Fig. 3. (A) Schematic of MUC aptamer-guided fusion of the DOX/UCNP loaded liposomes L₁ with HeLa cells functionalized with strand (3) consisting of the MUC-1 aptamer coupled to the domain “X” composed of the sequence (2). The strand (3) binds to the MUC-1 ligand associated with the HeLa cells and the hybridization of the free tether “X” with the photo-fragmented duplex (1′/1′′) leads to the fusion of liposomes L₁ with the respective cells and to the release of the UCNPs and DOX into the cells. (B) Confocal microscopy images corresponding to: panel I – blue channel of NIR-irradiated mixture of the UCNP/DOX-loaded liposomes L₁ and the (3)-functionalized HeLa cells. Panel II – red channel fluorescence of DOX released into the cells. Panel III – merged image demonstrating the overlap of fluorescence of UCNPs/DOX released into the cells. Panel IV and panel V – blue and red channel, respectively, of the NIR-irradiated mixture of UCNPs/DOX-loaded liposomes, L₁, and the hESC cells pretreated with (3). Panel VI – merged image of the blue/red channels and the bright field fluorescence image, demonstrating that no release of UCNPs or DOX into the hESC cells occurs (scale bar, 20 μm). (C) Confocal microscopy images of a mixture of HeLa cells and hESC normal cells treated with the UCNP/DOX-loaded liposomes, L₁ and subjected to NIR-irradiation. The shapes of the cells and the fluorescence features of the two types of cells are used to differentiate the fusion behavior of the different-shaped cells. Panel I – blue channel demonstrating the fusion of the UCNPs only into HeLa cells (cell marked with dotted rims correspond to hESC cells). Panel II – red channel-DOX fluorescence restricted to the HeLa cells (no fluorescence in the elongated hESC cells, marked with dashed boundaries). Panel III – merged image demonstrating the selective fusion of UCNPs and DOX with the HeLa cells (scale bar, 100 μm). (D) Viability of the HeLa/hESC cells subjected to the UCNP/DOX-loaded liposomes L₁: entry I – hESC cells treated with the NIR-irradiated UCNPs/DOX-loaded liposomes. Entry II – the HeLa cells subjected to UCNP-loaded liposomes L₁, lacking the DOX load. The results indicate that despite the fusion of the liposomes with HeLa cells proceeding, the viability of the cells is preserved, demonstrating that the UCNPs are non-toxic towards the HeLa cells. Entry III – the HeLa cells treated with the UCNP/DOX-loaded liposomes L₁ and subjected to NIR-irradiation. The results demonstrate that the fusion of the liposomes and the HeLa cells, and the release of DOX lead to selective cytotoxicity towards the HeLa cells.