Engineering the mechano-biological properties of ZnxFe3-xO4 nanoparticles for enhanced stiffness-associated magneto-mechanical therapeutic performance

Magneto-mechanical therapy (MMT) has emerged as a promising physical oncology strategy; however, the contribution of nanoparticle mechanical properties, particularly stiffness, to therapeutic performance remains insufficiently understood. Here, a series of zinc-doped ferrite nanoparticles (Zn_xFe_3-xO₄; x = 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.8, 1.0) was engineered to systematically investigate how compositional tuning modulates Young's modulus, magnetic properties, and associated biological responses under rotating magnetic field (RMF) stimulation. Single-molecule force spectroscopy atomic force microscopy (SMFS-AFM) revealed a stiffness range of 25-48 GPa, with a maximum at x = 0.2, while vibrating sample magnetometry (VSM) showed a corresponding peak magnetization (79.2 emu/g), indicating coupled optimization of mechanical rigidity and magnetic responsiveness. Transmission electron microscopy (TEM) and ImageJ analysis confirmed comparable particle size distributions, while zeta potential measurements in deionized water and serum-containing media indicated no significant differences in surface charge. Cellular uptake and intracellular distribution were further evaluated using confocal imaging and inductively coupled plasma mass spectrometry (ICP-MS), revealing non-monotonic uptake behavior with maximal internalization at intermediate compositions. Confocal immunofluorescence imaging further revealed stiffness-associated alterations in F-actin distribution, suggesting cytoskeletal adaptation following nanoparticle internalization. AFM nanoindentation measurements demonstrated measurable changes in cellular mechanical properties, indicating that nanoparticle uptake influences baseline biomechanical behavior. Upon RMF exposure, Zn₀.₂ induced the most pronounced reduction in cell viability and biomechanical modulations, including increased apoptotic and necrotic fractions. Comparison between Zn₀ and Zn₀.₄, which exhibited similar magnetization but different stiffness, suggests that nanoparticle rigidity contributes to therapeutic outcomes beyond magnetic strength alone. Collectively, these results indicate that nanoparticle stiffness is associated with variations in cellular internalization behavior and magneto-mechanical therapeutic response. This study highlights intrinsic mechanical properties as an additional design parameter in MMT and provides a framework for stiffness-informed optimization of magnetic nanotherapeutics. STATEMENT OF SIGNIFICANCE: In this work, we systematically engineer zinc-doped ferrite magnetic nanoparticles (Zn_xFe_3-xO₄, x = 0-1.0) to elucidate how nanoparticle stiffness, independent of and synergistic with magnetization, governs cellular mechanobiology and magneto-mechanical therapeutic efficacy. Our key findings include: (i) precise tuning of nanoparticle Young's modulus (25-48 GPa) via Zn²⁺ doping, with a pronounced stiffness maximum at x = 0.2; (ii) a unique co-optimization of mechanical rigidity and magnetic responsiveness at Zn₀.₂Fe₂.₈O₄; (iii) stiffness-dependent cellular uptake, cytoskeletal remodeling, and pre-conditioning of intracellular mechanics; and (iv) direct experimental evidence that nanoparticle stiffness, independent of magnetization, significantly amplifies magneto-mechanical force transmission, leading to enhanced apoptosis and necrosis under rotating magnetic field stimulation. Importantly, this study provides the first AFM-based quantitative linkage between nanoparticle stiffness, intracellular mechanical priming, and magneto-mechanical therapeutic outcome.