Investigation On Immunological Effects And Mechanism Of Activated Complement Induced By HAP-NPs And SiO₂-NPs

Objective:Nano drug carrier will eventually enter the blood when intravenously injected or in other ways.The complement could be activated as the first defensive reactions and has always been involved in every step of the immune response once foreign pathogen enters the body.Furthermore,nanoparticles are likely affected by the complement system resulting in a series of immunological response because of multiple immune cells in blood.So it is necessary to study the relationship with nanoparticles,complement and immune cells.But now it is not still clear about immune effects of activated complement induced by nanoparticles.In our studies,on the basis of complement activation effect and regulation mechanism of HAP-NPs and SiO₂-NPs,we try to reveal the damage effect of immune cells by activated complement.And the recognition mechanism of nanoparticles protein corona by mococyte will be discussed in order to illuminate the relationship with nanoparticles,complement and immune cells.So the study will be providing a scientific basis for avoiding the risk of nano drug carrier.Materials and Methods:?1?HAP-NPs,SiO₂-NPs and Fe₃O₄ were screened for biocompatibility evaluation according the adverse effects.?2?The absorbed complement was analyzed by proteomics with LC-MS,including qualitative,quantitative and biological information.?3?The activated complement was studied by iC3b/Sc5b-9/C3a/C4a/C5a with ELISA.The activated pathway was estimated by expression of iC3b in an inhibition of complement activation model through EDTA/EGTA/anti-C1q/anti-CFB,and soluble regulators and membrane regulators were analyzed in complement system.?4?In order to reveal the damage effect of immune cells by activated complement,the expression of ROS,cytokine,adhesion molecule,and arachidonic acid were measured when THP-1 and HUVECs were stimulated by activated complement of NPs.?5?In order to illuminate the recognition mechanism of nanoparticles protein corona by mococyte,expression of TLR4/CR1/CR were analyzed by FCM.Results:?1?HAP-NPs and SiO₂-NPs caused in lung and liver damage after injection for7d/14d continuous in vivo.And hemoglobin,mean corpuscular volume,hematocrit index was significantly lower than that of control.?2?HAP-NPs and SiO₂-NPs absorbed 21complement proteins and they are all Cellular Component.And HAP-NPs absorbed SP and CD59 obviously,while C1q family and CFD,CFB were in SiO₂-NPs.?3?HAP-NPs could result in a high production of iC3b/Sc5b-9/C3a/C4a/C5a,and SiO₂-NPs could activate complement,but there had an obviously absorption of Sc5b-9/C3a/C4a/C5a.?4?EDTA and EGTA can also inhibit complement activation of HAP-NPs,and there a high expression of C4bp,CFI and SP in the inhibition model.Only EGTA can inhibit complement activation of SiO₂-NPs,and there a high expression of CFH and CFP.?5?NF-?B,ROS,TNF-a,LTB and Histamine were stimulated by activated complement of NPs on THP-1.But HAP-NPs only result in a high expression of E-selectin,while ICAM-1 and VCAM-1 induced by SiO₂-NPs.?6?HAP-NPs-Pro could induce a CR3expressing on THP-1,but TLR4/CR1/CR3 could also had a up-regulation when THP-1was contact with SiO₂-NPs-Pro.Conclusions:?1?HAP-NPs and SiO₂-NPs were more toxic than Fe₃O₄-NPs.And they will cause changes in blood composition and histopathology after long contact in vivo.?2?Nanoparticles protein corona had been formed because of specific adsorption on HAP-NPs and SiO₂-NPs.?3?HAP-NPs can activate complement with classical pathway through the absorption of C1q and release of C1r/C1s;While SiO₂-NPs can activate complement with alternative pathway through the absorption of CFD and CFB.?4?It was considerate that there had damage on THP-1 and HUVECs by activation complement,including an oxidative stress,inflammation and increased vascular permeability.?5?HAP-NPs can be recognized by opsonin receptor?iC3b/CR3?model,while plasma protein,opsonin receptor and Toll-like receptors are all likely launch cell recognition of SiO₂-NPs.So surface of nano drug carrier can be modified to avoid being clear and reducing the efficacy according to the TLR4/CR1/CR3 three receptors.