Machine Learning Assisted Spectral Fingerprinting for Immune Cell Phenotyping.

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.

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.

Decoupling Individual Optical Nanosensor Responses Using a Spin-Coated Hydrogel Platform.

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.

Aggregation Reduces Subcellular Localization and Cytotoxicity of Single-Walled Carbon Nanotubes.

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.

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.

A Spin-Coated Hydrogel Platform Enables Accurate Investigation of Immobilized Individual Single-Walled Carbon Nanotubes.

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.

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.

Multispectral Fingerprinting Resolves Dynamics of Nanomaterial Trafficking in Primary Endothelial Cells.

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.

A Magnetically Responsive Hydrogel System for Controlling the Timing of Bone Progenitor Recruitment and Differentiation Factor Deliveries.

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.

Biomolecular Functionalization of a Nanomaterial To Control Stability and Retention within Live Cells.

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.

Enhancing the Thermal Stability of Carbon Nanomaterials with DNA.

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 (C60), 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.

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.

DNA Sequence Mediates Apparent Length Distribution in Single-Walled Carbon Nanotubes.

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.

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.

Quantitative self-assembly prediction yields targeted nanomedicines.

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.

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.