Study On Overcoming Oxaliplatin Resistance By Multifunctional Nanocarrier Targeting The DNA Hypermethylation Of SLC22A2 And Hypoxia In RCC

Abnormal DNA hypermethylation leads to high incidence of drug resistance and limits the effectiveness of renal cell carcinoma（RCC）therapy.One of the epigenetic mechanisms of chemoresistance in RCC has been reported by our previous research showed that epigenetic inactivate of SLC22A2 promotor contributed to some anti-cancer drugs resistance.The reversibility of epigenetic phenomena makes it an important target for RCC therapy.DNA methylation inhibitor such as5-aza-2’-deoxycytidine also known as decitabine（DAC）could reduce hypermethylation of SLC22A2 promoter,improve expression of organic cationic transporters 2（OCT2）and overcome RCC drug resistant of oxaliplatin（OHP）.However,DAC is a cytidine analogue which dependent equilibrative nucleoside transporters 1（ENT1）which is oxygen dependent gene,inhibited expression under hypoxia.This impede epigenetics treatment under hypoxia.Additionally,hypoxia induced the increase of expression of multidrug-resistant gene 1（MDR1）which encodes drug efflux P-glycoprotein（P-gp）leads to the failure of chemotherapy.Thus,the alleviation of tumor hypoxia becomes an important target of overcoming RCC drug resistance.Hemoglobin（HB）is an oxygen-transport metalloprotein which contains in red blood cells of all vertebrates and has high bio-compatibility and biosafety.HB based nano-platforms for alleviation of tumor hypoxia had been widely applied in enhanced photodynamic therapy and chemotherapy.HB is also a Fe²⁺rich containing metalloprotein,which can be used as an inducer of ferroptosis through intracellular fenton reaction.Ferroptosis which is caused by accumulations of oxidative stress and subsequent lipid peroxidation has become a novel therapeutic way for therapy-resistant cancer.Nevertheless,abnormal high expression of glutathione（GSH）in RCC increases detoxification of reactive molecules,limits the curative effect of ferroptosis.Hence development of a GSH consuming HB-based oxygen and epigenetic drug nanocarrier could enhance chemotherapy and ferroptosis with effect of cooperative treatment resulting in innovative therapeutic strategy in multidrug-resistant RCC therapy.Eventually,we developed a simple and efficient multi-functional oxygen nanocarrier based on hemoglobin,namely H-Nps which contains a Hb based core and coated with low molecular weight chitosan（CHS）.After characterizations,the effects of H-Nps on relieving hypoxia microenvironment and recovering OCT2 expression in combination with DAC,and its renal targeting ability were investigated.Results shown that H-Nps had ideal size dimension,stability and renal targeting ability which benfits from CHS.The loading oxygen of H-Nps gave it the ability to relieve hypoxia intracellular.The oxygen-dependent protein ENT1 had been improved expression in both mRNA and protein,which resulted in more uptake of DAC.H-Nps/DAC can reduce methylation level in promoter of SLC22A2 and improve OCT2 expressiom,increase the anti-RCC effect of OHP.Therefore,H-Nps combined with DAC could reverse RCC drug resistance to OHP.In order to further develop the ability of hemoglobin to induce ferroptosis and overcoming the problem of low expression of hypoxia mediated DAC transporter ENT1.H-Nps was further improved and prepared a multifunctional oxygen and epigenetic drug nanocarrier based on HB,namely CNDHNNPs with fluorescence imaging of renal neoplasm.Multiple emulsion method（W/O/W）was employed in construction of CNDHNNPs.Epigenetic drug DAC is modified on CHS by Schiff base reaction,targeted release by acidic microenvironment of tumor.CNDHNNPs utilize oxygen carrying capacity of HB to alleviate hypoxia.We modified GSH consumptive fluorescence probe 7-nitro-1,2,3-benzoxadiazole（NBD）on CHS together with encapsulated Nile red as a fluorescence co-localize reference to achieve fluorescence imaging of renal neoplasm.CNDHNNPs induce ferroptosis through iron center of porphyrin catalyzed increasing lipid peroxidation.The results shown that CHDHNNPs effectively recoverd expression of OCT2 by epigenetic demethylation of SLC22A2,enhanced chemotherapy via increasing OHP accumulation in RCC cells and alleviated hypoxia with downregulating expression of drug efflux transporter P-gp overcoming hypoxia induced RCC drug resistance.HB induced ferroptosis by consuming GSH,meanwhile,achieved fluorescence imaging.This study developed a therapeutic strategy to overcome RCC resistance to OHP and achieve fluorescence imaging for tumor detection by multifunctional nanocarrier targeting DNA hypermethylation of SLC22A2 and hypoxia,This new strategy could have a potencial of high-efficiency chemotherapy for multidrug-resistant tumors.