Identification of plasma proteins facilitated by enrichment on particulate surfaces: Analysis by two‐dimensional electrophoresis and N‐terminal microsequencing

Abstract

Plasma protein adsorption on intravenously injectable drug carriers is regarded as an important factor for the fate of the particles in the body after their administration. Therefore, the plasma protein adsorption patterns on a number of different carrier systems were analyzed in vitro employing two-dimensional electrophoresis (2-DE). The particulate systems presented in this study were polystyrene (PS) model particles, PS nanoparticles surface-modified by adsorption of a surfactant, a commercial fat emulsion, and magnetic iron oxide particles used as contrast agents in magnetic resonance imaging. Most of the spots in the plasma protein adsorption patterns could be identified by matching the resulting 2-DE gels with a reference map of human plasma proteins. Several other proteins that indicated preferentially adsorbed proteins on the surface of the particles investigated have either not been identified on the reference map, or their identity was found to be ambiguous. The relevant proteins are all present in plasma in low abundance. Since these proteins were strongly enriched on the surface of the particles, the resulting spots on the 2-DE gels were successfully identified by N-terminal microsequencing. With this approach, two chains of spots, designated PLS:6 and PLS:8, were determined on a plasma reference map: inter-alpha-trypsin inhibitor family heavy chain-related protein (also named PK-120) and a dimer of fibrinogen gamma, respectively. Plasma gelsolin is presented in a 2-DE adsorption pattern of PS model particles. One of the main proteins adsorbed by droplets of a commercial fat emulsion was identified as apoliprotein H. Moreover, the positions of apolipoproteins apoC-II and apoC-III were also verified on the 2-DE protein map of human plasma. Thus, protein adsorption experiments of the kind presented in this study are increasing our insight into human plasma proteins.

Stealth Properties to Improve Therapeutic Efficacy of Drug Nanocarriers

“…PEG-and poloxamine-coated nanocarriers have been demonstrated to undergo immunoglobulin, fibronectin, and apolipoprotein association [14, 29, 33, 118, 122–124, 147] as well as C3 opsonisation that mediates the biorecognition by macrophages through specific complement receptors (CR1 and CR3, CD11b/CD18) [18]. However, these systems possess long-lasting profiles in blood [148].…”

Analytical strategies for detecting nanoparticle–protein interactions

“…Spots on 2-DE can be used for protein sequencing using an automatic gas phase sequencer 74–79 . A successful sequence of a peptide of 10–30 N-terminal amino acids residues can determine the identity of the protein through database search 79 .…”

Functional groups on polystyrene model nanoparticles: Influence on protein adsorption

“…In previous studies a preferential adsorption of proteins from human plasma could be shown, i.e., enrichment of certain proteins on particulate surfaces while others are diminished compared with their concentration in the bulk medium. [7][8][9][10][11][12] Because the in vivo fate of intravenously administered nanoparticles is strongly influenced by interactions with blood components, enrichment of certain proteins might be able to direct the particles to specific target cells or tissues. Exemplarily, this could be shown for particles with ApoE enrichment on the surface to target drugs to the brain.…”

“…and the significance of protein adsorption for the behavior of, e.g., intravenously administered particulate drug carriers. [1][2][3][4][5][6][7][8][9] These carriers are one approach to the sitespecific delivery of drugs, and the immunological recognition by cells of the mononuclear phagocyte system (MPS) is a major obstacle for their application. In previous studies a preferential adsorption of proteins from human plasma could be shown, i.e., enrichment of certain proteins on particulate surfaces while others are diminished compared with their concentration in the bulk medium.…”