Construction Of The Tumor Microenvironment Responsive Multifunctional Nano-Delivery System For Enhanced Photodynamic Therapy

Background and objectiveAs a high-efficiency and low-toxicity local treatment strategy,photodynamic therapy（PDT）has gained great promise in cancer treatment owing to the advantages of strong controllability,good selectivity,high safety,and negligible invasiveness,which has been approved for polytype cancer treatment.However,hypoxia tumor microenvironment,GSH related antioxidant defense mechanism and the limited laser penetration greatly restrict the PDT efficiency,which remain a grand challenge for its clinical application.Photodynamic therapy is performed on the basis of local or systemic application of photosensitizers.Under specific laser irradiation,the photosensitizer will absorb the light energy and transition from the ground state to the excited state,after then the excess energy will be transferred to the surrounding oxygen to produce the cytotoxic reactive oxygen species（ROS）for tumor therapy.The generation efficiency of ROS is in direct relation to the treatment effect of PDT and the concentration of oxygen in the tumor microenvironment（TME）is a key factor in the process of ROS generation.However,the high oxygen consumption of tumor cells and abnormal tumor blood vessels often result in insufficient oxygen supply.Moreover,the oxygen consumption during the PDT process will further aggravate the tumor hypoxia.This severely reduces the efficiency of ROS generation and limits the PDT efficiency.Meanwhile,the inherent GSH-related cellular antioxidant defense mechanisms,which can effectively regulate the level of oxidative stress in tumor cells to resist the oxidative damage generated from ROS,greatly limits the clinical therapeutic effect of PDT.In addition,the limited laser penetration is also the main obstacle to the application of PDT for deep tumors.As for the overlarge or deep tumor,the laser would not able to penetrate the thick tissue for effective tumor treatment.The reflection loss of light and light-skin interaction severely weaken the laser energy for sufficient ROS generation,thus greatly lowering the PDT efficacy.Hence,how to improve the hypoxia and anti-oxidant tumor microenvironment and increase the ROS generation efficiency are the urgent issues and research hotspot for enhanced photodynamic therapy.In recent years,the development of intelligent activatable multifunctional nano-delivery system hold superb promise for PDT treatment.On the one hand,such nano-delivery system will improve the safety and controllability of photosensitizer by encapsulation or coupling method.On the other hand,the nano-delivery system can also makes up for the drawback of PDT through the way of improving the tumor microenvironment and introducing the therapeutic agent for synergistic treatment.In this way,the dissertation here constructs a series of tumor microenvironment responsive multifunctional nano-delivery system,aiming at developing a novel PDT strategy to improve the ROS generation efficiency for efficient tumor photodynamic therapy.We hope that this research can provide an experimental and theoretical evidence for applying the tumor microenvironment responsive multifunctional nano-delivery system in optimized photodynamic therapy;and,we also look foward that such strategy presented here can provide new ideas and new methods for overcoming the bottlenecks to achive highly effective photodynamic therapy.Main contents and resultsBased on the limitations of tumor hypoxia and GSH-related cellular antioxidant defense mechanisms on tumor photodynamic therapy,we designed and synthesized a new type of NO donor-nitrated mannan in the first part of this dissertation,and used it as a basic material to construct the GSH-activatable nitric oxide（NO）generating nanoplatform（Ce6-loaded NO-mannan）.According to the redox reaction between the nitrated mannan and GSH,we putted forward an“open up the source and regulate the flow”strategy with simultaneous oxygen supply and GSH depletion,and systematically explored its potential for improving the hypoxia/anti-oxidant tumor microenvironment and increasing the ROS generation efficiency for enhanced photodynamic therapy.On the ground of the results of the GSH-responsive behavior measurement,ROS generation assay and NO generation measurement,we proved the redox sensitivity of the nitrated mannan.Upon exposure to the high GSH environment,the Ce6-loaded NO-mannan continually underwent a structure disruption since the hydrophobic nitrate ester was transformed into the hydrophilic hydroxyl compound.The redox reaction triggered by the overexpressed GSH successfully consumed the intracellular GSH and caused the NO gas release.The great GSH scavenging ability of the nitrated mannan destructed the cellular antioxidant defense system thus protected the generated ROS from the clearance of GSH.Such“regulate the flow”strategy effectively increased the efficiency of ROS.Meanwhile,the in vivo laser speckle contrast images and the immunofluorescence staining of tumor hypoxia demonstrated the oxygen supply ability of the nanoplatform.The GSH triggered NO gas released at tumor site relaxed the tumor blood vessels and increased the blood perfusion,thereby significantly improved the blood oxygen supply and relieved the tumor hypoxia.Such“open up the source”strategy effectively overcame the limitations of tumor hypoxia.The in vitro cytotoxicity measurement further confirmed that the nitrated mannan significantly enhanced the PDT efficiency,which may account for the elevated ROS generation that surviving the elimination of GSH.In addition,the in vivo anti-tumor efficacy had again proved that the“open up the source and regulate the flow”strategy presented a superior anti-tumor efficacy than traditional PDT alone.To further handle out the limitation of laser penetration in PDT process,in the second part of this dissertation,we fabricated a copper peroxide-based tumor p H-responsive autocatalytic nanoreactor via an albumin-mediated biomimetic mineralization strategy from a pharmaceutics standpoint.The copper peroxide hold a CDT potential which is activated by endogenous chemical energy without the need of external laser irradiation and the supply of local oxygen.This performance of the copper peroxide ideally met the limitation of PDT which held great promise to combine the external energy-triggered PDT and the internal force-driven CDT to achieve an advanced anticancer effect.In addition,according to the capacity of low p H-triggered oxy-substrates（H₂O₂/O₂）self-supply of the copper peroxide,we putted forward a“H₂O₂ and O₂ self-supplying”strategy and systematically explored its potential for increasing ROS generation in multiple ways for enhanced ROS-based photodynamic/chemodynamic therapy.Based on the considerable hydrogen peroxide production,·OH generation,O₂ production and Cu ion release behavior in low p H condition,we confirmed the superior CDT performance and H₂O₂/O₂self-supplying ability of the autocatalytic nanoreactor.Upon exposure to the acidic tumor microenvironment,the nanoreactor achieved a p H-dependent H₂O₂ generation along with copper ions release.Then the Fenton-like reaction between the decomposition products produced the highly toxic hydroxyl radicals for chemodynamic therapy.The generated H₂O₂ met the oxy-substrate requirements of Fenton-like reaction,thus enhanced the CDT efficiency.Meanwhile,the catalytic decomposition reaction of conversion H₂O₂ into O₂ was proved to be accelerated by the Cu ions,thereby alleviating the tumor hypoxia and increasing ROS production to further improve the efficacy of PDT.The total ROS generation assay and the in vitro PDT/CDT antitumor efficiency confirmed that our designed nanoreactor combined the external energy-triggered PDT with the internal force-driven CDT achieved a multiple anticancer therapeutic effect with amplified tumor oxidative stress.The photoacoustic images and the immunofluorescence staining of tumor hypoxia demonstrated the O₂ self-supplying ability of our designed nanoreactor which significantly alleviated the tumor hypoxia.The in vivo antitumor efficacy suggested that the“H₂O₂ and O₂self-supplying”strategy presented here combining the external energy-triggered PDT and the internal force-driven CDT caused the most significant tumor apoptosis and necrosis compared with either single treatment alone,indicating the notably improvement of such ROS-mediated antitumor efficacy.ConclusionsIn this dissertation,we have systemically researched the construction of tumor microenvironment responsive multifunctional nano-delivery system and their capacity for enhanced photodynamic therapy.We found that the GSH-activatable nitric oxide generating nanoplatform could improve the hypoxia/anti-oxidant tumor microenvironment and increase the ROS generation efficiency for enhanced photodynamic therapy with simultaneous oxygen supply and GSH depletion.The copper peroxide-based tumor p H-responsive autocatalytic nanoreactor could combine the external energy-triggered PDT and the internal force-driven CDT to significantly increase the ROS generation efficiency for multiple anticancer effect.In addition,the p H-triggered oxy-substrates（H₂O₂/O₂）self-supply of the copper peroxide further amplified the anti-tumor effect of PDT and CDT with notably improvement of such ROS-mediated antitumor efficacy.Our dissertation demonstrated simple and efficient methods for overcoming the major bottlenecks in PDT treatment,which expanded the ideas and approaches for PDT research.Moreover,the strategies presented here paved a new way for the fabrication of other nanoagent for tumor treatment.