Nanomaterials have the potential to play a significant role in the development of new biotechnology for diverse applications, including agricultural genetic engineering, biological sensing, nanomedicine, optics, and more. Therefore, a robust, fundamental understanding of their material properties and the techniques to engineer these properties is a key area of scientific interest.

In this dissertation, we explore several key areas where the chemical engineering of nanotechnology, particularly single walled carbon nanotubes (SWNTs), can have a significant impact. We first broadly explore emerging trends in some of the most promising near-infrared luminescent materials and applications. In particular, we focus on how a more comprehensive understanding of their intrinsic material properties might allow researchers to better leverage these traits for innovative and robust applications.

Further, we focus on the ways in which nanomaterials can address some of the most critical challenges of CRISPR genome editing in plants through improvements in cargo delivery, species independence, germline transformation and gene editing efficiency. We identify major barriers preventing CRISPR-mediated plant genetic engineering from reaching its full potential, and discuss ways that nanoparticle technologies can lower or eliminate these barriers.

Next, we present mechanisms to optimize DNA loading on SWNTs with a library of polymer- SWNT constructs and assess DNA loading ability, polydispersity, and both chemical and colloidal stability. We demonstrate that polymer hydrolysis from nanomaterial surfaces can occur depending on polymer properties and attachment chemistries, and we describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the stress response of plants to polymer-based nanomaterials and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.

We then adapt our findings from the development of polymer-SWNTs towards the synthesis of protein-functionalized SWNTs and the covalent attachment of DNA cargo. Specifically, we explore the development of streptavidin-biotin chemistry as a potential alternative to other existing nanoparticle-biomolecule delivery systems and offer exciting opportunities for future research.

Finally, we explore the federal regulatory challenges in the United States that limit scientific innovation and unduly hinder the widespread production of genetically engineered crops, like those we propose to develop throughout this thesis. To address these shortcomings, we propose policy recommendations including the consolidation of federal regulatory communication and a unified web platform for commercial approval applications.

Single-walled carbon nanotubes (SWCNT) are an emerging nanomaterial platform enabling promising advances in biomolecular imaging. Noncovalent modification of SWCNTs with amphiphilic ligands reveals intrinsic SWCNT near-infrared fluorescence and imparts molecular recognition. In particular, SWCNTs wrapped with DNA oligonucleotides develop selective fluorescent response to catecholamine neurotransmitters such as dopamine. Consequently, these DNA-SWCNT probes have been applied for recording dopamine activity in brain tissue. However, two major barriers to in vivo application of SWCNT dopamine probes exist. Firstly, DNA-SWCNTs are highly susceptible to biofouling by protein adsorption reducing efficacy of sensors in vivo. Secondly, the impact of these artificial nanomaterials on biological environments remains poorly understood. Towards the first point, we develop a novel method for the study of biomolecular adsorption to the SWCNT surface utilizing the fluorescence quenching ability of carbon nanotubes to track adsorption of fluorophore-conjugated biomolecules. We leverage multiplexing using dissimilar dyes conjugated to each molecular species in order to simultaneously monitor the competitive adsorption/desorption of proteins and single-stranded DNA, respectively. Here we show that attenuation of dopamine response is largely due to protein adsorption rather than DNA desorption. Furthermore, we identify fibrinogen as a ubiquitous protein with high affinity to the SWCNT surface.

Next, we study the effects these SWCNT probes may elicit once implanted in brain tissue. Here we utilize microglial cells as a model for neuroinflammation. Microglia are the specialized immune cells of the central nervous system which are central in maintaining the homeostasis of the neuronal environment. Microglia are activated by interaction with foreign or pathogenic material, mediating and propagating inflammatory responses throughout the brain which can elicit downstream neurotoxicity and neurodegeneration. Examining the interactions between this cell type and carbon nanotube neuro-sensors is crucial in characterizing and quantifying the net biological impact of these probes on surrounding brain tissue. We utilize high-throughput sequencing and live-cell imaging to show that exposure to SWCNT probes induces significant upregulation of inflammatory signaling pathways and cell morphology change in SIM-A9 microglia. Although inflammation response is lower in magnitude than that provoked by inflammatory simulants such as lipopolysaccharide, SWCNTs uniquely induced drastic changes to cell morphology, where cells exposed to SWCNTs progressed from round and motile states to highly ramified and stationary within several hours. These effects suggest activation of microglia into pro-inflammatory phenotypes by SWCNTs. This must be minimized in order to accurately study chemical signaling in the brain.

Subsequently, we devise strategies for noncovalent passivation of exposed SWCNT graphene lattice to improve biocompatibility. We apply protein adsorption and microglial activation as metrics by which to test noncovalently modified SWCNT catecholamine sensors. We find that passivation of DNA-SWCNTs using PEGylated phospholipid significantly decreases nonspecific protein adsorption and SWCNT-induced microglial ramification. These passivated neuro-sensors are then applied to image striatal dopamine release and reuptake events in excised mouse brain tissue. We show that brain slices labeled with passivated neuro-sensor exhibited both improved diffusivity through tissue and higher fluorescence response to dopamine release over unmodified SWCNT probes. Hence, we present three stages of development and optimization of a carbon nanotube-based neuro-sensor through design of the nanoparticle corona phase. The methodologies presented here are readily translatable to other bionanotechnologies and represent an advancement in the field of nanomaterial biosensors for molecular imaging.

The implementation of nascent CRISPR-based gene reading and writing tools in plant systems has lagged relative to mammalian counterparts. Methods for direct delivery of pre-formed ribonucleoproteins (RNPs) mediated by nanocarriers such as peptides and polymers have been explored with limited success. In part, this is due to the plant cell wall, a unique barrier to exogenous biomolecule delivery absent in other cell types. As such, the dominant model for studying direct delivery of RNPs and RNP-nanoparticles are protoplasts - plant cells that have had their cell wall enzymatically degraded. However, insights gained from such studies are limited in their generalization across species and the ultimate usefulness of such a simplified model is a question of concern.

Here, we report on the results of our efforts to translate three preparation methods previously tested in mammalian cells for polymer- and peptide-based Cas9 and CasΦ RNP-nanoparticles to both plant protoplast and 7-day-old seedlings: i) non-covalent complexing with charged polymers, ii) non-covalent complexing, and iii) tyrosinase-mediated covalent conjugation of various relevant peptide motifs. We use next generation sequencing to evaluate the ability of these RNP-nanoparticles to generate edits in the PDS and PDS3 genes of model plant systems Nicotiana benthamiana and Arabidopsis thaliana respectively. In seedlings, we observe no editing and confirm the absence of successful delivery events using the quantitative microscopy tool DCIP. In protoplasts, the amphiphilic peptide A5K or covalent attachment of peptides had no positive effect on editing efficiencies. However, the inclusion of the anionic polymer polyglutamic acid significantly improved intended editing efficiencies in both species relative to plain RNPs without negatively impacting protoplast viability. Our results support previously published literature that suggests the mechanism of action is stabilization of the RNP rather than the polymer having orthogonal activity in vivo. We believe that this cheap, simple, and accessible method of stabilizing Cas9 RNPs can be easily adopted by others working on direct protein delivery to plant protoplasts to increase gene editing. Furthermore, we believe this multi-scale analysis provides helpful insight into the feasibility of translating nanocarriers methodologies from mammalian to plant systems.

Many diseases, such as neurodegenerative disorders, cancer, and autoimmune diseases are caused by or exhibit symptoms of abnormal regulation of signaling proteins. In order to obtain a molecular understanding of diseases, we require tools capable of probing the intricate signaling pathways that govern biological function. Understanding the role these proteins play as they circulate between cells will allow us to examine disease progression from an intercellular point of view. Currently, it is difficult to study these proteins in their natural environment, in vivo, because they are created and function on very broad time and length scales. Existing protein detection methods have several limitations as they are optimized for intracellular imaging, performed in vitro, require lengthy sample handling, function over short time scales, or have molecular recognition elements that are unstable in biological media.

This dissertation presents a modular platform to create optical nanosensors that are able to address current limitations in signaling protein detection. Nanosensor elements require optimization of both signal transduction and molecular recognition elements. Single-walled carbon nanotubes (SWCNTs) are nanoparticles that are ideal signal transducers for biological imaging. SWCNTs have optical properties well-suited for biological sensing such as infinite fluorescence lifetime, no blinking, small size, and fluorescence in the near-infrared region of the electromagnetic spectrum that is least attenuated by biological systems. Several SWCNT nanosensors have already been developed for signaling small molecule targets and peptides, but, to date, none have been created for signaling proteins.

In order to create a robust imaging platform based on SWCNTs for sensing signaling proteins, it is possible to couple molecular recognition elements to SWCNT signal transducers using dual noncovalent and covalent functionalization strategies. The development of noncovalent molecular recognition elements is explored using peptide mimetic polymers called peptoids. The discovery of a synthetic peptoid binding loop for wheat germ agglutinin protein as a proof-of-principle case study shows the ability to utilize diverse chemical materials to create a fully synthetic binding element for desired protein targets. Additionally, the development of covalently functionalized SWCNTs is explored for the creation of multifunctional optical SWCNT nanosensors. Here covalently functional groups provide functional handles that work synergistically with noncovalent passivation to enable the development of a diverse nanosensor toolbox.

The findings presented in this dissertation lay the foundation of valuable techniques and materials to optimize the binding to and study of signaling proteins. Future work to streamline the development of novel nanosensors is discussed, and the framework for the creation of other biological nanomaterial tools through these noncovalent and covalent techniques is also explored.

Introducing CRISPR machinery to a host plant genome may be accomplished either through expression of recombinant plasmids or direct delivery of the Cas9 ribonucleoprotein (RNP). However, introducing the biomolecular workhorses of CRISPR into model and crop species is hindered by the molecular delivery challenge across the plant cell wall. The cell wall presents a rigid barrier to biomolecule delivery which can be overcome by Agrobacterium or biolistic particle bombardment, the success of which is limited by host species, tissue type, and random transgene integration. Furthermore, the necessity of tissue culture introduces further limitations, as many crops are recalcitrant towards regeneration. RNP delivery simplifies the workflow of plant gene editing by circumventing the need for plasmid optimization and improves specificity by reduction of off-target cleavage. However, introducing Cas9 RNPs into plants is not easily addressed by current delivery technologies, as Agrobacterium is only amenable to DNA delivery and biolistics rely on protein dehydration which can result in loss of Cas9 activity. Nanomaterials offer an addition to the workhorses of plant genetic engineering due to their ability to load diverse cargo, traverse the cell wall and plasma membrane, and selectively localize in tissues and organelles. We have developed a polyamine-carbon nanotube conjugate (PEI-SWNT) which is loaded with DNA and administered by aqueous infiltration to the leaf abaxis. In Nicotiana benthamiana, we report indel generation in a GFP transgene via PEI-SWNT plasmid delivery. We also present the development of Cas9-SWNT conjugates for RNP delivery to mature plant tissue without biolistics – both through direct binding of an engineered Cas9 variant to the CNT surface and through the inclusion of a noncovalent peptoid intermediate for binding of WT Cas9 to the CNT surface. Peptoids, or poly-N-substituted glycines, possess remarkable biostability and peptide-like structural properties. We present the design and synthesis of modular peptoids with two domains, a CNT-binding domain and an RNP-binding domain, and demonstrate screening of a peptoid library based on these domains for non-covalent binding of Cas9 to peptoid-CNTs. We have identified charge and the protein:SWNT ratio as key variables in the design of a stable conjugate, where a stable RNP-peptoid-conjugate can then enable cytosolic delivery of preassembled Cas9 RNP to mature plant cells. Lastly, we report indel generation in the model gene target phytoene desaturase (PDS) from N. benthamiana via peptoid-SWNT mediated delivery of Cas9 RNPs.

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 study 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 spatiotemporal resolution and chemical selectivity are needed to facilitate

investigations of dopamine neurochemistry.

To address this need, this dissertation describes the synthesis and implementation of

an ultrasensitive near-infrared “turn-on” nanosensor (nIRCat) for the catecholamine

neuromodulators dopamine and norepinephrine. To guide probe development, we present

results from a computational model that offers insight into the spatiotemporal dynamics of

dopamine in the striatum, a subcortical structure that is enriched in dopamine. With this

model, we elucidated the kinetic requirements for a prototypical optical indicator as well as

optimal imaging frame rates needed for measuring dopamine neurochemical dynamics.

Stochastic modeling of dopamine dynamics, driven by kinetic phenomena of vesicular

release, diffusion and clearance, provide a platform to evaluate dopaminergic volume

transmission arising from a single terminal or ensemble terminal activity. With this work,

we illustrate that only probes with kinetic parameters in a particular range are feasible for

dopamine imaging at spatiotemporal scales likely to be encountered in brain tissue.

In two subsequent chapters, we describe the development and in vitro

characterization of nIRCats, synthesized from functionalized single wall carbon nanotubes

(SWCNT) that fluoresce in the near infrared range of the spectrum. We show that nIRCats

exhibit maximal relative change in fluorescence intensity (ΔF/F0) of up to 35-fold in

response to catecholamines and have optimal dynamic range that span physiological

concentrations of their target brain analytes. Through a combination of experimental and

molecular dynamics approaches, we elucidate the photophysical principles and intermolecular

interactions that govern the molecular recognition and fluorescence modulation of nIRCats by dopamine.

Finally, we demonstrate that nIRCat can be used to measure electrically and

optogenetically evoked release of dopamine in striatal brain slices, revealing hotspots of

activity with a median size of 2 μm, and exhibiting a log-normal size distribution that extends

up to 10 μm. Moreover, nIRCats are shown to be compatible with dopamine pharmacology

and permit studies of how receptor-targeting drugs modulate evoked dopamine release. Our

results suggest nIRCats 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 and

psychoactive molecules that modulate the release and clearance profiles of dopamine. We

conclude that nIRCats and other nanosensors of this class can serve as versatile synthetic

optical tools to monitor interneuronal chemical signaling in the brain extracellular space at

spatial and temporal scales pertinent to the encoded information.

Nanoparticles are nanometer-scale materials capable of interacting with biomolecules on various size scales from small molecules to oligonucleotides to proteins. Their unique ability to distribute throughout the body and internalize into cells has led to their recent use as signal transducers for analyte sensing and therapeutic cargo carriers. Current techniques for attaching biomolecular cargo to nanoparticles typically rely on affinity between the two species, especially noncovalent adsorption via hydrophobic interactions. Proteins in particular have complex tertiary structures that must be maintained for biosensing and delivery applications. This is difficult to achieve when attaching proteins to nanoparticles by noncovalent adsorption due to the propensity of proteins to denature and desorb in complex biological environments such as blood plasma. Additionally, the large variability among proteins and their affinity for nanoparticles makes it difficult to predict the successful generation of potential protein-nanoparticle systems. Control of the interactions between nanoparticles and proteins is thus crucial to the successful development of tools to understand and interface with biological systems.

Towards this end, exploring protein conjugation to single-walled carbon nanotubes (SWCNTs) could advance the study of nanoparticle-protein conjugates. SWCNTs have shown utility as biosensing signal transducers due to their intrinsic, photostable, and minimally attenuated fluorescence emission in biological environments. Furthermore, their small size and large surface area make them ideal vehicles for cellular cargo delivery. Several noncovalent protein-SWCNT constructs for sensing and delivery have been developed but are susceptible to instability and loss of function when applied in biological systems. Covalent conjugation of proteins to SWCNTs could mitigate these effects and produce stable constructs to monitor disease progression and deliver therapeutics.

This dissertation presents the development of an adaptable platform for the covalent attachment of proteins to SWCNTs for analyte sensing and protein delivery. We first optimized the platform and workflow by attaching a model enzyme, Horseradish Peroxidase, to SWCNTs for hydrogen peroxide (H2O2) sensing. We find that it is possible to maintain both intrinsic SWCNT fluorescence and peroxidase enzymatic activity via triazine-thiol-SMCC crosslinker chemistry, which are key to subsequent function as a sensor. The resulting sensor shows sensitive, stable, and repeatable fluorescence modulation when exposed to H2O2 in solution and immobilized on glass. After demonstrating the utility of the platform, we have applied this methodology to covalently conjugate the CRISPR-Cas9 ribonucleoprotein to SWCNTs for delivery to cells for targeted gene editing. Using an in vitro DNA cleavage assay, we verified that Cas9 activity is maintained after SWCNT conjugation and dependent on crosslinker:protein ratio. This also suggests that the covalent conjugation platform is adaptable to other proteins, but reaction optimization should be completed every time to elucidate the optimal conditions for each protein.

The findings presented in this dissertation review the creation and optimization of a platform for the development of covalent protein-SWCNT constructs for increased stability and function in biological environments. Prioritizing covalent attachment of recognition elements and functional groups will lead to nanoparticle-based sensors and delivery vehicles with enhanced efficiency compared to previous efforts. Future directions of inquiry are also discussed and guidelines for efficiently exploring these new directions are summarized within.

The fundamental properties of the stimluated Raman scattering (SRS) interaction make it attractive for application in biological microscopy. The signal is linear in both the concentration of scatterers, as well as the power of both excitation sources used. In turn, this allows for quantitative concentration measurements to be made within a sample. Furthermore, the SRS spectrum, an exact match to the spontaneous Raman spectrum, is chemically specific. I.e., individual molecules can be specifically and uniquely identified by their spectra. As a result, in principle, SRS microscopy allows for quantitative label-free measurements to be made of biological molecules.

In practice, however, outside of a few applications, SRS microscopy is neither widely adopted nor applied in biology. It is often thought that the specificity of fluorescence cannot be achieved in a label-free manner with SRS due to the complexity of the biological systems under investigation. Additionally, as the standard implementation of SRS microscopy requires laser scanning for the formation of an image, fundamental limits on acquisition speed are thought to make the application of SRS microscopy to the study of dynamic processes challenging, if not infeasible.

The following dissertation presents new experimental and computational methods to broaden the applications of SRS imaging within the life sciences. An overview of the fabrication of the standard SRS imaging system is provided, with attention to the implications of certain design choices. This helps establish the limits within which the SRS microscopy technique currently operates, as well as make the technique more accessible to a general audience. Best practices for performing measurements and analyzing the results are presented. The application of SRS microscopy to the imaging of small molecule neurotransmitters, in a specific manner, is demonstrated, highlighting the capability of SRS microscopy to achieve of the specificity of fluorescence imaging. Finally, extensions to the standard imaging system are presented which lay the groundwork for future applications with faster acquisition speeds. In aggregate, the work presented in this dissertation serves to extend the range of problems to which SRS microscopy can be applied in biology.

Engineered nanoparticles have emerged as a promising platform upon which to develop biological sensing, imaging, and delivery tools. Yet, a paramount challenge toward implementing such technologies is understanding how these engineered nanoparticles are affecting and being affected by the complex bioenvironments in which they are applied. Introducing a nanoparticle into a biological system rapidly establishes a nano-bio interface, as biomolecules, most notably proteins, adsorb to the nanoparticle surface to form the “corona”. The abruptness of protein adsorption on foreign nanoparticle surfaces causes proteins to interact in atypical modes, contrary to normal biomolecular interactions governed by precise genetic control, and often produces undesirable outcomes such as protein denaturation. Further, protein corona formation unpredictably changes the nanoparticle identity and fate, as the adsorbed proteins mask original surface characteristics and endow new biochemical properties to the nanoparticle. As a result, how the nanoparticle-corona complex interacts with biological machinery is impacted and in vivo circulation, bioaccumulation, and biocompatibility outcomes are drastically modified. Consequently, protein corona formation can disrupt the nanoparticle’s designated function by attenuating or eliminating nanoparticle efficacy relative to its in vitro performance. Conversely, the protein corona can be taken advantage of, with an engineered protein corona that facilitates new sensing modalities or stealth transport in targeted delivery applications, with improved nanoparticle functionality or therapeutic effect to follow. In both cases, the protein corona displayed on the nanoparticle surface is a principal design parameter for ensuring successful applications of nanotechnologies in biological systems.

In this dissertation, I develop a framework of analysis to quantitatively characterize protein corona formation that occurs on nanoparticle substrates in different biological environments. I adopt a primarily experimental approach, with complementary physics-based and statistical modeling, toward investigating these nano-bio interfacial interactions. I begin with a multimodal characterization of protein corona composition, driving forces of formation, and kinetics in relevant biological media. These studies primarily focus on protein adsorption to single-stranded DNA-functionalized single-walled carbon nanotube (ssDNA-SWCNT) probes. ssDNA-SWCNTs are an appealing tool for biological sensing and imaging because they operate at spatiotemporal scales necessary to capture information on complex biological systems, such as neurotransmitter signaling in the brain. However, it is of note that the methods of corona characterization and framework of understanding are generalizable to different nanoparticles in different bioenvironments. To probe the protein corona composition, I have optimized a platform to isolate nanoparticle-bound corona proteins and to determine abundance and differential enrichment vs. depletion of corona proteins by mass spectrometry-based proteomics. This analysis provides protein corona compositional maps for nanoparticles that underscore the selectivity of corona formation, reveal key proteins and protein functional roles implicated in corona formation, and enable analysis of protein physicochemical properties governing adsorption to nanoparticle surfaces. By varying incubation conditions of ssDNA-SWCNTs in biofluids, I investigate the role different interactions have on driving selective protein corona formation dependent on corona layer, including electrostatic and hydrophobic forces. These experimental studies are complemented by mathematical modeling, with a colloid thermodynamic framework to describe how the nanoparticle and protein interact in solution, and classification modeling, with a supervised learning approach to move toward predicting nano-bio interactions. To study the dynamic exchange of biomolecules on the SWCNT surface, I have developed a multiplexed fluorescence assay that enables real-time tracking of biomolecule adsorption and desorption events, with corresponding kinetic modeling of this exchange process. From these dynamic corona studies, I provide insight on the general translatability of whole-biofluid nano-bio interactions to single-protein experiments, while highlighting the role of cooperativity effects driving certain protein-nanoparticle interactions. Moreover, this corona exchange assay can be readily extended to examine various biomolecules binding on nanoparticles and enables study in solution rather than in a surface-immobilized, less biologically relevant, setting. Understanding the protein corona composition, driving forces of formation, and dynamics under relevant solution conditions informs design and synthesis of nanotechnology-based tools applied in protein-rich environments.

Although corona formation can impair nanobiotechnology efficacy, it also presents an opportunity to create improved protein-nanoparticle architectures by exploiting selective protein adsorption to the nanoparticle surface. Toward this end, I leverage the fundamental understanding of nano-bio interactions developed in the first portion of this dissertation to design and develop a novel nanosensor for viral protein detection. This nanosensor harnesses SWCNTs to provide the optical signal readout, together with proteins that possess intrinsic recognition ability for the analyte of interest: the ACE2 cell membrane protein is used to bind to the SARS-CoV-2 spike protein analyte. The resulting ACE2-SWCNT nanosensors are thus developed and validated in a proof-of-principle study to create hybrid nano-bio constructs for rapid, label-free protein detection. Notably, this sensing platform is intended to enable rapid detection of live virus (denoting current viral infection) with a near-infrared signal readout that is transmissible through biological media (detecting virus in unprocessed patient samples such as saliva and nasal fluid). As such, further development of this nanosensor construct is envisioned to yield an accessible, point-of-care testing platform.

In sum, this work develops techniques and analyses to characterize the protein corona and subsequently employs this knowledge toward rational design of nanobiotechnologies. The holistic approach developed herein represents a step toward generalized corona formation rules and understanding that will aid the translation of sensing, imaging, and delivery nanotechnologies into biological application.