Effects Of Different Sized Iron Oxide Nanoparticles On Cancer Cell Death

In recent years,superparamagnetic iron oxide nanoparticles?SPIONs?have gradually been highly recognized in the field of biomedicine due to their unique properties,so they are often used as contrast agents for medical diagnosis,drug delivery systems for treatment,magnetic hyperthermia systems and so on.However,there are many obstacles to clinical transformation of nano-drugs.The main challenges are:instability in vivo environment,safety issues,neglect of heterogeneity of disease and patient differences.Therefore,it is necessary to fully understand the in vivo and in vitro behavior of nanodrugs and accurately evaluate their safety and efficacy.Compared with the larger size of the same materials,nanomaterials exhibit significant differences in properties such as large specific surface area,which makes them have strong chemical reactivity.These properties may change their interactions with cells and biological molecules and their biological distribution,thus significantly affecting their safety and efficacy evaluation.SPIONs,on the other hand,are very small in size,comparable to biological molecules.Such a small size may cause nanomaterials to disperse into various systems of the body,which may interfere with the normal functions of various parts of the body.The intracellular metabolic pathways,metabolic burdens,scavenging methods and toxic and side effects of nanomaterials are closely related not only to the safety of nanomaterials,but also to their efficacy.Safety is the basis of the application of nanomaterials in clinical medicine.Quantifying the interaction between cells and nanoparticles and putting forward the scheme of optimizing the safe use of nanomaterials has become an urgent task at the present stage of research.At present,many research teams try to reveal the internal mechanism of cell death caused by nanomaterials,but there is no systematic information network between nanoparticles and cell death pathways.Therefore,this study is based on the biological safety and efficacy of SPIONs.On the one hand,a dynamic quantification model of physical and chemical parameters of nanoparticles and oxidative stress of human breast cancer cell MCF7 was constructed,and a safe use scheme of nanoparticles was proposed.On the other hand,the size effect of SPIONs on oxidative stress and activation of tumor cell death regulatory pathway were studied in depth,so as to give full play to the therapeutic effect of SPIONs on tumors.Three sizes of oil phase SPIONs?3nm,7nm,14nm?were prepared by thermaldecomposition.In order to disperse SPIONs in aqueous solution,we modified phosphorylated polyethylene glycol?PEG?on the surface of oil phase particles.The diameters are 7.3 nm,15.1 nm and 30 nm aqueous Fe₃O₄ nanoparticles,respectively.The SPIONs were co-cultured with human breast cancer cells?MCF7?to simulate the in vitro environment.Plasma inductively coupled emission spectroscopy?ICP-AES?was used to quantitatively analyze the amount of Fe₃O₄ nanoparticles in the aqueous phase,and the cell kinetic model was established.Methods such as flow cytometry and gene interference explore the mechanism by which particles cause cell death.Research indicates:?1?The size of SPIONs is an important determinant of endocytosis.SPION1?3 nm?represents the passive transport model of substances across the membrane.In 6 hours,the rate of cell entry is from fast to slow,and after 6 hours,the quantity of cell entry is basically stable.SPION 2?7 nm?and SPON 3?14 nm?represent the active transport model of substances across the membrane.At 8 h,SPION 2?7 nm?and SPION 3?14nm?reached the maximum rate of cell entry,and after that,SPION 2?7 nm?and SPION 3?14 nm?decreased the rate of cell entry,and the number of cell entry became stable after 24 hours.Different sizes of iron oxide nanoparticles have different endocytosis kinetics curves.The changing trend of the rate of three sizes of iron oxide nanoparticles is different.The rate of iron oxide nanoparticles entering cells is too fast to cause metabolic disorder and cell death.In the follow-up study,we tried to find the root cause of cell death and establish the relationship between the rate of nanoparticles entering cells and cell death.?2?Reactive oxygen species?ROS?are the direct factors associated with cell death.The production of ROS increases with the increase of the cell uptake rate of nanoparticles.ROS mainly comes from cytoplasm and mitochondria.Excessive ROS results in imbalance of intracellular oxidative stress,which leads to cell death.The oxidative damage of Fe₃O₄ nanoparticles in cells has obvious time effect,concentration effect and size effect.Through the analysis of oxidative stress indicators,it was found that the degree of intracellular oxidative stress caused by Fe₃O₄nanoparticles increased with the increase of concentration,increased first and then decreased with time,and reached its peak in 6 hours.3 nanoparticles caused the lowest content of reactive oxygen species in the cytoplasm,7 nanoparticles and 14nanoparticles caused the highest content of reactive oxygen species in the cytoplasm and basically the same.The polarization degree of mitochondria was the highest in SPIONS with different sizes at 6 hours,and the damage degree of mitochondria was the lowest in the other two sizes.According to the production of reactive oxygen species?ROS?in mitochondria and cytoplasm,we concluded that the total ROS content in cells was 7 nm,14 nm and 3 nm after incubation of nanoparticles with cells for 6 hours,which was due to the fact that the cell entry rates of 7 nm and 14 nm at 6hours had exceeded 3 nm.The degree of membrane peroxidation damage caused by three kinds of nanoparticles is 3,7 and 14 nm in turn,which indicates that within 6hours of incubation with nanoparticles,3 nm particles cause the most cell death and14 nm particles cause the least cell death.This is because the endocytosis rate of 3 nm particles increased too fast in 6 hours and was greater than 7 nm and 14 nm.?3?The production of cytoplasmic reactive oxygen species?ROS?and mitochondrial reactive oxygen species?mitochondrial ROS?is regulated by multiple oxidative stress pathways.Three nanoparticles activate five oxidative stress pathways,and seven nanoparticles and 14 nm activate three oxidative stress pathways,respectively.By comparing the data,we can see that the oxidative stress of cells induced by 3nanoparticles is mainly through the mitochondrial pathway,which is consistent with the data of the highest content of reactive oxygen species in mitochondria caused by 3nanoparticles under the same conditions.The oxidative stress of cells induced by 7nanoparticles and 14 nanoparticles is mainly through the cytoplasmic pathway,which is the same as that caused by 7 nanoparticles and 14 nanoparticles under the same conditions.The highest sexual oxygen content was consistent.?4?The oxidative damage caused by nanoparticles is the result of the combined action of the four cell death pathways.The results show that the flux of cell death pathways caused by nanoparticles of different sizes is quite different,which shows the size effect of nanoparticles.The necrosis pathway induced by 3 nm granules was inhibited,the apoptotic pathway induced by 7 nm granules was slightly inhibited,and the necrosis and apoptotic pathway induced by 14 nm granules were inhibited.The expression and interference analysis of four key cell death genes CASP3,RIPK3,ATG9B,ALOX5AP showed that the distribution of SPION1?3 nm?in iron death,necrosis,apoptosis,autophagy and these four pathways were 20%,10%,35%and35%,respectively;the distribution of SPION2?7 nm?in iron death,necrosis,apoptosis and autophagy were 25%,25%,20%,30%,SPIONs respectively.The distribution of SPION3?14 nm?in iron death,necrosis,apoptosis and autophagy were35%,0%,30%and 35%respectively.The difference of cell death pathway flow rate caused by different size nanoparticles was consistent with the data of key gene expression.