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Spectral fingerprinting has emerged as a powerful tool that is adept at identifying chemical compounds and deciphering complex interactions within cells and engineered nanomaterials. Using near-infrared (NIR) fluorescence spectral fingerprinting coupled with machine learning techniques, we uncover complex interactions between DNA-functionalized single-walled carbon nanotubes (DNA-SWCNTs) and live macrophage cells, enabling in situ phenotype discrimination. Utilizing Raman microscopy, we showcase statistically higher DNA-SWCNT uptake and a significantly lower defect ratio in M1 macrophages compared to M2 and naive phenotypes. NIR fluorescence data also indicate that distinctive intraendosomal environments of these cell types give rise to significant differences in many optical features, such as emission peak intensities, center wavelengths, and peak intensity ratios. Such features serve as distinctive markers for identifying different macrophage phenotypes. We further use a support vector machine (SVM) model trained on SWCNT fluorescence data to identify M1 and M2 macrophages, achieving an impressive accuracy of >95%. Finally, we observe that the stability of DNA-SWCNT complexes, influenced by DNA sequence length, is a crucial consideration for applications, such as cell phenotyping or mapping intraendosomal microenvironments using AI techniques. Our findings suggest that shorter DNA-sequences like GT 6 give rise to more improved model accuracy (>87%) due to increased active interactions of SWCNTs with biomolecules in the endosomal microenvironment. Implications of this research extend to the development of nanomaterial-based platforms for cellular identification, holding promise for potential applications in real time monitoring of in vivo cellular differentiation.

Spectral fingerprinting has emerged as a powerful tool, adept at identifying chemical compounds and deciphering complex interactions within cells and engineered nanomaterials. Using near-infrared (NIR) fluorescence spectral fingerprinting coupled with machine learning techniques, we uncover complex interactions between DNA-functionalized single-walled carbon nanotubes (DNA-SWCNTs) and live macrophage cells, enabling in situ phenotype discrimination. Through the use of Raman microscopy, we showcase statistically higher DNA-SWCNT uptake and a significantly lower defect ratio in M1 macrophages as compared to M2 and naïve phenotypes. NIR fluorescence data also indicate that distinctive intra-endosomal environments of these cell types give rise to significant differences in many optical features such as emission peak intensities, center wavelengths, and peak intensity ratios. Such features serve as distinctive markers for identifying different macrophage phenotypes. We further use a support vector machine (SVM) model trained on SWCNT fluorescence data to identify M1 and M2 macrophages, achieving an impressive accuracy of > 95%. Finally, we observe that the stability of DNA-SWCNT complexes, influenced by DNA sequence length, is a crucial consideration for applications such as cell phenotyping or mapping intra-endosomal microenvironments using AI techniques. Our findings suggest that shorter DNA-sequences like GT 6 give rise to more improved model accuracy (> 87%) due to increased active interactions of SWCNTs with biomolecules in the endosomal microenvironment. Implications of this research extend to the development of nanomaterial-based platforms for cellular identification, holding promise for potential applications in real time monitoring of in vivo cellular differentiation.

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.

Significant advances have been made in fields such as nanotechnology and biomedicine using the unique properties of single-walled carbon nanotubes (SWCNTs). Specifically, SWCNTs are used as near-infrared fluorescence sensors in the solution phase to detect a wide array of biologically relevant analytes. However, solution-based sensing has several limitations, including limited sensitivity and poor spatial resolution. We have therefore devised a new spin-coated poly(ethylene glycol) diacrylate (PEG-DA) hydrogel platform to examine individual DNA-functionalized SWCNTs (DNA-SWCNTs) in their native aqueous state and have subsequently used this platform to investigate the temporal modulations of each SWCNT in response to a model analyte. A strong surfactant, sodium deoxycholate (SDC), was chosen as the model analyte as it rapidly exchanges with DNA oligonucleotides on the SWCNT surface, modulating several optical properties of the SWCNTs and demonstrating multiparameter analyte detection. Upon addition of SDC, we observed time-dependent spectral modulations in the emission center wavelengths and peak intensities of the individual SWCNTs, indicative of a DNA-to-surfactant exchange process. Interestingly, we found that the modulations in the peak intensities, as determined by kinetic data, were significantly delayed when compared to their center wavelength counterparts, suggesting a potential decoupling of the response of these two spectral features. We used a 1-D diffusion model to relate the local SDC concentration to the spectral response of each SWCNT and created dose-response curves. The peak intensity shifts at a higher SDC concentration than the center wavelength, indicating a potential change in the conformation of the surfactant molecules adsorbed to the SWCNT sidewall after the initial exchange process. This platform allows for a unique single-molecule analysis technique that is significantly more sensitive and modifiable than utilizing SWCNTs in the solution phase.

The non-covalent biomolecular functionalization of fluorescent single-walled carbon nanotubes (SWCNTs) has resulted in numerous in vitro and in vivo sensing and imaging applications due to many desirable optical properties. In these applications, it is generally presumed that pristine, singly dispersed SWCNTs interact with and enter live cells at the so-called nano-biointerface, for example, the cell membrane. Despite numerous fundamental studies published on this presumption, it is known that nanomaterials have the propensity to aggregate in protein-containing environments before ever contacting the nano-biointerface. Here, using DNA-functionalized SWCNTs with defined degrees of aggregation as well as near-infrared hyperspectral microscopy and toxicological assays, we show that despite equal rates of internalization, initially aggregated SWCNTs do not further accumulate within individual subcellular locations. In addition to subcellular accumulations, SWCNTs initially with a low degree of aggregation can induce significant deleterious effects in various long-term cytotoxicity and real-time proliferation assays, which are markedly different when compared to those of SWCNTs that are initially aggregated. These findings suggest the importance of the aggregation state as a critical component related to intracellular processing and toxicological response of engineered nanomaterials.

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.

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.

Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials (ENMs) which gain cellular entry via active endocytic processes. A recent emphasis on routes of ENM uptake has established key physicochemical properties which direct certain mechanisms, yet relatively few studies have identified their effect on intracellular trafficking processes past entry and initial subcellular localization. Here, we developed and applied an approach where single-walled carbon nanotubes (SWCNTs) play a dual role-that of an ENM undergoing intracellular processing, in addition to functioning as the signal transduction element reporting these events in individual cells with single organelle resolution. We used the exceptional optical properties exhibited by noncovalent hybrids of single-stranded DNA and SWCNTs (DNA-SWCNTs) to report the progression of intracellular processing events via two orthogonal hyperspectral imaging approaches of near-infrared (NIR) fluorescence and resonance Raman scattering. A positive correlation between fluorescence and G-band intensities was uncovered within single cells, while exciton energy transfer and eventual aggregation of DNA-SWCNTs were observed to scale with increasing time after internalization. An analysis pipeline was developed to colocalize and deconvolute the fluorescence and Raman spectra of subcellular regions of interest (ROIs), allowing for single-chirality component spectra to be obtained with submicron spatial resolution. This approach uncovered correlations between DNA-SWCNT concentration, dielectric modulation, and irreversible aggregation within single intracellular vesicles. An immunofluorescence assay was designed to directly observe the DNA-SWCNTs in labeled endosomal vesicles, revealing a distinct relationship between the physical state of organelle-bound DNA-SWCNTs and the dynamic luminal conditions during endosomal maturation processes. Finally, we trained a machine learning algorithm to predict endosome type using the Raman spectra of the vesicle-bound DNA-SWCNTs, enabling major components in the endocytic pathway to be simultaneously visualized using a single intracellular reporter.

Single-walled carbon nanotubes (SWCNTs) have been used in a variety of sensing and imaging applications over the past few years due to their unique optical properties. In the solution phase, SWCNTs are employed as near-infrared (NIR) fluorescence-based sensors of target analytes via modulations in emission intensity and/or wavelength. In an effort to lower the limit of detection, research has been conducted into isolating SWCNTs adhered to surfaces for potential single molecule analyte detection. However, it is known that SWCNT fluorescence is adversely affected by the inherently rough surfaces that are conventionally used for their observation (e.g., glass coverslip), potentially interfering with fluorescence-based analyte detection. Here, using a spin-coating method with thin films of alginate and SWCNTs, we demonstrate that a novel hydrogel platform can be created to investigate immobilized individual SWCNTs without significantly perturbing their optical properties as compared to solution-phase values. In contrast to the glass coverslip, which red-shifted DNA-functionalized (6,5)-SWCNTs by an average of 3.4 nm, the hydrogel platform reported emission wavelengths that statistically matched the solution-phase values. Additionally, the heterogeneity in the wavelength measurements, as determined from the width of created histograms, was reduced nearly by a factor of 3 for the SWCNTs in the hydrogel platform when compared to glass coverslips. Using long SWCNTs, i.e., those with an average length above the diffraction limit of our microscope, we show that a glass coverslip can induce optical heterogeneity along the length of a single SWCNT regardless of its surface functionalization. This is again significantly mitigated when examining the long SWCNTs in the hydrogel platform. Finally, we show that upon the addition of a model analyte (calcium chloride), the optical response can be spatially resolved along the length of a single SWCNT, enabling localized analyte detection on the surface of a single nanoscale sensor.

The sequence and timing of growth factor delivery plays a crucial role in bone regeneration. While a variety of biomaterial scaffolds have been developed to provide multiple growth factor deliveries, there still exists a strong need for on-demand control over sequential delivery profiles to optimize regenerative outcomes. One particular growth factor, bone morphogenetic protein-2 (BMP-2), has established effects in the osteodifferentiation process; however, the optimal timing of its delivery is not yet known. Here, we investigate the effect of the timing of BMP-2 delivery on osteodifferentiation on both 2D and 3D cell cultures in vitro. It was shown that immediate BMP-2 delivery inhibited mouse mesenchymal stem cell (mMSC) proliferation and therefore resulted in suboptimal levels of mMSC osteodifferentiation (as measured by alkaline phosphatase activity) compared to mMSC cultures exposed to delayed BMP-2 delivery (4 day delay). Because of this, we aimed to develop a biomaterial system capable of rapidly recruiting mMSCs and exposing them to BMP-2 in a delayed manner (i.e., after a strong mMSC population has been established). This biomaterial system consisted of (i) an outer porous gelatin compartment that could be loaded with an mMSC recruitment factor (stromal cell-derived factor 1-α (SDF-1α)) for rapid establishment of a 3D mMSC culture and (ii) an inner ferrogel compartment that could deliver BMP-2 in an immediate or delayed manner, depending on when magnetic stimulation was applied. It was shown that the outer compartment was able to recruit and harbor mMSCs and that the rapidity of this recruitment could be enhanced by loading the compartment with SDF-1α. The inner ferrogel compartment enabled magnetically triggered release of BMP-2 where the timing of release could be remotely controlled from immediate to a delay of up to 11 days. This hydrogel system provides controllability over the timing between bone progenitor recruitment and osteodifferentiation factor release and can thus potentially enhance therapies that require new bone growth by optimizing the timing of these deliveries.

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 O 2 /H 2 O reactivity show correlation with the pH/p K 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.

Noncovalent hybrids of single-stranded DNA and single-walled carbon nanotubes (SWCNTs) have demonstrated applications in biomedical imaging and sensing due to their enhanced biocompatibility and photostable, environmentally responsive near-infrared (NIR) fluorescence. The fundamental properties of such DNA-SWCNTs have been studied to determine the correlative relationships between oligonucleotide sequence and length, SWCNT species, and the physical attributes of the resultant hybrids. However, intracellular environments introduce harsh conditions that can change the physical identities of the hybrid nanomaterials, thus altering their intrinsic optical properties. Here, through visible and NIR fluorescence imaging in addition to confocal Raman microscopy, we show that the oligonucleotide length controls the relative uptake, intracellular optical stability, and retention of DNA-SWCNTs in mammalian cells. Although the absolute NIR fluorescence intensity of DNA-SWCNTs in murine macrophages increases with increasing oligonucleotide length (from 12 to 60 nucleotides), we found that shorter oligonucleotide DNA-SWCNTs undergo a greater magnitude of spectral shift and are more rapidly internalized and expelled from the cell after 24 h. Furthermore, by labeling the DNA with a fluorophore that dequenches upon removal from the SWCNT surface, we found that shorter oligonucleotide strands are displaced from the SWCNT within the cell, altering the physical identity and changing the fate of the internalized nanomaterial. Finally, through a pharmacological inhibition study, we identified the mechanism of SWCNT expulsion from the cells as lysosomal exocytosis. These findings provide a fundamental understanding of the interactions between SWCNTs and live cells as well as evidence suggesting the ability to control the biological fate of the nanomaterials merely by varying the type of DNA wrapping.

Single-walled carbon nanotubes (SWCNTs) have recently been utilized as fillers that reduce the flammability and enhance the strength and thermal conductivity of material composites. Enhancing the thermal stability of SWCNTs is crucial when these materials are applied to high temperature applications. In many instances, SWCNTs are applied to composites with surface coatings that are toxic to living organisms. Alternatively, single-stranded DNA, a naturally occurring biological polymer, has recently been utilized to form singly-dispersed hybrids with SWCNTs as well as suppress their known toxicological effects. These hybrids have shown unrivaled stabilities in both aqueous suspension or as a dried material. Furthermore, DNA has certain documented flame-retardant effects due to the creation of a protective char upon heating in the presence of oxygen. Herein, using various thermogravimetric analytical techniques, we find that single-stranded DNA has a significant flame-retardant effect on the SWCNTs, and effectively enhances their thermal stability. Hybridization with DNA results in the elevation of the thermal decomposition temperature of purified SWCNTs in excess of 200 °C. We translate this finding to other carbon nanomaterials including multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (RGO) and fullerene (C 60 ), and show similar effects upon complexation with DNA. The rate of thermal decomposition of the SWCNTs was also explored and found to significantly depend upon the sequence of DNA that was used.

Single-walled carbon nanotubes (SWCNTs) functionalized with short single-stranded DNA have been extensively studied within the last decade for biomedical applications due to the high dispersion efficiency and intrinsic biocompatibility of DNA as well as the photostable and tunable fluorescence of SWCNTs. Characterization of their physical properties, particularly their length distribution, is of great importance regarding their application as a bioengineered research tool and clinical diagnostic agent. Conventionally, atomic force microscopy (AFM) has been used to quantify the length of DNA-SWCNTs by depositing the hybrids onto an electrostatically charged flat surface. Here, we demonstrate that hybrids of DNA-SWCNTs with different oligomeric DNA sequences ((GT) 6 and (GT) 30 ) differentially deposit on the AFM substrate, resulting in significant inaccuracies in the reported length distributions of the parent solutions. Using a solution-based surfactant exchange technique, we placed both samples into a common surfactant wrapping and found identical SWCNT length distributions upon surface deposition. Additionally, by spin-coating the surfactant-wrapped SWCNTs onto a substrate, thus mitigating effects of electrostatic interactions, we found length distributions that did not depend on DNA sequence but were significantly longer than electrostatic deposition methods, illuminating the inherent bias of the surface deposition method. Quantifying the coverage of DNA molecules on each SWCNT through both absorbance spectroscopy and direct observation, we found that the density of DNA per SWCNT was significantly higher in short (GT) 6 -SWCNTs (length < 100 nm) compared to long (GT) 6 -SWCNTs (length > 100 nm). In contrast, we found no dependence of the DNA density on SWCNT length in (GT) 30 -SWCNT hybrids. Thus, we attribute differences in the observed length distributions of DNA-SWCNTs to variations in electrostatic repulsion induced by sequence-dependent DNA density.

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., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection.

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.

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.

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 β without the aid of importin α and not by the more common importin α/β pathway. Additionally, the nanotube photoluminescence exhibited distinct red-shifting upon entry to the nucleus, potentially functioning as a reporter of the importin β-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.

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 sp 2 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.

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.

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., Competing Interests: Competing Financial Interests The authors have no competiting financial interests to declare.

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.

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.

The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species' fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo, and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration, and single-molecule resolution in vivo.

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.

Biological polymers hybridized with single-walled carbon nanotubes (SWCNTs) have elicited much interest recently for applications in SWCNT-based sorting as well as biomedical imaging, sensing, and drug delivery. Recently, de novo designed peptides forming a coiled-coil structure have been engineered to selectively disperse SWCNT of a certain diameter. Here we report on a study of the binding strength and structural stability of the hybrid between such a "HexCoil-Ala" peptide and the (6,5)-SWCNT. Using the competitive binding of a surfactant, we find that affinity strength of the peptide ranks in comparison to that of two single-stranded DNA sequences as (GT) 30 -DNA > HexCoil-Ala > (TAT) 4 T-DNA. Further, using replica exchange molecular dynamics (REMD), we show that the hexamer peptide complex has both similarities with and differences from the original design. While one of two distinct helix-helix interfaces of the original model was largely retained, a second interface showed much greater variability. These conformational differences allowed an aromatic tyrosine residue designed to lie along the solvent-exposed surface of the protein instead to penetrate between the two helices and directly contact the SWCNT. These insights will inform future designs of SWCNT-interacting peptides.

The single-stranded DNA to single-walled carbon nanotube (SWCNT) hybrid continues to attract significant interest as an exemplary biological molecule-nanomaterial conjugate. In addition to their many biomedical uses, such as in vivo sensing and delivery of molecular cargo, DNA-SWCNT hybrids enable the sorting of SWCNTs according to their chirality. Current experimental methods have fallen short of identifying the actual structural ensemble of DNA adsorbed onto SWCNTs that enables and controls several of these phenomena. Molecular dynamics (MD) simulation has been a useful tool for studying the structure of these hybrid molecules. In recent studies, using replica exchange MD (REMD) simulation we have shown that novel secondary structures emerge and that these structures are DNA-sequence and SWCNT-type dependent. Here, we use REMD to investigate in detail the structural characteristics of two DNA-SWCNT recognition pairs: (TAT)(4)-(6,5)-SWCNT, i.e., DNA sequence TATTATTATTAT bound to the (6,5) chirality SWCNT, and (CCG)(2)CC-(8,7)-SWCNT as well as off-recognition pairs (TAT)(4)-(8,7)-SWCNT and (CCG)(2)CC-(6,5)-SWCNT. From a structural clustering analysis, dominant equilibrium structures are identified and show a right-handed self-stitched motif for (TAT)(4)-(6,5) in contrast to a left-handed β-barrel for (CCG)(2)CC-(8,7). Additionally, characteristics such as DNA end-to-end distance, solvent accessible SWCNT surface area, DNA hydrogen bonding between bases, and DNA dihedral distributions have been probed in detail as a function of the number of DNA strands adsorbed onto the nanotube. We find that the DNA structures adsorbed onto a nanotube are also stabilized by significant numbers of non-Watson-Crick hydrogen bonds (intrastrand and interstrand) in addition to π-π stacking between DNA bases and nanotube surface and Watson-Crick pairs. Finally, we provide a summary of DNA structures observed for various DNA-SWCNT hybrids as a preliminary set of motifs that may be involved in the functional role of these hybrids.

Several applications of single-walled carbon nanotubes (SWCNT) as nanovectors in biological systems have been reported, and several molecular pathways of cellular entry have been proposed. We employed transmission electron microscopy, confocal fluorescent microscopy, and UV-vis spectroscopic analysis to confirm the internalization of DNA-SWCNT in human umbilical vein endothelial cells. Additionally, by using pharmacological inhibitors as well as genetic approaches, we have found that SWCNT is endocytosed through Rac1- GTPase mediated macropinocytosis in normal endothelial cells., (© 2012 American Chemical Society)

Hybrids of biological molecules and single-walled carbon nanotubes (SWCNT) have proven useful for SWCNT sorting and are enabling several biomedical applications in sensing, imaging, and drug delivery. In the DNA-SWCNT system, certain short (10-20mer) sequences of single-stranded DNA recognize specific SWCNT, allowing the latter to be sorted from a chirality diverse mixture. (1) However, little is known about the DNA secondary structures that underlie their recognition of SWCNTs. Using replica exchange molecular dynamics (REMD) of multiple strands on a single SWCNT, we report that DNA forms ordered structures on SWCNTs that are strongly DNA sequence and SWCNT dependent. DNA sequence (TAT)(4) on its recognition partner, the (6,5) SWCNT, (1) forms an ordered right-handed helically wrapped barrel, stabilized by intrastrand, self-stitching hydrogen bonds and interstrand hydrogen bonding. The same sequence on the larger diameter (8,7)-SWCNT forms a different and less-stable structure, demonstrating SWCNT selectivity. In contrast, homopolymer (T)(12), with weaker tendency for intrastrand hydrogen bonding, forms a distinctly left-handed wrap on the (6,5)-SWCNT, demonstrating DNA sequence specificity. Experimental measurements show that (TAT)(4) selectively disperses smaller diameter SWCNTs more efficiently than (T)(12), establishing a relationship between recognition motifs and binding strength. The developing understanding of DNA secondary structure on nanomaterials can shed light on a number of issues involving hybrids of nanomaterials and biological molecules, including nanomedicine, health-effects of nanomaterials, and nanomaterial processing., (© 2012 American Chemical Society)

The DNA-single-walled carbon nanotube (SWCNT) hybrid molecule has attracted significant attention recently for its ability to disperse and sort SWCNTs according to their chirality. Key for utilizing their unique properties is an understanding of the structure of DNA adsorbed on the SWCNT surface, which we study here using molecular simulations. Using replica exchange molecular dynamics (REMD), we explore equilibrium structures formed by single strands of 12-mer oligonucleotides, of varying sequence, adsorbed on a (6,5)-SWCNT. We find a consistent motif in which the DNA strand forms a right-handed helical wrap around the SWCNT, stabilized by "stitches" (hydrogen bonding between distant bases) to itself. Variability among equilibrium populations of DNA self-stitched structures was observed and shown to be directly influenced by DNA sequence and composition. Competition between conformational entropy and hydrogen bonding between bases is predicted to be responsible for the formation of random versus stitched configurations.

The ability to sort mixtures of carbon nanotubes (CNTs) based on chirality has recently been demonstrated using special short DNA sequences that recognize certain matching CNTs of specific chirality. In this work, we report on a study of the relationship between recognition sequences and the strength of their binding to the recognized CNT. We have chosen the (6,5) CNT and its corresponding DNA recognition sequences for investigation in this study. Binding strength is quantified by studying the kinetics of DNA replacement by a surfactant, which is monitored by following shifts in the absorption spectrum. We find that recognition ability correlates strongly with binding strength thus measured; addition or subtraction of just one base from the recognition sequence can enhance the kinetics of DNA displacement some 20-fold. The surfactant displaces DNA in two steps: a rapid first stage lasting less than a few seconds, followed by progressive removal lasting tens of minutes. The kinetics of the second stage is analyzed to extract activation energies. Fluorescence studies support the finding that the DNA sequence that recognizes the (6,5)-CNT forms a more stable hybrid than its close relatives., (© 2011 American Chemical Society)