De-Hao Tsai, Sherrie Elzey, Frank W. DelRio, Athena M. Keene, Katherine M. Tyner, Jeffrey D. Clogston, Robert I. MacCuspie, Suvajyoti Guha, Michael R. Zachariah, Vincent A. Hackley


The cytokine protein tumor necrosis factor (TNF) has substantial potential for applications in cancer therapy due to its ability to inhibit tumorigenesis; however, toxic effects have raised concern about the safety of TNF in drug delivery applications. Gold nanoparticle (AuNP)-based targeted drug delivery platforms confine TNF within the tumor, reducing collateral damage to healthy tissue and reducing the required systemic dose. The capacity to characterize the surface density, conformation, and functionality of TNF-AuNP conjugates is of paramount importance from a drug development and regulatory perspective. In this work, we used complementary physical and spectroscopic characterization methods to interrogate the conjugation of TNF onto AuNPs. Prior to conjugation, we used electrospray-differential mobility analysis (ES-DMA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis to obtain the average molecular mass and mass distribution of TNF. Based on the characterization results of unbound TNF, we investigated TNF conjugated on AuNPs. We evaluated changes in the molecular conformation of TNF protein conjugates in wet and dry states via particle size characterization using dynamic light scattering and ES-DMA. In addition, fluorescence assay, ES-DMA, and attenuated total reflectance-Fourier transform infrared spectroscopy were used as orthogonal methods to characterize the surface packing density of TNF proteins on AuNPs and also to provide a qualitative assessment of the binding affinity for TNF on AuNPs.