(123b) Novel Polymer Modification on Spion Coatings for Improved Colloidal Stability and Surface Chemistry Control for Use in Magnetic Particle Imaging (MPI)

Introduction: Monodisperse Fe₃O₄ superparamagnetic iron oxide nanoparticles (SPIONs) have gathered extreme interest in use as tracers for Magnetic Particle Imaging (MPI). For these nanoparticles to be useful in biomedical applications, a polymer coating surrounding the particles is necessary to provide steric repulsion and render them colloidally stable in biological media, including cell culture media and blood. Additionally, this polymer shell offers the ability to functionalize the surface of the nanoparticles with agents that can improve their ability to become internalized by cells for labeling purposes, or to attach agents that repel cells for improved circulation half-life by reducing interaction with clearance cells. For this functionalization, the polymer shell must be clearly defined to produce nanoparticles with consistent hydrodynamic and colloidal properties. One attractive route of SPION modification is ligand exchange using silane-functional polyethylene glycol (PEG) coatings (PEG-silane), where the silane group affords strong interactions with the SPION surface. There are several ways to obtain PEG-silanes. One route is oxidation of PEG terminal hydroxyl groups using Jones reagent, followed by reaction with amine-functional silanes. However, Jones oxidation has extreme safety concerns, can lead to inconsistent polymer oxidation and can affect PEG molecular weight due to uncontrollable cleavage of the polymer backbone. Thus, this project aims to eliminate Jones reagent from a safety point of view, in addition to improving understanding of the various steps in the ligand exchange process, and their influence on the size distribution and stability of the resulting particles using a well-defined polymer precursor.

Methods: Methyl ether poly(ethylene glycol) (mPEG) was modified to bear terminal carboxylic acids opposite the methyl terminal. Two oxidation methods were utilized: Jones reagent oxidation, using strong acids and oxidizers, and a ring-opening-procedure (ROP) to open and place a succinic anhydride on the polymer. Both methods result in carboxylic acid (COOH) bearing linear PEG with a methyl group opposite the acid. However, results show that during the Jones oxidation process, the strong acids and oxidizers unpredictably splice the polymer chain, leading to smaller polymer chains that now bear two COOH groups. On the other hand, the ROP process leads to consistent molecular weight chains with well-defined chemistry. To achieve this ROP oxidation, succinic anhydride was added to dichloromethane with PEG, using EDC/NHS chemistry and 4-dimethylaminopyridine (4-DMAP) as catalysts for 24 hours, then washed and precipitated with ethyl ether. The polymer was characterized by standard ¹H NMR and Heteronuclear Multiple Bond Correlation (HMBC) NMR to confirm successful oxidation and characterized by gel permeation chromatography (GPC) to confirm polymer molecular weight was not impacted by the oxidation process. These polymers were then reacted with 3-aminopropyltriethoxysilane (APTES), an amine bearing silane group, which serves as the anchoring group of the polymer to the particle. These amine-reacted polymers were introduced to the SPIONs under high temperature to exchange the oleic acid shell in the as-synthesized nanoparticles with a siloxane-grafted mPEG shell. This reaction is done in excess of free APTES to provide increased graft density of the siloxane groups near the particle surface, while also providing free amines near the surface of the nanoparticle for additional modification. These particles were washed of free polymer and suspended in water, completing the phase transfer to aqueous solutions. The particles were characterized by DLS to determine their hydrodynamic size and colloidal stability.

Results: Polymer modifications using Jones Oxidation and a novel ROP process have been developed and demonstrated. NMR and GPC measurements show that the Jones oxidation process leads to cleaving of the polymer chain and ‘over-oxidation’ due to excess oxidation sites after polymer cleavage. As shown in Figure 1a, increased molar ratios of Jones reagent led to polymer peak molecular weight decrease. Not shown, the NMR characterization for these polymers showed up to 150% oxidation, compared to the methyl group present. These results confirm that this route led to polymer scission and additional sites for oxidation. On the other hand, NMR and GPC measurements show that the ROP process leads to consistent polymer length and expected oxidation rates. Figure 1b shows that as increasing molar ratios of ROP oxidation reagents were added, the peak molecular weight of the polymer did not change. Additionally, NMR of these polymer showed between 90-100% oxidation, confirming that this route led to successful oxidation of the polymer without impacting molecular weight and providing predictable polymer chemistry modifications. These polymers were reacted with APTES to link the anchoring group to the polymer, confirmed by NMR measurements while GPC measurements showed that the polymers do not agglomerate during the process. Successful ligand exchange was demonstrated resulting in particles with hydrodynamic diameters near 60 nm. Additionally, this coating was confirmed by FTIR measurements indicative of siloxane chain formation and a successful phase transfer to water. Ongoing work is focused on size and surface chemistry control and improved colloidal stability by optimizing process conditions, such as reaction solvent, temperature of reaction, molecular weight of polymer, ratios of polymer and free APTES to particles, and forms of agitation.

Conclusions: This work demonstrates that careful consideration of polymer oxidation route is important when modifying polymers. The ability to modify PEG with a ring-opening process to produce PEG bearing carboxylic acid moieties without affecting molecular weight of the polymer was developed and demonstrated. Effective coating of the tracers in a dense polymer brush and colloidal stability through steric repulsion has been demonstrated in this work. This polymer has been tested against its Jones oxidation counterpart, and shows similar ligand exchange capabilities to SPION tracers, suggesting that this new oxidation route does not impede ligand exchange results. Future and ongoing work is focusing on the ability to control size and surface chemistry, improve colloidal stability of the particles, conjugate additional ligands to the surface of the nanoparticle, and attaching thiol-reactive, folic acid, and other functional groups to the nanoparticle surface, providing targeting agent addition to the nanoparticle surface. Finally, these functionalized nanoparticles will be cultured with cells of interest to demonstrate improved cell labeling capabilities in vitro and performing pharmacokinetic studies to demonstrate long circulating tracers in vivo.