Catch and Release Photosensitizers: Combining Dual-Action Ruthenium Complexes with Protease Inactivation for Targeting Invasive Cancers

Dual action agents containing a cysteine protease inhibitor and Ru-based photosensitizer for photodynamic therapy (PDT) were designed, synthesized, and validated in 2D culture and 3D functional imaging assays of triple-negative human breast cancer (TNBC). These combination agents deliver and release Ru-based PDT agents to tumor cells and cause cancer cell death upon irradiation with visible light, while at the same time inactivating cathespin B (CTSB), a cysteine protease strongly associated with invasive and metastatic behavior. In total five Ru-based complexes were synthesized with the formula [Ru­(bpy)₂(<b>1</b>)]­(O₂CCF₃)₂ (<b>3</b>), where bpy = 2,2′-bipyridine and <b>1</b> = a bipyridine-based epoxysuccinyl inhibitor; [Ru­(tpy)­(NN)­(<b>2</b>)]­(PF₆)₂, where tpy = terpiridine, <b>2</b> = a pyridine-based epoxysuccinyl inhibitor and NN = 2,2′-bipyridine (<b>4</b>); 6,6′-dimethyl-2,2′-bipyridine (<b>5</b>); benzo­[<i>i</i>]­dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine (<b>6</b>); and 3,6-dimethylbenzo­[<i>i</i>]­dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine (<b>7</b>). Compound <b>3</b> contains a [Ru­(bpy)₃]²⁺ fluorophore and was designed to track the subcellular localization of the conjugates, whereas compounds <b>4</b>–<b>7</b> were designed to undergo either photoactivated ligand dissociation and/or singlet oxygen generation. Photochemical studies confirmed that complexes <b>5</b> and <b>7</b> undergo photoactivated ligand dissociation, whereas <b>6</b> and <b>7</b> generate singlet oxygen. Inhibitors <b>1</b>–<b>7</b> all potently and irreversibly inhibit CTSB. Compounds <b>4</b>–<b>7</b> were evaluated against MDA-MB-231 TNBC and MCF-10A breast epithelial cells in 2D and 3D culture for effects on proteolysis and cell viability under dark and light conditions. Collectively, these data reveal that <b>4</b>–<b>7</b> potently inhibit dye-quenched (DQ) collagen degradation, whereas only compound <b>7</b> causes efficient cell death under light conditions, consistent with its ability to release a Ru­(II)-based photosensitizer and to also generate ¹O₂.