The Toxic Effects And Mechanisms Of SiO₂ Nanoparticles And Protein Corona Formation On Erythrocytes

In recent years,nanomaterials have been widely used in people’s production and life,such as biomedical reagents,cosmetics,paints,food additives and automobile industry.Nanoparticles（NPs）can enter the human body through various ways.Due to their small particle size,they are easy to cross biological barriers and distribute throughout the body.The interaction between NPs and blood components is inevitable and potentially dangerous.Red blood cells（RBCs）are the main cells in the blood.Once NPs enter the circulation system,they can directly interact with RBCs,induce hemolysis and eryptosis,and have a series of effects on the physiological activity of RBCs.When NPs in the environment enter the human body,they will be immediately encapsulated by the proteins contained in the surrounding biological environment,forming a"Protein corona（PC）"on the surface of NPs,giving NPs a new biological identity.At present,the ways of NPs contact with the human body are increasing,but there are few studies on the protein formation of NPs in the organism and the toxic effect of NPs on RBCs.Therefore,in this study,amorphous silica nanoparticles（Si O₂ NPs）were used as the research model to explore the effect and specific mechanism of their induction of eryptosis,and the damage effect of different protein crowns adsorption on erythrocytes in the body fluid environment was also evaluated.This study provides a reference for understanding the blood toxicity of NPs in the environment,and also lays a foundation for the safety evaluation and biological application of nanomaterials.PartⅠ:This part of the work evaluated the eryptosis induced by Si O₂ NPs in vitro and in vivo experiments.In addition,we compared the RBC toxicity of polyvinylpyrrolidone（PVP）and plasma proteins on the surface of Si O₂ NPs.Our findings confirm that Si O₂NPs induce eryptosis,underlining the protective role of PC as well as the grafted polymers on the NPs surface.The study found that after the encapsulation of plasma proteins and macromolecular PVP,a layer of protein layer or macromolecular layer with thickness was formed on the surface of Si O₂NPs,and the particle size of Si O₂NPs increased significantly.Si O₂ NPs can induce phosphatidylserine exposure and hemolysis of RBCs,and with the outflow of hemoglobin,decaying RBCs will be engulfed and degraded by macrophages.However,Si O₂@MP and Si O₂@PVP do not induce eryptosis or phagocytosis.TEM images of the interaction between Si O₂ NPs and RBCs showed that the bare Si O₂ NPs could damage the RBC membrane,resulting in the outflow of intracellular substances.Si O₂@MP and Si O₂@PVP are only distributed in the space of RBCs,not in direct contact with RBCs,protecting the integrity of the cell membrane.In order to further clarify the possible mechanism of Si O₂ NP-induced eryptosis,several triggering factors of eryptosis induced by Si O₂ NPs were also explored in this study.The study found that the release of reactive oxygen species（ROS）from RBCs increased with the increase of Si O₂ NPs concentration,and the oxidative stress indicators also changed accordingly.These results indicated that ROS was produced in RBCs under the stimulation of Si O₂ NPs,which induced oxidative stress and triggered peroxidation,and the antioxidant activity of RBCs also increased with the increase of material concentration.In addition,it was found that the intracellular Ca²⁺concentration increased with the increase of Si O₂NPs concentration,and the eryptosis was significantly alleviated when incubated in Ca²⁺-free Ringer solution,indicating that Ca²⁺was an important cause of eryptosis induced by Si O₂NPs.Finally,we found that with the increase of Si O₂ NPs concentration,the expression of platelet activating factor receptor increased,and the content of ceramide was significantly up-regulated,suggesting that ceramide is also a key molecule in eryptosis induced by Si O₂ NPs.This study further evaluated the in vivo toxicity of Si O₂ NPs.Single administration of Si O₂ NPs（20 mg/kg）in mice did not cause significant damage to RBCs,which may be due to the rapid formation of protein crowns and subsequent rapid clearance of bare Si O₂ NPs once they enter the blood circulation.However,after seven days of administration,it was found that there was obvious eryptosis in the Si O₂ NPs group,which may be due to the accumulation of Si O₂ NPs in the mice for a long time,resulting in toxicity to RBCs,but the specific mechanism needs to be further explored.The results in this chapter showed that Si O₂ NPs could directly contact with the erythrocyte membrane to induce eryptosis,and the cell membrane would be broken and the contents would flow out.The encapsulation of MP and PVP inhibited eryptosis by blocking the direct contact between Si O₂ NPs and RBCs.In addition,oxidative stress,intracellular calcium and ceramide are all important causes of eryptosis induced by Si O₂NPs.PartⅡ:In this part of the study,we found that Si O₂ NPs coated with mouse serum protein cap（Si O₂@MS）could damage RBCs by forming a complement attack complex,causing phosphatidylserine exposure of RBC membrane.To explore the mechanism of complement activation by Si O₂ NPs,we incubated Si O₂ NPs with mouse serum.LC-MS/MS was used to determine the types and abundance of complement related proteins in serum protein crowns on the surface of Si O₂ NPs.Western blotting was also used to detect the expression of different complement activation pathway markers,and it was found that the classical pathway marker C1q and alternative pathway marker factor B were expressed in different degrees.Moreover,the classical pathway（C1NH）and alternative pathway（LNP023）inhibitors were used to further prove the role of the two pathways in the activation of complement by Si O₂NPs.Our experiment revealed that Si O₂ NPs activated complement,leading to a significant up-regulation of IL-6,IL-1β,TNF-αand other inflammatory factors,thus suggesting that Si O₂ NPs may provoke an inflammatory response in RBCs.The results of this chapter show that Si O₂ NPs could activate the classical and alternative pathways of complement to form C5b-9 on RBCs,cause RBC damage,and induce the inflammatory response of RBCs.The above experimental results show that Si O₂ NPs can directly contact with RBCs to cause cell contents to flow out,induce hemolysis and eryptosis,cause phosphatidylserine exposure,cytoskeleton changes,and reduce oxygen transport ability.In addition,oxidative stress,intracellular Ca²⁺and ceramide are all important causes of eryptosis caused by Si O₂ NPs.Moreover,the encapsulation of mouse plasma protein coronas and the grafting of macromolecular polymers can inhibit the eryptosis by blocking the direct contact between Si O₂ NPs and RBCs,but the complement components in the Si O₂ NPs serum coronas can activate the complement cascade to form a membrane attack complex,and induce eryptosis and inflammation.