The unpredictable carbon nanotube biocorona and a functionalization method to prevent protein biofouling

Background: The intrinsic physicochemical properties of carbon nanotubes (CNTs) make them unique tools in nanotechnology. Their elemental composition, resilience, thermal properties, and surface reactivity make CNTs also of undisputed interest in biotechnology. In particular, their extraordinary ability to capture biomolecules on their surface makes them essential in this field. The proteins adsorbed on the CNTs create a biological coating that endows them the ability to interact with some cell receptors, penetrate membranes or interfere with cell biomechanics, thus behaving as an active bio-camouflage. But some of these proteins unfold, triggering an immune response that unpredictably changes the biological activity of CNTs. For this reason, the control of the biocorona is fundamental in the nanobiotechnology of CNTs. Results: Using TEM and AFM here we demonstrate a significant increase in CNTs diameter after protein functionalization. A quantitative analysis using TGA revealed that between 20 and 60% of the mass of functionalized nanotubes corresponds to protein, with single-walled CNTs capturing the highest amounts. To qualitatively/quantitatively characterize these biocoatings, we studied the biochemical "landscape" of the proteins captured by the different nanotubes after functionalization under various conditions. This study revealed a significant variability of the proteins in the corona as a function of the type of nanotube, the functionalization temperature, or the time after exposure to serum. Remarkably, the functionalization of a single type of CNT with sera from various human donors also resulted in different protein landscapes. Given the unpredictable assortment of proteins captured by the corona and the biological implications of this biocoating, we finally designed a method to genetically engineer and produce proteins to functionalize nanotubes in a controlled and customizable way. Conclusions: We demonstrate the high unpredictability of the spontaneous protein corona on CNTs and propose a versatile functionalization technique that prevents the binding of nonspecific proteins to the nanotube to improve the use of CNTs in biomedical applications.