Table of contents

Unique physical, chemical, and optical phenomena arise when materials are confined to the nano-scale. We are accustomed to making observations and predictions for the behavior of living systems on a macroscopic scale that is intuitive for the time and size scales of our day-to-day lives. However, the building blocks of life: proteins, nucleic acids, and cells, occupy different spatiotemporal scales. Our lab focuses on understanding and exploiting tunable optical and mechanical properties of nanomaterials to access information about biological systems stored at the nano-scale. In the context of leveraging nanomaterial optical properties, we present recent work on developing and implementing dopamine nanosensors to image dopamine volume transmission in the extracellular space of the brain striatum. We validate our dopamine nanosensor in acute striatal slices with electrical and optogenetic stimulation of dopamine release, and show disrupted dopamine release or reuptake kinetics when brain tissue is exposed to dopamine agonist or antagonist drugs [1,2]. In the context of leveraging nanomaterial chemical properties, we also discuss how high aspect ratio nanomaterials can be synthesized to carry biomolecular cargo to living systems. In particular, genetic engineering of plants is at the core of environmental sustainability efforts, but the physical barrier presented by the cell wall has limited the ease and throughput with which exogenous biomolecules can be delivered to plants. We will describe how nanomaterials engineering principles can be leveraged to manipulate living plants [3, 4], in efforts to reconcile the benefits of crop genetic engineering with the demand for non-GMO foods [5]. Our work in the agricultural space provides a promising tool for species-independent, targeted, and passive delivery of genetic material, without transgene integration, into plant cells for rapid and parallelizable testing of plant genotype-phenotype relationships.

1. Beyene, A.B., McFarlane, I.R., Pinals, R.L, Landry, M.P.^‡ Stochastic Simulation of Dopamine Neuromodulation for Implementation of Fluorescent Neurochemical Probes in the Striatal Extracellular Space. ACS Chemical Neuroscience 8 (10), 2275-2289 (2017).

2. Beyene, A. G., Delevich, K., O'Donnell, J.T.D., Piekarski, D.J., Lin, W.C., Thomas, A.W., Yang, S.J., Kosillo, P., Yang, D., Wilbrecht, L., Landry, M.P.^‡ Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor. Science Advances 5 (7), 1-11 (2019)

3. Demirer, G.S., Zhang, H., Matos, J., Goh, N., Cunningham, F.J., Sung, Y., Chang, R., Aditham, A.J., , Chio, L., Cho, M.J., Staskawicz, B., Landry, M.P.^‡ High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without DNA Integration in Mature Plants. Nature Nanotechnology (2019). DOI: 10.1038/s41565-019-0382-5NNANO-18081684

4. Demirer, G.S., Zhang, H., Goh, N.S., Grandio, E.G., Landry, M.P.^‡ Carbon nanotube-mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019). DOI: 0.1038/s41596-019-0208-9

5. Landry, M.P.^‡, Mitter, N.^‡ How nanocarriers delivering cargoes in plants can change the GMO landscape. Nature Nanotechnology 2019, 14; 512–514

Nanoscale materials provide new opportunities to interface solid-state electronics with biomolecules and biochemical activity. For example, single-walled carbon nanotubes (SWNTs) have the special property of electronic resistance that is sensitive to single electrons. We have exploited this sensitivity to build nanoelectronic biosensors that monitor the biochemical activity of individual proteins [1]. As an attached protein moves, binds, or performs catalysis, its charged amino acid sidechains induce resistance fluctuations in the SWNT device that may be monitored with microsecond resolution [2].

Recently, we have used this measurement platform for single-molecule measurements of DNA polymerases [3]. Polymerases are the key enzymes for converting single-stranded DNA to double-stranded helices, the primary step in DNA replication, amplification, and most sequencing technologies. When a polymerase processing DNA is also attached to a SWNT device, the electrical signal provides a high-resolution readout of single-nucleotide incorporations and exciting possibilities for high-density, high-throughput electronic DNA sequencing.

To investigate the feasibility of electronic DNA sequencing, we have compared single-molecule transduction by DNA polymerases from three different organisms. By working with multiple families of DNA polymerases, we have tested the applicability of the electronic technique while also generating detailed records of differences among the enzymes. For example, we observe an anomalous rate variability when measuring the polymerase from the bacillus phage φ29. Base incorporation rates average 20 s^-1 for most the enzymes processing single-stranded DNA templates, but rates up to 200 and 400 s^-1 occurred when φ29 encountered homopolymeric sequences of poly(dT) or poly(dC), respectively. When processing poly(dA) and poly(dG) sequences, on the other hand, φ29 had bursts of activity interrupted by pauses lasting 50 to 300 s. This sequence-dependent activity illustrates one way that single-molecule methods reveal information hidden in ensemble-based techniques.

Another workhorse protein in DNA sequencing technologies is the DNA polymerase derived from the thermophilic bacteria Thermus aquaticus (Taq). Anomalous stability at high temperatures makes Taq a unique enzyme for the polymerase chain reaction (PCR) and commercial amplification of DNA. In a first for single-molecule biophysics, SWNT devices have recorded Taq activity over a wide temperature range from 22 to 94 °C, including the typical PCR operating temperature of 72 °C. Even operating at this high temperature, the technique resolved Taq testing incoming nucleotides for complementarity and incorporating correct matches in the base-by-base construction of Watson-Crick pairs. The detailed recordings reveal the similarities of Taq's operation to other, room temperature polymerases.

[1] Y. Choi, et. al., Science335, 319 (2012). [2] M. V. Akhterov, et. al., ACS Chem. Biol.10, 1495 (2015). [3] T. J. Olsen et. al., JACS135, 7855 (2013); O. T. Gul et. al., Biosensors6, 29 (2016)

Interest in the use of engineered nanomaterials for biological sensing and imaging applications continues to grow due to the vast potential of tuning the surface chemistry of nanomaterials using various biomolecules. Carbohydrate recognitions of specific proteins play a crucial role in many biological processes, including cellular adhesion, cell-cell communication, immune response, cancer development and metastasis. In addition, the highly predictable electronic structures and optical properties of chirality-defined SWCNTs offer many advantages for biochemical sensing and imaging advancement. This work explored the photochemistry of pure-chirality single-wall carbon nanotubes (SWCNTs) using galactose-containing glycopolymers to create engineered fluorescent probes with precise optical and carbohydrate functionalities. These SWCNT-based fluorescent probes can be potentially utilized for profiling targeted carbohydrate-protein interactions in complicated biological samples and in vivo analysis.

The biodistribution of single-walled carbon nanotubes (CNTs) was quantified after intravenous administration to mice based on their absorption of near-infrared light. CNTs with two different diameters, 1–5 nm (Ld-CNT) or 0.7–0.9 nm (Sd-CNT), were compared. The accumulation of Ld-CNTs in the lung was less than that of Sd-CNTs, and both were cleared almost completely within 60 days. The rapid clearance of CNTs could be due to their degradation by macrophages and/or to their trafficking to other organs via the vasculature. In the liver and spleen, the accumulation of Ld-CNTs was greater than that of Sd-CNTs. The quantity of Ld- or Sd-CNTs in the liver decreased for about 10% of the dose in 60 days. These results were confirmed by histological analysis. Levels of the cytokines TGF-β1, IL-6, INF-γ, and TNF-α in the plasma and organs of mice injected with Ld- and Sd-CNTs were low, suggesting the low toxicity of Ld- and Sd-CNTs.

Single wall carbon nanotube (SWCNT) based biosensors provide opportunities for building an ultra-sensitive biosensing system due to their unique optical properties and strong sensitivity to changes in the local environment. Consequently, much effort has been made to develop SWCNT-based sensors. However, the usual method is based on one-to-one recognition which is a difficult way to detect various molecules since it requires the same number of highly specific receptors as the number of molecules one wishes to detect. To detect a combination of various analytes simultaneously, an effective and automatic data processing system is essential. In this study, we propose a new perception-based sensing system using weakly-specific sensor arrays that can be analyzed by an artificial perception model, which we call the Molecular Perceptron. We show how machine learning algorithms along with choice of feature representation is designed to predict presence and concentration of biomarkers or direct prediction of disease states. For example, we demonstrate that the Molecular Perceptron can detect Human epididymis protein 4 (HE4) in the presence or absence of other analytes; HE4 is one of two FDA-approved serum biomarkers for ovarian cancer which provides noticeable sensitivity and specificity for ovarian cancer diagnosis. DNA/SWCNT hybrids were utilized to optically detect the analytes by observing changes in the fluorescence spectra of each SWCNT. Using the experimental data, machine learning models were trained using three different algorithms: Support Vector Machine, Random Forest, and Artificial Neural Network. The models were then validated using new experimental data for different analyte concentrations. Overall, the machine learning models successfully predict the presence of HE4 at the concentrations of 10 nM or higher by giving F¹-scores of ~0.85. This is strongly suggestive of the idea that the perception mode of sensing can make accurate judgements in a noisy sensing environment.

Nanoscale field-effect transistors (FETs) embedded in microfluidics form a promising technology as compact and portable lab-on-a-chip biosensors for applications in biology and medicine. Such sensors can be designed to monitor a variety of biochemical mechanisms, at the ensemble or single-molecule scale, through fluctuations in the electrical conductance of the circuit. In particular, nanocarbon materials, i.e. carbon nanotubes and graphene, are materials of choice for FET biosensors, due to their high electrical conductance, the sensitivity of their electronic properties to the surrounding environment, and the versatility of their carbon-based surface chemistry. In this presentation, I will report on recent developments and key considerations in the design, fabrication and characterization of such nanocarbon-based biomolecular FET sensors. In particular, I will discuss approaches for chemical functionalization and structural patterning of nanocarbon materials to optimize their coupling with molecules. I will also describe hardware and software developments for the analysis of single-molecule measurements in hybrid biomolecule-nanocarbon FET devices.

Cells use small molecules to exchange information. A very prominent example is neurotransmitter signaling between neuronal cells or paracrine signaling between blood cells. However, it is up to date difficult to visualize signaling processes between cells with high spatial, temporal and chemical resolution. We are interested in developing fluorescent sensors/probes that overcome these limitations and answer novel biological questions.

For that purpose we use near infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs). SWCNTs fluoresce in the nIR tissue transparency window (900 nm -1700 nm) and do not bleach. They are chemically modified to render them selective for neurotransmitters such as dopamine and serotonin. In this context, we developed different chemical strategies that include screening for artificial binding motifs, aptamers and nanobodies. Using these strategies we created sensors for the neurotransmitters dopamine and serotonin that increase their fluorescence in the presence of the target molecule and detect down to single molecules. Fast parallel imaging of many of those sensors provides a nIR image (>900 nm) of the neurotransmitter concentration around cells. This technique was used to image hot spots of dopamine release from primary dopaminergic neurons. It enabled us to observe localized release sites and correlate it with the exocytosis machinery of the cells. Neurotransmitters are not only relevant in the neuronal system but as well in the periphery e.g. in blood cells. In this context, we imaged for the first time how human blood platelets release serotonin after activation and resolved the heterogeneity in release patterns. These results illustrate that tailored SWCNTs are powerful tools for quantitative chemical imaging of biological processes.

Nanocarbons such as photoluminescent single-walled carbon nanotubes are promising materials in biomedical field as molecular imaging agents, optical probes and biosensors. However, these carbon-only nanomaterials lack active surface functional moieties to interact effectively with select biomolecules of interest, which limits their potential applications. Tailored surface functionalization is essential for anticipated functions and bio-compatibility. Polymer adsorption on nanocarbons through multivalent interactions provides a versatile platform to develop functional materials with tunable surface chemistry. Herein, we investigate on biomimetic polymer scaffolds for carbon nanotubes to modulate their physiochemical properties and bio-molecular interactions.

Dopamine neurotransmission plays critical roles in brain function in both health and disease and aberrations in dopamine neurotransmission are implicated in several psychiatric and neurological disorders, including schizophrenia, depression, anxiety, and Parkinson's disease. Until recently, measuring the dynamics of dopamine and other neurotransmitters of this class could not be achieved at spatiotemporal resolutions necessary to understand how dopamine regulates the plasticity and function of neurons and neural circuits, and how dysfunctions in this regulation lead to disease. Probes that satisfy critical attributes in spatial and temporal resolution, and chemical selectivity are needed to facilitate investigations of brain neurochemistry.

To address this need, we developed an ultrasensitive near-infrared "turn-on" nanosensor (nIRCat) for the catecholamines dopamine and norepinephrine and implemented nIRCat to study endogenous dopamine dynamics in brain tissue. nIRCats are synthesized from photostable and near infrared emissive functionalized single wall carbon nanotubes (SWCNT) and exhibit maximal relative change in fluorescence intensity (ΔF/F₀) of up to 35-fold, with a dynamic range that spans physiological concentrations of their target brain analytes. We demonstrate that nIRCat can detect electrically and optogenetically evoked release of dopamine in brain tissue, revealing putative hotspots of activity that exhibited a log-normal distribution in size, ranging from 2 – 10 µm for mice brain tissue in the dorsomedial striatum. Furthermore, the synthetic nature of the molecular recognition platform afforded compatibility with dopamine-receptor targeting antipsychotics and psychoactive drugs and permitted studies of how such receptor-targeting drugs modulate evoked dopamine release. This assay revealed presynaptic correlates of drug activity at the level of putative dopamine release sites, which had heretofore been inaccessible to probe with existing tools. Our results suggest that nIRCat technology may uniquely support similar explorations of processes that regulate dopamine neuromodulation at the level of individual synapses, and exploration of the effects of receptor agonists and antagonists that are commonly used as psychiatric drugs as well as drugs that lead to substance use disorder. We conclude that SWCNTs can serve as versatile synthetic optical scaffolds for monitoring interneuronal chemical signaling in the brain extracellular space at spatial and temporal scales pertinent with the encoded neurochemical information.

Nanotechnology-based sensors are promising tools for optimizing plant performance and use of resources in agriculture by equipping plants with capabilities to report when they are under stress and require intervention. Herein, we interfaced near infrared (nIR) fluorescent nanosensors with plant leaves that report hydrogen peroxide (H₂O₂), a key signaling molecule associated with the onset of plant environmental and pathogen stresses. For this purpose, we used single-walled carbon nanotubes (SWCNT) as non-photobleaching fluorescent building blocks for H₂O₂ nanoscale sensors with a nIR fluorescent emission (> 800 nm) in a ultra-low background region that is ideal for high-sensitivity remote sensing and imaging. SWCNTs were functionalized with a DNA aptamer that binds to hemin, resulting in nIR fluorescence nanosensor response that is quenched by H₂O₂ in a dose dependent way within the plant physiological range (10-100 H₂O₂ µM). The in vivo sensor sensitivity for H₂O₂ (10 µM) allows it to reversibly report signs of stress in plants exposed to UV-B, high light, and a microbial pathogen related peptide (flg 22), but not mechanical leaf wounding. The sensor response was imaged in leaves by a standoff nIR camera, which reported remotely changes in sensor nIR emission up on stress. Such nanotechnology-based sensors report plant signaling molecules associated with early signs of stress and will impact our understanding of plant stress communication, provide novel tools for precision agriculture, and reduce undesirable losses of agrochemicals in the environment.

Implantable optical nanosensors hold the promise of rapid, minimally-invasive longitudal monitoring of analytes in a patient or animal. Such devices will allow for informed clinical intervention based on changes in drug, biomarker, or disease state profiles. We developed implantable nanosensors based on the optical properties of single-walled carbon nanotubes (SWCNT). SWCNT fluorescence is in the 'tissue-transparent' near-infrared spectrum, allowing for fluorescence measurements after implantation inside an animal. Further, SWCNT fluorescence is photostable, allowing for multiple measurements over time. We engineered separate SWCNT-based sensors such that their fluorescence emission responded to various analytes, including small molecule drugs as well as protein and nucleic acid biomarkers. Each sensor device was immobilized into a semi-permeable biocompatible membrane, allowing analyte penetration but retaining SWCNT inside. We implanted each sensor device into live mice, either in the peritoneal cavity or subcutaneously, and performed whole-mouse near-infrared fluorescence imaging to ensure the stability of the membrane and sensor brightness. We used a sensor implant specific for the chemotherapeutic doxorubicin to drug monitor transport within the animal. We used sensor implants specific for the ovarian cancer protein biomarker HE4 or for individual microRNA sequences to measure exogenous or cancer-generated quantities of each biomarker in mice. We expect these implantable sensors to have potential as research tools or clinical diagnostics to monitor pharmacokinetics or locally measure disease biomarkers in real-time.

Single-walled carbon nanotubes (SWCNTs) are advantageous signal transduction elements for biological imaging and sensing due to their notable fluorescence in the near-infrared (NIR) region with minimal light absorbance by water or scattering by tissue (1). Recently, exciting developments in SWCNT surface chemistry have shown the retention and improvement of fluorescence intensity in SWCNTs after covalent surface functionalization (2). These chemistries provide chemical handles for specific attachment of molecules of interest to the SWCNT surface while keeping the intrinsic SWCNT NIR fluorescence intact for bioimaging and sensing applications. However, despite their unperturbed fluorescence, it is unclear whether functionalized SWCNTs maintain their ability for molecular sensing, which necessitates a modulation in SWCNT fluorescence provided by the molecular recognition of a noncovalently surface-adsorbed polymer. We compare the photophysical properties of functionalized SWCNTs to their pristine counterparts, and show SWCNT optical properties remain available for sensing applications, however with noted attenuation of fluorescence modulation between certain surface coating and analyte pairs. Molecular recognition provided by phospholipid SWCNT surface coatings maintain their ability to detect molecular analytes such as fibrinogen and insulin. Conversely, DNA oligonucleotide surface coatings under-perform for molecular recognition of dopamine compared to sensors constructed from pristine SWCNTs. We also discuss the use of covalent handles to tune the intrinsic properties of the SWCNT surface for use as biological tools. Lastly, we explore the application of covalently functionalized SWCNTs as dual-functional nanoparticles with both targeting and sensing capabilities. Our work establishes the potential advantages and drawbacks of covalent SWCNT functionalization, despite preservation of SWCNT fluorescence, and implications for applications in molecular sensing.

(1) Bonis-O'Donnell, J. T. D.; Page, R. H.; Beyene, A. G.; Tindall, E. G.; McFarlane, I. R.; Landry, M. P. Dual Near-Infrared Two-Photon Microscopy for Deep-Tissue Dopamine Nanosensor Imaging. Adv. Funct. Mater.2017, 27 (39), 1–10.

(2) Setaro, A.; Adeli, M.; Glaeske, M.; Przyrembel, D.; Bisswanger, T.; Gordeev, G.; Maschietto, F.; Faghani, A.; Paulus, B.; Weinelt, M.; et al. Preserving π-Conjugation in Covalently Functionalized Carbon Nanotubes for Optoelectronic Applications. Nat. Commun.2017, 8, 1–7.

Luminescent single wall carbon nanotubes are now well established, as unique nanoreporters to probe the brain extracellular space[1-3]. On the imaging side, this comes from their rich near-infrared optical properties, which also eventually be improved by sp³-chemical functionalization. In addition, their uncommon 1D morphology is an important asset for tissue penetration, yet understanding their diffusion behavior in the complex brain extracellular network is challenging and necessitates dedicated analysis tools. To this aim, we propose novel approaches (i) based on the local analysis of trajectory contours to locally measure the nanoscale dimensions of the brain network [4] and (ii) based on the transient evaluation of carbon nanotube mean square displacement to delineate the ECS molecular diffusion landscape [5]. The application of these analytical tools to extract relevant biological parameters in physiological and pathological brain models will be presented [6].

References

[1] Godin et al Nat. Nanotechnol. 12 (2017) 238-243.

[2] Gao, et al, Nanomaterials 7, 11, (2017) 393.

[3] Danné et al ACS Photonics, 5, 2, (2018) 359-364

[4] Paviolo et al Methods (2019) in press

[5] in preparation

[6] Soria et al in revision

Fluorescent sensors are frequently not used for in vivo research due to photobleaching and difficulty in signal detection. Single walled carbon nanotubes (SWNT) circumvent these issues with near infrared fluorescent emission and long term stability; yet detection of carbon nanotube sensors has never before been performed in large animal models. The ability to place and read SWNT sensors in vivo for a large animal requires specialized instrumentation and research facilities. By teaming up with large animal veterinarians and meat scientists, our lab was able to surgically place, monitor, and recover SWNT sensors from 14 sheep. We examined various SWNT platforms, implantation locations, and detection methods to determine a strategy for frequent, consistent detection of SWNT sensors that serve as an indicator of animal stress and overall health.

Single wall carbon nanotubes (SWCNTs) have been rigorously studied and engineered for a myriad of applications including drug delivery due to their unique, desirable properties. Since SWCNTs bundle in aqueous media and the bundling substantially diminishes their desirable properties along with inducing cytotoxicity, strategies to disperse individually SWCNTs with therapeutic molecules and biologic excipients have been heavily investigated. However, a handful of studies has been performed to understand the interactions between therapeutic molecules and excipients with SWCNT interface as well as the mechanism of intracellular release for therapeutic effects. Additionally, little attention has been paid to how cell type and cell metabolic activity level influence internalization of SWCNTs with different lengths. In this talk, I will discuss cell type, cell metabolic activity, and SWCNT length dependent cellular processing of SWCNTs dispersed with diverse biocompatible dispersing agents. Our results demonstrate that proper choice of dispersing agent can allow for interaction with F-actin, accumulation in the endoplasmic reticulum for eventual expulsion, or accumulation in cell nucleus. Further, macrophages with relatively high cell metabolic activity level compared to other cell types such as fibroblasts have shown to internalize significantly higher amount of SWCNTs per cell. The total mass of SWCNTs internalized per cell increases with a decrease in SWCNT length. Lastly, I will discuss our recent effort to create a ternary complex of SWCNTs, drug, and protein for intracellular delivery and release of model drugs. To overcome the limitation of the existing methods being only plausible for model drugs that are soluble in aqueous solutions, we developed a facile schema to assemble stepwise various combinations of therapeutic molecules and excipients on SWCNTs in different solvents using a preformed SWCNT network. These ternary complexes allow dialing in delivery of therapeutic molecules and excipients to achieve desired cell viability reduction. These findings allow for the development of new SWCNT-based biological applications as they establish important parameters of biomolecule adsorption on SWCNTs, SWCNT internalization and subcellular processing, which are crucial for applications ranging from drug delivery to cell type-specific modulation.

Molecular recognition is fundamental to the design of therapeutics, diagnostics, and sensors. Many of these sensor technologies rely on natural or synthetic molecular recognition elements such as antibodies and aptamers, which have evolved to be highly selective for their molecular targets. However, there are numerous applications for which existing approaches to molecular recognition have been unable to generate optical probes, particularly for neuromodulators and neuropeptides. Herein, we describe a polymer evolution-based platform, in which 10^12 unique polynucleotide polymers are adsorbed to single-walled carbon nanotubes (SWNT). The SWNT-pinned polynucleotide polymers can be screened for their ability to bind a target analyte and provide a selective near-infrared fluorescence signal. Iterative selection of analyte-binding polymers that form a SWNT-surface-adsorbed phase for target recognition are identified through ionic desorption of sub-optimal polymers, and exponential amplification of synthetic polymers that recognize the target analyte. Next-generation sequencing identifies SWNT-polynucleotide conjugates with sequences selective and sensitive for the desired analyte. We demonstrate the utility of this platform for the evolution of SWNT-polymer nanosensors for neuromodulators [1] and neuropeptides, and discuss its potential for identification of chirality-specific SWNT separation. Our results suggest our platform is fundamentally generic in enabling the evolution of nanosensors from synthetic polymer-nanoparticle conjugates for any desired analyte.

1. Jeong, S., Yang, D., Beyene, A.G., O'Donnell, J.T.D., Gest, A. M., Navarro, N., Sun, X., Landry, M.P. High Throughput Evolution of Near Infrared Serotonin Nanosensors. Science Advances (2019). DOI: 10.1101/673152

Biosensors based on fluoro-labelled antibody fragments combine the recognition and transduction element into the same molecule leading to real-time result detection and reducing the need for laborious, multi-step assays. The key challenge is the efficient site-specific modification of antibodies with environmentally-sensitive fluorescent dyes, without affecting binding functionality. Fluorescence labelling via unnatural amino acids (UAAs) is a relatively new and highly efficient method for 100% efficient site-specific fluorescence labelling, and can be genetically incorporated into any permissible site during protein synthesis. Although over 100 UAAs have been incorporated into various proteins for diverse applications including antibody drug conjugates and bispecific antibody development using Fab, to date none of the UAAs has been incorporated into scFv for biosensing applications nor has this been used for detection of large biomolecules (e.g. protein).

We demonstrate that incorporation of environmentally sensitive fluorescent UAA (Anap) into a permissible site of antibody fragments (e.g. anti-EGFR scFv) can be used for detection of target binding by monitoring the wavelength and/or intensity changes in emission spectra. A mutation screen was initially performed in order to identify the Anap mutation site that yielded the largest spectral change. We found that, across two different protein/antibody case studies, that only relatively hydrophobic amino acids within the binding interface could be mutated to generate optically-reactive species, and that the affinity of the mutants was not significantly affected. When immobilising these antibody fragments onto various surfaces (e.g. silica, carbon nanotubes, polymeric nanoparticles) for solid-phase biosensor development, we also observed unique behaviour indicative of the local hydrophilicity of the surface environment, which may be advantageous in identifying optimal surfaces for high specificity and sensitivity in complex biological environments. Here we will present our latest results on generalising this approach for protein detection in complex biological environments, and also to outline the challenges and opportunities for future developments

Figure 1

Ovarian cancer patient prognosis and quality of life are significantly affected by the failure to accurately diagnose disease at early stage. Currently, less than 15% of ovarian cancer patients are diagnosed at stage 1, when 5-year survival rates are near 93%. Early detection may save more than 80% of women diagnosed with ovarian cancer. Our goal is to develop long-term implantable carbon nanotube-based sensors for ovarian cancer biomarkers to transiently detect and monitor their levels in patients. The proposed sensors will harness the unique optical properties and sensitivity of single-walled carbon nanotubes. To improve sensitivity and specificity of such sensors, previously developed to detect ovarian cancer biomarkers in vivo, we used molecular perceptron—data analytics-based methods to detect specific spectroscopic fingerprints from the binding of protein biomarkers to the nanotube surface.

Non-covalent functionalization of single-walled carbon nanotubes (SWCNTs) has enabled advances in biomolecule imaging. In particular, modification of SWCNTs with select sequences of single-stranded DNA (ssDNA) produces near infrared optically active sensors for neurotransmitters such as dopamine, enabling high spatiotemporally resolved optical recording of synaptic and extrasynaptic signaling. However, the carbon lattice surface of SWCNT induces activation of the innate immune system, leading to an inflammatory response. Activation of microglia, the brain's resident immune cells, is of particular relevance to imaging neuromodulation. Here, we show that non-covalent ssDNA-SWCNT nanosensor passivation with a PEGylated phospholipid can mitigate microglial activation while maintaining the nanosensor's response towards dopamine. To do so, we screen a candidate library of PEGylated biomolecules, nonionic surfactants, and block copolymers against their ability to mitigate the microglial immune response as measured by transcriptomic analysis. We find that PEGylated phospholipids are capable of adsorption to ssDNA-SWCNTs without significant ssDNA displacement, with moderate reduction in nonspecific protein adsorption. These trends are recapitulated by a reduction in inflammatory response of SIM-A9 microglial cells, marked by lowered expression of cytokines CXCL2 and IL-6, mitigated morphological change, and decreased cytotoxicity. Taken together, these results suggest noncovalent passivation improves biocompatibility of DNA-SWNCT nanosensors and provides the groundwork for implementing similar passivation methods for other optical probes of this class.

The modulation of single-walled carbon nanotube (SWCNT) photoluminescence has been under investigation for the unique potential to develop biosensors capable of detecting many classes of analytes. Covalent sp3 defects have introduced emissive color centers on SWCNTs, significantly enhancing photoluminescence quantum yield and chemical selectivity/sensitivity to certain analytes. The application of these organic color centers (OCCs) for biomedical applications requires new methods to both conduct covalent chemistry and to concomitantly control the surface chemistry and the resulting biological interactions/biocompatibility via their supramolecular interactions with polymers and other excipients. We will report specific utilities of OCCs for the detection of chemical phenomena where SWCNT photoluminescence is normally relatively insensitive, such as pH changes in the physiologic range of 4.5 - 8. We have developed OCCs to measure endolysosomal pH in live cells and in vivo, as well as other emerging applications. The results support the feasibility of using OCCs as biosensors for disease detection and biomedical research.

Carbon nanomaterials offer remarkable properties including high durability, conductivity, and versatility in modification. Carbon nanotubes and graphene oxide also exhibit fluorescence in the near-infrared (NIR) making those attractive for bioimaging and drug delivery applications due to the high tissue penetration depth of the NIR emission. However, despite these remarkable properties, the biocompatibility and degradability of carbon-based platforms still rise some unfortunate controversy that hampers their clinical utilization. In order to address this, we specifically develop NIR-emissive graphene-based quantum dots with high biocompatibility. We explore both the bottom-up and the top-down synthetic approaches together with a variety of doping strategies that yield 2 – 5 nm quasi-spherical quantum dots with graphitic lattice structure observable in TEM. In the bottom-up glucosamine-based synthesis the most prominent NIR emission is observed from graphene quantum dots doped by nitrogen, sulfur or rare-earth metals exhibiting transitions in that spectral region. These GQDs decorated with oxygen-containing functional groups identified with the FTIR have high water solubility and offer efficient cell internalization in HeLa and MCF-7 cells maximized at 12 h. Few percent doping with rare earth metals or nitrogen/sulfur heteroatoms as verified by the EDX does not substantially contribute to the toxic profile of the formulation: doped GQDs exhibit high biocompatibility up to 1 – 2 mg/mL concentrations and degradation in cell culture at 36 h. Finally, these GQDs exhibit emission in the visible with quantum yields up to 60% and also in the NIR, which is utilized for in vitro fluorescence imaging in both spectral regions.

Top-down synthesized GQDs are derived from graphitic materials via UV-assisted radical-based oxidation. Unlike their parent material, these few-layered GQDs containing oxygen addends are water-soluble and exhibit fluorescence in the visible and a wavelength-independent emission in the NIR with NIR quantum yields ranging from 1 to 8%. They are also biocompatible with cell viability over 80% with up to 1 mg/mL GQD concentrations and exhibit cellular internalization within several hours tracked with their NIR fluorescence excited by the 808 nm diode laser. The NIR imaging capabilities of both bottom-up and top-down synthesized GQDs are verified in vivo in live mouse models showing detectable fluorescence in spleen with some liver and kidney signal observed with 808 laser excitation through the tissues of live animals. Excised organs show NIR GQD emission from kidneys, liver, spleen and intestine with GQDs also detected in single organ slices indicating their location within the particular organ. As a result, we suggest that NIR-emissive biocompatible GQDs synthesized both via bottom-up and top-down approaches can be developed and utilized as imaging and, potentially, drug delivery agents both in vitro and in vivo in small animal models.

Introduction:

Graphene field-effect transistors (G-FETs) constitute an emerging platform for biosensing applications. Indeed, graphene is an ideal material for the detection of biomolecules: every atom of its monolayer structure is in contact with its environment, resulting in an electrical conductivity that is highly responsive to local electrostatic fluctuations from adjacent molecules. For genomic applications, most detection methods use DNA probe sequences bound to the graphene surface, in order to capture a specific target DNA sequence and detect the corresponding change in the electrical response of the sensor^1-2. However, the effect of interactions between DNA and graphene on the electrical conductance is still not fully understood. Here, we investigate specifically the adsorption of short DNA oligomers on graphene field-effect transistors, in order to model and control the effect of such interactions in biosensing applications with G-FETs.

Methods and Results:

First, we fabricated G-FET sensors as follow³: Using photolithography techniques, an array of source and drain electrodes were patterned in gold on a Si wafer with a SiO₂ insulator layer, as well as a common on-chip gate electrode in platinum. High-quality monolayer CVD-grown graphene was transferred onto the substrate and then etched to create 6 x 4 µm ribbons between each source-drain pair. Transfer curves (Isd vs. Vg) performed in saline buffer solution revealed a conductance minimum at the charge neutrality point of the graphene. Devices were then exposed to solutions of DNA, consisting in 22-single-stranded nucleotides (ssDNA) or double-stranded nucleotides (dsDNA) DNA oligomers diluted in 0,01X PBS buffer. Selected G-FET devices were exposed to different ssDNA concentrations during 15 min, followed by washing steps with 0,01X PBS. Electrical curves were recorded before, during and after each DNA exposure. In this presentation, we will present results showing that ssDNA exposure causes a left-shift of the charge neutrality point above a concentration threshold, and that this shift is proportional to ssDNA concentration. In addition, non-covalent adsorption of ssDNA on graphene appears to be reversible upon washing. Finally, we will discuss differences between the adsorption of dsDNA and ssDNA.

Conclusion and Relevance:

Our results suggest that unspecific DNA adsorption on graphene can lead to a G-FET response, which needs to be modeled, compensated and /or passivated in biosensing experiments, especially in order to achieve low detection limits for target sequences in complex biological media.

References:

1. Hwang MT, Landon PB, Lee J, et al. Highly specific SNP detection using 2D graphene electronics and DNA strand displacement. Proc Natl Acad Sci. 2016;113(26):7088-7093. doi:10.1073/pnas.1603753113

2. Cai B, Wang S, Huang L, Ning Y, Zhang Z, Zhang G. Ultrasensitive Label-Free Detection of PNA À DNA Hybridization by Reduced Graphene Oxide Field-E ff ect Transistor. 2014;(3):2632-2638. doi:Doi 10.1021/Nn4063424

3. Bazan CM, Bencherif A, Sauvage M, Huliganga E, Borduas G, Bouilly D.Fabrication of Nanocarbon-Based Field-Effect Transistor Biosensors for Electronic Detection of DNA Sequences. ECS Trans. 2018;85(13):499-507. doi:10.1149/08513.0499ecst

The ability to modulate cellular electrophysiology is a fundamental aspect for investigation of tissue development, maturation and function. Currently, there is a need for remote, non-genetic, light-induced control of cellular activity in native-like state, three-dimensional (3D) tissues such as spheroids and organoids. Here, we report a breakthrough hybrid-nanomaterial for remote, non-genetic photostimulation of both two-dimensional (2D) and 3D neural cellular systems. We combine one-dimensional (1D) nanowires (NWs) and 2D graphene flakes grown out-of-plane for highly controlled photostimulation at subcellular precision with laser energies lower than hundred nanojoules, 1-2 orders of magnitude lower than Au-, C- and Si-based photostimulation. Photostimulation using NW-templated 3D fuzzy graphene (NT-3DFG) is flexible due to its broadband absorption and does not generate cellular stress. Therefore, it serves a novel powerful toolset for studies of cell signaling within and between tissues and can enable new therapeutic interventions.

Graphene quantum dots (GQDs), the newest type of carbon nanoparticles (CNPs) with size <20 nm, have attracted many research attentions due to the highly tunable photoluminescence (PL) properties, biocompatibility, chemical and photo stability, and low toxicity. Doping GQDs with heteroatoms, such as nitrogen, can increase the PL quantum yield and has strong electron withdrawing ability. Therefore, nitrogen-doped GQDs as fluorescent pH monitor excels in sensitivity, selectivity, rapidity, and applicability comparing with the conventional electrochemical method, especially for bio-related and medical applications. Good biocompatibility and low toxicity also make N-GQDs more preferable to semiconductor quantum dots, organic fluorescent dyes, and noble-metal nanoparticles. As a matter of fact, pH value plays a very important and crucial role in all life forms. A small variations in pH not only endangers the lives of plants and animal but also human beings as it will mainly pollute our drinking water, which may cause some health issues, like physiological dysfunction or diseases. Furthermore, cancer and tumor cells can also be diagnosed through cellular imaging due to the abnormal pH inside human body.

Most of the approaches for synthesizing N-GQDs are complex, time-consuming, in need of harsh reaction condition, and costly. Hereby, microplasma emerges as one of the most promising approaches for nanomaterials processing due to its facile, stable, efficient, and relatively low cost. The high electron density provided are very energetic (~10eV), which allows non-thermal dissociation of molecular gases to form high concentrations of reactive radical species, e.g. OH˙, O˙, O₂^-˙, and NO˙. These reactive radical species along with electrons are brought into the precursor solutions and will interact with the solution chemical species to form GQDs in a cascaded chemical reactions.

Here we report a microplasma synthesis of N-GQDs for pH sensing. N-GQDs with high quantum yield up to 30% have been synthesized via microplasma approach from chitosan. This synthetic method leads to highly crystalline particles with easily tunable properties in high reproducibility from a simple biomass as the sole precursor. Based on protonation and deprotonation of N-GQDs surface functional groups, a PL based pH probe with broad linear range of 1.78-13.56 and high accuracy can be constructed. Moreover, two linear lines relationship between UV absorption and different pH value are also obtained, covering from pH 1.25-10.17 and pH 10.17-13.24. The dual optical sensing characteristics allow for almost covering the entire pH range and better accuracy.

Figure 1

Field-effect transistor devices based on functionalized graphene (G-FETs) are a promising technology for biomolecular sensing applications due to the several advantages they present, including the label-free detection of biomolecules with direct electrical read-out, real-time detection and multiplexing capability. The development of highly selective and sensitive sensors for protein biomarkers is especially desirable to open novel technological avenues for the early detection and monitoring of biomarkers associated with cancer diseases. In this presentation, I'll describe our recent progress on the design and development of a label-free immunosensor based on antibody-modified graphene field-effect transistors for protein biomarker detection. Specifically, monoclonal antibodies were selected to target a protein biomarker specific to MLL translocated acute myeloid leukemia and we tested approaches for their immobilization on graphene. We found that the antibodies can spontaneously adhere to the graphene surface but this adhesion is partially reversible under solution flow. In order to stabilize the immobilization of antibodies, we developed a protocol based on electrochemically-driven chemistry to form stable covalent anchor groups at the graphene surface which can then capture antibodies. We optimized the rate of formation of anchor groups at the surface in order to maximize the density of anchor groups while maintaining high electrical currents in the graphene. We were then able to record a specific electrical signature showing irreversible immobilization of antibodies on the graphene surface, thus allowing further optimization of target detection. Employing a combination of microfluidics and real-time electrical measurements, we then investigated the response of the sensors to non-specific interactions with graphene as well as their response to specific antibody-antigen interactions in phosphate buffer solution. Finally, the sensors response to different concentrations of target protein was characterized to assess their performance parameters.

Carbon-based nanomaterials have emerged as platforms for biomedical applications because of their low toxicity and ability to be internalized by cells [1]. The development of imaging probes and drug delivery devices based on carbon nanomaterials for biomedical studies requires the understanding of their biological response as well as the efficient and safety exposition of the nanomaterial to the cell compartment where it is designed to operate.

Multi-shell fullerenes, also known as carbon nano-onions (CNOs) are structured by concentric shells of carbon atoms and display several unique properties, such as a large surface area to volume ratio, low density and a graphitic multilayer morphology [2]. In my research group we have developed a versatile and robust approach for the functionalisation of CNOs, involving the facile introduction of a number of simple functionalities onto their surface. Our results have shown that chemical functionalization of the CNOs dramatically enhance their solubility and reduce their inflammatory properties in vitro and in vivo [3]. We reported the absence of adverse effects induced by CNOs on short and long term toxicity in Hydra [4] and in zebrafish [5] suggesting a reasonable degree of biosafety of this new class of nanomaterials. We have developed intracellular imaging systems based on CNOs functionalised with BODIPY derivatives [6], with special attention to the biologically important near-infra red (NIR) region [7] and pH dependent fluorescence on-off switching [8]. The on/off emission of the fluorescent CNOs is fast and reversible both in solution and in vitro, making this nanomaterial suitable as pH-dependent probes for diagnostic applications [8].

We have also developed a synthetic multi-functionalisation strategy for the introduction of different functionalities (receptor targeting unit and imaging unit) onto the surface of the CNOs The modified CNOs display high brightness and photostability in aqueous solutions and their selective and rapid uptake in two different cancer cell lines without significant cytotoxicity is demonstrated. The localization of the functionalized CNOs in late-endosomes cell compartments is revealed by a correlative approach with confocal and transmission electron microscopy [9].

To probe the possible applications of CNOs as a platform for therapeutic and diagnostic interventions on CNS diseases, we injected fluorescent CNOs in vivo in mice hippocampus. We analyzed ex vivo their diffusion within brain tissues and their cellular localization by confocal and electron microscopy. The subsequent fluorescent staining of hippocampal cells populations indicates they efficiently internalize the nanoparticles. Furthermore, the inflammatory potential of the CNOs injection was found comparable to sterile vehicle infusion, and it did not result in manifest neurophysiological and behavioral alterations of hippocampal-mediated functions [10]. These results encourage further development as brain disease-targeted diagnostics or therapeutics nanocarriers.

References

[1] J. Bartelmess et al., Chem Soc Rev 44 (14), 115 (2015).

[2] S. Giordani et al., Current Medicinal Chemistry, in press (2019).

[3] M. Yang et al., Small 9, 4194 (2013).

[4] V. Marchesano et al., Nanomaterials 5, 1331 (2015).

[5] M. d'Amora et al., Scientific Reports 6, 33923 (2016).

[6] J. Bartelmess et al. Nanoscale 6, 13761 (2014).

[7] S. Lettieri et al., RSC Advances 7, 45676 (2017).

[8] S. Lettieri et al., Beilstein J. Nanotechnol. 8, 1878 (2017).

[9] M. Frasconi et al., Chem Eur J 21 (52), 19071 (2015).

[10] M. Trusel et al., ACS Appl. Mat. & Inter. 10 (20), 16952 (2018).

Figure 1

The combination of biocompatibility and unique surface properties has led to the extensive study of the biological applications of carbon nanomaterials. We are interested in the application of single walled carbon nanohorns (CNH) for the development of tools for cellular imaging, diagnostics and therapeutics. CNHs are made up of sp2 conical shaped postulates, with diameters of 2–5 nm and lengths of 40–50 nm, assembled into robust bud-like spherical aggregates of between 80 and 100 nm. The key advantage of these materials is their low toxicity, high surface area, and ease of functionalisation.^1,2 In this presentation results from our recent work on the use of CNHs for enhanced biosensing of glutamate,³ to study of the cellular interactions of fluorescently labelled CNH systems and their use of CNHs to delivery photodynamic therapy agents to cells will be presented.

The poor cellular uptake of many porphyrins is a barrier to their application in phototherapeutic applications,⁴ biocompatible CNH carriers offer a means to overcome this. A spectroscopic study of the binding interactions of the PtTMPyP4 cationic porphyrin with oxidised carbon nanohorns through non-covalent, electrostatic interactions revealed a high a loading of 200 wgt%. Brightfield microscopy demonstrated the efficacy of sparsely loaded Porphyrin-CNH constructs to be internalised within HeLa cells, which were found to undergo rapid cell death upon visible light excitation.⁵

The ability to image processes in cells and track the interaction and efficacy of nanomaterial cellular uptake is of great interest. The use of an ON/OFF fluorophore whose emission is triggered upon cellular uptake will be described as a means to monitor time dependent uptake by live-imaging and also to report on the influence of functionalisation on the extent of cellular aggregation.⁶ Finally, some more recent results on tracking the destination of carbon nanohorns in cells will be reported.

1. Iijima, M. Yudasaka, R. Yamada, S. Bandow, K. Suenaga, F. Kokai, K. Takahashi, Chem. Phys. Lett. 1999, 309, 165−170.S.

2. Zhuab, G. Xu, Nanoscale, 2010, 2, 2538–2549.

3. R. Ford, S. J. Devereux, S. J. Quinn, R. D. O'Neill, Analyst, 2019, 144, 5299-5307

4. A. Rajora, J.W.H. Lou and G. Zheng, Chem. Soc. Rev., 2017, 46, 6433 – 6469.

5. J. Devereux, M. Massaro, A. Barker, D. T. Hinds, B. Hifni, J. C. Simpson and S. J. Quinn, J. Mater. Chem. B, 2019, 7, 3670–3678.

6. S. J. Devereux, S. Cheung, H. C. Daly, D. F. O'Shea and S J. Quinn, Chem. Eur. J., 2018, 24, 14162–14170.

The interest in application of carbon nanostructures for theranostics is growing, considering the importance this approach is assuming, as well as personalized medicine and nanomedicine.

The family of carbon nanostructures presents some components which are intrinsically fluorescent while in other case the functionalization with well-known tags, among which fluorescein, can supply to the lack of this properties. In this contribution we will explore some biological studies of materials belonging to the two classes of nanostructures.

To utilize single-wall carbon nanotubes (SWCNTs) for biomedical applications, individual dispersion and coating of SWCNTs with biocompatible molecules are important to avoid triggering cytotoxicity and increase their stability in serum-containing media. One of the common methods for producing SWCNT-based drug delivery is to individually disperse SWCNTs in aqueous solutions with various biocompatible molecules such as DNA, biopolymers, and proteins, followed by attachment of drugs and targeting moieties to the biologically dispersed nanotubes. Another approach that is less frequently used involves attaching drugs to biocompatible molecules prior to dispersing SWCNTs in a solution. Unfortunately, both approaches suffer from low SWCNT dispersion yield and lack adequate control of drug loading process, especially if the drugs are highly hydrophobic. In this talk, we present an extremely facile schema to generate SWCNT-based drug delivery systems via a controlled, stepwise assembly of drugs and biocompatible molecules on preformed SWCNT networks. We first created a three-dimensional freestanding network of SWCNTs in either aqueous or non-aqueous solvents to facilitate loading of drugs; many potent drugs are only soluble in non-aqueous solvents. SWCNTs in freestanding networks were then coated with drugs such as doxorubicin, which is a common model drug used for characterization of drug loading and release processes owing to its optical properties, and paclitaxel, which is a model drug representing highly lipophilic drugs. Drug-coated SWCNT networks were coated with proteins, such as albumins, followed by dispersing in water via gentle sonication with almost no loss of SWCNT dispersion yield. Optical characterization and imaging have shown that SWCNTs/drug/protein complexes are highly individualized in solution and readily internalized by cells upon exposure. The loaded drugs were efficiently released upon internalization and displayed larger reduction in cell viability compared to free drugs. Further, to demonstrate robustness of our approach and to assist with drug release, we coated SWCNTs with biocompatible polymers prior to decorating with drugs to serve as a sacrificial yet assistive layer. The polymer coating did not alter SWCNT dispersion and drug loading significantly and allowed for faster release of drugs upon internalization in the cells. Overall, the proposed method allows for precisely controlled, stepwise assembly of SWCNTs with any drug and biomolecule combinations and could be further developed for multifunctional drug delivery platform utilizing SWCNTs.

Cancer cells have altered metabolic activity that can be appropriated for delivery of drugs. Rapidly proliferating cancer cells internalize a high amount of protein-dispersed single-wall carbon nanotubes (SWCNTs) with increased endocytosis from deprivation of metabolic fuels, overexpression and increased recycling of membrane receptors, and mechanosensitivity. Differential uptake of protein-dispersed SWCNTs in various cell types have been reported previously, demonstrating that SWCNT uptake highly depends on uptake pathway and cell metabolism. However, there is still gap in understanding how metastatic potential and cell metabolism can differentiate SWCNT uptake and delivery of drugs in a type of cells. In this work, we present uptake and efficacy of SWCNTs/drug/protein complexes in three different epithelial cell lines, NRK-52E, HeLa, and Hs 578T, that present different metabolic levels from their metastatic potentials. We first create SWCNTs/drug/protein complexes with doxorubicin, a common model cancer drug, and bovine serum albumin (BSA), a model protein that has been extensively studied with SWCNT, using a previously developed unique drug loading system using stepwise assembly on SWCNT networks. The result presented that the cells takes up SWCNTs differently in accordance to their metabolic activity, where Hs 578T cells, with the highest metastatic potential and metabolism, take up ~ 10× more nanotubes compared to an epithelial monolayer of NRK-52E cells. Extent of reduction in cell viability from the delivered drugs were in line with the amount of SWCNT uptake. Our work provides further understanding of differential uptake of protein-dispersed SWCNTs with respect to metabolism and energy requirement of the cells. This suggests that protein-dispersed SWCNTs could preferentially target and deliver drugs to cancer cells in a local area with a combination of normal epithelial cells and cancerous cells.

Understanding the impact of single-wall carbon nanotube (SWCNT) lengths on cytotoxicity, uptake and intracellular processing is essential in realizing SWCNT-based drug delivery systems. In addition, cell type-specific response to SWCNT cannot be neglected since SWCNT uptake and processing vary across different cell types. Immune cell-specific processing of SWCNTs in different lengths is of particular interest because immune cells are capable of two distinct size-dependent cellular entry pathways; endocytosis and phagocytosis, and they engage in various physiological conditions such as wound healing, inflammation, and cancer. In this work, we present the impact of SWCNT lengths on uptake, inflammation, and intracellular processing of SWCNTs in macrophages. SWCNTs with average lengths of 50 nm (ultra-short), 145 nm (short), and 500 nm (long) were noncovalently dispersed with bovine serum albumin (BSA), which promotes cellular uptake without affecting cell viability. Interestingly, the amount of uptake was significantly higher for ultra-short SWCNTs compared to longer nanotubes. Moreover, short-SWCNTs became highly bundled in macrophages, which were mostly retained for at least 24 h. In contrast, most long- and ultra-short- SWCNTs remained individualized inside macrophages and were eventually exocytosed over time. Further, we observed pro-inflammatory behavior of macrophage upon exposure to ultra-short-SWCNTs. Overall, the length-dependent inflammation and intracellular processing in macrophages allow for tailoring SWCNT properties for biomedical therapy and imaging. High internalization and subsequent exocytosis of ultra-short-SWCNTs and the triggered pro-inflammatory behavior are more preferred for cancer therapy and drug delivery, while high retention of short-SWCNTs in macrophages could be more desirable for biomedical imaging and macrophage tracking in vivo.

Non-invasive temperature sensing is necessary for the analysis of biological processes occurring in the human body including cellular enzyme activity, protein expression, and ion regulation. Considering that a variety of such biological processes occur at the microscopic scale, a novel mechanism allowing for the detection of the temperature changes in microscopic environments is desired. One-dimensional graphene quantum dots can serve as agents for such detection: they are promising non-invasive probes that because of their 2-5 nm size and optical sensitivity to temperature change enable sub-cellular resolution imaging. Both biocompatible bottom-up synthesized nitrogen-doped graphene quantum dots and quantum dots produced from reduced graphene oxide via top-down approach exhibit temperature-induced fluorescence variations. This response observed for the first time is utilized for deterministic temperature sensing in bulk suspension as well as inside mammalian cells. Distinctive quenching of quantum dot fluorescence by up to 19.8% is observed, in a temperature range from 25℃ to 49℃, in aqueous solution, while the intensity is restored to the original values as the temperature decreases back to 25℃. A similar trend is observed in vitro in HeLa cells as the cellular temperature is increased from 25℃ to 41℃. Our findings suggest that the temperature-dependent fluorescence quenching of bottom-up and top-down-synthesized graphene quantum dots can serve as a non-invasive, reversible, and deterministic mechanism for temperature sensing in microscopic sub-cellular biological environments.

Single-Walled Carbon Nanotubes (SCWNTs) have attracted a lot of attention in biomedical fields. Their easily functionalised surface and ability to encapsulate different materials make them interesting not only for imaging, but also for other applications, such as drug delivery and cell targeting. This work concerns specifically the use of SWCNTs to fabricate Raman nanoprobes for bio-imaging. These nanoprobes are composed of dyes encapsulated inside the SWCNTs and the nanotubes are grafted with anti-bodies functionalized on the outer surface.

First, we aim to optimize the encapsulation process so that the dye/SWCNTs nanohybrid gives a strong, uniform and reproductible signal that is easily detectable by Raman imaging. This is done by varying the parameters of the liquid-phase encapsulation process and crosschecking the results by Raman imaging. We also work to optimize the stability of the nanohybrid in biological media. This parameter is important to determine the best conditions for antibody attachment and cell/tissue interactions. Using different biological buffers, we study the dispersion of the assemblies with Dynamic Light Scattering (DLS) and their stability with Electrophoretic Light Scattering (ELS). The results of the analysis on the nanohybrids show an average hydrodynamic radius of 160 nm and a Zeta potential at -45 mV in aqueous media, proving that the stability of the short nanohybrids in water is favourable to the development of nanoprobes.

Carbon nanotubes are among the most promising platforms for biological applications such as cancer targeting, medical imaging and drug delivery. Herein we present a novel approach to modify single-walled carbon nanotubes (SWCNTs) into Raman nanoprobes capable of targeting biomarkers on cancer cells. The process involves cleaning and shortening in acids followed by encapsulation of specific organic dyes. Subsequently the as-synthesized Raman nanoprobes are then covalently attached with modified NH₂-PEG-COOH to make a generic nanoprobe that is bio-compatible and highly dispersed in aqueous or buffer media. When covalently linked to specific antibodies anti-E-cadherin (monoclonal mouse anti-human) and CK19 (cytokeratin), such nanoprobes can be used to detect there bio-markers in the membrane of breast cancer cell lines.

Here, we will first discuss a modified synthetic approach to densify the covalent pegylation used to link bio-molecules on the SWCNTs and show results with nanoprobes incubated with T47D antibody (ab) and MDA231 cell lines. The immunofluorescence and Raman imaging data shows that dyes@SWCNT/PEG/ab composites can specifically target cancer cells overexpressing high affinity receptors of the specific biomarkers linked to the probes. By densifying the covalently attached PEGs, the results show higher stability and improved selectivity of the Raman nanoprobes towards bio-molecules on cancer cells.

Treatment of complex conditions, such as cancer, has been substantially advanced by a field of molecular therapeutics. However, many of these therapies are limited by the dose toxicity and lack the predictive power of tomography-guided approaches. This can be in part addressed by targeting approaches, focusing the therapy on a tumor site. On the other hand, more benign gene therapeutics often require in vivo delivery, so as to avoid degradation in blood. Additionally, monitoring the location of the therapeutic can help evaluate its targeted accumulation and in part, efficacy. Therefore, there is an open need for multifunctional therapies allowing for targeted delivery, imaging, and treatment. Nanomaterial platforms can provide these capabilities, safely delivering therapeutics, concomitantly imaging their delivery pathways, and presenting sites for targeting agent attachment. Within this scope, the applications of nanoscale graphene derivatives have been largely studied. However, despite their remarkable properties, the biocompatibility and degradability of carbon-based platforms still raises a lot of debate, while modifying those with targeting agents may negatively affect their optical imaging properties. In order to address these issues, we develop graphene quantum dots (GQDs) produced via biocompatible bottom-up synthetic route as attractive candidates for bioimaging and targeted drug delivery. These GQDs are biocompatible and biodegradable, exhibit high-yield intrinsic fluorescence in the visible/near-infrared, high water solubility, pH-based fluorescence response, and have smaller size for more efficient cellular internalization. We explore these properties to develop GQD-based targeted delivery/imaging/treatment agents for cancer therapeutics. Our work utilizes nitrogen-doped GQDs as an emissive platform for covalent attachment of a targeting agent (hyaluronic acid (HA) targeted to the CD44 receptors on several cancer cell types) and oxidative stress-based cancer therapeutic (ferrocene (Fc)). The synthesized multifunctional formulation is characterized and its efficacy evaluated in vitro. Elemental mapping indicates that the purified from reactants synthetic product has an average iron content of 0.64 atomic percent, suggesting the successful attachment of the therapeutic, while FFT analysis of TEM images confirms the crystalline structure of the GQDs. Although GQDs alone yield no cytotoxicity as quantified via the MTT assay up to the maximum imaging concentrations of 1 mg/mL, the Fc-HA-GQD formulation exhibits a higher cytotoxic response in the cancer cells (MCF-7) targeted by the HA as opposed to healthy ones (HEK-293) that do not overexpress CD44 suggesting cancer-selective targeted efficacy. As Fc induces oxidative stress that is less mitigated in cancer cells, we expect it to also contribute to the observed cancer-selective treatment response. We further use spectrally-resolved in vitro fluorescence imaging showing the efficient cellular internalization maximized in MCF-7 cells over the HEK-293, and thus verifying the possibility of successful image-guided drug delivery. As a result, we propose Fc-HA-GQD formulation as a multifunctional targeted delivery, imaging, and cancer-specific treatment agent further to be studied in vivo.

Objectives

Scaffolds for bone tissue engineering (BTE) are three-dimensional (3D) porous matrices that provide the necessary sites for cell adhesion and proliferation, where the architecture plays an important role. Ideally, BTE scaffolds should have an interconnected network of both large and small pores to facilitate the infiltration of cells and the diffusion of growth factors and nutrients¹. Scaffolds for BTE should also enhance osteogenic differentiation to improve bone regeneration. Graphene oxide (GO) can promote osteogenic differentiation of mesenchymal stem cells (MSCs) because it can provide biophysical cues and adsorb biological factors². However, due to the lack of fabrication strategies, it remains a challenge to develop GO and GO composite scaffolds with a hierarchical architecture for BTE. In this study, we aim to develop a dual-templating method to control the assembly of GO sheets, in order to design and achieve this architecture.

Experimental Methods

We modified the amphiphilicity of GO by adding different amounts of cetyltrimethylammonium bromide (CTAB), hydroxyapatite (HA), polyacrylic acid (PAA) and elastin from bovine neck ligament. Then we developed GO-CTAB, GO-CTAB-HA, GO-CTAB-PAA and GO-elastin stabilized oil in water emulsions. Afterwards, we froze the emulsions at different temperatures (-190 ^oC and -20 ^oC), and upon freeze-drying we produced free-standing scaffolds. We reduced GO-CTAB scaffolds in different conditions to obtain rGO scaffolds. We studied the formation of emulsions and the structural and chemical properties of scaffolds through optical microscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared, and compressive strength tests. We seeded mouse bone marrow MSCs on 2D GO substrates and 3D GO-based scaffolds. Then, we compared the cell proliferation after 10 days of incubation. We also analyzed the cells on GO-based scaffolds after 7 days of incubation using SEM and confocal microscopy, in order to study cell morphology, attachment and infiltration in the scaffolds.

Results and Discussion

The scaffolds based on GO, GO-HA, GO-GO-PAA, GO-elastin and rGO have interconnected primary pores of 150-300 µm in diameter (Figure 1 a-j), which matches the size of the hexane droplet templates. Freezing at -190 ^oC results in smaller secondary pores around 10 µm in diameter (Figure 1 a-e) while freezing at -20 ^oC results in larger secondary pores with diameters around 30-50 µm (Figure 1 f-j). This result shows that the secondary pore formation is controlled by ice nucleation and growth in the aqueous phase of the emulsions at different temperatures. Scaffolds based on GO-HA, GO-PAA and especially GO-elastin show an improved modulus of compression compared to GO-based scaffolds (Figure 1 k). The cell culture using MSCs reveals cell proliferation up to 10 days on 2D GO substrates and 3D GO-based scaffolds (Figure 1 l). Compared to 2D substrates, the large and interconnected pores in 3D scaffolds contribute to the higher cell proliferation result. The cells are well spread on the scaffolds with an elongated shape and filamentous extensions at day 7 (Figure 1m). They infiltrate all the way to the center of GO-based scaffolds through primary pores (Figure 1 m, n). These results indicate that primary pores can facilitate the cell infiltration.

Conclusions

The rapid development and broad application of graphene-based porous materials requires efficient and facile fabrication methods. We developed a novel strategy to control the assembly of GO sheets using emulsions and ice templates to fabricate GO-based scaffolds with an interconnected and hierarchical porous structure. The ability to fabricate rGO and GO composite scaffolds with similar architectures demonstrates the versatility of this strategy. The GO-based scaffolds show biocompatibility and allow cell infiltration. We expect that this strategy can lead to improvements in the fabrication of GO-type scaffolds for BTE.

References

1. Karageorgiou V et al. Biomaterials 26 (27):5474-91. 2005

2. Lee WC et al. Acs Nano 5 (9):7334-41. 2011

Acknowledgements

We acknowledge support from NSERC, the Canada Research Chair Foundation and McGill Engineering Doctoral Award.

Figure 1