Studies in MALDI-TOF/MS as a tool for the characterization of low molecular weight-branched polyethyleneimine

Gene therapy has become an essential tool of biomedical research. The replacement of defective nucleotides with corrected sequences has succeeded in treating diseases such as cancer and cystic fibrosis. Biotechnology researchers have explored various classes of carrier molecules, "vectors", with varying degrees of success. The earliest were viruses and cationic liposomes, both of which induce cell death and transmit only minimal amounts of "good" material. Synthetic polymers were then explored as potential vectors. Although several have been considered, polyethylenimine, "PEI", holds the most promise because of its unique chemistry. Polyethyleneimine has the ability to easily protonate. Every third atom is a nitrogen that can be protonated, giving PEI a high cationic charge density. PEI is water soluble and has a semi-crystalline characteristic, the degree of which depends upon the degree of hydration. Although both branched and linear polyethyleneimine have been used for in vivo and in vitro gene therapy, its success has been marred by inadequate characterization of its MMD and in the case of branched PEI, the degree of its branching. MALDI-TOF/MS has been chosen as the analytical method to characterize the MMD of branched PEI. However, because MALDI was originally developed to analyze large biomolecules the methodology had to be specifically optimized to analyze very low MW PEI. This study focuses first on the instrumental and sample preparation conditions required to optimize sample preparation and the methods to analyze low MW branched PEI. Second, this study provides detailed information about PEI's branching structure. Specifically, approximately every fourth atom is a tertiary amino nitrogen, indicating point of branching. Third, the branching structures imply that PEI is a cross-linked polymer. Additionally, this study contributes information about the MALDI desorption/ionization process. Finally, this study offers two unanticipated yet interesting observations. First, although polyethyleneimine is predominantly linear, cyclical structures exist. As the molecular mass increases, the abundance of cyclic structures decreases. Second, an unidentified isotopically resolved mass series occurs every 12 u from the oligomers, implying a causal factor in either the polymerization process or the desorption/ionization process.