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The interaction of liposomes with complement proteins and protein s

University of British Columbia

The use of liposomes as drug delivery units necessitates that they come into contact with a number of plasma proteins and cells. This dissertation investigates the interaction of liposomes with two plasma reaction cascades: the complement and the coagulant cascade. The first section of the studies involves the investigation of the contribution of immunoglobulin to the liposome-induced activation of complement in human serum. Liposomes containing the negatively charged phospholipids cardiolipin, phosphatidyiglycerol or phosphatidylinositol, in addition to the neutral charged lipids phosphatidylcholine and cholesterol, were used to activate complement in a whole serum system. The contribution of immunoglobulin was studied by comparing normal human serum to serum depleted of lgG and IgM. Using hemolytic assays of complement function, greater concentrations of phospholipids were required to activate complement in the absence of immunoglobulins. Activation of the classical pathway was confirmed using a C1q consumption assay which showed that activation was dependent on the presence of C1q and confirmed that greater concentrations of phospholipids were required to activate complement in the absence of immunoglobulins. Complement activation was also assessed using crossed immunoelectrophoresis of C3 activation fragments. Using immunoblot analysis, iC3b was detected on the surface of liposomes exposed to normal human serum or immunoglobulin-depleted serum. Having demonstrated that specific antibody was not required, the second section of the studies involves the investigation of the contribution of mannose binding protein to the liposome-induced activation of complement in human serum. The ligand to which mannose binding protein binds was also identified. Mannose binding protein is an activator of the classical complement pathway in the absence of immunoglobulin and C1q. Using immunoaffinity chromatography it was demonstrated that mannose binding protein copurified with beta-2-glycoprotein-1 when beta-2-glycoprotein-1 was immobilized by specific antibody. Using immunoblot analysis mannose binding protein was shown to bind to PC:CH:CL (35:45:20 mol%) through beta-2-glycoprotein-1 which in itself binds avidly to anionic liposomes. This binding was specific as it was inhibited using N-acetyl- mannosamine, an inhibitor of mannose binding protein. Using complement hemolytic assays in the presence of mannose binding protein inhibitors, it was demonstrated that the route of activation of the classical pathway was primarily through C1q in the absence or presence of immunoglobulins. Preliminary competitive binding studies confirmed the presence of the competition between C1q and mannose binding protein that was previously observed using the functional complement hemolytic assays. The competitive binding study demonstrated that various concentrations of mannose binding protein, including physiological concentration, could compete with and inhibit binding of ¹²⁵l-labeled C1q to PC:CH:CL (35:45:20 mol%). There was no binding of ¹²⁵l-labeled C1q detected with the neutral composition PC:CH (55:45 mol%). During the course of the immunoaffinity chromatography studies conducted in section two, it was demonstrated that a number of other proteins copurified with beta-2-glycoprotein-1. Protein S was one of five proteins that copurified with beta-2-glycoprotein-1 when the latter was immobilized using immunoaffinity chromatography. The final section of the studies deals with the investigation of the contribution of protein S on the anticoagulant ability of plasma as the removal of free protein S from circulation may disrupt the hemostatic balance. Using Western blot analysis, protein S was detected on the surface of anionic liposomes. Furthermore, free protein S was deposited on the surface of increasing concentrations of anionic liposomes, quantitated using immunological assays. This effect translated into a lower functional activity of protein S for plasma exposed to anionic liposomes, demonstrated using a commercial assay to measure the functional activity of protein S. The removal of free protein S from the plasma was due to the depletion of beta-2-glycoprotein-1. These studies suggest that the incorporation of glycosides and polyethyleneglycol into the liposomes would be beneficial since they may reduce the observed interactions demonstrated in this study. This in turn could potentially increase the half-life of the circulating liposomes.

Thesis/Dissertation

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2009-06-09

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http://hdl.handle.net/2429/8871

Pathology

University of British Columbia

UBCV

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