It is crucial for understanding mechanisms of drug action to quantify the three-dimensional (3D) drug distribution within a single cell at nanoscale resolution. Yet it remains a great challenge due to limited lateral resolution, detection sensitivities, and reconstruction problems. The preferable method is using X-ray nano-computed tomography (Nano-CT) to observe and analyze drug distribution within cells, but it is time-consuming, requiring specialized expertise, and often subjective, particularly with ultrasmall metal nanoparticles (NPs). Furthermore, the accuracy of batch data analysis through conventional processing methods remains uncertain. In this study, we used radioenhancer ultrasmall HfO₂ nanoparticles as a model to develop a modular and automated deep learning aided Nano-CT method for the localization quantitative analysis of ultrasmall metal NPs uptake in cancer cells. We have established an ultrasmall objects segmentation method for 3D Nano-CT images in single cells, which can highly sensitively analyze minute NPs and even ultrasmall NPs in single cells. We also constructed a localization quantitative analysis method, which may accurately segment the intracellularly bioavailable particles from those of the extracellular space and intracellular components and NPs. The high bioavailability of HfO₂ NPs in tumor cells from deeper penetration in tumor tissue and higher tumor intracellular uptake provide mechanistic insight into HfO₂ NPs as advanced radioenhancers in the combination of quantitative subcellular image analysis with the therapeutic effects of NPs on 3D tumor spheroids and breast cancer. Our findings unveil the substantial uptake rate and subcellular quantification of HfO₂ NPs by the human breast cancer cell line (MCF-7). This revelation explicates the notable efficacy and safety profile of HfO₂ NPs in tumor treatment. These findings demonstrate that this 3D imaging technique promoted by the deep learning algorithm has the potential to provide localization quantitative information about the 3D distributions of specific molecules at the nanoscale level. This study provides an approach for exploring the subcellular quantitative analysis of NPs in single cells, offering a valuable quantitative imaging tool for minute amounts or ultrasmall NPs.

Gold nanomaterials have garnered significant attention in biomedicine owing to their tunable size and morphology, facile surface modification capabilities, and distinctive optical properties. The surface functionalization of these nanoparticles can enhance their safety and efficacy in nanomedical applications. In this study, we examined the biological effects of gold nanoparticles (GNPs) with three distinct surface modifications (polyethylene glycol, chitosan, and polyethylenimine) in murine models, elucidating their mechanisms of action on hepatic tissue at both the transcriptomic and metabolomic levels. Our findings revealed that PEG-modified GNPs did not significantly alter any major metabolic pathway. In contrast, CS-GNPs markedly affected the metabolic pathways of retinol, arachidonic acid, linoleic acid, and glycerophospholipids (FDR < 0.05). Similarly, PEI-GNPs significantly influenced the metabolic pathways of retinol, arachidonic acid, linoleic acid, and sphingolipids (FDR < 0.05). Through a comprehensive analysis of the regulatory information within these pathways, we identified phosphatidylcholine compounds as potential biomarkers that may underlie the differential biological effects of the three functionalized GNPs. These findings provide valuable experimental data for evaluating the biological safety of functionalized GNPs.

Pre-coating with a protein corona on the surface of nanomaterials (NMs) is an important strategy for reducing non-specific serum protein absorption while maintaining targeting specificity. Here, we present lipoic acid-terminated polyethylene glycol and transferrin bi-functionalized MoS₂ nanosheets (Tf@MoS₂-PEG NSs) as a feasible approach to enhance cellular uptake. Tf@MoS₂-PEG NSs can maintain good dispersion stability in cell culture medium and effectively protect MoS₂ NSs from oxidation in ambient aqueous conditions. Competitive adsorption experiments indicate that transferrin was more prone to bind MoS₂ NSs than bovine serum albumin (BSA). It is noteworthy that single HepG2 cell uptake of Tf@MoS₂-PEG presented a heterogeneous distribution pattern, and the cellular uptake amount spanned a broader range (from 0.4 fg to 2.4 fg). Comparatively, the intracellular Mo masses in HepG2 cells treated with BSA@MoS₂-PEG and MoS₂-PEG showed narrower distribution, indicating homogeneous uptake in the single HepG2 cells. Over 5% of HepG2 cells presented uptake of the Tf@MoS₂-PEG over 1.2 fg of Mo, about three-fold that of BSA@MoS₂-PEG (0.4 fg of Mo). Overall, this work suggests that Tf coating enhances the cellular uptake of MoS₂ NSs and is a promising strategy for improving the intracellular uptake efficiency of cancer cells.