We described a facile method for assembly gold nanochains by using octreotide acetate as template in aqueous environment. In acidic solution, octreotide acetate was conferred positive charges and its structure changed to chain-like. The monodisperse negative gold nanoparticles were bound to the surface of octreotide acetate template by electrostatic attraction and the interaction of gold nanoparticles with amino acid residues (tryptophan and lysine). The fabricated gold nanostructure presented chain-like observed by transmission electron microscopy. The cytotoxicity of gold nanochains was examined by tetrazolium dye-based microtitration (MTT) assay, which demonstrated significantly less toxicity than that of octreotide acetate alone. The MTT assay also reflected the combinative action between the gold nanoparticles with octreotide acetate. Our work lays the groundwork for developing octreotide acetate-templated nanomaterials that can be used as a building block for the creation of nanomaterials. Meanwhile, the harmfulless gold nanochains have great application prospects in the biomedical filed.
Gold nanoparticles (AuNPs) have attracted increasing attention due to their great potential in biological and medical applications in recent years. AuNPs show excellent stability, universal biocompatibility, and unique optical, surface, electronic, and photocatalytic properties [
In various methods, biotemplate synthesis has emerged as an ideal encouraging approach to produce nanomaterials. The strategy can make AuNPs possessing excellent optical property by controlling the AuNPs at size and shape. Meanwhile, the biotemplate and AuNPs have high affinity, and the special morphologies of gold nanostructures were formed by different biotemplates. The high reproducibility and accuracy in controlling mineralizations and nanocrystal synthesis of various metals are also the merits of biotemplate method [
Therefore, we chose a neoteric strategy, using a new biomaterial as template to solve the problem of the chemical modifier. In numerous biomaterials, peptides have excellent potential as one of morphology-specific ligands due to their various amino acid sequences [
Octreotide acetate (OCT) is a synthetic octapeptide analogue of nature somatostatin [
In this study, a mild, versatile, and controlled methodology was used to form OCT-AuNPs chains by assembling AuNPs with OCT. The morphology of OCT-AuNPs chains was characterized by TEM, and UV-vis spectroscopy was used to measure the optical property of OCT-AuNPs chains. Finally, 3-(4,5-dimethylazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) array was measured to evaluate whether the OCT-AuNPs chains have the drug toxic effect of OCT. The result of MTT array demonstrated the potential biomedicine applications of the obtained OCT-AuNPs chains. Meanwhile, the OCT-Au complex as contrast was also studied prepared by reducing the solution of Au (III) precursor and OCT.
Octreotide acetate (C49H66N10O10S2) was purchased by Shanghai TASH Biotechnology Co., Ltd. (Shanghai, China). Gold (III) chloride (AuCl3) was obtained from Chengdu West Chemical Co., Ltd. (Chengdu, China). Sodium borohydride (NaBH4) was purchased from Tianjin chemistry factory (Tianjin, China). Hydrochloric acid (HCl) was supplied by Gu’an Chenmical plant (Langfang, China). 3-(4,5-dimeth-ylazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) got from Beyotime Institute of Biotechnology, Ltd. (Haimen, China). HeLa cells were provided by Cell Resource Center, IBMS, CAMS/PUMC. Deionized water prepared in our laboratory is used throughout all experiments.
OCT powder (0.3 mg) was dissolved into 2 mL of 0.01 mM HCl at room temperature, in the dark. OCT was denatured and obtained positive charges owing to the acidic environment. Then, some bond sites of OCT were exposed so that its structure converted from microspheres to chains. Ultimately, OCT nanochains were prepared.
The monodisperse AuNPs were synthesized by sodium borohydride reduction method at different Au : NaBH4 molar ratios. The AuCl3 (0.10, 0.25, 0.50, 0.75, and 1.00 mM, 1 mL), and freshly prepared NaBH4 (253.0 mM, 100
OCT solution (200
The OCT (0.15 mM, 200
Transmission electron microscope (TEM) images were obtained on a Hitachi model H-7650 instrument operated at an accelerating voltage of 80 kV. The specimens were prepared for analysis by evaporating a drop of aqueous product onto a carbon-coated 300 mesh TEM copper grid and then naturally dried in air. The dates of AuNP size were calculated from TEM images using particle size distribution calculation software. The TEM instrument was used for presenting the crystal spacing of the OCT-AuNPs (0.5 mM) chains (HRTEM) at an accelerating voltage of 200 KV. Meanwhile, its crystallizations were further identified by the selected area electron-diffraction (SAED) pattern on a JEM model 2010 instrument. For investigating the component elements of the OCT-AuNPs (0.5 mM) chains, an energy-dispersive X-ray spectroscopy (EDS) equipped on the TEM was used.
UV-vis absorption spectra of the samples were recorded on a Shimadzu model UV-2550 double-monochromator spectrophotometer with 1 cm quartz cuvette. The spectra were collected over the range of 200–800 nm. To study the interaction between AuNPs and OCT template, the absorption spectra of monodisperse AuNPs and OCT were measured to compare with these of OCT-AuNPs chains. For the OCT-AuNPs chains analysis, the original concentrations of Au element were 0.10, 0.25, 0.5, 0.75, and 1.00 mM and the samples were diluted with deionized water (1 : 4).
The human cervical carcinoma cell line (HeLa cell) was used to determine the cytotoxicity of OCT-AuNPS chains (0.50 mM) by a tetrazolium dye-based microtitration (MTT) assay. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal calf serum (10%) at 37°C in a 5% CO2 incubator. Cells were plated out in 96-well plates at a density of 2000 cells per well in 200
Dates are expressed as the mean and standard deviation (SD) in all experiments. Statistical analysis was performed using SPSS version 13.0. ANOVA was used to analyze statistical comparison between groups. The level of significance was set at
This procedure yielded AuNPs with different concentrations (1.0, 0.75, 0.5 mM) attached to the surface of the OCT scaffold was determined by TEM. Through the analysis of the particle size distribution calculation software, the mean AuNPs diameter and diameter distribution histogram were obtained. The dimensions of at least 200 AuNPs recorded on different locations of the TEM image have been compiled to obtain the date. Representative TEM images and the corresponding diameter distribution histograms of AuNPs were shown in Figure
Representative TEM images (left) and the corresponding diameter distribution hist-ograms (right) of OCT-AuNPs chains with different concentrations: (a) 1.0, (b) 0.75, and (c) 0.5 mM.
The variation in surface morphology before and after the mix of the AuNPs and OCT biotemplate was also examined by TEM. Figure
TEM images of dispersion AuNPs (a), before and after mixed with OCT template at pH 2.0 (b), and pH 8.0 (c).
OCT is a synthetic octapeptide analogue of nature somatostatin, cyclized by a disulfide bond between the two Cys residues (D-Phe-c(Cys-Phe-D-Trp-Lys-Thr-Cys)-Thr(ol)). OCT is a charged molecule, containing nonpolar amino acids (Phe, Trp), polar uncharged amino acids (Cys, Thr), and alkaline amino acid (Lys). In aqueous solution, OCT is ionized to form zwitterions. So, we can control the electronic charges of OCT by adjusting the pH of solution. According the report that OCT exhibits a net 2+ charge at pH 5.5 [
In addition, Tan et al. has reported that all the amino acids have different capping capability for the AuNPs. There are many other reports mentioned AuNPs can interact with many amino acid residues, such as tyrosine [
Preparation of OCT-Au complex as the control experiment was researched. The TEM image (Figure
TEM image of OCT-Au complex prepared by the co-incubation of OCT and AuCl3.
There has been reported some amino acids including Try own reductive capability [
The microstructure of AuNPs (0.5 mM) absorbing on the OCT nanochains was checked by HRTEM technique. The HRTEM image (Figure
HRTEM image of OCT-AuNPs (0.50 mM) chains, and the inset image shows the SAED pattern, corresponding to (111), (200), (220), (311), and (222) planes of Au fcc crystals.
The composition of OCT-AuNPs chains (0.5 mM) was analyzed by energy-dispersive X-ray spectroscopy (EDS). A typical EDS spectrum was shown in Figure
EDX analysis of OCT-AuNPs (0.50 mM) chains.
To confirm the interaction of OCT and AuNPs, the UV-Vis absorption spectra of OCT, AuNPs, and OCT-AuNPs were shown in Figure
UV-vis spectra of OCT, AuNPs, and OCT-AuNPs chains.
The UV-Vis absorption spectra of OCT-AuNPs chains with different concentrations are presented in Figure
UV-vis spectra of OCT-AuNPs chains with different concentrations.
On the other hand, the AuNPs absorption peaks displayed red shift along with the increase of concentrations in general except the sample (0.75 mM). The phenomenon is relative to the SPR of AuNPs. The AuNPs with different particle diameters produced different SPR effects, leading to the red shift in the absorption peak. The mean diameter of sample (0.75 mM) is slightly smaller than that of the sample (0.5 mM), so the sample (0.75 mM) became a special case. The special case also proved the theory that the surface plasmon resonance (SPR) property of AuNPs depended upon their particle characteristics (shape and size) [
Pictures of dispersion AuNPs solutions (a) and OCT-AuNPs solutions (b) with different concentrations. The concentration increased from left to right: 0.10, 0.25, 0.50, 0.75, 1.00 mM.
Just like other drugs, OCT has some side effects, including nausea, vomiting, loose/oily stools, and constipation. OCT-AuNPs chains need to be examined for biocompatibility if they are to be manufactured on a large scale for
Cytotoxicity of the monodisperse AuNPs, octreotide acetate (OCT), and OCT-AuNPs chains study by MTT assay. HeLa cells were incubated with samples with different concentrations (initial concentrations 1 = 0.25 mM),
OCT-AuNPs chains with lower toxicity than OCT can attribute the success to the interaction of AuNPs with the biological active sites of OCT. The essential amino acids of OCT are Phe3, Trp4, Lys5, and Thr6, in which Lys is the key active site of OCT peptide [
In summary, we have successfully demonstrated that octreotide acetate as biotemplate was used to assemble gold nanochains in aqueous environment for the first time. The significant findings include that a unique aspect of octreotide acetate is the ability to change its morphology to chain-like by adjusting pH value, which makes it attractive scaffolds for ordered arrays of nanochains. The formation mechanism of gold nanochains can be summarized as the electrostatic interaction between octreotide acetate and gold nanoparticles, conjugation of gold with special amino acids, Trp, and Lys. The [NaBH4] to [AuCl3] ratio determined the diameter of gold nanoparticles to change their surface plasmon resonance effects. The resulting OCT-AuNPs chains can have upstanding biomedicine applications because they did not have the evident toxic effect of octreotide acetate.
Jing Zhou and Zhanzhao Fu have equally contributed to this paper.
This work was financially supported by a research grant from the Chinese Ministry of Education Doctor Degree (20101333120011), grants from the Hebei Province Natural Science Fund (C2011203137, 11965152D), and a Chinese postdoctoral grant (480013).