Oxygen Delivery Combined With Chemotherapy Drugs To Overcome Tumor Hypoxia-induced Chemoresistance

As a global public health problem,cancer is still a disease that needs to be overcome.Hypoxic microenvironment,one of the critical features of tumors,can lead to tumor proliferation,invasion and metastasis,tolerance to chemotherapy,radiotherapy and photodynamic therapy.Chemotherapy is the most common and effective means of treating tumors.However,owing to hypoxia,tumor presents resistance to most of the current chemotherapy drugs.In addition,the limited drug accumulation in tumor sites also inhibits chemotherapy efficiency.Therefore,alleviating tumor hypoxia and increasing drug accumulation in tumor sites are important factors in enhancing chemotherapy effect.Various strategies for tumor hypoxia have been studied,such as hypoxia-activated prodrugs and hypoxic-responsive nanocarriers,nanosystems that produce oxygen or deliver oxygen.Among them,oxygen-carrying carriers can effectively alleviate tumor hypoxia,including perfluorocarbon(PFC),artificial red blood cells,modified hemoglobin,etc.Although PFC shows high oxygen solubility,it can only release and diffuse oxygen depending on oxygen concentration gradient,which is less efficient.In contrast,hemoglobin(Hb)can effectively bind oxygen under high oxygen conditions,and release oxygen rapidly under hypoxic conditions.In addition,hemoglobin liposome is a mature clinical blood substitute.Therefore,we prepared the liposome(DOX-Hb-lipo,DHL)co-loaded with the chemotherapy drug doxorubicin(DOX)and the oxygen-carrying carrier hemoglobin.The ammonium sulfate gradient method was finally determined and the best prescription was E80 phospholipid:cholesterol = 2:1(molar ratio)after comparing different prescriptions and preparation methods.The fabricated liposome DHL presented a uniform particle size which was about 130 nm,and good stability.Thanks to the self-oxygenation characteristics of hemoglobin,DHL can release oxygen to overcome hypoxia-induced chemoresistance.The ability of DHL to alleviate tumor hypoxia was detected by soluble oxygen analyzer,pimonidazole hypoxic probe and HIF-1α protein expression in vitro.The results showed that DHL can continuously release oxygen and effectively improve tumor hypoxia.Furthermore,due to the hydrophobic interaction between Hb and phospholipids,fractional Hb was bound to the surface of the liposome membrane,which was confirmed by particle size and transmission electron micrograph.Interestingly,we found that Hb on the surface of the liposomes presented a certain tumor targeting effect,which was verified by the uptake study in various tumor cells and normal cell lines and the biodistribution study.The results showed that compared to doxorubicin liposomes(DOX-lipo,DL),DHL can be more internalized by tumor cells and accumulated in tumor site.According to relevant literature and experimental research,tumor cells tend to take up iron and hemoglobin consists of iron,which may contribute to the targeting effect.Cytotoxicity and anti-tumor efficacy in vivo indicated that DHL can alleviate tumor hypoxia and overcome the chemoresistance induced by hypoxia,thereby enhancing the anti-tumor effect of doxorubicin.In anti-tumor efficacy experiments,tumor hypoxia was detected using HIF-1α,VEGF,and pimonidazole hypoxic probes.The results showed that DHL was also effective in improving tumor hypoxia in vivo.In addition,the reactive oxygen species(ROS)detection demonstrated that DHL significantly increased the production of ROS in hypoxic environments and promoted ROS-mediated DOX cytotoxicity.In summary,this study prepared a liposome carrying oxygenated hemoglobin and the chemotherapeutic drug doxorubicin.DHL presented increased drug accumulation in tumor site attributed to the tumor targeting effect of Hb on liposome surface.Besides,Hb could release oxygen to overcome tumor hypoxia-induced chemoresistance,thereby improving the anti-tumor efficacy of chemotherapy drugs.