Fig. 2: Establishment of an alive and multi-functional technical platform for determining cell cycle- and autophagy-associated nanoparticle accumulation.

a Schematic diagram shows the construction of cell lines stably expressing the PIP-FUCCI (cell cycle indicator) plasmid. Envelope plasmid PMD2.G, packing plasmid psPAX2 and transfer plasmid PIP-FUCCI were simultaneously added into 293 T cells. Afterwards, the culture medium was collected and concentrated, followed by application of lentiviral supernatant containing PIP-FUCCI to isogenic wild type (WT) and ATG7 knockout (ATG7 KO) U2OS cell lines. Subsequently, WT and ATG7 KO PIP-FUCCI cell lines stably expressing PIP-FUCCI were obtained after selection with G418 and flow cytometry (FACS). b Schematic diagram indicates the establishment of a multi-functional platform for determining cell cycle phase- and autophagy-associated nanoparticle accumulation. WT and ATG7 KO PIP-FUCCI cells were treated with DiD-labeled liposomes (DiD-LIP), at a dose equivalent to 5 μg/mL doxorubicin (DOX), or lipid nanoparticles (DiD-LNP), at a dose equivalent to 6 μg/mL nucleoside-modified mRNA (mRNA), for 2 h. The same volume of PBS was used as a control. During the final 30 min of this incubation period, FITC-EpCAM (green fluorescence) was added to label the cell membrane. Complete distinguishment and collection of cells at four distinct cell cycle phases was achieved through combining PIP-FUCCI expression to indicate G1-phase (mVenus positive, green nucleus), S-phase (mCherry positive, red nucleus) and G2-phase (mVenus and mCherry double-positive, yellow nucleus) cells attached to the culture dish, with mitotic shake-off to collect detached or floating M-phase cells (mVenus and mCherry double-positive, yellow nucleus). Further integration of confocal laser scanning microscopy (CLSM) and three-dimensional reconstruction techniques enabled detection and calculation of nanoparticle accumulation at both two-dimensional and three-dimensional levels. c Representative light microscopic and CLSM images selected from 15 randomly chosen cells, demonstrating the successful construction and application of a technical platform using DiD-LIP (blue fluorescence) and WT PIP-FUCCI cells as a model system. Scale bars, 20 μm.