Significant increase of anticancer efficacy in vitro and in vivo of liposome entrapped ruthenium(II) polypyridyl complexes

Two polypyridyl ruthenium(II) complexes [Ru(DIP) 2 (BIP)](PF 6 ) 2 (DIP = 4,7-diphenyl-1,10-phenanthrolie, BIP = 2-(1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f][1,10]phenanthroline, Ru1) and [Ru(DIP) 2 (CBIP)](PF 6 ) 2 (CBIP = 2-(4′-chloro-1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f][1,10]phenanthroline, Ru2) were synthesized. The cytotoxic activities in vitro of Ru1, Ru2 toward B16, A549, HepG2, SGC-7901, HeLa, BEL-7402, non-cancer LO2 were investigated using MTT method (3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide). Unexpectedly, Ru1, Ru2 can't prevent these cancer cells proliferation . To improve the anti-cancer effect, we used liposomes to entrap the complexes Ru1, Ru2 to form Ru1lipo, Ru2lipo. As expectation, Ru1lipo and Ru2lipo exhibit high anti-cancer efficacy, especially, Ru1lipo (IC 50 3.4 ± 0.1 μM), Ru2lipo (IC 50 3.5 ± 0.1 μM) display strong ability to block the cell proliferation in SGC-7901. The cell colony, wound healing, and cell cycle distribution show that the complexes can validly inhibit the cell growth at G2/M phase. Apoptotic studied with Annex V/PI doubling method showed that Ru1lipo and Ru2lipo can effectively induce apoptosis . Reactive oxygen species (ROS), malondialdehyde , glutathione and GPX4 demonstrate that Ru1lipo and Ru2lipo improve ROS and malondialdehyde levels, inhibit generation of glutathione, and finally result in a ferroptosis . Ru1lipo and Ru2lipo interact on the lysosomes and mitochondria and damage mitochondrial dysfunction. Additionally, Ru1lipo and Ru2lipo increase intracellular Ca 2+ concentration and induce autophagy. The RNA-sequence and molecular docking were performed, the expression of Bcl-2 family was investigated by Western blot analysis . Antitumor in vivo experiments confirm that 1.23 mg/kg, 2.46 mg/kg of Ru1lipo possesses a high inhibitory rate of 53.53% and 72.90% to prevent tumor growth, hematoxylin-eosin (H&E) results show that Ru1lipo doesn't cause chronic organ damage and strongly promotes the necrosis of solid tumor . Taken together, we conclude that Ru1lipo and Ru2lipo cause cell death through the following pathways: autophagy, ferroptosis, ROS-regulated mitochondrial dysfunction, and blocking the PI3K/AKT/mTOR.