Near-infrared light-activated membrane fusion for cancer cell therapeutic applications

The spatiotemporal stimulation of liposome-liposome or liposome-membrane fusion processes attracts growing interest as a means to mimic cell-cell interactions in nature and for using these processes for biomedical applications. We report the use of o-nitrobenzyl phosphate functionalized-cholesterol tethered nucleic acid-modified liposomes as functional photoresponsive units for inducing, by NIR-irradiation, spatiotemporal liposome-liposome or liposome-membrane fusion processes. The liposomes are loaded with upconversion nanoparticles (UCNPs) and their NIR irradiation (λ = 980 nm) yields luminescence at λ = 365 nm, providing a localized light-source to deprotect the o-nitrobenzyl phosphate groups and resulting in the fragmentation of the nucleic acid structures. In one system, the NIR-triggered fusion of two liposomes, L₁ and L₂, is exemplified. Liposome L₁ is loaded with UCNPs and Tb³⁺ ions, and the liposome boundary is functionalized with a cholesterol-tethered, o-nitrobenzyl phosphate caged hairpin nucleic acid structure. Liposome L₂ is loaded with 2,6-pyridinedicarboxylic acid, DPA, and its boundary is modified with a cholesterol-tethered nucleic acid, complementary to a part of the caged hairpin, associated with L₁. NIR-irradiation of the L₁/L₂ mixture resulted in the photocleavage of the hairpin structure, associated with L₁, and the resulting fragmented nucleic acid associated with L₁ hybridized with the nucleic acid linked to L₂, leading to the fusion of the two liposomes. The fusion process was followed by dynamic light scattering, and by monitoring the fluorescence of the Tb³⁺-DPA complex generated upon the fusion of the liposomes and their exchange of contents (fusion efficiency 30%). In a second system, the fusion of the liposomes L₁, loaded with UCNPs and doxorubicin (DOX), with HeLa cancer cells functionalized with nucleic acid tethers, complementary to the hairpin units associated with the boundary of L₁, and linked to the MUC-1 receptor sites associated with the HeLa cells, through a MUC-1 aptamer unit is exemplified. The effect of DOX-loaded L₁/HeLa cell fusion on the cytotoxicity towards HeLa cells is addressed. The NIR UCNP-stimulated cleavage of the o-nitrobenzyl phosphate caged hairpin units associated with L₁ leads to the fragmentation of the hairpin units and the resulting nucleic acid tethers hybridize with the nucleic acid-modified HeLa cells, resulting in the liposome-HeLa cell fusion and the release of DOX into the HeLa cells. Selective spatiotemporal cytotoxicity towards HeLa cells is demonstrated (ca. 40% cell killing within two days). The study presents a comprehensive stepwise set of experiments directed towards the development of NIR-driven liposome-liposome or liposome-membrane fusion processes.