Investigation Of Multi-functional Nanoenzymes For Synergistic Cancer Therapy

Compared with natural enzymes,artificial nanozymes have been widely used in catalysis,medicine,biology and other fields due to their higher catalytic activity and physical or chemical stability.With the rapid development of nanotechnology,the excellent physical and chemical properties of nanozymes make them not only substitutes of natural enzymes,but also multifunctional nanoplatforms responsive to many environmental stimuli.Recently,extensive efforts has been focused on designing nanozymes that can react with one or more tumor microenvironmental cues.For example,nanozymes can respond to variations in acidity,hydrogen peroxide and glutathione concentrations,and oxygen levels.In addition,nanozymes can be also excited with different external stimuli,such as magnetic field,light,ultrasound and microwaves for synergistic treatments.Taken together,tuning these factors properly may significantly increase the effectiveness of nanozymes for cancer diagnosis and therapy.Therefore,if the catalytic capability of nanoenzymes is introduced to a stimulus-responsive nanomaterial,it may open up new horizons for cancer diagnosis,therapy,and imaging.Nanozyme-mediated chemodynamic therapy（CDT）may produce cytotoxic^·OH to kill tumors through Fenton or Fenton-like reactions,independent of external stimulation or oxygen levels.Many CDT agents,such as the iron-based nanomaterials,transition metal ions(Mn²⁺,Cu²⁺and Co²⁺)and organic metal frameworks（MOF）,work only in strong acidic conditions（p H=3-5）,and the generation of^·OH is vulnerable because of high levels of glutathione（GSH）in tumor microenvironment.In addition,the efficiency of the CDT therapy is limited due to the lack of catalytic substrate H₂O₂.Therefore,efficient and stable nanozymes to domesticate the otherwise unfriendly tumor microenvironment are highly desirable to improve the therapeutic efficacy.Tumor microenvironment naturally promotes the proliferation and metastasis of tumor,making it difficult to cure cancer with monotherapeutic modality.Interestingly,recent studies have shown that nanomaterial-mediated light,sound and chemotherapy may induce immunogenic cell death and release tumor-associated antigens to dendritic cells.Many researchers are developing various"nano vaccines"hoping to realize artificial nanomaterial-mediated immunotherapy.Inspired by these findings,we designed and prepared a class of multifunctional nanozymes that can specifically respond to tumor microenvironment and improve the efficiency of cancer treatment by combining multiple therapeutic modalities.This thesis work includes two major aspects as described in the following:1.Application of a self-depolymerizing Co O@Au Pt nanozyme clusters for enhanced CDTWe designed and synthesized a novel hollow Co O@Au Pt nanozyme using Co as sacrificial templates.The resultant Co O@Au Pt exhibited high biocompatibility and stability in physiological conditions.After accumulation in the tumor site due to the enhanced permeability and retention（EPR）effect,Co O template can be gradually degraded into Co²⁺in the TME with high acidity and over expressed H₂O₂,inducing the release of Au/Pt nanosatellites.Decent stability and TME-clues responsive property of Co O@Au Pt not only protected the tiny Au/Pt nanosatellites from clearance during long-term circulation,but also increased their penetration depth in solid tumors after acidity-induced disintegration.while the released Au/Pt nanosatellites facilitated versatile enzymatic activities.Firstly,Au/Pt nanosatellites worked as a GSH peroxidase by converting GSH into GSH disulfide（GSSG）,which tremendously mitigated the issue of GSH-mediated ROS consumption for CDT.Secondly,a higher amount of intracellular H₂O₂was decomposed into O₂and^?OH through the catalase-like and peroxidase-like activities,respectively.Thirdly,the strong GOx-mimic activity of Au/Pt nanosatellites caused a dramatic decrease of intratumoral glucose level in the presence of O₂and produced a large amount of H₂O₂,which in turn enhanced the generation of O₂and^?OH.Such cycle-like glucose-depletion and H₂O₂-repletion mechanism not only boosted tumor starvation but also dramatically enhanced the CDT outcome by enzymatic self-supply of H₂O₂.These findings suggested that the hollow Co O@Au Pt had multiple enzyme-mimic functions to ameliorate TME for highly efficacious CDT,which may also inspire more strategic designs of inorganic nanozyme for biomedical applications.2.Applications of a self-enhancing Co Fe₂O₄nanoenzyme for combination SDT/CDT/ImmunotherapyIn this work,flower-like Co Fe₂O₄-PEG（CFP）was obtained by modifying PEG on the surface of Co Fe₂O₄,which was successfully designed and synthesized by solvent thermal method.After intravenous injection,the Co Fe₂O₄nanozyme reach to the tumor area through the EPR effect.Firstly,the cobalt ferrite nanozyme has peroxidase-like activity,which can response to the expressed hydrogen peroxide in tumor microenvironment to produce hydroxyl radicals for CDT,on the other hand,Co Fe₂O₄nanozyme also has catalase-like activity,which can be used to decomposed H₂O₂producing oxygen.More importantly,the Co Fe₂O₄can also be an ideal candidate for inorganic sonosensitizers due to its small band gap energy.In addition,its catalase-like activity can catalyze hydrogen peroxide in the tumor microenvironment to produce oxygen to realize self-enhancement of its sonodynamic therapy（SDT）efficiency,thus overcoming the problem of oxygen level limited the efficiency of traditional inorganic sonosensitizers.The synergistic therapy of self-enhanced SDT and CDT is realized by Co Fe₂O₄nanozyme with US irritation.We also found that this nanozyme-mediated therapy modulates metastase-associated proteins to inhibit tumor metastasis and induce cell immunogenic death.Therefore,the combination of PD-L1 antibody with Co Fe₂O₄nanozyme with US irritation can effectively inhibit tumor growth and metastasis.In summary,we firstly designed an multifunctional nanozyme Co O@Au Pt to address the low efficiency of many traditional CDT therapeutic agents.The enzyme-like activity of Co O@Au Pt can specifically respond to a variety of tumor microenvironmental cues,which can remarkably improve the efficiency of CDT treatment.We further designed an cobalt ferrite nanozyme,which is not only very sensitive to tumor microenvironment but also can take advantage of external acoustic stimulus and immune inhibitors to inhibit tumor growth and metastasis.Based on this novel nanozyme,we demonstrated an evident synergistic anti-tumor effect in a mouse model by combining CDT/SDT/immunotherapy.This thesis work presented an new strategy and solid experimental evidence for the design of a multifunctional nanozyme for tumor diagnosis and treatment.