Magnetic nanoparticles (MNPs) represent one of the most versatile platforms in nanomedicine, enabling drug delivery, imaging, and magnetically triggered therapeutic responses. We present herein a methodology to establish with reasonable accuracy the drug loading on MNPs based on Fe3O4 (magnetite). This method combines magnetometry and Mossbauer spectroscopy, and was exemplified for the first time on L-cysteine (or citric acid)-coated Fe₃O₄ further functionalized with Dox (doxorubicin).

The novelty of this approach resides in the utilizing the variation in magnetization of functionalized MNPs by low-temperature Mossbauer spectroscopy, when spontaneous magnetization of the magnetic core can be estimated. As a nondestructive methodology for quantitative evaluation of drug loading by combining SQUID magnetometry with low-temperature Mössbauer spectroscopy, this approach directly probes the magnetic core, allowing precise differentiation between intrinsic nanoparticle properties and the contribution of surface-bound organic molecules.

The method is reliable and easy to implement, as it uses the ratio between the spontaneous magnetization of the covered nanoparticles and that of the magnetic core, producing results that are less than 10% off the exact analytical result of drug loading. This method has a great advantage in offering the potential to expand the NPs scope to any Fe-containing magnetic core to which ⁵⁷Fe Mossbauer spectroscopy can be applied.