Unpredictable and uncontrollable protein adsorption on nanoparticles remains a considerable challenge to achieving effective application of nanotechnologies within biological environments. ...Nevertheless, engineered nanoparticles offer unprecedented functionality and control in probing and altering biological systems. In this review, we highlight recent advances in harnessing the "protein corona" formed on nanoparticles as a handle to tune functional properties of the protein-nanoparticle complex. Towards this end, we first review nanoparticle properties that influence protein adsorption and design strategies to facilitate selective corona formation, with the corresponding characterization techniques. We next focus on literature detailing corona-mediated functionalities, including stealth to avoid recognition and sequestration while in circulation, targeting of predetermined in vivo locations, and controlled activation once localized to the intended biological compartment. We conclude with a discussion of biocompatibility outcomes for these protein-nanoparticle complexes applied in vivo. While formation of the nanoparticle-corona complex may impede our control over its use for the projected nanobiotechnology application, it concurrently presents an opportunity to create improved protein-nanoparticle architectures by exploiting natural or guiding selective protein adsorption to the nanoparticle surface.
Optical nanoscale technologies often implement covalent or noncovalent strategies for the modification of nanoparticles, whereby both functionalizations are leveraged for multimodal applications but ...can affect the intrinsic fluorescence of nanoparticles. Specifically, single‐walled carbon nanotubes (SWCNTs) can enable real‐time imaging and cellular delivery; however, the introduction of covalent SWCNT sidewall functionalizations often attenuates SWCNT fluorescence. Recent advances in SWCNT covalent functionalization chemistries preserve the SWCNT's pristine graphitic lattice and intrinsic fluorescence, and here, such covalently functionalized SWCNTs maintain intrinsic fluorescence‐based molecular recognition of neurotransmitter and protein analytes. The covalently modified SWCNT nanosensor preserves its fluorescence response towards its analyte for certain nanosensors, presumably dependent on the intermolecular interactions between SWCNTs or the steric hindrance introduced by the covalent functionalization that hinders noncovalent interactions with the SWCNT surface. These SWCNT nanosensors are further functionalized via their covalent handles with a targeting ligand, biotin, to self‐assemble on passivated microscopy slides, and these dual‐functionalized SWCNT materials are explored for future use in multiplexed sensing and imaging applications.
Recent advances in covalent functionalization of single‐walled carbon nanotubes are explored to create intrinsically near‐infrared fluorescent carbon nanotube nanosensors with multimodal applications. Simultaneous covalent and noncovalent functionalizations on single‐walled carbon nanotube (SWCNT) surfaces interact through steric hindrance and intermolecular interactions. The ability to dual functionalize SWCNT nanoparticles has potential applications in diverse biotechnology applications including biomolecule imaging applications described herein.
Noncovalent adsorption of DNA on nanoparticles has led to their widespread implementation as gene delivery tools and optical probes. Yet, the behavior and stability of DNA-nanoparticle complexes once ...applied in biomolecule-rich, in vivo environments remains unpredictable, whereby biocompatibility testing usually occurs in serum. Here, we demonstrate time-resolved measurements of exchange dynamics between solution-phase and adsorbed corona-phase DNA and protein biomolecules on single-walled carbon nanotubes (SWCNTs). We capture real-time binding of fluorophore-labeled biomolecules, utilizing the SWCNT surface as a fluorescence quencher, and apply this corona exchange assay to study protein corona dynamics on ssDNA-SWCNT-based dopamine sensors. We study exchange of two blood proteins, albumin and fibrinogen, adsorbing to and competitively displacing (GT)6 vs (GT)15 ssDNA from ssDNA-SWCNTs. We find that (GT)15 binds to SWCNTs with a higher affinity than (GT)6 and that fibrinogen interacts with ssDNA-SWCNTs more strongly than albumin. Albumin and fibrinogen cause a 52.2% and 78.2% attenuation of the dopamine nanosensor response, coinciding with 0.5% and 3.7% desorption of (GT)6, respectively. Concurrently, the total surface-adsorbed fibrinogen mass is 168% greater than that of albumin. Binding profiles are fit to a competitive surface exchange model which recapitulates the experimental observation that fibrinogen has a higher affinity for SWCNTs than albumin, with a fibrinogen on-rate constant 1.61-fold greater and an off-rate constant 0.563-fold smaller than that of albumin. Our methodology presents a generic route to assess real-time corona exchange on nanoparticles in solution phase and more broadly motivates testing of nanoparticle-based technologies in blood plasma rather than the more ubiquitously tested serum conditions.
When nanoparticles enter biological environments, proteins adsorb to form the “protein corona” which alters nanoparticle biodistribution and toxicity. Herein, we measure protein corona formation on ...DNA‐functionalized single‐walled carbon nanotubes (ssDNA‐SWCNTs), a nanoparticle used widely for sensing and delivery, in blood plasma and cerebrospinal fluid. We characterize corona composition by mass spectrometry, revealing high‐abundance corona proteins involved in lipid binding, complement activation, and coagulation. We investigate roles of electrostatic and entropic interactions driving selective corona formation. Lastly, we study real‐time protein binding on ssDNA‐SWCNTs, obtaining agreement between enriched proteins binding strongly and depleted proteins binding marginally, while highlighting cooperative adsorption mechanisms. Knowledge of protein corona composition, formation mechanisms, and dynamics informs nanoparticle translation from in vitro design to in vivo application.
Introducing engineered nanoparticles into biosystems leads to formation of the interfacial protein corona, modifying nanoparticle identity and function. Herein, protein corona composition, interactions, and dynamics are analyzed on carbon nanotube‐based sensors. This generic framework will enhance our fundamental understanding of protein corona formation and enable effective translation of nanotechnologies from in vitro design to in vivo use.
To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube ...(SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.
Posttranscriptional gene silencing (PTGS) is a powerful tool to understand and control plant metabolic pathways, which is central to plant biotechnology. PTGS is commonly accomplished through ...delivery of small interfering RNA (siRNA) into cells. Standard plant siRNA delivery methods (
and viruses) involve coding siRNA into DNA vectors and are only tractable for certain plant species. Here, we develop a nanotube-based platform for direct delivery of siRNA and show high silencing efficiency in intact plant cells. We demonstrate that nanotubes successfully deliver siRNA and silence endogenous genes, owing to effective intracellular delivery and nanotube-induced protection of siRNA from nuclease degradation. This study establishes that nanotubes could enable a myriad of plant biotechnology applications that rely on RNA delivery to intact cells.
Rapidly growing interest in the nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into ...plant cells. However, little is known about how the size and shape of nanoparticles influence transport in plants and the delivery efficiency of their cargoes, limiting the development of nanotechnology in plant systems. In this study we employed non-biolistically delivered DNA-modified gold nanoparticles (AuNPs) of various sizes (5-20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana leaves. Generally, smaller AuNPs demonstrated more rapid, higher and longer-lasting levels of association with plant cell walls compared with larger AuNPs. We observed internalization of rod-shaped but not spherical AuNPs into plant cells, yet, surprisingly, 10 nm spherical AuNPs functionalized with small-interfering RNA (siRNA) were the most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Overall, our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious biocargo delivery for future developments in plant nanobiotechnology.
The global SARS-CoV-2 coronavirus pandemic has led to a surging demand for rapid and efficient viral infection diagnostic tests, generating a supply shortage in diagnostic test consumables including ...nucleic acid extraction kits. Here, we develop a modular method for high-yield extraction of viral single-stranded nucleic acids by using “capture” ssDNA sequences attached to carbon nanotubes. Target SARS-CoV-2 viral RNA can be captured by ssDNA-nanotube constructs via hybridization and separated from the liquid phase in a single-tube system with minimal chemical reagents, for downstream quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. This nanotube-based extraction method enables 100% extraction yield of target SARS-CoV-2 RNA from phosphate-buffered saline in comparison to ∼20% extraction yield when using a commercial silica-column kit. Notably, carbon nanotubes enable extraction of nucleic acids directly from 50% human saliva with a similar efficiency as achieved with commercial DNA/RNA extraction kits, thereby bypassing the need for further biofluid purification and avoiding the use of commercial extraction kits. Carbon nanotube-based extraction of viral nucleic acids facilitates high-yield and high-sensitivity identification of viral nucleic acids such as the SARS-CoV-2 viral genome with a reduced reliance on reagents affected by supply chain obstacles.
Cells rely on their cytoskeleton for key processes including division and directed motility. Actin filaments are a primary constituent of the cytoskeleton. Although actin filaments can create a ...variety of network architectures linked to distinct cell functions, the microscale molecular interactions that give rise to these macroscale structures are not well understood. In this work, we investigate the microscale mechanisms that produce different branched actin network structures using an iterative classification approach. First, we employ a simple yet comprehensive agent-based model that produces synthetic actin networks with precise control over the microscale dynamics. Then we apply machine learning techniques to classify actin networks based on measurable network density and geometry, identifying key mechanistic processes that lead to particular branched actin network architectures. Extensive computational experiments reveal that the most accurate method uses a combination of supervised learning based on network density and unsupervised learning based on network symmetry. This framework can potentially serve as a powerful tool to discover the molecular interactions that produce the wide variety of actin network configurations associated with normal development as well as pathological conditions such as cancer.
Single-walled carbon nanotubes (SWCNT) are used in neuroscience for deep-brain imaging, neuron activity recording, measuring brain morphology, and imaging neuromodulation. However, the extent to ...which SWCNT-based probes impact brain tissue is not well understood. Here, we study the impact of (GT)6-SWCNT dopamine nanosensors on SIM-A9 mouse microglial cells and show SWCNT-induced morphological and transcriptomic changes in these brain immune cells. Next, we introduce a strategy to passivate (GT)6-SWCNT nanosensors with PEGylated phospholipids to improve both biocompatibility and dopamine imaging quality. We apply these passivated dopamine nanosensors to image electrically stimulated striatal dopamine release in acute mouse brain slices, and show that slices labeled with passivated nanosensors exhibit higher fluorescence response to dopamine and measure more putative dopamine release sites. Hence, this facile modification to SWCNT-based dopamine probes provides immediate improvements to both biocompatibility and dopamine imaging functionality with an approach that is readily translatable to other SWCNT-based neurotechnologies.