POLYMER NANOPARTICLES WITH ARTIFICIAL CELL MEMBRANE SURFACE FOR HIGH-AFFINITY SEPARATION OF PROTEINS

conducting research in the life sciences, particularly proteomics, and also for the applications of these molecules in these fields. Adsorptive and chromatographic separation processes have played a major role in protein separation. Affinity separation is based on the natural biological affinity displayed between biological macromolecules and complementary ligands such as enzymes and coenzymes, antibodies and antigens, and receptors and hormones. A novel protein purification technique involves the use of affinity nanoparticles—an affinity purification method in which ligand-immobilized nanoparticles are used in a batchwise manner. Nanoparticles have a very high ratio of surface area to volume. However, the nonspecific binding of proteins onto nanoparticles has complicated the identification of the target protein. Thus, the most important property to be considered while developing affinity nanoparticles is low nonspecific adsorption of proteins and other biomolecules for achieving a high signal-to-noise (S/N) ratio (signal: the target protein, noise: the other proteins). Additionally, though the activity of the molecules immobilized on the particles decides the performance of the affinity particles, it has not ever been examined. Indeed, the surface simultaneously achieving reduction of non-specific adsorption and retention of the molecule’s activity should be necessary for an affinity nanoparticle to enhance its sensitivity. As new biocompatible polymers, phospholipid polymers have been synthesized from a methacrylate with a phosphorylcholine group, 2methacryloyloxyethyl phosphorylcholine (MPC), and other vinyl compounds by radical copolymerization.[1] Phospholipid polymers can form an artificial cell membrane structure by coating, blending with other polymers, thus they provide bioinert properties. By controlling the molecular structure of the polymer, water-soluble MPC polymers, including poly[MPC–co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)] (PMBN, Fig. 1), could be prepared. PMBN could suspend poorly water-soluble organic compounds in an aqueous medium due to its amphiphilic nature, and the MEONP unit could bind biomolecules covalently under very mild conditions. Phospholipid polymer nanoparticles (PMBN/PLA-NP) were prepared in an aqueous solution containing PMBN to cover the surfaces of hydrophobic poly(L-lactic acid) (PLA) by the solvent evaporation technique. We have reported the applications of various PMBN/PLA-NP-immobilized biomolecules, enzymes, and antibodies.[2,3] In this study, we evaluated the effects of phosphorylcholine groups on the suppression of protein adsorption and the retention of the antibody’s activity in the nanoparticle system. The affinity binding of the bovine serum albumin (BSA) and anti-BSA antibody was examined as a model antigenantibody interaction. An anti-BSA antibody was covalently immobilized onto PMBN/PLA-NP, and the performance of these immobilized nanoparticles in affinity separation was evaluated by using a plasma protein mixture solution. Experimental Preparation of phospholipid polymer nanoparticles. PMBNabc/PLANP (a,b,c represent mole ratio of each component in the PMBN) were prepared using a solvent evaporation technique. A brief explanation of the procedure is as follows. An aqueous solution (40 mL) containing 400 mg of PMBN (10 mg/mL) was placed in a glass bottle, and the solution was stirred at 400 rpm with cooling in an ice bath. PLA (20 mg) was dissolved in 2.0 mL of dichloromethane. The PLA solution was then added to the aqueous PMBN solution. The mixture was sonicated using a probe-type sonicator for 30 min under a cool condition and was maintained under reduced pressure for 1 h to evaporate the dichrolomethane. The formed nanoparticles were collected by centrifugation at 10,300 g at 4 °C for 30 min. To remove the excess PMBN in the solution, the nanoparticles were repeatedly washed with water by centrifugation and resuspended in water. Purified PMBN/PLA-NP were maintained at 4 °C at a concentration of 20 mg/mL before use. Evaluating protein adsorption onto various nanoparticles. The active ester groups on the PMBN/PLA-NP were reacted with glycine (3 mg/mL)