Study On Cascade Response Of Nitrobenzene Hypoxia-Sensitive Drug Delivery System For Enhanced Antitumor Therapy

Stimuli-responsive nano-drug delivery system can specifically respond to endogenous or exogenous stimuli at target site,effectively solving the dilemma of toxicity and low bioavailability of chemotherapy.Hypoxia is one of the characteristics of tumor pathological microenvironment.Reductive enzymes and reducing substances are highly expressed in hypoxic tumor environment,which provides opportunities for the designs of hypoxia-responsive nano drug delivery systems.Hypoxia is insufficient in tumors at early stage and hypoxia is heterogeneous in tumor sites,leading to low selectivity and specificity of hypoxia-sensitive nanoparticles,influencing curative effect.For the purpose of improving the selectivity and sensitivity of the stimuli-responsive nano-drug delivery system during responsive process,a cascade response strategy was proposed to amplify the difference between early stage tumors and normal tissue and achieve a rapid response at tumor site.This paper is devoted to the designs of nitrobenzene cascade-responsive hypoxia-sensitive nano drug delivery systems,Glucose oxidase（GOx）and photodynamic therapy（PDT）were adopted to aggravate hypoxia in tumors at early stage and activate the cascade hypoxia-responsive process,achieving specific rapid drug release at the tumor site or modulating cell sensitivity to ferroptosis,ultimately realizing enhanced therapeutic effect for tumors.Utilizing 6-aminocaproic acid as a bridging molecule,the amino terminal is connected to the hypoxia-responsive molecule p-nitrobenzyl chloroformate（PNZ-Cl）by acylation reaction,and the carboxyl terminal of 6-aminocaproic acid is grafted to the positively charged chitosan（CS）by amidation reaction,ultimately synthesizing hypoxia-responsive chitosan grafted nitrobenzene（CS-PNZ-Cl）.Taking the chemotherapy drug Mitoxantrone（MIT）as a model drug,the hydrophobic cavity of CS-PNZ-Cl micelles was used to encapsulate MIT,and at the same time,the positive charge electrostatic of CS-PNZ-Cl on the surface was utilized to immobilize the negatively charged GOx,preparing cascade-responsive hypoxia-sensitive drug-loaded nanoreactor（GOx/MIT@CS-PNZ-Cl）.When distributed to tumor sites through blood circulation,GOx served as the"key"to initiate the cascade reaction,which can catalyze the oxidation of glucose,consume oxygen and aggravate the hypoxic tumor microenvironment,ultimately up-regulating Nitroreductase（NTR）in the hypoxic microenvironment and activating the cascade responsive process of GOx/MIT@CS-PNZ-Cl to promote rapid release of MIT.H₂O₂produced at the same time can reduce GSH levels in tumor cells by regulating redox microenvironment,and reverse MIT efflux caused by GSH-dependent multidrug resistance protein（MRP）efflux,improving the efficacy of chemotherapy drugs.The GOx-initialized cascade-responsive drug release strategy realizes the combinational tumor therapy by"starvation"treatment and chemotherapy and achieve enhanced therapeutic effect.It was determined that the particle size of GOx/MIT@CS-PNZ-Cl was 157.0±5.2 nm,the zeta potential was 0.2±0.4 m V,and the drug loading and encapsulation efficiency of MIT were 16.37%and 86.19%,respectively.The dynamic changing size of CS-PNZ-Cl under hypoxic condition and in vitro drug release assay of drug-loaded nanoparticles showed that the CS-PNZ-Cl skeleton disintegrated rapidly under hypoxic condition,the particle size of CS-PNZ-Cl dramatically decreased and drug release was accelerated.By measuring the changes of H₂O₂ concentration and p H value in vitro,it was shown that the catalytic activity of GOx was not significantly affected when immobilized to the surface of CS-PNZ-Cl.GOx could catalyze the oxidation of glucose to generate H₂O₂,reduce intracellular GSH,and finally reversing GSH-dependent MIT efflux.In the same time,the biochemical reaction catalyzed by GOx would consumes oxygen,which aggravates hypoxia,up-regulates the reductive enzyme NTR and activates the cascade-responsive process of hypoxia-sensitive nanoparticles.Taking 4T1 orthotopic tumors bearing BALB/c mice as model animals,in vivo imaging showed that CS-PNZ-Cl could effectively distribute to tumors.GOx/CS-PNZ-Cl can construct hypoxic microenvironment in tumors at early stage and magnify the hypoxic difference between normal organs and tumors at early stages,effectively activating the in vivo cascade-responsive process of CS-PNZ-Cl and realize specific drugs release at tumor sites.In vivo antitumor experiments showed that GOx/MIT@CS-PNZ-Cl displayed the best anti-tumor effect,the tumor inhibition rate reached 89.45%.Besides,the final preparation possessed high biocompatibility and satisfactory in vivo.The hypoxia-responsive process catalyzed by NTR is a reaction receiving electrons, the nitrobenzene bond can be reduced,and at the same time consume a large amount of Nicotinamide adenine dinucleotide phosphate（NAPDH）,which further leads to the imbalance of intracellular antioxidant systems,including GSH and reduced thioredoxin（Trx）systems,reduce the contents of GSH and reduced Trx,eventually influencing GPx4/GSH and GPx4/Trx pathways,impairing the activity of GPx4 and sensitizing cells to ferroptosis.PDT consume oxygen and aggravate the hypoxic microenvironment,which can serve as the“Key”to initiate the cascade reaction.Further studies were carried by conjugating the photosensitizer chlorin e6（Ce6）onto chitosan of CS-PNZ-Cl via PEG,preparing Ce6modified chitosan grafted nitrobenzene（Ce6-CS-PNZ-Cl）.When receiving near-infrared laser light irradiation,Ce6-CS-PNZ-Cl can exert photodynamic effect,generating a large amount of ROS to kill tumor cells,which would deplete intracellular GSH content,inactive GPx4 activity,reduce the antioxidant capacity of cells and induce ferroptosis.PDT consume oxygen at the same time,resulting in anabatic tumor hypoxia,which activates cascade hypoxia-responsive process of Ce6-CS-PNZ-Cl.The hypoxia-responsive process catalyzed by NTR selectively deplete NAPDH,which further impaire the activity of GPx4,enhance cell sensitivity to ferroptosis.Ferroptosis induced Immunogenic cell death（ICD）,release Damage associated molecular patterns（DAMP）,activating tumor immune effect,the combinational therapy strategy of PDT and immunotherapy achieve enhanced therapeutic effect.In vitro particle size detection and intracellular fluorescence resonance energy transfer experiments proved that Ce6-CS-PNZ-Cl could response to hypoxia and disintegrate rapidly.Ce6-CS-PNZ-Cl was irradiated with near-infrared laser,and the ability of ROS generation in cells was detected by ROS probe,the results revealed that the graft of Ce6to CS-PNZ-Cl would not impair the photodynamic effect.The cytotoxicity of Ce6-CS-PNZ-Cl to 4T1 was much higher than that of Ce6 when receiving laser irradiation.The results of hypoxia probe detection showed that the photodynamic therapy of Ce6-CS-PNZ-Cl consumes oxygen and induce intracellular hypoxia.Investigating the mechanism of Ce6-CS-PNZ-Cl in modulating cells sensitivity to ferroptosis by hypoxia-responsive process,the results showed that hypoxia-responsive process catalyzed by NTR deplete intracellular NADPH,inhibit GSH and Trx antioxidant system,GSH,total thiol content and reducing Trx content were all down-regulated in cells,which finally impair the GPx4activity and enhance cells sensitivity to ferroptosis.By detecting lipid peroxide,GPx4expression,and mitochondrial membrane potential,proving that Ce6-CS-PNZ-Cl can generate a large amount of ROS through PDT,which would reduce GSH,impair GPx4activity and antioxidant capacity of cells,lead to lipid peroxide accumulation,finally induce ferroptosis.PDT would consume oxygen and aggravate hypoxia at the same time,the anabatic hypoxia activates the cascade hypoxia-responsive process of Ce6-CS-PNZ-Cl,selectively consuming NADPH,which further impair the activity of GPx4 enzyme and sensitize cell to ferroptosis.Ferroptosis can induce immunogenic cell death（ICD）,release High-mobility group box 1（HMGB1）and Calreticulin（CRT）,inducing follow-up tumor immune activation.Using 4T1 orthotopic tumors bearing BALB/c mice as animal model,in vivo imaging showed that Ce6-CS-PNZ-Cl could effectively distributed to tumors compared to free Ce6.In vivo antitumor studies showed that Ce6-CS-PNZ-Cl displayed the best anti-tumor effect.Besides,Ce6-CS-PNZ-Cl can effectively activate DCs,induce stronger T cell immune response,and alleviate the immunosuppressive microenvironment,where M2 macrophages and myeloid-derived suppressor cells（MDSCs）were significantly decreased in tumors.