(Invited) Non-Covalent Functionalization of Single Wall Carbon Nanotubes with Engineered Proteins for Targeted Subcellular Delivery
Authors: Dahl Kris Noel 
Published: February 2020

Single wall carbon nanotubes (SWCNTs) can be non-covalently dispersed with surfactants, polymers and proteins. We have observed that protein coated SWCNTs enter numerous cell types by the millions, more than an order of magnitude more than polymer coated SWCNTs. In addition to increasing uptake, protein coatings can impart function to nanomaterials. While many contemporary studies have focused on using proteins to target SWCNTs and nanomaterials to specific cells, we utilize proteins to target SWCNTs to regions within cells. Here, we will show the utility of modified albumin proteins and engineered native cellular proteins as dispersing agents for SWCNTs. To localize SWCNTs to the nucleus we dispersed SWCNTs with the tail domain of a nucleoskeletal protein lamin B1 (LB1) engineered from the full-length LMNB1 cDNA. The low molecular weight globular protein has a central hydrophobic core for nanotube association and stabilization and an exposed nuclear localization sequence to promote active nuclear import. SWCNTs-LB1 enter HeLa cells and localize to the nucleus of cells; we visualize localization with Raman spectroscopy and NIR fluorescence imaging of SWCNTs and interaction with DNA by fluorescence lifetime imaging microscopy.
We have used similar techniques to show cellular uptake of bovine serum albumin (BSA)-coated SWCNTs within cells. We see localization of SWCNTs-BSA within the endosomal and metabolic processing compartments. Similarly to LB1, BSA is a small globular protein, and the hydrophobic cleft of the BSA binds to SWCNTs. BSA has smaller hydrophobic regions, independent of the hydrophobic cleft, that can be loaded with drugs or fluorophores. In vitro, we show that denaturation or enzymatic processing of the BSA releases the small molecules. SWCNTs-BSA in which the BSA are pre-loaded with rhodamine drastically increases small molecule delivery in culture. We have determined spatial and concentration distribution of rhodamine within the cells, and signal is both coincident and distinct from SWCNTs as measured by NIR fluorescence and Raman. We demonstrate efficacy of this approach by delivering a fluorescent chemotherapeutic drug daunomycin that reduces proliferation in cancer cells. Together, our results demonstrates a pathway to increase the delivery of a wide variety of drugs to cells through SWCNTs coated with albumin pre-loaded with drug molecules.
The complexity of protein structures allows for multimodal modification and manipulation of cellular processes in addition to SWCNT dispersion. The modification of native cellular proteins as non-covalent dispersing agents to provide specific transport opens new possibilities to utilize both SWCNT and protein properties for multifunctional subcellular targeting applications. Specifically, nuclear targeting will allow delivery of anticancer therapies, genetic treatments, or DNA, which in turn will promote development of novel cellular therapies. Using the transport properties of albumins and intracellular processing by enzymes allows delivery of native molecules and drugs.