Facile Synthesis of Ligand-Free Iridium Nanoparticles and Their In Vitro Biocompatibility
- 532 Downloads
High-density inorganic nanoparticles have shown promise in medical applications that utilize radiation including X-ray imaging and as radiation dose enhancers for radiotherapy. We have developed an aqueous synthetic method to produce small (~ 2 nm) iridium nanoparticles (IrNPs) by reduction of iridium(III) chloride using a borohydride reducing agent. Unlike other solution-based synthesis methods, uniform and monodispersed IrNPs are produced without the use of surfactants or other solubilizing ligands. These nanoparticles are highly crystalline as observed by X-ray diffraction and high-resolution transmission electron microscopy (TEM). In vitro metabolic toxicity assays using hepatocyte and macrophage cells demonstrate that both IrNPs and iridium(III) chloride are well tolerated at concentrations of up to 10 μM iridium. Furthermore, the IrNPs were assessed in a hemolytic assay and found to have no significant impact on red blood cells when exposed to concentrations up to 100 μM. Overall, these results support the potential for the in vivo application of this nanomaterial.
KeywordsNanoparticle synthesis Nanocrystals Iridium Surface characterization Cellular toxicity
Dynamic light scattering
Inductively coupled plasma mass spectrometry
Powder diffraction file
X-ray photoelectron spectroscopy
Noble metal nanoparticles are a mainstay of emerging nanotechnologies due to their interesting optical, electronic, and surface catalytic properties. In nanomedicine, these unique biomaterials have drawn significant attention due the ability to tailor their biological interactions through surface modifications for a wide range of applications . Gold nanoparticles (AuNPs) have been investigated extensively for sensing and therapeutic applications [2, 3], while other noble metals, including silver, have found niche uses such as anti-microbials . However, nanoparticles composed of platinoid elements, which are commonly employed for their surface catalytic properties , have yet to be thoroughly examined for biomedical applications. The exceptional surface stability and known biological compatibility of these elements, as well as their potential novel physical properties on the nanoscale, make them unique alternatives to AuNPs.
High-energy radiation is utilized extensively in medicine including in diagnostic imaging and radiation therapy. Therefore, functional materials that interact with radiation, such as high atomic number and high-density nanoparticles, may improve the performance of these modalities. The majority of the chemical and engineering studies to date have focused on AuNPs to enhance radiation interactions, although bismuth and hafnium have been examined for diagnostic and therapeutic applications respectively [6, 7].
Here, we present a synthetic method to produce iridium nanoparticles (IrNPs), which are predicted to have strong radiation attenuation due to its high density. Iridium is one of the least reactive metals, considered generally biologically compatible, and has an elemental density of 22.56 g/cm3 (second only to osmium, which is known to be highly toxic). An isotope of iridium, 192Ir, is a commonly used brachytherapy gamma emitter, and part of the success of this material is due to the high density, i.e., the large number of atoms in a small volume of the material. In the current study, we present the synthesis of IrNPs and their in vitro biocompatibility as well as that of iridium ions, which has not previously been evaluated in the selected cell lines. These novel IrNPs have not been readily explored for medical purposes despite the material’s chemical inertness and superior density. Although iridium is a relatively expensive material like other noble metals, its current value as a commodity is approximately three quarters the price of gold and half that of rhodium, making it an interesting economic alternative.
Synthesis of IrNPs
All synthesis reactions were performed at room temperature under aerobic conditions in purified 18 MΩ water. A 20 mM iridium (III) chloride (Acros Organics) stock was prepared by bath sonication and stirred for at least 20 min to generate an optically clear solution. A solution of 1.0 M borane morpholine (Alfa Aesar) was also prepared by bath sonication. For larger scale syntheses of 500 mL total volume, 25 mL iridium (III) chloride solution was used (diluted to 1.0 mM) and 5.0 mL borane morpholine was added (final 10 mM concentration) with rapid stirring. The solution gradually turned from dark brown to black over 30 min. The nanoparticles were allowed to stabilize for at least 60 min. This colloidal solution was directly added to centrifugal spin filters (Amicon Ultra-4, 10k MWCO regenerated cellulose), and the nanoparticles were collected at 4000×g and washed in purified water. The nanoparticles were then suspended in water, passed through a syringe filter (Millex-MP 0.22 μm EO), and stored for quantification.
For X-ray photoelectron spectroscopy (XPS) analysis, nanoparticles were suspended in an equal volume nitric acid, collected by centrifugation in a microcentrifuge tube (5 min, 17 rcf), and suspended in water prior to analysis. Transmission electron microscopy (TEM) was performed on an FEI Tecnai F-20 TEM operating at 200 kV. Purified IrNPs were drop-cast on holey carbon Cu supported TEM grids (Ted Pella) and dried at room temperature overnight. Line diffraction analysis was performed using ImageJ software analysis. For X-ray diffraction (XRD) analysis, concentrated IrNPs were drop-cast on a glass slide and dried at room temperature. XRD data were collected in focused beam (Bragg–Brentano) geometry on a Rigaku Ultima IV X-ray diffraction system using graphite monochromatized Cu Kα radiation. Scans were performed over the angular range 20–80° 2θ at a scan rate of 0.1°/min at room temperature. Dynamic light scattering (DLS) was performed on a Malvern Nano ZSP in disposable polystyrene cuvettes. Nanoparticles were suspended in water, and data is reported as distributed by number. UV-Vis absorbance spectra were collected on a Tecan M200 Pro in a black 96-well plate and a total solution volume of 100 μL. Concentrations of iridium were adjusted to illustrate relative absorbance peaks. XPS analysis was carried out on a PHI Versaprobe II fitted with a hemispherical electron analyzer and aluminum Kɑ (1486.7 eV) X-ray source. Spectrum analysis was performed using the Multipak software suite. Binding energy calibration was performed using the C1s peak at 284.6 eV, and peak fitting was based on asymmetric peaks and an iterated Shirley background, resulting in a chi-squared value of 1.13. Inductively coupled plasma mass spectrometry (ICP-MS) of IrNPs and iridium(III) chloride solution were assessed prior to biological toxicity assays. Fifty microliters of each IrNP solution was digested in 50 μL aqua regia (3:1 M concentrated nitric acid to hydrochloric acid) overnight at 70 °C in a digestion tube. Samples were then diluted in 5.0 mL 1% nitric acid for analysis. ICP-MS was performed on an Agilent 7900 using helium as a collision gas. Calibration curves were prepared using 100–0.1 μg/mL iridium stock solutions (in 1% HCl), and all samples were diluted such that concentrations were measured in the tens of ppb range.
HepG2 and J774A.1 cell lines were seeded at 2 × 105 cells per well (100 μL) in a 96-well plate (DMEM with 10% FBS) and allowed to settle for 24 h. Iridium nanoparticles, iridium salt, water, or DMSO was added at 10% volume (10 μL additional volume). Cells were then incubated for 24 or 48 h. For viability analysis, media was removed, and cells were washed once in PBS. One hundred microliters of culture media with 10% Alamar Blue (Thermo Scientific) was incubated with cells for 2 h. Media was then re-plated into a black 96-well plate, and fluorescence was read (ex530/em590) on a Tecan M200 Pro. All data were performed in quadruplicate, and experiments were repeated on independent days to confirm general trends. A hemolytic assay was performed as previously reported .
Iridium Nanoparticle Synthesis and Characterization
Various synthetic processes have been examined to produce nanoscale iridium for catalytic applications, including reduction of iridium salts by hydrides and hydrogen gas [9, 10, 11, 12, 13], UV and gamma radiation [14, 15, 16, 17], and polyol or alcohol reduction [18, 19, 20]. However, many of these synthetic methods are designed for integration of iridium onto a substrate or support for chemical reactions and are not compatible with biological applications . Recently, aerosolized 192Ir was employed as model nanoscale materials for lung toxicity and was chosen for its exceptional inertness [22, 23]. The primary purpose of these studies was to examine the clearance and translocation of inhaled fine particulates from the lungs; however, it also highlights the biocompatibility of this element.
We evaluated the in vitro biological compatibility of the uncapped IrNPs and compared this to iridium(III) chloride salt in two types of mammalian cells that are expected to accumulate the highest concentrations of injected nanoparticles. Iridium(III) toxicity in J774A.1 cells follows a normal toxicity dose-response curve; 100 μM iridium(III) reduces cellular viability to 93 and 66%, and 500 μM results in a 40 and 10% cellular viability at 24 and 48 h respectively. This data reflect interesting cell-specific response to iridium(0) and iridium(III), and we anticipate further exploring these effects in vivo. Smaller IrNPs and other poorly soluble inorganic nanomaterials are expected to be translocated to the kidney and the liver, with a short temporal residence in the kidneys, and longer residence in the liver, which may further impact cell-specific toxicity profiles. Excretion is expected through the feces for larger iridium particles, although we expect the extremely small size of these IrNPs may be readily filtered through the renal system if colloidal stability in vivo can be maintained .
In preparation for in vivo applications, the blood compatibility of the IrNPs was evaluated by a hemolytic assay. Utilizing whole mouse blood, we evaluated the effect of these IrNPs on the rupture of erythrocytes and potential release of hemoglobin. Although in-depth studies of the final surface modified IrNP will need to be evaluated, the current IrNP building blocks do not elicit a detectable hemolytic response until extremely high concentrations (500 μM).
We conclude from these studies that iridium(0) nanocrystals can be readily synthesized by a simple aqueous borohydride reduction of iridium(III) chloride, which results in 2–3 nm highly crystalline nanoparticles that are colloidally stable in water with an approximately 5 nm hydrodynamic size. During acute exposure, these particles are non-toxic at concentrations up to 50 μM iridium (compared to 10 μM for iridium chloride) in hepatocytes, stimulate metabolic activity in macrophage cells, and do not elicit a hemolytic response at practical concentrations. These ligand-free nanoparticles may serve as building blocks or cores for subsequent surface-modified IrNPs for use in biological and medical applications. Further investigation of the functional properties of these high-density nanomaterials in the presence of X-rays or other radiation presents the opportunity for novel therapeutic and diagnostic agents.
We gratefully acknowledge the laboratory facilities and equipment support provided by the Portland State University (PSU) Center for Electron Microscopy & Nanofabrication.
This work was supported by the NIH NIGMS as a Maximizing Investigators’ Research Award, 1R35GM119839-01 (C.S.), NIH NIBIB 1R15EB021581-01 (G.S.), and Oregon State University College of Pharmacy Start-up Funds.
Availability of Data and Materials
All datasets on which the conclusions of the manuscript rely are presented in the main paper.
AB and CS conceived and designed the study. AB and HW carried out the experiments. All authors analyzed the data. AB and CS wrote the manuscript. All authors have given approval to the final version of the manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 6.Brown AL, Naha PC, Benavides-Montes V, Litt HI, Goforth AM, Cormode DP (2014) Synthesis, X-ray opacity, and biological compatibility of ultra-high payload elemental bismuth nanoparticle X-ray contrast agents. Chem Mater. https://doi.org/10.1021/cm500077z
- 10.Migowski P, Zanchet D, Machado G, Gelesky MA, Teixeira SR, Dupont J (2010) Nanostructures in ionic liquids: correlation of iridium nanoparticles’ size and shape with imidazolium salts’ structural organization and catalytic properties. Phys Chem Chem Phys 12:6826–6833. https://doi.org/10.1039/B925834E CrossRefGoogle Scholar
- 13.Fonseca GS, Umpierre AP, Fichtner PFP, Teixeira SR, Dupont J (2003) The use of imidazolium ionic liquids for the formation and stabilization of Ir0 and Rh0 nanoparticles: efficient catalysts for the hydrogenation of arenes. Chem Eur J 9:3263–3269. https://doi.org/10.1002/chem.200304753 CrossRefGoogle Scholar
- 17.Rojas, J. V.; Molina Higgins, M. C.; Toro Gonzalez, M.; Castano, C. E. Single step radiolytic synthesis of iridium nanoparticles onto graphene oxide. Appl Surf Sci 2015, 357, Part B, 2087–2093, doi: https://doi.org/10.1016/j.apsusc.2015.09.190
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.