- 期刊
Complement Activation on Surfaces Carrying Amino Groups
並列摘要
Introduction Most information of complement activation induced by polymeric materials was obtained from studies on interactions of blood with hemodialysis membranes [1, 21. Polymeric substances carrying hydroxyl groups, such as polysaccharides, poly(vinyl alcohol) and poly(ethylene-co-vinyl alcohol), are known to initiate the cascade reactions of the alternative pathway. The current understanding of the mechanism is that nuclephilic groups such as hydroxyl groups presented at polymer surfaces react with a highly reactive thioester group of a complement fragment C3b and then the surface-bound C3b forms C3 convertase which is a main enzyme of positive feed-back of the alternative pathway. Amine is stronger nucleophilic groups than hydroxyl groups. However, it is not yet clearly demonstrated that polymeric materials carrying amino groups is an activator of the complement system. The present study was conducted to investigate interactions of surfaces carrying primary amines with the complement system. For this purpose, model surfaces carrying amine groups were prepared on surfaces of a thin gold layer deposited to glass plates by self-assembling an amine-terminated alkanethiol. Protein adsorption on the surfaces was examined by a real-time surface plasmon resonance (SPR) analysis method after exposure to 10% whole serum, and then an amount of C3b in the protein adsorbed layer was analyzed using anti-C3b antibody. Materials and Methods A thin gold layer was deposited by thermal evaporation of gold onto the surface of a chlomium-primed BK7 glass plate. Then the gold-coated glass plate was immediately immersed in a 1 mM ethanol solution of 11-amino-1-mercaptoundecane [HS(CH2)11NH2] , and the formation of an alkanethiolate monolayer was allowed to proceed at room temperature for at lease 24 hr. Finally the glass plate having a monolayer was sequentially washed with water and 2-propanol and then dried under a stream of dried nitrogen gases. The glass plate with an amine-terminated monolayer was placed in a flow cell chamber of a home-made SPR apparatus. After equilibrated with veronal buffer (VB) solution, the sample surface was exposed to 10% normal human serum diluted with VB at 30 ºC for 90min. In another series of experiments, solutions of immunoglobulin and albumin were passed through the SPR follow cell to determine adsorbed amount of each protein on the amine surfaces. And then weakly adsorbed molecules were washed out with VB solution. During these procedures a SPR angle was continuously measured to determine the real-time surface density of adsorbed serum proteins. The amounts of adsorbed proteins were estimated under the assumption that 1.0 degree shift of a resonance angle corresponds to 0.5 μg/cm^2 of adsorbed proteins. To distinguish adsorbed C3b from other serum proteins, 1% anti-human C3b polyclonal antibody solution was circulated, while specific binding of the antibodies was evaluated from a SPR angle shift. Results and Discussion Figure 1 shows the surface density of proteins adsorbed onto the amine-terminated monolayer during the entire process of the adsorption experiment. As can be seen, protein adsorption from serum reached plateau within an hour, and 0.4 μg/cm^2 of proteins remained after washing with VB solution. Adsorption of anti-C3b antibody was taking place at the second step of the adsorption experiment. The surface density of the antibody was, however, about two times lower than that of hydroxyl-terminated monolayers [3]. This finding suggests that the amine-terminated monolayer surface is less potent for complement activation via the alternative pathway, although the amine present at the surface is highly neucleophilic. The observed discrepancy may be explained by the effect that serum proteins such as albumin and immunoglobulin are preferentially adsorbed to the amine surface (Fig.2).It is probable that the layer of other proteins hinders inhibit access of C3b to necleophilic amines. References 1. Wegmueler E, Montandon A, Nydegger U, Descoeudres C, Intern J Artif Organs, 1986; 8(3): 85. 2. Chenoweth DE, Artif Organs, 1984; 9(2): 281. 3. Hirata I, Morimoto Y, Murakami Y, Iwata H, Kitano E, Kitamura H, Ikada Y: Colloids Surf B, 2000; 18: 285.