Delivery Of Mitochondriotropic Doxorubicin Derivatives For Combined Therapy In Anti-tumor Research

Objective In this paper,Triphenylphosphine(TPP)and Doxorubicin(DOX)were conjugated to obtain Triphenylphosphine-Doxorubicin(TPP-DOX),which was used to promote the targeting of Doxorubicin to mitochondria in tumor cells.Using DOX as control,the synergies of TPP-DOX combined with Chloroquine,an autophagy inhibitor,were detected in terms of overcoming tumor multi-drug resistance(MDR).And its synergistic anti-tumor effect with radiotherapy was also discussed through cellular and molecular biology research in vitro,as well as in vivo studies in a nude mouse tumor heterograft model.Methods 1)TPP-DOX was synthesized by the carbodiimide method.The structure of TPP-DOX was detected by nuclear magnetic resonance,mass spectrometry and infrared spectroscopy to verify the successful synthesis of TPP-DOX.2)The cytotoxicity,cell uptake,caspase activity and cell cycle inhibition of K562/DOX cells induced by TPP-DOX,DOX and their combinations with chloroquine(an autophagy inhibitor)were detected by CCK8 and flow cytometry.3)The drug’s ability to induce apoptosis,mitochondrial targeting,ROS production,and mitochondrial membrane potential after TPP-DOX,DOX and their combinations with radiotherapy administration were investigated in 4T1 cells in vitro.4)A nude mouse tumor heterograft model was established to investigate the synergistic effect of TPP-DOX and radiotherapy.After administration,mice were dissected to obtain organs and tumors,and H&E staining,CYT C,Ki67,CD31,TUNEL fluorescence staining were performed to investigate the efficacy and organ toxicity.Results 1)TPP-DOX was successfully synthesized and verified by nuclear magnetic resonance,mass spectrometry and infrared spectroscopy.2)TPP-DOX combined with the autophagy inhibitor chloroquine can significantly inhibit K562/DOX cell growth,effectively overcome multi-drug resistance,highly increase the expression of Caspase-3 and Caspase-9,but it does not exert its effect by interfering with the cell replication through entering the nucleus.3)In vitro experiments showed that compared with DOX,TPP-DOX enhanced tumor cytotoxicity,effectively promoted its uptake in 4T1 cells,and induced tumor cell apoptosis.Combined with radiotherapy,TPP-DOX promoted mitochondrial ROS production,reduced mitochondrial membrane potential,and could effectively enhance its anti-tumor effect.4)In vivo pharmacodynamic experiment of nude mouse tumor heterograft model confirmed that TPP-DOX combined with radiotherapy exhibited better anti-tumor activity than DOX,significantly promoted tumor tissue apoptosis,inhibited tumor angiogenesis,and showed a good in vivo safety profile.Conclusion TPP-DOX,a mitochondria-targeted drug delivery system,was successfully prepared,which has a better anti-tumor effect than DOX.It was confirmed that in combination with the autophagy inhibitor chloroquine and radiotherapy,it could promote tumor cell apoptosis,effectively enhance the anti-tumor effect and better overcome the multidrug resistance.The research is promising to provide an efficient and safe therapeutic strategy for the treatment of tumors.