| Endotoxin, composed mainly of lipopolysaccharide,can elicit a wide varity of pathophysiological effects.Exploiting effective treatments for endotoxemia and its complications induced by excessive endotoxins level are always being a tough issue in the clinical research.Hemoperfusion technology, which involves passing patients'blood over an special adsorbent, can remove endotoxins in the blood directly by adsorption.In order to obtain efficient endotoxin adsorbents with high selectivity in human serum, this thesis established different methods for detection of endotoxins in different solutions firstly, and then a series of adsorbents with kinds of ligands were prepared. By means of multi-site interaction of molecular cluster, it may achieve endotoxin adsorption selectively. Systhesis conditions of adsorbent with higher endotoxin capacity was optimized and its adsorption mechanism and property were researched deeply.The adsorption performance in human serum could provide certain guidances for the design of novel adsorbents further. Experimental results showed:(1) Tris-HCl buffer solution with no interference was a better choice when endotoxins had to be detected in buffer solution by LAL test; new perchloric acid method was selected as pretreatment of serum, followed by adjusting the sample pH to 7.4 and the addition of Ca2+ and Mg2+with concentration of 2.5 mM and 80 mM respectively.(2) Endotoxin adsorption capacity of six adsorbents had no significant difference in Tris-HCl solution, however, adsorbents mainly relying on electrostatic interaction were superior to adsorbents mainly on hydrophobic interaction in human serum. Among these adsorbents, lysine or betaine as terminal functional group was much bettter.(3) Endotoxin adsorption capacity and clearance increased with increasing the ligand density, and by comparision, adsorbent coupled with lysine was better than betaine. Moreover, adsorbent with lysine density of 80μmol/mL could reach a high endotoxin clearance of 58.31% at an initial endotoxin concentration of 3.81 EU/mL. Non-specific adsorption for serum proteins was obvious with excess density. Additional spacer arm couldn't improve selective adsorption further for the linear large molecular s-polylysine.(4) Environmental factors had great influences on endotoxin adsorption of adsorbent with lysine molecular cluster:the effect of ionic strength on adsorption indicated the adsorption was possibly due to the synergism of electrostatic and hydrophobic interaction; neutral or weakly acidic condition was of benefit to adsorption; temperature mainly affected the initial rate of adsorption and higher temperature was more favorable to the adsorption of endotoxin.(5) Adsorption experiments of adsorbent with lysine molecular cluster in Tris-HCl buffer solution at 37℃revealed that the equilibrium can reach within 60min, the adsorption kinetic and the isotherm of the adsorbent were well accorded with the second order equation and Freundlich equation with maximal adsorption greater than 2000 EU/mL, respectively.(6) Further evaluation in human serum demonstrated that upon the same ligand density,adsorbent with lysine molecular cluster had parallel affinity with polymyxin B but higher maximal adsorption capacity(Qm were 35.25 EU/mL and 28.92 EU/mL,respectively), and they all displayed excellent adsorption potential than adsorbent with single lysine ligand (Qm was 19.38 EU/mL). |
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