title: Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.
abstract: Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the modification of native cellular proteins as noncovalent dispersing agents to provide specific transport will open new possibilities to utilize both SWCNT and protein properties for multifunctional subcellular targeting applications. Specifically, nuclear targeting could allow delivery of anticancer therapies, genetic treatments, or DNA to the nucleus.
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Boyer
authors.forename: Sairaam
authors.initials: S
authors.lastname: Ganesh
authors.affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Qin
authors.forename: Brian D
authors.initials: BD
authors.lastname: Holt
authors.affiliation: Department of Civil and Environmental Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Buehler
authors.forename: Mohammad F
authors.initials: MF
authors.lastname: Islam
authors.forename: Kris Noel
authors.initials: KN
authors.lastname: Dahl
mesh_terms: D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D034921:Lamin Type B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D037742:Nanotubes, Carbon / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013059:Spectrum Analysis, Raman; D014867:Water / Q000737:chemistry
chemical_list: D034921:Lamin Type B; D037742:Nanotubes, Carbon; D014867:Water
keywords: carbon nanotubes; lamin; nanomedicine; nuclear localization; nucleus; subcellular targeting