Hyperbaric Oxygen Potentiates Chemotherapy And Photodynamic Therapy Via Remodeling Tumor Microenvironment

Hypoxia is one of the main characteristics of tumor,which is caused by rapid growth of tumor.Hypoxia leads to drug resistance by regulation of a variety of genes and impairs photodynamic therapy?PDT?directly because of the shortage of oxygen.Thus,hypoxia poses a severer constraint to the treatment of tumor.Mechanistic investigation of the factors that hypoxia imposes to tumor therapy will be beneficial for finding effective solutions to attenuate tumor hypoxia and improve therapeutic efficacy.Hyperbaric oxygen?HBO?as an adjunct therapy in clinic,has been employed to cure various diseases,such as carbon monoxide poisoning,ischaemic,and so on,the combination of HBO and radiotherapy in clinical tumor therapy has been studied.However,combination of HBO and chemotherapy was trapped for the possible side effects,and limited penetration depth impacted combination of HBO and PDT.Characteristic effect and physicochemical property of nanomedicine offered new opportunity for application of HBO in tumor therapy.Here,we detailed studied the mechanism of hypoxia-induced resistance of5-fluorouracil?5-FU?,and HBO's effect on hypoxia-induced drug resistance.On that basis,we synthesized a new upconversion nanophotosensitizer that was excited by near-infrared light?NIR?,and the synergy of PDT with HBO was studied.Main results are as follows:?1?Hypoxia?1%O₂?induced universal but differential resistance to 6 anticancer drugs?sorafenib,5-FU,cisplatin,gemcitabine,6-thioguanine and adriamycin?in 3 HCC cell lines?BEL-7402,HepG2 and SMMC-7721?.The resistance factor of BEL-7402 cell to 5-FU reached 134.29.It was shown that hypoxia influenced 5-FU action mainly from reducing cell proliferation inhibition and cell apoptosis.On one hand,hypoxia reduced inhibition rate of 5-FU on thymidylate synthase?TS?,suppressed DNA replication and5-FU-induced S-phase arrest.BEL-7402 cells decreased TS expression by 15.31-fold and slowed down DNA replication to adapt to hypoxia.Hypoxia notably decreased inhibition rate of 5-FU on TS,and inhibited 5-FU-induced ATM/ChK1 activation,leading to reduced S-phase arrest and cell proliferation inhibition.On the other hand,hypoxia decreased DNA damage,and reduced apoptosis by regulating B-cell lymphoma-2?BCL-2?family proteins expression.According to comet assay results,hypoxia restrained both DNA single strands and DNA double strands induced by 5-FU.Hypoxia promoted expression of anti-apoptotic protein MCL-1 and inhibited expression of pro-apoptotic protein BAX and BIM,resulting in decreased apoptosis.Finally,with HBO administration during treatment of hypoxic BEL-7402 cell with 5-FU,the sensitization index was measured to be 7.88.All these results indicate the multiple effects of hypoxia on 5-FU,and HBO indeed improved hypoxia-induced resistance of 5-FU effectively.?2?Rare earth upconversion nanoparticles?UCNPs?has been used in bioimaging and therapy for their excellent physicochemical and fluorescent properties.Er³⁺doped UCNPs was synthesized by thermal decomposition method.The size of the nanoparticles was 10nm,and the nanoparticles had a strong green emission under 808 nm NIR light irradiation.After further modification of mesoporous SiO₂ and PEG,rose bengal?RB?was loaded via diffusion to afford water soluble nanophotosensitizers?UNPSs?,and the loading efficiency of photosensitizer was 4.28%.¹O₂ generation efficiency was determined to be 0.65 and0.98 under normoxia and HBO conditions,respectively.Cell experiments showed that HBO promoted reactive oxygen species?ROS?generation by UNPSs,and enhanced its cytotoxicity to tumor cells.The UNPSs were found to accumulated most at 48 h after i.v.injection into xenografts tumor bearing mice.It was found that UNPSs inhibited tumor growth significantly upon both HBO and NIR irradiation.Immunofluorescence staining and Western Blotting were both conducted to decipher the underlying mechanism.HBO was shown to relieve hypoxia and reduced HIF-1?level,as well as promoting penetration of UNPSs.It was also found that HBO+UNPSs+NIR group had the minimal hypoxia and deepest penetration among the treatment groups.Immunofluorescence images of collagen I and ex vivo study of breakage of collagen I indicated that the synergetic effect was due to degradation of extracellular matrix by amplified ROS in tumor microenvironment.The degradation of extracellular matrix promoted penetration of UNPSs and diffusion of oxygen in tumor.Such a synergic effect finally achieved a significantly enhanced PDT efficacy.Safety evaluation showed that no obvious toxicity of HBO and UNPSs on main organs was detected.Thus,HBO has the potential to improve PDT therapeutic outcome.With the study on mechanism of hypoxia-induced 5-FU resistance and the combination of HBO and 5-FU,improvement of HBO on hypoxia-induced chemotherapy resistance was confirmed.Simultaneously,the study of synergetic effect between HBO and nanophotosensitizers-mediated PDT verified that HBO could enhance PDT effect via remodeling tumor microenvironment.Studies here provide basis for drug discovery and strategies for improving the therapeutic effect of chemotherapy and PDT in clinic.