(117d) Multimodal Study of Biological Corona Structure and Dynamics on Single-Walled Carbon Nanotubes

Adsorption of polymers on single-walled carbon nanotubes (SWCNTs) has enabled developments in molecular sensing, in vivo imaging, gene and drug delivery, and chirality sorting applications. Noncovalent functionalization offers a modification route that preserves the pristine SWCNT atomic structure, thus retaining the intrinsic near-infrared SWCNT fluorescence for sensing or imaging functions, and offers a reversible binding mode for cargo delivery or chirality separation processes. However, noncovalently functionalized nanoparticles are inherently difficult to study due to the transient conformation of the solubilized complex. Moreover, noncovalent adsorption is a dynamic process, where exchange occurs between molecules in the bulk solution and molecules on the nanoparticle surface, into what is known as the ‘corona phase’. Prior studies of the SWCNT corona^1,2 require drying or surface-immobilizing the complex and thus changing the native corona conformation, or do not operate at the necessary spatiotemporal scale to capture time-dependent binding behavior. Yet, the intended end-use of polymer-SWCNTs is in aqueous environments, thus the nature, strength, and kinetics of polymers adsorbing in the SWCNT corona are important contributors to the success of polymer-SWCNT based technologies. Understanding these parameters is especially important for applying functionalized SWCNTs in biological environments, where native proteins compete with the original polymer to occupy the SWCNT surface. Binding of proteins to the SWCNT not only disrupts the intended functionality of the nanoparticle, but further leads to unpredictable biocompatibility and biodistribution outcomes³.

We present multimodal characterization of (i) the protein corona composition on polymer-SWCNTs in relevant biofluids, (ii) the structural conformation of the solubilized polymer-SWCNT complexes, and (iii) the kinetics of protein adsorption to the polymer-SWCNTs in solution. Single-stranded DNA-wrapped SWCNTs (ssDNA-SWCNTs) are chosen as the polymer-SWCNTs of interest due to their broad relevance in sensing⁴, neuro-imaging⁵, and chirality sorting⁶, though this platform is extendable to other nanoparticles. We determine the protein corona composition by a selective adsorption assay with characterization by protein mass spectrometry in both human blood plasma and cerebrospinal fluid. We next probe the morphology of ssDNA-SWCNT complexes using biological small angle x-ray scattering (BioSAXS). Differences in corona morphology are observed depending on solution ionic strength, ssDNA length, and exposure to key corona proteins. To characterize the dynamic exchange in the SWCNT corona phase, we develop a multiplexed fluorescence assay that enables real-time tracking of biomolecule adsorption and desorption events. Studies are conducted with systematic variation of the molecular entities (ssDNA, protein) and solution conditions, whereby binding profiles inform a kinetic model of the SWCNT system under exposure to different biological conditions. The binding kinetics are compared against an orthogonal platform monitoring solvatochromic shifting of the near-infrared fluorescent SWCNT spectrum as a proxy for SWCNT corona perturbations. The work presented herein develops an understanding of the fundamental corona exchange mechanism, contextualizes the nature of the ligand exchange process, and provides insight into performance of these SWCNT-based systems in biologically relevant, protein-rich conditions such as human blood plasma and cerebrospinal fluid.

References

[1] Bisker, G. et al. Protein-targeted corona phase molecular recognition. Nat. Commun. 7, 10241 (2016).

[2] Safaee, M. M., Gravely, M., Rocchio, C., Simmeth, M. & Roxbury, D. DNA Sequence Mediates Apparent Length Distribution in Single-Walled Carbon Nanotubes. ACS Appl. Mater. Interfaces 11, 2225–2233 (2019).

[3] Nel, A. E. et al. Understanding biophysicochemical interactions at the nano–bio interface. Nat. Mater. 8, 543–557 (2009).

[4] Kruss, S. et al. Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors. J. Am. Chem. Soc. 136, 713–724 (2014).

[5] Beyene, A. G. et al. Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor. bioRxiv 356543 (2018). doi:10.1101/356543

[6] Tu, X., Manohar, S., Jagota, A. & Zheng, M. DNA sequence motifs for structure-specific recognition and separation of carbon nanotubes. Nature 460, 250–253 (2009).