http://informahealthcare.com/nan ISSN: 1743-5390 (print), 1743-5404 (electronic) Nanotoxicology, 2015; 9(1): 116–125 ! 2014 Informa UK Ltd. DOI: 10.3109/17435390.2014.894151

ORIGINAL ARTICLE

Differential interferences with clinical chemistry assays by gold nanorods, and gold and silica nanospheres Georgia K. Hinkley1, Paul L. Carpinone2, John W. Munson1, Kevin W. Powers2, and Stephen M. Roberts1 Center for Environmental & Human Toxicology and 2Particle Engineering Research Center, University of Florida, Gainesville, FL, USA

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1

Abstract

Keywords

Nanomaterials are known to cause interference with several standard toxicological assays. As part of an in vivo study of PEG-coated gold nanorods in mice, nanorods were added to reference serum, and results for standard clinical chemistry parameters were compared with serum analyzed without nanorods. PEG-coated gold nanorods produced several concentrationdependent interferences. Comparisons were then made with PEG-coated gold and silica nanospheres. Interferences were observed for both materials that differed from gold nanorods. Removal of the particles from serum by centrifugation prior to analysis resolved most, but not all of the interferences. Additional clinical chemistry analyzers were used to further investigate trends in assay interference. We conclude that PEG-coated gold and silica nanoparticles can interfere with standard clinical chemistry tests in ways that vary depending upon material, shape, and specific assay methodology employed. Assay interferences by nanomaterials cannot always be predicted, underscoring the need to verify that nanomaterials under study do not interfere with methods used to evaluate potential biological effects.

Assay interference, clinical chemistry, in vivo, nanotoxicology, particle characterization

Introduction Although the toxicological evaluation of engineered nanomaterials has expanded considerably in recent years, understanding the potential hazards posed by the use of nanomaterials in medicine and consumer products remains quite limited (Boverhof & David, 2010; Calzolai et al., 2012; Dekkers et al., 2011). Most of the literature in the field consists of studies of effects of nanomaterials on cells in culture, and while these studies offer insight into possible mechanisms for adverse effects, the principal means of assessing hazard for regulatory purposes remains in vivo tests in laboratory animals (Fischer & Chan, 2007). There are several examples in the literature of in vivo toxicity tests involving nanomaterials (e.g. Baek et al., 2012; Hillyer & Albretch, 2001; Kim et al., 2010; Loeschner et al., 2011; Schleh et al., 2012; Wang et al., 2007), but progress in developing a robust database of information on engineered nanomaterial toxicity is hampered in part by uncertainty regarding the extent to which standard in vivo toxicity testing approaches may need to be modified for nanomaterials (Kroll et al., 2009, 2012; Landsiedel et al., 2009). Standard toxicity testing protocols evaluating systemic effects typically include batteries of clinical chemistry assays for blood and urine as sensitive indicators of organ/system function. Abnormalities indicated by these assays are often interpreted as indicating toxicity, even in the absence of accompanying

Correspondence: Georgia K. Hinkley, Center for Environmental & Human Toxicology, Box 110885, University of Florida, Gainesville, FL 32611, USA. Tel: (352) 294-4710. Fax: (352) 392-4707. E-mail: [email protected]

History Received 10 September 2013 Revised 7 February 2014 Accepted 9 February 2014 Published online 12 March 2014

histopathology. Nanomaterials possess a number of properties that can theoretically cause interference with these assays and, as a result, lead to incorrect conclusions regarding nanomaterial hazard. Possible mechanisms for interference include disruption of light absorption or fluorescence measurements, adsorption of reactants, oxidation of assay components, and interference with enzyme catalysis (Barrett et al., 1999; Dobrovolskaia et al., 2008, 2009). There is ample evidence of significant assay interferences from in vitro studies of nanomaterial effects in cell culture. For example, interference with the MTT cell viability assay has been demonstrated with carbon nanotubes and carbon black particles. These interferences have led to false negative results due to adsorption of the reporter dye (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) to the particle surface (Belyanskaya et al., 2007; Kroll et al., 2012). The limulus amoebocyte lysate assay for endotoxin detection has also shown interference from several nanomaterials causing both false positive and false negative results (Dobrovolskaia et al., 2010; McNeil, 2011). Nanoparticle interference with cytokine measurements has been reported for titanium dioxide, silicon dioxide, and carbon black nanoparticles, usually leading to artificially low measurements of cytokine concentrations (Brown et al., 2010; Kroll et al., 2012; Veranth et al., 2007). While the literature clearly demonstrates nanoparticle interference with several in vitro assays, there have been few empirical studies investigating potential interferences with standard clinical chemistry tests essential to in vivo toxicity testing. As part of a study of potential toxicity of PEG (polyethylene glycol)-coated gold nanorods administered intravenously to mice, the possibility of interference with serum clinical chemistry assays was assessed. PEG-coated gold nanorods were added in varying concentrations to laboratory quality control serum and a standard battery of assays was performed.

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Nanoparticle concentrations up to the highest observed in whole blood after gold nanorod administration were evaluated and compared with assay results when no particles were added. After observing concentration-dependent interference of the PEG-gold nanorods with several assays, the study was expanded to explore the effect of nanomaterial shape and composition on assay interference using PEG-gold nanospheres and PEG-silica nanospheres. Additional clinical chemistry analyzers were employed to help further elucidate any pattern of assay interference with nanomaterials, and the utility of centrifugation of blood to eliminate interferences by these nanomaterials was assessed.

Methods

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Materials All reagents used for synthesis of PEG-coated gold nanospheres and nanorods and PEG-coated silica nanospheres were purchased from Fisher Scientific (Waltham, MA) and were ACS/Reagent Grade. The only exception was ethanol (200 proof), which was purchased from Decon Laboratories (King of Prussia, PA). Assayed Human Sera Level 3 (Randox3) was purchased from Labsco (Louisville, KY) and was reconstituted in Milli-Q water in 5 mL quantities following the manufacturer’s instructions. Clinical chemistry analytes in Level 3 serum are moderately elevated, facilitating the detection of negative interferences as well as positive interferences (i.e. artificially low or high results, respectively). Particle synthesis Gold nanorods (approx. 11 nm  44 nm) were synthesized using the seed mediated, surfactant (CTAB; cetyltrimethylammonium bromide) assisted growth method as detailed by Jana et al. (2001). These particles were washed with filtered deionized water and concentrated by centrifugation and reacted with excess thiol-terminated PEG (5 kDa) to achieve PEG coating. Spherical gold particles with a nominal particle size of 22 nm were synthesized by citrate reduction of gold chloride as described previously (Frens et al., 1973). These particles were washed with filtered deionized water and concentrated by centrifugation and reacted with excess thiol-terminated PEG (5 kDa) to achieve PEG coating. Silica nanospheres (22 nm) were synthesized using the Sto¨ber method (Sto¨ber et al., 1968). Particle size was monitored during synthesis until the desired size was achieved, at which point water was added to quench the reaction. The suspension was then heated to precipitate any unreacted tetraethyl orthosilicate (TEOS) as large silica particles, which were subsequently removed by filtration through a 0.1 mm filter. During this heating process, the ethanol and ammonia present in the suspension were evaporated and replaced with filtered deionized water repeatedly. The particles were finally washed by centrifugation with filtered deionized water and were coated with PEG using an ethoxy-silane terminated 5 kDa PEG.

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Measuring absorption and fluorescence of nanomaterials and dyes The light absorption spectra of the PEG-coated gold nanorods and nanospheres and silica nanospheres and dyes were measured using a Perkin-Elmer Lambda 800 spectophotometer (Perkin Elmer, Waltham, MA). Fluorescence emission of PEG-coated gold nanorods at 560 nm was measured using an excitation scan from 400 nm to 550 nm on a HORIBA NanoLogÕ spectrophotofluorometer (HORIBA Scientific, Edison, NJ). Animals Adult (8 weeks of age) male ICR outbred mice were purchased from Harlan Laboratories (Pratville, AL). Body weights of mice used in this study were approximately 30 g. Mice were housed in cages (three mice per cage), with controlled light/dark cycles of 12 hours (08:00–20:00 light). Temperature (20–26  C) and humidity (30–70%) were also controlled. Animals had continuous access to water and food (Teklad Rodent Diet 7919, Harlan Laboratories). Three mice were injected via the tail vein with 5 mg of PEG-gold nanorods in 100 mL saline. This dose was selected based on the literature, with both higher and lower doses being used by other investigators, from 60 mg/mouse to 30 mg/mouse (Arnida et al., 2011; Sonavane et al., 2008; Zhang et al., 2010). Five minutes following injection, mice were euthanized by carbon dioxide asphyxiation and cervical dislocation. Whole blood was collected via cardiac puncture for measurement of PEG-gold nanorod concentration. This study was approved by the Institutional Animal Care and Use Committee, and all animals were treated humanely according to criteria provided in the NIH ‘‘Guide for the Care and Use of Laboratory Animals’’. Determination of particle concentration in whole blood and serum PEG-gold particle concentration was determined by sample digestion with aqua regia (3:1 ratio of 70% hydrochloric acid: 70% nitric acid) and 70% nitric acid for 1 hour at 140  C, followed by gold concentration measurement by inductively coupled plasma mass spectroscopy (ICP-MS; Thermo Electron X Series II, Thermo Scientific, West Palm Beach, FL). Samples were run in triplicate and quantified based on a 6-point standard curve with a limit of quantitation (LOQ) of 1 ppb. Indium was used as an internal standard. PEG-silica particle concentration was determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES; Perkin Elmer Optima 3200 RL, Perkin Elmer, Waltham, MA). Serum samples with silica particles were digested in nitric acid (trace metal grade 70% nitric acid from Fisher Scientific) and brought to dryness, followed by addition of 15% hydrofluoric acid (HF) to dissolve silica. The HF concentration was then diluted to 5% for analysis. All samples were run in triplicate, with an LOQ of 1 ppm. Serum incubation

Characterization of nanomaterials Particle suspensions were dried onto transmission electron microscopy (TEM) grids and imaged as prepared to verify primary particle size and particle morphology. Imaging was performed with a JEOL 2010F TEM (Tokyo, Japan). Ensemble particle size distribution measurements of the as-prepared spherical particle suspensions were analyzed by dynamic light scattering (Microtrac Nanotrac, Montgomeryville, PA). The zeta potential of the as-prepared particles was analyzed by dynamic light scattering (Brookhaven ZetaPlus, Brookhaven Instruments Corporation, Holtsville, NY).

Reconstituted Randox3 assayed serum was homogenized by pipetting and then separated into 900 mL aliquots for nanoparticle addition and clinical chemistry analysis. PEG-gold nanorods were added in 100 mL saline to produce final target nanoparticle concentrations of 125, 250, 500 and 1000 mg/L in serum. Measurement of gold content in the diluted samples indicated actual concentrations of 148, 297, 595, and 1190 mg/L. The same approach was used to create samples of serum with PEG-gold nanospheres (818 mg/L) and PEG-silica nanospheres (111 mg/L). These concentrations of PEG-gold and PEG-silica have similar calculated nanoparticle surface area per volume as the 1190 mg/L

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PEG-gold nanorod serum (Table 1). Eosin Y (283 ppm) and Indocyanine green (409 ppm) were incubated in serum in an identical fashion to mimic the spectral properties of the gold nanospheres and nanorods, respectively. The dye concentrations were selected to match the maximum absorbance of the particles. Saline (100 mL) without nanoparticles was added to Randox3 serum (900 mL,) to serve as negative controls. Samples were allowed to incubate at room temperature for 15 minutes before analysis or particle removal. Particle removal

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Particle removal was achieved by centrifugation of serum for 15 minutes at 20 000 RCF at room temperature. Particle removal was confirmed using ICP-MS for gold particles and with ICP-AES for silica particles. Following centrifugation, 500 mL of supernatant were removed for clinical chemistry analysis. Clinical chemistry analysis Samples were analyzed using Hitachi 912, Siemens Dimension, and Vitros systems by the University of Florida, College of Veterinary Medicine Clinical Pathology and Diagnostics lab. All samples and controls were prepared and analyzed in triplicate according to the manufacturers’ operating conditions for each instrument.

Results Image analysis of the transmission electron micrographs for primary particle size showed that the gold nanorods were 47 ± 9 nm in length and 13 ± 3 nm in width on average (±SD), the gold spheres were 16 ± 1.3 nm in diameter and the silica spheres were 31 ± 3 nm in diameter; 80–100 particles were measured for each particle type (Figure 1). Due to the narrow size distribution for all particle types, counting of additional particles was not necessary. Dynamic light scattering showed the particle size distribution (PSD) of the gold spheres to be centered on 20 nm with a left skewed distribution, while the silica sphere

PSD was centered over 24 nm with a slightly skewed distribution to the right (Figure 2). The zeta potential of the spherical gold and silica particles as prepared were negative at 34 ± 2 and 30 ± 3 mV, respectively. The zeta potential of the gold nanorods was measured at +33 ± 5 mV, with the difference in sign due to the adsorbed bilayer of the cationic CTAB surfactant. The gold nanorod zeta potential measurement should be interpreted with caution because the models employed by dynamic light scattering assume spherical particle morphology. Once coated, the particles demonstrated a high degree of stability in the reconstituted Randox3 serum, and no settling of particles was observed. In preparation for an in vivo toxicity study of intravenously administered PEG-coated gold nanorods, a group of mice were administered the highest dose planned for the study (5 mg) and whole blood was collected after 5 minutes. The concentration of PEG-gold nanorods observed in the blood was just over 1000 mg/mL. This served as the upper concentration for testing potential PEG-gold nanorod interference with clinical chemistry assays that would be used in part to assess potential toxicity. The effect of lower particle concentrations (125, 250, and 500 mg/mL) on a suite of clinical chemistry tests was also evaluated. Concentration-dependent interferences, both positive and negative, were observed for several clinical chemistry tests when analyzed with PEG-gold nanorods present (Table S1). Traditional statistical tests were not used for comparisons because the variances associated with each test were very small, making changes of even a few percent statistically significant. Instead, to identify changes that might be meaningful in a clinical context, we used an arbitrary cutoff of 10% or more difference compared to controls to determine which tests were significantly affected.

Table 1. Nominal concentrations of PEG-coated gold nanorods, gold nanospheres, and silica nanospheres.

Concentration expressed by:

Gold nanorods

Gold nanospheres

Silica nanospheres

Mass (mg/mL) Surface area (m2/mL) Particle number (number/mL)

1190 2.16e-2 9.88e12

818 1.63e-2 2.09e13

111 1.44e-2 6.7e12

Mass calculations are based on ICP-MS and ICP-AES data, while surface area calculations are based upon image analysis data.

Figure 2. Dynamic light scattering particle size distributions of spherical particles prior to PEG-coating.

Figure 1. Transmission electron micrographs of nanoparticles. (A) PEG-coated gold nanorods (nominal size 11 nm  44 nm), (B) PEG-coated gold nanospheres (22 nm), (C) PEG-coated silica nanospheres (22 nm).

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Figure 3. Concentration-dependent interferences of PEG-coated gold nanorods in Randox3 serum. PEG-coated gold nanorods were added to Randox3 assayed serum to achieve the indicated concentrations. Clinical chemistry parameters were measured on the Hitachi 912 as described in the ‘‘Methods’’ section. Results are expressed as mean (n ¼ 3) percent change from control (0 ppm) +/ SD calculated as [((sample value/control value)  1)  100].

Figure 4. Interference of PEG-coated gold nanorods, nanospheres and silica nanospheres with clinical chemistry assays in Randox3 serum. Nanoparticles were added to Randox3 serum to achieve the concentration of 1000, 818, and 111 ppm for PEG-gold nanorods, nanospheres and silica nanospheres, respectively. Clinical chemistry parameters were measured on the Hitachi 912 as described in the ‘‘Methods’’ section. Results are expressed as mean (n ¼ 3) percent change from control (0 ppm) + / SD calculated as [((sample value/control value)  1)  100].

Gold nanorods produced concentration-dependent errors in measurement of 10% or more for total protein, calcium, triglyceride, magnesium, GGT and, most substantially, total bilirubin (Figure 3). In the presence of 500 mg/mL PEG-coated gold nanorods, total bilirubin was decreased 97%, and there was complete failure to detect bilirubin at the highest particle concentration. The interferences observed with PEG-coated gold nanorods led to the investigation of additional materials in serum. PEGcoated gold nanospheres and PEG-coated silica nanospheres were both examined in an attempt to gain insight on the role of shape and material in assay interferences. It was hypothesized that for interferences based upon surface properties, gold nanorods and nanospheres should have similar effects, while the pattern of interferences by silica nanoparticles would be different. If, however, spectral properties were responsible for the observed interferences, we expected all three particles to have different interference profiles due to differences in light absorption. To minimize surface area as a variable, gold and silica nanospheres were examined at concentrations that would produce equivalent surface area per volume of serum as the target concentration of 1000 mg/mL of PEG-gold nanorods. Although not exact, the calculated surface areas of the three particle types

at the concentrations employed for this experiment were similar (Table 1). When results from the particle types were compared, significant positive interferences in total protein, glucose and triglyceride were observed for PEG-gold nanospheres, with measured total protein more than a 150% increase from the actual concentration as measured in particle-free controls and triglyceride measured a 74% increase from the concentration in controls (Figure 4). PEG-silica nanospheres had a single interference 410%, a falsely low total bilirubin measurement (Figure 4). The full list of results for all three particle types analyzed in this experiment is provided in Table S2. Due to the differences in interference produced by the three particle types, we investigated the spectral properties of the particles as a potential source of interference. A large majority of the assays are bichromatic, meaning they are measured at two wavelengths; one wavelength is used to measure absorption of the endpoint molecule (primary) and the second is used to measure background absorption (secondary). Light absorption by the particles at the primary wavelength leads to a positive interference and absorption at the secondary wavelength leads to a negative interference. In an attempt to mimic the spectral properties of the gold nanospheres and nanorods, eosin Y and indocyanine green,

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Figure 5. Absorbance spectra: gold spheres, eosin Y, silica spheres, randox serum. The absorption spectra for gold nanospheres, eosin Y, silica spheres and randox serum are shown. This illustrates that eosin Y should mimic the light absorption of gold nanospheres. The primary wavelength for the total protein, total bilirubin, triglyceride and magnesium assays are indicated along the X-axis.

Figure 6. Absorbance spectra: gold rods, indocyanine green, randox serum. The absorption spectra for gold nanorods, indocyanine green and randox serum are shown. This illustrates that indocyanine green should mimic the light absorption of gold nanorods. Several assays, indicated on the X-axis, have a secondary wavelength at 700 nm, providing a possible explanation for several negative interferences by gold nanorods.

respectively, were incubated with control serum and analyzed for interferences. Figure 5 shows the absorption spectrum for eosin Y and gold nanospheres and indicates the location of the primary wavelength of assays for which gold nanospheres have a positive interference (e.g. total protein, triglyceride, magnesium, total bilirubin). Figure 6 shows the absorption spectrum for indocyanine green and gold nanorods and has indicators for the secondary wavelength of several assays within the absorption region for gold nanorods. The interference results for eosin Y and indocyanine green are shown in Table S3, data are presented as percent change from controls. Table 2 lists the primary and secondary wavelengths for each assay, along with a potential explanation of observed and expected interferences.

In addition to light absorption, PEG-gold nanorods are also capable of fluorescence (Mohamed et al., 2000). Fluorescence by the particles at a primary wavelength would be able to cause a negative interference. Mohamed et al. have previously shown that PEG-gold nanorods with an aspect ratio similar to those used in this study have a fluorescence peak at 560 nm when excited at 480 nm. We analyzed fluorescence of the PEG-gold nanorods at 560 nm over a range of excitation wavelengths (400–550 nm) (Figure 7). We did not observe light emission above background that could account for any of the documented interferences. We also examined the possibility that some of the interferences might be an artifact of the presence of surfactant (viz., CTAB) introduced during PEG-gold nanorod synthesis. Addition of

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Table 2. Summary of interference explanations based on spectral properties of particles and dyes. Primary wavelength

Secondary wavelength

ALK phosphatase ALT w/P5P AST w/P5P

415 340 340

660 700 700

T Bilirubin L3K

546

600

Total protein

570

700

Albumin

600

700

Calcium L3K Phosphorus

600 340

700 700

Creatinine

505

570

BUN Glucose Triglyceride

340 334–365 (340) 505

376 380–410 (380) 700

Magnesium

546

700

GGT

415

700

Notes No interference expected or observed AuNR: () interference explained by secondary wavelength AuNR: should cause () interference, not observed ICG: should cause () interference, not observed AuNR: () interference not explained ICG: (+) interference not explained AuNS: (+) interference explained by primary wavelength Eosin Y: () interference not explained AuNR: (+) interference not explained ICG: () interference explained by secondary wavelength AuNS: (+) interference explained by primary wavelength Eosin Y: (+) interference explained by primary wavelength AuNR () interference explained by secondary wavelength ICG: () interference explained by secondary wavelength AuNR: () interference explained by secondary wavelength AuNR: should cause () interference, not observed ICG: should cause () interference, not observed AuNR: () interference not explained ICG: (+) interference not explained No interferences expected or observed No interferences expected or observed AuNR: (+) interference not explained ICG: () interference explained by secondary wavelength AuNS: (+) interference explained by primary wavelength Eosin Y: (+) interference explained by primary wavelength AuNR: () interference explained by secondary wavelength ICG: () interference explained by secondary wavelength AuNS: (+) interference explained by primary wavelength Eosin Y: (+) interference explained by primary wavelength AuNR: () interference explained by secondary wavelength

AuNR ¼ PEG-Coated Gold Nanorods, ICG ¼ Indocyanine Green, AuNS ¼ PEG-Coated Gold Nanospheres. Figure 7. Fluorescence spectra at 560 nm for PEG-coated gold nanorods excited from 400–550 nm. This spectra shows the fluorescence emission for PEG-coated gold nanorods at 560 nm for excitation wavelengths from 400 to 550 nm. Previous studies have shown that excitation at 480 nm causes light emission at 560 nm based on gold nanorod aspect ratio (Mohamed et al., 2000). No fluorescence peak larger than background was observed for any excitation wavelength from 400 to 550 nm.

CTAB (up to 1 mM) to Randox3 serum instead of nanoparticles failed to produce interferences in any of the assays (data not shown). In a follow-up experiment, high-speed centrifugation was used to remove particles from the Randox3 serum prior to measurement of clinical chemistry parameters. Particle removal was confirmed with ICP-MS for gold (499% removed) and ICP-AES for silica (96% removed). Serum samples incubated

with particles and centrifuged were then analyzed in comparison with controls. Full results are shown in Table S4. Figure 8 illustrates the effects of particle removal on serum clinical chemistry assays with interferences 410% for gold nanorods, gold nanospheres, and silica nanospheres. A majority of the affected assays returned to within +/ 10% of controls following particle removal, e.g. PEG-gold nanorod interference with total bilirubin and PEG-gold nanosphere interference with total

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Figure 8. Potential effects of particle removal on clinical chemistry interferences. Nanoparticles were added to Randox3 serum to achieve concentrations of 1000, 818, and 111 ppm for PEG-gold nanorods, nanospheres and silica nanospheres, respectively. Nanoparticles were incubated in Randox3 serum for 15 minutes and then centrifuged (20 000 RCF for 15 minutes at room temperature) for particle removal. Clinical chemistry parameters were measured on the Hitachi 912 as described in the ‘‘Methods’’ section. Results are expressed as mean (n ¼ 3) percent change from control (0 ppm) calculated as [((sample value/control value)  1)  100].

protein. Silica particle interference with total bilirubin and PEG-gold nanorod interference with calcium were the only affected assays that were not resolved by particle removal. While centrifugation made most affected assays more accurate, other assay interferences were only observed following centrifugation. For example, the phosphorous assay was not significantly affected by any of the three particle types before centrifugation. However, following particle removal, all three materials showed a negative interference with the phosphorus assay. Gold and silica nanospheres affected calcium measurement in a similar fashion, measuring within normal range when particles were present and exhibiting a negative interference following centrifugation. Interferences following this pattern may be explained by protein adsorption: for example, by adsorbing calcium or phosphorusbinding proteins. It appears that, in general, centrifugation of serum samples can be used to restore the validity of affected assays; however, it should be confirmed that additional interferences are not caused by particle removal. Clinical chemistry measurements for these experiments were made using the Hitachi 912 analyzer. Other clinical chemistry analyzing systems were examined for comparison to further investigate assay interference caused by these particles types. For most assays, the assay chemistry used by the analyzer systems (i.e. the reaction on which the assay is based) was the same, although there were a few exceptions (Table 3). The effects of PEG-gold nanorods, PEG-gold nanospheres and PEG-silica nanospheres on clinical chemistry tests were evaluated with Siemens Dimension and Vitros DT60 clinical chemistry analyzers using the same approach as previous experiments with the Hitachi 912, including assessment of the potential for improved accuracy with particle removal by centrifugation of serum. A summary of results for the Dimension and Vitros instruments is provided in Tables S5 and S6, respectively. Using the Siemens Dimension clinical chemistry analyzer, significant interferences (410% difference from control) in aspartate aminotransferase (AST), phosphorus, triglyceride,

gamma-glutamyl transpeptidase (GGT), bilirubin, and albumin were produced by PEG-gold nanorods. With particle removal, all of these interferences were within 10% of controls. PEG-gold nanorods also caused interferences that were not resolved by particle removal: alanine aminotransferase (ALT) 44% of controls, potassium 244% of controls, calcium 17% of controls, creatinine 11% of controls, blood urea nitrogen (BUN) 12% of controls and magnesium 17% of controls (values following centrifugation). PEG-gold nanospheres caused interferences with total protein and total bilirubin that were eliminated by centrifugation of serum. However, a large interference with triglyceride measurement was not resolved (+147% change from controls) after particle removal. All assays analyzed with PEG-silica nanospheres present were within +/12% of controls. Using the Vitros DT60 analyzer we investigated interference with seven assays that had significant problems on the Hitachi 912 analyzer: total bilirubin, calcium, total protein, triglycerides, magnesium, glucose, and albumin. Randox3 serum analyzed with PEG-gold nanorods present caused total bilirubin, calcium, total protein and albumin measurements to read above range (Table S6A), meaning that the instrument could not make a measurement. After centrifugation (Table S6B), albumin was 17% of controls, while the other assays were within 10% of the controls. PEG-gold nanospheres caused large interferences in total bilirubin, total protein, and albumin measurement that returned to within 10% of controls following centrifugation. PEG-gold spheres also caused a large positive interference in the triglyceride assay that was not resolved by centrifugation. All serum samples mixed or incubated with PEG-silica spheres were within 11% of controls on the Vitros DT60 analyzer. As shown by the examples in Figure 9, the pattern of interferences with clinical chemistry assays, as well as corrections provided by centrifugation of serum, were inconsistent among the three analyzers. These inconsistencies occurred even among assays based upon the same chemical reaction, e.g. PEG-gold nanosphere effects on total protein.

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Table 3. Differences in assay chemistry among analyzers. Assay

Instrument

Total bilirubin

Hitachi Siemens Vitros

Bilirubin + 2,4-dichlorophenyldiazonium salt (surfactant) ! azobilirubin Bilirubin + diazotized sulfanilic acid ! red chromophore Bilirubin + [4-(N-carboxymethylsulfonyl)benzenediazoniumhexafluorophosphate] ! azobilirubinchromophores Dyphylline is used to free conjugated bilirubin

546 nm 540 nma 555 nm

Calcium

Hitachi Siemens Vitros

Phosphonazo III + Ca+2 ! Ca-Phosphonazo Complex Ca2+ + OCPC ! Ca-OCPC complex; Mg+2 chelator is used. Ca2+ + Arsenazo III ! colored complex

600 nm 577 nmb 680 nm

Glucose

Hitachi

Glucose + ATP (HK, Mg2+) ! G-6-P + ADP, 2.G-6-P + NAD+ (G-6-PDH) ! 6-PG + NADH + H+ Glucose + ATP (HK, Mg2+) ! G-6-P + ADP, 2.G-6-P + NAD+ (G-6-PDH), ! 6-PG + NADH + H+ b-D-glucose + O2 + H2O (glucose oxidase) ! D-gluconic acid + H2O2. 2.H2O2 + 4-aminoantipyrine + POD ! red dye

340 nm

Siemens

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Vitros Magnesium

Hitachi Siemens Vitros

Assay chemistry

Mg2+ + magon (xylidyl blue 1) ! colored complex; Ca+2 chelator used Mg2+ + MTB ! Mg-MTB complex; Ca+2 chelator used Mg2+ + formazan dye derivative ! colored complex; Ca+2 chelator used

Wavelength

340 nmc 555 nm 520 nm 600 nmd 660 nm

OCPC ¼ O-Cresolphthalein complexone; G-6-P ¼ glucose 6 phosphate; POD ¼ 1,7-dihydroxynaphthalene, MTB ¼ methylthymol blue. Controls are measured at 700 nm. Controls are measured at 540 nm. c Controls are measured at 383 nm. d Controls are measured at 510 nm. a

b

Discussion Nanoparticle interference with clinical chemistry assays has not been extensively studied to date, perhaps due to the fact that a preponderance of nanotoxicology studies are based on in vitro systems. A small investigation of interference with clinical chemistry tests has been reported in which only a few interferences were observed with metal containing nutraceutical products (Banfi et al., 2006). Despite observing only a few interferences, Banfi et al. (2006) advised clinicians to be aware of the possibility of nanoparticle interference as the mechanisms are unknown. Outside of in vivo studies, there is substantial evidence to show that nanoparticles are capable of interfering with a number of standard assays. Many of these interferences have been summarized by the Nanotechnology Characterization Laboratory (Hall et al., 2007). Several other groups have published similar results for interference with colorimetric assays involved in genotoxicity and cell viability (Casey et al., 2007; Doak et al., 2009; Kroll et al., 2009; Oostingh et al., 2011). In this study, concentration-dependent interferences with several standard clinical chemistry assays were observed for gold nanorods in serum. The concentration range tested corresponded to nanoparticle concentrations observed in an in vivo toxicity study. When the study was extended to three nanoparticle types, interferences varied among the nanoparticles tested in terms of the assay affected, the magnitude of effect, and sometimes direction of effect (i.e. assay result was falsely elevated or lowered). The extent of error was sufficiently large for a number of assays that a wrong conclusion regarding toxicity could potentially be reached, and for a few assays, prevented any measurement from being made by the clinical chemistry analyzer. In a preliminary attempt to understand the mechanism(s) underlying the interferences, several hypotheses were examined. Spectral properties of the nanoparticles were able to explain several of the observed interferences, either through light absorption at the primary or secondary wavelength. Positive interferences by PEG-gold nanospheres in the total bilirubin, total protein, magnesium and triglyceride assays can be explained by light absorption at the primary wavelength (Figure 5, Table 2).

Negative interferences by the PEG-gold nanorods in the albumin, calcium, magnesium and ALT assays can be explained by light absorption at the secondary wavelength (Figure 6, Table 2). However, there were also assays in which interferences were expected based on spectral properties, but were not observed. For example, the AST and phosphorous assays both have a secondary wavelength at 700 nm, meaning that PEG-gold nanorods should cause a negative interference, but this was not observed. Other interferences, for example, the PEG-gold nanorod interference with the bilirubin assay, could not be explained by light absorption or emission at either wavelength. Due to the high surface area of nanomaterials, we also considered the possibility of protein adsorption as the source of this negative interference (Shinke et al., 2010). However, if the particles were adsorbing bilirubin, the assay interference would not be resolved following centrifugation. If the nanorods were adsorbing assay reagents or the azobilirubin end product a negative interference would be expected, however, the light absorption of the particles alone should theoretically cause a positive interference. In addition, the assay reagents are typically present in excess of a 70:1 ratio with the sample. These values make it unlikely that the particle concentrations used would be able to adsorb a sufficient amount of reagent to cause interference. Another possibility is interference in assay reactions due to catalytic activities of the nanoparticles. For this mechanism, we would have expected PEG-gold nanorods and nanospheres with the same surface area available for catalytic activity to produce the same interferences at about the same magnitude. As shown in Figure 4, this kind of comparable interference for these two nanoparticle types was not observed. Our initial efforts to explore mechanisms of clinical chemistry assay interferences by these three nanoparticles suggest that establishing the causes for all interferences will be a substantial undertaking. Several factors contribute to the complexity of the problem, including the number of assays where interference was observed, different chemistries used to measure a given parameter in different analyzer systems, and occasionally different interferences in commercial analyzers ostensibly using the same chemistry for a given parameter. In the absence of the ability to reliably

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Figure 9. Differential interference and recovery of clinical chemistry assays on the Hitachi 912, Siemens Dimension and Vitros DT60 Analyzers. Nanoparticles were added to Randox3 serum to achieve concentrations of 1000, 818, and 111 ppm for PEG-gold nanorods, nanospheres and silica nanospheres, respectively. Nanoparticles were incubated in Randox3 serum for 15 minutes and then centrifuged (20 000 RCF for 15 minutes at room temperature) for particle removal. Clinical chemistry parameters were measured on the Hitachi 912, Siemens Dimension and Vitros DT60 as described in the ‘‘Methods’’ section. Results are expressed as mean (n ¼ 3) percent change from control (0 ppm) calculated as [((sample value/control value)  1)  100]. The star indicates that this assay value was above the range of the instrument; the presented value indicates the top of the assay range.

predict clinical chemistry assay interferences by nanoparticles, it is important to evaluate the potential for error empirically. Although it is difficult to generalize based upon observations with only three nanoparticles, results of this study suggest that interferences with standard clinical chemistry tests are dependent upon nanomaterial composition, shape, and concentration, as well as the assay chemistry and perhaps the commercial analyzer system used. This in turn suggests that verification of the absence of interference should be done virtually on a study-by-study basis, with nanoparticle concentrations known or anticipated to be in serum at the time clinical chemistry parameters are assessed. The results also indicate that centrifugation of serum samples prior to clinical chemistry analysis to remove nanoparticles may eliminate many of the interferences, but not all, and the centrifugation process itself may introduce some error in the measurements.

Acknowledgements We would like to thank Tina Conrad at the University of Florida, College of Veterinary Medicine Clinical Pathology and Diagnostics lab for performing clinical chemistry analyses.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

References Arnida, Jana´t-Amsbury MM, Ray A, Peterson CM, Ghandehari H. 2011. Geometry and surface characteristics of gold nanoparticles influence their biodistribution and uptake by macrophages. Eur J Pharm Biopharm 77:417–23. Banfi G, Fabbro MD. 2006. Nanotechnologic nutraceuticals: nurturing or nefarious? Clin Chem 52:331–2. Barrett EG, Johnston C, Oberdorster G, Finkelstein JN. 1999. Silica binds serum proteins resulting in a shift of the dose-response for silica-induced chemokine expression in an alveolar Type II cell line. Toxicol Appl Pharmacol 161:111–22. Baek M, Chung HE, Yu J, Lee JA, Kim TH, Oh JM, et al. 2012. Pharmacokinetics, tissue distribution, and excretion of zinc oxide nanoparticles. Int J Nanomedicine 7:3081–97. Belyanskaya L, Manser P, Spohn P, Bruinink A, Wick P. 2007. The reliability and limits of the MTT reduction assay for carbon nanotubes-cell interaction. Carbon 45:2643–8. Boverhof DR, David RM. 2010. Nanomaterial characterization: considerations and needs for hazard assessment and safety evaluation. Anal Bioanal Chem 396:953–61. Brown DM, Dickson C, Duncan P, Al-Attili F, Stone V. 2010. Interaction between nanoparticles and cytokine proteins: impact of protein and particles functionality. Nanotechnology 21:1–9. Calzolai L, Gilliland D, Rossi F. 2012. Measuring nanoparticles size distribution in food and consumer products: a review. Food Addit Contam Part A Chem Anal Expo Risk Assess 29:1183–93. Casey A, Herzog E, Davoren M, Lyng FM, Byrne HJ, Chambers G. 2007. Spectroscopic analysis confirms the interactions between single

Interference with clinical chemistry assays

Nanotoxicology Downloaded from informahealthcare.com by University of Birmingham on 03/23/15 For personal use only.

DOI: 10.3109/17435390.2014.894151

walled carbon nanotubes and various dyes commonly used to assess cytotoxicity. Carbon 45:1425–32. Dekker S, Krystek P, Peters RJ, Lankveld DPK, Bokkers BGH, HoevenArentzen PHV, et al. 2011. Presence and risks of nanosilica in food products. Nanotoxicology 5:393–405. Doak SH, Griffiths SM, Manshian B, Singh N, Williams PM, Brown AP, Jenkins GJS. 2009. Confounding experimental considerations in nanogenotoxicology. Mutagenesis 24:285–93. Dobrovolskaia MA, Aggarwal P, Hall JB, McNeil SE. 2008. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Mol Pharm 5: 487–95. Dobrovolskaia MA, Neun BW, Clogston JD, Ding H, Ljubimova J, McNeil SE. 2010. Ambiguities in applying traditional Limulus Amebocyte Lysate tests to quantify endotoxin in nanoparticle formulations. Nanomedicine 5:555–62. Dobrovolskaia MA, Patri AK, Zheng J, Clogston JD, Ayub N, Aggarwal P, et al. 2009. Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles. Nanomed-Nanotechnol 5:106–17. Fischer HC, Chan WC. 2007. Nanotoxicity: the growing need for in vivo study. Curr Opin Biotechnol 18:565–71. Frens G. 1973. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature (London), Phys Sci 241: 20–2. Hall JB, Dobrovolskaia MA, Patri AK, McNeil SE. 2007. Characterization of nanoparticles for therapeutics. Nanomedicine-UK 2:789–803. Hillyer JF, Albretch RM. 2001. Gastrointestinal persorption and tissue distribution of differential sized colloidal gold nanoparticles. J Pharm Sci 90:1927–36. Jana NR, Gearheart L, Murphy CJ. 2001. Wet chemical synthesis of high aspect ratio cylindrical gold nanorods. J Phys Chem B 105:4065–7. Kim YS, Song MY, Park JD, Song KS, Ryu HR, Chung YH, et al. 2010. Subchronic oral toxicity of silver nanoparticles. Particle Fibre Toxicol 7:1–11. Kroll A, Pillukat MH, Hahn D, Schnekenburger J. 2009. Current in vitro methods in nanoparticle risk assessment: limitations and challenges. Eur J Pharm Biopharm 72:370–7. Kroll A, Pillukat MH, Hahn D, Schnekenburger J. 2012. Interference of engineered nanoparticles with in vitro toxicity assays. Arch Toxicol 86: 1123–36.

125

Landsiedel R, Kapp MD, Schulz M, Wiench K, Oesh F. 2009. Genotoxicity investigations on nanomaterials: methods, preparation and characterization of test material, potential artifacts and limitations – many questions, some answers. Mutation Res 681:241–58. Loeschner K, Hadrup N, Qvortrup K, Larsen A, Gao X, Vogel U, et al. 2011. Distribution of silver in rats following 28 days of repeated oral exposure to silver nanoparticles or silver acetate. Particle Fibre Toxicol 8:1–14. McNeil SE. 2011. Characterization of nanoparticles intended for drug delivery. In Methods in Molecular Biology 697. New York: Humana Press. Mohamed MB, Volkov V, Link S, El-Sayed MA. 2000. The ‘lightning’ gold nanorods: fluorescence enhancement of over a million compared to the gold metal. Chem Phys Lett 317:517–23. Oostingh GJ, Casals E, Italiani P, Colognato R, Stritzinger R, Ponti J, et al. 2011. Problems and challenges in the development and validation of human cell-based assays to determine nanoparticleinduced immunomodulatory effects. Particle Fibre Toxicol 8:1–21. Schleh C, Semmler-Behnke M, Lipka J, Wenk A, Hirn S, Scha¨ffler M, et al. 2012. Size and surface charge of gold nanoparticles determine absorption across intestinal barriers and accumulation in secondary target organs after oral administration. Nanotoxicology 6: 36–46. Shinke K, Ando K, Koyana T, Takai T, Nakaji S, Ogino T. 2010. Properties of various carbon nanomaterial surfaces in bilirubin adsorption. Colloids Surf B: Biointerf 77:18–21. Sonavane G, Tomoda K, Makino K. 2008. Biodistribution of colloidal gold nanoparticles after intravenous administration: effect of particle size. Colloids Surf B: Biointerf 66:274–80. Sto¨ber W, Fink A, Bohn E. 1968. Controlled growth of monodisperse silica spheres in the micron size range. J Colloid Interf Sci 26:62–9. Veranth JM, Kaser EG, Veranth MM, Koch M, Yost GS. 2007. Cytokine responses of human lung cells (BEAS-2B) treated with micron-sized and nanoparticles of metal oxides compared to soil dusts. Particle Fibre Toxicol 4:1–18. Wang J, Zhou G, Chen C, Yu H, Wang T, Ma Y, et al. 2007. Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administration. Toxicol Lett 168: 176–85. Zhang XD, Wu HY, Wu D, Wang YY, Chang JH, Zhai ZB, et al. 2010. Toxicologic effects of gold nanoparticles in vivo by different administration routes. Int J Nanomed 5:771–81.

Supplementary material available online Supplementary Tables S1–S6

Differential interferences with clinical chemistry assays by gold nanorods, and gold and silica nanospheres.

Nanomaterials are known to cause interference with several standard toxicological assays. As part of an in vivo study of PEG-coated gold nanorods in m...
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