Cross-reactivities in conjugation reactions involving iron oxide nanoparticles

The preparation of multimodal nanoparticles by capping magnetic iron oxide nanoparticles (IONPs) with functional organic molecules is a major area of research for biomedical applications. Conjugation reactions, such as carbodiimide coupling and the highly selective class of reactions known as "click chemistry", have been instrumental in tailoring the ligand layers of IONPs to produce functional biomedical nanomaterials. However, few studies report the controls performed to determine if the loading of molecules onto IONPs is due to the proposed coupling reaction(s) employed, or some other unknown interaction with the IONP surface. Herein, we use 3,4-dihydroxybenzoic acid-functionalized IONPs (IONP-3,4-DHBA) as a platform upon which carbodiimide coupling can be used to conjugate clickable small molecules for further functionalization using two common click reactions, namely, the copper-catalyzed azide-alkyne cycloaddition (CuAAC), and the thiol-maleimide Michael addition reactions. Through the judicious use of controls, we demonstrate significant cross-reactivities of amines, thiols, maleimides, and common disulfide reducing agents with surface Fe of IONPs and show how these unwanted interactions can produce false positive results. Without proper controls, these can lead to erroneous conclusions about the efficacy of conjugation reactions, which can have detrimental impacts on the functionality and safety of IONPs in biomedical applications.