Assistant Professor of Biomedical Engineering NYU, New York, United States
Introduction: Iron oxide nanoparticles (IONPs) are extensively studied for their potential biomedical applications, particularly in targeted drug delivery, magnetic resonance imaging, and hyperthermia treatment. When IONPs are administered systemically, they interact with plasma proteins, forming a protein “corona” that transforms their biological identity and can impact their functionality significantly. Studying the composition of the protein corona and the rules that govern its absorbance onto proteins is key to characterize IONPs in a physiologically relevant context. Here, our aim was to optimize the isolation of proteins adsorbed on the surface of IONPS for their downstream analysis. We synthetized IONPs, coated or not with different polymers, and subsequently optimized a method for isolating the protein corona upon incubation with human plasma.
Materials and
Methods: Bare IONPs were synthesized by iron salt co-precipitation and subsequently coated with dextran and carboxymethyl-dextran (CMD). Protein corona isolation was performed by three to five rounds of magnetic decantation. In this process, the following parameters were optimized: composition of the washing solution (water, 1M NaCl, 100mM NaCl, 1xPBS), incubation time (1hr and 24hr), IONP concentration (1, 5, 10, 50 mg/ml), heating temperature and time of protein denaturation in Laemmli buffer for SDS-PAGE, and protein corona stability in at 4°C (1, 2, and 3 days after formation). Nanoparticle size and charge was characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM) before and after incubation for plasma to confirm the formation of the protein corona, Bicinchoninic acid (BCA) assay was used for protein quantification, and SDS-PAGE for protein profiling.
Results, Conclusions, and Discussions: DLS and TEM analysis confirmed the formation of a protein corona in all the IONPs screened. Optimization of the protein corona isolation protocol revealed that 1xPBS was the most effective washing solution, yielding the highest protein recovery and purity. An incubation time of 24 hours showed no significant differences than 1 hour incubation. Increasing IONP concentrations yielded increasing amounts of recovered proteins. Even 3 days after incubation protein coronas were present, however same day intubation yielded the highest number of protein bands. Among the different coatings, CMD-coated IONPs exhibited the most stable and uniform protein coronas. BCA assays indicated a significant reduction in protein content from wash 0 to wash 3, demonstrating effective washing of the soft bound corona. SDS-PAGE profiles revealed diverse protein compositions depending on the washing solution and IONP type.
Our study successfully optimized an efficient method for protein corona isolation. The choice of washing solution, incubation time, IONP concentration, and particle type significantly affects the recovery of protein corona and its composition. These findings will contribute to the understanding of protein-IONP interactions and provide a foundation for further research into the biological behavior of IONPs.
The optimization of the protein corona isolation method is crucial for enhancing the biomedical applicability of IONPs. Our findings highlight the importance of selecting appropriate washing solutions and IONP concentrations to obtain the maximum information of the protein corona. Future research should focus on in vivo studies to further validate these findings and explore the impact of the protein corona on the therapeutic efficacy and safety of IONPs. Understanding the dynamic interactions between IONPs and plasma proteins will pave the way for the development of more effective nanoparticle-based therapies.
