Fig. 2. (A) Schematic of photochemically triggered fusion of the (1)-hairpin-functional liposomes L₁, loaded with doxorubicin (DOX) and UCNPs, with HeLa cells modified with the cholesterol-functionalized nucleic acid (2). The NIR-irradiation of the L₁/HeLa cell mixture leads to the UCNP-stimulated cleavage of the hairpins associated with L₁. The resulting (1′/1′′) duplexes associated with are displaced by the tethers (2) linked to the HeLa cells and the (1′/2) contacted liposome/HeLa cell assembly results in the fusion and the release of DOX (and UCNPs) into the cytoplasm. (B) Confocal microscopy images corresponding to NIR-stimulated fusion of the (1)-modified liposome/HeLa cell mixture: panel I – blue channel fluorescence of the exchanged and released UCNPs into HeLa cells. Panel II – red channel fluorescence of the DOX released into the cells. Panel III – merged image demonstrating the overlap of fluorescence of the UCNPs and DOX in the fused cells (scale bar, 20 μm). (C) Confocal microscopy images corresponding to the non-irradiated (1)-modified liposome, L₁/HeLa cell mixture: panel I and panel II show no fluorescence in the respective blue/red channels. Panel III – merged image demonstrating that no UCNPs and DOX were introduced into the HeLa cells (scale bar, 20 μm). From a set of N = 5 bright field images of the cells and the corresponding fluorescence responses of the overlapped confocal microscopy images, we estimate a fusion efficiency of DOX/UCNPs with the HeLa cells (or the normal hESC cells) of 85–90%. (D) Cytotoxicity of the UCNP and DOX loaded microcapsules L₁ towards HeLa cells and hESC normal cells: entry I – non-treated cells; entry II – cells treated with liposomes loaded with UCNPs and DOX without irradiation. Entry III – cells subjected to NIR-irradiated liposomes loaded with UCNPs and DOX. Viability of the cells monitored after a time-interval of two days. Results show ca. 50% residual cell viabilities implying non-selective fusion of the DOX/UCNP-loaded liposomes L₁ with the two types of cells: HeLa and hESC. Error bars derived from N = 3 experiments. It should be noted that the cytotoxicity experiments of photochemically fused DOX/UCNP-loaded liposomes into the HeLa cells (or hESC cells) were performed under conditions where cell cultures containing 7.3 × 10⁶ cells were subjected to 1.2 × 10^–9 moles of DOX incorporated into the liposomes. Considering the fusion efficiency of the liposomes with the cells, the average loading of DOX in the cells corresponded to 1.5 × 10^–16 moles per cell. The resulting 50% viability of the cells, after two days of interaction, could be attributed to insufficient loading of the cells with DOX. Thus, the cytotoxicity of the fused DOX/UCNPs could be enhanced by increasing the concentration of the liposomes, increasing the loading of DOX in the liposomes, and prolonging the time of interaction of the fused liposome/cell assemblies.