Construction Of CaO₂/Fe₃O₄@OA Nanocomposites And The Application In Hypoxic Tumor Therapy

Many tumor treatment methods rely on reactive oxygen species（ROS）to kill tumor cells.However,the hypoxic microenvironment at the tumor site makes it difficult to produce ROS,which seriously affects the therapeutic effect.In addition,although some ROS can be produced during treatment,such as H₂O₂,O₂^-,etc.,there are corresponding enzymes in the body to rapidly metabolize them,resulting in low toxicity.Therefore,there is an urgent need for a strategy that can generate strong toxic free radicals under anaerobic conditions to improve the efficiency of tumor treatment.CaO₂is a solid precursor to H₂O₂.When it encounters water,it can slowly react with water to form H₂O₂,therefore,it can be used for the treatment of hypoxic tumors.In the presence of Fe²⁺,H₂O₂can be transformed into highly toxic ·OH through Fenton reaction,further enhancing the therapeutic effect and realizing efficient chemodynamic therapy（CDT）for tumors.In this study,a nanocomposite（CaO₂/Fe₃O₄@OA）with CaO₂/Fe₃O₄as the core and oleylamine（OA）as the outer shell was prepared to achieve free radical therapy under anaerobic conditions.OA is an oil-soluble material,which can prevent CaO₂from reacting with water before reaching the target site.Under the mildly acidic environment of the tumor,the water solubility of OA increases,causing CaO₂ to react with water to generate free radicals.The specific content is as follows.1.Preparation and characterization of CaO₂nanoparticles（NPs）with controllable particle size.CaO₂NPs with controllable particle size were prepared using Ca Cl₂as the precursor,polyvinyl pyrrolidone（PVP）as the stabilizer,and ethanol as the solvent.It was found that the concentration of Ca Cl₂and PVP were the main factors affecting the particle size,and the concentration of other reactants had little effect.The prepared CaO₂NPs were spherical aggregates composed of numerous nanocrystals in structure,and the crystal structure was tetragonal crystals.Due to its good stability in ethanol,it can be stored for a long time.The rate of H₂O₂released by the reaction of CaO₂NPs with water was related to the particle size.The smaller the particle size,the faster the release.Under acidic conditions,CaO₂NPs can react quickly with water,and the impact of particle size on release rate was reduced.2.Construction and characterization of CaO₂/Fe₃O₄@OA nanocomposites.On the basis of the above preparation method of CaO₂NPs,pre-prepared Fe₃O₄NPs were added to construct a core-shell structured CaO₂/Fe₃O₄@OA composite nanocarrier with CaO₂/Fe₃O₄core and OA outer shell.The obtained nanocomposites were spherical in shape with uniform hydrated particle size of 120.2 nm,and they were owned good dispersibility.More importantly,the Fe₃O₄NPs in the core and the outer OA oil film can be clearly observed.As a protective layer,OA were owned good water resistance and p H sensitivity,and can effectively protect the CaO₂/Fe₃O₄core in neutral media.Under neutral conditions,the cumulative release of H₂O₂within 2 h was only 11.68%,while in acid conditions,OA had little effect on its release,and the cumulative release within 2 h reached 45.64%.The released H₂O₂can be effectively catalyzed by Fe²⁺produced by Fe₃O₄to generate·OH.The hemolysis experiments showed that the nanocomposites were owned ideal biocompatibility and stability,and they can be used for in vivo treatment.3.Evaluation of the intracellular behavior and in vitro anti-tumor effects of CaO₂/Fe₃O₄@OA nanocomposites using MCF-7 breast cancer cells as a model.The CaO₂/Fe₃O₄@OA nanocomposites were uptaken by tumor cells through the lysosomal pathway in a time-dependent manner.Under weakly acidic conditions,CaO₂/Fe₃O₄@OA exhibited stronger cytotoxicity.At a concentration of 200μg/m L,the cell inhibition rate reached 82%,while it was only 24%in a neutral environment.Intracellular oxidative stress studies showed that the CaO₂/Fe₃O₄@OA nanocomposites could produce high concentration of H₂O₂in the cells after interacting with cells under weakly acidic conditions,and its fluorescence intensity was 3.38 times that under neutral conditions.H₂O₂was converted into high levels of·OH in the cell through further reactions,and its fluorescence intensity was3.80 times that under neutral conditions.In addition,the changes in total ROS levels in cells were basically the same as·OH.Under weakly acidic conditions,CaO₂/Fe₃O₄@OA could also induce cells to produce more ROS,and its fluorescence intensity could reach 3.94 times that under neutral environment.4.Evaluation of the in vivo tumor treatment effect and safety of CaO₂/Fe₃O₄@OA nanocomposites taking S180 tumor-bearing mice as a model.The nanocomposites were owned good tumor targeting ability.After intravenous injection,the amount of enrichment in tumor tissue was the largest and reached the maximum at 12 h when the content of Ca was2.86 times that of the control group.At the same time,they were also owned ideal tumor retention ability.They can still maintain a high concentration in the tumor tissue after 24 h of injection,and the content of Ca was 2.30 times that of the control group.After reaching the tumor site,CaO₂/Fe₃O₄@OA nanocomposites could produce high levels of·OH in the tumor tissue,and the fluorescence intensity could reach 2.02 times that of CaO₂/Fe₃O₄NPs.CaO₂/Fe₃O₄@OA could significantly inhibit tumor growth.After 14 days of treatment,the tumor volume in the CaO₂/Fe₃O₄@OA group only grew to 2.24 times the initial volume and the tumor inhibition rate reached 83.07%,which was significantly better than other groups.The results of histopathological analysis showed that CaO₂/Fe₃O₄@OA could cause serious damage to tumor tissues,which further indicated its good anti-tumor effect,but it would not cause obvious damage to other major physiological tissues,which confirmed the good biocompatibility and safety of the nanocomposites.