Synthesis And Antitumor Effects Of Mitochondrial Targeting Drug Rhein-Ciprofloxacin

Cancer is a disease with high fatality rate,and chemotherapy is primarily utilized to treat it.Tumor recurrence caused by chemotherapeutic drug induced nuclear DNA lesions is a major issue in cancer therapy.Complex tumor microenvironment crosstalk can also intervene in the therapeutic effects of chemotherapeutic drug,which can reduce immune response and increase drug resistance.One of the most efficient ways to treat cancers is with antineoplastic agents that target mitochondria.A component of traditional Chinese medicine called Rhein has antitumor effects,the potential to target mitochondria in cells,and is higher biosafety than other drugs that target mitochondria.Ciprofloxacin requires relatively high doses to destroy tumor cells and regulate immunological reactions because of its non-specific distribution and lack of tumor-specific therapy.In order to increase Ciprofloxacin concentration in cancer cell mitochondria,decrease drug concentration,and improve antitumor effects,Ciprofloxacin is conjugated to Rhein.It is essential to develop an antitumor combination therapy utilizing nanoparticles as a platform that induces the ICD effects,reprogrammes the tumor microenvironment,and has good biological safety.In this context,this paper involves the following three aspects:(1)Synthesis of mitochondrial targeting drugs Rhein-Ciprofloxacin.N-Boc-1,6-hexanediamine,N-Boc-1,4-butanediamine,and N-Boc-1,2-ethylenediamine are three distinct linker adopts to synthesize the Rhein-2n C-Ciprofloxacin(n=1,2,3).Infrared spectroscopy,mass spectrometry,and hydrogen nuclear magnetic resonance spectroscopy were used to determine the structures of the three compounds and intermediates.(2)Investigation of the synergistic lethal effect,ICD effects,and immunological effects of the mitochondria targeting drugs Rhein-4C-Ciprofloxacin/Chloroquine in vitro.In the MTT assay,the IC50 values for Rhein-Ciprofloxacin against three cancer cell lines(4T1,MCF-7,A549)were lower than that of the monotherapy group and the mixed group.Rhein-4C-Ciprofloxacin is more cytotoxic than Rhein-2C-Ciprofloxacin(7.0μM)and Rhein-6C-Ciprofloxacin(8.3μM),with an IC50 of 5.9 M in the 4T1 cell line.Results from the CLSM demonstrated that Rhein-4C-Ciprofloxacin was highly correlated with mitochondria,indicating that it had the capacity to target mitochondria.In the 4T1 cell line,the Pearson coefficient for Rhein-4C-Ciprofloxacin was 0.8532.The MTT assay was carried out using Rhein-4C-Ciprofloxacin in various ratios with Chloroquine,Liensinine,RSL3,and Ferrocene.When the molar ratio of chloroquine to Rhein-4C-Ciprofloxacin was 1,the drug’s high synergistic lethal effect on the 4T1 cell line was evident.The results of CLSM,FCM,and WB showed that Rhein-4C-Ciprofloxacin produced a significant amount of ROS in the mitochondria,which damaged the mitochondrial membrane potential and activated apoptosis.Chloroquine blocked 4T1 cell autophagy,increased the expression of LC3 protein,and enhanced the apoptosis effect.The synergistic group had 69.5%of late apoptotic cells.In the synergistic group compared to the other groups,ICD effects of releasing DMAPs were found to include CRT exposure,intracellular HMGB1,and extracellular ATP.More than 40%of CRT-expressing cells were detected after 36 hours of incubation with 4μM Rhein-4C-Ciprofloxacin/Chloroquine.By detecting macrophage surface proteins,intracellular ROS and i NOS,NO and cytokines secreted by extracellular secretion,Rhein-4C-Ciprofloxacin/Chloroquine can re-educate M2 anti-inflammatory cells into M1 inflammatory macrophages,increase intracellular ROS and i NOS,promotes the extracellular release of TNF-αand IL-6 cytokine and NO.(3)Lecithin was loaded with Rhein-4C-Ciprofloxacin to prepare nanoparticles combined with Anti-PD-L1 and SDT for in vivo treatment.It was detected by ultraviolet spectrophotometer,Rhein-4C-Ciprofloxacin formed nanoparticles loaded with lecithin with a drug load of approximately 8.2%and an encapsulation rate of approximately 75%,showed good biosafety in hemolysis experiments.Rhein-4C-Ciprofloxacin@Lecithin and Rhein-4C-Ciprofloxacin@Lecithin@4T1 cytomembrane nanoparticles with low PDI and stable ZETA were successfully prepared and were characterized by TEM and DLS.According to in vivo bioluminescence imaging,Ce6@Lecithin enhanced the drug concentration at the tumor location by EPR effects.Establishing a subcutaneous 4T1 tumor in mice,under the treatment of Rhein-4C-Ciprofloxacin@lecithin/Anti-PD-L1+US,the relative tumor inhibition rate reached more than 80%.On the seventh day,the high expression of CD8~+T in the tumor was detected by FCM,and on day 23 of treatment,the fluorescent staining of the tumor sections showed that the expression of CD8 was obvious,and the proliferation of tumor cells was damage.The nanoparticles showed biosafety,and no cell damage or inflammation was shown in the tissue sections of H&E staining.Furthermore,in the combination group,tumor cell metastasis is prevented prevented metastasis of tumor cells in lung metastasis model.SDT raised the susceptibility of tumors to chemotherapeutic medicines,Rhein-4C-Ciprofloxacin@Lecithin could play an ideal tumor-cytotoxicity effect under the cooperation of Anti-PD-L1 and SDT,activates the immune effects,providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.