Carbon nanomaterial-derived lung burden analysis using UV-Vis spectrophotometry and proteinase K digestion.

BACKGROUND The quantification of nanomaterials accumulated in various organs is crucial in studying their toxicity and toxicokinetics. However, some types of nanomaterials, including carbon nanomaterials (CNMs), are difficult to quantify in a biological matrix. Therefore, developing improved methodologies for quantification of CNMs in vital organs is instrumental in their continued modification and application. RESULTS In this study, carbon black, nanodiamond, multi-walled carbon nanotube, carbon nanofiber, and graphene nanoplatelet were assembled and used as a panel of CNMs. All CNMs showed significant absorbance at 750 nm, while their bio-components showed minimal absorbance at this wavelength. Quantification of CNMs using their absorbance at 750 nm was shown to have more than 94% accuracy in all of the studied materials. Incubating proteinase K (PK) for 2 days with a mixture of lung tissue homogenates and CNMs showed an average recovery rate over 90%. The utility of this method was confirmed in a murine pharyngeal aspiration model using CNMs at 30 μg/mouse. CONCLUSIONS We developed an improved lung burden assay for CNMs with an accuracy > 94% and a recovery rate > 90% using PK digestion and UV-Vis spectrophotometry. This method can be applied to any nanomaterial with sufficient absorbance in the near-infrared band and can differentiate nanomaterials from elements in the body, as well as the soluble fraction of the nanomaterial. Furthermore, a combination of PK digestion and other instrumental analysis specific to the nanomaterial can be applied to organ burden analysis.