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Inhibition Mechanism of Calcium Oxalate Crystals Growth by Cooperation Influence of Colloidal Selenium Nanoparticles and Bovine Serum Albumin Cailing Zhong, Zixia Deng, Rui Wang, and Yan Bai Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/cg500880m • Publication Date (Web): 16 Mar 2015 Downloaded from http://pubs.acs.org on March 20, 2015
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Inhibition Mechanism of Calcium Oxalate Crystals Growth by Cooperation Influence of Colloidal Selenium Nanoparticles and Bovine Serum Albumin Cailing Zhong, Zixia Deng, Rui Wang, Yan Bai ∗ Chemistry Department of Jinan University, Guangzhou 510632, PR China
Abstract: This study investigated the effect of colloidal selenium nanoparticles modified with bovine serum albumin (nanoSe0-BSA) on the crystal phase and morphology of CaC2O4 and explained the cooperative inhibition mechanism of nanoSe0 and BSA on the crystallization of CaC2O4. The results were compared with that of nanoSe0 and BSA, respectively. NanoSe0 could induce the formation of oval or spherical calcium oxalate monohydrate (COM) crystals at high concentrations. BSA could induce the formation of hexagonal plate-shape COM crystals at low concentrations and the formation of thin diamond-shaped COM crystals and mixed hydrates with cracks at high concentrations. The nanoSe0-BSA showed additive effects on CaC2O4 crystals growth. The nanoSe0-BSA could induce the formation of mixed hydrates with more surface cracks and obvious voids in relatively low BSA concentrations. These mixed hydrates were considered to be the calcium oxalate trihydrate (COT) or calcium oxalate dihydrate (COD) crystals containing BSA or nanoSe0-BSA. These results resulted from the interaction of nanoSe0 with BSA. The UV-Vis, CD and FT-IR spectra indicated that the binding of nanoSe0 to BSA induced a change in secondary structure of BSA and formed the complex. The nanoSe0-BSA binding constant (2.257×104 L•mol-1) and number of binding sites (1.13) were calculated from the data of fluorescence spectra.
∗
Corresponding author: Yan Bai Address: Chemistry Department, College of Life Science and Technology, Jinan University, Guangzhou, China Tel.: +86-15818823611 E-mail address:
[email protected] 1
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Title Page
Inhibition Mechanism of Calcium Oxalate Crystals Growth by Cooperation Influence of Colloidal Selenium Nanoparticles and Bovine Serum Albumin
By Cailing Zhong, Zixia Deng, Rui Wang, Yan Bai* Chemistry Department of Jinan University, Guangzhou 510632, PR China ∗
E-mail address:
[email protected] 2
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Inhibition Mechanism of Calcium Oxalate Crystals Growth by Cooperation Influence of Colloidal Selenium Nanoparticles and Bovine Serum Albumin Cailing Zhong, Zixia Deng, Rui Wang, Yan Bai* Chemistry Department of Jinan University, Guangzhou 510632, PR China ∗
E-mail address:
[email protected] Abstract: This study investigated the effect of colloidal selenium nanoparticles modified with bovine serum albumin (nanoSe0-BSA) on the crystal phase and morphology of CaC2O4 and explained the cooperative inhibition mechanism of nanoSe0 and BSA on the crystallization of CaC2O4. The results were compared with that of nanoSe0 and BSA, respectively. NanoSe0 could induce the formation of oval or spherical calcium oxalate monohydrate (COM) crystals at high concentrations. BSA could induce the formation of hexagonal plate-shape COM crystals at low concentrations and the formation of thin diamond-shaped COM crystals and mixed hydrates with cracks at high concentrations. The nanoSe0-BSA showed additive effects on CaC2O4 crystals growth. The nanoSe0-BSA could induce the formation of mixed hydrates with more surface cracks and obvious voids in relatively low BSA concentrations. These mixed hydrates were considered to be the calcium oxalate trihydrate (COT) or calcium oxalate dihydrate (COD) crystals containing BSA or nanoSe0-BSA. These results resulted from the interaction of nanoSe0 with BSA. The UV-Vis, CD and FT-IR spectra indicated that the binding of nanoSe0 to BSA induced a change in secondary structure of BSA and formed the complex. The nanoSe0-BSA binding constant (2.257×104 L•mol-1) and number of binding sites (1.13) were calculated from the data of fluorescence spectra.
1. Introduction Several proteins have been shown to effect the formation of calcium oxalate (CaC2O4) crystals in vitro. For example, Nephrocalcin can inhibit the formation of CaC2O4 crystals,
1
while,
Tamm-Horsfall protein can both promote and inhibit calcium oxalate monohydrate (CaC2O4 •H2O, COM) crystallization and aggregation. 2 Interestingly, osteopontin and albumin not only inhibit the 3
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formation of COM crystals but also favor the formation of calcium oxalate dihydrate (CaC2O4•2H2O, COD) crystals. 3, 4 Among them, albumin is one of the most abundant proteins in urine and regarded as the major component of stones protein matrix, 5, 6 the role of albumin in the crystallization of CaC2O4 crystals has been studied by other groups. Some research works have indicated that albumin was an inhibitor of COM crystals aggregation
7
and growth,
8
and also
could promote the formation of COD and COT (calcium oxalate trihydrate, CaC2O4•3H2O) crystals.
1, 9, 10
However, the inhibition mechanisms of CaC2O4 crystals formation by proteins are
not well understood. Some studies showed that proteins inhibited the COM crystals growth through adsorbing or incorporating to the crystals. Hug and coworkers found that protein inhibited COM crystals by adsorbing to the terraces or the riser of growth hillocks and induced the formation of plate-shaped crystals with rounded ends and fan-shaped {100} face.
11
Fischer and
coworkers suggested that peptide was able to promote the formation of COD and COT crystals and make these metastable phases remain stabilized over very long periods of time by incorporating in the crystals. 12 On the other hand, mechanism of inhibition of CaC2O4 crystals by proteins also depended upon the protein structure, which includes the hydrophilicity, molecular charge, chain and the relationship between protein structure and the crystals lattice of CaC2O4 crystals. The results of study indicated that the proteins with highly hydrophilicity and net charge showed the strongest inhibition on COM crystals formation. 13 Jung and coworkers suggested that biopolymeric additives with the shortest side chains and smallest distance between neighboring carboxylate groups were advantageous for spatial matching with calcium ions arrayed on the COD crystals faces, resulting the formation of COD crystals during crystallization. 14 Recently, the interactions between nanoparticles and bovine serum albumin (BSA), a serum albumin protein, have been widely studied, as both have numerous biochemical and medicinal applications. The protein structure can be changed in the presence of nanoparticles. Liu and coworkers studied the interaction between BSA and doxorubicin-loaded superparamagnetic iron oxide nanoparticles (DOX-SPION). 15 Their research indicated that DOX-SPION could make the protein secondary structure change quantitatively. Chakraborty and coworkers discovered that gold nanorods could alter the structure of BSA and promote the formation of protein aggregate. 16 The change of protein structure might have an effect on CaC2O4 crystals growth. Cerini and coworkers indicated that the aggregation of albumin could induce CaC2O4 crystals nucleation 4
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much more efficient than that of monomers and increasing the number of crystal nuclei would decrease average crystals size and facilitate the elimination of crystals. 17 Selenium is essential for humans and animals. Colloidal selenium nanoparticles (nanoSe0) have high bioavailability, good bioactivity and low toxicity.
18, 19
For example, Kojouri and
coworkers indicated that oral nanoSe0 supplementation to donkey had the beneficial role in cell stability under intense exercise and would prevent further injury to the cell. 20 Moreover, nanoSe0 as a medicine carrier or its complex with medicine could improve the therapeutic effect.
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The
stability and biological behavior of nanoSe0 are closely related to its surface modifiers. Some studies have revealed that BSA could be used to stabilize nanoSe0 18, 24, 25 and also could be used as a shape-directing agent to synthesize fine crystalline nanobars and amorphous spheres of selenium. 25
Our previous study has shown that nanoSe0 with ascorbic acid (nanoSe0-Vc) prevented the
aggregation of COM crystals and induced the formation of spherical COD crystals containing Se. 26
In this study, we used nanoSe0 modified with BSA (nanoSe0-BSA) as a novel template for
CaC2O4 mimetic biomineralization. During the CaC2O4 crystals growth process, the crystal phase and morphology could be regulated by the template through the interaction of CaC2O4 crystals with nanoSe0 and BSA. Therefore, the template could inhibit CaC2O4 crystals growth by the cooperative influence of nanoSe0 and BSA. In order to understand the inhibition mechanism of nanoSe0-BSA on the crystallization of CaC2O4 crystals, we further investigated the interaction mechanism of nanoSe0 with BSA by spectroscopic techniques. This investigation corroborated the additive inhibitory effects on CaC2O4 crystals growth because of forming the complex of nanoSe0 with BSA and the partial conversion of secondary structure of BSA.
2. Experimental 2.1 Materials Selenium dioxide (SeO2), ascorbic acid (Vc), bovine serum albumin (BSA), sodium oxalate (Na2C2O4), anhydrous calcium chloride (CaCl2) and sodium chloride (NaCl) were all of analytical purity. All the chemicals were used as purchased without any further purification. Double distilled water was used throughout the experiment. Glass coverslips were thoroughly cleaned by treatment with piranha solution (1 h immersion in freshly prepared 3: 1 of H2SO4: H2O2), then sonicated and rinsed with double distilled water. The dialysis bag was boiled in the double distilled water with ethylenediaminetetraacetic acid 5
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(EDTA) for 30 min and stored at 277.15 K in the refrigerator. It was washed by double distilled water before use. 2.2 Preparation of nanoSe0 and nanoSe0-BSA NanoSe0 was prepared by mixing Vc solution (2 mL, 0.02 M) and SeO2 solution (2 mL, 0.01 M) and subsequently diluted to 10 mL. After the color of the solution changed from colorless to orange, the solution was dialyzed against double distilled water to separate Vc from nanoSe0 until no ultraviolet absorption of Vc could be found in the outer solutions. The mean diameter of nanoSe0 was 106 nm with a diameter distribution in the range of 37.8-115.7 nm. The nanoSe0-BSA was prepared by mixing Vc and SeO2 solution (2: 1 in molar ratio) in the presence of appropriate amount of BSA and subsequently diluted to required concentration. After the color of solution became orange, the solution was also dialyzed against double distilled water to separate Vc. 2.3 Preparation of CaC2O4 crystals Supersaturated CaC2O4 subphases were prepared by mixing appropriate amounts of CaCl2, NaCl and Na2C2O4 solution. The supersaturated CaC2O4 subphases were filtered through a 0.22 µm Millipore filter and transferred into the 25 mL beakers in which there were small pieces of glass coverslips. After that, different amounts of nanoSe0, BSA and nanoSe0-BSA were added into the supersaturated CaC2O4 subphases, respectively, and subsequently adjusted to pH 7 by adding appropriate amount of NaOH solution. The final concentration of NaCl was 2 mM and the final concentration of Ca2+ and C2O42- was 0.3 mM. Then the subphases were allowed to stand for 3 days. The temperature was controlled at 25±2℃. After 3 days, the CaC2O4 crystals grew in different systems and deposited on the glass coverslips. The small pieces of glass coverslips with the CaC2O4 crystals were transferred carefully in a silica-gel drier. Then CaC2O4 crystals were allowed to analyze by scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray analysis (EDX) and thermogravimetric analysis (TGA). 2.4 Methods for spectral measurements Solutions for spectral measurements were prepared as follows: in a series of 10 mL colorimetric tubes, 1 mL of 10 mg·mL-1 BSA, Tris-HCl buffer solution and appropriate nanoSe0 were added, respectively. So there were a series of solutions containing different concentration of 6
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nanoSe0 and the same concentration of BSA. Then the mixture was vibrated in order to make nanoSe0 and BSA combines completely. Ultraviolet-visible (UV-Vis) spectra and circular dichroism (CD) spectra of all the solutions were recorded in the range of 240-700 nm and 190-260 nm, respectively. For the fluorescence quenching phenomenon, the fluorescence spectra were measured (λex=278 nm) at three different temperatures (298 K, 304 K, and 310 K). 2.5 Instrumentation The morphologies of deposited CaC2O4 were observed by SEM (Pholips XL-30), and the CaC2O4 crystals were sputter-coated with gold before SEM analyzed. Elemental analysis of the crystals was measured by EDX (Zeiss Ultra55). The phase structure of CaC2O4 was determined by XRD (MSAL XD-2) using Cu Kα radiation at a scan rate of 8°/min, the accelerating voltage and applied current were 36 kV and 20 mA, respectively. The divergence and scattering slit was 1° in the range of 10°
