Ligand‑Free Iridium Nanoparticles: A Simple Aqueous Synthesis and Demonstrated In‑Vitro Biocompatibility

Background

Noble‑metal nanoparticles are central to nanomedicine thanks to their unique optical, electronic, and catalytic properties. While gold and silver nanoparticles have been extensively studied for sensing, therapy, and antimicrobial use, platinoid metals remain largely untapped in biomedical contexts. Iridium, with its exceptional density (22.56 g cm⁻³), chemical inertness, and known biocompatibility, offers a compelling alternative to gold for radiation‑enhancement applications. Existing studies on high‑Z nanoparticles have focused on gold, bismuth, and hafnium; iridium has not been explored in this regard despite its favorable physical and chemical traits.

Iridium’s high density underpins its utility in radiation physics—¹⁹²Ir, a common brachytherapy isotope, demonstrates how a densely packed element can deliver potent gamma emission. However, no systematic synthesis of ligand‑free iridium nanoparticles suitable for biological use has been reported. Our work fills this gap by providing a simple, scalable aqueous protocol and a comprehensive assessment of in‑vitro biocompatibility.

Methods

Synthesis of IrNPs

All reactions were carried out at ambient temperature in oxygen‑free, 18 MΩ deionized water. A 20 mM stock of IrCl₃ (Acros Organics) was sonicated and stirred for 20 min to clear the solution. A 1.0 M borane‑morpholine solution (Alfa Aesar) was likewise sonicated. For a 500 mL scale, 25 mL of 1.0 mM IrCl₃ was mixed with 5 mL of 10 mM borane‑morpholine under vigorous stirring. The mixture darkened from brown to black over 30 min, indicating nanoparticle formation. After an additional 60 min of stirring, the colloid was concentrated using 10 kDa MWCO centrifugal filters (Amicon Ultra‑4) at 4000 × g, washed with water, and filtered through a 0.22 µm syringe filter (Millex‑MP) before storage.

Nanoparticle Characterization

High‑resolution TEM (FEI Tecnai F‑20, 200 kV) was used to image the particles on holey carbon grids. Diffraction patterns were analyzed with ImageJ to confirm the 0.22 nm lattice spacing characteristic of metallic iridium. X‑ray diffraction (Rigaku Ultima IV, Cu Kα, 20–80° 2θ, 0.1° min⁻¹) matched the PDF card 9008470 for elemental iridium. Dynamic light scattering (Malvern Nano ZSP) reported a hydrodynamic diameter of ~5 nm. UV‑Vis spectra (Tecan M200 Pro) confirmed the broad, featureless absorption of IrNPs versus the sharp peaks of IrCl₃. X‑ray photoelectron spectroscopy (PHI Versaprobe II) revealed a dominant Ir(0) surface with ~20 % oxide contamination. Inductively coupled plasma mass spectrometry (Agilent 7900) quantified iridium concentrations in the ppb range after aqua regia digestion.

Cytotoxicity Analysis

HepG2 (human hepatocellular carcinoma) and J774A.1 (murine macrophage) cells were seeded at 2 × 10⁵ cells per well in 96‑well plates (DMEM + 10% FBS) and incubated 24 h. IrNPs, IrCl₃, water, or DMSO were added to a final volume of 110 µL (10% v/v). Viability was assessed after 24 or 48 h using Alamar Blue (10% v/v) and fluorescence readout (ex 530 nm/em 590 nm). All experiments were performed in quadruplicate and repeated on independent days. Hemolysis was evaluated by incubating erythrocytes in PBS with IrNPs at 37 °C for 1 h and measuring hemoglobin release.

Results

Iridium Nanoparticle Synthesis and Characterization

The reduction of IrCl₃ with a 10‑fold molar excess of borane‑morpholine in water yields monodispersed, 2–3 nm IrNPs within 30 min. TEM images (Fig. 1a) and XRD patterns (Fig. 1c) confirm a highly crystalline iridium core. The particles remain colloidally stable in water for months (Fig. 1d). In basic media, the nanoparticles convert to iridium oxide, presenting a blue hue; acidic conditions induce flocculation without disrupting crystal structure. XPS indicates a predominantly elemental surface with ~20 % surface oxide (Fig. 3). Attempts to use thiol surfactants during synthesis inhibit particle formation, underscoring the necessity of a ligand‑free environment for this approach.

a IrNPs are 2–3 nm in TEM; b show highly crystalline lattice; c XRD matches elemental iridium; d DLS indicates ~5 nm hydrodynamic size.

Iridium Cytotoxicity

In HepG2 cells, IrCl₃ increased metabolic activity to 115 % at 24 h but fell to 90 % at 48 h with 500 µM. IrNPs reduced viability to 78 % at 50 µM after 48 h. Macrophages (J774A.1) displayed a transient metabolic boost (122 % at 24 h) with 50 µM IrNPs, returning to ~98 % after 48 h. At 500 µM, IrNPs showed neutral viability at 24 h but reduced viability after 48 h, suggesting dose‑dependent effects. Hemolysis assays revealed no significant red‑blood‑cell lysis up to 100 µM IrNPs.

Cellular viability of HepG2 and J774A.1 after 24/48 h exposure to IrNPs or IrCl₃ (*p < 0.05 vs. untreated).

Discussion

Traditional iridium nanomaterial syntheses—hydride reduction, UV/gamma irradiation, or polyol routes—are tailored for catalytic support integration and are not suited for biological applications. Our aqueous, ligand‑free approach circumvents these limitations and produces particles amenable to further functionalization. In vitro, IrNPs exhibit low acute toxicity, with tolerable concentrations up to 50 µM in hepatocytes and minimal hemolytic activity. Macrophage stimulation suggests a potential for modulating immune responses, which warrants further mechanistic studies.

Given iridium’s high density, these nanoparticles are expected to enhance radiation attenuation, analogous to the performance of ¹⁹²Ir in brachytherapy. Future work will investigate in‑vivo biodistribution, renal clearance, and therapeutic efficacy when combined with X‑ray or gamma irradiation.

Conclusions

We have demonstrated that iridium(0) nanocrystals can be synthesized via a simple aqueous borohydride reduction of IrCl₃, yielding 2–3 nm, highly crystalline, ligand‑free IrNPs with ~5 nm hydrodynamic diameter. In acute exposure, these particles are non‑toxic up to 50 µM in hepatocytes, transiently stimulate macrophage metabolism, and do not provoke hemolysis at physiologically relevant concentrations. Their ligand‑free core offers a versatile platform for surface modification toward biomedically relevant applications, especially as high‑density radiographic or radiotherapeutic agents.

Abbreviations

AuNP

Gold nanoparticle

DLS

Dynamic light scattering

ICP-MS

Inductively coupled plasma mass spectrometry

IrNP

Iridium nanoparticle

PDF

Powder diffraction file

UV

Ultraviolet

XPS

X‑ray photoelectron spectroscopy

XRD

X‑ray diffraction