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Synthesis of highly stable and biocompatible gold nanoparticles for use as a new X-ray contrast agent

Synthesis of highly stable and biocompatible gold nanoparticles for use as a new X-ray contrast... 1 Introduction X-ray-computed tomography (CT) is an imaging technique which is applied for variety of clinical diagnostics and research. High-resolution technique is used for 3D imaging of different tissue types and organ structures. Commonly, Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10856-018-6053-5) contains supplementary materials containing high atomic number (Z) or higher material, which is available to authorized users. * Pooya Iranpour Department of Chemistry, College of Sciences, Shiraz University, [email protected] Shiraz 7194684795, Iran * Afsaneh Safavi Department of Biology, Faculty of Science, University of Guilan, [email protected] Rasht, Iran Medical Imaging Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 1234567890();,: 1234567890();,: 48 Page 2 of 9 Journal of Materials Science: Materials in Medicine (2018) 29:48 density (ρ) are sufficient for CT therapy due to better X-ray different ionic liquids. Therefore, these anions are good absorption. Consequently, X-ray attenuation elements such choices for producing polar and hydrogen bond–rich ionic as barium, iodine and gold are commonly used for clinical liquids [25–27]. tissue imaging [1–3]. In this work, we introduce the newly synthesized AuNPs Iodine molecules as contrast agents are mostly used for which have been reduced with glucosammonium formate CT imaging due to high X-ray absorption coefficient of ionic liquid as a reducer and are capped with GA as a iodine, but they have rapid pharmacokinetics and high stabilizer. viscosity for injection to body [4, 5]. Thus, recently, nanoparticles have been introduced as a new class of con- trast agents [6]. The great advantage of application of 2 Experimental nanoparticles is that they have a longer vascular half-life than other molecular contrast agents and they are prone to 2.1 Materials functionalization for combining several imaging techniques [1, 5]. Nowadays, different nanoparticles have been intro- Tetrachloroauric (III) acid trihydrate (HAuCl.3H O, 4 2 duced as imaging probes for biomedicine and therapy 99.5%), sodium dihydrogen phosphate monohydrate applications [5]. Therefore, to obtain biocompatible nano- (NaH PO .H O, 99%), disodium hydrogen phosphate dihy- 2 4 2 particles a variety of parameters such as their shape, size, drate (Na HPO .2H O, 99.5%), formic acid, Gum Arabic 2 4 2 and surface functionalities [7] should be controlled and and ethanol were obtained from Merck. Human serum optimized. Among the numerous nanoparticles, gold albumin (HSA), bovine serum albumin (BSA) and D- nanoparticles (AuNPs) as biocompatible nanomaterial have glucosamine hydrochloride (>99%, Bio Reagent) were pur- −1 been applied for contrast enhancement of CT imaging. chased from Sigma. Visipaque (Iodixanol) (320 mgI mL ) These nanoparticles have higher X-ray absorption coeffi- was used as a commercial contrast agent. All chemicals were cient compared to iodine-based commercial contrast agents analytical grade and used as received without further [8–10]. purification. A wide range of methods have been reported for synthesis and modification of AuNPs and for formation of stable 2.2 Preparation of ionic liquid (D-glucosammonium AuNPs in CT applications. Some of these reports study the formate) application of biomolecules such as DNA, proteins, drugs and antibodies for functionalization of AuNPs [11–14]. For D-glucosamine hydrochloride (1.07 g, 5 mmol) was dissolved in vivo biomedical applications, the use of stable and bio- in 4 ml of deionized water and then the solution was neu- compatible AuNPs is inevitable. For this purpose, nontoxic tralized with a mole equivalent of sodium hydroxide (0.2 g, and green materials such as Gum Arabic (GA), a plant 5 mmol). Then, the solution containing D-glucosamine was extract, have been introduced for stabilizing AuNPs [15, 16]. placed in a two-necked flask and kept in an ice bath for Ionic liquids (ILs) have unique chemical and physical 15 min in order to adjustment the temperature of the solution properties, which have found numerous applications in at 0 °C. 5 mmol of formic acid was added drop-wise to the different fields [17]. Due to their unique properties, they are stirring solution of D-glucosamine. Stirring was continued for suitable green-based alternative to the conventional organic 24 h at room temperature. The obtained clear solution was solvents. In recent years, the majority of the studies have then dried in vacuum for 24 h at room temperature to remove been used for identification of their various properties such excess of water. For removal of the inorganic salt (sodium as thermal stability, conductivity, non-volatility, non- chloride), ethanol was added to the synthesized crude ionic flammability and ionic character [18, 19]. ILs have sig- liquid at 60 °C and filtered. The obtained bright yellow liquid nificant roles in chemical reactions such as catalysis, was evaporated and further dried in vacuum to remove separation, electrochemical process and nanoparticles ethanol. The synthesized compound was characterized using 1 13 synthesis [20–23]. Recently, imidazolium-based ILs have FTIR, Mass and NMR ( H, C). Scheme 1 shows the been reported for synthesis of AuNPs [7, 24]. structure of the synthesized ionic liquid. Although imidazolium-based ionic liquids with chloride anion are used as solvent for variety of compounds, but the 2.3 Synthesis of AuNPs halogen containing ionic liquids have problem such as high viscosity as solvent. The ionic liquids having carboxylic GA solution was prepared by mixing 15 mg of GA in 5 ml of acid anion have been introduced as halogen-free with high deionized water. Different amounts of HAuCl .3H O 4 2 hydrogen-bonding character [25]. As reported in literature, (0.05 M) were added to this solution, then the prepared a variety of carboxylic acid anions such as amino acid, solution was placed under mild heat at 60 °C. 50 mg of the lactic acid and formic acid are used as anion species of synthesized ionic liquid was added to the stirring solution and Journal of Materials Science: Materials in Medicine (2018) 29:48 Page 3 of 9 48 exposed to different concentrations of GA-AuNPs for 24 h. Cells treated with medium only served as a negative control group. The supernatant of each well was removed and 20 µl −1 of MTT solution (5 mg mL in PBS) was introduced to each well. After incubation for another 4 h, the resultant formazan crystals were dissolved in dimethyl sulfoxide (100 µl) and the absorbance intensity was measured by a microplate reader at 570 nm. All experiments were per- formed in quadruplicate, and the relative cell viability (%) related to control wells (cell culture medium without nanoparticles) was calculated by [absorbance] /[absor- treated Scheme 1 Structure of the synthesized ILs: glucosammonium formate bance] × 100. Data are expressed as the mean ± SD of control stirring was continued for 5 to 20 min depending on the four replicates. Statistical analysis was performed using concentration of HAuCl .3H O (150–1000 µM). During the Prism Software version 5.0 (Graph Pad, USA). P values less 4 2 synthesis process, the color of the solution changed from light than 0.05 were considered as statistically significant dif- yellow to red color which indicates the formation of AuNPs. ferences from the least cytotoxic treatment. For investigation of stability of AuNPs, the aqueous solution of AuNPs was evaporated by heating in a water bath 2.6 Instrumentation and then the solid state product was re-constituted in blood serum media (without deproteinization and pre-treatment The FT-IR and NMR spectra of ionic liquid were obtained steps). The stability and identity of the AuNPs were mea- using PerkinElmer FTIR model spectrum RXI spectrometer sured by recording the UV-Vis absorbance during 2 weeks and Bruker advance DPX-250( H NMR at 250 MHz and [8, 15]. Furthermore, to evaluate the stability of the synthe- CNMR at 62.9 MHz), respectively. The absorbance spectra sized AuNPs in physiological media, 0.5 ml of the prepared were recorded on a Schimadzu spectrophotometer by using AuNPs solution was treated with 0.5 mL of each of the fol- a 1-cm quartz cell. pH values were measured with a −1 lowing proteins: 1 mg mL human serum albumin (HSA) Metrohm 780 pH-meter. and bovine serum albumin (BSA) in phosphate buffer (PBS, Dynamic light scattering (DLS) measurements were 0.1 M pH 7.4) solutions. The stability and identity of the operated with ZEN 3600, Malvern. Philips F20-200KV AuNPs were evaluated by recording the surface plasmon instrument was used to obtain the transmission electron resonance (SPR) spectrum for one week [8, 15]. microscope (TEM) images. X-ray diffraction (XRD) pattern AuNPs were prepared for CT analysis as follows. The was taken by using D8 ADVANCE type (BRUKER-Ger- synthesized AuNPs solutions with different concentrations many). Phillips brilliance 16 slice CT scanner at 130 kVp of HAuCl .3H O (0.05 M) were evaporated by using water was used for CT analysis. 4 2 bath. Then the solid state products were re-constituted in least amount of deionized water [15]. For comparison of the contrast enhancement ability of the synthesized AuNPs, the 3 Results samples of commercial contrast agent (Visipaque) were prepared by appropriate dilution in deionized water. 3.1 Characterization of ionic liquid 2.4 Cell culture The ionic liquid was formed by a simple acid-base reaction between glucosamine and formic acid as explained in Section Cellular studies were performed on hepatocyte cell line 2.2. For characterization of the obtained ionic liquid, the FT- 1 13 HepG2, obtained from Pasteur Institute (Tehran, Iran). The IR, mass and NMR ( H, C) spectral data were used. The cells were cultured in RPMI medium (PAA, Austria) sup- peaks features of H-NMR spectrum were δ: 8.22 ppm (s, 1 H, plemented with 10% fetal bovine serum (PAA, Austria) and H–COO ); 5.26ppm (d, 0.59 H, related to 1α proton in 1% Penicillin-Streptomycin (PAA, Austria) in the incubator glucosammonium cation); 4.79 ppm (d, 0.49 H, 1β proton in (Heraeus, Germany) at 37 °C and under 5% CO . glucosammonium cation); 2.78-3.78 ppm; (m, 6 H, protons in the ring of glucosammonium cation). As the results show 2.5 In vitro cytotoxicity assay (MTT assay) the sharp singlet peak at 8.22 ppm assigns to proton of for- myl group. Furthermore, the peak of formyl group at δ:169.2 To determine cell viability of GA-AuNPs, the colorimetric ppm in C-NMR spectrum confirms the presence of formate MTT metabolic activity assay was used. HepG2 cells (1.5 × (H–COO ) anion in the structure of ionic liquid. Also, in the 10 cells/well) were cultured in a 96-well plate at 37 °C, and FT-IR spectrum, the OH and ammonium stretching 48 Page 4 of 9 Journal of Materials Science: Materials in Medicine (2018) 29:48 Fig. 1 a Photographs and b Absorption spectra of the prepared GA-AuNPs in the presence of (a) glucosamine hydrochloride (b) IL (c) Sodium formate −1 vibrations are embedded in 2800–3400 cm range. A broad presence of 50 mg of the prepared ionic liquid. The resultant −1 peak at 1600 cm was assigned to vibrations of N-H plane UV-vis spectra of GA capped AuNPs were recorded. The bending and carbonyl stretching [26]. Additionally, mass optimum amount of GA for the synthesis of AuNPs was spectrum shows the M peak at m/z= 225 which is related 15 mg (Fig. S4, supporting information). to the synthesized ionic liquid. These spectral data have been shown in Fig.’s S1-3 (supporting information), which con- 3.3.2 Effect of different amounts of ionic liquid firm the formation of glucosammonium formate compound. In order to obtain sufficient reducing effect of gluco- 3.2 UV-Vis spectrum sammonium formate as the IL, different amounts of ionic liquid were added to 500 µM of HAuCl solution containing In order to study the effect of GA as a stabilizer in this 15 mg GA as capping agent. The resultant UV-Vis spectra work, the formation of AuNPs was evaluated in the absence were obtained and 50 mg of the IL gave the best result. of GA. Although AuNPs were formed in the presence of glucosammonium forrmate as a reducer, the yield of the 3.4 Characterization of the synthesized AuNPs synthesized dispersed AuNPs was low and precipitation occurred. Thus, to improve the stabilization effect and yield The synthesis of AuNPs was simple and fast as stated in of synthesis of AuNPs, GA was used. Also, it is important Section 2.3. They were characterized by using TEM, DLS to signify the role of the prepared ionic liquid. For this and XRD techniques. The morphologies of the AuNPs are purpose, in a preliminary test, glucosammonium hydro- shown in Fig. 2a. The average size of the synthesized chloride and formic acid which had been used as precursors nanoparticles was found as ~25 nm from TEM image. The for synthesis of the ionic liquid were separately treated with synthesized capped- AuNPs were also characterized with AuCl . In both cases, the reduction of gold ions occurred DLS analysis. Figure 2b shows the results of DLS analysis of after long times and there was not a clear surface plasmon the synthesized GA capped- AuNPs. The average size of resonance at 530 nm after 2 h, (Fig. 1). However, when the AuNPs was obtained as 24 nm from DLS analysis, showing a glucosammonium formate as the ionic liquid was used as a good agreement with the result obtained by TEM technique. reducing agent, synthesis of AuNPs was occurred after XRD analysis was also used for characterization of the 5–20 min depending on the concentration of HAuCl .3H O as-prepared AuNPs. Figure 2c shows the peaks of (111), 4 2 (150–1000 µM). (200), (220), (311), and (222) planes of fcc (face-centered- cubic) lattice of gold [28, 29]. 3.3 Optimization experiments 3.5 Stability of AuNPs 3.3.1 Effect of different amounts of Gum Arabic To study the stability of the resultant AuNPs in conven- In order to obtain stable AuNPs, different amounts of GA tional electrolyte media, the changes in their SPR band were (5–20 mg) were added to 500 µM of HAuCl solution in the examined in sodium chloride (NaCl) medium. The SPR 4 Journal of Materials Science: Materials in Medicine (2018) 29:48 Page 5 of 9 48 Fig. 2 a TEM image b DLS analysis and c XRD pattern of the synthesized GA-AuNPs solid product was then re-constituted in 5 ml of human serum blood instead of deionized water. As it is obvious from Fig. 4, the synthesized AuNPs show successful sta- bility in serum medium which lasted for at least two weeks. For evaluation of in vitro stability of the synthesized AuNPs, 0.5 ml of the prepared AuNPs solution was treated −1 with 0.5 mL each of the following proteins: 1 mg mL −1 human serum albumin (HSA) and 1 mg mL bovine serum albumin (BSA) in phosphate buffer (PBS, 0.1 M pH 7.4) solutions. The stability of the prepared AuNPs was studied by monitoring their SPR band in a seven-days period. As it is obvious in Fig. 5, the SPR of AuNPs has low variation after seven days and no precipitation was observed in the solution. Fig. 3 SPR wavelength shifts upon injections of NaCl 3.6 Cytotoxicity and biocompatibility studies of GA- AuNPs changes of AuNPs upon successive injections of 40 µL of MTT assay has been performed to determine the toxicity of NaCl (2 M) solution to 2 ml of AuNPs are obvious in Fig. 3. the GA-AuNPs in vitro. Since an appreciable amount of the It was found that the synthesized AuNPs are highly stable in nanoparticles accumulate in the liver, HepG2 hepatocyte the presence of NaCl medium. cell line was chosen. Figure 6 depicts the biocompatibility The stability of GA-capped AuNPs was further studied of GA-AuNPs with cellular environments. It is clearly by evaporation of AuNPs solution using a water bath. The observed that after 24 h, almost 100% of cells are viable 48 Page 6 of 9 Journal of Materials Science: Materials in Medicine (2018) 29:48 −1 with GA-AuNPs concentrations as high as 50 µg mL . attenuation of the prepared AuNPs and Visipaque as a Moreover, more than 80% of the cells (Fig. 6) were alive commercial contrast agent are compared in Fig. 7a at dif- −1 after 24 h of incubation with 100 µg mL of GA-AuNPs. ferent concentrations. Moreover, Fig. 7b shows X-ray CT These results are in good agreement with those obtained in vitro images of the prepared AuNPs and Visipaque at from Visipaque as conventional X-ray contrast agent equal concentrations. (Fig. 6). 3.7 Contrast Enhancement by GA Capped–AuNPs in 4 Discussion CT Imaging The characteristic surface plasmon resonance (SPR) band of Contrast enhancement by the prepared AuNPs was checked AuNPs is usually the first indicator of AuNPs formation in using phantoms from GA capped-AuNPs. The X-ray solution. Size and shapes of the prepared AuNPs directly depend on the concentration and nature of the protecting agents in the solution. In this work, glucosammonium for- mate ionic liquid was used as a nontoxic and biocompatible compound for reducing AuCl . GA has been previously a- AuNPs b- Visipaque 2 5 10 20 50 100 -1 Concentration (µg mL ) Fig. 6 Cell viability of HepG2 cells using MTT assay after 24 h of Fig. 4 Photograph images and UV-Vis spectra of GA-AuNPssolution incubation with increasing amounts of (a) GA-AuNPs and (b) Visi- with HAuCl concentration of 500 µM, 15.0 mg GA and 50 mg IL in paque. Data are expressed as the mean ± SD of 4 replicates. The 5 mL solution before evaporation (a) and after evaporation and re- symbols denote statistically significant differences from the least constitute in blood serum (b) after two weeks in blood serum media (c) cytotoxic treatment: * P < 0.05 Fig. 5 UV-Vis spectra of GA-AuNPs in the absence and presence of HSA and BSA solution after incubation times of 2 h (a) and one week (b), −1 respectively. [BSA] = [HSA] = 1.0 mg mL in phosphate buffer (pH = 7.4, 0.10 M) Relative cell viability (%) Journal of Materials Science: Materials in Medicine (2018) 29:48 Page 7 of 9 48 −1 Fig. 7 a Linear dependence of ΔHU on the (a) Visipaque and (b) AuNPs concentration (mg mL ) and b X-ray CT in vitro images, the vials −1 containing of 0.2 mg mL (a) GA–AuNPs and (b) Visipaque introduced as a nontoxic stabilizer for AuNPs [8, 15]. As aggregation of capped- AuNPs. Moreover, the obtained shown in Fig. 1, SPR spectrum of AuNPs with an absorption results indicate that the synthesized AuNPs are stable in maximum at about 530 nm shows the appropriate synthesis biological media at physiological pH value (Fig. 5). of AuNPs in the presence of glucosammonium formate and Therefore, the HSA and BSA were not adsorbed to the GA as reducing and stabilizer agents, respectively. surface of GA-capped AuNPs significantly. As reported In order to prepare the stable and disperse AuNPs, 15 mg previously [30], these proteins have extreme tendency to the of GA was selected as the optimum amount (Fig. S4, sup- surface of bare AuNPs. It can be concluded from the porting information). The data revealed that for low and high obtained results, that the GA molecule framework around amounts of GA, the SPR band changed from red to blue the synthesized AuNPs is an effective coating for producing region with a broad and weak absorption. This observation intact AuNPs. Another point that should be considered is suggested that for low amounts of GA as capping agent, the biocompatibility of GA and the prepared ionic liquid as protective shell was not effective for synthesis of stable capping and effective reducing agents, respectively. The AuNPs. Hence, by increasing the GA amounts from 5 to results of MTT assay clearly show the possible application 15 mg, sufficient protection of AuNPs was obtained and of GA-AuNPs as a biocompatible contrast agent in intra- absorption band became strong and sharp. But for high cellular environments. However, more detailed toxicity amounts of GA (20 mg), the band broadening occurred (Fig. studies to other cell lines in vitro and study of long-term S4). This could be due to the fact that in excess amounts of organ toxicity, are needed before these nanoparticles can be GA, the intermolecular forces of glycoprotein networks of used in clinical trials. GA molecules are increased by adding the GA amounts [16]. Various AuNPs were investigated as CT contrast agents Also, sufficient reducing effect of glucosammonium formate [31]. Among them, citrate-capped AuNPs is the most popular was achieved for 50 mg of IL. Small amounts of IL (<50 mg) for the reduction of HAuCl . However, the resulting citrate- were not sufficient to reduce all gold ions. Also, as the stabilized AuNPs are not stable in biological media [31, 32]. amount of IL was increased more than 50 mg, the color of the Thus, the introduction of a developed method for producing solution changed from red to purple. With increasing the stable AuNPs, especially in the field of CT, is significant. amount of IL, a broad particle size distribution can be hap- Therefore, in this work, the ability of the synthesized AuNPs pened due to broadening of SPR band. to absorb X-ray was evaluated. As shown in the Fig. 7a, the Generally, it has been known that AuNPs are unstable X-ray absorption of the prepared AuNPs and Visipaque and aggregated in electrolyte medium [30]. As shown in increases with increasing the concentration of both gold and Fig. 3, when NaCl concentration increased from 0 to 2 M, Visipaque. However, at similar concentrations, the X-ray the characteristic absorption band did not change sig- intensities of the prepared AuNPs are higher than those of nificantly, showing the high stability of AuNPs in NaCl Visipaque. Also, as shown in Fig. 7a, there is a linear rela- solution. This stability of the synthesized AuNPs in a tionship between the concentration of the prepared AuNPs complex matrix such as whole blood can be explained by and Visipaque with ΔHU (HU= Hounsfield units). The −1 the presence of GA as a capping agent which prevents the measurements show that 0.20 mg ml of the prepared 48 Page 8 of 9 Journal of Materials Science: Materials in Medicine (2018) 29:48 −1 AuNPs gives an equivalent X-ray absorption as 0.66 mg ml 7. Safavi A, Zeinali S, Yazdani M. Synthesis of biologically stable gold nanoparticles using imidazolium-based amino acid ionic of Visipaque. So that, at equal concentrations, the X-ray liquids. Amino Acids. 2012;43:1323–30. attenuation coefficient of the synthesized AuNPs is ~3.3 8. Shahidi S, Iranpour S, Iranpour P, Alavi AA, Mahyari FA, Tohidi times higher than that of Visipaque as a conventional iodine- M et al. A new X-ray contrast agent based on highly stable Gum based contrast agent (Fig. 7). This enhanced HU value of Arabic-gold nanoparticles synthesised in deep eutectic solvent. J Exp Nano 2014(ahead-of-print):1-14. GA–AuNPs is in good agreement with other reported AuNPs 9. Peng C, Zheng L, Chen Q, Shen M, Guo R, Wang H, et al. [33, 34]. The obtained results clearly indicate that the dis- PEGylated dendrimer-entrapped gold nanoparticles for in vivo persed and stable AuNPs have a high potential for use in X- blood pool and tumor imaging by computed tomography. Bio- ray imaging. materials. 2012;33:1107–19. 10. Xu C, Tung GA, Sun S. Size and concentration effect of gold nanoparticles on X-ray attenuation as measured on computed tomography. Chem Mater. 2008;20:4167–9. 5 Conclusion 11. Loo L, Guenther RH, Basnayake VR, Lommel SA, Franzen S. Controlled encapsidation of gold nanoparticles by a viral protein shell. J Am Chem Soc. 2006;128:4502–3. In conclusion, a new procedure has been reported for 12. 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Synthesis of highly stable and biocompatible gold nanoparticles for use as a new X-ray contrast agent

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Springer Journals
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Copyright © 2018 by Springer Science+Business Media, LLC, part of Springer Nature
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Materials Science; Biomaterials; Biomedical Engineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
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10.1007/s10856-018-6053-5
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Abstract

1 Introduction X-ray-computed tomography (CT) is an imaging technique which is applied for variety of clinical diagnostics and research. High-resolution technique is used for 3D imaging of different tissue types and organ structures. Commonly, Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10856-018-6053-5) contains supplementary materials containing high atomic number (Z) or higher material, which is available to authorized users. * Pooya Iranpour Department of Chemistry, College of Sciences, Shiraz University, [email protected] Shiraz 7194684795, Iran * Afsaneh Safavi Department of Biology, Faculty of Science, University of Guilan, [email protected] Rasht, Iran Medical Imaging Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 1234567890();,: 1234567890();,: 48 Page 2 of 9 Journal of Materials Science: Materials in Medicine (2018) 29:48 density (ρ) are sufficient for CT therapy due to better X-ray different ionic liquids. Therefore, these anions are good absorption. Consequently, X-ray attenuation elements such choices for producing polar and hydrogen bond–rich ionic as barium, iodine and gold are commonly used for clinical liquids [25–27]. tissue imaging [1–3]. In this work, we introduce the newly synthesized AuNPs Iodine molecules as contrast agents are mostly used for which have been reduced with glucosammonium formate CT imaging due to high X-ray absorption coefficient of ionic liquid as a reducer and are capped with GA as a iodine, but they have rapid pharmacokinetics and high stabilizer. viscosity for injection to body [4, 5]. Thus, recently, nanoparticles have been introduced as a new class of con- trast agents [6]. The great advantage of application of 2 Experimental nanoparticles is that they have a longer vascular half-life than other molecular contrast agents and they are prone to 2.1 Materials functionalization for combining several imaging techniques [1, 5]. Nowadays, different nanoparticles have been intro- Tetrachloroauric (III) acid trihydrate (HAuCl.3H O, 4 2 duced as imaging probes for biomedicine and therapy 99.5%), sodium dihydrogen phosphate monohydrate applications [5]. Therefore, to obtain biocompatible nano- (NaH PO .H O, 99%), disodium hydrogen phosphate dihy- 2 4 2 particles a variety of parameters such as their shape, size, drate (Na HPO .2H O, 99.5%), formic acid, Gum Arabic 2 4 2 and surface functionalities [7] should be controlled and and ethanol were obtained from Merck. Human serum optimized. Among the numerous nanoparticles, gold albumin (HSA), bovine serum albumin (BSA) and D- nanoparticles (AuNPs) as biocompatible nanomaterial have glucosamine hydrochloride (>99%, Bio Reagent) were pur- −1 been applied for contrast enhancement of CT imaging. chased from Sigma. Visipaque (Iodixanol) (320 mgI mL ) These nanoparticles have higher X-ray absorption coeffi- was used as a commercial contrast agent. All chemicals were cient compared to iodine-based commercial contrast agents analytical grade and used as received without further [8–10]. purification. A wide range of methods have been reported for synthesis and modification of AuNPs and for formation of stable 2.2 Preparation of ionic liquid (D-glucosammonium AuNPs in CT applications. Some of these reports study the formate) application of biomolecules such as DNA, proteins, drugs and antibodies for functionalization of AuNPs [11–14]. For D-glucosamine hydrochloride (1.07 g, 5 mmol) was dissolved in vivo biomedical applications, the use of stable and bio- in 4 ml of deionized water and then the solution was neu- compatible AuNPs is inevitable. For this purpose, nontoxic tralized with a mole equivalent of sodium hydroxide (0.2 g, and green materials such as Gum Arabic (GA), a plant 5 mmol). Then, the solution containing D-glucosamine was extract, have been introduced for stabilizing AuNPs [15, 16]. placed in a two-necked flask and kept in an ice bath for Ionic liquids (ILs) have unique chemical and physical 15 min in order to adjustment the temperature of the solution properties, which have found numerous applications in at 0 °C. 5 mmol of formic acid was added drop-wise to the different fields [17]. Due to their unique properties, they are stirring solution of D-glucosamine. Stirring was continued for suitable green-based alternative to the conventional organic 24 h at room temperature. The obtained clear solution was solvents. In recent years, the majority of the studies have then dried in vacuum for 24 h at room temperature to remove been used for identification of their various properties such excess of water. For removal of the inorganic salt (sodium as thermal stability, conductivity, non-volatility, non- chloride), ethanol was added to the synthesized crude ionic flammability and ionic character [18, 19]. ILs have sig- liquid at 60 °C and filtered. The obtained bright yellow liquid nificant roles in chemical reactions such as catalysis, was evaporated and further dried in vacuum to remove separation, electrochemical process and nanoparticles ethanol. The synthesized compound was characterized using 1 13 synthesis [20–23]. Recently, imidazolium-based ILs have FTIR, Mass and NMR ( H, C). Scheme 1 shows the been reported for synthesis of AuNPs [7, 24]. structure of the synthesized ionic liquid. Although imidazolium-based ionic liquids with chloride anion are used as solvent for variety of compounds, but the 2.3 Synthesis of AuNPs halogen containing ionic liquids have problem such as high viscosity as solvent. The ionic liquids having carboxylic GA solution was prepared by mixing 15 mg of GA in 5 ml of acid anion have been introduced as halogen-free with high deionized water. Different amounts of HAuCl .3H O 4 2 hydrogen-bonding character [25]. As reported in literature, (0.05 M) were added to this solution, then the prepared a variety of carboxylic acid anions such as amino acid, solution was placed under mild heat at 60 °C. 50 mg of the lactic acid and formic acid are used as anion species of synthesized ionic liquid was added to the stirring solution and Journal of Materials Science: Materials in Medicine (2018) 29:48 Page 3 of 9 48 exposed to different concentrations of GA-AuNPs for 24 h. Cells treated with medium only served as a negative control group. The supernatant of each well was removed and 20 µl −1 of MTT solution (5 mg mL in PBS) was introduced to each well. After incubation for another 4 h, the resultant formazan crystals were dissolved in dimethyl sulfoxide (100 µl) and the absorbance intensity was measured by a microplate reader at 570 nm. All experiments were per- formed in quadruplicate, and the relative cell viability (%) related to control wells (cell culture medium without nanoparticles) was calculated by [absorbance] /[absor- treated Scheme 1 Structure of the synthesized ILs: glucosammonium formate bance] × 100. Data are expressed as the mean ± SD of control stirring was continued for 5 to 20 min depending on the four replicates. Statistical analysis was performed using concentration of HAuCl .3H O (150–1000 µM). During the Prism Software version 5.0 (Graph Pad, USA). P values less 4 2 synthesis process, the color of the solution changed from light than 0.05 were considered as statistically significant dif- yellow to red color which indicates the formation of AuNPs. ferences from the least cytotoxic treatment. For investigation of stability of AuNPs, the aqueous solution of AuNPs was evaporated by heating in a water bath 2.6 Instrumentation and then the solid state product was re-constituted in blood serum media (without deproteinization and pre-treatment The FT-IR and NMR spectra of ionic liquid were obtained steps). The stability and identity of the AuNPs were mea- using PerkinElmer FTIR model spectrum RXI spectrometer sured by recording the UV-Vis absorbance during 2 weeks and Bruker advance DPX-250( H NMR at 250 MHz and [8, 15]. Furthermore, to evaluate the stability of the synthe- CNMR at 62.9 MHz), respectively. The absorbance spectra sized AuNPs in physiological media, 0.5 ml of the prepared were recorded on a Schimadzu spectrophotometer by using AuNPs solution was treated with 0.5 mL of each of the fol- a 1-cm quartz cell. pH values were measured with a −1 lowing proteins: 1 mg mL human serum albumin (HSA) Metrohm 780 pH-meter. and bovine serum albumin (BSA) in phosphate buffer (PBS, Dynamic light scattering (DLS) measurements were 0.1 M pH 7.4) solutions. The stability and identity of the operated with ZEN 3600, Malvern. Philips F20-200KV AuNPs were evaluated by recording the surface plasmon instrument was used to obtain the transmission electron resonance (SPR) spectrum for one week [8, 15]. microscope (TEM) images. X-ray diffraction (XRD) pattern AuNPs were prepared for CT analysis as follows. The was taken by using D8 ADVANCE type (BRUKER-Ger- synthesized AuNPs solutions with different concentrations many). Phillips brilliance 16 slice CT scanner at 130 kVp of HAuCl .3H O (0.05 M) were evaporated by using water was used for CT analysis. 4 2 bath. Then the solid state products were re-constituted in least amount of deionized water [15]. For comparison of the contrast enhancement ability of the synthesized AuNPs, the 3 Results samples of commercial contrast agent (Visipaque) were prepared by appropriate dilution in deionized water. 3.1 Characterization of ionic liquid 2.4 Cell culture The ionic liquid was formed by a simple acid-base reaction between glucosamine and formic acid as explained in Section Cellular studies were performed on hepatocyte cell line 2.2. For characterization of the obtained ionic liquid, the FT- 1 13 HepG2, obtained from Pasteur Institute (Tehran, Iran). The IR, mass and NMR ( H, C) spectral data were used. The cells were cultured in RPMI medium (PAA, Austria) sup- peaks features of H-NMR spectrum were δ: 8.22 ppm (s, 1 H, plemented with 10% fetal bovine serum (PAA, Austria) and H–COO ); 5.26ppm (d, 0.59 H, related to 1α proton in 1% Penicillin-Streptomycin (PAA, Austria) in the incubator glucosammonium cation); 4.79 ppm (d, 0.49 H, 1β proton in (Heraeus, Germany) at 37 °C and under 5% CO . glucosammonium cation); 2.78-3.78 ppm; (m, 6 H, protons in the ring of glucosammonium cation). As the results show 2.5 In vitro cytotoxicity assay (MTT assay) the sharp singlet peak at 8.22 ppm assigns to proton of for- myl group. Furthermore, the peak of formyl group at δ:169.2 To determine cell viability of GA-AuNPs, the colorimetric ppm in C-NMR spectrum confirms the presence of formate MTT metabolic activity assay was used. HepG2 cells (1.5 × (H–COO ) anion in the structure of ionic liquid. Also, in the 10 cells/well) were cultured in a 96-well plate at 37 °C, and FT-IR spectrum, the OH and ammonium stretching 48 Page 4 of 9 Journal of Materials Science: Materials in Medicine (2018) 29:48 Fig. 1 a Photographs and b Absorption spectra of the prepared GA-AuNPs in the presence of (a) glucosamine hydrochloride (b) IL (c) Sodium formate −1 vibrations are embedded in 2800–3400 cm range. A broad presence of 50 mg of the prepared ionic liquid. The resultant −1 peak at 1600 cm was assigned to vibrations of N-H plane UV-vis spectra of GA capped AuNPs were recorded. The bending and carbonyl stretching [26]. Additionally, mass optimum amount of GA for the synthesis of AuNPs was spectrum shows the M peak at m/z= 225 which is related 15 mg (Fig. S4, supporting information). to the synthesized ionic liquid. These spectral data have been shown in Fig.’s S1-3 (supporting information), which con- 3.3.2 Effect of different amounts of ionic liquid firm the formation of glucosammonium formate compound. In order to obtain sufficient reducing effect of gluco- 3.2 UV-Vis spectrum sammonium formate as the IL, different amounts of ionic liquid were added to 500 µM of HAuCl solution containing In order to study the effect of GA as a stabilizer in this 15 mg GA as capping agent. The resultant UV-Vis spectra work, the formation of AuNPs was evaluated in the absence were obtained and 50 mg of the IL gave the best result. of GA. Although AuNPs were formed in the presence of glucosammonium forrmate as a reducer, the yield of the 3.4 Characterization of the synthesized AuNPs synthesized dispersed AuNPs was low and precipitation occurred. Thus, to improve the stabilization effect and yield The synthesis of AuNPs was simple and fast as stated in of synthesis of AuNPs, GA was used. Also, it is important Section 2.3. They were characterized by using TEM, DLS to signify the role of the prepared ionic liquid. For this and XRD techniques. The morphologies of the AuNPs are purpose, in a preliminary test, glucosammonium hydro- shown in Fig. 2a. The average size of the synthesized chloride and formic acid which had been used as precursors nanoparticles was found as ~25 nm from TEM image. The for synthesis of the ionic liquid were separately treated with synthesized capped- AuNPs were also characterized with AuCl . In both cases, the reduction of gold ions occurred DLS analysis. Figure 2b shows the results of DLS analysis of after long times and there was not a clear surface plasmon the synthesized GA capped- AuNPs. The average size of resonance at 530 nm after 2 h, (Fig. 1). However, when the AuNPs was obtained as 24 nm from DLS analysis, showing a glucosammonium formate as the ionic liquid was used as a good agreement with the result obtained by TEM technique. reducing agent, synthesis of AuNPs was occurred after XRD analysis was also used for characterization of the 5–20 min depending on the concentration of HAuCl .3H O as-prepared AuNPs. Figure 2c shows the peaks of (111), 4 2 (150–1000 µM). (200), (220), (311), and (222) planes of fcc (face-centered- cubic) lattice of gold [28, 29]. 3.3 Optimization experiments 3.5 Stability of AuNPs 3.3.1 Effect of different amounts of Gum Arabic To study the stability of the resultant AuNPs in conven- In order to obtain stable AuNPs, different amounts of GA tional electrolyte media, the changes in their SPR band were (5–20 mg) were added to 500 µM of HAuCl solution in the examined in sodium chloride (NaCl) medium. The SPR 4 Journal of Materials Science: Materials in Medicine (2018) 29:48 Page 5 of 9 48 Fig. 2 a TEM image b DLS analysis and c XRD pattern of the synthesized GA-AuNPs solid product was then re-constituted in 5 ml of human serum blood instead of deionized water. As it is obvious from Fig. 4, the synthesized AuNPs show successful sta- bility in serum medium which lasted for at least two weeks. For evaluation of in vitro stability of the synthesized AuNPs, 0.5 ml of the prepared AuNPs solution was treated −1 with 0.5 mL each of the following proteins: 1 mg mL −1 human serum albumin (HSA) and 1 mg mL bovine serum albumin (BSA) in phosphate buffer (PBS, 0.1 M pH 7.4) solutions. The stability of the prepared AuNPs was studied by monitoring their SPR band in a seven-days period. As it is obvious in Fig. 5, the SPR of AuNPs has low variation after seven days and no precipitation was observed in the solution. Fig. 3 SPR wavelength shifts upon injections of NaCl 3.6 Cytotoxicity and biocompatibility studies of GA- AuNPs changes of AuNPs upon successive injections of 40 µL of MTT assay has been performed to determine the toxicity of NaCl (2 M) solution to 2 ml of AuNPs are obvious in Fig. 3. the GA-AuNPs in vitro. Since an appreciable amount of the It was found that the synthesized AuNPs are highly stable in nanoparticles accumulate in the liver, HepG2 hepatocyte the presence of NaCl medium. cell line was chosen. Figure 6 depicts the biocompatibility The stability of GA-capped AuNPs was further studied of GA-AuNPs with cellular environments. It is clearly by evaporation of AuNPs solution using a water bath. The observed that after 24 h, almost 100% of cells are viable 48 Page 6 of 9 Journal of Materials Science: Materials in Medicine (2018) 29:48 −1 with GA-AuNPs concentrations as high as 50 µg mL . attenuation of the prepared AuNPs and Visipaque as a Moreover, more than 80% of the cells (Fig. 6) were alive commercial contrast agent are compared in Fig. 7a at dif- −1 after 24 h of incubation with 100 µg mL of GA-AuNPs. ferent concentrations. Moreover, Fig. 7b shows X-ray CT These results are in good agreement with those obtained in vitro images of the prepared AuNPs and Visipaque at from Visipaque as conventional X-ray contrast agent equal concentrations. (Fig. 6). 3.7 Contrast Enhancement by GA Capped–AuNPs in 4 Discussion CT Imaging The characteristic surface plasmon resonance (SPR) band of Contrast enhancement by the prepared AuNPs was checked AuNPs is usually the first indicator of AuNPs formation in using phantoms from GA capped-AuNPs. The X-ray solution. Size and shapes of the prepared AuNPs directly depend on the concentration and nature of the protecting agents in the solution. In this work, glucosammonium for- mate ionic liquid was used as a nontoxic and biocompatible compound for reducing AuCl . GA has been previously a- AuNPs b- Visipaque 2 5 10 20 50 100 -1 Concentration (µg mL ) Fig. 6 Cell viability of HepG2 cells using MTT assay after 24 h of Fig. 4 Photograph images and UV-Vis spectra of GA-AuNPssolution incubation with increasing amounts of (a) GA-AuNPs and (b) Visi- with HAuCl concentration of 500 µM, 15.0 mg GA and 50 mg IL in paque. Data are expressed as the mean ± SD of 4 replicates. The 5 mL solution before evaporation (a) and after evaporation and re- symbols denote statistically significant differences from the least constitute in blood serum (b) after two weeks in blood serum media (c) cytotoxic treatment: * P < 0.05 Fig. 5 UV-Vis spectra of GA-AuNPs in the absence and presence of HSA and BSA solution after incubation times of 2 h (a) and one week (b), −1 respectively. [BSA] = [HSA] = 1.0 mg mL in phosphate buffer (pH = 7.4, 0.10 M) Relative cell viability (%) Journal of Materials Science: Materials in Medicine (2018) 29:48 Page 7 of 9 48 −1 Fig. 7 a Linear dependence of ΔHU on the (a) Visipaque and (b) AuNPs concentration (mg mL ) and b X-ray CT in vitro images, the vials −1 containing of 0.2 mg mL (a) GA–AuNPs and (b) Visipaque introduced as a nontoxic stabilizer for AuNPs [8, 15]. As aggregation of capped- AuNPs. Moreover, the obtained shown in Fig. 1, SPR spectrum of AuNPs with an absorption results indicate that the synthesized AuNPs are stable in maximum at about 530 nm shows the appropriate synthesis biological media at physiological pH value (Fig. 5). of AuNPs in the presence of glucosammonium formate and Therefore, the HSA and BSA were not adsorbed to the GA as reducing and stabilizer agents, respectively. surface of GA-capped AuNPs significantly. As reported In order to prepare the stable and disperse AuNPs, 15 mg previously [30], these proteins have extreme tendency to the of GA was selected as the optimum amount (Fig. S4, sup- surface of bare AuNPs. It can be concluded from the porting information). The data revealed that for low and high obtained results, that the GA molecule framework around amounts of GA, the SPR band changed from red to blue the synthesized AuNPs is an effective coating for producing region with a broad and weak absorption. This observation intact AuNPs. Another point that should be considered is suggested that for low amounts of GA as capping agent, the biocompatibility of GA and the prepared ionic liquid as protective shell was not effective for synthesis of stable capping and effective reducing agents, respectively. The AuNPs. Hence, by increasing the GA amounts from 5 to results of MTT assay clearly show the possible application 15 mg, sufficient protection of AuNPs was obtained and of GA-AuNPs as a biocompatible contrast agent in intra- absorption band became strong and sharp. But for high cellular environments. However, more detailed toxicity amounts of GA (20 mg), the band broadening occurred (Fig. studies to other cell lines in vitro and study of long-term S4). This could be due to the fact that in excess amounts of organ toxicity, are needed before these nanoparticles can be GA, the intermolecular forces of glycoprotein networks of used in clinical trials. GA molecules are increased by adding the GA amounts [16]. Various AuNPs were investigated as CT contrast agents Also, sufficient reducing effect of glucosammonium formate [31]. Among them, citrate-capped AuNPs is the most popular was achieved for 50 mg of IL. Small amounts of IL (<50 mg) for the reduction of HAuCl . However, the resulting citrate- were not sufficient to reduce all gold ions. Also, as the stabilized AuNPs are not stable in biological media [31, 32]. amount of IL was increased more than 50 mg, the color of the Thus, the introduction of a developed method for producing solution changed from red to purple. With increasing the stable AuNPs, especially in the field of CT, is significant. amount of IL, a broad particle size distribution can be hap- Therefore, in this work, the ability of the synthesized AuNPs pened due to broadening of SPR band. to absorb X-ray was evaluated. As shown in the Fig. 7a, the Generally, it has been known that AuNPs are unstable X-ray absorption of the prepared AuNPs and Visipaque and aggregated in electrolyte medium [30]. As shown in increases with increasing the concentration of both gold and Fig. 3, when NaCl concentration increased from 0 to 2 M, Visipaque. However, at similar concentrations, the X-ray the characteristic absorption band did not change sig- intensities of the prepared AuNPs are higher than those of nificantly, showing the high stability of AuNPs in NaCl Visipaque. Also, as shown in Fig. 7a, there is a linear rela- solution. This stability of the synthesized AuNPs in a tionship between the concentration of the prepared AuNPs complex matrix such as whole blood can be explained by and Visipaque with ΔHU (HU= Hounsfield units). 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