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.

Long-term in vivo biocompatibility of single-walled carbon nanotubes.

Over the past two decades, measurements of carbon nanotube toxicity and biodistribution have yielded a wide range of results. Properties such as nanotube type (single-walled vs. multi-walled), purity, length, aggregation state, and functionalization, as well as route of administration, greatly affect both the biocompatibility and biodistribution of carbon nanotubes. These differences suggest that generalizable conclusions may be elusive and that studies must be material- and application-specific. Here, we assess the short- and long-term biodistribution and biocompatibility of a single-chirality DNA-encapsulated single-walled carbon nanotube complex upon intravenous administration that was previously shown to function as an in-vivo reporter of endolysosomal lipid accumulation. Regarding biodistribution and fate, we found bulk specificity to the liver and >90% signal attenuation by 14 days in mice. Using near-infrared hyperspectral microscopy to measure single nanotubes, we found low-level, long-term persistence in organs such as the heart, liver, lung, kidney, and spleen. Measurements of histology, animal weight, complete blood count; biomarkers of organ function all suggest short- and long-term biocompatibility. This work suggests that carbon nanotubes can be used as preclinical research tools in-vivo without affecting acute or long-term health.

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.

Noninvasive ovarian cancer biomarker detection via an optical nanosensor implant.

Patients with high-grade serous ovarian carcinoma (HGSC) exhibit poor 5-year survival rates, which may be significantly improved by early-stage detection. The U.S. Food and Drug Administration-approved biomarkers for HGSC-CA-125 (cancer antigen 125) and HE4 (human epididymis protein 4)-do not generally appear at detectable levels in the serum until advanced stages of the disease. An implantable device placed proximal to disease sites, such as in or near the fallopian tube, ovary, uterine cavity, or peritoneal cavity, may constitute a feasible strategy to improve detection of HGSC. We engineered a prototype optical sensor composed of an antibody-functionalized carbon nanotube complex, which responds quantitatively to HE4 via modulation of the nanotube optical bandgap. The complexes measured HE4 with nanomolar sensitivity to differentiate disease from benign patient biofluids. The sensors were implanted into four models of ovarian cancer, within a semipermeable membrane, enabling the optical detection of HE4 within the live animals. We present the first in vivo optical nanosensor capable of noninvasive cancer biomarker detection in orthotopic models of disease.

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.

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.

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.

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.

Polymer cloaking modulates the carbon nanotube protein corona and delivery into cancer cells.

Carbon nanotube-based molecular probes, imaging agents, and biosensors in cells and in vivo continue to garner interest as investigational tools and clinical devices due to their unique photophysical properties. Surface chemistry modulation of nanotubes plays a critical role in determining stability and interaction with biological systems both in vitro and in vivo. Among the many parameters that influence the biological fate of nanomaterials, surface charge is particularly influential due to direct electrostatic interactions with components of the cell membrane as well as proteins in the serum, which coat the nanoparticle surface in a protein corona and alter nanoparticle-cell interactions. Here, we modulated functional moieties on a helical polycarbodiimide polymer backbone that non-covalently suspended the nanotubes in aqueous media. By derivatizing the polymer with either primary amine or carboxylic acid side chains, we obtained nanotube complexes that present net surface charges of opposite polarity at physiological pH. Using these materials, we found that the uptake of carbon nanotubes in these cells is highly dependent on charge, with cationic nanotubes efficiently internalized into cells compared to the anionic nanotubes. Furthermore, we found that serum proteins drastically influenced cell uptake of the anionic nanotubes, while the effect was not prominent for the cationic nanotubes. Our findings have implications for improved engineering of drug delivery devices, molecular probes, and biosensors.

Embryonically inspired scaffolds regulate tenogenically differentiating cells.

Tendon injuries heal as scar tissue with significant dysfunction and propensity to re-injure, motivating efforts to develop stem cell-based therapies for tendon regeneration. For these therapies to succeed, effective cues to guide tenogenesis are needed. Our aim is to identify these cues within the embryonic tendon microenvironment. We recently demonstrated embryonic tendon elastic modulus increases during development and is substantially lower than in adult. Here, we examined how these embryonic mechanical properties influence tenogenically differentiating cells, by culturing embryonic tendon progenitor cells (TPCs) within alginate gel scaffolds fabricated with embryonic tendon mechanical properties. We showed that nano- and microscale moduli of RGD-functionalized alginate gels can be tailored to that of embryonic tendons by adjusting polymer concentration and crosslink density. These gels differentially regulated morphology of encapsulated TPCs as a function of initial elastic modulus. Additionally, higher initial elastic moduli elicited higher mRNA levels of scleraxis and collagen type XII but lower levels of collagen type I, whereas late tendon markers tenomodulin and collagen type III were unaffected. Our results demonstrate the potential to engineer scaffolds with embryonic mechanical properties and to use these scaffolds to regulate the behavior of tenogenically differentiating cells.

Comparative analysis of mesenchymal stem cell and embryonic tendon progenitor cell response to embryonic tendon biochemical and mechanical factors.

INTRODUCTION: Advances in tendon engineering with mesenchymal stem cells (MSCs) are hindered by a need for cues to direct tenogenesis, and markers to assess tenogenic state. We examined the effects of factors involved in embryonic tendon development on adult MSCs, and compared MSC responses to that of embryonic tendon progenitor cells (TPCs), a model system of tenogenically differentiating cells. METHODS: Murine MSCs and TPCs subjected to cyclic tensile loading, transforming growth factor-ß2 (TGFß2), and fibroblast growth factor-4 (FGF4) in vitro were assessed for proliferation and mRNA levels of scleraxis, TGFß2, tenomodulin, collagen type I and elastin. RESULTS: Before treatment, scleraxis and elastin levels in MSCs were lower than in TPCs, while other tendon markers expressed at similar levels in MSCs as TPCs. TGFß2 alone and combined with loading were tenogenic based on increased scleraxis levels in both MSCs and TPCs. Loading alone had minimal effect. FGF4 downregulated tendon marker levels in MSCs but not in TPCs. Select tendon markers were not consistently upregulated with scleraxis, demonstrating the importance of characterizing a profile of markers. CONCLUSIONS: Similar responses as TPCs to specific treatments suggest MSCs have tenogenic potential. Potentially shared mechanisms of cell function between MSCs and TPCs should be investigated in longer term studies.