Abstract
Extensive biomedical applications of nanoparticles are mainly determined by their safety and compatibility in biological systems. The aim of this study was to compare the biosafety and biocompatibility of gold nanoparticles (GNPs) prepared with HEPES buffer, which is popular for cell culture, and sodium citrate, a frequent reducing agent. From experimental results on the body weight and organ coefficients of acute oral toxicity tests, it could be observed that HEPES-prepared GNPs are biologically safer than citric-prepared GNPs at the same dose of 500 μg/kg. The in vitro cell viability was higher for HEPES-prepared GNPs than citric-prepared GNPs at 5.0- and 10.0-ug/mL concentrations. More reactive oxygen species (ROS) were generated in the cell suspension when supplemented with citric-prepared GNPs than HEPES-prepared GNPs when their concentrations were higher than 20 μg/mL. The results stated that HEPES-prepared GNPs had better biosafety and biocompatibility than citric-prepared GNPs. This study not only revealed the influence of reducing agent on biosafety and biocompatibility of nanomaterials but also provided accumulative evidence for nanomaterials in biomedical applications.
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Introduction
Biosafety and biocompatibility are critical factors for almost all biological and medical applications. In 1997, International Standard ISO-10993 was formulated in order to provide a comprehensive and general guideline on biocompatibility of materials for medical applications, with priority on cell culture-based in vitro tests. For modern biotechnology, biosafety should be guaranteed so that the potential adverse impact could be minimized and managed in a manner without a negative impact on biodiversity and human health. The investigation on the biosafety of nanomaterials could effectively eliminate doubts from the public. More importantly, quantitative study of the biocompatibility of nanoparticles is fundamental for us to understand their interactions with biological systems. Therefore, biosafety and biocompatibility of nanomaterials have attracted more and more attention during the past years [1, 2].
Among a number of nanomaterials, gold nanoparticles (GNPs) have shown tremendous growth in various biomedical applications because of their convenient preparation, controllable sizes, and easy modification. Extensive uses of GNPs make it necessary and inevitable to assess their biosafety and biocompatibility [3]. Previous studies had revealed that gold ions were toxic on some types of cells, such as red blood cells [4]. In vivo studies revealed that the toxicity of GNPs was detectable in hematology, blood chemistry, and inflammatory and pathological response [5–8]. It is necessary to improve the safety of GNPs to avoid harming the human body.
More evidences have to be accumulated on the toxicity of GNPs for in vivo and in vitro applications. Previous investigations have revealed that the biosafety of GNPs was likely dependent on their composition, size, and surface characteristics [9, 10]. Semmler-Behnke et al. observed that a considerable percentage of GNPs with the size of 18 nm could be removed from the blood and trapped predominantly in the liver and spleen [11]. GNPs could be taken up by Kupffer cells in the liver or by macrophages in other places, regardless of the particle size [12].
In the preparation of GNPs, especially reducing agents could reduce gold ions and stable GNPs. In the past, sodium citrate has been commonly utilized as the reducing agent. Under physiological circumstances, however, citrate-prepared GNPs could form aggregates easily [13, 14]. In a biological system, this condition might be even worse since high-level accumulation of GNPs in liver and spleen might induce gene mutations and liver necrosis [15]. To date, various approaches on the fabrication of GNPs have been developed with the aim to improve their biocompatibility and biosafety for in vivo and in vitro applications [16, 17]. Proteins, peptides, DNA, and other chemical/biological polymers have been used to prepare GNPs in order to improve their biocompatibility [18]. Such studies suggested that surface modification could be essential for safe utilization of GNPs. Therefore, it might be possible to improve the biocompatibility of nanoparticles by using new reducing reagents [18].
Since many kinds of nanoparticles could interact with cells, it is of importance to ensure that their modifications do not cause adverse effects. It is desirable to develop biocompatible and green ways for preparing GNPs. Herein we studied the influence of reducing agents on the biosafety and biocompatibility of gold nanoparticles; we chose 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES), which is a popular buffer extensively applied for cell culture [19], and a commonly used sodium citrate as reducing agents. Our experimental results showed that HEPES-prepared GNPs were normally safer in acute toxicity studies under in vivo and in vitro conditions. It is estimated that our study could potentially extend applications of GNPs in biomedical areas.
Materials and Methods
Materials
AuCl3 · HCl · 4H2O was purchased from Shanghai No. 1 Reagent Factory, China. HEPES was generously provided by Nanjing SunShine Biotechnology Co., Ltd., China. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco, USA. MTT and 2,7-dichlorofluorescein diacetate (DCFH-DA) were purchased from Beyotime Institute of Biotechnology, China. All other chemical reagents were of analytical grade. Milli-Q water with electrical resistance of 18.2 MΩ cm was used in all of our experiments.
Preparation of HEPES-Prepared and Citric-Prepared GNPs
AuCl3 · HCl · 4H2O and HEPES solutions need to be filtered through 0.22-μm microporous membrane filters prior to use. HEPES-prepared GNPs were prepared by adding HEPES to boiling AuCl3 · HCl · 4H2O aqueous solution with different concentrations corresponding to the HEPES/Au molar ration of 1:10, 2:10, 4:10, 8:10, and 20:10 and stirred to be homogenous in order to form a stable mixed liquor.
Citric-prepared GNPs were prepared according to the report of Gu et al. [20]. Briefly, they were prepared by adding trisodium citrate solution to boiling AuCl3 · HCl · 4H2O aqueous solution at the molar ratio of 2:1. The final volume of citric-prepared GNPs was adjusted with de-ionized water, and the concentration was 100 ppm (0.1 μg/mL of gold).
UV–Vis Measurement
UV–vis measurement was performed with UV2450 UV–vis spectrophotometer (Shimadzu, Kyoto, Japan) in the absorbance mode and the range from 400 to 650 nm using deionized water as the reference. The fresh HEPES-prepared and citric-prepared GNP solution without any further treatment was used as samples for UV–vis measurement using deionized water as a standard for background correction.
Transmission Electron Microscopy Measurement
TEM measurements were performed in JEM-200 CX microscope (JEOL, Tokyo, Japan) operated at an accelerating voltage of 80 kV. The samples for TEM measurements were prepared by placing a drop of GNP solution onto copper grids and drying at room temperature. Micrographs at various positions were taken, and the sizes of the preferentially present particles were estimated based on several micrographs. The particle sizes were analyzed by software Image-pro plus 6.0.
FT-IR Analysis and Zeta Potential Measurement
FT-IR spectra were obtained using Nicolet Avater 370 spectrophotometer (Thermo, MA, USA). All the colloidal nanoparticles for FT-IR measurement were prepared by evaporating under reduced pressure to remove the solvent. The resulting precipitate was collected by centrifuging at 4,000 rpm for 5 min, rinsing periodically with alcohol, and then drying under vacuum. The samples were then mixed with potassium bromide, ground, and pelletized. Liquid specimen was prepared by coating a thin film of the pure ionic liquid on a KBr disk.
The zeta potential of HEPEs-prepared GNPs was measured on a Zetasizer (EEN3690, Malvern Instruments, Ltd., UK). GNPs were suspended in double-distilled water and placed in an electrophoretic cell (3 mL) and the electrophoretic mobility was measured at room temperature.
Biosafety Studies of In Vivo Applications
Specific pathogen free Spleen-Deficiency (SD) rats of both genders (male, 200-215 g; female, 175-190 g) were obtained from the Experimental Animal Center of Nantong University. For every cage about three rats were housed under controlled environmental conditions (22 ± 3 °C, 45-70 % relative humidity, and a 12 h light/dark cycle). All animal experiments were approved by the Care Committee of Nantong University which conducted the ethical review of the experiments.
The methods of acute toxicity studies were based on the OECD Guidelines for the Testing of Chemicals, “ROS Results”. The limited dose level of HEPES-prepared and citric-prepared GNPs at least 500 μg/kg body weight/day (adjusting the final volume with the animal weight for the given dose) was administered by gavage to six male and six female rats.
The surviving rats were then sacrificed by decapitation under ketamine anesthesia. All organs were observed macroscopically, and selected vital organs (including liver, spleen and kidney) were excised, blotted, and weighed. Organ coefficient was also measured. Hematoxylin–eosin staining was performed as described [7]. Histological examination was performed under light microscopy (Olympus CKX 41).
Biocompatibility Studies of In Vitro Applications
Cytotoxicity was evaluated by MTT assay using well-grown passage L929 mouse fibroblasts under the standard ISO 10993.5-1999. The cells were seeded in 25-cm2 plastic culture flasks and cultured in DMEM that was supplemented with 10 % fetal bovine serum. The medium was renewed every day, and cultures were maintained in a 5-% CO2 incubator at 37 °C. After 80 % confluence was reached, the cells were detached using 0.25 % trypsin-EDTA and transferred.
L929 mouse fibroblasts were seeded into 96-well plates at a density of 1 × 104 cells per well. After 12 h of preculture, the cells were treated with different concentrations of HEPES-prepared and citric-prepared GNPs (5.0, 10.0, 15.0, 20.0, and 25.0 ppm). Gold-free wells cultured under the same conditions were used as controls.
After 24 and 48 h of treatment, 20 μL MTT solution (5 mg/mL) was added to each well and incubated for 4 h in a humidified atmosphere (37 °C, 5 % CO2) to form formazan crystals. Then, the culture medium was removed, and 150 μL dimethyl sulfoxide was added to each well to solubilize formazan dye. After 15 min, the absorbance was measured at 490 nm in a microplate reader.
Measurement of ROS Production
The amount of ROS was utilizing the DCFH-DA assay as previously described [13]. After 24 h of treatment, cells were seeded into 96-well plates at a density of 20,000 cells per well. After recovery, cells were washed twice with Hank's balanced salt solution and incubated with 50 μM DCFH-DA for 15 min. Cells were then washed with Hank's balanced salt solution, with non-treated cells used as negative controls and with rosup-treated cells employed as positive controls. After exposure to the chemicals, the fluorescence intensity of a total of 5,000 cells was measured by the FACS Calibur and Cell Quest Pro software (BD Biosciences).
Statistical Analysis
The results were presented with mean ± SD and analyzed using SPSS 13.0. Experimental data were examined for equal variance and normal distribution prior to statistical analysis. One-way analysis of variance (ANOVA) was used for statistical analysis. Values were considered significantly different if P < 0.05.
Results
UV–Vis Spectra and TEM Results
Figure 1a illustrates the UV–vis spectra of GNPs prepared with different HEPES/Au ratios. The absorbance peak at the wavelength of around 525 nm in UV–vis spectra implied formation of GNPs. With the HEPES/Au ratio varied from 1:10 to 20:10, it could be found that the peak heights increased gradually. Figure 1b shows TEM images of GNPs prepared with the HEPES/Au ratio of 2:10, 4:10, and 8:10. There were some HEPES-prepared GNPs with triangle or polygon forms at the HEPES/Au ratio of 2:10. Sizes of GNPs were more uniform at the HEPES/Au ratio of 4:10 compared with citric-prepared GNPs (Fig. 1d). The inset shows the distribution diameter of the GNPs obtained. Table 1 lists the differences of the corresponding λmax of GNPs prepared with citrate and HEPES with different HEPES/Au ratios. Such results revealed that smaller GNPs with narrow and uniform distribution could be obtained at the HEPES/Au ratio of 4:10. By comparison, citrate-prepared GNPs showed a wider distribution at the Na3citrate/Au molar ratio of 20:10.
Since uniform GNPs could be obtained at the HEPES/Au ratio of 4:10, the as-prepared GNPs were then applied for in vitro and in vivo experiments.
FT-IR and Zeta Results
Figure 2a shows the FT-IR spectra of pure HEPES and powders of HEPES-prepared GNPs at the HEPES/Au ratio of 4:10. It could be found that both HEPES-prepared GNP powders and pure HEPES exhibited two IR bands at 3,134 and 1,338 cm−1, which could be attributed to the stretching bands of C–H and C–O, respectively. We can infer that the HEPES formed in the part of the prepared GNPs. Figure 2a also shows the difference of νS–H vibration band at 2,538 cm−1 between the GNPs and HEPES, which suggests that the S–H bond is broken upon binding to the gold particle surface to form the bond of S–Au [21]. The function of thiol groups in HEPES is to prevent Au(0) particles from aggregating and control the growth of nanoparticle in aqueous media [15].
Since the size of HEPES-prepared GNPs was greater than 10 nm, 50 ppm suspension of gold nanoparticles was used for the measurement of zeta potential. As can be seen in Fig. 2b, the gold nanoparticles displayed the highest count with a ζ value of −31.7 mV, indicating a moderately negative charge on the surface of the nanoparticles.
Biosafety Studies
The study of acute oral toxicity includes mortality, body weight variation, and histological examination. At a dose of 500 μg/kg, all treated rats grew normally and survived in the observation period. It could be found from Table 2 that the mean body gain decreased in the group fed with citric-prepared GNPs (male, P < 0.05; female, P < 0.01) after the test. By comparison, no significant change was observed on the mean body gain for the group fed with HEPES-prepared GNPs. As shown in Table 3, organ coefficients showed no considerable change in both sexes except the liver dosed with citric-prepared GNPs (compared with the control group, male, P < 0.05; female, P < 0.01). The organ coefficients of liver of both male and female rats in citric-prepared GNP group were significantly higher than HEPES-prepared GNP group (P < 0.05).
Histological examination of various tissues was carried out for other toxic effects. All of the specimens harvested during necropsy had no noteworthy reorganization in their histological structure. Rats exposed to HEPES-prepared and citric-prepared GNPs showed no tissue damage in any section from kidney and spleen except liver. As shown in Fig. 3, inflammatory liver injury could be observed in the group treated with citric-prepared treated GNPs but not with HEPES-prepared GNPs.
Biocompatibility Studies
Figure 4a illustrated the MTT reduction reaction of L929 cells cultured for 24 and 48 h in the medium supplemented with HEPES-prepared or citric-prepared GNPs at different concentrations, respectively. The lines mean the OD of control groups at 24 and 48 h. The L929 fibroblasts co-cultured with 5.0 and 10.0 ppm HEPES-prepared GNPs presented a high proliferation rate compared to the control group throughout the culture time (24 h, P < 0.05; 48 h, P < 0.01). Furthermore, cell viability was higher for 5.0 and 10.0 ppm HEPES-prepared GNPs than citric-prepared GNPs (P < 0.05). As GNP concentration increased, L929 cell proliferation was inhibited in varying degrees. Higher doses (20.0 and 25.0 ppm) of citric-prepared GNPs (48 h) showed a thorough inhibition on cell proliferation. Cell viability at 48 h was significantly higher with 20.0 and 25.0 ppm HEPES-prepared GNPs than those with citric-prepared GNPs (P < 0.01), although the HEPES GNPs also inhibit cell proliferation at 20 and 25 ppm at 48 h.
One noticeable effect on biocompatibility was different cell shapes and morphology in the monolayer. It could be found from Fig. 4b that the shapes of cells treated with 20.0 ppm HEPES-prepared GNPs were triangle, spindle, and extended pseudopodia to adhere to the culture flasks. By comparison, only a few cells treated with 20.0 ppm citric-prepared GNPs could survive and maintain the triangle and spindle shape.
ROS Results
In the following, we examined the dependence of ROS production of cells on treatment of HEPES-prepared and citric-prepared GNPs (Fig. 5). It could be found that the amount of ROS with HEPES-prepared and citric-prepared GNPs at the concentration of 5.0 and 10.0 ppm were similar to the negative group. With the increase of GNP concentration to 15.0 ppm, there were more ROS generated in the cells treated with citric-prepared GNPs than with HEPES-prepared GNPs (P < 0.01). Although the amounts of ROS increased with the increase of GNP concentration, they were significantly lower than the positive control (Fig. 5, inset). Overall, there were more ROS generated in citric-prepared GNPs than in HEPES-prepared GNPs.
Discussion
In this study, we prepared GNPs with HEPES, which is a popular buffer solution extensively used in biological laboratories for cell and tissue culture [16]. HEPES has a pKa close to physiological conditions (pH = 7.5), maximum aqueous solubility, low permeability to cell membrane, and high chemical stability [17]. It has been reported that a simple one-pot synthesis of three-dimensional gold nanocrystals with multi-tips could be fabricated by adding HAuCl4 solution into HEPES without shaking and heating [17], and HEPES is serving as both reducing and shape-directing agent. GNPs with polygon forms could also be prepared by mixing HEPES with an aqueous solution of 6 ppm Au (III) at room temperature [16]. Herein we mixed HEPES to boiling AuCl3 · HCl · 4H2O aqueous solution and optimized the conditions in order to obtain sphere GNPs to improve their biosafety and biocompatibility [18]. Our results showed that GNPs with sphere shape, uniform distribution, and well stability could be achieved at the HEPES/Au ratio of 4:10, which indicated that the number of thiol groups in ionic liquid plays an important role in the stabilization of gold nanoparticles. The interaction between gold atoms and thiol groups in HEPES could be further investigated by FT-IR spectra.
It is believed that the mechanism of GNPs formation in our approach was similar to that in previous reports [16]. The surface of GNPs could be modified by the thiol group in HEPES because of the strong connection between the gold surface and thiol/amine [19]. FT-IR results confirmed this step with the stretching band of methylene, which could be ascribed to the modification of the thiol group on the surface of gold clusters [20]. We think that the boiling process could facilitate the stabilization of GNPs and prevent the aggregation at higher temperature. The process of GNP formation was further proved by the characteristic bands in surface plasmon resonance (SPR) [22]. The moderate value of zeta potential might also reflect that the S–H bond is broken upon binding to the gold particle surface to form the bond of S–Au and the electrostatically stable form of HEPES-prepared GNPs.
The harmfulness of GNPs to mammalian cells was thought to depend on their sizes or residual hazardous chemicals. Pan et al. reported that GNPs of 1–2 nm in size were more toxic than 15-nm gold colloids in the study of the cytotoxicity of TPPMS/TPPTS-modified gold nanoparticles [23]. Their results suggested that the sizes of GNPs might play dominant roles in the metabolism of nanoparticles [9]. Shukla et al. found that Au (0) nanoparticles were not cytotoxic and might become a suitable candidate for nanomedicine. The reason is that GNPs could reduce the production of reactive oxygen and nitrite species without eliciting secretion of proinflammatory cytokines TNF-α and IL1-β [24].
We demonstrated that HEPES-prepared GNPs with a size of 26.01 nm exhibited improved biosafety compared with citric-prepared GNPs. For example, HEPES-prepared GNPs could not pass through the glomerular filtration of kidneys due to its size and negative electrostatic potential [25]. The bioaccumulation of HEPES-prepared GNPs may be because of their regulation in the reticulo-endothelial system, which is involved in the uptake and metabolism of exogenous nanoparticles in these tissues [26]. Interestingly, there were more viable cells in the groups treated with 5.0 and 10.0 ppm HEPES-prepared GNPs (48 h) than in the control, which was consistent with our previous study: a concentration of 5.0 ppm gold nanoparticle has the best effect on the promotion of cell growth [27]. This may be due to the fact that the small amount of gold nanoparticles could enter the cell and enhance DNA synthesis. Our in vitro results confirmed that HEPES-prepared GNPs showed better biocompatibility at the concentration of 5.0 to 10.0 ppm. Although GNPs have been considered bioinert and therapeutically used, GNPs might change the hematology and blood chemistry, indicating potential damage on liver and kidney as well as histological disorders, such as apoptosis and inflammation in various organs [6, 7, 28]. While administrated to rats, citric-prepared GNPs could disappear from the circulation and localized in the tissues, primarily the liver [26]. Our results indicate that the administration of HEPES-prepared GNPs could not induce extensive inflammation on rats, which are superior to citric-prepared GNPs. In liver (heavy metal detoxification organ), there were some inflammatory cells after injection of citric-prepared GNPs. It is probably caused by the reducing agent citric acid. Such experimental results agreed with the previous report from Lasagna-Reeves et al. [3].
The MTT and ROS assay were utilized to characterize the effect of HEPES-prepared and citric-prepared GNPs on L929 cell proliferation. Oxidative stress, which includes ROS generation, is currently the most popular theory in the study of nanoparticle toxicity [29]. Oxidative stress has been shown to occur in cells following exposure to gold nanoparticles [30]. Citric-prepared GNP was more effective in increasing ROS compared to the negative control as the concentration rises above 20.0 ppm, which was consistent with the fact that the proliferation of cells was significantly inhibited by citric-prepared GNPs at 20.0- and 25.0-ppm concentrations. Some cells began to shrink and disintegrate, which might be due to the increased ROS levels.
Interestingly, it was found that ROS production slightly decreased in groups treated with HEPES-prepared GNPs at the low concentrations. It might be possible that agents binding to the AuNPs through Au–S bonds could decrease the catalytic activity of the AuNPs. Pan et al. demonstrated that several thiol-containing antioxidants could significantly reduce their toxic effects [29]. MTT and ROS results showed that cytotoxicity might depend on both the concentration of GNPs and the type of reducing agent used.
Conclusions
In summary, our investigation demonstrated that HEPES could get a better reducing agent and stabilizer for GNPs from the view of “biocompatible”. HEPES-prepared GNPs showed better biosafety and biocompatibility than citric-prepared GNPs under both in vivo and in vitro circumstances. Preparation of GNPs with HEPES is convenient for biomedical applications. This study not only demonstrated the key fact of reducing agent but also accumulated more evidence for the study of biosafety and biocompatibility of nanoparticles.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 21175075, 21075070, and 21375066), the Natural Science Foundation of Jiangsu Province of China (Nos. BK2011047, BK2012651, and BK2012652), Social Development Item of Nantong City (Nos. BK 2013028 and BK 2012058), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) for their support of this research.
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Dong-Lin Xia and Yu-Fei Wang contributed equally to this work.
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Xia, DL., Wang, YF., Bao, N. et al. Influence of Reducing Agents on Biosafety and Biocompatibility of Gold Nanoparticles. Appl Biochem Biotechnol 174, 2458–2470 (2014). https://doi.org/10.1007/s12010-014-1193-7
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DOI: https://doi.org/10.1007/s12010-014-1193-7