The identification of effective polypeptide ligands for magnetic iron oxide nanoparticles

The identification of effective polypeptide ligands for magnetic iron oxide nanoparticles (IONPs) could considerably accelerate the high-throughput analysis of IONP-based reagents for imaging and cell labeling. as polyethylene glycol (PEG) or dextran, and then conjugated to functional moieties that promote target binding or detection of analytes. 2 The complexity of multistep synthesis and conjugation chemistry is an impediment to production of large collections of IONPs. In addition, traditional passivation and functionalization of IONPs adds substantial 585543-15-3 IC50 bulk to IONPs, limiting steric access to the mineral cores where magnetic fields are highest; this in turn limits the effectiveness and manipulability of IONPs for molecular-scale applications. In an effort to discover simple and versatile IONP modification strategies suitable for high-throughput analysis and identification of desirable IONP reagents, we explored the ability of short polypeptides to act as chemically tunable direct iron oxide ligands. Peptides that bind iron oxide cores could form an ideal basis for large-scale investigation of the determinants of IONP properties in applications such as magnetic resonance imaging (MRI) contrast manipulation. Effective stabilization of IONP cores by individual peptide sequences has not previously been demonstrated, but both functional groups and peptide sequences with iron oxide binding characteristics have been identified. We constructed a small initial set of 6C15 residue sequences using some of these moieties, including carboxylate3 and catechol groups,4,5 as well as peptide motifs derived from phage display screens for binding to magnetite6 (DSPHRHS) and hematite7 (LSTVQTISPSNH). Catechol-containing sequences were formulated with variations in net charge and with polyserine moieties to ensure hydrophilicity. In order to screen the peptide library for IONP binding and MRI contrast properties, we developed a procedure for parallel analysis of candidate peptideCIONP complexes. Iron oxide cores were prepared for complexation by exchanging oleate-stabilized iron oxide nanocrystals into aqueous solutions of tetramethylammonium 585543-15-3 IC50 hydroxide (TMA) at basic pH (Figure ?(Figure11a).8,9 Candidate peptides from the initial collection were added to the resulting TMA-associated cores and neutralized by dilution into phosphate-buffered saline (PBS). Peptides were assessed for their ability to stabilize IONPs in the resulting solutions by evaluating relative optical density at 450 nm (OD450) following formulation Rabbit polyclonal to AKAP5 (Figure ?(Figure1b).1b). Lower stability results in precipitation and lower OD450 (Figure ?(Figure1b1b images). Examination of results from the initial library showed that only an anionic l-3,4-dihydroxyphenylalanine (DOPA)-terminated peptide, SSSSSSDDZ, where Z = DOPA, provided significant enhancement of stability with respect to the negative control of no peptide addition (= 0.02, = 3). The stabilization afforded by SSSSSSDDZ was statistically indistinguishable from that provided by citrate, an established IONP ligand (= 0.4, = 3).10 Among peptides that did not appear to stabilize IONPs in PBS were the two phage display motifs, two polyglutamate-containing peptides, and additional sequences that lacked either acidic residues or a DOPA moiety. Some of these sequences, including polyacidic but 585543-15-3 IC50 not phage display peptides, were better able to stabilize IONPs under low salt 585543-15-3 IC50 conditions (Supporting Information), suggesting that poor performance 585543-15-3 IC50 of some of the candidate peptides in the absorbance assay of Figure ?Figure11 may result from a combination of low affinity for IONP cores and lack of electrostatic properties conducive to colloidal behavior. The assay conditions applied in our screening procedure are likely therefore to select simultaneously for effective peptideCIONP binding and complexes with favorable colloidal stability. Figure 1 Identification of stable peptideCIONP complexes. (a) Schematic of peptideCIONP library production. Oleate-stabilized iron oxide cores are exchanged into TMA/water mixtures, library peptides (50 M) are added to IONP aliquots (4.2 … To further probe molecular determinants of IONP stabilization by SSSSSSDDZ, a second set of peptides was constructed. OD450 results obtained using this library (Figure ?(Figure1b1b and Supporting Information) indicated that replacement of the catechol by phenol (Z to Y substitution) or addition of DOPA to a phage display-selected sequence failed to stabilize colloidal IONPs. Separation of aspartate.