tyr residue (at Δλ = 15 nm) was red-shifted slightly from 283 nm to 284 nm at the 7
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investigated concentrations range, which indicated that a decrease in the hydrophobicity
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surrounding Tyr residue occurred in the presence of SNPs. The results indicated that the
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conformation of BSA was changed with the insertion of SNPs. Cao et al. [29] suggested that the
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maximum emission wavelength (at Δλ = 15 nm) had an obvious red shift with the addition of
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Glipizide. However, the synchronous fluorescence spectra of BSA at Δλ= 60 nm with various
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amounts of SNPs illustrated that the emission peaks did not shift, indicating that SNPs had little
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effect on the microenvironment of Trp residue in BSA.
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3.4 Resonance Light Scattering (RLS) spectra
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The RLS spectra of BSA and the BSA–SNPs system are shown in Fig. 4. With increasing SNPs concentration, the RLS intensity of all samples increased markedly. The generation of RLS
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spectrum is correlated with the formation of aggregates. Our results indicated that the added BSA
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could interact with SNPs in solution, forming a BSA–SNPs complex that would be expected to be
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an aggregate. Simiarly, Bhogale et al. [31] reported that the RLS spectra intensity of BSA
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increased significantly with the increasing concentration of ZnO nanoparticles, indicating the
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formation of aggregates. Li et al. [32] also reported that the RLS spectra intensity of BSA increased apparently while increasing the concentration of single-walled carbon nanotubes. 3.5 Mechanism of fluorescence quenching Fluorescence quenching is described by the Stern–Volmer equation [33]: F0/F = 1+KSV[Q] = 1+Kqτ0[Q]
(2)
where F0 and F are the fluorescence intensities of BSA in the absence and presence of the
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quencher (SNPs), respectively. KSV is the Stern–Volmer quenching constant, and [Q] is the
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concentration of SNPs, Kq is the quenching rate constant, τ0 is the average life time of molecules 8
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in the absence of SNPs and its value is about 10 -8 s.
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As can be seen from Fig. 5, F0/F has a linear correlation to the concentration of SNPs. According to Eq. (2), KSV values and Kq values are shown in Table 1. Generally, collision constant
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of the quenching on the BSA of the large diffusion is 2.0×1010 L (mol s)-1. From Table 1, we can
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see that Kq is about six orders higher than 2.0×1010 L (mol s)-1, indicating that the quenching
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mechanism of SNPs binding is static quenching. Xiao et al. [34] also reported that BSA
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fluorescence was statically quenched by polymer-hyperbranched poly (amine) ester.
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3.6 Binding constant and binding sites
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Fluorescence quenching of BSA also provides the information on the binding constant (K)
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and the number of binding sites (n) between the quencher and BSA accordingly to the equation
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[14]:
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(3)
Where F0 and F are the fluorescence intensity without and with quencher (SNPs), respectively. K is the binding constant, n is the number of binding sites, and [Q] is the
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log[(F0–F)/F] = logK + nlog[Q]
concentration of SNPs. According to the plot of log[(F0-F)/F] against log[SNPs], we calculated the number of binding sites to be 1.15, whereas for the binding constant we obtained 4.0×10 8 L mol-1 (Table 1). The calculated results illustrated that the interaction between BSA and SNPs has only one binding site, which was in accordance with previous reports [35].
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3.7 Binding force
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Thermodynamic parameters are usually applied to judge the categories of binding force. Enthalpy
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changes (ΔH) in reactions can be considered as constants due to no obvious changes in
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temperature. The thermodynamic parameters could be calculated by the van’s Hoff equation: 9
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ΔG = –RTlnK
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(4)
lnK2/K1 = [(1/T1 – 1/T2)ΔH/R]
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(5)
ΔS = (ΔH – ΔG)/T
(6)
where ΔH, ΔG and ΔS denote enthalpy change, free enthalpy change and entropy change,
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respectively, K is the binding constant, at the corresponding temperature. R is the gas constant,
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8.314 Jmol-1 K-1, and T is experimental temperature (K). Generally speaking, when ΔH < 0 and
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ΔS > 0, the main force is due to electrostatic interactions; when ΔH < 0 and ΔS < 0, the main force
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is due to van der Waals or hydrogen bonding, and when ΔH > 0 and ΔS > 0, the main force is due
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to hydrophobic interactions. Values of ΔH, ΔG, and ΔS are shown in Table 1, and the negative
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values of ΔH and ΔS suggest that the hydrogen bond and van der Waals forces play major roles in
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the binding process.
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3.8 Morphology of SNPs and BSA-SNPs complex
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The size and morphology of SNPs and BSA-SNPs were characterized by TEM (Fig. 6). The TEM of the SNPs exhibited shapes such as oval and spherical, with a granule size between 40 and
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50 nm. The same results were demonstrated by Sun et al. [24] and Sun et al. [36]. In the TEM image, we noticed that the nanoparticles’ edges had different contrast and texture than the centers, which may be due to the BSA molecules being absorbed to the SNPs and forming BSA-SNPs coronae. The results were in agreement with Gebregeorgis et al. [37], who reported that Ag/BSA
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nanoparticle edges had a different contrast and texture than the centers. Bhunia et al. [38] has
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reported that BSA molecules were attached to the ZnO nanoparticle and formed BSA-SNPs
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coronae. Hu et al. [39] also reported that the plasma proteins would interact with Fe3O4
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nanoparticles to form the protein coronae. 10
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3.9 Fourier transform infrared spectroscopy (FTIR) The FTIR spectra of BSA in the absence and presence of SNPs are shown in Fig. 7. The
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amino group presents characteristic absorption at 3500–3000 cm-1 (N-H stretching). The
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characteristic absorption at 3287 cm-1 slightly shifted to 3431 cm-1, indicating the formation of
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BSA-SNPs complex. Usually the amide I peak position for BSA occurs in the region of
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1600–1700 cm-1 [40]. The amide I peak at 1642 cm-1 shifted to 1647 cm-1, indicating the
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conformation of BSA changed. Similarly, Yue et al. [41] reported that the bands shifted from
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3385.2 to 3439.9 cm-1 due to the formation of citrate-coated Au nanoparticles conjugates.
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Kathiravan et al. [42] also reported that the addition of ZnO nanoparticles affected the
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conformation of BSA.
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3.10 Circular dichroism analysis
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CD was carried out to study the protein conformations. The CD spectra of BSA and
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BSA-SNPs were recorded with a CD spectrometer at room temperature in the wavelength range
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between 190 and 260 nm. All the samples were analyzed in the far-UV region in a quartz cuvette
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with a path length of 1 mm. The second structure of BSA curve and data are presented in Fig. 8 and Table 2, respectively. The CD spectrum of BSA possesses two broad dip bands at 208 nm and 222 nm, which are characteristic of the transition of π–π* and n–π* of the α-helical structure of BSA molecule. Changes in the ellipticity at 208 and 222 nm are useful probes for visualizing
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varying α-helical content. The α-helix structure in BSA was 55%, which was in close agreement
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with Gebregeorgis et al. [37]. As the SNP concentration increased, the α-helix content of BSA was
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found to decrease compared with native BSA, suggesting that there was indeed change in the
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secondary structure of BSA due to the association of SNPs. In addition, the contents of the β-turn 11
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in BSA increased with the addition of SNPs. These results indicated that SNPs had an affinity for
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BSA and led to structural change. Similar behavior was reported by Saurabh et al. [25] who found
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that the CD spectra of BSA conjugated with silver nanoparticles showed considerable changes in
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the secondary structure of BSA, with an increase in β-sheet structure. Rajeshwari et al. [27] also
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reported that Al2O3 nanoparticles decreased the α-helix content of BSA from 47.0% to 23.6%
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when compared with native BSA.
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4. Conclusions
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In this article, the interaction of SNPs with BSA was investigated using UV-visible,
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fluorescence, TEM, and CD. The maximum spectrum of BSA at 278 nm increased with increasing
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concentration of SNPs. The fluorescence intensity of BSA decreased gradually following
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increased concentrations of SNPs. Synchronous fluorescence intensities decreased with increasing
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concentrations of SNPs. The maximum emission wavelength had red shift when the Δλ = 15 nm at
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the investigated concentrations range, indicating that the conformation of BSA changed. The
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quenching mechanism of SNPs binding was static quenching and the number of binding sites (n)
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was more than 1. Surface binding and reorganization of BSA on the surface of SNPs formed stable coronae. The FTIR and CD spectra confirmed the conformational changes in the secondary structure of BSA. The α-helix decreased from 55% to 28%, and β-sheet increased from 14% and 33%. The present study’s findings may provide more insight on the biocompatibility of SNPs for a
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variety medical application.
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Acknowledgements
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Financial support from the Qingdao Municipal Science and Technology Plan Project (14-2-3-48-nsh) is gratefully acknowledged. 12
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Pharm. Biomed. Anal. 67 (2012) 2018. [31]A. Bhogale, N. Patel, P. Sarpotdar, J. Mariam, P.M. Dongre, A. Miotello and D.C. Kothari, 14
Page 14 of 27
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Colloids Surf. B 102 (2013) 257. [32]L.L. Li, R. Lin, H. He, M.L. Sun, L. Jiang and M.M. Gao, Lumin. 145 (2014) 125.
311
[33]C. Qin, M.X. Xie, Y. Liu, Biomacromolecules 8 (2007) 2182.
312
[34]F.J. Xiao, M.Q. Gu, Y. Liang, L.L. Li and Y.J. Luo. Spectrochim Acta A, 118 (2014) 1106.
313
[35]B.K.P.D. Victor, Colloids Surf. B, 111 (2013) 71.
314
[36]Q.J. Sun, M. Gong, Y. Li and L. Xiong, Carbohyd Polym, 111 (2014b) 133.
315
[37]A. Gebregeorgis, C. Bhan, O. Wilson and D. Raghavan, J. Colloid Interface Sci. 389 (2013)
cr
us
31.
an
316
ip t
310
[38]A.K. Bhunia, P.K. Samanta, S. Saha and T. Kamilya, Appl Phys Lett, 103 (2013) 143701.
318
[39]Z.Y. Hu, H.Y. Zhang, Y. Zhang, R.A. Wu and H.F Zou, Colloids Surf. B 121 (2014) 354.
319
[40]P. Athina, J.G. Rebecca, A.F. Richard, J. Agric. Food Chem. 53 (2005) 158.
320
[41]H.L. Yue, Y.J. Hu, H.G. Huang, S. Jiang, and B. Tu, Spectrochim Acta A, 130 (2014) 402.
321
[42]A. Kathiravan, G. Paramaguru, and R. Renganathan, J Mol Struct, 934 (2009) 129.
d
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322
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317
15
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322
Table 1 Quenching parameters, binding constants and thermodynamic parameters for SNPs–BSA
323
at different temperatures K
n
ΔH
ΔG
ΔS
(L mol-1)
(L mol-1 S-1)
(L mol-1)
(KJ mol-1)
(KJ mol-1)
(J mol-1 K-1)
293
9.0×108
9.0×1016
1.9×108
1.05
-102.69
-33.45
-236
303
7.0×108
7.0×1016
1.8×108
1.18
-102.69
-30.37
-236
310
3.5×108
3.5×1016
1.6×108
1.47
-102.69
-43.28
-236
ip t
Kq
cr
KSV
us
T(K)
Ac ce p
te
d
M
an
324 325
16
Page 16 of 27
Table 2. The changes of BSA secondary-structure in the SNPs and BSA binding process. Sample BSA BSA+0.2SNPs BSA+0.4SNPs BSA+0.6SNPs BSA+0.8SNPs BSA+1.0SNPs
β-sheet
a
14 ± 1e 17 ± 1d 20 ± 1c 24 ± 1b 26 ± 1b 33 ± 2a
55 ± 2 48 ± 2b 42 ± 2c 35 ± 1d 33 ± 1d 28 ± 1e
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cr
Values mean ± SD indicates the replicates of three experiments. Means with the same letter in each column are not significantly different (p < 0.05).
Ac ce p
326 327 328
α-helix
ip t
325
17
Page 17 of 27
1.2
0.8
ip t
Absorbance
6
1
0.0 260
280
300
328
320
340
an
Wavelength(nm)
us
cr
0.4
Fig. 1. The UV-Vis spectra of BSA-conjugated SNPs from 1 to 6: BSA=3.0×10-6 mol/L;
330
SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
M
329
Ac ce p
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331
18
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400
Intensity
ip t
1
300
200
cr
6
0 300
400
Wavelength (nm)
500
an
331
us
100
Fig. 2. The fluorescence quenching spectra of BSA-conjugated SNPs from 1 to 6:
333
BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
M
332
Ac ce p
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334
19
Page 19 of 27
1
20
6
ip t
40
cr
Intensity
(a)
240
250
260
270
280
290
300
Wavelength (nm)
60
M
(b) 50
Ac ce p
10
320
330
340
1
d
30
te
Intensity
40
20
310
an
334
us
0
6
0
230
335 336 337
240
250
260
270
280
290
300
310
320
330
340
Wavelength (nm)
Fig. 3 The sychronous fluorescence of BSA-conjugated SNPs: (a) Δλ=15nm, (b) Δλ=60 nm BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L), 1→6: 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
338
20
Page 20 of 27
2500
2000
ip t
1000
500
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6
1
0 250
300
350
400
450
500
550
600
650
700
800
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d
M
Fig. 4. RLS spectra of BSA-conjugated SNPs from 1 to 6: BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
Ac ce p
338 339 340 341
750
an
Wavelength(nm)
us
Intensity
1500
21
Page 21 of 27
293K 303K
1.2 1 0.8 0
0.2
0.4
0.6
0.8
1
1.2
-9
us
[Q] (10 mol/L)
341
te
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Fig. 5. The Stern-Volmer curves of BSA quenched by SNPs at 293, 303, and 310 K. The error bar corresponds to the standard deviation for n = 3.
Ac ce p
342 343 344 345
ip t
310K
cr
F0 /F
1.4
22
Page 22 of 27
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a
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100 nm
345
100 nm
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b
Ac ce p
346 c
50 nm
347 348
Fig. 6. TEM images (a) pure SNPs (bar = 100 nm), (b) BSA and SNPs complex (bar = 100nm)
349
(c) BSA and SNPs complex (bar = 50nm).
350 23
Page 23 of 27
1647
ip t
BSA
4000
3500
3000
2500
2000
1500
1000
-1
M d te
352 353 354 355 356 357
Fig. 7. The FTIR spectra of BSA and BSA-SNPs complex.
Ac ce p
350 351
500
an
Wavenumber ( cm )
us
1642
cr
3287
Transmitance ( %)
3431
BSA-SNPs complex
24
Page 24 of 27
100
6
80 60
ip t
CD(mdeg)
40
1
20 0
cr
-20 -40
200
210
220
230
240
Wavelength( nm)
250
260
an
357
us
-60
Fig. 8. The circular dichroism spectra of the SNPs and BSA binding procedure from 1 to 6:
359
BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
M
358
Ac ce p
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360
25
Page 25 of 27
360 361
BSA
SNPs
362
ip t
363 364
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365
us
366 BSA-SNPs
367
an
368
100 nm
M
369 370
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371
BSA: bovine serum albumin, SNPs: starch nanoparticles
373
The BSA molecules was absorbed to the SNPs and formed BSA-SNP coronae.
Ac ce p
374
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372
26
Page 26 of 27
The starch nanoparticles (SNPs) was prepared by isoamylolysis and recrystallization.
375
There was an interaction between SNPs and bovine serum albumin (BSA).
376
BSA could bind to SNPs and form a BSA-SNP complex.
377
The BSA molecules was absorbed to the SNPs and formed BSA-SNP coronae.
378
The SNPs induced structural changes in the secondary structure of BSA.
cr
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379 380
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S0927-7765(15)00150-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.03.016 COLSUB 6956
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
16-10-2014 11-2-2015 5-3-2015
Please cite this article as: N. Ji, C. Qiu, X. Li, L. Xiong, Q. Sun, Study on the interaction between bovine serum albumin and starch nanoparticles prepared by isoamylolysis and recrystallization, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.03.016 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.
Study on the interaction between bovine serum albumin and starch nanoparticles prepared
2
by isoamylolysis and recrystallization
3
Na Ji Chao Qiu Xiaojing Li Liu Xiong Qingjie Sun*
4
School of Food Science and Engineering, Qingdao Agricultural University
5
(Qingdao, Shandong Province, 266109, China)
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*Correspondence
7
[email protected]), School of Food Science and Engineering, Qingdao Agricultural University,
8
266109, 700 Changcheng Road, Chengyang District, Qingdao, China.
9
Abstract: The current study primarily investigated the interaction of bovine serum albumin (BSA)
10
with starch nanoparticles (SNPs) prepared by isoamylolysis and recrystallization using UV-visible,
11
fluorescence, transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and
12
circular dichroism (CD). The enhanced absorbance observed by UV-visible spectroscopy and
13
decreased intensity of fluorescence spectroscopy suggested that BSA could bind to SNPs and form
14
a BSA-SNP complex. The synchronous fluorescence spectra revealed that the emission maximum
16 17 18
86-532-88030448,
Fax:
86-532-88030449,
e-mail:
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(Tel:
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15
author
of Tyr residue (at Δλ = 15 nm) was red-shifted at the investigated concentrations range, indicating that the conformation of BSA was changed. Quenching parameters showed that the quenching effect of SNPs was static quenching. TEM images showed that the SNPs were surrounded by protein coronae, indicating that nanoparticle-protein complexes had formed. The FTIR and CD
19
characterization indicated that the SNPs induced structural changes in the secondary structure of
20
BSA.
21
Keywords: starch nanoparticles, bovine serum albumin, interaction, spectroscopy
22
1. Introduction 1
Page 1 of 27
23
Nanomaterials are at the forefront of the swiftly-developing field of commercial exploration of nanotechnology [1]. The advent of nanotechnology has accelerated the evolution of materials
25
with many exceptional size-dependent properties for use in numerous biological and medical
26
applications [2-3]. When a nanomaterial enters a physiological environment, its surface is
27
immediately covered by a layer of proteins, forming what is known as the protein “corona” [4].
28
The adsorbed proteins on nanoparticle surfaces and the formation of the protein corona, have been
29
identified as determining their fate in vivo [5]. Therefore, it is essential to understand the
30
interactions of nanoparticles and proteins.
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As a water-soluble protein, serum albumin is the most abundant and basic protein of blood plasma. It is primarily involved in the transport of substances in the blood that are poorly soluble
33
in water, including drugs, and also in the disposition of endogenous and exogenous compounds
34
present in blood [6]. Albumin is also reported to associate with nanoscale particles to promote
35
their transportation through cells [7-8]. Bovine serum albumin (BSA) is one of the most
36
extensively-studied proteins and has structural homology to human serum albumin as a substitute
38 39 40
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for research [9]. As a model protein, BSA has two tryptophans, Trp-134 and Trp-212 [10] that possess intrinsic fluorescence and have commonly been utilized to study the interactions between BSA and nanoparticles [11-13].
The conformational behavior of albumin in conjugation with nanoparticles is of great
41
importance in numerous biomedical applications, particularly drug delivery and receptor targeting.
42
However, the structural changes of protein after conjugation with nanoparticles are not very clear
43
and require some basic understanding before they can be employed for biomedical applications.
44
At present, many researchers have reported their studies on the interaction of BSA with metal NPs 2
Page 2 of 27
such as Ag [14], and Au [15-16]. On the other hand, a certain amount of literature is available on
46
the interaction of metal oxide nanoparticles, such as ZnO [17-18] and TiO2 [19] with BSA. Starch
47
nanoparticles (SNPs), a novel product derived from starch, are widely used as a carrier in drug
48
delivery systems [20-22]. Recently, there has been growing interest in SNPs for their abundant
49
availability of starch, comparatively easy processability, biocompatibility, biodegradability, and
50
non-toxicity [23]. However, the interaction between SNPs and BSA has not been investigated. In
51
this study, we investigate on this interaction by UV-vis, fluorescence, transmission electron
52
microscopy (TEM), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic
53
techniques.
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The objective of this study is to investigate interaction between BSA and SNPs prepared by
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45
isoamylolysis and recrystallization; it is expected to provide novel insight into SNPs interaction
56
with BSA, and to provide theoretical guidance for their application in the field of biomedical
57
materials and nano-sized drug carriers.
58
2. Materials and Methods
60 61 62
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2.1. Materials
Waxy rice starch (approximately 1% amylose and 99% amylopectin) was obtained from
Zhucheng Xingmao Corn Development Co., Ltd. (Shangdong, China). Pullulanase (E.C.3.2.1.41, 6000 ASPU/g, 1.15 g/mL, where ASPU is defined as the amount of enzyme that liberates 1.0 mg
63
glucose from starch in 1 min at pH 4.4 and 60 °C) was supplied by Novozymes (China)
64
Investment Co. Ltd. (Bagsvaerd, Denmark). BSA pH 7.0 (MW 66 kDa and > 95% purity) was
65
procured from Sigma-Aldrich (St. Louis, MO). All other chemicals used were of analytical grade.
66
2.2. Preparation of SNPs 3
Page 3 of 27
The SNPs were prepared using the method described by Sun et al. [24], with some
68
modifications. Typically, waxy rice starch (15 g, db) was dispersed in 100 mL of disodium
69
hydrogen phosphate and citric acid buffer solution (pH 5.0). The starch slurry was cooked in
70
boiling water with vigorous stirring for 30 min to fully gelatinize the starch. The temperature of
71
the cooked waxy rice starch was adjusted to 58 °C and pullulanase (30 ASPU/g of dry starch) was
72
added. After an 8 h incubation period at 58 °C, isoamylolysis was stopped by heating at 100 °C for
73
30 min to inactivate the pullulanase and the slurry was centrifuged, followed by cooling to room
74
temperature. Then, the solutions were stored at 4 °C for 8 h and retrogradated to form
75
nanoparticles. The suspensions were washed several times with distilled water until neutrality was
76
achieved and then freeze-dried to obtain SNPs.
77
2.3. Interaction of BSA with SNPs
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Various concentrations of SNPs (0, 0.2, 0.4, 0.6, 0.8, and 1.0×10-9 mol/L) interacted with a
79
constant BSA (3.0×10-6 mol/L) concentration for 30 minutes on a rotary shaker (300 rpm) at room
80
temperature [25]. After the interaction period, characterization studies were performed with the
82 83 84 85
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help of UV-visible, fluorescence, TEM and CD spectroscopy. Molar concentration of SNPs is hypothesized from the molecular numbers of SNPs
multiplied by Avogadro's constant. The molecular numbers of SNPs were calculated by dividing the sample weight by the weight of one SNP. The weight of one SNP was calculated by the following equation:
86 87 88
m = ρv
(1),
Where ρ is the density of starch, v is the volume of SNPs, and the average particle size of SNPs is 50 nm. 4
Page 4 of 27
89
2.4. UV-visible spectroscopy After the interaction period, 3 mL of each sample was used to record UV-visible spectra
91
using a UV-visible spectrophotometer (Shimadzu-2600, Kyoto, Japan) from 260 to 340 nm. The
92
spectra obtained for the BSA-SNPs system were analyzed to evaluate the changes occurring in the
93
absorption peak of BSA at 278 nm, a characteristic peak of BSA [26].
94
2.5. Fluorescence spectra analysis
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Fluorescence of BSA after interaction with SNPs was measured using a F2500
96
Spectrofluorimeter (Hitachi, Tokyo, Japan). An aliquot of 3.0×10-6 mol/L of BSA interacted with
97
various concentrations of SNPs (0, 0.2, 0.4, 0.6, 0.8, and 1.0×10-9 mol/L) and 3 mL of the sample
98
was subjected to analysis at the excitation wavelength of 278 nm and emission spectra in the range
99
of 300-500 nm. A quartz cuvette (4 cm × 1 cm × 1 cm) with path length of 1 cm was used for
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measurements.
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2.6. Synchronous fluorescence measurement
103 104 105 106 107 108
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The synchronous fluorescence spectra of the mixture solutions of SNPs and BSA were
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recorded on a F2500 Spectrofluorimeter (Hitachi, Tokyo, Japan) at room temperature. For this measurement, the difference between excitation and emission wavelength (Δλ = λemission – λexcitation) was set at 15 nm for tyrosine residue or at 60 nm for tryptophan residue. Excitation and emission slit width was set at 2.5 nm. 2.7 Resonance light scattering (RLS) In the case of RLS scans, both excitation and emission wavelengths were scanned
109
simultaneously in the wavelength range of 220–800 nm with Δλ = 0.
110
2.8. Transmission electron microscopy (TEM) 5
Page 5 of 27
Transmission electron micrographs of SNPs and BSA-SNPs were taken with a 7650
112
transmission electron microscope (Hitachi, Tokyo, Japan) with an acceleration voltage of 80 kV. A
113
drop of BSA-conjugated SNPs dispersed in double-distilled water was placed on a copper grid and
114
lyophilized.
115
2.9 Fourier transform infrared (FTIR)
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The FTIR spectra of BSA and the mixture of BSA and SNPs were recorded on an FTIR
117
spectrophotometer (NEXUS-870; Thermo Nicolet Corporation, Madison, WI, USA) within the
118
wavenumber range of 4000–600 cm-1. The background obtained from the scan of KBr was
119
automatically subtracted from the sample spectra.
120
2.10. Circular dichroism (CD) spectroscopy
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Circular dichroism spectroscopy was performed to analyze the changes that occurred in the
122
secondary structure of BSA. For CD analysis, an aliquot of 3.0×10-6 mol/L of BSA interacted with
123
various concentrations of SNPs (0, 0.2, 0.4, 0.6, 0.8, and 1.0×10-9 mol/L). After 30 min incubation,
124
CD spectra were analyzed using JASCOJ-715 CD spectroscopy (Welltech Enterprises, Maryland,
126 127 128
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United States). Change in secondary structure of protein was confirmed by comparing the CD spectrum data of the control sample to the test sample. 3. Results and discussion 3.1 UV-visible spectra
129
The UV-visible absorption spectra of BSA in the absence and in presence of SNPs of
130
different concentration are shown in Fig. 1. The maximum spectrum of BSA at 278 nm is caused
131
by the π→π* transition of the aromatic amino acid residues [26]. With the increase in the
132
concentration of SNPs, the maximum peak at 278 nm was increased, clearly indicating the 6
Page 6 of 27
formation of a ground state complex of BSA-SNPs. Our results were in agreement with Ravindran
134
et al. [26], who reported that the ground state complex of Al2O3-BSA was formed by the
135
adsorption of some BSA molecules on the Al2O3 nanoparticles. Wu et al. [28] reported that the
136
interaction between BSA and ZnS quantum dots (QDs) led to the formation of QDs-BSA complex.
137
3.2 Steady state fluorescence spectra
cr
The fluorescence spectra of the SNPs-BSA system are shown in Fig. 2. Due to the presence
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of two aromatic amino acids (tryptophan (Trp) and tyrosine (Tyr)), proteins are considered to have
140
intrinsic fluorescence [29]. The fluorescence spectrum of BSA presented strong emission at 345
141
nm when excited at 278 nm. The fluorescence intensity of BSA showed a significant gradual
142
decrease accompanied by an increase in the concentration of SNPs and there was no shift of the
143
emission wavelength. This was due to the adsorption of some BSA molecules on the SNPs’
144
surface and the formation of the ground state complex of BSA-SNPs. This result showed that
145
SNPs could quench the intrinsic fluorescence of protein strongly, and that there was an interaction
146
between SNPs and BSA.
148 149 150
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3.3 Synchronous fluorescence spectra The synchronous fluorescence spectra were measured when the difference between excitation
wavelength and emission wavelength were stabilized at 15 or 60 nm, as shown in Fig. 3. Generally, the shift of the maximum emission wavelength reveals the alteration of polarity microenvironment
151
around Tyr and Trp residues [30]. The synchronous fluorescence characteristic of BSA protein was
152
studied at different scanning intervals. The fluorescence intensities of Tyr and Trp residues in BSA
153
decreased regularly with increased concentrations of SNPs. At the same time, the emission
154
maximum of Tyr residue (at Δλ = 15 nm) was red-shifted slightly from 283 nm to 284 nm at the 7
Page 7 of 27
investigated concentrations range, which indicated that a decrease in the hydrophobicity
156
surrounding Tyr residue occurred in the presence of SNPs. The results indicated that the
157
conformation of BSA was changed with the insertion of SNPs. Cao et al. [29] suggested that the
158
maximum emission wavelength (at Δλ = 15 nm) had an obvious red shift with the addition of
159
Glipizide. However, the synchronous fluorescence spectra of BSA at Δλ= 60 nm with various
160
amounts of SNPs illustrated that the emission peaks did not shift, indicating that SNPs had little
161
effect on the microenvironment of Trp residue in BSA.
162
3.4 Resonance Light Scattering (RLS) spectra
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The RLS spectra of BSA and the BSA–SNPs system are shown in Fig. 4. With increasing SNPs concentration, the RLS intensity of all samples increased markedly. The generation of RLS
165
spectrum is correlated with the formation of aggregates. Our results indicated that the added BSA
166
could interact with SNPs in solution, forming a BSA–SNPs complex that would be expected to be
167
an aggregate. Simiarly, Bhogale et al. [31] reported that the RLS spectra intensity of BSA
168
increased significantly with the increasing concentration of ZnO nanoparticles, indicating the
170 171 172 173 174
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formation of aggregates. Li et al. [32] also reported that the RLS spectra intensity of BSA increased apparently while increasing the concentration of single-walled carbon nanotubes. 3.5 Mechanism of fluorescence quenching Fluorescence quenching is described by the Stern–Volmer equation [33]: F0/F = 1+KSV[Q] = 1+Kqτ0[Q]
(2)
where F0 and F are the fluorescence intensities of BSA in the absence and presence of the
175
quencher (SNPs), respectively. KSV is the Stern–Volmer quenching constant, and [Q] is the
176
concentration of SNPs, Kq is the quenching rate constant, τ0 is the average life time of molecules 8
Page 8 of 27
177
in the absence of SNPs and its value is about 10 -8 s.
178
As can be seen from Fig. 5, F0/F has a linear correlation to the concentration of SNPs. According to Eq. (2), KSV values and Kq values are shown in Table 1. Generally, collision constant
180
of the quenching on the BSA of the large diffusion is 2.0×1010 L (mol s)-1. From Table 1, we can
181
see that Kq is about six orders higher than 2.0×1010 L (mol s)-1, indicating that the quenching
182
mechanism of SNPs binding is static quenching. Xiao et al. [34] also reported that BSA
183
fluorescence was statically quenched by polymer-hyperbranched poly (amine) ester.
184
3.6 Binding constant and binding sites
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Fluorescence quenching of BSA also provides the information on the binding constant (K)
186
and the number of binding sites (n) between the quencher and BSA accordingly to the equation
187
[14]:
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188
191 192 193 194
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(3)
Where F0 and F are the fluorescence intensity without and with quencher (SNPs), respectively. K is the binding constant, n is the number of binding sites, and [Q] is the
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log[(F0–F)/F] = logK + nlog[Q]
concentration of SNPs. According to the plot of log[(F0-F)/F] against log[SNPs], we calculated the number of binding sites to be 1.15, whereas for the binding constant we obtained 4.0×10 8 L mol-1 (Table 1). The calculated results illustrated that the interaction between BSA and SNPs has only one binding site, which was in accordance with previous reports [35].
195
3.7 Binding force
196
Thermodynamic parameters are usually applied to judge the categories of binding force. Enthalpy
197
changes (ΔH) in reactions can be considered as constants due to no obvious changes in
198
temperature. The thermodynamic parameters could be calculated by the van’s Hoff equation: 9
Page 9 of 27
199
ΔG = –RTlnK
200
(4)
lnK2/K1 = [(1/T1 – 1/T2)ΔH/R]
201
(5)
ΔS = (ΔH – ΔG)/T
(6)
where ΔH, ΔG and ΔS denote enthalpy change, free enthalpy change and entropy change,
203
respectively, K is the binding constant, at the corresponding temperature. R is the gas constant,
204
8.314 Jmol-1 K-1, and T is experimental temperature (K). Generally speaking, when ΔH < 0 and
205
ΔS > 0, the main force is due to electrostatic interactions; when ΔH < 0 and ΔS < 0, the main force
206
is due to van der Waals or hydrogen bonding, and when ΔH > 0 and ΔS > 0, the main force is due
207
to hydrophobic interactions. Values of ΔH, ΔG, and ΔS are shown in Table 1, and the negative
208
values of ΔH and ΔS suggest that the hydrogen bond and van der Waals forces play major roles in
209
the binding process.
210
3.8 Morphology of SNPs and BSA-SNPs complex
213 214 215 216
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The size and morphology of SNPs and BSA-SNPs were characterized by TEM (Fig. 6). The TEM of the SNPs exhibited shapes such as oval and spherical, with a granule size between 40 and
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ip t
202
50 nm. The same results were demonstrated by Sun et al. [24] and Sun et al. [36]. In the TEM image, we noticed that the nanoparticles’ edges had different contrast and texture than the centers, which may be due to the BSA molecules being absorbed to the SNPs and forming BSA-SNPs coronae. The results were in agreement with Gebregeorgis et al. [37], who reported that Ag/BSA
217
nanoparticle edges had a different contrast and texture than the centers. Bhunia et al. [38] has
218
reported that BSA molecules were attached to the ZnO nanoparticle and formed BSA-SNPs
219
coronae. Hu et al. [39] also reported that the plasma proteins would interact with Fe3O4
220
nanoparticles to form the protein coronae. 10
Page 10 of 27
221
3.9 Fourier transform infrared spectroscopy (FTIR) The FTIR spectra of BSA in the absence and presence of SNPs are shown in Fig. 7. The
223
amino group presents characteristic absorption at 3500–3000 cm-1 (N-H stretching). The
224
characteristic absorption at 3287 cm-1 slightly shifted to 3431 cm-1, indicating the formation of
225
BSA-SNPs complex. Usually the amide I peak position for BSA occurs in the region of
226
1600–1700 cm-1 [40]. The amide I peak at 1642 cm-1 shifted to 1647 cm-1, indicating the
227
conformation of BSA changed. Similarly, Yue et al. [41] reported that the bands shifted from
228
3385.2 to 3439.9 cm-1 due to the formation of citrate-coated Au nanoparticles conjugates.
229
Kathiravan et al. [42] also reported that the addition of ZnO nanoparticles affected the
230
conformation of BSA.
231
3.10 Circular dichroism analysis
M
an
us
cr
ip t
222
CD was carried out to study the protein conformations. The CD spectra of BSA and
233
BSA-SNPs were recorded with a CD spectrometer at room temperature in the wavelength range
234
between 190 and 260 nm. All the samples were analyzed in the far-UV region in a quartz cuvette
236 237 238
te
Ac ce p
235
d
232
with a path length of 1 mm. The second structure of BSA curve and data are presented in Fig. 8 and Table 2, respectively. The CD spectrum of BSA possesses two broad dip bands at 208 nm and 222 nm, which are characteristic of the transition of π–π* and n–π* of the α-helical structure of BSA molecule. Changes in the ellipticity at 208 and 222 nm are useful probes for visualizing
239
varying α-helical content. The α-helix structure in BSA was 55%, which was in close agreement
240
with Gebregeorgis et al. [37]. As the SNP concentration increased, the α-helix content of BSA was
241
found to decrease compared with native BSA, suggesting that there was indeed change in the
242
secondary structure of BSA due to the association of SNPs. In addition, the contents of the β-turn 11
Page 11 of 27
in BSA increased with the addition of SNPs. These results indicated that SNPs had an affinity for
244
BSA and led to structural change. Similar behavior was reported by Saurabh et al. [25] who found
245
that the CD spectra of BSA conjugated with silver nanoparticles showed considerable changes in
246
the secondary structure of BSA, with an increase in β-sheet structure. Rajeshwari et al. [27] also
247
reported that Al2O3 nanoparticles decreased the α-helix content of BSA from 47.0% to 23.6%
248
when compared with native BSA.
249
4. Conclusions
us
cr
ip t
243
In this article, the interaction of SNPs with BSA was investigated using UV-visible,
251
fluorescence, TEM, and CD. The maximum spectrum of BSA at 278 nm increased with increasing
252
concentration of SNPs. The fluorescence intensity of BSA decreased gradually following
253
increased concentrations of SNPs. Synchronous fluorescence intensities decreased with increasing
254
concentrations of SNPs. The maximum emission wavelength had red shift when the Δλ = 15 nm at
255
the investigated concentrations range, indicating that the conformation of BSA changed. The
256
quenching mechanism of SNPs binding was static quenching and the number of binding sites (n)
258 259 260
M
d
te
Ac ce p
257
an
250
was more than 1. Surface binding and reorganization of BSA on the surface of SNPs formed stable coronae. The FTIR and CD spectra confirmed the conformational changes in the secondary structure of BSA. The α-helix decreased from 55% to 28%, and β-sheet increased from 14% and 33%. The present study’s findings may provide more insight on the biocompatibility of SNPs for a
261
variety medical application.
262
Acknowledgements
263 264
Financial support from the Qingdao Municipal Science and Technology Plan Project (14-2-3-48-nsh) is gratefully acknowledged. 12
Page 12 of 27
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us
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an
316
ip t
310
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d
te
Ac ce p
322
M
317
15
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322
Table 1 Quenching parameters, binding constants and thermodynamic parameters for SNPs–BSA
323
at different temperatures K
n
ΔH
ΔG
ΔS
(L mol-1)
(L mol-1 S-1)
(L mol-1)
(KJ mol-1)
(KJ mol-1)
(J mol-1 K-1)
293
9.0×108
9.0×1016
1.9×108
1.05
-102.69
-33.45
-236
303
7.0×108
7.0×1016
1.8×108
1.18
-102.69
-30.37
-236
310
3.5×108
3.5×1016
1.6×108
1.47
-102.69
-43.28
-236
ip t
Kq
cr
KSV
us
T(K)
Ac ce p
te
d
M
an
324 325
16
Page 16 of 27
Table 2. The changes of BSA secondary-structure in the SNPs and BSA binding process. Sample BSA BSA+0.2SNPs BSA+0.4SNPs BSA+0.6SNPs BSA+0.8SNPs BSA+1.0SNPs
β-sheet
a
14 ± 1e 17 ± 1d 20 ± 1c 24 ± 1b 26 ± 1b 33 ± 2a
55 ± 2 48 ± 2b 42 ± 2c 35 ± 1d 33 ± 1d 28 ± 1e
te
d
M
an
us
cr
Values mean ± SD indicates the replicates of three experiments. Means with the same letter in each column are not significantly different (p < 0.05).
Ac ce p
326 327 328
α-helix
ip t
325
17
Page 17 of 27
1.2
0.8
ip t
Absorbance
6
1
0.0 260
280
300
328
320
340
an
Wavelength(nm)
us
cr
0.4
Fig. 1. The UV-Vis spectra of BSA-conjugated SNPs from 1 to 6: BSA=3.0×10-6 mol/L;
330
SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
M
329
Ac ce p
te
d
331
18
Page 18 of 27
400
Intensity
ip t
1
300
200
cr
6
0 300
400
Wavelength (nm)
500
an
331
us
100
Fig. 2. The fluorescence quenching spectra of BSA-conjugated SNPs from 1 to 6:
333
BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
M
332
Ac ce p
te
d
334
19
Page 19 of 27
1
20
6
ip t
40
cr
Intensity
(a)
240
250
260
270
280
290
300
Wavelength (nm)
60
M
(b) 50
Ac ce p
10
320
330
340
1
d
30
te
Intensity
40
20
310
an
334
us
0
6
0
230
335 336 337
240
250
260
270
280
290
300
310
320
330
340
Wavelength (nm)
Fig. 3 The sychronous fluorescence of BSA-conjugated SNPs: (a) Δλ=15nm, (b) Δλ=60 nm BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L), 1→6: 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
338
20
Page 20 of 27
2500
2000
ip t
1000
500
cr
6
1
0 250
300
350
400
450
500
550
600
650
700
800
te
d
M
Fig. 4. RLS spectra of BSA-conjugated SNPs from 1 to 6: BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
Ac ce p
338 339 340 341
750
an
Wavelength(nm)
us
Intensity
1500
21
Page 21 of 27
293K 303K
1.2 1 0.8 0
0.2
0.4
0.6
0.8
1
1.2
-9
us
[Q] (10 mol/L)
341
te
d
M
an
Fig. 5. The Stern-Volmer curves of BSA quenched by SNPs at 293, 303, and 310 K. The error bar corresponds to the standard deviation for n = 3.
Ac ce p
342 343 344 345
ip t
310K
cr
F0 /F
1.4
22
Page 22 of 27
cr
ip t
a
us
100 nm
345
100 nm
te
d
M
an
b
Ac ce p
346 c
50 nm
347 348
Fig. 6. TEM images (a) pure SNPs (bar = 100 nm), (b) BSA and SNPs complex (bar = 100nm)
349
(c) BSA and SNPs complex (bar = 50nm).
350 23
Page 23 of 27
1647
ip t
BSA
4000
3500
3000
2500
2000
1500
1000
-1
M d te
352 353 354 355 356 357
Fig. 7. The FTIR spectra of BSA and BSA-SNPs complex.
Ac ce p
350 351
500
an
Wavenumber ( cm )
us
1642
cr
3287
Transmitance ( %)
3431
BSA-SNPs complex
24
Page 24 of 27
100
6
80 60
ip t
CD(mdeg)
40
1
20 0
cr
-20 -40
200
210
220
230
240
Wavelength( nm)
250
260
an
357
us
-60
Fig. 8. The circular dichroism spectra of the SNPs and BSA binding procedure from 1 to 6:
359
BSA=3.0×10-6 mol/L; SNPs/ (×10-9 mol/L): 0, 0.2, 0.4, 0.6, 0.8, and 1.0.
M
358
Ac ce p
te
d
360
25
Page 25 of 27
360 361
BSA
SNPs
362
ip t
363 364
cr
365
us
366 BSA-SNPs
367
an
368
100 nm
M
369 370
d
371
BSA: bovine serum albumin, SNPs: starch nanoparticles
373
The BSA molecules was absorbed to the SNPs and formed BSA-SNP coronae.
Ac ce p
374
te
372
26
Page 26 of 27
The starch nanoparticles (SNPs) was prepared by isoamylolysis and recrystallization.
375
There was an interaction between SNPs and bovine serum albumin (BSA).
376
BSA could bind to SNPs and form a BSA-SNP complex.
377
The BSA molecules was absorbed to the SNPs and formed BSA-SNP coronae.
378
The SNPs induced structural changes in the secondary structure of BSA.
cr
ip t
374
us
379 380
Ac ce p
te
d
M
an
381
27
Page 27 of 27
