Study On Pamam Regulating Plasma Protein Behavior And Mediating In-Vivo&In-Situ Extracellular Microenvironment Formation Of Ti Surface

Stent implantation works as an effective strategy for treating atherosclerosis disease,which can open narrowed vessels and restore blood flow.The stent material contacts the blood and is in the blood environment for a long time.Once biomaterial enters into blood,plasma proteins reach the surface of material quickly.The amount,type and conformation of adsorbed proteins are determined by the surface properties of biomaterials（including electricity,roughness,etc.）.Since protein adsorption is an inevitable dynamic response process,for materials with surface function design,when cells arrive at the material,they cannot"see"the material itself,and their behavior is affected by surface adsorbed proteins.In this context,this paper aims to design material surface specifically to regulate the behavior of plasma proteins adsorbed on the surface,thus affecting the biological properties of the surface and mediating the formation of in-situ microenvironment on the surface of materials,providing a new way for the surface design and in-situ microenvironment construction of interventional materials/devices contacting with blood.PAMAM dendrimers are monodisperse,well-defined size nanoparticles with a large number of terminal functional groups.Their binding affinity to plasma proteins has been confirmed.In this work,three PAMAM dendrimers with different groups（-NH₂,-COOH and-OH）were applied to examining their interaction with three high abundance plasma proteins（albumin,immunoglobulin and fibrinogen）.After this,the dendrimers with appropriate functional groups（PAMAM-NH₂/-COOH）were selected and then introduced to the surface of Ti material.Finally,the biological function,microenvironment formation in vivo and mechanism of protein participation mediated by PAMAM-X had been investigated.The main contents of this study are listed as follows:（1）The effect of PAMAM’s charge characteristics on PAMAM-proteins interaction.Via fluorescence spectroscopy and circular dichroism,the molecular interaction between PAMAM-NH₂/-COOH/-OH and BSA/fibrinogen/γ-globulin were studied,which proved that protein binding to PAMAM molecule was a spontaneous process,and outer groups affected the affinity,kinetic characteristics and thermodynamic parameters of PAMAM-protein interaction.Moreover,it was found that PAMAM-NH₂ was more conducive to maintain the BFG conformation,while PAMAM-COOH was more advantageous to maintain the BSA conformation.（2）Exploring the amount of PAMAM-NH₂/-COOH fixed onto Ti surface and the effect of fixed amount on protein adsorption.Taking PAMAM-NH₂ as the model,the reaction time（more than 4 hours）and the applicable concentration range（0.01 to 5 mg/m L）were confirmed by colorimetry.Good stability of introduced dendrimers on Ti also had been proved.The effect of PAMAM immobilization on protein adsorption was studied in a variety of protein solution systems.It was found that PAMAM-NH₂/-COOH immobilized on Ti surface regulated the surface protein adsorption in a density dependent manner,further realizing the optimization of PAMMA concentration.（3）To investigate the biocompatibility of PAMAM-NH₂/-COOH modified Ti surface.Followed studying the effects of PAMAM-NH₂/-COOH-modified Ti surface on tissue cells,blood cell compatibility and blood coagulation,it was demonstrated that these modified surfaces could maintain the normal growth and proliferation of ECs and inhibit the adhesion of macrophages.And the immobilization of PAMAM-NH₂ on Ti at high density suppressed the adhesion and activation of platelets potently,showing better anticoagulant performance,but significantly stimulated the activation of neutrophils.（4）Functional design and evaluation of Ti-PAMAM-NH₂ surface based on Ac-SDKP grafting.The Ac-SDKP peptide was covalently grafted to the PAMAM-NH₂ modified Ti surface.It was found that the peptide release behavior mainly occurred in the first 8 days.Subsequently,the effect of modified surface on cell behavior was studied.It was found that the grafting of peptides promoted the activity and proliferation of ECs and inhibited TNF-α-induced ECs apoptosis and ICAM-1 expression,and inhibited macrophage dependent inflammation and neutrophil activation.These results suggested that Ac-SDKP peptide promoted endothelial activity and anti-inflammatory properties of Ti surface.（5）Ti-PAMAM-X mediated microenvironment formation,characterization and biological function in vivo.Through ex-vivo experiments,the formation of microenvironment mediated by PAMAM-X was studied,then the participation mechanism of plasma proteins was characterized.It was found that PAMAM-NH₂ resulted in less adsorption and conformational changes of absorbed fibrinogen,promoted albumin adsorption and maintained its conformation,which induced the formation of antithrombotic microenvironment on Ti surface.In vivo studies,through promoting the endothelialization,inhibiting proliferation and material-induced inflammation,Ti-PAMAM-Ac-SDKP would be expected to achieve better intimal repair.In conclusion,in this paper,PAMAM-NH₂/-COOH/-OH were selected to interact with albumin,gama globulin and fibrinogen,finding that PAMAM-NH₂/-COOH was conducive to the formation of PAMAM-protein complex and the maintenance of protein conformation.It was also proved that regulating the density of PAMAM-NH₂/-COOH on the Ti surface can adjust the adsorption amount of protein,and the PAMAM-NH₂ fixing led to better biocompatibility,which could mediate the formation of surface anticoagulation microenvironment in vivo and in situ by regulating the behavior of plasma protein.Meanwhile,PAMAM-NH₂-Ac-SDKP evoked excellent intimal repair effect in vivo.This paper provides a new idea for the surface modification of blood contact materials and the"in vivo formation"microenvironment.