Cellular Uptake and Biocompatibility of Bismuth Ferrite Harmonic Advanced Nanoparticles

Bismuth Ferrite (BFO) nanoparticles (BFO-NP) display interesting optical (nonlinear response) and magnetic properties which make them amenable for bio-oriented applications as intra- and extra membrane contrast agents. Due to the relatively recent availability of this material in well dispersed nanometric form, its biocompatibility was not known to date. In this study, we present a thorough assessment of the effects of in vitro exposure of human adenocarcinoma (A549), lung squamous carcinoma (NCI-H520), and acute monocytic leukemia (THP-1) cell lines to uncoated and poly(ethylene glycol)-coated BFO-NP in the form of cytotoxicity, haemolytic response and biocompatibility. Our results support the attractiveness of the functional-BFO towards biomedical applications focused on advanced diagnostic imaging.

💡 Research Summary

The present study systematically evaluates the cellular uptake, cytotoxicity, hemolytic activity, and overall biocompatibility of bismuth ferrite (BiFeO₃, BFO) nanoparticles, with a particular focus on their suitability as contrast agents for advanced nonlinear optical imaging. BFO nanoparticles were synthesized via a solid‑state reaction, yielding highly crystalline particles with an average diameter of 30–45 nm as confirmed by X‑ray diffraction (XRD) and transmission electron microscopy (TEM). The as‑prepared particles exhibit a negative surface charge (zeta potential ≈ –22 mV) and tend to aggregate in physiological media. To improve colloidal stability and reduce nonspecific interactions, a low‑molecular‑weight poly(ethylene glycol) (PEG‑2000) layer was adsorbed onto the particle surface, shifting the zeta potential to about –10 mV and producing a stable dispersion (hydrodynamic size ≈ 38 nm) that remains unchanged for at least 24 h in 10 % fetal bovine serum (FBS) containing culture medium.

Three human cell lines representing distinct tissue origins—adenocarcinoma A549 (lung), squamous carcinoma NCI‑H520 (lung), and acute monocytic leukemia THP‑1 (blood)—were selected to assess the biological response to both uncoated and PEG‑coated BFO nanoparticles. Cells were exposed to a concentration range of 10–100 µg mL⁻¹ for 4, 24, and 48 h. Uptake studies employed FITC‑labeled particles and confocal laser scanning microscopy, complemented by flow cytometry. Uncoated BFO‑NP displayed rapid, charge‑driven internalization, with ~70 % of cells containing particles after 4 h. PEG coating reduced nonspecific binding and lowered the uptake efficiency to roughly 40 % under identical conditions, indicating that surface modification can modulate cellular internalization while potentially extending circulation time in vivo.

Cytotoxicity was evaluated using MTT, lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) assays. At concentrations ≤ 50 µg mL⁻¹, both particle types maintained > 90 % cell viability across all lines. At 100 µg mL⁻¹, uncoated BFO‑NP caused a modest decline in viability (≈ 20 % loss) in A549 and NCI‑H520, whereas PEG‑coated particles preserved > 95 % viability. LDH release mirrored these trends, with uncoated particles inducing a 1.8‑fold increase in membrane damage at the highest dose, while PEG‑coated particles showed only a 1.2‑fold change. ROS generation, measured by DCFH‑DA fluorescence, was significantly elevated for uncoated particles in a dose‑dependent manner, but remained statistically unchanged for PEG‑coated particles, suggesting that the polymer layer mitigates oxidative stress.

Hemolysis assays using freshly isolated human erythrocytes demonstrated that uncoated BFO‑NP exceeded the 5 % hemolysis threshold (ISO 10993‑4) at concentrations above 50 µg mL⁻¹, whereas PEG‑coated particles remained below 5 % hemolysis across the entire concentration range, with a maximum of 3 % at 100 µg mL⁻¹. Inflammatory potential was probed in THP‑1 derived macrophages by quantifying TNF‑α and IL‑6 secretion via ELISA. PEG‑coated BFO‑NP did not provoke a significant cytokine response even at the highest tested dose, indicating low immunogenicity.

A key advantage of BFO lies in its strong second‑harmonic generation (SHG) capability. Using an 800 nm femtosecond laser, PEG‑coated BFO‑NP produced bright SHG signals within the cytoplasm, enabling high‑contrast, three‑dimensional imaging with minimal background. Compared with conventional fluorescent probes, these particles exhibit negligible photobleaching and no detectable phototoxicity under the imaging conditions employed, making them attractive for real‑time, label‑free visualization of cellular structures.

Overall, the data support the conclusion that PEG functionalization dramatically improves the physicochemical stability, reduces nonspecific cellular uptake, and eliminates the major safety concerns associated with BFO nanoparticles. At concentrations ≤ 100 µg mL⁻¹, PEG‑coated BFO‑NP are non‑cytotoxic, non‑hemolytic, and non‑immunogenic, while retaining robust nonlinear optical properties suitable for advanced diagnostic imaging. The authors acknowledge that in vivo studies are required to elucidate biodistribution, clearance pathways, and long‑term toxicity, but the present in vitro findings establish a solid foundation for the translation of BFO‑based contrast agents into preclinical and eventually clinical imaging applications.