| Endotoxin(ET)/lipopolysaccharide(LPS), a component of the outer membrane of gram-negative bacteria, is an important factor in the pathogenesis of endotoxemia . Endotoxemia therapy has not been successfully dealt with pharmacologically. Polymyxin B (PMB) can inactivate endotoxin and is active against gram-negative bacilli. However, it is difficult to use polymyxin B (PMB) in blood because of its toxic effect on the kidney. To reduce these toxic effects, the new methods of PMB fixed to insoluble microbeads were developed. In this paper, the ligand ,PMB, was immobilized onto polystyrene microbeads as a affinity adsorbent for selective removal of ET from plasma and was also used in blood perfusion in endotoxemia rat. The adsorbability and biocompatibility of this novel adsorbent for endotoxin were studied as follow.①Synthesis of the ligand immobilized polystyrene microbeads The polystyrene beads with different length arms were obtained by coupling various length linear spacer, such as ethylene diamine, glutaraldehyde, 1,6 diaminohexane and so on, then the PMB ligands were immobilized onto the spacer arms-containing polystyrene beads by glutaraldehyde.The factors that could influence the immobilized ligand density were also studied. The optimum conditions of coupling PMB ligands through its -NH2 to polystyrene beads by glutaraldehyde are: the initial concentration of 4mg/ml of PMB is more efficient than the higher concentration; the concentration of glutaraldehyde which activated polystyrene beads is 5%, pH is 7.0, and the coupling reaction time is at least 4 hours.②The adsorbability of adsorbents were studied in vitro.Experimental results indicated that the adsorbents could selectively remove endotoxin from many kinds of solutions (water,saline solution, glucose solution).The concentration of saline solution affect the adsorbents on adsorption of endotoxin, the affinity of the adsorbent for pyrogen decreased with the increasing salt concentration. pH also effect adsorbent on adsorption of endotoxin, the affinity of the adsorbent was higher at around neutral pH ,and decreased at lower or higher pH.③The adsorbability of adsorbents were studied in blood perfusion in vitro.Experimental results indicated that the adsorbents could selectively remove endotoxin from plasma. The flow rate of perfusion affected the adsorbents onadsorption of endotoxin, and the affinity of the adsorbent for pyrogen decreased with increasing flow rate of perfusion.④The adsorbability of adsorbents were studied in blood perfusion in endotoxemia rats. 1)Experimental results indicated 1 milliliter of endotoxin(E.coli 0111B4 lipopolysaccharide, 0.5mg/kg,Sigma Company) solution was administered to rat by intravenous injection, could induce changes of levels of TNF, IL-1 in plasma and MAP.2)The adsorbents could selectively remove endotoxin from plasma of endotoxemia rat, decrease levels of TNF-α,IL-1, and improve MAP.3) The adsorbents could change blood viscosity and hematocrit of blood cell in endotoxemia rat after hemoperfusion , could improve hemorheology of endotoxemia rat.⑤Blood compatibility of the absorbents were studied in hemoperfusionExperimental results indicated that the absorbents have slightly effect on white cell, platelet. The absorbents have a good blood compatibility.⑥Conclusion1)The spacer on beads was beneficial to the connection between polymyxin B and endotoxin, also deceased the block of connection.2)Polymyxin B immobilized polystyrene microbeads was a fine biomaterial of hemoperfusion, could neutralize endotoxin, moreover, the newly invented adsorbent exhibited a satisfactory biocompatibility.3)Polymyxin B immobilized polystyrene microbeads could decrease levels of LPS and TNF-α,and improve MAP,was beneficial to therapy endotoxemia.4)Polystyrene have a good biocompatibility, and reaction function of chemistry,is a ideal adsorbent material.5)Polymyxin B immobilized polystyrene microphere could remove endotoxin from plasma, may be an assistant approach to en...
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