Surface Modification Of Heparin As A Spacer With Effects On Adsorption And Anticoagulant Performance In Blood Purification

Blood purification technology was an extracorporeal circulation therapy that removed certain pathogenic substances from blood in the clinic.Blood contact materials such as adsorbents in hemoperfusion was required not only to be effective,but also safe.However,traditional adsorbents in hemoperfusion were prone to non-selective adsorption,leading to the loss of blood components.At the same time,blood-contact surface of materials might also face the risk of coagulation and thrombosis.Therefore,it was very necessary to improve the selective adsorption of adsorbents and the anticoagulant of hemoperfusion materials through surface modification.Heparin,a linear polysaccharide with a variety of functional groups,had good anticoagulant properties.It was widely used to improve the blood compatibility of blood contact materials,but the ability of heparin to graft molecules or extend their functions through groups was often overlooked,such as-NH₂,-COOH and-OH.There was no much attention to the design and role of heparin as a spacer in surface modification.Thus,in this study,the active groups,flexible molecular chains and anticoagulant activity of heparin were used to design and modify the surface of the adsorbent and the blood-contact materials for the different functions.Adsorbents modified with heparin as a spacer and phenylalanine as a ligand were constructed to improve the blood compatibility and the selective adsorption of endotoxin at the same time.In addition,co-deposition of dopamine and polyethyleneimine was used to provide a stable platform for secondary modification on a variety of blood-contact materials,and a heparin-based NO generating surface was constructed on p DA/PEI coating.The main research contents and conclusions are as follows:1.Heparin as a spacer immobilized on microspheres to improve blood compatibility for endotoxin adsorptionHeparin as a spacer was covalently immobilized on the chloromethylated polystyrene microspheres（Ps）and then connected with phenylalanine obtaining Ps-Hep-Phe,which was prepared for endotoxin adsorption in hemoperfusion.The successful modification was characterized by FTIR and XPS,and the properties of the adsorbent during modification were studied.The results showed that the morphology of the adsorbent microspheres were not changed after violent substitution reactions.Compared with Ps,the BET surface area and hydrophilicity of Ps-Hep-Phe was increased and the surface charge was closed to neutrality.Then the blood compatibility of the adsorbent was evaluated.The protein adsorption and hemolysis rate of Ps-Hep-Phe were reduced,and APTT was extended from 32.5 s to 101.2 s,indicating that the introduction of the heparin as spacers improved the blood compatibility of adsorbent.Finally,the endotoxin adsorption capacity was evaluated.Compared with Ps,the endotoxin adsorption capacity of Ps-Hep-Phe was increased.Moreover,it was found that Ps-Hep-Phe was more in line with the fitting of the pore diffusion equation during the kinetic adsorption process of Ps and Ps-Hep-Phe,indicating that the pore diffusion behavior was increased after surface modification.In addition,the adsorption capacity of Ps-Hep-Phe for endotoxin in plasma adsorption perfusion was～3 times that of Ps,while the adsorption capacity of albumin was only～1/2 of Ps,indicating the selective adsorption for endotoxin of Ps-Hep-Phe was improved.2.Co-deposition of polyethyleneimine and dopamine on surfaces with enhanced stability of polydopamine coatingIn addition to hemoperfusion absorbents,there are many other hemoperfusion materials that require surface modification to improve their desired properties.In order to avoid cumbersome and violent chemical reactions,the universal adhesive force of polydopamine（p DA）coatings was used to modify a variety of materials,mainly focusing on the effect of polyethyleneimine（PEI,Mw=600 Da）co-deposited with DA on stability of the p DA coating on the surface of PP,PTFE and PVC.The results showed that PEI and dopamine（DA）could be co-deposited on the surface of PP,PTFE and PVC through Michael addition and Schiff base reaction.Compared with the p DA coating,the detachment of p DA/PEI coating on the surface of PP,PTFE and PVC is in1.0 M HCl or 1.0 M Na OH solution was reduced.From statistical analysis of FTIR spectra,the rate of detachment of p DA/PEI coating on the PTFE surface was 14.6%and23.7%respectively after eluted with 1.0 M HCl or 1.0 M Na OH solution,while the detachment rate of p DA coating was 35.0%and 74.6%,respectively,suggesting that addition of PEI reduced the detachment of p DA coating in strong acid and strong alkali solution,especially in strong alkali conditions.Besides,the p DA/PEI coating could maintain a stable amino and oxygen-containing group content and keep the hydrophilicity unchanged after eluted with 1.0 M HCl or 1.0 M Na OH solution.In addition,p DA/PEI coating had a wider p H stability range than the p DA coating.And when the mass ratio of DA/PEI was 2:1-1:1,coatings with higher deposition density and more stable could be obtained.There results demonstrated that addition of PEI provides a more stable platform for the subsequent secondary grafting.3.Effects of different molecular weight of polyethyleneimine co-deposited with dopamine on surface heparinizationThe effect of different molecular weight PEI on DA/PEI co-deposition and heparin grafting was investigated.And protein asorption and clotting time of different coatings grafted with heparin was evaluated.It showed that the smaller molecular weight PEI（600 Da and 1800 Da）was beneficial to co-deposition with DA,while the larger molecular weight（10000 Da and 70000 Da）hindered the deposition of the coating,resulting in reduction of deposition density.For the grafting of heparin,heparin could be grafted on coatings successfully on PP-D/P600-H and reach saturated grafting amount when the initial concentration of heparin solution was 4-6 mg/m L.The heparin grafting density of PP-D/P600-H can reach about 26.97 ng/cm²,but heparin grafting density was gradually decreased with the molecular weight of PEI increased,and accordingly,the hydrophilicity was gradually decreased.Among them,PP-D/P600-H and PP-D/P1800-H had better hydrophilicity and anti-protein adsorption capacity,and the clotting time of PP-D/P600-H was longest.These results indicate that the smaller molecular weight PEI（600 Da and 1800 Da）co-deposited with DA was more conducive to heparin grafting and improvement of the blood compatibility.4.Construction of heparin-based NO generating surface with synergistic anticoagulant effectDual anticoagulant coating surface with heparin（Hep）as a spacer and cysteine（Cys）as ligand on the co-deposition coating of DA and PEI（600 Da）was constructed（PP-Hep-Cys）.The successful construction of the surface was characterized by EDS mapping,ATR-FTIR and XPS characterization methods.The sulfhydryl content was determined by Ellman reagent,and the results showed that the sulfhydryl content on PP-Hep-Cys could reached about 2.06 nmol/cm².PP-Hep-Cys could catalyze the generation of NO from 50μM SNAP evaluated by Griess regents,and found that the equilibrium could be reached within 60 minutes,and the amout of NO release after 2 h was about 0.2415 nmol/cm².Water contact angle and protein adsorption experiments showed that compared with PP,the hydrophilicity PP-Hep-Cys was improved,resluting in the reduction adsorption of BSA.And it was found that the introduction of heparin reduced the protein adsorption of PP-Cys.Then the platelet adhesion and activation of materials were studied.The results showed that the amount of platelet adhesion on PP/PVC-Hep-Cys and the amount of PF-4 release were less than those on PP/PVC without NO donor addition,mainly due to a large number of negatively charged groups of heparin.After the addition of NO donor,the platelet adhesion on PP/PVC-Hep-Cys and the amount of PF-4 release were further reduced,indicating that heparin and NO generation-surface played a synergistic effect on inhibiting platelet adhesion and activation.In addition,the NO generation-surface had little effect on clotting time and hemolysis rate.Compared with PP,PT,TT and APTT of PP-Hep-Cys were significantly prolonged and the hemolysis rate was reduced.