Delivering Single-Walled Carbon Nanotubes to the Nucleus Using

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Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains Patrick Boyer, Sairaam Ganesh, Zhao Qin, Brian D. Holt, Markus J. Buehler, Mohammad F. Islam, and Kris Noel Dahl ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12602 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 21, 2016

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ACS Applied Materials & Interfaces

Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains Patrick D. Boyer,┴ Sairaam Ganesh,◊ Zhao Qin,║ Brian D. Holt,† Markus J. Buehler,║ Mohammad F. Islam,†,* Kris Noel Dahl ┴,◊,* ┴

Department of Chemical Engineering, ◊Department of Biomedical Engineering, and

†

Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, United States ║

Department of Civil and Environmental Engineering, The Massachusetts Institute of Technology, Cambridge, MA 02139, United States

KEYWORDS: Nucleus, nuclear localization, lamin, carbon nanotubes, subcellular targeting, nanomedicine 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 non-covalently attaching the tail domain of the nuclear protein lamin B1 (LB1),

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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 non-covalent 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.


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1. INTRODUCTION Targeted delivery of nanoparticles to subcellular structures including actin filaments,1-2 mitochondria,3-4 and the nucleus5-7 has potential cellular and medical applications including organelle-specific modulation,8 in situ sensing,9 ablation,10 and drug delivery.11 Specifically, transport to the nucleus is desired for delivery of DNA for gene therapy, and many anti-cancer strategies include nuclear targeting of small molecular drugs to inhibit DNA duplication or induction of DNA damage by radiation or ablation. Indeed, numerous types of small nanoparticles (diameters 3–20 nm) including fullerenes, gold spheres, quantum dots and nanohorns have been attempted to deliver to the nucleus aided by covalently or noncovalently attached biocompatible molecules containing peptides and nuclear localization sequence (NLS) with mixed successes.5-7,12 The challenges originate, in part, from indirect optical imaging techniques that lack resolution in determining nanomaterial location within cells and nucleus, artifacts in images generated by transmission electron microscope that otherwise has necessary resolution, and sample heterogeneity, among many others, that complicate unambiguous quantification of nuclear uptake of nanomaterials. Further, while above mentioned small nanomaterials can easily diffuse through the large diameter nuclear pores, transport of much larger nanomaterials (≥100 nm) is sought after to deliver larger quantities of foreign cargo to the nucleus but such transport across nuclear pores is complicated because it requires active nuclear import. In this work, we consider targeted nuclear delivery of single-walled carbon nanotubes (SWCNTs) of length ≈ 150 nm facilitated by non-covalently attached, engineered nuclear protein. SWCNTs possess unique mechanical, thermal, electrical, and optical properties making them attractive materials for subcellular imaging, sensing, ablation, and modulation.13 Locations


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of SWCNTs can be precisely determined via direct visualization using confocal Raman and fluorescence lifetime imaging microscopes, and their uptake in cells can be quantified through intensities of characteristic Raman features of SWCNTs.14-16 Furthermore, the large surface areato-volume ratio of SWCNTs provides ample possibilities for targeted delivery to the nucleus via chemical functionalization and substantial molecular loading. Unfortunately, SWCNTs are hydrophobic and aggregate in water or cell media due to strong van der Waals forces, leading to formation of bundles that has diminished optical properties17 and deleterious cellular effects.18 Covalent modification of SWCNTs promotes dispersion into water and cell media while adding moieties for targeting to the nucleus,5,19-21 but alters the sp2 structure and diminishes inherent properties desired for biomedical applications.22 Non-covalent attachment of macromolecules to SWCNTs are preferred to retain SWCNT properties, and surfactant-like copolymers, lipids, DNA, proteins and other biomolecules have been reported to stably disperse SWCNTs into solution.23-24 In particular, surfactant-like proteins such as albumins have emerged as excellent dispersing agents for SWCNTs.14-16,25-32 Furthermore, we have shown that bovine serum albumin (BSA) proteins promote cellular entry of short SWCNTs at extremely high levels (up to millions per cell).14,31 However, the interfacial thermodynamic requirements for proteins to individually disperse SWCNTs at suitable concentrations for cellular therapy (≈ 1–100 µg/mL) limits the choice of proteins.16 The nuclear protein we have engineered for targeted delivery of short SWCNTs is the tail domain of the lamin B1 (LB1), has a globular shape, and is relatively small (molecular weight ≈ 22 kDa). LB1 lacks a three-dimensional crystal structure aside from small β-sheets formed by an immunoglobulin (Ig)-fold that also possesses a hydrophobic pocket.33-34 The small overall size along with the hydrophobic core makes LB1 highly suitable to disperse SWCNTs in water or cell


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media. Furthermore, LB1 also contains a nuclear localization sequence (NLS) at the C-terminus, which supports active import of proteins and larger materials (>40 kDa) into the nucleus through the nuclear pore complex (NPC).35 We find that LB1 can indeed disperse short SWCNTs in water (cell media), hereinafter referred to as SWCNTs-LB1, at high concentrations of ≥150 µg/mL (≥30 µg/mL) with a longterm stability of ≥75 days (≥3 days) at 4 °C (37 °C). We examine the association of LB1 with SWCNTs using molecular dynamics (MD) simulations, which reveal that the NLS exposure is unaffected by LB1 binding conformations on SWCNTs. We determine cellular and nuclear localization of SWCNTs-LB1 using near infrared (NIR) fluorescence imaging of the SWCNTs and examine molecular interactions between SWCNTs and DNA within the nucleus using fluorescence lifetime imaging microscopy (FLIM). These measurements show that SWCNTsLB1 enter HeLa cells by the millions and more importantly, translocate to the nucleus. In comparison, SWCNTs dispersed by the nonspecific protein BSA also enter cells at similarly high numbers but remain largely in the cytoplasm. This functional delivery of non-covalently dispersed SWCNTs holds the potential for other engineered protein fragments for targeted subcellular delivery.

2. EXPERIMENTAL SECTION 2.1. Protein Production and Characterization. LB1 cDNAs (from amino acid R386 to the C-terminus at amino acid M586; Figure S1, Supporting Information) were produced from full length cDNA (Accession number AAC37575) by PCR and subcloned into the Glutathione S Transferase (GST)-parallel vector, as described in previous work.36-39 The plasmids were expressed in E. coli BL21 Codon-Plus cells (Agilent) at 37 °C. Purification was performed with


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glutathione magnetic beads (Pierce) and the protein was cleaved enzymatically with proTEV cleavage enzyme (Promega) at 30 °C for 5–7 h. The cleaved protein was further purified by exposure to agarose glutathione beads (Pierce) to remove excess GST. Purified protein was dialyzed (Slide-A-Lyzer Dialysis Cassettes; Pierce) with deionized water. Concentrations were measured by Bradford assay, and protein concentration was adjusted to 5 mg/mL. Complete characterization of protein purity using mass spectroscopy and gel electrophoresis as well as structure using fluorescence spectroscopy, calorimetry, and circular dichroism have been reported elsewhere.36-39 2.2. SWCNT Dispersions. Unpurified HiPCO (high-pressure carbon monoxide conversion synthesis) SWCNTs (Carbon Nanotechnologies, Inc.) with diameters of 1.0 ± 0.3 nm and polydisperse lengths were purified according to previously established methods.40-41 The purified SWCNT sample contained

Delivering Single-Walled Carbon Nanotubes to the Nucleus Using

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