Quantitative Study on the Charge-Dependent Uptake of Ultrasmall Fluorescent Gold Nanoclusters in 3D Spheroids of Cancer Cells

Gold nanoclusters (AuNCs) have garnered significant attention in biomedical applications, particularly in biosensing, cancer therapy, and imaging, due to their unique optical property, good biocompatibility, and distinct bioactivity. Understanding the cellular uptake behavior of AuNCs is critical to improve the efficacy of their applications, whose mechanism has not been adequately validated. In this work, we synthesized AuNCs with varying surface modifications to quantify the exact law of surface charge on the cellular uptake of AuNCs in a multidimensional manner by using 3D multicellular tumor spheroids of both HeLa cells and MCF-7 cells as the model system. By the combined use of fluorescence live cell imaging and inductively coupled plasma–mass spectrometry, we systematically investigated the effect of surface charge on their uptake rate, intracellular versus intercellular distribution, and penetration depth in a quantitative manner. Our results showed that the cellular uptake of AuNCs was strongly charge dependent, with uptake efficiency increasing with the degree of surface positive charges. A similar charge-dependent uptake behavior was observed in both 2D cell cultures and 3D multicellular tumor spheroids, but the difference in 3D spheroids was less pronounced, in comparison to the 2D model. The effect of AuNCs' surface charge on the cellular uptake has been quantified in multiple dimensions in this work, which also provides crucial knowledge for effective cancer therapeutics and imaging applications based on AuNCs and other nanomaterials.