Disulfide cross-linking in protein microspheres.

摘要

Microencapsulation has diverse applications for drug delivery and targeting, medical imaging, and flavor and fragrance delivery. Many applications, especially biomedical, use liposomes to form an encapsulating lipid bilayer, ranging in size from 10 nm up to 100 microns. Lipid vesicles have limited stability since they are held together only by hydrophobic interactions. Microcapsules with polymeric shells are generally more robust, but are far less biocompatible.; Suslick and co-workers have used high-intensity ultrasound to produce stable, near uniform sized vesicles from several proteins that are more biocompatible than polymeric spheres. Formation of these vesicles via high-intensity ultrasound occurs in a three-step mechanism: (1) emulsification; (2) protein agglomeration; (3) interprotein cross-linking. Experimental evidence has indicated that the inter-protein disulfide bonds result from oxidation by the hydroperoxyl radical (HO2).; In this thesis, the emulsification and cross-linking steps have been further explored by separating them. Protein solutions of albumin (human and bovine), hemoglobin, and pepsin are first emulsified with a high speed blender, and then KO2 or KOH are added to initiate disulfide formation. Microspheres generated have a narrow size distribution (≈1--4 mum) and are stable for over six months at 4°C with minimal degradation (10%). Characterization performed via SEM, TEM, CD, PAGE and mechanistic controls indicate that these microspheres are identical to those formed ultrasonically, and are composed of free native protein and higher molecular weight disulfide cross-linked protein units.; Microspheres composed of either thiol-modified myoglobin or albumin have been extensively examined by a combination of techniques including SDS-PAGE, SEC, and MALDI mass spectrometry peptide mapping. Electrophoresis and chromatography confirm the presence of higher molecular weight protein units as the principle components of the microsphere shell. Treatment with a disulfide reductant established that disulfide bonds are the covalent cross-link between protein molecules. Mass spectrometry has shown that inter-protein disulfide bonding is not random. The disulfide bonds that do form reflect the electrostatic surfaces of the proteins as they approach each other during emulsification and agglomeration.