Density-gradient ultracentrifugation (UC) is a method widely used in biochemistry and lately has been exploited in nanotechnology to obtain monodispersed nanoparticle (NP) suspensions1. It is now known that when NPs come in contact with a biological environment they form protein corona complexes (PCC) 2. PCCs are new entities composed of NPs coated by multi-layers of proteins. The inner layer, closer to the NP’s surface, is the most tightly bound and generates stable objects with a so-called hard corona (HC). HC PCCs mediate the interaction with the cell membrane and affect NPs’ fate inside the cell3. Isolation and characterization of these NP-protein complexes are important for a rational design of future nanotherapeutics. Here we propose the application of UC as alternative methodology to isolate HC PCCs at a higher resolution than conventional methods, being able to separate HC PCCs of different size and composition present simultaneously in the biological milieu. We systematically studied several NPs different in size, material and surface functional groups in serum and other complex biological fluids in comparison to standard centrifugation protocols. Recovered HC PCCs were characterized by DLS, NTA and SDS-PAGE and applied on lipid bilayers. The interaction between the recovered HC PCCs and lipid bilayer was studied by QCM (Quartz microbalance) and fluorescence microscopy. The technology presented here can be a fundamental preliminary step to investigate further HC PCCs extrinsic behaviour in vivo on cells.
1Miller JB, Harris JM, Hobbie EK. Langmuir, 2014
2 Walczyk D et al. JACS, 2010, 132 (16), pp 5761–5768
3 Sherwood V, Di Silvio D, Bombelli FB. Topics in Medicinal Chemistry, 2014, Springer
D.Di-Silvio@uea.ac.uk
