An Investigation Into The In Vitro Anticancer Performance Of Ferrocene-Laden Ferroptotic Nanocarrier

Cancer severely threatens human health globally.Ferroptosis,an emerging cell death,has drawn great interest as a substitute for current apoptosis-based therapies and immunotherapy.Ferroptosis is characterized by the iron-dependent accumulation of lipid hydroperoxides（LPO）,which is different from traditional apoptosis,necrosis,and autophagy in morphological,biochemical,and genetic aspects.It has been reported that ferroptosis plays a significant role in cancer treatment.But the levels of intracellular iron ions in cancer are too low to meet the requirement of ferroptosis.Therefore,ferrocene（Fc）,a typical organic compound containing ferrous ions,was used to increase intracellular iron levels and synchronously avoid the disadvantages of low Fenton efficiency caused by the susceptibility of ferrous ions to oxidation.In addition,the intracellular glutathione peroxidase 4（GPX4）is primarily responsible for removing the lipid peroxides,thereby reducing the efficacy of ferroptosis.On the contrary,once the GPX4 activity is lost,it can promote the occurrence of ferroptosis.Besides,the degree of lipid peroxidation,which indicates the extent to ferroptosis,is determined by the abundance and localization of polyunsaturated fatty acids,among which phosphatidylethanolamines（PE）of arachidonic acid（AA）and adrenal acid（Ad A）are the preferred substrates for ferroptosis.It was postulated that the concurrent delivery of Fc,Ras-selective lethal 3（RSL3,a typical inhibitor of GPX4）,and AA could generate more LPO and thus induce ferroptosis.Based on this hypothesis,we contrasted a biocompatible nanosystem,named RSL3/Fc-SA@NCs,with multichannel amplification of ferroptosis for cancer ablation.Among this nanocarrier,Fc-SA could not only provide iron ions,but also reacted with H₂O₂ to generate·OH for lipid peroxidation of unsaturated fatty acids.RSL3 was imported to inhibit GPX4 activity,thereby reducing the consumption of lipid peroxides in tumor site and improving the efficiency of ferroptosis.At the same time,arachidonic acid was used to enhance the degree of lipid peroxidation in tumor cells.The loading of RSL3 and Fc-SA in the nanocarrier was determined at 1.0±0.2%and 10.0±0.5%,respectively.Dynamic light scattering（DLS）indicated a suitable size distribution（about 170 nm）for the EPR effect.The Fenton reaction of Fc-SA@NCs was evaluated using terephthalic acid（TA,an indicator of hydroxyl radical）and phenanthroline（an indicator of ferrous ion）,which showed a valid Fenton ability of Fc-SA@NCs.Thereafter,RSL3/Fc-SA@NCs showed significant inhibition of GPX4activity in vitro,which resulted from the presence of RSL3.The in vitro anticancer study revealed that high cytotoxicity with half-maximal inhibitory concentration(IC₅₀)of 1.5±0.2μM was achieved through the inhabitation of intracellular GPX4 activity and generation of more LPO.This work designed an all-in-one ferroptotic nanocarrier with multichannel amplification of ferroptosis for cancer ablation which was validated in vitro at cellular level.