Quantification of cellular uptake of gold nanoparticles <i>via</i> scattering intensity changes in flow cytometry

Quantifying cellular uptake of nanoparticles is critical for understanding their biological interactions and optimizing their applications in nanomedicine. In this study, we developed a flow cytometry-based method to quantify the uptake of gold nanoparticles (AuNPs) using A549 cells. Taking advantage of the scattering properties of AuNPs, this method uses side scatter intensity to estimate the number of nanoparticles internalized by cells. However, directly measuring the exact number of internalized nanoparticles remains challenging due to the tendency of AuNPs to aggregate within cells. To address this, we introduce a new unit, molecules of equivalent gold nanoparticle (MEAuNP), which expresses side scatter intensity as a standardized unit based on the scattering of a single AuNP. While this method does not directly solve the problem of accurately measuring the exact number of internalized nanoparticles, it provides a semi-quantitative approach for estimating nanoparticle uptake. The obtained MEAuNP values are consistent with literature reports, suggesting that the approach yields reliable and comparable data. Moreover, the use of calibrated values ensures that consistent results can be obtained across different acquisition settings and potentially across different instruments. We further examined uptake dynamics and validated the method across multiple cell lines including HeLa, Beas-2B, Jurkat, and RPMI8226. This approach provides a robust tool for quantifying metal nanoparticle uptake, supporting the standardization of estimating uptake levels in various biological systems.