The Preparation And Application Of Novel Co-delivery Nano-systems For Tumor Oxidation Therapy

Redox homeostasis regulation is essential for maintaining normal function of cells.Tumor cells always exhibit consistently high reactive oxygen species(ROS)level due to changes in genetic,metabolic and microenvironment.Moderate increase of ROS promote cell proliferation and differentiation,while excessive ROS lead to oxidative damage of lipids,proteins and DNA.Therefore,in order to maintain rapid proliferation without toxicity,tumor cells up-regulate antioxidant system to adapt to the high level of ROS.The feedback mechanism helps maintaining the"vulnerable redox homeostasis"characterized by high level of ROS and high level of antioxidant system.Maintaining"vulnerable redox homeostasis"is a unique biochemical change that tumor cells make to proliferate.Targeting such unique biochemical change is an effective and targeting tumor therapy strategy.Therefore,the concept of oxidation therapy came into being.Increasing the production of ROS or reducing the clearance of ROS by inhibiting antioxidant system in tumor cells will help break"vulnerable redox homeostasis",resulting in the increase of ROS level.When ROS exceeds the tolerance threshold of tumor cells,tumor cells will die.Ferroptosis is one of the most concerned oxidation therapy.It's a newly form of regulated cell-death pathway relying on lipid peroxide(LPO),which originates from hydroxyl radicals generated by Fenton reaction and accumulates from inhibition of the cellular antioxidant system.Low Fenton reaction efficiency and upregulated antioxidant system repairing LPO are the main factors limiting ferroptosis.Therefore,increasing the efficiency of Fenton reaction and inhibiting antioxidant system are key scientific issues to increase efficiency of ferroptosis.Radiotherapy is another widely used oxidation therapy.Radiotherapy relies on the photoelectron effects between radiation and substances in tumor sites,which generate large amounts of ROS and lead to DNA damage.Hypoxic tumor microenvironment and high expression of histone deacetylase limit effect of radiotherapy by inhibiting DNA oxidative damage and increasing DNA damage repair.Therefore,improving hypoxic microenvironment and inhibiting histone deacetylase activity are key scientific issues in radiotherapy sensitization.In this study,co-delivery nano-systems were constructed based on two key scientific issues above,including improving Fenton reaction efficiency and inhibiting the antioxidant system to increase efficiency of ferroptosis,improving tumor oxygenation and inhibiting activity of histone deacetylase to enhance the therapeutic efficacy of radiation.Main research contents are divided into two parts as follow.(1)Preparation of Fe and NQA(6-[2-(3-methyl)naphthoquinolyl]hexanoic acid)co-delivery nano-system and its anti-tumor application.Many nanomaterials,for instance,inorganic iron-based nanoparticles have been developed to promote ferroptosis of cancer cells.Nevertheless,it is far from satisfactory to induce ferroptosis by iron-based nanoparticles.On the one hand,high dosage of iron is required to cause effective Fenton reaction in tumor sites.On the other hand,although many types of tumors produce more endogenous H₂O₂ than normal tissues,H₂O₂concentrations are still insufficient to trigger an efficient Fenton reaction.In addition,high expression of glutathione peroxidase(GPX)repair the lipid peroxides caused by Fenton reaction,resulting in poor ferroptosis efficiency.Therefore,increasing Fenton reaction efficiency and inhibiting the activity of antioxidant enzymes simultaneously are conducive to inducing efficient ferroptosis.Based on coordination affinity between Fe³⁺and carboxyl-containing drug NQA,we constructed a Fe and NQA co-delivery nano-system(Fe-NQA NPs)through a facile"one-step coordination self-assembly"method.Firstly,various in vitro characterization methods were used to investigate the morphological structure,particle size,surface potential,and coordination self-assembly mechanism of Fe-NQA NPs.Results showed that stable Fe-NQA NPs nanoparticles are formed by the coordination of Fe³⁺with the carboxyl group of NQA.Besides,stability of nano-system and the pH-responsive drug release ability were investigated.It was demonstrated that Fe-NQA NPs were highly stable in storage and in vivo application.The nanoparticles decomposed and released in slightly acidic environment of tumor.Next,the oxidation therapy mechanisms of Fe-NQA NPs inducing in vitro ferroptosis were studied.The results showed that Fe-NQA NPs provided a large amount of both H₂O₂ and Fe²⁺to initiate Fenton reaction.In addition,NQA effectively inhibited the antioxidant system,leading to tumor cell lipid peroxide accumulation and tumor cell death.Then,the in vivo anti-tumor effect of Fe-NQA NPs was investigated.It was demonstrated that Fe-NQA NPs showed excellent anti-tumor therapeutic effects in CT26 colorectal cancer model,with a tumor inhibition rate of 73.67%,causing tumor necrosis and inhibiting tumor proliferation.Finally,the effects of Fe-NQA NPs in preventing metastasis and enhancing radiotherapy were investigated.The results showed that Fe-NQA NPs effectively inhibited tumor metastasis and enhanced the efficiency of radiotherapy by inducing ferroptosis and inhibiting multiple antioxidant systems in tumor cells.Notably,Fe-NQA NPs showed no systemic toxicity during combined radiotherapy,indicating the biosafety of Fe-NQA NPs.(2)Preparation of catalase and SAHA(Vorinostat)co-delivery nano-system and its anti-tumor application.Hypoxic tumor microenvironment is one of the direct factors leading to radioresistance.During radiotherapy,oxygen can stabilize DNA damage and improve the effect of radiotherapy.Therefore,the degree of cell damage caused by ionizing radiation is highly dependent on the oxygen concentration within tumor site.However,the abnormality of tumor metabolism and vascular distribution results in a hypoxic microenvironment in most solid tumors,which leads to radioresistance.On the other hand,the up-regulation of histone deacetylase(HDAC)in tumor cells usually leads to chromatin condensation and up-regulation of DNA repair mechanisms.Concentrated chromatin protect DNA molecules from being damaged by radiotherapy.The up-regulated DNA repair mechanisms will accelerate the repair of DNA damage and further reduce efficiency of radiotherapy.Therefore,overcoming tumor hypoxic microenvironment and inhibiting histone deacetylase activity can synergistically enhance radiotherapy.The catalase and SAHA(HDAC inhibitor)co-delivery nano-system(CAT-SAHA@PLGA)was constructed by the classic double emulsion method.Firstly,the CAT-SAHA@PLGA was characterized by transmission electron microscope(TEM)and dynamic light scattering(DLS)instrument in vitro.The diameters of CAT-SAHA@PLGA was uniform and measured to be about 144nm.In addition,the prepared CAT-SAHA@PLGA was stable and remained about 150 nm within 48 hours.Then,we investigated the catalase protection and oxygen production capacity of CAT-SAHA@PLGA.Results indicated that the PLGA encapsulation can protect catalase from protease hydrolysis,and the nanoparticle decompose hydrogen peroxide to produce oxygen effectively and continuously.Then,the ability of nanoparticles being internalized by tumor cells and the in vitro therapeutic efficiencies by combining radiotherapy were investigated at cellular level.The results showed that the nanoparticles effectively internalized into tumor cells and induced the most DNA double-strand breaks,inhibiting tumor cell proliferation.Finally,the pharmacokinetics,intratumoral accumulation,oxygen production and in vivo therapeutic efficiencies of CAT-SAHA@PLGA were investigated at animal level.These results showed that nanoparticles improved the pharmacokinetic behavior of SAHA and overcame the tumor hypoxic microenvironment.In CT26 tumor-bearing mice models,CAT-SAHA@PLGA induced significant tumor growth inhibition effect when combining with radiotherapy,and did not exhibit obvious biological toxicity.In summary,based on the key issues in oxidation therapy,we constructed two novel co-delivery nano-systems for regulating ROS production,inhibiting antioxidant systems,or inhibiting cell damage repair.Additionally,both co-delivery nano-systems exerted negligible toxicity on normal tissues.This study offered new strategies to develop nano-systems to improve the efficiency of oxidation therapy such as ferroptotic therapy and radiotherapy.