Enhancing Intracellular Optical Performance and Stability of Engineered Nanomaterials via Aqueous Two-Phase Purification.

Supramolecular hybrids of DNA and single-walled carbon nanotubes (SWCNTs) have been introduced in numerous biosensing applications due to their unique optical properties. Recent aqueous two-phase (ATP) purification methods for SWCNTs have gained popularity by introducing specificity and homogeneity into the sensor design process. Using murine macrophages probed by near-infrared and Raman microscopies, we show that ATP purification increases the retention time of DNA-SWCNTs within cells while simultaneously enhancing the optical performance and stability of the engineered nanomaterial. Over a period of 6 h, we observe 45% brighter fluorescence intensity and no significant change in emission wavelength of ATP-purified DNA-SWCNTs relative to as-dispersed SWCNTs. These findings provide strong evidence of how cells differentially process engineered nanomaterials depending on their state of purification, lending to the future development of more robust and sensitive biosensors with desirable in vivo optical parameters using surfactant-based ATP systems with a subsequent exchange to biocompatible functionalization.

Harnessing nanotechnology to expand the toolbox of chemical biology.

Although nanotechnology often addresses biomedical needs, nanoscale tools can also facilitate broad biological discovery. Nanoscale delivery, imaging, biosensing, and bioreactor technologies may address unmet questions at the interface between chemistry and biology. Currently, many chemical biologists do not include nanomaterials in their toolbox, and few investigators develop nanomaterials in the context of chemical tools to answer biological questions. We reason that the two fields are ripe with opportunity for greater synergy. Nanotechnologies can expand the utility of chemical tools in the hands of chemical biologists, for example, through controlled delivery of reactive and/or toxic compounds or signal-binding events of small molecules in living systems. Conversely, chemical biologists can work with nanotechnologists to address challenging biological questions that are inaccessible to both communities. This Perspective aims to introduce the chemical biology community to nanotechnologies that may expand their methodologies while inspiring nanotechnologists to address questions relevant to chemical biology.

Single-Chirality Near-Infrared Carbon Nanotube Sub-Cellular Imaging and FRET Probes.

Applications of single-walled carbon nanotubes (SWCNTs) in bioimaging and biosensing have been limited by difficulties with isolating single-chirality nanotube preparations with desired functionalities. Unique optical properties, such as multiple narrow near-infrared bands and several modes of signal transduction, including solvatochromism and FRET, are ideal for live cell/organism imaging and sensing applications. However, internanotube FRET has not been investigated in biological contexts. We developed single-chirality subcellular SWCNT imaging probes and investigated their internanotube FRET capabilities in live cells. To functionalize SWCNTs, we replaced the surfactant coating of aqueous two-phase extraction-sorted single-chirality nanotubes with helical polycarbodiimide polymers containing different functionalities. We achieved single-chirality SWCNT targeting of different subcellular structures, including the nucleus, to enable multiplexed imaging. We also targeted purified (6,5) and (7,6) chiralities to the same structures and observed internanotube FRET within these organelles. This work portends the use of single-chirality carbon nanotube optical probes for applications in biomedical research.

Nanoreporter of an Enzymatic Suicide Inactivation Pathway.

Enzymatic suicide inactivation, a route of permanent enzyme inhibition, is the mechanism of action for a wide array of pharmaceuticals. Here, we developed the first nanosensor that selectively reports the suicide inactivation pathway of an enzyme. The sensor is based on modulation of the near-infrared fluorescence of an enzyme-bound carbon nanotube. The nanosensor responded selectively to substrate-mediated suicide inactivation of the tyrosinase enzyme via bathochromic shifting of the nanotube emission wavelength. Mechanistic investigations revealed that singlet oxygen generated by the suicide inactivation pathway induced the response. We used the nanosensor to quantify the degree of enzymatic inactivation by measuring response rates to small molecule tyrosinase modulators. This work resulted in a new capability of interrogating a specific route of enzymatic death. Potential applications include drug screening and hit-validation for compounds that elicit or inhibit enzymatic inactivation and single-molecule measurements to assess population heterogeneity in enzyme activity.

Single Nanotube Spectral Imaging To Determine Molar Concentrations of Isolated Carbon Nanotube Species.

Electronic and biological applications of carbon nanotubes can be highly dependent on the species (chirality) of nanotube, purity, and concentration. Existing bulk methods, such as absorbance spectroscopy, can quantify sp2 carbon based on spectral bands, but nanotube length distribution, defects, and carbonaceous impurities can complicate quantification of individual particles. We present a general method to relate the optical density of a photoluminescent nanotube sample to the number of individual nanotubes. By acquiring 3-dimensional images of nanotubes embedded in a gel matrix with a reducing environment, we quantified all emissive nanotubes in a volume. Via spectral imaging, we assessed structural impurities and precisely determined molar concentrations of the (8,6) and (9,4) nanotube species. We developed an approach to obtain the molarity of any structurally enriched semiconducting single-walled carbon nanotube preparation on a per-nanotube basis.

Photoluminescent carbon nanotubes interrogate the permeability of multicellular tumor spheroids.

Nanomaterials have been extensively investigated for cancer drug delivery and imaging applications. Nanoparticles that show promise in two-dimensional cell culture systems often fail in more complex environments, possibly due to the lack of penetration in dense, three-dimensional structures. Multicellular tumor spheroids are an emerging model system to investigate interactions of nanoparticles with 3D in vitro cell culture environments. Using the intrinsic near-infrared emission of semiconducting carbon nanotubes to optically reconstruct their localization within a three-dimensional volume, we resolved the relative permeability of two different multicellular tumor spheroids. Nanotube photoluminescence revealed that nanotubes rapidly internalized into MCF-7 breast cancer cell-derived spheroids, whereas they exhibited little penetration into spheroids derived from SK-136, a cell line that we developed from murine liver cancer. Characterization of the spheroids by electron microscopy and immunohistochemistry revealed large differences in the extracellular matrix and interstitial spacing, which correlated directly with nanotube penetration. This platform portends a new approach to characterize the permeability of living multicellular environments.

Helical polycarbodiimide cloaking of carbon nanotubes enables inter-nanotube exciton energy transfer modulation.

The use of single-walled carbon nanotubes (SWCNTs) as near-infrared optical probes and sensors require the ability to simultaneously modulate nanotube fluorescence and functionally derivatize the nanotube surface using noncovalent methods. We synthesized a small library of polycarbodiimides to noncovalently encapsulate SWCNTs with a diverse set of functional coatings, enabling their suspension in aqueous solution. These polymers, known to adopt helical conformations, exhibited ordered surface coverage on the nanotubes and allowed systematic modulation of nanotube optical properties, producing up to 12-fold differences in photoluminescence efficiency. Polymer cloaking of the fluorescent nanotubes facilitated the first instance of controllable and reversible internanotube exciton energy transfer, allowing kinetic measurements of dynamic self-assembly and disassembly.

Targeting DNA G-quadruplexes with helical small molecules.

We previously identified quinoline-based oligoamide helical foldamers and a trimeric macrocycle as selective ligands of DNA quadruplexes. Their helical structures might permit targeting of the backbone loops and grooves of G-quadruplexes instead of the G-tetrads. Given the vast array of morphologies G-quadruplex structures can adopt, this might be a way to achieve sequence selective binding. Here, we describe the design and synthesis of molecules based on macrocyclic and helically folded oligoamides. We tested their ability to interact with the human telomeric G-quadruplex and an array of promoter G-quadruplexes by using FRET melting assay and single-molecule FRET. Our results show that they constitute very potent ligands--comparable to the best so far reported. Their modes of interaction differ from those of traditional tetrad binders, thus opening avenues for the development of molecules specific for certain G-quadruplex conformations.

Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles.

Nanomaterials are the subject of a range of biomedical, commercial, and environmental investigations involving measurements in living cells and tissues. Accurate quantification of nanomaterials, at the tissue, cell, and organelle levels, is often difficult, however, in part due to their inhomogeneity. Here, we propose a method that uses the distinct optical properties of a heterogeneous nanomaterial preparation in order to improve quantification at the single-cell and organelle level. We developed "hyperspectral counting", which employs diffraction-limited imaging via hyperspectral microscopy of a diverse set of fluorescent nanomaterials to estimate particle number counts in live cells and subcellular structures. A mathematical model was developed, and Monte Carlo simulations were employed, to improve the accuracy of these estimates, enabling quantification with single-cell and single-endosome resolution. We applied this nanometrology technique with single-walled carbon nanotubes and identified an upper limit of the rate of uptake into cells─approximately 3,000 nanotubes endocytosed within 30 min. In contrast, conventional region-of-interest counting results in a 230% undercount. The method identified significant heterogeneity and a broad non-Gaussian distribution of carbon nanotube uptake within cells. For example, while a particular cell contained an average of 1 nanotube per endosome, the heterogeneous distribution resulted in over 7 nanotubes localizing within some endosomes, substantially changing the accounting of subcellular nanoparticle concentration distributions. This work presents a method to quantify the cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for the nanotoxicology, drug/gene delivery, and nanosensor fields.

En route to single-step, two-phase purification of carbon nanotubes facilitated by high-throughput spectroscopy.

Chirality purification of single-walled carbon nanotubes (SWCNTs) is desirable for applications in many fields, but general utility is currently hampered by low throughput. We discovered a method to obtain single-chirality SWCNT enrichment by the aqueous two-phase extraction (ATPE) method in a single step. To achieve appropriate resolution, a biphasic system of non-ionic tri-block copolymer surfactant is varied with an ionic surfactant. A nearly-monochiral fraction of SWCNTs can then be harvested from the top phase. We also found, via high-throughput, near-infrared excitation-emission photoluminescence spectroscopy, that the parameter space of ATPE can be mapped to probe the mechanics of the separation process. Finally, we found that optimized conditions can be used for sorting of SWCNTs wrapped with ssDNA as well. Elimination of the need for surfactant exchange and simplicity of the separation process make the approach promising for high-yield generation of purified single-chirality SWCNT preparations.

G-quadruplex DNA bound by a synthetic ligand is highly dynamic.

Using single molecule fluorescence resonance energy transfer, we investigated the interaction between a quadruplex-binding ligand and the human telomeric G-quadruplex. The binding of quinolinecarboxamide macrocycle to telomeric DNA was essentially irreversible and selectively induced and favored one quadruplex conformation. The ligand-quadruplex complex displayed intramolecular dynamics including quadruplex folding and unfolding in the absence of ligand association and dissociation. We report that the G-quadruplex can be stabilized without preventing the intrinsic intramolecular dynamics of telomeric DNA.

Optical Voltammetry of Polymer-Encapsulated Single-Walled Carbon Nanotubes.

The semiconducting single-walled carbon nanotube (SWCNT), noncovalently wrapped by a polymeric monolayer, is a nanoscale semiconductor-electrolyte interface under investigation for sensing, photonics, and photovoltaic applications. SWCNT complexes are routinely observed to sensitize various electrochemical/redox phenomena, even in the absence of an external field. While the photoluminescence response to gate voltage depends on the redox potential of the nanotube, analogous optical voltammetry of functionalized carbon nanotubes could be conducted in suspension without applying voltage but by varying the solution conditions as well as the chemistry of the encapsulating polymer. Steady-state photoluminescence, absorbance, and in situ measurements of O2/H2O reactivity show correlation with the pH/pK a-dependent reactivity of π-rich coatings. The nanotube emission responses suggest that the presence of photogenerated potential may explain the observed coating electrochemical reactivity. This work finds that electronic and chemical interactions of the nanotube with the encapsulating polymer may play a critical role in applications that depend on radiative recombination, such as optical sensing.

Synthetic molecular recognition nanosensor paint for microalbuminuria.

Microalbuminuria is an important clinical marker of several cardiovascular, metabolic, and other diseases such as diabetes, hypertension, atherosclerosis, and cancer. The accurate detection of microalbuminuria relies on albumin quantification in the urine, usually via an immunoturbidity assay; however, like many antibody-based assessments, this method may not be robust enough to function in global health applications, point-of-care assays, or wearable devices. Here, we develop an antibody-free approach using synthetic molecular recognition by constructing a polymer to mimic fatty acid binding to the albumin, informed by the albumin crystal structure. A single-walled carbon nanotube, encapsulated by the polymer, as the transduction element produces a hypsochromic (blue) shift in photoluminescence upon the binding of albumin in clinical urine samples. This complex, incorporated into an acrylic material, results in a nanosensor paint that enables the detection of microalbuminuria in patient samples and comprises a rapid point-of-care sensor robust enough to be deployed in resource-limited settings.

An optical nanoreporter of endolysosomal lipid accumulation reveals enduring effects of diet on hepatic macrophages in vivo.

The abnormal accumulation of lipids within the endolysosomal lumen occurs in many conditions, including lysosomal storage disorders, atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and drug-induced phospholipidosis. Current methods cannot monitor endolysosomal lipid content in vivo, hindering preclinical drug development and research into the mechanisms linking endolysosomal lipid accumulation to disease progression. We developed a single-walled carbon nanotube-based optical reporter that noninvasively measures endolysosomal lipid accumulation via bandgap modulation of its intrinsic near-infrared emission. The reporter detected lipid accumulation in Niemann-Pick disease, atherosclerosis, and NAFLD models in vivo. By applying the reporter to the study of NAFLD, we found that elevated lipid quantities in hepatic macrophages caused by a high-fat diet persist long after reverting to a normal diet. The reporter dynamically monitored endolysosomal lipid accumulation in vivo over time scales ranging from minutes to weeks, indicating its potential to accelerate preclinical research and drug development processes.

Progress Towards Applications of Carbon Nanotube Photoluminescence.

In the fifteen years following the discovery of single-walled carbon nanotube (SWCNT) photoluminescence, investigators have made significant progress in their understanding of the phenomenon and towards the development of applications. The intrinsic potential of semiconducting carbon nanotubes - a family of bright, photostable near infrared (NIR) fluorophores (900-2100 nm) with tunable properties, has motivated their use as optical probes and sensors. In this perspective, we highlight the advances made in the synthesis, processing, modification, separation, and metrology of carbon nanotubes in the context of applications of their photoluminescence.

DNA-Carbon Nanotube Complexation Affinity and Photoluminescence Modulation Are Independent.

Short single-stranded DNA (ssDNA) has emerged as the natural polymer of choice for noncovalently functionalizing photoluminescent single-walled carbon nanotubes. In addition, specific empirically identified DNA sequences can be used to separate single species (chiralities) of nanotubes, with an exceptionally high purity. Currently, only limited general principles exist for designing DNA-nanotube hybrids amenable to separation processes, due in part to an incomplete understanding of the fundamental interactions between a DNA sequence and a specific nanotube structure, whereas even less is known in the design of nanotube-based sensors with determined optical properties. We therefore developed a combined experimental and analysis platform on the basis of time-resolved near-infrared fluorescence spectroscopy to extract the complete set of photoluminescence parameters that characterizes DNA-nanotube hybrids. Here, we systematically investigated the affinity of the d(GT)n oligonucleotide family for structurally defined carbon nanotubes by measuring photoluminescence response of the nanotube upon oligonucleotide displacement. We found, surprisingly, that the rate of displacement of the oligonucleotides is independent of the coverage on the nanotube, as inferred through the intrinsic optical properties of the hybrid. The kinetics of intensity modulation is essentially a single-exponential, and the time constants, which quantify the stability of DNA binding, span an order of magnitude. Surprisingly, these time constants do not depend on the intrinsic optical parameters within the hybrids, suggesting that the DNA-nanotube stability is not due to increased nanotube surface coverage by DNA. Further, a principal component analysis of the excitation and emission shifts along with intensity enhancement at equilibrium accurately identified the (8,6) nanotube as the partner chirality to (GT)6 ssDNA. When combined, the chirality-resolved equilibrium and kinetics data can guide the development of the DNA-nanotube pairs, with tunable stability and optical modulation. Additionally, this high-throughput optical platform could function as a primary screen for mapping the DNA-chirality recognition phase space.

A Carbon Nanotube Optical Sensor Reports Nuclear Entry via a Noncanonical Pathway.

Single-walled carbon nanotubes are of interest in biomedicine for imaging and molecular sensing applications and as shuttles for various cargos such as chemotherapeutic drugs, peptides, proteins, and oligonucleotides. Carbon nanotube surface chemistry can be modulated for subcellular targeting while preserving photoluminescence for label-free visualization in complex biological environments, making them attractive materials for such studies. The cell nucleus is a potential target for many pathologies including cancer and infectious diseases. Understanding mechanisms of nanomaterial delivery to the nucleus may facilitate diagnostics, drug development, and gene-editing tools. Currently, there are no systematic studies to understand how these nanomaterials gain access to the nucleus. Herein, we developed a carbon nanotube based hybrid material that elucidate a distinct mechanism of nuclear translocation of a nanomaterial in cultured cells. We developed a nuclear-targeted probe via cloaking photoluminescent single-walled carbon nanotubes in a guanidinium-functionalized helical polycarbodiimide. We found that the nuclear entry of the nanotubes was mediated by the import receptor importin ß without the aid of importin α and not by the more common importin α/ß pathway. Additionally, the nanotube photoluminescence exhibited distinct red-shifting upon entry to the nucleus, potentially functioning as a reporter of the importin ß-mediated nuclear transport process. This work delineates a noncanonical mechanism for nanomaterial delivery to the nucleus and provides a reporter for the study of nucleus-related pathologies.

A Carbon Nanotube Reporter of miRNA Hybridization Events In Vivo.

MicroRNAs and other small oligonucleotides in biofluids are promising disease biomarkers, yet conventional assays require complex processing steps that are unsuitable for point-of-care testing or for implantable or wearable sensors. Single-walled carbon nanotubes are an ideal material for implantable sensors, owing to their emission in the near-infrared spectral region, photostability and exquisite sensitivity. Here, we report an engineered carbon-nanotube-based sensor capable of real-time optical quantification of hybridization events of microRNA and other oligonucleotides. The mechanism of the sensor arises from competitive effects between displacement of both oligonucleotide charge groups and water from the nanotube surface, which result in a solvatochromism-like response. The sensor, which allows for detection via single-molecule sensor elements and for multiplexing by using multiple nanotube chiralities, can monitor toehold-based strand-displacement events, which reverse the sensor response and regenerate the sensor complex. We also show that the sensor functions in whole urine and serum, and can non-invasively measure DNA and microRNA after implantation in live mice.

A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux.

Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, that responds to lipid accumulation via modulation of the nanotube's optical band gap. The engineered nanomaterial, composed of short, single-stranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis, was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann-Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned subcellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone-marrow-derived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers additional capabilities for drug development processes and the investigation of lipid-linked diseases.

Cell Membrane Proteins Modulate the Carbon Nanotube Optical Bandgap via Surface Charge Accumulation.

Cell adhesion is a protein-mediated process intrinsic to most living organisms. Dysfunction in cell adhesion processes is implicated in various diseases, including thrombosis and metastatic cancers. Using an approach to resolve spectral features from cell membrane-associated photoluminescent single-walled carbon nanotubes, we found that nanotube optical bandgaps respond to the electrostatic potential of the cell surface, which corresponds to cell adhesion properties. We studied the carbon nanotube emission energy response to solution ionic potentials, which suggests sensitivity to local charge accumulation. We conclude that nanotubes respond to cell surface electrostatic potentials that are mediated by membrane proteins, which vary significantly across cell types. These findings portend the optical measurement of surface electrostatic potentials for biophysical measurements and biomedical applications.