Nanoformulation of therapeutic and diagnostic agents is beneficial as they accumulate at tumor sites due to enhanced permeability and retention effects. However, many in vitro studies performed on 2D cultures lack the realistic dimension of in vivo systems. In this work, a photosensitizer, acridine orange (AO), used in photodynamic therapy of cancer, is nanoformulated by entrapment in thermophilic ferritin. The efficacy is tested on 2D and 3D in vitro models and complemented with studies on the cellular uptake routes. Ferritin from the archaeon Archaeoglobus fulgidus (AfFtn) exhibits the unique property of divalent metal ion or ionic strength mediated assembly, making it an interesting nanocarrier for the entrapment of small molecules. The photosensitization and toxicity studies on 2D monolayers and 3D spheroid models of colorectal cancer cells show that AO loaded in AfFtn is functional. The killing efficiency in 2D monolayers reaches 50% while the growth of 3D spheroids is arrested after the first day with a subsequent 10% reduction of tumor diameter over the next 2 days. Results show that AO loaded AfFtn has better penetration ability than free AO alone in 3D spheroid models indicating that nanocage formulation provides for an efficient delivery vehicle into the tumor tissue.