MICROWAVE SYNTHESIS, CHARACTERIZATION AND TESTING OF ACUTE TOXICITY OF BORON NITRIDE NANOPARTICLES BY MONITORING OF BEHAVIORAL AND PHYSIOLOGICAL PARAMETERS

Abstract

Research presents a comprehensive study on the synthesis, physicochemical characterization, and toxicological evaluation of boron nitride (BN) nanoparticles synthesized via a novel low-temperature microwave-assisted route, along with a systematic investigation into their acute toxicity in warm-blooded mammals and avian embryos using advanced behavioral and physiological monitoring systems. The study addresses critical gaps in current nanomaterial research, particularly concerning the eco-friendly and cost-effective production of high-purity hexagonal boron nitride (h-BN) nanoparticles and their biocompatibility when considered for biomedical applications such as boron-neutron and boron-proton capture therapy, targeted hyperthermia, and drug delivery systems. Traditional methods for synthesizing h-BN commonly rely on high-temperature carbothermic reduction techniques involving the reduction and nitridation of boric oxide and related salts at temperatures exceeding 1500 °C. These approaches pose significant challenges, including high energy demands, environmental pollution due to carbon emissions, the use of toxic and expensive reagents such as boron tribromide (BBr₃), and limitations in nanoparticle size control and phase purity. The method developed in this study circumvents these limitations through a simple two-step process involving the reaction of boron trifluoride (BF₃) and ammonia (NH₃) at a significantly lower temperature of 150 °C, followed by thermal decomposition and purification. The resultant product comprises nano-sized particles of hexagonal boron nitride with structural features indicative of turbostratic disorder due to the low synthesis temperature, but still maintaining desirable physicochemical properties. Structural analysis via X-ray diffraction (XRD) confirmed the formation of h-BN, with characteristic peaks corresponding to hexagonal symmetry and no observable impurity phases. The broadening and slight shift in the (002) peak of the XRD pattern toward lower angles point to an increased interlayer spacing and lattice expansion, which is characteristic of nanocrystalline materials and turbostratic boron nitride (t-BN). The Scherrer equation estimated the average crystallite size along the c-axis to be approximately 3 nm, suggesting the formation of ultrathin h-BN nanosheets. Raman spectroscopy provided further confirmation of the nanocrystalline structure and purity of the synthesized h-BN. The Raman spectra exhibited a strong E₂g mode at approximately 1373 cm⁻¹, upshifted from the bulk h-BN peak position, which is consistent with literature reports on nanocrystalline h-BN synthesized at higher temperatures. This upshift, along with the broadening of the Raman band, reflects size confinement effects and structural disorder inherent to low-temperature synthesis methods. Scanning electron microscopy (SEM) revealed the morphology of the synthesized h-BN nanoparticles as predominantly flat platelets with a thickness less than 10 nm and lateral dimensions significantly larger, further affirming the formation of nanosheets. These nanosheets were dispersed on a gold-coated silicon substrate for enhanced imaging resolution. In addition to material characterization, the study placed significant emphasis on evaluating the biosafety of these nanomaterials. Toxicological assessments were carried out using two distinct yet complementary models: (1) non-lethal in vivo testing in warm-blooded mammals (Wistar rats) through behavioral and physiological monitoring and (2) embryo toxicity assays in avian models (Golden Wyandotte chick embryos) using ovoscopy and plethysmography. The in vivo rat experiments were conducted in strict compliance with the 4R principles (replace, reduce, refine, and responsibility), ensuring humane treatment of laboratory animals. Five different groups of rats received intramuscular injections of various boron-containing nanoparticles, including h-BN synthesized using BF₃ and NH₃, h-BN synthesized from boric acid, boron nitride nanotubes (BO-N-02-NT), BN-encapsulated copper nanoparticles, and a comparative group exposed to carbon nanotubes (C-E-015-NT), with additional control groups receiving iron oxide nanoparticles and saline. Toxicity was quantified using a complex toxicity index (CTI), a novel analytical metric integrating multiple physiological and behavioral parameters including maze navigation error rates, maze completion time, changes in blood pressure, body temperature, and reactive oxygen species (ROS) activity. This multidimensional approach provides a robust and sensitive measure of acute toxicity without resorting to lethal endpoints. The experimental results demonstrated that all BN-based nanoparticles exhibited significantly lower acute toxicity compared to carbon nanotubes, with differences in CTI becoming negligible between BN samples after 9 to 11 days and disappearing entirely by day 12. These findings highlight the relatively benign nature of BN nanoparticles, making them attractive candidates for biomedical applications. The highest toxicity was observed in rats exposed to carbon nanotubes, which demonstrated elevated CTI scores throughout the 15-day observation period. Conversely, BN-based particles, particularly those synthesized via the new low-temperature microwave-assisted method, showed comparatively mild and transient physiological disturbances. Additional tests involving whole-body hyperthermia at controlled temperatures of 40 °C and 43.5 °C further underscored the favorable toxicity profile of h-BN nanoparticles under stress-inducing conditions, reinforcing their safety margin for potential clinical applications such as hyperthermic cancer therapy. Notably, the use of advanced, non-invasive tools such as visual surveillance systems, pulse oximetry, non-contact thermometers, ROS analyzers, and blood pressure monitoring devices enhanced the reliability and reproducibility of data, allowing for real-time physiological tracking without undue stress to the animals. Parallel toxicity testing was performed using the chick embryo model, which is a widely accepted, ethical, and cost-effective alternative to mammalian testing. The combination of visible and green-light ovoscopy, along with plethysmographic monitoring of respiratory and heart rate signals, enabled comprehensive and continuous evaluation of embryonic development. Toxicity was calculated based on the survival rate of embryos in treated versus control groups using a standardized toxicity index. The experimental design allowed the researchers to visually and quantitatively assess developmental abnormalities, survival outcomes, and physiological disruptions induced by the tested nanomaterials. The consistency in toxicity outcomes between the rodent and chick embryo models validated the robustness and translatability of the employed methodology. Additionally, the study explored the potential application of the synthesized nanoparticles in proton and neutron capture therapies by evaluating the boron isotopic composition and considering the reactivity of the nanoparticles in fusion reactions. The unique combination of high neutron and proton capture cross-sections, bioavailability, and purity of the synthesized ¹⁰B and ¹¹B particles suggests that these nanomaterials could be utilized as cost-effective and safer alternatives in boron neutron capture therapy (BNCT) and proton boron fusion therapy, thus contributing to the development of new generations of radiopharmaceuticals. The researchers also proposed a new agarose-based phantom model to test the effectiveness of various nanoparticle-loaded layers under simulated thermal and proton beam conditions, showing encouraging preliminary results for targeted delivery and localized therapy. This study provides a compelling argument for the viability of a novel, environmentally sustainable, and cost-efficient method for synthesizing high-quality h-BN nanoparticles. The data indicate that these materials possess favorable structural properties, can be produced at scale using relatively simple and safe laboratory procedures, and exhibit a low toxicity profile in both mammalian and avian models. The synthesis route offers an attractive alternative to existing high-temperature, energy-intensive methods and supports the development of advanced nanomaterials for cancer therapy, diagnostic imaging, and future multi-modal clinical applications. The dual evaluation models used in this work not only reinforce the credibility of the findings but also establish a gold standard for future nanotoxicological research. The promising preclinical results justify further investigations into the in vitro and in vivo interactions of boron nitride-based nanoparticles with biological systems under varied pathological and physiological conditions, including their functionalization, biodistribution, immune response, clearance pathways, and long-term safety.