Light- and X-ray-Activated Liposomes for Controlled Gene Editing and Drug Delivery
Abstract
Photodynamic therapy (PDT) exploits photosensitizer activation to generate reactive oxygen species (ROS), principally singlet oxygen. Beyond direct cytotoxicity, this photochemistry can be repurposed for on-demand cargo release from lipid nanocarrier systems, enabling spatiotemporal control over therapeutic delivery that is not achievable with conventional lipid nanoparticles. This protocol presents methods for fabricating and characterising two distinct verteporfin (VP)-integrated lipid nanoparticle formulations: (1) light-triggered liposomes composed of DOTAP, DOPE, cholesterol, and VP for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes; and (2) X-ray-triggered liposomes composed of DOTAP, DOPC, VP, and gold nanoparticles for controlled chemotherapy drug release. Upon activation at 690 nm (visible light) or by clinical X-ray radiation (6 MeV), VP generates singlet oxygen that oxidises unsaturated lipid components, destabilising the nanoparticle membrane and releasing encapsulated cargos. Protocols are provided for liposome formulation by thin-film hydration and membrane extrusion, physicochemical characterisation, light- and X-ray-triggered cargo release assessment, in vitro gene knockout in human cells, and in vivo validation using a quantitative zebrafish visual reporter system and a mouse xenograft tumour model. Representative results demonstrate knockout of up to approximately 326 slow-muscle fibres per zebrafish embryo via light activation and significant…
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